中国猪源HSN1和H9N2亚型流感病毒的分离鉴定

猪是禽流感病毒"禽-猪-人"传播链中重要的中间宿主,了解猪流感的疫情动态将为动物流感及人流感的疾病预测及防制提供重要依据.1999～2001年间进行的血清学和病毒学监测发现我国猪群存在大范围的H1和H3亚型猪流感感染(李海燕等,2002).2002～2003年,我们进一步对来自全国14个省市的1936份血清进行了H9亚型猪流感的检测,同时在广东、福建等省进行了H5亚型猪流感的检测.2002年辽宁、广东、山东及重庆猪血清中出现H9亚型流感抗体,阳性率分别为7.3%、6.8%、5.1%和1.6%.2003年采集的猪血清H9亚型流感抗体均为阴性,同时发现广东、福建两省2003年出现H5亚型流感阳性猪群,阳性率分别为4.7%和8.2%.从2001～2003年收集和送检的样品中分离鉴定了6株H9N2亚型和2株H5N1亚型猪流感病毒,部分序列分析发现H9和H5亚型猪流感病毒均与我国分离的禽流感病毒高度同源.本研究进一步确证了我国猪群中存在H9N2亚型流感病毒,并且首次发现我国猪群已出现H5N1亚型流感病毒,为人类流感及动物流感的防制敲响了警钟.对这两个亚型流感病毒所具有的公共卫生和兽医公共卫生危害性应予以高度重视.     

Isolation and pathotyping of H9N2 avian influenza viruses in Indian poultry

A total of 1246 faecal and tissue samples collected/received from 119 farms located in various states of India were processed for isolation of avian influenza viruses (AIV) during 2003-2004 as part of a program to monitor AIV infection in Indian poultry population. Avian influenza virus was isolated for the first time in India from poultry farms with history of drop in egg production, respiratory illness and increased mortality in Haryana state. A total of 29 H9N2 AIV isolates were obtained from the states of Punjab, Haryana, Uttar Pradesh, Gujarat, and Orissa and Union Territory Delhi. Subtyping was done by HI, RT-PCR and neuraminidase inhibition assay. Pathotyping of six representative isolates by intravenous pathogenicity index (0.0/3.0) in 6-8 weeks old chicken, trypsin dependency in cell culture and HA cleavage site analysis (335RSSR*GLF341) confirmed that these isolates are low pathogenic. Nucleotide sequence analysis of the HA gene showed that the Indian isolates are very closely related (95.0-99.6%) and shared a homology of 92-96% with H9N2 isolates from Germany and Asian regions other than that of mainland China. Deduced amino acid sequences showed the presence of L226 (234 in H9 numbering) which indicates a preference to binding of alpha (2-6) sialic acid receptors. Two of the six isolates had 7 glycosylation sites in the HA1 cleaved protein and the remaining four had 5 sites. Phylogenetic analysis showed that they share a common ancestor Qa/HK/G1/97 isolate which had contributed internal genes of H5N1 virus circulating in Vietnam. Further characterization of Indian H9N2 isolates is required to understand their nature and evolution.     

Differential replication properties among H9N2 avian influenza viruses of Eurasian origin

Avian influenza H9N2 viruses have become panzootic in Eurasia causing respiratory manifestations, great economic losses and occasionally being transmitted to humans. To evaluate the replication properties and compare the different virus quantification methods, four Eurasian H9N2 viruses from different geographical origins were propagated in embryonated chicken egg (ECE) and Madin-Darby canine kidney epithelial cell systems. The ECE-grown and cell culture-grown viruses were monitored for replication kinetics based on tissue culture infectious dose (TCID₅₀), Hemagglutination (HA) test and quantitative real time RT-PCR (qRT-PCR). The cellular morphology was analyzed using immunofluorescence (IF) and cellular ELISA was used to screen the sensitivity of the viruses to amantadine. The Eurasian wild type-H9N2 virus produced lower titers compared to the three G1-H9N2 viruses at respective time points. Detectable titers were observed earliest at 16 h post inoculation (hpi), significant morphological changes on cells were first observed at 32 hpi. Few nucleotide and amino acid substitutions were noticed in the HA, NA and NS gene sequences but none of them are related to the known conserved region that can alter pathogenesis or virulence following a single passage in cell culture. All studied H9N2 viruses were sensitive to amantadine. The G1-H9N2 viruses have higher replication capabilities compared to the European wild bird-H9N2 probably due to their specific genetic constitutions which is prerequisite for a successful vaccine candidate. Both the ECE and MDCK cell system allowed efficient replication but the ECE system is considered as the better cultivation system for H9N2 viruses in order to get maximum amounts of virus within a short time period.     

