两株鸭源H5N1亚型禽流感病毒的序列分析及致病性研究

本研究对2010年在湖北活禽市场监测分离到的两株鸭源H5N1亚型禽流感病毒(AIV) (HuB/495/10和HuB/513/10)进行了序列分析和致病性试验研究,以了解湖北地区H5N1亚型AIV的生物学特性差异.序列分析显示:2株病毒全基因组核苷酸同源性在97.3 ％～98.6％,2株病毒的8个节段基因均与青海和香港分离的野鸟源病毒A/great crested-grebe/Qinghai/1/2009 (H5N1)和A/black-crowned night heron/Hong Kong/659/2008 (H5N1)的核苷酸高度同源,HA蛋白裂解位点序列基序为341RRRKR345,呈现典型的高致病力分子特征.以105 EID50/100 μL病毒剂量感染4周龄SPF鸭发现:HuB/495/10和HuB/513/10对鸭的致死率分别为100％和20％,病毒在鸭体内呈全身性复制并可通过呼吸道和消化道向外排毒;不同滴度的病毒感染6周龄BALB/c小鼠,HuB/495/10和HuB/513/10的MLD50分别为1.38 log10 EID50和1.68 log10 EID50,对小鼠表现为高致病力,均在肺脏中高拷贝复制.     

马流感病毒（H3N8亚型，Huabei株）对马的致病性研究

为研究从华北地区分离的H3N8亚型马流感病毒（Huabei株）对马的致病性,分别用E2、E3、E4、E5及E10代病毒液人工感染健康马,比较了不同代次毒株对马的致病性差异.研究表明,E2、E3代病毒以10^7.2～ 10^7.4EID50剂量经喷雾途径可使12 ～ 18个月龄的马出现持续性发热、咳嗽和流浆液性鼻液等典型马流感症状;同样剂量的E4、E5和E10代病毒感染马匹后仅表现为流浆液性鼻液.本研究确定了马流感病毒Huabei株的感染剂量及代次,为马流感灭活疫苗效果评价奠定了实验感染模型基础.     

一株重配型H1N2猪流感病毒的进化分析及对小鼠的致病性

为了进一步了解我国辽宁省猪流感(SI)的流行情况及病原的进化规律,对2016年年末在辽宁省某生猪屠宰场进行SI病原学监测时分离到的一株H1N2亚型猪流感病毒(SIV)A/swine/Liaoning/FX575/2016(简称FX575)进行全基因组序列测定,通过对其8个基因片段的基因来源进行分析,确定基因型;构建系统进化树;分析相关分子特征;进一步以6周龄雌性BALB/c小鼠为感染模型,通过测定感染后小鼠的体重变化和脏器病毒含量评估病毒的致病性。结果显示:FX575为一株三重重配型的H1N2病毒,遗传进化分析结果显示病毒的HA基因来自于欧亚类禽型H1N1(EA H1N1)分支的SIV,NA基因来自于北美三重配型H1N2分支的SIV,6个内部基因(PB2、PB1、PA、NP、M和NS)均来自于2009/H1N1分支的病毒。以10~6EID₅₀的剂量经鼻腔途径感染小鼠后,能够引起小鼠出现明显的体重下降,其中有1只小鼠在感染后第7天体重下降的比率超过25%,判为死亡;感染后第3天在小鼠肺及鼻甲骨中均检测到较高滴度的病毒,含量分别为4.82、4.20 log₁₀EID₅₀·mL^-1。结果表明SIV在不断流行的过程中,不同基因型病毒之间发生重组导致出现基因三重配的病毒,病毒对小鼠呈现中等致病力特征,提示应进一步加强对SI的主动监测,从而降低病毒对兽医及公共卫生学风险。     

