A comparative analysis of envelope and tegument proteins of suid herpesvirus 1, bovine herpesvirus 1 and bovine herpesvirus 5

The aim of this study was to analyze the disordered regions (DRs) in the envelope and tegument proteins of three closely related herpesviruses: bovine herpesvirus 1, bovine herpesvirus 5 and suid herpesvirus 1. Tegument proteins showed a greater percentage of DRs than the envelope proteins did. Regions of disorder were found in the less conserved portions of the proteins, N-terminal and C-terminal regions, and, in a few cases, functionally important sites. The presence of DRs is an important factor in the evolution of viruses, representing points where positive pressure led to structural changes.     

Comparison of a cheetah herpesvirus isolate to feline herpesvirus type 1

Summary A cytopathogenic virus with size and structural characteristics of a Herpesviridae was isolated from a cheetah with severe ulcerative dermatitis. Restriction endonuclease analysis and cross-hybridization studies revealed that the isolate was related to feline herpesvirus type 1 (FHV-1). Antigenic comparison studies using anti-FHV-1 serum demonstrated the presence of common antigens in the FHV-1 and the isolate from the cheetah.     

Comparative genome mapping of bovine encephalitis herpesvirus,bovine herpesvirus 1, and buffalo herpesvirus

Summary A clone library of 11 of 15BamHI fragments representing 81% of the 140 kilobase DNA genome of the prototype bovine encephalitis herpesvirus strain N569 (BEHV.N569) was constructed. The clones were used to verify theBamHI,BstEII,EcoRI, andHindIII genomic maps for BEHV.N569 published by Engels et al. [Virus Res 6: 57–73 (1986)] for the same virus although some amendments/variations to theBamHI map were found in that 3 previously unidentified restriction sites were identified. Restriction site maps forBglII andKpnI were also derived for BEHV.N569.Southern blot analysis using³²P-labelled BEHV DNA as probe indicated that bovine herpesvirus 1 (BHV1), buffalo herpesvirus 1 (BuHV1) and caprine herpesvirus 1 (CaHV1) were similar and that the similarity occurred throughout the entire length of the genomes; CaHV1 was more distantly related to the other 3 viruses. Because of the similarities BEHV.N569 and BHV1.Cooper cloned DNA fragments were used to constructBamHI,BglII,BstEII,EcoRI,KpnI, andHindIII restriction site maps for the genome of BuHV1 andBamHI,BglII, andKpnI maps for the genome of BHV1.V155, a genital strain.     

Cloning and sequencing of truncated gIV glycoprotein gene of an Indian isolate of bovine herpesvirus 1

Bovine herpesvirus 1 (BHV-1) has been reported from the Indian subcontinent in late 70's. In order to identify the origin of an Indian isolate of BHV-1 (IBR/H 167 VS) and its molecular relationship to known strains of BHV-1, a 680 bp region of the glycoprotein gene gIV was amplified by polymerase chain reaction (PCR), cloned and sequenced. Comparison of this sequences with the corresponding one of an European strain of BHV-1 (Cooper) revealed more than 99% nucleotide (nt) homology. We conclude that the Indian isolate under study does not differ from the Cooper strain regarding the gIV gene.     

Characterization of RKZ isolate of ovine herpesvirus 1

Cytopathic effect (CPE) characterized mainly by foci of rounded cells was observed in cultures of primary plexus choroideus cells from healthy lamb following cryopreservation. It was possible to transmit the infectious agent to other primary cells of ovine origin by co-cultivation with infected cells. By indirect immunofluorescence microscopy it was found that high percentage of sheep (65-80% in 3 different herds from Slovakia) are infected with this infectious agent. Electron microscopy of cells with CPE revealed the presence of herpesvirus particles. Viral DNA was isolated from infected cells using pulse-field gel electrophoresis and further used as probe in Southern blot analysis. The probe reacted specifically only with DNA from cells infected with Ovine herpesvirus 1 (OvHV-1) but not with DNA of other ruminant herpesviruses. Some of the HindIII restriction fragments of DNA of the obtained OvHV-1 isolate denominated RKZ were cloned. Part of the H9 clone was sequenced identifying a gene that encoded a polypeptide homologous to conserved herpesvirus VP23 structural protein. From comparison of the sequence of this clone with VP23 sequences of other herpesviruses it was deduced that OvHV-1 might be classified within the Rhadinovirus genus of the Gammaherpesvirinae subfamily. The sequencing of the H9 clone of DNA of RKZ isolate enabled establishment of sensitive and highly specific polymerase chain reaction (PCR) assay for detection of OvHV-1.     

