Immunization With Bovine Herpesvirus-4-Based Vector Delivering PPRV-H Protein Protects Sheep From PPRV Challenge.

The Morbillivirus peste des petits ruminants virus (PPRV) is the causal agent of a highly contagious disease that mostly affects sheep and goats and produces considerable losses in developing countries. Current PPRV control strategies rely on live-attenuated vaccines, which are not ideal, as they cannot differentiate infected from vaccinated animals (DIVA). Recombinant vector-based vaccines expressing viral subunits can provide an alternative to conventional vaccines, as they can be easily paired with DIVA diagnostic tools. In the present work, we used the bovine herpesvirus-4-based vector (BoHV-4-A) to deliver PPRV hemagglutinin H antigen (BoHV-4-A-PPRV-H-ΔTK). Vaccination with BoHV-4-A-PPRV-H-ΔTK protected sheep from virulent PPRV challenge and prevented virus shedding. Protection correlated with anti-PPRV IgGs, neutralizing antibodies and IFN-γ-producing cells induced by the vaccine. Detection of antibodies exclusively against H-PPRV in animal sera and not against other PPRV viral proteins such as F or N could serve as a DIVA diagnostic test when using BoHV-4-A-PPRV-H-ΔTK as vaccine. Our data indicate that BoHV-4-A-PPRV-H-ΔTK could be a promising new approach for PPRV eradication programs.

Epidemiological study of the association between bovine gammaherpesvirus type 4 and reproductive disease in dairy cattle from northwestern Spain.

Bovine gammaherpesvirus 4 (BoHV-4) has controversially been related with cattle reproductive disease. In the present study we analyze the relationship between exposure to BoHV-4 and reproductive performance in dairy cattle from northwestern Spain. A total of 2022 sera from 50 farms were examined to detect anti-BoHV-4 antibodies. Herd and individual reproductive records were collected to analyze association with exposure to BoHV-4. In addition, 52 abortion cases were examined to detect BoHV-4 DNA. An individual seroprevalence of 66.6 % and a herd prevalence of 98 % were found. Exposure to BoHV-4 increased with age, particularly in individuals between 26-36 months old (OR = 2.7; CI 95 %: 1.2-5.0, compared to animals < 26 months). Seroprevalence was not associated with herd fertility and herd abortion rate, but seropositive animals between 26-36 months presented prolonged calving to fertilizing insemination intervals (HR: 1.4; CI 95 %: 1.2-2.0) as well as higher odds of an unsuccessful 1st insemination (OR: 2.5; CI 95 %: 1.2-5.0). In abortion cases, BoHV-4 DNA was found in 12 vaginal swabs from 5 farms but not in any fetal tissue. Our results reveal an endemic, high and widespread exposure to BoHV-4 among dairy cattle from NW Spain with a limited impact in the reproductive performance of herds. The significantly worse reproductive performance of seropositive animals of 26-36 months of age may be the consequence of the establishment of primo-infections when moving heifers to lactation lots. Our findings may be useful to understand the potential population impact of BoHV-4.

Analysis of the transcripts encoding for antigenic proteins of bovine gammaherpesvirus 4.

The major glycoproteins of bovine gammaherpesvirus 4 (BoHV-4) are gB, gH, gM, gL, and gp180 with gB, gH, and gp180 being the most glycosylated. These glycoproteins participate in cell binding while some act as neutralization targets. Glycosylation of these envelope proteins may be involved in virion protection against neutralization by antibodies. In infected cattle, BoHV-4 induces an immune response characterized by low neutralizing antibody levels or an absence of such antibodies. Therefore, virus seroneutralization in vitro cannot always be easily demonstrated. The aim of this study was to evaluate the neutralizing capacity of 2 Argentine BoHV-4 strains and to associate those findings with the gene expression profiles of the major envelope glycoproteins. Expression of genes coding for the envelope glycoproteins occurred earlier in cells infected with isolate 10/154 than in cells infected with strain 07/435, demonstrating a distinct difference between the strains. Differences in serological response can be attributed to differences in the expression of antigenic proteins or to post-translational modifications that mask neutralizing epitopes. Strain 07/435 induced significantly high titers of neutralizing antibodies in several animal species in addition to bovines. The most relevant serological differences were observed in adult animals. This is the first comprehensive analysis of the expression kinetics of genes coding for BoHV-4 glycoproteins in 2 Argentine strains (genotypes 1 and 2). The results further elucidate the BoHV-4 life cycle and may also help determine the genetic variability of the strains circulating in Argentina.