H1N1亚型流感病毒鸡胚增殖参数的优化

为了研究最佳的H1N1亚型流感病毒鸡胚增殖参数,本试验进行了孵化前种蛋的选择与保存、鸡胚孵化中各参数设定等因素对H1N1亚型流感病毒产毒量影响的研究。其中种蛋的选择与保存,主要考察了蛋重、蛋形指数、保存期、消毒时间及方法等因素,结果显示,蛋重为55~65 g,蛋形指数为1.30~1.35,种蛋保存期为1~4 d,保存温度为16~18 ℃,保存湿度为70%~80%,保存期种蛋的甲醛熏蒸消毒时间为30 min时,可以为H1N1亚型流感疫苗生产提供最佳的种蛋。孵化过程中孵化参数对H1N1亚型流感病毒产毒量的影响,本试验主要将孵化温度、湿度、翻蛋、通风等参数作为研究对象,结果显示在生产H1N1亚型流感疫苗时,最佳孵化参数设定为:温度1~7 d为38.2 ℃、8~9 d为38.0 ℃、10 d为37.8 ℃,湿度1~10 d为65%~70%,翻蛋频率为1次/2 h,前后倾角各为45°,通风风门设定为1~5 d为4、6~10 d为5。本试验结果为H1N1亚型流感疫苗生产提供优质的鸡胚孵化技术,确保鸡胚尿囊液的质量和收获量。     

广东1株H3N2亚型犬流感病毒全基因组遗传进化分析

本试验对1株犬源H3N2亚型流感病毒(A/Canine/Guangdong/10/2014)进行了遗传进化分析。结果显示,本毒株8个基因片段与在中国和泰国分离的H3N2亚型犬流感的关系最近,与当前亚洲分离的毒株高度相似(99%)。基因亚型分析显示8个基因片段和目前流行的H3N2CIVs有相同的基因型(K,G,E,3B,F,2D,F,1E)。本毒株为低致病性的禽源H3N2亚型犬流感,从而证实了禽源犬流感亚型在中国的存在,对流感病毒实施广泛的血清学和流行病学检测,以及对该病的防控具有重要意义。     

广东1株H3N2亚型犬流感病毒的分离与鉴定

本试验采集一只具有流感临床症状的病犬鼻咽拭子经常规处理后接种SPF鸡胚,分离到一株流感病毒,并对其进行鉴定及生物学特性研究。结果表明,该毒株对1%鸡红细胞的血凝价为26,能被H3亚型流感病毒阳性血清中和,与H1、H5、H7、H9亚型阳性血清和阴性血清无交叉反应。序列分析显示,该毒株的HA和NA基因核苷酸序列分别与犬流感病毒(CIV)H3和N2亚型的病毒株同源性最高。确定该毒株为H3N2亚型CIV,并将其命名为A/canine/Guangdong/01/2011。     

H3N2亚型犬流感病毒重组HA1蛋白间接ELISA检测方法的建立

为建立一种H3N2亚型犬流感病-毒(CIV)血清学检测方法,本研究利用CIV重组HA1蛋白作为检测抗原,建立H3N2亚型CIV抗体的间接ELISA检测方法,并优化反应条件.通过检测阴性血清样本30份确定其临界值为0.228.该方法与抗猪流感病毒H1N1、H1N2、H6N6、H9N2的阳性血清和犬瘟热病、犬细小病、犬副流感的阳性血清均无交叉反应.变异系数在1.01％～7.52％之间,具有较好的重复性.通过对150份临床样品进行检测并与血凝抑制试验检测结果比较,总符合率为98.6％.本研究为CIV流行病学调查提供了一种快速、方便、敏感的抗体检测方法.     

