Circulation Of Avian Influenza Viruses Research Articles

First detection of influenza A virus subtypes H1N1 and H3N8 in the Antarctic region: King George Island, 2023.

Influenza A virus is characterized by a segmented single-stranded RNA genome. Such organization of the virus genome determines the possibility of reassortment, which can lead to the emergence of new virus variants. The main natural reservoir of most influenza A virus subtypes are wild waterfowl. Seasonal migrations gather waterfowl from all major migration routes to nesting areas near the northern and southern polar circles. This makes intercontinental spread of influenza A viruses possible. Objective ‒ to conduct molecular genetic monitoring and study the phylogenetic relationships of influenza A virus variants circulating in Antarctica in 2023. We studied 84 samples of biological material obtained from birds and marine mammals in April‒May 2023 in coastal areas of Antarctica. For 3 samples, sequencing was performed on the Miseq, Illumina platform and phylogenetic analysis of the obtained nucleotide sequences of the influenza A virus genomes was performed. The circulation of avian influenza virus in the Antarctic region was confirmed. Heterogeneity of the pool of circulating variants of the influenza A virus (H3N8, H1N1) was revealed. Full-length genomes of the avian influenza virus were sequenced and posted in the GISAID database (EPI_ISL_19032103, 19174530, 19174467). The study of the genetic diversity of influenza A viruses circulating in the polar regions of the Earth and the identification of the conditions for the emergence of new genetic variants is a relevant task for the development of measures to prevent biological threats.

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.

PB1-F2 of low pathogenicity H7N7 restricts apoptosis in avian cells

Avian influenza viruses (AIV) pose a continuous challenge to global health and economy. While countermeasures exist to control outbreaks in poultry, the persistent circulation of AIV in wild aquatic and shorebirds presents a significant challenge to effective disease prevention efforts. PB1-F2 is a non-structural protein expressed from a second open reading frame (+1) of the polymerase basic 1 (PB1) segment. The sequence and length of the PB1-F2 protein can vary depending on the host of origin. While avian isolates typically carry full-length PB1-F2, isolates from mammals, often express truncated forms. The selective advantage of the full-length PB1-F2 in avian isolates is not fully understood. Most research on the role of PB1-F2 in influenza virus replication has been conducted in mammalian systems, where PB1-F2 interfered with the host immune response and induced apoptosis. Here, we used Low Pathogenicity (LP) AIV H7N7 expressing full-length PB1-F2 as well as a knockout mutant. We found that the full-length PB1-F2 of LPAIV prolonged survival of infected cells by limiting apoptotic cell death. Furthermore, PB1-F2 knockout LPAIV significantly decreased MHC-I expression on fibroblasts, delayed tissue healing and increased phagocytic uptake of infected cells, whereas LPAIV expressing PB1-F2 has limited effects. These findings indicate that full-length PB1-F2 enables AIV to cause prolonged infections without severely harming the avian host. Our observations may explain maintenance of AIV in the natural bird reservoir in absence of severe clinical signs.

Effect of Nonpharmaceutical Interventions in Preventing COVID-19 on the Circulation of Avian Influenza Virus in Wuhan, Hubei Province, China

Background. In late 2019, several medical institutions in Wuhan, Hubei Province, China, reported cases of unexplained pneumonia. A novel coronavirus was isolated from human airway epithelial cells causing coronavirus disease-2019 (COVID-19). In recent years, many nonpharmaceutical interventions (NPIs) have been implemented to stop COVID-19 epidemic. This study aimed to explore the effect of NPIs on the circulation of avian influenza virus (AIV) in Wuhan. Materials and Methods. External environmental samples were collected and subjected to viral RNA extraction. Real-time quantitative polymerase chain reaction was used to detect the H5, H7, and H9 subtypes of AIV. Statistical analyses were performed using the chi-square test and binary logistic regression in SPSS 20.0 software. Results. A total of 2,451 external environmental samples were collected from seven districts from 2018 to 2022 in Wuhan, comprising 1,041 samples collected before COVID-19 and 1,410 samples after COVID-19. After COVID-19, the positive rate of AIV decreased significantly with the implementation of NPIs. The dominant subtype was the H9 subtype, followed by the H5 subtype. The positive rates of AIV in live poultry markets and poultry free-range sites were reduced significantly through the implementation of NPIs. Among the different sample types, higher positive rates of AIV were found in chopping boards, sewage, and cages. The positive rate of AIV was higher in trafficked source samples than that in autotrophic source samples. Conclusions. This study identified the characteristics of AIV in terms of different districts, surveillance sites, sample types, and bird sources in Wuhan. This study conducted a multifactorial analysis of the factors affecting AIV infection and provided a theoretical basis and guidance for the future prevention and control of AIV in Wuhan.

