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Validation of viral safety is essential in ensuring the safety of mammalian cell culture-derived biopharmaceuticals since numerous adventitious viruses get contaminated during the manufacturing process. In particular, Chinese hamster ovary (CHO) cells are highly susceptible to minute virus of mice (MVM), bovine parvovirus (BPV), and bovine herpesvirus (BHV). Therefore, viral detection during CHO cell culturing is necessary to ensure the safety of biopharmaceuticals against viruses. In this study, a multiplex PCR assay was developed and subsequently evaluated for its effectiveness to simultaneously detect MVM, BPV and BHV during the manufacture of cell culture-derived biopharmaceuticals. Specific primers for MVM, BPV, and BHV were selected, and a multiplex PCR was optimized. The sensitivity of the assay was 6.49 × 101 TCID50/mL for MVM, 7.23 × 102 TCID50/mL for BPV, and 5.80 × 101 TCID50/mL for BHV. The multiplex PCR assay was very specific to MVM, BPV, and BHV and was subsequently applied to the validation of CHO cells artificially infected with each virus. It could detect each viral DNA from CHO cells as well as culture supernatants. Therefore, we concluded that the multiplex PCR assay is invaluable for detecting adventitious viruses during the manufacture of cell culture-derived biopharmaceuticals.

BHV-1 is an important pathogen of cattle. Because of its ability to induce immune suppression, BHV-1 is an important agent in the multifactorial disorder, bovine respiratory disease complex (BRDC). BHV-1 encodes several proteins that inhibit various arms of the immune system suggesting that these proteins are important in the development of BRDC.

Os herpesvírus bovino tipos 1 (BoHV-1) e 5 (BoHV-5) são agentes virais genética e antigenicamente relacionados, associados com diversas manifestações clínicas em bovinos, incluindo doença respiratória, genital, neurológica e abortos. Estudos epidemiológicos indicam que esses vírus estão amplamente disseminados no rebanho bovino brasileiro. O diagnóstico sorológico, que permite identificar animais portadores da infecção latente, se constitui em importante ferramenta para monitoramento individual e de rebanho. O presente artigo relata a padronização de um teste imunoenzimático do tipo ELISA, com base em anticorpo monoclonal (AcM), para a detecção de anticorpos séricos que reagem contra BoHV-1 e/ou BoHV-5. Inicialmente, determinou-se o AcM mais adequado para a sensibilização das placas, as diluições apropriadas do antígeno e dos soros-teste e o ponto de corte do ensaio. Após a padronização, o ensaio foi validado testando-se 506 amostras de soro bovino, previamente testadas para anticorpos neutralizantes contra BoHV-1 e/ou BoHV-5 pela técnica de soroneutralização (SN). Comparando-se com os resultados da SN frente a BoHV-1, o teste de ELISA apresentou sensibilidade e especificidade de 96,6% e 98,3%, respectivamente. Os valores preditivos positivo e negativo foram de 97,6%, a concordância foi de 97,6% e o índice de correlação kappa entre os testes foi de 0,95, o que indica uma excelente concordância. Comparando-se com os resultados da SN frente o BoHV-5, o ELISA apresentou 94,3% de sensibilidade; 97,9% de especificidade; 97,1% de valor preditivo positivo e 95,9% de valor preditivo negativo. Para BoHV-5, a concordância entre os testes foi de 96,4% e o índice de correlação foi de 0,92, também excelente. Esses resultados demonstram que o teste padronizado apresenta sensibilidade e especificidade adequados para o diagnóstico sorológico das infecções por BoHV-1 e BoHV-5 em nível individual e de rebanho. Dessa forma, o ensaio pode se constituir em alternativa para o teste de SN e para os kits de ELISA importados. Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) are antigenic and genetically related viruses associated with different clinical syndromes in cattle, including respiratory, reproductive, neurological disease and abortion. Epidemiological studies indicate the widespread distribution of both viruses among Brazilian cattle. Serological diagnosis, that allows the identification of latently infected animals, represents an important tool for individual and herd monitoring. The present article describes the standardization of a monoclonal antibody (MAb)-based immunoenzymatic test (ELISA) for detection of antibodies to BoHV-1 and/or BoHV-5. The initial steps involved the determination of the most suitable MAb, the appropriate dilutions of viral antigen and serum samples, and the cut-off value of the assay. After standardization, the ELISA was validated by testing 506 cattle serum samples previously tested for neutralizing antibodies to BoHV-1 and BoHV-5 by virus neutralizing assay (VN). Comparing to the VN for BoHV-1 antibodies, the ELISA presented sensitivity and specificity of 96.6% and 98.3%, respectively. Positive and negative predictive values were 97.6%, the concordance between the tests was 97.6% and the coefficient of correlation k (kappa) was 0.95, demonstrating an excellent correlation. Comparing to the VN for BoHV-5 antibodies, the ELISA presented 94.3% of sensitivity, 97.9% of specificity, 97.1% of positive predictive value, 95.9% negative predictive value, concordance of 96.4% and kappa coefficient of 0.92. These results demonstrate that the ELISA presents suitable specificity and sensitivity to be used for individual and herd serological diagnosis of BoHV-1 and BoHV-5, thus, representing an alternative for VN assays and imported ELISA kits.

This study was carried out to determine whether the sensitivity of serum neutralization (SN) tests would be affected by the use of distinct subtypes of bovine herpesvirus 1 (BoHV1) and 5 (BoHV-5) as test challenge viruses. Bovine sera collected from a randomized sample (n = 287) were tested in a 24 h incubation SN against three type 1 viruses (BoHV1.1 strains ‘‘Los Angeles’’ (LA) and ‘‘EVI 123’’; BoHV-1.2a strain ‘‘SV 265’’) and three type 5 viruses (BoHV-5a strain ‘‘EVI 88’’; BoHV-5b strain ‘‘A 663’’ and BoHV-5c ‘‘ISO 97’’). SN sensitivity varied greatly depending on the test challenge virus used in the test, particularly when results against each virus were considered individually, where it ranged from 77% (detecting 80 out of 104 antibody-positive sera) with ISO 97 to 91% (95/104) with BoHV-1.1 strain LA. All tests to single viruses revealed a significantly low sensitivity (McNemar’s; p < 0.05). Maximum sensitivity (104/104) was achieved when positive results to a particular combination of four of the challenge viruses (LA + EVI 123 + SV 265 + A 663) or some combinations of five viruses (or all six viruses) were added cumulatively. These results provide evidence for no association between any particular virus type/subtype and higher SN sensitivity. In addition, it was clearly shown that when SN is performed with single test challenge viruses, sensitivity can vary so significantly that might compromise control or eradication efforts. Performing SN against a number of different viruses demonstrated to improve significantly the test’s sensitivity.

