DNA restriction enzyme analysis of a bovine herpesvirus 1 strain isolated from encephalitis in Hungary

The DNA of a bovine herpesvirus 1 (BHV-1) strain isolated from calf encephalitis in Hungary was analysed with restriction enzymes. The cleavage pattern of the encephalitis strain Na/67 differed from those of all the other Hungarian BHV-1 isolates investigated so far. The EcoRI and HindIII cleavage patterns of virus strain Na/67 were found to be similar to the patterns of two other encephalitis strains (N569 and A663 from Australia and Argentina, respectively) characterized earlier. Strain Na/67 is the first isolate in Europe which showed the restriction enzyme pattern of BHV-1.3 previously supposed to be characteristic of encephalitis strains.     

Molecular cloning of complementary DNA from a pneumopathic strain of bovine viral diarrhea virus and its diagnostic application.

A pneumopathic strain of bovine viral diarrhea virus was grown in cell culture and purified. Genomic ribonucleic acid was extracted, polyadenylated at the 3' end, and copied into complementary DNA after oligo-dT priming. Complementary DNA was male double stranded and cloned into the pUC9 plasmid. Approximately 200 complementary DNA clones varying in length from 0.5 to 2.5 kilobases were obtained. Hybridization assays indicated that the sequences isolated were specific for bovine viral diarrhea virus and that at least 5.5 kilobases of bovine viral diarrhea virus genome was represented in the library of complementary DNA clones, the majority of which may have originated from the 3' end of the virus genome. One cloned complementary DNA sequence was used as a 32P-labelled hybridization probe for bovine viral diarrhea virus detection. The probe hybridized with all cytopathic and noncytopathic strains of bovine viral diarrhea virus tested and was 100 times more sensitive than infectivity assays for the detection of bovine viral diarrhea virus. Hybridization did not occur with nucleic acids from bovine coronavirus, bluetongue virus, bovine adenovirus or uninfected cell cultures. Native plasmid DNA sequences, labelled with 32P, did not hybridize with bovine viral diarrhea virus ribonucleic acid.     

Restriction endonuclease patterns of bovine herpesvirus type 1 isolated from bovine mammary glands

Restriction endonuclease analysis was used, in conjunction with viral neutralization and growth-curve experiments, to compare a bovine herpesvirus type 1 (BHV-1) isolate, originally obtained from bovine mammary gland lesions, with a standard BHV-1 strain, infectious bovine rhinotracheitis virus. Although differences were not detected by viral neutralization or growth-curve experiments, restriction fragment patterns generated by Bam HI, Eco RI, Hind III, and Hpa I, revealed definite differences between the isolate and the prototype strain. Additionally, Eco RI, Hind III, and Hpa I patterns revealed that the mammary gland isolate had DNA-fragment patterns characteristic of infectious pustular vulvovaginitis strain of BHV-1, type 2b. Seemingly, type-2b isolates, similar to types 1 and 2a, may be capable of causing divergent types of infection of variable severity in cattle.     

A bovine herpesvirus isolated from sheep.

A viral agent was isolated from the trachea of a lamb that was suffering from a respiratory disorder. The physical and chemical properties of the isolates are characteristic of the herpesvirus group. It contains DNA in its virion, is ether sensitive, acid labile at pH 3.0 and heat labile at 56 degrees C after five minutes. The cytopathology observed provided further evidence of a herpesvirus isolate. The neutralization of the infectivity of the isolate with antiserum to bovine herpesvirus 1 is evidence that it should be considered an isolate of bovine herpesvirus 1. It is concluded that this is a report of a bovine herpesvirus infection in sheep.     

Isolation of a glycoprotein E-deleted bovine herpesvirus type 1 strain in the field

During a field trial to evaluate the efficacy of repeated vaccinations with bovine herpesvirus type 1 (BHV-1) marker vaccines, a glycoprotein E (gE)-negative BHV-1 strain was isolated from the nasal secretions of two cows, eight months after vaccination with a gE-negative live-attenuated vaccine, initially given intranasally, then intramuscularly. The strain isolated was characterised using immunofluorescence, restriction analysis and PCR. All the techniques used identified the isolated virus as a gE-negative BHV-1 phenotypically and genotypically identical to the Za strain used as a control.     