Association of biosecurity and hygiene practices with avian influenza A/H5 and A/H9 virus infections in turkey farms.

High pathogenicity avian influenza (HPAI) H5N1 outbreaks pose a significant threat to the health of livestock, wildlife, and humans. Avian influenza viruses (AIVs) are enzootic in poultry in many countries, including Bangladesh, necessitating improved farm biosecurity measures. However, the comprehension of biosecurity and hygiene practices, as well as the infection of AIV in turkey farms, are poorly understood in Bangladesh. Therefore, we conducted this study to determine the prevalence of AIV subtypes and their association with biosecurity and hygiene practices in turkey farms. We collected oropharyngeal and cloacal swabs from individual turkeys from 197 farms across 9 districts in Bangladesh from March to August 2019. We tested the swab samples for the AIV matrix gene (M gene) followed by H5, H7, and H9 subtypes using real-time reverse transcriptase-polymerase chain reaction (rRT-PCR). We found 24.68% (95% CI:21.54-28.04) of turkey samples were AIV positive, followed by 5.95% (95% CI: 4.33-7.97) for H5, 6.81% (95% CI: 5.06-8.93) for H9 subtype and no A/H7 was found. Using a generalized linear mixed model, we determined 10 significant risk factors associated with AIV circulation in turkey farms. We found that the absence of sick turkeys, the presence of footbaths, the absence of nearby poultry farms, concrete flooring, and the avoidance of mixing newly purchased turkeys with existing stock can substantially reduce the risk of AIV circulation in turkey farms (odds ratio ranging from 0.02 to 0.08). Furthermore, the absence of nearby live bird markets, limiting wild bird access, no visitor access, improved floor cleaning frequency, and equipment disinfection practices also had a substantial impact on lowering the AIV risk in the farms (odds ratio ranging from 0.10 to 0.13). The results of our study underscore the importance of implementing feasible and cost-effective biosecurity measures aimed at reducing AIV transmission in turkey farms. Particularly in resource-constrained environments such as Bangladesh, such findings might assist governmental entities in enhancing biosecurity protocols within their poultry sector, hence mitigating and potentially averting the transmission of AIV and spillover to humans.

Potential risk zones and climatic factors influencing the occurrence and persistence of avian influenza viruses in the environment of live bird markets in Bangladesh

Live bird markets (LBMs) are critical for poultry trade in many developing countries that are regarded as hotspots for the prevalence and contamination of avian influenza viruses (AIV). Therefore, we conducted weekly longitudinal environmental surveillance in LBMs to determine annual cyclic patterns of AIV subtypes, environmental risk zones, and the role of climatic factors on the AIV presence and persistence in the environment of LBM in Bangladesh. From January 2018 to March 2020, we collected weekly fecal and offal swab samples from each LBM and tested using rRT-PCR for the M gene and subtyped for H5, H7, and H9. We used Generalized Estimating Equations (GEE) approaches to account for repeated observations over time to correlate the AIV prevalence and potential risk factors and the negative binomial and Poisson model to investigate the role of climatic factors on environmental contamination of AIV at the LBM. Over the study period, 37.8% of samples tested AIV positive, 18.8% for A/H5, and A/H9 was, for 15.4%. We found the circulation of H5, H9, and co-circulation of H5 and H9 in the environmental surfaces year-round. The Generalized Estimating Equations (GEE) model reveals a distinct seasonal pattern in transmitting AIV and H5. Specifically, certain summer months exhibited a substantial reduction of risk up to 70–90% and 93–94% for AIV and H5 contamination, respectively. The slaughtering zone showed a significantly higher risk of contamination with H5, with a three-fold increase in risk compared to bird-holding zones. From the negative binomial model, we found that climatic factors like temperature and relative humidity were also significantly associated with weekly AIV circulation. An increase in temperature and relative humidity decreases the risk of AIV circulation. Our study underscores the significance of longitudinal environmental surveillance for identifying potential risk zones to detect H5 and H9 virus co-circulation and seasonal transmission, as well as the imperative for immediate interventions to reduce AIV at LBMs in Bangladesh. We recommend adopting a One Health approach to integrated AIV surveillance across animal, human, and environmental interfaces in order to prevent the epidemic and pandemic of AIV.