Experimental and field studies have proven that the bovine herpesvirus type-4 (BHV-4) infection leads to various reproductive system problems. In this study, the role of BHV-4 infection in repeat breeding was investigated serologically. Eighty-four samples were obtained from repeat-breeding diagnosed cows in two organized dairy herds; an equal number of healthy cows were sampled from the same farms. The rest of the samples (105) were obtained from reproductively normal cows that were breeding in 18 small enterprises as a control group. The seropositivity proportion in repeat-breeding diagnosed cows was found to be significantly higher (69% (58/84)) than other cows (44% (37/84)) on the same farms. The lowest antibody positivity value for BHV-4 was detected as 24.7% (26/105) in the samples from family-type small farms. The odds ratio (OR) value was calculated as 2.834 in repeat-breeding diagnosed and healthy cows on the same farms, while 6.778 was determined in cows with and without reproductive problems on organized farms compared to small farms. As a result, the BHV-4 infection can be considered one of the reasons for repeat breeding besides other reproductive disorders.

Summary Serum samples were collected from 254 yak (Poephagus grunniens, presently Bos grunniens) in different yak tracts of India. These samples were then screened by virus neutralisation test (VNT) and avidin-biotin enzyme-linked immunosorbent assay (AB-ELISA) to study the seroprevalence of antibodies against bovine herpesvirus type 1 (BHV-1). The overall seroprevalence in yak was found to be 41% (105) by VNT and AB-ELISA. The sex of the animal, whether it was on a farm or free-ranging and the location of the different yak tracts did not seem to have any effect on seroprevalence. However, seroprevalence was found to increase with the age of the animals, being highest in yak older than three years of age (49%). Yak generally share feeding, watering and grazing areas with other domestic and wild animals and this common ecological niche is thought to be a possible avenue of infection. This is the first time that the seroprevalence of antibodies against BHV-1 has been studied in yak in India.

Oncolytic virotherapy is a promising avenue of cancer gene therapy. Current vectors include human viruses that have been engineered to replicate in tumor cells or nonhuman viruses that are naturally oncotropic and preferentially replicate in tumor cells harboring defects in innate immune pathways such as the type 1 interferon (IFN) pathway. Bovine herpesvirus type 1 (BHV-1) is a species-specific herpesvirus closely related to the human herpes simplex virus type 1 (HSV-1). Although BHV-1 does not efficiently replicate in and affect cellular viability of normal human cells, it is capable of infecting and killing various immortalized and transformed human cell types. Surprisingly, BHV-1 infection of human cells fails to elicit IFN production at the mRNA or protein level and the ability of BHV-1 to kill immortalized and transformed human cells does not correlate with defects in IFN pathways. Furthermore, although some cross-reactivity between BHV-1 and HSV-1 exists, the majority of human antibody or serum samples tested failed to neutralize BHV-1 despite possessing HSV-1 neutralizing capacity. Thus, BHV-1 is a novel candidate oncolytic virus with a distinct mechanism of tumor targeting.

Sensory neurons within trigeminal ganglia (TG) are the primary site for bovine herpesvirus 1 (BHV-1) latency. During latency, viral gene expression is restricted to the latency-related (LR) gene and the open reading frame ORF-E. We previously constructed an LR mutant virus that expresses LR RNA but not any of the known LR proteins. In contrast to calves latently infected with wild-type (wt) BHV-1 or the LR rescued virus, the LR mutant virus does not reactivate from latency following dexamethasone (DEX) treatment. In this study, we demonstrated that bICP0, but not bICP4, transcripts were consistently detected in TG of calves infected with the LR mutant or LR rescued virus following DEX treatment. Calves latently infected with the LR rescued virus but not the LR mutant virus expressed late transcripts, which correlated with shedding of infectious virus following DEX treatment. The bICP4 and bICP0 genes share a common immediate-early promoter, suggesting that this promoter was not consistently activated during DEX-induced reactivation from latency. The bICP0 gene also contains a novel early promoter that was activated by DEX in mouse neuroblastoma cells. Expression of a cellular transcription factor, C/EBP-alpha, was stimulated by DEX, and C/EBP-alpha expression was necessary for DEX induction of bICP0 early promoter activity. C/EBP-alpha directly interacted with bICP0 early promoter sequences that were necessary for trans activation by C/EBP-alpha. In summary, DEX treatment of latently infected calves induced cellular factors that stimulated bICP0 early promoter activity. Activation of bICP0 early promoter activity does not necessarily lead to late gene expression and virus shedding. Fil: Workman, Aspen. University of Nebraska; Estados Unidos Fil: Perez, Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina. University of Nebraska; Estados Unidos Fil: Doster, Alan. University of Nebraska; Estados Unidos Fil: Jones, Clinton. University of Nebraska; Estados Unidos

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).

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 the eradication of the virus was followed in a large herd in Germany and one in Italy, and a major serological survey involving 147 farms was conducted in Hungary. Commercial batches of the same vaccine were used in all three studies. The herds were vaccinated according to agreed protocols and the animals' bhv-1 antibody status was determined at local institutes by using commercial glycoprotein B (gB)- and glycoprotein E (gE)-elisas. In all three studies, the seroprevalence of bhv-1 gE decreased progressively. Given the starting conditions and the long duration of the studies, reactivation events and virus circulation would have been more likely to have occurred if the herds had not been vaccinated.

The present study demonstrates the interest of two slow-release systems as vaccination tools in cattle. Two experiments show that a first intradermal administration of one DNA vaccine dose combined with the slow-release of a second dose conduct to a priming of the bovine herpesvirus 1-specific immune response similar to the one generated by two discrete administrations 4 weeks apart. The first experiment demonstrates the efficacy of the slow-release system with well-characterized Alzet® osmotic pumps, whereas the second experiment extends the same concept with innovative agarose hydrogel implants. These latter implants are cheaper and more convenient than the osmotic pumps or repeated intradermal administrations since they contribute to an efficient priming of the immune response in a single manipulation of the animals.

