1株猪源牛病毒性腹泻病毒的分离与鉴定

从BVDV阳性仔猪病料中分离病毒,为开展猪源BVDV病原学研究奠定基础。将处理后BVDV阳性仔猪组织样品接种MDBK细胞,分离到1株猪源BVDV,命名为SD0803株。通过细胞培养、直接免疫荧光、5′-UTR与Npro PCR扩增、电镜观察、TCID50测定及对其分子进化特征加以分析。结果表明,该毒株在MDBK细胞上盲传至13代未出现细胞病变。在直接免疫荧光试验中呈阳性荧光信号。PCR扩增分别获得5′-UTR与Npro预期大小DNA片段。电镜观察,病毒粒子略呈圆形,有囊膜,直径约50nm。病毒滴度为10-6.5 TCID50.0.2 mL-1。SD0803 5′-UTR、Npro序列进化分析显示,该分离株属于BVDV-1,与已知BVDV-1各亚型之间同源性较低,单独成一分支。结果表明,成功分离鉴定1株猪源BVDV SD0803,该毒株为非致病变型BVDV-1,极有可能为BVDV-1新的亚型。     

牛病毒性腹泻病毒新疆分离株E2基因的克隆及序列分析

应用RT-PCR方法从牛病毒性腹泻病毒（BVDV）新疆石河子分离株、玛纳斯不同年份两个分离株扩增获得的3株BVDV的E2基因序列,分别命名Shihezi148、Manasi和MNS2（GenBank登录号：EU159699、EU159703和EU169937）。序列分析结果表明3株E2基因长度分别为1121bp、1122bp和1122bp,分别编码373、374和374个氨基酸残基。核酸序列同源性和系统发生分析表明,3株病毒均属于BVDV基因1型（BVDV-1）,Shihezi 148株与澳大利亚Bega株亲缘关系最近,同处于BVDV-1c基因亚型群,其E2氨基酸同源性为90.11%。Manasi株和MNS2株E2基因核酸序列仅有4个碱基的差别,它们与Changchun 184和匈牙利VEDEVAC株亲缘关系最近,位于BVDV-1b基因亚型群。Manasi和MNS2株与Changchun 184株E2氨基酸同源性分别高达98.66%和98.93%。     

甘肃、青海和宁夏地区牛病毒性腹泻病毒Erns基因的变异分析

分析2013—2019年中国西北部分省区不同基因亚型牛病毒性腹泻病毒（BVDV）抗原基因E^rns的分子特征,了解其遗传演化规律。从甘肃、青海、宁夏规模化牛场送检的疑似牛病毒性腹泻发病牛150份EDTA抗凝血提取总RNA,利用RT-PCR扩增病毒基因组E^rns-E1区,克隆测序后比对,构建系统进化树进行遗传演化关系分析。利用牛肾细胞MDBK对检出的不同基因亚型BVDV进行分离,并鉴定其生物型。RT-PCR扩增结果表明,BVDV总体阳性率为37.33%,其中甘肃省、青海省、宁夏回族自治区BVDV阳性率分别为37.68%、35.71%、40.00%。获得56份E^rns-E1 DNA,克隆测序获得33条不同的E^rns序列,长度均为681 bp,分析表明流行株分属10个BVDV基因亚型：BVDV-1a （2株）、BVDV-1b （5株）、BVDV-1c （1株）、BVDV-1d （3株）、BVDV-1m （11株）、BVDV-1o （1株）、BVDV-1p （4株）、BVDV-1q （4株）、BVDV-1v （1株）、BVDV-2a （1株）。分离获得BVDV-1a亚型、BVDV-1b亚型、BVDV-1v亚型、BVDV-2a亚型分离株各1株,BVDV-1 d亚型分离株2株,均为非致细胞病变型。各亚型株间E^rns基因核苷酸相似性以BVDV-1a～1d经典亚型株（79.8%～85.9%）或1m～1q及1v新亚型株（81.0%～87.3%）较高,以BVDV-1 m和BVDV-1p流行株亚型间相似性最高（87.3%）。各亚型株E^rns基因编码蛋白的RNA酶活性位点以及双链RNA作用基序（₁₃₉KKGK₁₄₂）保守,但E^rns第26位糖基化位点（26 NRSL）在1m～1q、1v亚型株移位（24 NVSR）。首次以E^rns核苷酸序列构建系统进化树,结果显示1m～1q及1v等亚型BVDV株在进化上关系较为密切。本研究首次选用E^rns靶标基因对甘肃、青海、宁夏部分省区牛源BVDV株进行同源性及系统进化分析,发现10个基因亚型流行株,以1m亚型株最为普遍,1m～1q及1v等亚型株亲缘关系密切。     

