13. Virol J. 2013 Apr 10;10(1):112. [Epub ahead of print]

Molecular and epidemiological characterization of avian influenza viruses from
gulls and dabbling ducks in Norway.

Tønnessen R, Kristoffersen AB, Jonassen CM, Hjortaas MJ, Hansen EF, Rimstad E,
Germundsson Hauge A.

BACKGROUND: Wild aquatic birds constitute the natural reservoir for avian
influenza viruses (AIVs). Separate Eurasian and American AIV gene pools exist.
Here, the prevalence and diversity of AIVs in gulls and dabbling ducks in Norway 
were described. The influence of host species and temporal changes on AIV
prevalence was examined. Five AIVs from Norway, including three from common gull 
(Larus canus), were analyzed along with 10 available AIV genomes from gulls in
Eurasia to search for evidence of intracontinental and intercontinental
reassortment of gene segments encoding the internal viral proteins. METHODS:
Swabs collected from 2417 dabbling ducks and gulls in the south-west of Norway
during five ordinary hunting seasons (August-December) in the period 2005--2010
were analyzed for presence of AIV. Multivariate linear regression was used to
identify associations between AIV prevalence, host species and sampling time.
Five AIVs from mallard (Anas platyrhynchos) (H3N8, H9N2) and common gull (H6N8,
H13N2, H16N3) were full-length characterized and phylogenetically analyzed
together with GenBank reference sequences. RESULTS: Low pathogenic AIVs were
detected in 15.5% (CI: 14.1--17.0) of the samples. The overall AIV prevalence was
lower in December compared to that found in August to November (p = 0.003). AIV
was detected in 18.7% (CI: 16.8--20.6) of the dabbling ducks. A high AIV
prevalence of 7.8% (CI; 5.9--10.0) was found in gulls. A similar temporal pattern
in AIV prevalence was found in both bird groups. Thirteen hemagglutinin and eight
neuraminidase subtypes were detected. No evidence of intercontinental
reassortment was found. Eurasian avian (non H13 and H16) PB2 or PA genes were
identified in five reference Eurasian gull (H13 and H16) AIV genomes from
GenBank. The NA gene from the Norwegian H13N2 gull isolate was of Eurasian avian 
origin. CONCLUSIONS: The similar temporal pattern in AIV prevalence found in
dabbling ducks and gulls, the relatively high virus prevalence detected in gulls 
and the evidence of intracontinental reassortment in AIVs from gulls indicate
that gulls that interact with dabbling ducks are likely to be mixing vessels for 
AIVs from waterfowl and gulls. Our results support that intercontinental
reassortment is rare in AIVs from gulls in Eurasia.

PMID: 23575317  [PubMed - as supplied by publisher]
Þ

41. Asian Pac J Trop Biomed. 2012 Nov;2(11):858-62. doi:
10.1016/S2221-1691(12)60243-3.

Genetic analysis of polymerase complex (PA, PB1 and PB2) genes of H9N2 avian
influenza viruses from Iran (1999 to 2009).

Soltanialvar M, Goodarzi R, Akbarnejad F.

Department of Avian Diseases, Faculty of Agriculture, Islamic Azad University,
Shoushtar Branch, Khozestan, Iran.

OBJECTIVE: To determine the molecular characterization of Polymerase complex (PA,
PB1 and PB2) genes of H9N2 avian influenza viruses and the genetic relationship
of Iranian H9N2 viruses and other Asian viruses.
METHODS: The Polymerase complex (PA, PB1 and PB2) genes from seven isolates of
H9N2 viruses isolated from commercial chickens in Iran during 2008-2009 were
amplified (by RT-PCR method) and sequenced. Nucleotide sequences (Open Reading
Frame: orf) of the PA, PB1 and PB2 genes were used for phylogenetic tree
construction.
RESULTS: Most PB2 and PA genes of the H9N2 viruses isolated in 2008-2009 belonged
to the unknown avian sublineage which grouped with the 2004 Pakistani H7N3
viruses. The PB1 genes of Iranian viruses indicated greater genetic diversity and
shared a high level of similarity to PB1 genes from either H5 or H7 subtypes with
compared to established H9N2 Eurasian sublineages.
CONCLUSIONS: Our findings demonstrated that the H9N2 viruses in Iran exhibit
striking reassortment which has led to the generation of new genotypes.

PMCID: PMC3609240
PMID: 23569861  [PubMed]
Þ

44. J Wildl Dis. 2013 Apr;49(2):375-80. doi: 10.7589/2012-03-079.

Exposure to Swine h1 and h3 and avian h5 and h9 influenza a viruses among feral
Swine in southern china, 2009.

Luo J, Dong G, Li K, Lv Z, Huo X, He H.

National Research Center for Wildlife Borne Diseases, Key Laboratory of Animal
Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of
Sciences, Beijing 100101, People's Republic of China.

Swine play an important role in the disease ecology of influenza. Swine may
provide the potential for mixed infections and genetic reassortment between
avian, human, and porcine influenza viruses. We investigated the prevalence of
antibodies to swine H1 and H3 influenza viruses and avian H5 and H9 influenza
viruses in feral swine in southern China. Serum samples were collected from 31
feral swine harvested in 2009 in southern China. Of 31 serum samples tested, 14
(45%) had detectable antibody to H1 influenza virus and 23 (74%) were positive
for H3 subtype. The antibody prevalence against both the swine H1 virus and the
swine H3 virus was 45% (14/31). Five samples were reactive with both H1 and N1
subtype viruses, suggesting exposure to H1N1 viruses. All the sera tested were
negative for avian H5 and H9 influenza viruses. Further investigations of
influenza virus exposure of feral swine are needed to clarify their role in
influenza ecology.

PMID: 23568913  [PubMed - in process]
Þ

519. J Virol. 2013 May;87(9):5118-27. doi: 10.1128/JVI.02490-12. Epub 2013 Feb 28.

Influenza viruses with rearranged genomes as live-attenuated vaccines.

Pena L, Sutton T, Chockalingam A, Kumar S, Angel M, Shao H, Chen H, Li W, Perez
DR.

Department of Veterinary Medicine, University of Maryland, College Park, and
Virginia-Maryland Regional College of Veterinary Medicine, College Park,
Maryland, USA.

H5N1 and H9N2 avian influenza virus subtypes top the World Health Organization's 
list for the greatest pandemic potential. Inactivated H5N1 vaccines induce
limited immune responses and, in the case of live-attenuated influenza virus
vaccines (LAIV), there are safety concerns regarding the possibility of
reassortment between the H5 gene segment and circulating influenza viruses. In
order to overcome these drawbacks, we rearranged the genome of an avian H9N2
influenza virus and expressed the entire H5 hemagglutinin open reading frame
(ORF) from the segment 8 viral RNA. These vectors had reduced polymerase
activities as well as viral replication in vitro and excellent safety profiles in
vivo. Immunization with the dual H9-H5 influenza virus resulted in protection
against lethal H5N1 challenge in mice and ferrets, and also against a potentially
pandemic H9 virus. Our studies demonstrate that rearranging the influenza virus
genome has great potential for the development of improved vaccines against
influenza virus as well as other pathogens.

PMID: 23449800  [PubMed - in process]
Þ

580. PLoS One. 2013;8(2):e49597. doi: 10.1371/journal.pone.0049597. Epub 2013 Feb 20.

Quantitative description of glycan-receptor binding of influenza a virus h7
hemagglutinin.

Srinivasan K, Raman R, Jayaraman A, Viswanathan K, Sasisekharan R.

Harvard-MIT Division of Health Sciences and Technology, Koch Institute for
Integrative Cancer Research, Singapore-MIT Alliance for Research and Technology, 
Department of Biological Engineering, Massachusetts Institute of Technology
(MIT), Cambridge, Massachusetts, USA.

In the context of recently emerged novel influenza strains through reassortment, 
avian influenza subtypes such as H5N1, H7N7, H7N2, H7N3 and H9N2 pose a constant 
threat in terms of their adaptation to the human host. Among these subtypes, it
was recently demonstrated that mutations in H5 and H9 hemagglutinin (HA) in the
context of lab-generated reassorted viruses conferred aerosol transmissibility in
ferrets (a property shared by human adapted viruses). We previously demonstrated 
that the quantitative binding affinity of HA to α2→6 sialylated glycans (human
receptors) is one of the important factors governing human adaptation of HA.
Although the H7 subtype has infected humans causing varied clinical outcomes from
mild conjunctivitis to severe respiratory illnesses, it is not clear where the HA
of these subtypes stand in regard to human adaptation since its binding affinity 
to glycan receptors has not yet been quantified. In this study, we have
quantitatively characterized the glycan receptor-binding specificity of HAs from 
representative strains of Eurasian (H7N7) and North American (H7N2) lineages that
have caused human infection. Furthermore, we have demonstrated for the first time
that two specific mutations; Gln226→Leu and Gly228→Ser in glycan receptor-binding
site of H7 HA substantially increase its binding affinity to human receptor. Our 
findings contribute to a framework for monitoring the evolution of H7 HA to be
able to adapt to human host.

PMCID: PMC3577880
PMID: 23437033  [PubMed - in process]
Þ

1405. Virus Genes. 2013 Apr;46(2):323-9. doi: 10.1007/s11262-012-0868-9. Epub 2012 Dec 
21.

Surveillance and characterization of avian influenza viruses from migratory water
birds in eastern Hokkaido, the northern part of Japan, 2009-2010.

Abao LN, Jamsransuren D, Bui VN, Ngo LH, Trinh DQ, Yamaguchi E, Vijaykrishna D,
Runstadler J, Ogawa H, Imai K.

Research Center for Animal Hygiene and Food Safety, Obihiro University of
Agriculture and Veterinary Medicine, 2-11 Inada, Obihiro, Hokkaido, 080-8555,
Japan.

Avian influenza virus (AIV) surveillance was conducted around a small pond in
Obihiro, eastern Hokkaido, Japan. Eleven AIVs were isolated from a total of 1,269
fecal samples of migratory wild birds collected during 2009 and 2010. The sample 
number covered approximately 60 % of the total number of birds observed during
sampling periods. The subtypes of the isolates included H3N8 (4 isolates), H5N2
(3), H6N2 (2), H6N1 (1), and H11N2 (1). The H3N8 subtype was most prevalent as in
the previous studies performed in Hokkaido. The three H5N2 isolates genetically
characterized as low pathogenic AIV were closely related to the strains
previously isolated from aquatic wild birds in Japan and also to the Korean
strains isolated from aquatic birds in recent years. In Korea, H5N2 subtype virus
has often been isolated from poultry and wild birds, as well as reassortant
viruses generated from duck H5N2 viruses and chicken H9N2 virus, and
avian-swine-like reassortant H5N2 viruses. Considering the previous chicken
outbreaks caused by highly pathogenic H5N2 viruses, which affected many
countries, it should be an important priority to continue, monitoring the
evolution of H5N2 viruses circulating in the region.

PMID: 23264106  [PubMed - in process]
Þ

1877. J Virol. 2012 Dec;86(24):13836-7. doi: 10.1128/JVI.02595-12.

Genetic characterization of a novel recombinant H5N2 avian influenza virus
isolated from chickens in Tibet.

Zhao S, Suo L, Jin M.

Unit of Animal Infectious Disease, National State Key Laboratory of Agricultural 
Microbiology, Huazhong Agricultural University, Wuhan, People's Republic of
China.

In this report, a novel H5N2 avian influenza virus (AIV) was isolated from
chickens in Tibet in 2010, western China. Phylogenetic analysis demonstrated that
it was a natural reassortant between H9N2 and H5N1 subtypes. It is of note that
this virus has an HP genotype with HA, PB2, M, and NS genes homologous to those
of A/peregrine falcon/Hong Kong/2142/2008(H5N1)-like HPAIV isolated from dead
wild birds. Publishing this genome information will contribute to the
investigation of avian influenza epidemiology and to further research of AIV's
biological properties.

PMCID: PMC3503117 [Available on 2013/6/1]
PMID: 23166246  [PubMed - indexed for MEDLINE]
Þ

2239. J Virol. 2012 Nov;86(22):12443. doi: 10.1128/JVI.02236-12.

Complete genome sequence of a novel reassortant H11N2 avian influenza virus
isolated from a live poultry market in eastern China.

Zhang Y, Teng Q, Ren C, Li G, Li X, Li Z.

Department of Avian Infectious Disease, Shanghai Veterinary Research Institute,
Chinese Academy of Agricultural Sciences, Shanghai, China.

A/chicken/Nanjing/908/2009(H11N2) (CK908) was isolated from a live poultry market
in Nanjing, China. Using PCR and sequencing analysis, we obtained the complete
genome sequences of the CK908 virus. The sequence analysis demonstrated that this
H11N2 virus was a novel reassortant AIV whose PB1, PB2, PA, HA, NP, NA, M, and NS
genes originated from H9N2, H7N7, H5N2, H11N8, H3N6, H6N2, H1N1, and H5N1,
respectively. Knowledge regarding the complete genome sequences of the CK908
virus will be useful for epidemiological surveillance.

PMCID: PMC3486450 [Available on 2013/5/1]
PMID: 23087108  [PubMed - indexed for MEDLINE]
Þ

2414. PLoS One. 2012;7(9):e46183. doi: 10.1371/journal.pone.0046183. Epub 2012 Sep 25.

Novel reassortant highly pathogenic H5N2 avian influenza viruses in poultry in
China.

Zhao G, Gu X, Lu X, Pan J, Duan Z, Zhao K, Gu M, Liu Q, He L, Chen J, Ge S, Wang 
Y, Chen S, Wang X, Peng D, Wan H, Liu X.

College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China.

There has been multiple evidence that domestic poultry may act as a vessel for
the generation of novel influenza A viruses. In this study, we have analyzed the 
evolution and pathogenicity of 4 H5N2 avian influenza viruses isolated from
apparently healthy poultry from H5N1 virus endemic areas in China. Phylogenetic
analysis revealed that two of these viruses, A/duck/Eastern China/1111/2011
(DK/EC/1111/11) and A/goose/Eastern China/1112/2011 (GS/EC/1112/11) were derived 
from reassortment events in which clade 2.3.4 highly pathogenic avian influenza
(HPAI) H5N1 viruses acquired novel neuraminidase and nonstructural protein genes.
Another two isolates, A/chicken/Hebei/1102/2010 (CK/HB/1102/10) and
A/duck/Hebei/0908/2009 (DK/HB/0908/09), possess hemagglutinin (HA) gene belong to
clade 7 H5 viruses and other genes from endemic H9N2 viruses, or from viruses of 
various subtypes of the natural gene pool. All of these H5N2 isolates bear
characteristic sequences of HPAI virus at the cleavage site of HA, and animal
experiments indicated that all of these viruses but DK/HB/0908/09 is highly
pathogenic to chickens. In particular, DK/EC/1111/11 and GS/EC/1112/11 are also
highly pathogenic to ducks and moderately pathogenic to mice. All of these 4
viruses were able to replicate in domestic ducks and mice without prior
adaptation. The emergence of these novel H5N2 viruses adds more evidence for the 
active evolution of H5 viruses in Asia. The maintenance of the highly pathogenic 
phenotype of some of these viruses even after reassortment with a new NA
subtypes, their ability to replicate and transmit in domestic poultry, and the
pathogenicity in the mammalian mouse model, highlight the potential threat posed 
by these viruses to both veterinary and public health.

PMCID: PMC3458027
PMID: 23049973  [PubMed - indexed for MEDLINE]
Þ

2453. J Virol. 2012 Nov;86(21):11944. doi: 10.1128/JVI.02082-12.

Complete genome sequence of an H3N2 avian influenza virus isolated from a live
poultry market in eastern China.

Teng Q, Hu T, Li X, Li G, Li Z.

Department of Avian Infectious Disease, Shanghai Veterinary Research Institute,
Chinese Academy of Agricultural Sciences, Shanghai, China.

Previous studies indicate that the H3 influenza virus has the ability to
establish infection upon interspecies transmission and poses a threat to mammals.
Therefore, it is important to enhance the surveillance of H3 avian influenza
viruses (AIVs). In this study, A/duck/Shanghai/C84/2009(H3N2) (C84) was isolated 
from a live poultry market in Shanghai, China. Using PCR and sequencing analyses,
we obtained the whole-genome sequence of this virus. The H3N2 virus proved to be 
a novel multiple-gene reassortant AIV whose genes were derived from H3N2, H4N6,
H6N2, and H9N2. Knowledge regarding the complete genome sequence of the C84 virus
will be useful for epidemiological surveillance.

PMCID: PMC3486323 [Available on 2013/5/1]
PMID: 23043175  [PubMed - indexed for MEDLINE]
Þ

2454. J Virol. 2012 Nov;86(21):11939. doi: 10.1128/JVI.02042-12.

Complete genome sequence of an H9N2 avian influenza virus isolated from egret in 
Lake Dongting wetland.

Wang B, Chen Q, Chen Z.

State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of
Sciences, Wuhan, Hubei, China.

We isolated a recombinant H9N2 avian influenza virus (AIV) from fresh egret feces
in the Ardeidae protection region of the Dongting Lake wetland area in China, and
it was designated A/Egret/Hunan/1/2012(H9N2). This is the first report of
isolating H9N2 AIV from wild birds in the Dongting Lake wetland. Its eight gene
segments are generated by reassortment of gene segments of different AIV
subtypes. These results are helpful for understanding the epidemiology and
evolution of AIV in wild birds during migration.

PMCID: PMC3486334 [Available on 2013/5/1]
PMID: 23043171  [PubMed - indexed for MEDLINE]
Þ

2798. J Gen Virol. 2012 Nov;93(Pt 11):2326-36. doi: 10.1099/vir.0.044503-0. Epub 2012
Sep 12.

Estimating reassortment rates in co-circulating Eurasian swine influenza viruses.

Lycett SJ, Baillie G, Coulter E, Bhatt S, Kellam P, McCauley JW, Wood JL, Brown
IH, Pybus OG, Leigh Brown AJ; Combating Swine Influenza Initiative-COSI
Consortium.

Institute of Evolutionary Biology, University of Edinburgh, Kings Buildings, West
Mains Road, Edinburgh EH9 3JT, UK. samantha.lycett@ed.ac.uk

Swine have often been considered as a mixing vessel for different influenza
strains. In order to assess their role in more detail, we undertook a
retrospective sequencing study to detect and characterize the reassortants
present in European swine and to estimate the rate of reassortment between H1N1, 
H1N2 and H3N2 subtypes with Eurasian (avian-like) internal protein-coding
segments. We analysed 69 newly obtained whole genome sequences of subtypes
H1N1-H3N2 from swine influenza viruses sampled between 1982 and 2008, using
Illumina and 454 platforms. Analyses of these genomes, together with previously
published genomes, revealed a large monophyletic clade of Eurasian swine-lineage 
polymerase segments containing H1N1, H1N2 and H3N2 subtypes. We subsequently
examined reassortments between the haemagglutinin and neuraminidase segments and 
estimated the reassortment rates between lineages using a recently developed
evolutionary analysis method. High rates of reassortment between H1N2 and H1N1
Eurasian swine lineages were detected in European strains, with an average of one
reassortment every 2-3 years. This rapid reassortment results from co-circulating
lineages in swine, and in consequence we should expect further reassortments
between currently circulating swine strains and the recent swine-origin H1N1v
pandemic strain.

PMID: 22971819  [PubMed - indexed for MEDLINE]
Þ

3041. Virol J. 2012 Aug 27;9:173. doi: 10.1186/1743-422X-9-173.

Isolation and mutation trend analysis of influenza A virus subtype H9N2 in Egypt.

Abdel-Moneim AS, Afifi MA, El-Kady MF.

Department of Virology, Faculty of Veterinary Medicine, Beni-Suef University,
Beni-Suef 62511, Egypt. asa@bsu.edu.eg

BACKGROUND: Avian influenza virus H9N2 is a panzootic pathogen that affects
poultry causing mild to moderate respiratory distress but has been associated
with high morbidity and considerable mortality. Interspecies transmission of H9N2
from avian species to mammalian hosts does occur. The virus possesses human
virus-like receptor specificity and it can infect humans producing flu-like
illness.
METHODS: Recently, mild influenza like symptoms were detected in H5N1 vaccinated 
flocks. Influenza A subtype H9N2 was isolated from the infected flock. The virus 
evolution was investigated by sequencing the viral genes to screen the possible
virus recombination. The viral amino acid sequences from the isolated H9N2
strains were compared to other related sequences from the flu data base that were
used to assess the robustness of the mutation trend. Changes in the
species-associated amino acid residues or those that enabled virulence to mammals
were allocated.
RESULTS: Phylogenetic analyses of haemagglutinin and neuraminidase genes showed
that the recently isolated Egyptian strain belonged to the H9N2 sub-lineage that 
prevails in Israel. The six internal segments of the isolated virus were found to
be derived from the same sub-lineage with no new evidence of reassortment. The
results demonstrated conserved genetic and biological constitution of H9N2
viruses in the Middle East. The recently isolated H9N2 virus from chicken in
Egypt possessed amino acids that could enable the virus to replicate in mammals
and caused severe disease in domestic chickens.
CONCLUSION: The study highlights the importance of continuous monitoring of the
mutations evolved in avian influenza viruses and its impact on virulence to avian
species in addition to its importance in the emergence of new strains with the
capacity to be a pandemic candidate.

PMCID: PMC3492205
PMID: 22925485  [PubMed - in process]
Þ

3051. J Virol. 2012 Sep;86(18):10240-1. doi: 10.1128/JVI.01623-12.

Complete genome sequence of a novel H9N2 subtype influenza virus FJG9 strain in
China reveals a natural reassortant event.

Xie Q, Yan Z, Ji J, Zhang H, Liu J, Sun Y, Li G, Chen F, Xue C, Ma J, Bee Y.

College of Animal Science, South China Agricultural University, Tianhe District, 
Wushan Road, Guangzhou, Guangdong, People's Republic of China.

A/chicken/FJ/G9/09 (FJ/G9) is an H9N2 subtype avian influenza virus (H9N2 AIV)
strain causing high morbidity that was isolated from broilers in Fujian Province 
of China in 2009. FJ/G9 has been used as the vaccine strain against H9N2 AIV
infection in Fujian Province of China. Here, we report the complete genome
sequence of FJ/G9 with natural six-way reassortment, which is the most complex
genotype strain in China and even in the world so far. The present findings will 
aid in understanding the complexity and diversity of H9N2 subtype avian influenza
virus.

PMCID: PMC3446608
PMID: 22923800  [PubMed - indexed for MEDLINE]
Þ

3277. J Gen Virol. 2012 Nov;93(Pt 11):2337-45. doi: 10.1099/vir.0.044040-0. Epub 2012
Aug 8.

Pathogenicity and transmissibility of reassortant H9 influenza viruses with genes
from pandemic H1N1 virus.

Qiao C, Liu Q, Bawa B, Shen H, Qi W, Chen Y, Mok CK, García-Sastre A, Richt JA,
Ma W.

Department of Diagnostic Medicine/Pathobiology, Kansas State University,
Manhattan, KS 66506, USA.

Both H9N2 avian influenza and 2009 pandemic H1N1 viruses (pH1N1) are able to
infect humans and swine, which has raised concerns that novel reassortant H9
viruses with pH1N1 genes might be generated in these hosts by reassortment.
Although previous studies have demonstrated that reassortant H9 viruses with
pH1N1 genes show increased virulence in mice and transmissibility in ferrets, the
virulence and transmissibility of reassortant H9 viruses in natural hosts such as
chickens and swine remain unknown. This study generated two reassortant H9
viruses (H9N2/CA09 and H9N1/CA09) in the background of the pH1N1
A/California/04/2009 (CA09) virus by replacing either both the haemagglutinin
(HA) and neuraminidase (NA) genes or only the HA gene with the respective genes
from the A/quail/Hong Kong/G1/1997 (H9N2) virus and evaluated their replication, 
pathogenicity and transmission in chickens and pigs compared with the parental
viruses. Chickens that were infected with the parental H9N2 and reassortant H9
viruses seroconverted. The parental H9N2 and reassortant H9N2/CA09 viruses were
transmitted to sentinel chickens, but H9N1/CA09 virus was not. The parental H9N2 
replicated poorly and was not transmitted in pigs, whereas both H9N2/CA09 and
H9N1/CA09 viruses replicated and were transmitted efficiently in pigs, similar to
the pH1N1 virus. These results demonstrated that reassortant H9 viruses with
pH1N1 genes show enhanced replication and transmissibility in pigs compared with 
the parental H9N2 virus, indicating that they may pose a threat for humans if
such reassortants arise in swine.

PMID: 22875253  [PubMed - indexed for MEDLINE]
Þ

3780. Virus Genes. 2012 Oct;45(2):283-94. Epub 2012 Jun 30.

Complete genome characterization of avian influenza virus subtype H9N2 from a
commercial quail flock in Egypt.

Arafa AS, Hagag N, Erfan A, Mady W, El-Husseiny M, Adel A, Nasef S.

National Laboratory for Veterinary Quality Control on Poultry Production, Animal 
Health Research Institute, P.O. Box 264, Dokki, Giza 12618, Egypt.
abd.arafa@gmail.com

The suspected presence of avian influenza virus subtype H9N2 in poultry in Egypt 
is a major concern since this subtype is widely distributed in different
countries in the Middle East, here we describe the full genetic characterization 
of an avian influenza A virus (Qa/Egypt/11; H9N2) of subtype H9N2 that was
previously isolated from a clinically normal quail flock in Giza, Egypt in May
2011. The nucleotide sequence analysis of the hemagglutinin gene of the isolated 
Egyptian virus showed the highest similarity with one group of recent Israeli
strains (97 %) circulating from 2006-2010. Sequence homology and phylogenetic
analysis indicated that the Qa/Egypt/11 isolate belonged to the A/quail/Hong
Kong/G1/1997-like lineage with new mutations identified in all viral proteins.
The phylogenetic analysis for the eight genes indicated placement of the Egyptian
virus within the same lineage of H9N2 viruses that circulated in the region from 
2006, especially with one group of recent Israeli strains. However, phylogenetic 
analysis of the internal genes like PB2, NP, and PA genes identified possible
reassortment events for these genes with singular Israeli strains. This study
indicates progressive evolution of this subtype in the Middle East region and
possible mechanism of virus adaptation in land-based poultry like in quails.

PMID: 22752536  [PubMed - indexed for MEDLINE]
Þ

3802. Influenza Other Respi Viruses. 2012 Jul 2. doi: 10.1111/j.1750-2659.2012.00399.x.
[Epub ahead of print]

H9N2 influenza A virus circulates in H5N1 endemically infected poultry population
in Egypt.

Monne I, Hussein HA, Fusaro A, Valastro V, Hamoud MM, Khalefa RA, Dardir SN,
Radwan MI, Capua I, Cattoli G.

Research & Development Department, Istituto Zooprofilattico Sperimentale delle
Venezie, OIE/FAO and National Reference Laboratory for Newcastle Disease and
Avian Influenza, OIE Collaborating Centre for Diseases at the Human-Animal
Interface, Legnaro, Padova, Italy. Virology Department, Faculty of Veterinary
Medicine, Cairo University, Giza, Egypt. Poultry and Rabbit Diseases Department, 
Faculty of Veterinary Medicine, Cairo University, Giza, Egypt. Private Poultry
Consultant, Giza, Egypt. National Lab for Veterinary Quality Control on Poultry
Production, Animal Heath Research Institute, Agriculture Research Center, Giza,
Egypt.

Please cite this paper as: Monne et al. (2012) H9N2 influenza A virus circulates 
in H5N1 endemically infected poultry population in Egypt. Influenza and Other
Respiratory Viruses DOI: 10.1111/j.1750-2659.2012.00399.x. We describe the
identification and characterization of the H9N2 influenza subtype reported in
Egyptian broiler and broiler breeder farms for the first time. Circulation of
this subtype in a highly pathogenic H5N1 influenza virus endemic population
provides an opportunity for genetic reassortment and emergence of novel viruses.

© 2012 Blackwell Publishing Ltd.

PMID: 22747716  [PubMed - as supplied by publisher]
Þ

3817. Virology. 2012 Oct 10;432(1):91-8. doi: 10.1016/j.virol.2012.06.002. Epub 2012
Jun 27.

Surface glycoproteins of influenza A H3N2 virus modulate virus replication in the
respiratory tract of ferrets.

Cheng X, Zengel JR, Xu Q, Jin H.

MedImmune, Mountain View, CA 94043, USA.

The hemagglutinin (HA) genes of the influenza A H3N2 subtype viruses isolated
from 1968 to 2010 have evolved substantially but their neuraminidase (NA) genes
have been relatively less divergent. The H3N2 viruses isolated since 1995 were
found to replicate in the lower respiratory tract of ferrets less efficiently
than the earlier isolates. To evaluate whether the HA or/and NA or the internal
protein gene segments of the H3N2 virus affected viral replication in the
respiratory tract of ferrets, recombinant A/California/07/2004 (CA04) (H3N2)
virus and its reassortants that contained the same CA04 internal protein gene
segments and the HA and/or NA of A/Udorn/309/1972 (UD72) or A/Wuhan/359/1995
(WH95) H3N2 viruses were generated and evaluated for their replication in the
respiratory tract of ferrets. All the reassortant viruses replicated efficiently 
in the upper respiratory tract of ferrets, but their replication in the lower
respiratory tract of ferrets varied. In contrast to the UD72-HA reassortant virus
that replicated efficiently in the lungs of ferrets, the virus with the WH95-HA
or the CA04-HA either replicated modestly or did not replicate in the lungs of
ferrets. The reassortants with the WH95-HA and UD72-NA or CA04-NA had the
tendency to lose a N-linked glycosylation site at residue 246 in the HA,
resulting in viral lung titer of 100-fold higher than the virus with the HA and
NA from WH95. The UD72-NA had the highest neuraminidase activity and increased
viral replication by up to 100-fold in tissue culture cells during early
infection. Thus, our data indicate that both the HA and NA glycoproteins play
important roles in viral replication of the H3N2 influenza virus in ferrets.

Copyright © 2012 Elsevier Inc. All rights reserved.

PMID: 22743127  [PubMed - indexed for MEDLINE]
Þ

4547. Bioorg Med Chem Lett. 2012 Jun 1;22(11):3688-92. doi: 10.1016/j.bmcl.2012.04.028.
Epub 2012 Apr 12.

Xanthones from Polygala karensium inhibit neuraminidases from influenza A
viruses.

Dao TT, Dang TT, Nguyen PH, Kim E, Thuong PT, Oh WK.

BK21 Project Team, College of Pharmacy, Chosun University, Dong-gu, Gwangju,
Republic of Korea.

The emergence of the H1N1 swine flu pandemic has the possibility to develop the
occurrence of disaster- or drug-resistant viruses by additional reassortments in 
novel influenza A virus. In the course of an anti-influenza screening program for
natural products, 10 xanthone derivatives (1-10) were isolated by bioassay-guided
fractionation from the EtOAc-soluble extract of Polygala karensium. Compounds 1, 
3, 5, 7, and 9 with a hydroxy group at C-1 showed strong inhibitory effects on
neuraminidases from various influenza viral strains, H1N1, H9N2, novel H1N1 (WT),
and oseltamivir-resistant novel H1N1 (H274Y) expressed in 293T cells. In
addition, these compounds reduced the cytopathic effect of H1N1 swine influenza
virus in MDCK cells. Our results suggest that xanthones from P. karensium may be 
useful in the prevention and treatment of disease by influenza viruses.

Copyright © 2012 Elsevier Ltd. All rights reserved.