A recombinant bovine herpesvirus-4 vectored vaccine delivered via intranasal nebulization elicits viral neutralizing antibody titers in cattle.

Recombinant herpesvirus vaccine vectors offer distinct advantages in next-generation vaccine development, primarily due to the ability to establish persistent infections to provide sustainable antigen responses in the host. Recombinant bovine herpesvirus-4 (BoHV-4) has been previously shown to elicit protective immunity in model laboratory animal species against a variety of pathogens. For the first time, we describe the induction of antigen-specific immune responses to two delivered antigens in the host species after intranasal nebulization of recombinant BoHV-4 expressing the chimeric peptide containing the bovine viral diarrhea virus (BVDV) glycoprotein E2 and the bovine herpesvirus 1 (BoHV-1) glycoprotein D (BoHV-4-A-CMV-IgK-gE2gD-TM). In this study, four cattle were immunized via intranasal nebulization with the recombinant BoHV-4 construct. Two of the cattle were previously infected with wild-type BoHV-4, and both developed detectable serologic responses to BVDV and BoHV-1. All four immunized cattle developed detectable viral neutralizing antibody responses to BVDV, and one steer developed a transient viral neutralizing response to BoHV-1. Approximately one year after immunization, immunosuppressive doses of the glucocorticoid dexamethasone were administered intravenously to all four cattle. Within two weeks of immunosuppression, all animals developed viral neutralizing antibody responses to BoHV-1, and all animals maintained BVDV viral neutralizing capacity. Overall, nebulization of BoHV-4-A-CMV-IgK-gE2gD-TM persistently infects cattle, is capable of eliciting antigen-specific immunity following immunization, including in the presence of pre-existing BoHV-4 immunity, and recrudescence of the virus boosts the immune response to BoHV-4-vectored antigens. These results indicate that BoHV-4 is a viable and attractive vaccine delivery platform for use in cattle.

BoHV-4-Based Vector Single Heterologous Antigen Delivery Protects STAT1(-/-) Mice from Monkeypoxvirus Lethal Challenge.

Monkeypox virus (MPXV) is the etiological agent of human (MPX). It is an emerging orthopoxvirus zoonosis in the tropical rain forest of Africa and is endemic in the Congo-basin and sporadic in West Africa; it remains a tropical neglected disease of persons in impoverished rural areas. Interaction of the human population with wildlife increases human infection with MPX virus (MPXV), and infection from human to human is possible. Smallpox vaccination provides good cross-protection against MPX; however, the vaccination campaign ended in Africa in 1980, meaning that a large proportion of the population is currently unprotected against MPXV infection. Disease control hinges on deterring zoonotic exposure to the virus and, barring that, interrupting person-to-person spread. However, there are no FDA-approved therapies against MPX, and current vaccines are limited due to safety concerns. For this reason, new studies on pathogenesis, prophylaxis and therapeutics are still of great interest, not only for the scientific community but also for the governments concerned that MPXV could be used as a bioterror agent. In the present study, a new vaccination strategy approach based on three recombinant bovine herpesvirus 4 (BoHV-4) vectors, each expressing different MPXV glycoproteins, A29L, M1R and B6R were investigated in terms of protection from a lethal MPXV challenge in STAT1 knockout mice. BoHV-4-A-CMV-A29LgD106ΔTK, BoHV-4-A-EF1α-M1RgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK were successfully constructed by recombineering, and their capacity to express their transgene was demonstrated. A small challenge study was performed, and all three recombinant BoHV-4 appeared safe (no weight-loss or obvious adverse events) following intraperitoneal administration. Further, BoHV-4-A-EF1α-M1RgD106ΔTK alone or in combination with BoHV-4-A-CMV-A29LgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK, was shown to be able to protect, 100% alone and 80% in combination, STAT1(-/-) mice against mortality and morbidity. This work demonstrated the efficacy of BoHV-4 based vectors and the use of BoHV-4 as a vaccine-vector platform.

