Characterization of H7N9 avian influenza viruses isolated from duck meat products

Avian influenza H7N9 viruses have caused five epidemic waves of human infections since the first human cases were reported in 2013. In 2016, the initial low pathogenic avian influenza (LPAI) H7N9 viruses became highly pathogenic, acquiring multi‐basic amino acids at the haemagglutinin cleavage site. These highly pathogenic avian influenza (HPAI) H7N9 viruses have been detected in poultry and humans in China, causing concerns of a serious threat to global public health. In Japan, both HPAI and LPAI H7N9 viruses were isolated from duck meat products carried illegally and relinquished voluntarily at the border by passengers on flights from China to Japan between 2016 and 2017. Some of the LPAI and HPAI H7N9 viruses detected at the border in Japan were characterized previously in chickens and ducks; however, their pathogenicity and replicative ability in mammals remain unknown. In this study, we assessed the biological features of two HPAI H7N9 virus isolates [A/duck/Japan/AQ‐HE29‐22/2017 (HE29‐22) and A/duck/Japan/AQ‐HE29‐52/2017 (HE29‐52); both of these viruses were isolated from duck meat at the border)] and an LPAI H7N9 virus isolate [A/duck/Japan/AQ‐HE28‐3/2016 (HE28‐3)] in mice and ferrets. In mice, HE29‐52 was more pathogenic than HE29‐22 and HE28‐3. In ferrets, the two HPAI virus isolates replicated more efficiently in the lower respiratory tract of the animals than did the LPAI virus isolate. Our results indicate that HPAI H7N9 viruses with the potential to cause severe diseases in mammals have been illegally introduced to Japan.     

Characterization of avian influenza H5N3 reassortants isolated from migratory waterfowl and domestic ducks in China from 2015 to 2018

Wild and domestic aquatic birds are the natural reservoirs of avian influenza viruses (AIVs). All subtypes of AIVs, including 16 hemagglutinin (HA) and nine neuraminidase (NA), have been isolated from the waterfowls. The H5 viruses in wild birds display distinct biological differences from their highly pathogenic H5 counterparts. Here, we isolated seven H5N3 AIVs including three from wild birds and four from domestic ducks in China from 2015 to 2018. The isolation sites of all the seven viruses were located in the region of the East Asian‐Australasian Migratory Flyway. Phylogenetic analysis indicated that the surface genes of these viruses originated from the wild bird H5 HA subtype and the N3 Eurasian lineage. The internal genes of the seven H5N3 isolates are derived from the five gene donors isolated from the wild birds or ducks in Eastern‐Asia region. They were also divided into five genotypes according to their surface genes and internal gene combinations. Interestingly, two of the seven H5N3 viruses contributed their partial internal gene segments (PB1, M and NS) to the newly emerged H7N4 reassortants, which have caused first human H7N4 infection in China in 2018. Moreover, we found that the H5N3 virus used in this study react with the anti‐serum of the H5 subtype vaccine isolate (Re‐11 and Re‐12) and reacted well with the Re‐12 anti‐serum. Our findings suggest that worldwide intensive surveillance and the H5 vaccination (Re‐11 and Re‐12) in domestic ducks are needed to monitor the emergence of novel H5N3 reassortants in wild birds and domestic ducks and to prevent H5N3 viruses transmission from the apparently healthy wild birds and domestic ducks to chickens.     

The PB2 and M genes are critical for the superiority of genotype S H9N2 virus to genotype H in optimizing viral fitness of H5Nx and H7N9 avian influenza viruses in mice

Genotype S H9N2 avian influenza virus, which has been predominant in China since 2010, contributed its entire internal gene cassette to the genesis of novel reassortant influenza viruses, including H5Nx, H7N9 and H10N8 viruses that pose great threat to poultry and humans. A key feature of the genotype S H9N2 virus is the substitution of G1‐like M and PB2 genes for the earlier F/98‐like M and PB2 of genotype H virus. However, how this gene substitution has influenced viral adaptability of emerging influenza viruses in mammals remains unclear. We report here that reassortant H5Nx and H7N9 viruses with the genotype S internal gene cassette displayed enhanced replication and virulence over those with genotype H internal gene cassette in cell cultures as well as in the mouse models. We showed that the G1‐like PB2 gene was associated with increased polymerase activity and improved nuclear accumulation compared with the F/98‐like counterpart, while the G1‐like M gene facilitated effective translocation of RNP to cytoplasm. Our findings suggest that the genotype S H9N2 internal gene cassette, which possesses G1‐like M and PB2 genes, is superior to that of genotype H, in optimizing viral fitness, and thus have implications for assessing the potential risk of these gene introductions to generate emerging influenza viruses.     

