H5 Avian Influenza Viruses Research Articles

Serological and Molecular Detection of Avian Influenza Virus ‎Hemagglutinin (H5) in Wild Birds in and around Zaria, Nigeria

Avian influenza (AI) has a worldwide distribution and affects domestic and wild birds, thus causing great economic losses to the poultry industry. This study was carried out to detect avian influenza virus H5 antibodies and nucleic acidin some wild birds [Laughing doves (Spilolepia senegalensis), Speckled pigeons (Columba guinea), Cattle egrets (Bubulcus ibis), Senegalese parrots (Poicephalussenegalus), Mallards (Anas platyrhynchos) and Geese (Anseranserini)] in Zaria and its environs, Kaduna State Nigeria. Sera were tested for avian influenza virus (AIV) H5 antibody using competitive enzyme linked immunosorbent assay (cELISA). Pooled oropharyngeal and cloacal swabs of each bird species (8-10 samples) were tested for AIV nucleic acidusing one-stepreverse transcriptase polymerase chain reaction (RT-PCR). Results revealed overall prevalence of 6.62% and 3.85% for AIV antibody and nucleic acid respectively. Based on species, AIV antibody was detected in laughing dove (10%), speckled pigeon (13.64%) and mallard (19.05%).Also, AIV antigen was detected in Senegalese parrot (20%).In conclusion, AIV antibody and antigen were detected in wild birds in Zaria, Nigeria. Thus, these species of birds could play significant roles in the spread of this virus to chickens. Therefore, measures to limit the interactions of these wild birds with chickens should be implemented to minimize the spread of AI.

Antigenic Characterization of Low and Highly Pathogenic H5 Avian Influenza Viruses using Antigenic Cartography

Antigenic Characterization of Low and Highly Pathogenic H5 Avian Influenza Viruses using Antigenic Cartography

Antigenic Characterization of Low and Highly Pathogenic H5 Avian Influenza Viruses using Antigenic Cartography

Antigenic Characterization of Low and Highly Pathogenic H5 Avian Influenza Viruses using Antigenic Cartography

Non-competitive fluorescence polarization immunoassay for detection of H5 avian influenza virus using a portable analyzer.

Nowadays, the diagnosis of viral infections is receiving broad attention. We have developed a non-competitive fluorescence polarization immunoassay (NC-FPIA), which is a separation-free immunoassay, for a virus detection. H5 subtype avian influenza virus (H5-AIV) was used as a model virus for the proof of concept. The fluorescein-labeled Fab fragment that binds to H5 hemagglutinin was used for NC-FPIA. The purified H5-AIV which has H5 hemagglutinin was mixed with the fluorescein-labeled Fab fragment. After that, the degree of fluorescence polarization was measured with a portable FPIA analyzer. H5-AIV was successfully detected with an incubation time of 15 min. In addition, the portable FPIA analyzer enables performance of on-site NC-FPIA with a sample volume of 20 μL or less. This is the first research of detecting a virus particle by FPIA. This NC-FPIA can be applied to rapid on-site diagnosis of various viruses.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-021-03193-y.

The role of glycosylation in the N-terminus of the hemagglutinin of a unique H4N2 with a natural polybasic cleavage site in virus fitness in vitro and in vivo

ABSTRACT To date, only low pathogenic (LP) H5 and H7 avian influenza viruses (AIV) have been observed to naturally shift to a highly pathogenic (HP) phenotype after mutation of the monobasic hemagglutinin (HA) cleavage site (HACS) to polybasic motifs. The LPAIV monobasic HACS is activated by tissue-restricted trypsin-like enzymes, while the HPAIV polybasic HACS is activated by ubiquitous furin-like enzymes. However, glycosylation near the HACS can affect proteolytic activation and reduced virulence of some HPAIV in chickens. In 2012, a unique H4N2 virus with a polybasic HACS was isolated from quails but was LP in chickens. Whether glycosylation sites (GS) near the HACS hinder the evolution of HPAIV H4N2 remains unclear. Here, we analyzed the prevalence of potential GS in the N-terminus of HA1, 2NYT4 and 18NGT20, in all AIV sequences and studied their impact on H4N2 virus fitness. Although the two motifs are conserved, some non-H5/H7 subtypes lack one or both GS. Both sites were glycosylated in this H4N2 virus. Deglycosylation increased trypsin-independent replication in cell culture, cell-to-cell spread and syncytium formation at low-acidic pH, but negatively affected the thermostability and receptor-binding affinity. Alteration of 2NYT4 with or without 18NGT20 enabled systemic spread of the virus to different organs including the brain of chicken embryos. However, all intranasally inoculated chickens did not show clinical signs. Together, although the conserved GS near the HACS are important for HA stability and receptor binding, deglycosylation increased the H4N2 HA-activation, replication and tissue tropism suggesting a potential role for virus adaptation in poultry.

