Immunogenicity of an inactivated vaccine for intravaginal application against bovine alphaherpesvirus type 5 (BoHV-5).

The present study was carried out to evaluate the intravaginal vaccine potential against bovine alphaherpesvirus type 5 (BoHV-5). Sixty three cows were divided into seven groups (n: 9) and inoculated intravaginally (VA) or intramuscularly (IM) with inactivated BoHV-5, associated with the recombinant B subunit of the heat-labile enterotoxin of E. coli (rLTB), 2-hydroxyethylcellulose (Drug Delivery System A - DDS-A) or Poloxamer 407 (Drug Delivery System B - DDS-B) as follows: G1 (DDS-A + BoHV-5 + rLTB), G2 (DDS-A + BoHV-5), G3 (DDS-B + BoHV-5 + rLTB), G4 (DDS-B + BoHV-5), G5 (BoHV-5 + rLTB), G6 (Negative control) e G7 (Positive control). The local and systemic humoral responses were measured by indirect ELISA (IgA and IgG) and serum neutralization tests, and the cellular response was measured by a quantitative direct ELISA (IL-2 and IFN-Gamma). The results showed the group inoculated by the IM route, G5, demonstrated the highest levels of IgG in the vaginal mucosa among the experimental groups (p < 0.05). In the groups tested with polymers (G1 and G3) in the vaginal mucosa, even higher levels of IgG were seen in comparison to the positive control (G7; p < 0.01). Higher levels of IgA were also noted in relation to the other groups (p < 0.05) on days 30, 60 and 90 post-inoculations. The groups G1 and G3 also provided higher titers of neutralizing antibodies (Log2) in relation to other treatments (p < 0.01) 90 days after inoculation. In the nasal mucosa, there was an increase in the levels of IgA and IgG with the use of vaccines from groups G1 and G3, in relation to the positive control, G7 (p < 0.05) at 60 and 90 days after the first inoculation. Moreover, neutralizing antibodies titers were detected at 60 and 90 days by serum neutralization. The inclusion of the evaluated polymers resulted in a superior response (p < 0.05) of immunoglobulins and IL-2 and IFN-Gamma in relation to the treatment using only rLTB (G5). This data demonstrates the capabilities of a vaccine with an intravaginal application in cattle to stimulate a local and systemic immune response.

Whole-genome analysis of natural interspecific recombinant between bovine alphaherpesviruses 1 and 5.

Bovine alphaherpesviruses 1 and 5 (BoHV-1 and BoHV-5) are closely related viruses that co-circulate in South America and recombine in the field. The complete genomes of three natural gB gene recombinant viruses between BoHV-1 and BoHV-5 were obtained by Illumina next-generation sequencing. Complete genome sequences of the three recombinant strains (RecA1, RecB2, and RecC2) have a similar size of approximately 138.3kb and a GC content of 75%. The genome structure corresponds to herpesvirus class D, with 69 open reading frames (ORFs) arranged in the same order as other bovine alphaherpesviruses related to BoHV-1. Their genomes were included in recombination network studies indicating statistically significant recombination evidence both based on the whole genome, as well as in the sub-regions. The novel recombinant region of 3074 nt of the RecB2 and RecC2 strains includes the complete genes of the myristylated tegument protein (UL11) and the glycoprotein M (UL10) and part of the helicase (UL9) gene, and it seems to have originated independently of the first recombinant event involving the gB gene. Phylogenetic analyzes performed with the amino acid sequences of UL9, UL 10, and UL11 indicated that RecB2 and RecC2 recombinants are closely related to the minor parental virus (BoHV-1.2b). On the contrary, RecA1 groups with the major parental (BoHV-5), thus confirming the absence of recombination in this region for this recombinant. One breakpoint in the second recombinant region lies in the middle of the UL9 reading frame, originating a chimeric enzyme half encoded by BoHV-5 and BoHV-1.2b parental strains. The chimeric helicases of both recombinants are identical and have 96.8 and 96.3% similarity with the BoHV-5 and BoHV-1 parents, respectively. In vitro characterization suggests that recombinants have delayed exit from the cell compared to parental strains. However, they produce the similar viral titer as their putative parents suggesting the accumulation of viral particles for the cell exit delayed on time. Despite in vitro different behavior, these natural recombinant viruses have been maintained in the bovine population for more than 30 years, indicating that recombination could be playing an important role in the biological diversity of these viral species. Our findings highlight the importance of studying whole genome diversity in the field and determining the role that homologous recombination plays in the structure of viral populations. A whole-genome recombinant characterization is a suitable tool to help understand the emergence of new viral forms with novel pathogenic features.

Meningoencephalitis caused by Bovine alphaherpesvirus 5 in Pernambuco, Brazil / [Meningoencefalite causada pelo Bovine alphaherpesvirus 5 em Pernambuco, Brasil]

Objetivou-se descrever a ocorrência do Bovine alphaherpesvirus 5 (BoHV5) como causa de meningoencefalite não supurativa em bovinos do estado de Pernambuco, Brasil. Para tanto, 32 amostras de sistema nervoso embebidas em parafina foram obtidas de animais acometidos por doenças neurológicas atendidos na Clínica de Bovinos de Garanhuns da Universidade Federal Rural de Pernambuco (CBG-UFRPE), entre 2012 e 2016. As amostras foram analisadas quanto à presença do gene da glicoproteína C do BoHV5 por reação em cadeia da polimerase (PCR). Dois animais (6,25%) tiveram resultado positivo à PCR, e sua análise de sequenciamento indicou 100% de similaridade para o BoHV5. Os resultados histopatológicos desses dois animais revelaram lesões multifocais de meningoencefalite não supurativa associada à polioencefalomalácia, presença de corpúsculos de inclusão basofílicos, infiltração de células de Gitter e presença de manguitos perivasculares. A PCR se mostra uma importante ferramenta para diferenciação das infecções por BoHV5 de outras enfermidades neurológicas de bovinos, especialmente a raiva.(AU)

Association of Bacillus toyonensis spores with alum improves bovine herpesvirus 5 subunit vaccine immune response in mice.

