Bovine Herpesvirus Type Research Articles

Presence of Bovine herpesvirus type 4 (BHV-4) infection in bulls for artificial insemination in Serbia

Bovine herpesvirus type 4 (BHV-4) is a worldwide distributed gammaherpesvirus, with different antigenic and biologic properties from all the other bovine herpesviruses. In vivo distribution of BHV-4 was examined by testing various organs of experimentally infected calves. However, limited data regarding excretion and transmission of BHV-4 from naturally infected cattle are available. In this report, we describe the detection of BHV-4 in the semen of bulls used for artificial insemination. Blood and semen of bulls from two major centers for artificial insemination (A.I. center) were examined in this study. In order to detect the presence of BHV-4 infection in the studied herds, 35 serum samples from Center I and 15 from Center II were examined using indirect ELISA for detection of specific anti-BHV4 IgGl immunoglobulins. Serological examination showed that 18 out of 50 serum samples were positive for BHV-4 antibodies. Despite the fact that BHV-4 was not isolated from semen samples the, presence of BHV-4 was detected in one sample using the nested PCR. This fact can be used to presuppose that artificial insemination is a potential route for BHV-4 transmission. .

Caracterização clinicopatológica da mamilite aguda em ovelhas lactantes infectadas experimentalmente com o herpesvírus bovino 2

A mamilite causada pelo herpesvírus bovino tipo 2 (BoHV-2) possui importante repercussão sanitária em gado leiteiro de alguns países, mas a sua patogenia permanece pouco conhecida. Este trabalho descreve a reprodução e caracterização da mamilite aguda em ovelhas lactantes inoculadas com o BoHV-2 na pele do úbere e dos tetos. Cinco de oito ovelhas inoculadas desenvolveram extensas placas com necrose focal, pequenas vesículas e formação de crostas nos locais de inoculação. As lesões foram inicialmente observadas no 4º dia pós-inoculação (pi), progrediram em extensão e gravidade até os dias 7-8pi e posteriormente regrediram. O vírus foi isolado das lesões entre os dias 7 e 8 pi. Antígenos virais e partículas típicas de herpesvírus foram demonstrados por microscopia eletrônica em biópsias de lesões coletadas nos dias 5, 6 e 10pi. Os achados histológicos foram caracterizados por necrose epitelial, erosões e úlceras, com formação de células sinciciais e infiltrado inflamatório linfoplasmocitário na derme. Associadas a essas lesões observaram-se inclusões eosinofílicas intranucleares em células epiteliais, sinciciais e inflamatórias. Em um segundo experimento, 7 de 10 cordeiros inoculados na mucosa nasal desenvolveram hiperemia e descarga nasal, bem como vesículas e erosões no focinho. O vírus foi isolado das secreções nasais por até três dias e todos os cordeiros soroconverteram ao BoHV-2. Tentativas de reativar a infecção latente pela administração de dexametasona no 40º dia pi foram infrutíferas, pois não foram observados re-excreção viral, recrudescência clínica ou soroconversão. Esses resultados demonstram que ovelhas são susceptíveis à infecção experimental pelo BoHV-2 e desenvolvem lesões semelhantes às observadas em casos naturais de mamilite herpética. Além disso, esses resultados sugerem o uso dessa espécie animal como modelo para o estudo de vários aspectos da biologia da infecção pelo BoHV-2.

Respiratory Disease Associated with Bovine Coronavirus Infection in Cattle Herds in Southern Italy

Four outbreaks of bovine respiratory disease (BRD) associated with bovine coronavirus (BCoV) infection in Italian cattle herds were reported. In 3 outbreaks, BRD was observed only in 2-3-month-old feedlot calves, whereas in the remaining outbreak, lactating cows, heifers, and calves were simultaneously affected. By using reverse transcription polymerase chain reaction (RT-PCR), BCoV RNA was detected in all outbreaks without evidence of concurrent viral pathogens (i.e., bovine respiratory syncytial virus, bovine herpesvirus type 1, bovine viral diarrhea virus, bovine parainfluenza virus). Common bacteria of cattle were recovered only from 2 outbreaks of BRD: Staphylococcus spp. and Proteus mirabilis (outbreak 1) and Mannheimia haemolytica (outbreak 4). A recently established real-time RT-PCR assay showed that viral RNA loads in nasal secretions ranged between 3.10 x 10(2) and 7.50 x 10(7) RNA copies/microl of template. Bovine coronavirus was isolated from respiratory specimens from all outbreaks except outbreak 1, in which real-time RT-PCR found very low viral titers in nasal swabs.

