Characterization Of Avian Influenza Viruses Research Articles

Analyzing Molecular Traits of H9N2 Avian Influenza Virus Isolated from a Same Poultry Farm in West Java Province, Indonesia, in 2017 and 2023

Background Indonesia is one of the countries that is endemic to avian influenza virus subtype H9N2. This study aims to compare the molecular characteristics of avian influenza virus (AIV) subtype H9N2 from West Java. Methods Specific pathogen-free (SPF) embryonated chicken eggs were used to inoculate samples. RNA extraction and RT–qPCR confirmed the presence of H9 and N2 genes in the samples. RT–PCR was employed to amplify the H9N2-positive sample. Nucleotide sequences were obtained through Sanger sequencing and analyzed using MEGA 7. Homology comparison and phylogenetic tree analysis, utilizing the neighbor-joining tree method, assessed the recent isolate’s similarity to reference isolates from GenBank. Molecular docking analysis was performed on the HA1 protein of the recent isolate and the A/Layer/Indonesia/WestJava-04/2017 isolate, comparing their interactions with the sialic acids Neu5Ac2-3Gal and Neu5Ac2-6Gal. Results RT–qPCR confirmed the isolate samples as AIV subtype H9N2. The recent virus exhibited 11 amino acid residue differences compared to the A/Layer/Indonesia/WestJava-04/2017 isolate. Phylogenetically, the recent virus remains within the h9.4.2.5 subclade. Notably, at antigenic site II, the recent isolate featured an amino acid N at position 183, unlike A/Layer/Indonesia/WestJava-04/2017. Molecular docking analysis revealed a preference of HA1 from the 2017 virus for Neu5Ac2-3Gal, while the 2023 virus displayed a tendency to predominantly bind with Neu5Ac2-6Gal. Conclusion In summary, the recent isolate displayed multiple mutations and a strong affinity for Neu5Ac2-6Gal, commonly found in mammals.

Characterization of Avian Influenza Viruses Detected in Kenyan Live Bird Markets and Wild Bird Habitats Reveal Genetically Diverse Subtypes and High Proportion of A(H9N2), 2018-2020.

Following the detection of highly pathogenic avian influenza (HPAI) virus in countries bordering Kenya to the west, we conducted surveillance among domestic and wild birds along the shores of Lake Victoria. In addition, between 2018 and 2020, we conducted surveillance among poultry and poultry workers in live bird markets and among wild migratory birds in various lakes that are resting sites during migration to assess introduction and circulation of avian influenza viruses in these populations. We tested 7464 specimens (oropharyngeal (OP) and cloacal specimens) from poultry and 6531 fresh fecal specimens from wild birds for influenza A viruses by real-time RT-PCR. Influenza was detected in 3.9% (n = 292) of specimens collected from poultry and 0.2% (n = 10) of fecal specimens from wild birds. On hemagglutinin subtyping, most of the influenza A positives from poultry (274/292, 93.8%) were H9. Of 34 H9 specimens randomly selected for further subtyping, all were H9N2. On phylogenetic analysis, these viruses were genetically similar to other H9 viruses detected in East Africa. Only two of the ten influenza A-positive specimens from the wild bird fecal specimens were successfully subtyped; sequencing analysis of one specimen collected in 2018 was identified as a low-pathogenicity avian influenza H5N2 virus of the Eurasian lineage, and the second specimen, collected in 2020, was subtyped as H11. A total of 18 OP and nasal specimens from poultry workers with acute respiratory illness (12%) were collected; none were positive for influenza A virus. We observed significant circulation of H9N2 influenza viruses in poultry in live bird markets in Kenya. During the same period, low-pathogenic H5N2 virus was detected in a fecal specimen collected in a site hosting a variety of migratory and resident birds. Although HPAI H5N8 was not detected in this survey, these results highlight the potential for the introduction and establishment of highly pathogenic avian influenza viruses in poultry populations and the associated risk of spillover to human populations.