Detection of bovine herpesvirus type 4 antibodies and bovine lymphotropic herpesvirus in New Zealand dairy cows.

AIM: To detect the presence of bovine herpesvirus (BoHV) type 4 in New Zealand dairy cows with clinical metritis. METHODS: Serum samples taken from 92 dairy cows with clinical metritis, each from a different farm, were tested for the presence of antibodies against BoHV-4 using a commercially available, indirect ELISA. Peripheral blood mononuclear cells (PBMC) were collected from 10 BoHV-4 seropositive cows, and PBMC were examined by a pan-herpesvirus nested PCR to detect herpesvirus. PCR products were sequenced directly and a proportion of the PCR products were cloned and sequenced to identify the virus present. RESULTS: Antibodies to BoHV-4 were detected in 23/92 (25%) serum samples. The pan-herpesvirus PCR was positive in 8/10 PBMC samples. Cloning and sequencing identified that all of the eight PCR-positive PBMC contained bovine lymphotropic herpesvirus (BLHV); no BoHV-4 DNA was detected. CONCLUSIONS: This study reports the finding of the presence of apparent antibodies to BoHV-4, and BLHV DNA in New Zealand dairy cows affected by metritis. CLINICAL RELEVANCE: Bovine herpesvirus type 4 and BLHV are reported to have the potential to cause reproduction failure in cows. This is the first report of apparent BoHV-4 antibodies, and BLHV in New Zealand. The importance and epidemiology of these viruses in cattle in New Zealand requires further investigation.

A gammaherpesvirus uses alternative splicing to regulate its tropism and its sensitivity to neutralization.

Human gammaherpesviruses are associated with the development of lymphomas and epithelial malignancies. The heterogeneity of these tumors reflects the ability of these viruses to route infection to different cell types at various stages of their lifecycle. While the Epstein Barr virus uses gp42--human leukocyte antigen class II interaction as a switch of cell tropism, the molecular mechanism that orientates tropism of rhadinoviruses is still poorly defined. Here, we used bovine herpesvirus 4 (BoHV-4) to further elucidate how rhadinoviruses regulate their infectivity. In the absence of any gp42 homolog, BoHV-4 exploits the alternative splicing of its Bo10 gene to produce distinct viral populations that behave differently based on the originating cell. While epithelial cells produce virions with high levels of the accessory envelope protein gp180, encoded by a Bo10 spliced product, myeloid cells express reduced levels of gp180. As a consequence, virions grown in epithelial cells are hardly infectious for CD14+ circulating cells, but are relatively resistant to antibody neutralization due to the shielding property of gp180 for vulnerable entry epitopes. In contrast, myeloid virions readily infect CD14+ circulating cells but are easily neutralized. This molecular switch could therefore allow BoHV-4 to promote either, on the one hand, its dissemination into the organism, or, on the other hand, its transmission between hosts.

Efficient heterologous antigen gene delivery and expression by a replication-attenuated BoHV-4-based vaccine vector.

