Infectious bronchitis virus serotypes in poultry flocks in Jordan

Infectious bronchitis virus (IBV) causes respiratory disease in chickens all over the world. IBV has many serotypes that do not confer cross protection against each other. Hemagglutination inhibition (HI) test has been used to determine the serotypes of IBV as a substitute to the more laborious virus neutralization test and the more sophisticated restriction endonuclease digestion or sequencing of the S1 gene. In Jordan, no previous studies have been carried out to determine the involvement of IBV in respiratory disease in chickens, or the serotypes of IBV that possibly exist. In this study, serum from different chicken flocks (n = 20) that suffered from respiratory disease were tested for IBV antibodies using commercial IBV antibody ELISA at time of the initial signs of the respiratory disease and repeated on serum samples from the same flocks 10–14 days later. ELISA titer for IBV increased in 14 out of 20 flocks (70%) after 10–14 days of the initial signs of the respiratory disease and this indicates a recent exposure to IBV. The second serum samples from these 14 flocks were further examined against a panel of five IBV antigens (Ark, Conn, DE-072, JMK, and Mass) by HI test to determine the serotype(s) of IBV they have been exposed to. The HI test results indicated that the exposure of some of these flocks were to Ark, DE-072, and Mass like serotypes. However, the HI titers against the antigens used in this study were relatively similar in 10 out of the 14 flocks (71%) and the serotype of IBV that these flocks were exposed to could not be determined and the possible causes of this are discussed.     

一株H9N2亚型禽流感病毒全基因组分析

为了研究H9N2亚型AIV A/Chicken/Guangdong/333/2008的全基因组序列变异情况,试验利用RT-PCR方法扩增出该病毒的8个基因序列,并应用DNAStar和MEGA4.0软件分析所得基因序列。结果表明:该病毒HA基因的第226位氨基酸由Q(Gln)变为了L(Leu),其NP基因与Viet Nam/1203/2004(H5N1)的NP基因同源率最高。说明该病毒已具备了感染哺乳动物的分子特征,并可能在遗传演化过程中突变为高致病性的禽流感病毒(AIV)。     

广东一株重组H9N2亚型禽流感病毒的鉴定及全基因组序列分析

本研究于2013年从广东某发病鸡场分离到一株H9N2亚型AIV,命名为A/chicken/Guangdong/LG1/2013.对病毒进行全基因组克隆测序和进化分析,其HA基因裂解基序为333pSRSSR ↓ GLF341,呈典型低致病性AIV分子特征.其HA基因发生Q226突变和A316S突变,NA基因出现第63～65位氨基酸缺失.该病毒株HA、NA和NS基因属于类BJ/1/94分支,PB2属于类SD/H/09分支,PB1、PA、NP基因属于类SH/F/98分支,M基因属于类HK/G1/97分支,该基因型在广东地区未见报道,其内部基因与H7N9亚型人流感病毒SH/02/2013株核苷酸同源性在95.6 ％～98.6％之间.本研究表明我国H9N2亚型AIV呈现遗传演化的多样性及基因重组的复杂性,对该亚型病毒的监测和研究具有重要的兽医和公共卫生意义.     

Comparative molecular characterization,pathogenicity and seroprevalence of avian influenza virus H9N2 in commercial and backyard poultry flocks

This study was conducted to perform the comparative molecular characterization of avian influenza virus (AIV) H9N2, pathogenicity and seroprevalence in commercial and backyard poultry flocks. Fifty commercial poultry flocks were investigated between 2012 and 2015. Eighteen flocks (36%) out of 50 were positive HA. Seven (38.9%) out of 18 were positive by chromatographic strip test for AI common antigen. By Real-time RT-PCR, only two flocks were positive H9. The molecular characterization of two different AI-H9N2 viruses, one isolated from a broiler flock (A/chicken/Egypt/Mansoura-18/2013) and the other from a layer flock (A/chicken/Egypt/Mansoura-36/2015) was conducted on HA gene. Moreover, a higher seroprevalence, using the broiler strain as a known antigen, was shown in backyard chicken flocks 15/26 (57.7%) than duck flocks 9/74 (12.2%). Interestingly, the pathogenicity index (PI) of the H9N2 broiler strain in inoculated experimental chickens ranged from 1.2 (oculonasal route) to 1.9 (Intravenous route). The PI indicated a highly pathogenic effect, with high mortality (up to 100%) in the inoculated chickens correlated with the high mortality (80%) in the flock where the virus was isolated. The firstly recorded clinical signs, including cyanosis in the combs and wattles and subcutaneous haemorrhages in the leg shanks and lesions, as well as histopathology and immunohistochemistry, revealed a systemic infection of the high pathogenicity with the H9N2 virus. Conversely, the H9N2 layer strain showed a low pathogenicity. In conclusion, as a first report, the molecular analysis and pathogenicity of the tested strains confirmed the presence of a high pathogenicity AIV-H9N2 with systemic infections.     