RESUMOO objetivo deste trabalho foi determinar a prevalência das infecções latentes por BoHV-1 e por BoHV-5 em bovinos de corte criados no Estado do Paraná. Os gânglios do nervo trigêmeo foram coletados de 400 bovinos hígidos, entre 18 e 36 meses de idade, provenientes de 90 propriedades rurais localizadas em diferentes mesorregiões geográficas do Estado e abatidos em frigorífico com Serviço de Inspeção Federal. A reação em cadeia da polimerase com amplificação do gene que codifica a glicoproteína C foi empregada para a detecção do DNA viral. Cento e nove bovinos eram herpéticos (27,25%), sendo 14,25% (57/400) infectados com BoHV-1, 9,75% (39/400) infectados com BoHV-5 e 3,25% (13/400) portadores de infecção mista. A distribuição geográfica foi heterogênea e as infecções foram mais prevalentes nas mesorregiões localizadas ao norte do Estado. A vigilância para a encefalite por BoHV-5 deve ser intensificada na mesorregião Noroeste. ABSTRACTThe prevalence of latent infection with BoHV-1 or BoHV-5 in beef cattle raised in the state of Paraná, Brazil, was studied. The trigeminal ganglia were collected in a slaughterhouse from 400 healthy cattle, 18 to 36 months old, raised in 90 farms located in distinct geographical regions of the state. Polymerase chain reaction for amplification of the gene encoding C glycoprotein was performed to detect virus DNA. One hundred and nine (27.25%) animals were herpetic; 14.25% (57/400) were infected with BoHV-1, 9.75% (39/400) were infected with BoHV-5 and 3.25% (13/400) had mixed infection. The geographical distribution was heterogeneous and the infections were more prevalent in the north of the state. The surveillance for BoHV-5 encephalitis should be intensive in the Northwest region.

Este trabalho teve por objetivo estudar a interação entre moléculas orgânicas e anticorpos visando seu uso em sistemas de entrega de fármacos e diagnóstico fluorescente. Foram investigadas moléculas orgânicas de porfirinas e anticorpos reativos a Herpesvírus bovino, micro-organismo amplamente disseminado em rebanhos de corte e leite. Para o desenvolvimento deste projeto foram empregadas técnicas espectroscópicas de absorção UV/Vis, emissão fluorescente e espalhamento ressonante. Os resultados obtidos apresentaram indícios de que a ligação entre as porfirinas e os anticorpos ocorre. As constantes de ligação associadas à essa interação sugeriram alta afinidade das porfirinas pelos anticorpos. As investigações da conformação dos anticorpos indicaram que a sua estrutura enovelada não é significativamente afetada pelas porfirinas e os estudos de Transferência de Energia Ressonante de Förster sugeriram que esse mecanismo pode ser o principal responsável pela supressão da fluorescência dos anticorpos pelas porfirinas. (Sem resumo em outra língua) Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG

Neste trabalho foi avaliada a presença do DNA viral, por meio da Reação em Cadeia da Polimerase (PCR), no tecido ovariano, nos oócitos, líquido folicular e sangue de vacas naturalmente infectadas. Também foi avaliado o perfil sorológico dos animais amostrados. Foram coletadas amostras de soro, sangue, tecido ovariano, líquido folicular e complexo cumulus-oócitos de 147 animais abatidos em frigorífico não vacinados contra o herpesvirus bovino 1 (BoHV-1). Amostras tóxicas ou insuficientes foram descartadas. Os testes sorológicos foram realizados permitindo a identificação dos animais soropositivos para anticorpos neutralizantes contra o BoHV-1. Foram realizadas as PCRs onde foi observada a presença do DNA viral em 0,9% (1/115) dos oócitos, em 4,3% (5/117) do tecido ovariano e em 2,8% (3/108) do sangue. Em nenhuma das amostras de líquido folicular foi detectado o DNA viral. Nas amostras sorológicas observou-se 83,6% (117/140) de positividade para o BoHV-1. Dentre os animais positivos na PCR, independente das amostras, todos apresentavam positividade no teste de soroneutralização para detecção de anticorpos para BoHV-1. Conclui-se que em animais de diferentes rebanhos analisados foi detectada alta prevalência de anticorpos contra o BoHV-1 e que nos testes moleculares houve a presença do DNA viral em amostras de tecidos ovarianos de bovinos, evidenciando que estas estruturas poderiam ser sítios de infecção pelo vírus em animais naturalmente infectados. In this study, the polymerase chain reaction (PCR) was used to evaluate the presence of viral DNA in ovarian tissue, in the cumulus-oocyte complex (COC), follicular liquid, and blood of animals naturally infected with bovine herpesvirus-1 (BoHV-1). The serum profile of the sampled animals was also evaluated. Samples of serum, blood, ovarian tissue, follicular liquid, and COC were collected from 147 slaughterhouse animals that were not vaccinated against BoHV-1. Contaminated or insufficient samples were disregarded. Serological tests allowed the identification of serum-positive animals with neutralizing antibodies against BoHV-1. Analysis of samples by PCR revealed the presence of viral DNA in 0.9% (1/115) of the COC samples, in 4.3% (5/117) of the ovarian tissue samples, and in 2.8% (3/108) of the blood samples. Viral DNA was not detected in any of the follicular liquid samples. In serological samples, a positivity of 83.6% (117/140) was observed for BoHV-1. All PCR-positive animals, regardless of the samples analyzed, showed positivity in the serum neutralization test for the detection of BoHV-1-specific antibodies. According to these results, a high prevalence of antibodies against BoHV-1 was detected in naturally infected animals from different herds, and the molecular tests revealed the presence of viral DNA in bovine ovarian tissue, providing evidence that this might be a site of BoHV-1 infection in naturally infected animals.

During a series of experiments attempting to reproduce clinically apparent bovine herpesvirus type 5 (BoHV-5) infections, a group of calves was inadvertently infected with bovine viral diarrhoea virus (BVDV). Another group of calves was infected with BoHV-5 only. This paper reports the outcome of such infections. Two out of six calves solely infected with BoHV-5 displayed moderate to severe clinical signs. Three out of four calves of the group co-infected with BoHV-5 and BVDV developed severe clinical signs, two of them died. BoHV-5 virus was isolated to higher titres and for a longer period from the group of calves infected with both viruses. These results suggest that BVDV may enhance clinical signs induced by BoHV-5 and may also play a role in extending the period of BoHV-5 shedding.

An outbreak of contagious mastitis occurred among cattle on a farm, and bovine herpesviruses were isolated from the affected mammary tissues, scabs and abscess discharge of the cattle. A bovine herpesvirus type 4 (BoHV-4)-specific fragment was amplified from the isolates by polymerase chain reaction (PCR). Restriction endonuclease analyses demonstrated that the isolates were related to Movar-like European type BoHV-4. To determine the ratio of BoHV-4 subclinical infection in the cattle, a genomic survey was performed by PCR for cattle that were moved to the animal hygiene service station in Ibaraki prefecture. The BoHV-4 genome was occasionally detected in peripheral blood leukocytes, lymph nodes and nervous tissues. The rate of BoHV-4 subclinical infection was relatively high in the cattle.