Development and evaluation of a monoclonal antibody-based competitive ELISA for the detection of antibodies against H7 avian influenza virus

BackgroundH7 subtype avian influenza has caused great concern in the global poultry industry and public health. The conventional serological subtype-specific diagnostics is implemented by hemagglutination inhibition (HI) assay despite lengthy operation time. In this study, an efficient, rapid and high-throughput competitive enzyme-linked immunosorbent assay (cELISA) was developed for detection of antibodies against H7 avian influenza virus (AIV) based on a novel monoclonal antibody specific to the hemagglutinin (HA) protein of H7 AIV.ResultsThe reaction parameters including antigen coating concentration, monoclonal antibody concentration and serum dilution ratio were optimized for H7 antibody detection. The specificity of the cELISA was tested using antisera against H1 ~ H9, H11 ~ H14 AIVs and other avian viruses. The selected cut-off values of inhibition rates for chicken, duck and peacock sera were 30.11, 26.85 and 45.66% by receiver-operating characteristic (ROC) curve analysis, respectively. With HI test as the reference method, the minimum detection limits for chicken, duck and peacock positive serum reached 20, 21 and 2− 1 HI titer, respectively. Compared to HI test, the diagnostic accuracy reached 100, 98.6, and 99.3% for chicken, duck and peacock by testing a total of 400 clinical serum samples, respectively.ConclusionsIn summary, the cELISA assay developed in this study provided a reliable, specific, sensitive and species-independent serological technique for rapid detection of H7 antibody, which was applicable for large-scale serological surveillance and vaccination efficacy evaluation programs.

Genetic characteristics and pathogenicity of novel reassortant H6 viruses isolated from wild birds in China

During our routine surveillance, we isolated seven H6 avian influenza virus (AIV) strains, including three H6N1 strains, three H6N2 strains, and one H6N8 strain, from 3667 fresh fecal samples that were collected from wild bird habitats in China from March 2017 and May 2019. Phylogenetic analysis revealed that these viruses formed five different genotypes and have undergone complicate reassortment during their evolution by acquiring genes from AIVs of both Eurasian and North American lineages that have been previously detected in migrating waterfowl and poultry. Viral pathogenesis in mice showed that these H6 viruses replicated efficiently in both the nasal turbinates and lungs of mice without pre-adaptation, but none of them were lethal for mice. We studied the genetic characteristic and biological property of novel reassortant H6 viruses isolated from wild birds in China. It also highlights the need for continued surveillance of H6 AIVs circulating in nature.

A reassortant highly pathogenic avian influenza H5N6 virus originating from the wildbird-origin H5N6 and the poultry H9N2/H7N9 viruses in Xinjiang, China

The asian lineage of H5, H7, and H9 avian influenza viruses (AIVs) is the most prevalent in the world, and the co-existence of H5, H7, and H9 viruses provide an opportunity to generate some novel reassortants. Herein we report our isolation of two H5N6 virus strains (named XJ-H5N6) from domestic geese in live poultry markets (LPMs) in Urumqi during the winter of 2016-2017, and analysis of the viral genetic mutations, pathogenicity, reassortant, and origin. BLAST analysis revealed that the HA, NA, MP, NP, and NS of XJ-H5N6 had the highest sequence identity (98.5-99.5%) with those 5 genes from H5N6 highly pathogenic avian influenza virus (HPAIV) isolated from wild birds in Ningxia (NX-WB-H5N6); the viral PA and PB1 had the highest sequence identity (98.6% and 98.9%) with those 2 genes from HPAIV strain A/Environment/Chongqing/45373/2015 (H5N6) (CQ-EN-H5N6); while the viral PB2 had the highest sequence identity (99.0%) with this gene from low pathogenic avian influenza virus (LPAIV) strain A/environment/Jiangsu/01.20 TCCX004/2015 (H9N2). Except for the PB2, phylogenetic analysis showed that the other 7 viral genes were clustered together with NX-WB-H5N6 and CQ-EN-H5N6, while the viral PB2 was clustered together with the poultry H9N2/H7N9 viruses. The viral HA protein contained multiple basic amino acid residues (RRRKR/GL) at the cleavage site which represents HPAIV. Additionally, the viral multiple mutations could enhance the viral virulence and the capacity of cross-species transmission. Our data indicate that XJ-H5N6 has been generated by the reassortant of H5N6 HPAIV from wild birds in Ningxia and H9N2/H7N9 LPAIV in poultry. The results of this study may help us understand the viral adaptation and transmission patterns from wild birds to poultry.

