Isolation and characterization of avian influenza viruses, including highly pathogenic H5N1, from poultry in live bird markets in Hanoi, Vietnam, in 2001

Abstract

Since 1997, outbreaks of highly pathogenic (HP) H5N1 and circulation of H9N2 viruses among domestic poultry in Asia have posed a threat to public health. To better understand the extent of transmission of avian influenza viruses (AIV) to humans in Asia, we conducted a cross-sectional virologic study in live bird markets (LBM) in Hanoi, Vietnam, in October 2001. Specimens from 189 birds and 18 environmental samples were collected at 10 LBM. Four influenza A viruses of the H4N6 (n = 1), H5N2 (n = 1), and H9N3 (n = 2) subtypes were isolated from healthy ducks for an isolation frequency of over 30% from this species. Two H5N1 viruses were isolated from healthy geese. The hemagglutinin (HA) genes of these H5N1 viruses possessed multiple basic amino acid motifs at the cleavage site, were HP for experimentally infected chickens, and were thus characterized as HP AIV. These HA genes shared high amino acid identities with genes of other H5N1 viruses isolated in Asia during this period, but they were genetically distinct from those of H5N1 viruses isolated from poultry and humans in Vietnam during the early 2004 outbreaks. These viruses were not highly virulent for experimentally infected ducks, mice, or ferrets. These results establish that HP H5N1 viruses with properties similar to viruses isolated in Hong Kong and mainland China circulated in Vietnam as early as 2001, suggest a common source for H5N1 viruses circulating in these Asian countries, and provide a framework to better understand the recent widespread emergence of HP H5N1 viruses in Asia.

FIG. 1.

Phylogenetic relationships of hemagglutinin genes from (A) H4, (B) H9, and (C) H5 viruses isolated from LBM in Vietnam in 2001 and other representative AIV of these subtypes. The trees were drawn using the TreeView 1.6.6 program (45). Numbers below branches indicate the bootstrap values from 1,000 analyses; values less than 70% are not shown. The nucleotide sequence regions used for phylogenetic analysis were 20 to 1309 (1,290 bp), 179 to 1130 (952 bp), and 139 to 1068 (930 bp) for the H4, H5, and H9 genes, respectively. The H14N5 subtype virus A/Mallard/Gurjev/263/82, H12N5 virus A/Duck/Alberta/60/76, and H5N1 virus A/Chicken/Scotland/59 were used as outgroups for the H4, H9, and H5 genes, respectively. Eurasian and North American lineage and sublineages (Korea, G1, and Y280) are indicated. The viruses characterized in this study are indicated in boldface. The number of nucleotide changes represented by the scale bar is given for each figure.

FIG. 1.

Phylogenetic relationships of hemagglutinin genes from (A) H4, (B) H9, and (C) H5 viruses isolated from LBM in Vietnam in 2001 and other representative AIV of these subtypes. The trees were drawn using the TreeView 1.6.6 program (45). Numbers below branches indicate the bootstrap values from 1,000 analyses; values less than 70% are not shown. The nucleotide sequence regions used for phylogenetic analysis were 20 to 1309 (1,290 bp), 179 to 1130 (952 bp), and 139 to 1068 (930 bp) for the H4, H5, and H9 genes, respectively. The H14N5 subtype virus A/Mallard/Gurjev/263/82, H12N5 virus A/Duck/Alberta/60/76, and H5N1 virus A/Chicken/Scotland/59 were used as outgroups for the H4, H9, and H5 genes, respectively. Eurasian and North American lineage and sublineages (Korea, G1, and Y280) are indicated. The viruses characterized in this study are indicated in boldface. The number of nucleotide changes represented by the scale bar is given for each figure.

FIG. 1.

Phylogenetic relationships of hemagglutinin genes from (A) H4, (B) H9, and (C) H5 viruses isolated from LBM in Vietnam in 2001 and other representative AIV of these subtypes. The trees were drawn using the TreeView 1.6.6 program (45). Numbers below branches indicate the bootstrap values from 1,000 analyses; values less than 70% are not shown. The nucleotide sequence regions used for phylogenetic analysis were 20 to 1309 (1,290 bp), 179 to 1130 (952 bp), and 139 to 1068 (930 bp) for the H4, H5, and H9 genes, respectively. The H14N5 subtype virus A/Mallard/Gurjev/263/82, H12N5 virus A/Duck/Alberta/60/76, and H5N1 virus A/Chicken/Scotland/59 were used as outgroups for the H4, H9, and H5 genes, respectively. Eurasian and North American lineage and sublineages (Korea, G1, and Y280) are indicated. The viruses characterized in this study are indicated in boldface. The number of nucleotide changes represented by the scale bar is given for each figure.

FIG. 2.

Virus titers in tissues from (A) ducks and (B) ferrets infected with H5N1 viruses. (A) Ducks were inoculated i.n. with 10^6.0 EID₅₀ of Gs/VN/113/01 (shaded bars) or Eg/HK/757.2/02 (open bars) virus. The mean tissue titers plus standard errors (SE) from two birds on day 2 p.i. are shown. Titers are expressed as mean log₁₀ EID₅₀/g except for the oropharyngeal swab (OroPh), for which titers are expressed as mean log₁₀ EID₅₀/ml. (B) Ferrets were inoculated i.n. with 10^7.0 EID₅₀ of Gs/VN/113/01 (shaded bars) or Hong Kong/486/97 (open bars) virus. The mean tissue titers plus SE for three animals per group on day 3 p.i. are shown. Titers are expressed as mean log₁₀ EID₅₀/g except for the nasal turbinate samples (N.Turb), for which titers are expressed as mean log₁₀ EID₅₀/ml. Ol.Bulb, olfactory bulb; ND, not done.