New Introduction of Clade 2.3.2.1 Avian Influenza Virus (H5N1) into Bangladesh

Since the first outbreak of highly pathogenic H5N1 avian inafluenza (HPAI) in Bangladesh in February 2007, a total of 519 disease events have been reported till 22 October 2011. Partial HA gene sequences of 11 selected H5N1 HPAI isolates of 2007 to 2011 were determined and subjected to phylogenetic analysis. The study revealed a recent introduction of clade 2.3.2 and 2.3.4 viruses into Bangladesh in 2011 in addition to clade 2.2 viruses that had been in circulation since 2007. Clade 2.3.2 virus isolates from Bangladesh are phylogenetically related to the newly designated clade 2.3.2.1 viruses, reported recently from Asia and Eastern Europe.     

Experimental infection of H5N1 and H5N8 highly pathogenic avian influenza viruses in Northern Pintail (Anas acuta)

The wide geographic spread of Eurasian Goose/Guangdong lineage highly pathogenic avian influenza (HPAI) clade 2.3.4.4 viruses by wild birds is of great concern. In December 2014, an H5N8 HPAI clade 2.3.4.4 Group A (2.3.4.4A) virus was introduced to North America. Long‐distance migratory wild aquatic birds between East Asia and North America, such as Northern Pintail (Anas acuta ), were strongly suspected of being a source of intercontinental transmission. In this study, we evaluated the pathogenicity, infectivity and transmissibility of an H5N8 HPAI clade 2.3.4.4A virus in Northern Pintails and compared the results to that of an H5N1 HPAI clade 2.3.2.1 virus. All of Northern Pintails infected with either H5N1 or H5N8 virus lacked clinical signs and mortality, but the H5N8 clade 2.3.4.4 virus was more efficient at replicating within and transmitting between Northern Pintails than the H5N1 clade 2.3.2.1 virus. The H5N8‐infected birds shed high titre of viruses from oropharynx and cloaca, which in the field supported virus transmission and spread. This study highlights the role of wild waterfowl in the intercontinental spread of some HPAI viruses. Migratory aquatic birds should be carefully monitored for the early detection of H5 clade 2.3.4.4 and other HPAI 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.     

Experimental infection of clade 1.1.2 (H5N1), clade 2.3.2.1c (H5N1) and clade 2.3.4.4 (H5N6) highly pathogenic avian influenza viruses in dogs

Since the emergence of highly pathogenic avian influenza (HPAI) H5N1 in Asia, the haemagglutinin (HA) gene of this virus lineage has continued to evolve in avian populations, and H5N1 lineage viruses now circulate concurrently worldwide. Dogs may act as an intermediate host, increasing the potential for zoonotic transmission of influenza viruses. Virus transmission and pathologic changes in HPAI clade 1.1.2 (H5N1)‐, 2.3.2.1c (H5N1)‐ and 2.3.4.4 (H5N6)‐infected dogs were investigated. Mild respiratory signs and antibody response were shown in dogs intranasally infected with the viruses. Lung histopathology showed lesions that were associated with moderate interstitial pneumonia in the infected dogs. In this study, HPAI H5N6 virus replication in dogs was demonstrated for the first time. Dogs have been suspected as a “mixing vessel” for reassortments between avian and human influenza viruses to occur. The replication of these three subtypes of the H5 lineage of HPAI viruses in dogs suggests that dogs could serve as intermediate hosts for avian–human influenza virus reassortment if they are also co‐infected with human influenza viruses.     

Amino acid substitutions in antigenic region B of hemagglutinin play a critical role in the antigenic drift of subclade 2.3.4.4 highly pathogenic H5NX influenza viruses

As one of the important control strategies for highly pathogenic avian influenza (HPAI) in China, vaccination has been implemented compulsively in poultry flocks since 2004. However, the emergence and dominance of the circulating antigenic variants require the update of vaccines periodically. In order to investigate the key molecular sites responsible for the antigenic drift, a total of 13 amino acid positions divergent between clade 2.3.4 H5 viruses and their descendent subclade 2.3.4.4 variants in or around the recognized antigenic epitopes A–E were initially identified through inspecting a comprehensive HA sequence alignment of the H5 subtype HPAI viruses. Subsequently, a panel of single‐site or multi‐site HA mutants was constructed by reverse genetics with two H5N1 viruses of S (clade 2.3.4) and QD1 (subclade 2.3.4.4) as the HA backbone to study their antigenic variations, respectively. The hemagglutination–inhibition assay revealed an evident impact of mutations at sites 88, 156, 205, 208, 239 and 289 to the HA antigenicity and highlighted that the amino acid substitutions located in the antigenic region B, especially the combined mutations at sites 205 and 208, were the major antigenic determinant which was also consistent with results from flow cytometry and antigenic mapping. Our findings provided more insights into the molecular mechanism of antigenic drift of the H5 subtype HPAI virus, which would be helpful for the selection of vaccine candidates and accordingly for the prevention and control of this devastating viral agent.     

