Serologic and virologic studies of selected cattle with antibodies to bovine herpesviruses

In order to investigate the specificity of low titer antibodies to BHV 1, twelve cattle were subjected to stress and dexamethasone treatment. They were monitored virologically by inoculating cell cultures with naso-pharyngeal-, ocular- and vaginal- or preputial swabs and serologically by assessing the prevalence and incidence of antibodies to bovine, caprine-, porcine-, and equine herpesviruses and to bovine leukemia virus. Antibodies were classified as specific for BHV 1 if the animals excreted IBR virus, or if the antibodies neutralized BHV 1 and reacted with BHV 1 antigens, or if they reacted additionally with CapHV antigens. Animals whose sera recognized BHV 1 and BHV 2 but not other herpesviruses, were judged to have experienced both infections. Nine of the twelve animals had specific BHV 1 antibodies. With three animals the question for specificity of their antibodies remains open. Two animals experienced several herpesvirus infections. Therefore, the induction of crossreacting antibodies, directed against epitopes common to herpesviruses, could not be ruled out. The sera of one animal reacted with BHV 1 and BHV 4 antigens in ELISA tests. They did, however, not neutralize BHV 1.     

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.     

Antibodies against bovine herpesvirus (BHV) 5 may be differentiated from antibodies against BHV1 in a BHV1 glycoprotein E blocking ELISA

We examined whether antibodies against bovine herpesvirus (BHV) 5 cross-react with BHV1 antigens and whether they could interfere with BHV1 eradication programmes. Six calves were experimentally infected with different doses of BHV5 strain N569; homologous antibodies were first detectable on day 11 post infection; they cross-reacted in a BHV1 virus neutralisation test, in a BHV1-glycoprotein (g)-B blocking ELISA and in a BHV1-gE ELISA, but not in a BHV1-gE blocking ELISA. This study indicates that, in ongoing BHV1 eradication programmes, based on vaccines that lack gE, BHV5 infections may not lead to false-positive serological reactions in case cattle are tested for BHV1-gE antibodies by the BHV1-gE blocking ELISA; antibodies against BHV5 may be differentiated from antibodies against BHV1. The BHV1-gE blocking ELISA may, therefore, offer opportunities for the serological differentiation between BHV1 and BHV5 infections.     

Aujeszky's disease ELISA: cross-reactions with other herpesvirus antisera

The production of antibodies in pigs to 11 herpesviruses was investigated in relation to their ability to cross-react with Aujeszky's disease virus (suid herpesvirus 1--SHV1). Of the herpesviruses tested only two, sheep herpesvirus (caprine herpesvirus 1) and dog herpesvirus (canid herpesvirus 1), failed to produce homologous virus antibodies. Only the antibodies to bovine herpesvirus 1 (BHV1) produced a cross-reaction by SHV1 enzyme-linked immunosorbent assay (ELISA). No SHV1 neutralizing antibodies were detected in any of the herpesvirus antisera. A cross-reaction with SHV1 by a serum from a pig naturally infected with BHV1 or with any of the other herpesviruses tested was considered unlikely.     

甲型疱疹病毒亚科的疱疹病毒囊膜糖蛋白gC对病毒感染复制的影响

甲型疱疹病毒亚科的疱疹病毒宿主广泛,能感染哺乳类、两栖类和禽类,主要侵害宿主的皮肤、黏膜和神经组织,病毒还会在宿主神经组织潜伏感染,一经感染,难以清除。gC是甲型疱疹病毒亚科的病毒囊膜糖蛋白之一,在病毒感染复制过程中发挥了重要作用。gC与细胞上受体结合帮助病毒吸附至宿主细胞表面、介导低pH条件下病毒入侵上皮细胞、影响病毒基因组的复制。此外,gC帮助病毒逃避补体和抗体的中和,促进病毒的吸附入侵。本文就gC影响病毒感染复制的相关功能进行综述,旨在为研究gC和深入了解甲型疱疹病毒亚科病毒的生命周期,以及gC亚单位疫苗和mRNA疫苗的研究提供参考。     

Development and trial of a bovine herpesvirus 1 - thymidine kinase deletion virus as a vaccine

SUMMARY An Australian bovine herpesvirus 1 (BHV1) isolate with a defined (427 base pair) deletion in the protein coding region of the thymidine kinase gene was obtained by standard marker rescue procedures. After selection in the presence of the nucleotide analogue 5iodo-deoxy-uridine the virus was analysed by hybridisation with three differential oligonucleotide probes, restriction endonuclease profile studies and DNA sequence analysis. The virus elicited an immune response in recipient animals after either intramuscular or intravenous administration and produced no significant deleterious side-effects when administered at a dose sufficient to stimulate the host immune response. The safety and immunogenicity of the recombinant BHV1 virus 39B1 were similar to those reported for other registered BHV1 vaccines and the virus would appear to be suitable for the production of a vaccine seed lot and more exhaustive field trials as a prelude to commercial vaccine production and registration.     

