Generation and evaluation of an H9N1 influenza vaccine derived by reverse genetics that allows utilization of a DIVA strategy for control of H9N2 avian influenza

H9N2 avian influenza viruses have circulated widely in domestic poultry around the world, and their outbreaks have resulted in heavy morbidity and mortality. In addition, H9N2 avian influenza viruses were transmitted directly from birds to humans in Hong Kong and mainland China during 1998 and 2003, which prompted the public health authorities to seek protective strategies to control H9N2 influenza viruses. In this study, we attempted to develop a DIVA (differentiating infected and vaccinated animals) strategy for H9N2 avian influenza viruses. This strategy does not interfere with serological monitoring and allows effective control of H9N2 avian influenza. We generated a reassortant H9N1 influenza vaccine strain by reverse genetics and employed an enzyme-linked immunosorbent assay (ELISA) with a truncated N1 antigen expressed in E. coli to differentiate between vaccinated and naturally infected animals. Immunization of BALB/c mice with the inactivated reassortant H9N1 vaccine conferred protection against lethal challenge with H9N2 viruses. Meanwhile, the ELISA can be used to distinguish between vaccination and natural infection quickly and easily. Therefore, this study has opened up a new avenue for the control of H9N2 avian influenza.     

A cross-sectional serological survey of the Dutch commercial poultry population for the presence of low pathogenic avian influenza virus infections.

After the discovery of poultry infected with highly pathogenic avian influenza (HPAI) virus of subtype H7N7 in the central area of The Netherlands on 28 February 2003, the hypothesis was put forward that an outbreak of the low pathogenic (LP) variant of H7N7 had preceded, unnoticed, the occurrence of the HPAI virus. Consequently, a cross-sectional serological survey of the Dutch poultry population was executed in the second week of March 2003. The basic requirements set were detection of a 5% prevalence of flocks exposed to LPAI virus with 95% confidence within the production type stratification level within each province in The Netherlands. Because of supposed higher risk of avian influenza infections in ducks, turkeys and free-range poultry, all the commercial flocks of these production types present in The Netherlands were sampled. The serological screening of 28018 sera from 1193 randomly selected poultry farms, located outside surveillance zones showed that LPAI H7 virus infections had occurred on three neighbouring farms all located in the southwest of The Netherlands. No antibodies against the neuraminidase N7 subtype were detected in the sera of these farms, indicating that the subtype was different from the HPAI H7N7 subtype that caused the avian influenza epidemic in 2003. In addition, evidence of infections with non-H5 or non-H7 subtypes of influenza A virus were obtained in two other farms located in the northeast and the southeast of The Netherlands. It was concluded that the HPAI subtype H7N7 outbreak was most likely not preceded by a significant circulation of a LPAI subtype H7N7 virus. Based on the Dutch experience, recommendations are made to detect avian influenza infections faster in the future.     

Characterization of the M2e antibody response following highly pathogenic H5N1 avian influenza virus infection and reliability of M2e ELISA for identifying infected among vaccinated chickens

A surveillance method able to differentiate between vaccinated and infected poultry is required for those countries that practice vaccination against highly pathogenic avian influenza H5N1. The external domain of the M2 protein (M2e) of influenza virus is a potentially useful differentiating-infected from vaccinated animals (DIVA) antigen but little is known about the M2e antibody response and factors influencing its detection. In this study, the M2e antibody response was characterized in layer birds vaccinated and challenged with an Indonesian H5N1 virus isolate, using a single M2e peptide or four-branched multiple antigenic peptide form of M2e (MAP-M2e) as antigens in two separate ELISAs. Anti-M2e antibodies were absent in chicks with high level of maternal haemagglutination inhibition antibodies and also in all layers vaccinated once, twice or three times with an inactivated commercial H5N1 vaccine. In contrast, anti-M2e antibodies were detected in vaccinated layers challenged with H5N1 virus and their presence was associated with virus isolation and an increase in haemagglutination inhibition titres. The number of birds that developed M2e antibodies, as well as the strength and duration of the M2e antibody response were strongly influenced by the length of the interval between vaccination and challenge. Birds challenged at six weeks after vaccination all developed M2e antibodies by 14 days that lasted until at least 56 days after infection. In birds challenged at two weeks after vaccination, only a proportion of birds developed M2e antibodies by 14 days that lasted only until 28 days post-infection. Both single M2e peptide and MAP-M2e ELISAs had high diagnostic specificity but the diagnostic sensitivity of MAP-M2e ELISA was significantly higher and more effective in detecting M2e antibody in immune and infected birds. The results show that MAP-M2e ELISA would be useful for surveillance in countries using vaccination to control highly pathogenic avian influenza H5N1.     

