Genetic characterization of a novel influenza A virus H5N2 isolated from a dog in China

An influenza virus, A/canine/Shandong/JT01/2009, has been isolated from a dog exhibiting classical flu signs in China. HAI and NAI assays subtyped A/canine/Shandong/JT01/2009 as a H5N2 like virus. Phylogenetic reconstructions indicated strong relationships with viruses from various hosts and dispersed geographic locations. These analyses indicate A/canine/Shandong/JT01/2009 is a novel virus generated by complex reassortment of the viral segments.     

Antigenic characterization of an H3N2 swine influenza virus isolated from pigs with proliferative and necrotizing pneumonia in Quebec.

A new strain of swine influenza A virus, designated A/Swine/Saint-Hyacinthe/150/90 has been isolated from pigs with severe proliferative and necrotizing pneumonia in Quebec. The antigenic characterization of the hemagglutinin was performed by hemagglutination inhibition test, immunoblot and indirect immunoprecipitation using polyclonal antisera. Only the last test was able to detect an antigenic relationship between the hemagglutinin of this isolate and an H3 subtype influenza virus. The immunoprecipitation test was a useful alternative for determining the hemagglutinin of influenza A virus subtypes. The neuraminidase inhibition test demonstrated a reactivity between the A/Swine/Saint-Hyacinthe/150/90 and antiserum against a N2 subtype influenza virus. Our results indicate that this new strain isolated for the first time in the porcine population of Canada is related to A/Sw/Hong Kong/76 H3N2 swine influenza virus.     

一株鸟源H1N2型禽流感病毒全基因序列分析

2011—2012年,对我国广西活禽市场进行流行病学监测时从麻雀体内分离鉴定出1株H1亚型禽流感病毒(AIV),命名为A/Sparrow/Guangxi/GXs-1/2012(H1N2)。为了解该株H1亚型AIV的来源、特征及其分子演化规律,本研究对其全基因序列进行测定,并与GenBank登录的相关病毒进行遗传演化分析。结果表明:这株分离纯化的H1N2毒株HA基因切割位点附近的氨基酸序列为PSIQSR↓GLF,属于低致病力AIV的特征;NA基因与A/duck/Guangxi/GXd-2/2010(H6N2)在同一分支内,核苷酸同源性为98%;PB1与PB2基因与H5和H4亚型较为接近;PA基因与A/duck/Guangdong/E1/2012(H10N8)同源性最高;NP基因则与A/chicken/Pakistan/NARC-16945/2010(H3N1)同源性达97%;而NS与MP基因分别与A/wild duck/Korea/SH5-26/2008(H4N6)、A/duck/Shanghai/C84/2009(H3N2)同源性最高。因此,推测A/Sparrow/Guangxi/GXs-1/2012(H1N2)可能是不同来源的基因经过复杂重组演变后的1株重组病毒。     

Reassortant H1N1 influenza virus vaccines protect pigs against pandemic H1N1 influenza virus and H1N2 swine influenza virus challenge

Influenza A (H1N1) virus has caused human influenza outbreaks in a worldwide pandemic since April 2009. Pigs have been found to be susceptible to this influenza virus under experimental and natural conditions, raising concern about their potential role in the pandemic spread of the virus. In this study, we generated a high-growth reassortant virus (SC/PR8) that contains the hemagglutinin (HA) and neuraminidase (NA) genes from a novel H1N1 isolate, A/Sichuan/1/2009 (SC/09), and six internal genes from A/Puerto Rico/8/34 (PR8) virus, by genetic reassortment. The immunogenicity and protective efficacy of this reassortant virus were evaluated at different doses in a challenge model using a homologous SC/09 or heterologous A/Swine/Guangdong/1/06(H1N2) virus (GD/06). Two doses of SC/PR8 virus vaccine elicited high-titer serum hemagglutination inhibiting (HI) antibodies specific for the 2009 H1N1 virus and conferred complete protection against challenge with either SC/09 or GD/06 virus, with reduced lung lesions and viral shedding in vaccine-inoculated animals compared with non-vaccinated control animals. These results indicated for the first time that a high-growth SC/PR8 reassortant H1N1 virus exhibits properties that are desirable to be a promising vaccine candidate for use in swine in the event of a pandemic H1N1 influenza.     

