冠状病毒受体的研究进展

近年来多种感染人或家畜的冠状病毒病轮番出现,给人类社会的经济和公共卫生安全带来很大危害.特别是在2019年底出现并在全球迅速蔓延的SARS-CoV-2,由此引发的新型冠状病毒肺炎(COVID-19)在2019年12月至2020年3月之间在全球范围内造成超过7万人死亡.冠状病毒感染宿主的第一步是识别宿主细胞膜受体分子并与...     

犬瘟热病毒YD株的分离鉴定及其H基因序列分析

从疑似犬瘟热(CD)病犬的脏器中分离到1株病毒,经间接免疫荧光试验、RT-PCR鉴定、红细胞凝集试验和对不同动物致病性试验,证实该病毒为犬瘟热病毒(CDV)强毒株,命名为CDV YD株.对H基因序列的测定和分析表明,YD株H基因与国内强毒株更接近,核苷酸同源性为98.4％～99.7％,氨基酸同源性为97.8％～99.7...     

一株鸭源禽流感病毒（H9N2）全基因序列分析及其排毒规律的研究

为研究一株鸭源H9N2亚型禽流感病毒(JN1株)全基因组的分子特征和遗传进化情况以及感染雏鸭后的排毒规律,对其生物学特性、全基因组序列以及遗传进化进行了分析,通过点眼滴鼻方式感染3周龄健康雏鸭,利用建立的实时荧光定量PCR(RT-PCR)检测鸭的排毒规律,并测定血清抗体效价。JNI株的鸡胚半数感染量为10~(-7.54)/0.2mL,鸡胚最小致死量的平均死亡时间为72h,静脉致病指数为0.266,抗原相关系数为0.47。系统发育分析表明,JN1株的HA与CK/HK/G9/97和DK/HK/Y280/97在同一分支上,NA、M、NP、PA、PB1、PB2基因与SH/F/98同源性较高,NS基因则与H5N1亚型禽流感病毒进化关系较近。HA的裂解位点为PSRSSR↓GL,存在6个潜在糖基化位点,在234位的氨基酸残基为L,NA基因颈部存在缺失。雏鸭在感染该病毒后,饮食正常,精神良好,3~17d均有排毒,咽拭子和泄殖腔棉拭子排毒规律基本一致,分别在第3天和第13天出现排毒高峰。该H9N2亚型禽流感病毒属欧亚大陆分支,为低致病性禽流感,病毒可通过呼吸道和泄殖腔向外界排毒,排毒时间较长。     

IBV AH1-99分离株N基因的克隆及序列分析

利用RT-PCR技术成功扩增出IBV AH1-99分离株的N基因全长cNDA,将其克隆到pMD18-T载体上。经酶切分析、PCR鉴定及核苷酸序列测定,成功获得该分离株N基因的重组质粒。序列分析结果表明,分离株N基因全长1 230个核苷酸,编码409个氨基酸。同部分IBV参考毒株相比,核苷酸同源性为85.8%~89.9%,氨基酸同源性为86.6%~91.0%,在进化关系树中,AH1-99与国内分离株X、LX4亲缘关系较近。     

福建省规模猪场伪狂犬病流行病学调查与分析

作者分别应用血清中和试验（SNT）和PCR方法,对福建省近3年来56个规模猪场收集的1470份血清和71个规模猪场采集的136份病料进行了猪伪狂犬病的血清学调查和病原检测。结果表明：在2005年、2006年和2007年,猪伪狂犬病病毒中和抗体合格率：母猪分别为91.5％、89.2%和94.8%;6周龄前猪群分别为75.3%、56.6%和75.5%;6周龄后猪群分别为38%、33.9%和30.7%;被检病料中猪伪狂犬病病毒野毒阳性检出率分别为12.5%、17.4%和16.0%。由此可见,猪伪狂犬病在福建省规模猪场仍未得到完全控制,应加强防控。     

鸭肠炎病毒NP蛋白的原核表达及间接ELISA方法的建立

设计一对特异性引物扩增出鸭肠炎病毒(DEV)核衣壳蛋白(NP)基因,并将其定向插入到原核表达载体pET32a上,构建了NP基因的原核表达载体pET-NP;将重组载体pET-NP转化表达宿主菌BL21后,经SDS-PAGE分离后行Western blot显示,获得的表达产物具有良好的免疫原性;应用His.Bind亲和层析柱纯化重组NP蛋白,并以此作为包被抗原,初步建立了检测鸭肠炎病毒抗体的iNP-ELISA;经方阵滴定确定,重组蛋白抗原的最佳包被浓度为5.0μg/L,血清最佳稀释度为1∶80,阳性判定标准为:待检血清OD405值≥1.2,且待检血清OD405和阴性血清OD405的比值≥2.0;应用iNP-ELISA对450份鸭血清样本进行检测,结果iNP-ELISA与全病毒包被的iDEV-ELISA符合率达90.9%。     