PMID: 22552195  [PubMed - indexed for MEDLINE]
Þ

4575. Curr Genomics. 2011 Nov;12(7):466-74. doi: 10.2174/138920211797904052.

Prospective of Genomics in Revealing Transmission, Reassortment and Evolution of 
Wildlife-Borne Avian Influenza A (H5N1) Viruses.

Lei F, Shi W.

Key Laboratory of the Zoological Systematics and Evolution, Institute of Zoology,
Chinese Academy of Sciences, Beijing 100101, China.

The outbreak of highly pathogenic avian influenza (HPAI) H5N1 disease has led to 
significant loss of poultry and wild life and case fatality rates in humans of
60%. Wild birds are natural hosts for all avian influenza virus subtypes and
over120 bird species have been reported with evidence of H5N1 infection.
Influenza A viruses possess a segmented RNA genome and are characterized by
frequently occurring genetic reassortment events, which play a very important
role in virus evolution and the spread of novel gene constellations in
immunologically naïve human and animal populations. Phylogenetic analysis of
whole genome or sub-genomic sequences is a standard means for delineating genetic
variation, novel reassortment events, and surveillance to trace the global
transmission pathways. In this paper, special emphasis is given to the
transmission and circulation of H5N1 among wild life populations, and to the
reassortment events that are associated with inter-host transmission of the H5N1 
viruses when they infect different hosts, such as birds, pigs and humans. In
addition, we review the inter-subtype reassortment of the viral segments encoding
inner proteins between the H5N1 viruses and viruses of other subtypes, such as
H9N2 and H6N1. Finally, we highlight the usefulness of genomic sequences in
molecular epidemiological analysis of HPAI H5N1 and the technical limitations in 
existing analytical methods that hinder them from playing a greater role in
virological research.

PMCID: PMC3219842
PMID: 22547954  [PubMed]
Þ

5055. Virus Genes. 2012 Aug;45(1):38-47. doi: 10.1007/s11262-012-0737-6. Epub 2012 Mar 
28.

Genetic evolution of the H9N2 avian influenza virus in Korean poultry farms.

Lee CH, Byun SH, Lee YJ, Mo IP.

Avian Disease Laboratory, College of Veterinary Medicine, Chungbuk National
University, Cheongju 361-763, Korea.

We performed whole genome sequencing of 22 H9N2 avian influenza viruses (AIV)
isolated from domestic laying hens on farms between 2005 and 2008, and compared
the sequences with viruses previously reported in Asia. A previous study revealed
that two antigenically distinct sublineages were established within the MS96
lineage by antigenic drift since the first H9N2 AIV outbreak in South Korea. We
designated them as the 01310-like lineage and the 116/04-like lineage. Since late
2004, most identified isolates in Korea have belonged to the 116/04-like lineage,
however, in this study we found that six among twenty-two isolates were belonged 
to 01310-like lineage, indicating that the genetic divergence is still occurring 
after 2004. Furthermore, it is noteworthy that five isolates among the defined
01310-like lineage had a 24 amino acid deletion in the neuraminidase stalk
region, which were not in any other H9N2 isolates previously reported. The
internal genes analysis demonstrated extensive reassortment events among isolates
from poultry farms, live bird markets, and wild birds, and multiple new genotypes
were identified. We identified several features of gene evolution in H9N2 AIV
suggesting that the long-term H9N2 AIV surveillance study should be continued in 
South Korea.

PMID: 22453860  [PubMed - indexed for MEDLINE]
Þ

5233. Bing Du Xue Bao. 2012 Jan;28(1):7-14.

[Genome sequencing and phylogenetic analysis of avian influenza viruses subtype
H9N2].

[Article in Chinese]

Li SC, Li XH, Zhong SG, Sun HL, Pan JJ, Chen SJ, Peng DX, Liu XF.

Key Laboratory of Animal Infectious Disease, College of Veterinary Medicine,
Yangzhou University, Yangzhou 225009, China. lishuchun111@yahoo.com.cn

Samples of chicken, duck, quail, and pigeon were collected from Jiangsu, Anhui,
and Hebei in 2009-2011, and sixteen H9N2 subtype isolates of avian influenza
virus (AIV) were identified. The eight full-length genes of 16 AIV isolates were 
amplified by RT-PCR and sequenced. Genome sequence analysis showed that the amino
acid motif of cleavage sites in the HA gene was P-S-R/K-S-S-R, which was
consistent with the characterization of the LPAIV, and the Leucine (L) at the
amino acid position 226 in the HA genes of all isolates indicated the potential
of binding with SAalpha, 2-6 receptor. All isolates had a S to N substitution at 
residue 31 in the M2 gene, which is related to the resistance phenotype of
adamantanes. The key molecular features of 16 AIV isolates from different hosts
were same. Genome phylogenetic analysis revealed that all 16 H9N2 subtype AIVs
originated from F98-like virus as backbone and formed two new genotypes through
reassortment with HA gene of Y280-like virus and PB2 and M genes of G1-like
virus. Our findings suggest that more attention should be paid to the
surveillance of H9N2 influenza virus and its direction of reassortment.

PMID: 22416344  [PubMed - indexed for MEDLINE]
Þ

5270. Arch Virol. 2012 Jun;157(6):1193-8. doi: 10.1007/s00705-012-1266-2. Epub 2012 Mar
10.

Epidemiological survey and genetic evolution of H9 subtype influenza viruses in
Shanghai, China, from 2006 to 2010.

Zhou JP, Ge FF, Liu J, Ju HB, Yang DQ, Wang J, Zhang WY, Liu PH.

Shanghai Animal Disease Control Center, People's Republic of China.

The H9N2 influenza virus is endemic in poultry. We report its occurrence in
live-poultry markets, fair-trade markets and poultry farms in the Shanghai region
between September 2006 and December 2010. An analysis of partial sequences of the
HA, NA, PB1, PB2 and NP genes of eleven distinct H9N2 isolates revealed that all 
carried an RSSR motif at the cleavage site of HA, diagnostic of low pathogenicity
in chickens. A phylogenetic analysis indicated that these isolates are derived
from the lineage represented by Duck/HK/Y280/97, but they have evolved a range of
reassortments. Their PB1 and NP sequences resembled those of H5N1 strains,
indicating a hybrid origin involving both H9 and H5 strains. The HA and NA
sequences present in all eleven isolates resembled those of the
Duck/HK/Y280/97-like lineage. Infection by H9N2 is commonplace in Shanghai
live-poultry markets, allowing the viruses to have evolved rapidly.

PMID: 22407404  [PubMed - indexed for MEDLINE]
Þ

5376. Infect Genet Evol. 2012 Jul;12(5):933-46. doi: 10.1016/j.meegid.2012.02.001. Epub
2012 Feb 20.

Generation of reassortant influenza viruses within the non-industrial poultry
system.

Lee HJ, Lee DH, Lee YN, Kwon JS, Lee YJ, Lee JB, Park SY, Choi IS, Song CS.

College of Veterinary Medicine, Konkuk University, 1 Hwayang-dong, Gwangjin-gu,
Seoul 143-701, Republic of Korea.

We compared the genetic and biologic characteristics of 35 influenza viruses of
different epidemiological backgrounds in Korea, including H3N2 canine influenza
virus (CIV). Phylogenetic analysis revealed that chicken adapted H9N2 viruses
(A/chicken/Korea/96006/96 [CK/Kor/96006-like]) have acquired aquatic avian gene
segments through reassortment, and these reassorted H9N2 viruses were more
frequently detected from minor poultry species than from industrial poultry.
Conversely, gene segments from CK/Kor/96006-like viruses were also detected in
most of the viruses from domestic ducks. Interestingly, domestic ducks, rather
than wild aquatic birds, harbored close relatives of all eight gene segments of
H3N2 CIV, which preferred binding to avian receptors. Therefore, bidirectional
virus transmission events are assumed to have occurred between land-based poultry
and aquatic poultry, in particular within the non-industrial poultry system.
These events have contributed to the generation of a novel reassortant, H3N2 CIV.
To prevent generating other reassortants capable of interspecies transmission,
gene movements in the non-industrial poultry systems should be clarified and
managed.

Copyright © 2012 Elsevier B.V. All rights reserved.

PMID: 22386854  [PubMed - indexed for MEDLINE]
Þ

5790. Avian Dis. 2011 Dec;55(4):724-7.

Isolation and characterization of a novel H9N2 influenza virus in Korean native
chicken farm.

Lee YN, Lee DH, Park JK, Lim TH, Youn HN, Yuk SS, Lee YJ, Mo IP, Sung HW, Lee JB,
Park SY, Choi IS, Song CS.

Avian Disease Laboratory, College of Veterinary Medicine, Konkuk University,
Seoul, 143-701, Korea

An outbreak of avian influenza, caused by an H9N2 low-pathogenic avian influenza 
virus (AIV), occurred in a chicken farm and caused severe economic losses due to 
mortality and diarrhea. AIV was isolated and identified in a sample from an
affected native Korean chicken. Genetic analysis of the isolate revealed a high
sequence similarity to genes of novel reassortant H9N2 viruses isolated from
slaughterhouses and live bird markets in Korea in 2008 and 2009. Animal challenge
studies demonstrated that the replication kinetics and pathogenicity of the
isolate were considerably altered due to adaptation in chickens. Vaccine
protection studies indicated that commercial vaccine was not able to prevent
virus shedding and clinical disease when chickens were challenged with the
isolate. These results suggest that the novel H9N2 virus possesses the capacity
to replicate efficiently in the respiratory system against vaccination and to
cause severe disease in domestic chickens. The results also highlight the
importance of appropriate updating of vaccine strains, based on continuous
surveillance data, to prevent the possibility of a new H9N2 epidemic in Korea.

PMID: 22313001  [PubMed - indexed for MEDLINE]
Þ

5844. Proc Natl Acad Sci U S A. 2012 Feb 14;109(7):2579-84. doi:
10.1073/pnas.1109397109. Epub 2012 Jan 30.

Avian influenza virus hemagglutinins H2, H4, H8, and H14 support a highly
pathogenic phenotype.

Veits J, Weber S, Stech O, Breithaupt A, Gräber M, Gohrbandt S, Bogs J, Hundt J, 
Teifke JP, Mettenleiter TC, Stech J.

Institute of Molecular Biology, Friedrich-Loeffler-Institut, Federal Research
Institute for Animal Health, 17493 Greifswald-Insel Riems, Germany.

High-pathogenic avian influenza viruses (HPAIVs) evolve from low-pathogenic
precursors specifying the HA serotypes H5 or H7 by acquisition of a polybasic HA 
cleavage site. As the reason for this serotype restriction has remained unclear, 
we aimed to distinguish between compatibility of a polybasic cleavage site with
H5/H7 HA only and unique predisposition of these two serotypes for insertion
mutations. To this end, we introduced a polybasic cleavage site into the HA of
several low-pathogenic avian strains with serotypes H1, H2, H3, H4, H6, H8, H10, 
H11, H14, or H15, and rescued HA reassortants after cotransfection with the genes
from either a low-pathogenic H9N2 or high-pathogenic H5N1 strain. Oculonasal
inoculation with those reassortants resulted in varying pathogenicity in chicken.
Recombinants containing the engineered H2, H4, H8, or H14 in the HPAIV background
were lethal and exhibited i.v. pathogenicity indices of 2.79, 2.37, 2.85, and
2.61, respectively, equivalent to naturally occurring H5 or H7 HPAIV. Moreover,
the H2, H4, and H8 reassortants were transmitted to some contact chickens. The H2
reassortant gained two mutations in the M2 proton channel gate region, which is
affected in some HPAIVs of various origins. Taken together, in the presence of a 
polybasic HA cleavage site, non-H5/H7 HA can support a highly pathogenic
phenotype in the appropriate viral background, indicating requirement for further
adaptation. Therefore, the restriction of natural HPAIV to serotypes H5 and H7 is
likely a result of their unique predisposition for acquisition of a polybasic HA 
cleavage site.

PMCID: PMC3289369
PMID: 22308331  [PubMed - indexed for MEDLINE]
Þ

6916. Biomed Environ Sci. 2011 Oct;24(5):530-6. doi: 10.3967/0895-3988.2011.05.012.

Pathogenesis and immunogenicity of an avian H9N2 influenza virus isolated from
human.

Liu L, Zi L, Zhou J, Zhu Y, Dong J, Zhao X, Guo J, Shu Y.

State Key Laboratory for Molecular Virology and Genetic Engineering, Chinese
National Influenza Center, National Institute for Viral Disease Control and
Prevention, China CDC, Beijing 102206, China.

OBJECTIVE: To investigate the pathogenesis and immunogenicity of H9N2 influenza
virus A/Guangzhou/333/99 (a reassortant of G1 and G9 viruses isolated from a
female patient in 1999) in a mouse model of infection.
METHODS: Mice were infected with increasing virus titers. Viral load in the lungs
and trachea was determined by EID50 assay. Pulmonary histopathology was assessed 
by hematoxylin-eosin staining. Anti-HI antibody titers and T-cell responses to
viral HA were determined by ELISPOT and confirmed by flow cytometry.
RESULTS: Mice presented a mild syndrome after intranasal infection with
A/Guangzhou/333/99 (H9N2) influenza virus. Virus was detected in the trachea and 
lungs of mice harvested on days 3, 6, and 9 post-infection. A T-cell response to 
viral HA was detected on day 6 and H9 HA-specific CD(4+) T-cells predominated.
Seroconversion was detected after 14 days and antibody persisted for at least 28 
weeks.
CONCLUSION: Our results suggest that H9N2 (A/Guangzhou/333/99) can replicate in
the murine respiratory tract without prior adaptation, and both humoral and
cell-mediated immunity play an important role in the immune response.

Copyright © 2011 The Editorial Board of Biomedical and Environmental Sciences.
Published by Elsevier B.V. All rights reserved.

PMID: 22108420  [PubMed - indexed for MEDLINE]
Þ

7122. Arch Virol. 2012 Feb;157(2):247-57. doi: 10.1007/s00705-011-1152-3. Epub 2011 Nov
9.

Characterization of avian influenza viruses isolated from domestic ducks in
Vietnam in 2009 and 2010.

Nomura N, Sakoda Y, Endo M, Yoshida H, Yamamoto N, Okamatsu M, Sakurai K, Hoang
NV, Nguyen LV, Chu HD, Tien TN, Kida H.

Laboratory of Microbiology, Department of Disease Control, Graduate School of
Veterinary Medicine, Hokkaido University, Kita 18 Nishi 9, Kita-ku, Sapporo
060-0818, Japan.

In the surveillance of avian influenza in Vietnam, 26 H9N2, 1 H3N2, 1 H3N8, 7
H4N6, 3 H11N3, and 1 H11N9 viruses were isolated from tracheal and cloacal swab
samples of 300 domestic ducks in April 2009, and 1 H9N6 virus from 300 bird
samples in March 2010. Out of the 27 H9 virus isolates, the hemagglutinins of 18 
strains were genetically classified as belonging to the sublineage G1, and the
other nine belonged to the Korean sublineage. Phylogenetic analysis revealed that
one of the 27 H9 viruses was a reassortant in which the PB2 gene belonged to the 
Korean sublineage and the other seven genes belonged to the G1 sublineage. Three 
representative H9N2 viruses were intranasally inoculated into ducks, chickens,
pigs, and mice. On the basis of experimental infection studies, it was found that
each of the three viruses readily infected pigs and replicated in their upper
respiratory tracts, and they infected chickens with slight replication. Viruses
were recovered from the lungs of mice inoculated with two of the three isolates. 
The present results reveal that H9 avian influenza viruses are prevailing and
genetic reassortment occurs among domestic ducks in Vietnam. It is recommended
that careful surveillance of swine influenza with H9 viruses should be performed 
to prepare for pandemic influenza.

PMID: 22068881  [PubMed - indexed for MEDLINE]
Þ

7185. Virol J. 2011 Nov 4;8:505. doi: 10.1186/1743-422X-8-505.

Novel genetic reassortants in H9N2 influenza A viruses and their diverse
pathogenicity to mice.

Bi Y, Lu L, Li J, Yin Y, Zhang Y, Gao H, Qin Z, Zeshan B, Liu J, Sun L, Liu W.

Center for Molecular Virology, Key Laboratory of Pathogenic Microbiology and
Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing
100101, China.

BACKGROUND: H9N2 influenza A viruses have undergone extensive reassortments in
different host species, and could lead to the epidemics or pandemics with the
potential emergence of novel viruses.
METHODS: To understand the genetic and pathogenic features of early and current
circulating H9N2 viruses, 15 representative H9N2 viruses isolated from diseased
chickens in northern China between 1998 and 2010 were characterized and compared 
with all Chinese H9N2 viruses available in the NCBI database. Then, the
representative viruses of different genotypes were selected to study the
pathogenicity in mice with the aim to investigate the adaptation and the
potential pathogenicity of the novel H9N2 reassortants to mammals.
RESULTS: Our results demonstrated that most of the 15 isolates were reassortants 
and generated four novel genotypes (B62-B65), which incorporated the gene
segments from Eurasian H9N2 lineage, North American H9N2 branch, and H5N1
viruses. It was noteworthy that the newly identified genotype B65 has been
prevalent in China since 2007, and more importantly, different H9N2 influenza
viruses displayed a diverse pathogenicity to mice. The isolates of the 2008-2010 
epidemic (genotypes B55 and B65) were lowly infectious, while two representative 
viruses of genotypes B0 and G2 isolated from the late 1990s were highly
pathogenic to mice. In addition, Ck/SD/LY-1/08 (genotype 63, containing H5N1-like
NP and PA genes) was able to replicate well in mouse lungs with high virus titers
but caused mild clinical signs.
CONCLUSION: Several lines of evidence indicated that the H9N2 influenza viruses
constantly change their genetics and pathogenicity. Thus, the genetic evolution
of H9N2 viruses and their pathogenicity to mammals should be closely monitored to
prevent the emergence of novel pandemic viruses.

PMCID: PMC3236014
PMID: 22050764  [PubMed - indexed for MEDLINE]
Þ

7522. Virol J. 2011 Oct 12;8:467. doi: 10.1186/1743-422X-8-467.

Genetic evolution of low pathogenecity H9N2 avian influenza viruses in Tunisia:
acquisition of new mutations.

Tombari W, Nsiri J, Larbi I, Guerin JL, Ghram A.

Veterinary Microbiology laboratory, Pasteur Institute of Tunis, 1002
Tunis-Belvédère, Tunisia.

BACKGROUND: Since the end of 2009, H9N2 has emerged in Tunisia causing several
epidemics in poultry industry resulting in major economic losses. To monitor
variations of Influenza viruses during the outbreaks, Tunisian H9N2 virus
isolates were identified and genetically characterized.
METHODS: The genomic RNA segments of Tunisian H9N2 strains were subjected to
RT-PCR amplifications followed by sequencing analysis.
RESULTS: Phylogenetic analysis demonstrated that A/Ck/TUN/12/10 and A/Migratory
Bird/TUN/51/10 viruses represent multiple reassortant lineages, with genes coming
from Middle East strains, and share the common ancestor Qa/HK/G1/97 isolate which
has contributed internal genes of H5N1 virus circulating in Asia. Some of the
internal genes seemed to have undergone broad reassortments with other influenza 
subtypes. Deduced amino acid sequences of the hemagglutinin (HA) gene showed the 
presence of additional glycosylation site and Leu at position 234 indicating to
binding preference to α (2, 6) sialic acid receptors, indicating their potential 
to directly infect humans. The Hemagglutinin cleavage site motif sequence is 333 
PARSSR*GLF341 which indicates the low pathogenicity nature of the Tunisian H9N2
strains and the potential to acquire the basic amino acids required for the
highly pathogenic strains. Their neuraminidase protein (NA) carried substitutions
in the hemadsorption (HB) site, similar to those of other avian H9N2 viruses from
Asia, Middle Eastern and human pandemic H2N2 and H3N2 that bind to α -2, 6
-linked receptors. Two avian virus-like aa at positions 661 (A) and 702 (K),
similar to H5N1 strains, were identified in the polymerase (PB2) protein.
Likewise, matrix (M) protein carried some substitutions which are linked with
increasing replication in mammals. In addition, H9N2 strain recently circulating 
carried new polymorphism, "GSEV" PDZ ligand (PL) C-terminal motif in its non
structural (NS) protein.Two new aa substitutions (I) and (V), that haven't been
previously reported, were identified in the polymerase and matrix proteins,
respectively. Nucleoprotein and non-structural protein carried some substitutions
similar to H5N1 strains.
CONCLUSION: Considering these new mutations, the molecular basis of tropism, host
responses and enhanced virulence will be defined and studied. Otherwise,
Continuous monitoring of viral genetic changes throughout the year is warranted
to monitor variations of Influenza viruses in the field.

PMCID: PMC3223530
PMID: 21992186  [PubMed - indexed for MEDLINE]
Þ

7569. PLoS One. 2011;6(9):e25808. doi: 10.1371/journal.pone.0025808. Epub 2011 Sep 29.

Reassortant H9N2 influenza viruses containing H5N1-like PB1 genes isolated from
black-billed magpies in Southern China.

Dong G, Xu C, Wang C, Wu B, Luo J, Zhang H, Nolte DL, Deliberto TJ, Duan M, Ji G,
He H.

Key Laboratory of Animal Ecology and Conservation Biology, National Research
Center For Wildlife Born Diseases, Institute of Zoology, Chinese Academy of
Sciences, Beijing, China.

H9N2 influenza A viruses have become endemic in different types of terrestrial
poultry and wild birds in Asia, and are occasionally transmitted to humans and
pigs. To evaluate the role of black-billed magpies (Pica pica) in the evolution
of influenza A virus, we conducted two epidemic surveys on avian influenza
viruses in wild black-billed magpies in Guangxi, China in 2005 and characterized 
three isolated black-billed magpie H9N2 viruses (BbM viruses). Phylogenetic
analysis indicated that three BbM viruses were almost identical with 99.7 to 100%
nucleotide homology in their whole genomes, and were reassortants containing
BJ94-like (Ck/BJ/1/94) HA, NA, M, and NS genes, SH/F/98-like (Ck/SH/F/98) PB2,
PA, and NP genes, and H5N1-like (Ck/YN/1252/03, clade 1) PB1 genes. Genetic
analysis showed that BbM viruses were most likely the result of multiple
reassortments between co-circulating H9N2-like and H5N1-like viruses, and were
genetically different from other H9N2 viruses because of the existence of
H5N1-like PB1 genes. Genotypical analysis revealed that BbM viruses evolved from 
diverse sources and belonged to a novel genotype (B46) discovered in our recent
study. Molecular analysis suggested that BbM viruses were likely low pathogenic
reassortants. However, results of our pathogenicity study demonstrated that BbM
viruses replicated efficiently in chickens and a mammalian mouse model but were
not lethal for infected chickens and mice. Antigenic analysis showed that BbM
viruses were antigenic heterologous with the H9N2 vaccine strain. Our study is
probably the first report to document and characterize H9N2 influenza viruses
isolated from black-billed magpies in southern China. Our results suggest that
black-billed magpies were susceptible to H9N2 influenza viruses, which raise
concerns over possible transmissions of reassortant H9N2 viruses among poultry
and wild birds.

PMCID: PMC3183077
PMID: 21980538  [PubMed - indexed for MEDLINE]
Þ

7584. Acta Virol. 2011;55(3):219-26.

Molecular characterization and pathogenicity of swine influenza H9N2 subtype
virus A/swine/HeBei/012/2008/(H9N2).

Rui-Hua Z, Hong-Yu C, Ming-Ju X, Kai L, Hua-Lan C, Cun-Lian W, Dong W, Cun-Xin L,
Tong X.

Department of Veterinary Medicine, HeBei North University, Hebei, People's
Republic of China.

The H9N2 subtype influenza virus (IV) is a remarkable member of the influenza A
viruses because it can infect not only chickens, ducks and pigs, but also humans.
Pigs are susceptible to both human and avian influenza viruses and have been
proposed to be intermediate hosts for the generation of pandemic influenza
viruses through reassortment or adaptation to the mammalian host. To further
understand the genetic characteristics and evolution, we investigated the source 
and molecular characteristics of the H9N2 subtype swine influenza virus (SIV),
and observed its pathogenicity in BALB/c mice. The BALB/c mice were inoculated
intranasally with 100 median mouse infectious dose of
A/swine/HeBei/012/2008/(H9N2) viruses to observe the pathogenicity. The HA, NP,
NA and M gene were cloned, sequenced and phylogenetically analyzed with related
sequences available in GenBank. The infected mice presented with inactivity,
weight loss and laboured respiration, while the pathological changes were
characterized by diffuse alveolar damage in the lung. The nucleotide and deduced 
amino acid sequence of HA, NP, NA and M gene was similar with that of
A/chicken/Hebei/4/2008(H9N2). The HA protein contained 6 glycosylation sites and 
the motif of HA cleavage site was PARSSR GLF, which is characteristic of low
pathogenic IV. In the HA, NP, M and NA gene phylogenetic trees, the isolate
clustered with A/chicken/Hebei/4/2008(H9N2). The isolate possibly came from
A/chicken/Hebei/4/2008(H9N2) and was partially varied during its cross-species
spread.

PMID: 21978155  [PubMed - indexed for MEDLINE]
Þ

8240. Vaccine. 2011 Oct 19;29(45):8032-41. doi: 10.1016/j.vaccine.2011.08.054. Epub
2011 Aug 22.

Increase in viral yield in eggs and MDCK cells of reassortant H5N1 vaccine
candidate viruses caused by insertion of 38 amino acids into the NA stalk.

Zhang W, Xue T, Wu X, Zhang P, Zhao G, Peng D, Hu S, Wang X, Liu X, Liu W, Liu X.

Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou
University, 12 East Wenhui Road, Yangzhou, Jiangsu 225009, China.

BACKGROUND: The H5N1 subtype of highly pathogenic avian influenza viruses has
spread to over 63 countries in Asia, Europe, and Africa and has become endemic in
poultry. Since 2004, vaccination against H5N1 influenza has become common in
domestic poultry operations in China. Most influenza vaccines have been produced 
in embryonated chicken eggs. High yield is the essential feature of a good
vaccine candidate virus.
OBJECTIVE: Therefore, the large-scale manufacture of such a vaccine requires that
the viral yield of H5N1 reassortant vaccine viruses in eggs and MDCK cells be
increased.
METHODS: We generated two sets of reassortant H5N1 viruses based on backbone
viruses A/Chicken/F/98 (H9N2) and A/Puerto Rico/8/34 (H1N1) using reverse
genetics. The HAs and NAs of the reassortants were derived from the three
epidemic H5N1 strains found in China. We compared the replication properties of
these recombinant H5N1 viruses in embryonated chicken eggs and MDCK cells after
inserting either 20 or 38 amino acids into their NA stalks.
RESULTS: In this study, we demonstrated that inserting 38 amino acids into the NA
stalks can significantly increase the viral yield of H5N1 reassortant viruses in 
both embryonated chicken eggs and MDCK cells, while inserting only 20 amino acids
into the same NA stalks does not. Hemagglutinin inhibition testing and protection
assays indicated that recombinant H5N1 viruses with 38 aa inserted into their NA 
stalks had the same antigenicity as the viruses with wt-NA.
CONCLUSION: These results suggest that the generation of an H5N1 recombinant
vaccine seed by the insertion of 38 aa into the NA stalk may be a suitable and
more economical strategy for the increase in viral yield in both eggs and MDCK
cells for the purposes of vaccine production.

Copyright © 2011 Elsevier Ltd. All rights reserved.

PMID: 21864614  [PubMed - indexed for MEDLINE]
Þ

9096. Proc Natl Acad Sci U S A. 2011 Jul 19;108(29):12084-8. doi:
10.1073/pnas.1108058108. Epub 2011 Jul 5.

Compatibility of H9N2 avian influenza surface genes and 2009 pandemic H1N1
internal genes for transmission in the ferret model.

Kimble JB, Sorrell E, Shao H, Martin PL, Perez DR.

Department of Veterinary Medicine, University of Maryland, College Park, MD
20742, USA.

In 2009, a novel H1N1 influenza (pH1N1) virus caused the first influenza pandemic
in 40 y. The virus was identified as a triple reassortant between avian, swine,
and human influenza viruses, highlighting the importance of reassortment in the
generation of viruses with pandemic potential. Previously, we showed that a
reassortant virus composed of wild-type avian H9N2 surface genes in a seasonal
human H3N2 backbone could gain efficient respiratory droplet transmission in the 
ferret model. Here we determine the ability of the H9N2 surface genes in the
context of the internal genes of a pH1N1 virus to efficiently transmit via
respiratory droplets in ferrets. We generated reassorted viruses carrying the HA 
gene alone or in combination with the NA gene of a prototypical H9N2 virus in the
background of a pH1N1 virus. Four reassortant viruses were generated, with three 
of them showing efficient respiratory droplet transmission. Differences in
replication efficiency were observed for these viruses; however, the results
clearly indicate that H9N2 avian influenza viruses and pH1N1 viruses, both of
which have occasionally infected pigs, have the potential to reassort and
generate novel viruses with respiratory transmission potential in mammals.

PMCID: PMC3141953
PMID: 21730147  [PubMed - indexed for MEDLINE]
Þ

9108. J Infect Dev Ctries. 2011 Jul 4;5(6):413-24.

Comparative sequence, antigenic and phylogenetic analysis of avian influenza
(H9N2) surface proteins isolated in Pakistan between 1999 and 2008.

Butt AM, Siddique S, Tahir S, Nasrullah I, Hussain M, Idrees M, Lu J, Tong Y.

National Centre of Excellence in Molecular Biology (CEMB), University of the
Punjab, Lahore, Pakistan. azeem@cemb.edu.pk

INTRODUCTION: Influenza A viruses possess a unique genomic structure which leads 
to genetic instability, especially in products of neuraminidase and hemagglutinin
genes. These surface proteins play major roles in viral entry and release, and in
the activation of the host immune system.
METHODOLOGY: This study involved an in silico sequence, phylogenetic and
antigenic analyses of hemagglutinin and neuraminidase proteins of avian influenza
A (H9N2) strains that circulated in Pakistan's poultry flocks from 1999 to 2008
and determined variations among these sequences at different levels.
RESULTS: Sequence and phylogenetic analysis revealed a large number of similar
substitution mutations and close evolutionary relation among sequences of both
proteins. Changes were observed in the N-glycosylation sites of both surface
proteins, along with the appearance of a new glycosylation site in the
neuraminidase sequence isolated in 2007. Epitopes for hemagglutinin remained
conserved, whereas for neuraminidase, epitopes from older strains reappeared in
present sequences.
CONCLUSIONS: Because of the rapid mutating nature of avian influenza subtype
H9N2, constant surveillance of annual sequence variations is important.
Preventive measures and vaccine products can be evaluated by keeping track of
changes that may lead to reassortment among different circulating strains in
Pakistan's commercial poultry flocks or in humans.

PMID: 21727639  [PubMed - indexed for MEDLINE]
Þ

9331. J Virol. 2011 Aug;85(16):8413-21. doi: 10.1128/JVI.00219-11. Epub 2011 Jun 15.

Phylogeography and evolutionary history of reassortant H9N2 viruses with
potential human health implications.

Fusaro A, Monne I, Salviato A, Valastro V, Schivo A, Amarin NM, Gonzalez C,
Ismail MM, Al-Ankari AR, Al-Blowi MH, Khan OA, Maken Ali AS, Hedayati A, Garcia
Garcia J, Ziay GM, Shoushtari A, Al Qahtani KN, Capua I, Holmes EC, Cattoli G.