Identification of two small RNAs within the first 1.5-kb of the herpes simplex virus type 1–encoded latency-associated transcript

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is abundantly expressed in latently infected neurons. In the rabbit or mouse ocular models of infection, expression of the first 1.5 kb of LAT coding sequences is sufficient for and necessary for wild-type levels of spontaneous reactivation from latency. The antiapoptosis functions of LAT, which maps to the same 1.5 kb of LAT, are important for the latency-reactivation cycle because replacement of LAT with other antiapoptosis genes (the baculovirus IAP gene or the bovine herpesvirus type 1 latency-related gene) restores wild-type levels of reactivation to a LAT null mutant. A recent study identified a micro-RNA within LAT that can inhibit apoptosis (Gupta et al, Nature 442: 82-85). In this study, the authors analyzed the first 1.5 kb of LAT for additional small RNAs that may have regulatory functions. Two LAT-specific small RNAs were detected in productively infected human neuroblastoma cells within the first 1.5 kb of LAT, in a region that is important for inhibiting apoptosis. Although these small RNAs possess extensive secondary structure and a stem-loop structure, bands migrating near 23 bases were not detected suggesting these small RNAs are not true micro-RNAs. Both of the small LAT-specific RNAs have the potential to base pair with the ICP4 mRNA. These two small LAT RNAs may play a role in the latency-reactivation cycle by reducing apoptosis and/or by reducing ICP4 RNA expression.

Bovine herpesvirus type 1 induces cell death by a cell-type-dependent fashion

Bovine herpesvirus 1 (BHV-1), a member of the alpha-herpesvirinae sub-family, causes significant losses to the cattle industry. BHV-1 establishes latency in trigeminal ganglionic sensory neurons, but periodically reactivates from latency. Previous studies suggested that infection with BHV-1-induced novel morphological changes in rabbit skin (RS) cells versus bovine kidney cells (MDBK). Consequently, we hypothesized that viral infection led to a novel form of cell death in RS cells compared to MDBK cells. To test this hypothesis, we examined the levels of apoptosis in these cell types following infection with BHV-1. Infection of RS, but not MDBK, cells leads to high levels of apoptosis compared to mock-infected cells. Previous studies indicated that a BHV-1 recombinant virus that does not express the bICP0 protein grows poorly in permissive cells and induces a persistent-like infection. This suggested that bICP0 played an important role in regulating cell death following infection. To test this hypothesis, we compared the levels of apoptosis in cells infected with the bICP0 null mutant versus viral strains that expressed bICP0. The bICP0 null mutant induces low levels of apoptosis in RS or MDBK cells. When MDBK cells are treated with UV light prior to infection, bICP0 expressing viral strains, but not the bICP0 null mutant, inhibited UV-induced apoptosis. Infection of MDBK cells with the bICP0 null mutant, leads to an accumulation of autophagosomes that are not detected following infection with bICP0 expressing viruses. These studies suggest that the bICP0 null mutant induces autophagy in MDBK cells, and bICP0 protein expression mediates cell-type specific cytotoxicity.

