Lack of transmission of H5N1 avian-human...

sharon sanders · Aug 2, 2006

Lack of transmission of H5N1 avian?human<?xml:namespace prefix = o ns = "urn:schemas-microsoft-comfficeffice" /><o></o>
reassortant influenza viruses in a ferret model<o></o>
Taronna R. Maines*, Li-Mei Chen*, Yumiko Matsuoka*, Hualan Chen*?, Thomas Rowe*?, Juan Ortin?, Ana Falco? n??,<o></o>
Nguyen Tran Hien_, Le Quynh Mai_, Endang R. Sedyaningsih**, Syahrial Harun**, Terrence M. Tumpey*,<o></o>
Ruben O. Donis*, Nancy J. Cox*, Kanta Subbarao*??, and Jacqueline M. Katz*??<o></o>
*Influenza Branch, Division of Viral and Rickettsial Diseases, <?xml:namespace prefix = st1 ns = "urn:schemas-microsoft-com

ffice:smarttags" /><st1

lace><st1

laceName>National</st1

laceName> <st1

laceType>Center</st1

laceType></st1

lace> for Infectious Diseases, Centers for Disease Control and Prevention,<o

></o

>
Atlanta, GA 30333; ?Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientı?ficas, 28049 Madrid, Spain; _National Institute<o

></o

>
of Hygiene and Epidemiology, <st1

lace><st1:City>Hanoi</st1:City>, <st1:country-region>Vietnam</st1:country-region></st1

lace>; and **Center for Biomedical and Pharmaceutical Research and Development, Ministry of Health,<o

></o

>
<st1

lace><st1:City>Jakarta</st1:City> <st1

ostalCode>10560</st1

ostalCode>, <st1:country-region>Indonesia</st1:country-region></st1

lace><o

></o

>
Communicated by Peter Palese, Mount Sinai School of Medicine, New York, NY, <st1:date Year="2006" Day="23" Month="6">June 23, 2006</st1:date> (received for review <st1:date Year="2006" Day="23" Month="5">May 23, 2006</st1:date>)<o

></o

>
Avian influenza A H5N1 viruses continue to spread globally among<o></o>
birds, resulting in occasional transmission of virus from infected<o></o>
poultry to humans. Probable human-to-human transmission has<o></o>
been documented rarely, but H5N1 viruses have not yet acquired<o></o>
the ability to transmit efficiently among humans, an essential<o></o>
property of a pandemic virus. The pandemics of 1957 and 1968<o></o>
were caused by avian?human reassortant influenza viruses that<o></o>
had acquired human virus-like receptor binding properties. However,<o></o>
the relative contribution of human internal protein genes or<o></o>
other molecular changes to the efficient transmission of influenza<o></o>
viruses among humans remains poorly understood. Here, we<o></o>
report on a comparative ferret model that parallels the efficient<o></o>
transmission of H3N2 human viruses and the poor transmission of<o></o>
H5N1 avian viruses in humans. In this model, an H3N2 reassortant<o></o>
virus with avian virus internal protein genes exhibited efficient<o></o>
replication but inefficient transmission, whereas H5N1 reassortant<o></o>
viruses with four or six human virus internal protein genes exhibited<o></o>
reduced replication and no transmission. These findings indicate<o></o>
that the human virus H3N2 surface protein genes alone did not<o></o>
confer efficient transmissibility and that acquisition of human virus<o></o>
internal protein genes alone was insufficient for this 1997 H5N1<o></o>
virus to develop pandemic capabilities, even after serial passages<o></o>
in a mammalian host. These results highlight the complexity of the<o></o>
genetic basis of influenza virus transmissibility and suggest that<o></o>
H5N1 viruses may require further adaptation to acquire this essential<o></o>
pandemic trait.<o></o>
transmissibility _ pandemic virus properties _ pandemic influenza _<o

></o

>
animal model _ receptor specificity<o

></o

>
Highly pathogenic avian influenza (HPAI) H5N1 viruses are<o

></o

>
now enzootic in several countries and are presently undergoing<o

></o

>
unprecedented geographic expansion among wild and<o

></o

>
domestic birds. Since 1997, when HPAI H5N1 viruses first<o

></o

>
emerged in <st1

lace>Hong Kong</st1

lace> to cause human respiratory illness and<o

></o

>
death, _200 laboratory-confirmed human infections have been<o

></o

>
reported, primarily as a result of transmission of the H5N1 virus<o

></o

>
from domestic poultry to humans (1?4). Despite limited instances<o

></o

>
of probable human-to-human transmission (5, 6), H5N1<o

></o

>
viruses have not yet acquired the ability to transmit efficiently<o

></o

>
among humans. The viral properties that confer transmissibility<o

></o

>
of influenza viruses among humans, and mammalian species in<o

></o

>
general, remains poorly understood, yet are clearly key factors<o

></o

>
that determine whether a novel subtype introduced into an<o

></o

>
immunologically naı?ve human population will result in a pandemic.<o

></o

>
The continuing occurrence of human H5N1 infections<o

></o

>
underscores the ongoing public health threat and the urgent<o

></o

>
need to better understand the potential of H5N1 viruses to<o

></o

>
acquire properties that would confer efficient transmissibility<o

></o

>
among humans.<o

></o

>
Pandemic viruses of the 20th century arose in two ways. The<o

></o

>
1918 H1N1 virus likely derived all eight genes from an avian<o

></o

>
virus and accumulated mutations during adaptation in a mammalian<o

></o

>
host (7, 8). In contrast, the H2N2 and H3N2 viruses that<o

></o

>
caused pandemics in 1957 and 1968, respectively, were the result<o

></o

>
of reassortment between avian and human influenza A viruses,<o

></o

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acquiring the neuraminidase (NA) and_or hemagglutinin (HA),<o

></o

>
and PB1 gene from an avian virus and other genes from the<o

></o

>
previously circulating human strain (9). However, the earliest<o

></o

>
human H2 and H3 isolates differed from their avian counterparts<o

></o

>
in key receptor-binding residues that resulted in preferred<o

></o

>
binding of the human virusHA to sialic acid (SA) _2,6 receptors,<o

></o

>
suggesting that a shift from avian virus SA _2,3 to human virus<o

></o

>
SA _2,6 receptor-binding specificity is critical for efficient<o

></o

>
replication and spread of a pandemic strain (10). H5N1 viruses<o

></o

>
isolated from humans to date possess all eight gene segments<o

></o

>
that are entirely avian in origin, and with only a few exceptions,<o

></o

>
have retained a receptor-binding site sequence in the HA that is<o

></o

>
typical of the avian virus preference for binding SA _2,3 (11?13).<o

></o

>
Therefore, despite considerable genetic diversity in H5N1 viruses<o

></o

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isolated from humans since 1997, no consistent adaptation<o