Novel reassortant 2.3.4.4B H5N6 highly pathogenic avian influenza viruses circulating among wild, domestic birds in Xinjiang, Northwest China.

BackgroundThe H5 avian influenza viruses (AIVs) of clade 2.3.4.4 circulate in wild and domestic birds worldwide. In 2017, nine strains of H5N6 AIVs were isolated from aquatic poultry in Xinjiang, Northwest China.ObjectivesThis study aimed to analyze the origin, reassortment, and mutations of the AIV isolates.MethodsAIVs were isolated from oropharyngeal and cloacal swabs of poultry. Identification was accomplished by inoculating isolates into embryonated chicken eggs and performing hemagglutination tests and reverse transcription polymerase chain reaction (RT-PCR). The viral genomes were amplified with RT-PCR and then sequenced. The sequence alignment, phylogenetic, and molecular characteristic analyses were performed by using bioinformatic software.ResultsNine isolates originated from the same ancestor. The viral HA gene belonged to clade 2.3.4.4B, while the NA gene had a close phylogenetic relationship with the 2.3.4.4C H5N6 highly pathogenic avian influenza viruses (HPAIVs) isolated from shoveler ducks in Ningxia in 2015. The NP gene was grouped into an independent subcluster within the 2.3.4.4B H5N8 AIVs, and the remaining six genes all had close phylogenetic relationships with the 2.3.4.4B H5N8 HPAIVs isolated from the wild birds in China, Egypt, Uganda, Cameroon, and India in 2016–2017, Multiple basic amino acid residues associated with HPAIVs were located adjacent to the cleavage site of the HA protein. The nine isolates comprised reassortant 2.3.4.4B HPAIVs originating from 2.3.4.4B H5N8 and 2.3.4.4C H5N6 viruses in wild birds.ConclusionsThese results suggest that the Northern Tianshan Mountain wetlands in Xinjiang may have a key role in AIVs disseminating from Central China to the Eurasian continent and East African.

Comparative pathogenicity and environmental transmission of recent highly pathogenic avian influenza H5 viruses

ABSTRACT Strategies to control spread of highly pathogenic avian influenza (HPAI) viruses by wild birds appear limited, hence timely characterization of novel viruses is important to mitigate the risk for the poultry sector and human health. In this study we characterize three recent H5-clade 2.3.4.4 viruses, the H5N8-2014 group A virus and the H5N8-2016 and H5N6-2017 group B viruses. The pathogenicity of the three viruses for chickens, Pekin ducks and Eurasian wigeons was compared. The three viruses were highly pathogenic for chickens, but the two H5N8 viruses caused no to mild clinical symptoms in both duck species. The highest pathogenicity for duck species was observed for the most recent H5N6-2017 virus. For both duck species, virus shedding from the cloaca was higher after infection with group B viruses compared to the H5N8-2014 group A virus. Higher cloacal virus shedding of wild ducks may increase transmission between wild birds and poultry. Environmental transmission of H5N8-2016 virus to chickens was studied, which showed that chickens are efficiently infected by (fecal) contaminated water. These results suggest that pathogenicity of HPAI H5 viruses and virus shedding for ducks is evolving, which may have implications for the risk of introduction of these viruses into the poultry sector.

Annual Report on surveillance for Avian Influenza in poultryand wild birds in Member States of the European Union in 2019.