Determinants for the presence of avian influenza virus in live bird markets in Bangladesh: Towards an easy fix of a looming one health issue

Highly pathogenic avian influenza virus subtype H5N1 endangers poultry, wildlife, and human health and is enzootic in large parts of Asia, with live bird markets (LBMs) as putative hotspots for their maintenance, amplification, and spread. To mitigate the extent of these and other avian influenza viruses (AIV) of concern, we aimed to increase our quantitative understanding of the factors determining the presence of avian influenza virus in LBM stalls. Between 2016 and 2017, we collected fecal or offal samples from 1008 stalls in 113 LBMs across the Dhaka and Rajshahi districts in Bangladesh. For each stall, samples were pooled and tested for the AIV matrix gene, followed by H5 and H9 subtyping using rRT-PCR. We detected Influenza A viral RNA in 49% of the stalls. Of the AIV positive samples, 52% and 24% were determined to be H5 and H9 viruses, respectively, which are both subtypes of considerable health concern. We used generalized linear mixed effect modelling to study AIV presence in individual stalls within LBMs as a function of 13 out of the 20 risk factors identified by FAO. We found that small and feasible improvements in cleaning and disinfection frequency, installing running water in stalls, and not mixing different breeds of chicken in the same cages had large impacts on the presence of AIV in stalls (Odds ratios 0.03–0.05). Next, cleaning vehicles used in poultry transport, not selling waterfowl with chickens in the same stall, buying stock directly from commercial farms, separating sick birds from healthy ones, and avoiding access by wild birds like house crows, also had major effects on lowering the risk of stalls having AIV (Odds ratios 0.16–0.33). These findings can be directly used in developing practical and affordable measures to reduce the prevalence of AIV in LBMs. Also, in settings with limited resources like Bangladesh, such mitigation may significantly contribute to reducing AIV circulation amongst poultry and spillover to wildlife and humans.

Annual trading patterns and risk factors of avian influenza A/H5 and A/H9 virus circulation in turkey birds (Meleagris gallopavo) at live bird markets in Dhaka city, Bangladesh.