Spores of the genus Bacillus are molecules capable of increasing the vaccine adjuvanticity. Bovine herpesvirus type 5 (BoHV-5) is responsible for meningoencephalitis that causes important economic losses in cattle. BoHV-5 glycoprotein D (gD) is a target of vaccine antigen and plays an important role in host cell penetration. The present study aimed to evaluate the adjuvanticity of Bacillus toyonensis (B.t) spores, live and heat-killed, associated with a vaccine formulated with aluminum hydroxide (alum) and the recombinant BoHV-5 glycoprotein D (rgD) in an experimental murine model. Six experimental groups of mice were subcutaneously vaccinated on day 0 and received a booster on day 21 of the experiment, with the following vaccine formulations: rgD (40 µg) + live spores (2 × 109 CFU); rgD + killed spores; rgD + live spores + alum (2.0 mg); rgD + killed spores + alum; rgD + alum, and rgD + PBS. Mice from rgD + live spores group showed an increase in rgD IgG titers from the 21st day until the end of the experiment. The groups of live and killed spores, associated to alum, had similar levels of IgG titers with no significant difference between each other; however, by the 14th and 28th day until the end of the experiment, presented higher IgG titers in comparison to the rgD + alum group. Moreover, increased serum levels of IgG1, IgG2a, and IgG2b were detected in mice that received spores in the vaccine formulation. The spores associated with alum groups showed neutralizing BoHV-5 antibodies and high mRNA transcription of the cytokines IFN-γ (66-fold), IL-17 (14-fold), and IL-12 (2.8-fold). In conclusion, our data demonstrated that the B. toyonensis spores, live or killed, associated with alum increased the adjuvanticity for BoHV-5 rgD in mice, suggesting the use of B. toyonensis spores as a promising component for vaccine formulations.

Diagnóstico diferencial das doenças neurológicas dos bovinos no estado do Paraná / Differential diagnosis of neurologic diseases of cattle in the state of Paraná

O objetivo deste trabalho foi identificar as doenças neurológicas que acometeram bovinos no estado do Paraná entre os anos de 2009 e 2015. A investigação aconteceu, preferencialmente, nas propriedades rurais onde os casos ocorreram. Foram registradas as informações sobre a evolução das doenças nos bovinos afetados do rebanho, e os prováveis fatores de risco foram identificados. Todos os procedimentos de exame físico geral e neurológico foram realizados sistematicamente para a caracterização da síndrome neurológica presente. Amostras de sangue e de líquor foram colhidas para a realização de exames laboratoriais. De acordo com o tempo de evolução e com a gravidade dos sinais clínicos observados, os bovinos doentes eram mantidos vivos para acompanhamento da evolução ou da resposta ao tratamento, ou eram submetidos à eutanásia seguida de necropsia. Fragmentos do sistema nervoso e dos demais órgãos foram colhidos para exame histopatológico. O exame de imunofluorescência direta e a prova biológica em camundongos foram realizados em todos os bovinos que morreram, com a finalidade de confirmar ou descartar o diagnóstico de raiva. Métodos laboratoriais específicos das rotinas de virologia, bacteriologia e toxicologia foram empregados, como complementares, para o estabelecimento do diagnóstico diferencial. Foram investigados 236 bovinos com doença neurológica, sendo 85 casos de ocorrência individual e 151 casos distribuídos por surtos que ocorreram em 79 rebanhos. As encefalopatias (180/236; 76,2%) predominaram sobre as mielopatias (27/236; 11,4%). As doenças inflamatórias determinadas por infecções (98/236; 41,5%) e as doenças tóxicas (91/236; 38,6%) foram as principais, enquanto as causas degenerativas (10/236; 4,2%), metabólicas (9/236; 3,8%), físicas (9/236; 3,8%), neoplásicas (4/236; 1,7%), e os defeitos congênitos (1/236; 0,4%) ocorreram menos frequentemente. Os casos inconclusivos somaram 5,9% (14/236). A meningoencefalite por BoHV-5 e a raiva foram as doenças de frequência maior e podem ser consideradas as mais importantes. Dentre as causas tóxicas, as intoxicações por plantas se destacaram (63/91; 69,2%) e foram responsáveis por 26,6% de todos os casos. Destacaram-se ainda a polioencefalomalácia, a meningoencefalite trombótica por Histophilus somni e o botulismo. Essas informações contribuem para que os médicos veterinários adotem condutas mais efetivas de diagnóstico e de prevenção, e são valiosas para o sistema oficial de vigilância epidemiológica do estado.(AU)

The aim of this study was to identify the neurological diseases that affected cattle in Paraná state between the years 2009 and 2015. The investigation took place, preferably, in the farms where cases occurred. Information on the evolution of the diseases in the affected cattle of the herd was recorded, and the probable risk factors were identified. All general and neurological examination procedures were performed systematically for the characterization of the neurological syndrome in each case. Samples of blood and CSF for laboratory exams were also collected. According to the evolution features and the severity of the observed clinical signs, the diseased cattle were kept alive to follow the progress of the disease, or were submitted to euthanasia followed by necropsy. Fragments of tissues from nervous system and other organs were collected for histopathological examination. Direct immunofluorescence test and biological test were performed on all the cattle that died, in order to confirm or rule out the diagnosis of rabies. Specific virology, bacteriology and toxicology laboratory methods were used as complementary exams in order to establish differential diagnosis. A total of 236 cattle with neurological disease were investigated, 85 cases of individual occurrence and 151 cases distributed by outbreaks that occurred in 79 herds. Encephalopathies (180/236, 76.2%) predominated over mielopathies (27/236, 11.4%). Inflammatory diseases caused by infections (98/236, 41.5%) and the toxic diseases (91/236, 38.6%) were the main causes, while degenerative (10/236, 4.2%), metabolic (9/236; 3.8%), physical (9/236, 3.8%), neoplastic (4/236, 1.7%), and congenital defects (1/236, 0.4%) occurred less often. The inconclusive cases were 5.9% (14/236). BoHV-5 meningoencephalitis and rabies were diseases of higher frequency and may be considered the most important. Among the toxic causes, plant poisonings were highlighted (63/91, 69.2%) and were responsible for 26.6% of all cases. Polioencephalomalacia, thrombotic meningoencephalitis caused by Histophilus somni and botulism were also highlighted. This information helps veterinarians to adopt more effective diagnostic and preventive approaches and is valuable to the state's official epidemiological surveillance system.(AU)

Evidence of natural interspecific recombinant viruses between bovine alphaherpesviruses 1 and 5.