Influenza virus subtypes in aquatic birds of eastern Germany

Summary We report the findings of a 12-year surveillance study (1977–89) of avian influenza A viruses in eastern Germany. Viruses were isolated directly from feral ducks (n=236) and other wild birds (n=89); from domestic ducks (n=735) living on a single farm; and from white Pekin ducks (n=193) used as sentinels for populations of wild aquatic birds; mainly sea birds. The efficiency of virus isolation was 9.9% overall, with considerable variability noted among species: 8.7% in wild ducks, 0.9% in other feral birds and 38% in Pekin ducks. Use of sentinel ducks in wild pelagic bird colonies improved virus detection rates fivefold, suggesting that this approach is advantageous in ecological studies. Among the 40 different combinations of hemagglutinin (HA) and neuraminidase (NA) subtypes we identified, H6N1 predominated (23.6% for all avian species), followed by H4N6 (11%). Among individual species, the frequency profiles favored H2N3 (20.8%) and H4N6 (20.3%) in feral ducks; H7N7 (22.3%), H4N6 (24.4%) and H2N3 (10.4%) in Pekin ducks used as sentinels; and H6N1 (34.8%) and H6N6 (15.1%) in domestic ducks maintained on a single farm. By relying on sentinel birds for serological assays, it was possible to trace an influenza season in feral swan populations, beginning in August and continuing through the winter months. Comparison of subtype distribution of influenza viruses for Europe and North American showed significant differences. This supports the fact of two geographically distinct gene pools of influenza viruses in birds connected with their distinct flyways of each hemisphere. The high frequency of isolation of H2 influenza viruses is of considerable interest to those interested in the recycling of this subtype in humans. Similarly the frequent isolation of H7N7 influenza viruses raises concern about reservoirs of potentially pathogenic influenza virus for domestic poultry. Our results confirm the existence of a vast reservoir of influenza A viruses in European aquatic birds, which possesses sufficient diversity to account for strains that infect lower animals and humans.This article is dedicated to the memory of Dr. Herbert Sinnecker who died in 1991 at the age of 61. He was the Director of the Institute of Viral Zoonosis in the former German Democratic Republic (GDR). It was Herbert Sinnecker's forsight and understanding of the need to resolve the origin of human and animal influenza pandemics that initiated the studies described in this article. He developed novel epidemiological and ecological methods that permitted definition of the influenza virus gene pool in central Europe. The unification of Germany made it possible to publish this article; otherwise, the studies encouraged and organized by H. Sinnecker would have been lost to the scientific community.     

广州地区禽H9N2亚型流感病毒的发现及感染人调查

目的 了解广州地区禽流感病毒在家禽中的流行及感染人的情况，防止香港H5N1禽流感在广州地区流行。方法 对广州地区的主要鸡场和农贸市场的家禽和密切接触家禽的职业人群进行病原学和血清学的检测。病毒分离同时采用MDCK细胞和鸡胚双腔接种法；采用微量血凝抑制半致敏法进行血清学检测。结果 从54份鸡咽拭液中分离到1株H9N2亚型流感病毒；鸡及职业人群血对分离的H9N2毒株的血抑抗体阳性率分别为12．8％和15．1％。结论 广州地区鸡群中有H9N2，亚型流感病毒存在，禽H9N2亚型流感病毒能感染人。     

