Unusual characteristics of a parvovirus isolated from a clinically ill steer

A parvovirus was isolated from the feces of an 8- and 9-month-old steer that died acutely with hemorrhagic diarrhea and microscopic evidence of a coccidial infection. The concurrent intestinal parasitism in this steer appeared to play a role in the development of clinical disease. The viral isolate was identified as a bovine parvovirus (BPV) on the basis of its size (22 nm) and icosahedral morphology, the neutralization of viral cytopathology by antiserum to BPV, a strong immunofluorescent reaction with fluorescein-labeled antiserum to BPV, and the inhibition of viral hemagglutination of guinea-pig erythrocytes by antiserum to BPV. Cell cultures infected with this isolate showed a slight nuclear fluorescent reaction with fluorescein-labeled antiserum to canine parvovirus, suggesting an antigenic relationship to canine parvovirus. Patterns of hemagglutination for this isolate with human erythrocytes from 20 donors of various blood types differed from those obtained with the reference Abinanti strain of BPV. These results indicate that blood from multiple donors of a species may be necessary to confirm the presence or absence of viral hemagglutinating activity with clinical isolates of BPV.     

Isolation of equine herpesvirus-2 from the lung of an aborted fetus.

This study describes the isolation of equine herpesvirus-2 (EHV-2) from the lung of an aborted equine fetus in Argentina. The isolated virus was confirmed as EHV-2 by indirect immunofluorescence using a rabbit anti-EHV-2 polyclonal antiserum and by virus-neutralization test using an equine polyclonal antibody against EHV-2. Restriction endonuclease DNA fingerprinting with BamHI also confirmed the identity of the virus as EHV-2. Furthermore, viral nucleic acid was detected by polymerase chain reaction from the original lung sample and from the DNA obtained from cells infected with the virus isolate. This work constitutes the first reported isolation of EHV-2 from an aborted equine fetus. The presence of EHV-2 in the lung of the aborted fetus would indicate that this virus is capable of crossing the placental barrier. However, no cause-effect relationship was established between the EHV-2 isolate and the abortion.     

Virological and molecular characterization of a mammalian orthoreovirus type 3 strain isolated from a dog in Italy

A mammalian orthoreovirus (MRV) strain was isolated from a pup with fatal diarrhea, which had a concurrent infection by canine parvovirus type 2. The reovirus isolate showed an atypical hemagglutination pattern and a retarded electrophoretic mobility of the S1 segment, which is characteristic of MRV type 3 (MRV-3). Assignment of the isolated virus to MRV-3 was confirmed by type-specific RT-PCR assays, targeting the S1 gene, and by subsequent sequence analysis of the PCR product. By phylogeny based on the S1 gene of several MRVs, the isolate fell into lineage E, along with the murine strain T3C9/61 and the bovine strains T3C18/61 and T3C31/59. Conversely, L1 sequences were found to segregate regardless of the viral type. A total of 110 fecal samples, 56 nasal and 31 ocular swabs from dogs with diarrhea or nasal/ocular discharge were tested by a nested-PCR assay specific for reoviruses, and no sample was found to contain MRV RNA, a finding that is apparently in contrast with the seroprevalence (25.77%) observed in dogs.     

Effect of influenza A/equine/H3N8 virus isolate variation on the measurement of equine antibody responses.

This study has tested the effect of using homologous or heterologous equine influenza A virus isolates to evaluate serum antibody levels to influenza A virus in vaccinated and naturally-infected horses. In addition, the potential effect of antigenic selection of virus variants in egg versus tissue culture propagation systems was studied. Serum antibody levels in samples from horses recently infected with a local influenza A virus isolate (A/equine 2/Saskatoon/1/90) or recently vaccinated with a prototype isolate (A/equine 2/Miami/1/63) were assessed by hemagglutination inhibition and by single radial hemolysis using cell or egg-propagated A/equine 2/Saskatoon/1/90, A/equine 2/Miami/1/63 or A/equine 2/Fontainebleau/1/79. There were no significant differences in hemagglutination inhibition or single radial hemolysis antibody levels obtained with homologous or heterologous isolates or between viruses propagated in either eggs or cell culture. However there was a trend to higher titers in the hemagglutination inhibition assay when cell-propagated virus was used. These results suggest that antigenic variation in equine influenza A virus isolates and host-cell selection of antigenic variants during virus propagation may not be of sufficient magnitude to influence serological evaluation of antibody responses by hemagglutination inhibition or single radial hemolysis.     

