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

161. Vet Res. 2013 Mar 28;44(1):23. [Epub ahead of print]

Pathobiology and transmission of highly and low pathogenic avian influenza
viruses in European quail (Coturnix c. coturnix).

Bertran K, Dolz R, Busquets N, Gamino V, Vergara-Alert J, Chaves AJ, Ramis A,
Abad FX, Höfle U, Majó N.

European quail (Coturnix c. coturnix) may share with Japanese quail (Coturnix c. 
japonica) its potential as an intermediate host and reservoir of avian influenza 
viruses (AIV). To elucidate this question, European quail were experimentally
challenged with two highly pathogenic AIV (HPAIV) (H7N1/HP and H5N1/HP) and one
low pathogenic AIV (LPAIV) (H7N2/LP). Contact animals were also used to assess
the viral transmission among birds. Severe neurological signs and mortality rates
of 67% (H7N1/HP) and 92% (H5N1/HP) were observed. Although histopathological
findings were present in both HPAIV-infected groups, H5N1/HP-quail displayed a
broader viral antigen distribution and extent of microscopic lesions. Neither
clinical nor pathological involvement was observed in LPAIV-infected quail.
Consistent long-term viral shedding and effective transmission to naive quail was
demonstrated for the three studied AIV. Drinking water arose as a possible
transmission route and feathers as a potential origin of HPAIV dissemination. The
present study demonstrates that European quail may play a major role in AI
epidemiology, highlighting the need to further understand its putative role as an
intermediate host for avian/mammalian reassortant viruses.

PMID: 23537387  [PubMed - as supplied by publisher]
Þ

456. J Virol. 2013 Mar 6. [Epub ahead of print]

Reassortment of NS-segments modifies highly-pathogenic avian influenza virus
interaction with avian hosts and host cells.

Petersen H, Wang Z, Lenz E, Pleschka S, Rautenschlein S.

Clinic for Poultry, University of Veterinary Medicine Hannover, Bünteweg 17,
D-30559 Hannover, Germany.

Highly pathogenic avian influenza viruses (HPAIV) of subtypes H5 and H7 have
caused numerous outbreaks in diverse poultry species and rising numbers of human 
infections. Both HPAIV subtypes support a growing concern of a pandemic outbreak 
specifically via the avian-human link. Natural reassortment of both HPAIV
subtypes is a possible event with unpredictable outcome for virulence and host
specificity of the progeny virus for avian and mammalian species. NS-reassortment
of H5N1 HPAIV viruses in the background of A/FPV/Rostock/1934 (H7N1) HPAIV has
been shown to change virus replication kinetics and host cell response in
mammalian cells. However, not much is known about virus-host interaction of such 
viruses in avian species. In the present study, we show that the NS-segment of
A/Vietnam/1203/2004 (FPV NS VN, H5N1) HPAIV significantly altered the
characteristics of the H7-prototype HPAIV in tracheal organ cultures (TOC) of
chicken and turkey in vitro, with decreased replication efficiency accompanied by
increased induction of type I interferon (IFN) and apoptosis. Furthermore,
species-specific differences between chicken and turkey were demonstrated.
Interestingly, NS-reassortant FPV NS VN showed an overall highly-pathogenic
phenotype with increased virulence and replication potential compared to the
wild-type virus after systemic infection of chicken and turkey embryos. Our data 
demonstrate that single reassortment of an H5-type NS into an H7-type HPAIV
significantly changed virus replication abilities and influenced the avian host
cell response without prior adaptation.

PMID: 23468508  [PubMed - as supplied by publisher]
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

589. Virus Res. 2013 Feb 19. pii: S0168-1702(13)00050-6. doi:
10.1016/j.virusres.2013.02.001. [Epub ahead of print]

Proteins of duck influenza virus responsible for acquisition of pathogenicity in 
chickens.

Yamamoto N, Soda K, Sakoda Y, Okamatsu M, Kida H.

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

Influenza virus rgVac1sub-P0 (H5N1) (rgVac1-P0), in which a pair of dibasic amino
acid residues was introduced at the cleavage site of the HA of a reassortant of
H5N2 and H7N1 viruses of duck origin, was low pathogenic in chickens. Vac1sub-P3 
(H5N1) (Vac1-P3) was selected as a highly pathogenic avian influenza virus by 3
consecutive passages in chickens from low pathogenic strain rgVac1-P0. Comparison
of amino acid sequences of the virus proteins and experimental infection of
chickens with a series of recombinant viruses demonstrated that in addition to
the HA, each of the PA, NP, M1, and M2 of Vac1-P3 are responsible for the
acquisition of pathogenicity in chickens. These 4 proteins of Vac1-P3
synergistically contributed to efficient virus replication in chickens.

Copyright © 2013 Elsevier B.V. All rights reserved.

PMID: 23434880  [PubMed - as supplied by publisher]
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

3252. J Virol. 2012 Sep;86(17):9553-4. doi: 10.1128/JVI.01523-12.

Genome sequence of a novel reassortant H3N2 avian influenza virus in southern
China.

Tian J, Zhang C, Qi W, Xu C, Huang L, Li H, Liao M.

College of Veterinary Medicine, South China Agricultural University, People's
Republic of China.

The distribution and prevalence of H3 subtype influenza viruses in avian and
mammalian hosts constitutes a potential threat to both human and avian health. We
report a complete genome sequence of a novel reassortant H3N2 avian influenza
virus. Phylogenetic analysis showed that HA and NA showed the highest sequence
homologies with those of A/white-backed munia/Hong Kong/4519/2009 (H3N2).
However, the internal genes had the highest sequence homologies with those of H6 
and H7 subtypes. The data provide further evidence of the existence of a natural 
reassortant H3N2 strain in southern China.

PMCID: PMC3416133
PMID: 22879622  [PubMed - indexed for MEDLINE]
Þ

3458. Arch Virol. 2012 Dec;157(12):2257-64. doi: 10.1007/s00705-012-1415-7. Epub 2012
Jul 26.

The nucleoprotein is responsible for intracerebral pathogenicity of
A/duck/Mongolia/47/2001 (H7N1) in chicks.

Isoda N, Tsuda Y, Asakura S, Okamatsu M, Sakoda Y, Kida H.

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

Avian influenza viruses A/duck/Mongolia/47/2001 (H7N1) (47/01) and
A/duck/Mongolia/867/2002 (H7N1) (867/02) were defined as low-pathogenic avian
influenza viruses (LPAIVs) using an intravenous pathogenicity test in chickens.
On the other hand, the intracerebral pathogenicity indices of 47/01 and 867/02
were 1.30 and 0.00, respectively. A series of reassortant viruses were generated 
between 47/01 and 867/02, and their intracerebral pathogenicity was compared in
one-day-old chicks to identify the protein(s) responsible for the intracerebral
pathogenicity of 47/01. The results indicate that the amino acids at positions 50
and 98 of the nucleoprotein are related to the pathogenicity of 47/01 in chicks
by intracerebral inoculation. A significant association was found between
mortality of the chicks inoculated intracerebrally with 47/01 and virus
replication in the lungs and/or brain. These results indicate that the NP of
avian influenza viruses may be responsible for intracerebral pathogenicity in the
host.

PMID: 22833102  [PubMed - indexed for MEDLINE]
Þ

5201. J Gen Virol. 2012 Jun;93(Pt 6):1278-87. doi: 10.1099/vir.0.041269-0. Epub 2012
Mar 14.

Low pathogenic H7 subtype avian influenza viruses isolated from domestic ducks in
South Korea and the close association with isolates of wild birds.

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

Animal Disease Diagnosis Division, Animal, Plant and Fisheries Quarantine and
Inspection Agency, 175 Anyangro, Manangu, Anyangsi, Gyeonggido 430-757, Republic 
of Korea.

We characterized low pathogenic avian influenza (LPAI) viruses of the H7 subtype 
that were isolated from domestic ducks and wild birds in South Korea from 2008 to
2011. A total of 20 H7 viruses were collected from live-bird markets (LBMs), duck
farms and wild-bird habitats using avian influenza (AI) surveillance and
epidemiological approaches. A phylogenetic analysis of the H7 viruses that were
isolated from domestic ducks and wild birds demonstrated that they were separated
into 12 genotypes (A-D and Wb-1-8, respectively), indicating genetic diversity.
These H7 viruses were related to the recently isolated Eurasian LPAI H7 viruses
and various influenza viruses that are circulating in Asia, including southern
China and South Korea. The same genotype was not found between domestic poultry
and wild-bird isolates; however, most of the H7 viruses in poultry (genotypes B
and C) were closely related to the H7 virus isolated from a wild bird (genotype
Wb-3). Animal-challenge studies revealed that certain H7 AI viruses replicated
well only in chickens or ducks depending on the genotype, indicating that the
pathogenicity of H7 viruses has the potential to be altered due to multiple
reassortments, and these viruses can potentially expand their host range. Our
results are evidence of abundant and frequent reassortment between H7 viruses in 
poultry and wild birds and emphasize the continuing need to monitor the
evolutionary genetics of the influenza virus in poultry and wild birds.

PMID: 22422062  [PubMed - indexed for MEDLINE]
Þ

5839. Zh Mikrobiol Epidemiol Immunobiol. 2011 Nov-Dec;(6):72-6.

[Formation of antibodies against neuraminidase of A/California/07/2009 (H1N1)
influenza virus after immunization with live monovalent influenza vaccine].

[Article in Russian]

Smolonogina TA, Desheva IuA, Shaldzhian AA, Grudinin MP, Rudenko LG.

AIM: Detection of antibodies against neuraminidase (NA) of A/California/07/2009
(H1N1) influenza virus in blood sera of volunteers after the immunization with
live monovalent influenza vaccine (LIV).
MATERIALS AND METHODS: Neuraminidase enzyme activity inhibition by antibodies
test with reassortant strain A(H7N1) containing NA of pandemic strain was used.
Anti-neuraminidase IgG antibodies against whole reassortant virus A(H7N1) and
purified NA of A/California/07/2009 (H1N1) strains were determined by enzyme
immunoassay (EIA).
RESULTS: After two immunizations with LIV of seronegative individuals a 1.5 times
mean increase of antibodies against homologous neuraminidase was detected (by
hemagglutinin inhibition reaction). The high level of anti-neuraminidase
antibodies were detected in individuals that had been naturally infected. A
correlation between anti-neuraminidase IgG antibody titers obtained in EIA with
whole reassortant virus A(H7N1) and purified protein was demonstrated.
CONCLUSION: Modified sialidase activity inhibition method and EIA with
reassortant diagnostic strain can be applied to evaluate anti-neuraminidase
antibodies.

PMID: 22308734  [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

6924. Avian Pathol. 2011 Dec;40(6):613-28. doi: 10.1080/03079457.2011.621410.

Genetic characterization of low pathogenic H5N1 and co-circulating avian
influenza viruses in wild mallards (Anas platyrhynchos) in Belgium, 2008.

Van Borm S, Vangeluwe D, Steensels M, Poncin O, van den Berg T, Lambrecht B.

Department of Virology, Veterinary and Agrochemical Research Center, Ukkel,
Belgium. stevenvanborm@var.fgov.be

As part of a long-term wild bird monitoring programme, five different low
pathogenic (LP) avian influenza viruses (AIVs) were isolated from wild mallards
(subtypes H1N1, H4N6, H5N1, H5N3, and H10N7). A LP H5N1 and two co-circulating
(same location, same time period) viruses were selected for full genome
sequencing. An H1N1 (A/Anas platyrhynchos/Belgium/09-762/2008) and an H5N1 virus 
(A/Anas platyrhynchos/Belgium/09-762-P1/2008) were isolated on the same day in
November 2008, then an H5N3 virus (A/Anas platyrhynchos/09-884/2008) 5 days later
in December 2008. All genes of these co-circulating viruses shared common
ancestors with recent (2001 to 2007) European wild waterfowl influenza viruses.
The H5N1 virus shares genome segments with both the H1N1 (PB1, NA, M) and the
H5N3 (PB2, HA) viruses, and all three viruses share the same NS sequence. A
double infection with two different PA segments from H5N1 and from H5N3 could be 
observed for the H1N1 sample. The observed gene constellations resulted from
multiple reassortment events between viruses circulating in wild birds in
Eurasia. Several internal gene segments from these 2008 viruses and the N3
sequence from the H5N3 show homology with sequences from 2003 H7 outbreaks in
Italy (LP) and the Netherlands (highly pathogenic). These data contribute to the 
growing sequence evidence of the dynamic nature of the avian influenza natural
reservoir in Eurasia, and underline the importance of monitoring AIV in wild
birds. Genetic information of potential hazard to commercial poultry continues to
circulate in this reservoir, including H5 and H7 subtype viruses and genes
related to previous AIV outbreaks.

PMID: 22107096  [PubMed - indexed for MEDLINE]
Þ

8013. MMWR Morb Mortal Wkly Rep. 2011 Sep 9;60(35):1213-5.

Swine-origin influenza A (H3N2) virus infection in two children--Indiana and
Pennsylvania, July-August 2011.

Centers for Disease Control and Prevention (CDC).

Influenza A viruses are endemic in many animal species, including humans, swine, 
and wild birds, and sporadic cases of transmission of influenza A viruses between
humans and animals do occur, including human infections with avian-origin
influenza A viruses (i.e., H5N1 and H7N7) and swine-origin influenza A viruses
(i.e., H1N1, H1N2, and H3N2). Genetic analysis can distinguish animal origin
influenza viruses from the seasonal human influenza viruses that circulate widely
and cause annual epidemics. This report describes two cases of febrile
respiratory illness caused by swine-origin influenza A (H3N2) viruses identified 
on August 19 and August 26, 2011, and the current investigations. No
epidemiologic link between the two cases has been identified, and although
investigations are ongoing, no additional confirmed human infections with this
virus have been detected. These viruses are similar to eight other swine-origin
influenza A (H3N2) viruses identified from previous human infections over the
past 2 years, but are unique in that one of the eight gene segments (matrix [M]
gene) is from the 2009 influenza A (H1N1) virus. The acquisition of the M gene in
these two swine-origin influenza A (H3N2) viruses indicates that they are
"reassortants" because they contain genes of the swine-origin influenza A (H3N2) 
virus circulating in North American pigs since 1998 and the 2009 influenza A
(H1N1) virus that might have been transmitted to pigs from humans during the 2009
H1N1 pandemic. However, reassortments of the 2009 influenza A (H1N1) virus with
other swine influenza A viruses have been reported previously in swine.
Clinicians who suspect influenza virus infection in humans with recent exposure
to swine should obtain a nasopharyngeal swab from the patient for timely
diagnosis at a state public health laboratory and consider empiric neuraminidase 
inhibitor antiviral treatment to quickly limit potential human transmission.

PMID: 21900876  [PubMed - indexed for MEDLINE]
Þ

8969. Vet Microbiol. 2011 Dec 15;153(3-4):393-7. doi: 10.1016/j.vetmic.2011.06.011.
Epub 2011 Jun 15.

Genetic characterization of H7N2 influenza virus isolated from pigs.