Isolation of avian influenza virus (H9N2) from emu in China

This is the first reported isolation of avian influenza virus (AIV) from emu in China. An outbreak of AIV infection occurred at an emu farm that housed 40 four-month-old birds. Various degrees of haemorrhage were discovered in the tissues of affected emus. Cell degeneration and necrosis were observed microscopically. Electron microscopy revealed round or oval virions with a diameter of 80 nm to 120 nm, surrounded by an envelope with spikes. The virus was classified as low pathogenic AIV (LPAIV), according to OIE standards. It was named A/Emu/HeNen/14/2004(H9N2)(Emu/HN/2004). The HA gene (1683bp) was amplified by RT-PCR and it was compared with other animal H9N2 AIV sequences in GenBank, the US National Institutes of Health genetic sequence database. The results suggested that Emu/HN/2004 may have come from an avian influenza virus (H9N2) from Southern China.     

基因重排H1N2亚型猪流感病毒的分离鉴定和生物学特性的研究

本研究对2005年～2006年广西和海南省疑似猪流感(SD)病猪的组织病料进行了病毒分离,并对分离毒株进行了亚型鉴定和生物学特性的研究.结果显示:分离的3株流感病毒均为H1N2亚型猪流感病毒(SIV).能凝集多种动物的红细胞,但凝集谱与以往报道略有不同;为热不稳定型病毒;在电镜下可观察到典型流感病毒粒子.动物试验显示:小白鼠、大白鼠和家兔对分离毒株不敏感,而豚鼠较敏感.能较好地复制出流感症状和病理变化;本体试验动物仅有轻微的临床症状,但能检测到抗体升高的变化,并能从鼻腔和上呼吸道检测和分离到SIV.核苷酸同源性分析显示:分离毒株Sw/GX/17/05、Sw/GX/13/06和Sw/HN/1/05的血凝素(HA)基因分别与基因重排H1N2 亚型SIV A/Swine/lndiana 9K035/99、A/SW/MN/23124-T/01和A/swine/Zhejiang/1/04的核苷酸同源性最高,分别达97.4%、97.0%和95.7%;神经氨酸酶(NA)基因均与基因重排H1N2 亚型流感病毒A/Trurkey/MO/24093/99 的核苷酸同源性最高,达97.2%～98.0%.核苷酸同源性分析进一步证实了分离毒株为基因重排H1N2亚型SIV.     

H9N2亚型鸡源禽流感病毒分离与鉴定

禽流感 (AI)又名真性鸡瘟或欧洲鸡瘟 ,是由正粘病毒科 A型流感病毒引起的一种传染病 ,是国际兽医局规定的 A类烈性传染病。鸡、火鸡、鸭和鹌鹑等家禽及其他野禽均可感染。我们从新乡市某蛋鸡场分离鉴定 1株低致病力的 H9N2亚型的禽流感病毒毒株 ,现报告如下。1　材料与方法1.1　材料　病料来自河南职业技术师范学院禽病研究所接诊检验的病、死鸡。禽流感 A型琼扩抗原、标准阳性血清以及抗 HA、NA分型血清购自中国农科院哈尔滨兽医研究所 ;抗新城疫 (ND)血清和抗减蛋综合征 (EDS- 76 )血清由本院禽病研究所提供。 SPF鸡胚和雏鸡购自…     