果子狸源细小病毒分离鉴定及VP2基因遗传进化分析

【目的】探究果子狸源细小病毒流行特点及其VP2基因遗传变异情况,为细小病毒防治提供理论依据和技术支撑。【方法】采用猫肾细胞(CRFK细胞)对患有肠炎的果子狸肠道组织样品进行病毒分离,通过血凝试验、免疫荧光试验鉴定分离的病毒,并对病毒VP2基因进行PCR扩增和遗传进化分析。【结果】分离到的毒株在CRFK细胞中培养出现细胞脱落、崩解和破碎等典型细小病毒细胞病变效应(CPE);血凝试验结果显示,此病毒可凝集猪红细胞,血凝效价为1∶512;免疫荧光试验结果显示,在接种分离毒株的CRFK细胞内可观察到亮绿色荧光,而对照细胞没有荧光。将分离毒株命名为MPCPV-SX。VP2基因进化树分析发现,其与犬细小病毒处于同一分支,位于CPV-2和CPV-2a型之间,同时VP2氨基酸的87和101位显示为CPV-2型,而300和305位氨基酸则与CPV-2a型一致。【结论】本研究成功从果子狸肠道组织样品中分离出1株细小病毒MPCPV-SX,其VP2基因是介于CPV-2和CPV-2a之间的中间型,表明细小病毒通过果子狸等野生动物跨越宿主流行传播,可能在细小病毒遗传进化方面起到一定的过渡作用。     

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.     

Isolation and characterization of an adenovirus and isolation of its adenovirus-associated virus in cell culture from foals with respiratory tract disease.

An adenovirus was isolated from a foal with respiratory tract disease. The virus produced cytopathic effects (CPE) in equine embryo kidney (EEK) cell culture, contained deoxyribonucleic acid (DNA), was resistant to chloroform and pH 3, and was moderately resistant to heat. The virus caused hemagglutination of human (type O) erythrocytes. Viral density was 1.34 g/cm,3 and diameter was 75 nm. An adenovirus-associated virus (AAV) isolated from the infected cell culture was 22 nm in diameter. These viruses are classified as equine adenovirus and equine AAV.     

Desorption of porcine parvovirus from aluminum hydroxide adjuvant with subsequent viral immunoassay or hemagglutination assay

Cell culture fluids containing porcine parvovirus were adjuvanted with varying concentrations of aluminum hydroxide gel. Adsorption of virus and total protein to adjuvant was proportional to adjuvant concentration. Desorption of virus and protein from the adjuvant in substantial, reproducible quantities was achieved by washing adjuvanted preparations with 1.2 M potassium phosphate, followed by dialysis and concentration of wash fluids. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of unadjuvanted viral fluids, supernatants of adjuvanted mixtures, and desorptive washings of the corresponding adjuvant pellets revealed no qualitative differences in protein banding patterns. Desorbed virus was quantitated by a hemagglutination assay, or by an enzymelinked immunoassay employing a monoclonal anti-porcine parvovirus antibody. Virus desorption and subsequent assays permitted in vitro estimation of virus content in adjuvanted porcine parvovirus preparations. This approach may be useful in estimating the antigen content of inactivated aluminum hydroxide adjuvanted veterinary vaccines and reducing the extent of required in vivo testing.     

一株内源性猪细小病毒的分离与鉴定

本研究从不明病毒污染的ＰＫ１５细胞培养物中分离到了一株猪细小病毒,从形态学、血清学、分子生物学几个方面对其进行了鉴定,同时克隆了该毒株ＶＰ２基因,并测定了其核苷酸序列。将该毒株命名为ＳＹ－９９株。     

Isolation of bovine herpesvirus III from diarrheic feces

A herpesvirus was isolated from the feces of a cow with diarrhea. The viral isolate was identified as a herpesvirus on the basis of morphology and chloroform sensitivity. It was serologically distinct from bovine Herpesvirus I (IBR) and II (mammillitis virus) as well as equine herpesviruses and pseudorabies virus. It was serologically related to members of the bovine Herpesvirus III group.Experimentally inoculated calves developed a transient fever but no other clinical signs or lesions. The virus could be re-isolated from the calves and significant levels of virus neutralizing antibodies were present in the serum 40 days after inoculation.     

