Identification and characterization of pestiviruses isolated from individual fetal bovine serum samples originated in Rio Grande do Sul state, Brazil / Identificação e caracterização de pestivírus isolados de amostras individuais de soro fetal bovino originárias do Rio Grande do Sul, Brasil

The identification of diversity of bovine pestiviruses circulating in the field is fundamental for continuous evaluation of diagnostic tests and vaccine composition. In this article we performed the genetic and antigenic characterization of twelve bovine pestiviruses isolated in the western region of Rio Grande do Sul, Brazil. The viruses were isolated from sera of bovine fetuses or from animals with clinical presentations suggestive of pestivirus infection. Genetic characterization by sequencing and phylogenetic analysis of the 5'UTR region of the viral genome allowed for the identification of bovine viral diarrhea virus (BVDV-1a, 4/12, 33.3%), BVDV-1b (6/12, 50%) and BVDV-2 (2/12, 16.7%). The reactivity of the isolates with a panel of monoclonal antibodies raised against envelope proteins (Erns, E1 and E2) demonstrated a high antigenic variability among isolates. Thus, the active circulation of bovine pestivirus infection, with high genetic and antigenic variability, in cattle on the western border of RS was confirmed, demonstrating the importance of continuous characterization of the pestiviruses circulating in the cattle herds to keep the diagnostic and control measures up to date.(AU)

A identificação da diversidade de pestivírus bovinos que circulam no campo é fundamental para a avaliação contínua dos testes de diagnóstico e composição de vacina. Neste artigo, realizamos a caracterização genética e antigênica de doze pestivírus bovinos isolados na região oeste do Rio Grande do Sul, Brasil. Os vírus foram isolados de soros de fetos bovinos ou de animais com apresentações clínicas sugestivas de infecção por pestivírus. A caracterização genética por sequenciamento e análise filogenética da região 5'UTR do genoma viral permitiu a identificação do vírus da diarréia viral bovina (BVDV-1a, 4/12, 33,3%), BVDV-1b (6/12, 50%) e BVDV-2 (2/12, 16,7%). A reatividade dos isolados com um painel de anticorpos monoclonais criados contra proteínas do envelope (Erns, E1 e E2) demonstrou uma alta variabilidade antigênica entre os isolados. Assim, confirmou-se a circulação ativa da infecção por pestivírus bovino, com alta variabilidade genética e antigênica, em bovinos na fronteira oeste do RS, demonstrando a importância da contínua caracterização dos pestivírus circulantes em bovinos para manter atualizadas as medidas de diagnóstico e controle.(AU)

Partial sequence analysis of B2L gene of Brazilian orf viruses from sheep and goats.

We herein describe the partial nucleotide sequencing and phylogenetic analysis of the B2L gene of seventeen Brazilian orf viruses (ORFV). Seventeen viruses were recovered from outbreaks of contagious ecthyma in sheep and goats in four states in Southern and Northeast country, and three from commercial vaccines. Most analyzed viruses were associated with outbreaks of classical contagious ecthyma, with lip, nostrils and labial commissure involvement, yet udder/teat, feet, vulvar and disseminated lesions were also reported in some cases. Nucleotide sequence analysis revealed a high degree of B2L similarity among sheep sequences (>99%) regardless the geographic origin, and a remarkable high identity for the two goat isolates (>99.8%), with similarity dropping to below 99% when comparing viruses from the two species. A phylogenetic tree grouped most sheep and goat viruses on different branches. In addition, sequence alignment allowed the identification of up to six scattered nucleotide changes that were predominant and more consistent in goat isolates, including a number of sequences from other continents. Thus, in spite of the high nucleotide similarity, different degrees of similarity and discrete nucleotide changes in the B2L gene may help in grouping ORFV viruses according to host species.

Assessing the variability of Brazilian Vaccinia virus isolates from a horse exanthematic lesion: coinfection with distinct viruses.