Context. Avian influenza (AI) is an infectious viral disease that affects all species of domestic and wild birds. The viruses causing this disease can be of high (HPAI) or low (LPAI) pathogenicity and represent a continuous threat to poultry in Europe. Council Directive 2005/94/EC requires EU Member States (MSs) to carry out surveillance in poultry and wild birds and notify the results to the responsible authority. Therefore, MSs, Iceland, Norway, Switzerland and the United Kingdom have implemented ongoing surveillance programmes to monitor incursions of AI viruses in poultry and wild birds. EFSA received a mandate from the European Commission to collate, validate, analyse and summarise the data resulting from the avian influenza surveillance programmes in an annual report. Poultry. Overall 24,419 poultry establishments (PEs) were sampled, of which 87 were seropositive for H5 virus strains and 22 for H7 strains. Seropositive PEs were found in eight MSs (Belgium, Bulgaria, Denmark, France, Germany, the Netherlands, Poland, Spain) and the United Kingdom (also a MS at the time of collection). The highest percentage of seropositive PEs was found in establishments raising waterfowl game birds and breeding geese. Out of the 109 PEs with positive serological tests for H5/H7, only two tested positive in PCR and virology for H5/H7 virus strains, both of which were LPAI strains (H5N1 and H7N7, respectively) and were reported by Denmark. In addition, 12 countries also reported PCR results from 653 PEs carried out either as a screening test or subsequent to a negative serological test result. Five of these PEs were found positive for AI viral RNA: four H5N8 HPAI in Bulgaria and one H7N3 LPAI in Italy. Wild birds. A total of 19,661 dead/moribund wild birds were sampled, with one bird testing positive to HPAI virus H5N6, which was reported by Denmark. In addition, there were 84 birds testing positive for LPAI H5 or H7 virus and 848 birds testing positive for non‐H5/H7 AI virus, reported by 30 countries. The surveillance findings for poultry and wild birds for 2019 are discussed in relation to findings from previous years and current knowledge of the epidemiology of AI in Europe.

Highly Pathogenic Avian Influenza H5N8 in Poland in 2019-2020.

IntroductionRepeated incursions of highly pathogenic avian influenza virus (HPAIV) H5 subtype of Gs/GD lineage pose a serious threat to poultry worldwide. We provide a detailed analysis of the spatio-temporal spread and genetic characteristics of HPAIV Gs/GD H5N8 from the 2019/20 epidemic in Poland.Material and methodsSamples from poultry and free-living birds were tested by real-time RT-PCR. Whole genome sequences from 24 (out of 35) outbreaks were generated and genetic relatedness was established. The clinical status of birds and possible pathways of spread were analysed based on the information provided by veterinary inspections combined with the results of phylogenetic studies.ResultsBetween 31 December 2019 and 31 March 2020, 35 outbreaks in commercial and backyard poultry holdings and 1 case in a wild bird were confirmed in nine provinces of Poland. Most of the outbreaks were detected in meat turkeys and ducks. All characterised viruses were closely related and belonged to a previously unrecognised genotype of HPAIV H5N8 clade 2.3.4.4b. Wild birds and human activity were identified as the major modes of HPAIV spread.ConclusionThe unprecedentedly late introduction of the HPAI virus urges for re-evaluation of current risk assessments. Continuous vigilance, strengthening biosecurity and intensifying surveillance in wild birds are needed to better manage the risk of HPAI occurrence in the future.

Equine-Like H3 Avian Influenza Viruses in Wild Birds, Chile.

Since their discovery in the United States in 1963, outbreaks of infection with equine influenza virus (H3N8) have been associated with serious respiratory disease in horses worldwide. Genomic analysis suggests that equine H3 viruses are of an avian lineage, likely originating in wild birds. Equine-like internal genes have been identified in avian influenza viruses isolated from wild birds in the Southern Cone of South America. However, an equine-like H3 hemagglutinin has not been identified. We isolated 6 distinct H3 viruses from wild birds in Chile that have hemagglutinin, nucleoprotein, nonstructural protein 1, and polymerase acidic genes with high nucleotide homology to the 1963 H3N8 equine influenza virus lineage. Despite the nucleotide similarity, viruses from Chile were antigenically more closely related to avian viruses and transmitted effectively in chickens, suggesting adaptation to the avian host. These studies provide the initial demonstration that equine-like H3 hemagglutinin continues to circulate in a wild bird reservoir.

Evolution and pathogenicity of H6 avian influenza viruses isolated from Southern China during 2011 to 2017 in mice and chickens

H6 subtype avian influenza viruses spread widely in birds and pose potential threats to poultry and mammals, even to human beings. In this study, the evolution and pathogenicity of H6 AIVs isolated in live poultry markets from 2011 to 2017 were investigated. These H6 isolates were reassortant with other subtypes of influenza virus with increasing genomic diversity. However, no predominant genotype was found during this period. All of the H6N2 and most of the H6N6 isolates replicated efficiently in lungs of inoculated mice without prior adaptation. All of the H6N2 and two H6N6 isolates replicated efficiently in nasal turbinates of inoculated mice, which suggested the H6N2 viruses were more adaptive to the upper respiratory tract of mice than the H6N6 viruses. One of H6N2 virus caused systemic infection in one out of three inoculated mice, which indicated that H6 avian influenza virus, especially the H6N2 viruses posed a potential threat to mammals. Five H6 strains selected from different genotypes caused no clinical signs to inoculated chickens, and their replication were limited in chickens since the viruses have been detected only from a few tissues or swabs at low titers. Our study strongly suggests that the H6 avian influenza virus isolated from live poultry markets pose potential threat to mammals.