></o

>
to the human host has been identified (14).<o

></o

>
Person-to-person transmission of human influenza viruses<o

></o

>
occurs either through contact (direct or indirect) or respiratory<o

></o

>
droplet (droplet or droplet nuclei) transmission (15). Although<o

></o

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the exact contribution of each of these modes of transmission is<o

></o

>
not known, transmission of respiratory droplets, expelled when<o

></o

>
infected persons cough or sneeze, is likely the major mode of<o

></o

>
sustained viral spread in community settings during epidemics<o

></o

>
and pandemics (15). Several animal models have been used to<o

></o

>
investigate factors that contribute to influenza virus transmissibility<o

></o

>
(16?19). Ferrets, like humans, have a predominance of _2,6<o

></o

>
SA and a lesser amount of _2,3 SA on respiratory tract epithelial<o

></o

>
cells, exhibit preferential binding of H5N1 viruses in the lower<o

></o

>
respiratory tract similar to humans (20?24), and have been used<o

></o

>
previously to investigate transmissibility of human influenza<o

></o

>
viruses (17?19, 25). Here, we conduct both contact and respi-<o

></o

>
ratory droplet transmission studies in ferrets to establish a model<o

></o

>
that reflects the relative transmissibility of H3N2 and H5N1<o

></o

>
viruses in humans. We use this model to evaluate the transmissibility<o

></o

>
of various avian H5N1 and human H3N2 reassortant<o

></o

>
viruses in ferrets to assess the pandemic potential of such viruses.<o

></o

>
All H5N1 avian?human reassortant viruses generated exhibited<o

></o

>
reduced replication efficiency compared with the parental H5N1<o

></o

>
virus and were not capable of efficient respiratory droplet<o

></o

>
transmission between ferrets.<o

></o

>
Results<o></o>
Transmissibility of H3N2 Human Influenza Viruses. To establish our<o