Analyzing Molecular Traits of H9N2 Avian Influenza Virus Isolated from a Same Poultry Farm in West Java Province, Indonesia, in 2017 and 2023

Background Indonesia is one of the countries that is endemic to avian influenza virus subtype H9N2. This study aims to compare the molecular characteristics of avian influenza virus (AIV) subtype H9N2 from West Java. Methods Specific pathogen-free (SPF) embryonated chicken eggs were used to inoculate samples. RNA extraction and RT–qPCR confirmed the presence of H9 and N2 genes in the samples. RT–PCR was employed to amplify the H9N2-positive sample. Nucleotide sequences were obtained through Sanger sequencing and analyzed using MEGA 7. Homology comparison and phylogenetic tree analysis, utilizing the neighbor-joining tree method, assessed the recent isolate’s similarity to reference isolates from GenBank. Molecular docking analysis was performed on the HA1 protein of the recent isolate and the A/Layer/Indonesia/WestJava-04/2017 isolate, comparing their interactions with the sialic acids Neu5Ac2-3Gal and Neu5Ac2-6Gal. Results RT–qPCR confirmed the isolate samples as AIV subtype H9N2. The recent virus exhibited 11 amino acid residue differences compared to the A/Layer/Indonesia/WestJava-04/2017 isolate. Phylogenetically, the recent virus remains within the h9.4.2.5 subclade. Notably, at antigenic site II, the recent isolate featured an amino acid N at position 183, unlike A/Layer/Indonesia/WestJava-04/2017. Molecular docking analysis revealed a preference of HA1 from the 2017 virus for Neu5Ac2-3Gal, while the 2023 virus displayed a tendency to predominantly bind with Neu5Ac2-6Gal. Conclusion In summary, the recent isolate displayed multiple mutations and a strong affinity for Neu5Ac2-6Gal, commonly found in mammals.

Control of highly pathogenic avian influenza through vaccination

The stamping-out strategy has been used to control highly pathogenic avian influenza viruses in many countries, driven by the belief that vaccination would not be successful against such viruses and fears that avian influenza virus in vaccinated birds would evolve more rapidly and pose a greater risk to humans. In this review, we summarize the successes in controlling highly pathogenic avian influenza in China and make suggestions regarding the requirements for vaccine selection and effectiveness. In addition, we present evidence that vaccination of poultry not only eliminates human infection with avian influenza virus, but also significantly reduces and abolishes some harmful characteristics of avian influenza virus.

Differential Impact of Specific Amino Acid Residues on the Characteristics of Avian Influenza Viruses in Mammalian Systems.

Avian influenza virus (AIV) H9N2 was declared to be endemic in birds of the Middle East, in particular in Egypt, with multiple cases of human infections. Despite concerns about the pandemic threat posed by H9N2 AIV, due to the fact that its receptor specificity is similar to that of human influenza viruses, its morbidity and mortality rates in humans are so far negligible. However, the acquisition of specific adaptive amino acid (aa) mutations in the viral polymerase can enhance cross-species transmission of the virus itself or of reassortants, which gained these changes. The polymerase basic protein 2 (PB2) is one of the key determinants for AIV adaptation towards mammals. Although mammalian pathogenicity-related mutations (MPMs) in PB2 genes were identified in different AIVs, the specific effect of single or multiple mutations on viral fitness has not been compared so far. Here, we studied the effect of the aa K at position 591, which was frequently reported in the PB2 of Egyptian H9N2 isolates, on the proliferation efficiency and polymerase activity of an H5N1 (clade 2.2.1.2) AIV already carrying the mammalian adaptive mutation 627K. Using reverse genetics, we generated a set of recombinant parental strains and H5N1 variants carrying the avian-like 591Q/627E or mammalian-like adaptive mutations 591K/627K (H5N1EGY, H9N2EGY, H5N1PB2-H9N2EGY, H5N1H9N2_PB2_K591Q, H5N1PB2_K627E, H5N1PB2_K627E/591K, H5N1PB2_627K/591K). Regardless of the avian-like 627E or the mammalian-adaptive 627K, both variants carrying the 591K (H5N1PB2_K627E/591K, H5N1PB2_627K/591K) and the reassortant H5N1PB2-H9N2EGY replicated to significantly higher levels in mammalian continuous MDCK and Calu-3 cell lines and primary normal human bronchial epithelial cells than the parental H5N1EGY virus (carrying solely the 627K adaptive mutation). Expectedly, the H5N1 variants carrying avian-like PB2 mutations (H5N1H9N2_PB2_K591Q, H5N1PB2_K627E) replicated to significantly lower levels than the parental H5N1EGY virus in the predefined primary and continuous mammalian cell line systems. Consistently, the activity of H5N1 subtype AIV polymerase complexes comprising PB2 segments with singular 591K or combined with 627K was significantly enhanced when compared to parental H5N1EGY and H9N2EGY. This study emphasizes the significant impact of 591K containing PB2 segments in the background of H5N1 polymerase on viral fitness in addition to the well-known MPM 627K in vitro.