During the last bovine vaccinia (BV) outbreaks, several Vaccinia virus (VACV) strains were isolated and characterised, revealing significant polymorphisms between strains, even within conserved genes. Although the epidemiology of VACV has been studied in BV outbreaks, there is little data about the circulation of the Brazilian VACV isolates. This study describes the genetic and biological characterisation of two VACV isolates, Pelotas 1 virus (P1V) and Pelotas 2 virus (P2V), which were obtained concomitantly from a horse affected by severe cutaneous disease. Despite being isolated from the same exanthematic clinical sample, P1V and P2V showed differences in their plaque phenotype and in one-step growth curves. Moreover, P1V and P2V presented distinct virulence profiles in a BALB/c mouse model, as observed with other Brazilian VACV isolates. Sequencing and phylogenetic analysis of four different genes demonstrated that the isolates are segregated in different VACV clusters. Our results raise interesting questions about the diversity of VACV isolates in Brazil.

Cepa recombinante do herpesvírus bovino tipo 5 defectiva na timidina quinase e glicoproteína E é atenuada e imunogênica para bezerros / A recombinant bovine herpesvirus 5 defective in thymidine kinase and glycoprotein E is attenuated and immunogenic for calves

Bovine herpesvirus 5 (BoHV-5) is an important pathogen of cattle in South America and efforts have been made to produce safer and more effective vaccines. In addition to afford protection, herpesvirus vaccines should allow serological differentiation of vaccinated from naturally, latently infected animals. We previously reported the construction and characterization in vitro of a double mutant BoHV-5 (BoHV-5gE/TK􀀀Δ) lacking the genes encoding thymidine kinase (tk) for attenuation, and glycoprotein E (gE) as the antigenic marker, as a vaccine candidate strain (Brum et al. 2010a). The present article reports an investigation on the attenuation and immunogenicity of this recombinant in calves. In a first experiment, 80 to 90-day-old seronegative calves (n=6) inoculated intranasally with the recombinant (titer of 107.5TCID50) shed virus in low to moderate titers in nasal secretions for up to 6 days, yet did not develop any respiratory, systemic or neurological signs of infection. At day 30 post-infection (pi) all calves had BoHV-5 specific neutralizing (VN) antibodies in titers of 4 to 8 and were negative for anti-gE antibodies in a commercial ELISA test. Administration of dexamethasone (0.1mg/kg/day during 5 days) to four of these calves at day 42 pi did not result in virus shedding or increase in VN titers, indicating lack of viral reactivation. Secondly, a group of 8-month-old calves (n=9) vaccinated intramuscularly (IM) with the recombinant virus (107.5TCID50/animal) did not shed virus in nasal secretions, remained healthy and developed VN titers from 2 to 8 at day 42 post-vaccination (pv), remaining negative for gE antibodies. Lastly, 21 calves (around 10 months old) maintained under field conditions were vaccinated IM with the recombinant virus (titer of 107.3TCID50)...

O herpesvírus bovino tipo 5 (BoHV-5) é um importante patógeno de bovinos na América do Sul e tem suscitado esforços para o desenvolvimento de vacinas mais seguras e eficazes. Além de conferirem proteção, vacinas contra os herpesvírus animais devem permitir a diferenciação sorológica entre animais vacinados e infectados naturalmente. Foi recentemente relatada a construção e caracterização in vitro de um recombinante do BoHV-5 defectivo na enzima timidina quinase (tk) para atenuação e, na glicoproteína E (gE) como marcador antigênico, como potencial cepa vacinal (Brum et al. 2010a). O presente artigo relata uma investigação sobre da atenuação e imunogenicidade do recombinante BoHV-5gE/TKΔ em bezerros. Em um primeiro experimento, bezerros soronegativos, com 80 a 90 dias de idade (n=6), inoculados pela via intranasal (IN) com o vírus recombinante (título de 107,5TCID50) excretaram baixos títulos de vírus nas secreções nasais por até seis dias, mas não desenvolveram sinais sistêmicos, respiratórios ou neurológicos. No dia 30 pós-infecção (pi), todos os animais possuíam anticorpos neutralizantes contra o BoHV-5, em títulos entre 4 e 8, mas permaneceram negativos para anticorpos contra a gE. Administração de dexametasona (Dx) a quatro desses bezerros no dia 42 pi (0.1mg/kg/dia durante 5 dias) não resultou em excreção viral ou em aumento dos títulos de anticorpos, indicando ausência de reativação viral. Em um segundo experimento, vacinação intramuscular (IM) de bezerros com 8 meses de idade (n=9) com o recombinante (107,5TCID50/animal) não resultou em excreção viral ou em manifestações clínicas. Os animais vacinados desenvolveram anticorpos neutralizantes em títulos de 2 a 8 no dia 42 pós-vacinação (PV) e permaneceram negativos para anticorpos anti-gE. Finalmente, 21 bezerros (aproximadamente 10 meses de idade) mantidos a campo foram vacinados com o recombinante (107,3TCID50) pela via IM...

