新疆南疆某规模牛场轮状病毒基因分型鉴定与分析

新疆南疆某规模奶牛场部分犊牛出生后1周左右出现严重腹泻,严重地影响了犊牛健康。为查明该牛场犊牛腹泻是否存在牛轮状病毒感染以及病毒基因类型,以及更好地防治犊牛腹泻发生。试验采集了部分腹泻犊牛的粪便样本,提取粪样病毒核酸、设计病毒特异性扩增引物,采用RT-PCR法、基因测序、核苷酸同源性分析及构建遗传进化树。结果显示,待检样本通过VP7特异性引物(VP7-1/VP7/2)和基因分型鉴定引物(VP7-1/G10-2)的RT-PCR扩增得到大小1 062 bp、715 bp目的片段,经测序及核苷酸同源性比对分析,确定该样本为轮状病毒A群G10型,结合临床诊断、病理变化确定该牛场疑似患牛存在牛轮状病毒感染,为该病防控提供了重要的理论依据。     

我国部分地区牛轮状病毒的病原学调查及一株G10P[11]型牛轮状病毒的分离鉴定

应用轮状病毒RNA电泳方法,对内蒙古、黑龙江、新疆、北京、安徽等地共90份犊牛腹泻粪便样品进行检测.在内蒙古和新疆采集的粪样中共检出11份轮状病毒阳性样品,平均阳性率为12%(11/90).阳性样品经RT-PCR分型鉴定,新疆2份和内蒙古5份样品属于G10型,新疆另一牛场的4份样品属于G6型.对所有阳性样品进行病毒分离,获得1株能在MA104细胞上稳定传代的病毒株,经鉴定该分离株属于G10P[11]型轮状病毒,命名为HM26.G10型牛轮状病毒的分离在国内尚属首次.     

我国部分地区牛轮状病毒的病原学调查及一株G1OP[11]型牛轮状病毒的分离鉴定

应用轮状病毒RNA电泳方法，对内蒙古、黑龙江、新疆、北京、安徽等地共90份犊牛腹泻粪便样品进行检测。在内蒙古和新疆采集的粪样中共检出11份轮状病毒阳性样品，平均阳性率为12％（11／90）。阳性样品经RT-PCR分型鉴定，新疆2份和内蒙古5份样品属于G10型，新疆另一牛场的4份样品属于G6型。对所有阳性样品进行病毒分离，获得1株能在MA104细胞上稳定传代的病毒株，经鉴定该分离株属于G10P[11]型轮状病毒，命名为HM26。G10型牛轮状病毒的分离在国内尚属首次。     

仔猪感染猪轮状病毒的鉴定及基因分析

为弄清2013年年初上海某猪场发生的仔猪腹泻疫情病因,本研究随机采集发病猪场的仔猪腹泻样品9份,利用RT-PCR方法对采集样品进行病原检测。结果显示,9份临床样品中有4份为猪轮状病毒阳性,而猪流行性腹泻病毒和猪传染性胃肠炎病毒均为阴性。根据GenBank公布的猪轮状病毒参考株序列设计两对引物,对其中阳性样品的VP6和VP7基因进行RT-PCR扩增、基因克隆和序列测定分析。结果显示,4份阳性样品的VP6基因大小为1356 bp,VP7基因大小为1100 bp;序列分析结果显示,VP6基因与A群代表株OSU的氨基酸同源性为64.1%~87.0%,与代表株Gottfriend的氨基酸同源性为26.3%~60.8%;VP7基因与不同G型代表株的氨基酸同源性为71.8%~97.3%。根据上述结果分析,我们推测引起此次仔猪腹泻疫情中的主要病原是由属于A群G9血清型的猪轮状病毒引起,本研究为此次仔猪腹泻疫情的诊断提供了依据。     

新疆部分地区致犊牛腹泻轮状病毒的检测及VP6基因序列分析

为了调查新疆部分地区规模化奶牛场致犊牛腹泻轮状病毒的感染情况,查明该病毒在致犊牛腹泻中的作用,试验采用双抗体夹心ELISA、RT-PCR的方法检测轮状病毒抗原及VP6基因,并对该基因进行序列分析以比较其同源性。结果表明:各牛场腹泻犊牛粪便的轮状病毒阳性检出率为23.08%~90.91%;在健康犊牛粪便中,仅从石河子某奶牛场检出轮状病毒,检出率为38.89%;RTPCR扩增得到大小为383 bp的片段;8份克隆毒株序列的同源性为97.9%,同源性较高。说明新疆部分地区犊牛感染轮状病毒十分普遍,轮状病毒是引起犊牛腹泻的主要病原之一,部分健康犊牛带毒但未发病。     