Lymph nodes (Ln) are the preferred samples for virus isolation and detection. Inthe present study, carcass and visceral Ln of apparently healthy cattle were screened for the presence of bovine herpes virus type 1 (BHV-1) and bovine ephemeral fever virus (BEFV) antigens. A total of 198 Ln (114 carcasses Ln and 84 visceral Ln) were collected. Lymph node homogenates were assayed by agar gel precipitation test (AGPT), rapid Staphyloccocal protein A (SPA) agglutination test and Dot-ELISA. The overall results revealed that BHV-1 antigens were detected in 43.9%, 56.1% and in 68.4% of carcass Ln, and in 29.8%, 47.6% and 57.1% of visceral Ln collected from slaughtered cattle by AGPT, SPA agglutination test and Dot-ELISA respectively. On the other hand, BEFV antigens were detected in 5.3%, 38.6% and 52.6% of carcass Ln, and in 6%, 41.7% and 54.8 % of visceral Ln collected from slaughtered animals by AGPT, rapid SPA agglutination test and Dot-ELISA respectively. The results showed high percentage of positive samples with SPA agglutination test and Dot- ELISA in comparison to AGPT for both BHV-1 and BEF.

Bovine herpesvirus type 5 (BHV-5) is the causative agent of bovine herpetic encephalitis, a major concern for cattle farming in Brazil and Argentina. We recently developed a differential, gE-negative vaccine (265 gE-), based on a Brazilian BHV-1 strain. The present study was carried out to examine whether such a vaccine would confer protection to BHV-5 infections. It was concluded that the recombinant BHV-1 vaccine tested here is not capable of conferring full protection to BHV-5 challenge.

Newborn calves received a low dose of bovine interferon-tau (boIFN-tau) orally for 4 weeks and calves that had developed diarrhea received a low dose of boIFN-tau orally for 5 days. No effects of boIFN-tau were seen in the duration of the diarrhea, or in daily weight gain. Calves received a high dose of boIFN-tau subcutaneously 3 times and they were then stimulated with bovine herpesvirus type 1 vaccine. No adverse effects were observed after the administration of boIFN-tau and lymphocyte subsets from calves did not change after the stimulation. Our results suggest that boIFN-tau does not seem protecting for preventing calves from diarrhea, recovering the health of calves with diarrhea or immunomodulation, although the treatment itself is not toxic.

Anticorpos monoclonais (AcMs) produzidos com uma amostra brasileira do herpesvírus bovino tipo 5 (BHV-5) foram utilizados na adaptação e padronização de técnicas rápidas para o diagnóstico de infecções pelo BHV-1 e BHV-5. A detecção de antígenos virais por imunofluorescência em células descamativas (IFCD) foi comparada com o isolamento viral, em secreções nasais de 16 bezerros, sendo 6 inoculados com o BHV-1 e 10 com o BHV-5. De 203 amostras testadas, 182 (89,6%) apresentaram resultados concordantes nos dois testes (143 positivas; 39 negativas). Comparando-se com o isolamento, a IFCD apresentou sensibilidade de 96,6%, especificidade de 70,9%, e precisão de 89,6%. Os AcMs também foram utilizados para detectar antígenos do BHV-5 em impressões frescas do cérebro de bezerros acometidos de enfermidade neurológica e em células de cultivo utilizadas na técnica de soroneutralização (SN), permitindo a obtenção dos resultados em 24 horas. A técnica, denominada de soroneutralização rápida (SNR), apresentou sensibilidade de 97,3%; especificidade de 95,5% e precisão de 96,2% em comparação com a SN tradicional; e sensibilidade de 94,7%, especificidade de 97,3% e precisão de 96,1% em comparação com um ELISA comercial. Esses resultados demonstram que esses AcMs podem ser muito úteis para uso em técnicas rápidas de diagnóstico de infecções suspeitas de BHV-1 ou de BHV-5, sobretudo em situações de surto, quando a tomada de decisões adequadas depende de um diagnóstico rápido e confiável. Monoclonal antibodies (mAbs) produced to a Brazilian isolate of bovine herpesvirus type 5 (BHV-5) were used to standardize rapid tests for the diagnostic of BHV-1 and BHV-5 infections. The detection of viral antigens by immunofluorescence in descamative cells (IFDC) was compared to virus isolation in nasal secretions of 16 calves inoculated with BHV-1 (n=6) or BHV-5 (n=10). One hundred and sixty two out of 203 specimens (89.6%) had identical results in both assays (143 positive; 39 negative); the IFDC assay showing a sensitivity of 96.6%, specificity of 70.9% and precision of 89.6%. The mAbs were also used to detect BHV-5 antigens in fresh brain smears of calves from outbreaks of neurological disease; and in cell cultures used in a virus neutralizing (VN) test, allowing the obtention of results in 24 hours. The rapid virus neutralizing test (RVN) showed a sensitivity of 97.3%, specificity of 95.5% and precision of 96.2% comparing to the standard VN test; and a sensitivity of 94.7%, specificity of 97.3% and precision of 96.1% as compared to a commercial ELISA. These results demonstrate that these mAbs are useful in rapid diagnostic tests, which may be very important in field outbreaks, when taking adequate decisions depends on rapid and reliable diagnostic tests.

Bovine herpesvirus 4 (BoHV-4) is increasingly considered as responsible for various problems of the reproductive tract. The virus infects mainly blood mononuclear cells and displays specific tropism for vascular endothelia, reproductive and fetal tissues. Epidemiological studies suggest its impact on reproductive performance, and its presence in various sites in the reproductive tract highlights its potential transmission in transfer-stage embryos. This work describes the biological and genetic characterization of BoHV-4 strains isolated from an in vitro bovine embryo production system. BoHV-4 strains were isolated in 2011 and 2013 from granulosa cells and bovine oocytes from ovary batches collected at a local abattoir, used as “starting material” for in vitro production of bovine embryos. Compatible BoHV-4-CPE was observed in the co-culture of granulosa cells and oocytes with MDBK cells. The identity of the isolates was confirmed by PCR assays targeting three ORFs of the viral genome. The phylogenetic analyses of the strains suggest that they were evolutionary unlinked. Therefore it is possible that BoHV-4 ovary infections occurred regularly along the evolution of the virus, at least in Argentina, which can have implications in the systems of in vitro embryo production. Thus, although BoHV-4 does not appear to be a frequent risk factor for in vitro embryo production, data are still limited. This study reveals the potential of BoHV-4 transmission via embryo transfer. Moreover, the high variability among the BoHV-4 strains isolated from aborted cows in Argentina highlights the importance of further research on the role of this virus as an agent with the potential to cause reproductive disease in cattle. The genetic characterization of the isolated strains provides data to better understand the pathogenesis of BoHV-4 infections. Furthermore, it will lead to fundamental insights into the molecular aspects of the virus and the means by which these strains circulate in the herds. EEA Balcarce Fil: Gonzalez Altamiranda, Erika Analia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina Fil: Manrique, Julieta Marina. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Perez, Sandra. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Rios, Glenda Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina Fil: Odeón, Anselmo Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina Fil: Leunda, Maria Rosa. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina Fil: Jones, Leandro Roberto. Universidad Nacional de la Patagonia "San Juan Bosco". Facultad de Ciencias Naturales - Sede Trelew; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Verna, Andrea Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina

