RESUMO
Rabies and herpetic encephalitis are the main viral infections in bovines with neurological symptoms. Bovine rabies has a high prevalence in Central and South America, while bovine encephalitis associated with herpesvirus is especially important in South America. Viral isolation is the classical way to confirm herpesvirus infection, but molecular evidence of the presence of the virus in affected animals is gaining importance in the diagnosis of the disease in the laboratory. This study investigated the presence of herpesvirus type 1 and 5 (BoHV-1 and BoHV-5) in 182 encephalon of rabies-suspected cattle in Rio Grande do Sul state (RS), Brazil using multiplex real-time polymerase chain reaction (mRT-PCR). The rabies virus was investigated by direct fluorescent antibody assay and intracerebral suckling mouse inoculation. The genomes of BoHV-1 and BoHV-5 were detected in 17% of samples. BoHV-5 and BoHV-1 were detected in 100% and 19% of BoHV positive samples, respectively, indicating the circulation of the pathogens in cattle herds in RS. The high Ct values and the absence of isolation suggest viral latency. Coinfection of herpesvirus and the rabies virus was detected in 28% of samples, although no significant association between pathogens was observed. Rabies was detected in 57.7% of suspected samples, confirming the importance of the disease in the state. Concerning the method by which samples were conserved, no significant difference was observed between the number of positive results in frozen and refrigerated samples.
Assuntos
Doenças dos Bovinos/virologia , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 5/genética , Raiva/veterinária , Animais , Encéfalo/virologia , Brasil/epidemiologia , Bovinos , Doenças dos Bovinos/epidemiologia , Criopreservação/veterinária , DNA Viral/isolamento & purificação , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/virologia , Camundongos , Reação em Cadeia da Polimerase Multiplex/veterinária , Raiva/epidemiologia , Refrigeração/veterináriaRESUMO
Wild birds carry a number of infectious agents, some of which may have pathogenic potential for the host and others species, including humans. Domestic pigeons (Columba livia) are important targets of study since these increasingly cohabit urban spaces, being possible spillover sources of pathogens to humans. In the present study, two genomes (PiGyV_Tq/RS/Br and PiGyV_RG/RS/Br), representative of Gyrovirus genus, family Anelloviridae, were detected in sera of free-living pigeons collected in Southern Brazil. The genomes exhibit less than 50% identity to previously described members of Gyrovirus genus, suggesting that they constitute a new viral species circulating in pigeons, to which the name "pigeon gyrovirus (PiGyV)" is proposed. The current study characterizes these two PiGyV genomes which, to date, are the first gyrovirus species identified in domestic pigeons.
Assuntos
Animais Selvagens/virologia , Doenças das Aves/virologia , Columbidae/virologia , Gyrovirus/isolamento & purificação , Animais , Brasil , Genoma Viral , Gyrovirus/classificação , Gyrovirus/genéticaRESUMO
Unfortunately, the word "evolution" was found missing in title of the original article which is corrected here by this erratum. The original article has been corrected.
RESUMO
Pigeon circovirus (PiCV) is taxonomically classified as a member of the Circovirus genus, family Circoviridae. The virus contains a single stranded DNA genome of approximately 2 kb, with minor length variations among different isolates. The occurrence of PiCV infections in pigeons (Columba livia) has been documented worldwide over the past 20 years; however, in Brazil there were still no reports on PiCV detection. This study identifies seven PiCV genomes recovered from domestic pigeons of South Brazil through high-throughput sequencing and shows a high frequency of PiCV infection, through quantitative real-time PCR. Phylogenetic classification was performed by maximum likelihood analysis of the full genomes, ORF V1 (Rep) and ORF C1 (Cap). The results show that either full genome or Cap based analysis allowed PiCV classification into five major clades (groups A to E), where Brazilian sequences were classified as A, C or D. Recombination analyses were carried out with Simplot and RDP4 and the results show that both Rep and Cap ORFs contain several recombination hotspots, pointing to an important role for such events in PiCV evolution.
Assuntos
Doenças das Aves/virologia , Infecções por Circoviridae/veterinária , Circovirus/isolamento & purificação , Columbidae/virologia , Evolução Molecular , Animais , Brasil , Infecções por Circoviridae/virologia , Circovirus/classificação , Circovirus/genética , Sequenciamento de Nucleotídeos em Larga Escala , Fases de Leitura Aberta , FilogeniaRESUMO
Two full-genome sequences of porcine circovirus type 3 (PCV3) are reported. The genomes were recovered from pooled serum samples from sows who had just delivered litters with variable numbers of stillbirths. The two circular genomes (PCV3-BR/RS/6 and PCV3-BR/RS/8) are 2,000 nucleotides long and contain two open reading frames (ORFs) oriented in opposite directions that encode the putative capsid (Cap) and replicase (Rep) proteins. The intergenic region contains a stem-loop motif, as reported for other circoviruses. Rolling circle replication motifs and putative helicase domains were identified in the Rep coding region. The degree of overall nucleotide similarity between the genomes reported here and those available at GenBank was higher than 97%. No PCV3 sequence was detected in pooled serum samples from sows which had no stillbirths on the same farms. However, further studies are necessary to confirm the association between PCV3 and the occurrence of stillbirths.