Intracontinental and intercontinental dissemination of Asian H5 highly pathogenic avian influenza virus (clade 2.3.4.4) in the winter of 2014–2015

Asian H5 highly pathogenic avian influenza viruses (HPAIVs) that possess the clade 2.3.4.4 HA gene have been identified in wild birds and poultry since late 2014 in both Europe and North America (N. America). Clade 2.3.4.4 H5 HPAIVs of the H5N8 subtype have been isolated in both regions, whereas reassortment viruses with NA N1 and N2 subtypes of the North American (N. American). avian lineage have only been identified in N. America. The HA genes of those isolates were closely related to genes of the HPAIVs that have caused massive outbreaks in poultry in Korea since January 2014. The outbreaks caused by those viruses and the genetic relatedness of their HA and NA genes are reviewed in this study. Although the illegal movement of poultry and poultry products cannot be ruled out as a cause of intercontinental and intracontinental dissemination of clade 2.3.4.4 H5 HPAIVs during the winter of 2014–2015, transmission of the viruses by infected migratory birds appears to be a more plausible mechanism for their dissemination. In particular, the involvement of migratory birds in HPAIV transmission between Asia and N. America is highly likely because of the reassortments between H5N8 HPAIV and the N. American lineage avian influenza viruses. Copyright © 2015 John Wiley & Sons, Ltd.     

Pandemic threat posed by avian influenza A viruses

Influenza pandemics, defined as global outbreaks of the disease due to viruses with new antigenic subtypes, have exacted high death tolls from human populations. The last two pandemics were caused by hybrid viruses, or reassortants, that harbored a combination of avian and human viral genes. Avian influenza viruses are therefore key contributors to the emergence of human influenza pandemics. In 1997, an H5N1 influenza virus was directly transmitted from birds in live poultry markets in Hong Kong to humans. Eighteen people were infected in this outbreak, six of whom died. This avian virus exhibited high virulence in both avian and mammalian species, causing systemic infection in both chickens and mice. Subsequently, another avian virus with the H9N2 subtype was directly transmitted from birds to humans in Hong Kong. Interestingly, the genes encoding the internal proteins of the H9N2 virus are genetically highly related to those of the H5N1 virus, suggesting a unique property of these gene products. The identification of avian viruses in humans underscores the potential of these and similar strains to produce devastating influenza outbreaks in major population centers. Although highly pathogenic avian influenza viruses had been identified before the 1997 outbreak in Hong Kong, their devastating effects had been confined to poultry. With the Hong Kong outbreak, it became clear that the virulence potential of these viruses extended to humans.     

Seroprevalence of avian influenza H9N2 among poultry workers in Shandong Province, China

H9N2 avian influenza virus has been circulating widely in birds, with occasional infection among humans. Poultry workers are considered to be at high risk of infection with avian influenza due to their frequent exposure to chickens, but the frequency of H9N2 avian influenza virus infections among them is still indistinct. This study was carried out in order to identify the seroprevalence of H9N2 avian influenza virus among poultry workers in Shandong, China. During the period from December 2011 to February 2012, a total of 482 subjects took part in this study, including 382 poultry workers and 100 healthy residents without occupational poultry exposure. Serum samples were collected and tested for the presence of antibodies against H9N2 avian influenza virus by hemagglutination inhibition (HI) and microneutralization (MN) assays. Nine subjects (9/382?=?2.3 %) were positive for antibodies against H9N2 avian influenza virus among poultry workers by either HI or MN assays using ≥40 cut-off, while none of the 100 healthy residents were seropositive. In conclusion, our study identified H9N2 avian influenza infections among poultry workers in Shandong, China, and continuous surveillance of H9N2 avian influenza virus infection in humans should be carried out to evaluate the threat to public health.     

