Bovine Herpesvirus 1 Isolates Research Articles

Determination of protection provided by two modified live bovine herpesvirus-1 vaccines against bovine herpesvirus-1 conjunctivitis in calves

A study was performed in 2 phases to determine the ability of 2 different modified live (MLV) bovine herpesvirus-1 (BoHV-1)vaccines to protect against conjunctivitis caused by a recent field isolate of BoHV-1. In phase I, BoHV-1 isolates were used todetermine the ability of the isolates to cause conjunctivitis and to establish a challenge model for phase II. Fifteen calves wererandomly assigned to 1 of 3 groups: 5 calves received 5 mL containing 107 tissue culture infectious dose (TCID)50/mL of BoHV-1adminisered intraocularly (topically) for 2 days, 5 calves received 5 mL of inoculum consisting of 107TCID50/mL of BoHV-1 administered intranasally for 2 days, and 5 calves served as unchallenged sentinels and received phosphatebuffered saline, 5 mL administered by the intranasal and 5 mL by the intraocular route for 2 days. All 15 calves developedpyrexia (rectal temperature greater than 103.5°F (39.7°C)), were BoHV-1 virus isolation positive from ocular swabsand 10/15 calves had increased ocular discharge and conjunctivitis. In phase II, 10 calves were intranasally vaccinated witha MLV BoHV-1, 10 calves were parenterally vaccinated with a MLV BoHV-1, and 5 calves were unvaccinated. All 25 calveswere challenged by receiving 5 mL ntraocularly of 107 TCID50/mL of BoHV-1 for 2 consecutive days. Following challenge, calves were examined daily, and blood (serum), nasal swabs, and conjunctival swab samples were obtained on study days 0, 3-7, 10, 14 and 28. Both vaccines significantly decreased post challenge ocular lesions, clinical scores, and quantity of virus isolated.

Investigation of viral pathogens in cattle with bovine respiratory disease complex in Inner Mongolia, China

As a multifactor disease, the bovine respiratory disease complex (BRDC) causes high morbidity and mortality that is devastating to the cattle industry. To assess viral infections in beef cattle suffering from respiratory diseases in Inner Mongolia, 302 nasal swabs and serum samples were randomly collected from cattle with mild respiratory symptoms between March 2018 and May 2019. Our results showed that the rate of RT-PCR results positive for nucleic acids of viral pathogens in 6 cities was between 54 and 80%.The rates of bovine viral diarrhea virus (BVDV), bovine herpesvirus 1 (BHV-1), bovine parainfluenza virus type 3(BPIV3), and bovine respiratory syncytial virus(BRSV)infections were 44.70% (135/302), 24.83% (75/302), 5.63% (17/302), and 6.95% (21/302),respectively. There are also 8.94% (27/302) of samples were positive for BVDV and BHV-1, and 3.97% (12/302) of samples were positive for BPIV3 and BRSV. In addition, the RT-PCR products were sequenced, and phylogenetic analysis based on these sequences was performed. The results indicated that: a) all of the BVDV isolates were BVDV-1 and were classified as BVDV-1a (66.67%) and BVDV-1b (33.33%); b) all of the BHV-1 isolates were classified as subtype 1.1; 44.44% of the isolates were closely related to modified live viral vaccine strains, and 55.56% of the isolates were closer to epidemic strains; c) all of the BPIV3 isolates belonged to BPIV3c; d) all of the BRSV isolates were classified into subgroup III. It is suggested that an important cause of respiratory diseases for beef cattle is viral infection, and phylogenetic analysis can help us choose the proper strain to develop a vaccine.

A new molecular method for the rapid subtyping of bovine herpesvirus 1 field isolates.

Bovine herpesvirus 1 (BoHV-1) causes several clinical syndromes in cattle worldwide. There are 3 subtypes of BoHV-1: 1.1, 1.2a, and 1.2b. Several molecular methods are commonly used in the detection and characterization of BoHV-1. Among them, restriction endonuclease analysis (REA) and single-nucleotide polymorphism (SNP) analysis of the complete viral genome allow classification of BoHV-1 into different subtypes. However, developing countries need simpler and cheaper screening assays for routine testing. We designed a standard multiplex PCR followed by a REA assay allowing straightforward subclassification of all BoHV-1 isolates tested into 1.1, 1.2a, and 1.2b subtypes based on the analysis of fragment length polymorphism. Our standard multiplex PCR-REA was used to analyze 33 field strains of BoHV-1 isolated from various tissues. The assay confirmed the subtype identified previously by REA. In addition, non-polymorphic or undigested fragments were sequenced in order to confirm the mutation affecting the RE HindIII site. Our PCR-REA method is an affordable and rapid test that will subtype all BoHV-1 strains.