川藏地区牦牛牛病毒性腹泻病毒分子流行病学调查及分离鉴定

为了解川藏高原地区牦牛牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)的感染情况和牦牛BVDV的主要流行亚型及遗传变异情况,采用RT-PCR方法,对四川和西藏部分高原地区采集的149份临床腹泻牦牛粪便开展分子流行病学调查,根据5′-UTR、Npro和E2基因的变异研究该地区牦牛BVDV的遗传变异情况。同时,通过RT-PCR、病毒分离培养和间接免疫荧光检测等方法鉴定牦牛BVDV分离株。结果表明,149份粪便中,BVDV阳性检出率为19.46%[95%置信区间(CI)=13.4%~26.7%],其中西藏地区牦牛BVDV阳性检出率为27.14%(95%CI=17.2%~39.1%),四川地区牦牛BVDV阳性检出率为12.66%(95%CI=6.2%~22.0%)。随机抽取10份阳性样本测序,根据5′-UTR、Npro和E2基因序列建立系统发育进化树分析,10份样本为BVDV-1型,包括BVDV-1a(n=4)和1d(n=6)亚型。此外,成功分离鉴定出两株致细胞病变型(cytopathic,cp)BVDV,分别属于牦牛BVDV-1a和1d亚型,命名为SWU-Z10和SWU-L8。研究结果提示,川藏部分高原地区牦牛存在BVDV感染,BVDV-1a和1d为牦牛感染的主要亚型。本研究丰富了BVDV的分子流行病学资料,也为后续研制牦牛专用BVDV疫苗提供理论基础。     

猪源牛病毒性腹泻病毒的分离与鉴定

为了对临床疑似牛病毒性腹泻病毒(BVDV)感染病料进行确诊,并掌握致病原的生物学和遗传变异特征,试验通过临床样品PCR检测、病毒分离培养与细胞病变特征观察、理化特性试验、5′UTR基因序列分析等方法对临床病料进行了BVDV的分离和鉴定。结果表明:临床病料BVDV特异性引物PCR扩增为阳性,可引起单层MDBK细胞产生细胞病变,分离毒株的TCID_(50)高达1×10~(-5.23)/0.1 mL,其对氯仿、乙醚、胰酶、酸、热均敏感,在基于5′UTR基因的遗传进化树中其位于BVDV-1型的分支中,归属于BVDV-1a型,与登录号为JN400273.1的猪源BVDV-1a型毒株的亲缘关系最近,同源性达100%。说明试验获得了1株猪源BVDV分离毒株,属于BVDV-1a型,并掌握了该毒株的生物学特性和遗传特征。     

牦牛病毒性腹泻病毒全基因测序及生物信息学分析

从牦牛(Yak)体内分离出牛病毒性腹泻/黏膜病病毒(BVDV/MDV),参考GenBank中已收录的BVDV-Ⅰ型的全基因组序列,设计了19对引物,通过RT-PCR方法,对牛病毒性腹泻病毒牦牛株进行克隆及测序,得到其全基因组序列(GenBank登录号:JQ799141),序列全长12214 nt,其中5'-UTR长288 nt,3'-UTR长232 nt。将牦牛株BVDV全基因序列与GenBank中登录的其他8株BVDV病毒全基因序列进行同源性比对及系统进化分析,结果表明,牦牛株BVDV与BVDV-Ⅰ型毒株出于同一分支,但与其他8株BVDV毒株全基因序列同源性均不高,有可能属于新的独立基因型或基因亚型,仍需进一步研究证实。     

