水貂肠炎细小病毒的分离鉴定及其VP2基因的序列分析

本试验从疑似细小病毒感染的病死水貂中分离到1株病毒。经PCR鉴定、细胞培养、蛋白质电泳鉴定最终确定为水貂肠炎细小病毒(MEV),命名为MEV-WFD。对该分离病毒的衣壳蛋白VP2基因进行克隆测序分析,结果表明此病毒VP2基因3处碱基发生点突变,其中一处的突变导致第328处氨基酸残基由疏水性丙氨酸(Ala)变为亲水性苏氨酸(Thr)。将此株病毒VP2基因与GenBank上公布的所有MEV的VP2基因碱基序列进行同源性比较及进化树分析,结果表明该病毒与ZYL-1、MEV/LN/-10和Manzhouli的VP2基因同源率最高,为99.8%;进化树构建结果表明,该病毒与6株已公布的病毒属于同一进化分支。     

Detection of mink enteritis virus in mink feces, using enzyme-linked immunosorbent assay, hemagglutination, and electron microscopy

Twenty-five mink were inoculated with mink enteritis virus (MEV). Fecal specimens were collected daily and were simultaneously evaluated for MEV antigen by use of a direct enzyme-linked immunosorbent assay (ELISA), hemagglutination (HA), and electron microscopy. Results of the evaluations indicated that MEV was shed in the feces on postinoculation days 5 and 6. The virus was not detectable by ELISA or HA after postinoculation day 6, although viruses were found in reduced numbers by use of electron microscopy. The ELISA was specific for MEV, and the sensitivity of the ELISA for MEV was comparable with that of HA.     

The Dynamic of Maternal Antibody Against Enteritis Virus in Newborn Minks and Immune Efficacy of the Subunit Vaccine

The purpose of this study was to investigate the dynamic of mink enteritis virus (MEV) maternal antibody and the immune effects of subunit vaccines.The offspring of immunized female mink were studied,serum of 21,30,45 and 60 days old mink were collected to determine the HI titer of maternal antibody of MEV.25 healthy minks aged 47~52 days were selected to be inoculated with MEV genetic engineering subunit vaccine,and then blood samples were collected 14 d before and after immunization to determine MEV HI titer.The clinical symptoms and the titer of feces HA of mink enteritis virus were observed 14 d after immunization.Dying and surviving minks were euthanized 14 d after challenge,the duodenum,jejunum and ileum were collected for histopathological observation and immunohistochemical detection.The results showed that the MEV HI of the offspring of immunized female mink decreased gradually with the increase of day age,it was higher at 21 d of age,the MEV HI titer of some minks at 45 d was <1:32 and ≤ 1:4 at 60 d.The MEV HI titer of the control mink was not higher than 1:4 on 14 d after the preparation of qualified vaccine,while it was increased to 1:64~1:512 in immune group.Challenge protection tests showed that the immuned minks were 100% resistant to the attack of mink enteritis virus,and there was no abnormality in the mink's mental state,diet and feces,the HA titer of feces matter after challenge was 1:8~1:16.Histopathological and immunohisto-chemical tests showed that MEV genetic engineering subunit vaccine could well prevent the replication of virus in intestinal mucosal epithelial cells and the damage to intestinal epithelial cells.Therefore,the antibody titer was highest at 21 day-old after immunizing female mink,and the prepared vaccine could break through maternal antibody interference and produce high levels of antibodies when it immunized minks at about 50 d of age,and could resist the attack of mink enteritis virus virulent strain.     

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.     

新生水貂肠炎病毒母源抗体消长规律及其亚单位疫苗免疫效果研究

本研究旨在探索水貂肠炎病毒（mink enteritis virus,MEV）的母源抗体消长规律及其亚单位疫苗免疫效果。以免疫母貂所生仔貂为研究对象,采集21、30、45、60日龄仔貂血清,测定水貂病毒性肠炎母源抗体HI效价（MEV HI）;选取25只47~52日龄健康水貂接种制备MEV基因工程亚单位疫苗,免疫前后14 d采血测定MEV HI效价;免疫后14 d进行水貂肠炎病毒攻毒临床症状观察,并测定粪便HA效价;攻毒后14 d对濒死和存活水貂安乐死,采集十二指肠、空肠和回肠进行病理组织学观察和免疫组化检测。结果显示,免疫母貂所产仔貂的MEV HI效价随着日龄的增加逐渐降低,在21日龄时较高,45日龄时少部分仔貂MEV HI效价<1：32,60日龄时大部分仔貂HI效价≤ 1：4;使用制备合格疫苗免疫后14 d对照组水貂的MEV HI效价均不高于1：4,免疫组MEV HI效价升高至1：64~1：512;攻毒保护试验表明,免疫组水貂100%抵抗水貂肠炎病毒强毒的攻击,水貂的精神、饮食、粪便等均未见异常,且攻毒后粪便HA效价为1：8~1：16;病理组织学和免疫组化检测结果表明,MEV基因工程亚单位疫苗能够很好地阻止病毒在肠黏膜上皮细胞的复制和对肠黏膜上皮细胞的损伤。因此,免疫母貂所生仔貂21日龄时体内抗体效价最高,制备的疫苗免疫50日龄左右的水貂时能够突破母源抗体干扰并产生高水平的抗体,能够抵抗水貂肠炎病毒强毒株的攻击。     

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.     