Stress due to vaccination may induce re-activation of latent bovine herpesvirus-1 in cattle

This study evidences that, stress due to vaccination can cause re-activation and clinical outbreak of bovine herpesvirus-1 in latently infected cattle. Three to four days following mass-vaccination of cattle against Foot and Mouth Disease in Karnataka of India, symptoms of acute respiratory distress, conjunctivitis and vulvo-vaginitis were observed in vaccinated cattle in many villages of the state. Nasal and ocular swabs were collected from 25 ailing cattle in six villages of Hassan district of Karnataka. Upon bacteriological analysis, the samples were found negative for Pasteurella multocida. The serum samples from ailing animals were positive for bovine herpesvirus- 1 (BoHV-1) antibodies by Indirect-ELISA. The swab samples were found for BoHV-1 by PCR targeting glycoprotein- C gene of BoHV-1. The swab samples when subjected to virus isolation in MDBK cells yielded characteristic CPE of bunch of grape like clustering of cells by fifth passage. PCR targeting conserved region on glycoprotein-C gene on DNA extracted from cell-culture supernatants showing CPE confirmed the presence of BoHV-1. Nucleotide sequencing of the PCR amplicon showed that the BoHV-1 isolated during this study shared 100% sequence identity with BoHV-1 isolates from India, Switzerland and Brazil. This study emphasizes that stress due to vaccination can cause re-activation and clinical outbreak of bovine herpesvirus-1 in latently infected cattle.

Whole-genome sequence analysis reveals unique SNP profiles to distinguish vaccine and wild-type strains of bovine herpesvirus-1 (BoHV-1)

Bovine herpesvirus-1 (BoHV-1) is a major pathogen affecting cattle worldwide causing primarily respiratory illness referred to as infectious bovine rhinotracheitis (IBR), along with reproductive disorders including abortion and infertility in cattle. While modified live vaccines (MLVs) effectively induce immune response against BoHV-1, they are implicated in disease outbreaks in cattle. Current diagnostic methods cannot distinguish between MLVs and field strains of BoHV-1. We performed whole genome sequencing of 18 BoHV-1 isolates from Pennsylvania and Minnesota along with five BoHV-1 vaccine strains using the Illumina Miseq platform. Based on nucleotide polymorphisms (SNPs) the sequences were clustered into three groups with two different vaccine groups and one distinct cluster of field isolates. Using this information, we developed a novel SNP-based PCR assay that can allow differentiation of vaccine and clinical strains and help accurately determine the incidence of BoHV-1 and the association of MLVs with clinical disease in cattle.

Phylogenetic analysis of glycoprotein B gene sequences of bovine herpesvirus 1 isolates from India reveals the predominance of subtype 1.1.

Aim:This study was conducted for the isolation and molecular characterization of bovine herpesvirus 1 (BoHV-1) isolated from the nasal and vaginal swabs collected from naturally infected cattle showing clinical symptoms of the respiratory disease.Materials and Methods:Isolation of BoHV-1 virus performed on clinical samples collected from 65 cattle from five states of India. The BoHV-1 isolates were further confirmed by polymerase chain reaction (PCR) using primers specific for glycoprotein B (gB) genomic region. PCR amplification was performed using previously published gB gene-specific primer pairs. gB PCR amplicons obtained from all isolates were sequenced, and phylogenetic analysis was performed using software.Results:A total of 12 samples were found positive in cell culture isolation. 11 isolates showed the visible cytopathic effect on Madin-Darby bovine kidney after 72 h. Partial sequence analysis of gB gene of all isolates revealed 99.0-100% homology between them. All isolates showed 99.2-99.8% homology with Cooper stain.Conclusion:BoHV-1.1 is the predominant circulating subtype of BoHV in India, and all isolates have homology with Cooper stain.

