Phylogenetic trees for the HA (A), NA (B), and PA (C) genes of the H5N1 influenza A viruses analyzed.

<p>The trees were generated by using CLUSTALx1.83 and MEGA4.0 software by the NJ method (Bootstrap test:1000 replicates, seed = 64238 ) on the basis of the following gene sequences: nucleotides 29–1,695 (1,667 bp) of HA, 21–1,358 (1,338 bp) of NA, and 25–2,163 (2,139 bp) of PA. The length of each pair of branches represents the distance between the sequence pairs, and the units at the bottom of the tree indicate the number of substitution events. The 15 H5N1 isolates from Vietnam are marked in bold italic. Abbreviations: CK, chicken; DK, duck; MDK, Muscovy duck; QL, quail; GS, goose; GD, Guangdong; GX, Guangxi; HK, Hong Kong; VN, Vietnam; SX, Shanxi.</p

Genotypic evolution of H5N1 viruses isolated from poultry in Vietnam in 2006 and 2007.

<p>The eight gene segments are indicated at the top of each bar. The number in each bar shows the group of genes indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050959#pone-0050959-g001" target="_blank">Figure 1</a>. DK/VN208/05 was used in this analysis because it represents the earliest clade 2.3.4 isolate in Vietnam in the public databases to date. † The letters S and N denote southern Vietnam and northern Vietnam, respectively.</p

Replication and virulence of H5N1 viruses in mice.^a.

a<p>Six-week-old BALB/c mice were used for this study.</p>b<p>Standard deviation.</p>c<p>The data were acquired when mice were inoculated intranasally with 10⁶ EID₅₀ of H5N1 virus in a volume of 50 µL.</p>d<p>The titer shown are the means ± standard deviations of the mice inoculated.</p>e<p>+, Viruses were only detected from undiluted samples; -, the viruses were not detected in the organs.</p>f<p>The data were not acquired.</p

Replication and virulence of H5N1 influenza viruses in mice.

<p>(A) Weight changes of mice inoculated with different H5N1 viruses. Groups of five mice were intranasally inoculated with 10⁶ EID₅₀ (50 µL) or with PBS as a control and weighed daily for 14 days. (B) Survival percentage of mice inoculated with H5N1 viruses.</p

Western blot analyses of H5N1 avian influenza HA1 protein.

<p>Lysates of H5N1 viruses treated with or without PNGase F were incubated with chicken anti-H5N1 antiserum. Binding was visualized with 3,30-diaminobenzidine after incubation with peroxidase-conjugated secondary antibodies. The locations of marker proteins are indicated on the left.</p

Replication of H5N1 avian influenza viruses in guinea pigs.

a<p>Data shown are summarized from previous reports <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000709#ppat.1000709-Chen1" target="_blank">[2]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000709#ppat.1000709-Chen2" target="_blank">[7]</a>. Six-week-old BALB/c mice were infected i.n. with 10⁶ EID₅₀ of each virus in a 50-µl volume. Organs were collected on day 3 p.i., and clarified homogenates were titrated for virus infectivity in eggs at initial dilutions of 1∶10 (lung), 1∶2 (other tissues), or undiluted if negative at the lowest dilution. + and −, virus was detected or not detected, respectively, in the undiluted samples.</p>b<p>Groups of four guinea pigs were slightly anesthetized and intranasally inoculated with 10⁶EID₅₀ of test virus in a 300µl volume, 150 µl per nostril. Two animals from each group were euthanized on day 3 p.i. and samples, including nasal wash, trachea, lung, spleen, kidney, colon and brain, were collected for virus titration in eggs. The remaining two animals were observed for two weeks and sera were collected at the end of the observation period. Virus was not detected in the spleen, kidney, colon and brain of any animals inoculated with the six viruses, therefore, the data from these samples are not shown in the table. −, virus was not detected in the undiluted sample.</p>c<p>Data shown are log₁₀EID₅₀/ml.</p>d<p>Virus was only detected in one of the two animals inoculated.</p>e<p>Seroconversion was confirmed by hemagglutination inhibition (HI) assay.</p

Receptor-binding preference and transmission of BHGQH/3 and its HA mutant.

<p>Receptor-binding preference of r-BHGQH/3 (<b>A</b>) and BHGQH/3-160T (<b>B</b>) were performed by dose-dependent direct binding assay as described in the text. (<b>C</b>) and (<b>D</b>) Transmisson of H5N1 duck viruses in guinea pigs. (C) r-BHGQH/3-inoculated group. (D) BHGQH/3-160T-inoculated group. The dashed blue lines in these panels indicate the lower limit of detection.</p

Glycan binding specificity of H1N1 and H5N1 viruses.

<p>(<b>A</b>) H1N1 human influenza BC/05 virus. (<b>B</b>) DKGX/35 virus. (<b>C</b>) 35/HA-226L/228S. (<b>D</b>) 35/HA-160T.</p

Seroconversion of the guinea pigs in our H5N1 avian influenza virus transmission studies.

a<p>Sera were collected from guinea pigs on day 14 p.i. and treated overnight with <i>Vibrio cholerae</i> receptor-destroying enzyme. Seroconversion was confirmed by hemagglutination inhibition (HI) assay.</p

Transmisson of H5N1 avian influenza viruses in guinea pigs.

<p>Groups of three guinea pigs were inoculated i.n. with 10⁶EID₅₀ of test virus and, 24 hours after the inoculation, three contact guinea pigs were placed in each cage. Nasal washes were collected every two days from all animals beginning 2 days p.i. for detection of virus shedding. (<b>A</b>) DKGX/22 virus; (<b>B</b>) DKGX/17 virus; (<b>C</b>) DKSH/13 virus; (<b>D</b>) DKGX/35 virus; (<b>E</b>) DKGD/22 virus; and (<b>F</b>) BHGQH/3 virus. Each color bar represents the virus titer from an individual animal. The dashed blue lines in these panels indicate the lower limit of detection.</p