Bovine Herpesvirus 4 (BoHV-4) is a gammaherpesvirus belonging to the Rhadinovirus genus and due to its biological characteristics has been proposed as a vaccine vector for veterinary vaccines. Because viral vector-associated risk is a major concern for viral vector applications, attenuation is a desirable feature. Therefore, efforts are directed toward the development of highly attenuated viral vectors. BoHV-4 naturally exhibits limited pathogenicity and a further attenuation, in terms of replication, was obtained by disrupting the late gene encoding the 1.7-kb polyadenylated RNA (L1.7). An L1.7 deleted mutant BoHV-4 (BoHV-4-A-KanaGalKΔL1.7), as well as its revertant (BoHV-4-A-Rev), was generated by homologous recombination from the genome of a BoHV-4 isolate (BoHV-4-A) cloned as a bacterial artificial chromosome (BAC). BoHV-4-A-KanaGalKΔL1.7 showed attenuation in terms of competence to reconstitute infectious virus, viral replication, and plaque size when compared to BoHV-4-A, BoHV-4-A-Rev, and BoHV-4-A-KanaGalKΔTK, a recombinant control virus where the KanaGalK selectable marker was inserted into the thymidine kinase open reading frame. The capability of BoHV-4-A-KanaGalKΔL1.7 to deliver and express a heterologous antigen was investigated by replacing the KanaGalK cassette with a vesicular stomatitis virus glycoprotein (VSVg) expression cassette to generate BoHV-4-A-EF1αVSVgΔL1.7. BoHV-4-A-EF1αVSVgΔL1.7 infected cells robustly expressed VSVg, thus confirming that the replication deficiency resulting from L1.7 disruption did not prevent heterologous gene delivery and expression. Although further work is needed to identify the specific function of the BoHV-4 L1.7 gene, the L1.7 gene may represent an ideal targeting locus for the integration of a heterologous antigen expression cassette, resulting in attenuation of the viral vector.

Antibody evasion by a gammaherpesvirus O-glycan shield.

All gammaherpesviruses encode a major glycoprotein homologous to the Epstein-Barr virus gp350. These glycoproteins are often involved in cell binding, and some provide neutralization targets. However, the capacity of gammaherpesviruses for long-term transmission from immune hosts implies that in vivo neutralization is incomplete. In this study, we used Bovine Herpesvirus 4 (BoHV-4) to determine how its gp350 homolog--gp180--contributes to virus replication and neutralization. A lack of gp180 had no impact on the establishment and maintenance of BoHV-4 latency, but markedly sensitized virions to neutralization by immune sera. Antibody had greater access to gB, gH and gL on gp180-deficient virions, including neutralization epitopes. Gp180 appears to be highly O-glycosylated, and removing O-linked glycans from virions also sensitized them to neutralization. It therefore appeared that gp180 provides part of a glycan shield for otherwise vulnerable viral epitopes. Interestingly, this O-glycan shield could be exploited for neutralization by lectins and carbohydrate-specific antibody. The conservation of O-glycosylation sites in all gp350 homologs suggests that this is a general evasion mechanism that may also provide a therapeutic target.

Immunization of knock-out α/ß interferon receptor mice against lethal bluetongue infection with a BoHV-4-based vector expressing BTV-8 VP2 antigen.

New effective tools for vaccine strategies are necessary to limit the spread of bluetongue, an insect-transmitted viral disease of domestic and wild ruminants. In the present study, BoHV-4-based vector cloned as a bacterial artificial chromosome (BAC) was engineered to express the bluetongue virus (BTV) immune-dominant glycoprotein VP2 provided of a heterologous signal peptide to its amino terminal and a trans-membrane domain to its carboxyl terminal (IgK-VP2gDtm), to allow the VP2 expression targeting to the cell membrane fraction. Based on adult α/ß interferon receptor knockout (IFNAR(-/-)) mice, a newly generated bluetongue laboratory animal model, a pre-challenge experiment was performed to test BoHV-4 safety on such immune-compromised animal. BoHV-4 infected IFNAR(-/-) mice did not show clinical signs even following the inoculation of BoHV-4 intra-cerebrally, although many areas of the brain got transduced. IFNAR(-/-) mice intraperitoneally inoculated twice with BoHV-4-A-IgK-VP2gDtm at different time points developed serum neutralizing antibodies against BTV and showed a strongly reduced viremia and a longer survival time when challenged with a lethal dose of BTV-8. The data acquired in this pilot study validate BoHV-4-based vector as a safe and effective heterologous antigen carrier/producer for the formulation of enhanced recombinant immunogens for the vaccination against lethal bluetongue.