></o

>
ferret transmission model, we first evaluated the ability of two<o

></o

>
human H3N2 influenza viruses, A_Panama_2007_99 (Pan99)<o

></o

>
and A_Victoria_3_75 (Vic75), to undergo efficient respiratory<o

></o

>
droplet transmission by housing ferrets in adjacent cages, each<o

></o

>
with a perforated side wall that prevented direct contact but<o

></o

>
allowed spread of virus through the air. Three ferrets were<o

></o

>
inoculated intranasally (i.n.) with 104 50% ferret infectious dose<o

></o

>
(FID50) of Pan99 [105.2 50% egg infectious dose (EID50)] or<o

></o

>
Vic75 (104.5 EID50), and 24 h later three naive ferrets were each<o

></o

>
placed in a transmission cage adjacent to an inoculated ferret.<o

></o

>
Inoculated ferrets achieved peak mean titers in nasal washes of<o

></o

>
6.9 _ 1.5 or 7.8 _ 1.1 log10 EID50_ml, respectively on day 1<o

></o

>
postinoculation (p.i.) (Fig. 1 A and B); and exhibited modest<o

></o

>
signs of illness, including sneezing beginning on day 2 p.i. (Table<o

></o

>
1). Both viruses were efficiently transmitted to each of the three<o

></o

>
contact ferrets by day 3 or 5 postcontact (p.c.) as demonstrated<o

></o

>
by detection of virus in nasal secretions and seroconversion for<o

></o

>
hemagglutination inhibition (HI) antibody 14 days p.c. in all<o

></o

>
contact animals (Fig. 1 A and B and Table 1). These data indicate<o

></o

>
that both human H3N2 viruses were efficiently transmitted from<o

></o

>
inoculated to naive ferrets in a situation that did not allow for<o

></o

>
direct contact between animals or indirect contact with viruscontaminated<o

></o

>
food or surfaces. These results reflect the general<o

></o

>
transmissibility of human H3N2 influenza viruses in humans by<o

></o

>
respiratory droplets.<o

></o

>
Transmissibility of H5N1 Avian Influenza Viruses. We next evaluated<o

></o

>
the ability of avian H5N1 influenza viruses to undergo respiratory<o

></o

>
droplet transmission between ferrets by inoculating three<o

></o

>
ferrets i.n. with 104 FID50 (106 EID50) of A_Hong Kong_486_97<o

></o

>
(HK486), an H5N1 virus isolated from a human case patient in<o

></o

>
1997. Mean nasal wash virus titers of the HK486-infected ferrets<o

></o

>
reached a maximal of 7.0 _ 1.1 log10 EID50_ml on day 5 p.i. (Fig.<o

></o

>
1C), and signs of severe illness were observed (Table 1). The<o

></o

>
mean maximum weight loss in HK486-infected ferrets was 16.5%<o

></o

>
on days 2?7 p.i.; sneezing was observed only in one of three<o

></o

>
ferrets on day 4 p.i. (Table 1), and one of the ferrets was<o

></o

>
humanely killed on day 7 p.i. because of the onset of neurological<o

></o

>
symptoms. Virus was not detected in the nasal washes from any<o

></o

>
of the contact ferrets through day 9 p.c. (Fig. 1C). However,<o

></o

>
anti-HK486 HI antibody titers of 160 or 320 were detected in two<o

></o

>
of the three contact ferrets 17 days p.c., indicating that HK486<o

></o

>
virus was transmitted to two of the contact ferrets in the absence<o

></o

>
of detectable virus in nasal secretions (Table 1). To determine<o

></o

>
whether HK486 virus would transmit more efficiently between<o

></o

>
cohoused ferrets, we next inoculated two animals i.n. with the<o

></o

>
same dose of HK486, and 24 h later we placed a naive ferret in<o

></o

>
the same cage with each inoculated animal. A low level of virus<o

></o

>
(102.3 EID50_ml) was recovered on day 1 p.c. from one of the<o

></o

>
contact ferrets that had been cohoused with an inoculated<o

></o

>
animal that shed peak virus titers of 106.5 EID50_ml (Fig. 2A),<o

></o

>
although both contact ferrets seroconverted with titers of 320 or<o

></o

>
640 (data not shown). Two additional ferrets inoculated with a<o

></o

>
lower dose of HK486 virus (103 EID50) shed peak virus titers<o

></o

>
approximately three logs lower than the ferrets inoculated with<o

></o

>
the higher dose and failed to transmit virus to their cage mates,<o

></o

>
because the contact animals did not shed virus and did not<o

></o

>
seroconvert (data not shown).<o

></o

>
The HK486 virus is genetically distinct from H5N1 viruses<o

></o

>
isolated since 1997; therefore, we also evaluated a 2003 and two<o

></o

>
2005 H5N1 viruses for their ability to transmit between ferrets.<o

></o

>
A_Hong Kong_213_03 (HK213) contains a mutation at residue<o

></o

>
223 (H5 numbering; residue 227 by H3 numbering) within the<o

></o

>
receptor-binding domain of the HA and was shown to bind to<o

></o

>
both _2,6 and _2,3 SA in vitro (13). Therefore, three ferrets were<o

></o

>
inoculated i.n. with 106 EID50 of HK213, and the next day three<o

></o

>
naı?ve ferrets were placed in adjacent transmission cages. HK213-<o

></o

>
infected ferrets achieved mean virus titers of 5.5 _ 0.3 log10<o

></o

>
EID50_ml on days 1 and 3 p.i. (Fig. 1D) and exhibited some<o

></o

>
weight loss and lethargy but no sneezing (Table 1). Virus was not<o

></o

>
detected in the nasal washes from the contact ferrets nor was<o

></o

>
seroconversion detected by HI analysis of convalescent sera.<o

></o

>
A_Indonesia_5_05 (Indon05) and A_Vietnam_HN30408_05<o

></o

>
(VN30408) are representative of the two distinct genetic clades<o

></o

>
of H5N1 viruses (clades 2 and 1, respectively) (14) that circulated<o

></o

>
in <st1

lace>Asia</st1

lace> during 2004?2005 and were isolated from human cases<o

></o

>
that had been associated with family clusters of H5N1 illness (4).<o

></o

>
In a contact transmission experiment, ferrets infected with 106<o

></o

>
EID50 of Indon05 virus exhibited severe illness with substantial<o

></o

>
weight loss (18.8% mean maximum 7 days p.i.) and dyspnea but<o

></o

>
no sneezing; none of the ferrets survived past day 7 p.i. (Table<o

></o

>
1). Although high titers of infectious virus were detected in the<o

></o

>
upper respiratory tract of Indon05-inoculated ferrets, virus was<o

></o

>
not detected in any of the nasal washes from the contact ferrets<o

></o

>
through day 9 p.c. (Fig. 2B), and convalescent sera collected<o

></o

>
from the contact animals lacked anti-Indon05 HI antibodies<o

></o

>
(Table 1). Ferrets infected with the same dose of VN30408 virus<o

></o

>
exhibited less severe illness and also failed to transmit the virus<o

></o

>
to contact animals (Table 1 and Fig. 2C). Taken together, these<o

></o

>
data demonstrate the lack of efficient transmission of H5N1<o

></o

>
viruses in ferrets.<o

></o

>
Characterization of Avian?Human Reassortant Influenza Viruses. To<o

></o

>
evaluate the pandemic potential of H5N1 reassortant viruses and<o

></o

>
better understand the relative contribution of avian or human<o

></o

>
virus surface and internal protein genes to transmissibility,<o

></o

>
reassortant viruses containing various gene constellations from<o

></o

>
the avian H5N1 virus, HK486, and the human H3N2 virus,<o

></o

>
Vic75, were generated by using plasmid-based reverse genetics<o

></o

>
(rg). The parental viruses, containing all eight genes of either<o

></o

>
Vic75 (rgVic) or HK486 (rg486), were generated and their<o

></o

>
genetic identities were confirmed; the parental rg viruses exhibited<o

></o

>
virulence and transmissibility properties in ferrets similar to<o

></o

>
their WT counterparts (Table 2 and data not shown). To assess<o

></o

>
the contribution of the influenza virus internal protein genes, we<o

></o

>
first generated the reassortant virus, rgVic:486HANA, containing<o

></o

>
all six human virus internal protein genes and the avian virus<o

></o

>
H5 and N1 surface protein genes, and the reciprocal,<o

></o

>
rg486:VicHANA, containing avian virus internal protein genes<o

></o

>
and human H3 and N2 surface protein genes, and evaluated their<o

></o

>
infectivity for Madin-Darby canine kidney (MDCK) cells and<o

></o

>
eggs (Table 2). Although these two reassortant viruses had<o

></o

>
