Characterization of recent H5 subtype avian influenza viruses from US poultry.

In the US, the isolation of H5 subtype avian influenza (AI) viruses has been uncommon in commercial chickens and turkeys, although sporadic isolations have been made from the live bird markets or its supply chain since 1986. In 2002, two different outbreaks of H5 AI occurred in commercial chicken or turkey operations. The first occurred in Texas and was identified as a H5N3 subtype AI virus. The second outbreak was caused by a H5N2 virus isolated from a turkey farm in California. In this study we analyzed recent H5 subtype AI viruses from different avian species and different sources in the US. Most recent H5 subtype isolates shared a high sequence identity and phylogenetically assorted into a separate clade from the Pennsylvania/83 lineage isolates. However, no established lineage was found within this clade and the recent H5 subtype isolates seemed to be the result of separate introductions from the wild bird reservoir. The Texas H5N3 isolate shared the lowest homology with the other recent isolates in the haemagglutinin gene and had a unique haemagglutinin cleavage site sequence of REKR/G (other recent isolates have the typical avirulent motif, RETR/G). Furthermore, this isolate had a 28 amino acid deletion in the stalk region of the neuraminidase protein, a common characteristic of chicken adapted influenza viruses, and may indicate that this virus had actually been circulating in poultry for an extended period of time before it was isolated. In agreement with genetic evidence, the Texas H5N3 isolate replicated better than other H5 isolates in experimentally infected chickens. The outbreak in Texas with a more chicken-adapted H5N3 virus underscores the importance of ongoing surveillance and control efforts regarding the H5 subtype AI virus in the US.     

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.     

Efficacy of avian influenza Neuraminidase-specific vaccines in chickens

This paper investigates the protection induced in chickens by live neuraminidase (N)-specific vaccines against highly pathogenic avian influenza. Vaccination with viruses belonging to N1 and N8 subtypes protected against challenge with virulent viruses of the same N subtype and irrelevant haemagglutinin (H) subtypes. The main advantage of N-specific vaccines is that they do not confuse serological diagnosis by haemagglutination inhibition tests. The anomalous cross-protection between influenza viruses of H1 and H5 subtypes could be exploited to control outbreaks caused by highly pathogenic H5 viruses by using vaccines based on H1 and the appropriate N.     

Continuing evolution of H9 influenza viruses in Korean poultry

We analyzed the evolution of H9 influenza viruses isolated from Korean chicken farms from 2002 to 2004. Korean H9 viruses formed two antigenically distinct groups: those isolated from 1996 to mid-2003, and those isolated from late 2003 through 2004. Most of the 2004 isolates showed greater cross-reactivity with the second group than with the first group. Phylogenetic analysis of the 12 viruses studied revealed three genotypes of H9N2 viruses and showed that reassortment had occurred. One isolate, Ck/Kor/164/04, belonged to the H9N8 subtype. Its HA and PB1 genes were similar to those of the H9N2 viruses, but its other genes were closely related to H3N8 viruses. This report is the first (to our knowledge) of H9N8 infection in this host. The pathogenicity of the early isolates altered due to antigenic drift and reassortment, leading to H9 avian influenza viruses in Korea that potentially can expand their host range to mammalians.     

Pathogenicity of a goose isolate of highly pathogenic H5N1 influenza A virus for chickens, mice, and pigs

In recent years, a highly pathogenic avian influenza virus (AIV) H5N1 subtype has emerged as a pathogenic agens for a variety of species including humans. To investigate the pathogenicity of AIV, the isolate ZFE/2004 (H5N1) was used to infect chickens, mice, and pigs. The results showed that the isolate showed attributes of the highly pathogenic AIV such as high pathogenicity index (intravenous pathogenicity index (IVPI) of 2.85 and intracerebral pathogenicity index (ICPI) of 3.00) and a specified hemagglutinin (HA) cleavage site. In vivo experiments indicated that this isolate was highly lethal to the Balb/c mice. In addition, it was able to infect pigs, elicit specific antibodies, and cause "flu-like" clinical symptoms. Typical pathological changes were observed in the chickens as well. In vitro experiments showed that the isolate ZFE/2004 was able to induce apoptosis of infected MDCK cells that was detected by the DNA fragmentation analysis and electron microscopy. Key words: apoptosis; avian Influenza virus A; H5N1 subtype; pathogenicity.     