新城疫病毒贵州不同鸡源分离株F基因的克隆与序列分析

应用PCR技术扩增NDV贵州不同鸡源分离株,包括肉鸡源P1株与BY株、蛋鸡源H2株与FW株、七彩山鸡源N98株和越南斗鸡源DQ株的F蛋白基因,将该基因片段分别进行克隆和测序,并与国内外NDV参考株的对应序列进行比较分析。结果表明:6株NDV贵州不同鸡源分离株的F基因长度均为1 662 bp,编码553个氨基酸;分离株间核苷酸同源性为86.9%~99.7%,氨基酸同源性为91.0%~99.3%;与国内外NDV代表株(LaSota株、B1株、F48E9株、CH2000株和TW 2000株)的核苷酸同源性为84.0%~98.9%,氨基酸同源性为87.2%~98.6%;经系统发育进化树分析,DQ株、N98株、P1株和H2株为基因VIId型,而BY株和FW株为基因IX型。这些结果提示贵州省不同鸡群间存在相同NDV毒株感染的可能性,不同年份间NDV毒株发生基因型改变,而近年来贵州省流行的ND疫情主要是由NDV基因VII型引起。     

Molecular characterization and phylogenetic analysis of new Newcastle disease virus isolates from the mainland of China

Seventy-nine velogenic Newcastle disease virus (NDV) isolates were obtained from infected chicken flocks during the outbreaks of Newcastle disease (ND) in various regions of the mainland of China in 2006. The F gene fragment (535 bp, from nt 47 to 581 of the F gene) which codes the main functional region of the F protein was obtained by RT-PCR and sequenced. All sequences obtained in this study have been submitted to GenBank. All the isolates have the motif ¹¹²R-R-Q/R-K/R-R-F¹¹⁷ at the cleavage site of the fusion protein, which is typical of velogenic NDV isolates. For genotyping, a phylogenetic tree based on nucleotides 47–435 of the F gene was constructed, and the 79 isolates could be divided into two genotypes, namely VIId and III. Most of the isolates proved to be of genotype VIId; only two isolates were of genotype III. Genotype VIId NDV has been the predominant pathogen responsible for most Newcastle disease outbreaks in China. The proportion of isolates of genotype VIId NDV shows an increasing trend, according to studies on the molecular epidemiology of NDV in China from 2002 to 2006.     

Outbreak of acute bovine viral diarrhea in Brazilian beef cattle: clinicopathological findings and molecular characterization of a wild-type BVDV strain subtype 1b

When first described in 1946, bovine viral diarrhea (BVD) was characterized as an acute transmissible disease associated with severe leucopenia, high fever, depression, diarrhea, gastrointestinal erosions, and hemorrhages. Recently the severe acute form has been related only to some hypervirulent BVDV-2 strains. This article reports the detection of BVDV-1b associated with an acute and fatal outbreak of BVD in a Brazilian beef cattle herd. Depression, anorexia, watery diarrhea, sialorrhea, and weakness were observed in six steers. One of these animals was evaluated for laboratorial, clinical, and pathological alterations. Laboratory findings were non-specific; clinically, the animal was weak, with dehydration and erosive oral lesions. Pathological alterations were predominant at the tongue, esophagus, and rumen. A RT-PCR assay using primers to partially amplify the 5′ untranslated region (5′UTR) of the BVDV genome was performed and identified BVDV in all clinical samples analyzed. Phylogenetic analysis of BVDV derived from lymph node revealed that this strain was clustered within the BVDV subtype 1b. This differentiating was only possible to be performed by molecular characterization since both clinical presentation and pathologic findings were similar to BVDV-2 infection.     

用地高辛标记的核酸探针检测猪伪狂犬病毒野毒感染的研究

利用PCR技术从带有伪狂犬病毒（PRV）gE基因的重组质粒pMD18-T-gE中扩增回收约304bp大小的片段,并制备出地高辛标记的gE基因核酸探针。特异性检测结果表明,该探针能与重组质粒DNA发生特异性杂交,而与对照的PRVBartha-k61株疫苗毒DNA、猪细小病毒（PPV）DNA、猪圆环病毒（PCV）DNA、猪繁殖与呼吸综合征病毒（PRRsV）cDNA、猪瘟病毒（CSFV）cDNA的杂交反应均为阴性;敏感性检测结果表明,该探针对PRV野毒的最低检出量为4pg。应用该探针对11份繁殖障碍病料进行了杂交检测,共检出4份阳性病料,该结果与PCR检测结果一致,表明该核酸探针可用于猪伪狂犬病野毒感染的临床诊断。