Diagnosis and prophylaxis of infectious bovine rhinotracheitis: the role of virus latency

Efficient methods of diagnosis and prophylaxis of infectious bovine rhinotracheitis must consider the concept of latency of the etiological agent, infectious bovine rhinotracheitis virus (Bovine herpesvirus 1; BHV 1). The identification of BHV 1 in nasal mucus samples or a rise in specific antibodies have to be cautiously interpreted, because they can signify either a primary infection or a reexcretion of the virus after reactivation. The isolated virus can also either be a vaccine or a virulent strain. Another aspect of BHV 1 infection diagnosis is the detection of latent carriers, which are able to transmit the virus to uninfected animals; delayed hypersensitivity test seems to be a good candidate. The classical methods of prophylaxis protect the animal against the disease, but they should also impede the reexcretion of virulent strains by latent carriers. Since, in several countries, attenuated viruses are used as vaccines, a special emphasis has to be laid on the persistence of these vaccine viruses in a latent form in the bovine population.     

Eradication of infectious bovine rhinotracheitis in Switzerland: review and prospects

Ten years after the first outbreak of infectious bovine rhinotracheitis (IBR) in Swiss dairy cows, the national cattle herd is almost free from infection with IBR virus (bovine herpesvirus 1, BHV 1). The national programme for the eradication of IBR was divided into four phases: (1) Prevention of transmission of the infection by restrictions on trade of bovines and assessment of the prevalence of cattle with antibodies to BHV 1. (2) Slaughtering animals with antibodies to BHV 1 in order to eradicate BHV 1 from breeding herds. (3) Detection and eradication of further BHV 1 reservoirs (e.g. fattening cattle). (4) Monitoring programme and legal actions in order to maintain the favourable situation. Approximately 50,000 animals were slaughtered in the course of the eradication of IBR. The total costs amounted to approximately SFr. 110,000,000 over 10 years. The costs for maintaining the situation are estimated at approximately SFr. 5,000,000 per annum.     

Encephalitis induced by bovine herpesvirus 5 and protection by prior vaccination or infection with bovine herpesvirus 1.

Calves were intranasally challenged with bovine herpesvirus 5 (BHV5) and followed for the development of viral infection, clinical encephalitis, histologic lesions in the brain, and viral sequences in the trigeminal ganglia. Calves that were previously vaccinated with bovine herepesvirus 1 (BHV1, n = 4) or previously infected with BHV1 (n = 5) or that had not been exposed to either virus (n = 4) were compared. No calf developed signs of encephalitis, although all calves developed an infection as indicated by nasal secretion of BHV5 and seroconversion to the virus. Histologic lesions of encephalitis consisting of multifocal gliosis and perivascular cuffs of lymphocytes were observed in calves not previously exposed to BHV1. BHV5 sequences were amplified from the trigeminal ganglia of calves previously vaccinated and from calves not previously exposed to BHV1; calves sequentially challenged with BHV1 and later BHV5 had exclusively BHV1 sequences in their trigeminal ganglia. Administration of dexamethasone 28 days after BHV5 challenge did not influence clinical disease or histologic lesions in either previously unexposed calves (n = 2) or previously immunized calves (n = 2), although it did cause recrudescence of BHV5, as detected by nasal virus secretion.     

The glycoproteins of the human herpesviruses.

The herpesvirus family contains several important human pathogens. Human herpesviruses include herpes simplex virus type 1 and 2, varicella-zoster virus, human cytomegalovirus, Epstein-Barr virus and human T-cell lymphotropic virus. The general property of herpesviruses is their ability to establish latency and to be periodically reactivated. All human herpesviruses contain a subset of genes encoding viral glycoproteins that are clearly homologous, and their similarity is significantly greater among members of the same subfamily. Membrane glycoproteins specified by human herpesviruses are important determinants of viral pathogenicity. They are exposed on the viral envelope and on the surface of infected cells. They mediate entry of the virus into cells and cell-to-cell spread of infection and also influence tissue tropism and host range. Viral membrane glycoproteins are also the most important elicitors of protective immune response and are therefore the best candidates for subunit vaccines.     