Detection and characterization of viruses as field and vaccine strains in feedlot cattle with bovine respiratory disease

This study investigated viruses in bovine respiratory disease (BRD) cases in feedlots, including bovine herpesvirus-1 (BoHV-1), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine coronaviruses (BoCV) and parainfluenza-3 virus (PI3V). Nasal swabs were collected from 114 cattle on initial BRD treatment. Processing included modified live virus (MLV) vaccination. Seven BRD necropsy cases were included for 121 total cases. Mean number of days on feed before first sample was 14.9 days. Swabs and tissue homogenates were tested by gel based PCR (G-PCR), quantitative-PCR (qPCR) and quantitative real time reverse transcriptase PCR (qRT-PCR) and viral culture. There were 87/114 (76.3%) swabs positive for at least one virus by at least one test. All necropsy cases were positive for at least one virus. Of 121 cases, positives included 18/121 (14.9%) BoHV-1; 19/121 (15.7%) BVDV; 76/121 (62.8%) BoCV; 11/121 (9.1%) BRSV; and 10/121 (8.3%) PI3V. For nasal swabs, G-PCR (5 viruses) detected 44/114 (38.6%); q-PCR and qRT-PCR (4 viruses) detected 81/114 (71.6%); and virus isolation detected 40/114 (35.1%). Most were positive for only one or two tests, but not all three tests. Necropsy cases had positives: 5/7 G-PCR, 5/7 q-PCR and qRT-PCR, and all were positive by cell culture. In some cases, G-PCR and both real time PCR were negative for BoHV-1, BVDV, and PI3V in samples positive by culture. PCR did not differentiate field from vaccines strains of BoHV-1, BVDV, and PI3V. However based on sequencing and analysis, field and vaccine strains of culture positive BoHV-1, BoCV, BVDV, and PI3V, 11/18 (61.1%) of BoHV-1 isolates, 6/17 (35.3%) BVDV isolates, and 1/10 (10.0%) PI3V identified as vaccine. BRSV was only identified by PCR testing. Interpretation of laboratory tests is appropriate as molecular based tests and virus isolation cannot separate field from vaccine strains. Additional testing using sequencing appears appropriate for identifying vaccine strains.

Bovine herpesvirus-1: Evaluation of genetic diversity of subtypes derived from field strains of varied clinical syndromes and their relationship to vaccine strains

Bovine herpesvirus-1 (BoHV-1) causes significant disease in cattle. Control programs in North America incorporate vaccination with modified live viral (MLV) or killed (KV) vaccine. BoHV-1 strains are isolated from diseased animals or fetuses after vaccination. There are markers for differentiating MLV from field strains using whole-genome sequencing and analysis identifying single nucleotide polymorphisms (SNPs). Using multiple primer sets and sequencing of products permits association of BoHV-1 isolates with vaccines. To determine association between vaccine virus and strains isolated from clinical cases following vaccination, we analyzed 12 BoHV-1 isolates from animals with various clinical syndromes; 9 corresponded to BoHV-1.1 respiratory group. The remaining three corresponded to BoHV-1.2b, typically found in genital tracts of cattle. Four BoHV-1 isolates were identical to a vaccine strain; three were from post-vaccination abortion episodes with typical herpetic lesions whose dams had received MLV vaccine during pregnancy, and one from a heifer given a related MLV vaccine; Sequences of two respiratory isolates perfectly matched mutations characterizing RLB106 strain, a temperature sensitive mutant used in intranasal and parenteral vaccines. The last three respiratory strains clearly appeared related to a group of MLV vaccines. Previously the MLV vaccines were grouped into four groups based on SNPs patterns. In contrast with above-mentioned isolates that closely matched SNP patterns of their respective MLV vaccine virus, these 3 strains both lacked some and possessed a number of additional mutations compared to a group of MLV vaccine viral genome. Finding BoHV-1.2b in respiratory cases indicates focus should be given BoHV-1.2b as an emerging virus or a virus not recognized nor fully characterized in BRD.

Kennedy, the early sixties, and visitation by the angel of death.

The inaugural issue of Pathologia Veterinaria in 1964 contained the first detailed account of lesions in aborted fetuses following natural, experimental, and postvaccinal infection with bovine herpesvirus 1 (BoHV-1). The article, written by pathologists Kennedy and Richards, described diagnostic gross and histologic features in 13 bovine fetuses. The authors provided clinical and epidemiologic features of 1 postvaccination outbreak, including the absence of clinical signs in infected dams and the propensity for abortions to occur after 6 months' gestation. Subsequent field and experimental studies corroborated and expanded these observations. As a result of this and later reports, veterinarians became alert to the association between infectious bovine rhinotracheitis and abortion, including the risks of exposing pregnant cattle to live vaccinal BoHV-1. Methods were developed to corroborate a morphologic diagnosis of herpetic abortion in cattle, including immunofluorescence, immunohistochemistry, and polymerase chain reaction methods. Outbreaks of postvaccinal BoHV-1 abortion in the United States began to be reported with apparently increased frequency in the early 2000s. This coincided with licensure in 2003 of modified live BoHV-1 vaccines intended for use in pregnant cattle, which are now sold by 3 manufacturers. Ten recent herd episodes of postvaccinal BoHV-1 abortion are reported. All 10 BoHV-1 isolates had single-nucleotide polymorphism (SNPs) profiles previously identified in a group of BoHV-1 isolates that contains vaccine strains, based on a BoHV-1 SNP classification system. They lacked SNP features typical of those in characterized field-type strains of BoHV-1.