Avian influenza H9N2 subtype in Poland – characterization of the isolates and evidence of concomitant infections

In April/May 2013, four outbreaks of avian influenza virus (AIV) infections caused by H9N2 subtype were diagnosed in Poland in fattening turkey flocks exhibiting a drop in feed and water intake, depression, respiratory signs and mortality. The subsequent serological survey carried out on samples collected between June 2012 and September 2013 from 92 poultry flocks detected positive sera in two additional meat turkey flocks located in the same province. The analysis of amino acids in the haemagglutinin and neuraminidase glycoproteins revealed that the detected H9N2 viruses possessed molecular profiles suggestive of low pathogenicity, avian-like SAα2,3 receptor specificity and adaptation to domestic poultry. Phylogenetic studies showed that these H9N2 AIVs grouped within the Eurasian clade of wild bird-origin AIVs and had no relationship with H9N2 AIV circulating in poultry in the Middle East and Far East Asia over the past decade. Experimentally infected SPF chickens with the index-case H9N2 virus remained healthy throughout the experiment. On the other hand, ten 3-week-old commercial turkeys infected via the oculonasal route showed respiratory signs and mortality (2/10 birds). Additional diagnostic tests demonstrated the consistent presence of DNA/RNA of Ornithobacterium rhinotracheale, Bordetella avium and, less frequently, of astro-, rota-, reo-, parvo- and adenoviruses in turkeys both from field outbreaks and laboratory experiment. Although no microbiological culture was performed, we speculate that these secondary pathogens could play a role in the pathogenicity of the current H9N2 infections.     

Genetic analysis of nonstructural genes (NS1 and NS2) of H9N2 and H5N1 viruses recently isolated in Israel

The avian influenza virus subtype H9N2 affects wild birds, domestic poultry, swine, and humans; it has circulated amongst domestic poultry in Israel during the last 6 years. The H5N1 virus was recorded in Israel for the first time in March 2006. Nonstructural (NS) genes and NS proteins are important in the life cycle of the avian influenza viruses. In the present study, NS genes of 21 examples of H9N2 and of two examples of H5N1 avian influenza viruses, isolated in Israel during 2000–2006, were completely sequenced and phylogenetically analyzed. All the H9N2 isolates fell into a single group that, in turn, was subdivided into three subgroups in accordance with the time of isolation; their NS1 and NS2 proteins possessed 230 and 121 amino acids, respectively. The NS1 protein of the H5N1 isolates had five amino acid deletions, which was typical of highly pathogenic H5N1 viruses isolated in various countries during 2005–2006. Comparative analysis showed that the NS proteins of the H9N2 Israeli isolates contained few amino acid sequences associated with high pathogenicity or human host specificity.     

Effect of low-pathogenicity influenza virus H3N8 infection on Mycoplasma gallisepticum infection of chickens

Mycoplasma infection is still very common in chicken and turkey flocks. Several low-pathogenicity avian influenza (LPAI) viruses are circulating in wild birds that can be easily transmitted to poultry flocks. However, the effect of LPAI on mycoplasma infection is not well understood. The aim of the present study was to investigate the infection of LPAI virus H3N8 (A/mallard/Hungary/19616/07) in chickens challenged with Mycoplasma gallisepticum. Two groups of chickens were aerosol challenged with M. gallisepticum. Later one of these groups and one mycoplasma-free group were aerosol challenged with the LPAI H3N8 virus. The birds were observed for clinical signs for 8 days, then euthanized, and examined for the presence of M. gallisepticum in the trachea, lung, air sac, liver, spleen, kidney and heart, and for developing anti-mycoplasma and anti-viral antibodies. The LPAI H3N8 virus did not cause any clinical signs but M. gallisepticum infection caused clinical signs, reduction of body weight gain and colonization of the inner organs. These parameters were more severe in the birds co-infected with M. gallisepticum and LPAI H3N8 virus than in the group challenged with M. gallisepticum alone. In addition, in the birds infected with both M. gallisepticum and LPAI H3N8 virus, the anti-mycoplasma antibody response was reduced significantly when compared with the group challenged with M. gallisepticum alone. Co-infection with LPAI H3N8 virus thus enhanced pathogenesis of M. gallisepticum infection significantly.     