Bulk milk from 60 herds of dairy cattle in a rural region in the central highlands of Peru was tested for antibodies to bovine viral-diarrhoea virus (BVDV) and bovine herpesvirus type 1 (BHV-1). None of the herds had been vaccinated against BVDV or BHV-1. Commercially available indirect ELISA-kits were used for antibody detection. True prevalences of BVDV and BHV-1 antibody-positive herds were 96 and 51%, respectively. A relatively low proportion of strongly positive herds suggests, however, a low prevalence of active BVDV infection. BVDV optical densities (ODs) in bulk milk increased with herd size—indicating a higher within-herd prevalence in the larger herds (probably, in part a consequence of a higher rate of animal movement into these herds). For BHV-1, this pattern was not found; a relatively high proportion of the herds was free from BHV-1 infection in each size category. This could indicate a low rate of reactivation of latent BHV-1 infection.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2014-02-26T17:13:41Z No. of bitstreams: 0 Made available in DSpace on 2014-02-26T17:13:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2002-04-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:25:35Z No. of bitstreams: 0 Made available in DSpace on 2014-05-20T15:25:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2002-04-01 This paper reports the detection of bovine herpesvirus type 5 (BoHV-5) by a specific nested PCR assay. Samples were collected from the central nervous system (CNS) of cattle from Minas Gerais and São Paulo States, Brazil. All animals died presenting neurological symptoms. Nineteen frozen CNS samples analyzed had been previously tested by fluorescence antibody test for rabies virus and showed negative results. Three paraffin-embedded brain tissue samples were examined by histopatology and the observed alterations suggested nonsuppurative meningoencephalitis. BoHV-5 was detected in five (22.7%) among 22 tested samples. The occurrence of BoHV-5 infection is reported in the Southeast region of Brazil, indicating that epidemiological studies should be carried out. Univ Fed Minas Gerais, Inst Ciências Biol, BR-31270901 Belo Horizonte, MG, Brazil Univ Fed Minas Gerais, Escola Vet, BR-31270901 Belo Horizonte, MG, Brazil UNESP, Dept Clin Cirurgia & Reprod Anim, São Paulo, Brazil UNESP, Dept Clin Cirurgia & Reprod Anim, São Paulo, Brazil

The specificity of monoclonal antibodies (mAbs) to desired targets makes these molecules suitable for therapeutic and diagnostic uses against a wide range of pathogens. Phage display antibody libraries offer one method by which mAbs can be selected for, without the use of conventional hybridoma technology. In this work, phage display technology was used to construct, select and characterize a combinatorial single chain fragment variable (scFv) antibody library against bovine herpesvirus type 1 (BoHV-1) from the immune repertoire of chickens immunized with the virus. In silico analysis of the hypervariable domains of the antibody heavy chains revealed a high frequency of scFv fragments with low variability, suggesting that selection had probably been carried out and favored by a few im-munogenic viral antigens. The reactivity of the scFv fragments selected against BoHV-1 was demon-strated by Phage-ELISA. A significant increase in antibody reactivity to the target was observed after six rounds of library selection, showing its potential use as a molecule for BoHV-1 diagnosis. The strategy described here opens up a field for the use of phage display as a tool for selection of mono-clonal antibodies that could be used for theranostic applications against infectious and parasitic dis-eases of veterinary interest.

O perfil antigênico de 45 herpesvírus (44 de bovinos, sendo seis amostras de referência de BHV-1 e 15 prováveis BHV-1; três amostras de referência de BHV-5 e 20 prováveis BHV-5) e uma amostra de herpesvírus bubalino (BuHV) foi examinado com um painel de anticorpos monoclonais (Acms) produzidos contra antígenos de herpesvírus bovinos. Para os exames, foi utilizada a prova de imunoperoxidase (IPX) sobre cultivos de células infectadas, tendo os Acms como anticorpos primários. A determinação dos padrões de reatividade das amostras de vírus frente aos Acms permitiu a diferenciação entre os tipos 1 e 5. Todas as amostras isoladas de casos de encefalite apresentaram perfil de BHV-5. Quatro amostras de BHV-5 isoladas de áreas geograficamente distintas apresentaram perfís de reatividade diferenciados em relação às demais amostras do tipo 5. Duas amostras de vírus com perfil antigênico de BHV-5 foram isoladas de sêmen de animais infectados. Estes resultados comprovam a utilidade da caracterização antigênica com este painel de Acms na tipagem de amostras de BHV-1 e BHV-5.The antigenic profile of 45 herpesviruses (44 viruses from cattle, including six reference BHV-1 strains and 15 putative BHV-1; three reference BHV-5 strains and 20 putative BHV-5) and one buffalo isolate (BuHV) were examined with a panel of monoclonal antibodies (Mabs) prepared against bovine herpesvirus antigens. Tests were performed by immunoperoxidase (IPX) on infected cell cultures, with the Mabs as primary antibodies. Immunostaining allowed the differentiation between types 1 and 5 viruses. All isolates from cases of encephalitis displayed BHV-5 profiles. Four BHV-5 isolates obtained from geographically distinct areas displayed different and highly variable IPX patterns of reactivity. Two viruses with BHV-5 antigenic profile were isolated from semen of asymptomatic bulls. The results showed that the antigenic characterization with the Mab panel employed here is useful for typing BHV-1 and BHV-5 isolates.