Closely related bovine alphaherpesviruses 1 (BoHV-1) and 5 (BoHV-5) co-circulate in certain countries, rendering cattle co-infection possible. This is a prerequisite for BoHV recombination. Here, we report the first identification of homologous recombination between field isolates of BoHV-1 and BoHV-5, two alphaherpesviruses belonging to two distinct species with an average genomic similarity of 82.3%. Three isolates of BoHV-5, previously classified as subtype "BoHV-5b", were phylogenetically studied and analyzed via eight PCR sequencing assays dispersed at regular intervals throughout the genome to discriminate between BoHV-1 and BoHV-5. In the phylogenetic analysis, differences of clustering were found in the UL27 gene which encodes the glycoprotein B (gB). We detected two recombination breakpoints in the open reading frame of the UL27 gene. We compared the amino acid sequences of the gB of BoHV-1.1 and 1.2, BoHV-5a and recombinant formerly named BoHV-5b (chimeric gB) and subsequently performed molecular modeling. All structures were alike and, simultaneously, similar to the chimeric gB. Neutralizing antibodies against BoHV-1, BoHV-5 and recombinant viruses were analyzed via serum virus neutralization test using polyclonal sera and a monoclonal antibody against gB to demonstrate an absence of viral escape for both assays. Our results show that homologous recombination between two related species of ruminant alphaherpesviruses can occur in natural field conditions. We found three recombinant field isolates, previously classified as BoHV-5b subtypes, between BoHV-1 and BoHV-5.

Susceptibility of mice to bovine herpesvirus type 5 infection in the central nervous system.

Bovine herpesvirus type 5 (BoHV-5) is an important pathogen that causes meningoencephalitis in cattle. Few studies have used the mouse as a model for BoHV-5 infection. Despite the fact that BoHV-5 can infect mice with immune deficiencies, little is known about viral replication, immune response, and the course of infection in the central nervous system (CNS) of wild-type mice. Therefore, the aim of this study was to evaluate the response in the CNS of BALB/c mice acutely infected with BoHV-5 at different days post-inoculation (dpi). BoHV-5, when inoculated intracranially, was able to infect and replicate within the CNS of BALB/c mice. Until 15 dpi, the mice were able to survive without showing prominent neurological signs. The infection was accompanied by a Th1 immune response, with a significant expression of the cytokines IFN-γ and TNF-α and chemokine CCL-2. The expression of these cytokines and chemokines was most significant in the early course of infection (3 and 4 dpi), and it was followed by meningoencephalitis with perivascular cuffing and periventriculitis, composed mainly of macrophages and lymphocytes. After the expression of cytokines and chemokine, the mice were able to curb BoHV-5 acute infection in the brain, since there was a decrease in the number of BoHV-5 DNA copies after 3 dpi and viable viral particles were not detected after 6 dpi. Importantly, BoHV-5 was able to infect the trigeminal ganglia during acute infection, since a large number of BoHV-5 DNA copies were detected on 1 and 2 dpi.

Latency of bovine herpesvirus type 5 (BoHV-5) in tonsils and peripheral blood leukocytes.

Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) can both establish latency in the trigeminal ganglion. Non-neural sites of latency have been described for BoHV-1 but not for BoHV-5. The aim of this study was to determine whether peripheral blood leukocytes and tonsils are targets for BoHV-5 infection and to establish whether all stages of that virus's infectious cycle can occur in those cell types. Comparisons with BoHV-1 infection of these tissues were also made in order to better understand the pathogenesis of both viruses. BoHV-1 and BoHV-5 were isolated from tonsils of acutely-infected calves. BoHV-5 was also isolated from a tonsil homogenate after dexamethasone-induced reactivation. During latency, infectious virus was recovered from a tonsil explant of one BoHV-5-infected calf. The genomes of BoHV-5 and BoHV-1 were detected in tonsils from acutely-infected calves although were not detected in tonsils from latently-infected calves or from calves treated with dexamethasone. Virus DNA was intermittently detected in leukocytes. The study has shown that BoHV-5 can establish latency in bovine tonsils and peripheral white blood cells, and that it can be reactivated from latently-infected tonsils, which might contribute to viral transmission. The titres of BoHV-1 and BoHV-5 in tonsils were similar, suggesting that replication at this site is a common feature for both viruses.

The constitutive expression of the V gene of Parainfluenza virus 5 affects the growth properties of bovine herpesvirus 5

This study aimed to analyze the effect of the expression of Parainfluenza virus 5 (PIV5) V protein in bovine cells on the replication of Bovine herpesvirus 5 (BoHV-5). Growth properties of BoHV-5 were evaluated in parental and PIV5 transfected cells. In one-step growth experiments, the BoHV-5 reached higher titers at earlier time points in the transfected cells when compared to the parental cells. The mean plaque size produced by the BoHV-5 in transfected cells was larger than the parental cells. This indicated that the expression of the PIV5 V gene facilitated the release and cell-to-cell spread of BoHV-5 in bovine cells.

Mapping the sites of latency and reactivation by bovine herpesvirus 5 (BoHV-5) and a thymidine kinase-deleted BoHV-5 in lambs / Mapeamento dos sítios de latência e reativação pelo herpesvírus bovino tipo 5 (BoHV-5) e por mutante deletado no gene da timidina quinase em ovinos

A thymidine kinase (tk)-deleted bovine herpesvirus 5 (BoHV-5tkΔ) was previously shown to establish latent infection and reactivate - even poorly - in a sheep model (Cadore et al. 2013). As TK-negative alphaherpesviruses are unlike to reactivate in neural tissue, this study investigated the sites of latency and reactivation by this recombinant in lambs. For this, groups of lambs were inoculated intranasally with the parental BoHV-5 strain (SV-507/99) or with the recombinant BoHV-5tkΔ. During latent infection (40 days post-inoculation, pi), the distribution of recombinant virus DNA in neural and non-neural tissues was similar to that of the parental virus. Parental and recombinant virus DNA was consistently detected by PCR in trigeminal ganglia (TGs); frequently in palatine and pharyngeal tonsils and, less frequently in the retropharyngeal lymph nodes. In addition, latent DNA of both viruses was detected in several areas of the brain. After dexamethasone (Dx) administration (day 40pi), the recombinant virus was barely detected in nasal secretions contrasting with marked shedding of the parental virus. In tissues of lambs euthanized at day 3 post-Dx treatment (pDx), reverse-transcription-PCR (RT-PCR) for a late viral mRNA (glycoprotein D gene) demonstrated reactivation of parental virus in neural (TGs) and lymphoid tissues (tonsils, lymph node). In contrast, recombinant virus mRNA was detected only in lymphoid tissues. These results demonstrate that BoHV-5 and the recombinant BoHV-5tkΔ do establish latent infection in neural and non-neural sites. Reactivation of the recombinant BoHV-5tkΔ, however, appeared to occur only in non-neural sites. In anyway, the ability of a tk-deleted strain to reactivate latent infection deserves attention in the context of vaccine safety.