Comparative pathogenicity of H5N6 subtype highly pathogenic avian influenza viruses in chicken,Pekin duck and Muscovy duck

In Japan during the 2016–2017 winter season, clade 2.3.4.4 highly pathogenic avian influenza viruses (HPAIVs) of the H5N6 subtype caused 12 outbreaks in chicken and Muscovy duck farms. These viruses have been circulating in Vietnam and China since 2014. In this study, we evaluated the susceptibility of chicken, Pekin duck (Anas platyrhynchos domesticus) and Muscovy duck (Cairina moschata) to H5N6 HPAIVs that originated in Japan, Vietnam and China. The H5N6 HPAIVs examined in this study were highly lethal to chickens compared with their pathogenicity in Pekin duck and Muscovy duck. One of five chickens infected with A/Muscovy duck/Aomori/1‐3T/2016 (MusDk/Aomori) survived despite viral shedding, although all of the chickens infected with the other viruses died. The 50% chicken lethal dose differed among the Japanese strains that shared the same gene constellation indicating that gene constellation was not a major determinant of pathogenicity in chicken. MusDk/Aomori, A/chicken/Niigata/1‐1T/2016 (Ck/Niigata) and A/duck/Hyogo/1/2016 (Dk/Hyogo) infected all Muscovy ducks inoculated; Ck/Niigata killed 50% of the ducks it infected whereas the other two did not kill any ducks. A/chicken/Japan/AnimalQuarantine‐HE144/2016 (HE144) isolated from chicken meat that originated in China was highly pathogenic to Pekin duck: all of the ducks died within 3.75 days of inoculation. This study shows that the pathogenicity of the clade 2.3.4.4 H5N6 HPAIVs differs not only between hosts but also within the same host species.     

Prevalence of H7N9 subtype avian influenza viruses in poultry in China, 2013–2018

H7N9 subtype avian influenza viruses (AIV) circulating in China over recent years have had an enormous impact on public health and economy. During the period between November 2016 and April 2017, an increase in human infections caused by these viruses was reported, with rapid emergence and spread of variants in China. Consequently, the government of China implemented a controversial vaccination strategy in September 2017. Here, we provide evidence of the prevalence of H7N9 AIVs in China based on systematic large‐scale surveillance in poultry during 2013–2018. Emerging variants were confirmed as highly pathogenic in chickens using the intravenous pathogenicity index (IVPI) test. The currently available vaccine provided complete protection against the H7N9 HPAIV challenge in chickens. The collective findings clearly indicate that the vaccination strategy implemented not only significantly decreases the prevalence of H7N9 AIVs in poultry but also effectively prevents human infection with H7N9 viruses.     

A new reassortant clade 2.3.2.1a H5N1 highly pathogenic avian influenza virus causing recent outbreaks in ducks,geese, chickens and turkeys in Bangladesh

A total of 15 dead or sick birds from 13 clinical outbreaks of avian influenza in ducks, geese, chickens and turkeys in 2017 in Bangladesh were examined. The presence of H5N1 influenza A virus in the affected birds was detected by RT‐PCR. Phylogenetic analysis based on full‐length gene sequences of all eight gene segments revealed that these recent outbreaks were caused by a new reassortant of clade 2.3.2.1a H5N1 virus, which had been detected earlier in 2015 during surveillance in live bird markets (LBMs) and wet lands. This reassortant virus acquired PB2, PB1, PA, NP and NS genes from low pathogenic avian influenza viruses mostly of non‐H9N2 subtypes but retained HA, NA and M genes of the old clade 2.3.2.1a viruses. Nevertheless, the HA gene of these new viruses was 2.7% divergent from that of the old clade 2.3.2.1a viruses circulated in Bangladesh. Interestingly, similar reassortment events could be traced back in four 2.3.2.1a virus isolates of 2013 from backyard ducks. It suggests that this reassortant virus emerged in 2013, which took two years to be detected at a broader scale (i.e. in LBMs), another two years until it became widely spread in poultry and fully replaced the old viruses. Several mutations were detected in the recent Bangladeshi isolates, which are likely to influence possible phenotypic alterations such as increased mammalian adaptation, reduced susceptibility to antiviral agents and reduced host antiviral response.     