Molecular characterisation of a field strain of bubaline herpesvirus isolated from buffaloes (Bubalus bubalis) after pharmacological reactivation

Two healthy buffaloes (Bubalus bubalis) in a herd which had not been vaccinated against infectious bovine rhinotracheitis (IBR), were selected for their seropositivity for anti-bovine herpesvirus type 1 (BoHV-1) glycoprotein E antibodies, and injected intramuscularly daily with dexamethasone for five consecutive days (day 1 to day 5) to reactivate any latent herpesvirus. Blood samples and nasal and vaginal swabs were collected daily from day 5 to day 15 from each buffalo for virological examination. All the vaginal swabs and blood samples were negative, but 13 of the 22 nasal swabs were positive; a cytopathic effect was observed in primary cultures of bovine fetal lung cells, and the viral isolates were identified as a herpesvirus by PCR. The viral strains were characterised by the sequence analysis of the genes coding for glycoproteins D and B, and the gene sequences were then used for phylogenetic analysis. The isolates from both buffaloes appeared identical at the level of the two genes, and were more closely related to bovine herpesvirus type 5 than to BoHV-1.     

Molecular cloning and characterization of bovine P-selectin glycoprotein ligand-1

Human P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin expressed on leukocytes that binds selectins. Here, we report that the open reading frame (ORF) of bovine PSGL-1 (bPSGL-1) cDNA is 1284 base pairs in length, predicting a protein of 427 amino acids including an 18-amino-acid signal peptide, an extracellular region with a mucin-like domain, and transmembrane and cytoplasmic domains. The amino acid sequence of bPSGL-1 demonstrated 52, 49 and 40% overall homology to equine, human and mouse, respectively. A single extracellular cysteine, at the transmembrane and extracellular domain junction, suggests a disulfide-bonding pattern. Alignment of bovine with equine, human and mouse PSGL-1 demonstrates high conservation of transmembrane and cytoplasmic domains, but diversity of the extracellular domain, especially in the anionic NH(2)-terminal of PSGL-1, the putative P-selectin binding domain. In the NH(2)-terminal of bPSGL-1, there are three potential tyrosine sulfation sites and three potential threonine O-glycosylation sites, all of which are required for P-selectin binding in human PSGL-1 (hPSGL-1). bPSGL-1 shares only 57% homology in amino acid sequence with the corresponding epitope region which binds the monoclonal antibody PL1 for hPSGL-1, and no cross-reactivity was found in bovine leukocytes. In summary, bPSGL-1 shares homology with hPSGl-1, but has differences in the putative extracellular P-selectin binding domain.     

Isolation of a herpesvirus serologically related to bovine herpesvirus 1 from a reindeer (Rangifer tarandus)

Serological evidence of exposure of reindeer (Rangifer tarandus) to a virus related to bovine herpesvirus 1 (BHV1) (Synonym: Infectious bovine rhinotracheitis (IBR) virus) has been reported in Canada (El Azhary 1979) and the USA (Dieterich 1981). A serological survey conducted in Finnish Lapland also detected neutralising antibodies to BHV1 in reindeer sera; 23 % of 300 reindeer had detectable antibodies, whereas none of 300 cattle sera from the same region contained antibodies to BHV1 (Ek-Kommonen et al. 1982). There is currently no evidence of BHV1 infection of cattle in Finland, so the isolation and characterisation of the reindeer herpesvirus was of considerable interest. This short communication describes the isolation and preliminary characterisation of a herpesvirus from a reindeer following the administration of dexamethasone.     

一株分离自中国鹅源黄病毒的分子鉴定

为了分析2011年分离到的1株(ZZ株)引起产蛋量下降的鹅源黄病毒的全基因组序列及病原的分子特性,试验采用PCR技术对其全基因组进行PCR扩增、克隆和测序,之后对多聚蛋白的剪切位点、潜在的糖基化位点、半胱氨酸的分布及3’UTR的二级结构进行分析。结果表明:该病毒由10986个核苷酸组成,并具有典型的黄病毒基因组结构;开放阅读框(ORF)编码3 425个氨基酸(95～10 372 nt);与其他黄病毒比较,该病毒的5’UTR(94 nt)大小居中,而3’UTR(614 nt)相对较长;对多聚蛋白及3’UTR的系统进化树分析显示,ZZ分离株与恩塔亚病毒(Ntaya virus)的巴格扎病毒(Bagazavirus)亲缘关系最近。     

Molecular biology of bovine herpesvirus type 4.