The impacts of the avian influenza virus (AIV) on farmed poultry and wild birds affect human health, livelihoods, food security, and international trade. The movement patterns of turkey birds from farms to live bird markets (LBMs) and infection of AIV are poorly understood in Bangladesh. Thus, we conducted weekly longitudinal surveillance in LBMs to understand the trading patterns, temporal trends, and risk factors of AIV circulation in turkey birds. We sampled a total of 423 turkeys from two LBMs in Dhaka between May 2018 and September 2019. We tested the swab samples for the AIV matrix gene (M-gene) followed by H5, H7, and H9 subtypes using real-time reverse transcriptase-polymerase chain reaction (rRT-PCR). We used exploratory analysis to investigate trading patterns, annual cyclic trends of AIV and its subtypes, and a generalized estimating equation (GEE) logistic model to determine the factors that influence the infection of H5 and H9 in turkeys. Furthermore, we conducted an observational study and informal interviews with traders and vendors to record turkey trading patterns, demand, and supply and turkey handling practices in LBM. We found that all trade routes of turkey birds to northern Dhaka are unidirectional and originate from the northwestern and southern regions of Bangladesh. The number of trades from the source district to Dhaka depends on the turkey density. The median distance that turkey was traded from its source district to Dhaka was 188 km (Q1 = 165, Q3 = 210, IQR = 45.5). We observed seasonal variation in the median and average distance of turkey. The qualitative findings revealed that turkey farming initially became reasonably profitable in 2018 and at the beginning of 2019. However, the fall in demand and production in the middle of 2019 may be related to unstable market pricing, high feed costs, a shortfall of adequate marketing facilities, poor consumer knowledge, and a lack of advertising. The overall prevalence of AIV, H5, and H9 subtypes in turkeys was 31% (95% CI: 26.6-35.4), 16.3% (95% CI: 12.8-19.8), and 10.2% (95% CI: 7.3-13.1) respectively. None of the samples were positive for H7. The circulation of AIV and H9 across the annual cycle showed no seasonality, whereas the circulation of H5 showed significant seasonality. The GEE revealed that detection of AIV increases in retail vendor business (OR: 1.71; 95% CI: 1.12-2.62) and the bird's health status is sick (OR: 10.77; 95% CI: 4.31-26.94) or dead (OR: 11.33; 95% CI: 4.30-29.89). We also observed that winter season (OR: 5.83; 95% CI: 2.80-12.14) than summer season, dead birds (OR: 61.71; 95% CI: 25.78-147.75) and sick birds (OR 8.33; 95% CI: 3.36-20.64) compared to healthy birds has a higher risk of H5 infection in turkeys. This study revealed that the turkeys movements vary by time and season from the farm to the LBM. This surveillance indicated year-round circulation of AIV with H5 and H9 subtypes in turkey birds in LBMs. The seasonality and health condition of birds influence H5 infection in birds. The trading pattern of turkey may play a role in the transmission of AIV viruses in the birds. The selling of sick turkeys infected with H5 and H9 highlights the possibility of virus transmission to other species of birds sold at LBMs and to people.

Prevalence and risk factors for avian influenza virus (H5 and H9) contamination in peri-urban and rural live bird markets in Bangladesh.

Avian influenza viruses (AIV) have been frequently detected in live bird markets (LBMs) around the world, primarily in urban areas, and have the ability to spillover to other species, including humans. Despite frequent detection of AIV in urban LBMs, the contamination of AIV on environmental surfaces in rural and peri-urban LBMs in Bangladesh is poorly documented. Therefore, we conducted this study to determine the prevalence of AIV subtypes within a subset of peri-urban and rural LBMs in Bangladesh and to further identify associated risk factors. Between 2017 and 2018, we collected faecal and offal samples from 200 stalls in 63 LBMs across four sub-districts. We tested the samples for the AIV matrix gene (M-gene) followed by H5, H7, and H9 subtypes using real-time reverse transcriptase-polymerase chain reaction (rRT-PCR). We performed a descriptive analysis of market cleanliness and sanitation practices in order to further elucidate the relationship between LBM biosecurity and AIV subtypes by species, sample types, and landscape. Subsequently, we conducted a univariate analysis and a generalized linear mixed model (GLMM) to determine the risk factors associated with AIV contamination at individual stalls within LBMs. Our findings indicate that practices related to hygiene and the circulation of AIV significantly differed between rural and peri-urban live bird markets. 42.5% (95% CI: 35.56-49.67) of stalls were positive for AIV. A/H5, A/H9, and A HA/Untyped were detected in 10.5% (95% CI: 6.62-15.60), 9% (95% CI: 5.42-13.85), and 24.0% (95% CI: 18.26-30.53) of stalls respectively, with no detection of A/H7. Significantly higher levels of AIV were found in the Sonali chicken strain compared to the exotic broiler, and in offal samples compared to fecal samples. In the GLMM analysis, we identified several significant risk factors associated with AIV contamination in LBMs at the stall level. These include: landscape (AOR: 3.02; 95% CI: 1.18-7.72), the number of chicken breeds present (AOR: 2.4; 95% CI: 1.01-5.67), source of birds (AOR: 2.35; 95% CI: 1.0-5.53), separation of sick birds (AOR: 3.04; 95% CI: 1.34-6.92), disposal of waste/dead birds (AOR: 3.16; 95% CI: 1.41-7.05), cleaning agent (AOR: 5.99; 95% CI: 2.26-15.82), access of dogs (AOR: 2.52; 95% CI: 1.12-5.7), wild birds observed on site (AOR: 2.31; 95% CI: 1.01-5.3). The study further revealed a substantial prevalence of AIV with H5 and H9 subtypes in peri-urban and rural LBMs. The inadequate biosecurity measures at poultry stalls in Bangladesh increase the risk of AIV transmission from poultry to humans. To prevent the spread of AIV to humans and wild birds, we suggest implementing regular surveillance at live bird markets and enhancing biosecurity practices in peri-urban and rural areas in Bangladesh.