Poultry trading behaviours in Vietnamese live bird markets as risk factors for avian influenza infection in chickens

Vietnamese poultry are host to co‐circulating subtypes of avian influenza viruses, including H5N1 and H9N2, which pose a great risk to poultry productivity and to human health. AIVs circulate throughout the poultry trade network in Vietnam, with live bird markets being an integral component to this network. Traders at LBMs exhibit a variety of trading practices, which may influence the transmission of AIVs. We identified trading practices that impacted on AIV prevalence in chickens marketed in northern Vietnamese LBMs. We generated sequencing data for 31 H9N2 and two H5N6 viruses. Viruses isolated in the same LBM or from chickens sourced from the same province were genetically closer than viruses isolated in different LBMs or from chickens sourced in different provinces. The position of a vendor in the trading network impacted on their odds of having AIV‐infected chickens. Being a retailer and purchasing chickens from middlemen was associated with increased odds of infection, whereas odds decreased if vendors purchased chickens directly from large farms. Odds of infection were also higher for vendors having a greater volume of ducks unsold per day. These results indicate how the spread of AIVs is influenced by the structure of the live poultry trading network.     

Domestic ducks play a major role in the maintenance and spread of H5N8 highly pathogenic avian influenza viruses in South Korea

The H5N8 highly pathogenic avian influenza viruses (HPAIVs) belonging to clade 2.3.4.4 spread from Eastern China to Korea in 2014 and caused outbreaks in domestic poultry until 2016. To understand how H5N8 HPAIVs spread at host species level in Korea during 2014–2016, a Bayesian phylogenetic analysis was used for ancestral state reconstruction and estimation of the host transition dynamics between wild waterfowl, domestic ducks and chickens. Our data support that H5N8 HPAIV most likely transmitted from wild waterfowl to domestic ducks, and then maintained in domestic ducks followed by dispersal of HPAIV from domestic ducks to chickens, suggesting domestic duck population plays a central role in the maintenance, amplification and spread of wild HPAIV to terrestrial poultry in Korea.     

A Systematic Review of the Comparative Epidemiology of Avian and Human Influenza A H5N1 and H7N9 – Lessons and Unanswered Questions

The aim of this work was to explore the comparative epidemiology of influenza viruses, H5N1 and H7N9, in both bird and human populations. Specifically, the article examines similarities and differences between the two viruses in their genetic characteristics, distribution patterns in human and bird populations and postulated mechanisms of global spread. In summary, H5N1 is pathogenic in birds, while H7N9 is not. Yet both have caused sporadic human cases, without evidence of sustained, human‐to‐human spread. The number of H7N9 human cases in the first year following its emergence far exceeded that of H5N1 over the same time frame. Despite the higher incidence of H7N9, the spatial distribution of H5N1 within a comparable time frame is considerably greater than that of H7N9, both within China and globally. The pattern of spread of H5N1 in humans and birds around the world is consistent with spread through wild bird migration and poultry trade activities. In contrast, human cases of H7N9 and isolations of H7N9 in birds and the environment have largely occurred in a number of contiguous provinces in south‐eastern China. Although rates of contact with birds appear to be similar in H5N1 and H7N9 cases, there is a predominance of incidental contact reported for H7N9 as opposed to close, high‐risk contact for H5N1. Despite the high number of human cases of H7N9 and the assumed transmission being from birds, the corresponding level of H7N9 virus in birds in surveillance studies has been low, particularly in poultry farms. H7N9 viruses are also diversifying at a much greater rate than H5N1 viruses. Analyses of certain H7N9 strains demonstrate similarities with engineered transmissible H5N1 viruses which make it more adaptable to the human respiratory tract. These differences in the human and bird epidemiology of H5N1 and H7N9 raise unanswered questions as to how H7N9 has spread, which should be investigated further.     

