Long-Term Epidemiology and Evolution of Swine Influenza Viruses, Vietnam.

Influenza A viruses are a One Health threat because they can spill over between host populations, including among humans, swine, and birds. Surveillance of swine influenza virus in Hanoi, Vietnam, during 2013-2019 revealed gene pool enrichment from imported swine from Asia and North America and showed long-term maintenance, persistence, and reassortment of virus lineages. Genome sequencing showed continuous enrichment of H1 and H3 diversity through repeat introduction of human virus variants and swine influenza viruses endemic in other countries. In particular, the North American H1-δ1a strain, which has a triple-reassortant backbone that potentially results in increased human adaptation, emerged as a virus that could pose a zoonotic threat. Co-circulation of H1-δ1a viruses with other swine influenza virus genotypes raises concerns for both human and animal health.

SARS-CoV-2 Infection in a Hippopotamus, Hanoi, Vietnam.

While investigating the death of a hippopotamus at a zoo in Hanoi, Vietnam, we isolated SARS-CoV-2 and sequenced the RNA-dependent RNA polymerase gene from different organs. Phylogenetic analysis showed that the SARS-CoV-2 strain was closely related to 3 human SARS-CoV-2 strains in Vietnam.

Duck circovirus in northern Vietnam: genetic characterization and epidemiological analysis.

In the present study, tissue samples collected from 130 ducks from clinically suspected commercial flocks and diseased birds in six provinces of northern Vietnam were tested for duck circovirus (DuCV) infection. The DuCV genome was detected in 56 out of 130 (43.08%) duck samples by PCR. Of 38 tested farms, 26 (68.42%) were positive for the DuCV genome. The rate of the DuCV genome detection in ducks at 3-4 weeks of age (54.17%) was significantly higher (p < 0.05) than that at <3 (32.43%) and >7 (33.33%) weeks of age and insignificantly higher than that at 5-7 weeks of age (43.33%) (p = 0.11). The genomes of six Vietnamese DuCV isolates were determined. They ranged in length from 1,988 to 1,995 nucleotides, and their nucleotide sequences were 83.24% to 99.69% identical to each other. Phylogenetic analysis based on the complete genome sequences indicated that the DuCV strains circulating in northern Vietnam can be divided into two main genotypes (I and II) and several subgenotypes. The Vietnamese DuCV isolates were closely related to Chinese, Taiwanese, and Korean strains. One positively selected site was detected in the capsid protein.

Respiratory microbes detected in hospitalized adults with acute respiratory infections: associations between influenza A(H1N1)pdm09 virus and intensive care unit admission or fatal outcome in Vietnam (2015-2017).

BACKGROUND: Acute respiratory tract infection (ARI) is a leading cause of hospitalization, morbidity, and mortality worldwide. Respiratory microbes that were simultaneously detected in the respiratory tracts of hospitalized adult ARI patients were investigated. Associations between influenza A(H1N1)pdm09 virus (H1N1pdm) detection and intensive care unit (ICU) admission or fatal outcome were determined. METHODS: This prospective observational study was conducted between September 2015 and June 2017 at Bach Mai Hospital, Hanoi, Vietnam. Inclusion criteria were hospitalized patients aged ≥15 years; one or more of symptoms including shortness of breath, sore throat, runny nose, headache, and muscle pain/arthralgia in addition to cough and fever > 37.5 °C; and ≤ 10 days from the onset of symptoms. Twenty-two viruses, 11 bacteria, and one fungus in airway specimens were examined using a commercial multiplex real-time PCR assay. Associations between H1N1pdm detection and ICU admission or fatal outcome were investigated by univariate and multivariate logistic regression analyses. RESULTS: The total of 269 patients (57.6% male; median age, 51 years) included 69 ICU patients. One or more microbes were detected in the airways of 214 patients (79.6%). Single and multiple microbes were detected in 41.3 and 38.3% of patients, respectively. Influenza A(H3N2) virus was the most frequently detected (35 cases; 13.0%), followed by H1N1pdm (29 cases; 10.8%). Hematological disease was associated with ICU admission (p < 0.001) and fatal outcomes (p < 0.001) using the corrected significance level (p = 0.0033). Sex, age, duration from onset to sampling, or number of detected microbes were not significantly associated with ICU admission or fatal outcomes. H1N1pdm detection was associated with ICU admission (odds ratio [OR] 3.911; 95% confidence interval [CI] 1.671-9.154) and fatal outcome (OR 5.496; 95% CI 1.814-16.653) after adjusting for the confounding factors of comorbidities, bacteria/Pneumocystis jirovecii co-detection, and age. CONCLUSIONS: H1N1pdm was associated with severe morbidity and death in adult patients hospitalized with respiratory symptoms. The diagnosis of subtype of influenza virus may be epidemiologically important.