comparable infectivity titers in eggs, rgVic:486HANA achieved<o

></o

>
a titer that was 100-fold lower than that for the parental rg486<o

></o

>
in MDCK cells, whereas rg486:VicHANA replicated as efficiently<o

></o

>
as the parental rgVic. An additional H5N1 reassortant<o

></o

>
virus, rg486:VicRNP, which contained the human virus ribonucleoprotein<o

></o

>
(RNP) genes (PB2, PB1, PA, NP), was generated<o

></o

>
and found to replicate to a higher titer in MDCK cells than<o

></o

>
rgVic:486HANA, although the infectivity of the two reassortants<o

></o

>
in embryonated eggs was similar (Table 2). Reassortant viruses,<o

></o

>
rg486:VicHANA and rg486:VicRNP, also exhibited higher infectivity<o

></o

>
for ferrets than rgVic:486HANA virus, and therefore<o

></o

>
had lower FID50 values (1.5 and 1.5 compared with 3.0, respectively;<o

></o

>
expressed as the log10 EID50 required to give 1 FID50). For<o

></o

>
comparison, WT Vic75 had an FID50 value of 0.5, whereas<o

></o

>
HK486 had an FID50 of 2.0. The data suggest that the infectivity<o

></o

>
for ferrets and MDCK cells of reassortant viruses bearing the<o

></o

>
HK486 HA and NA genes may be enhanced by the presence of<o

></o

>
the HK486 M and NS gene. In contrast, the reassortant virus<o

></o

>
bearing the Vic HA and NA genes replicated efficiently even<o

></o

>
when all internal genes, including the M and NS genes, were of<o

></o

>
avian virus origin. Certain gene constellations, such as<o

></o

>
rgVic:486HANAPB1 and rgVic:486HAPB1, which reflect the<o

></o

>
gene constellations of the viruses responsible for the 1957 and<o

></o

>
1968 pandemics, respectively, could not be rescued even after<o

></o

>
multiple attempts, although the same plasmids were used to<o

></o

>
successfully rescue other reassortant combinations. These results<o

></o

>
suggest that certain combinations of genes from the avian and<o

></o

>
human influenza viruses used in this study may not be compatible<o

></o

>
for virus viability.<o

></o

>
Transmissibility of Avian?Human Reassortant Influenza Viruses. We<o

></o

>
next assessed the ability of the avian?human reassortant viruses<o

></o

>
to undergo respiratory droplet transmission in ferrets, because<o

></o

>
this is a key property of a pandemic virus. Three ferrets were<o

></o

>
inoculated i.n. with 104 FID50 of rgVic:486HANA or<o

></o

>
rg486:VicRNP (107 or 105.5 EID50, respectively), and the next<o

></o

>
day, three naı?ve ferrets were placed in adjacent transmission<o

></o

>
cages. Although all ferrets inoculated with either<o

></o

>
rgVic:486HANA or rg486:VicRNP shed virus in the upper<o

></o

>
respiratory tract for at least 5 days p.i. (Table 2), there was no<o

></o

>
evidence of transmission because contact animals did not shed<o

></o

>
virus or seroconvert (Table 3). In fact, the mean virus titers of<o

></o

>
each reassortant in nasal washes from ferrets were lower than<o

></o

>
titers observed with either parental strain (Table 2). Furthermore,<o

></o

>
the inoculated ferrets exhibited only minor weight loss,<o

></o

>
indicating that both reassortant viruses were substantially attenuated<o

></o

>
for ferrets compared with the rg486 parental strain, which<o

></o

>
caused a mean weight loss of 21% (Table 2).<o

></o

>
Because none of the reassortants possessing the HA and NA<o

></o

>
of the avian HK486 virus demonstrated the ability to transmit,<o

></o

>
we next determined whether the human virus HA and NA were<o

></o

>
required for transmission. Ferrets were inoculated with the same<o

></o

>
dose of the reassortant virus, rg486:VicHANA [104 FID50 (105.5<o

></o

>
EID50)], and virus was recovered from all three infected animals<o

></o

>
with a mean peak virus titer of 5.2 _ 0.6 log10 EID50_ml, which<o

></o

>
was similar to titers achieved in ferrets inoculated with the rg<o

></o

>
parental virus, rgVic (Table 2). Nevertheless, the<o></o>
rg486:VicHANA virus failed to transmit as efficiently as rgVic;<o

></o

>
virus was not detected in the nasal washes collected from any of<o

></o

>
the rg486:VicHANA contact ferrets, and only one of three<o

></o

>
contact ferrets exhibited a modest rise in serum HI antibody on<o

></o

>
day 20 p.c., indicating that some transmission of the reassortant<o

></o

>
had occurred (Table 3). Interestingly, the contact animal that<o

></o

>
seroconverted was exposed to an inoculated ferret that had shed<o

></o

>
the highest level of virus detected from any of the reassortantinoculated<o

></o

>
ferrets evaluated (105.5 EID50_ml). These data suggest<o

></o

>
that the avian internal protein genes of rg486:VicHANA<o

></o

>
virus reduce the transmission efficiency of the human H3N2<o

></o

>
virus in ferrets without affecting the replication efficiency.<o

></o

>
The acquisition of mutations, such as those that confer the<o

></o

>
human _2,6 SA receptor-binding preference, is likely to be<o

></o

>
important for an avian influenza virus or an avian?human<o

></o

>
reassortant virus to transmit efficiently among humans and is<o

></o

>
thought to occur during adaptation in the new host (10). To<o

></o

>
determine whether an H5N1 reassortant virus could acquire<o

></o

>
mutations that would enhance transmissibility through multiple<o

></o

>
rounds of replication in a mammalian host, we passaged<o

></o

>
rg486:VicRNP virus (the H5N1 reassortant that had replicated<o

></o

>
most efficiently in ferrets) five times in ferrets and tested the<o

></o

>
resulting virus, rg486:VicRNPF5, for its ability to undergo<o

></o

>
respiratory droplet transmission. Although peak nasal wash<o

></o

>
titers of rg486:VicRNPF5-inoculated animals were similar to<o

></o

>
those for ferrets inoculated with rgVic (5.1_0.5 log10 EID50_ml)<o

></o

>
(Table 2), transmission of rg486:VicRNPF5 was not detected<o

></o

>
(Table 3). Sequence analysis of the complete rg486:VicRNPF5<o

></o

>
genome revealed only a single amino acid change (S353F) within<o

></o

>
the NP gene. These data and the observation that most avian<o

></o

>
viruses isolated from humans retain their receptor preference<o

></o

>
for _2,3 SA (13, 26) suggest that there is a level of stability of<o

></o

>
receptor specificity among circulating H5N1 viruses.<o

></o

>
Discussion<o></o>
If H5N1 viruses acquire the ability to undergo efficient and<o

></o

>
sustained transmission among humans, a pandemic would be<o

></o

>
inevitable. An understanding of the molecular and biologic<o

></o

>
requirements for efficient transmissibility is critical for the early<o

></o

>
identification of a potential H5N1 pandemic virus and the<o

></o

>
application of optimal control measures. The results of this study<o

></o

>
demonstrate, that unlike human H3N2 viruses, avian H5N1<o

></o

>
viruses isolated from humans in 1997, 2003, or 2005 lack the<o

></o

>
ability to transmit efficiently in the ferret model. Furthermore,<o

></o

>
reassortant viruses bearing 1997 avian H5N1 surface glycoproteins<o

></o

>
with four or six human virus internal protein genes do not<o

></o

>
transmit efficiently in ferrets and thus lack the key property that<o

></o

>
predicts pandemic spread.<o

></o

>
We found that the rgVic:486HANA reassortant bearing the<o

></o

>
H5 HA and human virusMand NS exhibited reduced infectivity<o

></o

>
forMDCKcells and 10-fold lower viral titers in ferrets compared<o

></o

>
with rg486:VicRNP that possessed avian virus M and NS as well<o

></o

>
as HA and NA. Another reassortant that possessed seven avian<o

></o

>
virus genes and only the human virus M gene also exhibited<o

></o

>
reduced infectivity for MDCK cells (data not shown). These<o

></o

>
results suggest that the infectivity of reassortant viruses bearing<o

></o

>
the HK486 HA and NA genes may be enhanced by the presence<o

></o

>
of the HK486 M gene, a finding supported by Scholtissek et al.<o></o>
(27), who showed that reassortants bearing an avian virus HA<o

