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.     

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。     

Immunohistochemical detection of Influenza virus infection in formalin-fixed tissues with anti-H5 monoclonal antibody recognizing FFWTILKP

The worldwide outbreak of avian influenza among poultry species and humans is associated with the H5N1 subtype of avian influenza A virus (AIV). This highlighted the need to develop safe H5 AIV diagnostic methods. 7H10, an H5-specific monoclonal antibody (Mab), can be used for immunohistochemical (IHC) staining for formalin-fixed tissue. An assortment of H5N1 tissue specimens infected naturally in paraffin sections from Asia, between years 2002-2006, including one human specimen, were tested. 7H10 detected H5 infection in all of these tissue samples infected naturally. In addition, 24 different human H5N1 isolates from Indonesia, 5 avian H5 isolates and 3 non-H5 isolates from Asia were inoculated into BALB/C mice and chicken embryos. Among these influenza viruses, 7H10 detected 28 of the 29 H5 virus strains by immunohistochemical staining, while none of non-H5 strains used in this study could be detected by 7H10, confirming its specificity to H5. Further, the eight-residue-long linear epitope, "FFWTILKP", identified through epitope mapping, enables 7H10 to detect >98.3% of H5 subtype viruses reported worldwide before 2007. This study describes a specific H5 diagnostic system with minimal possibility of exposure to live virus based on immunochemical staining.     

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.     

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.     

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.     

Human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals

Viral attachment to the host cell is critical for tissue and species specificity of virus infections. Recently, pattern of viral attachment (PVA) in human respiratory tract was determined for highly pathogenic avian influenza virus of subtype H5N1. However, PVA of human influenza viruses and other avian influenza viruses in either humans or experimental animals is unknown. Therefore, we compared PVA of two human influenza viruses (H1N1 and H3N2) and two low pathogenic avian influenza viruses (H5N9 and H6N1) with that of H5N1 virus in respiratory tract tissues of humans, mice, ferrets, cynomolgus macaques, cats, and pigs by virus histochemistry. We found that human influenza viruses attached more strongly to human trachea and bronchi than H5N1 virus and attached to different cell types than H5N1 virus. These differences correspond to primary diagnoses of tracheobronchitis for human influenza viruses and diffuse alveolar damage for H5N1 virus. The PVA of low pathogenic avian influenza viruses in human respiratory tract resembled that of H5N1 virus, demonstrating that other properties determine its pathogenicity for humans. The PVA in human respiratory tract most closely mirrored that in ferrets and pigs for human influenza viruses and that in ferrets, pigs, and cats for avian influenza viruses.     

中国职业暴露人群感染H6N6禽流感病毒血清学调查

目的 系统评估我国职业暴露人群感染H6N6禽流感病毒的状况.方法 本研究利用我国2009-2011年开展的高致病性H5N1禽流感病毒职业暴露人群血清学监测所采集的近15 000份血清标本,开展H6N6禽流感病毒血清学调查.结果 本研究中检测到H6N6禽流感病毒阳性血清共10份,分别来自不同的职业暴露人群,包括活禽市场、家禽规模养殖场、家禽散养户、屠宰加工场和野生候鸟栖息地.从地域上看该10份阳性血清来自8个不同的省份,分布在我国的南北方.结论 这是我国大陆地区首次报道人感染H6亚型禽流感病毒.     

禽H9N2亚型流感病毒能感染人的发现

目的了解禽（Ｈ９Ｎ２）亚型流感病毒是否能感染人。方法对人、鸡和猪进行Ｈ９亚型毒株血清流行病学调查。对流感样患者和鸡咽喉部采样,用常规鸡胚双腔法分离流感病毒并进行毒株鉴定。对分离出Ｈ９Ｎ２亚型毒株的患者进行个案调查。结果约１９％的人含有对Ｈ９Ｎ２毒株的抗体,其ＨＩ滴度为≥２０,从流感样患者中分离到５株Ｈ９Ｎ２病毒。结论Ｈ９Ｎ２亚型毒株能自然感染人。     

Single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) for influenza A virus subtype H5N1 detection