H9亚型禽流感病毒血凝素特异性单因子血清制备

为制备特异性的H9亚型禽流感病毒(AIV)单因子血清,本研究分别将6株不同亚群的H9亚型AIV的血凝素(HA)基因以鸡偏嗜的密码子进行优化,经全基因合成插入高效真核表达载体pCAGGS中,构建的真核重组质粒转染293T细胞进行瞬时表达,间接免疫荧光试验结果表明,重组质粒中的HA目的基因获得表达.将重组质粒以200μg/只的剂量免疫1月龄SPF鸡,6周后采血分离血清.交叉微量血凝抑制试验结果表明,血凝抑制效价可达8 long2～12 log2,灵敏度高,与其他AIV亚型抗原无交叉反应,型特异性强.     

鸭源H9亚型禽流感病毒的分离与鉴定

从广东各地鸭群的392份泄殖腔样本中分离到3株病毒。用琼脂扩散试验(AGP)及血凝抑制试验(HI)证实3个分离株均为H9亚型禽流感病毒(AIV)。3个AIV分离株都能凝集人、鸡、山羊、豚鼠、兔的红细胞,但不能凝集猪红细胞;所有分离株血凝素(HA)对热不稳定;60℃加热处理10 m in后,病毒失去对鸡胚的感染性;3个AIV分离株的半数鸡胚感染剂量(E ID50)分别为107.2/0.2 mL、105.8/0.2 mL和106.1/0.2 mL。     

禽流感(H9亚型)油乳剂灭活疫苗的血凝抑制(HI)抗体动态变化的研究

从市场上随机购得不同厂家各两批次的商品化禽流感(H9)油乳剂灭活疫苗，按瓶签说明免疫禽流感母源抗体为阴性的蛋鸡。当疫苗抗体下降至5log2左右时部分鸡再二免，并做田间试验。结果表明，免疫1周出现血清抗体，并逐日上升，3周达高峰。首次免疫8周后抗体下降至5log2左右，二次免疫15周后抗体下降至5log2左右。这四批疫苗的免疫原性较高，安全可靠，两次免疫保护期可保护至开产后。     

利用反向遗传技术拯救H9N2重组病毒

为研制高效特异的H9N2亚型AI疫苗,选取我国具有代表性的毒株A/chicken/Shanghai/10/01(H9N2)(简称SH10),以12质粒系统为基础,利用反向遗传操作技术构建了1株表面基因由SH10提供、内部基因由12质粒系统提供的重组病毒SH/PR8,为新型疫苗的研制提供了新的毒株。     

H5、H9亚型禽流感病毒和新城疫病毒的多重RT-PCR检测

根据Genbank注册发表的H5、H9亚型禽流感病毒(AIV)的HA基因和新城疫病毒(NDV)的HA基因序列,设计多对引物,每种病毒各筛选出一对特异性好、灵敏度高的引物,其中FP1/FP2扩增H5亚型AIVHA基因片断长为545bp;P3/P4扩增H9亚型AIVHA基因片断长为321bp;P11/P22扩增NDVHA基因片断长为672bp。用RT-PCR方法,通过特异性试验、灵敏度试验,H5、H9亚型AIV和NDV引物的灵敏度分别达到了1:104、1:108、1:104稀释度,与传染性支气管炎(IB)、传染性法氏囊病(IBD)、传染性喉气管炎(ILTV)、传染性鼻炎(IC)、霉形体(MS)、H1N1猪流感等抗原无交叉反应。实验结果表明,本研究建立了检测H5、H9亚型AIV和NDV的多重RT-PCR方法,混合引物的最佳退火温度为50℃,鉴定检测仅需4小时。     

Sero-prevalence of avian influenza among broiler-breeder flocks in Jordan

Thirty blood samples were collected randomly from each of the 38 breeder-broiler farms in Jordan. Serum samples were examined using indirect ELISA for specific antibodies to avian influenza virus. The overall true flock-level sero-prevalence of avian influenza was 71% (95% CI: 55,83). Positive flocks had 2-30 sero-positive chickens and half of flocks had >20 sero-positive birds. The number of sero-positive flocks varied in the studied localities with more sero-positives in farms located within the migratory route of migratory wild fowl. The examined broiler-breeder flocks had no clinical signs, or noticeable decrease in egg production; mortalities were within the normal range (0.1-1%). The number of positive sera/flock correlated with flock size. There were a no significant (Pearsons r=0.21, p=0.21) correlation between positive flocks and age. A non-pathogenic AI virus infects broiler-breeder farms in Jordan. Wild local and migrating birds might promote the further spread of this virus in Jordan and other countries.     