Restriction maps of the DNA of cervid herpesvirus 1 and cervid herpesvirus 2, two viruses related to bovine herpesvirus 1

Summary Restriction maps of cervid herpesviruses 1 and 2 which are antigenically related to bovine herpesvirus 1, were deduced from Southern blot hybridization withHin dIII restriction fragments of BHV-1 DNA as probes.     

Epitope specificity of the bovine antibody response to the gIII glycoprotein of bovine herpesvirus type 1

A panel of monoclonal antibodies (MAbs) to BHV-1, specific for gI and gIII glycoproteins and for a nonglycosylated core protein p100, was used to examine the epitope specificity of the immune response to the virus in naturally exposed and experimentally infected cattle. Sera from experimentally infected calves were analyzed in a competition ELISA (C-ELISA). Antibody to an epitope on gIII appeared as early as 4 days post infection, although virus-neutralizing antibody did not appear until day 8 or later. Antibody production peaked at 13 to 18 days post infection but the level of antibody to each gIII epitope varied. Competition by sera from cattle naturally exposed to BHV-1 was analyzed as a function of the virus neutralization (VN) titer of these sera. Competition with most of the MAbs correlated linearly with neutralization titer. However, competition with 2 of the MAbs, one specific for gIII and one specific for gI, was maximal even at the lowest neutralization titer, and competition with another MAb, specific for a non-glycosylated core protein, p100, was negative. Selected sera from the naturally exposed group were also examined by radioimmunprecipitation, and were shown to react predominantly with gI, gIII and gIV glycoproteins and in a few shown to react predominantly with gI, gIII and gIV glycoproteins and in a few MAbs were determined, and it was found that neutralization was enhanced by certain combinations. A mutually exclusive relationship between neutralization enhancement and competition for binding by MAbs (as determined by reciprocal C-ELISA) indicated an epitope-specific, rather than antibody-specific, mechanism for neutralization enhancement.     

Nonspecific suppressive effect of bovine herpesvirus type 1 on bovine leukocyte functions.

The effect of bovine herpesvirus type 1 on the specific and nonspecific immune response of calves was examined. Animals with or without prior aerosol exposure to Pasteurella haemolytica serotype A1 were aerosol challenged with 10(8) PFU of virus. Blood and serum samples were taken before and after virus challenge for determining cell-mediated, humoral, and neutrophil responses. A significant depression of the blastogenic responses to phytohemagglutinin, P. haemolytica, and Pasteurella multocida and of neutrophil chemotactic response was observed 4 to 7 days after challenge. However, the antibacterial activity of neutrophils was not significantly affected by virus exposure. Anti-bovine herpesvirus type 1 antibody responses were detected 11 days postchallenge. A significant elevation of the anti-P. haemolytica antibody response (day 0 versus day +11) was detected in animals previously exposed to P. haemolytica.     

Validation of a real-time PCR assay for the detection of bovine herpesvirus 1 in bovine semen

A real-time polymerase chain reaction (PCR) assay was developed for detection of the presence of bovine herpesvirus type 1 (BoHV-1) in extended bovine semen. The assay detects a region encoding a highly conserved glycoprotein B gene. The real-time PCR assay was validated for specificity, sensitivity and repeatability using spiked semen and semen from naturally infected animals. The real-time PCR was very rapid, highly repeatable and more sensitive (lower detection limits) than conventional virus isolation method for the detection of BoHV-1 in extended semen. The specificity of the assay is as expected. The assay had an analytical sensitivity of 0.38 TCID(50) virus spiked into negative semen. The second real-time PCR system for the detection of the bovine growth hormone (bGH) gene was applied as an internal control for the DNA extraction and PCR. The bGH PCR can be performed separately to BoHV-1 PCR, or in a duplex format. The real-time PCR assay is intended for use in international trade. The complete validation dossier based on this study and an international inter-laboratory ring trial has been accredited by the Office International des Epizooties (OIE) and has been recommended to be adopted as a prescribed test for international trade.     