Swine adipose stromal cells loaded with recombinant bovine herpesvirus 4 virions expressing a foreign antigen induce potent humoral immune responses in pigs.

Increasingly effective vaccination strategies are needed to counteract the high incidence of contagious diseases associated with intensive swine breeding. Recombinant viral vaccines are a promising new avenue in this direction. Key features of viral vectors suitable for immunoprophylaxis are safety, ease of manipulation and the ability to replicate in a variety of hosts. Most of the above requirements are met by bovine herpesvirus 4 (BoHV-4), a non-pathogenic dsDNA virus capable of infecting a broad range of cell types in vitro. Here we report the results of an exploratory study using an engineered BoHV-4 virus (eBoHV-4) expressing two unrelated glycoprotein antigens from bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1), to assess the potential of recombinant BoHV-4 as a self-adjuvanted immunogen in pigs. Free eBoHV-4 virions and virions preloaded into homologous swine adipose-derived stromal cells (SADSC) were tested. Neither virus formulation elicited neutralizing anti-BoHV-4 antibodies, nor any disease symptom, yet both induced specific immune responses against the heterologous antigens. However, a much earlier (18 vs 28 days post-infection) and more robust neutralizing response against BVDV and BoHV-1 viruses was elicited by eBoHV-4-preinfected SADSCs compared to free virions. The data validate BoHV-4 as a safe and effective heterologous antigen carrier/producer and identify SADSCs as helpful tools for the formulation of increasingly efficacious recombinant immunogens for pig vaccination.

Cellular targeting of engineered heterologous antigens is a determinant factor for bovine herpesvirus 4-based vaccine vector development.

In a previous study, an apathogenic strain of bovine herpesvirus 4 (BoHV-4) cloned as a bacterial artificial chromosome and expressing a chimeric peptide (gE2/gD) as a secreted form was described. Recombinant virus-inoculated animals produced antibodies against bovine viral diarrhea virus (BVDV) gE2 and BoHV-1 gD. However, neutralizing antibodies were produced only against BVDV, not against BoHV-1. In the present work a recombinant BoHV-4 expressing a membrane-linked form of gE2/gD chimeric peptide was constructed, and inoculated rabbits produced serum-neutralizing antibodies against both BVDV and BoHV-1. Protein cell sorting and targeting are a very important issue when immunodominant antigens are engineered for recombinant virus vaccine development.

Double immunization strategy with a BoHV-4-vectorialized secreted chimeric peptide BVDV-E2/BoHV-1-gD.

A bovine herpesvirus 4 was isolated from the milk cell fraction of a healthy cow and his full genome cloned as a bacterial artificial chromosome. So cloned viral genome was used as a vector platform to deliver in vitro and in vivo an optimized secreted chimeric peptide obtained by the fusion of the bovine viral diarrhoea virus glycoprotein E2 ectodomain with the bovine herpesvirus 1 glycoprotein D ectodomain. Recombinant virus infected cells robustly expressed and secreted the chimeric peptide into the culture medium and inoculated animals with the recombinant virus successfully responded toward antigens, gE2 and gD. Thus, this work has implications for the development of safe and effective polyvalent vaccines.

Assessment of bovine herpesvirus 4 based vector in chicken.