Phylogenetic variations of highly pathogenic H5N6 avian influenza viruses isolated from wild birds in the Izumi plain,Japan, during the 2016–17 winter season

During the 2016–2017 winter season, we isolated 33 highly pathogenic avian influenza viruses (HPAIVs) of H5N6 subtype and three low pathogenic avian influenza viruses (LPAIVs) from debilitated or dead wild birds, duck faeces, and environmental water samples collected in the Izumi plain, an overwintering site for migratory birds in Japan. Genetic analyses of the H5N6 HPAIV isolates revealed previously unreported phylogenetic variations in the PB2, PB1, PA, and NS gene segments and allowed us to propose two novel genotypes for the contemporary H5N6 HPAIVs. In addition, analysis of the four gene segments identified close phylogenetic relationships between our three LPAIV isolates and the contemporary H5N6 HPAIV isolates. Our results implied the co‐circulation and co‐evolution of HPAIVs and LPAIVs within the same wild bird populations, thereby highlighting the importance of avian influenza surveillance targeting not only for HPAIVs but also for LPAIVs.     

A Meta‐Analysis of the Prevalence of Influenza A H5N1 and H7N9 Infection in Birds

Despite a much higher rate of human influenza A (H7N9) infection compared to influenza A (H5N1), and the assumption that birds are the source of human infection, detection rates of H7N9 in birds are lower than those of H5N1. This raises a question about the role of birds in the spread and transmission of H7N9 to humans. We conducted a meta‐analysis of overall prevalence of H5N1 and H7N9 in different bird populations (domestic poultry, wild birds) and different environments (live bird markets, commercial poultry farms, wild habitats). The electronic database, Scopus, was searched for published papers, and Google was searched for country surveillance reports. A random effect meta‐analysis model was used to produce pooled estimates of the prevalence of H5N1 and H7N9 for various subcategories. A random effects logistic regression model was used to compare prevalence rates between H5N1 and H7N9. Both viruses have low prevalence across all bird populations. Significant differences in prevalence rates were observed in domestic birds, farm settings, for pathogen and antibody testing, and during routine surveillance. Random effects logistic regression analyses show that among domestic birds, the prevalence of H5N1 is 47.48 (95% CI: 17.15–133.13, P < 0.001) times higher than H7N9. In routine surveillance (where surveillance was not conducted in response to human infections or bird outbreaks), the prevalence of H5N1 is still higher than H7N9 with an OR of 43.02 (95% CI: 16.60–111.53, P < 0.001). H7N9 in humans has occurred at a rate approximately four times higher than H5N1, and for both infections, birds are postulated to be the source. Much lower rates of H7N9 in birds compared to H5N1 raise doubts about birds as the sole source of high rates of human H7N9 infection. Other sources of transmission of H7N9 need to be considered and explored.     

Detection of reassortant avian influenza A (H11N9) virus in wild birds in China

Human infectious avian influenza virus (AIV) H7N9 emerged in China in 2013. The N9 gene of H7N9, which has the ability to cause death in humans, originated from an H11N9 influenza strain circulating in wild birds. To investigate the frequency and distribution of the N9 gene of the H11N9 and H7N9 influenza virus circulating in wild birds between 2006 and 2015, 35,604 samples were collected and tested. No H7N9 but four strains of the H11N9 subtype AIV were isolated, and phylogenetic analyses showed that the four H11N9 viruses were intra‐subtype and inter‐subtype reassortant viruses. A sequence analysis revealed that all six internal genes of A/wild bird/Anhui/L306/2014 (H11N9) originated from an H9N2 AIV isolated in Korea. The H9N2 strain, which is an inner gene donor reassorted with other subtypes, is a potential threat to poultry and even humans. It is necessary to increase monitoring of the emergence and spread of H11N9 AIV in wild birds.     