Genetic and pathogenic characteristics of clade 2.3.2.1c H5N1 highly pathogenic avian influenza viruses isolated from poultry outbreaks in Laos during 2015–2018

Since 2004, there have been multiple outbreaks of H5 highly pathogenic avian influenza (HPAI) viruses in Laos. Here, we isolated H5N1 HPAI viruses from poultry outbreaks in Laos during 2015–2018 and investigated their genetic characteristics and pathogenicity in chickens. Phylogenetic analysis revealed that the isolates belonged to clade 2.3.2.1c and that they differed from previous Laos viruses with respect to genetic composition. In particular, the isolates were divided into two genotypes, each of which had a different NS segments. The results of possible migration analysis suggested a high likelihood that the Laos isolates were introduced from neighbouring countries, particularly Vietnam. The recent Laos isolate, A/Duck/Laos/NL‐1504599/2018, had an intravenous pathogenicity index score of 3.0 and showed a 50% chicken lethal dose of 10^2.5 EID₅₀/0.1 ml, indicating high pathogenicity. The isolated viruses exhibited no critical substitution in the markers associated with mammalian adaptation, but possess markers related to neuraminidase inhibitor resistance. These results emphasize the need for ongoing surveillance of circulating influenza virus in South‐East Asia, including Laos, to better prepare for and mitigate global spread of H5 HPAI.     

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.     

Genetic relationship between poultry and wild bird viruses during the highly pathogenic avian influenza H5N6 epidemic in the Netherlands, 2017–2018

In the Netherlands, three commercial poultry farms and two hobby holdings were infected with highly pathogenic avian influenza (HPAI) H5N6 virus in the winter of 2017–2018. This H5N6 virus is a reassortant of HPAI H5N8 clade 2.3.4.4 group B viruses detected in Eurasia in 2016. H5N6 viruses were also detected in several dead wild birds during the winter. However, wild bird mortality was limited compared to the caused by the H5N8 group B virus in 2016–2017. H5N6 virus was not detected in wild birds after March, but in late summer infected wild birds were found again. In this study, the complete genome sequences of poultry and wild bird viruses were determined to study their genetic relationship. Genetic analysis showed that the outbreaks in poultry were not the result of farm‐to‐farm transmissions, but rather resulted from separate introductions from wild birds. Wild birds infected with viruses related to the first outbreak in poultry were found at short distances from the farm, within a short time frame. However, no wild bird viruses related to outbreaks 2 and 3 were detected. The H5N6 virus isolated in summer shares a common ancestor with the virus detected in outbreak 1. This suggests long‐term circulation of H5N6 virus in the local wild bird population. In addition, the pathogenicity of H5N6 virus in ducks was determined, and compared to that of H5N8 viruses detected in 2014 and 2016. A similar high pathogenicity was measured for H5N6 and H5N8 group B viruses, suggesting that biological or ecological factors in the wild bird population may have affected the mortality rates during the H5N6 epidemic. These observations suggest different infection dynamics for the H5N6 and H5N8 group B viruses in the wild bird population.     

Experimental infection of highly pathogenic avian influenza viruses,Clade 2.3.4.4 H5N6 and H5N8, in Mandarin ducks from South Korea

Outbreaks of highly pathogenic avian influenza (HPAI ) have been reported worldwide. Wild waterfowl play a major role in the maintenance and transmission of HPAI . Highly pathogenic avian influenza subtype H5N6 and H5N8 viruses simultaneously emerged in South Korea. In this study, the comparative pathogenicity and infectivity of Clade 2.3.4.4 Group B H5N8 and Group C H5N6 viruses were evaluated in Mandarin duck (Aix galericulata ). None of the ducks infected with H5N6 or H5N8 viruses showed clinical signs or mortality. Serological assays revealed that the HA antigenicity of H5N8 and H5N6 viruses was similar to each other. Moreover, both the viruses did not replicate after cross‐challenging with H5N8 and H5N6 viruses, respectively, as the second infection. Although both the viruses replicated in most of the internal organs of the ducks, viral replication and shedding through cloaca were higher in H5N8‐infected ducks than in H5N6‐infected ducks. The findings of this study provide preliminary information to help estimate the risks involved in further evolution and dissemination of Clade 2.3.4.4 HPAI viruses among wild birds.     