O perfil antigênico de 45 herpesvírus (44 de bovinos, sendo seis amostras de referência de BHV-1 e 15 prováveis BHV-1; três amostras de referência de BHV-5 e 20 prováveis BHV-5) e uma amostra de herpesvírus bubalino (BuHV) foi examinado com um painel de anticorpos monoclonais (Acms) produzidos contra antígenos de herpesvírus bovinos. Para os exames, foi utilizada a prova de imunoperoxidase (IPX) sobre cultivos de células infectadas, tendo os Acms como anticorpos primários. A determinação dos padrões de reatividade das amostras de vírus frente aos Acms permitiu a diferenciação entre os tipos 1 e 5. Todas as amostras isoladas de casos de encefalite apresentaram perfil de BHV-5. Quatro amostras de BHV-5 isoladas de áreas geograficamente distintas apresentaram perfís de reatividade diferenciados em relação às demais amostras do tipo 5. Duas amostras de vírus com perfil antigênico de BHV-5 foram isoladas de sêmen de animais infectados. Estes resultados comprovam a utilidade da caracterização antigênica com este painel de Acms na tipagem de amostras de BHV-1 e BHV-5. The antigenic profile of 45 herpesviruses (44 viruses from cattle, including six reference BHV-1 strains and 15 putative BHV-1; three reference BHV-5 strains and 20 putative BHV-5) and one buffalo isolate (BuHV) were examined with a panel of monoclonal antibodies (Mabs) prepared against bovine herpesvirus antigens. Tests were performed by immunoperoxidase (IPX) on infected cell cultures, with the Mabs as primary antibodies. Immunostaining allowed the differentiation between types 1 and 5 viruses. All isolates from cases of encephalitis displayed BHV-5 profiles. Four BHV-5 isolates obtained from geographically distinct areas displayed different and highly variable IPX patterns of reactivity. Two viruses with BHV-5 antigenic profile were isolated from semen of asymptomatic bulls. The results showed that the antigenic characterization with the Mab panel employed here is useful for typing BHV-1 and BHV-5 isolates.

The studies described in this report were performed to determine, whether it is possible to produce live virus vaccines without serum or fractions thereof used during any cell or virus passage, thus completely serum-free. Two viruses were included in the experiments: Bovine Herpesvirus 1 (BHV-1) and Bovine Parainfluenza type 3 virus (PI3). Both viruses were found to grow to satisfactory titers, and to be stable after freeze-drying and subsequent storage at temperatures of +4 °C and −20 °C for at least one year. Moreover, a vaccine containing serum free produced BHV-1 was tested in a vaccination-challenge experiment. For comparison, a vaccine batch with BHV-1 grown in serum-containing cell culture medium was included in the study. Both vaccine preparations performed equally well and both met the strict requirements as laid down in the European Phamacopeia. Moreover, in two separate experiments the safety of serum-free produced BHV-1 and PI3 after overdose and repeated administration even in very young calves and even after four administrations has been demonstrated. This report is the first, which to our knowledge demonstrates the safety and efficacy of serum-free produced live vaccines in the target animal as well as the stability of these products.

Objective—To evaluate and analyze data from controlled studies on the effectiveness of vaccinating cattle with commercially available viral antigen vaccines for mitigation of the effects of bovine respiratory disease complex (BRDC). Design—Systematic review and meta-analysis. Sample—31 studies comprising 88 trials. Procedures—Studies that reported the effectiveness of commercially available bovine herpesvirus-1 (BHV-1), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), and parainfluenza type 3 virus (PI3) vaccines for protection of cattle against BRDC or its components were included in the analysis. Studies or trials were categorized as natural exposure or experimental challenge and were further divided by the viral antigen evaluated and vaccine type (modified-live virus [MLV] or inactivated vaccine). Meta-analysis was performed; summary Mantel-Haenszel risk ratios were determined, and Forest plots were generated. Results—In natural exposure trials, beef calves vaccinated with various antigen combinations had a significantly lower BRDC morbidity risk than did nonvaccinated control calves. In trials evaluating BHV-1 and MLV BVDV vaccines in experimental challenge models, vaccinated calves had a lower BRDC morbidity risk than did control calves; however, in experimental challenge trials evaluating MLV BRSV and PI3 vaccines, no significant difference in morbidity or mortality risk was found between vaccinated and control calves. Conclusions and Clinical Relevance—Estimating clinical efficacy from results of experimental challenge studies requires caution because these models differ substantially from those involving natural exposure. The literature provides data but does not provide sufficiently strong evidence to guide definitive recommendations for determining which virus components are necessary to include in a vaccination program for prevention or mitigation of BRDC in cattle.

Multiple commercial vaccines are available with the goal of decreasing risk and severity of bovine respiratory disease complex (BRDC). The objective was to perform a systematic review of published literature and meta-analysis to evaluate the efficacy of vaccinating cattle with available viral vaccine antigens to mitigate the effects of BRDC compared to unvaccinated controls., American Association of Bovine Practitioners Proceedings of the Annual Conference, 2014

The antiviral effect of polysaccharides has been known for many years. Carrageenans are considered a good alternative for the prevention of a wide range of diseases, mainly caused by enveloped viruses. The advantages lie on their high availability, low cost and low induction of resistance. The aim of this study was to evaluate the sensitivity of two viral pathogens of veterinary interest to the presence of lambda-carrageenan. This is the first report of a lambda-carrageenan having antiviral activity against animal viruses belonging to the Alphaherpesvirinae subfamily, BoHV-1 (bovine herpesvirus type 1) strain Cooper and SuHV-1 (suid herpesvirus type 1) strain Bartha. Lambda-carrageenan was able to reduce infectivity of both viruses with a more pronounced effect against BoHV-1. These results proved, as previously shown for human herpes virus type 1, that these compounds could be used as potential antiviral agents in the veterinary field. Fil: Diogo, Jésica Victoria. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento Virologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Galdo Novo, Sabrina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento Virologia; Argentina Fil: Gonzalez, Marcelo Jose. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento Virologia; Argentina Fil: Ciancia, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Bratanich, Ana Cristina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento Virologia; Argentina

This study presents the first description of Bovine herpesvirus 6 (BoHV-6) that was isolated from buffaloes of Amazon region in Brazil. Phylogenetic analysis showed that the BoHV-6 Brazilian strains clustered with the sequence of BoHV-6 from elsewhere available at the GenBank. It was observed in some buffaloes with lymphoproliferative disease in one herd, thus the animals were also tested for Bovine leukemia virus (BLV), which has been associated to lymphoma in bovines. All animals were negative to BLV. These results indicate that BoHV-6 is present in buffaloes in Brazil, but the importance and impact of this infection and its association with any illness is still undefined.

Viral disease is one of the major causes of financial loss and animal suffering in today's cattle industry. Increases in global commerce and average herd size, urbanization, vertical integration within the industry and alterations in global climate patterns have allowed the spread of pathogenic viruses, or the introduction of new viral species, into regions previously free of such pathogens, creating the potential for widespread morbidity and mortality in naïve cattle populations. Despite this, no antiviral products are currently commercially licensed for use in bovine medicine, although significant progress has been made in the development of antivirals for use against bovine viral diarrhea virus (BVDV), foot and mouth disease virus (FMDV) and bovine herpesvirus (BHV). BVDV is extensively studied as a model virus for human antiviral studies. Consequently, many compounds with efficacy have been identified and a few have been successfully used to prevent infectionin vivoalthough commercial development is still lacking. FMDV is also the subject of extensive antiviral testing due to the importance of outbreak containment for maintenance of export markets. Thirdly, BHV presents an attractive target for antiviral development due to its worldwide presence. Antiviral studies for other bovine viral pathogens are largely limited to preliminary studies. This review summarizes the current state of knowledge of antiviral compounds against several key bovine pathogens and the potential for commercial antiviral applications in the prevention and control of several selected bovine diseases.