广西牛病毒性腹泻病毒GX4分离株全基因测序及序列分析

牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)属于瘟病毒属,是养牛业中常见的病原体。通过MDBK细胞首次从广西分离获得1株牛源牛病毒性腹泻病毒,命名为GX4。设计引物对其全基因组进行扩增,获得其全基因序列,测序结果表明,GX4全基因组为12 218bp,编码3 898个氨基酸,GenBank登录号JN704144.1。对全基因序列进行分析,根据其5′-UTR,确定GX4属于BVDV-1b基因亚型;与BVDV其他参考毒株的比对发现,GX4与巴西分离株IBSP4ncp同源性最高,核苷酸同源性为94.4%,推导氨基酸同源性为96.2%,并且P125基因无外源序列插入,属于非细胞病变型。分子流行病学的结果,揭示了目前流行株的差异性,为该病的防控措施的制定提供参考。     

牦牛病毒性黏膜病病毒P125基因的克隆及序列分析

试验参考GenBank中发表的牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)毒株基因组序列设计了5对引物,利用PCR扩增出预期的3475 bp目的片段;将扩增产物连接至pMD19-T 载体,经质粒PCR鉴定及双酶切鉴定获得阳性重组质粒并进行核苷酸序列测定。经BLAST同源性分析表明,牦牛BVDV P125基因与BVDV-Ⅰ型的NADL毒株P125基因的同源性为63.4%。系统分析结果表明,牦牛P125基因与BVDV标准毒株P125基因在遗传进化与亲缘关系上均较远,这可能是因为环境诱变的结果或该毒株具有新的遗传衍化来源。     

犬的病料中检测到猪圆环病毒3型

为进一步弄清我国犬是否感染猪圆环病毒3型(porcine circovirus 3,PCV3),本试验对山东省青岛市某宠物医院采集发病犬的48份样品进行PCV3荧光PCR检测,发现7份病料呈现明显阳性,对这7株阳性样品继续用普通PCR扩增,有3份可以扩增出228 bp的特异性条带。这些条带测序后与圆环病毒的其他参考序列进行同源性分析,结果显示这3份样品的病毒序列与其他PCV3参考序列处于同一个进化分支,与PCV3的同源性为93.3%～99.5%,表明我国犬确实存在PCV3感染。     

牛病毒性腹泻病毒TaqMan探针荧光RT-PCR方法的建立和临床应用

为建立一种牛病毒性腹泻病毒(BVDV)的快速检测方法,本研究根据国内BVDV流行毒株5’-UTR序列保守区域设计1对特异性引物和TaqMan探针,并优化了反应条件,最终建立了一种检测BVDV基因1型(BVDV-1)的TaqMan探针荧光RT-PCR检测方法,并开展7省份BVDV-1型检测和流行情况分析。结果显示：所建立方法能够特异性检测出BVDV-1,与基因2型BVDV、猪瘟病毒、边界病毒、牛冠状病毒、牛传染性鼻气管炎病毒、牛轮状病毒等病原无交叉反应;灵敏度高,最低检测下限为4.3 copies/μL;批内和批间变异系数均小于2%,重复性良好。7省份规模化牛养殖场BVDV-1型平均阳性率为17.40%(161/925),序列分析表明我国主要流行的为BVDV-1a,BVDV-1c亚型。本研究成功建立了一种良好的诊断BVDV的方法,监测地区优势流行毒株为BVDV-1a与1c亚型,为临床中BVDV早期诊断和流行病学调查监测提供了技术和数据支持。     

猪瘟疫苗中牛病毒性腹泻病毒2型的检测

为对上海某猪场送检的一份猪瘟疫苗进行牛病毒性腹泻病毒(BVDV)检测,本研究将猪瘟疫苗样品接种于MDBK细胞,盲传15代后仍无致细胞病变效应,但间接免疫荧光试验表明接种该疫苗后的MDBK细胞能够被单克隆抗体BZ-53(BVDV-2)识别。采用BVDV-1和BVDV-2的5’-UTR的通用检测引物和针对BVDV E2的引物,对样品RNA进行RT-PCR检测,结果显示,样品能够扩增出约288 bp的BVDV特异性片段;此外,5’-UTR和E2基因片段的测序分析结果表明分离株属于BVDV-2,并且其E2基因与牛源XJ-04株(BVDV-2)的E2基因同源性最高(92.3%),而与猪源ZM-95株(BVDV-1)的E2基因同源性较低(64.5%)。由此证明,该猪瘟疫苗中的确污染有一株BVDV-2株。     