Cloning of partial glycoprotein B gene and molecular epidemiological studies of bovine herpes virus -1 isolates

The study describes genetic grouping and molecular epidemiology of bovine herpes virus-1 (BoHV-1) through cloning of partial glycoprotein B gene of BoHV-1 isolates, with special reference to isolates recovered from cattle breeding stations. Samples were collected from 212 animals (91 bulls and 121 female cattle). Avidin-biotin ELISA employed on serum samples found 74 animals as seropositive for BoHV-1. On inoculation of 212 semen/swab samples to MDBK cell line for virus isolation, samples of 4 seropositive and 5 seronegative animals yielded cytopathic changes characteristic of BoHV-1. Partial gB gene of these isolates were cloned in pGEM T vector, nucleotide sequences were deduced and phylogenetic tree was constructed. Sequence analysis grouped 5 of these isolates under BoHV-1.1 cluster having highest sequence identities with previously described Indian, European and Brazilian isolates of BoHV-1.1. The other 4 isolates were clustered as BoHV-1.2 subtypes having 100% sequence identity with European strain of BoHV-1.2. We found that, apparently healthy, seronegative animals can be sources of BoHV-1, attributable to the unique pathogenesis/ latency of BoHV-1. This finding necessitates mandatory culling of breeding animals which are positive either in antigen detection or by serology, especially in countries which do not practice vaccination but report high seroprevalance of BoHV-1.

Genetic characterization of complete open reading frame of glycoprotein C gene of bovine herpesvirus 1

Aim: To characterize one of the major glycoprotein genes viz., glycoprotein C (gC; UL44, unique long region 44) of bovine herpesvirus 1(BoHV1) of Indian origin at genetic and phylogenetic level. Materials and Methods: A bovine herpesvirus 1 isolate viz., (BoHV1/IBR 216 II/ 1976/ India) maintained at Division of Virology, IVRI, Mukteswar was used for the current study. The DNA was extracted using commercial kit and the complete ORF of gC gene was amplified, cloned, and sequenced by conventional Sanger sequencing method. The sequence was genetically and phylogenetically analysed using various bioinformatic tools. The sequence was submitted in the Genbank with accession number Kc756965. Results: The complete ORF of gC gene was amplified and sequenced. It showed 100% sequence homology with reference cooper strain of BoHV1 and divergence varied from 0% to 2.7% with other isolates of BoHV1. The isolate under study had divergence of 9.2%, 13%, 26.6%, and 9.2% with BoHV5 (Bovine herpesvirus 5), CvHV1 (Cervid herpesvirus 1), CpHV1 (Caprine herpesvirus 1), and BuHV1 (Bubaline herpesvirus 1), respectively. Conclusion: This is the first genetic characterization of complete open reading frame (ORF) of glycoprotein C gene (UL44) of Indian isolate of BoHV1. The gC gene of BoHV1 is highly conserved among all BoHV1 isolates and it can be used as a target for designing diagnostic primers for the specific detection of BoHV1.

Bovine herpesvirus-1: Comparison and differentiation of vaccine and field strains based on genomic sequence variation

Bovine herpesvirus-1 (BoHV-1) causes significant disease in cattle including respiratory, fetal diseases, and reproductive tract infections. Control programs usually include vaccination with a modified live viral (MLV) vaccine. On occasion BoHV-1 strains are isolated from diseased animals or fetuses postvaccination. Currently there are no markers for differentiating MLV strains from field strains of BoHV-1. In this study several BoHV-1 strains were sequenced using whole-genome sequencing technologies and the data analyzed to identify single nucleotide polymorphisms (SNPs). Strains sequenced included the reference BoHV-1 Cooper strain (GenBank Accession JX898220), eight commercial MLV vaccine strains, and 14 field strains from cases presented for diagnosis. Based on SNP analyses, the viruses could be classified into groups having similar SNP patterns. The eight MLV strains could be differentiated from one another although some were closely related to each other. A number of field strains isolated from animals with a history of prior vaccination had SNP patterns similar to specific MLV viruses, while other field isolates were very distinct from all vaccine strains. The results indicate that some BoHV-1 isolates from clinically ill cattle/fetuses can be associated with a prior MLV vaccination history, but more information is needed on the rate of BoHV-1 genome sequence change before irrefutable associations can be drawn.

