A comparison of polypeptides and restriction endonuclease sites of BHV-1 isolates and identification of IPV virus in Japan.

Bovine herpesvirus 1 (BHV-1) isolates from respiratory tract and from vagina of bovine in Japan were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and the DNA restriction endonuclease cleavage pattern, and compared with European BHV-1 strains. Both protein profile and DNA cleavaged pattern of BHV-1 isolates from respiratory tract were the same as those of European infectious bovine rhinotracheitis (IBR) virus, whereas the protein profile and DNA cleavage patterns of one isolate (M1) from vagina was the same as those of the European infectious pustular vulvovaginitis (IPV) virus. The facts indicate that IPV virus has existed in Japan.

Further biological, serological and biochemical characterization of North American, European and Southeast Asian strains of bovine herpesvirus 1 compared with other alphaherpesvirinae members.

Hemagglutination activity, structural protein profiles and neutralization assays were used in a comparative study of bovine herpesvirus 1 strains from the U.S.A., Canada, Great Britain, Denmark and Malaysia with equine, feline and human herpesviruses in order to further characterize the bovine herpesvirus 1 hemagglutinin. Bovine herpesvirus 1 strains of different geographical origins all showed hemagglutinating activity for mouse erythrocytes; furthermore, feline herpesvirus 1 was also shown to hemagglutinate mouse erythrocytes. Analyses of partly purified viruses showed that a distinctive and specific polypeptides profile is associated with each species of herpesviruses used in our study; strains of bovine herpesvirus 1 from North America, Europe and Southeast Asia however, presented a remarkable similarity as to their electrophoretic protein patterns. A protein similar to the 97-kDa bovine viral hemagglutinin was not identified with the hemagglutinating feline herpesvirus. An important neutralization epitope on the bovine viral hemagglutinin was also not found on feline, equine and human herpesviruses but was identified on all bovine strains tested from North America, Europe and Southeast Asia stressing the importance of the bovine hemagglutinin for eventual prophylactic purposes.

Detection and purification of bovine herpesvirus 1 glycoproteins by lectin affinity.

A procedure for detection and purification of bovine herpesvirus 1 glycoproteins by their affinity for lectins is described. Wheat germ lectin, Limulus polyphemus lectin and concanavalin A, radioactive or biotin-labelled, have been used for the in vitro characterization of the glycoproteins. These lectins have also been used for affinity-purification of sets of glycoproteins. The procedure is easy, rapid, cheap and, when biotin is used, non-radioactive.

Occurrence of bovine herpesvirus-1 DNA in nucleosomes and chromatin of bovine herpesvirus-1-infected cells: identification of a virion-associated protein in chromatin of infected cells.

During virus replication a fraction of the intranuclear DNA of bovine herpesvirus-1 (BHV-1) was present in the nucleosomal structure of infected eukaryotic cells, and virion proteins were associated with the chromatin of virus infected cells. Synthesis of BHV-1 DNA in bovine embryonic lung (BEL) cells was found to begin four to six hours post-infection (p.i.) and to continue until at least 24 hours p.i. Chromatin isolated from infected cell nuclei at ten hours p.i. contained both BHV-1 viral and cell DNA. No BHV-1 DNA was found in mock-infected cell chromatin. Micrococcal nuclease cleavage products of both mock-infected and BHV-1-infected BEL cell nuclei produced monomers and multimers of unit fragment size which were indistinguishable from each other and displayed a typical nucleosome pattern on agarose gels. Southern analyses of micrococcal nuclease digests of infected cell nuclei indicated that some of the intranuclear BHV-1 DNA was present in a nucleosomal form. Three new proteins (with approximate molecular weights: 125,000, 42,000, and 17,000) were identified in chromatin isolated from BHV-1-infected BEL cells at ten hours p.i. These proteins were not present in mock-infected BEL cell chromatin. The 17,000 molecular weight protein was recognized by BHV-1 virion specific antisera. Neither of the two larger proteins appear to bind DNA from BHV-1. The smallest protein co-migrates with cellular histones, but no DNA binding proteins with the same molecular weight were found in the virion.

Bovine herpesvirus 1: strain comparison of polypeptides and identification of a neutralization epitope on the 90-kilodalton hemagglutinin.

The intracellular and structural polypeptides of the Los Angeles and Cooper 1 reference strains of bovine herpesvirus 1, together with 12 other Canadian field isolates, were analyzed by polyacrylamide gel electrophoresis. Although a few minor differences were noted among some isolates in regard to intracellular viral protein content, analysis of partly purified virus showed strikingly similar polypeptide profiles among 19 proteins with molecular masses of 14 to 145 kilodaltons (kDa). Moreover, a neutralizing monoclonal antibody produced against the Cooper 1 strain also neutralized all of the other 13 strains tested in this study and immunoprecipitated the major 90-kDa glycoprotein. A second monoclonal antibody with a high hemagglutination inhibition titer prevented hemagglutination of other strains tested and also reacted against the 90-kDa glycoprotein by immunoprecipitation, indicating that this glycoprotein is responsible for the hemagglutinating activity of the viral particle and carries an important neutralization epitope.