Establishment of latent infection within specific tissues in the host is a common biological feature of the herpesviruses. In the case of bovine herpesvirus 2 (BoHV-2), latency is established in neuronal tissues, while bovine herpesvirus 4 (BoHV-4) and ovine herpesvirus 2 (OvHV-2) latent virus targets on cells of the monocytic lineage. This study was conducted in quest of BoHV-2, BoHV-4 and OvHV-2 DNA in two hundred trigeminal ganglia (TG) specimens, derived from one hundred clinically healthy cattle, majority of them naturally infected with bovine herpesvirus 1 (BoHV-1) and bovine herpesvirus 5 (BoHV-5). Total DNA extracted from ganglia was analyzed by polymerase chain reaction (PCR) designed to amplify part of the genes coding for BoHV-2, and BoHV-4 glycoprotein B and, for OvHV-2, the gene coding for phosphoribosylformylglycinamidine synthase-like protein. BoHV-2 DNA was detected in TG samples of two (2%) and BoHV-4 DNA in nine (9%) of the animals, whereas OvHV-2 DNA could not be detected in any of the TG DNA. The two animals in which BoHV-2 DNA was identified were also co-infected with BoHV-1 and BoHV-5. Within the nine animals in which BoHV-4 DNA was detected, six were also co-infected with BoHV-1 and BoHV-5. This report provides for the first time evidence that viral DNA from BoHV-2 and BoHV-4 can be occasionally detected in TG of naturally infected cattle. Likewise, in this report we provided for the first time evidence that the co-infection of cattle with three distinct bovine herpesviruses might be a naturally occurring phenomenon. Fil: Campos, F. S.. Universidade Federal do Rio Grande do Sul; Brasil Fil: Franco, A. C.. Universidade Federal do Rio Grande do Sul; Brasil Fil: Oliveira, M. T.. Universidade Federal do Rio Grande do Sul; Brasil Fil: Firpo, R.. Universidade Federal do Rio Grande do Sul; Brasil Fil: Strelczuk, G.. Universidade Federal do Rio Grande do Sul; Brasil Fil: Fontoura, F. E.. Universidade Federal do Rio Grande do Sul; Brasil Fil: Kulmann, M. I. R.. Universidade Federal do Rio Grande do Sul; Brasil Fil: Maidana, Silvina Soledad. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina Fil: Romera, Sonia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Spilki, F. R.. Universidade Feevale; Brasil Fil: Silva, A. D.. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; Brasil Fil: Hübner, S. O.. Universidade Federal de Pelotas; Brasil Fil: Roehe, P. M.. Universidade Federal do Rio Grande do Sul; Brasil. Instituto de Pesquisa Veterinária Desiderio Finamor; Brasil

Artificial insemination is widely used in the cattle industry and a major challenge is to ensure that semen is free of infectious agents. A healthy donor bull was tested for freedom from infectious agents. A bovine herpesvirus was isolated in testis cells and identified as bovine herpesvirus type 5 (BoHV-5) by polymerase chain reaction and by direct amplicon sequencing. The amplicon sequence shared 100% similarity with the published sequence of BoHV-5. This is the first report in Australia of BoHV-5 in semen. The implications of this finding are discussed.

Avaliou-se o desempenho de um ensaio imunoenzimático, obtido de fonte comercial, na identificação de anticorpos contra herpesvírus bovino tipo 1 (BHV-1), induzidos tanto por infecção natural quanto por vacinação, em 1000 amostras de soros sangüíneos de bovinos. A análise comparativa dos resultados obtidos no sistema avaliado e na técnica padrão de soroneutralização mostrou uma concordância de 97,05% (K=0,94) entre as duas metodologias de diagnóstico sorológico. The performance of a commercial immune assay in the identification antibody of natural infection or vaccination against bovine herpesvirus type 1 (BHV-1) in 1000 samples of bovine serum was evaluated. The comparative analysis from the result of the evaluated system and standard serum neutralization technique showed a rate of agreement of 97.05% (K=0.94) between the two serologic diagnotic methods.

The linear virion DNA of bovine herpesvirus type 4 (BHV-4) is flanked by tandem repeats designated polyrepetitive DNA (prDNA). To investigate the structure and functional role of the prDNA for cleavage/packaging of progeny viral DNA, the complete nucleotide sequence (2267 bp) of a cloned prDNA unit of BHV-4 was determined. Moreover, the terminal fragments of the genome and the junctions between prDNA and the central unique DNA were analysed. In order to characterize the function of the prDNA of BHV-4, a transient packaging assay was developed. The prDNA has a G+C content of 71.1%. Its structure is composed of numerous internal repeats and every unit contains the conserved sequence of the cleavage/packaging signal. A fragment of 443 bp comprising the cleavage/packaging signal was found to be sufficient for cleavage and encapsidation of replicated concatemeric viral DNA. These results suggest that prDNA is a functionally important region of the genome of BHV-4.

Samples of 114 bovine fetuses and 10 calves, which dead in perinatal period, were examined for detection of DNA. The most common detected agent was Brucella spp. in 17 samples (13.7%) followed by Leptospira spp. in 4 cases (3.2%),bovine herpesvirus (BHV) and bovine viral diarrhea (BVDV) in 3 animals (2.4%) each, and 1 for the association of BVDV and BHV. In 77.4 % (96/124) of the samples it was not possible to detect any agent.