></o

>
and a human virus M gene were substantially compromised in<o

></o

>
their replication.<o

></o

>
Transmissibility of influenza viruses in this model is likely<o

></o

>
associated with virus infectivity for ferrets, which is reflected in<o

></o

>
the FID50, and efficiency of virus replication in the upper<o

></o

>
respiratory tract, which directly affects the amount of virus<o

></o

>
shedding. Mutations in the NA that affect enzymatic activity and<o

></o

>
replication efficiency were shown to reduce direct contact<o

></o

>
transmission in ferrets (18, 28). In this study, WT or rg H3N2<o

></o

>
parental viruses that achieved peak nasal wash viral titers<o

></o

>
ranging from 104.3 to 108.5 EID50_ml all exhibited efficient<o

></o

>
respiratory droplet transmission. The lack of SA _2,3 receptors<o

></o

>
in the upper respiratory tract of humans has been presented as<o

></o

>
a possible explanation for the lack of transmissibility of the H5N1<o

></o

>
viruses (23, 24). However, high titers of virus (ranging from 106.3<o

></o

>
to 107.8 EID50_ml) were recovered in nasal washes from ferrets<o

></o

>
inoculated with two of four WT H5N1 viruses evaluated in this<o

></o

>
study, yet efficient respiratory droplet or direct contact transmission<o

></o

>
was not observed. It is noteworthy that the HK213 H5N1<o

></o

>
virus, which has been shown to have a moderate affinity for the<o

></o

>
SA _2,6 receptor (13), did not replicate as efficiently as the other<o

></o

>
H5N1 viruses tested and showed no enhanced respiratory droplet<o

></o

>
transmission. Likewise, rg486:VicHANA virus, possessing<o

></o

>
human H3N2 surface genes, replicated to a mean peak nasal<o

></o

>
wash virus titer comparable to that of the parental rgVic virus,<o

></o

>
but failed to transmit as efficiently, suggesting that although the<o

></o

>
amount of virus shed by an infected animal may enhance the<o

></o

>
likelihood of transmission, other molecular or biologic properties<o

></o

>
are critical for efficient transmissibility.<o

></o

>
Other biologic properties that may influence the ability of a<o

></o

>
virus to undergo efficient respiratory droplet transmission include<o

></o

>
the ability to elicit symptoms that promote expulsion from<o

></o

>
the host. Sneezing was consistently observed in all H3N2-<o

></o

>
infected ferrets, including those infected with rgVic and<o

></o

>
rg486:VicHANA, starting 2 days p.i. (Tables 1 and 2), but was<o

></o

>
only rarely observed in ferrets infected with WT or rg parental<o

></o

>
H5N1 viruses and was not detected in any ferrets infected with<o

></o

>
the H5N1 reassortant viruses. It is yet to be determined whether<o

></o

>
the molecular factors enhancing this biologic property also<o

></o

>
enhance efficient transmissibility in the ferret model.<o

></o

>
Although these findings do not identify the precise genetic<o

></o

>
determinants responsible for influenza virus transmissibility,<o

></o

>
they provide an assessment of the risk of an H5N1 pandemic<o

></o

>
strain emerging through reassortment with a human influenza<o

></o

>
virus. Our results indicate that, within the context of the viruses<o

></o

>
used in this study, H5N1 avian?human reassortant viruses did<o

></o

>
not exhibit properties that would initiate a pandemic. Nevertheless,<o

></o

>
H5N1 viruses continue to spread geographically, infect a<o

></o

>
variety of mammals, and evolve rapidly. Therefore, further<o

></o

>
evaluation of the efficiency of replication and transmissibility of<o

></o

>
reassortants between contemporary H5N1 viruses and circulating<o

></o

>
human influenza viruses is an ongoing public health need.<o

></o

>
The ferret transmission model serves as a valuable tool for this<o

></o

>
purpose and the identification of molecular and biologic correlates<o

></o

>
of efficient transmissibility that may be used for early<o

></o

>
detection of a novel virus with pandemic capability.<o

></o

>
Methods<o></o>
Viruses. The human H3N2 influenza A viruses Vic75 and Pan99<o

></o

>
and the avian H5N1 influenza A viruses HK486, HK213, Indo05,<o

></o

>
and VN30408 were used in this study. Virus stocks were prepared<o

></o

>
in 10-day-old eggs as described (29) except that human viruses<o

></o

>
were incubated at 33.5?C for 48 h, whereas reassortant viruses<o

></o

>
were incubated at 37?C for 48 h. All research with H5N1<o

></o

>
viruses or reassortant viruses was conducted under biosafety<o

></o

>
level 3 containment, including enhancements required by the<o

></o

>
<st1:country-region><st1

lace>U.S.</st1

lace></st1:country-region> Department of Agriculture and the Select Agent Program<o

></o

>
(see interim guidance at www.cdc.gov_flu_h2n2bsl3.htm).<o

></o

>
Plasmids and Rescue of Reassortant Viruses. Plasmid-based rg was<o

></o

>
used to generate the reassortant viruses used in this study. As<o

></o

>
described (30), all eight Vic75 genes were amplified and cloned<o

></o

>
into a viral RNA (vRNA) expression plasmid, and Vic75 PB2,<o

></o

>
PB1, PA, and NP genes were cloned into an mRNA expression<o

></o

>
plasmid. All eight genes of HK486 were amplified from vRNA<o

></o

>
by RT-PCR with a One-Step RT-PCR kit (<st1

lace><st1:City>Qiagen</st1:City>, <st1:country-region>Valencia</st1:country-region></st1

lace>,<o

></o

>
CA) and cloned into a bidirectional plasmid (pBD) that possesses<o

></o

>
vRNA expression elements from pPolISapIRib (31, 32)<o

></o

>
and mRNA expression elements from pCI (Promega, Madison,<o

></o

>
WI). Reassortant viruses were rescued as described (33) by using<o

></o

>
the 12-plasmid Vic75 system, the eight-plasmid HK486 system,<o

></o

>
or a combination of the two. The genetic makeup of each<o

></o

>
reassortant virus was confirmed by sequencing as described (29).<o

></o

>
Inoculation of Ferrets. Male Fitch ferrets, 6?12 months of age<o

></o

>
(Triple F Farms, <st1

lace><st1:City>Sayre</st1:City>, <st1:State>PA</st1:State></st1

lace>), that were serologically negative by<o

></o

>
HI assay for currently circulating influenza viruses were used in<o

></o

>
this study. Ferrets were housed throughout each experiment in<o

></o

>
cages within a Duo-Flo Bioclean mobile clean room (Lab<o

></o

>
Products, <st1

lace><st1:City>Seaford</st1:City>, <st1:State>DE</st1:State></st1

lace>). Baseline serum, temperature, and<o

></o

>
weight measurements were obtained before infection. Temperatures<o

></o

>
were measured with an <st1:State><st1

lace>s.c.</st1

lace></st1:State> implantable temperature<o

></o

>
transponder (BioMedic Data Systems, <st1

lace><st1:City>Seaford</st1:City>, <st1:State>DE</st1:State></st1

lace>). Ferrets<o

></o

>
were inoculated, and nasal washes were collected and analyzed<o></o>
as described (29). FID50 titers were determined by inoculating<o