Pneumonia induced byEndobronchial inoculation of calves with bovine herpesvirus 1

Each of six calves inoculated endobronchially with bovine herpesvirus 1 (BHV-1) by means of a bronchoscope developed viral pneumonia. Gross and histopathological lesions were mainly localized to the right diaphragmatic lobe (middle to caudal region) of the lung and were closely associated with the site of the deposition of the inoculum. The lesions were characterized by intranuclear inclusion bodies associated with focal necrosis of the epithelium in the lower respiratory tract. BHV-1 antigen and BHV particles were detected in the degenerating bronchial, bronchiolar and alveolar epithelial cells. After infection, the total cell count in the broncho-alveolar lavage (BAL) fluid increased. In addition, BHV-1 antigen and virus were detected in the desquamated cells and macrophages of BAL fluid from the right diaphragmatic lobe, but not from the left diaphragmatic lobe. It is concluded that examination of BAL fluid is valuable for immunohistopathological and virological confirmation of BHV-1 infection.     

Rapid and efficient construction of recombinant bovine herpesvirus 1 genomes

Bovine herpesvirus 1 (BoHV-1) is an important pathogen of cattle. Recombinant bovine herpesvirus 1 viruses (rBoHV) have been studied extensively as potential vaccines for BoHV-1 associated diseases. A method is described which advances protocols used currently for constructing rBoHV by producing recombinant viruses free of parent virus. The method, restriction endonuclease mediated recombination (REMR), utilises a unique NsiI site in the BoHV-1 genome. Following NsiI digestion the two genomic fragments are prevented from recombining by dephosphorylation. However, when the genomic fragments are co-transfected into a susceptible cell-line with a third DNA fragment (DNA bridge), which encodes DNA homologous to the digested viral termini, the three DNA molecules are able to undergo homologous recombination and produce infectious BoHV-1. During the recombination process foreign DNA within the DNA bridge is incorporated into the BoHV-1 genome, producing rBoHV. In the absence of the DNA bridge virus reconstitution does not occur thus eliminating contamination by the nonrecombinant parent virus. As REMR used an NsiI site occurring naturally in the BoHV-1 genome it can be used for the insertion of foreign DNA into the genome without any prior modifications. REMR could also be applied to any herpesvirus for which the genome sequence is known.     

Induction of nitric oxide in bovine peripheral blood mononuclear cells by bovine herpesvirus 1

Bovine peripheral blood mononuclear cells (PBMCs) and monocytes were stimulated with bovine herpesvirus 1 (BHV-1), LPS and concanavalin A (Con A) to produce L-arginine-dependent nitric oxide (NO) in vitro. NO was detected as early as 12 hrs and up to 72 hrs post stimulation (p.s.). The NO from lipopolysacharide (LPS)-stimulated PBMCs and monocytes was found to exhibit antiviral effect against BHV-1. The anti-BHV-1 effect was inhibited with N(omega)-methyl L-arginine indicating involvement of inducible NO synthase (iNOS) in NO production.     

Pathogenetic characterization of a mouse herpesvirus isolate Sumava

BALB/c mice inoculated intranasally (i.n.) with the mouse herpesvirus isolate Sumava (MHV-Sumava) did not show apparent symptoms of illness. However, they showed an increase in the number of leukocytes and appearance of atypical leukocytes in peripheral blood. The infiltration of spleen with atypical cells resulted in splenomegaly. In the course of the infection the virus persisted in lungs, spleen, thymus, bone marrow, mammary glands, peritoneal macrophages and liver. We regard MHV-Sumava as one of the eight isolates of MHV-68 (a virus and species Murid herpesvirus 4, genus Rhadinovirus, subfamily Gammaherpesvirinae, family Herpesviridae (van Regenmortel et al., 2000)) so far obtained. MHV-Sumava differs from MHV-68, MHV-72 and MHV-76 in some virological and pathogenetical features.