Epidemiology and molecular characterization of avian influenza virus in backyard poultry of Chattogram, Bangladesh.

Ducks, the natural reservoir of avian influenza virus (AIV), act as reassortment vessels for HPAI and low pathogenic avian influenza (LPAI) virus for domestic and wild bird species. In Bangladesh, earlier research was mainly focused on AIV in commercial poultry and live bird markets, where there is scanty literature reported on AIV in apparently healthy backyard poultry at the household level. The present cross-sectional study was carried out to reveal the genomic epidemiology of AIV of backyard poultry in coastal (Anowara) and plain land (Rangunia) areas of Bangladesh. We randomly selected a total of 292 households' poultry (having both chicken and duck) for sampling. We administered structured pre-tested questionnaires to farmers through direct interviews. We tested cloacal samples from birds for the matrix gene (M gene) followed by H5 and H9 subtypes using real-time reverse transcriptase-polymerase chain reaction (rRT-PCR). All AIV-positive samples were subjected to four-gene segment sequencing (M, PB1, HA, and NA gene). We found that the prevalence of AIV RNA at the household level was 6.2% (n=18; N=292), whereas duck and chicken prevalence was 3.6% and 3.2%, respectively. Prevalence varied with season, ranging from 3.1% in the summer to 8.2% in the winter. The prevalence of subtypes H5 and H9 in backyard poultry was 2.7% and 3.3%, respectively. The phylogenetic analysis of M, HA, NA, and PB1 genes revealed intra-genomic similarity, and they are closely related to previously reported AIV strains in Bangladesh and Southeast Asia. The findings indicate that H5 and H9 subtypes of AIV are circulating in the backyard poultry with or without clinical symptoms. Moreover, we revealed the circulation of 2.3.2.1a (new) clade among the chicken and duck population without occurring outbreak which might be due to vaccination. In addition to routine surveillance, molecular epidemiology of AIV will assist to gain a clear understanding of the genomic evolution of the AIV virus in the backyard poultry rearing system, thereby facilitating the implementation of effective preventive measures to control infection and prevent the potential spillover to humans.

Genetic Characterization of Avian Influenza Viruses Isolated from the Izumi Plain, Japan in 2019/20 Winter Season.

The Izumi plain in the Kagoshima Prefecture, Japan, is known as an overwintering site for more than 30,000 migratory waterfowl, including endangered crane species. We previously reported that environmental water samples, from artificial wet paddies created as crane roost sites on the Izumi plain, are useful for avian influenza virus (AIV) surveillance. During the 2019/20 winter season, we collected 238 water samples from the crane roost sites and isolated 22 AIVs of six subtypes: one H1N1, one H3N2, seven H3N8, four H4N6, nine H6N6, and one H11N2 subtypes. Genetic analyses revealed that AIVs of the same subtype isolated from the Izumi plain during a single winter season exhibited multiple genetic constellations. Furthermore, phylogenetic analyses suggested that our H3N2 isolate may be a genetic reassortant between close relatives to our H3N8 and H11N2 isolates. Our study highlighted the importance of monitoring AIV circulation to better understand AIV ecology in migratory waterfowl populations.

Genetic and Pathogenic Characterization of Avian Influenza Virus in Migratory Birds between 2015 and 2019 in Central China.