Um recombinante do herpesvírus bovino tipo 5 com deleção no gene da timidina quinase (BoHV-5tkΔ) foi capaz de estabelecer latência e reativar - embora ineficientemente - em modelo experimental em ovinos (Cadore et al. 2013). Como a reativação de alfaherpesvírus defectivos na TK em tecido neural é improvável, o presente estudo investigou os sítios de latência e reativação por esse recombinante em ovinos. Para isso, grupos de ovinos foram inoculados com a cepa de BoHV-5 parental (SV-507/99) ou com o recombinante BoHV-5tkΔ. Durante a infecção latente (dia 40 pós-infecção, pi) a distribuição do DNA do vírus recombinante no encéfalo de ovinos infectados experimentalmente foi similar ao do vírus parental (SV-507/99). O DNA de ambos os vírus foi detectado consistentemente por PCR nos gânglios trigêmeos (TGs), frequentemente nas tonsilas faríngeas e palatinas e, com menos frequência, nos linfonodos retrofaríngeos. Após administração de dexametasona (Dx), o vírus recombinante foi raramente detectado nas secreções nasais, contrastando com excreção abundante do vírus parental. RT-PCR para mRNA de um gene tardio (glicoproteína D) realizado em tecidos de animais eutanasiados 3 dias pós-Dx demonstrou reativação do vírus parental em tecido neural (TGs) e não-neural (tonsilas, linfonodo). Em contraste, a reativação do vírus recombinante ficou restrita ao tecido linfoide. Esses resultados demonstram que tanto o BoHV-5 parental quanto o recombinante estabelecem latência em sítios neurais e não-neurais. No entanto, o recombinante BoHV-5tkΔ parece reativar apenas nos tecidos não-neurais (linfoide). De qualquer forma, a capacidade do recombinante reativar a infecção latente deve ser considerada no contexto de segurança vacinal.

Mapping the sites of latency and reactivation by bovine herpesvirus 5 (BoHV-5) and a thymidine kinase-deleted BoHV-5 in lambs / Mapeamento dos sítios de latência e reativação pelo herpesvírus bovino tipo 5 (BoHV-5) e por mutante deletado no gene da timidina quinase em ovinos

A thymidine kinase (tk)-deleted bovine herpesvirus 5 (BoHV-5tkΔ) was previously shown to establish latent infection and reactivate - even poorly - in a sheep model (Cadore et al. 2013). As TK-negative alphaherpesviruses are unlike to reactivate in neural tissue, this study investigated the sites of latency and reactivation by this recombinant in lambs. For this, groups of lambs were inoculated intranasally with the parental BoHV-5 strain (SV-507/99) or with the recombinant BoHV-5tkΔ. During latent infection (40 days post-inoculation, pi), the distribution of recombinant virus DNA in neural and non-neural tissues was similar to that of the parental virus. Parental and recombinant virus DNA was consistently detected by PCR in trigeminal ganglia (TGs); frequently in palatine and pharyngeal tonsils and, less frequently in the retropharyngeal lymph nodes. In addition, latent DNA of both viruses was detected in several areas of the brain. After dexamethasone (Dx) administration (day 40pi), the recombinant virus was barely detected in nasal secretions contrasting with marked shedding of the parental virus. In tissues of lambs euthanized at day 3 post-Dx treatment (pDx), reverse-transcription-PCR (RT-PCR) for a late viral mRNA (glycoprotein D gene) demonstrated reactivation of parental virus in neural (TGs) and lymphoid tissues (tonsils, lymph node). In contrast, recombinant virus mRNA was detected only in lymphoid tissues. These results demonstrate that BoHV-5 and the recombinant BoHV-5tkΔ do establish latent infection in neural and non-neural sites. Reactivation of the recombinant BoHV-5tkΔ, however, appeared to occur only in non-neural sites. In anyway, the ability of a tk-deleted strain to reactivate latent infection deserves attention in the context of vaccine safety.

Um recombinante do herpesvírus bovino tipo 5 com deleção no gene da timidina quinase (BoHV-5tkΔ) foi capaz de estabelecer latência e reativar - embora ineficientemente - em modelo experimental em ovinos (Cadore et al. 2013). Como a reativação de alfaherpesvírus defectivos na TK em tecido neural é improvável, o presente estudo investigou os sítios de latência e reativação por esse recombinante em ovinos. Para isso, grupos de ovinos foram inoculados com a cepa de BoHV-5 parental (SV-507/99) ou com o recombinante BoHV-5tkΔ. Durante a infecção latente (dia 40 pós-infecção, pi) a distribuição do DNA do vírus recombinante no encéfalo de ovinos infectados experimentalmente foi similar ao do vírus parental (SV-507/99). O DNA de ambos os vírus foi detectado consistentemente por PCR nos gânglios trigêmeos (TGs), frequentemente nas tonsilas faríngeas e palatinas e, com menos frequência, nos linfonodos retrofaríngeos. Após administração de dexametasona (Dx), o vírus recombinante foi raramente detectado nas secreções nasais, contrastando com excreção abundante do vírus parental. RT-PCR para mRNA de um gene tardio (glicoproteína D) realizado em tecidos de animais eutanasiados 3 dias pós-Dx demonstrou reativação do vírus parental em tecido neural (TGs) e não-neural (tonsilas, linfonodo). Em contraste, a reativação do vírus recombinante ficou restrita ao tecido linfoide. Esses resultados demonstram que tanto o BoHV-5 parental quanto o recombinante estabelecem latência em sítios neurais e não-neurais. No entanto, o recombinante BoHV-5tkΔ parece reativar apenas nos tecidos não-neurais (linfoide). De qualquer forma, a capacidade do recombinante reativar a infecção latente deve ser considerada no contexto de segurança vacinal.

Validation of a reference control for an SYBR-Green fluorescence assay-based real-time PCR for detection of bovine herpesvirus 5 in experimentally exposed bovine embryos.