An outbreak of orthopoxvirus-associated disease in horses in southern Brazil.

An outbreak of severe cutaneous disease associated with an orthopoxvirus in horses in southern Brazil is described. Fourteen Crioulo mares and foals from a husbandry farm developed papules, and vesicles progressing to proliferative and exudative lesions on the muzzle, external nares, and external and internal lips. The vesicles eroded, and the proliferative lesions eventually bled and progressed to moist crusts and scars. The clinical signs lasted approximately 6-12 days, after which the animals progressively recovered. Direct electron microscopy of skin biopsies revealed brick-shaped, 250-300-nm virus particles with orthopoxvirus morphology. Histological examination of the lesions revealed vacuolar degeneration of the cells of the stratum spinosum and the presence of large intracytoplasmic, eosinophilic inclusion bodies. Polymerase chain reaction amplification of poxvirus A-type inclusion body gene confirmed the presence of orthopoxvirus DNA in horse tissues. Inoculation of tissue homogenates into the chorioallantoic membrane of chicken embryonated eggs and intraperitoneally in mice resulted in pock-like lesions with the microscopic appearance of poxvirus-induced histopathology. Taken together, these results demonstrate the association of an orthopoxvirus with the outbreak of cutaneous disease in horses. The origin of the agent causing the outbreak is uncertain because no similar condition has been reported in Brazil.

Bovine herpesvirus type 1 (BoHV-1) anterograde neuronal transport from trigeminal ganglia to nose and eye requires glycoprotein E.

The requirement of bovine herpesvirus type 1 (BoHV-1) envelope protein gE (Us8 homolog) for establishment of latency and reactivation in trigeminal ganglia (TG) was examined. Although BHV-1 gE-rescued and gE-deleted viruses were isolated from nasal or ocular swabs during primary infection, only the gE-rescued virus was isolated following dexamethasone-induced reactivation. Furthermore, gC protein expression, which requires viral DNA replication for its expression, was detected in TG of calves infected with either virus following reactivation. These studies suggest that gE is required for anterograde transport of BoHV-1 from neuronal cell bodies in the TG to their nerve processes.

Immunopotentiation of a foot-and-mouth disease virus subunit vaccine by interferon alpha.

The adjuvant effect of porcine interferon alpha (pIFN-alpha) was examined in swine vaccinated with a replication-defective adenovirus containing foot-and-mouth disease virus (FMDV) A24 capsid and 3C proteinase coding regions (Ad5-A24). Groups of swine were inoculated with either high or low doses of Ad5-A24 in the presence or absence of Ad5-pIFNalpha or with a control Ad5 and challenged by intradermal inoculation in the heel bulb with FMDV at 42 days post-vaccination. After challenge all control animals developed viremia and lesions. Animals receiving low-dose Ad5-A24 had similar clinical disease, but only three of five animals developed viremia, while addition of IFN resulted in a delayed onset of lesions in three animals and only one animal had detectable viremia. Animals vaccinated with high-dose Ad5-A24 had no viremia, significantly fewer lesions and delayed onset of disease compared to the control and low-dose vaccine groups. Four of five pigs vaccinated with high-dose Ad5-A24 plus IFN were completely protected from disease and only one animal had a lesion which was restricted to the site of challenge. Thus, pIFN-alpha enhances the long-term level of protection induced by the Ad5-FMD vaccine, supporting its use as a potential adjuvant in FMD vaccination strategies.