两株H9亚型鹅流感病毒的分离和鉴定

禽类流感病毒（Avian influenza viruses）与人类流感病毒都属于正黏病毒科，二者关系密切。一方面，禽流感病毒为20世纪暴发的3次人类流感大流行提供丁所必需的基因。另一方面，禽流感病毒也可以直接感染人。例如：1997年香港和2004年初的泰国、越南H5N1亚型禽流感导致多人死亡。H9亚型流感病毒1966年首次从北美的火鸡分离。H9亚型禽流感虽然尚未有导致人类死亡的报道，但是该亚型病毒对动物和人的威胁正在增加。     

H5N1亚型流感病毒分子生物学研究进展

H5N1亚型流感病毒是目前在亚太地区蔓延的人禽流感病毒，在给家禽养殖业造成巨大经济损失的同时也威胁到了人类社会的公共健康卫生。笔者综述了近年来对H5N1流感病毒的分子生物学研究。以助于更好地预防禽流感病毒传播、预测未来大流行病毒的变异和走向。     

两株鸭源H1N3亚型流感病毒的分离鉴定和遗传进化分析

从2008年10月至2010年7月,每月定期在华东地区扬州活禽市场采集健康家鸭泄殖腔拭子,并从中分离鉴定了14株H1亚型禽流感病毒。对其中的2株病毒进行全基因测序和遗传演化分析,发现2株病毒均属于H1N3低致病性禽流感病毒。各基因遗传进化分析均与类禽猪流感的分离株的亲缘关系相对较近;除血凝素(haemagglutini,HA)基因外,其它所有的基因均发生了不同程度基因重组。其中H5N1病毒参与了多聚酶蛋白PB1与PB2基因的重组,H9N2病毒参与了PA基因的重组。     

Serological Evidence of Pandemic H1N1 Influenza Virus Infections in Greek Swine

The introduction of the 2009 pandemic H1N1 (pH1N1) influenza virus in pigs changed the epidemiology of influenza A viruses (IAVs) in swine in Europe and the rest of the world. Previously, three IAV subtypes were found in the European pig population: an avian‐like H1N1 and two reassortant H1N2 and H3N2 viruses with human‐origin haemagglutinin (HA) and neuraminidase proteins and internal genes of avian decent. These viruses pose antigenically distinct HAs, which allow the retrospective diagnosis of infection in serological investigations. However, cross‐reactions between the HA of pH1N1 and the HAs of the other circulating H1 IAVs complicate serological diagnosis. The prevalence of IAVs in Greek swine has been poorly investigated. In this study, we examined and compared haemagglutination inhibition (HI) antibody titres against previously established IAVs and pH1N1 in 908 swine sera from 88 herds, collected before and after the 2009 pandemic. While we confirmed the historic presence of the three IAVs established in European swine, we also found that 4% of the pig sera examined after 2009 had HI antibodies only against the pH1N1 virus. Our results indicate that pH1N1 is circulating in Greek pigs and stress out the importance of a vigorous virological surveillance programme.     

Detection of Novel Reassortant Influenza A (H3N2) and H1N1 2009 Pandemic Viruses in Swine in Hanoi,Vietnam

From May to September 2013, monthly samples were collected from swine in a Vietnamese slaughterhouse for influenza virus isolation and serological testing. A(H1N1)pdm09 viruses and a novel H3N2 originating from reassortment between A(H1N1)pdm09 and novel viruses of the North American triple reassortant lineage were isolated. Serological results showed low seroprevalence for the novel H3N2 virus and higher seroprevalence for A(H1N1)pdm09 viruses. In addition, serology suggested that other swine influenza viruses are also circulating in Vietnamese swine.     

1株H1N1亚型猪流感病毒的全基因组特征及其对小鼠的致病性

旨在了解河南省猪流感病毒的流行情况及其遗传进化和基因组特征。2018年4月,从河南省某一出现疑似流感症状猪群中采集鼻拭子样品150份用于分离病毒,对分离病毒的全基因组进行序列测定和分析。同时感染6周龄BALB/c小鼠,研究其对小鼠的致病性。结果显示,获得1株H1N1亚型病毒[命名为A/swine/Henan/NY20/2018（H1N1）]。遗传进化表明,其HA和NA基因属于欧亚类禽H1N1分支,PB2、PB1、PA、NP和M基因属于2009甲型H1N1分支,NS基因属于经典H1N1分支。HA蛋白的裂解位点序列为PSIQSR↓GL,具有低致病性流感病毒的分子特征,在小鼠肺和鼻甲有效复制并能引起肺组织病理学变化。本研究分离到1株3源重排H1N1亚型病毒,对小鼠呈现一定致病力,提示应进一步加强对SIV的监测。     

