The characterization of low pathogenic avian influenza viruses isolated from wild birds in northern Vietnam from 2006 to 2009.

Due to concerns that wild birds could possibly spread H5N1 viruses, surveillance was conducted to monitor the types of avian influenza viruses circulating among the wild birds migrating to or inhabiting in northern Vietnam from 2006 to 2009. An H5N2 virus isolated from a Eurasian woodcock had a close phylogenetic relationship to H5 viruses recently isolated in South Korea and Japan, suggesting that H5N2 has been shared between Vietnam, South Korea, and Japan. An H9N2 virus isolated from a Chinese Hwamei was closely related to two H9N2 viruses that were isolated from humans in Hong Kong in 2009, suggesting that an H9N2 strain relevant to the human isolates had been transmitted to and maintained among the wild bird population in Vietnam and South China. The results support the idea that wild bird species play a significant role in the spread and maintenance of avian influenza and that this also occurs in Vietnam.

Molecular epidemiology of avian influenza viruses circulating among healthy poultry flocks in farms in northern Vietnam.

Repeated epizootics of highly pathogenic avian influenza (HPAI) virus subtype H5N1 were reported from 2003 to 2005 among poultry in Vietnam. More than 200 million birds were killed to control the spread of the disease. Human cases of H5N1 infection have been sporadically reported in an area where repeated H5N1 outbreaks among birds had occurred. Subtype H5N1 strains are established as endemic among poultry in Vietnam, however, insights into how avian influenza viruses including the H5N1 subtype are maintained in endemic areas is not clear. In order to determine the prevalence of different avian influenza viruses (AIVs), including H5N1 circulating among poultry in northern Vietnam, surveillance was conducted during the years 2006-2009. A subtype H5N1 strain was isolated from an apparently healthy duck reared on a farm in northern Vietnam in 2008 and was identified as an HPAI. Although only one H5N1 virus was isolated, it supports the view that healthy domestic ducks play a pivotal role in maintaining and transmitting H5N1 viruses which cause disease outbreaks in northern Vietnam. In addition, a total of 26 AIVs with low pathogenicity were isolated from poultry and phylogenetic analysis of all the eight gene segments revealed their diverse genetical backgrounds, implying that reassortments have occurred frequently among strains in northern Vietnam. It is, therefore, important to monitor the prevalence of influenza viruses among healthy poultry between epidemics in an area where AIVs are endemic.

Possible circulation of H5N1 avian influenza viruses in healthy ducks on farms in northern Vietnam.

To estimate the prevalence of influenza A subtype H5N1 viruses among domestic ducks in the period between October and November 2006 when H5N1 outbreaks had been absent, 1106 healthy ducks raised in northern Vietnam were collected. Inoculation of all throat and cloacae samples into embryonated eggs resulted in the isolation of subtype H3N8 in 13 ducks, but not H5N1 viruses. Serological analyses demonstrated that five ducks (0.45%) solely developed H5N1 subtype-specific hemagglutinin-inhibiting and neuraminidase-inhibiting antibodies together with anti-non-structural protein 1 antibodies. The results suggested that the ducks were naturally infected with H5N1 viruses when obvious H5N1 outbreaks were absent.

Genetic analysis of avian influenza A viruses isolated from domestic waterfowl in live-bird markets of Hanoi, Vietnam, preceding fatal H5N1 human infections in 2004.

The first known cases of human infection with highly pathogenic avian influenza (HPAI) H5N1 viruses in Vietnam occurred in late 2003. However, HPAI H5N1 and low-pathogenic avian influenza (LPAI) H5N2 and H9N3 viruses were isolated from domestic waterfowl during live-bird market (LBM) surveillance in Vietnam in 2001 and 2003. To understand the possible role of these early viruses in the genesis of H5N1 strains infecting people, we performed sequencing and molecular characterization. Phylogenetic analysis revealed that the hemagglutinin (HA) genes of two geese HPAI H5N1 strains belonged to clade 3, and their surface glycoprotein and replication complex genes were most closely related (98.5-99.7% homologous) to A/duck/Guangxi/22/01 (H5N1) virus, detected contemporarily in southern China, whilst the M and NS genes were derived from an A/duck/Hong Kong/2986.1/00 (H5N1)-like virus. The H5 HA gene of the duck HPAI H5N1 strain belonged to clade 5 and acquired a gene constellation from A/quail/Shantou/3846/02 (H5N1), A/teal/China/2978.1/02 (H5N1) and A/partridge/Shantou/2286/03 (H5N1)-like viruses. The phylogenetic analysis further indicated that all eight gene segments of goose and duck HPAI H5N1 and LPAI H5N2 viruses were distinct from those of H5N1 clade-1 viruses known to have caused fatal human infections in Vietnam since late 2003. The duck H9N3 isolates derived genes from aquatic-bird influenza viruses, and their H9 HA belonged to the Korean lineage. The PB2 gene of A/duck/Vietnam/340/01 (H9N3) virus had lysine at position 627. Based on the molecular characterization of specific amino acid residues in the surface and relevant internal protein-coding genes, the Vietnamese H5N1 and H9N3 virus isolates indicated specificity to avian cell surface receptor and susceptibility for currently licensed anti-influenza A virus chemotherapeutics. Our findings suggest that the H5N1 and H5N2 viruses that circulated among geese and ducks in LBMs in Hanoi, Vietnam, during 2001 and 2003 were not the immediate ancestors of the clade-1 viruses associated with fatal human infections in Vietnam. The clade-1 HPAI H5N1 viruses were independently introduced into Vietnam.

Avian influenza (H5N1) viruses isolated from humans in Asia in 2004 exhibit increased virulence in mammals.

The spread of highly pathogenic avian influenza H5N1 viruses across Asia in 2003 and 2004 devastated domestic poultry populations and resulted in the largest and most lethal H5N1 virus outbreak in humans to date. To better understand the potential of H5N1 viruses isolated during this epizootic event to cause disease in mammals, we used the mouse and ferret models to evaluate the relative virulence of selected 2003 and 2004 H5N1 viruses representing multiple genetic and geographical groups and compared them to earlier H5N1 strains isolated from humans. Four of five human isolates tested were highly lethal for both mice and ferrets and exhibited a substantially greater level of virulence in ferrets than other H5N1 viruses isolated from humans since 1997. One human isolate and all four avian isolates tested were found to be of low virulence in either animal. The highly virulent viruses replicated to high titers in the mouse and ferret respiratory tracts and spread to multiple organs, including the brain. Rapid disease progression and high lethality rates in ferrets distinguished the highly virulent 2004 H5N1 viruses from the 1997 H5N1 viruses. A pair of viruses isolated from the same patient differed by eight amino acids, including a Lys/Glu disparity at 627 of PB2, previously identified as an H5N1 virulence factor in mice. The virus possessing Glu at 627 of PB2 exhibited only a modest decrease in virulence in mice and was highly virulent in ferrets, indicating that for this virus pair, the K627E PB2 difference did not have a prevailing effect on virulence in mice or ferrets. Our results demonstrate the general equivalence of mouse and ferret models for assessment of the virulence of 2003 and 2004 H5N1 viruses. However, the apparent enhancement of virulence of these viruses in humans in 2004 was better reflected in the ferret.