Istituto Zooprofilattico Sperimentale delle Venezie, Research & Development
Department, OIE/FAO and National Reference Laboratory for Newcastle Disease and
Avian Influenza, OIE Collaborating Center for Diseases at the Human-Animal
Interface, Viale dell'Università 10, 35020 Legnaro, Padua, Italy.
afusaro@izsvenezie.it

Avian influenza viruses of the H9N2 subtype have seriously affected the poultry
industry of the Far and Middle East since the mid-1990s and are considered one of
the most likely candidates to cause a new influenza pandemic in humans. To
understand the genesis and epidemiology of these viruses, we investigated the
spatial and evolutionary dynamics of complete genome sequences of H9N2 viruses
circulating in nine Middle Eastern and Central Asian countries from 1998 to 2010.
We identified four distinct and cocirculating groups (A, B, C, and D), each of
which has undergone widespread inter- and intrasubtype reassortments, leading to 
the generation of viruses with unknown biological properties. Our analysis also
suggested that eastern Asia served as the major source for H9N2 gene segments in 
the Middle East and Central Asia and that in this geographic region
within-country evolution played a more important role in shaping viral genetic
diversity than migration between countries. The genetic variability identified
among the H9N2 viruses was associated with specific amino acid substitutions that
are believed to result in increased transmissibility in mammals, as well as
resistance to antiviral drugs. Our study highlights the need to constantly
monitor the evolution of H9N2 viruses in poultry to better understand the
potential risk to human health posed by these viruses.

PMCID: PMC3147996
PMID: 21680519  [PubMed - indexed for MEDLINE]
Þ

10010. PLoS One. 2011 Apr 26;6(4):e19311. doi: 10.1371/journal.pone.0019311.

Live bird markets of Bangladesh: H9N2 viruses and the near absence of highly
pathogenic H5N1 influenza.

Negovetich NJ, Feeroz MM, Jones-Engel L, Walker D, Alam SM, Hasan K, Seiler P,
Ferguson A, Friedman K, Barman S, Franks J, Turner J, Krauss S, Webby RJ, Webster
RG.

St. Jude Children's Research Hospital, Memphis, Tennessee, United States of
America.

Avian influenza surveillance in Bangladesh has been passive, relying on poultry
farmers to report suspected outbreaks of highly pathogenic H5N1 influenza. Here, 
the results of an active surveillance effort focusing on the live-bird markets
are presented. Prevalence of influenza infection in the birds of the live bird
markets is 23.0%, which is similar to that in poultry markets in other countries.
Nearly all of the isolates (94%) were of the non-pathogenic H9N2 subtype, but
viruses of the H1N2, H1N3, H3N6, H4N2, H5N1, and H10N7 subtypes were also
observed. The highly pathogenic H5N1-subtype virus was observed at extremely low 
prevalence in the surveillance samples (0.08%), and we suggest that the current
risk of infection for humans in the retail poultry markets in Bangladesh is
negligible. However, the high prevalence of the H9 subtype and its potential for 
interaction with the highly pathogenic H5N1-subtype, i.e., reassortment and
attenuation of host morbidity, highlight the importance of active surveillance of
the poultry markets.

PMCID: PMC3082571
PMID: 21541296  [PubMed - indexed for MEDLINE]
Þ

10103. J Gen Virol. 2011 Aug;92(Pt 8):1843-53. doi: 10.1099/vir.0.031591-0. Epub 2011
Apr 27.

H9 avian influenza reassortant with engineered polybasic cleavage site displays a
highly pathogenic phenotype in chicken.

Gohrbandt S, Veits J, Breithaupt A, Hundt J, Teifke JP, Stech O, Mettenleiter TC,
Stech J.

Friedrich Loeffler Institute, Institute of Molecular Biology, Greifswald-Insel
Riems, Germany.

In the field, highly pathogenic avian influenza viruses (HPAIV) originate from
low-pathogenic strains of the haemagglutinin (HA) serotypes H5 and H7 that have
acquired a polybasic HA cleavage site. This observation suggests the presence of 
a cryptic virulence potential of H5 and H7 low-pathogenic avian influenza viruses
(LPAIV). Among all other LPAIV, the H9N2 strains are of particular relevance as
they have become widespread across many countries in several avian species and
have been transmitted to humans. To assess the potential of these strains to
transform into an HPAIV, we introduced a polybasic cleavage site into the HA of a
contemporary H9N2 isolate. Whereas the engineered polybasic HA cleavage site
mutant remained a low-pathogenic strain like its parent virus, a reassortant
expressing the modified H9 HA with engineered polybasic cleavage site and all the
other genes from an H5N1 HPAIV became highly pathogenic in chicken with an
intravenous pathogenicity index of 1.23. These results suggest that an HPAIV with
a subtype other than H5 or H7 would only emerge under conditions where the HA
gene could acquire a polybasic cleavage site and the other viral genes carry
additional virulence determinants.

PMID: 21525207  [PubMed - indexed for MEDLINE]
Þ

10838. PLoS One. 2011 Feb 28;6(2):e17212. doi: 10.1371/journal.pone.0017212.

Phylogenetic diversity and genotypical complexity of H9N2 influenza A viruses
revealed by genomic sequence analysis.

Dong G, Luo J, Zhang H, Wang C, Duan M, Deliberto TJ, Nolte DL, Ji G, He H.

Key Laboratory of Animal Ecology and Conservation Biology, National Research
Center for Wildlife Born Diseases, Institute of Zoology, Chinese Academy of
Sciences, Beijing, China.

H9N2 influenza A viruses have become established worldwide in terrestrial poultry
and wild birds, and are occasionally transmitted to mammals including humans and 
pigs. To comprehensively elucidate the genetic and evolutionary characteristics
of H9N2 influenza viruses, we performed a large-scale sequence analysis of 571
viral genomes from the NCBI Influenza Virus Resource Database, representing the
spectrum of H9N2 influenza viruses isolated from 1966 to 2009. Our study provides
a panoramic framework for better understanding the genesis and evolution of H9N2 
influenza viruses, and for describing the history of H9N2 viruses circulating in 
diverse hosts. Panorama phylogenetic analysis of the eight viral gene segments
revealed the complexity and diversity of H9N2 influenza viruses. The 571 H9N2
viral genomes were classified into 74 separate lineages, which had marked host
and geographical differences in phylogeny. Panorama genotypical analysis also
revealed that H9N2 viruses include at least 98 genotypes, which were further
divided according to their HA lineages into seven series (A-G). Phylogenetic
analysis of the internal genes showed that H9N2 viruses are closely related to
H3, H4, H5, H7, H10, and H14 subtype influenza viruses. Our results indicate that
H9N2 viruses have undergone extensive reassortments to generate multiple
reassortants and genotypes, suggesting that the continued circulation of multiple
genotypical H9N2 viruses throughout the world in diverse hosts has the potential 
to cause future influenza outbreaks in poultry and epidemics in humans. We
propose a nomenclature system for identifying and unifying all lineages and
genotypes of H9N2 influenza viruses in order to facilitate international
communication on the evolution, ecology and epidemiology of H9N2 influenza
viruses.

PMCID: PMC3046171
PMID: 21386964  [PubMed - indexed for MEDLINE]
Þ

10962. Proc Natl Acad Sci U S A. 2011 Mar 8;108(10):4164-9. doi:
10.1073/pnas.1019109108. Epub 2011 Feb 28.

High genetic compatibility and increased pathogenicity of reassortants derived
from avian H9N2 and pandemic H1N1/2009 influenza viruses.

Sun Y, Qin K, Wang J, Pu J, Tang Q, Hu Y, Bi Y, Zhao X, Yang H, Shu Y, Liu J.

Key Laboratory of Zoonosis of Ministry of Agriculture, College of Veterinary
Medicine, China Agricultural University, Beijing 100193, China.

H9N2 influenza viruses have been circulating worldwide in multiple avian species 
and repeatedly infecting mammals, including pigs and humans, posing a significant
threat to public health. The coexistence of H9N2 and pandemic influenza H1N1/2009
viruses in pigs and humans provides an opportunity for these viruses to reassort.
To evaluate the potential public risk of the reassortant viruses derived from
these viruses, we used reverse genetics to generate 127 H9 reassortants derived
from an avian H9N2 and a pandemic H1N1 virus, and evaluated their compatibility, 
replication ability, and virulence in mice. These hybrid viruses showed high
genetic compatibility and more than half replicated to a high titer in vitro. In 
vivo studies of 73 of 127 reassortants revealed that all viruses were able to
infect mice without prior adaptation and 8 reassortants exhibited higher
pathogenicity than both parental viruses. All reassortants with higher virulence 
than parental viruses contained the PA gene from the 2009 pandemic virus,
revealing the important role of the PA gene from the H1N1/2009 virus in
generating a reassortant virus with high public health risk. Analyses of the
polymerase activity of the 16 ribonucleoprotein combinations in vitro suggested
that the PA of H1N1/2009 origin also enhanced polymerase activity. Our results
indicate that some avian H9-pandemic reassortants could emerge with a potentially
higher threat for humans and also highlight the importance of monitoring the
H9-pandemic reassortant viruses that may arise, especially those that possess the
PA gene of H1N1/2009 origin.

PMCID: PMC3054021
PMID: 21368167  [PubMed - indexed for MEDLINE]
Þ

11566. Virol J. 2011 Jan 27;8:42. doi: 10.1186/1743-422X-8-42.

Characterization of an H10N8 influenza virus isolated from Dongting lake wetland.

Zhang H, Xu B, Chen Q, Chen J, Chen Z.

State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of
Sciences, Wuhan 430071, PR China.

BACKGROUND: Wild birds, especially those in wetlands and aquatic environments,
are considered to be natural reservoirs of avian influenza viruses. It is
accepted that water is an important component in the transmission cycle of avian 
influenza virus. Monitoring the water at aggregation and breeding sites of
migratory waterfowl, mainly wetland, is very important for early detection of
avian influenza virus. The epidemiology investigation of avian influenza virus
was performed in Dongting lake wetland which is an international important
wetland.
RESULTS: An H10N8 influenza virus was isolated from Dongting Lake wetland in
2007. Phylogenetic analysis indicated that the virus was generated by multiple
gene segment reassortment. The isolate was lowly pathogenic for chickens.
However, it replicated efficiently in the mouse lung without prior adaptation,
and the virulence to mice increased rapidly during adaptation in mouse lung.
Sequence analysis of the genome of viruses from different passages showed that
multiple amino acid changes were involved in the adaptation of the isolates to
mice.
CONCLUSIONS: The water might be an important component in the transmission cycle 
of avian influenza virus, and other subtypes of avian influenza viruses (other
than H5, H7 and H9) might evolve to pose a potential threat to mammals and even
humans.

PMCID: PMC3038951
PMID: 21272297  [PubMed - indexed for MEDLINE]
Þ

12051. Influenza Res Treat. 2011;2011:702092. doi: 10.1155/2011/702092. Epub 2010 Oct
18.

Changing patterns of h6 influenza viruses in Hong Kong poultry markets.

Ozaki H, Guan Y, Peiris M, Webster R, Webby R.

Laboratory of Veterinary Microbiology, Faculty of Agriculture, Tottori
University, Tottori 680-8553, Japan.

Until 2001, H6N1 influenza viruses in the Hong Kong bird markets were represented
by a single stable A/teal/Hong Kong/W312/97-like lineage. Beginning in 2001,
despite a reduction in overall prevalence, an increase was observed in the number
of H6 viruses isolated from chickens and other hosts. To assess any changes in H6
viruses, we characterized 18 H6 viruses isolated in the Hong Kong bird markets
from 2001 to 2003. Experimental data showed that the 2003 H6 viruses had similar 
infectivity for chickens as did A/teal/HK/W312/97, and they were unable to
transmit. Although all hemagglutinin genes were closely related to
A/teal/HK/W312/97, 7 isolates were reassortant viruses containing similar gene
segments of co-circulating H9N2 or H5N1 viruses. The receptor specificity was
different from that of A/teal/Hong Kong/W312/97. Interestingly, similar
observations have been documented in H9N2 viruses in Hong Kong. This evolution
strongly suggests that some change in the ecology of influenza in the region
selected for these changes. Taken together, these findings suggest that the H6
influenza viruses isolated in the Hong Kong markets are not well adapted to
chickens and that the likely continued source of these viruses are other "minor" 
poultry species in which they are undergoing genetic and biologic evolution.

PMCID: PMC3447289
PMID: 23074660  [PubMed]
Þ

12561. Vet Microbiol. 2011 Apr 21;149(1-2):254-61. doi: 10.1016/j.vetmic.2010.11.008.
Epub 2010 Nov 9.

Genetic diversity of H9N2 influenza viruses from pigs in China: a potential
threat to human health?

Yu H, Zhou YJ, Li GX, Ma JH, Yan LP, Wang B, Yang FR, Huang M, Tong GZ.

Division of Swine Infectious Diseases, Shanghai Veterinary Research Institute,
Chinese Academy of Agricultural Sciences, No. 518, Ziyue Road, Minhang District, 
Shanghai 200241, China.

Pandemic strains of influenza A virus might arise by genetic reassortment between
viruses from different hosts. Pigs are susceptible to both human and avian
influenza viruses and have been proposed to be intermediate hosts or mixing
vessels, for the generation of pandemic influenza viruses through reassortment or
adaptation to the mammalian host. In this study, we summarize and report for the 
first time the coexistence of 10 (A-J) genotypes in pigs in China by analyzing
the eight genes of 28 swine H9N2 viruses isolated in China from 1998 to 2007.
Swine H9N2 viruses in genotype A and B were completely derived from Y280-like and
Shanghai/F/98-like viruses, respectively, which indicated avian-to-pig
interspecies transmission of H9N2 viruses did exist in China. The other eight
genotype (C-J) viruses might be double-reassortant viruses, in which six genotype
(E-J) viruses possessed 1-4 H5-like gene segments indicating they were
reassortants of H9 and H5 viruses. In conclusion, genetic diversity of H9N2
influenza viruses from pigs in China provides further evidence that avian to pig 
interspecies transmission of H9N2 viruses did occur and might result in the
generation of new reassortant viruses by genetic reassortment with swine H1N1,
H1N2 and H3N2 influenza viruses, therefore, these swine H9N2 influenza viruses
might be a potential threat to human health and continuing to carry out swine
influenza virus surveillance in China is of great significance.

Copyright © 2010 Elsevier B.V. All rights reserved.

PMID: 21115230  [PubMed - indexed for MEDLINE]
Þ

12754. Virol J. 2010 Nov 15;7:319. doi: 10.1186/1743-422X-7-319.

Avian influenza A (H9N2): computational molecular analysis and phylogenetic
characterization of viral surface proteins isolated between 1997 and 2009 from
the human population.

Butt AM, Siddique S, Idrees M, Tong Y.

State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of
Microbiology and Epidemiology, Beijing, 100071, PR China. tong.yigang@gmail.com.

BACKGROUND: H9N2 avian influenza A viruses have become panzootic in Eurasia over 
the last decade and have caused several human infections in Asia since 1998. To
study their evolution and zoonotic potential, we conducted an in silico analysis 
of H9N2 viruses that have infected humans between 1997 and 2009 and identified
potential novel reassortments.
RESULTS: A total of 22 hemagglutinin (HA) and neuraminidase (NA) nucleotide and
deduced amino acid sequences were retrieved from the NCBI flu database. It was
identified that mature peptide sequences of HA genes isolated from humans in 2009
had glutamine at position 226 (H3) of the receptor binding site, indicating a
preference to bind to the human α (2-6) sialic acid receptors, which is different
from previously isolated viruses and studies where the presence of leucine at the
same position contributes to preference for human receptors and presence of
glutamine towards avian receptors. Similarly, strains isolated in 2009 possessed 
new motif R-S-N-R in spite of typical R-S-S-R at the cleavage site of HA, which
isn't reported before for H9N2 cases in humans. Other changes involved loss,
addition, and variations in potential glycosylation sites as well as in predicted
epitopes. The results of phylogenetic analysis indicated that HA and NA gene
segments of H9N2 including those from current and proposed vaccine strains belong
to two different Eurasian phylogenetic lineages confirming possible genetic
reassortments.
CONCLUSIONS: These findings support the continuous evolution of avian H9N2
viruses towards human as host and are in favor of effective surveillance and
better characterization studies to address this issue.

PMCID: PMC2994543
PMID: 21078137  [PubMed - indexed for MEDLINE]
Þ

13145. J Virol. 2011 Jan;85(1):456-69. doi: 10.1128/JVI.01503-10. Epub 2010 Oct 20.

Modifications in the polymerase genes of a swine-like triple-reassortant
influenza virus to generate live attenuated vaccines against 2009 pandemic H1N1
viruses.

Pena L, Vincent AL, Ye J, Ciacci-Zanella JR, Angel M, Lorusso A, Gauger PC, Janke
BH, Loving CL, Perez DR.

Department of Veterinary Medicine, University of Maryland, VA-MD Regional College
of Veterinary Medicine, 8075 Greenmean Dr., College Park, MD 20742, USA.

On 11 June 2009, the World Health Organization (WHO) declared that the outbreaks 
caused by novel swine-origin influenza A (H1N1) virus had reached pandemic
proportions. The pandemic H1N1 (H1N1pdm) virus is the predominant influenza virus
strain in the human population. It has also crossed the species barriers and
infected turkeys and swine in several countries. Thus, the development of a
vaccine that is effective in multiple animal species is urgently needed. We have 
previously demonstrated that the introduction of temperature-sensitive mutations 
into the PB2 and PB1 genes of an avian H9N2 virus, combined with the insertion of
a hemagglutinin (HA) tag in PB1, resulted in an attenuated (att) vaccine backbone
for both chickens and mice. Because the new pandemic strain is a
triple-reassortant (TR) virus, we chose to introduce the double attenuating
modifications into a swine-like TR virus isolate, A/turkey/OH/313053/04 (H3N2)
(ty/04), with the goal of producing live attenuated influenza vaccines (LAIV).
This genetically modified backbone had impaired polymerase activity and
restricted virus growth at elevated temperatures. In vivo characterization of two
H1N1 vaccine candidates generated using the ty/04 att backbone demonstrated that 
this vaccine is highly attenuated in mice, as indicated by the absence of signs
of disease, limited replication, and minimum histopathological alterations in the
respiratory tract. A single immunization with the ty/04 att-based vaccines
conferred complete protection against a lethal H1N1pdm virus infection in mice.
More importantly, vaccination of pigs with a ty/04 att-H1N1 vaccine candidate
resulted in sterilizing immunity upon an aggressive intratracheal challenge with 
the 2009 H1N1 pandemic virus. Our studies highlight the safety of the ty/04 att
vaccine platform and its potential as a master donor strain for the generation of
live attenuated vaccines for humans and livestock.

PMCID: PMC3014183
PMID: 20962084  [PubMed - indexed for MEDLINE]
Þ

13149. BMC Genomics. 2010 Oct 20;11:586. doi: 10.1186/1471-2164-11-586.

Use of consensus sequences for the design of high density resequencing
microarrays: the influenza virus paradigm.

Leclercq I, Berthet N, Batéjat C, Rousseaux C, Dickinson P, Old IG, Kong K,
Kennedy GC, Cole ST, Manuguerra JC.

Institut Pasteur, Laboratory for Urgent Response to Biological Threats (CIBU),
Paris, France.

BACKGROUND: A resequencing microarray called PathogenID v2.0 has been developed
and used to explore various strategies of sequence selection for its design. The 
part dedicated to influenza viruses was based on consensus sequences specific for
one gene generated from global alignments of a large number of influenza virus
sequences available in databanks.
RESULTS: For each HA (H1, H2, H3, H5, H7 and H9) and NA (N1, N2 and N7) molecular
type chosen to be tested, 1 to 3 consensus sequences were computed and tiled on
the microarray. A total of 12 influenza virus samples from different host origins
(humans, pigs, horses and birds) and isolated over a period of about 50 years
were used in this study. Influenza viruses were correctly identified, and in most
cases with the accurate information of the time of their emergence.
CONCLUSIONS: PathogenID v2.0 microarray demonstrated its ability to type and
subtype influenza viruses, often to the level of viral variants, with a minimum
number of tiled sequences. This validated the strategy of using consensus
sequences, which do not exist in nature, for our microarray design. The
versatility, rapidity and high discriminatory power of the PathogenID v2.0
microarray could prove critical to detect and identify viral genome reassortment 
events resulting in a novel virus with epidemic or pandemic potential and
therefore assist health authorities to make efficient decisions about patient
treatment and outbreak management.

PMCID: PMC3091733
PMID: 20961419  [PubMed - indexed for MEDLINE]
Þ

13160. Virol Sin. 2010 Oct;25(5):329-40. doi: 10.1007/s12250-010-3123-3. Epub 2010 Oct
8.

Analysis of synonymous codon usage bias in 09H1N1.

Li ZP, Ying DQ, Li P, Li F, Bo XC, Wang SQ.

Beijing Institute of Radiation Medicine, Beijing, 100850, China.

A novel subtype of influenza A virus 09H1N1 has rapidly spread across the world. 
Evolutionary analyses of this virus have revealed that 09H1N1 is a triple
reassortant of segments from swine, avian and human influenza viruses. In this
study, we investigated factors shaping the codon usage bias of 09H1N1 and carried
out cluster analysis of 60 strains of influenza A virus from different subtypes
based on their codon usage bias. We discovered that more preferentially used
codons of 09H1N1 are A-ended or U-ended, and the intra-genomic codon usage bias
of 09H1N1 is quite low. Base composition constraint, dinucleotide biases and
translational selection are the main factors influencing the codon usage bias of 
09H1N1. At the genome level, we find that the codon usage bias of 09H1N1 is
similar to H1N1 (A/swine/Kansas/77778/2007H1N1), H9N2 from Asia, H1N2 from Asia
and North America and H3N2 from North America. Our results provide insight for
understanding the processes governing evolution, regulation of gene expression,
and revealing the evolution of 09H1N1.

PMID: 20960179  [PubMed - indexed for MEDLINE]
Þ

13379. PLoS One. 2010 Sep 29;5(9). pii: e13063. doi: 10.1371/journal.pone.0013063.

Phylogenetic and molecular characterization of H9N2 influenza isolates from
chickens in Northern China from 2007-2009.

Bi J, Deng G, Dong J, Kong F, Li X, Xu Q, Zhang M, Zhao L, Qiao J.

Department of Pathophysiology, College of Veterinary Medicine, China Agricultural
University, Beijing, People's Republic of China.

The repeated transmission to pigs and humans, and the long-term endemicity in
terrestrial poultry of H9N2 viruses in China lend urgency to the study of their
ecology and pathogenicity. In the present paper, we reported an H9N2 virus
sublineage isolated from chickens in northern China from 2007 to 2009 has high
lethality for mice. Phylogenetic analysis of the full genome indicated that six
representative H9N2 isolates shared high homology to each other, and they
clustered in the same sublineage with other H9N2 viruses isolated recently in
northern China. The isolates were double-reassortant viruses containing M genes
similar to A/Quail/Hong Kong/G1/97 (H9N2) and the other seven gene segments from 
A/Chicken/Shanghai/F/98 (H9N2). These six isolates were capable of replicating in
the lungs of infected chickens without producing observable clinical signs of
disease or death. However, they were highly lethal to mice with mortality rates
as high as 100% (14/14) without prior adaptation. The affected mice exhibited
severe respiratory syndromes and diffuse lung injury. The H9N2 viruses could be
detected in multiple organs of the infected mice, including hearts, livers,
spleens, lungs and kidneys. Our findings demonstrated that H9N2 viruses isolated 
from the chickens in northern China have established a stable sublineage with
enhanced pathogenicity to mice, suggesting that urgent attention will need to be 
paid to the transmission of H9N2 viruses from chickens to mammals.

PMCID: PMC2947496
PMID: 20927364  [PubMed - indexed for MEDLINE]
Þ

13597. J Gen Virol. 2011 Jan;92(Pt 1):36-50. doi: 10.1099/vir.0.024992-0. Epub 2010 Sep 
22.

Rapid evolution of low-pathogenic H9N2 avian influenza viruses following poultry 
vaccination programmes.

Park KJ, Kwon HI, Song MS, Pascua PN, Baek YH, Lee JH, Jang HL, Lim JY, Mo IP,
Moon HJ, Kim CJ, Choi YK.

College of Medicine and Medical Research Institute, Chungbuk National University,
12 Gaeshin-Dong Heungduk-Ku, Cheongju 361-763, Republic of Korea.

To investigate whether currently circulating H9N2 avian influenza viruses (AIVs) 
in domestic poultry have evolved in Korean poultry since 2007, genetic and
serological comparisons were conducted of H9N2 isolates from poultry
slaughterhouses from January 2008 to December 2009. The isolation rate was
relatively low in 2008 but increased gradually from January 2009 onwards. Genetic
and phylogenetic analyses revealed that reassortant viruses had emerged,
generating at least five novel genotypes, mostly containing segments of a
previously prevalent domestic H9N2 virus lineage (Ck/Korea/04116/04-like). It was
noteworthy that the N2 genes of some H9N2 isolates (genotypes D, E and F) were
derived from those of H3N2-like viruses commonly isolated among domestic ducks in
live-poultry markets. Animal challenge studies demonstrated that the
pathogenicity of Ck/Korea/SH0906/09 (genotype B) and Ck/Korea/SH0912/09 (genotype
F) in domestic avian species was altered due to reassortment. Furthermore,
serological analysis revealed that the isolates were antigenically distinct from 
previous Korean H9N2 viruses including Ck/Korea/01310/01. Such antigenic
diversity was illustrated further in experiments using H9N2-immunized chickens,
which could not inhibit the replication and transmission of challenge viruses
from each genotype. These results suggest that H9N2 viruses from domestic poultry
have undergone substantial evolution since 2007 by immune selection as a result
of vaccinal and natural immunity, coupled with reassortment. Taken together, this
study demonstrates that periodical updating of vaccine strains, based on
continuous surveillance data, is an important issue in order to provide
sufficient protectivity against AIV infections.

PMID: 20861321  [PubMed - indexed for MEDLINE]
Þ

13775. Bing Du Xue Bao. 2010 Jul;26(4):298-304.

[Genome sequencing and genetic analysis of a natural reassortant H5N1 subtype
avian influenza virus possessing H9N2 internal genes].

[Article in Chinese]

Gu M, Liu WB, Cao JP, Cao YZ, Zhang XR, Peng DX, Liu XF.

Key Laboratory of Animal Infectious Diseases, Yang Zhou University, Yang Zhou
225009, China. lucygu083@yahoo.com.cn

Abstract:One H5N1 subtype avian influenza virus, A/duck/Shandong/009/2008
(Dk/SD/009/08), was isolated from apparently healthy domestic ducks in some live 
bird market in East China during our epidemiological surveillance. To investigate
the genetic composition, Dk/SD/009/08 was subjected to genome sequencing. The
amino acid motif of cleavage site was "PLRERRRK-R/GL", which was consistent with 
the characterization of the HPAIV. According to the newest unified nomenclature
system of H5N1, Dk/SD/ 009/08 was classified into Clade 2.3.4. The BLAST results 
showed that four gene segments (HA, NA, NP and NS) had the highest nucleotide
identities with H5N1 subtype AIVs whereas the remaining four (PB2, PB1, PA and M)
displayed the closest relationship with H9N2 subtype. Therefore, Dk/SD/009/08
might be a natural reassortant virus. The phylogenetic analysis further indicated
that G1-like H9N2 subtype AIVs which was prevalent mainly in quails of Southern
China might provide the internal genes for Dk/ SD/009/08.

PMID: 20836384  [PubMed - indexed for MEDLINE]
Þ

14327. Vet Microbiol. 2010 Dec 15;146(3-4):215-25. doi: 10.1016/j.vetmic.2010.05.010.
Epub 2010 May 7.

Genotypic evolution and antigenic drift of H9N2 influenza viruses in China from
1994 to 2008.

Sun Y, Pu J, Jiang Z, Guan T, Xia Y, Xu Q, Liu L, Ma B, Tian F, Brown EG, Liu J.

Key Laboratory of Zoonosis of Ministry of Agriculture, College of Veterinary
Medicine, China Agricultural University, Beijing 100193, PR China.

H9N2 influenza viruses have been circulating in China since 1994, but a
systematic investigation of H9N2 in northern China has not been undertaken since 
2004. Here, using the sequences of 22 viruses we isolated from poultry and pigs
in northern China during 2003-2008, in combination with sequences available in a 
public database, we analyzed the evolution of H9N2 influenza viruses in China
from 1994 to 2008. Our findings demonstrated that the H9N2 viruses in China
underwent extensive reassortment, and novel genotypes continued to emerge. Among 
330 viruses, 54 genotypes were observed including 19 novel genotypes that have
not been recognized before, and major genotypes were further divided into five
series (BJ/94-, G1-, BG-, F/98- and Aq-series). Different epidemiological and
biological features among these series were recognized. The BJ/94- and
F/98-series viruses were circulating in both southern and northern China, while
the other three series viruses were mainly detected in southern China.
BJ/94-series influenza viruses predominated in China before 2000 and were
gradually replaced by F/98-series viruses that became the predominant viruses
since 2004. At least five antigenic groups could be identified over the study
period, during which a significant antigenic drift likely occurred between 2002
and 2003. Animal experiments demonstrated that F/98-series viruses were able to
replicate and transmit more effectively in chickens than BJ/94-series viruses.
The continuing evolution of H9N2 influenza viruses in China emphasizes the
importance of H9N2 influenza virus surveillance throughout this region to aid
pandemic prediction and prevention.

Copyright © 2010 Elsevier B.V. All rights reserved.

PMID: 20685047  [PubMed - indexed for MEDLINE]
Þ

14945. Virol J. 2010 Jun 24;7:137. doi: 10.1186/1743-422X-7-137.

Sequence and phylogenetic analysis of H7N3 avian influenza viruses isolated from 
poultry in Pakistan 1995-2004.

Abbas MA, Spackman E, Swayne DE, Ahmed Z, Sarmento L, Siddique N, Naeem K, Hameed
A, Rehmani S.

National Reference Laboratory for Poultry Diseases, ASI, NARC, Islamabad 45500,
Pakistan.

BACKGROUND: Avian influenza virus (AIV) infections have caused heavy economic
losses to the poultry industry in Pakistan as well as numerous other regions
worldwide. The first introduction of H7N3 AIV to Pakistan occurred during 1995,
since then H7N3, H9N2 and H5N1 AIVs have each been sporadically isolated. This
report evaluates the genetic origin of the H7N3 viruses from Pakistan collected
1995-2004 and how they disseminated within the country. To accomplish this we
produced whole genome sequences for 6 H7N3 viruses and data for the HA and NA
genes of an additional 7 isolates. All available sequence from H7N3 AIV from
Pakistan was included in the analysis.
RESULTS: Phylogenetic analysis revealed that there were two introductions of H7
into Pakistan and one N3 introduction. Only one of the H7 introductions appears
to have become established in poultry in Pakistan, while the other was isolated
from two separate outbreaks 6 years apart. The data also shows that reassortment 
has occurred between H7N3 and H9N2 viruses in the field, likely during
co-infection of poultry. Also, with the exception of these few reassortant
isolates, all 8 genes in the predominant H7N3 virus lineage have evolved to be
phylogenetically distinct.
CONCLUSIONS: Although rigorous control measures have been implemented in
commercial poultry in Pakistan, AIV is sporadically transmitted to poultry and
among the different poultry industry compartments (broilers, broiler breeders,
table egg layers). Since there is one primary H7 lineage which persists and that 
has reassorted with the H9N2 AIV in poultry, it suggests that there is a
reservoir with some link commercial poultry. On a general level, this offers
insight into the molecular ecology of AIV in poultry where the virus has
persisted despite vaccination and biosecurity. This data also illustrates the
importance of sustained surveillance for AIVs in poultry.