Virological and Genetic Characterization of the Unusual Avian Influenza H14Nx Viruses in the Northern Asia.

Wild aquatic birds are generally identified as a natural reservoir of avian influenza viruses (AIVs), where a high diversity of subtypes has been detected. Some AIV subtypes are considered to have relatively low prevalence in wild bird populations. Six-year AIV surveillance in Siberia revealed sporadic cases of the rarely identified H14-subtype AIV circulation. Complete genome sequencing of three H14 isolates were performed, and the analysis indicated interconnections between low pathogenic avian influenza (LPAI) viruses. We conducted hemagglutination inhibition and virus neutralization assays, estimated the susceptibility of isolates to neuraminidase inhibitors, and characterized receptor specificity. Our study revealed circulation of a new H14N9 subtype described for the first time. However, the low prevalence of the H14-subtype AIV population may be the reason for the underestimation of the diversity of H14-subtype AIVs. According to the available data, a region in which H14-subtype viruses were detected several times in 2007-2022 in the Eastern Hemisphere is Western Siberia, while the virus was also detected once in South Asia (Pakistan). Phylogenetic analysis of HA segment sequences revealed the circulation of two clades of H14-subtype viruses originated from initial 1980s Eurasian clade; the first was detected in Northern America and the second in Eurasia.

Analysis of Avian Influenza (H5N5) Viruses Isolated in the Southwestern European Part of the Russian Federation in 2020-2021.

In 2021, several isolates of the H5N5 avian influenza virus (AIV) were detected in Europe and the Russian Federation, which differed from those detected in 2020. Genetic analysis revealed a relationship between the highly pathogenic avian influenza H5N5 subtype, detected in Europe, and some isolates detected in the Russian Federation territory in 2020-2021: it was shown that both originated in the Caspian Sea regions around the autumn of 2020. The appearance of H5N5 subtype viruses in the spring of 2021 in Europe and the Russian Federation was not associated with the mass migration of birds from Africa. The results of the analysis revealed the presence of a deletion in the stem of a neuraminidase between bp 139 and 204 (open reading frame). It has been shown that AIVs of the H5N5 subtype are capable of long-term circulation in wild bird populations with the possibility of reassortment. The results also highlighted the need for careful monitoring of the circulation of AIVs in the Caspian Sea region, the role of which, in the preservation and emergence of new antigenic variants of such viruses in Eurasia, is currently underestimated.

Aerosol exposure of live bird market workers to viable influenza A/H5N1 and A/H9N2 viruses, Cambodia.

Live bird markets (LBMs) have been identified as key factors in the spread, persistence and evolution of avian influenza viruses (AIVs). In addition, these settings have been associated with human infections with AIVs of pandemic concern. Exposure to aerosolised AIVs by workers in a Cambodian LBM was assessed using aerosol impact samplers. LBM vendors were asked to wear an air sampler for 30 min per day for 1week while continuing their usual activities in the LBM during a period of high AIV circulation (February) and a period of low circulation (May). During the period of high circulation, AIV RNA was detected from 100% of the air samplers using molecular methods and viable AIV (A/H5N1 and/or A/H9N2) was isolated from 50% of air samplers following inoculation into embryonated chicken eggs. In contrast, AIV was not detected by molecular methods or successfully isolated during the period of low circulation. This study demonstrates the increased risk of aerosol exposure of LBM workers to AIVs during periods of high circulation and highlights the need for interventions during these high-risk periods. Novel approaches, such as environmental sampling, should be further explored at key high-risk interfaces as a potentially cost-effective alternative for monitoring pandemic threats.

Assessment of poultry rearing practices and risk factors of H5N1 and H9N2 virus circulating among backyard chickens and ducks in rural communities.