H5N1亚型高致病性禽流感病毒对鸽的致病性分析

为评估H5N1亚型禽流感病毒对鸽的致病性,用我国2004年分离的一株H5NI亚型禽流感病毒A/pigeon/GD/C2/2004( H5NI)人工感染4周龄鸽,进行了致病性试验.结果表明,该株病毒以106EID50/I00μL剂量感染能致死鸽,致死率为22.2％,病毒能在感染鸽体内广泛分布,其中肺脏和肾脏中病毒含量达到...     

Comparative study of pandemic (H1N1) 2009, swine H1N1, and avian H3N2 influenza viral infections in quails

Quail has been proposed to be an intermediate host of influenza A viruses. However, information on the susceptibility and pathogenicity of pandemic H1N1 2009 (pH1N1) and swine influenza viruses in quails is limited. In this study, the pathogenicity, virus shedding, and transmission characteristics of pH1N1, swine H1N1 (swH1N1), and avian H3N2 (dkH3N2) influenza viruses in quails was examined. Three groups of 15 quails were inoculated with each virus and evaluated for clinical signs, virus shedding and transmission, pathological changes, and serological responses. None of the 75 inoculated (n = 45), contact exposed (n = 15), or negative control (n = 15) quails developed any clinical signs. In contrast to the low virus shedding titers observed from the swH1N1-inoculated quails, birds inoculated with dkH3N2 and pH1N1 shed relatively high titers of virus predominantly from the respiratory tract until 5 and 7 DPI, respectively, that were rarely transmitted to the contact quails. Gross and histopathological lesions were observed in the respiratory and intestinal tracts of quail inoculated with either pH1N1 or dkH3N2, indicating that these viruses were more pathogenic than swH1N1. Sero-conversions were detected 7 DPI in two out of five pH1N1-inoculated quails, three out of five quails inoculated with swH1N1, and four out of five swH1N1-infected contact birds. Taken together, this study demonstrated that quails were more susceptible to infection with pH1N1 and dkH3N2 than swH1N1.     

Experimental infection with H1N1 European swine influenza virus protects pigs from an infection with the 2009 pandemic H1N1 human influenza virus

The recent pandemic caused by human influenza virus A(H1N1) 2009 contains ancestral gene segments from North American and Eurasian swine lineages as well as from avian and human influenza lineages. The emergence of this A(H1N1) 2009 poses a potential global threat for human health and the fact that it can infect other species, like pigs, favours a possible encounter with other influenza viruses circulating in swine herds. In Europe, H1N1, H1N2 and H3N2 subtypes of swine influenza virus currently have a high prevalence in commercial farms. To better assess the risk posed by the A(H1N1) 2009 in the actual situation of swine farms, we sought to analyze whether a previous infection with a circulating European avian-like swine A/Swine/Spain/53207/2004 (H1N1) influenza virus (hereafter referred to as SwH1N1) generated or not cross-protective immunity against a subsequent infection with the new human pandemic A/Catalonia/63/2009 (H1N1) influenza virus (hereafter referred to as pH1N1) 21 days apart. Pigs infected only with pH1N1 had mild to moderate pathological findings, consisting on broncho-interstitial pneumonia. However, pigs inoculated with SwH1N1 virus and subsequently infected with pH1N1 had very mild lung lesions, apparently attributed to the remaining lesions caused by SwH1N1 infection. These later pigs also exhibited boosted levels of specific antibodies. Finally, animals firstly infected with SwH1N1 virus and latter infected with pH1N1 exhibited undetectable viral RNA load in nasal swabs and lungs after challenge with pH1N1, indicating a cross-protective effect between both strains.     