PMCID: PMC2901269
PMID: 20576101  [PubMed - indexed for MEDLINE]
Þ

15243. Avian Dis. 2010 Mar;54(1 Suppl):738-48.

Continuing evolution and interspecies transmission of influenza viruses in live
bird markets in Korea.

Lee HJ, Kwon JS, Lee DH, Lee YN, Youn HN, Lee YJ, Kim MC, Jeong OM, Kang HM, Kwon
JH, Lee JB, Park SY, Choi IS, Song CS.

Avian Disease Laboratory, College of Veterinary Medicine, Konkuk University, 1
Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea.

Live bird markets (LBMs) provide an ideal environment for the evolution and
interspecies transfer of avian influenza viruses (AIVs). In this study, we
analyzed AIVs present in LBMs in Korea during the winter seasons of 2006-08.
Sixty-five AIVs that belong to four hemagglutination (HA) subtypes ofAIV (H3, H4,
H6, and H9) were isolated from 644 pooled tissue or swab samples collected in
LBMs. Most H9 subtypes of AIVs were isolated from Galliformes (chickens, silky
fowls, pheasants, and guinea fowls), and other subtypes were isolated from
Anseriformes (Pekin ducks and mallards). In addition, we obtained a single H3N2
virus from nasal swabs of dogs sold in LBMs, and the virus was genetically
identical to the canine influenza virus (CIV) isolated from pet dogs in Korea.
Phylogenetic analysis suggests that the Korean H9N2 viruses prevalent in chickens
have provided their gene segments to AIVs circulating in ducks. These gene
transfers facilitated reassortment events among AIVs and likely generated the
ancestors of CIV in Korea. An animal challenge study using chickens, quail, mice,
and dogs had shown that the H4 and H6 subtypes could replicate in mice and that
some H4 and H6 viruses could replicate in chickens without preadaptation. In
addition, two H3 subtype viruses (H3N2 and H3N8) induced interstitial pneumonia
that accompanied clinical signs and seroconversion in dogs. Our findings indicate
that the newly evolved AIVs have been continuously generated by reassortment in
ducks, and these reassortments could result in expanding the host range of AIVs.

PMID: 20521725  [PubMed - indexed for MEDLINE]
Þ

15316. Avian Dis. 2010 Mar;54(1 Suppl):313-22.

Phylogenetic analysis of influenza A viruses (H6N8, H1N8, H4N2, H9N2, H10N7)
isolated from wild birds, ducks, and ostriches in South Africa from 2007 to 2009.

Abolnik C, Gerdes GH, Sinclair M, Ganzevoort BW, Kitching JP, Burger CE, Romito
M, Dreyer M, Swanepoel S, Cumming GS, Olivier AJ.

Agricultural Research Council-Onderstepoort Veterinary Institute, Private Bag
X05, Onderstepoort, Pretoria 0110, South Africa. abolnikc@arc.agric.za

Influenza A strains emerging from wild birds are a constant threat to South
Africa's valuable ostrich industry. In 2004 and again in 2006, low pathogenicity 
avian influenza H5N2 strains introduced from a wild bird reservoir mutated in
ostriches to high pathogenicity avian influenza (HPAI), with serious economic
consequences and export bans imposed by the European Union. Although no outbreaks
of notifiable avian influenza have occurred in South Africa since 2006, the H9N2 
virus caused a localized outbreak where ostriches displayed symptoms of green
urine, depression, and mild morbidity. Most recently, an outbreak of H10N7 in
farmed Pekin ducks (Anas platyrhynchos domestica) caused increased mortalities,
but this was exacerbated by a secondary Escherichia coli infection, because an
intravenous pathogenicity index of 0.00 was recorded. Each of the eight gene
segments of the five strains isolated from 2007 to 2009 from farmed ostriches in 
the Oudtshoorn region (H6N8, H9N2), Pekin ducks (H10N7, Joostenburgvlakte
region), and wild Egyptian geese (Alopochen aegypticus; H1N8, Baberspan wetlands;
H4N2, Oudtshoorn region) were sequenced, genetically analyzed, and compared to
previous South African isolates and viruses in the public data banks. An H5N8
strain was also detected by reverse-transcription PCR in cloacal swabs from swift
terns (Sterna bergii) in the Mosselbaai region during 2007, although a virus
could not be isolated. Initial phylogenetic results indicate that H6N8 and H9N2
ostrich and H10N7 Pekin duck viruses originated in the wild bird population that 
is geographically dispersed throughout southern Africa, based on the reassortment
of viral genes from birds sampled outside of the ostrich farming areas. No
evidence of internal genes associated with Asian HPAI H5N1 strains were detected 
in the South African isolates.

PMID: 20521652  [PubMed - indexed for MEDLINE]
Þ

15996. Avian Dis. 2010 Mar;54(1):22-7.

Evaluation of transmission route and replication efficiency of H9N2 avian
influenza virus.

Shi H, Ashraf S, Gao S, Lu J, Liu X.

Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou
University, Yangzhou, Jiangsu 225009, People's Republic of China.

A/Chicken/Beijing/1/94 (Ck/BJ/1/94) avian influenza virus (AIV), a prototype of
the H9N2 subtype, is phylogenetically similar in its hemagglutinin (HA) and
neuraminidase (NA) genes to A/Chicken/Shanghai/F/98 (Ck/SH/F/98; H9N2) AIV, a
natural reassortant between different sublineages. To understand the role of HA
and NA genes in the airborne transmission of H9N2 AIV, we compared the
transmission route and the relative replication efficiency of these strains in
specific-pathogen-free chickens. Three recombinant viruses were generated by
reverse genetics, containing the HA and NA genes (or both) from
A/Chicken/Guangdong/SS/94 (Ck/GD/SS/94), in a background of internal genes
derived from Ck/SH/F/98. Inoculated chickens were kept in either direct or
indirect contact with uninoculated chickens, and viral shedding and titers were
monitored. The results showed that Ck/GD/SS/94 lacks the ability to be
transmitted through indirect contact, while Ck/SH/F/98 could be transmitted
indirectly. Recombinant virus (RF/SSHA), containing the internal genes of
Ck/SH/F/98 and the HA gene of Ck/GD/ SS/94, resulted in decreased viral titers in
lung tissue as compared to the parental strain. Interestingly, substituting the
NA gene, or both the NA and HA genes, of Ck/SH/F/98 with that of Ck/GD/SS/94
completely abolished the airborne transmission of the recombinant RF/SSNA and
RF/SSHA/SSNA. In conclusion, Ck/SH/F/98 acquired the ability of airborne
transmission and replicated with a higher efficiency in the respiratory tract of 
the chickens. Our data indicated that the NA gene of Ck/SH/F/98 can affect virus 
replication and, therefore, indirectly affect the transmission for the gene
constellations of these viruses.

PMID: 20408394  [PubMed - indexed for MEDLINE]
Þ

16071. J Gen Virol. 2010 Aug;91(Pt 8):1978-83. doi: 10.1099/vir.0.021238-0. Epub 2010
Apr 14.

Characterization of H5N2 influenza viruses isolated in South Korea and their
influence on the emergence of a novel H9N2 influenza virus.

Kim HR, Park CK, Oem JK, Bae YC, Choi JG, Lee OS, Lee YJ.

Animal Disease Diagnosis Center, National Veterinary Research and Quarantine
Service, 335 Joongangro, Manangu, Anyangsi, Gyeonggido 430-824, Republic of
Korea.

We characterized low pathogenic avian influenza (LPAI) H5N2 and H9N2 viruses
isolated in South Korea from 2008 to 2009. Genetic analysis of the H5N2 viruses
isolated from wild birds and domestic ducks demonstrated that they were related
to the recently isolated southern Chinese LPAI H5 viruses and various influenza
viruses circulating in Eurasia. Three H9N2 viruses obtained at live bird markets 
and duck farms were reassortant viruses generated from the H5N2 viruses of
domestic ducks and the H9N2 virus endemic in Korean chickens. The H5N2 viruses
did not replicate well in experimentally infected chickens and mice, but novel
H9N2 viruses, without pre-adaptation, were recovered at high titres in chickens. 
Our results show that reassortment between H5N2 and H9N2 viruses must have
occurred in domestic ducks and may have contributed to the diversity expansion of
the gene pool, which has potential to alter the pathogenicity and host range of
the influenza virus.

PMID: 20392898  [PubMed - indexed for MEDLINE]
Þ

16387. Virus Res. 2010 Jul;151(1):26-32. doi: 10.1016/j.virusres.2010.03.010. Epub 2010 
Mar 25.

Diversified reassortant H9N2 avian influenza viruses in chicken flocks in
northern and eastern China.

Huang Y, Hu B, Wen X, Cao S, Gavrilov BK, Du Q, Khan MI, Zhang X.

Institute of Animal Science and Veterinary Medicine, Shandong Academy of
Agricultural Science, 10 Sang Yuan Road, Jinan, Shandong 250001, China.

According to our previous study of the M genes of H9N2 avian influenza viruses
(AIV) in infected chickens, A/Quail/Hong Kong/G1/97 (G1 97)-like M genes newly
emerged in northern and eastern China in addition to the existing A/chicken/Hong 
Kong/Y280/97 (Y280)-like lineage M genes. To systematically track the genesis and
evolution of H9N2 viruses in this region, whole genome sequences of seventeen
H9N2 isolates were obtained and their phylogenetic properties were determined.
Phylogenetic analysis revealed several newly emerged lineages of gene segments in
addition to the Y280-like and A/chicken/Shanghai/F/98(F 98)-like lineages, which 
are prevailing in northern and eastern China according to previous reports.
Reassortments among these gene segments generated five novel genotypes of H9N2
viruses that have not been reported before in China. The emerging genotypes of
H9N2 viruses in this region indicate that H9N2 virus genes undergo active
evolution, particularly their internal genes, which raises concern for their
likely contribution to gene reassortment and production of AIVs with new
properties. Our study provides valuable insight into the prevalence of H9N2
viruses in northern and eastern China and demonstrates the need of long-term
monitoring of the evolution of H9N2 AIV.

PMID: 20347894  [PubMed - indexed for MEDLINE]
Þ

17746. Exp Mol Med. 2010 Feb 28;42(2):116-21. doi: 10.3858/emm.2010.42.2.013.

Reverse genetic platform for inactivated and live-attenuated influenza vaccine.

Jung EJ, Lee KH, Seong BL.

Department of Biotechnology, College of Bioscience and Biotechnology, Translation
Research Center for Protein Function Control, Yonsei University, Seoul 120-749,
Korea.

Influenza vaccine strains have been traditionally developed by annual
reassortment between vaccine donor strain and the epidemic virulent strains. The 
classical method requires screening and genotyping of the vaccine strain among
various reassortant viruses, which are usually laborious and time-consuming. Here
we developed an efficient reverse genetic system to generate the 6:2 reassortant 
vaccine virus from cDNAs derived from the influenza RNAs. Thus, cDNAs of the two 
RNAs coding for surface antigens, haemagglutinin and neuraminidase from the
epidemic virus and the 6 internal genes from the donor strain were transfected
into cells and the infectious viruses of 6:2 defined RNA ratio were rescued. X-31
virus (a high- growth virus in embryonated eggs) and its cold-adapted strain X-31
ca were judiciously chosen as donor strains for the generation of inactivated
vaccine and live-attenuated vaccine, respectively. The growth properties of these
recombinant viruses in embryonated chicken eggs and MDCK cell were
indistinguishable as compared to those generated by classical reassortment
process. Based on the reverse genetic system, we generated 6+2 reassortant avian 
influenza vaccine strains corresponding to the A/Chicken/Korea/ MS96 (H9N2) and
A/Indonesia/5/2005 (H5N1). The results would serve as technical platform for the 
generation of both injectable inactivated vaccine and the nasal spray live
attenuated vaccine for the prevention of influenza epidemics and pandemics.

PMCID: PMC2827828
PMID: 20054235  [PubMed - indexed for MEDLINE]
Þ

17937. Arch Virol. 2010 Feb;155(2):229-41. doi: 10.1007/s00705-009-0577-4. Epub 2009 Dec
22.

Active reassortment of H9 influenza viruses between wild birds and live-poultry
markets in Korea.

Moon HJ, Song MS, Cruz DJ, Park KJ, Pascua PN, Lee JH, Baek YH, Choi DH, Choi YK,
Kim CJ.

College of Veterinary Medicine, Chungnam National University, 220 Gung-Dong,
Yuseong-Gu, Daejeon 305-764, Republic of Korea.

Surveillance of H9 avian influenza viruses in Korean live-poultry markets from
September 2004 through October 2007 was carried out to investigate active
reassortment between wild migratory birds and domestic poultry in Korea.
Antigenic and phylogenetic analyses showed that most of the isolates belong to
the previous Korean H9N2-like lineage and differ from the southeastern Chinese
strains. Interestingly, the Ck/Korea/LPM77/06 group (genotype B) and
Dk/Korea/LPM248/07 group (genotype C) showed unique properties distinct from
those of other Korean H9N2 strains. Although the HA genes of these two groups
belong to Korean H9N2-like lineage, the PA genes closely resemble those of the
Chinese Y280-like lineage. In addition, the PB2 genes of the Dk/Korea/LPM248/07
group were closely related to those isolated from migratory birds. Several other 
isolates also clustered within the H9N2 B genotype, an indication that there are 
at least two predominant H9N2 influenza genotypes in Korea. Another isolate,
Dk/Korea/LPM71/06, was identified as an H9N1 subtype, the first ever discovered
in Korean live-poultry markets. These findings reveal that reassortment of Korean
H9 influenza viruses has occurred frequently in live-poultry markets and may have
been mediated by introduction of genetic material from viruses circulating among 
migratory wild birds to domestic birds. Consequently, the new dominant H9N2
genotypes have become established in Korean live-poultry markets through
continued reassortment.

PMID: 20033463  [PubMed - indexed for MEDLINE]
Þ

18428. Arch Virol. 2010;155(1):107-10. doi: 10.1007/s00705-009-0535-1. Epub 2009 Nov 28.

Readaptation of a low-virulence influenza H9 escape mutant in mice: the role of
changes in hemagglutinin as revealed by site-specific mutagenesis.

Ilyushina NA, Rudneva IA, Khalenkov AM, Timofeeva TA, Krylov PS, Webster RG,
Kaverin NV.

The D.I. Ivanovsky Institute of Virology, 16 Gamaleya Str., 123098 Moscow,
Russia.

In our earlier studies, we showed that an escape mutant of mouse-adapted H9N2
influenza virus carrying a T198N amino acid change in heamagglutinin (HA) has a
lowered virulence for mice. The readaptation of this mutant to mice was
associated with N198S or N198D reverse mutations. In this study, single-gene
reassortants having HA gene of the wild-type virus, its low-virulence escape
mutant, or a readapted variant were generated by site-specific mutagenesis and
assayed for virulence. The results showed that antibody-selected mutations in the
HA of H9 influenza virus can decrease mortality and virus accumulation in mouse
lungs, though not in nasal turbinates, and the effect may be compensated by
reverse mutations in the course of passaging.

PMCID: PMC3117582
PMID: 19946717  [PubMed - indexed for MEDLINE]
Þ

18932. Vopr Virusol. 2009 Sep-Oct;54(5):14-9.

[Amino acid substitutions in the hemagglutinin of H5 influenza virus changing the
antigenic specificity and virus virulence].

[Article in Russian]

Krylov PS, Rudneva IA, Timofeeva TA, Shilov AA, Ignat'eva AV, Govorkova EA,
Kaverin NV.

In our earlier studies, we mapped the hemagglutinin antigenic epitopes of H5
influenza virus by selecting mutants resistant to the neutralizing effect of the 
antibody (escape mutants). Several escape mutants were shown to have a lowered
virulence for mice. The readaptation of low-virulent escape mutants to mice
resulted in the restoration of virulence. In the present communication. We
present data on the assay of virulence of single-gene reassortants containing HA 
genes of the wild-type virus, low-virulent escape mutant, or re-adapted variant, 
and the other genes of a mouse-adapted H9N2 Influenza virus. The results
demonstrate that the amino acid change S145F (H3 numbering) in the hemagglutinin 
ensuring the resistance to a monoclonal antibody can be deleterious to virulence,
and that the damaging effect on virulence may be compensated for by additional
amino acid changes in position 186 in the hemagglutinin arising in the course of 
virus passaging in mice. The data indicate that the compensational mutations
restoring the pathogenic potential of antigenic variants may be regarded as an
additional factor in the evolution of influenza virus hemagglutinin.

PMID: 19882897  [PubMed - indexed for MEDLINE]
Þ

19344. J Gen Virol. 2010 Jan;91(Pt 1):208-19. doi: 10.1099/vir.0.015800-0. Epub 2009 Oct
7.

Genetic relatedness of H6 subtype avian influenza viruses isolated from wild
birds and domestic ducks in Korea and their pathogenicity in animals.

Kim HR, Lee YJ, Lee KK, Oem JK, Kim SH, Lee MH, Lee OS, Park CK.

Animal Disease Diagnosis Center, National Veterinary Research and Quarantine
Service, 335 Joongangro, Manangu, Anyangsi, Gyeonggido 430-824, Republic of
Korea.

We report the genetic characterization of H6 avian influenza (AI) viruses
isolated from domestic ducks and wild birds in Korea between April 2008 and April
2009. A phylogenetic analysis showed that the H6N1 viruses of wild birds and
domestic ducks were of the same genotype (K-1) and were similar to the H6N1 virus
isolated from a live poultry market in 2003, as six of the eight gene segments of
those viruses had a common source. However, the H6N2 viruses of domestic poultry 
were separated into four genotypes (K-2a, K-2b, K-2c and K-2d) by at least a
triple reassortment between influenza viruses of low pathogenicity from Korean
poultry (H9N2 and H3N2) and viruses from aquatic birds. In an experimental
infection of animals, certain H6 AI viruses replicated well in chickens and mice 
without pre-adaptation, indicating that H6 virus pathogenicity has the potential 
to be altered due to multiple reassortments, and that these reassortments could
result in interspecies transmission to mammals.

PMID: 19812266  [PubMed - indexed for MEDLINE]
Þ

19545. Vet Microbiol. 2010 Jan 27;140(3-4):237-45. doi: 10.1016/j.vetmic.2009.08.022.
Epub 2009 Aug 26.

(Highly pathogenic) avian influenza as a zoonotic agent.

Kalthoff D, Globig A, Beer M.

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut Insel Riems,
Südufer 10, 17493 Greifswald-Insel Riems, Germany.

Zoonotic agents challenging the world every year afresh are influenza A viruses. 
In the past, human pandemics caused by influenza A viruses had been occurring
periodically. Wild aquatic birds are carriers of the full variety of influenza
virus A subtypes, and thus, most probably constitute the natural reservoir of all
influenza A viruses. Whereas avian influenza viruses in their natural avian
reservoir are generally of low pathogenicity (LPAIV), some have gained virulence 
by mutation after transmission and adaptation to susceptible gallinaceous
poultry. Those so-called highly pathogenic avian influenza viruses (HPAIV) then
cause mass die-offs in susceptible birds and lead to tremendous economical losses
when poultry is affected. Besides a number of avian influenza virus subtypes that
have sporadically infected mammals, the HPAIV H5N1 Asia shows strong zoonotic
characteristics and it was transmitted from birds to different mammalian species 
including humans. Theoretically, pandemic viruses might derive directly from
avian influenza viruses or arise after genetic reassortment between viruses of
avian and mammalian origin. So far, HPAIV H5N1 already meets two conditions for a
pandemic virus: as a new subtype it has been hitherto unseen in the human
population and it has infected at least 438 people, and caused severe illness and
high lethality in 262 humans to date (August 2009). The acquisition of efficient 
human-to-human transmission would complete the emergence of a new pandemic virus.
Therefore, fighting H5N1 at its source is the prerequisite to reduce pandemic
risks posed by this virus. Other influenza viruses regarded as pandemic
candidates derive from subtypes H2, H7, and H9 all of which have infected humans 
in the past. Here, we will give a comprehensive overview on avian influenza
viruses in concern to their zoonotic potential.

Copyright 2009 Elsevier B.V. All rights reserved.

PMID: 19782482  [PubMed - indexed for MEDLINE]
Þ

19640. Curr Top Microbiol Immunol. 2009;333:3-24. doi: 10.1007/978-3-540-92165-3_1.

Pandemic influenza as a current threat.

Yen HL, Webster RG.

Division of Virology, Department of Infectious Diseases, St. Jude Children's
Research Hospital, 262, Danny Thomas Place, Memphis, TN 38105, USA.

Pandemics of influenza emerge from the aquatic bird reservoir, adapt to humans,
modify their severity, and cause seasonal influenza. The catastrophic Spanish
H1N1 virus may have obtained all of its eight gene segments from the avian
reservoir, whereas the Asian H2N2 and the Hong Kong H3N2 pandemics emerged by
reassortment between the circulating human virus and an avian H2 or H3 donor. Of 
the 16 hemagglutinin subtypes, the H2, H5, H6, H7, and H9 viruses are considered 
to have pandemic potential. While this chapter focuses on the evolution of the
Asian highly pathogenic (HP) H5N1 influenza virus, other subtypes are also
considered. The unique features of the HP H5N1 viruses that have devastated the
domestic poultry of Eurasia are discussed. Although they transmit poorly to
humans, they continue to kill more than 60% of infected persons. It is unknown
whether HP H5N1 will acquire human pandemic status; if it does not, another
subtype eventually will do so, for a future influenza pandemic is inevitable.

PMID: 19768398  [PubMed - indexed for MEDLINE]
Þ

20207. J Clin Microbiol. 2009 Oct;47(10):3294-300. doi: 10.1128/JCM.00355-09. Epub 2009 
Aug 5.

Genetic evolution of H9 subtype influenza viruses from live poultry markets in
Shanghai, China.

Ge FF, Zhou JP, Liu J, Wang J, Zhang WY, Sheng LP, Xu F, Ju HB, Sun QY, Liu PH.

Shanghai Animal Disease Control Center, Shanghai 201103, People's Republic of
China.

H9N2 influenza viruses have become established and maintain long-term endemicity 
in poultry. The complete genomes of seven avian H9N2 influenza viruses were
characterized. These seven influenza virus isolates were obtained from live
poultry markets in Shanghai, China, in 2002 and from 2006 to 2008. Genetic
analysis revealed that all seven isolates had an RSSR motif at the cleavage site 
of hemagglutinin (HA), indicating low pathogenicity in chickens. Phylogenetic
analyses indicated that the seven avian H9N2 viruses belonged to the lineage
represented by Duck/Hong Kong/Y280/97 (H9N2), a virus belonging to the
Chicken/Beijing/1/94-like (H9N2) lineage, and that they are all quadruple
reassortants consisting of genes from different lineages. The six internal genes 
of the isolates possessed H5N1-like sequences, indicating that they were
reassortants of H9 and H5 viruses. All of the viruses had nonstructural (as well 
as HA and neuraminidase) genes derived from the Duck/Hong Kong/Y280/97-like virus
lineage but also had other genes of mixed avian virus origin, including genes
similar to those of H5N1 viruses (Gs/GD-like). The infected chickens showed no
signs of disease. These results show the genetic and biological diversity of H9N2
viruses in Shanghai and support their potential role as pandemic influenza
agents.

PMCID: PMC2756938
PMID: 19656985  [PubMed - indexed for MEDLINE]
Þ

20783. J Virol. 2009 Sep;83(17):8428-38. doi: 10.1128/JVI.00659-09. Epub 2009 Jun 24.

A novel genotype H9N2 influenza virus possessing human H5N1 internal genomes has 
been circulating in poultry in eastern China since 1998.

Zhang P, Tang Y, Liu X, Liu W, Zhang X, Liu H, Peng D, Gao S, Wu Y, Zhang L, Lu
S, Liu X.

Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou
University, 48 East Wenhui Road, Yangzhou, Jiangsu 225009, China.

Many novel reassortant influenza viruses of the H9N2 genotype have emerged in
aquatic birds in southern China since their initial isolation in this region in
1994. However, the genesis and evolution of H9N2 viruses in poultry in eastern
China have not been investigated systematically. In the current study, H9N2
influenza viruses isolated from poultry in eastern China during the past 10 years
were characterized genetically and antigenically. Phylogenetic analysis revealed 
that these H9N2 viruses have undergone extensive reassortment to generate
multiple novel genotypes, including four genotypes (J, F, K, and L) that have
never been recognized before. The major H9N2 influenza viruses represented by
A/Chicken/Beijing/1/1994 (Ck/BJ/1/94)-like viruses circulating in poultry in
eastern China before 1998 have been gradually replaced by
A/Chicken/Shanghai/F/1998 (Ck/SH/F/98)-like viruses, which have a genotype
different from that of viruses isolated in southern China. The similarity of the 
internal genes of these H9N2 viruses to those of the H5N1 influenza viruses
isolated from 2001 onwards suggests that the Ck/SH/F/98-like virus may have been 
the donor of internal genes of human and poultry H5N1 influenza viruses
circulating in Eurasia. Experimental studies showed that some of these H9N2
viruses could be efficiently transmitted by the respiratory tract in chicken
flocks. Our study provides new insight into the genesis and evolution of H9N2
influenza viruses and supports the notion that some of these viruses may have
been the donors of internal genes found in H5N1 viruses.

PMCID: PMC2738149
PMID: 19553328  [PubMed - indexed for MEDLINE]
Þ

20835. Arch Virol. 2009;154(8):1203-10. doi: 10.1007/s00705-009-0425-6. Epub 2009 Jun
19.

Generation and evaluation of an H9N1 influenza vaccine derived by reverse
genetics that allows utilization of a DIVA strategy for control of H9N2 avian
influenza.

Wu R, Chen Q, Zheng L, Chen J, Sui Z, Guan Y, Chen Z.

State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of
Sciences, 430071 Wuhan, Hubei, China. wurui1977@126.com

H9N2 avian influenza viruses have circulated widely in domestic poultry around
the world, and their outbreaks have resulted in heavy morbidity and mortality. In
addition, H9N2 avian influenza viruses were transmitted directly from birds to
humans in Hong Kong and mainland China during 1998 and 2003, which prompted the
public health authorities to seek protective strategies to control H9N2 influenza
viruses. In this study, we attempted to develop a DIVA (differentiating infected 
and vaccinated animals) strategy for H9N2 avian influenza viruses. This strategy 
does not interfere with serological monitoring and allows effective control of
H9N2 avian influenza. We generated a reassortant H9N1 influenza vaccine strain by
reverse genetics and employed an enzyme-linked immunosorbent assay (ELISA) with a
truncated N1 antigen expressed in E. coli to differentiate between vaccinated and
naturally infected animals. Immunization of BALB/c mice with the inactivated
reassortant H9N1 vaccine conferred protection against lethal challenge with H9N2 
viruses. Meanwhile, the ELISA can be used to distinguish between vaccination and 
natural infection quickly and easily. Therefore, this study has opened up a new
avenue for the control of H9N2 avian influenza.

PMID: 19543688  [PubMed - indexed for MEDLINE]
Þ

20976. PLoS One. 2009 Jun 11;4(6):e5788. doi: 10.1371/journal.pone.0005788.

Novel genotypes of H9N2 influenza A viruses isolated from poultry in Pakistan
containing NS genes similar to highly pathogenic H7N3 and H5N1 viruses.

Iqbal M, Yaqub T, Reddy K, McCauley JW.

Division of Microbiology, Institute for Animal Health, Compton Laboratory,
Compton, Newbury, Berkshire, UK. munir.iqbal@bbsrc.ac.uk

The impact of avian influenza caused by H9N2 viruses in Pakistan is now
significantly more severe than in previous years. Since all gene segments
contribute towards the virulence of avian influenza virus, it was imperative to
investigate the molecular features and genetic relationships of H9N2 viruses
prevalent in this region. Analysis of the gene sequences of all eight RNA
segments from 12 viruses isolated between 2005 and 2008 was undertaken. The
hemagglutinin (HA) sequences of all isolates were closely related to H9N2 viruses
isolated from Iran between 2004 and 2007 and contained leucine instead of
glutamine at position 226 in the receptor binding pocket, a recognised marker for
the recognition of sialic acids linked alpha2-6 to galactose. The neuraminidase
(NA) of two isolates contained a unique five residue deletion in the stalk (from 
residues 80 to 84), a possible indication of greater adaptation of these viruses 
to the chicken host. The HA, NA, nucleoprotein (NP), and matrix (M) genes showed 
close identity with H9N2 viruses isolated during 1999 in Pakistan and clustered
in the A/Quail/Hong Kong/G1/97 virus lineage. In contrast, the polymerase genes
clustered with H9N2 viruses from India, Iran and Dubai. The NS gene segment
showed greater genetic diversity and shared a high level of similarity with NS
genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian
lineages. These results indicate that during recent years the H9N2 viruses have
undergone extensive genetic reassortment which has led to the generation of H9N2 
viruses of novel genotypes in the Indian sub-continent. The novel genotypes of
H9N2 viruses may play a role in the increased problems observed by H9N2 to
poultry and reinforce the continued need to monitor H9N2 infections for their
zoonotic potential.

PMCID: PMC2690689
PMID: 19517011  [PubMed - indexed for MEDLINE]
Þ

21353. Vaccine. 2009 May 21;27(24):3189-94. doi: 10.1016/j.vaccine.2009.03.065. Epub
2009 Apr 10.

Novel use of a N2-specific enzyme-linked immunosorbent assay for differentiation 
of infected from vaccinated animals (DIVA)-based identification of avian
influenza.

Kwon JS, Kim MC, Jeong OM, Kang HM, Song CS, Kwon JH, Lee YJ.

Avian Disease Division, National Veterinary Research and Quarantine Service,
Gyeonggi-do, 430-824, Republic of Korea.

Proper vaccination with validated companion differentiation of infected from
vaccinated animals (DIVA) tests using a vaccine containing a heterologous
neuraminidase to the field virus can be effective to control avian influenza
(AI). However, indirect immunofluorescent assay, the only field validated DIVA
test, has limitations to be set up as high throughput screening test and the
assay requires subjective interpretation of the results. To apply the DIVA
strategy to the Korean H9N2 low pathogenic AI (LPAI) vaccine program and overcome
these limitations, we generated a reassortant H9N8 virus (rgH9N8) vaccine using
plasmid-based reverse genetics and developed a novel N2-specific enzyme-linked
immunosorbent assay (N2-ELISA). The rgH9N8 vaccine showed adequate immunogenicity
and protection, and the optimized N2-ELISA showed that the sensitivity was 97.0% 
and specificity was 96.4% compared with a hemagglutination inhibition test. In
vaccination-challenge experiments in specific pathogen-free chickens, the sera of
chickens vaccinated with rgH9N8 vaccine and uninfected were negative by the
N2-ELISA (S/P< or =0.4), whereas infected sera with H9N2 were positive (S/P>0.4).
These results suggest that the rgH9N8 vaccine and the companion DIVA test,
N2-ELISA, allow the utilization of the DIVA strategy for the control of H9N2 LPAI
infections in Korea.

PMID: 19446190  [PubMed - indexed for MEDLINE]
Þ

21667. Proc Natl Acad Sci U S A. 2009 May 5;106(18):7565-70. doi:
10.1073/pnas.0900877106. Epub 2009 Apr 20.

Minimal molecular constraints for respiratory droplet transmission of an
avian-human H9N2 influenza A virus.

Sorrell EM, Wan H, Araya Y, Song H, Perez DR.