Genetic characteristics,pathogenicity and transmission of H5N6 highly pathogenic avian influenza viruses in Southern China

Since 2014, H5 highly pathogenic avian influenza viruses (HPAIVs) from clade 2.3.4.4 have been persistently circulating in Southern China. This has caused huge losses in the poultry industry. In this study, we analysed the genetic characteristics of seven H5N6 HPAIVs of clade 2.3.4.4 that infected birds in Southern China in 2016. Phylogenetic analysis grouped the HA, PB2, PA, M and NS genes as MIX‐like, and the NA genes grouped into the Eurasian lineage. The PB1 genes of the GS24, GS25, CK46 and GS74 strains belonged to the VN 2014‐like group and the others were grouped as MIX‐like. The NP genes of GS24 and GS25 strains belonged to the ZJ‐like group, but the others were MIX‐like. Thus, these viruses came from different genotypes, and the GS24, GS25, CK46 and GS74 strains displayed genotype recombination. Additionally, our results showed that the mean death time of all chickens inoculated with 10⁵ EID₅₀ of CK46 or GS74 viruses was 3 and 3.38 days, respectively. The viruses replicated at high titers in all tested tissues of the inoculated chickens. They also replicated in all tested tissues of naive contact chickens, but their replication titers in some tissues were significantly different (p < 0.05). Thus, the viruses displayed high pathogenicity and variable transmission in chickens. Therefore, it is necessary to focus on the pathogenic variation and molecular evolution of H5N6 HPAIVs in order to prevent and control avian influenza in China.     

Genetic characterization of an H13N2 low pathogenic avian influenza virus isolated from gulls in China

Low pathogenic avian influenza viruses circulate in wild birds but are occasionally transmitted to other species, including poultry, mammals and humans. To date, infections with low pathogenic avian influenza viruses of HA subtype 6, HA subtype 7, HA subtype 9 and HA subtype 10 among humans have been reported. However, the epidemiology, genetics and ecology of low pathogenic avian influenza viruses have not been fully understood thus far. Therefore, persistent surveillance of low pathogenic avian influenza virus infections in wild birds and other species is needed. Here, we found a low pathogenic avian influenza virus of the subtype H13N2 (abbreviated as WH42) in black‐tailed gulls in China. All gene sequences of this H13N2 virus were determined and used for subsequent analysis. Phylogenetic analysis of the HA gene and NA gene indicated that WH42 was derived from the Eurasian lineage. We analysed the timing of the reassortment events and found that WH42 was a reassortant whose genes were transferred from avian influenza viruses circulating in Asia, Europe and North America. Additionally, WH42 possessed several molecular markers associated with mammalian virulence and mammalian transmissibility. Interestingly, we also found low but detectable haemagglutination inhibition antibodies against H13N2 low pathogenic avian influenza virus in serum samples collected from chickens. Taken together, our findings show that the H13 virus may have been introduced into poultry and that sustainable surveillance in gulls and poultry is required.     

Highly Pathogenic Avian Influenza H5N8 Clade 2.3.4.4 Virus: Equivocal Pathogenicity and Implications for Surveillance Following Natural Infection in Breeder Ducks in the United Kingdom

Since early 2014, several outbreaks involving novel reassortant highly pathogenic avian influenza (HPAI) A(H5N8) viruses have been detected in poultry and wild bird species in Asia, Europe and North America. These viruses have been detected in apparently healthy and dead wild migratory birds, as well as in domestic chickens, turkeys, geese and ducks. In this study, we describe the pathology of an outbreak of H5N8 HPAIV in breeder ducks in the UK. A holding with approximately 6000 breeder ducks, aged approximately 60 weeks, showed a gradual reduction in egg production and increased mortality over a 7‐day period. Post‐mortem examination revealed frequent fibrinous peritonitis, with severely haemorrhagic ovarian follicles and occasional splenic and pancreatic necrosis and high incidence of mycotic granulomas in the air sacs and lung. Low‐to‐moderate levels of HPAI H5N8 virus were detected mainly in respiratory and digestive tract, with minor involvement of other organs. Although histopathological examination confirmed the gross pathology findings, intralesional viral antigen detection by immunohistochemistry was not observed. Immunolabelled cells were rarely only present in inflamed air sacs and serosa, usually superficial to granulomatous inflammation. Abundant bacterial microcolonies were observed in haemorrhagic ovaries and oviduct. The limited viral tissue distribution and presence of inter‐current fungal and bacterial infections suggest a minor role for HPAIV H5N8 in clinical disease in layer ducks.     