></o

>
groups of nine ferrets i.n. with serial 10-fold dilutions of virus,<o

></o

>
calculated by using the method of Reed and Muench (34), and<o

></o

>
expressed as the EID50 value corresponding to 1 FID50.<o

></o

>
Transmission Experiments. For the respiratory droplet transmission<o

></o

>
experiments, ferrets were housed in adjacent transmission<o

></o

>
cages, each modified so that a side wall was replaced with a<o

></o

>
stainless-steel, perforated wall with holes 1?5 mm in diameter<o

></o

>
and spaced 3 mm apart to facilitate the transfer of respiratory<o

></o

>
droplets through the air while preventing direct contact between<o

></o

>
ferrets and indirect contact with the bedding and food of<o

></o

>
neighboring ferrets. The use of the term ??respiratory droplet<o

></o

>
transmission?? throughout this article refers to transmission in<o

></o

>
the absence of direct or indirect contact and does not imply an<o

></o

>
understanding of the droplet size involved in virus spread<o

></o

>
between ferrets. A total of six ferrets were used for each<o

></o

>
respiratory droplet transmission experiment. Three ferrets were<o

></o

>
inoculated with 104 FID50 of virus, unless otherwise stated, and<o

></o

>
each was placed in a separate cage. Twenty-four hours later (day<o

></o

>
1 p.i. for the inoculated ferrets and day 0 p.c. for the contact<o

></o

>
ferrets), three naive ferrets were each placed in a cage adjacent<o

></o

>
to an inoculated ferret. To prevent inadvertent physical transmission<o

></o

>
of virus by the investigators, the contact ferrets were<o

></o

>
always handled first, and all items that came into contact with the<o

></o

>
ferrets or their bedding were decontaminated between each<o

></o

>
ferret. For contact transmission experiments, two or three ferrets<o

></o

>
were each placed in an unmodified cage with solid walls and<o

></o

>
inoculated i.n. with 104 FID50 of virus, unless otherwise stated.<o

></o

>
Twenty-four hours later, a naive ferret was placed in the same<o

></o

>
cage with each inoculated ferret. Clinical signs were monitored<o

></o

>
daily in all ferrets for at least 14 days p.i._p.c. If any ferret lost<o

></o

>
_25% of its body weight or exhibited neurologic symptoms, it<o

></o

>
was humanely killed.<o

></o

>
HI Assays. HI analysis was conducted as described (35) by using<o

></o

>
convalescent sera collected from ferrets 14?33 days p.i._p.c. Sera<o

></o

>
collected from ferrets included in the transmission experiments<o

></o

>
with Vic75, Pan99, or reassortant H3N2 viruses were tested for<o

></o

>
H3-specific antibodies by using turkey RBCs and virus bearing<o

></o

>
the homologous HA. Ferret sera from the transmission experiments<o

></o

>
with HK486, Indon05, VN30408, or reassortant H5N1<o

></o

>
viruses were tested for H5-specific antibodies by using virus<o

></o

>
bearing the homologousHA and horse RBCs as a means of more<o

></o

>
sensitive detection (35).<o

></o

>
We thank Neal Van Hoeven for assistance with serological screening of<o

></o

>
ferrets, James Mitchell and Eddie Jackson for exceptional care of<o

></o

>
animals used in this study, and Patrick Blair for facilitating access to the<o

></o

>
clade 2 virus.<o

></o

>
1. de Jong, J. C., Claas, E. C., Osterhaus, A. D., Webster, R. G. & Lim, W. L.<o

></o

>
(1997) Nature 389, 554.<o

></o

>
2. Subbarao, K., Klimov, A., Katz, J., Regnery, H., Lim, W., Hall, H., Perdue, M.,<o

></o

>
Swayne, D., Bender, C., Huang, J., et al. (1998) Science 279, 393?396.<o

></o

>
3. Peiris, J. S., Yu, W. C., Leung, C. W., Cheung, C. Y., Ng, W. F., Nicholls, J. M.,<o

></o

>
Ng, T. K., Chan, K. H., Lai, S. T., Lim, W. L., et al. (2004) Lancet 363, 617?619.<o

></o

>
4. World Health Organization (2006) Epidemic and Pandemic Alert and Response:<o></o>
Pandemic Influenza (W.H.O., <st1:City><st1

lace>Geneva</st1

lace></st1:City>).<o

></o

>
5. Ungchusak, K., Auewarakul, P., Dowell, S. F., Kitphati, R., Auwanit, W.,<o

></o

>
Puthavathana, P., Uiprasertkul, M., Boonnak, K., Pittayawonganon, C., Cox,<o

></o

>
N. J., et al. (2005) N. Engl. J. Med. 352, 333?340.<o

></o

>
6. Olsen, S. J., Ungchusak, K., Sovann, L., Uyeki, T. M., Dowell, S. F., Cox, N. J.,<o

></o

>
Aldis, W. & Chunsuttiwat, S. (2005) Emerg. Infect. Dis. 11, 1799?1801.<o

></o

>
7. Reid, A. H., Taubenberger, J. K. & Fanning, T. G. (2004) Nat. Rev. Microbiol.<o></o>
2, 909?914.<o

></o

>
8. Taubenberger, J. K., Reid, A. H., Lourens, R. M., Wang, R., Jin, G. & Fanning,<o

></o

>
T. G. (2005) Nature 437, 889?893.<o

></o

>
9. Kawaoka, Y., Krauss, S. & Webster, R. G. (1989) J. Virol. 63, 4603?4608.<o

></o

>
10. Matrosovich, M., Tuzikov, A., Bovin, N., Gambaryan, A., Klimov, A., Castrucci,<o

></o

>
M. R., Donatelli, I. & Kawaoka, Y. (2000) J. Virol. 74, 8502?8512.<o

></o

>
11. Matrosovich, M., Zhou, N., Kawaoka, Y.&Webster, R. (1999) J. Virol. 73, 1146?1155.<o

></o

>
12. Shinya, K., Hatta, M., Yamada, S., Takada, A., Watanabe, S., Halfmann, P.,<o

></o

>
Horimoto, T., Neumann, G., Kim, J. H., Lim, W., et al. (2005) J. Virol. 79,<o></o>
9926?9932.<o

></o

>
13. Gambaryan, A., Tuzikov, A., Pazynina, G., Bovin, N., Balish, A. & Klimov, A.<o