Department of Veterinary Medicine, University of Maryland, College Park, and
Virginia-Maryland Regional College of Veterinary Medicine, 8075 Greenmead Drive, 
College Park, MD 20742, USA.

Pandemic influenza requires interspecies transmission of an influenza virus with 
a novel hemagglutinin (HA) subtytpe that can adapt to its new host through either
reassortment or point mutations and transmit by aerosolized respiratory droplets.
Two previous pandemics of 1957 and 1968 resulted from the reassortment of low
pathogenic avian viruses and human subtypes of that period; however, conditions
leading to a pandemic virus are still poorly understood. Given the endemic
situation of avian H9N2 influenza with human-like receptor specificity in Eurasia
and its occasional transmission to humans and pigs, we wanted to determine
whether an avian-human H9N2 reassortant could gain respiratory transmission in a 
mammalian animal model, the ferret. Here we show that following adaptation in the
ferret, a reassortant virus carrying the surface proteins of an avian H9N2 in a
human H3N2 backbone can transmit efficiently via respiratory droplets, creating a
clinical infection similar to human influenza infections. Minimal changes at the 
protein level were found in this virus capable of respiratory droplet
transmission. A reassortant virus expressing only the HA and neuraminidase (NA)
of the ferret-adapted virus was able to account for the transmissibility,
suggesting that currently circulating avian H9N2 viruses require little
adaptation in mammals following acquisition of all human virus internal genes
through reassortment. Hemagglutinin inhibition (HI) analysis showed changes in
the antigenic profile of the virus, which carries profound implications for
vaccine seed stock preparation against avian H9N2 influenza. This report
illustrates that aerosolized respiratory transmission is not exclusive to current
human H1, H2, and H3 influenza subtypes.

PMCID: PMC2670882
PMID: 19380727  [PubMed - indexed for MEDLINE]
Þ

22075. Virus Genes. 2009 Jun;38(3):398-403. doi: 10.1007/s11262-009-0339-0. Epub 2009
Feb 27.

Evolution analysis of the matrix (M) protein genes of 17 H9N2 chicken influenza
viruses isolated in northern China during 1998-2008.

Huang Y, Hu B, Wen X, Cao S, Xu D, Zhang X, Khan MI.

Institute of Animal Science and Veterinary Medicine, Shandong Academy of
Agricultural Science, Jinan, Shandong, 250100, China.

Matrix (M) protein genes of 17 H9N2 avian influenza viruses (AIVs) isolated from 
chickens in northern China during the last 10 years were completely sequenced and
phylogenetically analyzed. Homology of nucleotide sequences in the M gene of 17
isolates was 92.7-99.9%. Phylogenetic analysis showed that 11 of the tested M
genes belong to the A/chicken/HongKong/Y280/97 (Y280)-like lineage, while the
other six belong to the A/Quail/HongKong/G1/97 (G1)-like lineage. This is also
the first time that a G1-like M gene of a H9N2 virus was detected in chicken
flocks in northern China. These newly appearing changes in M genes may be due to 
reassortment events of AIVs, or they may have come from the H9N2 strains of
southern China which surged in northern China after translocation. An analysis of
the viral amino acid sequence of M2 protein has revealed substitution of S31N in 
two isolates, which is the molecular characterization of amantadine resistance in
AIVs. Results of this study suggest that long-term monitoring should be continued
to track the transmission and evolution of H9N2 AIVs in chickens in China.

PMID: 19247825  [PubMed - indexed for MEDLINE]
Þ

22968. J Gen Virol. 2008 Dec;89(Pt 12):3102-12. doi: 10.1099/vir.0.2008/005652-0.

Characterization of H9N2 influenza viruses isolated from vaccinated flocks in an 
integrated broiler chicken operation in eastern China during a 5 year period
(1998-2002).

Zhang P, Tang Y, Liu X, Peng D, Liu W, Liu H, Lu S, Liu X.

Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou
University, Yangzhou 225009, PR China.

In the current study, we characterized H9N2 influenza viruses isolated from
vaccinated flocks in an integrated broiler chicken operation during a 5 year
period (1998-2002). Phylogenetic analysis of the 8 genes of 11 representative
viruses showed that they all shared high similarity to that of the first isolate,
A/Chicken/Shanghai/F/1998 (Ck/SH/F/98), and clustered to the same lineages.
Furthermore, all 11 viruses had a 9 nt deletion between positions 206 and 214 of 
the neuraminidase gene. These genetic characteristics strongly suggest that these
viruses are descendants of the first isolate. In addition, our study also showed 
that the H9N2 viruses circulating in the operation during this 5 year period were
evolving, as shown by antigenic variations between viruses manifested by
reactivity with polyclonal antisera and monoclonal antibodies, by
haemagglutination with erythrocytes from different animals, by amino acid
differences in haemagglutinin and neuraminidase proteins, and by variation in
their ability to replicate in the respiratory and intestinal tract and to be
transmitted by aerosol. Phylogenetic analysis revealed that the internal genes
from some H5N1 viruses of duck origin clustered together with those from H9N2
virus and that the RNP genes of these H5N1 viruses isolated after 2001 are more
closely related to the genes of the Ck/SH/F/98-like H9N2 viruses, indicating more
recent reassortment events between these two subtypes of viruses. Continuous
surveillance of influenza virus in poultry and waterfowl is critical for
monitoring the genesis and emergence of potentially pandemic strains in this
region.

PMID: 19008399  [PubMed - indexed for MEDLINE]
Þ

23246. J Clin Microbiol. 2008 Nov;46(11):3788-90. doi: 10.1128/JCM.01427-08. Epub 2008
Sep 24.

Emergence of amantadine-resistant H3N2 avian influenza A virus in South Korea.

Lee J, Song YJ, Park JH, Lee JH, Baek YH, Song MS, Oh TK, Han HS, Pascua PN, Choi
YK.

College of Medicine and Medical Research Institute, Chungbuk National University,
12 Gaeshin-Dong Heungduk-Ku, Cheongju 361-763, Republic of Korea.

We found a relatively high frequency of unique amantadine-resistant H3N2 and H9N2
avian influenza viruses (Val27Ile on M2 protein) isolated from live poultry
markets in South Korea and confirmed that a Val27Ile single substitution in the
M2 protein is enough to acquire the amantadine resistance phenotype by using
reverse-genetically created human-avian reassortant viruses.

PMCID: PMC2576612
PMID: 18845822  [PubMed - indexed for MEDLINE]
Þ

23666. PLoS One. 2008 Aug 13;3(8):e2923. doi: 10.1371/journal.pone.0002923.

Replication and transmission of H9N2 influenza viruses in ferrets: evaluation of 
pandemic potential.

Wan H, Sorrell EM, Song H, Hossain MJ, Ramirez-Nieto G, Monne I, Stevens J,
Cattoli G, Capua I, Chen LM, Donis RO, Busch J, Paulson JC, Brockwell C, Webby R,
Blanco J, Al-Natour MQ, Perez DR.

Department of Veterinary Medicine, University of Maryland, College Park,
Maryland, United States of America.

H9N2 avian influenza A viruses are endemic in poultry of many Eurasian countries 
and have caused repeated human infections in Asia since 1998. To evaluate the
potential threat of H9N2 viruses to humans, we investigated the replication and
transmission efficiency of H9N2 viruses in the ferret model. Five wild-type (WT) 
H9N2 viruses, isolated from different avian species from 1988 through 2003, were 
tested in vivo and found to replicate in ferrets. However these viruses achieved 
mild peak viral titers in nasal washes when compared to those observed with a
human H3N2 virus. Two of these H9N2 viruses transmitted to direct contact
ferrets, however no aerosol transmission was detected in the virus displaying the
most efficient direct contact transmission. A leucine (Leu) residue at amino acid
position 226 in the hemagglutinin (HA) receptor-binding site (RBS), responsible
for human virus-like receptor specificity, was found to be important for the
transmission of the H9N2 viruses in ferrets. In addition, an H9N2 avian-human
reassortant virus, which contains the surface glycoprotein genes from an H9N2
virus and the six internal genes of a human H3N2 virus, showed enhanced
replication and efficient transmission to direct contacts. Although no aerosol
transmission was observed, the virus replicated in multiple respiratory tissues
and induced clinical signs similar to those observed with the parental human H3N2
virus. Our results suggest that the establishment and prevalence of H9N2 viruses 
in poultry pose a significant threat for humans.

PMCID: PMC2500216
PMID: 18698430  [PubMed - indexed for MEDLINE]
Þ

24125. Arch Virol. 2008;153(8):1549-55. doi: 10.1007/s00705-008-0139-1. Epub 2008 Jun
24.

Characterization of a pathogenic H9N2 influenza A virus isolated from central
China in 2007.

Wu R, Sui ZW, Zhang HB, Chen QJ, Liang WW, Yang KL, Xiong ZL, Liu ZW, Chen Z, Xu 
DP.

State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of
Sciences, Hubei, Wuhan, China.

The entire genome of the A/Chicken/Hubei/C1/2007 (H9N2) virus, isolated from
central China in 2007, was completely sequenced and phylogenetically analyzed.
Phylogenetic analysis demonstrated that A/Chicken/Hubei/C1/2007 (H9N2) virus
represents multiple reassortant lineages, with genes coming from the early
mainland China strain (Ck/Beijing/1/94), an H9N2 virus with special genotype
(Ck/shanghai/F/98) and other lineages from poultry in Asia. Infection studies
indicated that A/Chicken/Hubei/C1/2007 (H9N2) virus replicated efficiently in
MDCK cells and in BALB/c mice. The H9N2 virus also replicated to high titers in
chicken respiratory tracts and caused overt clinical signs in chickens. Our
results suggest that attention should be paid to the natural evolution of H9N2
influenza viruses and to the control of H9N2 influenza viruses in animals.

PMID: 18574551  [PubMed - indexed for MEDLINE]
Þ

24146. Arch Virol. 2008;153(8):1433-9. doi: 10.1007/s00705-008-0131-9. Epub 2008 Jun 22.

Genetic analysis of H9N2 avian influenza viruses isolated from India.

Tosh C, Nagarajan S, Behera P, Rajukumar K, Purohit K, Kamal RP, Murugkar HV,
Gounalan S, Pattnaik B, Vanamayya PR, Pradhan HK, Dubey SC.

High Security Animal Disease Laboratory, Indian Veterinary Research Institute,
Anand Nagar, Bhopal, India.

H9N2 avian influenza viruses are endemic in domestic poultry in Asia and are
grouped into three major sublineages represented by their prototype strains
A/Duck/Hong Kong/Y280/97 (Y280-like), A/Quail/Hong Kong/G1/97 (G1-like) and
A/Chicken/Korea/38349-p96323/96 (Korean-like). To understand the genetic
relationship of Indian viruses, we determined the partial nucleotide sequence of 
five H9N2 avian influenza viruses isolated from chicken in India during 2003-2004
and compared them with H9N2 sequences available in GenBank. Deduced amino acid
sequence analysis revealed that four isolates shared an R-S-S-R/G motif at the
cleavage site of HA, representing low pathogenicity in chickens, while one virus 
harbors an R-S-N-R/G motif at the same position. All the viruses maintained the
human-like motif 226Lysine (H3 numbering) at the HA receptor binding site.
Phylogenetic analysis showed that 50% of the genes (HA, NA, NP and M) were
similar to G1-like viruses, whereas the remaining genes of the Indian isolates
formed a separate, not yet defined, sublineage in the Eurasian lineage. Our
finding provides evidence of a novel reassortant H9N2 genotype of G1-like viruses
circulating in India.

PMID: 18568381  [PubMed - indexed for MEDLINE]
Þ

24485. Vaccine. 2008 Jun 2;26(23):2912-8. doi: 10.1016/j.vaccine.2008.03.048. Epub 2008 
Apr 11.

Generation and evaluation of the trivalent inactivated reassortant vaccine using 
human, avian, and swine influenza A viruses.

Du N, Li W, Li Y, Liu S, Sui Y, Qu Z, Wang Y, Du Y, Xu B.

College of Veterinary Medicine, China Agricultural University, No. 2 Yuanmingyuan
West Road, Haidian District, Beijing 100094, China.

Reassortant technology was used to obtain three interspecific reassortant
influenza viruses using three influenza viruses of A/Puerto Rico/8/34(H1N1),
A/swine/Hebei/1/2005(H3N2) and A/chicken/Guangdong/126/2002(H9N2). The
high-growth reassortant strains were H9/PR8, H3/H9N2 and H1/H9N2 that contained
hemagglutinin (HA) and neuraminidase (NA) genes from the inactivated parental
viruses and the other 6 internal genes from the live parental viruses. The
trivalent formalin-inactivated vaccine, containing H1, H3 and H9 subtype antigens
from human, swine and avian influenza viruses respectively, was prepared using
these reassortant viruses. Animal studies showed that the vaccine was safe and
immunogenic. Two-dosing regimen of the influenza vaccine induced high titers of
hemagglutination inhibiting (HI) antibodies and influenza-specific IgG antibodies
without antigenic cross-interference. It protected 100% chickens from challenge
of A/chicken/Guangdong/126/2002 virus and protected 100% mice against challenges 
with different combinations of the three infective parental viruses. These
results indicated that the trivalent vaccine could offer multi-protection against
multi-influenza viruses synchronously. This kind of multivalent inactivated
reassortant influenza vaccine maybe enlightens the pandemic influenza
preparedness as the emergency measure.

PMID: 18448208  [PubMed - indexed for MEDLINE]
Þ

24610. Nature. 2008 May 29;453(7195):615-9. doi: 10.1038/nature06945. Epub 2008 Apr 16.

The genomic and epidemiological dynamics of human influenza A virus.

Rambaut A, Pybus OG, Nelson MI, Viboud C, Taubenberger JK, Holmes EC.

Institute of Evolutionary Biology, University of Edinburgh, Ashworth
Laboratories, Edinburgh EH9 3JT, UK. a.rambaut@ed.ac.uk

The evolutionary interaction between influenza A virus and the human immune
system, manifest as 'antigenic drift' of the viral haemagglutinin, is one of the 
best described patterns in molecular evolution. However, little is known about
the genome-scale evolutionary dynamics of this pathogen. Similarly, how genomic
processes relate to global influenza epidemiology, in which the A/H3N2 and A/H1N1
subtypes co-circulate, is poorly understood. Here through an analysis of 1,302
complete viral genomes sampled from temperate populations in both hemispheres, we
show that the genomic evolution of influenza A virus is characterized by a
complex interplay between frequent reassortment and periodic selective sweeps.
The A/H3N2 and A/H1N1 subtypes exhibit different evolutionary dynamics, with
diverse lineages circulating in A/H1N1, indicative of weaker antigenic drift.
These results suggest a sink-source model of viral ecology in which new lineages 
are seeded from a persistent influenza reservoir, which we hypothesize to be
located in the tropics, to sink populations in temperate regions.

PMCID: PMC2441973
PMID: 18418375  [PubMed - indexed for MEDLINE]
Þ

24780. Vet Microbiol. 2008 Aug 25;130(3-4):268-76. doi: 10.1016/j.vetmic.2008.02.005.
Epub 2008 Feb 16.

Generation and evaluation of reassortant influenza vaccines made by reverse
genetics for H9N2 avian influenza in Korea.

Song JM, Lee YJ, Jeong OM, Kang HM, Kim HR, Kwon JH, Kim JH, Seong BL, Kim YJ.

National Veterinary Research & Quarantine Service, Anyang6dong, Manangu, Anyang
City, Gyeonggi-do 430-824, Republic of Korea.

The prevalence and continuous evolution of H9N2 avian influenza viruses in
poultry have necessitated the use of vaccines in veterinary medicine. Because of 
the inadequate growth properties of some strains, additional steps are needed for
producing vaccine seed virus. In this study, we generated three H9N2/PR8
reassortant viruses using a total cDNA plasmid-transfection system, as an
alternative strategy for developing an avian influenza vaccine for animals. We
investigated the vaccine potency of the reassortant viruses compared with the
existing vaccine strain which was adapted by the 20th serial passages in
embryonated eggs with A/Ck/Kor/01310/01 (H9N2). The H9N2/PR8 reassortant viruses,
containing the internal genes of the high-yielding PR8 strain and the surface
gene of the A/Ck/Kor/01310/01 strain, could be propagated in eggs to the same
extent as existing vaccine strain without additional processing. Similar to
vaccine strain, the H9N2/PR8 reassortant viruses induced
hemagglutination-inhibiting antibodies in chickens and prevented virus shedding
and replication in multiple organs in response to homologous infection. However, 
due to the continuing evolution and increasing biologic diversity of H9N2
influenza in Korea, the vaccine provided only partial protection against
currently isolates. Taken together, our results suggest that the H9N2/PR8
reassortant virus can be used as a seed virus for avian influenza vaccines in
poultry farm. Considering the constant genetic changes in H9 strains isolated in 
Korea, this reverse genetic system may offer a prompt and simple way to change
the vaccine seed virus and mitigate the impact of unexpected influenza outbreaks.

PMID: 18374520  [PubMed - indexed for MEDLINE]
Þ

24866. Mol Phylogenet Evol. 2008 Jun;47(3):1100-10. doi: 10.1016/j.ympev.2008.02.003.
Epub 2008 Feb 14.

Origin of the 1918 Spanish influenza virus: a comparative genomic analysis.

Vana G, Westover KM.

Department of Biology, Winthrop University, Rock Hill, SC 29733, USA.

To test the avian-origin hypothesis of the 1918 Spanish influenza virus we
surveyed influenza sequences from a broad taxonomic distribution and collected 65
full-length genomes representing avian, human and "classic" swine H1N1 lineages
in addition to numerous other swine (H1N2, H3N1, and H3N2), human (H2N2, H3N2,
and H5N1), and avian (H1N1, H4N6, H5N1, H6N1, H6N6, H6N8, H7N3, H8N4, H9N2, and
H13N2) subtypes. Amino acids from all eight segments were concatenated, aligned, 
and used for phylogenetic analyses. In addition, the genes of the polymerase
complex (PB1, PB2, and PA) were analyzed individually. All of our results showed 
the Brevig-Mission/1918 strain in a position basal to the rest of the clade
containing human H1N1s and were consistent with a reassortment hypothesis for the
origin of the 1918 virus. Our genome phylogeny further indicates a sister
relationship with the "classic" swine H1N1 lineage. The individual PB1, PB2, and 
PA phylogenies were consistent with reassortment/recombination hypotheses for
these genes. These results demonstrate the importance of using a complete-genome 
approach for addressing the avian-origin hypothesis and predicting the emergence 
of new pandemic influenza strains.

PMID: 18353690  [PubMed - indexed for MEDLINE]
Þ

24877. Vaccine. 2008 Apr 16;26(17):2127-34. doi: 10.1016/j.vaccine.2008.02.001. Epub
2008 Feb 22.

A vaccine prepared from a non-pathogenic H7N7 virus isolated from natural
reservoir conferred protective immunity against the challenge with lethal dose of
highly pathogenic avian influenza virus in chickens.

Sakabe S, Sakoda Y, Haraguchi Y, Isoda N, Soda K, Takakuwa H, Saijo K, Sawata A, 
Kume K, Hagiwara J, Tuchiya K, Lin Z, Sakamoto R, Imamura T, Sasaki T, Kokumai N,
Kawaoka Y, Kida H.

Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido
University, Sapporo, Hokkaido 060-0818, Japan.

During 2001-2004, 41 H7 influenza viruses (2 H7N1 and 39 H7N7 strains) were
isolated from fecal samples of migratory ducks that flew from Siberia in the
autumn of each year to Japan and Mongolia. A phylogenetic analysis of the
hemagglutinin (HA) genes of the nine representative isolates revealed that they
belonged to the Eurasian lineage and the deduced amino acid sequence at the
cleavage site of the HAs represented apathogenic profiles. One of the H7 isolates
A/duck/Mongolia/736/02 (H7N7) was chosen from these H7 isolates for the
preparation of the test vaccine. To improve the growth potential of
A/duck/Mongolia/736/02 (H7N7) in chicken embryos, A/duck/Hokkaido/Vac-2/04 (H7N7)
was generated by genetic reassortment between A/duck/Mongolia/736/02 (H7N7) as
the donor of the PB2, PB1, PA, HA, NA, and NS genes and A/duck/Hokkaido/49/98
(H9N2) as that of NP and M genes. The test vaccine was prepared as follows;
A/duck/Hokkaido/Vac-2/04 (H7N7) was propagated in chicken embryos and the virus
in the allantoic fluid was inactivated and adjuvanted to form an oil-in-water
emulsion. The test vaccine conferred immunity to chickens, completely protecting 
the manifestation of clinical signs against the challenge with lethal dose of H7 
highly pathogenic avian influenza virus. These results indicate that influenza
viruses isolated from natural reservoirs are useful for vaccine strains.

PMID: 18346824  [PubMed - indexed for MEDLINE]
Þ

24882. J Gen Virol. 2008 Apr;89(Pt 4):949-57. doi: 10.1099/vir.0.83462-0.

Ecology of H3 avian influenza viruses in Korea and assessment of their pathogenic
potentials.

Song MS, Oh TK, Moon HJ, Yoo DW, Lee EH, Lee JS, Kim CJ, Yoo GJ, Kim H, Choi YK.

College of Medicine, Chungbuk National University, 12 Gaeshin-Dong, Heungduk-Ku, 
Cheongju 361-763, Republic of Korea.

To determine the genetic origins of novel H3 avian influenza viruses of chickens 
and ducks in Korea, genetic characterization of H3 avian influenza viruses
isolated from live poultry markets and migratory aquatic birds in South Korea
during 2004-2006 was conducted. Phylogenetic analysis revealed that at least four
novel genotypes of H3N2 and two genotypes of H3N6 avian influenza viruses were
co-circulating in backyard poultry of Korea. The viruses were reassortants
between H9N2 viruses of Korean chickens and unknown influenza viruses of
migratory birds. Genetic comparison of H3 viruses from live bird markets with
those from wild bird isolates revealed that certain gene segments of wild bird
isolates are related closely to those of Korean group H9N2 viruses isolated from 
live poultry markets in 2003. Furthermore, animal-challenge studies demonstrated 
that the pathogenicity of certain avian H3 influenza viruses was altered due to
reassortment, leading to H3 avian influenza viruses in Korea that can potentially
expand their host range to include mammals. These studies emphasize the
continuing need to monitor backyard poultry at live poultry markets to better
understand interspecies transmission and the emergence of novel influenza viruses
that have the potential to infect humans.

PMID: 18343836  [PubMed - indexed for MEDLINE]
Þ

24983. Jpn J Vet Res. 2008 Jan;55(2-3):93-8.

Development of vaccine strains of H5 and H7 influenza viruses.

Soda K, Sakoda Y, Isoda N, Kajihara M, Haraguchi Y, Shibuya H, Yoshida H, Sasaki 
T, Sakamoto R, Saijo K, Hagiwara J, Kida H.

Laboratory of Microbiology, Department of Disease Control, Graduate School of
Veterinary Medicine, Hokkaido University, Sapporo, 060-0818, Japan.

To establish vaccine strains of H5 and H7 influenza viruses,
A/duck/Hokkaido/Vac-1/04 (H5N1) [Vac-1/04 (H5N1)], A/duck/Hokkaido/Vac-3/07
(H5N1) [Vac-3/07 (H5N1)], and A/duck/Hokkaido/ Vac-2/04 (H7N7) [Vac-2/04 (H7N7)] 
were generated from non-pathogenic avian influenza viruses isolated from
migratory ducks. Vac-1/04 (H5N1) and Vac-3/07 (H5N1) were generated by genetic
reassortment between H5N2 or H5N3 virus as an HA gene provider and H7N1 or H6N1
viruses as an NA gene provider. Vac-2/04 (H7N7) was a genetic reassortant
obtained using H7N7 and H9 N2 viruses to give high growth character of the H9N2
virus in chicken embryonated eggs. The results of sequence analyses and
experimental infections revealed that these H5N1 and H7N7 reassortant viruses
were non-pathogenic in chickens and embryos, and had good growth potential in
embryonated eggs. These viruses should be useful to develop vaccines against H5
and H7 highly pathogenic avian influenza viruses.

PMID: 18318111  [PubMed - indexed for MEDLINE]
Þ

25870. Arch Virol. 2008;153(1):211-7. Epub 2007 Nov 15.

Genetic analysis of four porcine avian influenza viruses isolated from Shandong, 
China.

Shi WF, Gibbs MJ, Zhang YZ, Zhang Z, Zhao XM, Jin X, Zhu CD, Yang MF, Yang NN,
Cui YJ, Ji L.

Institute of Life Sciences, Taishan Medical College, Shandong Tai'an, China.
weifeng_cau@sina.com

A Bayesian phylogenetic analysis of eight separate gene segments indicated
A/Swine/Shandong/2/2003 (H5N1), A/Swine/Shandong/na/2003 (H9N2),
A/Swine/Shandong/nb/2003 (H9N2) and A/Swine/Shandong/nc/2005 (H9N2) probably
represent two multiple reassortant lineages, that had not been described before, 
with genes coming from H5N1, H9N2 and other lineages from poultry in Asia. Amino 
acid motifs within the haemagglutinin sequence of A/Swine/Shandong/nb/2003
suggested it may be able to infect people, whereas the sequences of the other
three isolates suggested they would not have had that capability. Our analysis
emphasizes the need for a comprehensive study of the interactions between H5N1
and H9N2 viruses in Asia that includes sequencing and phylogenetic investigation.

PMID: 18000640  [PubMed - indexed for MEDLINE]
Þ

26036. Virol J. 2007 Oct 23;4:102.

Cloning of the canine RNA polymerase I promoter and establishment of reverse
genetics for influenza A and B in MDCK cells.

Wang Z, Duke GM.

MedImmune, 297 North Bernardo Avenue, Mountain View, CA 94043, USA.
wangz@medimmune.com

BACKGROUND: Recent incidents where highly pathogenic influenza A H5N1 viruses
have spread from avian species into humans have prompted the development of
cell-based production of influenza vaccines as an alternative to or replacement
of current egg-based production. Madin-Darby canine kidney (MDCK) cells are the
primary cell-substrate candidate for influenza virus production but an efficient 
system for the direct rescue of influenza virus from cloned influenza cDNAs in
MDCK cells did not exist. The objective of this study was to develop a highly
efficient method for direct rescue of influenza virus in MDCK cells.
RESULTS: The eight-plasmid DNA transfection system for the rescue of influenza
virus from cloned influenza cDNAs was adapted such that virus can be generated
directly from MDCK cells. This was accomplished by cloning the canine RNA
polymerase I (pol I) promoter from MDCK cells and exchanging it for the human RNA
pol I promoter in the eight plasmid rescue system. The adapted system retains
bi-directional transcription of the viral cDNA template into both RNA pol I
transcribed negative-sense viral RNA and RNA pol II transcribed positive-sense
viral mRNA. The utility of this system was demonstrated by rescue in MDCK cells
of 6:2 genetic reassortants composed of the six internal gene segments (PB1, PB2,
PA, NP, M and NS) from either the cold-adapted (ca) influenza A vaccine strain
(ca A/Ann Arbor/1/60) or the ca influenza B vaccine strain (ca B/Ann Arbor/1/66) 
and HA and NA gene segments from wild type influenza A and B strains.
Representative 6:2 reassortants were generated for influenza A (H1N1, H3N2, H5N1,
H6N1, H7N3 and H9N2) and for both the Victoria and Yamagata lineages of influenza
B. The yield of infectious virus in the supernatant of transfected MDCK cells was
106 to 107 plaque forming units per ml by 5 to 7 days post-transfection.
CONCLUSION: This rescue system will enable efficient production of both influenza
A and influenza B vaccines exclusively in MDCK cells and therefore provides a
tool for influenza pandemic preparedness.

PMCID: PMC2241602
PMID: 17956624  [PubMed - indexed for MEDLINE]
Þ

26267. Am J Ther. 2007 Sep-Oct;14(5):449-54.

Limitations of current prophylaxis against influenza virus infection.

Guralnik M, Rosenbloom RA, Petteruti MP, Lefante C.

Synergy Research, Teaneck, NJ; and 2Quigley Pharma, Doylestown, PA, USA.
Mario.guralnik@synergy-cro.com

Avian influenza has been a source of worldwide concern since Hong Kong
authorities detected the first outbreak in 1997. Mainly as a result of
poultry-to-human transmission, more than 200 cases of infection in humans have
been attributed to the A/H5, A/H7, and A/H9 viral subtypes, with a case fatality 
rate for A/H5N1 infections exceeding 50%. A mutant or reassortant virus capable
of efficient human-to-human transmission can set off a pandemic. Increased
attention to prophylaxis against viral infection has identified several
potentially complementary approaches: nonpharmacologic measures (eg, travel
restrictions), vaccination, chemotherapeutic agents, and herbal/natural products.
All have significant limitations that point out the need for additional
modalities. Herbal/natural products, particularly those based on green tea
extract, offer promise as adjuncts or alternatives to current interventions and
warrant further evaluation in well-controlled human trials.

PMID: 17890934  [PubMed - indexed for MEDLINE]
Þ

26327. Vaccine. 2007 Oct 16;25(42):7379-84. Epub 2007 Aug 24.

Generation of an attenuated H5N1 avian influenza virus vaccine with all eight
genes from avian viruses.

Shi H, Liu XF, Zhang X, Chen S, Sun L, Lu J.

Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou
University, 225009 Yangzhou, Jiangsu, People's Republic of China.
hyshi@yzu.edu.cn

In the face of disease outbreaks in poultry and the potential pandemic threat to 
humans caused by the highly pathogenic avian influenza viruses (HPAIVs) of H5N1
subtype, improvement in biosecurity and the use of inactivated vaccines are two
main options for the control of this disease. Vaccine candidates of influenza A
viruses of H5N1 subtype have been generated in several laboratories by
plasmid-based reverse genetics with hemagglutinin (HA) and neuraminidase (NA)
genes from the epidemic strains of avian viruses in a background of internal
genes from the vaccine donor strain of human strains, A/Puerto Rico/8/34 (PR8).
These reassortant viruses containing genes from both avian and human viruses
might impose biosafety concerns, also may be do if C4/F AIV would be a live
attenuated vaccine or cold-adaptive strain vaccine. In order to generate better
and safer vaccine candidate viruses, we genetically constructed attenuated
reassortant H5N1 influenza A virus, designated as C4/F AIV, by plasmid-based
reverse genetics with all eight genes from the avian strains. The C4/F AIV virus 
contained HA and NA genes from an epidemic strain A/Chicken/Huadong/04 (H5N1)
(C4/H5N1) in a background of internal genes derived from a low pathogenic strain 
of A/Chicken/F/98(H9N2). The reassortant virus was attenuated by removal of the
multibasic amino acid motif in the HA gene by mutation and deletion (from
PQRERRRKKR (downward arrow) G to PQIETR (downward arrow) G). The intravenous
pathogenicity index (IVPI) of C4/F AIV virus was 0, whereas that of the donor
virus C4/H5N1 was 3.0. The virus HA titer of C4/H5N1 in the allantoic fluid from 
infected embryonated eggs was as high as 1:2048. The inactivated vaccine prepared
from the reassortant virus C4/F AIV-induced high HI titer in vaccinated chickens 
and gave 100% protection when challenged with highly pathogenic avian influenza
virus of H5N1 subtype.

PMID: 17870216  [PubMed - indexed for MEDLINE]
Þ

26670. J Virol. 2007 Oct;81(19):10389-401. Epub 2007 Jul 25.

The genesis and evolution of H9N2 influenza viruses in poultry from southern
China, 2000 to 2005.