Granulocyte-macrophage colony-stimulating factor does not increase the potency or efficacy of a foot-and-mouth disease virus subunit vaccine

A febre aftosa é uma das doenças mais temidas nos rebanhos em todo o mundo. A vacinação tem sido uma arma eficiente no controle da doença, no entanto há preocupações com as vacinas atualmente utilizadas incluindo a necessidade de instalações de alta segurança para a produção dessas vacinas e a falta de um teste de diagnóstico aprovado que faça distinção precisa entre animais vacinados dos infectados. Várias vacinas têm sido testadas contra a febre aftosa e uma dessas utiliza como vetor um vírus defectivo para replicação, derivado do adenovírus humano tipo 5 (Ad5), o qual contém as proteínas que compõe capsídeo do vírus da febre aftosa (P1-2A) e a protease 3C, protegeu completamente suínos contra o desafio de uma cepa homóloga (A12 e A24). Uma vacina com o Ad5-P1-2A+3C proveniente da cepa O1 Campos (Ad5-O1C), no entanto, somente induziu um baixo título de anticorpos neutralizantes específicos em testes de potência vacinal em suínos. O fator estimulante de colônias de granulócitos e macrófagos (GM-CSF) tem sido utilizado com sucesso na formulação de vacinas para estimular a resposta imune contra inúmeras doenças, incluindo HIV, Hepatite C e B. Na tentativa de melhorar a resposta imune específica contra a febre aftosa induzida pelo Ad5-O1C, suínos foram vacinados com Ad5-O1C juntamente com Ad5-GM-CSFporcino. Entretanto nas, condições utilizadas nesse teste, o GM-CSF suíno não melhorou a resposta imune do Ad5-O1C e adversamente afetou o nível de proteção de suínos desafiados com o vírus homólogo da febre aftosa.

Rapid protection of cattle from direct challenge with foot-and-mouth disease virus (FMDV) by a single inoculation with an adenovirus-vectored FMDV subunit vaccine.

We have previously demonstrated that swine vaccinated with one dose of a replication-defective human adenovirus type 5 (Ad5) vector containing the capsid and 3C proteinase coding regions of foot-and-mouth disease virus (FMDV) were protected when challenged 7 days later with homologous virus. In the current study, we have extended this approach to cattle, the most economically important animals susceptible to FMD. Five cattle were vaccinated with the Ad5-FMDV subunit vaccine and these animals and 2 co-housed control animals were challenged intradermolingually 7 days later. Both control animals developed typical signs of FMD including fever and vesicular lesions on all 4 feet. All 5 vaccinated animals were protected against disseminated disease.

Constitutive expression of alpha interferon by skin dendritic cells confers resistance to infection by foot-and-mouth disease virus.

The role of dendritic cells (DC) in the initiation of immune responses against foot-and-mouth disease virus (FMDV) is poorly understood. We analyzed the innate response of freshly isolated swine skin DC to the virus and show a rapid induction of beta interferon (IFN-beta) mRNA but not IFN-alpha mRNA. However, these DC secreted both IFN-alpha and IFN-beta proteins in response to live virus but not killed virus. Furthermore, the surface expression of swine major histocompatibility complex class II (SLA II) or CD80/CD86 molecules and antigen processing functions were not affected by FMDV exposure. Given the demonstrated sensitivity of FMDV to IFN-alpha/beta, there was no productive or nonproductive infection of these cells. Finally, freshly isolated skin DC constitutively expressed intracellular IFN-alpha protein in the absence of stimulation, with no detectable secretion of the cytokine until virus exposure. In situ analysis of these DC showed that these cells express and store IFN-alpha in uninfected animals. This is the first demonstration of the constitutive expression of IFN-alpha in resident, tissue-derived DC and indicates that skin DC can play an important role in the innate immune response of swine to viral infections.

Immediate protection of swine from foot-and-mouth disease: a combination of adenoviruses expressing interferon alpha and a foot-and-mouth disease virus subunit vaccine.

We have previously shown that swine inoculated with recombinant, replication-defective human adenovirus type 5 containing the porcine interferon alpha gene (Ad5-pIFNalpha) are completely protected when challenged 1 day later with virulent foot-and-mouth disease virus (FMDV). In the current study, we examined the duration of protection afforded swine by Ad5-pIFNalpha and the ability of a combination of Ad5-pIFNalpha and a FMDV subunit vaccine delivered by Ad5-A24 (an Ad5 vector containing the capsid coding region of FMDV serotype A24 Cruzeiro and the 3C proteinase coding region of FMDV serotype A12) to induce immediate as well as long-lasting protection against homologous FMDV challenge. Groups of swine were inoculated with Ad5-pIFNalpha and challenged with virulent FMDV A24 1, 3, 5, and 7 days postinoculation (dpi) or 1 day preinoculation. All animals challenged 1 and 3dpi were completely protected from disease. The animals in the remaining groups had either no clinical signs of disease or clinical signs were delayed and less severe compared to the control group. Swine inoculated with a combination of Ad5-pIFNalpha and Ad5-A24 and challenged 5dpi were all completely protected from disease and developed a significant FMDV-specific neutralizing antibody response.