Influenza A virus subtype H5N1 causes a rapidly fatal systemic disease in domestic poultry and spreads directly from poultry to humans. The aim of this study was to develop a rapid, cost-saving and effective method for influenza A virus subtype H5N1 detection. The selected primer set was used in single-step RT-PCR for simultaneous detection in multiplex format of the 276-, 189-, and 131-bp fragments, corresponding to sequences specific for M, H5 and N1. The amplified DNA fragments were clearly separated by agarose gel electrophoresis. The sensitivity of this assay was about 10(3) copies/microL. Moreover, this method can be applied to detect not only avian but also human influenza A virus subtype H5N1. In conclusion, the highlights of this particular method are its rapidity and cost-effectiveness, thus rendering it feasible and attractive for large-scale screening at times of influenza A virus subtype H5N1 outbreak.     

H9N2禽流感病毒反向遗传系统的构建和鉴定

目的 建立H9N2亚型禽流感病毒反向遗传系统,为人禽流感疫苗研制以及传播和致病机制等方面的研究提供技术平台.方法 使用RT-PCR方法获得禽流感H9N2亚型病毒A/Guangzhou/333/99(H9N2)的8条全长基因节段,然后克隆到双表达载体pCI-pol Ⅰ中,获得H9N2禽流感病毒的8个基因节段的8质粒系统.将构建好的8质粒共转染293T细胞后,收获上清接种鸡胚,然后对鸡胚尿囊液进行鉴定;对拯救的病毒进行鉴定.结果 8质粒系统转染293T细胞后可以成功拯救出H9N2禽流感病毒,血凝效价可达到29/50μl,生长特性与野生型病毒类似.结论 成功建立了H9N2禽流感病毒反向遗传系统.     

Complete genome characterization of avian influenza virus subtype H9N2 from a commercial quail flock in Egypt

The suspected presence of avian influenza virus subtype H9N2 in poultry in Egypt is a major concern since this subtype is widely distributed in different countries in the Middle East, here we describe the full genetic characterization of an avian influenza A virus (Qa/Egypt/11; H9N2) of subtype H9N2 that was previously isolated from a clinically normal quail flock in Giza, Egypt in May 2011. The nucleotide sequence analysis of the hemagglutinin gene of the isolated Egyptian virus showed the highest similarity with one group of recent Israeli strains (97?%) circulating from 2006-2010. Sequence homology and phylogenetic analysis indicated that the Qa/Egypt/11 isolate belonged to the A/quail/Hong Kong/G1/1997-like lineage with new mutations identified in all viral proteins. The phylogenetic analysis for the eight genes indicated placement of the Egyptian virus within the same lineage of H9N2 viruses that circulated in the region from 2006, especially with one group of recent Israeli strains. However, phylogenetic analysis of the internal genes like PB2, NP, and PA genes identified possible reassortment events for these genes with singular Israeli strains. This study indicates progressive evolution of this subtype in the Middle East region and possible mechanism of virus adaptation in land-based poultry like in quails.     

Experimental infection of non-human primates with avian influenza virus (H9N2)

Several cases of humans infected with the H9N2 avian influenza virus (AIV) have been described since 1999; however, the infectivity and pathogenicity of H9N2 in humans is not well defined. A non-human primate model in rhesus macaques was developed to study H9N2 virus infections as a means of better understanding the pathogenesis and virulence of this virus, in addition to testing antiviral drugs. Rhesus macaques inoculated with H9N2 AIV presented with biphasic fever and viral pneumonia. H9N2 was recovered from nasal washes and pharyngeal samples up to days 7-9 postinfection, followed by an increase in HI (hemagglutination inhibition) antibody titers. Tissue tropism and immunohistochemistry indicated that H9N2 AIV replicated in the upper respiratory tract (turbinate, trachea, and bronchus) and in all lobes of the lung. Our data suggest that rhesus macaques are a suitable animal model to study H9N2 influenza virus infections, particularly in the context of viral evolution and pathogenicity.     