Xu KM, Smith GJ, Bahl J, Duan L, Tai H, Vijaykrishna D, Wang J, Zhang JX, Li KS, 
Fan XH, Webster RG, Chen H, Peiris JS, Guan Y.

International Institute of Infection and Immunity, Shantou University, Guangdong,
China, and Department of Infectious Diseases, St. Jude Children's Research
Hospital, Memphis, TN 38105, USA.

H9N2 influenza viruses have become established in terrestrial poultry in
different Asian countries over the last 2 decades. Our previous study
demonstrated that quail harbor increasingly diverse novel H9N2 reassortants,
including both Chicken/Beijing/1/94 (Ck/Bei-like) and Quail/Hong Kong/G1/97
(G1-like) viruses. However, since 1999, the genesis and evolution of H9N2 viruses
in different types of poultry have not been investigated systematically. In the
present study, H9N2 viruses isolated from chickens, ducks, and other minor
poultry species were characterized genetically and antigenically. Our findings
demonstrate that Ck/Bei-like H9N2 viruses have been introduced into many
different types of poultry in southern China, including quail, partridges,
chukar, pheasant, guinea fowl, and domestic ducks, while G1-like viruses were
commonly detected in quail, less frequently detected in other minor poultry
species, and not detected in chickens and ducks. Genetic analysis revealed 35
genotypes of H9N2 viruses, including 14 novel genotypes that have not been
recognized before. Our results also suggested that two-way interspecies
transmission exists between different types of poultry. Our study demonstrates
that the long-term cocirculation of multiple virus lineages (e.g., H5N1 and H9N2 
viruses) in different types of poultry has facilitated the frequent reassortment 
events that are mostly responsible for the current great genetic diversity in
H9N2 and H5N1 influenza viruses in this region. This situation favors the
emergence of influenza viruses with pandemic potential.

PMCID: PMC2045440
PMID: 17652402  [PubMed - indexed for MEDLINE]
Þ

26671. J Virol. 2007 Oct;81(19):10402-12. Epub 2007 Jul 25.

Establishment of influenza A virus (H6N1) in minor poultry species in southern
China.

Cheung CL, Vijaykrishna D, Smith GJ, Fan XH, Zhang JX, Bahl J, Duan L, Huang K,
Tai H, Wang J, Poon LL, Peiris JS, Chen H, Guan Y.

State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology,
The University of Hong Kong, Li Ka Shing Faculty of Medicine Building, 21 Sassoon
Road, Pokfulam, Hong Kong, SAR, China.

An H6N1 virus, A/teal/Hong Kong/W312/97 (W312), was isolated during the "bird
flu" incident in Hong Kong in 1997. Genetic analysis suggested that this virus
might be the progenitor of the A/Hong Kong/156/97 (HK/97) H5N1 virus, as seven of
eight gene segments of those viruses had a common source. Continuing surveillance
in Hong Kong showed that a W312-like virus was prevalent in quail and pheasants
in 1999; however, the further development of H6N1 viruses has not been
investigated since 2001. Here we report influenza virus surveillance data
collected in southern China from 2000 to 2005 that show that H6N1 viruses have
become established and endemic in minor poultry species and replicate mainly in
the respiratory tract. Phylogenetic analysis indicated that all H6N1 isolates had
W312-like hemagglutinin and neuraminidase genes. However, reassortment of
internal genes between different subtype virus lineages, including H5N1, H9N2,
and other avian viruses, generated multiple novel H6N1 genotypes in different
types of poultry. These novel H6N1/N2 viruses are double, triple, or even
quadruple reassortants. Reassortment between a W312-like H6N1 virus and an
A/quail/Hong Kong/G1/97 (HK/97)-like H9N2 virus simultaneously generated novel
H6N2 subtype viruses that were persistent in poultry. Molecular analyses suggest 
that W312-like viruses may not be the precursors of HK/97 virus but reassortants 
from an HK/97-like virus and another unidentified H6 subtype virus. These results
provide further evidence of the pivotal role of the live poultry market system of
southern China in generating increased genetic diversity in influenza viruses in 
this region.

PMCID: PMC2045442
PMID: 17652385  [PubMed - indexed for MEDLINE]
Þ

26996. J Gen Virol. 2007 Jul;88(Pt 7):2035-41.

Antigenic and genetic characterization of H9N2 swine influenza viruses in China.

Cong YL, Pu J, Liu QF, Wang S, Zhang GZ, Zhang XL, Fan WX, Brown EG, Liu JH.

Laboratory of Infectious Diseases, College of Veterinary Medicine, State Key
Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, 
PR China.

As pigs are susceptible to infection with both avian and human influenza A
viruses, they have been proposed to be an intermediate host for the generation of
pandemic virus through reassortment. Antigenic and genetic characterization was
performed for five swine H9N2 influenza viruses isolated from diseased pigs from 
different farms. The haemagglutinin (HA) antigenicity of swine H9N2 viruses was
different from that of chicken H9N2 viruses prevalent in northern China. Genetic 
analysis revealed that all five isolates had an RLSR motif at the cleavage site
of HA, which was different from those of A/duck/Hong Kong/Y280/97
(Dk/HK/Y280/97)-like viruses established in chickens in China. Phylogenetic
analyses indicated that the five swine H9N2 viruses formed novel HA and
neuraminidase sublineages that were related closely to those of earlier chicken
H9 viruses and were also consistent with the extent of the observed antigenic
variation. The six internal genes of the isolates possessed H5N1-like sequences, 
indicating that they were reassortants of H9 and H5 viruses. The present results 
indicate that avian to porcine interspecies transmission of H9N2 viruses might
have resulted in the generation of viruses with novel antigenic and genetic
characteristics; therefore, surveillance of swine influenza should be given a
high priority.

PMID: 17554038  [PubMed - indexed for MEDLINE]
Þ

27280. Avian Dis. 2007 Mar;51(1 Suppl):279-84.

Phylogenetic analysis of low-pathogenicity avian influenza H6N2 viruses from
chicken outbreaks (2001-2005) suggest that they are reassortants of historic
ostrich low-pathogenicity avian influenza H9N2 and H6N8 viruses.

Abolnik C, Bisschop SP, Gerdes GH, Olivier AJ, Horner RF.

Agricultural Research Council, Onderstepoort Veterinary Institute, Private Bag
X5, Pretoria 0110, South Africa.

Low-pathogenicity (LPAI) and high-pathogenicity (HPAI) avian influenza viruses
are periodically isolated from South African ostriches, but during 2002 the first
recorded outbreak of LPAI (H6N2) in South African chickens occurred on commercial
farms in the Camperdown area of KwaZulu/Natal (KZN) Province. Sequence analysis
of all eight genes were performed and phylogenetic analysis was done based on the
hemagglutinin and neuraminidasc sequences. Results from phylogenetic analyses
indicated that the H6N2 chicken viruses most likely arose from a reassortment
between two South African LPAI ostrich isolates: an H9N2 virus isolated in 1995
and an H6N8 virus isolated in 1998. Two cocirculating sublineages of H6N2 viruses
were detected, both sharing a recent common ancestor. One of these sublineages
was restricted to the KZN province. The neuraminidase gene contained a 22-amino
acid deletion in the NA-stalk region, which is associated with adaptation to
growth in chickens, whereas the other group, although lacking the NA-stalk
deletion, spread to commercial farms in other provinces. The persistence of
particular H6N2 types in some regions for at least 2 yr supports reports from
Asia and southern California suggesting that H6N2 viruses can form stable
lineages in chickens. It is probable that the ostrich H6N8 and H9N2 progenitors
of the chicken H6N2 viruses were introduced to ostriches by wild birds.
Ostriches, in which AI infections are often subclinical, may serve as mixing
vessels for LPAI strains that occasionally spill over into other poultry.

PMID: 17494567  [PubMed - indexed for MEDLINE]
Þ

28312. J Virol. 2007 Mar;81(6):2635-45. Epub 2006 Dec 27.

Evolution and molecular epidemiology of H9N2 influenza A viruses from quail in
southern China, 2000 to 2005.

Xu KM, Li KS, Smith GJ, Li JW, Tai H, Zhang JX, Webster RG, Peiris JS, Chen H,
Guan Y.

State Key Laboratory of Emerging Infectious Disease, Department of Microbiology, 
The University of Hong Kong, Li Ka Shing Faculty of Medicine Building, 21 Sassoon
Road, Pokfulam, Hong Kong SAR, China.

H9N2 influenza viruses have become established and maintain long-term endemicity 
in terrestrial poultry in Asian countries. Occasionally these viruses transmit to
other mammals, including humans. Increasing epidemiological and laboratory
findings suggest that quail may be an important host, as they are susceptible to 
different subtypes of influenza viruses. To better understand the role of quail
in influenza virus ecology and evolution, H9N2 viruses isolated from quail during
2000 to 2005 were antigenically and genetically characterized. Our results showed
that H9N2 viruses are prevalent year-round in southern China and replicate mainly
asymptomatically in the respiratory tract of quail. Genetic analysis revealed
that both the G1-like and Ck/Bei-like H9N2 lineages were cocirculating in quail
since 2000. Phylogenetic analyses demonstrated that most of the isolates tested
were double- or multiple-reassortant variants, with four G1-like and 16
Ck/Bei-like genotypes recognized. A novel genotype of G1-like virus became
predominant in quail since 2003, while multiple Ck/Bei-like genotypes were
introduced into quail, wherein they incorporated G1-like gene segments, but none 
of them became established in this host. Those Ck/Bei-like reassortants generated
in quail have then been introduced into other poultry. These complex interactions
form a two-way transmission system between quail and other types of poultry. The 
present study provides evidence that H9N2 and H5N1 subtype viruses have also
exchanged gene segments to generate currently circulating reassortants of both
subtypes that have pandemic potential. Continuing influenza virus surveillance in
poultry is critical to understanding the genesis and emergence of potentially
pandemic strains in this region.

PMCID: PMC1865985
PMID: 17192315  [PubMed - indexed for MEDLINE]
Þ

28361. Acta Virol. 2006;50(4):243-9.

Reassortment and modification of hemagglutinin cleavage motif of avian/WSN
influenza viruses generated by reverse genetics that correlate with attenuation.

Lu JH, Long JX, Jia LJ, Liu YL, Shao WX, Zhang YM, Liu XF.

Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou
University, Yangzhou, Jiangsu 225009, Jiangsu Province, P.R. China.

Avian influenza associated with H9N2 and H5N1 subtypes of avian influenza viruses
(AIVs) has raised great concerns in China. To study this problem, reverse
genetics has been employed. Three reassortants, rgH9N2, rgH5N1 and rgH5N2, were
prepared and compared. Their hemagglutinin (HA) and neuraminidase (NA) genes
originated from Chinese AIV isolates of H9N2 or H5N1 subtype, while the rest of
their genes were derived from A/WSN/33(H1N1) virus (WSN). In the H5 HA
reassortants, the multibasic cleavage site was converted to a monobasic one. The 
results demonstrated that the reassortants did not produce CPE on MDCK cells in
the absence of trypsin, showed egg-adaptation phenotype and stability of HA and
NA during consecutive egg passages, and were not lethal to chickens and mice.
However, the rgH5N1 reassortant exhibited a residual virulence in terms of
lethality to chick embryos and pathogenesis in chickens. It can be concluded that
(i) the genetic modification of H5 HA attenuated the H5 reassortants, (ii) the
presence of internal WSN proteins contributed to the attenuated properties of the
reassortants independently on H5 HA, and (iii) also the overall genome
composition contributed to virulence differences. This report provides further
contribution of reverse genetics to the knowledge of virulence of influenza
viruses.

PMID: 17177609  [PubMed - indexed for MEDLINE]
Þ

28614. Avian Pathol. 2006 Dec;35(6):443-7.

An outbreak of avian influenza subtype H9N8 among chickens in South Korea.

Kwon YK, Lee YJ, Choi JG, Lee EK, Jeon WJ, Jeong OM, Kim MC, Joh SJ, Kwon JH, Kim
JH.

National Veterinary Research and Quarantine Service, MAF, Anyang 430-824, Korea.

Low pathogenic avian influenza subtype H9N8 was diagnosed on a Korean native
chicken farm in Gyeonggi province, South Korea, in late April 2004. Clinical
signs included moderate respiratory distress, depression, mild diarrhoea, loss of
appetite and a slightly elevated mortality (1.4% in 5 days). Pathologically,
mucopurulent tracheitis and air sacculitis were prominently found with urate
renal deposition. The isolated A/chicken/Kr/164/04 (H9N8) had an Ala-Ser-Gly-Arg 
(A/S/G/R) motif at the cleavage site of haemagglutinin, which has been commonly
found in H9N2 isolated from Korean poultry. Phylogenetic analysis of the
haemagglutinin and neuraminidase genes of the H9N8 avian influenza virus (AIV)
isolate showed that reassortment had occurred. Its haemagglutinin gene was
similar to that of Korean H9N2 AIVs, but its neuraminidase gene was closely
related to that of A/WBF/Kr/KCA16/03 (H3N8) isolated from the faeces of wild
birds in Korea. The pathogenicity of the isolate was tested on 6-week-old
specific pathogen free chickens. The inoculated virus (H9N8) was recovered from
most tested organs, including the trachea, lung, kidney, spleen, and caecal
tonsil. This is the first report of an outbreak of low pathogenic avian influenza
in chickens caused by AIV subtype H9N8.

PMID: 17121732  [PubMed - indexed for MEDLINE]
Þ

28814. Med Pregl. 2006 Jan-Feb;59(1-2):29-32.

[Avian influenza viruses--new causative agents of human infections].

[Article in Serbian]

Hrnjaković-Cvjetković I, Cvjetković D, Jerant-Patić V, Milosević V,
Tadić-Radovanov J, Kovacević G.

Institut za zastitu zdravlja Novi Sad. ivacvj@neobee.net

INTRODUCTION: Influenza A viruses can infect humans, some mammals and especially 
birds. Subtypes of human influenza A viruses: A(H1N1), A(H2N2) and A(H3N2) have
caused pandemics. Avian influenza viruses vary owing to their 15 hemagglutinins
(H) and 9 neuraminidases (N). HUMAN CASES OF AVIAN INFLUENZA A: In the
Netherlands in 2003, there were 83 human cases of influenza A (H7N7). In 1997, 18
cases of H5N1 influenza A, of whom 6 died, were found among residents of Hong
Kong. In 2004, 34 human cases (23 deaths) were reported in Viet Nam and Thailand.
H5N1 virus-infected patients presented with fever and respiratory symptoms.
Complications included respiratory distress syndrome, renal failure, liver
dysfunction and hematologic disorders. Since 1999, 7 cases of human influenza
H9N2 infection have been identified in China and Hong Kong. THE IMPORTANCE OF
HUMAN INFECTION WITH AVIAN INFLUENZA VIRUSES: H5N1 virus can directly infect
humans. Genetic reassortment of human and avian influenza viruses may occur in
humans co-infected with current human A(H1N1) or A(H3N2) subtypes and avian
influenza viruses. The result would be a new influenza virus with pandemic
potential. All genes of H5N1 viruses isolated from humans are of avian origin.
PREVENTION AND CONTROL: The reassortant virus containing H and N from avian and
the remaining proteins from human influenza viruses will probably be used as a
vaccine strain. The most important control measures are rapid destruction of all 
infected or exposed birds and rigorous disinfection of farms. Individuals exposed
to suspected animals should receive prophylactic treatment with antivirals and
annual vaccination.

PMID: 17068888  [PubMed - indexed for MEDLINE]
Þ

28865. Virology. 2007 Mar 15;359(2):313-23. Epub 2006 Oct 23.

Continuing evolution of H9 influenza viruses in Korean poultry.

Lee YJ, Shin JY, Song MS, Lee YM, Choi JG, Lee EK, Jeong OM, Sung HW, Kim JH,
Kwon YK, Kwon JH, Kim CJ, Webby RJ, Webster RG, Choi YK.

Avian Disease division, National Veterinary Research and Quarantine Service, 480 
Anyang, Anyang city, 430-824, Korea.

We analyzed the evolution of H9 influenza viruses isolated from Korean chicken
farms from 2002 to 2004. Korean H9 viruses formed two antigenically distinct
groups: those isolated from 1996 to mid-2003, and those isolated from late 2003
through 2004. Most of the 2004 isolates showed greater cross-reactivity with the 
second group than with the first group. Phylogenetic analysis of the 12 viruses
studied revealed three genotypes of H9N2 viruses and showed that reassortment had
occurred. One isolate, Ck/Kor/164/04, belonged to the H9N8 subtype. Its HA and
PB1 genes were similar to those of the H9N2 viruses, but its other genes were
closely related to H3N8 viruses. This report is the first (to our knowledge) of
H9N8 infection in this host. The pathogenicity of the early isolates altered due 
to antigenic drift and reassortment, leading to H9 avian influenza viruses in
Korea that potentially can expand their host range to mammalians.

PMID: 17056087  [PubMed - indexed for MEDLINE]
Þ

29380. Virus Genes. 2007 Jan;34(1):37-45. Epub 2006 Aug 22.

Outbreaks of avian influenza H6N2 viruses in chickens arose by a reassortment of 
H6N8 and H9N2 ostrich viruses.

Abolnik C, Bisschop S, Gerdes T, Olivier A, Horner R.

ARC-Onderstepoort Veterinary Institute, Private Bag X5, Pretoria, 0110, South
Africa. abolnikc@arc.agric.za

The first recorded outbreak of avian influenza (AI) in South African chickens
(low pathogenicity H6N2) occurred at Camperdown, KwaZulu/Natal Province (KZN) in 
June 2002. To determine the source of the outbreak, we defined the phylogenetic
relationships between various H6N2 isolates, and the previously unpublished gene 
sequences of an H6N8 virus isolated in 1998 from ostriches in the Leeu Gamka
region (A/Ostrich/South Africa/KK98/98). We demonstrated that two distinct
genetic H6N2 lineages (sub-lineages I and II) circulated in the Camperdown area, 
which later spread to other regions. Sub-lineages I and II shared a recent common
H6N2 ancestor, which arose from a reassortment event between two South African
ostrich isolates A/Ostrich/South Africa/9508103/95 and (H9N2) A/Ostrich/South
Africa/KK98/98 (H6N8). Furthermore, the H6N2 sub-lineage I viruses had several
molecular genetic markers including a 22-amino acid stalk deletion in the
neuraminidase (NA) protein gene, a predicted increased N-glycosylation, and a
D144 mutation of the HA protein gene, all of which are associated with the
adaptation of AI viruses to chickens. The H6N2 NS1 and PB1 genes shared recent
common ancestors with those of contemporary Asian HPAI H5N1 viruses. Our results 
suggest that ostriches are potential mixing vessels for avian influenza viruses
(AIV) outbreak strains and support other reports that H6 viruses are capable of
forming stable lineages in chickens.

PMID: 16927114  [PubMed - indexed for MEDLINE]
Þ

30144. Vaccine. 2006 Jun 12;24(24):5158-68. Epub 2006 May 2.

Protection against H1, H5, H6 and H9 influenza A infection with liposomal matrix 
2 epitope vaccines.

Ernst WA, Kim HJ, Tumpey TM, Jansen AD, Tai W, Cramer DV, Adler-Moore JP, Fujii
G.

Molecular Express, Inc. 13310 S. Figueroa Street, Los Angeles, CA 90061, USA.

The recent emergence of multiple avian influenza A subtypes that cause human
disease (i.e., H5N1, H9N2 and H7N7), coupled with the fear that one of these
strains might precipitate a new pandemic, underscores the need to develop new
technological approaches to immunization which elicit protective immune responses
against multiple subtypes of influenza A. In response to this demand, several
matrix 2 protein ectodomain segments (M2eA) corresponding to the H1N1, H5N1 and
H9N2 influenza strains were formulated using a novel liposome-based vaccine
technology and evaluated as potential immunogens for developing a "universal"
influenza vaccine. Mice immunized with liposomal M2eA survived homologous
challenges with H1N1 (100% survival) or H9N2 (80% survival) influenza strains.
There were significant reductions in their lung viral load as well as in
immunized mice challenged with the H5N1 subtype. The mice vaccinated with an M2eA
segment corresponding to the H1N1 and H6N2 (a reassortant influenza A virus
carrying the M2eA from PR8/34) strains elicited elevated IgG ELISA antibody
titers to this M2eA epitope segment and antiserum from these immunized mice
provided passive protection (100% survival) to naïve mice receiving a lethal dose
of H6N2 influenza virus. These results provide the first evidence that
recombinant M2eA epitopes to multiple subtypes elicited immune protection against
a homologous challenge and provides further evidence in favor of the development 
of a "universal" influenza vaccine based on M2eA.

PMID: 16713037  [PubMed - indexed for MEDLINE]
Þ

31101. Dev Biol (Basel). 2006;124:189-99.

Phylogenetic analyses of genes from South African LPAI viruses isolated in 2004
from wild aquatic birds suggests introduction by Eurasian migrants.

Abolnik C, Cornelius E, Bisschop SP, Romito M, Verwoerd D.

Biotechnology Division, Onderstepoort Veterinary Institute, South Africa.
abolnikc@arc.agric.za

In 2004, South Africa experienced its first recorded outbreak of a highly
pathogenic notifiable avian influenza (HPNAI) viral strain of the H5N2 subtype in
ostriches in the Eastern Cape province. The traditional ostrich-farming areas in 
the Western Cape province report almost yearly outbreaks of low pathogenicity
avian influenza (LPAI) in ostriches, which is attributed to introduction by wild 
birds and certain climatic patterns. During the winter of 2004, LPAI H3N8, H4N8, 
H5N2 and H5N1 avian influenza viruses were isolated from wild aquatic birds. All 
eight genes of the H3N8, H4N8 and H5N1 viruses were analysed. The results show
that the H5N1 virus does not belong to the HPAI Z/Z+N genotype currently
circulating in Asia, but that the most recent common ancestors are Russian H5N2
and H5N3 viruses. The N1 gene lacks the stalk deletion associated with virulence.
Internal genes probably originate from a pool containing Chinese, Middle Eastern 
and Italian viruses. The South African H3N8 and H4N8 viruses appear to have
derived their genes from an ecosystem where Asian H5N1, H6N9 and H9N2, Russian
H4, and Danish H3N8 viruses have been circulating since 1997. All three viruses
share recent nucleoprotein common ancestors with the German and Dutch HPNAI H7N7 
viruses from 2003. The diverse pool of genes from which local viruses are derived
suggests that reassortment occurred at the Siberian breeding grounds where
migratory paths cross, or within the South African ecosystem. This data
highlights the importance of surveillance in aquatic migratory birds,
particularly members of the Charadriidae, for their potential roles in the
introduction of avian diseases to South African poultry and especially ostriches 
in the case of avian influenza.

PMID: 16447511  [PubMed - indexed for MEDLINE]
Þ

31115. Dev Biol (Basel). 2006;124:77-84.

Avian influenza viruses and human health.

Alexander DJ.

Virology Department, Veterinary Laboratories Agency Weybridge, Addlestone,
Surrey, United Kingdom. d.j.alexander@vla.defra.gsi.gov.uk

Influenza A viruses cause natural infections of humans, some other mammals and
birds. Few of the 16 haemagglutinin and nine neuraminidase subtype combinations
have been isolated from mammals, but all subtypes have been isolated from birds. 
In the 20th century, there were four pandemics of influenza as a result of the
emergence of antigenically different strains in humans: 1918 (H1N1), 1957 (H2N2),
1968 (H3N2) and 1977 (H1N1). Influenza A viruses contain eight distinct RNA genes
and reassortment of these can occur in mixed infections with different viruses.
The 1957 and 1968 pandemic viruses differed from the preceding viruses in humans 
by the substitution of genes that came from avian viruses, suggesting they arose 
by genetic reassortment of viruses of human and avian origin. Up to 1995, there
had been only three reports of avian influenza viruses infecting humans, in 1959,
1977 and 1981 (all H7N7), but, since 1996, there have been regular reports of
natural infections of humans with avian influenza viruses: in England in 1996
(H7N7), Hong Kong 1997 (H5N1), 1999 (H9N2), and 2003 (H5N1), in The Netherlands
2003 (H7N7), Canada 2004 (H7N3), Vietnam 2004 (H5N1) and Thailand 2004 (H5N1).
The H5N1 virus is alarming because 51 (64 %) of the 80 people confirmed as
infected since 1997 have died.

PMID: 16447497  [PubMed - indexed for MEDLINE]
Þ

31201. Chest. 2006 Jan;129(1):156-68.

Avian influenza virus infections in humans.

Wong SS, Yuen KY.

Department of Microbiology, Research Centre of Infection and Immunology, State
Key Laboratory of Emerging Infectious Disease, Faculty of Medicine, The
University of Hong Kong.

Seroepidemiologic and virologic studies since 1889 suggested that human influenza
pandemics were caused by H1, H2, and H3 subtypes of influenza A viruses. If not
for the 1997 avian A/H5N1 outbreak in Hong Kong of China, subtype H2 is the
likely candidate for the next pandemic. However, unlike previous poultry
outbreaks of highly pathogenic avian influenza due to H5 that were controlled by 
depopulation with or without vaccination, the presently circulating A/H5N1
genotype Z virus has since been spreading from Southern China to other parts of
the world. Migratory birds and, less likely, bird trafficking are believed to be 
globalizing the avian influenza A/H5N1 epidemic in poultry. More than 200 human
cases of avian influenza virus infection due to A/H5, A/H7, and A/H9 subtypes
mainly as a result of poultry-to-human transmission have been reported with a >
50% case fatality rate for A/H5N1 infections. A mutant or reassortant virus
capable of efficient human-to-human transmission could trigger another influenza 
pandemic. The recent isolation of this virus in extrapulmonary sites of human
diseases suggests that the high fatality of this infection may be more than just 
the result of a cytokine storm triggered by the pulmonary disease. The emergence 
of resistance to adamantanes (amantadine and rimantadine) and recently
oseltamivir while H5N1 vaccines are still at the developmental stage of phase I
clinical trial are causes for grave concern. Moreover, the to-be pandemic strain 
may have little cross immunogenicity to the presently tested vaccine strain. The 
relative importance and usefulness of airborne, droplet, or contact precautions
in infection control are still uncertain. Laboratory-acquired avian influenza
H7N7 has been reported, and the laboratory strains of human influenza H2N2 could 
also be the cause of another pandemic. The control of this impending disaster
requires more research in addition to national and international preparedness at 
various levels. The epidemiology, virology, clinical features, laboratory
diagnosis, management, and hospital infection control measures are reviewed from 
a clinical perspective.

PMID: 16424427  [PubMed - indexed for MEDLINE]
Þ

31230. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2005 Dec;19(4):358-61.

[Origin of internal genes of two strains of swine influenza A (H1N1) virus].

[Article in Chinese]

Guo YJ, Wen LY, Zhang Y, Wang M, Guo JF, Li Z, Shu YL.

Department of Influenza, Institute of Viral Disease Control and Prevention,
Chinese Center for Disease Control and Prevention, Beijing 100052, China.

BACKGROUND: Through the analysis of internal genes of viruses to understand
whether the internal genes of two strains of swine influenza A(H1N1) virus
contain the gene segment deriving from avian influenza A viruses, and whether the
reassortment of the internal genes occurred between swine H1N1 and swine H9N2
viruses.
METHODS: Viruses were passaged in embryonated hen eggs and virion RNA was
extracted from allantoic fluids and reverse transcribed to synthesize cDNA. cDNA 
was amplified by PCR and the PCR product was purified with a purification kit.
Afterwards, RNA sequence analysis was performed by dideoxynucleotide chain
termination and a cloning method. Finally, phylogenetic analysis of the
sequencing data was performed with MegAlign (version 1.03).
RESULTS: All the six, but PB2 gene segment internal gene segments were the same
between A/swine/Beijing/1/2002(H1N1) and A/Swine/Beijing/3/2002(H1N1) viruses.
Whereas all the six internal gene segments in two trains of swine (H1N1) virus
were similar to those of swine H1N1 viruses, but different from those of
classical strain of swine (H1N1) virus.
CONCLUSION: Two strains of Beijing swine H1N1 influenza A virus were not
reassortant, All the six internal gene segments were closely related to swine
influenza A (H1N1) viruses.

PMID: 16415995  [PubMed - indexed for MEDLINE]
Þ

31720. Wei Sheng Wu Xue Bao. 2003 Aug;43(4):434-41.

[Genetic characterization of the entire genome of an H9N2 avian influenza virus
A/Chicken/Shanghai/F/98].

[Article in Chinese]

Lu J, Liu X, Shao W, Zhang P, Wei D.

Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou
University, Yangzhou 225009, China. jianhl@sohu.com

Eight full-length genes of an H9N2 subtype avian influenza virus
A/Chicken/Shanghai/F/ 98(H9N2), were amplified by RT-PCR and rapid amplification 
of cDNA ends (RACE) and sequenced. The results of phylogenetic analysis showed
that A/Chicken/Shanghai/F/98 was entirely different from Quail/Hong Kong/G1/97
which was directly related to human infection with avian influenza virus in Hong 
Kong, whereas its HA, NA, M and NS genes belonged to A/Chicken/Beijing/ 1/94-like
sublineage with the nucleotide homology of 96.7%, 96.4%, 97.5% and 98.0%
respectively. The homology of NA gene between A/Chicken/Shanghai/F/98 and
A/Duck/Hong Kong/Y280/ 97 was 97.4% and they both lost 9 nucleotides between nt
205 to 206. However, the PB2, PB1, PA, NP genes did not show any close
relationship with those of the three known sublineages and the later three didn't
share immediate ancestors with available isolates whose sequences had been
reported. Therefore, A/Chicken/Shanghai/F/98 is a product of natural reassortment
between H9N2 avian influenza viruses from different sublineages.

PMID: 16276916  [PubMed - indexed for MEDLINE]
Þ

31813. Avian Dis. 2005 Sep;49(3):317-27.

Public health risk from avian influenza viruses.

Perdue ML, Swayne DE.

Department of Communicable Disease Surveillance and Response, World Health
Organization, Global Influenza Programme, 1211 Geneva 27, Switzerland.

Since 1997, avian influenza (AI) virus infections in poultry have taken on new
significance, with increasing numbers of cases involving bird-to-human
transmission and the resulting production of clinically severe and fatal human
infections. Such human infections have been sporadic and are caused by H7N7 and
H5N1 high-pathogenicity (HP) and H9N2 low-pathogenicity (LP) AI viruses in Europe
and Asia. These infections have raised the level of concern by human health
agencies for the potential reassortment of influenza virus genes and generation
of the next human pandemic influenza A virus. The presence of endemic infections 
by H5N1 HPAI viruses in poultry in several Asian countries indicates that these
viruses will continue to contaminate the environment and be an exposure risk with
human transmission and infection. Furthermore, the reports of mammalian
infections with H5N1 AI viruses and, in particular, mammal-to-mammal transmission
in humans and tigers are unprecedented. However, the subsequent risk for
generating a pandemic human strain is unknown. More international funding from
both human and animal health agencies for diagnosis or detection and control of
AI in Asia is needed. Additional funding for research is needed to understand why
and how these AI viruses infect humans and what pandemic risks they pose.

PMID: 16252482  [PubMed - indexed for MEDLINE]
Þ

32407. Virology. 2005 Sep 15;340(1):70-83.

Evolution of H9N2 influenza viruses from domestic poultry in Mainland China.

Li C, Yu K, Tian G, Yu D, Liu L, Jing B, Ping J, Chen H.

Animal Influenza Laboratory of the Ministry of Agriculture and National Key
Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute,
Chinese Academy of Agricultural Sciences, Harbin 150001, People's Republic of
China.