The objective of this study was to optimize an internal control to improve SYBR-Green-based qPCR to amplify/detect the BoHV-5 US9 gene in bovine embryos produced in vitro and experimentally exposed to the virus. We designed an SYBR-Green-based binding assay that is quick to perform, reliable, easily optimized and compares well with the published assay. Herein we demonstrated its general applicability to detect BoHV-5 US9 gene in bovine embryos produced in vitro experimentally exposed to BoHV-5. In order to validate the assay, three different reference genes were tested; and the histone 2a gene was shown to be the most adequate for normalizing the qPCR reaction, by considering melting and standard curves (p < 0.05). On the other hand, no differences were found in the development of bovine embryos in vitro whether they were exposed to BoHV-5 reference and field strains comparing to unexposed embryos. The developed qPCR assay may have important field applications as it provides an accurate BoHV-5 US9 gene detection using a proven reference gene and is considerably less expensive than the TaqMan qPCR currently employed in sanitary programs.

A thymidine kinase-deleted bovine herpesvirus 5 establishes latent infection but reactivates poorly in a sheep model / Recombinante do herpesvírus bovino tipo 5 com deleção na timidina quinase estabelece infecção latente porém reativa ineficientemente em ovinos

The ability of thymidine kinase (tk)-deleted recombinant bovine herpesvirus 5 (BoHV-5tkΔ) to establish and reactivate latent infection was investigated in lambs. During acute infection, the recombinant virus replicated moderately in the nasal mucosa, yet to lower titers than the parental strain. At day 40 post-infection (pi), latent viral DNA was detected in trigeminal ganglia (TG) of all lambs in both groups. However, the amount of recombinant viral DNA in TGs was lower (9.7-fold less) than that of the parental virus as determined by quantitative real time PCR. Thus, tk deletion had no apparent effect on the frequency of latent infection but reduced colonization of TG. Upon dexamethasone (Dx) administration at day 40 pi, lambs inoculated with parental virus shed infectious virus in nasal secretions, contrasting with lack of infectivity in secretions of lambs inoculated with the recombinant virus. Nevertheless, some nasal swabs from the recombinant virus group were positive for viral DNA by PCR, indicating low levels of reactivation. Thus, BoHV-5 TK activity is not required for establishment of latency, but seems critical for efficient virus reactivation upon Dx treatment.

A capacidade de um recombinante do herpesvírus bovino tipo 5 com deleção no gene da timidina quinase (BoHV-5tk∆) em estabelecer e reativar infecção latente foi investigada em cordeiros. Durante a infecção aguda, o vírus recombinante replicou na mucosa nasal em títulos moderados, porém menores do que os da cepa parental. Aos 40 dias pós-infecção (pi) DNA viral latente foi detectado no gânglio trigêmeo (TG) de todos os cordeiros em ambos os grupos. No entanto, a quantidade de DNA do vírus recombinante nos TGs foi 9,7 vezes menor do que do vírus parental, segundo determinação por PCR em tempo real. Assim, a deleção do gene tk (timidina quinase) não produziu efeito aparente sobre a frequência da infecção latente, porém reduziu a colonização do TG. Após a administração de dexametasona (Dx) no dia 40pi, os cordeiros inoculados com o vírus parental excretaram partículas virais infecciosas, contrastando com a falta de infectividade nas secreções nasais dos animais inoculados com o vírus recombinante. Entretanto, alguns suabes nasais dos cordeiros do grupo do vírus recombinante foram positivos para o DNA viral por PCR, indicando baixos níveis de reativação. Assim, a atividade da enzima timidina quinase não é requerida para o estabelecimento de latência pelo BoHV-5, mas parece fundamental para reativação eficiente da infecção latente após tratamento com Dx.

Prokaryotic expression of a truncated form of bovine herpesvirus 1 glycoprotein E (gE) and its use in an ELISA for gE antibodies / Expressão procariota de uma forma truncada da glicoproteína E (gE) do herpesvírus bovino tipo 1 e uso em ELISA para anticorpos contra a gE

This article describes the expression of a truncated form of bovine herpesvirus 1 (BoHV-1) glycoprotein E (gE) for use as immunodiagnostic reagent. A 651 nucleotide fragment corresponding to the amino-terminal third (217 amino acids) of BoHV-1 gE - that shares a high identity with the homologous BoHV-5 counterpart - was cloned as a 6×His-tag fusion protein in an Escherichia coli expression vector. A soluble protein of approximately 25 kDa purified from lysates of transformed E. coli was recognized in Western blot (WB) by anti-6xHis-tag and anti-BoHV-1 gE monoclonal antibodies. In addition, the recombinant protein was specifically recognized in WB by antibodies present in the sera of cattle seropositive to BoHV-1 and BoHV-5. An indirect ELISA using the expressed protein as coating antigen performed comparably to a commercial anti-gE ELISA and was able to differentiate serologically calves vaccinated with a gE-deleted BoHV-5 strain from calves infected with BoHV-1. Thus, the truncated gE may be useful for serological tests designed to differentiate BoHV-1/BoHV-5 infected animals from those vaccinated with gE-negative marker vaccines.

Este trabalho relata a expressão de uma forma truncada da glicoproteína E (gE) do herpesvírus bovino tipo 1 (BoHV-1) para uso em imunodiagnóstico. Um fragmento de 651 pares de bases (pb) correspondente ao terço amino-terminal (217 aminoácidos) da gE do BoHV-1 - que compartilha uma alta identidade com a gE do BoHV-5 - foi clonada como proteína de fusão com cauda 6x de histidina em um vetor de expressão em Escherichia coli. Uma proteína solúvel de aproximadamente 25 kDa purificada de lisados de E.coli foi reconhecida em Western blot (WB) por anticorpos monoclonais anti-6xHis-tag e anti-gE. Além disso, a proteína recombinante purificada foi reconhecida em WB por anticorpos presentes no soro de animais soropositivos ao BoHV-1 e BoHV-5. Um ELISA indireto utilizando a proteína recombinante como antígeno apresentou performance comparável a um ELISA gE comercial e foi capaz e diferenciar sorologicamente animais vacinados com uma cepa gE-negativa de BoHV-5 de animais infectados com o BoHV-1. Portanto, a gE truncada pode ser útil em testes sorológicos diferenciais para uso conjunto com vacinas com marcador antigênico gE para o BoHV-1 e BoHV-5.