Molecular and in vitro characterization of field isolates of bovine herpesvirus-1

Bovine Herpesvirus-1 (BoHV-1) is distributed worldwide and is a major pathogen in cattle, being the causal agent of a variety of clinical syndromes. The aim of this study was to isolate and to characterize (molecular and biological characterization) BoHV-1 from 29 immunosuppressed animals. It was possible to obtain 18 isolates, each from a different animal, such as from the respiratory and reproductive tracts. In some cases the cytopathic effect was visible 12 hours post-inoculation, and became characteristic after 36-48 hours. Biological characteristics were evaluated and compared with Iowa and Colorado-1 reference strains, and differences were found in plaque size, virus titer measured by TCID50 and PFU/mL, and one step virus curves. These results showed that some isolates had a highly virulent-like behavior in vitro, compared to the reference strains, with shorter eclipse periods, faster release of virus into the supernatants, and higher burst size and viral titer. There were no differences in glycoprotein expression of BoHV-1 isolates, measured by Western blot on monolayers. Moreover, using restriction endonucleases analysis, most of the viruses were confirmed as BoHV-1.1 and just one of them was confirmed as BoHV-1.2a subtype. These findings suggest that some wild-type BoHV-1 isolates could be useful as seeds to develop new monovalent vaccines.

Rhino-trachéite infectieuse bovine (IBR) dans le canton du Jura: enquête épidémiologique

Following an abortion in a beef herd in the summer of 2009, three outbreaks of infectious bovine rhinotracheitis (IBR) were diagnosed in the cantons of Jura and Neuchatel. An epidemiological outbreak investigation was conducted with the aims to identify the source of introduction of the bovine herpes virus 1 (BoHV-1) into the affected herds and to prevent further spread of the disease. The attack rates in the three outbreak farms were 0.89, 0.28 and 0, respectively. BoHV-1 could be isolated from nasal swabs of two animals originating from one of the affected farms. Comparative restriction enzyme analysis revealed slight differences between the isolates of the two animals, but a high similarity to previous BoHV-1 isolates from the canton of Jura, as well as to a French BoHV-1 isolate. This IBR outbreak has shown the importance of reporting and analyzing abortions. The current disease outbreaks recall the main risk factors for the spread of IBR in Switzerland: purchase and movement of bovines and semen of often unknown IBR status.

Epitopes on glycoprotein E and on the glycoprotein E/glycoprotein I complex of bovine herpesvirus 1 are expressed by all of 222 isolates and 11 vaccine strains.

Glycoprotein E (gE) of bovine herpesvirus 1 (BHV1) forms a complex with glycoprotein I (gI) and plays an important role in cell-to-cell spread mechanisms of the virus, but is not essential for propagation of the virus. To study the antigenic variability of BHV1 glycoprotein E, a set of six well characterised monoclonal antibodies (MAbs) was established using BHV1 gE and gI deletion mutants, eukaryotically expressed gE and gI and pepscan analysis. Two of these MAbs reacted with a linear gE epitope (MAbs 3 and 52), two reacted with a more conformation dependent gE epitope (MAbs 61 and 81) and two reacted with epitopes formed by a complex formed between gE and glycoprotein I (MAbs 67 and 75). With these six MAbs the gE expression of 222 BHV1 isolates and 11 BHV1 modified-live vaccine strains was studied in vitro, using an immunoperoxidase monolayer assay. All 222 BHV1 isolates and 11 vaccine strains were found to react with MAbs 61, 81 and 75. Three of the 222 isolates failed to react with MAb 67 and two of the vaccines reacted very weakly with MAbs 3 and 52. Analysis of the gE genes of these five aberrant isolates and the gE glycoproteins they expressed, did not show obvious size differences compared to wild-type BHV1. We conclude that the tested gE epitopes are highly conserved, including the epitopes formed by the gI/gE complex.