Dominant subtype switch in avian influenza viruses during 2016\u20132019 in China

We have surveyed avian influenza virus (AIV) genomes from live poultry markets within China since 2014. Here we present a total of 16,091 samples that were collected from May 2016 to February 2019 in 23 provinces and municipalities in China. We identify 2048 AIV-positive samples and perform next generation sequencing. AIV-positive rates (12.73%) from samples had decreased substantially since 2016, compared to that during 2014–2016 (26.90%). Additionally, H9N2 has replaced H5N6 and H7N9 as the dominant AIV subtype in both chickens and ducks. Notably, novel reassortants and variants continually emerged and disseminated in avian populations, including H7N3, H9N9, H9N6 and H5N6 variants. Importantly, almost all of the H9 AIVs and many H7N9 and H6N2 strains prefer human-type receptors, posing an increased risk for human infections. In summary, our nation-wide surveillance highlights substantial changes in the circulation of AIVs since 2016, which greatly impacts the prevention and control of AIVs in China and worldwide.

Identification of Efficacious Vaccines Against Contemporary North American H7 Avian Influenza Viruses

Five vaccines, including four inactivated, whole-virus water-in-oil adjuvanted vaccines and a commercial non-replicating alphavirus vectored RNA particle (RP) vaccine were evaluated in chickens for their ability to provide protection against challenge with a recent H7 highly pathogenic avian influenza virus (AIV) from the US (A/turkey/IN/1403-1/2016 H7N8). One of the inactivated vaccines and the RP vaccine were prepared with A/turkey/IN/16-01571-6/2016 H7N8 low pathogenic AIV (TK/IN/16), which is identical to the challenge virus, except for the proteolytic cleavage site of the HA protein. The remaining 3 inactivated vaccines were prepared with other North American H7 LPAIV viruses. Hemagglutination inhibition assay was used to evaluate the antigenic relationships among the vaccines and selected recent H7 AIV isolates. All five vaccines provided protection against mortality. The inactivated vaccines reduced virus shedding significantly at 2 and 4 days post challenge compared to sham vaccinated chickens. In contrast, the RP vaccine did not significantly reduce virus shedding. The inactivated vaccine prepared with TK/IN/16 elicited the highest antibody responses, which suggests this is a strong candidate for use as an antigen for North American H7 AIVs. Antigenic distance calculations showed that the four inactivated vaccine strains and other recent North American H7 isolates are antigenically similar, which suggests that the vaccines evaluated here would be similar enough to provide protection to other North American H7 AIVs. If future H7 outbreaks in poultry warrant vaccination, the field strain can be rapidly evaluated with these antigens and if adequately related, one of these characterized strains may be utilized.

Antibody Response of an H9 Subtype Avian Influenza Poultry Vaccine on Three Kinds of Wild Birds in Shanghai Zoo.

A semiannual immunization with a commercial inactivated H9 subtype avian influenza virus (AIV) vaccine developed for poultry has been used to prevent and control the avian influenza (AI) infections among captured wild birds in Shanghai Zoo. However, the overall safety and effectiveness of the poultry vaccine for housed birds in the zoo remain unclear. To verify the safety and efficacy of the commercial inactivated H9 AI vaccine on zoo birds and to explore a more reasonable and effective immunization procedure, 48 zoo birds, including 11 Oriental white storks, 25 peafowl, and 12 silver pheasants, were administered the AI vaccine developed for poultry use. Then, the clinical signs of the immunized birds were observed for 2 weeks, and the antibodies against H9 AI were determined via the hemagglutination inhibition test. Results showed that no harmful effects related to the vaccination were observed, and the antibody titers of the Oriental white stork, peafowl, and silver pheasants were all higher than 7 log 2 at 21 days, 30 days, 60 days, 120 days, and 180 days postimmunization. For further study, the H9 AIV titers of 11 peafowls and 6 Oriental storks, which were raised in the nursing ground, were continuously monitored for 15 months. All of their antibody titers were above the national standards of China (5 log 2; GB/T18936-2003), even at 12 months and 15 months postimmunization. We concluded that the commercial inactivated H9 AI vaccine used at the present time in Shanghai Zoo can induce high and prolonged immune responses in vaccinated birds.