Avian Influenza H5N1 Surveillance and its Dynamics in Poultry in Live Bird Markets,Egypt

H5N1, a highly pathogenic avian influenza (H5N1 HPAI), is an endemic disease that is significant for public health in Egypt. Live bird markets (LBMs) are widespread in Egypt and play an important role in HPAI disease dynamics. The aim of the study was to evaluate the H5N1 HPAI prevalence in representative LBMs from 2009 to 2014, assess the effects of other variables and evaluate past outbreaks and human cases. It was found that ducks and geese are high‐risk species and that the prevalence of H5N1 HPAI was higher immediately after the political crises of 2011. The end of a calendar year (June to December) was a high‐risk period for positive samples, and the risk in urban LBMs was twice the risk in rural LBMs. Winter and political unrest was associated with higher H5N1 HPAI prevalence. Both human and poultry populations will continue to rise in Egypt, so continued poultry outbreaks are likely to be linked to more human cases. LBMs will continue to play a role in the dynamics of poultry disease in Egypt, and there is a need to reorganize markets in terms of biosecurity and traceability. It may also be beneficial to reduce inter‐governorate inter‐regional movements associated with poultry trade through promotion of regional trade or in the alternative provide sanitary features along the poultry market chain to reduce the speed of H5N1 HPAI infections. Policy formulation, design and enforcement must be pro‐poor, and consideration of the sociocultural and economic realities in Egypt is important. The LBMs provide ideal platforms to carry out sound surveillance plans and mitigate zoonotic risks of H5N1 HPAI to humans.     

Co‐circulation and characterization of HPAI‐H5N1 and LPAI‐H9N2 recovered from a duck farm,Yogyakarta, Indonesia

In July 2016, an avian influenza outbreak in duck farms in Yogyakarta province was reported to Disease Investigation Center (DIC), Wates, Indonesia, with approximately 1,000 ducks died or culled. In this study, two avian influenza (AI) virus subtypes, A/duck/Bantul/04161291‐OR/2016 (H5N1) and A/duck/Bantul/04161291‐OP/2016 (H9N2) isolated from ducks in the same farm during an AI outbreak in Bantul district, Yogyakarta province, were sequenced and characterized. Our results showed that H5N1 virus was closely related to the highly pathogenic AI (HPAI) H5N1 of clade 2.3.2.1c, while the H9N2 virus was clustered with LPAI viruses from China, Vietnam and Indonesia H9N2 (CVI lineage). Genetic analysis revealed virulence characteristics for both in avian and in mammalian species. In summary, co‐circulation of HPAI‐H5N1 of clade 2.3.2.1c and LPAI‐H9N2 was identified in a duck farm during an AI outbreak in Yogyakarta province, Indonesia. Our findings raise a concern of the potential risk of the viruses, which could increase viral transmission and/or threat to human health. Routine surveillance of avian influenza viruses should be continuously conducted to understand the dynamic and diversity of the viruses for influenza prevention and control in Indonesia and SEA region.     

Isolation and genetic characterization of H13N8 low pathogenic avian influenza virus from migratory birds in eastern China

Low pathogenic avian influenza virus (LPAIV) is an important zoonotic pathogen. Migratory birds are the natural reservoir for all 16 haemagglutinin (HA) and nine neuraminidase (NA) subtypes of LPAIV. Surveillance of LPAIV in migratory waterfowl and poultry is important for animal and public health. An understanding of the ecology and epidemiology of LPAI viruses in their reservoirs is beneficial for routine surveillance projects. Here, we report the isolation of an H13N8 LPAIV from black‐tailed gulls in eastern China. Full genome sequences of this isolate were determined. Genetic analysis of the HA and NA segments of this isolate showed that this H13N8 LPAIV was derived from the Eurasian lineage. Additionally, we speculate that this H13N8 LPAIV was a reassortant between the North American and Eurasian lineages. Interestingly, we identified amino acid motifs responsible for increased virulence or transmission of influenza viruses in mammals. We also found weak but measurable haemagglutination inhibition antibody titers against H13N8 virus in serum samples collected from chickens. These results suggest that continued surveillance for LPAI viruses in migratory birds and poultry is required.