Adenovirus-mediated type I interferon expression delays and reduces disease signs in cattle challenged with foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is an economically important disease of livestock. Eliminating FMD outbreaks in previously disease-free countries often relies on restriction of animal movement and massive slaughter of infected and in-contact susceptible animals. To develop a more effective and humane FMD control strategy, we explored the possibility of using type I interferon (IFN-alpha/beta) as a novel anti-FMD agent. We have demonstrated previously that swine inoculated with replication-defective human adenovirus type 5 (Ad5) vector expressing porcine IFN-alpha (Ad5-PoIFN-alpha) were completely protected from FMD virus (FMDV) challenge. To extend this approach to bovines, we constructed Ad5 vectors that express bovine IFN-alpha or IFN-beta (Ad5-BoIFN-alpha and Ad5-BoIFN-beta). Cells infected with these viruses produced high levels of biologically active BoIFN-alpha/beta, but despite expression in vitro, no detectable IFN-induced biologic activity was found in cattle inoculated with Ad5-BoIFN-alpha. Because PoIFN-alpha inhibits FMDV replication in bovine cells, we evaluated the potential use of PoIFN-alpha against FMD in cattle. In cattle inoculated with Ad5-PoIFN-alpha, the appearance of vesicles was delayed after challenge with FMDV and disease was less severe than in control animals. One Ad5-PoIFN-alpha-inoculated animal never developed clinical disease. Similarly, although all the Ad5-PoIFN-alpha-inoculated animals developed viremia, it was delayed for 1 day as compared with the control group. These results suggest that in vivo expression of PoIFN-alpha partially protected cattle from FMD.

Proteçäo fetal frente a desafio com o vírus da Diarréia Viral Bovina (BVDV) em ovelhas imunizadas com duas amostras de vírus modificadas experimentalmente / Fetal protection against challenge with bovine viral diarrhea virus (BVDV) in pregnant ewes immunized with two strains experimentally attenuated

Duas amostras do vírus da Diarréia Viral Bovina (BVDV) submetidas a múltiplas passagens em cultivo celular e exposiçäo à radiaçäo ultravioleta (UV) a cada passagem foram avaliadas como candidatos a vírus vacinais. As amostras foram testadas quanto à sua atenuaçäo para bezerros e fetos ovinos, reatividade antigênica contra isolados de campo, e capacidade de induzir proteçäo fetal em ovelhas prenhes. Inoculaçäo intramuscular (IM) dos vírus modificados em quatro bezerros produziu apenas uma elevaçäo discreta e passageira da temperatura corporal, seguida de produçäo de altos títulos de anticorpos neutralizantes. O vírus näo foi detectado em secreçöes nasais ou sangue nos dias seguintes à inoculaçäo. Porém, a inoculaçäo IM desses vírus em quatro ovelhas prenhes foi seguida de transmissäo transplacentária e infecçäo em todos os fetos. Para os testes de proteçäo fetal, ovelhas prenhes previamente imunizadas com duas doses vacinais, foram inoculadas por via intranasal com amostras de BVDV-1 (SV-126.8, n=6) ou BVDV-2 (SV-260, n=5). No dia do desafio (134 dias após a segunda dose), todos os animais apresentavam altos títulos de anticorpos neutralizantes (256 a >4096) contra os vírus vacinais; além de títulos variados (8 a >4096) contra várias isolados brasileiros de BVDV-1 e BVDV-2. Quinze dias após o desafio, as ovelhas foram sacrificadas e os tecidos fetais foram examinados para a presença de vírus. Todos os fetos das ovelhas controle näo-vacinadas apresentaram-se (n=4) positivos para os vírus utilizados no desafio. Em contraste, nenhum feto das ovelhas imunizadas (n=11) foi positivo para vírus, indicando que a resposta imunológica induzida pela vacinaçäo com os vírus modificados foi capaz de prevenir a infecçäo fetal. Estes resultados indicam que é possível obter-se forte resposta imunológica e proteçäo fetal contra o BVDV com o uso de vacinas vivas modificadas