鸿雁源鸭甲肝炎病毒Ⅰ型的分离与鉴定

为了对一起死亡率高达91．3％、急性死亡的鸿雁病例进行病原学分析,通过细菌分离排除法和病毒分离方法获得致鸭胚死亡病毒（暂命名为FJ-017株）。该病毒无血凝活性,用鹅细小病毒、番鸭细小病毒、鸭呼肠孤病毒、鸭甲肝炎病毒1型、鸭瘟病毒、鸭坦布苏病毒特异引物分别进行扩增,电泳结果显示,仅鸭甲肝炎病毒1型引物可扩增出条带。将扩增产物回收后克隆,序列分析表明FJ-017株与22株DHAV-1参考株的同源率为93．5％-99％,而与DHAV-2和DHAV-3参考株的同源性均为79．9％。遗传进化分析表明FJ-017株与DHAV-1关系密切,在进化树中共处一分支,表明FJ-017株为鸭甲肝炎病毒1型,此为国内外首次报道。     

貉细小病毒分离鉴定及其VP2基因序列分析

本研究从山东省某貉养殖场发病貉粪便样品中分离到一株貉细小病毒(raccoon dog parvovirus,RDPV)。为了解该毒株的特性及其致病特点,对发病貉粪便处理后接种F81细胞,收获病毒液进行VP2基因序列分析、理化特性研究以及貉人工感染试验。结果显示:该分离株与RDPV参考株核苷酸同源性高达99.4%,属于犬细小病毒2型(CPV-2);对氯仿等不敏感,耐酸耐热,符合细小病毒特征;分离毒株对貉具有较强的致病力。结果表明,山东省仍存在RDPV流行且致病性有增强风险,须针对性研发RDPV疫苗,加强对RDPV流行的控制。本试验为RDPV流行病学研究提供了数据支持。     

犬呼肠孤病毒3型（AMMS株）的分离与鉴定

从 １ 只临床表现呼吸道症状的濒死病犬肺脏分离到 １ 株病毒,经理化特性、血凝性、核酸型、血凝抑制试验和电镜观察,鉴定为犬呼肠孤病毒 ３ 型。这是国内首次分离到此病毒。     

鹅细小病毒BC1105株的分离鉴定

2011年5月中旬在吉林省白城地区的某养鹅场疑似小鹅瘟病死雏鹅的肾脏、肝脏、脑和脾脏组织中分离到1株病毒,选用未免疫小鹅瘟疫苗的健康母鹅所产的14日龄鹅胚,进行鹅胚接种试验,连续传代、血凝试验、琼脂扩散试验、电镜观察、PCR鉴定和动物回归试验,初步确定该病毒为鹅细小病毒,命名为BC1105株。     

鸭疱疹病毒Ⅱ型（暂定名）的分离鉴定

自1990年以来，在福建、浙江和广东等省发生一种以双翅羽毛管淤血呈紫黑色、断裂和脱落以及皮下、脏器（肝脏、胰脏）和肠道（十二指肠、直肠和盲肠）出血为主要特征的新的鸭传染病，暂定名为鸭出血症。我们对从自然感染该病典型病死番鸭脏器中获得的1株含2种病毒粒子（直径大小分别为30－40mm和80－120mm)的分离物，以Ⅰ型雏鸭病毒性肝炎标准阳性血清进行连续4代中和后接种番鸭胚，收集死亡番鸭胚胚液进行病毒纯化、负染、电镜观察和SPF鸡胚接种试验证明获得单一病毒分离株（定名为鸭出血症病毒）。该病毒为不耐酸、不耐碱、不耐热、对氯仿处理敏感、核酸类型为双股DNA的有囊膜病毒，直径为80－150nm；对番鸭胚、鹅胚、麻鸭胚、半番鸭胚、北京鸭胚和SPF鸡胚的致 死率分别为100％、100％、78．3％、60％、82．1％和0％；对10－11日龄番鸭胚的ELD50为10^-7.78/0.2ml；无血凝活性，不凝集“O”型人、鸭、鸡、鹅、家兔、小鼠，豚鼠、猪和绵羊红细胞；经血清中和试验证明该病毒与Ⅰ型雏鸭肝炎病毒、雏番鸭细小病毒、雏鹅细小病毒、鸭瘟病毒无血清学相关性。据以上结果可将该病毒确定为疱疹病毒科成员，但鉴于其与鸭瘟病毒（鸭疱疹病毒Ⅰ型）无血清学相关性，则暂定名为鸭疱疹病毒Ⅱ型，此为国内外首次报道。     

小鹅瘟病毒的分离与鉴定

用接种鹅胚的病毒增殖法,从具有小鹅瘟典型症状和病理变化的病死雏鹅的脏器中分离到一株病毒,并进行了鹅胚致病性试验、鹅胚中和试验、雏鹅感染试验和琼脂扩散试验.结果表明,所分离的毒株为小鹅瘟病毒.     