A review of the biology of bovine herpesvirus type 1 (BHV-1), its role as a cofactor in the bovine respiratory disease complex and development of improved vaccines

Infection of cattle by bovine herpesvirus type 1 (BHV-1) can lead to upper respiratory tract disorders, conjunctivitis, genital disorders and immune suppression. BHV-1-induced immune suppression initiates bovine respiratory disease complex (BRDC), which costs the US cattle industry approximately 3 billion dollars annually. BHV-1 encodes at least three proteins that can inhibit specific arms of the immune system: (i) bICP0 inhibits interferon-dependent transcription, (ii) the UL41.5 protein inhibits CD8+ T-cell recognition of infected cells by preventing trafficking of viral peptides to the surface of the cells and (iii) glycoprotein G is a chemokine-binding protein that prevents homing of lymphocytes to sights of infection. Following acute infection of calves, BHV-1 can also infect and induce high levels of apoptosis of CD4+ T-cells. Consequently, the ability of BHV-1 to impair the immune response can lead to BRDC. Following acute infection, BHV-1 establishes latency in sensory neurons of trigeminal ganglia (TG) and germinal centers of pharyngeal tonsil. Periodically BHV-1 reactivates from latency, virus is shed, and consequently virus transmission occurs. Two viral genes, the latency related gene and ORF-E are abundantly expressed during latency, suggesting that they regulate the latency-reactivation cycle. The ability of BHV-1 to enter permissive cells, infect sensory neurons and promote virus spread from sensory neurons to mucosal surfaces following reactivation from latency is also regulated by several viral glycoproteins. The focus of this review is to summarize the biology of BHV-1 and how this relates to BRDC.

A live bovine herpesvirus-1 marker vaccine is not shed after intramuscular vaccination.

It should be established, whether animals vaccinated intramuscularly (IM) with a live Bovine herpesvirus type 1 (BHV-1) marker vaccine become viremic and/or excrete vaccine virus with nasal discharge. Five cattle, seronegative for BHV-1, were vaccinated with an overdose of the vaccine (Bovilis IBR marker live) via the IM route. Nasal swabs and blood samples were taken at regular intervals and tested for BHV-1 in a virus infectivity assay. In addition, a polymerase chain reaction (PCR) specific for BHV-1 DNA was performed on the blood samples. BHV-1 neutralizing antibody titres were determined in the sera taken prior to the vaccination and four weeks after immunisation. AIl animals were successfully vaccinated as judged by the development of BHV-1 neutralising antibodies. However, all nasal swab samples were tested negative for vaccine virus, and all blood samples were found negative for BHV-1 vaccine virus and BHV-1 specific DNA. From these data it can be concluded that the vaccine virus was not excreted with nasal discharge after IM vaccination and that the vaccinated animals did not have a detectable viremia. Therefore, it is recommended to apply the tested BHV-1 marker live vaccine by the IM route in situations where it is undesirable that the vaccine virus is excreted.

EVALUATION OF THE ANTIVIRAL ACTIVITY OF BRAZILIAN CERRADO PLANTS AGAINST ANIMAL VIRUSES

Medicinal plants are abundant in the Brazilian flora including the Cerrado region. Extracts of sixteen plant species of the Brazilian Cerrado were investigated in vitro for antiviral activity against bovine herpesviruses type 1 (BoHV-1), infectious bursal disease virus (IBDV) and avian reovirus. Eight plants presented potent antiviral activity. Erythroxylum deciduum, Lacistema hasslerianum and Xylopia aromatica against BoHV-1; Gochnatia polymorpha and Lithraea molleoides against avian reovirus; and Banisteriopsis variabilis, Byrsonima intermedia and Campomanesia xanthocarpa against both viruses. No extract was active against IBDV. DOI: http://dx.doi.org/10.17525/vrr.v12i1-2.13

Identificação e diferenciação de herpesvírus bovino tipos 1 e 5 isolados de amostras clínicas no Centro-Sul do Brasil, Argentina e Uruguai (1987-2006)