Seroprevalences of multi-pathogen and description of farm movement in pigs in two provinces in Vietnam.

BACKGROUND: In Vietnam, lack of animal health information is considered a major challenge for pig production. The main objective of this study was to assess the seroprevalences of five pathogens [porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), mycoplasma hyopneumoniae (M. hyo), Japanese encephalitis virus (JEV) and leptospirosis] and to better characterize the farm movements through a survey. RESULTS: A total of 600 samples were collected from 120 farms from Bac Giang and Nghe An. Among unvaccinated herds, the highest seroprevalence was found for JE with 73.81% (95% CI: 68.39-78.74) in Bac Giang and 53.51% (95% CI 47.68-59.27) in Nghe An. Seroprevalences for PCV2 and M.hyo were 49.43% (95% CI: 45.06-53.80) and 46.06% (95% CI: 41.48-50.69) among unvaccinated animals. Accumulative co-infections for JE (86.25%) showed the highest level followed by M. hyo (66.25%) and PCV2 (62.50%). Three co-infections with JE had the highest positive rate (28.75%) followed by four co-infections (25.0%). Medium farms had relatively higher herd prevalences for all pathogens, except from leptospirosis. Overall, farmers exported/imported their pigs at the most 1-2 times every 6 months. Some respondents (5% for exportation and 20% for importation) had moved pigs more than 6 times over the last 6 months. CONCLUSIONS: Our study provided another pool of evidence that showed that PCV2, PRRS and H. hyo are endemic in pigs in Vietnam. Given the economic impacts of these pathogens elsewhere, the findings confirm the need for studies to evaluate the association between antibody response and clinical relevance as well as to assess the economic impact of co-infections at farm level. We also found that high seroprevalences of JE and leptospirosis were detected in pigs. From a pubic health point of view, it is crucial to raise public awareness especially for high risk occupations (mainly pig farm workers).

Prevalence and phylogenetic analysis of hepatitis E virus in pigs in Vietnam.

BACKGROUND: Hepatitis E virus (HEV) is a zoonotic disease and has been reported around the world. The main objective of this study was to evaluate the sero-prevalence and phylogenetic analysis of HEV in Vietnam. Pig blood and fecal pooled samples were collected to assess the prevalence of HEV. We assessed the true prevalence (TP) of HEV from apparent prevalence (AP) by taking into account the sensitivity and specificity of diagnostic tests using a Bayesian approach. For phylogenetic analysis, the data compared with worldwide HEV reference strains including all eight genotypes (G1-G8) which were identified in previous study. RESULTS: A total of 475 sera and 250 fecal pooled samples were collected at slaughterhouses and pig farms from five provinces, in Viet Nam. Overall, the sero-AP of HEV was 58.53% (95% confidence interval: 53.95-62.70) while the sero-TP was slightly higher (65.43, 95% credible interval: 47.19-84.70). In terms of pooled samples, overall, the RNA-AP was 6.80% (95% confidence interval: 4.01-10.66). One strain in Hanoi, two strains in Dak Lak, seven strains in An Giang, four strains in Son La and two strains in Nghe An were isolated. The phylogenetic tree demonstrated that 19 Vietnamese strains were clustered into HEV 3 and 4. CONCLUSIONS: This study provided evidence that HEV is circulating in domestic pigs in Vietnam. From a public health perspective, it is very important to raise public awareness for high-risk groups (e.g. slaughterhouse workers, pig traders, farmers and market sellers) who have more opportunities to come in contact with pig and contaminated meats.

Bioaerosol Sampling to Detect Avian Influenza Virus in Hanoi's Largest Live Poultry Market.