H9N2亚型禽流感病毒抗原性变异的研究

对1998—2002年间在河南省豫北地区分离到的5株H9N2亚型禽流感病毒的抗原性变异进行了研究。经HI试验、鸡胚中和试验、细胞中和试验及攻毒保护试验证明，5株H9N2亚型间已经发生了抗原性漂移。98A5和99S毒株间的保护力接近100％，HI试验、鸡胚中和试验、细胞中和试验的相关性均在0．74以上。表明2毒株间的抗原性相近；用00Y毒株攻击其他4株免疫的鸡，其保护率仅为60％～80％；而02Y株对除00Y株外的4株的免疫保护率分别为60％、75％、80％、100％，与分离年代呈负相关性，HI、鸡胚中和试验、细胞中和试验也取得类似结果，说明2000年后的毒株间已发生抗原性变异。     

鸭源H9亚型禽流感病毒的分离及其生物学特性的研究

从南京某肉鸭养殖场用灭菌棉拭共采集肉鸭的泄殖腔样品20份,通过SPF鸡胚尿囊腔传代接种法分离到1株病毒。HA-HI试验表明:该病毒可凝集鸡红细胞,不能被ND、EDS-76阳性血清抑制,但能被H9阳性血清所抑制,为H9亚型AI。该分离株生物学特性研究结果表明,对鸡表现为弱致病性,而对鸭无致病性。能在小鼠体内良好的增殖,并且可引起发病和死亡。     

一株鸡源H9N2亚型禽流感病毒的分离鉴定

从福州市某活禽市场采集的鸡泄殖腔棉拭子样品中分离出1株病毒,经血凝抑制试验(HI)和聚合酶链反应(PCR)方法鉴定为H9N2亚型禽流感病毒。在GenBank基因库中对该病毒株的3个基因片段的测序结果进行BLAST比对分析表明,3个基因片段均属于H9N2亚型禽流感病毒的基因。HA基因遗传进化分析表明,该病毒分离株与代表株DK/HK/Y280/97处于同一分支,与上海的鸡源分离株A/chicken/Shanghai/06/2015(H9N2)同源性最高,同源性为99.5%。     

Molecular characterization of H5N2 avian influenza viruses isolated from South African ostriches in 2006

Highly pathogenic avian influenza (HPAI) H5N2 reemerged in ostriches in South Africa during 2006, and a low-pathogenic AI H5N2 virus was also isolated. Molecular and phylogenetic characterization was performed to determine whether the outbreak strains were genetically derived from the supposedly eradicated Eastern Cape ostrich outbreak HPAI H5N2 strain of 2004. It was demonstrated that although the 2004 and 2006 South African H5N2 strains shared a common ancestor, the two outbreaks were not related. Not only were extensive reassortments with wild bird viruses involved in the evolution of the 2006 strains, but the precursor HA molecule HA0 cleavage site sequence of the 2006 HPAI H5N2 virus also contained fewer basic amino-acid insertions. Multiple transmission events occurred from wild birds to ostriches in 2006, and it appears that a reservoir of H5N2 with pathogenic potential for poultry is established in the South African wild duck population.     

Evolutionary characterization of hemagglutinin gene of H9N2 influenza viruses isolated from Asia

The full length hemagglutinin (HA) genes of 287 H9N2 AI strains isolated from chickens in Asia during the period 1994-2009 were genetically analyzed. Phylogenetic analysis showed that G1-like viruses circulated in the Middle East and Indian sub-continent countries, whereas other sublineages existed in Far East countries. It also revealed G1-like viruses with an average 96.7% identity clustered into two subgroups largely based on their time of isolation. The Ka/Ks ratio was calculated 0.34 for subgroup 1 and 0.57 for subgroup 2 indicates purifying/stabilizing selection, but despite this there is evidence of localized positive selection when comparing the subgroups 1 and 2 protein sequences. Five sites in HA H9N2 viruses had a posterior probability >0.5 using the Bayesian method, indicating these sites were under positive selection. These sites were found to be associated with the globular head region of HA. To identify sites under positive selection; amino acid substitution classified depends on their radicalism and neutrality. The results indicate that, although most positions in HAs were under purifying selection and can be eliminated, a few positions located in the antigenic regions and receptor binding sites were subject to positive selection.     