The biological characteristics of BoHV-4 make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the ability to accommodate large amounts of foreign genetic material, the ability to infect several cell types from different animal species, such as sheep, goats, swine, cats, dogs, rabbits, mink, horses, turkeys, ferrets, monkeys, hamsters, rats, mice, and chickens. In this report, the feasibility to use BoHV-4 based vector in chicken was investigated. Although BoHV-4 was able to replicate, leading to a cytopathic effect in a chicken cell line and infect the chorion allantoic membrane of embryonated eggs, however it was not pathogenic even when a large dose of virus was injected into the chicken. An immune response could be produced against heterologous antigen delivered by a recombinant BoHV-4. These data suggest the feasibility of using BoHV-4 based vector for vaccination purposes in chickens.

Natural antibody--complement dependent neutralization of bovine herpesvirus 4 by human serum.

In contrast to most gammaherpesviruses, Bovine herpesvirus 4 (BoHV-4) has a broad range of host species both in vitro and in vivo. Several in vitro studies demonstrated that some human cell lines are sensitive or even permissive to BoHV-4. These observations led to the hypothesis that cross-species transmission of BoHV-4 could lead to human infections. In the present study, we investigate the sensitivity of BoHV-4 to neutralization by naïve human sera in order to determine if humans exhibit innate anti-viral activities against this virus. Our results demonstrate that human sera from naïve individuals, in contrast to the sera of naïve subjects from various animal species, neutralize BoHV-4 efficiently. A series of complementary experiments were performed to unravel the mechanism(s) of this neutralization. The data obtained in this study demonstrates that human serum neutralizes BoHV-4 in a complement dependent manner activated by natural antibodies raised against the Galalpha1-3Galbeta1-4GlcNAc-R epitope expressed by bovine cells.

First Serbian isolates of bovine herpesvirus 4 (BoHV-4) from a herd with a history of postpartum metritis.

Two farms in the Belgrade area have experienced serious problems with postpartal metritis. Serological examination of BoHV-4 infection was done using ELISA test and vaginal swabs were used for virus isolation. Average seroprevalence of BoHV-4 in these farms was 84.37%. BoHV-4 isolation was successful from three vaginal swabs on the MDBK cell line. Rising values of BoHV-4 antibodies were recorded in nine of ten cows with clinical signs of postpartal metritis. PCR and restriction analysis were used for better characterisation and isolate classification. Two isolates showed similarity with MOVAR 33/63 virus type, but one differed in polyrepetitive and other parts of DNA. This was the first isolation and characterisation of BoHV4 from Serbian herds.

Recombinant bovine herpesvirus 4 (BoHV-4) expressing glycoprotein D of BoHV-1 is immunogenic and elicits serum-neutralizing antibodies against BoHV-1 in a rabbit model.

Several biological characteristics of bovine herpesvirus 4 (BoHV-4) make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the capability to accommodate large amounts of foreign genetic material, the ability to infect several cell types coming from different animal species, and the ability to maintain transgene expression in both undifferentiated and differentiated cells. Starting from BoHV-4 cloned as a bacterial artificial chromosome (BAC), we used MuA transposase-mediated in vitro transposition to generate recombinant BoHV-4 expressing the immunodominant glycoprotein D (gD) of BoHV-1, one of the most important pathogens of cattle. Although a cis-acting element from woodchuck hepatitis virus (the woodchuck hepatitis virus posttranscriptional regulatory element [WPRE]) in the 3' end of the gD expression cassette was required for maximal gD expression from plasmids in transient transfection assays, this element was not necessary for efficient expression of gD from recombinant BoHV-4 genomes. BoHV-4 recombinants containing gD expression cassettes with or without the WPRE expressed gD at similarly high levels. Several cell lines originating from different animal species expressed gD when infected with BoHV-4 recombinants. When rabbits were immunized with one of the recombinants, high levels of serum neutralizing antibodies against BoHV-1 were generated. This work is one of the first demonstrations of the use BoHV-4 as a vector for vaccine purposes and may provide the basis for BoHV-1 vaccination of cattle with recombinant BoHV-4.