一株G6P[5]型牛轮状病毒的分离鉴定与全基因组序列分析

为了对患腹泻疾病的犊牛进行病原学鉴定,并进一步丰富我国牛轮状病毒(BRV)的流行病学数据,本研究采用RT-PCR方法对8份腹泻犊牛的粪便样品进行BRV VP7基因检测,结果显示有1份样品为阳性。将该阳性病料样品接种Marc-145细胞。对产生细胞病变(CPE)的阳性样品连续传5代后,经间接免疫荧光试验(IFA)和电镜观察,结果表明从粪便样品中分离的病毒为BRV,命名为DZ株。对DZ株的11个基因节段进行RTPCR扩增、测序、拼接后对11个基因节段进行同源性及分型分析;构建分离病毒VP7、VP4基因进化树,分析其遗传进化关系及其氨基酸序列的变异情况。结果显示,DZ株基因组10个基因节段分别与来源于牛(VP2、VP7、NSP1、NSP3)、犬(VP3、NSP5)、羔羊(NSP2)、马(VP6)、猕猴(VP1)、人(NSP4)以及人-牛基因重配(VP4)的轮状病毒株。其中VP1基因与猕猴轮状病毒(RRV)的同源性最高,为81.3%,但低于VP1基因分型的临界值83%,因此DZ株的VP1为新出现的基因型,命名为R17;分离株NSP3基因与法国RF株NSP3基因的同源性高达98%,DZ株与RF株的NSP3属于同一基因型,命名为T18型。所以,本研究分离的BRV DZ株的基因型为G6-P5-I2-R17-C2-M2-A3-N2-T18-E2-H5。VP7和VP4基因遗传进化分析结果显示,DZ株VP7基因来源于韩国KJ69-1株,VP4基因来源于美国疫苗株RotaTeq-SC2-9。与同源性最高的病毒株相比,DZ株VP4、VP7蛋白氨基酸序列分别发生了9处和8处突变。这些突变可能会引起VP7和VP4蛋白的抗原性和组织嗜性变化,进而影响病毒的免疫原性、宿主嗜性以及致病性。综上所述,DZ株是多宿主来源的基因重配病毒株,且主要抗原蛋白VP7和VP4发生了较大变异。本研究为我国BRV遗传进化及其分子流行病学和疫苗的研究奠定了实验基础。     

中国不同地区猪轮状病毒的分离鉴定及分子流行病学分析

为鉴定国内不同地区猪轮状病毒(Porcine rotavirus,PRo V)并分析其分子流行病学特征,本研究将从我国不同地区收集病料中检测到的16份PRoV阳性仔猪粪便样品和小肠内容物处理后接种MA-104细胞,传代后进行荧光定量PCR (qPCR)和间接免疫荧光试验(IFA)鉴定,并经电镜观察。结果显示,其中7份样品F1～F3代病毒液qPCR Ct值均随着传代次数的升高而逐渐降低,IFA鉴定结果显示均能在细胞中观察到特异性荧光,电镜观察到直径约70 nm无囊膜的病毒粒子,表明本实验分离到7株PRoV。利用RT-PCR扩增7株分离株和其余9份PRoV的阳性样品的VP7基因并测序分析基因型,基于VP7基因进行同源性和遗传进化分析。结果显示,16份阳性样品包含3种G型,即G9(7/16)、G4 (6/16)和G3 (3/16),同时G9型FJ-2021分离株和G4型JS01-2021分离株与近两年流行株亲缘关系较近。统计16份样品中G型PRoV的地域分布,结果显示华东地区有G9、G4和G3型的流行,四川有G3和G9型流行,广东有G9型的流行,分别与华东地区、四川地区和广西地区报道的流行G...     