Bovine herpesviruses do not play a major role in the differential diagnosis of rabies in cattle in southern Brazil

Background: Rabies has long been recognized as the major cause of encephalitis in cattle in Latin American countries. It has been estimated that nearly 50.000 cattle heads per year are lost due to encephalitis in that subcontinent, with a significant economic impact on cattle productive chains. In Brazil only, 2.500 to 3.000 cattle heads are estimated to be lost every year due to rabies. However, it is believed that rabies incidence in cattle is much larger, since usually only a few samples from affected animals in disease outbreaks are submitted to diagnostic laboratories. Rabies encephalitis is promptly and accurately diagnosed; however, particularly when rabies is excluded as causa mortis, the agent responsible for neurological disease of infectious origin often remains undetermined. Two bovine herpesviruses (BoHVs), bovine herpesvirus type 1 (BoHV-1) and bovine herpesvirus type 5 (BoHV-5) are major pathogens of cattle which are widely disseminated in Brazil. As usual in herpesvirus' biology, these tend to infect a large number of hosts and establish lifelong latent infections which may occasionally be reactivated. Both viruses, particularly BoHV-5, are often recovered from cases of neurological disease in cattle. The participation of BoHVs in the differential diagnosis of rabies must be evaluated. Besides, there might be associations between the occurrence of rabies and BoHV infections that deserve investigation. The aim of this study was to investigate whether bovine herpesvirus 1 and 5 would play a significant role in cases of neurological disease where rabies was the presumptive clinical diagnosis. In addition, associations between the occurrence of rabies and BoHV infections were searched for. The approach adopted for conducting such investigations was based on the search for viral nucleic acids as well as classical virus isolation on tissues of cattle submitted to rabies diagnosis over a two-year period, including rabies-positive and rabies-negative specimens. Materials, Methods & Results: Brain tissue samples of 101 cattle originally submitted to rabies diagnosis were collected over a two year period (2009-2010) from various municipalities within the state of Rio Grande do Sul, Brazil. Thirty nine of these samples had the diagnosis of rabies confirmed by standard laboratory diagnostic methods. Aliquots of tissues were submitted to DNA extraction and examined in search for genomes of bovine herpesviruses (BoHV) types 1 (BoHV-1) and 5 (BoHV-5) by as well as for infectious virus. Bovine herpesvirus genomes were detected in 78/101 (77.2%) samples, in which BoHV-1 genomes were detected in 26/78 (25.7%), BoHV-5 genomes in 22/78 (21.8%) and mixed BoHV infections (BoHV-1 and BoHV-5 genomes) were detected in 30/101 (29.7%) samples. In the 39 samples with confirmed rabies diagnosis, BoHV-1 DNA was detected in 9/39 (23%), BoHV-5 DNA in 6/39 (15.4%) and mixed infections with both BoHV types in 16/39 (41%) samples. However, no infectious herpesvirus was recovered from any of the specimens examined. Discussion: The high prevalence of BoHV1 and BoHV-5 infections was evidenced in the sampled population, but the absence of infectious BoHVs indicate that these were not associated to the occurrence of the cases of encephalitis where rabies was the primary suspicion. In addition, no association was detected between occurrence of rabies and detection of BoHVs, since the frequency of detection of herpesvirus genomes did not significantly differ between rabies-positive and rabies-negative samples. The detection of BoHV DNA in scattered areas of the brain with no infectious virus suggests that latency may take place in different regions of the brain.

Role of bovine herpesvirus type 5 (BoHV-5) in diseases of cattle. Recent findings on BoHV-5 association with genital disease.

Bovine herpesvirus type 5 (BoHV-5) belongs to the family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus. This virus is a major causative agent of non-suppurative meningoencephalitis in young cattle. It was first isolated in 1962 from a neurological disease outbreak in Australia. BoHV-5 is genetically and antigenically related to bovine herpesvirus type 1 (BoHV-1), a highly prevalent virus responsible for respiratory and genital disease in cattle. Initially, BoHV-5 was considered a subtype of BoHV-1 (BoHV-1.3). However, the exclusive presentation of outbreaks of neurological disease suggested that the virus was a new agent with characteristics of neuropathogenicity. Even though both are neurotropic viruses, only BoHV-5 is capable of replicating extensively in the central nervous system and inducing neurological disease. Occasionally, encephalitis caused by BoHV-1 has been reported. Like other alpha-herpesviruses, BoHV-5 can establish latency in nervous ganglia and, by stress factors or glucocorticoid treatment, latent virus can be reactivated. During episodes of reactivation, the virus is excreted in nasal, ocular and genital secretions and transmitted to other susceptible hosts. Recently, BoHV-5 has been associated with infection of the reproductive tract. The virus has been isolated and the presence of viral DNA has been demonstrated in semen samples from Brazil and Australia and natural transmission of the virus through contaminated semen has also been described. Embryos and oocytes are permissive for BoHV-5 infection and BoHV-5 DNA has been detected in the central nervous system of aborted fetuses. The objective of this review is to compile the limited information on the recent association between BoHV-5 and reproductive disorders in cattle.

Reativação e distribuição do DNA latente do herpesvírus bovino tipo 5 no encéfalo de ovinos infectados experimentalmente / Reactivation and distribution of bovine herpesvirus 5 DNA in the brain of latently infected sheep

A biologia da infecção latente pelo herpesvírus bovino tipo 5 (BoHV-5) tem sido estudada em bovinos e coelhos, mas vários aspectos permanecem desconhecidos. Este artigo relata uma avaliação de ovinos jovens como modelo para o estudo da infecção latente pelo BoHV-5. Treze cordeiros com idade entre seis e sete meses, inoculados pela via intranasal (IN) com a cepa SV-507/99 do BoHV5 (título de 10(6,8) DICC50/mL) excretaram o vírus em secreções nasais em títulos de até 10(5,5) DICC50/mL, com duração de até 11 dias, desenvolvendo anticorpos neutralizantes em títulos de 16 a 128 no dia 30 pós-inoculação (pi). Os ovinos inoculados apresentaram apenas secreção nasal serosa leve e hipertermia transitória. O PCR de secções do encéfalo de cinco animais inoculados no dia 30 pi revelou a presença de DNA viral latente nos gânglios trigêmeos (TG, 5 de 5 animais), bulbo olfatório (BO, 5/5), ponte (2/5), cerebelo (2/5), córtex cerebral (1/5). Administração de dexametasona (Dx, n=4) ou flumetasona (FluM, n=4) a oito ovinos no dia 65 pi resultou em reativação e excreção viral por 3 de 4 animais de cada grupo. A excreção viral nas secreções nasais iniciou no dia 3 pós-tratamento e durou entre 1 e 5 dias nos ovinos tratados com Dx (títulos até 10(2,8)TCID50/mL) e foi mais tardia, durando entre 1 e 3 dias nos animais tratados com FluM (títulos de 10(2,1) TCID50/mL). Uma análise por PCR do encéfalo dos animais submetidos à reativação, no dia 65 pós-infecção, revelou uma distribuição do DNA latente semelhante àquela observada nos animais não submetidos à reativação. Em resumo, a capacidade do BoHV-5 estabelecer infecção latente, a colonização dos TGs a BOs com DNA viral latente e a reativação induzida por corticoides são achados promissores para o uso de cordeiros como modelo para a infecção latente pelo BoHV-5.