></o

>
(2006) Virology 344, 432?438.<o

></o

>
14. World Health Organization (2005) Emerg. Infect. Dis. 11, 1515?1521.<o

></o

>
<st1

lace><st1

laceName>15.</st1

laceName> <st1

laceType>Bridges</st1

laceType></st1

lace>, C. B., Kuehnert, M. J. & Hall, C. B. (2003) Clin. Infect. Dis. 37,<o></o>
1094?1101.<o

></o

>
16. Schulman, J. L. & Kilbourne, E. D. (1962) Nature 195, 1129?1130.<o

></o

>
17. Herlocher, M. L., Elias, S., Truscon, R., <st1

lace>Harrison</st1

lace>, S., Mindell, D., Simon, C.<o

></o

>
& Monto, A. S. (2001) J. Infect. Dis. 184, 542?546.<o

></o

>
18. Herlocher, M. L., Carr, J., Ives, J., Elias, S., Truscon, R., Roberts, N. & Monto,<o

></o

>
A. S. (2002) Antiviral Res. 54, 99?111.<o

></o

>
19. Herlocher, M. L., Truscon, R., Elias, S., Yen, H. L., Roberts, N. A., Ohmit, S. E.<o

></o

>
& Monto, A. S. (2004) J. Infect. Dis. 190, 1627?1630.<o

></o

>
20. Baum, L. G. & Paulson, J. C. (1990) Acta Histochem. Suppl. 40, 35?38.<o

></o

>
21. Matrosovich, M. N., Matrosovich, T. Y., Gray, T., Roberts, N. A. & Klenk,<o

></o

>
H. D. (2004) Proc. Natl. Acad. Sci. USA 101, 4620?4624.<o

></o

>
22. Leigh, M. W., Connor, R. J., Kelm, S., Baum, L. G. & Paulson, J. C. (1995)<o

></o

>
Vaccine 13, 1468?1473.<o

></o

>
23. Shinya, K., Ebina, M., Yamada, S., Ono, M., Kasai, N. & Kawaoka, Y. (2006)<o

></o

>
Nature 440, 435?436.<o

></o

>
24. van Riel, D., Munster, V. J., de Wit, E., Rimmelzwaan, G. F., Fouchier, R. A.,<o

></o

>
Osterhaus, A. D. & Kuiken, T. (2006) Science 312, 399.<o

></o

>
25. Andrewes, C. H. & Glover, R. E. (1941) Br. J. Exp. Pathol. 22, 91?97.<o

></o

>
26. Stevens, J., Blixt, O., Tumpey, T. M., Taubenberger, J. K., Paulson, J. C. &<o

></o

>
Wilson, <st1

lace>I.</st1

lace> A. (2006) Science 312, 404?410.<o

></o

>
27. Scholtissek, C., Stech, J., Krauss, S. & Webster, R. G. (2002) J. Virol. 76,<o></o>
1781?1786.<o

></o

>
28. Yen, H. L., Herlocher, L. M., Hoffmann, E., Matrosovich, M. N., Monto, A. S.,<o

></o

>
Webster, R. G. & Govorkova, E. A. (2005) Antimicrob. Agents Chemother. 49,<o></o>
4075?4084.<o

></o

>
29. Maines, T. R., Lu, X. H., Erb, S. M., Edwards, L., Guarner, J., Greer, P. W.,<o

></o

>
Nguyen, D. C., Szretter, K. J., Chen, L. M., Thawatsupha, P., et al. (2005)<o

></o

>
J. Virol. 79, 11788?11800.<o

></o

>
30. Falcon, A. M., Marion, R. M., Zurcher, T., Gomez, P., Portela, A., Nieto, A.<o

></o

>
& Ortin, J. (2004) J. Virol. 78, 3880?3888.<o

></o

>
31. Fodor, E., Devenish, L., Engelhardt, O. G., Palese, P., Brownlee, G. G. &<o

></o

>
Garcia-Sastre, A. (1999) J. Virol. 73, 9679?9682.<o

></o

>
32. Li, Z., Chen, H., Jiao, P., Deng, G., Tian, G., Li, Y., Hoffmann, E., Webster,<o

></o

>
R. G., Matsuoka, Y. & Yu, K. (2005) J. Virol. 79, 12058?12064.<o

></o

>
33. Subbarao, K., Chen, H., Swayne, D., Mingay, L., Fodor, E., Brownlee, G., Xu,<o

></o

>
X., Lu, X., Katz, J., Cox, N., et al. (2003) Virology 305, 192?200.<o

></o

>
34. Reed, L. J. & Muench, H. (1938) Am. J. Hyg. 27, 493?497.<o

></o

>
35. Stephenson, <st1

lace>I.</st1

lace>, Wood, J. M., Nicholson, K. G., Charlett, A. & Zambon, M. C.<o

></o

>
(2004) Virus Res. 103, 91?95.<o

></o

>
12126 _ www.pnas.org_cgi_doi_10.1073_pnas.0605134103 Maines et al.<o

></o

>
<o

> </o

>

sharon sanders · Aug 2, 2006

Re: Lack of transmission of H5N1 avian-human...

I was not able to get the tables and figures so I did not post on main forum.

Shannon Bennett · Aug 2, 2006

Re: Lack of transmission of H5N1 avian-human...

Thanks Sharon.

sharon sanders · Mar 27, 2007

Important Follow-up Comments by Dr. Gerberding -

Important Follow-up Comments by Dr. Gerberding -

hat-tip to Steveosteen.

<HR style="COLOR: #cccccc" SIZE=1>

http://www.nytimes.com/2007/03/27/he...ce&oref=slogin

Le NY Times, tout en soulignant les mutations permanentes du virus H5N1, se fait l'echo du site Recombinomics du Dr Henry Niman.

Quote:
<TABLE cellSpacing=0 cellPadding=6 width="100%" border=0><TBODY><TR><TD class=alt2 style="BORDER-RIGHT: 1px inset; BORDER-TOP: 1px inset; BORDER-LEFT: 1px inset; BORDER-BOTTOM: 1px inset">Scientists Hope Vigilance Stymies Avian Flu Mutations

Published: March 27, 2007

Just exactly what is the bird flu virus doing?

Dr. Mitch Cohen and Julie L. Gerberding of the Centers for Disease Control and Prevention discussing pandemic flu.

The virus, H5N1, which was first isolated in humans in 1997, has not started a pandemic in a full decade of trying, so a few flu experts think it never will.

But the mainstream view is less optimistic. Viruses mutate constantly, many experts point out. And when one has already acquired the ability to jump species, occasionally spread from human to human and kill 60 percent of the people who catch it, it is far too early to dismiss it.

So even though the human death toll from H5N1 is still below 200, scientists around the world are racing to study the ways in which it might mutate to spread easily among humans.