一株猪轮状病毒的分离与鉴定

将1份经RT-PCR检测猪轮状病毒阳性的临床腹泻粪样感染Vero细胞,连续盲传8代,出现病变,成功分离到1株病毒,经RT-PCR检验和电镜观察,鉴定该病毒为轮状病毒,命名为GD-01-2015。扩增该病毒的VP4和VP7基因进行分型和系统进化分析,结果发现,其VP4基因为P[7]型,与来自韩国的猪源毒株K71同源性达到99.8%;其VP7基因为G5型,与来自韩国的猪源毒株06-6-1同源性达到99.7%。由此可以推测GD-01-2015株与韩国猪源毒株有相似的进化来源。根据轮状病毒分类委员会(RCWG)提出的A群轮状病毒的最新分类方法,GD-01-2015株基因型为G5P[7]。本研究为监测轮状病毒的流行状况及其疫苗的研制提供了理论依据。     

甘孜州牦牛源A群轮状病毒的分离和鉴定

本实验的目的是分离甘孜州牦牛源A群轮状病毒(Bovine Rotavirus A,BRVA)并调查其基因型。将BRVA阳性牦牛粪便样本接种于MA-104细胞,用间接免疫荧光和RT-PCR进行病毒的鉴定,并扩增分离毒株VP4和VP7基因片段,测序确定其基因型。结果显示:6份样本盲传至第3代后均出现了细胞病变,连续传至7代后细胞病变稳定,表现为细胞圆缩,细胞大面积脱落;间接免疫荧光和RT-PCR鉴定结果显示成功分离到6株牦牛源BRVA。6株病毒的G型均为G6型,其中3个毒株的P型为P [1]型,另外3株未能确定P型。本研究为甘孜州牦牛BRVA的分子流行病学调查及其防控提供了参考。     

2021—2022年我国部分地区猪轮状病毒分子流行病学调查

为了解我国部分地区猪场猪轮状病毒(porcine rotavirus, PoRV)的流行情况及分子特征,对2021—2022年我国部分省份的86个规模化猪场的6 472份腹泻样品进行检测,用RT-PCR方法调查PoRV的流行率,并对PoRV阳性样本进行VP7和VP4基因扩增、测序,使用MegAlign软件进行同源性分析,利用MEGA11.0构建系统进化树。结果显示,PoRV的样品阳性率为27.89%(1 805/6 472),猪场阳性率为65.12%(56/86);从98份阳性PoRV样本中扩增出76个VP7片段,G9为优势基因型(42.11%),基因型G5、G26、G3、G4、G1、G2和G11各占26.32%、9.21%、6.58%、6.58%、3.95%、2.63%和2.63%。扩增出的35个VP4基因片段,以P[13]型为主,占57.14%;其次为P[7]、P[23]、P[3]和P[6]型,分别占20%、14.29%、5.71%和2.86%。35个毒株成功鉴定出G/P基因型,G9P[13](28.57%)为优势组合基因型,其他基因型为G5P[13](11.43%)、G4P[13...     

规模化奶牛场犊牛腹泻的病原检测与分析报告

为了调查某规模化奶牛场犊牛腹泻的发病原因,试验采用血清抗体检测、细菌分离培养、寄生虫卵镜检和病毒核酸检测方法对腹泻犊牛的血样和粪便进行了检查。结果表明,该奶牛场牛病毒性腹泻/黏膜病病毒抗体阳性率为65.00%,牛轮状病毒抗体阳性率25.00%,牛冠状病毒抗体阳性率15.00%;分离到1 株结肠弯曲杆菌和1 株空肠弯曲杆菌;所检腹泻犊牛粪便和血液样品中,4 份粪便样品和1 份血液样品检出牛冠状病毒,2 份粪便样品检出轮状病毒,1 份粪便样品和1 份血液样品检出牛病毒性腹泻/黏膜病病毒,同一份粪便样品中同时检出牛病毒性腹泻/黏膜病病毒和牛冠状病毒;通过镜检,球虫卵囊检出率77.78%,在1 份粪样中发现蛔虫卵,2 份粪样中发现其他线虫卵。该牛场流行性腹泻的原因可能为夏季高温潮湿引起犊牛免疫力下降导致的多病原体感染。     