Reactivation of Bovid herpesvirus 1 and 2 and Parainfluenza-3 virus in calves latently infected

Two experiments were carried out to determine whether Bovid herpsvirus (BHV) 2 is able to induce a recurrent infection in experimentally infected calves. In the first experiment the stress induced by dexamethasone (DMS) treatment failed to reactivate the clinical condition or to induce shedding of BHV2. However, treatment with DMS reactivated a latent BHV1 infection in all calves previously inoculated with BHV2 and also in two noninoculated controls. Probably, because of the interference by BHV1 the study failed to resolve the question as to whether BHV2 could induce a recurrent infection. Consequently, a second experiment was performed using calves devoid of antibody to BHV1 and, therefore, probably, free of virus. By this study it was demonstrated that BHV2 can remain as a latent infection in cattle, which, when immunosuppressed as with DMS, can be reactivated. A finding of considerable interest in this experiment was that in 1 calf a concurrent piroplasma infection was also, unexpectedly, discovered.Recrudescence of latent BHV1 infection was induced by DMS treatment of calves possessing antibody to the virus. The infection once reactivated, was readly transmitted by contact to three other calves devoid of antibody to BHV1. In the same experiment Parainfluenza-3 (PI-3) virus was unexpectedly isolated from all calves. It was speculated that all calves were latently infected with PI-3 virus with concurrent infection by BHV1 acting as a stress inducing PI-3 reactivation.These studies seem to indicate that mixed infections could have an important role in the mechanism involved in the establishment of latent infections and viral reactivation.     

Infection of sheep and goats with bovid herpesvirus 2

Sheep and goats were shown to be susceptible to experimental infection with bovid herpesvirus 2 (BHV2), administered by either the intradermal or intravenous route. Lesions developing in sheep following intradermal inoculation of virus were similar to those in cattle inoculated intradermally, whereas the lesions in goats resolved without ulceration or scabbing. Disseminated circumscribed skin lesions developed in sheep and goats given BHV2 by the intravenous route. These lesions resolved in four to eight days without significant effect on the skin. BHV2 was isolated from skin lesions of sheep, goats and cattle that were infected intravenously, from sheep and cattle infected intradermally and from the leucocytes of the three species following intravenous inoculation of virus. Latent infection of sheep and goats was demonstrated following corticosteroid treatment 60 days after infection.     

A polymerase chain reaction (PCR) assay for the detection of bovine herpesvirus 1 (BHV1) in selectively digested whole bovine semen

A PCR assay for the detection of bovine herpesvirus type 1 (BHV1) DNA in selectively digested whole bovine semen was developed and evaluated. A brief treatment with proteinase-K was used to lyse free virus, virus present in non-sperm cells and virus adhering to the spermatozoa. Genomic bovine DNA was not released by this treatment. Primers and probes were based on the nucleotide sequence of the gD gene. BHV1 virus-spiked split samples were used as positive controls and the PCR products were detected by eye in ethidium bromide-stained agarose gels. Sequentially collected non-extended semen samples from experimentally infected bulls were used to compare this assay with virus isolation. Of a total of 162 ejaculates, 51 were found positive by virus isolation, whereas PCR detected BHV1 DNA in 73. PCR detected BHV1 DNA for a longer period after infection and reaction. Apart from its superior sensitivity, this PCR assay also has the advantage of being a relatively simple procedure, providing results within 24 h.Abbreviations AI artificial insemination - BHV1 bovine herpesvirus type 1 - PCR polymerase chain reaction - TCID₅₀ tissue culture infective dose, 50%     

Humoral Immune Response and Assessment of Vaccine Virus Shedding in Calves Receiving Modified Live Virus Vaccines Containing Bovine Herpesvirus‐1 and Bovine Viral Diarrhoea Virus 1a

Susceptible calves were administered modified live virus (MLV) vaccines containing bovine herpesvirus‐1 (BHV1) and bovine viral diarrhoea type 1 (BVDV1a) strains intramuscularly, with one vaccine containing both MLV and inactivated BHV‐1 and inactivated BVDV1a. There was no evidence of transmission of vaccine (BHV‐1 and BVDV1a) strains to susceptible non‐vaccinated controls commingled with vaccinates. No vaccinates had detectable BHV‐1 in peripheral blood leucocytes (PBL) after vaccination. Each of three vaccines containing an MLV BVDV1a strain caused a transient BVDV vaccine induced viremia in PBL after vaccination, which was cleared as the calves developed serum BVDV1 antibodies. The vaccine containing both MLV and inactivated BHV‐1 induced serum BHV‐1 antibodies more rapid than MLV BHV‐1 vaccine. Two doses of MLV BHV‐1 (days 0 and 28) in some cases induced serum BHV‐1 antibodies to higher levels and greater duration than one dose.     