BackgroundThe avian influenza virus (AIV) causes significant economic losses by infecting poultry and occasional spillover to humans. Backyard farms are vulnerable to AIV epidemics due to poor health management and biosecurity practices, threatening rural households’ economic stability and nutrition. We have limited information about the risk factors associated with AIV infection in backyard poultry in Bangladesh. Hence, we conducted a cross-sectional survey comprising epidemiological and anthropological investigations to understand the poultry rearing practices and risk factors of AIV circulation among backyard poultry in selected rural communities.MethodsWe sampled 120 poultry from backyard farms (n = 30) of the three selected communities between February 2017 and January 2018. We tested swab samples for the matrix gene (M gene) followed by H5, H7, and H9 subtypes using real-time reverse transcriptase-polymerase chain reaction (rRT-PCR). We applied multivariable logistic regression for risk factor analysis. Furthermore, we conducted an observational study (42 hours) and informal interviews (n = 30) with backyard farmers to record poultry-raising activities in rural communities.ResultsWe detected that 25.2% of the backyard poultry tested positive for AIV, whereas 5% tested positive for H5N1 and 10.8% tested positive for H9N2. Results showed that scavenging in both household garden and other crop fields has higher odds of AIV than scavenging in the household garden (AOR: 24.811; 95% CI: 2.11–292.28), and keeping a cage inside the house has higher odds (AOR:14.5; 95% CI: 1.06–198.51) than keeping it in the veranda, cleaning the cage twice a week or weekly has a higher risk than cleaning daily (AOR: 34.45; 95% CI: 1.04–1139.65), dumping litter or droppings (AOR: 82.80; 95% CI: 3.91–1754.59) and dead birds or wastage (AOR: 109.92, 95% CI: 4.34–2785.29) near water bodies and bushes have a higher risk than burring in the ground, slaughtering and consuming sick birds also had a higher odd of AIV (AOR: 73.45, 95% CI: 1.56–3457.73) than treating the birds. The anthropological investigation revealed that household members had direct contact with the poultry in different ways, including touching, feeding, slaughtering, and contacting poultry feces. Poultry is usually kept inside the house, sick poultry are traditionally slaughtered and eaten, and most poultry raisers do not know that diseases can transmit from backyard poultry to humans.ConclusionsThis study showed the circulation of H5N1 and H9N2 virus in backyard poultry in rural communities; associated with species, scavenging area of the poultry, location of the poultry cage, the practice of litter, wastage, droppings, and dead bird disposal, and practice of handling sick poultry. We suggest improving biosecurity practices in backyard poultry and mass awareness campaigns to reduce incidences of AIV in household-level poultry farms in rural communities in Bangladesh.

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.

Improved method for avian influenza virus isolation from environmental water samples.

Environmental water-targeted surveillance of migratory aquatic birds at overwintering sites is potentially one of the most effective approaches for understanding the ecology of avian influenza viruses (AIVs). In this study, we improved the method for AIV isolation from environmental water samples by making a minor modification to our previously reported process. We experimentally demonstrated that the AIV recovery efficiency of the modified method was 10-100-fold higher than that of the original method. This improved isolation method allowed us to isolate a considerably larger number of AIV isolates from environmental water samples collected at an overwintering site for tens of thousands of migratory aquatic birds in Japan during the 2018/2019 winter season, compared with those during previous winter seasons. Genetic and phylogenetic analyses revealed that AIVs of the same subtypes with multiple genetic constellations were circulating in a single overwintering site during a single winter season. These findings indicate that our improved isolation method contributes to enhance environmental water-targeted surveillance and to a better understanding of AIV ecology in migratory aquatic bird populations by monitoring ongoing AIV circulation.

Novel Low Pathogenic Avian Influenza H6N1 in Backyard Chicken in Easter Island (Rapa Nui), Chilean Polynesia.