Selection and antigenic characterization of immune-escape mutants of H7N2 low pathogenic avian influenza virus using homologous polyclonal sera

Understanding the dynamics of the selection of influenza A immune escape variants by serum antibody is critical for designing effective vaccination programs for animals, especially poultry where large populations have a short generation time and may be vaccinated with high frequency. In this report, immune-escape mutants of A/turkey/New York/4450/1994 H7N2 low pathogenic avian influenza virus, were selected by serially passaging the virus in the presence of continuously increasing concentrations of homologous chicken polyclonal sera. Amino acid mutations were identified by sequencing the parental hemagglutinin (HA) gene and every 10 passages by both Sanger and deep sequencing, and the antigenic distance of the mutants to the parent strain was determined. Progressively, a total of five amino acid mutations were observed over the course of 30 passages. Based on their absence from the parental virus with deep sequencing, the mutations appear to have developed de novo. The antigenic distance between the selected mutants and the parent strain increased as the number of amino acid mutations accumulated and the concentration of antibodies had to be periodically increased to maintain the same reduction in virus titer during selection. This selection system demonstrates how H7 avian influenza viruses behave under selection with homologous sera, and provides a glimpse of their evolutionary dynamics, which can be applied to developing vaccination programs that maximize the effectiveness of a vaccine over time.

Characterization of H3 subtype avian influenza viruses isolated from poultry in Vietnam.

To date, avian influenza viruses (AIVs) have persisted in domestic poultry in wet markets in East Asian countries. We have performed ongoing virus surveillance in poultry populations in Vietnam since 2011, with the goal of controlling avian influenza. Throughout this study, 110 H3 AIVs were isolated from 2760 swab samples of poultry in markets and duck farms. H3 hemagglutinin (HA) genes of the isolates were phylogenetically classified into eight groups (I-VIII). Genetic diversity was also observed in the other seven gene segments. Groups I-IV also included AIVs from wild waterbirds. The epidemic strains in poultry switched from groups I-III and VI to groups I, IV, V, and VIII around 2013. H3 AIVs in groups I and V were maintained in poultry until at least 2016, which likely accompanied their dissemination from the northern to the southern regions of Vietnam. Groups VI-VIII AIVs were antigenically distinct from the other groups. Some H3 AIV isolates had similar N6 neuraminidase and matrix genes as H5 highly pathogenic avian influenza viruses (HPAIVs). These results reveal that genetically and antigenically different H3 AIVs have been co-circulating in poultry in Vietnam. Poultry is usually reared outside in this country and is at risk of infection with wild waterbird-originating AIVs. In poultry flocks, the intruded H3 AIVs must have experienced antigenic drift/shift and genetic reassortment, which could contribute to the emergence of H5 HPAIVs with novel gene constellations.

The Single E627K Amino Acid Substitution in PB2 Enhances the Pathogenicity of Wild-Bird-Origin H6N6 Subtype Avian Influenza Virus in Mice

Avian Influenza Viruses (AIVs) are harbored by wild waterfowl as a natural host, and there is a species barrier restricting virus transmission from birds to mammals, including humans. However, it has been reported that, through genetic mutations, AIVs occasionally infect mammals and acquire high pathogenicity. The Amino Acid (aa) substitution of glutamic acid to lysine at position 627 (E627K) in polymerase basic protein 2 (PB2) is one of the wellknown factors underlying mammalian adaptation. Although this substitution was previously observed in mammalian-adapted H5, H7, and H9 AIV subtypes, the impact of this mutation on the mammalian adaptation of other AIV subtypes is not fully verified. Here, we isolated the low pathogenic AIV subtype H6N6 from a wild bird fecal sample in Tokachi Subprefecture, Hokkaido, Japan. We passaged this H6N6 subtype in BALB/c mice four times and acquired the mouse-adapted virus. Whole-genome sequence analysis showed that the adapted virus had only one aa substitution (E627K) in PB2. The adapted virus-inoculated mice tended to show increased weight loss and mortality compared with the original virus-inoculated mice. The viral titer in the lungs of the adapted virus-inoculated mice was significantly higher than that of the original virus-inoculated mice. Additionally, the virus isolated from the lung of the original virus-inoculated mice with serious symptoms harbored the E627K substitution. Our findings indicate the possibility that the PB2 E627K substitution in H6N6 subtype AIV rapidly appears in mammalian hosts and contributes to the enhanced pathogenicity of this virus.