The biology of latent infection by bovine herpesvirus type 5 (BoHV-5) has been studied in cattle and rabbits, yet many aspects remain poorly understood. We herein investigated the suitability of lambs to investigate aspects of BoHV-5 latency. Thirteen six-month-old lambs inoculated intranasally (IN) with BoHV-5 strain SV-507/99 (titer of 10(6.8) TCID50/ mL) shed the virus in nasal secretions in titers up 10(5.5) TCID50/mL, during up to 11 days, developing virus neutralizing (VN) titers of 16 to 128 at day 30 post-inoculation (pi). The inoculated animals developed only a mild serous nasal secretion and transient hyperthermia. Examination of brain sections of five lambs euthanized at day 30 pi by PCR revealed the presence of latent DNA in the trigeminal ganglia (TG, 5 out of five), olfactory bulbs (OB, 5/5), pons (2/5), cerebellum (2/5) and cerebral cortex (1/5). Administration of dexamethasone (Dx, n=4) or flumethasone (FluM, n=4) to eight latently infected lambs at day 65 pi resulted in virus reactivation and shedding by 3 out of 4 individuals in each group. Virus shedding in nasal secretions started at day 3 post-treatment and lasted up to five days (1-5) in Dx treated lambs (titers up to 10(2.8)TCID50/mL), was delayed and lasted up to three days (1-3) in FluM-treated lambs (titers up to 10(2.1) TCID50/mL). PCR examination of the brains of animals submitted to reactivation, at day 30 post-treatment, showed a pattern of distribution of latent viral DNA fairly similar to that found in those not submitted to reactivation. In summary, the ability of BoHV-5 to establish latent infection, the consistent colonization of TGs and OBs by latent viral DNA and virus reactivation induced by corticosteroid treatment are promising findings towards the use of lambs to study selected aspects of BoHV-5 latency.

A thymidine kinase-negative bovine herpesvirus 5 is highly attenuated for rabbits, but is neuroinvasive and establishes latent infection / Cepa do herpesvírus bovino tipo 5 defectiva na timidina quinase é altamente atenuada para coelhos mas é neuroinvasiva e estabelece infecção latente

Mutant viral strains deleted in non-essential genes represent useful tools to study the function of specific gene products in the biology of the virus. We herein describe an investigation on the phenotype of a bovine herpesvirus 5 (BoHV-5) recombinant deleted in the gene encoding the enzyme thymidine kinase (TK) in rabbits, with special emphasis to neuroinvasiveness and the ability to establish and reactivate latent infection. Rabbits inoculated with the parental virus (SV-507/99) (n=18) at a low titer (10(5.5)TCID50) shed virus in nasal secretions in titers up to 10(4.5)TCID50 for up to 12 days (average: 9.8 days [5-12]) and 5/ 16 developed neurological disease and were euthanized in extremis. Rabbits inoculated with the recombinant BoHV-5TKΔ at a high dose (10(7.1)TCID50) also shed virus in nasal secretions, yet to lower titers (maximum: 10(2.3)TCID50) and for a shorter period (average: 6.6 days [2-11]) and remained healthy. PCR examination of brain sections of inoculated rabbits at day 6 post-infection (pi) revealed a widespread distribution of the parental virus, whereas DNA of the recombinant BoHV-5TKΔ-was detected only in the trigeminal ganglia [TG] and olfactory bulbs [OB]. Nevertheless, during latent infection (52pi), DNA of the recombinant virus was detected in the TGs, OBs and also in other areas of the brain, demonstrating the ability of the virus to invade the brain. Dexamethasone (Dx) administration at day 65 pi was followed by virus reactivation and shedding by 5/8 rabbits inoculated with the parental strain (mean duration of 4.2 days [1 - 9]) and by none of seven rabbits inoculated with the recombinant virus. Again, PCR examination at day 30 post-Dx treatment revealed the presence of latent DNA in the TGs, OBs and in other areas of the brain of both groups. Taken together, these results confirm that the recombinant BoHV-5TKΔ is highly attenuated for rabbits. It shows a reduced ability to replicate in the nose but retains the ability...

Cepas virais mutantes defectivas em genes não essenciais se constituem em ferramentas úteis para o estudo da função de proteínas virais na biologia dos vírus. Este estudo relata uma investigação do fenótipo, em coelhos, de uma cepa recombinante do herpesvírus bovino tipo 5 (BoHV-5) defectiva na enzima timidina quinase (TK), com ênfase para a neuroinvasividade e capacidade de estabelecer e reativar a infecção latente. Coelhos inoculados com o vírus parental (SV-507/99, n=18) em baixo título (10(5,5)TCID50) excretaram o vírus nas secreções nasais em títulos de até 10(4,5)TCID50/ mL por até 12 dias (média: 9,8 dias [5-12]) e 5/16 desenvolveram doença neurológica e morreram ou foram eutanasiados in extremis. Em contraste, coelhos inoculados com o recombinante BoHV-5TKΔ em alto título (10(7,1)TCID50) excretaram o vírus em títulos inferiores (máximo 10(2,3)TCID50/ mL), por um período menor (média: 6,6 days [2-11]) e permaneceram saudáveis. A realização de PCR em seções do encéfalo no dia 6 pós-infecção (pi) revelou uma ampla distribuição do DNA do vírus parental, enquanto o DNA do vírus recombinante foi detectado apenas nos gânglios trigêmeos [TGs] e nos bulbos olfatórios [OBs]. Não obstante, durante a infecção latente (52pi), o DNA do vírus recombinante foi detectado nos TGs, OBs e em outros locais do encéfalo, demonstrando que o vírus recombinante mantém a neuroinvasividade. Tratamento com dexamethasona (Dx) no dia 65 pi resultou em reativação e excreção viral por 5/8 dos coelhos inoculados com o vírus parental (duração média de 4,2 dias [1-9]) e por nenhum dos sete coelhos inoculados com o vírus recombinante. No entanto, PCR realizado no dia 30 pós-Dx revelou a presença de DNA latente do BoHV-5TKΔ nos TGs, OBs e em outras áreas do encéfalo. Esses resultados confirmam que o recombinante BoHV-5TKΔ é altamente atenuado para coelhos. A sua capacidade de replicação na mucosa nasal é reduzida, mas mantém a capacidade de invadir o encéfalo e estabelecer infecção...

