Wild waterfowl, including ducks, are natural hosts of influenza A viruses. These viruses rarely caused disease in ducks until 2002, when some H5N1 strains became highly pathogenic. Here we show that these H5N1 viruses are reverting to nonpathogenicity in ducks. Ducks experimentally infected with viruses isolated between 2003 and 2004 shed virus for an extended time (up to 17 days), during which variant viruses with low pathogenicity were selected. These results suggest that the duck has become the “Trojan horse” of Asian H5N1 influenza viruses. The ducks that are unaffected by infection with these viruses continue to circulate these viruses, presenting a pandemic threat.

Several novel anti-influenza compounds are in various phases of clinical development. One of these, T-705 (favipiravir), has a mechanism of action that is not fully understood but is suggested to target influenza virus RNA-dependent RNA polymerase. We investigated the mechanism of T-705 activity against influenza A (H1N1) viruses by applying selective drug pressure over multiple sequential passages in MDCK cells. We found that T-705 treatment did not select specific mutations in potential target proteins, including PB1, PB2, PA, and NP. Phenotypic assays based on cell viability confirmed that no T-705-resistant variants were selected. In the presence of T-705, titers of infectious virus decreased significantly (P< 0.0001) during serial passage in MDCK cells inoculated with seasonal influenza A (H1N1) viruses at a low multiplicity of infection (MOI; 0.0001 PFU/cell) or with 2009 pandemic H1N1 viruses at a high MOI (10 PFU/cell). There was no corresponding decrease in the number of viral RNA copies; therefore, specific virus infectivity (the ratio of infectious virus yield to viral RNA copy number) was reduced. Sequence analysis showed enrichment of G→A and C→T transversion mutations, increased mutation frequency, and a shift of the nucleotide profiles of individual NP gene clones under drug selection pressure. Our results demonstrate that T-705 induces a high rate of mutation that generates a nonviable viral phenotype and that lethal mutagenesis is a key antiviral mechanism of T-705. Our findings also explain the broad spectrum of activity of T-705 against viruses of multiple families.

Wild waterfowl are the natural reservoir of all influenza A viruses, and these viruses are usually nonpathogenic in these birds. However, since late 2002, H5N1 outbreaks in Asia have resulted in mortality among waterfowl in recreational parks, domestic flocks, and wild migratory birds. The evolutionary stasis between influenza virus and its natural host may have been disrupted, prompting us to ask whether waterfowl are resistant to H5N1 influenza virus disease and whether they can still act as a reservoir for these viruses. To better understand the biology of H5N1 viruses in ducks and attempt to answer this question, we inoculated juvenile mallards with 23 different H5N1 influenza viruses isolated in Asia between 2003 and 2004. All virus isolates replicated efficiently in inoculated ducks, and 22 were transmitted to susceptible contacts. Viruses replicated to higher levels in the trachea than in the cloaca of both inoculated and contact birds, suggesting that the digestive tract is not the main site of H5N1 influenza virus replication in ducks and that the fecal-oral route may no longer be the main transmission path. The virus isolates' pathogenicities varied from completely nonpathogenic to highly lethal and were positively correlated with tracheal virus titers. Nevertheless, the eight virus isolates that were nonpathogenic in ducks replicated and transmitted efficiently to naïve contacts, suggesting that highly pathogenic H5N1 viruses causing minimal signs of disease in ducks can propagate silently and efficiently among domestic and wild ducks in Asia and that they represent a serious threat to human and veterinary public health.

There is no question that there will be another influenza pandemic someday. We simply don't know when it will occur or whether it will be caused by the H5N1 avian influenza virus. But given the number of cases of H5N1 influenza that have occurred in humans to date (251 as of late September 2006) and the rate of death of more than 50%, it would be prudent to develop robust plans for dealing with such a pandemic.

H5N1 influenza viruses transmitted from poultry to humans in Asia cause high mortality and pose a pandemic threat. Viral genes important for cell tropism and replication efficiency must be identified to elucidate and target virulence factors. We applied reverse genetics to generate H5N1 reassortants combining genes of lethal A/Vietnam/1203/04 (VN1203), a fatal human case isolate, and nonlethal A/chicken/Vietnam/C58/04 (CH58) and tested their pathogenicity in ferrets and mice. The viruses' hemagglutinins have six amino acids differences, identical cleavage sites, and avian-like α-(2,3)–linked receptor specificity. Surprisingly, exchanging hemagglutinin and neuraminidase genes did not alter pathogenicity, but substituting CH58 polymerase genes completely attenuated VN1203 virulence and reduced viral polymerase activity. CH58's NS gene partially attenuated VN1203 in ferrets but not in mice. Our findings suggest that for high virulence in mammalian species an avian H5N1 virus with a cleavable hemagglutinin requires adaptive changes in polymerase genes to overcome the species barrier. Thus, novel antivirals targeting polymerase proteins should be developed.