Os herpesvírus bovino tipos 1 e 5 (BoHV-1; BoHV-5) são genética e antigenicamente muito semelhantes e por isso são indistinguíveis pela maioria dos testes diagnósticos. Como o BoHV-1 tem sido classicamente associado com doença respiratória e genital, os herpesvírus isolados dessas enfermidades têm sido provisoriamente - e às vezes definitivamente - identificados como BoHV-1. Da mesma forma, os casos de infecção neurológica por herpesvírus em bovinos têm sido atribuídos em sua totalidade ao BoHV-5. Este trabalho relata a identificação de 40 amostras de herpesvírus isoladas de diferentes casos clínicos na região Centro-Sul do Brasil, Argentina e Uruguai entre 1987 e 2006, pelo uso de um PCR capaz de diferenciar esses vírus. As amostras identificadas como BoHV-1 (n=16) foram isoladas de doença respiratória (n=3), balanopostite e/ou vulvovaginite (n=3), do sêmen de touros saudáveis (n=5) e de casos doença neurológica (n=5). As amostras virais identificadas como BoHV-5 (n=24) foram em sua maioria isoladas de doença neurológica (n=21), mas também do sêmen de touros saudáveis (n=2) e do baço de um bezerro com doença sistêmica (n=1). Esses resultados demonstram que tanto o BoHV-1 como o BoHV-5 não estão estritamente associados às suas respectivas síndromes clínicas e que podem estar freqüentemente envolvidos em casos clínicos classicamente atribuídos ao outro vírus. Esses achados também reforçam a necessidade de se identificar corretamente os isolados de herpesvírus para um melhor conhecimento da sua patogenia e epidemiologia.

Reactivation of latent bovine herpesvirus type 5 in cattle with polioencephalomalacia induced by ammonium sulphate

In the state Mato Grosso do Sul, Brazil, outbreaks of meningoencephalitis by BoHV-5 and polioencephalomalacia (PEM) display similar epidemiological features, suggesting that meningoencephalitis may be associated with reactivation of a latent BoHV-5 infection, during the development of PEM. To test this hypothesis, four 7-8 months old steers negative for BoHV-5 antibodies were inoculated intranasally with BoHV-5 and received amprolium from day 35 to day 105 after inoculation. Because PEM was not produced during this period, ammonium sulphate was given from day 114 to day 180 after inoculation. Two uninfected control steers received amprolium and ammonium sulphate for the same periods. All inoculated cattle developed antibodies against BoHV-5 after inoculation and the virus was isolated from nasal swabs, indicating that they were infected. Two inoculated steers had clinical signs of PEM after 118 and 146 days after virus inoculation. One was euthanized after a clinical manifestation period of seven days and had severe lesions of PEM and meningoencephalitis. BoHV-5 was isolated from the central nervous system of this animal. The other animal recovered but continued to manifest chronic signs of PEM and was euthanatized. On histological examination, the cerebral cortex, caudate nucleus and thalamus had multifocal areas of malacia and mild meningoencephalitis of the cortex. BoHV-5 was not isolated from the brain. One uninfected control steer had signs of neurological disease on day 158 and had lesions of PEM without meningoencephalitis at necropsy. The simultaneous production of PEM and diffuse meningoencephalitis, with isolation of BoHV-5, in one steer treated with ammonium sulphate, 118 days after BoHV-5 inoculation, suggests that latent BoHV-5 was reactivated in this animal submitted to experimental induction of PEM.

Molecular and antigenic characterization of Brazilian bovine herpesvirus type 1 isolates recovered from the brain of cattle with neurological disease

Bovine herpesviruses type 1 and 5 (BoHV-1 and BoHV-5) are closely related yet differ markedly in their neuropathogenic potential. BoHV-1 isolates have been associated with respiratory and genital disease whereas BoHV-5 has been consistently isolated from neurological infection. We report the characterization of five Brazilian BoHV-1 isolates associated with neurological disease, an unusual finding. All five viruses were isolated from the brain of cattle presenting neurological disease, yet prominent histological encephalitis was not observed in three cases. The isolated viruses were identified as BoHV-1 by a glycoprotein C gene-based PCR able to differentiate BoHV-1 from BoHV-5. The identity of the isolates was confirmed by nucleotide sequencing of the amplicons and by restriction analysis of PCR products from another gC region. Monoclonal antibody binding and cross-neutralization assays with BoHV-1 and BoHV-5 antisera showed a typical BoHV-1 antigenic profile. Lastly, inoculation of rabbits with these five BoHV-1 isolates did not result in neurological disease, contrasting with fatal meningoencephalitis produced by BoHV-5. Thus, the involvement of BoHV-1 in neurological disease of cattle is more frequent than previously reported, indicating the need for fast and precise means of differentiating it from BoHV-5. Likewise, the potential role of BoHV-1 in neurological infection in cattle should be further investigated.