BACKGROUND: Newly emergent and virulent strains of H7N9 avian influenza virus are rapidly spreading in China and threaten to invade Vietnam. We sought to introduce aerosol sampling for avian influenza viruses in Vietnam. METHODS: During October 2017, National Institute for Occupational Safety and Health 2-stage aerosol samplers were assembled on a tripod and run for 4 hours. Concomitantly, up to 20 oropharyngeal (OP) swab samples were collected from chickens and ducks distanced at 0.2-1.5 m from each sampler. RESULTS: The 3 weeks of sampling yielded 30 aerosol samples that were 90% positive for influenza A, by quantitative reverse-transcription polymerase chain reaction, and 116 OP swab sample pools (5 samples per pool) that were 47% positive. Egg cultures yielded 1 influenza A virus (not H5 or H7) from aerosol and 25 influenza A viruses from OP swab sample pools (5 were H5 positive). The association between positive sample types (over time and position) was strong, with 91.7% of positive OP pooled swab samples confirmed by positive aerosol samples and 81% of influenza A positive aerosol samples confirmed by positive OP swab samples. CONCLUSIONS: We posit that aerosol sampling might be used for early warning screening of poultry markets for novel influenza virus detection, such as H7N9. Markets with positive aerosol samples might be followed up with more focused individual bird or cage swabbing, and back-tracing could be performed later to locate specific farms harboring novel virus. Culling birds in such farms could reduce highly pathogenic avian influenza virus spread among poultry and humans.

Chicken anemia virus in northern Vietnam: molecular characterization reveals multiple genotypes and evidence of recombination.

Chicken anemia virus (CAV) has a ubiquitous and worldwide distribution in the chicken production industry. Our group previously reported a high seroprevalence of CAV in chickens from northern Vietnam. In the present study, tissue samples collected from a total of 330 broiler and breeder commercial chickens in eleven provinces of northern Vietnam were tested for CAV infection. All samples were collected from clinically suspected flocks and diseased birds. The CAV genome was detected in 157 out of 330 (47.58%) chicken samples by real-time PCR. The rate of CAV genome detection in young chickens at 2-3 weeks of age (61.43%), which had not been previously reported in Vietnam, was significantly higher than that in older chickens at 4-11 (44.83%) and 12-28 (35.71%) weeks of age. For nine representative CAV strains from broiler chickens, analysis of the entire protein-coding region of the viral genome was conducted. Phylogenetic analysis of the VP1 gene indicated that the CAVs circulating in northern Vietnam were divided into three distinct genotypes: II, III, and V. Only one of the nine Vietnamese CAV strains clustered with a vaccine strain (Del-Ros), whereas the other eight strains did not cluster with any vaccine strains. Among the three genotypes, genotype III was most widely found in northern Vietnam and this included three sub-genotypes (IIIa, IIIb, and IIIc). The Vietnamese CAV strains were closely related to the Chinese, Taiwanese, and USA strains. One strain was defined to be of genotype V, which is a newly reported CAV genotype. Moreover, recombination analysis suggests that this novel genotype V was generated by recombination between genotype II and sub-genotype IIIc.

Application of the SureSelect target enrichment system for next-generation sequencing to obtain the complete genome sequence of bovine leukemia virus.

In this study, the SureSelect target enrichment system for Illumina Multiplexed Sequencing was applied to proviral DNA sequencing of bovine leukemia virus (BLV). The complete genomic DNA sequences of four Vietnamese BLV strains were successfully obtained with high read depth values and a genome coverage of 100% across all sequenced samples, in less than one week. This study provides the first complete Vietnamese BLV genome sequences. Their genetic variability and phylogenetic relationship were also analyzed and compared with those of 28 whole BLV genome sequences from different parts of the world. The results obtained provided new insights into the genetic diversity of the BLV tax gene, and further enabled us to identify nucleotide mutations in the gene that might not have been detected with the commercial detection kit that is currently available.

Correction to: Application of the SureSelect target enrichment system for next-generation sequencing to obtain the complete genome sequence of bovine leukemia virus.

Unfortunately, Figure 1 was incorrectly published in the original publication and the correct version is updated here.

Evaluation of the replication and pathogenicity of a variant avian paramyxovirus serotype 6 in mice.

Avian paramyxoviruses (APMVs) have been evaluated for their potential use as vaccine vectors, sparking research efforts leading to a better understanding of APMVs' replication and pathogenicity. However, within APMV serotypes, significant genetic diversity exists, and the infectivity of variant strains in mammals has not been studied. We utilized a mouse model to evaluate the pathogenicity of a variant strain of APMV-6 (APMV-6/red-necked stint/Japan/8KS0813/2008) in comparison with the prototype APMV-6 strain (APMV-6/duck/Hong Kong/18/199/1977). Although the two viruses differ substantially, both genetically and antigenically, we found that the variant and prototype strains could similarly replicate in respiratory tissues of infected mice and induce respiratory disease, sometimes resulting in death of the mice. Both viruses induced a humoral immune response that could be clearly detected by ELISA but which was poorly recognized by the hemagglutination inhibition test.