Kwon TY, Lee SS, Kim CY, Shin JY, Sunwoo SY, Lyoo YS.

Immunopathology Laboratory, College of Veterinary Medicine, Konkuk University, 1 
Hwayang-dong, Kwangjin-gu, Seoul 143-701, South Korea.

Because pigs have respiratory epitheliums which express both α2-3 and α2-6 linked
sialic acid as receptors to influenza A viruses, they are regarded as mixing
vessel for the generation of pandemic influenza viruses through genetic
reassortment. A H7N2 influenza virus (A/swine/KU/16/2001) was isolated from pig
lungs collected from the slaughterhouse. All eight genes of the influenza virus
were sequenced and phylogenetic analysis indicated that A/swine/KU/16/2001
originated in Hong Kong and genetic reassortment had occurred between the avian
H7N2 and H5N3 influenza viruses. The first isolation of H7 influenza virus in
pigs provides the opportunity for genetic reassortment of influenza viruses with 
pandemic potential and emphasizes the importance of surveillance for atypical
swine influenza viruses.

Copyright © 2011 Elsevier B.V. All rights reserved.

PMID: 21741185  [PubMed - indexed for MEDLINE]
Þ

9089. PLoS Pathog. 2011 Jun;7(6):e1002094. doi: 10.1371/journal.ppat.1002094. Epub 2011
Jun 23.

Evolutionary analysis of inter-farm transmission dynamics in a highly pathogenic 
avian influenza epidemic.

Bataille A, van der Meer F, Stegeman A, Koch G.

Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht
University, Utrecht, The Netherlands.

Phylogenetic studies have largely contributed to better understand the emergence,
spread and evolution of highly pathogenic avian influenza during epidemics, but
sampling of genetic data has never been detailed enough to allow mapping of the
spatiotemporal spread of avian influenza viruses during a single epidemic. Here, 
we present genetic data of H7N7 viruses produced from 72% of the poultry farms
infected during the 2003 epidemic in the Netherlands. We use phylogenetic
analyses to unravel the pathways of virus transmission between farms and between 
infected areas. In addition, we investigated the evolutionary processes shaping
viral genetic diversity, and assess how they could have affected our phylogenetic
analyses. Our results show that the H7N7 virus was characterized by a high level 
of genetic diversity driven mainly by a high neutral substitution rate, purifying
selection and limited positive selection. We also identified potential
reassortment in the three genes that we have tested, but they had only a limited 
effect on the resolution of the inter-farm transmission network. Clonal
sequencing analyses performed on six farm samples showed that at least one farm
sample presented very complex virus diversity and was probably at the origin of
chronological anomalies in the transmission network. However, most virus
sequences could be grouped within clearly defined and chronologically sound
clusters of infection and some likely transmission events between farms located
0.8-13 Km apart were identified. In addition, three farms were found as most
likely source of virus introduction in distantly located new areas. These long
distance transmission events were likely facilitated by human-mediated transport,
underlining the need for strict enforcement of biosafety measures during
outbreaks. This study shows that in-depth genetic analysis of virus outbreaks at 
multiple scales can provide critical information on virus transmission dynamics
and can be used to increase our capacity to efficiently control epidemics.

PMCID: PMC3121798
PMID: 21731491  [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

10230. Avian Pathol. 2011 Apr;40(2):131-8. doi: 10.1080/03079457.2010.537304.

Evidence of persistence and multiple genetic modifications of H7N7 low-pathogenic
avian influenza virus in wild mallards in Poland provided by phylogenetic
studies.

Smietanka K, Pikuła A, Minta Z, Meissner W.

National Veterinary Research Institute, Department of Poultry Diseases, Al.
Partyzantów 57, Pulawy, Poland.

Genetic characterization of the whole genome of four avian influenza H7N7 viruses
isolated in three successive winter seasons (2007 to 2009) from wild mallards in 
three cities in Poland was performed. All of the tested strains were of low
pathogenicity and no molecular marker associated with an increased adaptation to 
poultry, mammals or resistance to antiviral drugs was found. The major outcome of
the phylogenetic studies was that the isolate A/mallard/Poland/446/09 (detected
in December 2009) shared a recent common ancestor with A/mallard/Poland/41/09
(isolated in February 2009) in relation to HA and PB1 genes, with
A/mallard/Poland/16/09 (found in January 2009) regarding NA and NS genes, and
with A/mallard/Poland/01/08 (recovered in December 2007) as regards the NS gene. 
Interestingly, A/mallard/Poland/16/09 and A/mallard/Poland/446/09 were isolated
at the same sampling site almost exactly 1 year apart, which points to resident
population of mallards (and other resident waterfowl) as responsible for the
perpetuation of avian influenza virus (AIV) in the given area between successive 
winters. On the other hand, the ornithological data discussed in detail in the
paper strongly suggest that the virus transmission between close sites but
located in different urban areas is most probably achieved by migratory birds, a 
fact additionally supported by a close relatedness between different gene
segments of Polish H7N7 and AIV detected in wild birds in Europe. A high
heterogeneity of the gene pool found in the study is indicative of frequent
reassortment events. Additionally, two H7N7 isolates were shown to possess
selected genes closely related to AIV detected in domestic poultry in Italy and
the Czech Republic. The present study corroborates the importance of active
surveillance in wild birds as a valuable tool for early warning of avian
influenza in poultry.

PMID: 21500032  [PubMed - indexed for MEDLINE]
Þ

10604. J Virol. 2011 Jun;85(11):5312-22. doi: 10.1128/JVI.02619-10. Epub 2011 Mar 23.

Genome-scale evolution and phylodynamics of equine H3N8 influenza A virus.

Murcia PR, Wood JL, Holmes EC.

Cambridge Infectious Diseases Consortium, Department of Veterinary Medicine,
University of Cambridge, Cambridge, England, UK. prm42@cam.ac.uk

Equine influenza viruses (EIVs) of the H3N8 and H7N7 subtypes are the causative
agents of an important disease of horses. While EIV H7N7 apparently is extinct,
H3N8 viruses have circulated for more than 50 years. Like human influenza
viruses, EIV H3N8 caused a transcontinental pandemic followed by further
outbreaks and epidemics, even in populations with high vaccination coverage.
Recently, EIV H3N8 jumped the species barrier to infect dogs. Despite its
importance as an agent of infectious disease, the mechanisms that underpin the
evolutionary and epidemiological dynamics of EIV are poorly understood,
particularly at a genomic scale. To determine the evolutionary history and
phylodynamics of EIV H3N8, we conducted an extensive analysis of 82 complete
viral genomes sampled during a 45-year span. We show that both intra- and
intersubtype reassortment have played a major role in the evolution of EIV, and
we suggest that intrasubtype reassortment resulted in enhanced virulence while
heterosubtypic reassortment contributed to the extinction of EIV H7N7. We also
show that EIV evolves at a slower rate than other influenza viruses, even though 
it seems to be subject to similar immune selection pressures. However, a
relatively high rate of amino acid replacement is observed in the polymerase
acidic (PA) segment, with some evidence for adaptive evolution. Most notably, an 
analysis of viral population dynamics provided evidence for a major population
bottleneck of EIV H3N8 during the 1980s, which we suggest resulted from changes
in herd immunity due to an increase in vaccination coverage.

PMCID: PMC3094979
PMID: 21430049  [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

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

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

13927. J Virol. 2010 Nov;84(22):11950-60. doi: 10.1128/JVI.01305-10. Epub 2010 Sep 1.

A live attenuated H7N7 candidate vaccine virus induces neutralizing antibody that
confers protection from challenge in mice, ferrets, and monkeys.

Min JY, Vogel L, Matsuoka Y, Lu B, Swayne D, Jin H, Kemble G, Subbarao K.

LID, NIAID, NIH, Bethesda, MD 20892, USA.

A live attenuated H7N7 candidate vaccine virus was generated by reverse genetics 
using the modified hemagglutinin (HA) and neuraminidase (NA) genes of highly
pathogenic (HP) A/Netherlands/219/03 (NL/03) (H7N7) wild-type (wt) virus and the 
six internal protein genes of the cold-adapted (ca) A/Ann Arbor/6/60 ca (AA ca)
(H2N2) virus. The reassortant H7N7 NL/03 ca vaccine virus was temperature
sensitive and attenuated in mice, ferrets, and African green monkeys (AGMs).
Intranasal (i.n.) administration of a single dose of the H7N7 NL/03 ca vaccine
virus fully protected mice from lethal challenge with homologous and heterologous
H7 viruses from Eurasian and North American lineages. Two doses of the H7N7 NL/03
ca vaccine induced neutralizing antibodies in serum and provided complete
protection from pulmonary replication of homologous and heterologous wild-type H7
challenge viruses in mice and ferrets. One dose of the H7N7 NL/03 ca vaccine
elicited an antibody response in one of three AGMs that was completely protected 
from pulmonary replication of the homologous wild-type H7 challenge virus. The
contribution of CD8(+) and/or CD4(+) T cells to the vaccine-induced protection of
mice was evaluated by T-cell depletion; T lymphocytes were not essential for the 
vaccine-induced protection from lethal challenge with H7 wt viruses.
Additionally, passively transferred neutralizing antibody induced by the H7N7
NL/03 ca virus protected mice from lethality following challenge with H7 wt
viruses. The safety, immunogenicity, and efficacy of the H7N7 NL/03 ca vaccine
virus in mice, ferrets, and AGMs support the evaluation of this vaccine virus in 
phase I clinical trials.

PMCID: PMC2977864
PMID: 20810733  [PubMed - indexed for MEDLINE]
Þ

14020. J Virol. 2010 Nov;84(21):11323-35. doi: 10.1128/JVI.01034-10. Epub 2010 Aug 25.

NS reassortment of an H7-type highly pathogenic avian influenza virus affects its
propagation by altering the regulation of viral RNA production and antiviral host
response.

Wang Z, Robb NC, Lenz E, Wolff T, Fodor E, Pleschka S.

Institute for Medical Virology, Justus Liebig University Giessen, Frankfurter
Str. 107, D-35392 Giessen, Germany.

Highly pathogenic avian influenza viruses (HPAIV) with reassorted NS segments
from H5- and H7-type avian virus strains placed in the genetic background of the 
A/FPV/Rostock/34 HPAIV (FPV; H7N1) were generated by reverse genetics.
Virological characterizations demonstrated that the growth kinetics of the
reassortant viruses differed from that of wild-type (wt) FPV and depended on
whether cells were of mammalian or avian origin. Surprisingly, molecular analysis
revealed that the different reassortant NS segments were not only responsible for
alterations in the antiviral host response but also affected viral genome
replication and transcription as well as nuclear ribonucleoprotein (RNP) export. 
RNP reconstitution experiments demonstrated that the effects on accumulation
levels of viral RNA species were dependent on the specific NS segment as well as 
on the genetic background of the RNA-dependent RNA polymerase (RdRp). Beta
interferon (IFN-β) expression and the induction of apoptosis were found to be
inversely correlated with the magnitude of viral growth, while the NS allele,
virus subtype, and nonstructural protein NS1 expression levels showed no
correlation. Thus, these results demonstrate that the origin of the NS segment
can have a dramatic effect on the replication efficiency and host range of HPAIV.
Overall, our data suggest that the propagation of NS reassortant influenza
viruses is affected at multiple steps of the viral life cycle as a result of the 
different effects of the NS1 protein on multiple viral and host functions.

PMCID: PMC2953208
PMID: 20739516  [PubMed - indexed for MEDLINE]
Þ

14367. PLoS One. 2010 Jul 27;5(7):e11826. doi: 10.1371/journal.pone.0011826.

Highly pathogenic H5N1 influenza viruses carry virulence determinants beyond the 
polybasic hemagglutinin cleavage site.

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

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

Highly pathogenic avian influenza viruses (HPAIV) originate from avirulent
precursors but differ from all other influenza viruses by the presence of a
polybasic cleavage site in their hemagglutinins (HA) of subtype H5 or H7. In this
study, we investigated the ability of a low-pathogenic avian H5N1 strain to
transform into an HPAIV. Using reverse genetics, we replaced the monobasic HA
cleavage site of the low-pathogenic strain A/Teal/Germany/Wv632/2005 (H5N1)
(TG05) by a polybasic motif from an HPAIV (TG05(poly)). To elucidate the
virulence potential of all viral genes of HPAIV, we generated two reassortants
carrying the HA from the HPAIV A/Swan/Germany/R65/06 (H5N1) (R65) plus the
remaining genes from TG05 (TG05-HA(R65)) or in reversed composition the mutated
TG05 HA plus the R65 genes (R65-HA(TG05poly)). In vitro, TG05(poly) and both
reassortants were able to replicate without the addition of trypsin, which is
characteristic for HPAIV. Moreover, in contrast to avirulent TG05, the variants
TG05(poly), TG05-HA(R65), and R65-HA(TG05poly) are pathogenic in chicken to an
increasing degree. Whereas the HA cleavage site mutant TG05(poly) led to
temporary non-lethal disease in all animals, the reassortant TG05-HA(R65) caused 
death in 3 of 10 animals. Furthermore, the reassortant R65-HA(TG05poly) displayed
the highest lethality as 8 of 10 chickens died, resembling "natural" HPAIV
strains. Taken together, acquisition of a polybasic HA cleavage site is only one 
necessary step for evolution of low-pathogenic H5N1 strains into HPAIV. However, 
these low-pathogenic strains may already have cryptic virulence potential.
Moreover, besides the polybasic cleavage site, the additional virulence
determinants of H5N1 HPAIV are located within the HA itself and in other viral
proteins.

PMCID: PMC2910732
PMID: 20676399  [PubMed - indexed for MEDLINE]
Þ

14422. J Virol. 2010 Oct;84(19):9957-66. doi: 10.1128/JVI.00930-10. Epub 2010 Jul 28.

Molecular analysis of H7 avian influenza viruses from Australia and New Zealand: 
genetic diversity and relationships from 1976 to 2007.

Bulach D, Halpin R, Spiro D, Pomeroy L, Janies D, Boyle DB.

CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong,
Victoria 3220, Australia. dieter.bulach@csiro.au

Full-genome sequencing of 11 Australian and 1 New Zealand avian influenza A virus
isolate (all subtype H7) has enabled comparison of the sequences of each of the
genome segments to those of other subtype H7 avian influenza A viruses. The
inference of phylogenetic relationships for each segment has been used to develop
a model of the natural history of these viruses in Australia. Phylogenetic
analysis of the hemagglutinin segment indicates that the Australian H7 isolates
form a monophyletic clade. This pattern is consistent with the long-term,
independent evolution that is, in this instance, associated with geographic
regions. On the basis of the analysis of the other H7 hemagglutinin sequences,
three other geographic regions for which similar monophyletic clades have been
observed were confirmed. These regions are Eurasia plus Africa, North America,
and South America. Analysis of the neuraminidase sequences from the H7N1, H7N3,
and H7N7 genomes revealed the same region-based relationships. This pattern of
independent evolution of Australian isolates is supported by the results of
analysis of each of the six remaining genomic segments. These results, in
conjunction with the occurrence of five different combinations of neuraminidase
subtypes (H7N2, H7N3, H7N4, H7N6, H7N7) among the 11 Australian isolates, suggest
that the maintenance host(s) is nearly exclusively associated with Australia. The
single lineage of Australian H7 hemagglutinin sequences, despite the occurrence
of multiple neuraminidase types, suggests the existence of a genetic pool from
which a variety of reassortants arise rather than the presence of a small number 
of stable viral clones. This pattern of evolution is likely to occur in each of
the regions mentioned above.

