水貂细小病毒MEV-LT18株的分离鉴定及遗传进化分析

为了解河北地区水貂肠炎病毒(mink enteritis virus,MEV)的生物学特性,阐明其基因组与遗传进化等特征,采集疑似感染MEV致死貂的肠道组织,PCR鉴定为MEV阳性后,接种F81细胞进行病毒分离鉴定,通过电镜形态学观察、理化特性试验、血清学及分子生物学等试验方法进行验证。结果表明,成功鉴定并分离出1株MEV,命名为MEV-LT18株;电镜观察病毒粒子形态符合细小病毒形态特征;血凝试验发现该分离株不具有血凝性。对NS基因和VP2基因进行序列比对与遗传进化树分析,与Abashiri株进行氨基酸序列对比,结果显示在NS基因上有3处氨基酸发生非同义替换,分别为aa10(Val→Ile)、aa540(Val→Ala)、aa574(Val→Ile),在VP2基因上共有4处氨基酸发生非同义替换,分别为aa232(Ile→Val)、aa236(Thr→Ser)、aa300(Ala→Val)、aa411(Ala→Glu);系统进化树结果显示MEV-LT18株的NS基因和VP2基因分别与MEV-SDNH株和MEV-HLJ株亲缘关系最近,可能处于两者进化过程的过渡阶段。本研究对该地区水貂细小病毒的进化特征提供了一定的参考价值。     

水貂肠炎病毒LN-10分离株的分离鉴定

为了解我国水貂肠炎病毒(MEV)的流行情况,本研究采用F81细胞从疑似患有肠炎的水貂粪便样品中分离出一株病毒,经形态学、血清学、动物回归试验和分子生物学鉴定,分离的病毒为MEV,命名为LN-10。对该病毒主要结构蛋白VP2基因进行克隆测序和基因进化分析表明,LN-10分离株VP2基因与GenBank中的其他18株MEV株核苷酸和氨基酸均有较高的同源性,分别为99.3%～100%和99%～100%,其中核苷酸同源性与ZYL-1株为100%,而氨基酸同源性与ZYL-1株和Manzhouli株均为100%。本研究为MEV分子流行病学调查和疫苗的研究奠定了基础。     

貉细小病毒LN10-1株分离鉴定及其免疫原性研究

为研制貉细小病毒性肠炎疫苗,筛选出针对貉细小病毒性肠炎免疫原性好、安全高效的疫苗备选株,应用CRFK细胞从辽宁省发病貉的粪便中分离病毒,并通过形态学、血清学、分子生物学、动物回归及免疫接种等方法对分离株进行鉴定。鉴定结果表明成功分离出1株貉细小病毒,命名为LN10-1株。其VP2基因核苷酸序列与猕猴源猫泛白细胞综合征病毒株(BJ-22/2008/CHN株)相似性高达99.7%。VP2蛋白上决定宿主范围的2个氨基酸位点发生了突变。VP2基因种系发生分析显示,LN10-1株位于猫泛白细胞综合征病毒(Feline panleukopenia virus,FPLV)、蓝狐细小病毒(Blue fox parvovirus,BFPV)、水貂肠炎病毒(Mink enteritis virus,MEV)组成的食肉类动物细小病毒聚类分支与由犬细小病毒(Canine parvovirus,CPV)组成的聚类分支。由LN10-1株制备的灭活疫苗免疫结果显示,接种28d细小病毒中和抗体滴度可达到1∶256以上。推测LN10-1株可能正处于FPLV与CPV进化的中间状态,或是CPV适应新宿主(貉)而形成的一种新病毒,可以作为针对貉细小病毒性肠炎灭活疫苗的候选株。     

蓝狐细小病毒分离鉴定及VP2基因遗传进化分析

为了鉴定从蓝狐粪便中分离的病毒是否为细小病毒,试验采用病毒培养、电镜观察、中和试验、间接免疫荧光试验和分子生物学试验对其进行鉴定。结果表明:分离毒株在F81细胞中培养96 h后出现细胞病变(CPE);电镜观察可见直径为20 nm左右的病毒粒子;该病毒能被水貂肠炎细小病毒(MEV)阳性血清中和;PCR检测可扩增得到大小为570 bp的特异性条带,并证明分离毒株为细小病毒,将其命名为BFPV-HeB05/16;VP2全基因遗传进化与氨基酸序列分析显示,分离毒株BFPV-HeB05/16与MEV和BFPV处在同一分支上,具有较高的同源性,其关键氨基酸位点(80,300,426,564,568位)与BFPV和MEV保持一致。     