一株H9N2亚型猪流感病毒的遗传进化和致病性分析

本研究从有流感症状的病猪中分离到一株H9N2亚型猪流感病毒(SIV),命名为A/swine/Jiangsu/1/2015(SW/JS/1/15)。为探究其遗传特征和生物学特性,本研究采用RT-PCR技术扩增其全部基因节段后测序并进行遗传分析,并研究了其对鸡和豚鼠的致病特性。遗传进化分析显示,分离病毒SW/JS/1/15株是由BJ/94系、DK1系、G1系和F/98系4个分支病毒重组而成,8个基因节段均属于G57基因型。分离株HA蛋白裂解位点为PSRSSR*GL,符合低致病性流感病毒的特征。HA蛋白有9个潜在糖基化位点,其中218位糖基化位点缺失,145位与313位各新增一个糖基化位点。与疫苗株SH/F/98、SD/6/96、GD/SS/94相比,分离病毒HA抗原位点发生了G^90E、S^127R、S^145N、D^153G、N^167S、A^168N、A^198T、T^200R、N^201D、和Q^235M(H9numbering)突变;NA蛋白发生6个氨基酸突变:K^367R、K/E^368N、D^369N、D^401E、K^143N和T^434P。同时NA蛋白颈部缺失aa63~aa65。分离病毒的8个基因节段与2株禽源H9N2病毒的相应基因高度同源,其6个内部基因与两株人源H7N9病毒的内部基因高度同源。致病性试验结果显示分离病毒可以感染鸡和豚鼠,但不能在豚鼠群内水平传播,且可能作为H7N9等新型流感病毒内部基因供体,同时表明猪可以感染禽流感病毒(AIV),且可能是AIV获得感染哺乳动物能力的过渡宿主。本研究为H9N2亚型SIV的致病性以及遗传特征的研究提供科学依据。     

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.     

Genetic characterization and susceptibility on poultry and mammal of H7N6 subtype avian influenza virus isolated in Japan in 2009

From February to March 2009, six strains of H7N6 subtype avian influenza virus were isolated from quails in three farms in Aichi prefecture in Japan. The isolates were shown to be low pathogenic for chicken by the examination performed using the "Manual of Standards for Diagnostic Tests and Vaccines" by World organisation for Animal Health (OIE). The deduced amino acid sequence at the cleavage site was PE (I/Q/L) PKRR (nucleotide sequences were cct gaa (a/c) (t/a) a cc (a/g) aaa aga aga), suggesting persistence in domestic poultry for some time. The direct putative ancestor strain could not be elucidated by phylogenetic analysis of all genome segments of the quail isolates. Diverged date from a putative common ancestor in a non-rooted phylogenetic tree among quail viruses was estimated between March 2002 and July 2004. Three putative N-linked glycosylation sites resided in the vicinity of the receptor binding pocket of HA1 region. They are considered to decrease the reactivity of neutralizing antibody against the virus. Experiments for the infectivity and pathogenicity of a quail strain to poultry indicated that the quail isolate had higher infectivity to quails than chickens and ducks. Direct and dust-borne and/or droplet-borne transmissions among quail were proven in quails with and without direct contact with experimentally infected quails. The virus is seldom transmitted among chickens either directly or indirectly, and indirect transmission from infected quails to chickens was not observed. The pathogenicity of the quail strain for mammalian, pig and mouse was low, although it could replicate in those animals.     

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.     

Identification and characterization of H2N3 avian influenza virus from backyard poultry and comparison with novel H2N3 swine influenza virus

In early 2007, H2N3 influenza virus was isolated from a duck and a chicken in two separate poultry flocks in Ohio. Since the same subtype influenza virus with hemagglutinin (H) and neuraminidase (N) genes of avian lineage was also identified in a swine herd in Missouri in 2006, the objective of this study was to characterize and compare the genetic, antigenic, and biologic properties of the avian and swine isolates. Avian isolates were low pathogenic by in vivo chicken pathogenicity testing. Sequencing and phylogenetic analyses revealed that all genes of the avian isolates were comprised of avian lineages, whereas the swine isolates contained contemporary swine internal gene segments, demonstrating that the avian H2N3 viruses were not directly derived from the swine virus. Sequence comparisons for the H and N genes demonstrated that the avian isolates were similar but not identical to the swine isolates. Accordingly, the avian and swine isolates were also antigenically related as determined by hemagglutination-inhibition (HI) and virus neutralization assays, suggesting that both avian and swine isolates originated from the same group of H2N3 avian influenza viruses. Although serological surveys using the HI assay on poultry flocks and swine herds in Ohio did not reveal further spread of H2 virus from the index flocks, surveillance is important to ensure the virus is not reintroduced to domestic swine or poultry. Contemporary H2N3 avian influenza viruses appear to be easily adaptable to unnatural hosts such as poultry and swine, raising concern regarding the potential for interspecies transmission of avian viruses to humans.     