Detection of bovine Herpesvirus type 5 in formalin-fixed, paraffin-embedded bovine brain by PCR: a useful adjunct to conventional tissue-based diagnostic test of bovine encephalitis

The aim of this study was to evaluate the application of PCR technique for the detection of BoHV-5 in routinely formalin-fixed, paraffin-embedded brain tissues in 20 naturally infected calves affected by fatal meningoencephalitis. Brains were divided into two halves, one kept fresh for virus isolation and PCR assay, targeting the glycoprotein C gene from BoHV-5 genome. The other half brain, corresponding to posterior cortex region, was submitted to formalin fixation and embedded into paraffin blocks for microscopic evaluation and total DNA isolation. Most of the slides showed severe multifocal non-supurative encephalitis with neuronal degeneration, neurophagia, and no acidophilic intranuclear inclusions could be found in neurons and glial. The 20 fresh samples were confirmed, by virus isolation and PCR assay, as having the BoHV-5 virus and, respective glicoprotein C sequence, while 15 of 20 formalin-fixed, paraffin-embedded samples were considered positive for the same analysis. The results revealed the first description of PCR efficiency, applied to formalin-fixed, paraffin-embedded brain collected from naturally infected calves, improving the detection of BoHV-5 from archival samples in South America.

Efficacy of a live bovine herpesvirus type 1 marker vaccine under field conditions in three countries

The performance of a live marker vaccine for bovine herpesvirus type 1 (bhv-1) was studied in the field in three European Union countries with different farming conditions. The progress in...

Serological survey of bovine herpesvirus type 1 infection in China

To understand the nationwide seroprevalence of bovine herpesvirus type 1 (BoHV-1) infection of cows in China, 1344 sera of dairy cows from 29 provinces and 765 sera from 6 herds in Hubei province were collected with stratified random sampling. Another 483 sera from imported cows were included. The serum antibody was tested by BoHV-1 gG ELISA. The results demonstrated that the overall nationwide seroprevalence was 35.8% (481/1344), while the prevalence for individual province ranged from 12.1% to 77.8%. Although each province had positive samples, the prevalence was clustered in areas based on the cow population size. In Hubei Province, the overall seroprevalence was 22.2% (170/765) while the prevalence for individual farms varied greatly from 0.0% to 41.5%. The sera from imported cows had a moderate prevalence of 21.7% (105/483).

Produção e caracterização de anticorpos monoclonais contra uma cepa do herpesvírus bovino tipo 1 defectiva na glicoproteína C (gC)