Characterization of cross-clade monoclonal antibodies against H5N1 highly pathogenic avian influenza virus and their application to the antigenic analysis of diverse H5 subtype viruses.

H5N1 highly pathogenic avian influenza viruses (HPAIVs) are a threat to both animal and public health and require specific and rapid detection for prompt disease control. We produced three neutralizing anti-hemagglutinin (HA) monoclonal antibodies (mAbs) using two clades (2.2 and 2.5) of the H5N1 HPAIV isolated in Japan. Blocking immunofluorescence tests showed that each mAb recognized different epitopes; 3B5.1 and 3B5.2 mAbs against the clade 2.5 virus showed cross-clade reactivity to all 26 strains from clades 1, 2.2, 2.3.2.1, 2.3.2.1a, b, c and 2.3.4, suggesting that the epitope(s) recognized are conserved. Conversely, the 1G5 mAb against the clade 2.2 virus showed reactivity to only clades 1, 2.3.4 and 2.5 strains. An analysis of escape mutants, and some clades of the H5N1 viruses recognized by 3B5.1 and 3B5.2 mAbs, suggested that the mAbs bind to an epitope, including amino acid residues at position 162 in the HA1 protein (R162 and K162). Unexpectedly, however, when five Eurasian-origin H5 low-pathogenic AIV (LPAIV) strains with R162 were examined (EA-nonGsGD clade) as well as two American-origin strains (Am-nonGsGD clade), the mAb recognized only EA-nonGsGD clade strains. The R162 and K162 residues in the HA1 protein were highly conserved among 36 of the 43 H5N1 clades reported, including clades 2.3.2.1a and 2.3.2.1c that are currently circulating in Asia, Africa and Europe. The amino acid residues (158-PTIKRSYNNTNQE-170) in the HA1 protein are probably an epitope responsible for the cross-clade reactivity of the mAbs, considering the epitopes reported elsewhere. The 3B5.1 and 3B5.2 mAbs may be useful for the specific detection of H5N1 HPAIVs circulating in the field.

Genetic characterization of H5N2 influenza viruses isolated from wild birds in Japan suggests multiple reassortment.

Low-pathogenic avian influenza viruses (LPAIVs) of the H5 subtype can mutate to highly pathogenic forms, potentially destabilizing the poultry industry. Wild migratory birds are considered a natural reservoir of LPAIVs capable of dispersing both high- and low-pathogenic forms of the virus. Therefore, surveillance and characterization of AIV in wild birds are essential. Here, we report on the isolation and genetic characterization of 10 AIVs of the H5N2 subtype obtained through surveillance in Hokkaido, Japan, during 2009 and 2011. Full-genome sequencing revealed that the H5 and N2 genes of these isolates are all closely related to each other, belonging to the Eurasian avian-like lineage, but they are unrelated to H5 highly pathogenic strains of clade 2.3.4.4. The internal genes of the isolates were found to be diverse, consistent with our hypothesis that these H5N2 strains have undergone multiple reassortment events. Even though all of the H5N2 isolates were characterized as LPAIV based on the amino acid sequences at the HA cleavage site, this analysis demonstrates a diverse pool of precursors that may seed future outbreaks in poultry and possible human transmissions, suggesting the need for high-quality surveillance.

Characterization of mAbs to chicken anemia virus and epitope mapping on its viral protein, VP1.