ABSTRACTActive surveillance of avian influenza virus (AIV) in wetlands and lakes is important for exploring the gene pool in wild birds. Through active surveillance from 2015 through 2019, 10,900 samples from wild birds in central China were collected, and 89 AIVs were isolated, including 2 subtypes of highly pathogenic AIV and 12 of low-pathogenic AIV; H9N2 and H6Ny were the dominant subtypes. Phylogenetic analysis of the isolates demonstrated that extensive intersubtype reassortments and frequent intercontinental gene exchange occurred in AIVs. AIV gene segments persistently circulated in several migration seasons, but interseasonal persistence of the whole genome was rare. The whole genomes of one H6N6 and polymerase basic 2 (PB2), polymerase acidic (PA), hemagglutinin (HA), neuraminidase (NA), M, and nonstructural (NS) genes of one H9N2 virus were found to be of poultry origin, suggesting a spillover of AIVs from poultry to wild birds. Importantly, one H9N2 virus only bound to human-type receptor, and one H1N1, four H6, and seven H9N2 viruses possessed dual receptor-binding capacity. Nineteen of 20 representative viruses tested could replicate in the lungs of mice without preadaptation, which poses a clear threat of infection in humans. Together, our study highlights the need for intensive AIV surveillance.IMPORTANCE Influenza virus surveillance in wild birds plays an important role in the early recognition and control of the virus. However, the AIV gene pool in wild birds in central China along the East Asian-Australasian flyway has not been well studied. Here, we conducted a 5-year AIV active surveillance in this region. Our data revealed the long-term circulation and prevalence of AIVs in wild birds in central China, and we observed that intercontinental gene exchange of AIVs is more frequent and continuous than previously thought. Spillover events from poultry to wild bird were observed in H6 and H9 viruses. In addition, in 20 representative viruses, 12 viruses could bind human-type receptors, and 19 viruses could replicate in mice without preadaption. Our work highlights the potential threat of wild bird AIVs to public health.

Estimation of Avian Influenza Viruses in Water Environments of Live Poultry Markets in Changsha, China, 2014 to 2018.

In routine surveillance for avian influenza viruses (AIVs) in the environments of live poultry markets (LPMs), certain samples were positive for AIVs type A while negative for subtypes (e.g., H5, H7, and H9). However, little attention has been paid to these unsubtyped AIVs samples. To reveal the dynamic distribution and molecular characteristics of AIVs, especially the unsubtyped AIVs, we reported and analyzed 1969 samples collected from the water environments of LPMs in Changsha, China, from January 2014 to November 2018. Our results revealed that 1504 (76.38%) samples were positive for AIV type A. Of these samples, the predominant hemagglutinin (HA) subtype was H9, followed by H5 and H7 (P < 0.05). The positive rate of H5 subtype in water environmental samples exhibited seasonality, which reached a peak in each winter-spring season from January 2014 to March 2017. The positive rates of AIVs (including type A, subtype H9, and mixed subtype H5/H7/H9) in non-central-city regions were higher than that in the central-city regions (P < 0.05). Notably, 161 unsubtyped AIVs samples were detected during the routine surveillance. However, subtyping with the commercial kit further identified eight different HA and seven different neuraminidase subtypes. Analyses unraveled that further subtyped AIVs H1, H6, and H11 had only one basic amino acid (R or K) at the cleavage site and residues Q226 and G228 at the receptor-binding associated sites. Overall, in addition to H5, H7, and H9 subtypes, we should also pay attention to unsubtyped AIVs samples during the routine surveillance for AIVs in the environments of LPMs.

Genetic Characteristics of Avian Influenza Virus Isolated from Wild Birds in South Korea, 2019–2020