Experimental infection of calves with recombinants of bovine herpesvirus 5 defective in glycoprotein E (gE), thymidine kinase (TK) and both, gE/TK / Infecção experimental de bezerros com recombinantes do herpesvírus bovino tipo 5 defectivos na glicoproteína E (gE), timidina quinase (TK) e ambos, gE/TK

This article describes an investigation on the virulence/attenuation of bovine herpesvirus type 5 (BoHV-5) recombinants deleted in the genes encoding glycoprotein E (BoHV-5gEΔ), thymidine kinase (BoHV-5TKΔ), and both gE and TK (BoHV-5gEΔTKΔ). Seronegative calves (80 to 90 days-old) inoculated with the parental strain (SV-507/99, n=5) shed virus in nasal secretions for up to 15 days (average 10.8 days). Duration of virus shedding was 11 days for BoHV-5gΔ, 9.6 days for BoHV-5TKΔ and 6.2 days for BoHV-5gEΔTKΔ groups. The highest titers were observed between days 1 and 6 post-infection (pi) for SV-507/99 (10(6.8)TCID50/mL), 10(5.1)TCID50/mL (BoHV-5gEΔ), 10(5.9)TCID50/mL (BoHV-5TKΔ) and 10(4.7)TCID50/mL (BoHV-5gEΔTKΔ). Calves inoculated with the parental virus presented anorexia, profound apathy and loss of body condition. Two calves were euthanized in extremis on days 10 and 11 pi; infectious virus was recovered from several areas of the brain. In contrast, calves inoculated with the recombinants remained healthy and a few presented a mild and transient nasal secretion. Dexamethasone (Dx) administration at day 42 pi resulted in virus shedding by all controls calves (mean duration 3.7 days), by 2/5 of BoHV-5TKΔ calves (two days) and 2/5 of BoHV-5gEΔ (one day). No virus shedding was detected in BoHV-5gEΔTKΔ calves upon Dx treatment. PCR examination of brain sections of calves euthanized at day 30 post Dx treatment revealed the presence of latent viral DNA widely distributed in the brain of SV-507/99 calves. Latent viral DNA was detected in a few sections (3/30) of the brains of BoHV-5gEΔ calves and was not detected in the brains of calves inoculated with BoHV-5TKΔ and BoHV-5gEΔTKΔ. These results show that the single BoHV-5 mutants (gE and tk-deleted) are attenuated for calves and establish and/or reactivate latent infection inefficiently. The double mutant BoHV-5gEΔTKΔ is fully attenuated and appears not to establish or not reactivate efficiently from latent infection. Thus, these recombinants, especially the double mutant BoHV-5gEΔTKΔ, display an adequate phenotype for use in modified-live vaccine formulations.

Este artigo descreve uma investigação da virulência/atenuação de recombinantes do herpesvírus bovino tipo 5 (BoHV-5) com deleções nos genes da glicoproteína E (BoHV-5gEΔ), timidina quinase (BoHV-5TKΔ), e ambos gE e TK (BoHV-5gEΔTKΔ). Bezerros soronegativos (80-90 dias de idade) inoculados com o vírus parental SV-507/99 (n=5) excretaram o vírus em secreções nasais por até 15 dias (média de 10,8 dias). Nos animais inoculados com os recombinantes, a duração da excreção viral foi de 11 dias (BoHV-5gEΔ), 9,6 dias (BoHV-5TKΔ) e 6,2 dias (BoHV-5gEΔTKΔ). Os maiores títulos foram observados entre os dias 1 e 6 pós-inoculação (pi), sendo de 10(6,8)TCID50/mL para o SV-507/99, 10(5,1)TCID50/mL (BoHV-5gEΔ), 10(5,9)TCID50/mL (BoHV-5TKΔ) e 10(4,7)TCIΔ50/mL (BoHV-5gEΔTKΔ). Os bezerros inoculados com o vírus parental apresentaram anorexia e apatia; três deles mostraram apatia profunda e perda da condição corporal. Dois bezerros foram eutanasiados in extremis nos dias 10 e 11 pi, respectivamente e o vírus foi isolado de várias regiões do encéfalo. Já os bezerros inoculados com os recombinantes permaneceram saudáveis; alguns apresentaram uma secreção nasal serosa transitória. Administração de dexametasona (Dx) no dia 42 pi resultou em excreção viral por todos os bezerros inoculados com o vírus parental (duração média de 3,7 dias), por 2 de 5 bezerros dos grupos BoHV-5TKΔ (dois dias) e BoHV-5gEΔ (um dia). Os bezerros inoculados com o duplo mutante BoHV-5gEΔTKΔ não excretaram o vírus após o tratamento com Dx. Pesquisa de DNA viral por PCR no dia 30 pós-Dx revelou uma ampla distribuição do DNA do vírus parental no encéfalo; poucas seções (3/30) foram positivas no encéfalo dos animais do grupo BoHV-5gEΔ, e não detectou-se DNA latente no encéfalo dos animais dos grupos BoHV-5TKΔ e BoHV-5gEΔTKΔ. Esses resultados demonstram que os mutantes simples (gE and tk-deletados) são atenuados para bezerros e estabelecem e/ou reativam infecção latente ineficientemente. Já o duplo mutante BoHV-5gEΔTKΔ é atenuado e parece não estabelecer e/ou não reativar eficientemente a infecção latente. Portanto, os vírus recombinantes, e em especial o duplo mutante BoHV-5gEΔTKΔ apresentam um fenótipo compatível com a sua inclusão em vacinas vivas modificadas.