Isolation and characterization of H6N1 and H9N2 avian influenza viruses from Ducks in Hanoi, Vietnam

We report the genetic characterization of low pathogenic avian influenza (LPAI) viruses isolated from domestic ducks in northern Vietnam in 2009. In total, 22 influenza A viruses consisting of 21 H6N1 subtypes and one H9N2 subtype were isolated from 1488 ducks collected in February, March, and April 2009, accounting the overall virus isolation rate for 1.5%. No H5N1 strain was isolated in this study. Phylogenetic analysis indicated that all the eight genes of the H6N1 and H9N2 subtypes analyzed in this study were similar to those isolated in Korea, southeast China and northern Japan, and wild birds which migrate along the coastal East Asian Flyway are estimated to transmit these viruses. There was no evidence that the H6N1 and H9N2 subtypes share the gene segments with H5N1 subtypes. However, it is important to monitor the prevalence and genetical backgrounds of LPAI viruses among poultry in an area where several different influenza A subtypes are in circulation.     

Serologic evidence of avian influenza virus infections among Nigerian agricultural workers

Nigeria has had multiple incursions of highly pathogenic avian influenza A (HPAI) H5N1 virus into its poultry population since 2006. This study aimed to determine if Nigerians exposed to poultry had evidence of avian influenza virus transmission to man. Between 2008 and 2010, 316 adult farmers and open market workers and 54 age‐group matched, non‐animal exposed controls were enrolled in a prospective, population‐based study of zoonotic influenza transmission in four towns in southeastern Nigeria. Questionnaire data and sera obtained at the time of enrollment were examined for evidence of previous infection with 10 avian influenza virus strains. Serologic studies on sera collected at the time of enrollment showed modest evidence of previous infection with three avian‐origin influenza viruses (H5N1, H5N2, and H11N1) and one avian‐like H9N2 influenza virus, with eight (2.4%) of animal‐exposed subjects and two (3.7%) unexposed subjects having elevated microneutralization assay antibody titer levels (ranging from 1:10 to 1:80). Statistical analyses did not identify specific risk factors associated with the elevated antibody titers observed for these zoonotic influenza viruses. These data suggested only occasional virus transmission to humans in areas thought to have been enzootic for avian influenza virus. Prospective data from this cohort will help the authors to better understand the occurrence of zoonotic infections due to avian influenza viruses in Nigeria. J. Med. Virol. 85:670–676, 2013. © 2013 Wiley Periodicals, Inc.     

Genetic variation of the hemagglutinin of avian influenza virus H9N2

Avian influenza virus H9N2 has become the dominant subtype of influenza which is endemic in poultry. The hemagglutinin, one of eight protein-coding genes, plays an important role during the early stage of infection. The adaptive evolution and the positively selected sites of the HA (the glycoprotein molecule) of H9N2 subtype viruses were investigated. Investigating 68 hemagglutinin H9N2 avian influenza virus isolates in China and phylogenetic analysis, it was necessary that these isolates were distributed geographically from 1994, and were all derived from the Eurasian lineage. H9N2 avian influenza virus isolates from domestic poultry in China were distinct phylogenetically from those isolated in Hong Kong, including viruses which had infected humans. Seven amino acid substitutions (2T, 3T, 14T, 165D, 197A, 233Q, 380R) were identified in the HA possibly due to positive selection pressure. Apart from the 380R site, the other positively selected sites detected were all located near the receptor-binding site of the HA1 strain. Based on epidemiological and phylogenetics analysis, the H9N2 epidemic in China was divided into three groups: the 1994-1997 group, the 1998-1999 group, and the 2000-2007 group. By investigating these three groups using the maximum likelihood estimation method, there were more positive selective sites in the 1994-1997 and 1998-1999 epidemic group than the 2000-2007 groups. This indicates that those detected selected sites are changed during different epidemic periods and the evolution of H9N2 is currently slow. The antigenic determinant or other key functional amino acid sites should be of concern because their adjacent sites have been under positive selection pressure. The results provide further evidence that the pathogenic changes in the H9N2 subtype are due mainly to re-assortment with other highly pathogenic avian influenza viruses.