Biosecurity and Circulation of Influenza A (H5N1) Virus in Live‐Bird Markets in Bangladesh, 2012

Bangladesh has been considered as one of the five countries endemic with highly pathogenic avian influenza A subtype H5N1 (HPAI H5N1). Live‐bird markets (LBMs) in south Asian countries are believed to play important roles in the transmission of HPAI H5N1 and others due to its central location as a hub of the poultry trading. Food and Agriculture Organization (FAO) of the United Nations has been promoting improved biosecurity in LBMs in Bangladesh. In 2012, by enrolling 32 large LBMs: 10 with FAO interventions and 22 without assistance, we assessed the virus circulation in the selected LBMs by applying standard procedures to investigate market floors, poultry stall floors, poultry‐holding cases and slaughter areas and the overall biosecurity using a questionnaire‐based survey. Relative risk (RR) was examined to compare the prevalence of HPAI H5N1 in the intervened and non‐intervened LBMs. The measures practised in significantly more of the FAO‐intervened LBMs included keeping of slaughter remnants in a closed container; decontamination of poultry vehicles at market place; prevention of crows’ access to LBM, market/floor cleaning by market committee; wet cleaning; disinfection of floor/poultry stall after cleaning; and good supply of clean water at market (P < 0.05). Conversely, disposal of slaughter remnants elsewhere at market and dry cleaning were in operation in more of the FAO non‐intervened LBMs (P < 0.05). The RR for HPAI H5N1 in the intervened and non‐intervened LBMs was 1.1 (95% confidence interval 0.44–2.76), suggesting that the proportion positive of the virus in the two kinds of LBM did not vary significantly (P = 0.413). These observations suggest that the viruses are still maintained at the level of production in farms and circulating in LBMs in Bangladesh regardless of interventions, albeit at lower levels than in other endemic countries.     

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.     

Outbreaks of Clade 2.3.4.4 H5N8 highly pathogenic avian influenza in 2018 in the northern regions of South Africa were unrelated to those of 2017

Asian‐origin H5N8 highly pathogenic avian influenza (HPAI) viruses of the H5 Goose/Guangdong/96 lineage, clade 2.3.4.4 group B, reached South Africa by June 2017. By the end of that year, 5.4 million layers and broiler chickens died or were culled, with total losses in the poultry industry estimated at US$ 140 million, and thousands of exotic birds in zoological collections, endangered endemic species and backyard poultry and pet birds also perished. The 2017 H5N8 HPAI outbreaks were characterized by two distinct spatial clusters, each associated with specific reassortant viral genotypes. Genotypes 1, 2, 3 and 5 were restricted to the northern regions, spanning the provinces of Limpopo, Gauteng, North West, Mpumalanga, KwaZulu‐Natal and Free State. The second, much larger cluster of outbreaks was in the south, in the Western and Eastern Cape provinces, wherein 2017 and 2018 outbreaks were caused solely by genotype 4. The last confirmed case of H5N8 HPAI in the northern region in 2017 was in early October, and the viruses seemed to disappear over the summer. However, starting in mid‐February 2018, H5N8 HPAI outbreaks resurged in the north. Viruses from two of the eight outbreaks were sequenced, one from an outbreak in quails (Coturnix japonica) in the North West Province, and another from commercial pullets in the Gauteng province. Phylogenetic analysis identified the viruses as a distinct sixth genotype that was most likely a new introduction to South Africa in early 2018.     

Risk of Introduction of H5N1 HPAI from Europe to Spain by Wild Water Birds in Autumn

Highly pathogenic avian influenza (HPAI) subtype H5N1 continues to circulate across Eurasia and Africa since its unprecedented rapid spread in 2005. Diffusion by wild bird movements has been evidenced in the European Union in 2006 and 2007. Spain is an important wintering quarter for aquatic birds from northern latitudes, so identifying the critical areas and species where an outbreak is prone to happen is necessary. This work presents an assessment of the risk of introduction of H5N1 HPAI in Spain by aquatic wild birds estimating a relative risk value per province. For this purpose, an assessment of the release and exposure to the risk of infection with H5N1 HPAI of 25 selected water bird species has been carried out. Parameters considered in the assessment include H5N1 HPAI notifications from 2006 to 2008 and factors that favour the occurrence or persistence of H5N1 HPAI (wetlands’ surface, low temperatures), together with aquatic wild birds’ movements parameters (departure, destination, stop‐overs, abundance) and parameters relative to the susceptible population in Spain: poultry density and wild aquatic abundance. Results show the relative risk for each Spanish province of experiencing H5N1 HPAI introduced by wild aquatic birds helping to identify higher risk areas.     