Little is known about the prevalence of avian influenza viruses (AIV) in wildlife and domestic animals in Polynesia. Here, we present the results of active AIV surveillance performed during two sampling seasons in 2019 on Easter Island (Rapa Nui). Tracheal and cloacal swabs as well as sera samples were obtained from domestic backyard poultry, while fresh faeces were collected from wild birds. In addition to detecting antibodies against AIV in 46% of the domestic chickens in backyard production systems tested, we isolated a novel low pathogenic H6N1 virus from a chicken. Phylogenetic analysis of all genetic segments revealed that the virus was closely related to AIV’s circulating in South America. Our analysis showed different geographical origins of the genetic segments, with the PA, HA, NA, NP, and MP gene segments coming from central Chile and the PB2, PB1, and NS being closely related to viruses isolated in Argentina. While the route of introduction can only be speculated, our analysis shows the persistence and independent evolution of this strain in the island since its putative introduction between 2015 and 2016. The results of this research are the first evidence of AIV circulation in domestic birds on a Polynesian island and increase our understanding of AIV ecology in region, warranting further surveillance on Rapa Nui and beyond.

Longitudinal Analysis of Influenza A(H5) Sero-Surveillance in Myanmar Ducks, 2006-2019.

Between 2006 and 2019, serological surveys in unvaccinated domestic ducks reared outdoors in Myanmar were performed, using a haemagglutination inhibition (HI) test, to confirm H5 avian influenza virus circulation and assess temporal and spatial distribution. Positive test results occurred every year that samples were collected. The annual proportion of positive farms ranged from 7.1% to 77.2%. The results revealed silent/sub-clinical influenza A (H5) virus circulation, even in years and States/Regions with no highly pathogenic avian influenza (HPAI) outbreaks reported. Further analysis of the 2018/19 results revealed considerable differences in seroconversion rates between four targeted States/Regions and between years, and showed seroconversion before and during the sampling period. By the end of the trial, a high proportion of farms were seronegative, leaving birds vulnerable to infection when sold. Positive results likely indicate infection with Gs/GD/96-lineage H5Nx HPAI viruses rather than other H5 subtype low-pathogenicity avian influenza viruses. The findings suggested persistent, but intermittent, circulation of Gs/GD/96-lineage H5Nx HPAI viruses in domestic ducks, despite the veterinary services’ outbreak detection and control efforts. The role of wild birds in transmission remains unclear but there is potential for spill-over in both directions. The findings of this study assist the national authorities in the design of appropriate, holistic avian influenza control programs.

Surveillance on respiratory diseases reveals enzootic circulation of both H5 and H9 avian influenza viruses in small-scale commercial layer farms of Bangladesh.

Poultry production in Bangladesh has been experiencing H5N1 highly pathogenic avian influenza (HPAI) and H9N2 low pathogenic avian influenza (LPAI) for the last 14years. Vaccination of chickens against H5 HPAI is in practice since the end of 2012. Subsequently, the official reporting of HPAI outbreaks gradually decreased. However, the true extent of circulation of avian influenza virus (AIV) in commercial poultry production is not clear. To explore this, we conducted active surveillance in 422 small-scale commercial layer farms in 20 villages of Mymensingh and Tangail districts of Bangladesh during 2017 and 2018 for the presence of diseases with respiratory signs. A total of 88 farms with respiratory disease problems were identified and investigated during the surveillance. In addition, 22 small-scale commercial layer farms in the neighbouring areas with respiratory disease problem were also investigated on request from the farmers. Pooled samples of oropharyngeal swabs from live birds or respiratory tissues from dead birds of the farm suffering from respiratory disease problem were tested for molecular detection of avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum and Avibacterium paragallinarum. A total of 110 farms (88 in the surveillance site and 22 in the neighbouring region) were investigated, and one or more respiratory pathogens were detected from 89 farms. AIV was detected in 57 farms often concurrently with other pathogens. Among these 57 farms, H5, H9, both H5 and H9 or non-H5 and non-H9 AIV were detected in 28, 9, 13 or 7 farms, respectively. Birds of most of the H5 AIV-positive farms did not present typical clinical signs or high mortality. Twenty such farms were observed longitudinally, which had only 1.05%-5.50% mortality but a marked drop in egg production. This widespread circulation of H5 AIV along with H9 AIV and other pathogens in small-scale commercial layer farms, often with low mortality, reaffirms the enzootic circulation of AIV in Bangladesh, which may escape syndromic surveillance focused on unusual mortality only. To reduce public health risks, strengthening of the control programme with comprehensive vaccination, enhanced biosecurity, improved surveillance and outbreak response is suggested.

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.