山东地区水貂肠炎病毒VP_2基因的克隆和测序分析

本研究对潍坊、烟台、威海和青岛地区的疑似水貂病毒性肠炎病毒(MEV)粪便样品进行检测,并在9份样品QD1309、QD1407、WH1407、WH1408、WH1508、WH1509、WF1310、WF1408、YT1408中扩增出VP2基因,和Gen Bank上已经公布的4株MEV参考株的VP_2基因进行序列分析,结果显示,13份MEV VP2基因的核苷酸和氨基酸均有较高的同源性,分别为97.5%~99.8%和96.9%~99.7%。系统发育进化树表明,QD1309、YT1408、MEV-Beregovoj-Bincentr(MEV-BB)、MEV-e属于同一个组群,不同毒株主要在第5、62、87、101、232、236、279、300、534位氨基酸发生了替换。本研究对水貂细小病毒流行株的主要衣壳蛋白VP_2的编码蛋白进行遗传变异分析,为更好地预防和控制貂源细小病毒提供基础。     

江苏地区犬细小病毒的分离与变异分析

2007-2013年,在江苏省扬州市和苏州市的病犬临床样本中共分离鉴定了11株犬细小病毒(CPV)。经过VP2特定位置氨基酸的比较分析,证明其中9株为CPV-2a,1株为CPV-2b,1株为CPV-2。部分CPV-2a/b分离毒株在VP2蛋白氨基酸位点300位或440位有新的突变,VP2基因进化树分析表明,大部分江苏毒株在同1个独立分支中。     

2008年-2009年广东番鸭细小病毒分子流行病学研究

VP2基因是番鸭细小病毒(MDPV)的主要免疫原性基因,其编码的VP2蛋白能够诱导机体产生中和抗体。本文对2008年～2009年从广东各县市分离、鉴定的11个MDPV野毒株VP2基因进行克隆测序,并对获得的VP2基因序列进行比较和分析。结果表明:11个不同分离株之间核苷酸序列同源性为98.7%-100%;与6个国内外参考毒株同源性为97.7%-99.6%;所有毒株都含有4个完全相同的潜在糖基化位点;说明该病毒保守度高,仅有个别的点突变。将分离株SL2连续传70代获得致弱毒株SL-70,动物实验结果表明SL70株对雏鸭无致病力;经测序发现SL-70与其祖代分离株SL2的VP2基因序列同源性高达99.9%,仅发生了18个碱基的点突变。     

犬细小病毒CPV-SD03/19株的分离鉴定及遗传演化分析

为了解山东地区犬细小病毒(canine parvovirus,CPV)的生物学特性,阐明其基因组与遗传演化等特征,采集疑似感染CPV死亡犬的小肠,经PCR鉴定为CPV阳性,无菌处理病料后接种F81细胞进行病毒分离鉴定,通过电镜形态学观察及理化特性、血清学、分子生物学等试验进行验证。结果:成功鉴定、分离出1株CPV,命名为CPV-SD03/19株;电镜观察病毒粒子形态符合细小病毒形态特征;血凝试验效价为2;对NS基因和VP2基因进行序列比对与遗传进化树分析,结果显示CPV-SD03/19株抗原型为CPV-2a型,与CPV-LZ1株对比,在VP2基因上共有3处氨基酸发生非同义替换,分别为aa267(苯丙氨酸→酪氨酸)、aa426(天冬酰胺→赖氨酸)、aa440(苏氨酸→丙氨酸)。本研究对分析山东地区犬细小病毒的进化特征具有一定的参考价值。     

貉细小病毒HLJ11-1株的分离鉴定及VP2基因序列分析

为了研究貉细小病毒,试验从来自黑龙江省的疑似貉细小病毒感染貉的病料分离出1株貉细小病毒(Raccoon dog parvovirus,RDPV),进行了形态学、血清学、生物学和病毒分子生物学鉴定,并对分离株VP2基因进行克隆和序列分析。结果表明:分离的病毒为CPV突变株;该株病毒与肉食动物细小病毒有很高的同源性,属于CPV-2a突变株,命名为RDPV HLJ11-1,接近于CPV-2型毒株。说明RDPV HLJ11-1株可能正处于猫泛白细胞减少症病毒(FPLV)与犬细小病毒(CPV)进化的中间状态,或是为CPV适应新宿主(貉)而形成的一种新病毒,可以作为针对貉细小病毒性肠炎灭活疫苗的候选株。     