3株H9亚型禽流感病毒的分离与鉴定

为了获得H9亚型禽流感病毒(AIV)流行毒株并掌握流行毒株的分子特征和致病性,采用病毒分离、血凝性试验、鸡胚半数感染量(EID50)测定、HA基因序列分析、致病性试验、交叉保护性试验等对3份临床疑似H9亚型AIV感染病料进行了研究.结果:3份临床病料样品可引起10日龄SPF鸡胚规律性死亡,3株分离毒株对1％鸡红细胞的凝...     

Studies on influenza viruses H10N4 and H10N7 of avian origin in mink

An influenza A virus, A/mink/Sweden/84 (H10N4), was isolated from farmed mink during an outbreak of respiratory disease, histopathologically characterised by severe interstitial pneumonia. The virus was shown to be of recent avian origin and closely related to concomitantly circulating avian influenza virus. Serological investigations were used to link the isolated virus to the herds involved in the disease outbreak. Experimental infection of adult mink with the virus isolate from the disease outbreak reproduced the disease signs and pathological lesions observed in the field cases. The mink influenza virus also induced an antibody response and spread between mink by contact. The same pathogenesis in mink was observed for two avian influenza viruses of the H10N4 subtype, circulating in the avian population. When mink were infected with the prototype avian H10 influenza virus, A/chicken/Germany/N/49, H10N7, the animals responded with antibody production and mild pulmonary lesions but neither disease signs nor contact infections were observed. Detailed studies, including demonstration of viral antigen in situ by immunohistochemistry, of the sequential development of pathological lesions in the mink airways after aerosol exposure to H10N4 or H10N7 revealed that the infections progress very similarly during the first 24h, but are distinctly different at later stages. The conclusion drawn is that A/mink/Sweden/84, but not A/chicken/Germany/N/49, produces a multiple-cycle replication in mink airways. Since the viral distribution and pathological lesions are very similar during the initial stages of infection we suggest that the two viruses differ in their abilities to replicate and spread within the mink tissues, but that their capacities for viral adherence and entry into mink epithelial cells are comparable.     

Genetic characterization of H9N2 avian influenza virus previously unrecognized in Korea

In this study, we describe the isolation and characterization of previously unreported Y280-lineage H9N2 viruses from two live bird markets in Korea in June 2020. Genetic analysis revealed that they were distinct from previous H9N2 viruses circulating in Korea and had highest homology to A/chicken/Shandong/1844/2019(H9N2) viruses. Their genetic constellation showed they belonged to genotype S, which is the predominant genotype in China since 2010, where genotype S viruses have infected humans and acted as internal gene donors to H5 and H7 zoonotic influenza viruses. Active surveillance and control measures need to be enhanced to protect the poultry industry and public health.     

近年来中国H9亚型禽流感分离株谱系分析

从GenBank中下载所有来自中国（含港、澳、台）的H9亚型禽流感病毒血凝素基因885条核苷酸序列（长度≥900bp）,用MEGA5.0软件进行谱系分析。结果表明我国近年来H9亚型禽流感病毒以第h9.4.2.5分支为主（代表株为A/chicken/Guangxi/55/2005）,而不是WHO新近报告所列出的4株病毒（A/Quail/HongKong/G1/97、A/chicken/HongKong/G9/97、A/duck/HongKong/Y280/97、A/HongKong/33982/2009）所代表的分支。此分析结果对于研制针对这一病毒感染的疫苗有重要指导意义。     

Serologic and genetic characterization of North American H3N2 swine influenza A viruses

The H3N2 subtype of influenza A viruses isolated from pigs in the United States and Canada has shown both genetic and antigenic diversity. The objective of this study was to determine the serologic and genetic characteristics of contemporary strains of these viruses. Genetic analysis of 18 reference strains and 8 selected strains demonstrated differences in 1% to 9% of the nucleotides of the hemagglutinin (HA) gene. Phylogenetic analysis of the HA gene revealed 3 genetic clusters, as well as divergence of cluster III viruses from a cluster III prototype virus (A/Swine/Illinois/21587/99). By means of 1-way cross-hemagglutination inhibition with antiserum against 5 field isolates and 3 vaccine viruses, most of 97 isolates tested could be placed in 1 of 3 serogroups. The several isolates that did not react with any antiserum were in genetic cluster III, which suggests that continuous antigenic drift in cluster III may have resulted in virus variants. The efficacy of commercial vaccines against these virus variants should be evaluated with vaccination and challenge studies.     