PMCID: PMC2937813
PMID: 20668069  [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

14960. BMC Infect Dis. 2010 Jun 23;10:187. doi: 10.1186/1471-2334-10-187.

Mapping the risk of avian influenza in wild birds in the US.

Fuller TL, Saatchi SS, Curd EE, Toffelmier E, Thomassen HA, Buermann W, DeSante
DF, Nott MP, Saracco JF, Ralph C, Alexander JD, Pollinger JP, Smith TB.

Center for Tropical Research, Institute of the Environment, University of
California, Los Angeles, La Kretz Hall, Suite 300, Box 951496, Los Angeles, CA
90095-1496, USA. fullertl@ucla.edu

BACKGROUND: Avian influenza virus (AIV) is an important public health issue
because pandemic influenza viruses in people have contained genes from viruses
that infect birds. The H5 and H7 AIV subtypes have periodically mutated from low 
pathogenicity to high pathogenicity form. Analysis of the geographic distribution
of AIV can identify areas where reassortment events might occur and how high
pathogenicity influenza might travel if it enters wild bird populations in the
US. Modelling the number of AIV cases is important because the rate of
co-infection with multiple AIV subtypes increases with the number of cases and
co-infection is the source of reassortment events that give rise to new strains
of influenza, which occurred before the 1968 pandemic. Aquatic birds in the
orders Anseriformes and Charadriiformes have been recognized as reservoirs of AIV
since the 1970s. However, little is known about influenza prevalence in
terrestrial birds in the order Passeriformes. Since passerines share the same
habitat as poultry, they may be more effective transmitters of the disease to
humans than aquatic birds. We analyze 152 passerine species including the
American Robin (Turdus migratorius) and Swainson's Thrush (Catharus ustulatus).
METHODS: We formulate a regression model to predict AIV cases throughout the US
at the county scale as a function of 12 environmental variables, sampling effort,
and proximity to other counties with influenza outbreaks. Our analysis did not
distinguish between types of influenza, including low or highly pathogenic forms.
RESULTS: Analysis of 13,046 cloacal samples collected from 225 bird species in 41
US states between 2005 and 2008 indicates that the average prevalence of
influenza in passerines is greater than the prevalence in eight other avian
orders. Our regression model identifies the Great Plains and the Pacific
Northwest as high-risk areas for AIV. Highly significant predictors of AIV
include the amount of harvested cropland and the first day of the year when a
county is snow free.
CONCLUSIONS: Although the prevalence of influenza in waterfowl has long been
appreciated, we show that 22 species of song birds and perching birds (order
Passeriformes) are influenza reservoirs in the contiguous US.

PMCID: PMC2912310
PMID: 20573228  [PubMed - indexed for MEDLINE]
Þ

16358. Antimicrob Agents Chemother. 2010 Jun;54(6):2517-24. doi: 10.1128/AAC.01739-09.
Epub 2010 Mar 29.

In vitro antiviral activity of favipiravir (T-705) against drug-resistant
influenza and 2009 A(H1N1) viruses.

Sleeman K, Mishin VP, Deyde VM, Furuta Y, Klimov AI, Gubareva LV.

Virus Surveillance and Diagnosis Branch, Influenza Division, National Center of
Immunization and Respiratory Diseases, Centers for Disease Control and
Prevention, Atlanta, GA 30329-4018, USA.

Favipiravir (T-705) has previously been shown to have a potent antiviral effect
against influenza virus and some other RNA viruses in both cell culture and in
animal models. Currently, favipiravir is undergoing clinical evaluation for the
treatment of influenza A and B virus infections. In this study, favipiravir was
evaluated in vitro for its ability to inhibit the replication of a representative
panel of seasonal influenza viruses, the 2009 A(H1N1) strains, and animal viruses
with pandemic (pdm) potential (swine triple reassortants, H2N2, H4N2, avian H7N2,
and avian H5N1), including viruses which are resistant to the currently licensed 
anti-influenza drugs. All viruses were tested in a plaque reduction assay with
MDCK cells, and a subset was also tested in both yield reduction and focus
inhibition (FI) assays. For the majority of viruses tested, favipiravir
significantly inhibited plaque formation at 3.2 muM (0.5 microg/ml) (50%
effective concentrations [EC(50)s] of 0.19 to 22.48 muM and 0.03 to 3.53
microg/ml), and for all viruses, with the exception of a single dually resistant 
2009 A(H1N1) virus, complete inhibition of plaque formation was seen at 3.2 muM
(0.5 microg/ml). Due to the 2009 pandemic and increased drug resistance in
circulating seasonal influenza viruses, there is an urgent need for new drugs
which target influenza. This study demonstrates that favipiravir inhibits in
vitro replication of a wide range of influenza viruses, including those resistant
to currently available drugs.

PMCID: PMC2876376
PMID: 20350949  [PubMed - indexed for MEDLINE]
Þ

17138. PLoS One. 2010 Feb 5;5(2):e9097. doi: 10.1371/journal.pone.0009097.

Antigenic characterization of recombinant hemagglutinin proteins derived from
different avian influenza virus subtypes.

Mueller M, Renzullo S, Brooks R, Ruggli N, Hofmann MA.

Institute of Virology and Immunoprophylaxis (IVI), Mittelhaeusern, Switzerland.
matthias.mueller@ivi.admin.ch

Since the advent of highly pathogenic variants of avian influenza virus (HPAIV), 
the main focus of avian influenza research has been the characterization and
detection of HPAIV hemagglutinin (HA) from H5 and H7 subtypes. However, due to
the high mutation and reassortation rate of influenza viruses, in theory any
influenza strain may acquire increased pathogenicity irrespective of its subtype.
A comprehensive antigenic characterization of influenza viruses encompassing all 
16 HA and 9 neuraminidase subtypes will provide information useful for the design
of differential diagnostic tools, and possibly, vaccines. We have expressed
recombinant HA proteins from 3 different influenza virus HA subtypes in the
baculovirus system. These proteins were used to generate polyclonal rabbit
antisera, which were subsequently employed in epitope scanning analysis using
peptide libraries spanning the entire HA. Here, we report the identification and 
characterization of linear, HA subtype-specific as well as inter
subtype-conserved epitopes along the HA proteins. Selected subtype-specific
epitopes were shown to be suitable for the differentiation of anti-HA antibodies 
in an ELISA.

PMCID: PMC2816723
PMID: 20140098  [PubMed - indexed for MEDLINE]
Þ

18175. J Virol. 2010 Feb;84(4):2122-33. doi: 10.1128/JVI.01668-09. Epub 2009 Dec 9.

The NS segment of an H5N1 highly pathogenic avian influenza virus (HPAIV) is
sufficient to alter replication efficiency, cell tropism, and host range of an
H7N1 HPAIV.

Ma W, Brenner D, Wang Z, Dauber B, Ehrhardt C, Högner K, Herold S, Ludwig S,
Wolff T, Yu K, Richt JA, Planz O, Pleschka S.

Institute of Medical Virology, Justus Liebig University, D-35392 Giessen,
Germany.

A reassortant avian influenza virus (designated FPV NS GD), carrying the
NS-segment of the highly pathogenic avian influenza virus (HPAIV) strain
A/Goose/Guangdong/1/96 (GD; H5N1) in the genetic background of the HPAIV strain
A/FPV/Rostock/34 (FPV; H7N1), was rescued by reverse genetics. Remarkably, in
contrast to the recombinant wild-type FPV (rFPV), the reassortant virus was able 
to replicate more efficiently in different human cell lines and primary mouse
epithelia cells without prior adaptation. Moreover, FPV NS GD caused disease and 
death in experimentally infected mice and was detected in mouse lungs; in
contrast, rFPV was not able to replicate in mice effectively. These results
indicated an altered host range and increased virulence. Furthermore FPV NS GD
showed pronounced pathogenicity in chicken embryos. In an attempt to define the
molecular basis for the apparent differences, we determined that NS1 proteins of 
the H5N1 and H7N1 strains bound the antiviral kinase PKR and the F2F3 domain of
cleavage and polyadenylation specificity factor 30 (CPSF30) with comparable
efficiencies in vitro. However, FPV NS GD infection resulted in (i) increased
expression of NS1, (ii) faster and stronger PKR inhibition, and (iii) stronger
beta interferon promoter inhibition than rFPV. Taken together, the results shed
further light on the importance of the NS segment of an H5N1 strain for viral
replication, molecular pathogenicity, and host range of HPAIVs and the possible
consequences of a reassortment between naturally occurring H7 and H5 type HPAIVs.

PMCID: PMC2812369
PMID: 20007264  [PubMed - indexed for MEDLINE]
Þ

18229. Berl Munch Tierarztl Wochenschr. 2009 Nov-Dec;122(11-12):440-5.

[Bird populations--hatching grounds of pandemic influenza viruses?].

[Article in German]

Harder T, Grund C, Beer M, Staubach C, Schoene C, Wilking H, Conraths FJ; EU
Monitoringprogramm für AIV beteiligten Untersuchungseinrichtungen der
Bundesländer.

Institut für Virusdiagnostik, Friedrich-Loeffler Institut, Greifswald-Insel
Riems. timm.harder@fli.bund.de

Aquatic wild birds constitute the natural reservoir of influenza A viruses. Under
appropriate selection pressure these viruses display a remarkable genetic
flexibility which is based on their high mutation rate. At least 16 subtypes of
the viral hemagglutinin glycoprotein (HA) und 9 of the viral neuraminidase (NA)
are distinguishable as a result of natural evolution. Due to the segmentation of 
the viral genome, genetic reassortments and, thus, a theoretically unrestricted
combination of HA and NA subtypes is possible. Transspecies-specific
transmissions of viruses from the natural reservoirs to mammals (horses, swine,
humans) led to the establishment of several influenza virus lineages which are
circulating stably and independently from the previous reservoir. Introduction of
viruses featuring new HA/NA combinations into immunologically naive populations
can cause rapid and widespread, even pandemic, dissemination of such viruses. The
(highly pathogenic avian influenza, HPAI). These mutants constitute the causative
agents of classical fowl plague. HPAI viruses of subtypes H5 and H7 generally
share a polybasic endoproteolytical cleavage site in the HA protein which allows 
for systemic replication in the avian host. Subtype H5 and H7 viruses which are
maintained in the natural reservoir hosts, in contrast, are of low pathogenicity 
(LPAI) and harbour monobasic HA cleavage sites restricting their tissue tropism
to epithelia of the respiratory and gastronintestinal tracts. This context forms 
the basis of all legal measures targeting the monitoring, prevention and
eradication of HPAIV and LPAIV of subtypes H5 and H7 in poultry. Results of such 
measures in Germany in 2008 are presented and discussed.

PMID: 19999379  [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

20217. Arch Virol. 2009;154(9):1517-22. doi: 10.1007/s00705-009-0467-9. Epub 2009 Aug 5.

Characterization of H3N6 avian influenza virus isolated from a wild white pelican
in Zambia.

Simulundu E, Mweene AS, Tomabechi D, Hang'ombe BM, Ishii A, Suzuki Y, Nakamura I,
Sawa H, Sugimoto C, Ito K, Kida H, Saiwana L, Takada A.

Department of Global Epidemiology, Hokkaido University Research Center for
Zoonosis Control, Kita-20, Nishi-10, Kita-ku, Sapporo, 001-0020, Japan.

We characterized an influenza virus isolated from a great white pelican in
Zambia. Phylogenetic analysis showed that all of its gene segments belonged to
the Eurasian lineage and that they appear to have evolved in distinct
geographical regions in Europe, Asia, and Africa, suggesting reassortment of
virus genes maintained in wild aquatic birds whose flyways overlap across these
continents. It is notable that this virus might possess some genes of the same
origin as those of highly pathogenic H7 and H5 viruses isolated in Eurasia. The
present study underscores the need for continued monitoring of avian influenza
viruses in Eurasia and Africa.

PMID: 19655084  [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

21743. J Virol. 2009 May;83(9):4205-15. doi: 10.1128/JVI.02403-09.

Isolation and genetic characterization of H5N2 influenza viruses from pigs in
Korea.

Lee JH, Pascua PN, Song MS, Baek YH, Kim CJ, Choi HW, Sung MH, Webby RJ, Webster 
RG, Poo H, Choi YK.

College of Medicine and Medical Research Institute, Chungbuk National University,
Cheongju, Republic of Korea.

Due to dual susceptibility to both human and avian influenza A viruses, pigs are 
believed to be effective intermediate hosts for the spread and production of new 
viruses with pandemic potential. In early 2008, two swine H5N2 viruses were
isolated from our routine swine surveillance in Korea. The sequencing and
phylogenetic analysis of surface proteins revealed that the Sw/Korea/C12/08 and
Sw/Korea/C13/08 viruses were derived from avian influenza viruses of the Eurasian
lineage. However, although the Sw/Korea/C12/08 isolate is an entirely avian-like 
virus, the Sw/Korea/C13/08 isolate is an avian-swine-like reassortant with the
PB2, PA, NP, and M genes coming from a 2006 Korean swine H3N1-like virus. The
molecular characterization of the two viruses indicated an absence of significant
mutations that could be associated with virulence or binding affinity. However,
animal experiments showed that the reassortant Sw/Korea/C13/08 virus was more
adapted and was more readily transmitted than the purely avian-like virus in a
swine experimental model but not in ferrets. Furthermore, seroprevalence in swine
sera from 2006 to 2008 suggested that avian H5 viruses have been infecting swine 
since 2006. Although there are no known potential clinical implications of the
avian-swine reassortant virus for pathogenicity in pigs or other species,
including humans, at present, the efficient transmissibility of the swine-adapted
H5N2 virus could facilitate virus spread and could be a potential model for
pandemic, highly pathogenic avian influenza (e.g., H5N1 and H7N7) virus outbreaks
or a pandemic strain itself.

PMCID: PMC2668473
PMID: 19359528  [PubMed - indexed for MEDLINE]
Þ

21809. Zh Mikrobiol Epidemiol Immunobiol. 2009 Jan-Feb;(1):31-6.

[Study of biological properties of cold-adapted reassortant strain of influenza
virus subtype H7N3].

[Article in Russian]

Desheva IuA, Smolonogina TA, Sergeeva MV, Rekstin AR, Swayne D, Klimov AI,
Rudenko LG.