Surveillance and characterization of avian influenza viruses from migratory water birds in eastern Hokkaido, the northern part of Japan, 2009–2010

Avian influenza virus (AIV) surveillance was conducted around a small pond in Obihiro, eastern Hokkaido, Japan. Eleven AIVs were isolated from a total of 1,269 fecal samples of migratory wild birds collected during 2009 and 2010. The sample number covered approximately 60 % of the total number of birds observed during sampling periods. The subtypes of the isolates included H3N8 (4 isolates), H5N2 (3), H6N2 (2), H6N1 (1), and H11N2 (1). The H3N8 subtype was most prevalent as in the previous studies performed in Hokkaido. The three H5N2 isolates genetically characterized as low pathogenic AIV were closely related to the strains previously isolated from aquatic wild birds in Japan and also to the Korean strains isolated from aquatic birds in recent years. In Korea, H5N2 subtype virus has often been isolated from poultry and wild birds, as well as reassortant viruses generated from duck H5N2 viruses and chicken H9N2 virus, and avian-swine-like reassortant H5N2 viruses. Considering the previous chicken outbreaks caused by highly pathogenic H5N2 viruses, which affected many countries, it should be an important priority to continue, monitoring the evolution of H5N2 viruses circulating in the region.     

Deduced amino acid sequences at the haemagglutinin cleavage site of avian influenza A viruses of H 5 and H 7 subtypes

Summary The amino acid sequences at the haemagglutinin cleavage sites of 9 avian influenza A viruses of H 5 subtype (5 high and 4 low pathogenicity for chickens) and 21 of H 7 subtype (13 high and 8 low pathogenicity for chickens) were determined by direct RNA sequencing, PCR amplification sequencing or both. None of the viruses of low pathogenicity had multiple basic amino acids at the cleavage site. All highly pathogenic viruses had an insert of basic amino acids at the cleavage site, except A/chicken/Scotland/59 (H5N1) for which the multiple basic amino acids appeared as substitutions and not insertions. All highly pathogenic viruses examined conformed to the amino acid motif of R-X-R/K-R at the cleavage site which is considered to be essential for high pathogenicity in chickens, with the notable exception of highly pathogenic virus A/turkey/England/50-92/91 (H5N1) which had the sequence R-K-R-K-T-R adjacent to the cleavage site.     

Avian influenza viruses in Korean live poultry markets and their pathogenic potential

We surveyed live-poultry markets in Korea in 2003 and isolated 9 H9N2, 6 H3N2, and 1 H6N1 influenza viruses. Antigenic and phylogenetic analyses showed that all 9 H9N2 isolates were of A/Chicken/Korea/25232-96006/96-like lineage (which caused disease in chickens in Korea in 1996) but were different from H9N2 viruses of southeastern China. They had at least 4 genotypes and replicated in chickens but not in mice. The H3N2 and H6N1 viruses were new to Korea and were probably reassortants of avian influenza viruses from southeastern China and recent Korean H9N2 viruses. All 8 segments of the H3N2 viruses formed a single phylogenetic cluster with 99.1 to 100% homology. The H3N2 viruses replicated in chickens and mice without preadaptation, but the H6N1 virus did not. Our results show an increasingly diverse pool of avian influenza viruses in Korea that are potential pandemic influenza agents.     

Protection of chickens against highly lethal H5N1 and H7N1 avian influenza viruses with a recombinant fowlpox virus co-expressing H5 haemagglutinin and N1 neuraminidase genes

Inactivated whole avian influenza virus (AIV) vaccine provides protection against homologous haemagglutinin (HA) subtype virus, but poor protection against a heterologous HA virus. Moreover, it induces chickens to produce antibodies to cross-reactive antigens, especially nucleoprotein, which is limits AIV serological surveillance. In this study, a recombinant fowlpox virus co-expressing HA (H5 subtype) and NA (N1 subtype) genes of AIV was evaluated for its ability to protect chickens against intramuscular challenge with a lethal dose of highly pathogenic (HP) AIV. Susceptible chickens were also vaccinated by wing-web puncture with the parent fowlpox vaccine virus. Following challenge 4 weeks later with HPAIV, all chickens vaccinated with recombinant virus were protected, while the chickens vaccinated with either the unaltered parent fowlpox vaccine virus or unvaccinated controls experienced 100% mortality following challenge. This protection was accompanied by the high levels of specific antibody to the respective components of the recombinant vaccine. The above results showed that rFPV-HA-NA could be a potential vaccine to replace current inactivated vaccines for preventing AI.     