Improvement of serological discrimination between herpesvirus-infected animals and animals vaccinated with marker vaccines

Control/eradication plans of bovine herpesvirus 1 (BHV1) and suid herpesvirus 1 (SHV1) infections involve vaccination with inactivated or attenuated gE-deleted marker vaccines and associated companion serological tests to discriminate naturally infected from vaccinated animals. Blocking or competitive enzyme-linked immunosorbent assays (ELISAs) have been designed for the detection of specific antibodies against BHV1 or SHV1 gE glycoprotein. The antigen source usually consists of a crude viral preparation in which gE is associated with other envelope glycoproteins. Such assays suffer from a lack of specificity which is not due to serological cross-reactions with other pathogens. Interestingly, false-positive results occur with sera collected from multivaccinated cattle or pigs. After multivaccination with a marker vaccine, the binding of the conjugated monoclonal antibody used as a tracer, could be hampered by antibodies directed against the other viral glycoproteins.In order to validate the steric hindrance hypothesis, a simple preadsorption of such samples was carried out with a preparation of antigen devoid of gE, prior to the blocking ELISA itself. The decrease in antibody concentrations against the major glycoproteins, clearly leads to a better discrimination between positive and negative samples; that is between infected and multivaccinated animals, without significant loss of sensitivity. This experiment confirms the steric hindrance hypothesis, therefore serum preadsorption could be an easy way to improve the specificity of currently available diagnostic tests.     

A rapid and sensitive PCR-based diagnostic assay to detect bovine herpesvirus 1 in routine diagnostic submissions

We describe a rapid, sensitive and specific polymerase chain reaction (PCR) assay for the detection of BHV1 DNA in a range of routine diagnostic submissions without the need for prior virus isolation. The assay, which is based on the selected amplification of a portion of the viral tk gene, detected both BHV1.1 and BHV1.2 subtypes in a panel of 15 characterised field isolates, and its sensitivity was estimated to be <0.125 TCID(50). BHV2, alcephaline herpesvirus, BHV4, equine herpesvirus 1 (EHV1), EHV4 and pseudorabies virus were not detected confirming the specificity of the assay. One hundred and five diagnostic submissions, including tissues, nasal secretions and nasal swabs were taken from cattle with respiratory disease and tested using the routine methods of virus isolation (VI) and the fluorescent antibody test (FAT), and the results were compared with those obtained by PCR. The PCR assay detected BHV1 DNA in all samples that were positive by VI. BHV1 DNA was also detectable by PCR in raw and extended semen samples at a sensitivity of 1 TCID(50) per 50microl. The assay also detected BHV5, permitting differentiation between it and BHV1 by virtue of the size of the amplified PCR product. The PCR assay is more sensitive and independent of sample quality than either virus isolation or FAT, and it is faster than virus isolation. The sample preparation method is simple with few steps involved. There are no extra post-amplification blotting/hybridisation steps and the assay is not based on a nested PCR strategy that might otherwise exacerbate the problem of oversensitivity/contamination in the routine use of such a test in a diagnostic laboratory. This assay would permit discrimination between those animals naturally infected with wild type BHV1 and those vaccinated with tk-BHV1 strains.     

An evaluation of five serological tests for the detection of antibody to bovine herpesvirus 1 in vaccinated and experimentally infected cattle

More than 300 bovine sera from a previously reported vaccination and challenge trial were tested for antibodies to bovine herpesvirus 1 (BHV1) by five serological assays: enzyme-linked immunosorbent assay (ELISA) for IgM and IgG, passive haemagglutination (PHA), and two methods of virus neutralisation (VN). In a statistical comparison of ELISA (IgG), PHA and VN results, the assays showed highly significant correlations (P less than 0.01). The sensitivities of ELISA and 24-hour neutralisation tests were similar, in contrast to passive haemagglutination and one hour neutralisation which failed to detect BHV1 antibodies in some low titre sera.     

Homologous and heterologous antibody responses of mice immunized with purified feline herpesvirus type 1 and canine herpesvirus glycoproteins.