Epitopes on glycoprotein C of bovine herpesvirus-1 (BHV-1) that allow differentiation between BHV-1.1 and BHV-1.2 strains.

In cattle, bovine herpesvirus-1 (BHV-1) can cause a mild genital disease known as infectious pustular vulvovaginitis (IPV) and a more severe respiratory disease known as infectious bovine rhinotracheitis (IBR). On the basis of epidemiological data, it has been proposed that these diseases are caused by strains with different genotypes (IBR by BHV-1.1 and IPV by BHV-1.2 strains). By using a panel of 237 BHV-1 isolates, a monoclonal antibody (MAb 71) was found that failed to react with all 54 putative IPV strains in the panel, and another MAb (77) was found that did not react with 16 of these 54 IPV strains. Because MAbs 71 and 77 also failed to react with a BHV-1.1 glycoprotein C (gC)-deletion mutant, it was hypothesized that both MAbs recognize BHV-1.1 gC. By marker-rescue experiments and by expressing fragments of the BHV-1.1 gC gene in recombinant baculoviruses, it was shown that both MAbs indeed recognize BHV-1.1 gC. MAb 71 recognizes the N-terminal half and MAb 77 recognizes the C-terminal half of BHV-1.1 gC. In a PEPSCAN analysis with 12-mer oligopeptides, MAb 71 reacted with overlapping peptides containing gC amino acid residues 75-80 and MAb 77 did not react in this analysis. The differences in gC found in this study may contribute to the biological differences between BHV-1.1 and BHV-1.2.

Characterization of the bovine herpesvirus 1 UL8 gene and gene products.

The bovine herpesvirus 1 (BHV-1) strain Schönböken UL8 gene and the 5' flanking region were sequenced. Comparison of the UL8 ORF with the previously reported UL8 ORF of BHV-1 strain Cooper revealed significant differences that were mainly due to three frame-shifted segments. Reanalysis of the Cooper sequence after isolation of the respective segments from genomic DNA by PCR did not confirm the discrepancies; on the contrary, our results indicate a high degree of sequence conservation between the UL8 proteins of different BHV-1 isolates. A monospecific antiserum, raised against a bacterially expressed TrpE-UL8 fusion protein, identified the 80 kDa apparent molecular mass UL8 polypeptide which is localized in the nucleus of infected cells. Analysis of transcripts and time-course studies demonstrated that the UL8 protein is translated from a delayed-early expressed 3.1 kb polyadenylated mRNA which initiates within the UL9 ORF.

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.

Purification and characterization of bovine herpesvirus-1 isolates and virus DNA utilizing bovine embryonic lung cells

Techniques are described for the culture of bovine embryonic lung (BEL) cells by the primary explant technique and mild enzymatic disaggregation of lung tissue. Preparations of bovine herpesvirus-1 (BHV-1) isolates with titers of 109 plaque-forming units/ml are purified by sucrose and cesium chloride gradient centrifugation after replication in primary cultures of BEL cells. BHV-1 isolated by velocity sedimentation bands in 35% sucrose and sediments at a density of 1.23 g/cc in cesium chloride density gradients. The DNAs of all virus isolates have a velocity sedimentation coefficient of approximately 54S. Virus DNAs from all isolates sediment to equilibrium in cesium chloride at 1.730 g/cc. Differences between virus isolates can be demonstrated by digestion of virus DNA with the restriction endonucleaseKpnI and by polyacrylamide gel electrophoresis of virion polypeptides. These techniques are useful for production of purified high titer virus free of host cell contaminants.

Patterns of bovine T cell-mediated immune responses to bovine herpesvirus 1

Lymphocyte proliferation was used to evaluate T cell-mediated immune responses to different isolates of Bovine Herpesvirus 1 (BHV-1). Groups of high, moderate, and low responses were observed when lymphocytes from three breeds of dairy cattle were stimulated with each of the BHV-1 isolates. Proliferation of cells in the low responding group could be augmented by exogenous IL-2. The mechanism of unresponsiveness by cells from one individual whose response was not altered by IL-2 supplementation was further investigated. The patterns of response by limiting dilution frequency analysis eliminated the possibility that this individual lacked responsive cells but suggested the presence of regulatory cell interactions which resulted in the observed low proliferative response. These results show that animals exposed to the same environment can vary greatly in their ability to respond to BHV-1. At least two mechanisms may be responsible for low proliferative responses in vitro : inadequate levels of IL-2 and the presence of suppressor cells.