Development of an antigen-capture ELISA for detection of H7 subtype avian influenza from experimentally infected chickens

Emergence of highly pathogenic avian influenza H7N1 was due to mutation of low pathogenic avian influenza H7N1 strain, which caused outbreaks in Italy between 1999 and 2000, and resulted in complete mortality of infected poultry. This outbreak places increased importance on the early detection of H7N1 AIV. Here we describe the development of a detection method for H7N1 virus from infected chickens using a specific antigen-capture-ELISA (AC-ELISA). A panel of mAbs was developed against the surface antigen HA of H7N1 AIV strain A/chicken/Singapore/94. The mAbs were screened by immunofluorescence assays, ELISA and immunoblotting. Selected mAbs 5E5 and 8F10 were of isotypes IgM and IgG and were conformation- or linear epitope-specific, respectively. These mAbs were used as capture antibodies for AC-ELISA development. The detection limit was as little as 10(2)-10(3) TCID(50) units of virus derived from tissue culture supernatants. Virus from the tracheal swab samples of experimentally infected chickens was detected from days 3 to 7 post-infection using the AC-ELISA, with results being confirmed by RT-PCR. AIV subtypes H4N1, H5N3 H9N2 and H10N5 did not react in the AC-ELISA but were RT-PCR positive, indicating that this AC-ELISA is specific for H7N1 strains.     

Replication and pathogenesis associated with H5N1, H5N2, and H5N3 low-pathogenic avian influenza virus infection in chickens and ducks

A comparative study examining replication and disease pathogenesis associated with low-pathogenic H5N1, H5N2, or H5N3 avian influenza virus (AIV) infection of chickens and ducks was performed. The replication and pathogenesis of highly pathogenic AIV (HPAIV) has received substantial attention; however, the behavior of low-pathogenic AIVs, which serve as precursors to HPAIVs, has received less attention. Thus, chickens or ducks were inoculated with an isolate from a wild bird [A/Mute Swan/MI/451072/06 (H5N1)] or isolates from chickens [A/Ck/PA/13609/93 (H5N2), A/Ck/TX/167280-4/02 (H5N3)], and virus replication, induction of a serological response, and disease pathogenesis were investigated, and the hemagglutinin and neuraminidase (NA) gene sequences of the isolates were determined. Virus isolated from tracheal and cloacal swabs showed that H5N1 replicated better in ducks, whereas H5N2 and H5N3 replicated better in chickens. Comparison of the NA gene sequences showed that chicken-adapted H5N2 and H5N3 isolates both have a deletion of 20 amino acids in the NA stalk region, which was absent in the H5N1 isolate. Histopathological examination of numerous organs showed that H5N2 and H5N3 isolates caused lesions in chickens in a variety of organs, but to a greater extent in the respiratory and intestinal tracts, whereas H5N1 lesions in ducks were observed mainly in the respiratory tract. This study suggests that the H5N1, H5N2, and H5N3 infections occurred at distinct sites in chicken and ducks, and that comparative studies in different model species are needed to better understand the factors influencing the evolution of these viruses.     

Active reassortment of H9 influenza viruses between wild birds and live-poultry markets in Korea

Surveillance of H9 avian influenza viruses in Korean live-poultry markets from September 2004 through October 2007 was carried out to investigate active reassortment between wild migratory birds and domestic poultry in Korea. Antigenic and phylogenetic analyses showed that most of the isolates belong to the previous Korean H9N2-like lineage and differ from the southeastern Chinese strains. Interestingly, the Ck/Korea/LPM77/06 group (genotype B) and Dk/Korea/LPM248/07 group (genotype C) showed unique properties distinct from those of other Korean H9N2 strains. Although the HA genes of these two groups belong to Korean H9N2-like lineage, the PA genes closely resemble those of the Chinese Y280-like lineage. In addition, the PB2 genes of the Dk/Korea/LPM248/07 group were closely related to those isolated from migratory birds. Several other isolates also clustered within the H9N2 B genotype, an indication that there are at least two predominant H9N2 influenza genotypes in Korea. Another isolate, Dk/Korea/LPM71/06, was identified as an H9N1 subtype, the first ever discovered in Korean live-poultry markets. These findings reveal that reassortment of Korean H9 influenza viruses has occurred frequently in live-poultry markets and may have been mediated by introduction of genetic material from viruses circulating among migratory wild birds to domestic birds. Consequently, the new dominant H9N2 genotypes have become established in Korean live-poultry markets through continued reassortment.     