Immunologic comparison of the proteins of pseudorabies (Aujeszky's disease) virus and bovine herpesvirus-1.

The immunologic relationship between bovine herpesvirus-1 and pseudorabies virus was examined by 80% serum cross-neutralization test, enzyme-linked immunosorbent assays, and Western immunoblotting procedures. Immunogenic cross reactivity between the 2 viruses was observed with both the serum-neutralization test and the enzyme-linked immunosorbent assay. A probing of viral Western immunoblots with rabbit hyperimmune antiserum showed that there were a number of viral-specific cross-reactive proteins between bovine herpesvirus-1 and pseudorabies virus.

Characterization of envelope proteins of infectious bovine rhinotracheitis virus (bovine herpesvirus 1) by biochemical and immunological methods.

Ten glycoproteins of molecular weights of 180,000, 150,000, 130,000, 115,000, 97,000, 77,000, 74,000, 64,000, 55,000, and 45,000 (designated as 180K, 150K, etc.) and a single nonglycosylated 107,000-molecular-weight (107K) protein were quantitatively removed from purified bovine herpesvirus 1 (BHV-1) virions by detergent treatment. Immunoprecipitations with monospecific and monoclonal antibodies showed that three sets of coprecipitating glycoproteins, 180K/97K, 150K/77K, and 130K/74K/55K, were the major components of the BHV-1 envelope. These glycoproteins were present in the envelope of the virion and on the surface of BHV-1-infected cells and reacted with neutralizing monoclonal and monospecific antibodies. Antibodies to 150K/77K protein had the largest proportion of virus-neutralizing antibodies, followed by antibodies to 180K/97K protein. Monoclonal antibodies to 130K/74K/55K protein were neutralizing but only in the presence of complement; however, monospecific antisera produced with 55K protein did not have neutralizing activity. Analysis under nonreducing conditions showed that the 74K and 55K proteins interact through disulfide bonds to form the 130K molecule. Partial proteolysis studies showed that the 180K protein was a dimeric form of the 97K protein and that the 150K protein was a dimer of the 77K protein, but these dimers were not linked by disulfide bonds. The 107K protein was not glycosylated and induced antibodies that did not neutralize BHV-1. The 64K protein was not precipitated by anti-BHV-1 convalescent antisera, and monospecific antisera to this protein precipitated several polypeptides from uninfected cell lysates, suggesting that 64K is a protein of cellular origin associated with the BHV-1 virion envelope.

Ovarian lesions induced in heifers by intravenous inoculation with modified-live infectious bovine rhinotracheitis virus on the day after breeding.

Four commercially available vaccine strains of modified-live infectious bovine rhinotracheitis virus were passaged once in embryonic bovine kidney cells. Heifers were inoculated IV on the day after breeding with 5.0 ml of nondiluted cell culture fluid of each of the 4 strains. Virus was reisolated from nasal swabs and blood collected during the week after inoculation. The heifers were killed 9 to 14 days after inoculation. Mild-to-marked inflammatory and necrotic lesions were seen in the corpora lutea and ovaries of the heifers. The lesions were similar to, and almost as severe as, those resulting from the inoculation of virulent strains of infectious bovine rhinotracheitis virus. Adrenal lesions were also found in all heifers examined. Virus was reisolated from the ovaries of only 4 of the 8 heifers. However, virus was confirmed in the ovaries of all 8 heifers, using immunofluorescent or ultrastructural studies. Heifers with severe luteal damage had abnormally low plasma progesterone concentrations.

European isolates of bovine herpesvirus 1: a comparison of restriction endonuclease sites, polypeptides, and reactivity with monoclonal antibodies.

Eleven European isolates of bovine herpesvirus 1 (BHV-1), together with two reference virus strains were compared by restriction endonuclease digestion, by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and by their reactivity with a panel of monoclonal antibodies (McAb's). Based on the cleavage pattern of viral DNA with the restriction endonuclease Hind III the strains could be assigned to one of two established major virus types. Analysis by SDS-PAGE of viral polypeptides revealed that four protein species either displayed virus type or subtype specific minor variation of migration characteristics. Of 43 McAb's tested all reacted with all type 1 strains, whereas five antibodies failed to recognize some of the type 2 viruses. The existence of type specific variations among virus specified proteins was further evidenced by the recovery of one McAb recognizing type 1 viruses only. The data show that BHV-1 isolates can be assigned to established virus types according to the SDS-PAGE profile of viral proteins or the selective reactivity with type specific McAb's.

Identification of envelope and nucleocapsid proteins of infectious bovine rhinotracheitis virus by SDS-polyacrylamide gel electrophoresis.