For the development of live attenuated influenza vaccine (LAIV) against influenza
virus strains with pandemic potential, method of classic genetic reassortment of 
donor of attenuation A/Leningrad/134/17/57 (H2N2) [Len/17] with avian apathogenic
influenza viruses of different subtypes was used. Strain with genome formula 6:2,
which contains HA and NA genes from avian apathogenic virus A/wild
duck/Netherlands/12/00 (H7N3) [N7N3-wt] and 6 other genes--from Len/17, was
studied. Reassortant strain A/17/ wild duck/Netherlands/00/84 (H7N3) [Lenl7/ H7] 
exhibited ts- and ca- phenotype specific for cold-adapted strains. Reassortant
was identical on antigenic profile to parent avian virus H7N3-wt. Like
cold-adapted donor strain Len/17, Len17/H7 was attenuated for chickens, whereas
wild-type parent strain was lethal in 60% of birds after its intravenous
challenge. Reassortant strain Len17/H7 was attenuated during intranasal
inoculation of 6 EID50 to white mice, which was confirmed by absence of its
isolation from the lungs, actively reproduced on nasal mucosa and stimulated
specific systemic and local antibody response.

PMID: 19340965  [PubMed - indexed for MEDLINE]
Þ

22874. Antiviral Res. 2009 Feb;81(2):132-40. doi: 10.1016/j.antiviral.2008.10.009. Epub 
2008 Nov 24.

Characteristics of arbidol-resistant mutants of influenza virus: implications for
the mechanism of anti-influenza action of arbidol.

Leneva IA, Russell RJ, Boriskin YS, Hay AJ.

National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
wnyfd385@yandex.ru

The antiviral drug arbidol (ARB), which is licensed in Russia for use against
influenza, is known to inhibit early membrane fusion events in influenza A and B 
virus replication. To investigate in more detail the target and mechanism of ARB 
action we generated and studied the characteristics of ARB-resistant influenza
virus mutants. Observations of the ARB susceptibility of reassortants between
A/Singapore/1/57(H2N2) and A/chicken/Germany/27(H7N7, "Weybridge" strain) and of 
mutants of the latter virus identified the virus haemagglutinin (HA) as the major
determinant of ARB sensitivity. ARB-resistant mutants, selected from the most
sensitive reassortant, possessed single amino acid substitutions in the HA2
subunit which caused an increase in the pH of fusion and the associated
conformational change in HA. ARB was shown to stabilize the HA by causing a 0.2
pH unit reduction in the pH of the transition to the low pH form, which was
specifically abrogated by the resistance mutations. Some of the resistance
mutations, which reduce acid stability and would disrupt ARB-HA interactions, are
located in the vicinity of a potential ARB binding site identified using the
docking programme Gold. Together, the results of these investigations indicate
that ARB falls within a class of inhibitor which interacts with HA to stabilize
it against the low pH transition to its fusogenic state and consequently inhibit 
HA-mediated membrane fusion during influenza virus infection.

PMID: 19028526  [PubMed - indexed for MEDLINE]
Þ

23187. Arch Virol. 2008;153(11):2041-8. doi: 10.1007/s00705-008-0226-3. Epub 2008 Oct
21.

Antigenic and genetic analysis of H5 influenza viruses isolated from water birds 
for the purpose of vaccine use.

Soda K, Ozaki H, Sakoda Y, Isoda N, Haraguchi Y, Sakabe S, Kuboki N, Kishida N,
Takada A, Kida H.

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

In order to prepare H5N1 influenza virus vaccine, the hemagglutinins (HAs) of 14 
H5 virus isolates from water birds in Asia were antigenically and genetically
analyzed. Phylogenetic analysis of the H5 HA genes revealed that 13 isolates
belong to Eurasian and the other one to North American lineages. Each of the
deduced amino acid sequences of the HAs indicated a non-pathogenic profile.
Antigenic analysis using a panel of monoclonal antibodies recognizing six
different epitopes on the HA of A/duck/Pennsylvania/10218/1984 (H5N2) and chicken
antiserum to an H5N1 reassortant strain generated between A/duck/Mongolia/54/2001
(H5N2) and A/duck/Mongolia/47/2001 (H7N1), [R(Dk/Mong-Dk/Mong) (H5N1)] showed
that the HAs of highly pathogenic avian influenza (HPAI) viruses currently
circulating in Asia were antigenically closely related to those of the present
isolates from water birds. Mice subcutaneously injected with formalin-inactivated
R(Dk/Mong-Dk/Mong) were protected from challenge with 100 mouse lethal dose of
A/Viet Nam/1194/2004 (H5N1). The present results support the notion that the H5
isolates and the reassortant H5N1 strain should be useful for vaccine
preparation.

PMID: 18932020  [PubMed - indexed for MEDLINE]
Þ

23317. Virus Genes. 2009 Feb;38(1):66-73. doi: 10.1007/s11262-008-0289-y. Epub 2008 Sep 
30.

Characterization of H5N1 influenza A viruses isolated from domestic green-winged 
teal.

Chen J, Yang Z, Chen Q, Liu X, Fang F, Chang H, Li D, Chen Z.

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

Two avian influenza virus strains, A/domestic green-winged teal/Hunan/67/2005
(H5N1) (D-GWT/67) and A/domestic green-winged teal/Hunan/79/2005 (H5N1)
(D-GWT/79), were isolated from healthy domestic green-winged teals (Anas crecca) 
in Hunan Province, South China. Genomic analysis showed that both isolates were
reassortants. The hemagglutinin (HA) genes of the two isolates were closely
related to that of an H5N1 strain isolated from tree sparrow (A/tree
sparrow/Henan/1/04). The neuraminidase (NA) genes and the internal protein genes 
of both isolates were closely related to those from
A/chicken/Shantou/4231/2003-like (H5N1) viruses, with exception of the matrix (M)
gene of D-GWT/79, which was closely related to that of the H7N3 strain
A/mallard/Netherlands/12/2000 isolated from wild mallard duck. The virulence of
the two isolates was examined in chickens, ducks, and mice. Both strains were
found to be highly pathogenic in chickens and ducks, but showed low pathogenicity
in mice. These findings contribute to the realization that domestic green-winged 
teals carrying the H5N1 virus may play an important role in transmitting the
virus among birds.

PMID: 18825495  [PubMed - indexed for MEDLINE]
Þ

23365. J Virol. 2008 Dec;82(23):11880-8. doi: 10.1128/JVI.01445-08. Epub 2008 Sep 24.

Compatibility among polymerase subunit proteins is a restricting factor in
reassortment between equine H7N7 and human H3N2 influenza viruses.

Li C, Hatta M, Watanabe S, Neumann G, Kawaoka Y.

Department of Pathobiological Sciences, School of Veterinary Medicine, University
of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706, USA.

Reassortment is an important driving force for influenza virus evolution, and a
better understanding of the factors that affect this process could improve our
ability to respond to future influenza pandemics and epidemics. To identify
factors that restrict the generation of reassortant viruses, we cotransfected
human embryonic kidney cells with plasmids for the synthesis of viral RNAs of
both A/equine/Prague/1/56 (Prague; H7N7) and A/Yokohama/2017/03 (Yokohama; H3N2) 
viruses together with the supporting protein expression plasmids. Of the possible
256 genotypes, we identified 29 genotypes in 120 randomly plaque-picked
reassortants examined. Analyses of these reassortants suggested that the
formation of functional ribonucleoprotein (RNP) complexes was a restricting
factor, a finding that correlated with the activities of RNP complexes composed
of different combinations of the proteins from the two viruses, as measured in a 
minigenome assay. For at least one nonfunctional RNP complex (i.e., Prague PB2,
Prague PB1, Yokohama PA, and Prague NP), the lack of activity was due to the
inability of the three polymerase subunit proteins to form a heterotrimer.
Adaptation of viruses possessing a gene encoding a chimera of the PA proteins of 
the two viruses and the remaining genes from Prague virus resulted in
compensatory mutations in the PB2 and/or PA protein. These results indicate
substantial incompatibility among the gene products of the two test viruses, a
critical role for the RNP complex in the generation of reassortant viruses, and a
functional interaction of PB2 and PA.

PMCID: PMC2583690
PMID: 18815312  [PubMed - indexed for MEDLINE]
Þ

23605. J Virol. 2008 Nov;82(21):10854-63. doi: 10.1128/JVI.01206-08. Epub 2008 Aug 20.

Avian influenza h6 viruses productively infect and cause illness in mice and
ferrets.

Gillim-Ross L, Santos C, Chen Z, Aspelund A, Yang CF, Ye D, Jin H, Kemble G,
Subbarao K.

Laboratory of Infectious Diseases, NIAID, NIH, Bethesda, Maryland 20892, USA.

Influenza pandemic preparedness has focused on influenza virus H5 and H7
subtypes. However, it is not possible to predict with certainty which subtype of 
avian influenza virus will cause the next pandemic, and it is prudent to include 
other avian influenza virus subtypes in pandemic preparedness efforts. An H6
influenza virus was identified as a potential progenitor of the H5N1 viruses that
emerged in Hong Kong in 1997. This virus continues to circulate in the bird
population in Asia, and other H6 viruses are prevalent in birds in North America 
and Asia. The high rate of reassortment observed in influenza viruses and the
prevalence of H6 viruses in birds suggest that this subtype may pose a pandemic
risk. Very little is known about the replicative capacity, immunogenicity, and
correlates of protective immunity for low-pathogenicity H6 influenza viruses in
mammals. We evaluated the antigenic and genetic relatedness of 14 H6 influenza
viruses and their abilities to replicate and induce a cross-reactive immune
response in two animal models: mice and ferrets. The different H6 viruses
replicated to different levels in the respiratory tracts of mice and ferrets,
causing varied degrees of morbidity and mortality in these two models. H6 virus
infection induced similar patterns of neutralizing antibody responses in mice and
ferrets; however, species-specific differences in the cross-reactivity of the
antibody responses were observed. Overall, cross-reactivity of neutralizing
antibodies in H6 virus-infected mice did not correlate well with protection
against heterologous wild-type H6 viruses. However, we have identified an H6
virus that induces protective immunity against viruses in the North American and 
Eurasian lineages.

PMCID: PMC2573192
PMID: 18715930  [PubMed - indexed for MEDLINE]
Þ

23901. J Microbiol Immunol Infect. 2008 Jun;41(3):231-42.

Surveillance of avian and swine influenza in the swine population in Taiwan,
2004.

Shieh HK, Chang PC, Chen TH, Li KP, Chan CH.

Department of Veterinary Medicine, National Chung Hsing University, Taichung,
Taiwan.

BACKGROUND AND PURPOSE: We conducted serological and virological surveillance of 
pig farms in Taiwan from areas epidemic for low pathogenic avian influenza virus 
(AIV), H5N2 subtype, in order to determine the prevalence of AIV and swine
influenza virus (SIV) in 2004.
METHODS: Pig sera from 9833 animals from 1974 farms in 9 counties were examined
using agar gel precipitation (AGP) to screen for the presence of antibody against
influenza A virus. AGP-positive sera were subjected to
hemagglutination-inhibition test against H1, H3, H5 and H7 AIV subtypes and H1
and H3 SIV subtypes. Nasal swabs from 881 pigs were also examined for the
presence of SIV by virus isolation in specific pathogen-free embryonated chicken 
eggs. Virus isolates were identified by reverse transcriptase-polymerase chain
reaction followed by DNA sequencing of hemagglutinin and neuraminidase genes.
RESULTS: The AGP test on sera revealed the presence of antibodies against
influenza A virus in 62.6% of farms and in 37.7% of pig sera. SIV antibodies to
subtype H1 and H3 were found in 10.8% and 65.8% of sera, respectively. There were
two peaks of the serological prevalence of SIV in pigs: one between January and
February, and the other in October. By contrast, hemagglutinin tests against H5
and H7 AIV subtypes were negative in all sera, while there was a very low
positive rate against H1 and H3 AIV subtypes. One H1N2 and one H3N1 viral isolate
were obtained from nasal swabs of pigs. Phylogenetic analysis of hemagglutinin
and neuraminidase genes revealed both isolates were reassortants of both
classical and recent SIVs.
CONCLUSIONS: Different subtypes of SIV co-circulate among swine from different
farms within the same county and may cause clinical outbreaks of the disease in
Taiwan.

PMID: 18629419  [PubMed - indexed for MEDLINE]
Þ

24080. Virology. 2008 Aug 15;378(1):123-32. doi: 10.1016/j.virol.2008.05.021. Epub 2008 
Jun 27.

A live attenuated cold-adapted influenza A H7N3 virus vaccine provides protection
against homologous and heterologous H7 viruses in mice and ferrets.

Joseph T, McAuliffe J, Lu B, Vogel L, Swayne D, Jin H, Kemble G, Subbarao K.

Laboratory of Infectious Diseases, NIAID, NIH, Bethesda, MD 20892, USA.

The appearance of human infections caused by avian influenza A H7 subtype viruses
underscores their pandemic potential and the need to develop vaccines to protect 
humans from viruses of this subtype. A live attenuated H7N3 virus vaccine was
generated by reverse genetics using the HA and NA genes of a low pathogenicity
A/chicken/BC/CN-6/04 (H7N3) virus and the six internal protein genes of the
cold-adapted A/Ann Arbor/6/60 ca (H2N2) virus. The reassortant H7N3 BC 04 ca
vaccine virus was temperature sensitive and showed attenuation in mice and
ferrets. Intranasal immunization with one dose of the vaccine protected mice and 
ferrets when challenged with homologous and heterologous H7 viruses. The
reassortant H7N3 BC 04 ca vaccine virus showed comparable levels of attenuation, 
immunogenicity and efficacy in mice and ferret models. The safety,
immunogenicity, and efficacy of this vaccine in mice and ferrets support the
evaluation of this vaccine in clinical trials.

PMCID: PMC2586151
PMID: 18585748  [PubMed - indexed for MEDLINE]
Þ

24225. Vaccine. 2008 Jul 4;26(29-30):3580-6. doi: 10.1016/j.vaccine.2008.05.001. Epub
2008 May 22.

Characterization of influenza virus variants with different sizes of the
non-structural (NS) genes and their potential as a live influenza vaccine in
poultry.

Wang L, Suarez DL, Pantin-Jackwood M, Mibayashi M, García-Sastre A, Saif YM, Lee 
CW.

Food Animal Health Research Program, Ohio Agricultural Research and Development
Center, The Ohio State University, Wooster, OH 44691, United States.

From a stock of A/turkey/Oregon/71-delNS1 (H7N3) virus, which has a 10 nucleotide
deletion in the coding region of the NS1 gene, we found that several variants
with different sizes of NS genes could be produced by passaging the virus in 10- 
and 14-day-old embryonating chicken eggs (ECE), but not in 7-day-old ECE or Vero 
cells. We were able to rescue the reassortant virus that has different sizes of
the NS genes and confirmed that those NS genes are genetically stable. By
conducting in vivo studies in 2-week-old chickens, we found two plaque purified
variants (D-del pc3 and pc4) which can be used as a potential live-attenuated
vaccine. The variants were highly attenuated in chickens and did not transmit the
virus from infected chickens to uninoculated cage mates. At the same time, the
variants induced relatively high antibody titers which conferred good protection 
against a high dose heterologous virus challenge. Our study indicates that
naturally selected NS1 deletion variants might be useful in the development of
live-attenuated influenza vaccines in poultry. Furthermore, deletion in the NS1
protein can be potentially useful as a negative marker for a differentiating
infected from vaccinated animals (DIVA) approach.