关于水貂肠炎病毒结构蛋白基因5'-非翻译区调控其基因表达的评述

正水貂肠炎病毒(MEV)是细小病毒科细小病毒属的一种病毒,感染水貂引起水貂病毒性肠炎。作为感染真核细胞的最小DNA病毒之一,细小病毒基因组DNA仅有5 kb左右。为了利用有限的基因序列来完成病毒的复制周期,病毒利用了一些特殊的表达调控方式。比如:该属病毒成员(如:MVM,CPV)利用非结构蛋白NS1基因编码区的基因内启动子来调控结构蛋白VP1和VP2的表达;腺依赖病毒和浓核病毒利用leaky-scanning机制来表达衣壳蛋     

Comparisons of feline panleukopenia virus, canine parvovirus, raccoon parvovirus, and mink enteritis virus and their pathogenicity for mink and ferrets

Parvoviruses from mink (mink enteritis virus [MEV]), cats (feline panleukopenia virus [FPV]), raccoons (raccoon parvovirus [RPV]), and dogs (canine parvovirus [CPV]) were compared. Restriction enzyme analysis of the viral replicative-form DNA revealed no consistent differences between FPV and RPV isolates, but CPV and MEV isolates could be distinguished readily from other virus types. Feline panleukopenia virus, RPV, and MEV, but not CPV, replicated to high titers in mink. However, on the first passage, disease and microscopic lesions were observed only in mink inoculated with MEV. Feline panleukopenia virus and RPV isolates replicated in ferrets, but disease or microscopic lesions were not observed. Feline panleukopenia virus and RPV isolates could be passaged repeatedly in mink and ferrets. Virulence of FPV and RPV isolates was low compared with that of MEV, and only a single mink inoculated with FPV or with RPV developed clinical disease on the sixth passage of virus.     

Characterization of biological and antigenic properties of raccoon dog and blue fox parvoviruses: a monoclonal antibody study

Parvovirus isolates from blue foxes and raccoon dogs were characterized by studying their haemagglutination properties, host range in vitro and antigenic structure. In all 3 characters, raccoon dog parvovirus resembled canine parvovirus (CPV), while blue fox parvovirus was similar to mink enteritis virus (MEV). Monoclonal antibodies (MAbs) were prepared against both viruses. Raccoon dog parvovirus, while resembling CPV, had a unique antigenic site which could be specified by MAbs. The pattern of MAbs prepared against blue fox parvovirus indicated that it is a member of Type 2 MEV.     

水貂病毒性肠炎巢式PCR,PCR-RFLP联合诊断方法的建立

根据水貂肠炎细小病毒的VP2基因序列设计4条特异性引物,建立了检测水貂肠炎细小病毒的巢式PCR方法。采用一次扩增的敏感性来检测5个TCID50/mL的细小病毒MEVB株,二次扩增的敏感性来检测0.05个TCID50/mL的细小病毒MEVB株。同时,利用该方法第一轮PCR产物酶切,能够区分犬细小病毒与水貂肠炎细小病毒,具有重要的实用价值和应用的前景。     

Antigenic and genomic comparisons of some feline parvovirus subspecies strains.

Fourteen feline parvovirus (FPV) strains isolated from cats, mink and dogs were comparatively examined on their antigenic and genetic diversities by using monoclonal antibodies against feline panleukopenia virus (FPLV) and restriction enzyme analysis of viral DNA. Mink enteritis virus (MEV) strains recently isolated in the northeastern area of the People's Republic of China were found to possess more similar antigenic and genetic properties to the antigenic variant virus of canine parvovirus (CPV) ("new" antigenic type CPV), than to FPLV strains and MEV Abashiri strain of Japan. A feline isolate detected in normal cat feces was considered to be rather CPV because of its antigenic and genetic characteristics. An early isolate of "new" antigenic type CPV strains showed a similar cleavage pattern to those of "old" antigenic type CPV strains when digested with HinfI. The results including some features above-mentioned suggest the presence of antigenic heterogeneities and genomic polymorphisms among FPV subspecies viruses.     

A non-haemagglutinating isolate of mink enteritis virus

A virus was isolated from mink showing clinical and pathological signs of mink enteritis. This virus was identified as mink enteritis virus (MEV) from results of serological tests, determination of its density in CsCl (1.415 g cm^?3), and morphology, including size (20 nm in diameter). The isolate was designated MEV-S. In contrast to other known MEV strains, the MEV-S isolate has no haemagglutinating (HA) activity with swine red blood cells (RBCs) at 4°C and pH 6.8.Neither was there any HA at other pH values and temperatures, or when worse, bovine and rhesus monkey RBC's were used.     