一例冠状病毒、轮状病毒混合感染引起犊牛腹泻的诊治

2022年3月,河南省某规模化奶牛养殖场发生一起新生犊牛腹泻病例,在流行病学调查、临床症状观察、病理剖检的基础上,采集6头腹泻犊牛的新鲜粪便进行了寄生虫卵检查、小球隐孢子虫、牛轮状病毒、冠状病毒、大肠杆菌K99抗体检测,血液样品进行了牛病毒性腹泻病毒抗原检测,组织样品进行了细菌分离。结果表明牛轮状病毒抗原6头阳性,阳性率100%;冠状病毒抗原4头阳性、2头阴性,阳性率66.7%;其他病原均为阴性。根据临床症状、病理变化和实验室检测结果,确诊该病例为牛冠状病毒和轮状病毒混合感染引起的犊牛腹泻。根据诊断结果,采取了改善饲养管理、补液、收敛、止泻等综合性防治措施,疫情得到了有效的控制,为临床防治犊牛腹泻提供了参考。     

Detection of group a rotavirus strains circulating in calves in Tunisia

Faecal samples were collected from 89 dairy calves to determine the prevalence of rotavirus infection in Tunisia and the genomic diversity of bovine rotavirus strains. After screening of all faecal samples by enzyme-linked immunosorbent assay, rotavirus strains were analysed by RNA polyacrylamide gel electrophoresis and characterized antigenically by monoclonal antibodies to the VP6 subgroup. The VP7 genotype was determined by nested RT-PCR. Of the 89 calves tested, 27 (30%) were positive for rotavirus antigen. Four different long electrophoretypes were identified. All VP6 typeable strains carried the subgroup I specificity. G8 genotype was the most prevalent, but G6 and mixed strains G(6 + 8) were also detected.     

Detection and differentiation of bovine group A rotavirus serotypes using polymerase chain reaction-generated probes to the VP7 gene.

Dot and Northern blot hybridization assays were developed to detect and differentiate group A bovine rotavirus serotypes using radiolabeled serotype 6 (Nebraska calf diarrhea virus [NCDV] and United Kingdom [UK] strains) or serotype 10 (Crocker [Cr] strain) VP7 gene probes. Partial length VP7-specific cDNA encompassing areas of major sequence diversity were generated by the polymerase chain reaction (PCR) using either cloned VP7 genes (NCDV and UK strains) or reverse transcribed mRNA (Cr strain) as templates. Radiolabeled probes prepared from the PCR-generated cDNA were tested at various stringency conditions to optimize the hybridization assays. At high stringency conditions (52 C, 50% formamide, 5 x standard saline citrate), the NCDV, UK, and Cr probes serotypically differentiated bovine rotavirus isolates in RNA samples prepared from cell culture propagated viruses or in fecal specimens from infected gnotobiotic calves. The sensitivity and specificity of NCDV and Cr VP7 probes were characterized in dot blot hybridization assays, and the probes were estimated to detect at least 1 ng of viral RNA. The serotyping results obtained using VP7 probes were similar to those obtained using serologic assays. Further development of these assays may provide a useful means for the rapid detection and differentiation of bovine rotavirus serotypes in fecal samples from calves in the field.     

Bovine rotavirus in turkeys with enteritis

Rotaviruses are the main agents responsible for diarrhea in different animal species and for infantile gastroenteritis. These viruses have been isolated from various avian species and have often been associated with poult enteritis and mortality syndrome. Nevertheless, the knowledge of rotavirus infection in turkeys is scarce. Six group A rotavirus strains obtained from pooled enteric contents of diarrheic turkeys were isolated in MA-104 cell culture and typed as G(6)P(1), a typical bovine rotavirus genotype. Additionally, the electropherotypes showed a migration pattern identical to the Nebraska calf diarrhea virus, and the complete NSP4 gene phylogeny showed that all six strains segregated in the genotype E2. Taken together, these results point toward a cattle-to-turkey rotavirus transmission. As a conclusion, bovine-origin rotavirus can be found in turkeys, and this transmission route must now be considered for the improvement of the health status in turkey farms.     