PMCID: PMC2785844
PMID: 18539366  [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

24911. Vaccine. 2008 Mar 25;26(14):1742-50. doi: 10.1016/j.vaccine.2008.01.036. Epub
2008 Feb 13.

Development of Eurasian H7N7/PR8 high growth reassortant virus for clinical
evaluation as an inactivated pandemic influenza vaccine.

Jadhao SJ, Achenbach J, Swayne DE, Donis R, Cox N, Matsuoka Y.

Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton
Road, NE, Atlanta, GA 30333, USA. Samadhan.Jadhao@ars.usda.gov

Avian-to-human transmission of the high pathogenicity (HP) H7N7 subtype avian
influenza viruses in the Netherlands during 2003 caused zoonotic infections in 89
people, including a case of acute fatal respiratory distress syndrome. Public
health emergency preparedness against H7N7 avian influenza viruses with pandemic 
potential includes the development of vaccine candidate viruses. In order to
develop a high growth reassortant vaccine candidate virus, low pathogenicity (LP)
A/mallard/Netherlands/12/2000 (H7N3) and A/mallard/Netherlands/2/2000 (H10N7)
strains were selected as donors of the H7 haemagglutinin and N7 neuraminidase
genes, respectively. The donor viruses exhibited high amino acid sequence
homology with the surface glycoproteins of A/Netherlands/219/03 H7N7 virus
(NL219), an isolate recovered from the fatal human case. Adhering to the seasonal
influenza vaccine licensure regulations, we generated a H7N7/PR8 reassortant
containing desired surface glycoprotein genes from the mallard viruses and
internal genes of A/Puerto Rico/8/34 human vaccine strain (H1N1). Antigenic
analysis revealed that the vaccine candidate virus confers broad antigenic
cross-reactivity against contemporary Eurasian and the North American H7 subtype 
human isolates. Mice immunized with formalin inactivated (FI) H7N7/PR8 whole
virus vaccine with or without aluminum hydroxide adjuvant conferred clinical
protection from mortality and reduced pulmonary replication of the NL219
challenge virus. The FI H7N7/PR8 whole virus vaccine also afforded
cross-protection in mice at the pulmonary level against antigenically distinct
North American LP A/Canada/444/04 (H7N3) human isolate. The vaccine candidate
virus satisfied the agricultural safety requirements for chickens, proved safe in
mice, and has entered in phase-I human clinical trial in the United States.

PMID: 18336962  [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

25476. Vaccine. 2008 Feb 13;26(7):956-65. doi: 10.1016/j.vaccine.2007.11.052. Epub 2007 
Dec 10.

The potential of a protease activation mutant of a highly pathogenic avian
influenza virus for a pandemic live vaccine.

Gabriel G, Garn H, Wegmann M, Renz H, Herwig A, Klenk HD, Stech J.

Institut für Virologie, Fachbereich Medizin, Philipps-Universität Marburg,
Germany. guelsah.gabriel@path.ox.ac.uk

The most effective countermeasure against a pandemic originating from a highly
pathogenic avian influenza virus (HPAIV) is immunoprophylaxis of the human
population. We present here a new approach for the development of a pandemic
HPAIV live vaccine. Using reverse genetics, we replaced the polybasic
hemagglutinin cleavage site of an H7N7 HPAIV with an elastase motif. This mutant 
was strictly elastase-dependent, grew equally well as the wild-type in cell
culture and was attenuated in mice unlike the lethal wild-type. Immunization at
10(6)pfu dosage protected mice against disease and induced sterile immunity;
vaccination with homosubtypic or heterosubtypic reassortants led to
cross-protection. These observations demonstrate that a mutated hemagglutinin
requiring elastase cleavage can serve as an attenuating component of a live
vaccine against HPAIV.

PMID: 18164519  [PubMed - indexed for MEDLINE]
Þ

25585. J Gen Virol. 2008 Jan;89(Pt 1):48-59.

Molecular analysis of avian H7 influenza viruses circulating in Eurasia in
1999-2005: detection of multiple reassortant virus genotypes.

Campitelli L, Di Martino A, Spagnolo D, Smith GJ, Di Trani L, Facchini M, De
Marco MA, Foni E, Chiapponi C, Martin AM, Chen H, Guan Y, Delogu M, Donatelli I.

Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto
Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
laura.campitelli@iss.it

Avian influenza infections by high and low pathogenicity H7 influenza viruses
have caused several outbreaks in European poultry in recent years, also resulting
in human infections. Although in some cases the source of H7 strains from
domestic poultry was shown to be the viruses circulating in the wild bird
reservoir, a thorough characterization of the entire genome of H7 viruses from
both wild and domestic Eurasian birds, and their evolutionary relationships, has 
not been conducted. In our study, we have analysed low pathogenicity H7 influenza
strains isolated from wild and domestic ducks in Italy and southern China and
compared them with those from reared terrestrial poultry such as chicken and
turkey. Phylogenetic analysis demonstrated that the H7 haemagglutinin genes were 
all closely related to each other, whereas the remaining genes could be divided
into two or more phylogenetic groups. Almost each year different H7 reassortant
viruses were identified and in at least two different years more than one H7
genotype co-circulated. A recent precursor in wild waterfowl was identified for
most of the gene segments of terrestrial poultry viruses. Our data suggest that
reassortment allows avian influenza viruses, in their natural reservoir, to
increase their genetic diversity. In turn this might help avian influenza viruses
colonize a wider range of hosts, including domestic poultry.

PMID: 18089728  [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

26061. J Gen Virol. 2007 Nov;88(Pt 11):3094-9.

Origin of highly pathogenic H5N1 avian influenza virus in China and genetic
characterization of donor and recipient viruses.

Mukhtar MM, Rasool ST, Song D, Zhu C, Hao Q, Zhu Y, Wu J.

State Key Laboratory of Virology, College of Life Sciences, Wuhan University,
Wuhan 430072, P.R. China.

Genetic analysis of all eight genes of two Nanchang avian influenza viruses,
A/Duck/Nanchang/1681/92 (H3N8-1681) and A/Duck/Nanchang/1904/92 (H7N1-1904),
isolated from Jiangxi province, China, in 1992, showed that six internal genes of
H3N8-1681 virus and five internal (except NS gene) genes of H7N1-1904 virus were 
closely similar to A/Goose/Guangdong/1/96 (H5N1) virus, the first highly
pathogenic avian influenza (HPAI) virus of subtype H5N1 isolated in Asia. The
neuraminidase (NA) gene of Gs/Gd/1/96 had the highest genetic similarity with
A/Duck/Hokkaido/55/96 (H1N1-55) virus. The haemagglutinin (HA) gene of Gs/Gd/1/96
virus might have originated as a result of mutation of H5 HA gene from
A/Swan/Hokkaido/51/96 (H5N3-51)-like viruses. The PA gene of H5N3-51 virus had
the highest similarity with Gs/Gd/1/96. This study explains the origin of first
Asian HPAI H5N1 virus in Guangdong by the reassortment of Nanchang (close to
Guangdong) and Hokkaido (Japan) (H1N1-55 and H5N3-51) viruses. Genetic
characteristics of donor and recipient viruses were also studied.

PMID: 17947535  [PubMed - indexed for MEDLINE]
Þ

26175. Clin Vaccine Immunol. 2007 Nov;14(11):1425-32. Epub 2007 Oct 3.

Development and evaluation of an Influenza virus subtype H7N2 vaccine candidate
for pandemic preparedness.

Pappas C, Matsuoka Y, Swayne DE, Donis RO.

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

Influenza virus of the H7N2 subtype has been introduced into noncommercial
poultry in the United States, and this probably resulted in incidents of
transmission of H7N2 virus to humans, documented in 2002 and 2003. This virus
could be considered a potential threat to public health if it acquired
person-to-person transmissibility. A favored approach for global pandemic
preparedness includes development of prepandemic vaccines for any potential
pandemic virus. To this end, we created a high-growth reassortant virus
(H7N2-PR8) containing the genes for the hemagglutinin and the neuraminidase from 
a low-pathogenicity (H7N2) virus strain and the remaining six genes from a human 
vaccine strain (H1N1). The reassortant strain was evaluated to assess its
antigenicity, safety, and protective efficacy using a mouse model. Antigenicity
studies using ferret antibodies raised against H7N2-PR8 indicated that this virus
confers broad cross-reactivity with divergent H7 viruses of different years and
lineages. Mice and chickens inoculated with high doses of H7N2-PR8 supported
virus replication but survived, indicating that this virus is comparable to other
avian viruses of low pathogenicity. To assess the protective efficacy of
H7N2-PR8, mice were immunized with two doses of formalin-inactivated H7N2-PR8,
alone or with alum. Vaccinated mice subsequently challenged with highly
pathogenic viruses from homologous and heterologous lineages A/Canada/444/04
(H7N3) and A/Netherlands/219/03 (H7N7) showed pronounced reduction of wild-type
virus replication. These studies indicate that H7N2-PR8 is immunogenic, safe, and
protective in animal models; these are the essential attributes to qualify for
phase I human clinical trials as a prepandemic vaccine.

PMCID: PMC2168170
PMID: 17913860  [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

26460. J Virol Methods. 2007 Dec;146(1-2):293-7. Epub 2007 Aug 28.

The cleavage of the hemagglutinin protein of H5N2 avian influenza virus in yeast.

Wang CY, Luo YL, Chen YT, Li SK, Lin CH, Hsieh YC, Liu HJ.

Department of Life Science, National Pingtung University of Science and
Technology, Neipu, Pingtung 912, Taiwan.

Influenza viruses belonging to the Orthomyxoviridae family are enveloped viruses 
with segmented negative sense RNA genome surrounded by a helical symmetry shell. 
Influenza viruses, especially the highly pathogenic avian influenza virus (HPAI) 
such as H5 or H7 subtype are important pathogens for the poultry industry. Due to
genetic reassortments between avian and human influenza viruses, global pandemics
may emerge and the naive human immunity could not be ready for them. The
full-length HA-encoding gene of H5N2 AIV was inserted into a secretory
pPICZalphaA vector and integrated into the genome of Pichia pastoris by
heterologous recombination. The HA protein secretion into the medium was induced 
with methanol. Besides the expected 69kDa protein, another smaller fragment about
47kDa was recognized by an anti-AIV-HA monoclonal antibody in Western blot assay.
This is the first report on the cleavage of HA(0) into HA(1) and HA(2) in the
methylotrophic yeast P. pastoris. This possibly was due to digestion by proteases
from P. pastoris based on the amino acid sequences at the predicted cleavage
site, (326)R-X-K-R(329). With similar modifications to the eukaryotes, large
quantity, proper antigenicity, and low cost, this expression system may provide a
simple tool to produce HA proteins for further use in preparation of ELISA kits
and subunit vaccines.

PMID: 17727967  [PubMed - indexed for MEDLINE]
Þ

27234. Avian Dis. 2007 Mar;51(1 Suppl):479-80.

Generation of avian influenza reassortant viruses of the H7N5 subtype as
potential vaccine candidates to be used in the framework of a "DIVA" vaccination 
strategy.

Beato MS, Rigoni M, Milani A, Capua I.

OIE/FAO and National Reference Laboratory for Avian Influenza and Newcastle
Disease Istituto Zooprofilattico Sperimentale delle Venezie, Viale
dell'Università 10, Legnaro, 35020 Padova, Italy.

The recent outbreaks of avian influenza (AI) worldwide have highlighted the
difficulties in controlling this disease. Vaccination has become a recommended
tool to support the eradication efforts and to limit the economic losses due to
AI. A vaccination system based on the use of a vaccine containing a heterologous 
neuraminidase to the field virus has been shown to be efficacious in reducing the
viral shedding and clinical symptoms and in differentiating vaccinated from
infected animals. To further develop this so called differentiating infected from
vaccinated animal vaccination system, two reassortant avian influenza viruses of 
the H7N5 subtype have been generated. The aim of this study was to generate a
prototype strain with a rare N subtype to avoid interference with the anti-N
discriminatory test.

PMID: 17494613  [PubMed - indexed for MEDLINE]
Þ

27379. Influenza Other Respi Viruses. 2007 May;1(3):95-104. doi:
10.1111/j.1750-2659.2007.00015.x.

Preparation of genetically engineered A/H5N1 and A/H7N1 pandemic vaccine viruses 
by reverse genetics in a mixture of Vero and chicken embryo cells.

Legastelois I, Garcia-Sastre A, Palese P, Tumpey TM, Maines TR, Katz JM, Vogel
FR, Moste C.

Research and Development, sanofi pasteur, 1541 Avenue Marcel Mérieux, Marcy
L'Etoile, France. isabelle.legastelois@sanofipasteur.com

BACKGROUND: In case of influenza pandemic, a robust, easy and clean technique to 
prepare reassortants would be necessary.
OBJECTIVES: Using reverse genetics, we prepared two vaccine reassortants (A/H5N1 
x PR8 and A/H7N1 x PR8) exhibiting the envelope glycoproteins from non-pathogenic
avian viruses, A/Turkey/Wisconsin/68 (A/H5N9) and A/Rhea/New Caledonia/39482/93
(A/H7N1) and the internal proteins of the attenuated human virus A/Puerto
Rico/8/34 (H1N1).
METHODS: The transfection was accomplished using a mixture of Vero and chicken
embryo cells both of which are currently being used for vaccine manufacturing.
RESULTS: This process was reproducible, resulting in consistent recovery of
influenza viruses in 6 days. Because it is mainly the A/H5N1 strain that has
recently crossed the human barrier, it is the A/PR8 x A/H5N1 reassortant (RG5)
that was further amplified, either in embryonated hen eggs or Vero cells, to
produce vaccine pre-master seed stocks that met quality control specifications.
Safety testing in chickens and ferrets was performed to assess the non-virulence 
of the reassortant, and finally analysis using chicken and ferret sera immunized 
with the RG5 virus showed that the vaccine candidate elicited an antibody
response cross-reactive with the Hong Kong 1997 and 2003 H5N1 strains but not the
Vietnam/2004 viruses.
CONCLUSIONS: The seeds obtained could be used as part of a pandemic vaccine
strain 'library' available in case of propagation in humans of a new highly
pathogenic avian strain.

PMID: 19453414  [PubMed - indexed for MEDLINE]
Þ

28649. Arch Virol. 2007;152(3):585-93. Epub 2006 Nov 20.

Genome characterisation of the newly discovered avian influenza A H5N7 virus
subtype combination.

Bragstad K, Jørgensen PH, Handberg KJ, Fomsgaard A.

Department of Virology, Statens Serum Institut, Copenhagen, Denmark.