Bovine herpesvirus type 4 (BHV-4) is a ubiquitous virus of cattle. Its genome is a 144 +/- 6 kb double-stranded DNA consisting of a unique central part (L-DNA) flanked at both ends by tandem repeats called polyrepetitive DNA (prDNA or H-DNA). The overall arrangement of genes has been obtained by the analysis of homologies between short BHV-4 DNA sequences and corresponding genes of Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS). The gene expression is temporally regulated. Glycoprotein precursor p (gp10/gp17) is expressed as gamma 1 polypeptide. Glycoproteins gp1, gp8, gp11 and their precursors are gamma 2 proteins. The analysis of strain variations allows the definition of two types of strains, based on the DNA patterns: the Movar 33/63-like and the DN 599-like strains. Only the M40 strain, isolated in India, fails to fit this classification. The genomic variations have been compiled to build a dendrogram showing three levels of divergence between BHV-4 strains or isolates. The available molecular data indicate that the BHV-4 genome shares much similarity with the DNA of EBV and HVS, two representative members of the gammaherpesvirinae. BHV-4 may therefore be classified in the subfamily gammaherpesvirinae.     

Identification of an introduced bovine herpesvirus type 1 strain in a closed dairy herd by experimental virus reactivation followed by DNA restriction enzyme analysis

In a closed dairy herd in the province of Utrecht in 1995, nine replacement heifers were erroneously intramuscularly vaccinated with Tracherine, a live virus IBR vaccine. More than 18 months later, serology of the herd revealed that a large part of the herd had developed an antibody response towards BHV1 (62 of 87 animals). To investigate whether Tracherine had recirculated on the farm, four BHV1 antibody positive animals, of which two had been vaccinated with Tracherine, were treated with corticosteroids to reactivate latent BHV1. Two virus isolates were obtained and subsequently analysed by resctriction enzyme analysis. Both isolates were identified as BHV1.1 subtypes. One of the isolates was clearly distinct from Tracherine and was most likely a BHV1 field virus. A BHV1 field virus was most likely introduced into the farm even though the herd was closed, the animals had not been in contact with other cattle, and preventive hygienic measures had been implemented. There was no indication that Tracherine had recirculated.     

Molecular confirmation of an abortigenic strain of equine herpesvirus 1 (subtype 1) in a pregnant mare study

Four pregnant mares were inoculated intranasally and/or intravenously with equine herpesvirus 1 (EHV-1), subtype 1 during the third trimester of gestation. One mare aborted on postinfection day 15, one mare delivered a sick, weak full term foal, and two mares delivered healthy, full term foals. EHV-1, subtype 1 was isolated from several tissues of the aborted fetus and from the thymus of the sick foal. DNA restriction endonuclease patterns of the recovered EHV-1 viruses were identical to those of the EHV-1 challenge strain, documenting the origin of the abortigenic viruses.     

A combined bovine herpesvirus 1 gB-gD DNA vaccine induces immune response in mice

Although DNA vaccines have several advantages over conventional vaccines, antibody production and protection are often not adequate, particularly in single plasmid vaccine formulations. Here we assessed the potential for a combined vaccine based on plasmids encoding the membrane-anchored or secreted forms of bovine herpesvirus type 1 (BHV-1) glycoprotein B and D (gB and gD) to induce neutralizing and cell mediated immune responses in mice. Animals were injected by intramuscular, subcutaneous and intranasal routes. Mice immunized with the combined vaccine containing the secreted forms of BHV-1 glycoproteins developed higher titers of anti-BHV-1 neutralizing antibodies, compared to wild type gB/gD combined plasmids and to single plasmid injected groups. Cellular immunity was also developed in mice immunized with combined vaccines, whereas low or no response were observed in single plasmid injected animals. The data suggest the potential use of this combined vaccine in in vivo trials of calves, in order to evaluate its protective efficacy.     

DNA of bovine herpesvirus type 1 in the trigeminal ganglia of latently infected calves

Twelve calves infected with bovine herpesvirus type 1 (BHV-1) were killed when in a latent state of infection. Latency was verified 30 days after virus inoculation of the calves by seroconversion, absence of virus shedding, and in 2 calves, by recrudescence of the infection after they were treated with dexamethasone. By in situ hybridization techniques and autoradiography, DNA of BHV-1 was detected in 13 of 23 trigeminal ganglia of latently infected calves. Viral DNA was restricted to the nucleus of nerve cells. Single neurons harboring BHV-1 DNA were observed in 4.9% of the sections (n = 325) of the trigeminal ganglia. The results obtained correspond to those known from herpes simplex virus infections in mice. The implications for the virus-host relationship are discussed.