Wild aquatic birds, a natural reservoir of avian influenza viruses (AIVs), transmit AIVs to poultry farms, causing huge economic losses. Therefore, the prevalence and genetic characteristics of AIVs isolated from wild birds in South Korea from October 2019 to March 2020 were investigated and analyzed. Fresh avian fecal samples (3256) were collected by active monitoring of 11 wild bird habitats. Twenty-eight AIVs were isolated. Seven HA and eight NA subtypes were identified. All AIV hosts were Anseriformes species. The HA cleavage site of 20 representative AIVs was encoded by non-multi-basic amino acid sequences. Phylogenetic analysis of the eight segment genes of the AIVs showed that most genes clustered within the Eurasian lineage. However, the HA gene of H10 viruses and NS gene of four viruses clustered within the American lineage, indicating intercontinental reassortment of AIVs. Representative viruses likely to infect mammals were selected and evaluated for pathogenicity in mice. JB21-58 (H5N3), JB42-93 (H9N2), and JB32-81 (H11N2) were isolated from the lungs, but JB31-69 (H11N9) was not isolated from the lungs until the end of the experiment at 14 dpi. None of infected mice showed clinical sign and histopathological change in the lung. In addition, viral antigens were not detected in lungs of all mice at 14 dpi. These data suggest that LPAIVs derived from wild birds are unlikely to be transmitted to mammals. However, because LPAIVs can reportedly infect mammals, including humans, continuous surveillance and monitoring of AIVs are necessary, despite their low pathogenicity.

Surveillance of avian influenza virus in external environment in Jieyang, Guangdong, 2016-2020

Objective To understand the dynamic distribution and epidemic characteristics of avian influenza virus in external environment in Jieyang, assess the risk of human infection with avian influenza, and we provide scientific basis for prevention and control of human avian influenza. Methods According to the random sampling method, relevant specimens were collected from poultry markets in 5 counties (county-level cities and districts) in Jieyang, 2016-2020. Real-time fluorescence quantitative RT-PCR method was used to detect the nucleic acid of influenza virus Type A. Positive specimens were then tested for the nucleic acid of H5N6, H7N9 and H9N2 subtypes of avian influenza virus. Results From 2016 to 2020, a total of 1 798 environmental samples were collected, of which 660 samples were positive for influenza A virus (FluA), with a positive rate of 36.71%. The positive rates of H5N6, H7N9 and H9N2 subtypes were 1.61%, 1.28% and 21.91%, respectively. The positive rate of avian influenza virus was higher in Puning county-level city (54.14%) and Huilai county (51.43%), and lowest in Jiexi county (4.84%). The highest positive rate of different types of samples (47.09%) was the washing of poultry sewage. Conclusion There are H5N6, H7N9, H9N2 and various subtypes of avian influenza virus contamination in external environment of poultry markets in Jieyang. The avian influenza virus has a wide epidemic area and there is a risk of human infection. It is necessary to continue to strengthen the real-time monitoring of avian influenza in external environment and the health supervision of urban and rural poultry market. 摘要：目的 了解揭阳市外环境禽流感病毒动态分布情况和流行特点, 评估人感染禽流感病毒的风险, 为人禽流 感防控提供科学依据。 方法 按随机抽样的方法, 对揭阳市2016—2020年5个县 (市、区)禽类市场进行相关标本采 集, 用实时荧光定量(RT-PCR)对标本进行流感病毒A型检测, 阳性标本再进行H5N6、H7N9、H9N2亚型禽流感病毒核 酸检测。 结果 揭阳市2016—2020年共采集外环境标本1 798份, 甲型流感病毒(FluA)阳性标本660份, 阳性率为 36.71%;其中H5N6、H7N9、H9N2亚型阳性率分别为1.61%、1.28%和21.91%。普宁市和惠来县的外环境禽流感病毒阳 性率较高, 分别为54.14%和51.43%。揭西县的外环境禽流感病毒阳性率最低, 为4.84%。不同类型标本检测阳性率最 高为清洗禽类污水(47.09%)。 结论 揭阳市禽类市场外环境中存在H5N6、H7N9、H9N2及多种亚型混合的禽流感病 毒污染, 禽流感病毒流行区域较广, 存在感染人的风险, 应继续加强外环境禽流感实时监测和城乡禽类市场卫生监管。

Variation and Molecular Basis for Enhancement of Receptor Binding of H9N2 Avian Influenza Viruses in China Isolates.