The three glycoproteins each of feline herpesvirus type 1 (FHV-1) and canine herpesvirus (CHV) were purified by affinity chromatography using glycoprotein-specific monoclonal antibodies and used individually or in combination in immunizing mice to determine their relative immunogenicity. All the glycoproteins induced detectable virus neutralizing antibodies to the homologous virus but FHV-1 gp143/108 and its cross-reacting counterpart, CHV gp145/112, elicited the highest titers not only to the homologous virus but to the heterologous virus as well. The production of ELISA antibodies after glycoprotein immunization was variable, while hemagglutination-inhibiting antibodies were produced by only 1 out of 10 FHV-1 gp60-inoculated mice. In general, the antibody titers induced by CHV glycoproteins were lower than those by FHV-1 glycoproteins. These results indicate that these glycoproteins may be useful as subunit vaccines against FHV-1 and CHV infections.     

Development of a Multiplex Polymerase Chain Reaction for the Differentiation of Bovine Herpesvirus‐1 and ‐5

Bovine herpesvirus‐1 (BHV‐1) and bovine herpesvirus‐5 (BHV‐5) are closely related viruses which exhibit some important differences at the genetic and immunogenic levels which may explain the differences in their pathogenicity and epidemiological characteristics. A multiplex polymerase chain reaction (M‐PCR) was developed to detect and differentiate between BHV‐1 and BHV‐5. In this M‐PCR two pairs of primers (TK1, TK2 and GD1, GD2) were used in the same reaction mix to amplify a thymidine kinase genomic region (183 bp) of BHV‐1 and one genomic region of the glycoprotein D (564 bp) of BHV‐5. The specificity of the M‐PCR was demonstrated when using both primers pairs simultaneously with BHV‐1 and BHV‐5 templates. The two expected bands were amplified without the apparition of non‐specific products. However, when other herpesvirus strains were used, there was no amplification. To evaluate the sensitivity of the assay, dilutions of purified viral DNA were made for M‐PCR amplification. The detection limit was 7 pg for BHV‐1 and 22 pg for BHV‐5. It was also determined by comparing the M‐PCR with viral isolation. M‐PCR was able to detect one log₁₀ more than viral isolation for BHV‐1 and for BHV‐5 was two logarithms lower. The applicability of M‐PCR was demonstrated on different specimens. Twenty isolates from field samples (11 BHV‐1 and nine BHV‐5) were positive by M‐PCR, and the results were completely coincident with previous characterization using the immunoperoxidase assay. M‐PCR could detect viral DNA in organ samples from natural infections, such as semen and brain. In addition, M‐PCR detected more positive samples than observation of the citophatic effect in cell culture of nasal swabs from experimentally infected animals in two different assays. Owing to the difference in size of the M‐PCR products which allows easy identification in an electrophoretic run, it is not necessary to use extra blotting and hybridization steps or a second round of amplification to differentiate clearly between BHV‐1 and BHV‐5.     

Immune production of interferon by cultured peripheral blood mononuclear cells from calves infected with BHV1 and PI3 viruses

Peripheral blood mononuclear cells (PBMC) from calves infected with bovine herpesvirus type 1 (BHV1) or parainfluenza 3 virus (PI3) were cultured in vitro in the presence of inactivated specific antigen presented on MDBK cells. In the presence of inactivated antigen, PBMC from both BHV1-infected and control calves produced interferon (IFN)-alpha in 24 hour cultures. Altering the culture conditions did not result in the detection of immune-specific IFN produced by mononuclear cells from BHV1-infected calves. However, spontaneous IFN was detected in the absence of antigen in 24 hour cultures from infected animals: this IFN was pH 2 labile and completely neutralised by antiserum to recombinant bovine IFN-gamma. Spontaneous IFN-gamma production was only seen in calves following a second BHV1 inoculation, given four to seven weeks after the primary dose. In contrast PBMC cultures from PI3 virus-infected calves did not produce IFN-gamma spontaneously, but did so in cultures which contained inactivated PI3 antigen. Mononuclear cells from control animals failed to produce either IFN-alpha or -gamma when cultured with inactivated PI3 virus. IFN-gamma was detected in PBMC cultures after the primary infection, with no increase in production occurring following subsequent PI3 virus inoculations. Immunospecific production of IFN-gamma provides a simple method for monitoring cell-mediated immunity in BHV1- and PI3 virus-infected calves and can be used for evaluating the efficacy of vaccines against these viruses.