Pathogenesis and Subsequent Cross‐Protection of Influenza Virus Infection in Pigs Sustained by an H1N2 Strain

The H1N1, H3N2 and, more recently, H1N2 subtypes of influenza A virus are presently co‐circulating in swine herds in several countries. The objectives of this study were to investigate the pathogenesis of Sw/Italy/1521/98 (H1N2) influenza virus, isolated from respiratory tissues of pigs from herds in Northern Italy, and to evaluate its potential cross‐protection against the Sw/Fin/2899/82 (H1N1) strain. In the pathogenesis test, eight pigs were intranasally infected with H1N2 virus; at pre‐determined intervals, these animals were killed and necropsied, along with eight uninfected animals. In the cross‐protection test, sixteen pigs were infected by intranasal (i.n.) and intratracheal (i.t.) routes with either H1N2 or H1N1 virus. Twenty days later, all pigs were challenged (by the same route), with either the homologous H1N2 or heterologous H1N1 virus strains. Control group was inoculated with culture medium alone. On post‐challenge days (PCD) 1 and 3, two pigs from each infected group, along with one control pig, were killed. Clinical, virological, serological and histopathological investigations were performed in both the pathogenicity and cross‐protection tests. In the pathogenicity test, mild clinical signs were observed in two pigs during 3 and 4 days, respectively. Virus was isolated from two pigs over 6 days and from lung samples of pigs killed on post‐infection days 2 and 4. Seroconversion was detected in the two infected animals killed 15 days after infection. In the cross‐protection study, mild clinical respiratory signs were detected in all pigs infected with either the H1N2 or H1N1 virus. The virus was isolated from nasal swabs of almost all pigs till 6 days. After the challenge infection, the pigs remained clinically healthy and virus isolation from the nasal secretions or lung samples was sporadic. Antibody titres in H1N1 or H1N2 infected groups were similar, whereas the H1N2 sub‐type induced less protection against re‐infection by homologous and heterologous virus than H1N1 sub‐type. The controls had no signs of the disease. In the H1N2 infected pigs, a reduced number of goblet cells in nasal and tracheal mucosa and small foci of lymphomononuclear cell infiltrates in the submucosa were detected. Furthermore, the goblet cell reduction was related to the time of infection. Diffuse mild interstitial pneumonia was also recorded in pigs infected with the H1N2 virus and challenged with either H1N1or H1N2 pigs. These studies showed the moderate virulence of the H1N2 virus and a partial cross‐protection against heterologous infection.     

Optimization of Proliferation Parameter of H1N1 Influenza Virus in Chicken Embryo

In order to study the best propagation parameters of H1N1 subtype influenza virus in chicken embryo,this study was conducted to research the effects of egg selection and preservation before hatching and hatching parameters setting on production of H1N1 subtype influenza virus.In the egg selection and preservation,we mainly investigated the egg weight,egg shape index,storage period,disinfection time and method and other factors,the results showed that egg weight of 55 to 65 g,egg shape index of 1.30 to 1.35,egg storage period of 1 to 4 d,storage temperature of 16 to 18 ℃,humidity of 70% to 80%,during storage period,eggs formaldehyde fumigation time 30 min,we could provide the best eggs for H1N1 subtype influenza vaccine production.The effect of hatching parameters on production of H1N1 subtype influenza virus,this experiment mainly investigaed incubation temperature,humidity,times of turning over eggs,aeration and other parameters,the results showed that in the production of H1N1 subtype influenza virus,the optimal parameter set to hatch:temperature 1 to 7 d was 38.2 ℃,8 to 9 d was 38.0 ℃,10 d was 37.8 ℃;Humidity was 65% to 70% from 1 to 10 d;Turning over eggs frequency was 1 times/2 h,before and after each dip 45°;1 to 5 d aeration set to 4,6 to 10 d set to 5.The results provided quality chicken hatchery technology for H1N1 subtype influenza vaccine production in order to ensure the quality and yield of allantoic fluid.