免疫鸡群中分离的H9N2亚型禽流感病毒的分子特征研究

为研究浙江省禽流感病毒(AIV)的流行病学情况,本实验应用鸡胚传代方法从AIV疫苗免疫鸡群中表现典型呼吸症状的产蛋鸡体内分离1株H9N2亚型AIV A/Chicken/Jiande/01/2009(H9N2)。氨基酸序列分析显示,HA受体结合位点出现了人流感病毒结合位点226L,M基因出现S31N的突变。遗传进化分析显示,A/Chicken/Jiande/01/2009的M基因和PB2基因属于G1-Like谱系,HA基因、NA基因和NS基因属于Ck/BJ/1/94-Like分支,而NP基因、PA基因和PB1基因属于Ck/SH/F/98-Like谱系。这些资料表明,A/Chicken/Jiande/01/2009(H9N2)为一株重排病毒。     

Genetic characterization of H7N2 influenza virus isolated from pigs

Because pigs have respiratory epitheliums which express both α2-3 and α2-6 linked sialic acid as receptors to influenza A viruses, they are regarded as mixing vessel for the generation of pandemic influenza viruses through genetic reassortment. A H7N2 influenza virus (A/swine/KU/16/2001) was isolated from pig lungs collected from the slaughterhouse. All eight genes of the influenza virus were sequenced and phylogenetic analysis indicated that A/swine/KU/16/2001 originated in Hong Kong and genetic reassortment had occurred between the avian H7N2 and H5N3 influenza viruses. The first isolation of H7 influenza virus in pigs provides the opportunity for genetic reassortment of influenza viruses with pandemic potential and emphasizes the importance of surveillance for atypical swine influenza viruses.     

H5N1和H9N2亚型禽流感病毒型特异性抗原捕捉ELISA检测方法的建立

采用禽流感病毒多克隆抗体及型特异性单克隆抗体，研究建立了型特异性抗原捕捉ELISA检测方法，用于检测H5N1和H9N2亚型禽流感病毒。优化了反应条件，确定了包被抗体、检测抗体及酶结合物的最佳工作浓度，对该方法的敏感性、特异性、重复性及稳定性进行了分析，并与病毒分离鉴定的结果进行了比较。结果表明，该方法敏感、特异，具有良好的重复性和稳定性，可用于临床样品及鸡胚、细胞培养物中禽流感病毒的检测。     

Molecular characterization of low pathogenicity H7N3 avian influenza viruses isolated in Italy

The complete coding regions of the surface glycoproteins, nucleoprotein (NP), polymerase 2 (PB2), and matrix (M) of A/turkey/214845/02 and A/turkey/220158/99 (H7N3) low pathogenicity avian influenza (LPAI) viruses isolated in October 2002 in Italy were amplified and sequenced to determine the epidemiologic relationships with an A/turkey/Italy/4603/99 (H7N1/4603/99) LPAI virus isolated during the 1999-2001 epizootic in Italy. The hemagglutinin (HA) of H7N3 viruses showed 97.8% nucleotide similarity with A/turkey/Italy/4603/99 (H7N1), and NP, M, and PB2 gene similarities were 93.6%, 98.2%, and 96.2%, respectively. Phylogenetic analyses of HA, PB2, and M genes showed that H7N3 and H7N1 viruses were closely related. Sequence analysis revealed a 23 amino acid deletion in the stalk of the neuraminidase of H7N3 viruses and a unique deletion of amino acid glycine in position 17 in the NP gene of H7N1 virus.