Genetic diversity of ruminant <Emphasis Type="Italic">Pestivirus</Emphasis> strains collected in Northern Ireland between 1999 and 2011 and the role of live ruminant imports

Background

The genus pestivirus within the family Flaviviridae includes bovine viral diarrhoea virus (BVDV) types 1 and 2, border disease virus (BDV) and classical swine fever virus. The two recognised genotypes of BVDV are divided into subtypes based on phylogenetic analysis, namely a-p for BVDV-1 and a-c for BVDV-2.

Methods

Three studies were conducted to investigate the phylogenetic diversity of pestiviruses present in Northern Ireland. Firstly, pestiviruses in 152 serum samples that had previously tested positive for BVDV between 1999 and 2008 were genotyped with a RT-PCR assay. Secondly, the genetic heterogeneity of pestiviruses from 91 serum samples collected between 2008 and 2011 was investigated by phylogenetic analysis of a 288 base pair portion of the 5’ untranslated region (UTR). Finally, blood samples from 839 bovine and 4,437 ovine animals imported in 2010 and 2011 were tested for pestiviral RNA. Analysis of animal movement data alongside the phylogenetic analysis of the strains was carried out to identify any links between isolates and animal movement.

Results

No BVDV-2 strains were detected. All of the 152 samples in the first study were genotyped as BVDV-1. Phylogenetic analysis indicated that the predominant subtype circulating was BVDV-1a (86 samples out of 91). The remaining five samples clustered close to reference strains in subtype BVDV-1b. Out of the imported animals, 18 bovine samples tested positive and 8 inconclusive (Ct ≥36), while all ovine samples were negative. Eight sequences were obtained and were defined as BVDV-1b. Analysis of movement data between herds failed to find links between herds where BVDV-1b was detected.

Conclusion

Given that only BVDV-1a was detected in samples collected between 1968 and 1999, this study suggests that at least one new subtype has been introduced to Northern Ireland between 1999 and 2011 and highlights the potential for importation of cattle to introduce new strains.

     

Genotypic characterization of Chilean llama (Lama glama) and alpaca (Vicugna pacos) pestivirus isolates

Llamas and alpacas are domesticated South American camelids (SACs) important to ancestral population in the Altiplano region, and to different communities worldwide where they have been introduced. These ungulates have shown to be susceptible to several livestock viral pathogens such as members of the Pestivirus genus, in particular Bovine Viral Diarrhea (BVDV), but there is little data available on Pestivirus infections in SACs. In this study we aimed to detect and identify Pestivirus genotypes and subgroups infecting SACs in both wild and confined environments. Samples were collected from 136 llamas and 30 alpacas from different areas in the Chilean Altiplano (wild animals), and from 22 llamas and 26 alpacas diagnosed as Pestivirus positive from the Metropolitana region in Chile (confined animals). Seroneutralization tests showed titers lower than 2 in all 166 samples from Chilean Altiplano. These samples were also negative to BVDV isolation, indicating that these animals have not been exposed to Pestivirus. After reactivation of positive samples from the Metropolitana region, the 5′ non-codifying region (5′NCR) and E2 glycoprotein were amplified by RT-PCR from the Pestivirus genome. Viral sequences were pairwise compared and phylogenetic trees were constructed. The 5′NCR analysis showed that all 12 sequenced isolates belonged to BVDV-1. Of particular interest, isolates from eight llama and two alpaca were BVDV-1j and two alpacas were BVDV-1b. In agreement with these results, E2 phylogenetic analysis rendered a similar grouping indicating that all 16 isolates belong to BVDV-1. However, the lower availability of E2 sequences determines the creation of a smaller number of sub-groups than the 5′NCR sequences. Based on the E2 sequences, the 5′NCR BVDV 1j group consisting of all the llamas and 3 alpacas are completely included in the E2 BVDV 1e group. Due to the universal availability of the 5′NCR segment, we propose the classification of these Chilean llamas and alpacas Pestivirus isolates as BVDV 1j and BVDV 1b respectively. Thus, this is the first time BVDV-1j is obtained in SACs. In addition, these results indicate Pestivirus infection in llamas and alpacas is associated with bovine population as genotypes and sub-groups are the same as those affecting Chilean livestock.     