Submitted by Ubirajara Cruz (ubirajara.cruz@gmail.com) on 2016-09-21T16:14:51Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_daiana_medeiros.pdf: 661783 bytes, checksum: b730e6b69207fcfbd6fb978846974286 (MD5) Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2016-09-21T16:46:53Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_daiana_medeiros.pdf: 661783 bytes, checksum: b730e6b69207fcfbd6fb978846974286 (MD5) Made available in DSpace on 2016-09-21T16:46:53Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_daiana_medeiros.pdf: 661783 bytes, checksum: b730e6b69207fcfbd6fb978846974286 (MD5) Previous issue date: 2014-02-21 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES Infecções pelo herpesvírus bovino tipo 1 (BoHV-1) têm sido amplamente detectadas em rebanhos bovinos brasileiros, e são responsáveis por grandes prejuízos econômicos na bovinocultura. Entre as doenças causadas por esse vírus estão a Rinotraqueíte infecciosa bovina (IBR), Vulvovaginite Pustular Infeciosa (IPV) e Balanopostite Pustular Infecciosa (IBP). O impacto econômico dessa enfermidade é devido principalmente a falhas reprodutivas, como retorno ao estro e abortamentos. A espécie bovina é hospedeira natural do BoHV-1, no entanto, estudos sorológicos realizados em diversos países têm sugerido que búfalos (Bubalus bubalis) podem ser suscetíveis ao herpesvírus bovino tipo 1 (BoHV-1), e outros alfaherpesvirus geneticamente relacionados. Apesar de os bovinos se infectarem naturalmente pelo BoHV-1, a distribuição dessa enfermidade na espécie bubalina não tem sido estudada no Brasil, e as informações são limitadas e suportadas apenas por inquéritos sorológicos. Dessa forma, o objetivo desse trabalho foi detectar a presença de DNA viral de BoHV-1 em 202 gânglios trigêmeos de bubalinos coletados em um abatedouro localizado no município de Pelotas através de uma Semi-Nested PCR (SN-PCR) para detecção parcial do gene da glicoproteína D (gD) do envelope do BoHV-1. Além disso, 242 amostras de soro foram testadas pela técnica de soroneutralização (SN) para a detecção de anticorpos neutralizantes frente a diferentes amostras de herpesvírus de ruminantes: BoHV-1 (cepa Los Angeles), BoHV-5 (cepa Riopel - RP), e BuHV (cepa B6). O DNA de BoHV-1 foi detectado em 30,1% dos animais, e os resultados da SN mostraram que 27,6% dos animais apresentaram anticorpos neutralizantes contra, pelo menos, um dos vírus testados. Os resultados demonstram que o BoHV-1 circula em rebanhos bubalinos na região sul do Brasil, e apesar das reatividade cruzada, anticorpos anti-BoHV-5 e anti-BuHV-B6 também foram identificados na população estudada. Este é o primeiro estudo que utiliza testes moleculares para a detecção de infecções latentes por BoHV-1 na espécie bubalina no Brasil. Infections caused by the bovine herpesvirus type 1 (BoHV-1) have been widely detected in Brazilian cattle, and they are responsible for major economic losses to the cattle industry. Among the diseases caused by this virus there are infectious rhinotracheitis (IBR), Infectious Pustular vulvovaginitis (IPV) and balanoposthitis Infectious Pustular (IBP). The economic impact of this disease is mainly due to reproductive failures, such as return to estrus and abortions. The bovine is a natural host of BoHV-1, however, serologic studies in several countries have suggested that buffaloes (Bubalus bubalis) may be susceptible to bovine herpesvirus type 1 (BoHV-1), and other genetically related alphaherpesvirus. Despite the cattle become infected naturally by BoHV-1, the distribution of this disease in the buffalo species has not been studied in Brazil, and the information is limited and only supported by serological surveys. Thus, the aim of this study was to detect the presence of viral DNA of BHV - 1 in trigeminal ganglia of 202 buffaloes collected in a slaughterhouse located in the city of Pelotas through a semi -nested PCR (SN - PCR) for detection of partial gene glycoprotein D of BoHV-1. In addition, 242 serum samples were obtained and tested by the serum neutralization (SN ) technique for the detection of neutralizing antibodies against different samples of herpesvirus: BHV -1 ( Los Angeles strain ) , BHV -5 (strain Riopel - RP ) and BuHV (strain B6 ). The BHV -1 DNA was detected in 30.1 % of the animals and the results of SN showed that 27.68 % of the animals had neutralizing antibodies against at least one of the tested viruses. This is the first study that uses molecular tests for the detection of latent BoHV - 1 in buffalo species in Brazil.

Bovine herpesvirus type 5 (BoHV-5) is an etiologic agent of meningoencephalitis in cattle. The aim of this study was to evaluate the antiviral potential of a series of synthetic Mannich bases derived from lawsone and to investigate at which stage of the BoHV-5 replicative cycle the compounds might be acting. The most potent and selective inhibitor exhibited CC50 and EC50 values of 1867 μM ± 8.3 and 3.8 μM ± 1.2, respectively (ACV: 989 μM ± 2 and 166 μM ± 2, respectively).

The involvement of Toll-like receptors (TLRs) in bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) infections has not been analyzed. In this study, the role of TLR signaling on virus replication was investigated. Blood leukocytes consistently express TLRs. Thus, our approach was to study in vitro the effects of agonist stimulation of TLRs expressed by peripheral blood leukocytes on BoHV-1 and BoHV-5 replication. Furthermore, the patterns of TLRs 3, 7–9 expression on virus-infected-bovine leukocytes were analyzed. Only Imiquimod (TLR7/8 agonist) showed anti-viral activity on infected MDBK cells. This is the first evidence that the timely activation of TLR7/8 signaling is effective in impairing BoHV-1 and 5 replication, thereby providing an experimental indication that Imiquimod may be a promising immune modulator. This work describes, for the first time, the expression patterns of TLRs in BoHV-1- or BoHV-5-infected-bovine leukocytes, suggesting the involvement of TLR7 and TLR9 in the recognition of these viruses. Fil: Marin, Maia Solange. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Quintana, Silvina. Instituto de Análisis Fares Taie. Mar del Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Faverin, Claudia. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina Fil: Leunda, M. R.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina Fil: Odeón, Anselmo Carlos. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina Fil: Perez, Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Bovine herpesvirus 1 (BHV1) and bovine viral diarrhea virus 2 (BVD2) are endemic in India although no mixed infection with these viruses has been reported from India. We report first mixed infection of these viruses in cattle during routine screening with a microarray chip. 62 of the 69 probes of BHV1 and 42 of the 57 BVD2 probes in the chip gave positive signals for the virus. The virus infections were subsequently confirmed by RT-PCR. We also discuss the implications of these findings.

The expression kinetics of an essential trans -regulatory protein, ICP27, from herpes simplex virus type 1 correspond to that of an immediate-early gene whose expression increases rapidly upon infection and then decreases as of 7 hr postinfection. In contrast, here we report that the bovine herpesvirus type 1 (BHV-1) homolog BICP27, a 50-kDa protein, is expressed as an early gene. Both the transcript and protein accumulated gradually reaching peak levels at approximately 12 hr postinfection, after which point steady state levels were maintained up to 24 hr. Thus the expression profiles of ICP27 and BICP27 are significantly different, suggesting that they may possess different functions.