Currently, H9N2 avian influenza viruses (H9N2 AIVs) globally circulate in poultry and have acquired some adaptation to mammals. However, it is not clear what the molecular basis is for the variation in receptor-binding features of the H9N2 AIVs. The receptor-binding features of 92 H9N2 AIVs prevalent in China during 1994–2017 were characterized through solid-phase ELISA assay and reverse genetics. H9N2 AIVs that circulated in this period mostly belonged to clade h9.4.2. Two increasing incidents occurred in the ability of H9N2 AIVs to bind to avian-like receptors in 2002–2005 and 2011–2014. Two increasing incidents occurred in the strength of H9N2 AIVs to bind to human-like receptors in 2002–2005 and 2011–2017. We found that Q227M, D145G/N, S119R, and R246K mutations can significantly increase H9N2 AIVs to bind to both avian- and human-like receptors. A160D/N, Q156R, T205A, Q226L, V245I, V216L, D208E, T212I, R172Q, and S175N mutations can significantly enhance the strength of H9N2 AIVs to bind to human-like receptors. Our study also identified mutations T205A, D208E, V216L, Q226L, and V245I as the key sites leading to enhanced receptor binding of H9N2 AIVs during 2002–2005 and mutations S119R, D145G, Q156R, A160D, T212I, Q227M, and R246K as the key sites leading to enhanced receptor binding of H9N2 AIVs during 2011–2017. These findings further illustrate the receptor-binding characteristics of avian influenza viruses, which can be a potential threat to public health.

Comparative molecular characterization, pathogenicity and seroprevalence of avian influenza virus H9N2 in commercial and backyard poultry flocks

This study was conducted to perform the comparative molecular characterization of avian influenza virus (AIV) H9N2, pathogenicity and seroprevalence in commercial and backyard poultry flocks. Fifty commercial poultry flocks were investigated between 2012 and 2015. Eighteen flocks (36%) out of 50 were positive HA. Seven (38.9%) out of 18 were positive by chromatographic strip test for AI common antigen. By Real-time RT-PCR, only two flocks were positive H9. The molecular characterization of two different AI-H9N2 viruses, one isolated from a broiler flock (A/chicken/Egypt/Mansoura-18/2013) and the other from a layer flock (A/chicken/Egypt/Mansoura-36/2015) was conducted on HA gene. Moreover, a higher seroprevalence, using the broiler strain as a known antigen, was shown in backyard chicken flocks 15/26 (57.7%) than duck flocks 9/74 (12.2%). Interestingly, the pathogenicity index (PI) of the H9N2 broiler strain in inoculated experimental chickens ranged from 1.2 (oculonasal route) to 1.9 (Intravenous route). The PI indicated a highly pathogenic effect, with high mortality (up to 100%) in the inoculated chickens correlated with the high mortality (80%) in the flock where the virus was isolated. The firstly recorded clinical signs, including cyanosis in the combs and wattles and subcutaneous haemorrhages in the leg shanks and lesions, as well as histopathology and immunohistochemistry, revealed a systemic infection of the high pathogenicity with the H9N2 virus. Conversely, the H9N2 layer strain showed a low pathogenicity. In conclusion, as a first report, the molecular analysis and pathogenicity of the tested strains confirmed the presence of a high pathogenicity AIV-H9N2 with systemic infections.

The molecular characteristics of avian influenza viruses (H9N2) derived from air samples in live poultry markets