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。     

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.     

Genetic characterization and pathogenicity assessment of highly pathogenic H5N1 avian influenza viruses isolated from migratory wild birds in 2011, South Korea

The continued spread of a highly pathogenic avian influenza (HPAI) H5N1 virus among wild birds and poultry has posed a potential threat to human public health. In the present study, we report the isolation of HPAI H5N1 viruses (A/Md/Korea/W401/11 and A/Md/Korea/W404/11) from fecal samples of migratory birds. Genetic and phlyogenetic analyses demonstrated that these viruses are genetically identical possessing gene segments from avian virus origin and showing highest sequence similarities (as high as 99.8%) to A/Ws/Hokkaido/4/11 and 2009-2010 Mongolian-like clade 2.3.2 isolates rather than previous Korean H5N1 viruses. Both viruses possess the polybasic motif (QRERRRK/R) in HA but other genes did not bear additional virulence markers. Pathogenicity of A/Md/Korea/W401/11 was assessed and compared with a 2006 clade 2.2 HPAI H5N1 migratory bird isolate (A/EM/Korea/W149/06) in chickens, ducks, mice and ferrets. Experimental infection in these hosts showed that both viruses have high pathogenic potential in chickens (2.3-3.0 LD(50)s) and mice (3.3-3.9 LD(50)s), but A/Md/Korea/W401/11 was less pathogenic in duck and ferret models. Despite recovery of both infection viruses in the upper respiratory tract, efficient ferret-to-ferret transmission was not observed. These data suggest that the 2011 Korean HPAI wild bird H5N1 virus could replicate in mammalian hosts without pre-adaptation but could not sustain subsequent infection. This study highlights the role of migratory birds in the perpetuation and spread of HPAI H5N1 viruses in Far-East Asia. With the changing pathobiology caused by H5N1 viruses among wild and poultry birds, continued surveillance of influenza viruses among migratory bird species remains crucial for effective monitoring of high-pathogenicity or pandemic influenza viruses.     

Preparation of anti-idiotypic antibody against avian influenza virus subtype H9

To generate monoclonal anti-idiotypic antibodies(mAb2)against avian influenza virus subtype H9(H9 AⅣ),BALB/c mice were immunized with purified chicken anti-H9-AⅣ IgG and the splenocytes of immunized mice werefused with myeloma cells NS-1.Hybridoma cells were screened by indirect enzyme-linked immunosorbent assayswith both chicken and rabbit anti-H9-AⅣ IgG as coating antigens.One hybridoma cell clone secreting monoclonalantibody against idiotypes shared by both chicken and rabbit anti-H9-AⅣ IgG was established.Experimentsdemonstrated the mAb2 was able to inhibit the binding of hemagglutinin to anti-H9-AⅣ IgG and to inducechickens to generate hemagglutination inhibition antibodies,indicating this anti-species-sharing-idiotypic antibodybore the internal image of hemagglutinin on avian influenza virus.Cellular & Molecular Immunology.2005;2(2):155-157.     

Highly pathogenic avian influenza (H5N1) in the commercial domestic ducks of South Korea.

The present study reports the clinical, virological and pathological findings observed in a natural outbreak of highly pathogenic avian influenza in farmed commercial ducks. The ducks developed clinical signs, including mild respiratory distress, depression, mild diarrhoea, loss of appetite and increasing mortality (up to 12%). At necropsy, multifocal mottled necrosis was commonly found in the pancreas with splenomegaly, hepatomegaly, and swollen kidneys. Microscopically, there was necrotized pancreatitis and hepatitis, and lymphocytic meningoencephalitis and myocarditis. Influenza viral antigen was demonstrated in areas closely associated with histopathological lesion. Avian influenza virus was isolated from the caecal tonsil, faeces, and kidney of the domestic ducks. The isolated virus was identified as a highly pathogenic H5N1, with a haemagglutinin proteolytic cleavage site deduced amino acid sequence of ... QREKRKKR/GLFGAIAG ... In order to determine the pathogenicity of the isolate, eight 6-week-old specific pathogen free chickens were inoculated intravenously with the virus, and all birds died within 24 h after inoculation. This is the first report of an outbreak of highly pathogenic avian influenza with clinical signs in commercial domestic ducks in South Korea.