In Denmark, in 2003, a previously unknown subtype combination of avian influenza 
A virus, H5N7 (A/Mallard/Denmark/64650/03), was isolated from a flock of 12,000
mallards. The H5N7 subtype combination might be a reassortant between recent
European avian influenza A H5, H7, and a third subtype, possibly an H6. The
haemagglutinin and the acidic polymerase genes of the virus were closely related 
to a low-pathogenic Danish H5N2 virus A/Duck/Denmark/65041/04 (H5N2). The
neuraminidase gene and the non-structural gene were most similar to the highly
pathogenic A/Chicken/Netherlands/1/03 (H7N7) and the human-fatal
A/Netherlands/219/03 (H7N7), respectively. The basic polymerase 1 and 2 genes
were phylogenetically equidistant to both A/Duck/Denmark/65047/04 (H5N2) and
A/Chicken/Netherlands/1/03 (H7N7). The nucleoprotein and matrix gene had highest 
nucleotide sequence similarity to the H6 subtypes A/Duck/Hong Kong/3096/99 (H6N2)
and A/WDk/ST/1737/2000 (H6N8), respectively. All genes of the H5N7 strain were of
avian origin, and no further evidence of pathogenicity to humans has been found.

PMID: 17115305  [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

29675. Virology. 2006 Sep 30;353(2):388-95. Epub 2006 Jul 10.

Pathogenic potential of North American H7N2 avian influenza virus: a mutagenesis 
study using reverse genetics.

Lee CW, Lee YJ, Senne DA, Suarez DL.

Southeast Poultry Research Laboratory, USDA-ARS, 934 College Station Road,
Athens, GA 30605, USA.

An H7N2 subtype avian influenza virus (AIV) first appeared in the live bird
marketing system (LBMS) in the Northeastern United States in 1994. Since then
this lineage of virus has become the predominant subtype of AIV isolated from the
LBMS and has been linked to several costly commercial poultry outbreaks. Concern 
for this low pathogenicity isolate mutating to the highly pathogenic form has
remained high because of the increasing number of basic amino acids at the
hemagglutinin (HA) cleavage site, which is known to be associated with increased 
pathogenicity of AIV. To address the risk of low pathogenic LBMS-lineage H7N2
virus mutating to the highly pathogenic form of the virus, we generated a series 
of mutant viruses that have changes in the sequence at the HA cleavage site by
using plasmid-based reverse genetics. We confirmed that a conserved proline at -5
position from the HA cleavage site could be changed to a basic amino acid,
producing a virus with five basic amino acids in a row at the cleavage site, but 
with no increase in virulence. Increased virulence was only observed when
additional basic amino acids were inserted. We also observed that the virus
preferred the arginine instead of lysine at the -4 position from the cleavage
site to manifest increased virulence both in vitro and in vivo. Using helper
virus-based reverse genetics, where only one transcription plasmid expressing a
mutated HA vRNA is used, we identified specific HA cleavage site sequences that
were preferentially incorporated into the low pathogenic wild-type virus. The
resultant reassortant viruses were highly pathogenic in chickens. This study
provides additional evidence that H7 avian influenza viruses require an
insertional event to become highly pathogenic, as compared to H5 viruses that can
become highly pathogenic strictly by mutation or by insertions.

PMID: 16828833  [PubMed - indexed for MEDLINE]
Þ

29747. Vaccine. 2006 Nov 10;24(44-46):6736-41. Epub 2006 Jun 6.

An emerging avian influenza A virus H5N7 is a genetic reassortant of highly
pathogenic genes.

Bragstad K, Jørgensen PH, Handberg KJ, Fomsgaard A.

Department of Virology, Statens Serum Institut, 5 Artillerivej, DK-2300
Copenhagen S, Denmark. kbr@ssi.dk

We full genome characterised the newly discovered avian influenza virus H5N7
subtype combination isolated from a stock of Danish game ducks to investigate the
composition of the genome and possible features of high pathogenicity. It was
found that the haemagglutinin and the acidic polymerase genes were closely
related to a low pathogenic H5 strain (A/Duck/Denmark/65047/04 H5N2). The
neuraminidase and the non-structural genes were closely related to the highly
pathogenic H7N7 strains from The Netherlands 2003. The basic polymerase genes 1
and 2 were shared between the Danish H5N7 and H5N2 and the H7N7 from The
Netherlands. The nucleoprotein and the matrix genes were closely related to H6
strains. Thus, the new H5N7 subtype share genes with H5, H7 and H6 subtypes and
possesses internal genes originating from highly pathogenic strains. The findings
emphasize the need for surveillance presumed low pathogenic avian influenza A
viruses.

PMID: 16814904  [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

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

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

34068. Avian Pathol. 2004 Aug;33(4):393-404.

Avian influenza: recent developments.

Capua I, Alexander DJ.

OIE and National Reference Laboratory for Avian Influenza, Istituto
Zooprofilattico Sperimentale delle Venezie, Viale dell'Università 10, 35020
Legnaro, Padova, Italy. icapua@izsvenezie.it

This paper reviews the worldwide situation regarding avian influenza infections
in poultry from 1997 to March 2004. The increase in the number of primary
introductions and the scientific data available on the molecular basis of
pathogenicity have generated concerns particularly for legislative purposes and
for international trade. This has led to a new proposed definition of 'avian
influenza' to extend all infections caused by H5 and H7 viruses regardless of
their virulence as notifiable diseases, although this has encountered some
difficulties in being approved. The paper also reviews the major outbreaks caused
by viruses of the H5 or H7 subtype and the control measures applied. The zoonotic
aspects of avian influenza, which until 1997 were considered to be of limited
relevance in human medicine, are also discussed. The human health implications
have now gained importance, both for illness and fatalities that have occurred
following natural infection with avian viruses, and for the potential of
generating a reassortant virus that could give rise to the next human influenza
pandemic.

Copyright 2004 Houghton Trust Ltd

PMID: 15370036  [PubMed - indexed for MEDLINE]
Þ

34257. Vaccine. 2004 Aug 13;22(23-24):3175-81.

Generation of reassortant influenza vaccines by reverse genetics that allows
utilization of a DIVA (Differentiating Infected from Vaccinated Animals) strategy
for the control of avian influenza.

Lee CW, Senne DA, Suarez DL.

Southeast Poultry Research Laboratory, USDA-ARS, 934 College Station Road,
Athens, GA 30605, USA.

Vaccination of poultry with inactivated influenza vaccine can be an effective
tool in the control of avian influenza (AI). One major concern of using
inactivated vaccine is vaccine-induced antibody interference with serologic
surveillance and epidemiology. In the United States, low pathogenicity H5 and H7 
subtype AI viruses have caused serious economic losses in the poultry industry.
Most of these viruses also have the accompanying N2 subtype and no H5N1 or H7N8
subtype AI viruses have been identified in poultry in the US. In order to allow
the Differentiation of Infected from Vaccinated Animals (DIVA) while maintaining 
maximum efficacy of the vaccine, we generated reassortant viruses by reverse
genetics that contained the same H5 and H7 hemagglutinin (HA) gene as the
challenge virus, but a heterologous N1 or N8 neuraminidase (NA) gene. In
vaccination-challenge experiments in 2-week-old specific pathogen free chickens, 
reassortant influenza vaccines (rH5N1 and rH7N8) demonstrated similar antibody
profiles and comparable protection rates as vaccines prepared with parent H5N2
and H7N2 viruses. Further, we were able to differentiate the sera from infected
and vaccinated birds by neuraminidase inhibition test and indirect
immunofluorescent antibody assay on the basis of different antibodies elicited by
their NA proteins. These results demonstrate the usefulness of a reverse genetics
system for the rapid generation of reassortant AI virus that allows utilization
of the DIVA strategy for the control of AI infections in poultry.

PMID: 15297071  [PubMed - indexed for MEDLINE]
Þ

35412. Eur J Clin Microbiol Infect Dis. 2004 Jan;23(1):1-6. Epub 2003 Dec 9.

Human health implications of avian influenza viruses and paramyxoviruses.

Capua I, Alexander DJ.

Office International des Epizooties and National Reference Laboratory for Avian
Influenza, Istituto Zooprofilattico Sperimentale delle Venezie, Viale
dell'Università 10, 35020, Legnaro, Padova, Italy. icapua@izsvenezie.it

Among avian influenza viruses and avian paramyxoviruses are the aetiological
agents of two of the most devastating diseases of the animal kingdom: (i). the
highly pathogenic form of avian influenza, caused by some viruses of the H5 and
H7 subtypes, and (ii). Newcastle disease, caused by virulent strains of APMV type
1. Mortality rates due to these agents can exceed 50% in naïve bird populations, 
and, for some strains of AI, nearly 100%. These viruses may also be responsible
for clinical conditions in humans. The virus responsible for Newcastle disease
has been known to cause conjunctivitis in humans since the 1940s. The
conjunctivitis is self-limiting and does not have any permanent consequences.
Until 1997, reports of human infection with avian influenza viruses were sporadic
and frequently associated with conjunctivitis. Recently, however, avian influenza
virus infections have been associated with fatalities in human beings. These
casualties have highlighted the potential risk that this type of infection poses 
to public health. In particular, the pathogenetic mechanisms of highly pathogenic
avian influenza viruses in birds and the possibility of reassortment between
avian and human viruses in the human host represent serious threats to human
health. For this reason, any suspected case should be investigated thoroughly.

PMID: 14663646  [PubMed - indexed for MEDLINE]
Þ

35686. Avian Dis. 2003;47(3 Suppl):888-97.

Update on molecular epidemiology of H1, H5, and H7 influenza virus infections in 
poultry in North America.

Suarez DL, Spackman E, Senne DA.

Southeast Poultry Research Laboratory, Agriculture Research Service, U.S.
Department of Agriculture, 934 College Station Road, Athens, GA 30605, USA.

Avian influenza is endemic in wild birds in North America, and the virus
routinely has been transmitted from this reservoir to poultry. Influenza, once
introduced into poultry, can become endemic within the poultry population. It may
be successfully eradicated by human intervention, or the virus may fail to
successfully spread on its own. In the last 5 yr, influenza virus has been
isolated from poultry in the United States on numerous occasions, and, with the
use of molecular epidemiology, the relationships of these different viruses can
be determined. There are 15 different hemagglutinin subtypes of avian influenza
viruses, but infections with virus of H5 and H7 subtypes are of the most concern 
because of the potential for these viruses to mutate to the highly pathogenic
form of the virus. Most of the influenza isolations in the United States have
been associated with the live-bird markets (LBMs) in the Northeast. This has
included primarily H7N2 influenza viruses, but also H7N3, H5N2, and other
subtypes. Most of the H7N2 viruses were part of a single lineage that was first
observed in 1994, but new introductions of H7N2 and H7N3 were also observed. The 
predominant H7N2 LBM lineage of virus spread to large commercial poultry
operations on at least three occasions since 1997, with the largest outbreak
occurring in Virginia in 2002. The H5N2 viruses in the LBMs included viruses from
domestic ducks, gamebirds, and environmental samples. Some H5N2 viruses isolated 
in different years and in different locations had a high degree of sequence
relatedness, although the reservoir source, if it is endemic, has not been
identified. Finally, an H1N2 virus, associated with a drop in egg production, was
isolated from turkeys in Missouri in 1999. This virus was a complex reassortant
with swine, human, and avian influenza genes that was similar to recent swine
isolates from the Midwest. Additional serologic evidence suggests that flocks in 
other states were infected with a H1N2 virus.

PMID: 14575082  [PubMed - indexed for MEDLINE]
Þ

36927. Vet J. 2002 Nov;164(3):202-15.

Recent developments in avian influenza research: epidemiology and
immunoprophylaxis.

Tollis M, Di Trani L.

Istituto Superiore di Sanità, Laboratory of Veterinary Medicine, Vle Regina Elena
299, 00161 Rome, Italy. mtollis.iss.iy

Comment in
    Vet J. 2002 Nov;164(3):173-5.

Influenza A viruses have been isolated from humans, from several other mammalian 
species and a wide variety of avian species, among which, wild aquatic birds
represent the natural hosts of influenza viruses. The majority of the possible
combinations of the 15 haemagglutinin (HA) and nine neuraminidase (NA) subtypes
recognized have been identified in isolates from domestic and wild birds.
Infection of birds can cause a wide range of clinical signs, which may vary
according to the host, the virus strain, the host's immune status, the presence
of any secondary exacerbating microorganisms and environmental factors. Most
infections are inapparent, especially in waterfowl and other wild birds. In
contrast, infections caused by viruses of H5 and H7 subtypes can be responsible
for devastating epidemics in poultry. Despite the warnings to the poultry
industry about these viruses, in 1997 an avian H5N1 influenza virus was directly 
transmitted from birds to humans in Hong Kong and resulted in 18 confirmed
infections, thus strengthening the pandemic threat posed by avian influenza (AI).
Indeed, reassortant viruses, harbouring a combination of avian and human viral
genomes, have been responsible for major pandemics of human influenza. These
considerations warrant the need to continue and broaden efforts in the
surveillance of AI. Control programmes have varied from no intervention, as in
the case of the occurrence of low pathogenic (LP) AI (LPAI) viruses, to extreme, 
expensive total quarantine-slaughter programmes carried out to eradicate highly
pathogenic (HP) AI (HPAI) viruses. The adoption of a vaccination policy, targeted
either to control or to prevent infection in poultry, is generally banned or
discouraged. Nevertheless, the need to boost eradication efforts in order to
limit further spread of infection and avoid heavy economic losses, and advances
in modern vaccine technologies, have prompted a re-evaluation of the potential
use of vaccination in poultry as an additional tool in comprehensive disease
control strategies. This review presents a synthesis of the most recent research 
on AI that has contributed to a better understanding of the ecology of the virus 
and to the development of safe and efficacious vaccines for poultry.

Copyright 2002 Elsevier Science Ltd.

PMID: 12505393  [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

37729. Virology. 2002 Mar 30;295(1):44-53.

Reassortment and interspecies transmission of North American H6N2 influenza
viruses.

Webby RJ, Woolcock PR, Krauss SL, Webster RG.

Division of Virology, St. Jude Children's Research Hospital, Memphis, Tennessee
38105, USA.

H6N2 influenza viruses were isolated from California chickens in 2000 and 2001.
Here we report the characterization of these H6N2 viruses, one of the few
descriptions of non-H5, non-H7 subtype influenza viruses in this host. The H6N2
viruses were nonpathogenic in experimentally infected chickens and could be
divided into three genotypes. All three genotypes of virus had similar surface
glycoproteins and all contained an 18 amino acid deletion in the neuraminidase, a
characteristic of other chicken influenza viruses. Differences were apparent,
however, in the complement of replicative protein genes between the genotypes.
The presence of multiple H6N2 genotypes suggests that independent transmission
and/or reassortment events may have taken place between aquatic bird and chicken 
influenza viruses.

PMID: 12033764  [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

40087. J Virol. 2000 Sep;74(17):8018-27.

Targeted infection of endothelial cells by avian influenza virus A/FPV/Rostock/34
(H7N1) in chicken embryos.

Feldmann A, Schäfer MK, Garten W, Klenk HD.

Institut für Virologie, Philipps-Universität, Marburg, Germany.