Serological survey of avian H5N2-subtype influenza virus infections in human populations

To investigate the distribution of antibodies against H5N2 influenza virus in humans living in Ibaraki prefecture, Japan, 266 single serum samples were collected to perform serological tests. Results were compared to investigate the relationship between positive results and several factors. The number of positive serum neutralization antibody titers (≥40) against avian influenza virus A/H5N2 was significantly greater (P < 0.05) among poultry workers, in comparison to a Japanese healthy population. The geometric mean titers of serum neutralization antibody against A/H5N2 were significantly higher (P < 0.05) among Ibaraki inhabitants and poultry workers (P < 0.0001) when compared to a Japanese healthy population. Seropositivity against A/H5N2 virus was significantly (P < 0.05) associated with age (≥50 years old) in poultry workers. These results suggest that seropositivity against H5N2 virus in Ibaraki specimens is significantly higher than those of a Japanese healthy population and that the surveillance of avian influenza viruses is very important to evaluate the invasion or emergence of new pandemic influenza viruses from species other than humans.     

An outbreak of avian influenza subtype H9N8 among chickens in South Korea.

Low pathogenic avian influenza subtype H9N8 was diagnosed on a Korean native chicken farm in Gyeonggi province, South Korea, in late April 2004. Clinical signs included moderate respiratory distress, depression, mild diarrhoea, loss of appetite and a slightly elevated mortality (1.4% in 5 days). Pathologically, mucopurulent tracheitis and air sacculitis were prominently found with urate renal deposition. The isolated A/chicken/Kr/164/04 (H9N8) had an Ala-Ser-Gly-Arg (A/S/G/R) motif at the cleavage site of haemagglutinin, which has been commonly found in H9N2 isolated from Korean poultry. Phylogenetic analysis of the haemagglutinin and neuraminidase genes of the H9N8 avian influenza virus (AIV) isolate showed that reassortment had occurred. Its haemagglutinin gene was similar to that of Korean H9N2 AIVs, but its neuraminidase gene was closely related to that of A/WBF/Kr/KCA16/03 (H3N8) isolated from the faeces of wild birds in Korea. The pathogenicity of the isolate was tested on 6-week-old specific pathogen free chickens. The inoculated virus (H9N8) was recovered from most tested organs, including the trachea, lung, kidney, spleen, and caecal tonsil. This is the first report of an outbreak of low pathogenic avian influenza in chickens caused by AIV subtype H9N8.     

Potency of an inactivated avian influenza vaccine prepared from a non-pathogenic H5N1 reassortant virus generated between isolates from migratory ducks in Asia

A reassortant influenza virus, A/duck/Hokkaido/Vac-1/2004 (H5N1) (Dk/Vac-1/04), was generated between non-pathogenic avian influenza viruses isolated from migratory ducks in Asia. Dk/Vac-1/04 (H5N1) virus particles propagated in embryonated chicken eggs were inactivated with formalin and adjuvanted with mineral oil to form a water-in-oil emulsion. The resulting vaccine was injected intramuscularly into chickens. The chickens were challenged with either of the highly pathogenic avian influenza virus strains A/chicken/Yamaguchi/7/2004 (H5N1) or A/swan/Mongolia/3/2005 (H5N1) at 21 days post-vaccination (p. v.), when the geometric mean serum HI titers of the birds was 64 with the challenge virus strains. The vaccinated chickens were protected from manifestation of disease signs upon challenge with either of the highly pathogenic avian influenza viruses. However, challenge virus was recovered at low titers from the birds at 2 and 4 days post-challenge (p.c.). All 3 chickens challenged at 6 days p.v. died, whereas 3 chickens challenged at 8 days p.v. survived. These results indicate that the present vaccine confers clinical protection and reduction of virus shedding against highly pathogenic avian influenza virus challenge and should be useful as an optional tool in emergency cases.     