果子狸源H5N1亚型禽流感病毒的分离及生物学特性鉴定

2011年,对广西自治区进行禽流感流行病学调查时从野生哺乳动物果子狸体内分离到一株H5N1亚型禽流感病毒(AⅣ),命名为A/palm-civet/Guangxi/26/2011 (H5N1) (PC/GX/26/11).本研究对其全基因组序列进行测定及其对BALB/c鼠的致病性试验.全基因序列分析表明:该病毒属于Clade 2.3.2分支,其血凝素(HA)蛋白裂解位点存在多个连续的碱性氨基酸(-RRRR-),具有高致病性AIV (HPAIV)的典型分子特征,其HA、NA、PA、NS基因节段与A/muscovy duck/Vietnam/LBM57/2011 (MD/VN/LBM57/11) (H5N1)有较高的同源性,为99.2％～99.6％;PB2、PB1、M、NP4个基因节段与人源AIV A/Hubei/1/2010 (HuB/1/10) (H5N1)的同源性最高,为99.0％～99.5％.动物试验显示:该病毒对小鼠的半数致死量为4.9 log EID50,对小鼠具有较高的致病性.在小鼠的肺和鼻甲骨中均能够进行良好的复制.以上结果表明,该分离株未经适应便具备感染哺乳动物并有较高致死的能力.     

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.     

1株猪源2009/H1N1流感病毒反向遗传系统的建立

为建立1株猪源2009/H1N1流感病毒A/swine/Heilongjiang/44/2009(HLJ44)的反向遗传系统,利用双向表达质粒p HW2000,分别构建了该病毒株8个基因节段的重组质粒,将其共转染于293T和MDCK混合培养的细胞,拯救出重组病毒R-HLJ44。测序结果表明,R-HLJ44与亲本病毒HLJ44的核苷酸序列完全一致;二者在细胞上具有相似的增殖特性;抗原性未发生变化;分别以106TCID50的剂量鼻腔感染BALB/c小鼠,结果显示R-HLJ44与亲本HLJ44在小鼠脏器中的复制滴度也基本一致。以上结果表明拯救的重组病毒保持了与亲本病毒一致的生物学特性。该病毒反向遗传系统的建立,为进一步研究H1N1亚型流感病毒的致病分子机制及新型疫苗研制等奠定了基础。     

2017年国内大型鸡场分离的H9N2禽流感病毒的遗传进化和分子特征分析

H9N2禽流感病毒自1994年在广东首次报道暴发以来,已在国内广泛地流行,给养禽业带来了极大的损失,甚至威胁着人类的健康。为了解2017年国内H9N2亚型禽流感病毒的遗传进化和分子特征变化,本研究从江苏、安徽、山东等地区的大型鸡场中分离到了10株H9N2亚型禽流感病毒。对10株分离株的基因分析表明,所有分离株均属于在2013年后占主导地位的G57型,由BJ/94系的HA、NA和NS基因,DK1系的PB2基因,F/98系的PB1,PA和NP基因以及G1/97系的M基因的四重组病毒组成,并且分离株的EID_(50)显著低于早期分离的H9N2禽流感病毒。10株分离株中有7株受体结合位点的198位发生了A到T,或者A到V的替换,并发生糖基化位点在218位的缺失,313位的增加。与近5年已经发表的流行株HA蛋白比较,所有分离株的抗原位点均发生了S127R、S183N、D216E、T220I、Q235M、S283R突变,NA基因在61～63位缺失NIT;表明分离株的抗原发生了变异。而分离株内部基因PB2上的K318R突变,PB1上的L13P突变,PA上的K356R突变、Q/T/S400P突变,NS上的E227K突变,NP上的D34S、K398Q突变,M1上的V15I突变以及M2上的I28V、L55F突变,使分离株更易感染哺乳动物,说明必需要继续加强H9N2禽流感病毒的监测,密切关注其对人类可能造成的影响。特别是10株分离株中首次出现的NA基因的E433D突变和PB2基因的E627V突变是否会给H9N2禽流感病毒增加新的生物学特性还有待进一步研究。     

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.