抗H9N2亚型禽流感病毒单克隆抗体的制备及初步鉴定

利用纯化的H9N2亚型禽流感尿囊液病毒免疫原免疫Balb/c小鼠,应用杂交瘤技术将免疫鼠脾细胞与小鼠骨髓瘤细胞(SP2/0)融合,用间接ELISA方法检测培养上清是否对H5N1亚型AIV及NDV反应,筛选只针对H9N2亚型AIV的阳性细胞株,经克隆获得2株较高亲和力的杂交瘤细胞株,用其制备的腹水ELISA效价达到2×105。     

禽流感病毒H5、H9亚型的多重RT-PCR鉴别诊断

根据禽流感病毒H5、H9亚型的HA基因序列,设计了两对RT-PCR引物,同时对H5、H9亚型进行了多重RT-PCR扩增,得到了两条清晰的目的带.将目的带回收并进行测序,同源性比较结果证明其为禽流感H5、H9亚型的HA基因序列,作者对多重RT-PCR进行反应条件优化以及敏感性与特异性测定,证明该方法的敏感性和特异性都比较好.初步应用于30份临床病料,其检测结果与病毒的分离培养一致,比电镜和琼扩的检出率高,与其他NDV、IBV的病料无交叉反应,可用于禽流感病毒H5、H9亚型的鉴别诊断.     

H9N2亚型猪流感病毒对小鼠的致病性及其分子特性研究

本实验从河北地区疑似流感发病猪体内分离到一株病毒,经鉴定为H9N2亚型猪流感(SIV)病毒.将该分离株经滴鼻、点眼途径感染小鼠,观察临床症状和病理变化,同时对血凝素(HA)、神经氨酸酶(NA)、核蛋白(NP)和基质蛋白基因(M)进行克隆和序列测定,与GenBank中登录的相关序列进行比对并绘制系统发育进化树.致病性结果显示:感染小鼠出现精神不振,体重下降,并引起以弥漫性肺泡损伤为主的临床症状和病理变化.序列分析结果显示:该分离株与禽流感病毒(AW) A/chicken/Hebei/4/2008 (H9N2)(简称CK/HB/4/08)参考株的HA、NA、NP和M基因的核苷酸序列和推导的氨基酸序列的同源性最高.HA蛋白的裂解位点序列为PARSSR↓GLF,属于低致病性流感病毒的裂解位点.HA、NP、NA和M基因的遗传进化分析均显示该分离株与AIV的CK/HB/4/08株位于同一分支,具有较近的亲缘关系;由此推测该分离株可能是由CK/HB/4/08演化而来,并在跨物种传播的过程中发生了部分变异.     

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。     

H9亚型HP株禽流感病毒繁殖规律的探索

为了研究H9亚型HP株禽流感病毒接种非免疫鸡胚后病毒的繁殖规律,试验用H9亚型HP株禽流感病毒接种非免鸡胚,记录不同时间段死亡的鸡胚数量,并分别收获各时间段死亡鸡胚的尿囊液,测定不同时间段尿囊液的病毒效价。结果表明,接种H9亚型HP株禽流感病毒后,非免鸡胚死亡高峰期出现在60～84 h,死亡数量占接种鸡胚数的80%以上,而该时间段死亡鸡胚尿囊液的病毒滴度也处于最高峰,最高到达10^9.63EID₅₀/mL,直到96 h病毒仍维持较高水平(10^9.50EID₅₀/m L),而96 h后病毒滴度开始衰减。     

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…     

一株H9亚型禽流感病毒的分离鉴定及免疫试验

为了解养禽业禽病流行病毒株的基本情况,本研究对2014年从山东某鸡场采集的疑似流感的病料样品采用鸡胚尿囊腔接种进行病毒的分离,并经血凝试验(HA试验)、血凝抑制试验(HI试验)及分子生物学试验对其病原学进行鉴定。结果显示,分离鉴定到一株病毒分离株。HA试验结果表明该分离株具有血凝活性,经病毒亚型鉴定为一株H9亚型禽流感病毒(AIV),该病毒株为欧亚分支的BJ亚分支,将该病毒株筛选纯化后作为毒种,制备了禽流感油乳剂灭活疫苗,将该疫苗免疫SPF鸡,21 d后对其HI抗体效价进行检测,结果显示其平均效价达到9.1 log2,免疫后第35 d HI抗体平均效价达到峰值10.3 log2,至免疫后第10周,HI抗体平均效价仍达8.5 log2。本研究分离的病毒制备成疫苗后具有较好的免疫效果,可以作为疫苗候选株。