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.     

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.     

Genetic Diversity and Phylogenetic Analysis of Highly Pathogenic Avian Influenza (HPAI) H5N1 Viruses Circulating in Bangladesh from 2007–2011

Highly pathogenic avian influenza (HPAI) H5N1 virus has been endemic in Bangladesh since its first isolation in February 2007. Phylogenetic analysis of the haemagglutinin (HA) gene of HPAI H5N1 viruses demonstrated that 25 Bangladeshi isolates including two human isolates from 2007–2011 along with some isolates from neighbouring Asian countries (India, Bhutan, Myanmar, Nepal, China and Vietnam) segregate into two distinct clades (2.2 and 2.3). There was clear evidence of introduction of clade 2.3.2 and 2.3.4 viruses in 2011 in addition to clade 2.2 viruses that had been in circulation in Bangladesh since 2007. The data clearly demonstrated the movement of H5N1 strains between Asian countries included in this study due to migration of wild birds and/or illegal movement of poultry across borders. Interestingly, the two human isolates were closely related to the clade 2.2 Bangladeshi chicken isolates indicating that they have originated from chickens. Furthermore, comparative amino acid sequence analysis revealed several substitutions (including 189R>K and 282I>V) in HA protein of some clade 2.2 Bangladeshi viruses including the human isolates, suggesting there was antigenic drift in clade 2.2.3 viruses that were circulating between 2008 and 2011. Overall, the data imply genetic diversity among circulating viruses and multiple introductions of H5N1 viruses with an increased risk of human infections in Bangladesh, and establishment of H5N1 virus in wild and domestic bird populations, which demands active surveillance.     

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.     

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.     

Highly Pathogenic Avian Influenza Virus A/H5N1 Infection in Vaccinated Meat Duck Flocks in the Mekong Delta of Vietnam

We investigated episodes of suspected highly pathogenic avian influenza (HPAI)‐like illness among 12 meat duck flocks in two districts in Tien Giang province (Mekong Delta, Vietnam) in November 2013. In total, duck samples from 8 of 12 farms tested positive for HPAI virus subtype A/haemagglutinin 5 and neuraminidase 1 (H5N1) by real‐time RT‐PCR. Sequencing results confirmed clade of 2.3.2.1.c as the cause of the outbreaks. Most (7/8) laboratory‐confirmed positive flocks had been vaccinated with inactivated HPAI H5N1 clade 2.3.4 vaccines <6 days prior to onset of clinical signs. A review of vaccination data in relation to estimated production in the area suggested that vaccination efforts were biased towards larger flocks and that vaccination coverage was low [21.2% ducks vaccinated with two shots (range by district 7.4–34.9%)]. The low‐coverage data, the experimental evidence of lack of cross‐protection conferred by the currently used vaccines based on clade 2.3.4 together with the short lifespan of meat duck flocks (60–70 days), suggest that vaccination is not likely to be effective as a tool for control of H5N1 infection in meat duck flocks in the area.     

Re‐Emergence of a Novel H5N1 Avian Influenza Virus Variant Subclade 2.2.1.1 in Egypt During 2014

Large‐scale surveillance is crucial for understanding the evolution and the emergence of avian influenza viruses (AIVs) in endemic areas. Circulation of highly pathogenic avian influenza (HPAI) subtype H5N1 is continuously causing significant economic losses to the Egyptian poultry industry and is a threat to public health. In this report, a HPAI H5N1 strain (A/chicken/Egypt/Fadllah‐7/2014) was detected from a vaccinated flock showing clinical signs of infection. Genetic characterization of the isolate indicated a high level of nucleotide identity (95–98%) with variant and classical groups of H5N1. Moreover, multiple‐nucleotide and amino acid alignments revealed several prominent and characteristic substitutions in the surface glycoprotein, which may have biological relevance to the pathobiology of the virus. Phylogenetic analysis demonstrated that the reported isolate closely relates to H5N1 AIVs subclade 2.2.1.1 in spite of no reports of this subclade since 2011 from AI reported cases in Egyptian avian species. In conclusion, our results highlight the re‐emergence of a novel H5N1 AIV variant subclade 2.2.1.1 that could escape immunity induced by vaccines. This discovery illustrates the importance of continuous monitoring of poultry in this country for controlling AIV including identifying sources of vaccine seed viruses.     

Isolation and characterization of equine influenza virus (H3N8) from an equine influenza outbreak in Malaysia in 2015

Equine influenza is a major cause of respiratory infections in horses and can spread rapidly despite the availability of commercial vaccines. In this study, we carried out molecular characterization of Equine Influenza Virus (EIV) isolated from the Malaysian outbreak in 2015 by sequencing of the HA and NA gene segments using Sanger sequencing. The nucleotide and amino acid sequences of HA and NA were compared with representative Florida clade 1 and clade 2 strains using phylogenetic analysis. The Florida clade 1 viruses identified in this outbreak revealed numerous amino acid substitutions in the HA protein as compared to the current OIE vaccine strain recommendations and representative strains of circulating Florida sub‐lineage clade 1 and clade 2. Differences in HA included amino acids located within antigenic sites which could lead to reduced immune recognition of the outbreak strain and alter the effectiveness of vaccination against the outbreak strain. Detailed surveillance and genetic information sharing could allow genetic drift of equine influenza viruses to be monitored more effectively on a global basis and aid in refinement of vaccine strain selection for EIV.     

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.     

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.     

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.     

Epidemiological and Molecular Analysis of an Outbreak of Highly Pathogenic Avian Influenza H5N8 clade 2.3.4.4 in a German Zoo: Effective Disease Control with Minimal Culling

Outbreaks of highly pathogenic avian influenza A virus (HPAIV) subtype H5N8, clade 2.3.4.4, were first reported in January 2014 from South Korea. These viruses spread rapidly to Europe and the North American continent during autumn 2014 and caused, in Germany, five outbreaks in poultry holdings until February 2015. In addition, birds kept in a zoo in north‐eastern Germany were affected. Only a few individual white storks (Ciconia ciconia) showed clinical symptoms and eventually died in the course of the infection, although subsequent in‐depth diagnostic investigations showed that other birds kept in the same compound of the white storks were acutely positive for or had undergone asymptomatic infection with HPAIV H5N8. An exception from culling all of the 500 remaining zoo birds was granted by the competent authority. Restriction measures included grouping the zoo birds into eight epidemiological units in which 60 birds of each unit tested repeatedly negative for H5N8. Epidemiological and phylogenetical investigations revealed that the most likely source of introduction was direct or indirect contact with infected wild birds as the white storks had access to a small pond frequented by wild mallards and other aquatic wild birds during a period of 10 days in December 2014. Median network analysis showed that the zoo bird viruses segregated into a distinct cluster of clade 2.3.4.4 with closest ties to H5N8 isolates obtained from mute swans (Cygnus olor) in Sweden in April 2015. This case demonstrates that alternatives to culling exist to rescue valuable avifaunistic collections after incursions of HPAIV.     

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.     

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.     

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 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.