水貂肠炎病毒分子检测技术与基因工程疫苗研究进展

水貂肠炎病毒（MEV）是一种自主复制性的最小的动物DNA病毒。研究表明,MEV是引起水貂病毒性肠炎的病原体,能够引起水貂剧烈腹泻,具有较高的发病率和死亡率。随着对水貂肠炎病毒研究的不断深入,目前已经在病毒病原学、结构与非结构蛋白、感染机制、诊断与疫苗防控等方面取得很大进展。文章主要针对MEV分子检测新技术与基因工程疫苗等方面进行了综述。     

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.     

Comparison of feline parvovirus subspecific strains using monoclonal antibodies against a feline panleukopenia virus

Four monoclonal antibodies (mAb) against a feline panleukopenia virus (FPLV) TU 1 strain, one of the host range variants of feline parvovirus (FPV), were produced and applied for antigenic analysis of FPLV, canine parvovirus (CPV) and mink enteritis virus (MEV). All mAbs were considered to be directed at epitopes on the virus capsid surface because they neutralized the infectivity and inhibited the hemagglutination (HA) of the homologous virus as well as other FPV strains. They were of the mouse IgG1 type. High antigenic homogeneity among FPLV strains was confirmed by HA-inhibition (HI) test with the mAbs and polyclonal immune sera against FPLV or CPV. But the TU 11 strain of FPLV was antigenically distinguished from the remaining 14 FPLV strains by both the HI test and the micro-neutralization test with one of the mAbs produced. MEV Abashiri strain was found to be antigenically indistinguishable from FPLV. Most of the CPV strains isolated after 1981 were considered to be antigenically different from earlier CPV isolates when some mAbs were applied in the serological tests, confirming the replacement of CPV by an antigenic variant in Japan. However, antigenically different CPVs were detected at the end of 1984 from unrelated epizootics occurred a month apart in the same area.     

致病性水貂肠炎病毒MEV-ZJ1株的分离与鉴定

自临床疑似水貂病毒性肠炎发病貂的粪便中分离出1株病毒,经病毒形态观察、理化特性检测、血清学、聚合酶链式反应（PCR）和动物试验鉴定表明,该分离毒为水貂肠炎病毒（mink enteritis virus,MEV）,命名为MEV-ZJ1株。用分离毒接种水貂,试验动物均表现出典型水貂病毒性肠炎临床症状。研究表明,MEV-ZJ1分离株对水貂具有致病性,是1株强毒,为进一步开展该病毒流行病学、致病机理、疫苗免疫与诊断检测的研究奠定了基础。     

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.     

Neutralizing antibodies against feline parvoviruses in nondomestic felids inoculated with commercial inactivated polyvalent vaccines

The virus neutralization (VN) antibody titers of serum samples from 18 individuals representing 8 carnivore species vaccinated with commercial polyvalent vaccines optimized for domestic cats containing inactivated feline panleukopenia virus (FPLV) were evaluated against canine parvovirus type 2 (CPV2). In addition, the titers among 5 individuals from 4 carnivore were evaluated against antigenic variants of feline parvoviruses; FPLV, CPV2, CPV2a, CPV2b, CPV2c, mink enteritis virus type 1 (MEV1) and MEV2. The polyvalent vaccines induced cross-reactive VN titers against antigenic variants of feline parvoviruses in nondomestic felids. However, we observed very low cross-reactive VN antibody in lions and Siberian tigers, therefore we should pay attention to CPV infections in these animals even if they were vaccinated with inactivated FPLV vaccines.     