猪细小病毒的分离与鉴定

本试验从福建省某猪场疑似猪细小病毒病死胎的淋巴结、肝脏中分离到1 株病毒。病料接种PK-15细胞36 h后出现了圆缩、集聚、脱落等细胞病变,猪细小病毒阳性血清能特异性地中和该分离病毒。根据已发表的细小病毒(PPV) VP2基因的序列设计并合成了一对引物,采用PCR方法可扩增531 bp DNA片段。测序结果表明,分离株VP2基因与NCBI公布的NADL-2株的同源性高达99.2％,证实分离的病毒株为猪细小病毒。为进一步开展该病毒致病机理、流行病学、诊断研究与疫苗免疫等奠定了基础。     

Demonstration of parvovirus in Canadian swine and antigenic relationships with isolates from other countries.

A Canadian isolate of porcine parvovirus, isolated from cultured pig thyroid cells, was shown to be antigenically indistinguishable from a British (59e/63) and a German (G10/1) strain when treated by the modified direct complement-fixation, the hemagglutination-inhibition and the fluorescent antibody tests. These tests also revealed that antibodies to parvoviruses were detectable in a large proportion of the conventionally raised pigs in the provinces of Quebec and Ontario. Cell cultures, prepared from tissues collected in a slaughterhouse, were often found to be infected with parvovirus. In cell cultures the infection was demonstrated more effectively by immunofluorescence than by the hemagglutination test.     

Isolation of an equine coronavirus from adult horses with pyrogenic and enteric disease and its antigenic and genomic characterization in comparison with the NC99 strain

A new equine coronavirus was isolated from the feces of adult horses with pyrogenic and enteric disease. The disease outbreak was mainly observed among 2- to 4-year-old horses living in stables of a draft-horse racetrack in Japan. On comparing the isolated virus (isolate Tokachi09) with the equine coronavirus NC99 strain, no significant differences were observed in several biological properties such as hemagglutinating activity, antigenicity (in indirect immunofluorescence and neutralization tests), and one-step growth (in cell culture). The sequences of the nucleocapsid and spike genes of isolate Tokachi09 showed identical size (1341 and 4092 nucleotides, 446 and 1363 amino acids, respectively) and high similarity (98.0% and 99.0% at the nucleotides, 97.3% and 99.0% at the amino acids, respectively) to those of strain NC99. However, the isolate had a 185-nucleotide deletion from four bases after the 3'-terminal end of the spike gene, resulting in the absence of the open reading frame predicted to encode a 4.7-kDa nonstructural protein in strain NC99. These results suggest that the 4.7-kDa nonstructural protein is not essential for viral replication, at least in cell culture, and that the Japanese strain probably originated from a different lineage to the North American strain. This is the first equine coronavirus to be isolated from adult horses with pyrogenic and enteric disease.     

Properties of a coronavirus isolated from a cow with epizootic diarrhea

A coronavirus (Kakegawa isolate) isolated from a cow with epizootic diarrhea was grown in BEK-1 cells and examined for biophysical and biochemical properties. The Kakegawa isolate was able to replicate in the presence or absence of 5-iodo-2′-deoxy-uridine, indicating that its viral nucleic acid was RNA. It was highly sensitive to ether and chloroform, and moderately sensitive to trypsin and heat. It was, however, readily stabilized by treatment with cation at 50°C for 1 h. Its infectivity was slightly reduced at pH 3.0. The virus passed through a membrane filter of 200 nm pore size, but not through one of 100 nm pore size. The buoyant density of the virus was determined in a sucrose density gradient. The peak of infectivity and hemagglutinin activity was found at a density of 1.182. Neutralization and hemagglutination inhibition tests showed a close serological relationship between the Kakegawa isolate and the American strain of calf diarrhea coronavirus.