ObjectiveTo study the molecular characteristics of H9N2-subtype avian influenza viruses (AIVs) isolated from air samples collected in live poultry markets (LPMs) and explore their sequence identities with AIVs that caused human infection. MethodsWeekly surveillance of H9N2-subtype AIVs in the air of LPMs was conducted from 2015 to 2016. H9-positive samples were isolated from chicken embryos. Whole genome sequences of the isolated AIVs were obtained through high-throughput sequencing. Phylogenetic analysis and key loci variations of the sequences were further analyzed. ResultsA total of 327 aerosol samples were collected from LPMs. Nine samples were positive for H9-subtype AIVs based on quantitative real-time reverse transcription polymerase chain reaction (qRRT-PCR). According to the whole genome sequence analysis and phylogenetic analysis, except for the A/Environment/Zhongshan/ZS201505/2015 (ZS201505) strain, 8 gene segments of 8 aerosol H9N2 isolates and 2 H9N2 human isolates in 2015 were located in the same clade. Among key loci variations, except for the ZS201505 strain, H9N2-subtype AIVs had no mutations in eight receptor binding sites of hemagglutinin (HA), and stalks of neuraminidase (NA) proteins exhibited a deletion site of three bases. The PA gene of ZS201503 and ZS201602 exhibited an L336M mutation. The N30D and T215A mutations in the M1 gene and amino acid residues L89V in PB2, P42S in NS1 and S31N in M2 were retained in these 9 strains of H9N2 isolates, which could enhance the virus's virulence. ConclusionLive H9N2 AIVs survived in the aerosol of LPMs in Zhongshan City. The aerosol viruses had a close evolutionary relationship with human epidemic strains, indicating that there might be a risk of AIV transmission from polluted aerosols in LPMs to humans. Mutations in H9N2-subtype AIVs isolated from air samples collected from LPMs suggested their pathogenicity was enhanced to infect humans.

Isolation and characterization of avian influenza viruses from raw poultry products illegally imported to Japan by international flight passengers.

The transportation of poultry and related products for international trade contributes to transboundary pathogen spread and disease outbreaks worldwide. To prevent pathogen incursion through poultry products, many countries have regulations about animal health and poultry product quarantine. However, in Japan, animal products have been illegally introduced into the country in baggage and confiscated at the airport. Lately, the number of illegally imported poultry and the incursion risk of transboundary pathogens through poultry products have been increasing. In this study, we isolated avian influenza viruses (AIVs) from raw poultry products illegally imported to Japan by international passengers. Highly (H5N1 and H5N6) and low (H9N2 and H1N2) pathogenic AIVs were isolated from raw chicken and duck products carried by flight passengers. H5 and H9 isolates were phylogenetically closely related to viruses isolated from poultry in China, and haemagglutinin genes of H5N1 and H5N6 isolates belonged to clades 2.3.2.1c and 2.3.4.4, respectively. Experimental infections of H5 and H9 isolates in chickens and ducks demonstrated pathogenicity and tissue tropism to skeletal muscles. To prevent virus incursion by poultry products, it is important to encourage the phased cleaning based on the disease control and eradication and promote the reduction in contamination risk in animal products.

Genome Sequence of an H9N2 Avian Influenza Virus Strain with Hemagglutinin-Neuraminidase Combination, Isolated from a Quail in Guangxi, Southern China.

ABSTRACTWe isolated a strain of H9N2 avian influenza virus from a quail in southern China in May 2015 and named it A/quail/Guangxi/198Q39/2015. All eight gene segments of the strain were sequenced. Sequence analysis indicated that the amino acid motif of the hemagglutinin cleavage site of this H9N2 virus was RSSR↓GLF, which is a typical characteristic of the low pathogenic avian influenza virus. This study will help in better understanding the epidemiology and molecular characteristics of avian influenza virus in wild birds.

Genetic Characterization of an Avian Influenza Virus H4N6 Strain Isolated from a Guangxi Pockmark Duck.

ABSTRACTAn H4N6 subtype avian influenza virus was isolated from a pockmark duck in southern China in November 2013 and named A/duck/Guangxi/149D24/2013 (H4N6). All eight gene segments of the strain were sequenced. Sequence analysis indicated that this H4N6 virus was a natural reassortant virus. This H4N6 virus has two basic amino acids in the cleavage site of hemagglutinin 1 (HA1) and HA2, and the amino acid motif of cleavage site was PEKASRGLF, which is the typical characteristic of the low-pathogenic avian influenza virus. This study will help understand the epidemiology and molecular characteristics of avian influenza virus in pockmark ducks.

Avian influenza in Latin America: A systematic review of serological and molecular studies from 2000-2015.