Genetic diversity and frequency of bovine viral diarrhea virus (BVDV) detected in cattle in Turkey

The aim of this study was to investigate the frequency and diversity of bovine viral diarrhea viruses (BVDV) infecting cattle in Turkey. A total of 1124 bovine blood samples from 19 farms in 4 different Turkish regions were tested by antigen capture ELISA (ACE). BVDV antigen was found in 26 samples from 13 farms. Only 20 of the 26 initial test positive cattle were available for retesting. Of these, 6 of 20 tested positive for BVDV, by ACE and real-time RT-PCR, one month after initial testing. Phylogenetic analysis, based on comparison of the E2 or the 5'UTR coding regions, from 19 of the 26 initial positive samples, indicated that 17 belonged to the BVDV-1 genotype and 2 to the BVDV-2 genotype. Comparison of 5'UTR sequences segregated 8 BVDV-1 strains (strains 5, 6, 10, 11, 12, 13, 17, and 19) to the BVDV1f, 1 strain (strain 8) to the BVDV1i and 1 strain (strain 14) to the BVDV1d subgenotypes. One strain (strain 4) did not group with other subgenotypes but was closer to the BVDV1f. The remaining 6 BVDV-1 strains (strains 1, 2, 3, 7, 9, and 18) segregated to a novel subgenotype. The E2 sequence comparison results were similar, with the exception that strain 5 grouped with the novel subgenotype rather than BVDV1f subgenotype. It appears that among the diverse BVDV strains in circulation there may be a subgenotype that is unique to Turkey. This should be considered in the design of diagnostics and vaccines to be used in Turkey.     

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.     

牛病毒性腹泻病毒BVDV-JL分离株全基因序列测定与分析

为了解中国农业科学院特产研究所分子生物学重点实验室前期分离的牛病毒性腹泻病毒BVDV-JL毒株完整的基因序列信息,本试验对BVDV-JL F6代毒株进行了完整基因序列测定。利用反转录—聚合酶链式反应(RT-PCR)方法分段扩增了BVDV-JL分离毒株的7段cDNA片段,分别克隆于pMD18-T载体并进行测序。BVDV-JL株基因组序列全长为12276 bp,编码3901个氨基酸。基因组两端为非编码区5'UTR和3'UTR。基因比对及进化树分析结果表明BVDV-JL与BVDV CP7同源性最高,核苷酸同源性为93.2%,归类于BVDV-1b2基因亚型。BVDV-JL是第1个在中国报道的BVDV-1b2亚型毒株。了解BVDV-JL完整基因序列有利于中国BVDV流行病学调查。     

A survey for BVDV antibodies in cattle farms in Slovakia and genetic typing of BVDV isolates from imported animals

A serological survey for bovine viral diarrhoea virus (BVDV) antibodies on a collection of 1295 serum samples obtained from 6-12 months old cattle originating from 45 farms in Slovakia was carried out. On 13 farms more than 90% of the examined animals were seropositive, on 14 farms 71-90% seroprevalence was observed, on 13 farms only 50-70% animals were found to be positive for BVDV antibodies, while the remaining 5 farms showed fewer than 50% seropositive animals. The average incidence of BVDV antibodies (around 70%) was similar as determined 30 years ago. Of 84 serum samples from seronegative animals originating from 14 farms in which 70-98% seropositivity was observed, six were positive in Ag-BVDV ELISA indicating persistently infected (PI) cattle. On a farm to which animals were imported from abroad, a BVD outbreak was observed. Of 110 animals tested, four were positive in Ag-ELISA indicating the presence of PI cattle on this farm. Genetic typing of two isolates from imported animals performed by RT-PCR (324/326 primers from 5'-UTR), sequencing of PCR products and computer-assisted phylogenetic analysis revealed that they belong to BVDV-1 h group.