Three (MoCAV/F2, MoCAV/F8 and MoCAV/F11) of four mouse mAbs established against the A2/76 strain of chicken anemia virus (CAV) showed neutralization activity. Immunoprecipitation showed a band at ~50 kDa in A2/76-infected cell lysates by neutralizing mAbs, corresponding to the 50 kDa capsid protein (VP1) of CAV, and the mAbs reacted with recombinant VP1 proteins expressed in Cos7 cells. MoCAV/F2 and MoCAV/F8 neutralized the 14 CAV strains tested, whereas MoCAV/F11 did not neutralize five of the strains, indicating distinct antigenic variation amongst the strains. In blocking immunofluorescence tests with the A2/76-infected cells, binding of MoCAV/F11 was not inhibited by the other mAbs. MoCAV/F2 inhibited the binding of MoCAV/F8 to the antigens and vice versa, suggesting that the two mAbs recognized the same epitope. However, mutations were found in different parts of VP1 of the escape mutants of each mAb: EsCAV/F2 (deletion of T89+A90), EsCAV/F8 (I261T) and EsCAV/F11 (E144G). Thus, the epitopes recognized by MoCAV/F2 and MoCAV/F8 seemed to be topographically close in the VP1 structure, suggesting that VP1 has at least two different neutralizing epitopes. However, MoCAV/F8 did not react with EsCAV/F2 or EsCAV/F8, suggesting that binding of MoCAV/F8 to the epitope requires coexistence of the epitope recognized by MoCAV/F2. In addition, MoCAV/F2, with a titre of 1 : 12 800 to the parent strain, neutralized EsCAV/F2 and EsCAV/F8 with low titres of 32 and 152, respectively. The similarity of the reactivity of MoCAV/F2 and MoCAV/F8 to VP1 may also suggest the existence of a single epitope recognized by these mAbs.

Parrot bornavirus-2 and -4 RNA detected in wild bird samples in Japan are phylogenetically adjacent to those found in pet birds in Japan.

Bornaviruses (family Bornaviridae) are non-segmented negative-strand RNA viruses. Avian bornaviruses (ABVs), which are causative agents of proventricular dilatation disease, are a genetically diverse group with at least 15 genotypes, including parrot bornaviruses (PaBVs) and aquatic bird bornavirus 1(ABBV-1). Borna disease virus 1(BoDV-1), which infects mammals and causes neurological diseases, has also been reported to infect avian species, although the numbers of the cases have been markedly fewer than those of ABVs. In this study, we conducted genetic surveillance to detect ABVs (PaBV-1 to -5 and ABBV-1) and BoDV-1 in wild birds in Japan. A total of 2078 fecal or cloacal swab samples were collected from wild birds in 2006, 2007, 2008, and 2011, in two regions of Japan. The results demonstrated the presence of PaBV-2 and -4 RNA, while no positive results for other PaBVs, ABBV-1, and BoDV-1 were obtained. PaBV-2 and -4 RNA were detected in 18 samples (0.9 %) of the genera Anas, Grus, Larus, Calidris, Haliaeetus, and Emberiza, in which either PaBV-2 RNA or PaBV-4 RNA, or both PaBV-2 and -4 RNA were detected in 15 (0.7 %), 5 (0.2 %), and 2 (0.1 %) samples, respectively. The nucleotide sequences of PaBV-2 and -4 detected in these samples from wild birds are phylogenetically close to those found in samples from pet birds in Japan, with identities ranging from 99.8 to 100 % and from 98.2 to 99.4 %, respectively. To the best of our knowledge, this is the first report on the detection of PaBV-2 and -4 RNA detected in samples from wild birds.

Genetic characterization of a rare H12N3 avian influenza virus isolated from a green-winged teal in Japan.

This study reports on the genetic characterization of an avian influenza virus, subtype H12N3, isolated from an Eurasian green-winged teal (Anas crecca) in Japan in 2009. The entire genome sequence of the isolate was analyzed, and phylogenetic analyses were conducted to characterize the evolutionary history of the isolate. Phylogenetic analysis of the hemagglutinin and neuraminidase genes indicated that the virus belonged to the Eurasian-like avian lineage. Molecular dating indicated that this H12 virus is likely a multiple reassortant influenza A virus. This is the first reported characterization of influenza A virus subtype H12N3 isolated in Japan and these data contribute to the accumulation of knowledge on the genetic diversity and generation of novel influenza A viruses.

Influence of olive-derived hydroxytyrosol on the toll-like receptor 4-dependent inflammatory response of mouse peritoneal macrophages.

Macrophages play important roles in the host innate immune response and are involved in the onset of diseases caused by inflammation. Toll-like receptor 4 (TLR4)-mediated inflammatory responses of macrophages may be associated with diseases such as diabetes and diseases of the cardiovascular system. Hydroxytyrosol (HT) exerts strong antioxidant and anti-inflammatory effects and may be applied in the treatment of inflammatory diseases. In the present study conducted in vitro, we investigated the effects of the TLR4-dependent anti-inflammatory effect of HT on peritoneal macrophage of BALB/c mice. We show here that the elevated levels of iNOS gene expression and nitric oxide production induced by lipopolysaccharide (LPS) (0.25 µg/ml) were suppressed by HT (12.5 µg/ml). LPS-dependent NF-κB gene expression and phosphorylation of NF-κB were not affected by HT under these conditions. In contrast, the expression of TNF-α was significantly increased in the presence of LPS and HT. These results suggest that HT suppressed nitric oxide production by decreasing iNOS gene expression through a mechanism independent of the NF-κB signaling pathway. These novel findings suggest that the modulation by HT of the expression of genes involved in inflammation may involve multiple mechanisms.