Characterization of highly pathogenic avian influenza H5N8 virus from Egyptian domestic waterfowl in 2017

In 2016, the highly pathogenic avian influenza (HPAI) H5N8 virus was detected in wild birds for the first time in Egypt. In the present study, we identified the HPAI virus H5N8 of clade 2.3.4.4 from domestic waterfowl in Egypt, suggesting its transmission to the domestic poultry from the migratory birds. Based on partial haemagglutinin gene sequence, this virus has a close genetic relationship with subtype H5N8 viruses circulating in Asia and Europe. Pathologically, H5N8 virus in hybrid duck induced nervous signs accompanied by encephalomalacia, haemorrhages, nonsuppurative encephalitis and nonsuppurative vasculitis. The granular layer of cerebellum showed multifocal areas of hydropic degeneration and the Purkinje cell neurons were necrotized or lost. Additionally, the lung, kidney and spleen were congested, and necrotizing pancreatitis was also observed. The co-circulation of both HPAI H5N1 and H5N8 subtypes with the low pathogenic avian influenza H9N2 subtype complicate the control of avian influenza in Egypt with the possibility of emergence of new reassortant viruses. Therefore, continuous monitoring with implementation of strict control measures is required.

Research highlights

• HPAI H5N8 virus clade 2.3.4.4 was detected in domestic ducks and geese in Egypt in 2017.

• Phylogenetically, the virus was closely related to HPAI H5N8 viruses identified in Asia and Europe

• Nonsuppurative encephalitis was widely observed in HPAI H5N8 virus-infected ducks.

• Degeneration of the cerebellar granular layer was found in most of the brain tissues examined.

     

Evaluation of the protection induced by avian influenza vaccines containing a 1994 Mexican H5N2 LPAI seed strain against a 2008 Egyptian H5N1 HPAI virus belonging to clade 2.2.1 by means of serological and in vivo tests

Since 2006 Egypt has been facing an extensive epidemic of H5N1 highly pathogenic avian influenza (HPAI) with a huge number of outbreaks both in rural and intensively reared poultry areas. The use of efficacious vaccines in this country has been, and still remains, essential for the control and possible eradication of HPAI. The present study was performed to establish whether the administration of inactivated vaccines containing an H5 virus belonging to a different lineage to the Eurasian H5N1 HPAI viruses guarantees protection from clinical signs, provides significant immune response and is able to achieve a reduction of viral shedding in the face of a challenge with a contemporary H5N1 virus isolated in Egypt. Despite the genetic and antigenic differences between the vaccine strain (H5N2/Mexico) and the challenge strain (H5N1/Egypt), confirmed by molecular and serological (haemagglutination inhibition) tests, it was established that the immune response induced by these conventional vaccines is sufficient to prevent infection in the majority of birds challenged with a contemporary H5N1 Egyptian strain. The data reported in this study also indicate that there may be a low degree of correlation between haemagglutination inhibition titres, clinical protection and reduction of shedding.     

Susceptibility of wild passerines to subtype H5N1 highly pathogenic avian influenza viruses

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have spread throughout many areas of Asia, Europe and Africa, and numerous cases of HPAI outbreaks in domestic and wild birds have been reported. Although recent studies suggest that the dissemination of H5N1 viruses is closely linked to the migration of wild birds, information on the potential for viral infection in species other than poultry and waterfowl is relatively limited. To investigate the susceptibility of terrestrial wild birds to infection with H5N1 HPAI viruses, common reed buntings (Emberiza schoeniclus), pale thrushes (Turdus pallidus) and brown-eared bulbuls (Hypsipetes amaurotis) were infected with A/mountain hawk-eagle/Kumamoto/1/07(H5N1) and A/whooper swan/Aomori/1/08(H5N1). The results showed that common reed buntings and brown-eared bulbuls were severely affected by both virus strains (100% mortality). While pale thrushes did not exhibit any clinical signs, seroconversion was confirmed. In common reed buntings, intraspecies-transmission of A/whooper swan/Aomori/1/08 to contact birds was also confirmed. The findings show that three passerine species; common reed buntings, brown-eared bulbuls and pale thrushes are susceptible to infection by H5N1 HPAI viruses, which emphasizes that continued surveillance of species other than waterfowl is crucial for effective monitoring of H5N1 HPAI virus outbreaks.     