Emergence and spread of highly pathogenic avian influenza A(H5N8) in Europe in 2016‐2017

Circulation of highly pathogenic avian influenza (HPAI ) viruses poses a continuous threat to animal and public health. After the 2005–2006 H5N1 and the 2014–2015 H5N8 epidemics, another H5N8 is currently affecting Europe. Up to August 2017, 1,112 outbreaks in domestic and 955 in wild birds in 30 European countries have been reported, the largest epidemic by a HPAI virus in the continent. Here, the main epidemiological findings are described. While some similarities with previous HPAI virus epidemics were observed, for example in the pattern of emergence, significant differences were also patent, in particular the size and extent of the epidemic. Even though no human infections have been reported to date, the fact that A/H5N8 has affected so far 1,112 domestic holdings, increases the risk of exposure of humans and therefore represents a concern. Understanding the epidemiology of HPAI viruses is essential for the planning future surveillance and control activities.     

Phylogeography of H5N1 avian influenza virus in Indonesia

Highly pathogenic avian influenza (HPAI ) viruses of the H5N1 subtype are a major concern to human and animal health in Indonesia. This study aimed to characterize transmission dynamics of H5N1 over time using novel Bayesian phylogeography methods to identify factors which have influenced the spread of H5N1 in Indonesia. We used publicly available hemagglutinin sequence data sampled between 2003 and 2016 to model ancestral state reconstruction of HPAI H5N1 evolution. We found strong support for H5N1 transmission routes between provinces in Java Island and inter‐island transmissions, such as between Nusa Tenggara and Kalimantan Islands, not previously described. The spread is consistent with wild bird flyways and poultry trading routes. H5N1 migration was associated with the regions of high chicken densities and low human development indices. These results can be used to inform more targeted planning of H5N1 control and prevention activities in Indonesia.     

Evolutionary and Ecological Dynamics of Transboundary Disease Caused by H5N1 Virus in Southeast Asia

Southeast Asia has been the breeding ground for many emerging diseases in the past decade, and it is in this region that new genetic variants of HPAI H5N1 viruses have been emerging. Cross‐border movement of animals accelerates the spread of H5N1, and the changing environmental conditions also exert strong selective pressure on the viruses. The transboundary zoonotic diseases caused by H5N1 pose a serious and continual threat to global economy and public health. Here, we divided the H5N1 viruses isolated in Southeast Asia during 2003–2009 into four groups according to their phylogenetic relationships among HA gene sequences. Molecular evolution analysis suggests populations in expansion rather than a positive selection for group 2 and group 3, yet group 4 is under strong positive selection. Site 193 was found to be a potential glycosylation site and located in receptor‐binding domain. Note that site 193 tends to appear in avian isolates instead of human strains. Population dynamics analysis reveals that the effective population size of infections in Southeast Asia has undergone three obvious increases, and the results are consistent with the epidemiological analysis. Ecological and phylogeographical analyses show that agro‐ecological environments, migratory birds, domestic waterfowl, especially free‐ranging ducks, are crucial in the occurrence, maintenance and spread of H5N1 virus. The epidemiological links between Indonesia and Suphanburi observed suggest that viruses in Indonesia were originated from multiple introductions.     

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.     

Respiratory disease due to mixed viral infections in poultry flocks in Egypt between 2017 and 2018: Upsurge of highly pathogenic avian influenza virus subtype H5N8 since 2018

For several years, poultry production in Egypt has been suffering from co‐circulation of multiple respiratory viruses including highly pathogenic avian influenza virus (HPAIV) H5N1 (clade 2.2.1.2) and low pathogenic H9N2 (clade G1‐B). Incursion of HPAIV H5N8 (clade 2.3.4.4b) to Egypt in November 2016 via wild birds followed by spread into commercial poultry flocks further complicated the situation. Current analyses focussed on 39 poultry farms suffering from respiratory manifestation and high mortality in six Egyptian governorates during 2017–2018. Real‐time RT‐PCR (RT‐qPCR) substantiated the co‐presence of at least two respiratory virus species in more than 80% of the investigated flocks. The percentage of HPAIV H5N1‐positive holdings was fairly stable in 2017 (12.8%) and 2018 (10.2%), while the percentage of HPAIV H5N8‐positive holdings increased from 23% in 2017 to 66.6% during 2018. The proportion of H9N2‐positive samples was constantly high (2017:100% and 2018:63%), and H9N2 co‐circulated with HPAIV H5N8 in 22 out of 39 (56.8%) flocks. Analyses of 26 H5, 18 H9 and 4 N2 new sequences confirmed continuous genetic diversification. In silico analysis revealed numerous amino acid substitutions in the HA and NA proteins suggestive of increased adaptation to mammalian hosts and putative antigenic variation. For sensitive detection of H9N2 viruses by RT‐qPCR, an update of primers and probe sequences was crucial. Reasons for the relative increase of HPAIV H5N8 infections versus H5N1 remained unclear, but lack of suitable vaccines against clade 2.3.4.4b cannot be excluded. A reconsideration of surveillance and control measures should include updating of diagnostic tools and vaccination strategies.