A maioria dos anticorpos monoclonais (AcMs) já produzidos contra o herpesvírus bovino tipo 1 (BoHV-1) reage com a glicoproteína C (gC), um antígeno abundante e imunodominante presente no envelope viral. Com o objetivo de produzir AcMs com outras especificidades protéicas, antígenos de uma cepa do BoHV-1 defectiva no gene da gC foram utilizados para a imunização de camundongos BALB/c. Após fusão e seleção de 54 hibridomas resistentes ao meio seletivo HAT, foram obtidos três clones (1F1, 2H4 e 4D7) secretores de imunoglobulinas da classe IgG2a, que reagiram com antígenos da cepa homóloga. Os AcMs reagiram com antígenos virais nas técnicas de imunofluorescência (IFA) e imunoperoxidase (IPX) em diluições de até 1:640 (sobrenadante de cultivo) e 1:20.000 (fluído ascítico). Os três AcMs apresentaram um espectro amplo de reatividade, reagindo com antígenos de 14 herpesvírus isolados de doença respiratória ou genital (provavelmente BoHV-1) e com 17 isolados de doença neurológica (supostamente BoHV-5), e apresentaram atividade neutralizante em níveis variáveis contra todos esses isolados. A especificidade protéica dos AcMs não pode ser determinada diretamente, pois nenhum deles reagiu com proteínas virais na técnica de Western blot. Por outro lado, os três AcMs reagiram em IFA com células infectadas com uma cepa do BoHV-5 defectiva nas glicoproteínas E, I e proteína US9, o que exclui estes antígenos como possíveis alvos dos AcMs. Por exclusão (gC, gE, gI) e pela sua forte atividade neutralizante, os AcMs são provavelmente direcionados contra epitopos conservados de outras glicoproteínas do envelope viral que contêm epitopos neutralizantes: a gB e/ou gD. Pelo seu alto título de reação e pelo amplo espectro de reatividade, esses AcMs possuem potencial aplicação em técnicas diagnósticas. Além disso, podem ser úteis para o mapeamento de epitopos neutralizantes conservados nas glicoproteínas do envelope.

Cobaias como modelo para teste de vacinas inativadas contra o herpesvírus bovino tipo 1 e o vírus da diarréia viral bovina

O presente trabalho relata a avaliação de cobaias como modelo para testes de imunogenicidade de vacinas inativadas contra o herpesvírus bovino tipo 1 (BoHV-1) e o vírus da diarréia viral bovina (BVDV). Para isso, cobaias (n=60) e bovinos (n=10) foram imunizados duas vezes, com intervalo de 28 dias, com uma vacina experimental contendo antígenos dos dois vírus, e testados para anticorpos neutralizantes 28 dias após a segunda dose. Os bovinos foram vacinados com a dose recomendada para a espécie (5mL); as cobaias foram distribuídas em seis grupos e imunizadas com doses fracionadas (0,005mL a 1,6mL). Os grupos de cobaias imunizadas com doses equivalentes a 1/16 (0,320mL) e 1/8 (0,640mL) da dose bovina desenvolveram títulos médios geométricos (GMTs) de 6,46 e 7,56, respectivamente, estatisticamente semelhantes aos dos bovinos (GMT=8) (P>0,05). Uma alta correlação dose-resposta (R²=0,95) foi observada entre as doses vacinais e os títulos de anticorpos neutralizantes anti-BoHV-1 nos grupos de cobaias. Por outro lado, não foi possível o estabelecimento de uma dose vacinal que induzisse em cobaias uma resposta neutralizante anti-BVDV em níveis semelhantes à induzida em bovinos. Apenas as cobaias imunizadas com as doses maiores (0,640 e 1,6mL) desenvolveram títulos neutralizantes de magnitude moderada (GMTs de 8 e 9, respectivamente), porém estatisticamente inferiores ao GMT dos bovinos (GMT=34,9) (P<0,05). Esses resultados demonstram que cobaias podem ser utilizadas como modelo para estudos da imunogenicidade de vacinas inativadas contra o BoHV-1. Volumes entre 1/8 e 1/16 da dose bovina são suficientes para induzir nesta espécie uma resposta neutralizante de magnitude equivalente à de bovinos.

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

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

2,3,7,8‐tetrachlorodibenzo‐p‐dioxin increases bovine herpesvirus type‐1 (BHV‐1) replication in madin‐darby bovine kidney (MDBK) cells in vitro