水貂肠炎病毒VP2基因的原核表达及鉴定

在分析水貂肠炎病毒VP2基因主要抗原位点分布的基础上,设计了1对特异性引物,克隆一段长为846 bp,编码主要抗原位点的基因片段。将该基因片段定向插入表达载体PET-32a中,转化BL21(DE3)菌株,IPTG诱导实现高效表达。诱导表达比较实验结果表明,在37℃,IPTG浓度为1.0 mM,诱导时间为6 h,诱导表达达到最大效率。重组蛋白以包涵体形式存在,大小约为50 KD,应用兔抗水貂MEV抗体,经Western-blotting验证该重组蛋白具有MEV的抗原性,可为今后抗MEV单克隆抗体制备及相关免疫学检测方法的建立提供良好的抗原物质。     

Hemagglutination by canine parvovirus: serologic studies and diagnostic applications

Conditions for canine parvoviral hemagglutination (HA) and hemagglutination-inhibition (HI) reactions were defined. The HA phenomena were used to differentiate canine parvovirus (CPV) from feline panleukopenia virus (FPV), mink enteritis virus (MEV), and minute virus of canines. Serologic comparisons of the CPV, FPV, and MEV by HA-HI and serum-neutralization tests indicated that CPV, FPV, and MEV were antigenically similar but were different from minute virus of canines. Diagnostic application of HA tests to fecal samples from acute cases of enteritis was discussed. Combinating HA tests with HI tests on fecal samples provided a rapid and specific diagnostic method for CPV infection. Secular seroprevalence studies indicated the emergence of CPV infeciton in the United States dog population-at-large in 1978.     

水貂肠炎病毒VP2基因原核表达及病毒样颗粒制备

试验旨在制备水貂肠炎病毒（Mink enteritis parvovirus,MEV）可溶性VP2蛋白及其病毒样颗粒（virus-like particles,VLPs）。优化并合成MEV VP2基因,克隆至原核表达载体pET-30a（+）,将重组质粒pET-30a-VP2与分子伴侣pTf16共同转化到宿主菌ER2566中,以不同培养温度、L-阿拉伯糖浓度和IPTG浓度进行诱导表达,收集样品进行SDS-PAGE和Western blotting分析。通过硫酸铵沉淀结合蔗糖密度梯度离心法对表达产物进行纯化,由SDS-PAGE进行鉴定,透析去除蔗糖,通过动态光散射技术（DLS）和透射电子显微镜（TEM）观察不同组装条件下VLPs的粒径和形态。用制备的MEV VLPs疫苗免疫水貂评价其免疫原性。结果显示,以2 g/L L-阿拉伯糖、0.2 mmol/L IPTG、25 ℃培养16 h为诱导条件时,VP2蛋白在大肠杆菌中可溶性表达,分子质量约为65 ku。经Western blotting鉴定VP2蛋白具有良好的抗原特异性。将此表达产物利用硫酸铵沉淀结合蔗糖密度梯度离心法纯化后,纯度可达90%以上。在pH 8.0,150 mmol/L NaCl的透析液中,VP2经自组装可形成与天然MEV形状和粒径相似且具有血凝特性（1：2¹⁴）的VLPs。VLPs疫苗免疫水貂21 d后,测得水貂血清中的血凝抑制（HI）抗体水平均相对最高,可达1：2¹¹,说明该VLP疫苗有较好的免疫原性,能有效预防MEV的感染。本试验结果表明,将pET-30a-VP2与pTf16在原核表达系统中共表达,可获得具有高免疫原性的VLPs,为后期MEV VLPs疫苗的研发奠定基础。     

Prokaryotic Expression and Immunogenicity Analysis of Mink Parvovirus NS1 and VP2 Protein

In order to develop subunit vaccine of mink enteritis virus,the immunogenicity of mink parvovirus protein NS1 and VP2 had been evaluated.Two pairs of primers were designed,and the full-length NS1 and VP2 genes had been amplificated,and then prokaryotic expression vector pET-32a-NS1,PET-32a-VP2 were constructed.After the analysis of SDS-PAGE and Western blotting,target proteins had been purified by His-Bind affinity chromatography.The immunogenicity of purified protein NS1 and VP2 were evaluated by serum ELISA testing,after inoculated BALB/c mouse.The results showed that the molecular mass of NS1 and VP2 protein were 83 and 67 ku by SDS-PAGF and Western blotting;Although both target protein NS1 and VP2 had the ability to induce BALB/c mouse to produce anti-MEV specific antibodies,the level of antibodies induced by the protein VP2 was higher than protein NS1.Mink parvovirus protein VP2 was more suitable for the development of subunit vaccine.