Infectious bovine rhinotracheitis (IBR) virus was purified by rate zonal and isopycnic centrifugation in potassium tartrate gradients. Viral nucleocapsids were isolated from purified virions by treatment with the nonionic detergent Triton X-100 followed by high speed centrifugation. This treatment was shown to produce a suspension of 74% completely de-enveloped nucleocapsids, 24% incompletely de-enveloped nucleocapsids, and 2% whole virions. The viral nucleocapsids contained DNA and banded at a density of 1.25 g/cm3. Analysis of the viral polypeptides by gradient SDS-polyacrylamide gel electrophoresis revealed that 33 virion proteins, ranging in molecular weight from 13,000 to 275,000 dalton, were present in the complete virus particle. Detergent treatment of the virus quantitatively removed two of the major proteins (vp8, 90,000 dalton, and vp13, 73,000 dalton) and partially removed eleven other proteins. Fifteen viral polypeptides appeared to remain firmly associated with the viral nucleocapsids.

Analysis of bovine herpes virus-type 1 isolates by restriction endonuclease fingerprinting.

In an effort to determine whether distinct types of bovine herpesvirus 1 are responsible for causing specific syndromes, the polypeptides and DNA of 93 BHV 1 isolates from the province of Alberta as well as vaccine strains and numerous other Canadian, U.S. and European isolates were analyzed by PAGE and restriction endonuclease fingerprinting, respectively. The polypeptide patterns showed only slight variations: only six isolates contained polypeptides that varied from the norm in their molecular weight, or were absent. Although on the basis of endonuclease patterns the isolates could be categorized into three "strains" and nine "sub-strains", we were unable to associate any of the strains with specific clinical signs. This suggests that the type of disease caused may be determined more by the route of infection and animal management practices than by the inherent properties of certain types of BHV 1. Most of the Albertan isolates were of one BHV 1 type and sub-type.

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.

Proteins Specified by bovine herpesvirus 1 (infectious bovine rhinotracheitis virus).

An electrophoretic analysis of radioactively labeled, purified, "empty" and DNA-containing infectious bovine rhinotracheitis virions revealed the presence of 25 to 33 structural (virion) polypeptides. A total of 11 of these polypeptides could be labeled with [3H]glucosamine and were identified as glycoproteins. In addition to the 25 structural polypeptides, infectious bovine rhinotracheitis virus infected cells also contained at least 15 nonstructural (nonvirion) polypeptides that were not present in purified virions. Expression of the viral polypeptides in infected cells was controlled temporally. Thus, most viral polypeptides could be categorized as "alpha" (immediate early), "beta" (early), or "gamma" (late) on the basis of their order of appearance in infected cells and whether their syntheses were dependent upon prior viral protein or DNA synthesis. None of the glycoproteins belongs to the alpha class, although at least one (GVP11) was synthesized in the absence of viral DNA synthesis. Serum from a cow in which infectious bovine rhinotracheitis virus lesions were reactivated by dexamethasone precipitated both structural and nonstructural polypeptides.

Structural proteins of infectious bovine rhinotracheitis virus.

Infectious bovine rhinotracheitis (IBR) virus grown in bovine embryo kidney cell cultures was concentrated and purified in Ficoll density gradients. The polypeptide composition of the virus was studied by polyacrylamide gel electrophoresis. The mature virion was found to contain 18 structural proteins with molecular weights from 250,000 to 29,000 daltons; 8 of them were glycosylated. The similarity of IBR virus protein composition to proteins of other herpetoviruses is discussed.

Ribonucleotides in infectious bovine rhinotracheitis virus DNA.

Infectious bovine rhinotracheitis (IBR) virus was grown in the presence of 5-3H-uridine in a continuous line of bovine kidney cells. 5-3H-uridine was found to be associated with viral nucleocapsids. Furthermore, purification of the viral nucleic acid present in nucleocapsids illustrated that 5-3H-uridine was part of the viral nucleic acid. Purification of viral DNA from infected cells also indicated that 5-3H-uridine was associated with viral nucleic acid possibly as ribonucleotides. The label was identified as RNA by measuring its susceptibility to RNase and analysis of the bases. Short pulses with 5-3H-uridine, resulted in labelled nucleic acid which was extremely sensitive to RNase and alkali but resistant to DNase. Nucleotide analysis indicated that after short pulses all the radioactivity was associated with the base uracil whereas upon longer labelling periods a large percentage of the label was associated with cytosine. However even if viral DNA was isolated from nucleocapsids there was still some radioactivity associated with uracil. Sedimentation of heat denatured 5-3H-uridine label viral nucleic acid in CS2SO4 indicated that the label sedimented at a density of single stranded DNA suggesting that the ribonucleotides are covalently linked to the viral DNA.

Purification and buoyant density of infectious bovine rhinotracheitis virus.

A purification scheme for infectious bovine rhinotracheitis virus utilizing rate-zonal centrifugation in a 10-40% potassium tartrate gradient was described. The density of IBRV in the potassium tartrate gradient was found to be 1.22 g/cm3. Electron microscopic examination of purified virus preparations revealed homogeneous populations of enveloped virions with minute projections on the envelope surface.