H9N2 viruses have circulated in domestic poultry in Mainland China since 1994,
and an inactivated vaccine has been used in chickens to control the disease since
1998. The present study analyzed 27 H9N2 avian influenza viruses that were
isolated from chickens and ducks from 1996 to 2002. Infection studies indicated
that most of the viruses replicate efficiently but none of them is lethal for SPF
chickens. However, these viruses exhibit different phenotypes of replication in a
mouse model. Five viruses, including 4 early isolates and one 2000 isolate, are
not able to replicate in mice; 14 viruses replicate to moderate titers in mouse
lungs and cause less than 5% weight loss, while other 8 viruses could replicate
to high titers in the lungs and 7 of them induce 10-20% weight loss of the mice
on day 5 after inoculation. Most of the viruses isolated after 1996 are
antigenically different from the vaccine strain that is currently used in China. 
Three viruses isolated in central China in 1998 are resistant to adamantanes.
Phylogenetic analysis revealed that all of the viruses originated from
CK/BJ/1/94-like virus and formed multiple genotypes through complicated
reassortment with QA/HK/G1/97-, CK/HK/G9/97-, CK/SH/F/98-, and TY/WI/66-like
viruses. This study is a description of the previously uncharacterized H9N2 avian
influenza viruses recently circulating in chickens and ducks in Mainland China.
Our findings suggest that urgent attention should be paid to the control of H9N2 
influenza viruses in animals and to the human's influenza pandemic preparedness.

PMID: 16026813  [PubMed - indexed for MEDLINE]
Þ

32410. Virus Genes. 2005 Oct;31(2):163-9.

Phylogenetic analysis of eight genes of H9N2 subtype influenza virus: a mainland 
China strain possessing early isolates' genes that have been circulating.

Lu JH, Liu XF, Shao WX, Liu YL, Wei DP, Liu HQ.

Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou
University, 225009 Yangzhou, Jiangsu, People's Republic of China.

H9N2 subtype influenza virus has become worldwide and prevalent in China.
Previous studies illustrated that at least three sublineages had been established
in terrestrial poultry of Eurasian avian. In this presentation, eight full-length
genes of an H9N2 strain, A/Chicken/Shanghai/F/98 (Ck/SH/F/98) were obtained.
Sequence analysis and phylogenetic studies were conducted by comparing eight
genes with those of all the available H9N2 strains from the GenBank. The results 
showed that four genes (HA, NA, M and NS genes) of Ck/SH/F/98 were incorporated
into the sublineage represented by the early mainland China strain, Ck/BJ/1/94.
However, the other four of RNP genes of Ck/SH/F/98 did not show close
relationship with those of the three known sublineages' viruses. Therefore,
Ck/SH/F/98 was a natural reassortant between different sublineages. In addition, 
comparison showed that Ck/SH/F/98 could be a putative precursor of a later
isolate from southern China, Dk/ST/1605/01, with at least six genes of both
closely related, indicating genes of Ck/SH/F/98 and early isolates had ever been 
circulating. Further comparison in terms of molecular markers of species
specificity of HA1 revealed that DK/ST/1605/01 also resembled Ck/SH/F/98 more
than a common earlier duck strain. The results supported the idea of two-way
transmission between terrestrial and aquatic birds that emphasized the importance
to raise concerns on the natural evolution of all the eight genes of H9N2 avian
influenza viruses.

PMID: 16025241  [PubMed - indexed for MEDLINE]
Þ

33268. Rev Sci Tech. 2004 Aug;23(2):453-65.

Microbial adaptation and change: avian influenza.

Webster RG, Hulse DJ.

Saint Jude Children's Research Hospital, Department of Infectious Diseases,
Division of Virology, 332 North Lauderdale, Memphis, TN 38105, USA.

The evolution of influenza is a continuing process involving viral and host
factors. The increasing frequency of emergence of the highly pathogenic H5N1,
H7N3 and H7N7 influenza viruses and the panzootic spread of H9N2 influenza virus,
all of which can be potentially transmitted to humans, are of great concern to
both veterinary and human public health officials. The question is how soon the
next pandemic will emerge. A convergence of factors, including the population
densities of poultry, pigs and humans, are likely factors affecting the evolution
of the virus. Highly concentrated poultry and pig farming, in conjunction with
traditional live animal or 'wet' markets, provide optimal conditions for
increased mutation, reassortment and recombination of influenza viruses.
Strategies to reduce the evolution of influenza and the emergence of pandemics
include the separation of species, increased biosecurity, the development of new 
vaccine strategies and better basic knowledge of the virus. More effective
co-operation between scientists and veterinary and public health officials is
required to achieve these goals.

PMID: 15702713  [PubMed - indexed for MEDLINE]
Þ

33331. Virology. 2005 Feb 20;332(2):529-37.

Avian influenza viruses in Korean live poultry markets and their pathogenic
potential.

Choi YK, Seo SH, Kim JA, Webby RJ, Webster RG.

Division of Virology, Department of Infectious Diseases, Mail Stop 330, St. Jude 
Children's Research Hospital, 332 N. Lauderdale Street, Memphis TN 38105-2794,
USA.

We surveyed live-poultry markets in Korea in 2003 and isolated 9 H9N2, 6 H3N2,
and 1 H6N1 influenza viruses. Antigenic and phylogenetic analyses showed that all
9 H9N2 isolates were of A/Chicken/Korea/25232-96006/96-like lineage (which caused
disease in chickens in Korea in 1996) but were different from H9N2 viruses of
southeastern China. They had at least 4 genotypes and replicated in chickens but 
not in mice. The H3N2 and H6N1 viruses were new to Korea and were probably
reassortants of avian influenza viruses from southeastern China and recent Korean
H9N2 viruses. All 8 segments of the H3N2 viruses formed a single phylogenetic
cluster with 99.1 to 100% homology. The H3N2 viruses replicated in chickens and
mice without preadaptation, but the H6N1 virus did not. Our results show an
increasingly diverse pool of avian influenza viruses in Korea that are potential 
pandemic influenza agents.

PMID: 15680418  [PubMed - indexed for MEDLINE]
Þ

34209. Microbes Infect. 2004 Aug;6(10):919-25.

Isolation and identification of swine influenza recombinant
A/Swine/Shandong/1/2003(H9N2) virus.

Xu C, Fan W, Wei R, Zhao H.

College of Animal Science, Shandong Agriculture University, Shandong Province,
Taian City 271018, People's Republic of China. xcttaian2002@163.com

Ten influenza virus isolates were obtained from infected pigs from different
places in Shandong province showing clinical symptoms from October 2002 to
January 2003. All 10 isolates were identified in China's National Influenza
Research Center as influenza A virus of H9N2 subtype. The complete genome of one 
isolate, designated A/Swine/Shandong/1/2003(H9N2), was sequenced and compared
with sequences available in GenBank. The results of analyses indicated that the
sequence of A/Swine/Shandong/1/2003(H9N2) was similar to those of several chicken
influenza viruses and duck influenza viruses recently prevalent in South China.
According to phylogenetic analysis of the complete gene sequences,
A/Swine/Shandong/1/2003(H9N2) possibly originated from the reassortment of
chicken influenza viruses and duck influenza viruses. It was found that the amino
acid sequence at the HA cleavage site in Sw/SD/1/2003 is R-S-L-R-G, differing
clearly from that of other H9N2 subtype isolates of swine influenza and avian
influenza, which is R-S-S-R-G.

PMID: 15310468  [PubMed - indexed for MEDLINE]
Þ

34308. J Gen Virol. 2004 Aug;85(Pt 8):2327-37.

Influenza A viruses in feral Canadian ducks: extensive reassortment in nature.

Hatchette TF, Walker D, Johnson C, Baker A, Pryor SP, Webster RG.

Division of Virology, Department of Infectious Diseases, St Jude Children's
Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105-2794, USA.

The current dogma of influenza accepts that feral aquatic birds are the reservoir
for influenza A viruses. Although the genomic information of human influenza A
viruses is increasing, little of this type of data is available for viruses
circulating in feral waterfowl. This study presents the genetic characterization 
of 35 viruses isolated from wild Canadian ducks from 1983 to 2000, as the first
attempt at a comprehensive genotypic analysis of influenza viruses isolated from 
feral ducks. This study demonstrates that influenza virus genes circulating in
Canadian ducks have achieved evolutionary stasis. The majority of these duck
virus genes are clustered in distinct North American clades; however, some H6 and
H9 genes are clustered with those from Eurasian viruses. Genes appeared to
reassort in a random fashion. None of the genotypes identified remained present
throughout all of the years examined and most PA and PB2 genes that crossed over 
into swine were clustered in one phylogenetic grouping. Additionally, matrix
genes were identified that branch very early in the evolutionary tree. These
findings demonstrate the diversity of the influenza virus gene pool in Canadian
ducks, and suggest that genes which cluster in specific phylogenetic groupings in
the PB2 and PA genes can be used for markers of viruses with the potential for
crossing the species barrier. A more comprehensive study of this important
reservoir is needed to provide further insight into the genomic composition of
viruses that crossover the species barrier, which would be a useful component to 
pandemic planning.

PMID: 15269374  [PubMed - indexed for MEDLINE]
Þ

34419. Arch Virol. 2004 Jul;149(7):1397-407. Epub 2003 Dec 8.

Attenuation of a human H9N2 influenza virus in mammalian host by reassortment
with an avian influenza virus.

Saito T, Lim W, Tashiro M.

Department of Virology III, National Institute of Infectious Diseases, Tokyo,
Japan. taksaito@nih.go.jp

In order to develop a surrogate virus strain for production of an inactivated
influenza vaccine against a human H9N2 virus, A/Hong Kong/1073/99 (HK1073: H9N2) 
was co-infected in embryonated chicken eggs with an apathogenic avian influenza
virus, A/Duck/Czechoslovakia/56 (Dk/Cz: H4N6), for gene segment reassortment.
Multiple-gene reassortants obtained were examined for replication in mammalian
hosts in vitro and in vivo by infecting MDCK cells and by intranasal
administration to hamsters, respectively. A 2-6 gene reassortant with both
surface glycoproteins of HK1073 origin and the rest of Dk/Cz origin, HK/CZ-13,
was shown to replicate poorly in the mammalian hosts both in vivo and in vitro
comparing with HK1073, although this reassortant replicated as efficiently as
each parental strain in embryonated eggs. No sequence difference was observed in 
the HA1 region between HK1073 and HK/CZ-13, indicating that the reassortant would
be equivalent in its immunogenicity to the parental HK1073 strain when it is used
as an inactivated vaccine. A virus strain with attenuation in mammalian hosts is 
preferable for production of an H9 vaccine, since it should reduce the risk of
manufacturing-related infections of employees during the vaccine production.
HK/CZ-13 can therefore be a surrogate strain for production of an inactivated
vaccine as well as diagnostic antigens in case of a possible future pandemic
caused by an HK1073-like H9 influenza virus.

PMID: 15221539  [PubMed - indexed for MEDLINE]
Þ

35182. J Virol. 2004 Feb;78(4):1851-7.

Generation of high-yielding influenza A viruses in African green monkey kidney
(Vero) cells by reverse genetics.

Ozaki H, Govorkova EA, Li C, Xiong X, Webster RG, Webby RJ.

Departments of Infectious Disease, St. Jude Children's Research Hospital,
Memphis, Tennessee 38105, USA.

Influenza A viruses are the cause of annual epidemics of human disease with
occasional outbreaks of pandemic proportions. The zoonotic nature of the disease 
and the vast viral reservoirs in the aquatic birds of the world mean that
influenza will not easily be eradicated and that vaccines will continue to be
needed. Recent technological advances in reverse genetics methods and limitations
of the conventional production of vaccines by using eggs have led to a push to
develop cell-based strategies to produce influenza vaccine. Although cell-based
systems are being developed, barriers remain that need to be overcome if the
potential of these systems is to be fully realized. These barriers include, but
are not limited to, potentially poor reproducibility of viral rescue with reverse
genetics systems and poor growth kinetics and yields. In this study we present a 
modified A/Puerto Rico/8/34 (PR8) influenza virus master strain that has improved
viral rescue and growth properties in the African green monkey kidney cell line, 
Vero. The improved properties were mediated by the substitution of the PR8 NS
gene for that of a Vero-adapted reassortant virus. The Vero growth kinetics of
viruses with H1N1, H3N2, H6N1, and H9N2 hemagglutinin and neuraminidase
combinations rescued on the new master strain were significantly enhanced in
comparison to those of viruses with the same combinations rescued on the standard
PR8 master strain. These improvements pave the way for the reproducible
generation of high-yielding human and animal influenza vaccines by reverse
genetics methods. Such a means of production has particular relevance to epidemic
and pandemic use.

PMCID: PMC369478
PMID: 14747549  [PubMed - indexed for MEDLINE]
Þ

35641. Avian Dis. 2003;47(3 Suppl):1127-30.

Generation and characterization of an H9N2 cold-adapted reassortant as a vaccine 
candidate.

Chen H, Matsuoka Y, Chen Q, Cox NJ, Murphy BR, Subbarao K.

Influenza Branch, Division of Viral and Rickettsial Diseases, National Center for
Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton
Road, G-16, Atlanta, GA 30333, USA.

H9N2 subtype avian influenza viruses have been identified in avian species
worldwide, and infections in pigs were confirmed in Hong Kong in 1998.
Subsequently, H9N2 viruses were isolated from two children in Hong Kong in 1999, 
and five human infections were reported from China, raising the possibility that 
H9N2 viruses pose a potential pandemic threat for humans. These events prompted
us to develop a vaccine candidate to protect humans against this subtype of
influenza A viruses. Reassortant H1N1 and H3N2 human influenza A viruses with the
six internal gene segments of A/Ann Arbor/6/60 (H2N2)(AA) cold-adapted (ca) virus
have been tested extensively in humans and have proved to be attenuated and safe 
as live virus vaccines. Using classical genetic reassortment, we generated a
reassortant that contains the hemagglutinin and neuraminidase genes from
A/chicken/Hong Kong/G9/97 (H9N2) and six internal gene segments from the AAca
virus. The G9/AAca reassortant virus exhibits the ca phenotype and the
temperature-sensitive phenotypes of the AAca virus and was attenuated in mice.
The reassortant virus was immunogenic and protected mice from wild-type H9N2
virus challenge. The G9/AAca virus bears the in vitro and in vivo phenotypes
specified by the AAca virus and will be evaluated as a potential vaccine
candidate in humans.

PMID: 14575127  [PubMed - indexed for MEDLINE]
Þ

35644. Avian Dis. 2003;47(3 Suppl):1114-7.

Land-based birds as potential disseminators of avian mammalian reassortant
influenza A viruses.

Perez DR, Webby RJ, Hoffmann E, Webster RG.

Division of Virology, Department of Infectious Diseases, St. Jude Children's
Research Hospital, Memphis, TN 38105, USA.

Chickens, quail, and other land-based birds are extensively farmed around the
world. They have been recently implicated in zoonotic outbreaks of avian
influenza in Hong Kong. The possibility that land-based birds could act as mixing
vessels or disseminators of avian/mammalian reassortant influenza A viruses with 
pandemic potential has not been evaluated. In this report, we investigated
whether chickens and Japanese quail are susceptible to a mammalian influenza
virus (A/swine/Texas/4199-2/98 [H3N2]). This virus did not grow in chickens and
replicated to low levels in Japanese quail but did not transmit. Replacing the H3
gene of this virus for one of the avian H9 viruses resulted in transmission of
the avian/swine reassortant virus among quail but not among chickens. Our
findings demonstrated that Japanese quail could provide an environment in which
viruses like the A/swine/Texas/4199-2/98 [H3N2] virus could further reassort and 
generate influenza viruses with pandemic potential.

PMID: 14575124  [PubMed - indexed for MEDLINE]
Þ

35680. Avian Dis. 2003;47(3 Suppl):926-30.

Safety evaluation in chickens of candidate human vaccines against potential
pandemic strains of influenza.

Matsuoka Y, Chen H, Cox N, Subbarao K, Beck J, Swayne D.

Influenza Branch, Division of Viral and Rickettsial Diseases, National Center for
Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton
Road, G-16, Atlanta, GA 30333, USA.

Two candidate formalin-inactivated vaccines, made from high-growth reassortant
viruses with the HA and NA genes from avian viruses in a background of genes
derived from A/Puerto Rico/8/34 (PR8), were prepared against H5N1 and H9N2
subtypes (designated as H5N1/PR8 and H9N2/PR8, respectively). These viruses bear 
the genotypes, antigenicity, and attenuation in mouse models that are desirable
in candidate vaccines. The pathogenicity of the newly generated avian-human
reassortant vaccine viruses was also evaluated in chickens. Neither H5N1/PR8 nor 
H9N2/PR8 were highly pathogenic for chickens. No clinical signs, gross legions,
or histological lesions were observed in chickens that were administered H5N1/PR8
either intranasally (i.n.) or intravenously (i.v.), and virus was not detected in
oropharyngeal or cloacal swabs. When H9N2/PR8 was administered i.n., no clinical 
signs, gross lesions, or histological lesions were observed and no virus was
detected in cloacal swabs. However, virus was isolated at low titer from
oropharyngeal swabs of all eight chickens. Although no clinical signs were
observed when H9N2/PR8 was administered i.v., mild tracheitis was seen in one of 
two chickens. Moderate amounts of antigen were observed in tracheal respiratory
epithelium, and low titers of virus were recovered from oropharyngeal and cloacal
swabs of some chickens. In summary, both reassortant vaccine viruses replicated
poorly in chickens. These studies suggest that these candidate vaccine viruses
carry a low risk of transmission to chickens.

PMID: 14575088  [PubMed - indexed for MEDLINE]
Þ

35682. Avian Dis. 2003;47(3 Suppl):914-20.

The impact of avian influenza viruses on public health.

Katz JM.

Influenza Branch, Division of Viral and Rickettsial Diseases, National Center for
Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton
Road, G-16, Atlanta, GA 30333, USA.

In the late 1990s, H5N1 and H9N2 avian influenza viruses caused respiratory
infections in humans in Hong Kong. Exposure to domestic poultry in live-bird
markets was significantly associated with human H5N1 disease. Seroepidemiologic
studies conducted among contacts of H5N1-infected persons determined that
human-to-human transmission of the avian H5N1 viruses occurred but was rare. The 
relatively high rates of H5 and H9 antibody seroprevalence among Hong Kong
poultry workers in 1997 highlight the potential for avian viruses to transmit to 
humans, particularly those with occupational exposure. Such transmission
increases the likelihood of reassortment between a currently circulating human
virus and an avian virus and thus the creation of a strain with pandemic
potential.

PMID: 14575086  [PubMed - indexed for MEDLINE]
Þ

35851. Vaccine. 2003 Oct 1;21(27-30):4430-6.

Generation and characterization of a cold-adapted influenza A H9N2 reassortant as
a live pandemic influenza virus vaccine candidate.

Chen H, Matsuoka Y, Swayne D, Chen Q, Cox NJ, Murphy BR, Subbarao K.

Influenza Branch, CDC, Atlanta, GA, USA.

H9N2 subtype influenza A viruses have been identified in avian species worldwide 
and were isolated from humans in 1999, raising concerns about their pandemic
potential and prompting the development of candidate vaccines to protect humans
against this subtype of influenza A virus. Reassortant H1N1 and H3N2 human
influenza A viruses with the internal genes of the influenza A/Ann Arbor/6/60
(H2N2) (AA) cold-adapted (ca) virus have proven to be attenuated and safe as live
virus vaccines in humans. Using classical genetic reassortment, we generated a
reassortant virus (G9/AA ca) that contains the hemagglutinin and neuraminidase
genes from influenza A/chicken/Hong Kong/G9/97 (H9N2) (G9) and six internal gene 
segments from the AA ca virus. When administered intranasally, the reassortant
virus was immunogenic and protected mice from subsequent challenge with wild-type
H9N2 viruses, although it was restricted in replication in the respiratory tract 
of mice. The G9/AA ca virus bears properties that are desirable in a vaccine for 
humans and is available for clinical evaluation and use, should the need arise.

PMID: 14505926  [PubMed - indexed for MEDLINE]
Þ

36046. Vopr Virusol. 2003 May-Jun;48(3):4-10.

[Interspecies transmission of influenza A viruses and influenza pandemics].

[Article in Russian]

Kaverin NV, Smirnov IuA.

Molecular and genetic data are summarized on the origin of influenza A virus
pandemic variants. Conceptual modifications of the reassortment theory of the
origin of pandemic strains are discussed in connection with the appearance of new
H5 and H9 avian influenza viruses, which caused the respiratory infection in man 
and which are presently in the focus of attention as possible agents of future
pandemic.

PMID: 12894472  [PubMed - indexed for MEDLINE]
Þ

36296. J Virol. 2003 Jun;77(12):6988-94.

Characterization of H9 subtype influenza viruses from the ducks of southern
China: a candidate for the next influenza pandemic in humans?

Li KS, Xu KM, Peiris JS, Poon LL, Yu KZ, Yuen KY, Shortridge KF, Webster RG, Guan
Y.

Joint Influenza Research Centre (SUMC & HKU), Department of Microbiology and
Immunology, Shantou University Medical College, Shantou, Guangdong 515031, P. R. 
China.

A current view of the emergence of pandemic influenza viruses envisages a gene
flow from the aquatic avian reservoir to humans via reassortment in pigs, the
hypothetical "mixing vessel." Understanding arising from recent H5N1 influenza
outbreaks in Hong Kong since 1997 and the isolation of avian H9N2 virus from
humans raises alternative options for the emergence of a new pandemic virus. Here
we report that H9N2 influenza viruses established in terrestrial poultry in
southern China are transmitted back to domestic ducks, in which the viruses
generate multiple reassortants. These novel H9N2 viruses are double or even
triple reassortants that have amino acid signatures in their hemagglutinin,
indicating their potential to directly infect humans. Some of them contain gene
segments that are closely related to those of A/Hong Kong/156/97 (H5N1/97, H5N1) 
or A/Quail/Hong Kong/G1/97 (G1-like, H9N2). More importantly, some of their
internal genes are closely related to those of novel H5N1 viruses isolated during
the outbreak in Hong Kong in 2001. This study reveals a two-way transmission of
influenza virus between terrestrial and aquatic birds that facilitates the
generation of novel reassortant H9N2 influenza viruses. Such reassortants may
directly or indirectly play a role in the emergence of the next pandemic virus.

PMCID: PMC156195
PMID: 12768017  [PubMed - indexed for MEDLINE]
Þ

36453. Vaccine. 2003 May 16;21(17-18):1974-9.

Generation and evaluation of a high-growth reassortant H9N2 influenza A virus as 
a pandemic vaccine candidate.

Chen H, Subbarao K, Swayne D, Chen Q, Lu X, Katz J, Cox N, Matsuoka Y.

Influenza Branch, Mailstop G-16, Centers for Disease Control and Prevention, 1600
Clifton Road, Atlanta, GA 30333, USA.

H9N2 subtype avian influenza viruses (AIVs) are widely distributed in avian
species and were isolated from humans in Hong Kong and Guangdong province, China 
in 1999 raising concern of their potential for pandemic spread. We generated a
high-growth reassortant virus (G9/PR8) that contains the hemagglutinin (HA) and
neuraminidase (NA) genes from the H9N2 avian influenza virus A/chicken/Hong
Kong/G9/97 (G9) and six internal genes from A/Puerto Rico/8/34 (PR8) by genetic
reassortment, for evaluation as a potential vaccine candidate in humans.
Pathogenicity studies showed that the G9/PR8 reassortant was not highly
pathogenic for mice or chickens. Two doses of a formalin-inactivated G9/PR8 virus
vaccine induced hemagglutination inhibiting antibodies and conferred complete
protection against challenge with G9 and the antigenically distinct H9N2 A/Hong
Kong/1073/99 (G1-like) virus in a mouse model. These results indicate that the
high growth G9/PR8 reassortant has properties that are desirable in a vaccine
seed virus and is suitable for evaluation in humans for use in the event of an H9
pandemic.

PMID: 12706686  [PubMed - indexed for MEDLINE]
Þ

36513. J Appl Microbiol. 2003;94 Suppl:70S-79S.

The next influenza pandemic: lessons from Hong Kong.

Shortridge KF, Peiris JS, Guan Y.

Department of Microbiology, The University of Hong Kong, Queen Mary Hospital,
Hong Kong SAR, China. kennedyfs@xtra.co.nz

Pandemic influenza is a zoonosis. Studies on influenza ecology conducted in Hong 
Kong since the 1970s in which Hong Kong essentially functioned as an influenza
sentinel post indicated that it might be possible, for the first time, to have
influenza preparedness at the baseline avian level. This appreciation of
influenza ecology facilitated recognition of the H5N1 'bird flu' incident in Hong
Kong in 1997 in what was considered to be an incipient pandemic situation, the
chicken being the source of virus for humans and, if so, was the first instance
where a pandemic may have been averted. The 2001 and 2002 H5N1 incidents
demonstrated that it was possible to have an even higher order of baseline
preparedness with the recognition in chicken of a range of genotypes of H5N1-like
viruses before they had the opportunity to infect humans. Investigations of these
incidents revealed a complex ecology involving variously precursor avian H5N1
virus in geese and ducks, and H9N2 and H6N1 viruses in quail, the quail possibly 
functioning as an avian 'mixing vessel' for key genetic reassortment events for
onward transmission of H5N1 viruses highly pathogenic for chicken and humans.
These findings highlight the importance of systematic virus surveillance of
domestic poultry in recognizing changes in virus occurrence, host range and
pathogenicity as signals at the avian level that could presage a pandemic. For
example, there is now an increasing prevalence of avian influenza viruses in
terrestrial (in contrast to aquatic) poultry. Prior to 1997, no particular virus 
subtype other than H4N6 would have been considered a candidate for pandemicity
and this was based, in the absence of any other data, on its high frequency of
occurrence in ducks in southern China. Now,with the isolation of H5N1 and H9N2
viruses from humans supported by genetic, molecular and biological studies on
these and other avian isolates, there is credible evidence for the candidacy, in 
order, of H5N1, H9N2 and H6N1 viruses. These viruses have been made available for
the production of diagnostic reagents and exploratory vaccines. The 1997 incident
upheld the hypothesis that southern China is an epicentre for the emergence of
pandemic influenza viruses. However, the intensification of the poultry (chicken)
industry worldwide coupled with the spread of viruses such as the Eurasian
lineage of H9N2 suggest that the genesis of a pandemic could take place elsewhere
in the world. This re-emphasizes the importance of systematic virus surveillance 
of poultry globally for international public health and for economic and food
concerns. Faced with an incipient pandemic in 1997, Hong Kong brought in
international experts to join the investigative effort. Good teamwork at all
levels is essential in dealing with the many facets. The threat of a pandemic
should not be minimized, nor should governments be lulled into a sense of false
security. The media is a powerful channel and has the responsibility and the
avenues to convey and influence public perception of events. Close liaison
between the media and those on the operational side ensures effective, accurate
and timely dissemination of information. This will enhance public confidence in
the investigative process and in steps taken for its safety and health.

PMID: 12675938  [PubMed - indexed for MEDLINE]
Þ

36903. Kaohsiung J Med Sci. 2002 Sep;18(9):421-34.

Influenza: prospect for prevention and control.

Williams JR, Chen PY, Cho CT, Chin TD.

Department of Pediatrics, University of Kansas School of Medicine, Kansas City,
Kansas 66160, USA.

Influenza is an emerging and re-emerging disease. Since the late 1930s influenza 
viruses have been isolated yearly from different parts of the world during
epidemics and pandemics. The "epidemiologic success" of influenza is due largely 
to rapid and unpredictable antigenic changes (antigenic drift) among human
influenza viruses, and the emergence of new subtypes (antigenic shift), mostly
from reassortment between human and avian influenza viruses. Antigenic shifts
were attributed to the global pandemic viruses of 1957 (H2N2 Asian flu) and 1968 
(H3N2 Hong Kong flu). Concern over possible new pandemics has been heightened by 
recent reports of human infection in Asia in 1997 with avian viruses (H5N1) and
in 1999 (H9N2) and isolation of human-avian reassorted viruses from pigs and
humans in Europe. Influenza has a high rate of inapparent infection, short
incubation and high infectivity; epidemics usually start abruptly and spread
rapidly to neighboring communities and countries. Isolation and quarantine are
often unsuccessful in preventing the spread of the infection. Although not
perfect, immunization and chemoprophylaxis are highly effective at minimizing the
spread of influenza and reducing morbidity and mortality, social disruption and
economic loss. Plans for future influenza epidemics and pandemics require
national and international programs to be in place for the monitoring of
influenza activity, the dissemination and exchange of information and the
provision and delivery of sufficient quantities of vaccines and antiviral agents.
This paper reviews and discusses the antigenic variations of the influenza virus,
potential influenza pandemics, protective efficacy of inactivated vaccines and
antiviral agents and preparation for control of future epidemics and pandemics.

PMID: 12515400  [PubMed - indexed for MEDLINE]
Þ

37413. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2002 Jun;16(2):142-5.

[Characterization of genome of A/Guangzhou/333/99(H9N2) virus].

[Article in Chinese]

Guo Y, Xie J, Wu K, Dong J, Wang M, Zhang Y, Guo J, Chen J, Chen Z, Li Z.

Institute of Virology, Chinese Academy of Preventive Medicine, Beijing 100052,
China.

BACKGROUND: To understand the characterization of genome of a strain of avian
influenza A H9N2 virus repeatedly isolated from a child with influenza illness.
Thereafter to reveal the origin of this H9N2 virus.
METHODS: Viruses were passed in embryonated hen eggs and virion RNA was extracted
from allantoic fluid and reverse transcribed to synthesize cDNA. cDNA was
amplified by PCR and the PCR product was purified with a purification kit.
Afterwards RNA sequence analysis was performed by dideoxynucleotide chain
termination and a cloning method. Finally, phylogenetic analysis of the
sequencing data was performed with MegAlign (Version 1.03) and Editseg (Version
3.69) softwares.
RESULTS: Genome of A/Guangzhou/333/99 (H9N2) virus was closely related to avian
influenza A H9N2 virus, but obvious difference from that of A/Duck/Hong
Kong/Y439/97(H9N2) virus, as well as its genome did not include any RNA segment
derived from human influenza A virus. However, the genes encoding the HA,NA,NP
and NS proteins of A/Guangzhou/333/99 virus were derived from those of G9 lineage
virus, the rest genes encoding the M and three polymerase (PB2,PB1 and PA)
proteins were derived from G1 lineage strain.
CONCLUSIONS: A/Guangzhou/333/99 virus was a reassortant derived from reassortment
betweenG9 and G1 lineages of avian influenzaA(H9N2) viruses. Therefore, the most 
possibility is that it is derived from avian influenza A virus directly. The
results do not only demonstrate that avian influenza A (H9N2) virus could infect 
men, but also firstly prove that the genetic reassortment could be occurred
between different genetic lineages of avian influenza A (H9N2) viruses in the
nature.

PMID: 12196825  [PubMed - indexed for MEDLINE]
Þ

37474. Vaccine. 2002 Aug 19;20(25-26):3165-70.

Eight-plasmid system for rapid generation of influenza virus vaccines.

Hoffmann E, Krauss S, Perez D, Webby R, Webster RG.

Department of Virology and Molecular Biology, St. Jude Children's Research
Hospital, 332 North Lauderdale, Memphis, TN 38105-2794, USA.