犬轮状病毒荧光定量RT—PCR检测方法的建立

目的:建立轮状病毒快速准确的检测方法,为研发诊断试剂盒和疫苗奠定基础。方法:用Primer5软件设计VP7引物,从采集的腹泻犬粪便样品抽提病毒RNA,用反转录聚合酶链式反应(RT-PCR)技术,将RNA反转录为cDNA,并进行PCR扩增,对扩增产物进行非变性聚丙烯酰胺凝胶电泳和测序,与代表株的VP7进行同源性比较。结果:从腹泻病犬粪便样品中抽提到了轮状病毒RNA,反转录后扩增产物为51bp的基因片段,经核苷酸序列分析,其PCR序列与犬轮状病毒VP7基因编码源序列的同源性为86.3%。结论:正确克隆了轮状病毒主要保护性抗VP7基因中抗原表位区,建立了犬轮状病毒实时定量诊断方法,为研制实时定量诊断试剂盒奠定了基础。     

Determination of bovine rotavirus G serotype by polymerase chain reaction

A total of 113 diarrheic samples comprising of 68 buffalo calves and 45 cow calves were screened by RNA-PAGE for the detection of presence of rotavirus. RNA-PAGE analysis of these samples revealed 11 (9.73%) was found positive for rotavirus. Out of 68 faecal samples of buffalo calves tested for viral gastroenteritis, 8 (11.76%) were found positive for rotavirus. Similarly, out of 45 faecal samples of cattle calves tested for viral gastroenteritis, 3 (6.66%) was found positive for rotavirus. Rotavirus-positive samples represented long electropherotype. All RNA-PAGE-positive faecal samples for rotavirus subjected to RT-PCR for VP7 gene, ten samples yielded a specific product of 1,013 bp of VP7 gene. All the PCR-positive samples of the present study were subjected to genotyping with primers for G6, G8 and G10 genotype. All positive samples showed G10 genotype. This indicates that G10 may be predominant genotype among bovine calves in Mumbai region in India.     

猪A群轮状病毒的分离与鉴定

目前国内对猪轮状病毒的研究较少,作者旨在对猪A群轮状病毒进行分离与鉴定,为后续猪轮状病毒致病机理和分子生物学特性研究奠定基础.用胰蛋白酶处理RT-PCR检测猪A群轮状病毒病原阳性的临床腹泻粪样,然后接种长成单层的MA-104细胞,进行病毒分离传代.再对分离毒株进行常规RT-PCR鉴定和电镜观察,并对分离毒株的VP6、VP7和VP8基因进行测序及序列分析.结果成功分离猪A群轮状病毒TM-a株,经序列同源性分析发现,与TM-a株VP6、VP7和VP8基因同源性最高的毒株分别为中国北京人源LL3354株、印度人源RMC321株和日本野猪源GUB-71株,其相似性为96.0％、95.1％和97.0％.该TM-a株轮状病毒不同基因表现出与人源轮状病毒和猪源轮状病毒的高度同源性,推测猪A群轮状病毒可能在人畜间传播,发生基因重组现象.     

Longitudinal study of bovine rotavirus group A in newborn calves from vaccinated and unvaccinated dairy herds

Reports of rotavirus excretion in calves usually result from cross-sectional studies, and in face of the conflicting results regarding protection of calves born to vaccinated dams against diarrhea, the aim of the present study was to evaluate rotavirus excretion in dairy calves born to vaccinated or unvaccinated dams, to identify the genotypes of bovine rotavirus group A (RVA) strains isolated from these animals as well as to investigate characteristics of the disease in naturally occurring circumstances throughout the first month of life. Five hundred fifty-two fecal samples were taken from 56 calves, 28 from each farm and, in the vaccinated herd, 11/281 samples (3.91%) taken from six different calves tested positive for RVA while in the unvaccinated herd, 3/271 samples (1.11%) taken from 3 different calves tested positive. The genotyping of the VP7 genes showed 91.2% nucleotide sequence identity to G6 genotype (NCDV strain), and for the VP4 gene, strains from the vaccinated herd were 96.6% related to B223 strain, while strains from the unvaccinated herd were 88% related to P[5] genotype (UK strain). Genotypes found in this study were G6P[11] in the vaccinated herd and G6P[5] in the unvaccinated herd. All calves infected with rotavirus presented an episode of diarrhea in the first month of life, and the discrepancy between the genotypes found in the commercial vaccine (G6P[1] and G10P[11]) and the rotavirus strains circulating in both vaccinated and unvaccinated herds show the importance of keeping constant surveillance in order to avoid potential causes of vaccination failure.