抗猫泛白细胞减少症病毒单克隆抗体的制备及其生物学特性鉴定

用 PEG60 0 0沉淀和蔗糖密度梯度离心从细胞培养物中纯化猫泛白细胞减少症病毒 ( FPV) ,以纯化FPV免疫 BALB/c小鼠 ,运用淋巴细胞杂交瘤技术 ,获得了 4株抗 FPV的特异性单克隆抗体 ( Mc Ab)。其腹水 Mc Ab的 ELISA效价在 1 0 - 4～ 1 0 - 5之间 ,其中 1株具有血凝抑制能力。经 ELISA阻断试验及 ELISA交叉反应性试验测定 ,这 4株 Mc Ab可与 FPV、犬细小病毒 ( CPV)和水貂肠炎病毒 ( MEV)呈特异性反应 ,因此可作为检测 FPV、CPV和 MEV共同抗原的通用试剂     

The emergence of parvoviruses of carnivores

The emergence of canine parvovirus (CPV) represents a well-documented example highlighting the emergence of a new virus through cross-species transmission. CPV emerged in the mid-1970s as a new pathogen of dogs and has since become endemic in the global dog population. Despite widespread vaccination, CPV has remained a widespread disease of dogs, and new genetic and antigenic variants have arisen and sometimes reached high frequency in certain geographic regions or throughout the world. Here we review our understanding of this emergence event and contrast it to what is known about the emergence of a disease in mink caused by mink enteritis virus (MEV). In addition, we summarize the evolution of CPV over the past 30 years in the global dog population, and describe the epidemiology of contemporary parvovirus infections of dogs and cats. CPV represents a valuable model for understanding disease emergence through cross-species transmission, while MEV provides an interesting comparison.     

水貂细小病毒性肠炎灭活疫苗保存期试验

对2~8℃条件下保存90,120,150,180,210,240,300,360 d的水貂细小病毒性肠炎灭活疫苗接种水貂进行免疫试验,结果表明:保存300 d的疫苗接种水貂30 d后血清中水貂肠炎细小病毒(MEV)HI抗体均在1∶32以上,免疫水貂强毒攻击均获得100%保护,确定水貂细小病毒细胞灭活疫苗保存期为300 d。该结果为水貂细小病毒细胞灭活疫苗运输和保存提供了理论依据。     

Feline panleukopaenia virus and mink enteritis virus. A serological study.

The serological relationship of Danish feline panleukopaenia virus and mink enteritis virus and strains from Great Britain, USA, Germany and Canada was examined in neutralization tests using a direct immunofluorescence technique. Vaccine strains of the virus were used representing virus strains from the different countries. It was found that all Danish feline panleukopaenia virus strains and the mink enteritis strain belong to the same serotype and further that they are of similar antigenicity as feline panleukopaenia virus strains and mink enteritis strains isolated in other countries.     

Comparison of the viral proteins of canine parvovirus-2, mink enteritis virus and feline panleukopenia virus

Canine parvovirus-2 (CPV-2), Mink enteritis virus (MEV) and feline panleukopenia virus (FPV) were produced using identical cell culture and purification techniques. The distributions of the haemagglutinating activity of the three different parvoviruses in a CsCl gradient were similar with haemagglutinating peaks identified at 1.48–1.49, 1.42, 1.36 and 1.30–1.31 g cm^?3. The number and distribution of the viral proteins and the equivalent protein molecular weights are similar for all three viruses in SDS-polyacrylamide gels (10%). Four viral proteins were identified and their molecular weights were determined: protein A (77 500–79 500), protein B (63 000–63 500), protein C (61 500–63 000) and protein D (50 000–55 000). The viral protein D although reported for some other parvoviruses has not previously been demonstrated in CPV-2, MEV or FPV.     

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。     

A solid-phase fluorescent immunoassay for detecting canine or mink enteritis parvoviruses in faecal samples

Mink enteritis virus (MEV) and canine parvovirus (CPV) were detected in faecal samples from experimentally or naturally infected minks and dogs, respectively, using antibody-coated polyacrylamide beads (immunobeads, IB) as the solid phase for immunofluorescence (IF) tests. The specificity and sensitivity of the immunobead assay (IBA) were studied by comparing it with an enzyme-linked immunoassay (ELISA), a haemagglutination (HA) test and an IF test using tissue cultures. The IBA was as sensitive as ELISA, but more sensitive than the HA test and the IF test. Furthermore, the use of IB as the matrix for the immunological reactions allows FITC- or enzyme-conjugated antibodies to be used as indicators of the reactions and a simultaneous investigation of several pathogenic agents.     

MEV、ADV和CAV三种病毒多重PCR检测方法的建立

建立一种同时检测貂肠炎病毒(MEV)、貂阿留申病毒(ADV)和犬腺病毒(CAV)的多重PCR诊断方法.引用已有的CAV引物,并根据GenBank发表的MEV、ADV序列保守区域设计特异性引物进行PCR扩增,可同时得到扩增长度为795(MEV)、451(ADV)、1 019 bp(CAV)3奈特异性片段,对猪细小病毒(PPV),犬瘟热病毒(CDV)进行PCR检测结果为阴性.各种模板、引物之间相互不构成干扰.敏感性试验证明,可以检测到模板中MEV 101.5 TCID50和CAV 100.5 TCID50的病毒含量,对ADV检测的敏感性更高.