The antigenic variation of influenza A virus hemagglutinin (HA) and neuraminidase
(NA) glycoproteins requires frequent changes in vaccine formulation. The
classical method of creating influenza virus seed strains for vaccine production 
is to generate 6 + 2 reassortants that contain six genes from a high-yield virus,
such as A/PR/8/34 (H1N1) and the HA and NA genes of the circulating strains. The 
techniques currently used are time-consuming because of the selection process
required to isolate the reassortant virus. We generated the high-yield virus
A/PR/8/34 (H1N1) entirely from eight plasmids. Its growth phenotype in
embryonated chicken eggs was equivalent to that of the wild-type virus. By using 
this DNA-based cotransfection technique, we generated 6 + 2 reassortants that had
the antigenic determinants of the influenza virus strains A/New Caledonia/20/99
(H1N1), A/Panama/2007/99 (H3N2), A/teal/HK/W312 (H6N1), and A/quail/HK/G1/97
(H9N2). Our findings demonstrate that the eight-plasmid system allows the rapid
and reproducible generation of reassortant influenza A viruses for use in the
manufacture of vaccines.

Copyright 2002 Elsevier Science Ltd.

PMID: 12163268  [PubMed - indexed for MEDLINE]
Þ

37572. Vaccine. 2002 May 15;20 Suppl 2:S28-35.

Influenza activity in China: 1998-1999.

Yuanji G.

Institute of Virology, Chinese Academy of Preventive Medicine, 100 Ying Xin Jie, 
Xuan Wu Qu, Beijing, PR China. cnic_gyj@263.net

During 1989-1999, influenza A H3N2 and H1N1 subtypes and B type viruses were
still co-circulating in human population in China, while influenza A (H3N2) virus
was predominant strain. The two antigenically and genetically distinguishable
strains of influenza B virus were also still co-circulating in men in southern
China. The antigenic analysis indicated that most of the H3N2 viruses were
A/Panama/2007/99 (H3N2)-like strain, the most of the H1N1 viruses were
antigenically similar to A/Beijing/262/95 (H1N1) virus. However, most of the
influenza B viruses were B/Beijing/184/93-like strain, but few of them were
antigenically similar to B/Shandong/7/97 virus. In the summer of 1998, the
influenza outbreaks caused by H3N2 subtype of influenza A virus occurred widely
in southern China. Afterwards, during 1998-1999 influenza season, a severe
influenza epidemic caused by H3N2 virus emerged in northern China. The morbidity 
was reached as high as 10% in Beijing area. It was interesting that during
influenza, surveillance from 1998 to 1999, five strains of avian influenza A
(H9N2) virus were isolated from outpatients with influenza-like illness in
July-August of 1998, and another one was repeatedly isolated from a child
suffering from influenza-like disease in November of 1999 in Guangdong province. 
The genetic analysis revealed that the five strains isolated in 1998 were
genetically closely related to H9N2 viruses being isolated from chickens (G9
lineage virus), whereas, A/Guangzhou/333/99 (H9N2) virus was a reassortant
derived from reassortment between G9 and G1 lineage of avian influenza A (H9N2)
viruses due to its genes encoding the HA, NA, NP and NS proteins, closely related
to G9 lineage virus, the rest of the genes encoding the M and three polymerase
(PB2, PB1 and PA) were closely related to G1 lineage strain of H9N2 virus.
However, no avian influenza A (H5N1) virus has so far been isolated neither from 
in or outpatients with influenza-like disease in mainland China. Unfortunately,
where did the reassortment occur and how did the reassortant transmit to men?
These questions are still unknown.

PMID: 12110253  [PubMed - indexed for MEDLINE]
Þ

37666. Acta Trop. 2002 Jul;83(1):1-6.

Avian influenza and human health.

Capua I, Alexander DJ.

National Reference Laboratory for Avian Influenza, Istituto Zooprofilattico
Sperimentale delle Venezie, Via Romea 14/A, 35020 Legnaro, Padua, Italy.
icapua@izsvenezie.it

Natural infections with influenza A viruses have been reported in a variety of
animal species including humans, pigs, horses, sea mammals, mustelids and birds. 
Occasionally devastating pandemics occur in humans. Although viruses of
relatively few HA and NA subtype combinations have been isolated from mammalian
species, all 15 HA subtypes and all 9 NA subtypes, in most combinations, have
been isolated from birds. In the 20th century the sudden emergence of
antigenically different strains transmissible in humans, termed antigenic shift, 
has occurred on four occasions, 1918 (H1N1), 1957 (H2N2), 1968 (H3N2) and 1977
(H1N1), each time resulting in a pandemic. Genetic analysis of the isolates
demonstrated that 'new' strains most certainly emerged after reassortment of
genes of viruses of avian and human origin in a permissive host. The leading
theory is that the pig represents the 'mixing vessel' where this genetic
reassortment may occur. In 1996, an H7N7 influenza virus of avian origin was
isolated from a woman with a self-limiting conjunctivitis. During 1997 in Hong
Kong, an H5N1 avian influenza virus was recognised as the cause of death of 6 of 
18 infected patients. Genetic analysis revealed these human isolates of H5N1
subtype to be indistinguishable from a highly pathogenic avian influenza virus
that was endemic in the local poultry population. More recently, in March 1999,
two independent isolations of influenza virus subtype H9N2 were made from girls
aged one to four who recovered from flu-like illnesses in Hong Kong.
Subsequently, five isolations of H9N2 virus from humans on mainland China in
August 1998 were reported. H9N2 viruses were known to be widespread in poultry in
China and other Asian countries. In all these cases there was no evidence of
human to human spread except with the H5N1 infections where there was evidence of
very limited spread. This is in keeping with the finding that all these viruses
possessed all eight genes of avian origin. It may well be that infection of
humans with avian influenza viruses occurs much more frequently than originally
assumed, but due to their limited effect go unrecognised. For the human
population as a whole the main danger of direct infection with avian influenza
viruses appears to be if people infected with an 'avian' virus are infected
simultaneously with a 'human' influenza virus. In such circumstances reassortment
could occur with the potential emergence of a virus fully capable of spread in
the human population, but with antigenic characteristics for which the human
population was immunologically naive. Presumably this represents a very rare
coincidence, but one which could result in a true influenza pandemic.

PMID: 12062786  [PubMed - indexed for MEDLINE]
Þ

38288. Philos Trans R Soc Lond B Biol Sci. 2001 Dec 29;356(1416):1817-28.

Emergence of influenza A viruses.

Webby RJ, Webster RG.

Department of Virology and Molecular Biology, St Jude Children's Research
Hospital, 332 North Lauderdale, Memphis, TN 38105, USA.

Pandemic influenza in humans is a zoonotic disease caused by the transfer of
influenza A viruses or virus gene segments from animal reservoirs. Influenza A
viruses have been isolated from avian and mammalian hosts, although the primary
reservoirs are the aquatic bird populations of the world. In the aquatic birds,
influenza is asymptomatic, and the viruses are in evolutionary stasis. The
aquatic bird viruses do not replicate well in humans, and these viruses need to
reassort or adapt in an intermediate host before they emerge in human
populations. Pigs can serve as a host for avian and human viruses and are logical
candidates for the role of intermediate host. The transmission of avian H5N1 and 
H9N2 viruses directly to humans during the late 1990s showed that land-based
poultry also can serve between aquatic birds and humans as intermediate hosts of 
influenza viruses. That these transmission events took place in Hong Kong and
China adds further support to the hypothesis that Asia is an epicentre for
influenza and stresses the importance of surveillance of pigs and live-bird
markets in this area.

PMCID: PMC1088557
PMID: 11779380  [PubMed - indexed for MEDLINE]
Þ

38372. J Med Virol. 2002 Jan;66(1):107-14.

Molecular changes associated with the transmission of avian influenza a H5N1 and 
H9N2 viruses to humans.

Shaw M, Cooper L, Xu X, Thompson W, Krauss S, Guan Y, Zhou N, Klimov A, Cox N,
Webster R, Lim W, Shortridge K, Subbarao K.

Influenza Branch, Division of Viral and Rickettsial Diseases, Centers for Disease
Control and Prevention, Atlanta, GA 30333, USA. mws2@cdc.gov

In order to identify molecular changes associated with the transmission of avian 
influenza A H5N1 and H9N2 viruses to humans, the internal genes from these
viruses were compared to sequences from other avian and human influenza A
isolates. Phylogenetically, each of the internal genes of all sixteen of the
human H5N1 and both of the H9N2 isolates were closely related to one another and 
fell into a distinct clade separate from clades formed by the same genes of other
avian and human viruses. All six internal genes were most closely related to
those of avian isolates circulating in Asia, indicating that reassortment with
human strains had not occurred for any of these 18 isolates. Amino acids
previously identified as host-specific residues were predominantly avian in the
human isolates although most of the proteins also contained residues observed
previously only in sequences of human influenza viruses. For the majority of the 
nonglycoprotein genes, three distinct subgroups could be distinguished on
bootstrap analyses of the nucleotide sequences, suggesting multiple introductions
of avian virus strains capable of infecting humans. The shared nonglycoprotein
gene constellations of the human H5N1 and H9N2 isolates and their detection in
avian isolates only since 1997 when the first human infections were detected
suggest that this particular gene combination may confer the ability to infect
humans and cause disease. J. Med. Virol. 66:107-114, 2002. Published 2002
Wiley-Liss, Inc.

PMID: 11748666  [PubMed - indexed for MEDLINE]
Þ

38397. J Virol. 2002 Jan;76(1):118-26.

Characterization of H5N1 influenza viruses that continue to circulate in geese in
southeastern China.

Webster RG, Guan Y, Peiris M, Walker D, Krauss S, Zhou NN, Govorkova EA, Ellis
TM, Dyrting KC, Sit T, Perez DR, Shortridge KF.

Department of Virology and Molecular Biology, St. Jude Children's Research
Hospital, Memphis, Tennessee 38105, USA. robert.webster@stjude.org

The H5N1 influenza virus, which killed humans and poultry in 1997, was a
reassortant that possibly arose in one type of domestic poultry present in the
live-poultry markets of Hong Kong. Given that all the precursors of H5N1/97 are
still circulating in poultry in southern China, the reassortment event that
generated H5N1 could be repeated. Because A/goose/Guangdong/1/96-like (H5N1;
Go/Gd) viruses are the proposed donors of the hemagglutinin gene of the H5N1
virus, we investigated the continued circulation, host range, and
transmissibility of Go/Gd-like viruses in poultry. The Go/Gd-like viruses caused 
weight loss and death in some mice inoculated with high virus doses. Transmission
of Go/Gd-like H5N1 viruses to geese by contact with infected geese resulted in
infection of all birds but limited signs of overt disease. In contrast, oral
inoculation with high doses of Go/Gd-like viruses resulted in the deaths of up to
50% of infected geese. Transmission from infected geese to chickens occurred only
by fecal contact, whereas transmission to quail occurred by either aerosol or
fecal spread. This difference is probably explained by the higher susceptibility 
of quail to Go/Gd-like virus. The high degree of susceptibility of quail to Go/Gd
(H5N1)-like viruses and the continued circulation of H6N1 and H9N2 viruses in
quail support the hypothesis that quail were the host of origin of the H5N1/97
virus. The ease of transmission of Go/Gd (H5N1)-like viruses to land-based birds,
especially quail, supports the wisdom of separating aquatic and land-based
poultry in the markets in Hong Kong and the need for continued surveillance in
the field and live-bird markets in which different types of poultry are in
contact with one another.

PMCID: PMC135698
PMID: 11739677  [PubMed - indexed for MEDLINE]
Þ

38489. J Virol. 2001 Dec;75(23):11773-80.

Amino acids responsible for the absolute sialidase activity of the influenza A
virus neuraminidase: relationship to growth in the duck intestine.

Kobasa D, Wells K, Kawaoka Y.

Department of Pathobiological Sciences, School of Veterinary Medicine, University
of Wisconsin-Madison, 53706, USA.

The 1957 human pandemic strain of influenza A virus contained an avian virus
hemagglutinin (HA) and neuraminidase (NA), both of which acquired specificity for
the human receptor, N-acetylneuraminic acid linked to galactose of cellular
glycoconjugates via an alpha2-6 bond (NeuAcalpha2-6Gal). Although the NA retained
considerable specificity for NeuAcalpha2-3Gal, its original substrate in ducks,
it lost the ability to support viral growth in the duck intestine, suggesting a
growth-restrictive change other than a shift in substrate specificity. To test
this possibility, we generated a panel of reassortant viruses that expressed the 
NA genes of human H2N2 viruses isolated from 1957 to 1968 with all other genes
from the avian virus A/duck/Hong Kong/278/78 (H9N2). Only the NA of
A/Singapore/1/57 supported efficient viral growth in the intestines of orally
inoculated ducks. The growth-supporting capacity of the NA correlated with a high
level of enzymatic activity, comparable to that found to be associated with avian
virus NAs. The specific activities of the A/Ann Arbor/6/60 and A/England/12/62
NAs, which showed greatly restricted abilities to support viral growth in ducks, 
were only 8 and 5%, respectively, of the NA specific activity for
A/Singapore/1/57. Using chimeric constructs based on A/Singapore/1/57 and
A/England/12/62 NAs, we localized the determinants of high specific NA activity
to a region containing six amino acid substitutions in A/England/12/62:
Ser331-->Arg, Asp339-->Asn, Asn367-->Ser, Ser370-->Leu, Asn400-->Ser, and
Pro431-->Glu. Five of these six residues (excluding Asn400) were required and
sufficient for the full specific activity of the A/Singapore/1/57 NA. Thus, in
addition to a change in substrate specificity, a reduction in high specific
activity may be required for the adaptation of avian virus NAs to growth in
humans. This change is likely needed to maintain an optimal balance between NA
activity and the lower affinity shown by human virus HAs for their cellular
receptor.

PMCID: PMC114763
PMID: 11689658  [PubMed - indexed for MEDLINE]
Þ

38651. J Virol. 2001 Oct;75(20):9679-86.

Cocirculation of avian H9N2 and contemporary "human" H3N2 influenza A viruses in 
pigs in southeastern China: potential for genetic reassortment?

Peiris JS, Guan Y, Markwell D, Ghose P, Webster RG, Shortridge KF.

Department of Microbiology, The University of Hong Kong, Pokfulam, Hong Kong
Special Administrative Region, People's Republic of China. malik@hkucc.hku.hk

Pigs are permissive to both human and avian influenza viruses and have been
proposed to be an intermediate host for the genesis of pandemic influenza viruses
through reassortment or adaptation of avian viruses. Prospective virological
surveillance carried out between March 1998 and June 2000 in Hong Kong, Special
Administrative Region, People's Republic of China, on pigs imported from
southeastern China, provides the first evidence of interspecies transmission of
avian H9N2 viruses to pigs and documents their cocirculation with contemporary
human H3N2 (A/Sydney/5/97-like, Sydney97-like) viruses. All gene segments of the 
porcine H9N2 viruses were closely related to viruses similar to
chicken/Beijing/1/94 (H9N2), duck/Hong Kong/Y280/97 (H9N2), and the descendants
of the latter virus lineage. Phylogenetic analysis suggested that repeated
interspecies transmission events had occurred from the avian host to pigs. The
Sydney97-like (H3N2) viruses isolated from pigs were related closely to
contemporary human H3N2 viruses in all gene segments and had not undergone
genetic reassortment. Cocirculation of avian H9N2 and human H3N2 viruses in pigs 
provides an opportunity for genetic reassortment leading to the emergence of
viruses with pandemic potential.

PMCID: PMC114539
PMID: 11559800  [PubMed - indexed for MEDLINE]
Þ

39450. Rev Sci Tech. 2000 Apr;19(1):197-225.

Recent zoonoses caused by influenza A viruses.

Alexander DJ, Brown IH.

Virology Department, Veterinary Laboratories Agency-Weybridge, New Haw,
Addlestone, Surrey KT15 3NB, United Kingdom.

Influenza is a highly contagious, acute illness which has afflicted humans and
animals since ancient times. Influenza viruses are part of the Orthomyxoviridae
family and are grouped into types A, B and C according to antigenic
characteristics of the core proteins. Influenza A viruses infect a large variety 
of animal species, including humans, pigs, horses, sea mammals and birds,
occasionally producing devastating pandemics in humans, such as in 1918, when
over twenty million deaths occurred world-wide. The two surface glycoproteins of 
the virus, haemagglutinin (HA) and neuraminidase (NA), are the most important
antigens for inducing protective immunity in the host and therefore show the
greatest variation. For influenza A viruses, fifteen antigenically distinct HA
subtypes and nine NA subtypes are recognised at present; a virus possesses one HA
and one NA subtype, apparently in any combination. Although viruses of relatively
few subtype combinations have been isolated from mammalian species, all subtypes,
in most combinations, have been isolated from birds. In the 20th Century, the
sudden emergence of antigenically different strains in humans, termed antigenic
shift, has occurred on four occasions, as follows, in 1918 (H1N1), 1957 (H2N2),
1968 (H3N2) and 1977 (H1N1), each resulting in a pandemic. Frequent epidemics
have occurred between the pandemics as a result of gradual antigenic change in
the prevalent virus, termed antigenic drift. Currently, epidemics occur
throughout the world in the human population due to infection with influenza A
viruses of subtypes H1N1 and H3N2 or with influenza B virus. The impact of these 
epidemics is most effectively measured by monitoring excess mortality due to
pneumonia and influenza. Phylogenetic studies suggest that aquatic birds could be
the source of all influenza A viruses in other species. Human pandemic strains
are thought to have emerged through one of the following three mechanisms:
genetic reassortment (occurring as a result of the segmented genome of the virus)
of avian and human influenza A viruses infecting the same host direct transfer of
whole virus from another species the re-emergence of a virus which may have
caused an epidemic many years earlier. Since 1996, the viruses H7N7, H5N1 and
H9N2 have been transmitted from birds to humans but have apparently failed to
spread in the human population. Such incidents are rare, but transmission between
humans and other animals has also been demonstrated. This has led to the
suggestion that the proposed reassortment of human and avian viruses occurs in an
intermediate animal with subsequent transference to the human population. Pigs
have been considered the leading contender for the role of intermediary because
these animals may serve as hosts for productive infections of both avian and
human viruses and, in addition, the evidence strongly suggests that pigs have
been involved in interspecies transmission of influenza viruses, particularly the
spread of H1N1 viruses to humans. Global surveillance of influenza is maintained 
by a network of laboratories sponsored by the World Health Organization. The main
control measure for influenza in human populations is immunoprophylaxis, aimed at
the epidemics occurring between pandemics.

PMID: 11189716  [PubMed - indexed for MEDLINE]
Þ

39571. Clin Microbiol Rev. 2001 Jan;14(1):129-49.

Pandemic threat posed by avian influenza A viruses.

Horimoto T, Kawaoka Y.

Department of Veterinary Microbiology, Osaka Prefecture University, Sakai Osaka
599-8531, Japan.

Influenza pandemics, defined as global outbreaks of the disease due to viruses
with new antigenic subtypes, have exacted high death tolls from human
populations. The last two pandemics were caused by hybrid viruses, or
reassortants, that harbored a combination of avian and human viral genes. Avian
influenza viruses are therefore key contributors to the emergence of human
influenza pandemics. In 1997, an H5N1 influenza virus was directly transmitted
from birds in live poultry markets in Hong Kong to humans. Eighteen people were
infected in this outbreak, six of whom died. This avian virus exhibited high
virulence in both avian and mammalian species, causing systemic infection in both
chickens and mice. Subsequently, another avian virus with the H9N2 subtype was
directly transmitted from birds to humans in Hong Kong. Interestingly, the genes 
encoding the internal proteins of the H9N2 virus are genetically highly related
to those of the H5N1 virus, suggesting a unique property of these gene products. 
The identification of avian viruses in humans underscores the potential of these 
and similar strains to produce devastating influenza outbreaks in major
population centers. Although highly pathogenic avian influenza viruses had been
identified before the 1997 outbreak in Hong Kong, their devastating effects had
been confined to poultry. With the Hong Kong outbreak, it became clear that the
virulence potential of these viruses extended to humans.

PMCID: PMC88966
PMID: 11148006  [PubMed - indexed for MEDLINE]
Þ

39623. Cell Mol Life Sci. 2000 Nov;57(12):1770-84.

Avian influenza viruses infecting humans.

Subbarao K, Katz J.

Influenza Branch, Centers for Disease Control, Atlanta, Georgia 30333, USA.
ksubbarao@cdc.gov

Avian species, particularly waterfowl, are the natural hosts of influenza A
viruses. Influenza viruses bearing each of the 15 hemagglutinin and nine
neuraminidase subtypes infect birds and serve as a reservoir from which influenza
viruses or genes are introduced into the human population. Viruses with novel
hemagglutinin genes derived from avian influenza viruses, with or without other
accompanying avian influenza virus genes, have the potential for pandemic spread 
when the human population lacks protective immunity against the new
hemagglutinin. Avian influenza viruses were thought to be limited in their
ability to directly infect humans until 1997, when 18 human infections with avian
influenza H5N1 viruses occurred in Hong Kong. In 1999, two human infections with 
avian influenza H9N2 viruses were also identified in Hong Kong. These events
established that avian viruses could infect humans without acquiring human
influenza genes by reassortment in an intermediate host and highlighted
challenges associated with the detection of human immune responses to avian
influenza viruses and the development of appropriate vaccines.

PMID: 11130181  [PubMed - indexed for MEDLINE]
Þ

39843. J Gen Virol. 2000 Nov;81(Pt 11):2689-96.

Heterologous protection against lethal A/HongKong/156/97 (H5N1) influenza virus
infection in C57BL/6 mice.

O'Neill E, Krauss SL, Riberdy JM, Webster RG, Woodland DL.

Department of Virology and Molecular Biology and Department of Immunology, St
Jude Children's Research Hospital, Memphis, TN 38105-2794, USA.

The continual threat posed by newly emerging influenza virus strains is
demonstrated by the recent outbreak of H5N1 influenza virus in Hong Kong.
Currently, immunization against influenza virus infection is fairly adequate, but
it is imperative that improved vaccines are developed that can protect against a 
variety of strains and be generated rapidly. Since humoral immunity is
ineffective against serologically distinct viruses, one strategy would be to
develop vaccines that emphasize cellular immunity. Here we report the successful 
protection of C57BL/6 mice from a lethal A/HK/156/97 (HK156) infection by
immunizing first with an H9N2 isolate, A/Quail/HK/G1/97 (QHKG1), that harbours
internal genes 98% homologous to HK156. This strategy also protected mice that
are deficient in antibody production, indicating that the immunity is
T-cell-mediated. In the course of these studies, we generated a highly pathogenic
H5N1 reassortant which implicated NP and PB2 as having an important contribution 
to pathogenesis when present with a highly cleavable H5. These results provide
the first demonstration that protective cell-mediated immunity can be established
against the highly virulent HK156 virus and have important implications for the
development of novel strategies for the prevention and treatment of HK156
infection and the design of future influenza vaccines.

PMID: 11038381  [PubMed - indexed for MEDLINE]
Þ

39943. J Virol. 2000 Oct;74(20):9372-80.

H9N2 influenza viruses possessing H5N1-like internal genomes continue to
circulate in poultry in southeastern China.

Guan Y, Shortridge KF, Krauss S, Chin PS, Dyrting KC, Ellis TM, Webster RG,
Peiris M.

Department of Virology and Molecular Biology, St. Jude Children's Research
Hospital, Memphis, Tennessee 38105-2794, USA.

The transmission of H9N2 influenza viruses to humans and the realization that the
A/Hong Kong/156/97-like (H5N1) (abbreviated HK/156/97) genome complex may be
present in H9N2 viruses in southeastern China necessitated a study of the
distribution and characterization of H9N2 viruses in poultry in the Hong Kong SAR
in 1999. Serological studies indicated that H9N2 influenza viruses had infected a
high proportion of chickens and other land-based birds (pigeon, pheasant, quail, 
guinea fowl, and chukka) from southeastern China. Two lineages of H9N2 influenza 
viruses present in the live-poultry markets were represented by A/Quail/Hong
Kong/G1/97 (Qa/HK/G1/97)-like and A/Duck/Hong Kong/Y280/97 (Dk/HK/Y280/97)-like
viruses. Up to 16% of cages of quail in the poultry markets contained
Qa/HK/G1/97-like viruses, while about 5% of cages of other land-based birds were 
infected with Dk/HK/Y280/97-like viruses. No reassortant between the two H9N2
virus lineages was detected despite their cocirculation in the poultry markets.
Reassortant viruses represented by A/Chicken/Hong Kong/G9/97 (H9N2) were the
major H9N2 influenza viruses circulating in the Hong Kong markets in 1997 but
have not been detected since the chicken slaughter in 1997. The Qa/HK/G1/97-like 
viruses were frequently isolated from quail, while Dk/HK/Y280/97-like viruses
were predominately associated with chickens. The Qa/HK/G1/97-like viruses were
evolving relatively rapidly, especially in their PB2, HA, NP, and NA genes,
suggesting that they are in the process of adapting to a new host. Experimental
studies showed that both H9N2 lineages were primarily spread by the aerosol route
and that neither quail nor chickens showed evidence of disease. The high
prevalence of quail infected with Qa/HK/G1/97-like virus that contains six gene
segments genetically highly related to HK/156/97 (H5N1) virus emphasizes the need
for surveillance of mammals including humans.

PMCID: PMC112365
PMID: 11000205  [PubMed - indexed for MEDLINE]
Þ

40216. J Clin Microbiol. 2000 Jul;38(7):2579-83.

A simple restriction fragment length polymorphism-based strategy that can
distinguish the internal genes of human H1N1, H3N2, and H5N1 influenza A viruses.

Cooper LA, Subbarao K.

Influenza Branch, Division of Viral and Rickettsial Diseases, National Center for
Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
30333, USA. lcooperbiol@earthlink.net

A simple molecular technique for rapid genotyping was developed to monitor the
internal gene composition of currently circulating influenza A viruses. Sequence 
information from recent H1N1, H3N2, and H5N1 human virus isolates was used to
identify conserved regions within each internal gene, and gene-specific PCR
primers capable of amplifying all three virus subtypes were designed. Subtyping
was based on subtype-specific restriction fragment length polymorphism (RFLP)
patterns within the amplified regions. The strategy was tested in a blinded
fashion using 10 control viruses of each subtype (total, 30) and was found to be 
very effective. Once standardized, the genotyping method was used to identify the
origin of the internal genes of 51 influenza A viruses isolated from humans in
Hong Kong during and immediately following the 1997-1998 H5N1 outbreak. No
avian-human or H1-H3 reassortants were detected. Less than 2% (6 of 486) of the
RFLP analyses were inconclusive; all were due to point mutations within a
restriction site. The technique was also used to characterize the internal genes 
of two avian H9N2 viruses isolated from children in Hong Kong during 1999.

PMCID: PMC86974
PMID: 10878047  [PubMed - indexed for MEDLINE]
Þ

40255. J Virol. 2000 Jul;74(14):6592-9.

Continued circulation in China of highly pathogenic avian influenza viruses
encoding the hemagglutinin gene associated with the 1997 H5N1 outbreak in poultry
and humans.

Cauthen AN, Swayne DE, Schultz-Cherry S, Perdue ML, Suarez DL.

Southeast Poultry Research Laboratory, USDA, Agricultural Research Service,
Athens, Georgia 30605, USA.

Since the outbreak in humans of an H5N1 avian influenza virus in Hong Kong in
1997, poultry entering the live-bird markets of Hong Kong have been closely
monitored for infection with avian influenza. In March 1999, this monitoring
system detected geese that were serologically positive for H5N1 avian influenza
virus, but the birds were marketed before they could be sampled for virus.
However, viral isolates were obtained by swabbing the cages that housed the
geese. These samples, known collectively as A/Environment/Hong Kong/437/99
(A/Env/HK/437/99), contained four viral isolates, which were compared to the 1997
H5N1 Hong Kong isolates. Analysis of A/Env/HK/437/99 viruses revealed that the
four isolates are nearly identical genetically and are most closely related to
A/Goose/Guangdong/1/96. These isolates and the 1997 H5N1 Hong Kong viruses encode
common hemagglutinin (H5) genes that have identical hemagglutinin cleavage sites.
Thus, the pathogenicity of the A/Env/HK/437/99 viruses was compared in chickens
and in mice to evaluate the potential for disease outbreaks in poultry and
humans. The A/Env/HK/437/99 isolates were highly pathogenic in chickens but
caused a longer mean death time and had altered cell tropism compared to A/Hong
Kong/156/97 (A/HK/156/97). Like A/HK/156/97, the A/Env/HK/437/99 viruses
replicated in mice and remained localized to the respiratory tract. However, the 
A/Env/HK/437/99 isolates caused only mild pathological lesions in these tissues
and no clinical signs of disease or death. As a measure of the immune response to
these viruses, transforming growth factor beta levels were determined in the
serum of infected mice and showed elevated levels for the A/Env/HK/437/99 viruses
compared to the A/HK/156/97 viruses. This study is the first to characterize the 
A/Env/HK/437/99 viruses in both avian and mammalian species, evaluating the H5
gene from the 1997 Hong Kong H5N1 isolates in a different genetic background. Our
findings reveal that at least one of the avian influenza virus genes encoded by
the 1997 H5N1 Hong Kong viruses continues to circulate in mainland China and that
this gene is important for pathogenesis in chickens but is not the sole
determinant of pathogenicity in mice. There is evidence that H9N2 viruses, which 
have internal genes in common with the 1997 H5N1 Hong Kong isolates, are still
circulating in Hong Kong and China as well, providing a heterogeneous gene pool
for viral reassortment. The implications of these findings for the potential for 
human disease are discussed.

PMCID: PMC112169
PMID: 10864673  [PubMed - indexed for MEDLINE]
Þ

41368. Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9363-7.

Molecular characterization of H9N2 influenza viruses: were they the donors of the
"internal" genes of H5N1 viruses in Hong Kong?

Guan Y, Shortridge KF, Krauss S, Webster RG.

Department of Virology/Molecular Biology, St. Jude Children's Research Hospital, 
Memphis, TN 38105, USA.

The origin of the H5N1 influenza viruses that killed six of eighteen infected
humans in 1997 and were highly pathogenic in chickens has not been resolved.
These H5N1 viruses transmitted directly to humans from infected poultry. In the
poultry markets in Hong Kong, both H5N1 and H9N2 influenza viruses were
cocirculating, raising the possibility of genetic reassortment. Here we analyze
the antigenic and genetic features of H9N2 influenza viruses with different
epidemiological backgrounds. The results suggest that the H9N2 influenza viruses 
of domestic ducks have become established in the domestic poultry of Asia.
Phylogenetic and antigenic analyses of the H9N2 viruses isolated from Hong Kong
markets suggest three distinct sublineages. Among the chicken H9N2 viruses, six
of the gene segments were apparently derived from an earlier chicken H9N2 virus
isolated in China, whereas the PB1 and PB2 genes are closely related to those of 
the H5N1 viruses and a quail H9N2 virus-A/quail/Hong Kong/G1/97
(Qa/HK/G1/97)-suggesting that many of the 1997 chicken H9 isolates in the markets
were reassortants. The similarity of the internal genes of Qa/HK/G1/97 virus to
those of the H5N1 influenza viruses suggests that the quail virus may have been
the internal gene donor. Our findings indicate that the human and poultry H5N1
influenza viruses in Hong Kong in 1997 were reassortants that obtained internal
gene segments from Qa/HK/G1/97. However, we cannot be certain whether the
replicate complex of H5N1 originated from Qa/HK/G1/97 or whether the reverse
transfer occurred; the available evidence supports the former proposal.

PMCID: PMC17788
PMID: 10430948  [PubMed - indexed for MEDLINE]
Þ