The tissue tropism and spread of infection of the highly pathogenic avian
influenza virus A/FPV/Rostock/34 (H7N1) (FPV) were analyzed in 11-day-old chicken
embryos. As shown by in situ hybridization, the virus caused generalized
infection that was strictly confined to endothelial cells in all organs. Studies 
with reassortants of FPV and the apathogenic avian strain A/chick/Germany/N/49
(H10N7) revealed that endotheliotropism was linked to FPV hemagglutinin (HA). To 
further analyze the factors determining endotheliotropism, the HA-activating
protease furin was cloned from chicken tissue. Ubiquitous expression of furin and
other proprotein convertases in the chick embryo indicated that proteolytic
activation of HA was not responsible for restriction of infection to the
endothelium. To determine the expression of virus receptors in embryonic tissues,
histochemical analysis of alpha2,3- and alpha2,6-linked neuraminic acid was
carried out by lectin-binding assays. These receptors were found on endothelial
cells and on several epithelial cells, but not on tissues surrounding endothelia.
Finally, we analyzed the polarity of virus maturation in endothelial cells.
Studies on cultured human endothelial cells employing confocal laser scanning
microscopy revealed that HA is specifically targeted to the apical surface of
these cells, and electron microscopy of embryonic tissues showed that virus
maturation occurs also at the luminar side. Taken together, these observations
indicate that endotheliotropism of FPV in the chicken embryo is determined, on
one hand, by the high cleavability of HA, which mediates virus entry into the
vascular system, and, on the other hand, by restricted receptor expression and
polar budding, which prevent spread of infection into tissues surrounding
endothelia.

PMCID: PMC112334
PMID: 10933711  [PubMed - indexed for MEDLINE]
Þ

40257. J Virol. 2000 Jul;74(14):6316-23.

Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of
influenza virus growth: a study by reverse genetics.

Wagner R, Wolff T, Herwig A, Pleschka S, Klenk HD.

Institut für Virologie, Philipps-Universität, 35011 Marburg, Germany.

The hemagglutinin (HA) of fowl plague virus A/FPV/Rostock/34 (H7N1) carries two
N-linked oligosaccharides attached to Asn123 and Asn149 in close vicinity to the 
receptor-binding pocket. In previous studies in which HA mutants lacking either
one (mutants G1 and G2) or both (mutant G1,2) glycosylation sites had been
expressed from a simian virus 40 vector, we showed that these glycans regulate
receptor binding affinity (M. Ohuchi, R. Ohuchi, A. Feldmann, and H. D. Klenk, J.
Virol. 71:8377-8384, 1997). We have now investigated the effect of these
mutations on virus growth using recombinant viruses generated by an RNA
polymerase I-based reverse genetics system. Two reassortants of influenza virus
strain A/WSN/33 were used as helper viruses to obtain two series of HA mutant
viruses differing only in the neuraminidase (NA). Studies using N1 NA viruses
revealed that loss of the oligosaccharide from Asn149 (mutant G2) or loss of both
oligosaccharides (mutant G1,2) has a pronounced effect on virus growth in MDCK
cells. Growth of virus lacking both oligosaccharides from infected cells was
retarded, and virus yields in the medium were decreased about 20-fold. Likewise, 
there was a reduction in plaque size that was distinct with G1,2 and less
pronounced with G2. These effects could be attributed to a highly impaired
release of mutant progeny viruses from host cells. In contrast, with recombinant 
viruses containing N2 NA, these restrictions were much less apparent. N1
recombinants showed lower neuraminidase activity than N2 recombinants, indicating
that N2 NA is able to partly overrule the high-affinity binding of mutant HA to
the receptor. These results demonstrate that N-glycans flanking the
receptor-binding site of the HA molecule are potent regulators of influenza virus
growth, with the glycan at Asn149 being dominant and that at Asn123 being less
effective. In addition, we show here that HA and NA activities need to be highly 
balanced in order to allow productive influenza virus infection.

PMCID: PMC112137
PMID: 10864641  [PubMed - indexed for MEDLINE]
Þ

41695. J Virol. 1999 May;73(5):3567-73.

Phylogenetic analysis of H7 avian influenza viruses isolated from the live bird
markets of the Northeast United States.

Suarez DL, Garcia M, Latimer J, Senne D, Perdue M.

Southeast Poultry Research Laboratory, Agricultural Research Service, U.S.
Department of Agriculture, Athens, Georgia 30605, USA. dsuarez@asrr.arsusda.gov

The presence of low-pathogenic H7 avian influenza virus (AIV), which is
associated with live-bird markets (LBM) in the Northeast United States, was first
detected in 1994 and, despite efforts to eradicate the virus, surveillance of
these markets has resulted in numerous isolations of H7 AIVs from several states 
from 1994 through 1998. The hemagglutinin, nonstructural, and matrix genes from
representative H7 isolates from the LBM and elsewhere were sequenced, and the
sequences were compared phylogenetically. The hemagglutinin gene of most LBM
isolates examined appeared to have been the result of a single introduction of
the hemagglutinin gene. Evidence for evolutionary changes were observed with
three definable steps. The first isolate from 1994 had the amino acid threonine
at the -2 position of the hemagglutinin cleavage site, which is the most commonly
observed amino acid at this site for North American H7 AIVs. In January 1995 a
new genotype with a proline at the -2 position was detected, and this genotype
eventually became the predominant virus isolate. A third viral genotype, detected
in November 1996, had an eight-amino-acid deletion within the putative receptor
binding site. This viral genotype appeared to be the predominant isolate,
although isolates with proline at the -2 position without the deletion were still
observed in viruses from the last sampling date. Evidence for reassortment of
multiple viral genes was evident. The combination of possible adaptive evolution 
of the virus and reassortment with different influenza virus genes makes it
difficult to determine the risk of pathogenesis of this group of H7 AIVs.

PMCID: PMC104129
PMID: 10196246  [PubMed - indexed for MEDLINE]
Þ

42301. Virus Res. 1998 Jun;55(2):143-56.

Influence of host species on the evolution of the nonstructural (NS) gene of
influenza A viruses.

Kawaoka Y, Gorman OT, Ito T, Wells K, Donis RO, Castrucci MR, Donatelli I,
Webster RG.

Department of Virology/Molecular Biology, St. Jude Children's Research Hospital, 
Memphis, TN 38105, USA. kawaokay@svm.vetmed.wisc.edu

The matrix (M) and nonstructural (NS) genes of influenza A viruses each encode
two overlapping proteins. In the M gene, evolution of one protein affects that of
the other. To determine whether or not this evolutionary influence operating
between the two M proteins also occurs in the NS gene, we sequenced the NS genes 
of 36 influenza A viruses isolated from a broad spectrum of animal species (wild 
and domestic birds, horses, pigs, humans, and sea mammals) and analyzed them
phylogenetically, together with other previously published sequences. These
analyses enabled us to conclude the following host species-related points that
are not found in the other influenza A virus genes and their gene products. (1)
The evolution of the two overlapping proteins encoded by the NS gene are
lineage-dependent, unlike the M gene where evolutionary constraints on the Ml
protein affect the evolution of the M2 protein (Ito et al.. J. Virol. 65 (1991)
5491 5498). (2) The gull-specific lineage contained nonH13 gull viruses and the
non-gull avian lineage contained H13 gull viruses, indicating that the
gull-specific lineage does not link to the H13 HA subtype in the NS gene unlike
findings with other genes. (3) The branching topology of the recent equine
lineage (H7N7 viruses isolated after 1973 and H3N8) indicates recent introduction
of the NS, M, and PB2 genes into horses from avian sources by genetic
reassortment.

PMID: 9725667  [PubMed - indexed for MEDLINE]
Þ

42642. Vopr Virusol. 1998 Jan-Feb;43(1):24-9.

[Role of certain factors in intracellular oligomerization of influenza virus
nucleoproteins].

[Article in Russian]

Semenova NP, Chumakov VM, Rudneva IA, Prokudina EN.

Oligomerization of influenza virus nucleoprotein (NP) depends on the virus
strain. NP monomers of viruses A/Duck/Ukraine/63 (H3N8) and
A/Seal/Massachusets/1/80/ (H7N7) are oligomerized completely. The A/USSR/90/77
virus (H1N1) NP is characterized by just partial oligomerization, similarly as a 
reassortant containing surface protein genes of virus A/Duck/Ukraine and internal
protein genes of A/USSR/90 virus. Hence, it is probable that NP gene controls the
type of NP oligomerization. NP oligomerization is shown to depend on the
temperature, the optimal t = 37 degrees C, but not on the type of cells or
intracellular concentration of total NP. NP oligomers forming in vivo are
believed to differ from NP oligomers formed in vitro described previously.

PMID: 9559532  [PubMed - indexed for MEDLINE]
Þ

43204. J Infect Dis. 1997 Aug;176 Suppl 1:S14-9.

Predictions for future human influenza pandemics.

Webster RG.

Department of Virology/Molecular Biology, St. Jude Children's Research Hospital, 
Memphis, Tennessee 38101, USA.

Will there be another human influenza pandemic? The certainty is that there will 
be, and the probability is that the virus will emerge from Eurasian aquatic bird 
reservoirs and involve reassortment between a human and avian strain, with
accumulation of mutations or true recombinational events (or both) that will
permit spread and pathogenicity among humans. This process will probably occur in
pigs because they possess receptors for both avian and human influenza viruses,
and emergence may occur in southern China. Prediction of the subtype is
impossible, but there is a hypothesis based on seroarcheology that only H1, H2,
and H3 subtypes can infect humans; however, it is arguable that H7 or H2 strains 
might be equally capable of infecting humans.

PMID: 9240688  [PubMed - indexed for MEDLINE]
Þ

43667. Arch Virol Suppl. 1997;13:105-13.

Influenza virus: transmission between species and relevance to emergence of the
next human pandemic.

Webster RG.

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

Although influenza viruses are not spread from human to human through the
conventional food chain, this is not necessarily the case for the transmission of
the precursors of the human pandemic influenza viruses. Aquatic birds of the
world are the reservoirs for all influenza A viruses; the virus is spread by
fecal-oral transmission in untreated water. Influenza A viruses are frequently
transmitted to domestic poultry and two of the 15 subtypes H5 and H7 can become
highly pathogenic and have the capacity to decimate commercial poultry flocks.
Less frequently, avian influenza viruses are transmitted between species-to pigs,
horses and sea mammals. This transmission involves mutational, reassortant or
recombinational events and can occur through fecal contamination of unprocessed
avian protein or through the water. The transmission of avian influenza viruses
or virus genes to humans is postulated to occur through pigs that act as the
intermediate host. This involves either multiple mutational or reassortant events
and is believed to occur by airborne transmission. Once avian influenza viruses
are established in mammals, they are transmitted from animal to animal by the
respiratory airborne route. The transmission of avian influenza virus from their 
reservoir in wild aquatic birds to domestic poultry and to mammalian species
including humans can be prevented by treatment of the water supply and of avian
protein sources with disinfectants or by heating. Agricultural authorities have
recommended the separation of wild aquatic and domestic poultry and of pig and
poultry farming. It is theoretically possible to reduce the possibility of the
next pandemic of influenza in humans by changes in agricultural practices so that
ducks are separated from pigs and people.

PMID: 9413531  [PubMed - indexed for MEDLINE]
Þ

44595. Arch Virol. 1996;141(3-4):649-61.

Influenza infection in humans and pigs in southeastern China.

Zhou N, He S, Zhang T, Zou W, Shu L, Sharp GB, Webster RG.

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

The three last pandemic strains of influenza A virus-Asian/57, Hong Kong/68 and
Russian/77-are believed to have originated in China. The strains responsible for 
the 1957 and 1968 human pandemics were reassortants incorporating both human and 
avian influenza viruses, which may have arisen in pigs. We therefore undertook a 
population-based study in the Nanchang region of Central China to establish the
prevalence, types and seasonal pattern of human influenza infection and to screen
serum samples from animals and humans for evidence of interspecies transmission
of influenza viruses. Two definite influenza seasons were demonstrated, one
extending from November to March and the other July to September. The profile of 
antibodies to commonly circulating human influenza viruses was no different in
Nanchang and neighboring rural communities than in Memphis, Tennessee, USA. In
particular, Chinese women who raised pigs in their homes were no more likely to
have been exposed to influenza virus than were subjects who seldom or never had
contact with pigs. However, we did obtain evidence using isolated H7 protein in
an enzyme-linked immunoabsorbent assay for infection of pig farmers by an avian
H7 influenza virus suggesting that influenza. A viruses may have been transmitted
directly from ducks to humans. The results of the serological survey also
indicated that pigs in or near Nanchang were infected by human H1N1 and H3N2
influenza viruses, but not with typical swine viruses. We found no serological
evidence for H2 influenza viruses in humans after 1968.

PMID: 8645101  [PubMed - indexed for MEDLINE]
Þ

45994. Virus Res. 1994 Jun;32(3):391-9.

A rapid method for the analysis of influenza virus genes: application to the
reassortment of equine influenza virus genes.

Adeyefa CA, Quayle K, McCauley JW.

AFRC Institute for Animal Health, Pirbright Laboratory, Woking, Surrey, UK.

We describe a rapid method for genetic characterisation of influenza virus genes 
using reverse transcription and amplification by polymerase chain reaction
(RT/PCR) of all virus segments simultaneously (multiplex RT/PCR) using primers
based on the conserved terminal sequences. The product has been shown to be
suitable for determination of partial nucleotide sequences which can be used to
search nucleotide sequence databases and rapidly map the genetic origin of each
segment. We illustrate the use of the method by analysing genetic reassortment in
H7N7 equine influenza viruses.

PMID: 7521550  [PubMed - indexed for MEDLINE]
Þ

46871. Vopr Virusol. 1993 May-Jun;38(3):122-6.

[The mechanisms of the antiviral action of the bora-adamantane derivative
preparation BG-12].

[Article in Russian]

Markushin SG, Sinitsyn BV, Ginzburg VP, KhaÄ­der AM, Klimov AI.

The effect of one of the derivatives of boraadamantane, preparation BG-12, on
reproduction of influenza type A and B viruses was studied. This preparation was 
shown to inhibit multiplication of a wide range of influenza type A and B virus
strains. It is important that BG-12 inhibits in cell culture the replication of a
mutant of fowl plague virus A/FPV/Weibridge resistant to remantadine. BG-12 was
found to exert a marked viricidal effect, to inhibit the hemolytic activity of
both influenza A and influenza B viruses, but not the neuraminidase activity of
these viruses. The synthesis of some virus-specific proteins was found to be
disturbed in influenza B/Ann Arbor/86-infected cells in the presence of BG-12. A 
FPVBAR mutant of influenza A/FPV/Weibridge (H7N7) virus resistant to BG-12 was
obtained. All the BG-12-resistant reassortants inherited genes 4 and 7 coding for
hemagglutinin (HA) and membrane proteins (M) from FPVBAR mutant, respectively,
and the remaining genes from BG-12-sensitive A/Krasnodar/101/59 (H2N2) virus. The
sequencing of a region of the M gene encoding for the transmembrane protein M2
revealed the substitution of Ala30-Thre in this protein. Multiple attempts at
generating a mutant of influenza type B virus resistant to BG-12 failed.