The 1918 Spanish flu, they argue, was not even noticed until it had killed thousands. It might have been gathering virulence for years, hidden in the background of seasonal flu deaths.

Today?s H5N1 flu is probably changing more slowly, because health officials have been vigilant about attacking clusters of cases, which presumably wipes out the most dangerous strains. Whenever several human cases appear, even in remote villages in Indonesia or Egypt, local officials and World Health Organization teams move in to kill all the local poultry and dose all the humans with antiviral drugs ? the so-called Tamiflu blanket strategy.

Each stifled outbreak robs the virus of the chance to carom wildly through dozens of human hosts as it does in a flock of chickens or ducks. That fends off what virologists most fear: gene-swapping in people infected with both human and avian flu.

But the Tamiflu blanket may not be able to smother every spark, especially if countries cannot get their poultry epidemics under control.

A human-bird hybrid strain has not yet been seen in nature. But if it did surface, that ?would mean we might have a big problem on our hands,? said Dr. Nancy Cox, chief of the influenza branch of the Centers for Disease Control and Prevention.

Last year, Dr. Cox and colleagues created a hybrid in their lab between a human flu of the H3N2 strain and samples of the H5N1 virus collected from 1997 to 2004. They infected ferrets with it to see if it would spread to ferrets in the same cage or those in nearby cages. The hybrid strain proved less lethal and was transmitted only once after long contact.

But nature has a bigger laboratory than the C.D.C. does, and the agency?s director, Dr. Julie L. Gerberding, says the results do not mean that H5N1 cannot become more infectious. ?They mean it?s probably not a simple process,? she said.

Dr. Anne Moscona, a flu expert at Weill Cornell Medical Center, was more emphatic in arguing that there is still reason to worry. ?It would have been truly ominous if a monster virus had been created by these supersimple swaps,? she said. ?But of three ferrets, one got the virus. Is 33 percent nothing??

Geneticists at the University of California, Irvine, concluded that the H5N1 flu originated in Guangdong Province in Southern China, where millions of people and chickens live in close proximity. Guangdong is also believed to be the likely birthplace of previous flu strains ? even if they later picked up names like ?Hong Kong flu? ? and to be where the SARS virus jumped from horseshoe bats to masked palm civets to humans.

But flus mutate incessantly wherever they move, and in viral samples from Asia, the Middle East and Africa, many individual changes that look potentially dangerous have been spotted.

In May 2005, for example, the virus in China escaped in migratory birds going north and traveled across Russia, Europe and Africa. It became known as the Qinghai strain after the lake in Northern China where thousands of ducks and geese were found dead. (The older strain in Southern China and Southeast Asia is sometimes called the Fujian strain.)

The Qinghai strain has a mutation known as PB2 E627K. (The abbreviation can be read this way: at position No. 627 on polymerase basic protein 2, the amino acid called glutamic acid, abbreviated by scientists as E, has been replaced by lysine, known as K.)

The change helps the virus grow at the temperatures found in human noses, which are cooler than the insides of birds? intestines.

It is ?characteristic of a gene that?s been in mammals,? said Dr. Robert G. Webster, a virologist at St. Jude Children?s Research Hospital in Memphis. ?It says to me that it was in a mammalian species in China, and got back into ducks. But what species? We don?t know.?

The Qinghai strain has now reached about 50 countries.

To give a sense of how important such a tiny change can be: switching just one of the 1,255 amino acids in the SARS virus protein that attached to cells in the masked palm civet, a relative of the mongoose that is sold in wild-meat markets in Asia, allowed it to attach to human cells.

After that discovery, the Chinese government ordered that all the 10,000 civets in captivity in Guangdong be killed, thus probably wiping out the disease everywhere except in bats.

In avian flu, two mutations known to help viruses spread more easily ? because they attach to the receptors in human noses and throats instead of those deep in the lungs ? were found in outbreaks in Azerbaijan and Iraq in 2006. But those outbreaks were snuffed out.

Another mutation, increasingly common in Egypt, where the disease is still raging through poultry and occasionally infecting humans, is called M230I. Scientists do not know what it does, but its persistence is worrisome, says Henry L. Niman, a Pittsburgh biochemist who runs a Web site tracking the genetics of flu cases.

M230I is also found in typical annual flu strains like H1N1, H3N2 and influenza B; in H7 flus, which pass easily from birds to humans but usually cause nothing more serious than pinkeye; and in H3N8, the flu that has spread from dog to dog in many American kennels, often fatally.

All the human cases in Egypt with M230I have been fatal, Dr. Niman said, and those without it have not been, although that may be coincidence.

Mutations that confer resistance to Tamiflu have also been found in Egypt.

Any antiviral resistance is worrisome because the world still has very few weapons against the flu. H5N1 long ago became resistant to older ?M2 inhibitors? like amantadine, possibly because farmers in China are suspected of feeding those drugs to their chickens in the late 1990s.

Tamiflu is in another class, known as neuraminidase inhibitors, including Relenza and peramivir.

After Tamiflu resistance was found in Egypt, the World Health Organization, moving to stave off panic, said the same change was seen in Vietnam years before. Still, the Vietnam cases led doctors to start doubling the typical Tamiflu dose, effectively halving the world?s stockpiles of it.

An American Navy research lab in Cairo found that two Egyptian cases had a dangerous mutation known as N294S even before they got Tamiflu. That implies that it exists in Egyptian poultry, though it has not been found yet.

Every flu virus is different, and it is impossible to predict exactly what constellation of changes will turn one into a pandemic strain.

Dr. Cox and Dr. Ruben Donis, the influenza branch?s chief virologist, said they would be most worried if they saw spontaneous human-avian crossovers like those they created, or if they saw multiple changes in the virus?s hemagglutinin gene, the attachment ?spike? on the virus?s shell.

?We?re looking very, very carefully at the viruses that exhibit changes at the receptor binding pocket,? Dr. Cox said. ?But it?s clear that these single changes don?t allow the virus to move from person to person efficiently.?

And even if H5N1 fails to become a plague, Dr. Webster of St. Jude in Memphis has what he styles his ?hit list? of others waiting their turn.

They include H7N7, which infected 89 chicken industry workers in the Netherlands in 2003 but killed only one veterinarian; H9N2, which he says is in ?every poultry house in Eurasia? and causes no symptoms but every once in a while jumps into immuno-suppressed people; and H2N2, which is in the wild bird population in the United States. </TD></TR></TBODY></TABLE>

Lack of transmission of H5N1 avian-human...

sharon sanders

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sharon sanders

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sharon sanders

Editor-in-Chief & President