Dioxin-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a common environmental toxin of current interest. In the last years, higher levels of TCDD than those permitted in UE [European Commission. 2002. European Commission Recommendation 2002/201/CE. Official Gazette, L 67/69] were detected in milk samples from cow, water buffalo, goat, and sheep raised on some areas of Campania Region (South Italy). Dioxin often causes immunosuppression and might render the animal liable to viral infections. In addition, viral infections are able to alter the pattern of dioxin distribution in different organs of the exposed animals. Bovine Herpesvirus type-1 (BHV-1) is a widespread pathogen, which causes infectious rhinotracheitis and infectious pustular vulvovaginitis in cattle. Herein, we have studied the effects of TCDD and BHV-1 infection, in Madin-Darby Bovine Kidney (MDBK) cells, alone as well as in association, so as cellular proliferation, apoptosis, and virus replication. We have observed an increase in cell viability of confluent monolayers at low TCDD concentrations. TCDD treated cells demonstrated increased viability compared to controls as evaluated by MTT test. TCDD exposure increased cell proliferation but induced no changes on apoptosis. Cells exposed to TCDD along with BHV-1 showed a dose-dependent increase in cytopathy, represented by ample syncytia formation with the elimination of the cellular sheets and increased viral titer. These results suggest that TCDD increases viral replication in MDBK cells while BHV-1 further decreases viability of TCDD exposed cells. Since very low concentrations (0.01 pg/ml) are sufficient to augment BHV-1 titer, TCDD may contribute to reactivate BHV-1 from latency, leading to recurrent disease and increase virus transmission.

Glycoprotein E (gE) specified by bovine herpesvirus type 5 (BHV-5) enables trans-neuronal virus spread and neurovirulence without being a structural component of enveloped virions

Bovine herpesvirus 5 (BHV-5) is a neurovirulent alpha-herpesvirus that causes fatal encephalitis in calves. We previously demonstrated that deletion of a glycine-rich epitope in the gE ectodomain dramatically reduced BHV-5 neurovirulence. To investigate the role of gE cytoplasmic tail sequences in the neuropathogenesis of BHV-5 in rabbits, we constructed a BHV-5gE recombinant virus with a short residual cytoplasmic domain lacking the YXXL motifs and the acidic (BHV-5gEAm480). In vitro, BHV-5gEAm480 produced on the average smaller plaques, compared with wild-type BHV-5, but it produced on the average substantially larger plaques than the gE ORF-deleted BHV-5. The truncated gE was not phosphorylated, and was not endocytosed from the cell surface. Importantly, the truncated gE was not incorporated into enveloped infectious virions, but its glycosylation and interaction with gI were not affected. In a rabbit model of infection, the BHV-5gEAm480 remained highly virulent, while the gE-null virus was avirulent. The gEAm480 mutant virus invaded most of the central nervous system (CNS) structures that are invaded by the wild-type BHV-5. The number of neurons infected by BHV-5gEAm480 was very similar to the number infected by BHV-5 wild-type and gEAm480-rescued viruses. Collectively, the results suggest that gE functions in transsynaptic transmission of BHV-5 and neurovirulence without being a structural component of the virion particle.

Bio-Safety Technology in Production of Bovine Herpesvirus Type 5 (BoHV-5) Using an Alternative Serum-Free Medium

The detection and growth of bovine herpes virus type 5 (BoHV-5) was evaluated in three different media without foetal bovine serum or animal protein by infecting three different cell lines. The OPTI-PRO, VP-SFM and RPMI 1640 media were supplemented by L-glutamine, antibiotics added by 5µg of insulin-like growth factor (IGF-I) and used to adapt CER, MDBK and CRIB cells in a statistic continuous culture system. The results obtained by CRIB and MDBK cells adaptation showed steady growth after 10 th passages in OPTI-PRO medium, 20 th passages in VP-SFM medium and 30 th passages in RPMI 1640 medium, respectively. The OPTI-PRO and RPMI 1640 media were not able to support CER growth, even supplemented by IGF-1 and present apoptotic cells at 72h post-infection. The CER cells seeded after adaptation by 10 th passages in VP-SFM medium added by 5µg/20ml of IGF-1 growth factor revealed the best virus titres compared to MDBK and CRIB cell lines. It was concluded that CER, MDBK and CRIB cells infected by BoHV-5 serotype could be used for laboratory diagnosis propagated on much safer culture system of high biotechnology advances.