PMID: 8073750  [PubMed - indexed for MEDLINE]
Þ

48229. Virology. 1991 Sep;184(1):469-71.

Reassortants with equine 1 (H7N7) influenza virus hemagglutinin in an avian
influenza virus genetic background are pathogenic in chickens.

Banbura MW, Kawaoka Y, Thomas TL, Webster RG.

Department of Virology and Molecular Biology, St. Jude Children's Research
Hospital, Memphis, Tennessee 38101.

Reassortants possessing the hemagglutinin (HA) gene from A/Equine/London/1416/73 
(H7N7) [Eq/Lond] and five or more genes from A/Chicken/Pennsylvania/1370/83
(H5N2) [Ck/Penn] were lethal in chickens. This result demonstrates that horses
can maintain influenza viruses whose HAs are capable of promoting virulence.
Thus, reassortment of equine and avian influenza virus genes could generate
viruses that might be lethal in domestic poultry.

PMID: 1871981  [PubMed - indexed for MEDLINE]
Þ

48948. Virus Res. 1990 Nov;17(3):191-8.

The critical cut-off temperature of avian influenza viruses.

McCauley JW, Penn CR.

Division of Molecular Biology, AFRC Institute for Animal Health, Pirbright
Laboratory, Nr. Woking, Surrey, U.K.

We have measured the pathogenicity for 6-week-old chicks of infection by H7 avian
influenza viruses. One virus, strain S3 from A/FPV/Rostock/34(H7N1) showed a
temperature sensitive phenotype at 41.5 degrees C and reduced pathogenicity. By
analysis of reassortants made between virus S3 and A/FPV/Dobson/27(H7N7), a fully
pathogenic virus, two conclusions arise. (1) The critical cut-off temperature for
avian influenza virus in 6-week-old chicks is 41.5 degrees. (2) RNA segment 1 of 
virus S3 is responsible for the lack of pathogenicity in reassortant viruses.
Nucleotide sequencing of RNA segment 1 from S3 and its parent, A/FPV/Rostock/34
has revealed a single mutation at nucleotide 1561. This results in a substitution
of isoleucine for leucine at amino acid position 512 in the cap-binding protein, 
PB2.

PMID: 2077784  [PubMed - indexed for MEDLINE]
Þ

51225. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6869-73.

Antigen-presenting B cells and helper T cells cooperatively mediate intravirionic
antigenic competition between influenza A virus surface glycoproteins.

Johansson BE, Moran TM, Kilbourne ED.

Department of Microbiology, Mount Sinai School of Medicine, City University of
New York, NY 10029.

Parenteral vaccination of BALB/c mice primed by infection with H3N2 variants of
influenza A virus results in a reduced production of N2 antibody in response to
homologous (H3N2) vaccine compared with the response to an H7N2 vaccine equal in 
N2 immunologenicity. We now have studied the interaction in vitro of purified
splenic B and T lymphocytes from variably immunized mice to ascertain the
cellular basis of the hemagglutinin (HA)-influenced antibody response to
neuraminidase (NA). Assay of the proliferative response of T cells in B/T-cell
mixtures stimulated by H3N1 (HA-specific) and H6N2 (NA-specific) reassortant
(recombinant) viruses in vitro has enabled us to differentiate cellular responses
to HA and NA antigens. Using a factorial design in analysis of B/T-cell mixtures,
we have shown that: (i) intravirionic HA is dominant over NA in both B- and
T-cell priming; (ii) an increase in H3-specific B cells occurs in mice
administered boosters of H3N2 vaccine, and an increase in N2-specific B cells
occurs in those given a booster of H7N2 vaccine; and (iii) memory B cells
function as antigen-presenting cells and interact with memory helper T cells in
the mediation of intravirionic HA-NA antigenic competition in favor of HA. The
damping of response to the NA antigen in favor of HA with reinfection prohibits
balanced immunologic response to the two antigens. The present studies define
further the complex immunology of influenza virus infection.

PMCID: PMC299186
PMID: 2958849  [PubMed - indexed for MEDLINE]
Þ

51474. J Immunol. 1987 May 1;138(9):3010-3.

Immunologic response to the influenza virus neuraminidase is influenced by prior 
experience with the associated viral hemagglutinin. I. Studies in human
vaccinees.

Kilbourne ED, Cerini CP, Khan MW, Mitchell JW Jr, Ogra PL.

Analysis of an earlier study of H3N2 and H7N2 inactivated influenza vaccines in
schoolchildren demonstrated a greater viral neuraminidase (NA) immunogenicity of 
the vaccine containing the H7 hemagglutinin (HA) antigen to which they had not
been primed, despite the lesser NA antigen content of that vaccine. Thus, prior
experience with the influenza viral HA appeared to have a negative influence on
immune response to NA, the associated external glycoprotein, presumably on the
basis of intermolecular antigenic competition. In a second study, sequential
immunologic response to influenza viral NA was compared in college students who
were immunized with either conventional commercial vaccine or an antigenic
reassortant H7N1 vaccine, and who subsequently experienced natural infection with
an H1N1 influenza virus. Although both vaccines were only marginally immunogenic 
in inducing NA antibody response in seronegative subjects, in vaccinees initially
seropositive for HA antibody significant NA antibody titer increases occurred
with H7N1 vaccine. Subsequent natural infection boosted NA antibody less
effectively in the population previously primed by natural infection than in
initially seronegative subjects primed by H7N1 vaccination. It is suggested that 
primary immunization monospecific for influenza viral NA may alter the subsequent
pattern of immune response to one more favorable to the induction of NA antibody 
when virus is encountered.

PMID: 3571981  [PubMed - indexed for MEDLINE]
Þ

52092. Virus Res. 1986 Jul;5(1):27-42.

Studies on the temperature sensitivity of influenza A virus reassortants
nonpathogenic for chicken.

Giesendorf B, Bosch FX, Orlich M, Scholtissek C, Rott R.

Influenza A virus reassortants which are nonpathogenic for chickens are like
mammalian influenza A viruses in that they are temperature sensitive for growth
at 41 degrees C. We have investigated the mechanism of this temperature
sensitivity using reassortants between the two highly pathogenic strains
A/FPV/Rostock/34 (FPV, H7N1) and A/turkey/England/63 (TE, H7N3). These
reassortants show a strict correlation between the pathogenicity for chickens and
the constellation of the genes coding for the ribonucleoprotein complex, RNP.
Evidence is presented which shows that all viral components are synthesized in
sufficient amounts and that the block in the viral replication cycle at the
nonpermissive temperature is a late one affecting virus maturation. It is
suggested that the RNP, although still enzymatically functional, may lose its
ability to interact normally with viral surface components, thus interfering with
the process of virus maturation. Some of the nonpathogenic reassortants which
possessed the neuraminidase of TE showed an interesting temperature-dependent
phenomenon: the haemagglutinin synthesized at the elevated temperature could only
agglutinate erythrocytes at 20 degrees C, when the neuraminidase was inhibited or
the infected cells vigorously disrupted by ultrasonication. This phenomenon is
possibly not directly related to the temperature-sensitive block.

PMID: 3751286  [PubMed - indexed for MEDLINE]
Þ

52238. Virology. 1986 Apr 15;150(1):65-74.

A reassortant between influenza A viruses (H7N2) synthesizing an enzymatically
inactive neuraminidase at 40 degrees which is not incorporated into infectious
particles.

Breuning A, Scholtissek C.

Cells infected with a reassortant (113/Ho, H7N2) between A/fowl plague/Rostock/34
(FPV, H7N1) and A/Hong Kong/1/68 (H3N2) carrying RNA segments 1 and 6 of the Hong
Kong virus and the residual genes of FPV, synthesized at 40 degrees a
neuraminidase (NA) which is enzymatically not active and which is not
incorporated into infectious particles. At 40 degrees NA accumulates in the rough
endoplasmic reticulum. It contains mainly carbohydrate side chains of the mannose
type, and fucose is only scarcely incorporated. At 33 degrees NA of the
reassortant is overproduced, and at least some of it is active and is
incorporated into viral particles. Under nonreducing conditions during PAGE its
NA migrates to the same position as after heating with mercaptoethanol, in
contrast to the Hong Kong parent virus. It is speculated that at 40 degrees the
tetramerization of the NA in the rough endoplasmic reticulum does not function,
and in this way its migration to the cytoplasmic membrane and its incorporation
into infectious particles does not occur. Since 113/Ho is as pathogenic for the
chicken (body temperature of 41 degrees) as is FPV, the question arises which
role the NA plays in virus replication and spread in the infected organism.

PMID: 3952990  [PubMed - indexed for MEDLINE]
Þ

52303. Virology. 1986 Mar;149(2):165-73.

Molecular changes in A/Chicken/Pennsylvania/83 (H5N2) influenza virus associated 
with acquisition of virulence.

Webster RG, Kawaoka Y, Bean WJ Jr.

One of the unresolved questions concerning the acquisition of virulence by the
A/Chicken/Pennsylvania/83 (H5N2) influenza virus is which gene segments other
than the hemagglutinin (HA) showed changes that were relevant. To answer this
question, reassortants were made possessing the hemagglutinin gene of the
virulent virus and the seven other genes from the avirulent parent. Since both
the virulent and avirulent H5N2 strains are antigenically almost
indistinguishable, it was necessary to transfer the genes of interest to a
"carrier" virus before the appropriate reassortment could be selected. The gene
compositions of the reassortants was established by a combination of sequence
analysis and migration on polyacrylamide gels. These analyses established that
the avirulent influenza virus present in April 1983 possessed seven of the eight 
gene segments necessary for virulence; mutation(s) in the HA gene were required
for acquisition of virulence. Other viruses such as A/Seal/Mass/1/80 (H7N7) could
provide the other genes necessary for virulence. Two changes in the HA have been 
associated with the acquisition of virulence; these are at amino acid residues 23
and 78 (H3 numbering) (Y. Kawaoka and R.G. Webster, Virology, 146, 130-137
(1985]. Isolation of an amantadine-resistant avirulent revertant virus provided
the opportunity to determine which of the two amino acid changes in HA is
critical. Sequence analysis of the revertant virus revealed amino acid changes at
residues 23 in HA1 and 40 in HA2 (H3 numbering). The change at residue 23 of HA1 
is probably associated with reversion to avirulence, of cleavability of HA, and
inability to plaque in tissue culture without trypsin; while the change at
residue 40 of HA2 may be associated with the amantadine-resistant phenotype.
These studies establish that a single critical point mutation in the
hemagglutinin gene of the avirulent A/Chicken/Pennsylvania/1/83 (H5N2) was
probably all that was required to produce the highly virulent
Chicken/Pennsylvania virus; the avirulent virus already possessed the other genes
necessary for virulence.

PMID: 3946082  [PubMed - indexed for MEDLINE]
Þ

52842. Virus Res. 1985 Jul;3(1):35-40.

Sequence of the nucleoprotein gene of influenza A/parrot/Ulster/73.

Steuler H, Schröder B, Bürger H, Scholtissek C.

The nucleotide sequence of the nucleoprotein (NP) gene of the avian influenza A
virus strain A/parrot/Ulster/73 (H7N1) has been determined. The gene (RNA segment
5) consists of 1565 bases. The only large open reading frame of the complementary
RNA codes for a protein of 498 amino acids. A comparison of its sequence with
that of three other influenza virus NPs shows that the NP of the parrot Ulster
strain, although closely related to the NP of the other avian strain
(A/FPV/Rostock/34), is definitely more closely related genetically to the NPs of 
the two human influenza strains, A/PR/8/34 and A/NT/60/68 than that of FPV. This 
raises the question how far the NP gene can cross the species barrier by
reassortment and become adapted by mutation to the new host.

PMID: 4024728  [PubMed - indexed for MEDLINE]
Þ

53193. Arch Virol. 1985;85(1-2):1-11.

Stability of infectious influenza A viruses to treatment at low pH and heating.

Scholtissek C.

We have measured the infectivity of influenza A virus strains grown either in
embryonated eggs or in chick embryo cells in culture after treatment at low pH.
At pH values at which hemolysis occurs there was an irreversible loss of
infectivity. The threshold pH, at which the infectivity was lost, depended on the
hemagglutinin subtype of the virus strain. All H5 and H7 strains tested were
extremely labile at low pH. In contrast, all H3 strains were relatively stable,
independent of the species from which the viruses were isolated. With several H1 
viruses the hemagglutination (HA) activity was irreversibly lost at intermediate 
pH values causing inactivation of infectivity. Strains with noncleaved
hemagglutinins were much more stable. These observations might explain why duck
influenza viruses can easily survive in lake water and wet faeces, and multiply
in the intestinal tract, where trypsin is present. There are also significant
differences in heat stability exhibited by influenza A strains. In contrast to pH
stability this is not a specific trait of the hemagglutinin, since it can be
influenced by reassortment. There is no correlation between the stability of
infectivity at low pH and heat.

PMID: 4015405  [PubMed - indexed for MEDLINE]
Þ

54945. Infect Immun. 1982 Sep;37(3):1119-26.

Virulence of avian influenza A viruses for squirrel monkeys.

Murphy BR, Hinshaw VS, Sly DL, London WT, Hosier NT, Wood FT, Webster RG, Chanock
RM.

Ten serologically distinct avian influenza A viruses were administered to
squirrel monkeys and hamsters to compare their replication and virulence with
those of human influenza A virus, A/Udorn/307/72 (H3N2). In squirrel monkeys, the
10 avian influenza A viruses exhibited a spectrum of replication and virulence.
The levels of virus replication and clinical response were closely correlated.
Two viruses, A/Mallard/NY/6874/78 (H3N2) and A/Pintail/Alb/121/79 (H7N8),
resembled the human virus in their level and duration of replication and in their
virulence. At the other end of the spectrum, five avian viruses were restricted
by 100- to 10,000-fold in replication in the upper and lower respiratory tract
and were clearly attenuated compared with the human influenza virus. In hamsters,
the 10 viruses exhibited a spectrum of replication in the nasal turbinates,
ranging from viruses that replicated as efficiently as the human virus to those
that were 8,000- fold restricted. Since several avian viruses were closely
related serologically to human influenza viruses, studies were done to confirm
the avian nature of these isolates. Each of the avian viruses plaqued efficiently
at 42 degrees C, a restrictive temperature for replication of human influenza A
viruses. Avian strains that had replicated either very efficiently or very poorly
in squirrel monkeys still grew to high titer in the intestinal tracts of ducks, a
tropism characteristic of avian, but not mammalian, influenza viruses. These
observations indicate that some avian influenza A viruses grow well and cause
disease in a primate host, whereas other avian viruses are very restricted in
this host. These findings also provide a basis for determining the gene or genes 
involved in the restriction of replication that is observed with the attenuated
avian viruses. Application of such information may allow the preparation of
reassortant viruses derived from a virulent human influenza virus and an
attenuated avian virus for possible use in a live attenuated vaccine for
prevention of influenza in humans.

PMCID: PMC347656
PMID: 7129631  [PubMed - indexed for MEDLINE]
Þ