Virological and serological evidence of bovine herpesvirus type 4 in cattle in Northern Ireland.

Bovine herpesvirus type 4 (BHV-4), a member of the genus Rhadinovirus, subfamily Gammaherpesvirinae, within the family Herpesviridae, was isolated in fetal bovine lung cells from samples of vaginal discharge taken from a dairy herd in which approximately 50 per cent of the cattle developed metritis after calving. The identity of the isolate was confirmed by immunofluorescent staining with a BHV-4-specific monoclonal antibody and partial sequencing of a portion of the glycoprotein B gene. Serological testing failed to demonstrate a significant association between the exposure of the cattle to BHV-4 and the metritis, but several cattle seroconverted during the periparturient period, consistent with the recrudescence and shedding of virus associated with the stresses of parturition and the onset of lactation. Despite the previous failure to detect BHV-4 in Northern Ireland, a serological survey of 999 cattle in 49 dairy herds and 51 beef herds found widespread evidence of exposure: 29 of the dairy herds and 35 of the beef herds contained one or more seropositive cattle, and 33.3 per cent of the dairy cattle and 23.3 per cent of the beef cattle were positive.

Detection of bovine herpesvirus 4 in CD11b+ leukocytes of experimentally infected rabbits.

The presence and numbers of bovine herpesvirus 4 (BoHV-4) infected CD11b+ leukocytes were investigated during experimental infections of New Zealand White rabbits by Fluorescence Activated Cell Sorter (FACS) analysis. Peripheral blood leukocytes (PBL) were collected every second day, and the cells were stained with phycoerythrin-labelled CD11b-specific mouse monoclonal antibody and fluorescein-conjugated bovine herpesvirus 4-specific mouse monoclonal antibody. The numbers of double-stained cells from PBLs of the control and inoculated groups were measured and compared in FACSTREK analyser. Double-stained cells were detected in the virus-inoculated group on postinoculation days (PID) 2-5 and 9-12. The results indicated that CD11b+ PBLs were permissive for BoHV-4 infection, and are probably the main reservoir of the virus during the latent period. The data did not indicate production of infectious viral particles, but virus-specific proteins were expressed on the surface of CD11b+ cells. The two waves of double-stained cells gave similar results to the PCR assays from serum samples, which showed the presence of viral DNA in the serum on the same days when virus-infected CD11b cells were also present. Productive BoHV-4 infection of mast cells or undifferentiated leukocytes in the bone marrow and the antiviral immune response might be responsible for this periodic appearance of the virus in CD11b+ PBLs and in the serum. The paper provides evidence that CD11b+ PBLs are the main target cell populations in the blood for BoHV-4.

Latency and persistence of bovine herpesvirus type 4, strain B11-41, in bovine nervous tissues.

Three cattle were experimentally infected with bovine herpesvirus type 4 (BoHV-4), strain B11-41, isolated from the spinal cord of a cow, and monitored for clinical symptoms. None of them showed any clinical signs except increases of leukocyte numbers in two of them, and the body temperature remained normal throughout the experiment. Antibody titers against BoHV-4 continuously increased for one month and were maintained at a high level for more than 1 year by enzyme-linked immunosorbent assay (ELISA). The virus was isolated only from serum and peripheral blood leukocytes (PBL) of one cow in the early stage of infection, but the viral genome was detected in PBL continuously by PCR. When they were euthanized, the viral genome was detected in the lymph nodes and nervous tissues such as medulla, spinal cord, and trigeminal ganglion. These results indicate that cattle are infected with the virus latently and persistently, and the latency site would be in the tissues of the central nervous system as well as lymphoid tissues. When a seroepidemiological survey was performed on antibodies to BoHV-4 among cattle in Japan by ELISA, the rate of antibody-positive cattle was 8.9% and they were found irregularly on certain farms.