Emerging influenza.

In 1918 the Spanish influenza pandemic, caused by an avian H1N1 virus, resulted in over 50 million deaths worldwide. Several outbreaks of H7 influenza A viruses have resulted in human cases, including one fatal case. Since 1997, the outbreaks of highly pathogenic avian influenza (HPAI) of the H5N1 subtype have affected a wide variety of mammals in addition to poultry and wild birds. Here, we give an overview of the current knowledge of the determinants of pathogenicity of these three subtypes of avian influenza A virus in mammals. Common mechanisms for acquisition of virulence and replication of these avian influenza viruses in mammals are becoming apparent. Therefore, monitoring these and additional genetic changes upon zoonotic infections is important. Identification of genetic changes responsible for transmission between mammals will be an important task for the near future.     

Novel reassortant H10N7 avian influenza viruses isolated from chickens in Eastern China

BackgroundSince 2004, the H10N7 subtype avian influenza virus (AIV) has caused sporadic human infections with variable clinical symptoms world-wide. However, there is limited information pertaining to the molecular characteristics of H10N7 AIVs in China.ObjectiveTo more fully characterize the genetic relationships between three novel H10N7 strains isolated from chickens in Eastern China and the strains isolated from birds throughout Asia, and to determine the pathogenicity of the H10N7 isolates in vivo.Study designAll eight gene segments from the Chinese H10N7 strains were sequenced and compared with AIV strains available in GenBank. The virulence of the three isolates was determined in chickens and mice.ResultsThree H10N7 subtype avian influenza viruses were isolated from chickens in live poultry markets in Eastern China in 2014: (1) A/chicken/Zhejiang/2C66/2014(H10N7) (ZJ-2C66), (2) A/chicken/Zhejiang/2CP2/2014(H10N7) (ZJ-2CP2), and (3) A/chicken/Zhejiang/2CP8/2014(H10N7) (ZJ-2CP8). Phylogenetic analysis indicated that the viruses contained genetic material from H10, H2, H7, and H3 AIV strains that were circulating at the same time. The reassortant H10N7 viruses were found to be minimally pathogenic in chickens and moderately pathogenic in mice. The viruses were able to replicate in mice without prior adaptation.ConclusionThese results suggest that H10N7 surveillance in poultry should be used as an early warning system for avian influenza outbreaks. The novel strains identified here may post a threat to human health in the future if they continue to circulate.     

Genetic characterization of highly pathogenic H5N1 avian influenza virus from live migratory birds in Bangladesh

Since the first outbreak of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in Bangladesh in 2007, the virus has been circulating among domestic poultry causing severe economic losses. To investigate the presence of HPAIV H5N1 in migratory birds and their potential role in virus spread, 205 pools of fecal samples from live migratory birds were analyzed. Here, the first virus isolation and genome characterization of two HPAIV H5N1 isolates from migratory birds (A/migratory bird/Bangladesh/P18/2010 and A/migratory bird/Bangladesh/P29/2010)are described. Full-length amplification, sequencing, and a comprehensive phylogenetic analysis were performed for HA, NA, M, NS, NP, PA, PB1, and PB2 gene segments. The selected migratory bird isolates belong to clade 2.3.2.1 along with recent Bangladeshi isolates from chickens, ducks, and crows which grouped in the same cluster with contemporary South and South-East Asian isolates. The studied isolates were genetically similar to other H5N1 isolates from different species within the respective clade although some unique amino acid substitutions were observed among them. Migratory birds remain a real threat for spreading pathogenic avian influenza viruses across the continent and introduction of new strains into Bangladesh.