Virucidal effect of acidic electrolyzed water and neutral electrolyzed water on avian influenza viruses.

The virucidal effects of two types of electrolyzed water, acidic electrolyzed water (AEW) and neutral electrolyzed water (NEW), on avian influenza viruses were studied. Virus titers of the highly pathogenic H5N1 virus and the low-pathogenic H9N2 virus irreversibly decreased by >5-log at 1 min after the viruses were mixed with NEW containing ≥43 ppm free available chlorine (FAC), but not with NEW containing <17 ppm FAC. The minimum concentration of FAC for a virucidal effect of NEW was estimated at around 40 ppm. In contrast, the virus titers decreased by >5 log at 1 min after the viruses were mixed with AEW, in which the concentration of the FAC ranged from 72 to 0 ppm. Thus, the virucidal effect of AEW did not depend on the presence of FAC. Reverse transcription polymerase chain reaction amplified fragments of the M and NP genes, but not the complete M gene, from RNA extracted from the AEW-inactivated virus. Moderate morphological changes were found under the electron microscope, although no changes were observed in the electrophoresed proteins of the AEW-inactivated virus. No viral genes were amplified from the RNA extracted from the NEW-inactivated virus, regardless of the length of the targeted genes. No viral particles were detected under the electron microscope and no viral proteins were detected by electrophoresis for the NEW-inactivated virus. Thus, this study demonstrated potent virucidal effects of AEW and NEW and differences in the virucidal mechanism of the two types of electrolyzed water.

Characterization of a genetic and antigenic variant of avian paramyxovirus 6 isolated from a migratory wild bird, the red-necked stint (Calidris ruficollis).

A hemagglutinating virus (8KS0813) was isolated from a red-necked stint. Hemagglutination inhibition and neutralization tests indicated that 8KS0813 was antigenically related to a prototype strain, APMV-6/duck/Hong Kong/18/199/77, but with an 8- and 16-fold difference, respectively, in their titers. The full genome sequence of 8KS0813 showed 98.6 % nucleotide sequence identity to that of APMV-6/duck/Italy/4524-2/07, which has been reported to belong to an APMV-6 subgroup, and showed less similarity to that of the prototype strain (70.6 % similarity). The growth of 8KS0813 and the prototype strain in four different cell cultures was greatly enhanced by adding trypsin. Interestingly, this virus induced syncytia only in Vero cells. 8KS0813 was identified as APMV-6/red-necked stint/Japan/8KS0813/08, but it is antigenically and genetically distinguishable from the prototype strain, suggesting that variant APMV-6 is circulating in migratory birds.

Genetic characterization of an H5N1 avian influenza virus from a vaccinated duck flock in Vietnam.

This study reports the genetic characterization of a highly pathogenic avian influenza virus subtype H5N1 isolated from a moribund domestic duck in central Vietnam during 2012. In the moribund duck's flock, within 6 days after vaccination with a commercial H5N1 vaccine (Re-5) to 59-day-old birds, 120 out of 2,000 ducks died. Genetic analysis revealed a substantial number of mutations in the HA gene of the isolate in comparison with the vaccine strains, Re-1 and Re-5. Similar mutations were also found in selected Vietnamese H5N1 strains isolated since 2009. Mutations in the HA gene involved positions at antigenic sites associated with antibody binding and also neutralizing epitopes, with some of the mutations resulting in the modification of N-linked glycosylation of the HA. Those mutations may be related to the escape of virus from antibody binding and the infection of poultry, interpretations which may be confirmed through a reverse genetics approach. The virus also carried an amino acid substitution in the M2, which conferred a reduced susceptibility to amantadine, but no neuraminidase inhibitor resistance markers were found in the viral NA gene. Additional information including vaccination history in the farm and the surrounding area is needed to fully understand the background of this outbreak. Such understanding and expanded monitoring of the H5N1 influenza viruses circulating in Vietnam is an urgent need to provide updated information to improve effective vaccine strain selection and vaccination protocols, aiding disease control, and biosecurity to prevent H5N1 infection in both poultry and humans.