Avian influenza or bird flu is a highly contagious acute viral disease that can occur in epidemics and cross-border forms in poultry and wild birds. The characteristics of avian influenza viruses (AIVs) allow the emergence of new viral variants, some with zoonotic and pandemic potential. AIVs have been identified in Latin America; however, there is a lack of understanding of these viruses at the regional level. We performed a systematic literature review on serological or molecular evidence of AIVs circulation in Latin America. Methods were designed based on the PRISMA and STROME guidelines. Only peer-reviewed studies published between 2000 to 2015 and data was analysed based on country, viral subtype, avian species, and phylogenetic origins. From 271 studies initially found only twenty-six met our inclusion criteria. Evidence of AIVs infection was found in most Latin American countries, with Mexico as the country with the largest number of conducted studies and reported cases during the period analysed, followed by Chile and Argentina. Most of the AIVs were early reported through surveillance systems and at least 14 different subtypes of influenza viruses were reported in birds, and the presence of both low (92.9%) and high (7.1%) pathogenic AIVs was shown in Latin America. Of the reported AIVs in Latin America, 43.7% belong to migratory birds, 28.1% to local wild birds, and 28.1% to poultry. The migratory bird population mainly comprises families belonging to the orders Anseriformes and Charadriformes. We highlight the importance of epidemiological surveillance systems and the possible role of different migratory birds in the transmission of AIVs within the Americas. Our findings demonstrate the limited information on AIVs in Latin America and highlight the need of more studies on AIVs at the regional level, particularly those focused on identifying the endemic subtypes in regional wild birds.

First Characterization of Avian Influenza Viruses from Greenland 2014.

In late February 2014, unusually high numbers of wild thick-billed murres (Uria lomvia) were found dead on the coast of South Greenland. To investigate the cause of death, 45 birds were submitted for laboratory examination in Denmark. Avian influenza viruses (AIVs) with subtypes H11N2 and low pathogenic H5N1 were detected in some of the birds. Characterization of the viruses by full genome sequencing revealed that all the gene segments belonged to the North American lineage of AIVs. The seemingly sparse and mixed subtype occurrence of low pathogenic AIVs in these birds, in addition to the emaciated appearance of the birds, suggests that the murre die-off was due to malnutrition as a result of sparse food availability or inclement weather. Here we present the first characterization of AIVs isolated in Greenland, and our results support the idea that wild birds in Greenland may be involved in the movement of AIV between North America and Europe.

Isolation and Genetic Characterization of Avian Influenza Viruses Isolated from Wild Birds in the Azov-Black Sea Region of Ukraine (2001-2012).

Wild bird surveillance for avian influenza virus (AIV) was conducted from 2001 to 2012 in the Azov - Black Sea region of the Ukraine, considered part of the transcontinental wild bird migration routes from northern Asia and Europe to the Mediterranean, Africa, and southwest Asia. A total of 6281 samples were collected from wild birds representing 27 families and eight orders for virus isolation. From these samples, 69 AIVs belonging to 15 of the 16 known hemagglutinin (HA) subtypes and seven of nine known neuraminidase (NA) subtypes were isolated. No H14, N5, or N9 subtypes were identified. In total, nine H6, eight H1, nine H5, seven H7, six H11, six H4, five H3, five H10, four H8, three H2, three H9, one H12, one H13, one H15, and one H16 HA subtypes were isolated. As for the NA subtypes, twelve N2, nine N6, eight N8, seven N7, six N3, four N4, and one undetermined were isolated. There were 27 HA and NA antigen combinations. All isolates were low pathogenic AIV except for eight highly pathogenic (HP) AIVs that were isolated during the H5N1 HPAI outbreaks of 2006-08. Sequencing and phylogenetic analysis of the HA genes revealed epidemiological connections between the Azov-Black Sea regions and Europe, Russia, Mongolia, and Southeast Asia. H1, H2, H3, H7, H8, H6, H9, and H13 AIV subtypes were closely related to European, Russian, Mongolian, and Georgian AIV isolates. H10, H11, and H12 AIV subtypes were epidemiologically linked to viruses from Europe and Southeast Asia. Serology conducted on serum and egg yolk samples also demonstrated previous exposure of many wild bird species to different AIVs. Our results demonstrate the great genetic diversity of AIVs in wild birds in the Azov-Black Sea region as well as the importance of this region for monitoring and studying the ecology of influenza viruses. This information furthers our understanding of the ecology of avian influenza viruses in wild bird species.