Assuntos
Infecções por Circoviridae/virologia , Circovirus/genética , Genoma Viral/genética , Natimorto/veterinária , Doenças dos Suínos/virologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Brasil , Proteínas do Capsídeo/genética , Circovirus/isolamento & purificação , Feminino , Fases de Leitura Aberta/genética , Filogenia , Gravidez , Suínos , Replicação ViralRESUMO
Abstract The human polyomaviruses JC and BK (JCPyV and BKPyV) are ubiquitous, species-specific viruses that belong to the family Polyomaviridae. These viruses are known to be excreted in human urine, and they are potential indicators of human wastewater contamination. In order to assess the distribution of both JCPyV and BKPyV in urban water samples collected from a sewage treatment plant (STP) and from a canalized water stream of Porto Alegre, Brazil, two nested-PCR assays were optimized and applied to the samples collected. The amplicons obtained were submitted to sequencing, and the sequences were analyzed with sequences of human polyomaviruses previously deposited in GenBank. Twelve out of 30 water samples (40%) were JCPyV positive, whereas six samples (20%) were BKPyV positive. The sequencing results confirmed the presence of JCPyV subtypes 1 and 3, whereas only BKPyV Ia and Ib were found. This study shows for the first time the presence of human polyomaviruses in surface water and in samples collected in a sewage treatment plant in southern Brazil.
Resumo Os poliomavírus humanos JC e BK (JCPyV e BKPyV) são virus ubíquos, espécie-específicos, pertencentes à família Polyomaviridae. Estes vírus são conhecidos por serem excretados pela urina humana, sendo considerados potenciais indicadores de contaminação por águas residuais urbanas. Buscando acessar a distribuição de JCPyV e BKPyV em amostras de águas coletadas de uma estação de tratamento de esgoto e de um arroio canalizado de Porto Alegre, Brasil, duas técnicas de nested-PCR foram otimizadas e aplicadas às amostras coletadas. Os amplificados obtidos foram submetidos ao sequenciamento e suas sequências analisadas com base em sequências de poliomavírus humanos previamente depositadas no GenBank. Doze de 30 amostras de água (40%) foram positivas para JCPyV, enquanto 6 amostras (20%) foram positivas para BKPyV. Os resultados do sequenciamento confirmaram a presença dos subtipos 1 e 3 de JCPyV, enquanto apenas os BKPyV Ia e Ib foram encontrados. Este estudo demonstra pela primeira vez a presença de poliomavírus humanos em águas superficiais e em amostras coletadas em uma estação de tratamento de esgoto na região sul do Brasil.
Assuntos
Esgotos/virologia , Vírus BK/isolamento & purificação , Vírus BK/genética , Vírus JC/isolamento & purificação , Vírus JC/genética , Água Doce/virologia , Variação Genética , Brasil , Reação em Cadeia da PolimeraseRESUMO
The human polyomaviruses JC and BK (JCPyV and BKPyV) are ubiquitous, species-specific viruses that belong to the family Polyomaviridae. These viruses are known to be excreted in human urine, and they are potential indicators of human wastewater contamination. In order to assess the distribution of both JCPyV and BKPyV in urban water samples collected from a sewage treatment plant (STP) and from a canalized water stream of Porto Alegre, Brazil, two nested-PCR assays were optimized and applied to the samples collected. The amplicons obtained were submitted to sequencing, and the sequences were analyzed with sequences of human polyomaviruses previously deposited in GenBank. Twelve out of 30 water samples (40%) were JCPyV positive, whereas six samples (20%) were BKPyV positive. The sequencing results confirmed the presence of JCPyV subtypes 1 and 3, whereas only BKPyV Ia and Ib were found. This study shows for the first time the presence of human polyomaviruses in surface water and in samples collected in a sewage treatment plant in southern Brazil.
Assuntos
Vírus BK/genética , Vírus BK/isolamento & purificação , Água Doce/virologia , Vírus JC/genética , Vírus JC/isolamento & purificação , Esgotos/virologia , Brasil , Variação Genética , Reação em Cadeia da PolimeraseRESUMO
Bovine herpesvirus 1 (BoHV-1) is an alphaherpesvirus with a genome of 135 kb. Some BoHV-1 genes are nonessential and may be deleted from the viral genome. Here, a spontaneous gene deletion was identified in the BoHV-1 strain Cooper. Genes of the US1.67/US2 region were absent, as determined by next-generation sequencing.
RESUMO
A complete genomic sequence of porcine parvovirus 2 (PPV-2) was detected by viral metagenome analysis on swine sera. A phylogenetic analysis of this genome reveals that it is highly similar to previously reported North American PPV-2 genomes. The complete PPV-2 sequence is 5,426 nucleotides long.
RESUMO
Despite the putative endemic status of swine influenza A virus (swIAV) infections, data on the occurrence of swine influenza outbreaks are scarce in Brazil. The aim of this study was to detect and subtype swIAVs from six outbreaks of porcine respiratory disease complex (PRDC) in southern Brazil. Nasal swabs were collected from 66 piglets with signs of respiratory disease in six herds. Lung tissue samples were collected from six necropsied animals. Virus detection was performed by PCR screening and confirmed by virus isolation and hemagglutination (HA). Influenza A subtyping was performed by a real-time reverse transcriptase PCR (rRT-PCR) to detect the A(H1N1)pdm09; other swIAV subtypes were determined by multiplex RT-PCR. In lung tissues, the major bacterial and viral pathogens associated with PRDC (Pasteurella multocida, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Haemophilus parasuis and PCV2) were investigated. In some affected pigs, clinico-pathological evaluations were conducted. Influenza A was detected by screening PCR in 46 of 66 swab samples and from five of six lungs. Virus was recovered from pigs of all six herds. Subtype A(H1N1)pdm09 was detected in four of six herds and H1N2 in the other two herds. In lung tissues, further agents involved in PRDC were detected in all cases; Pasteurella multocida was identified in five of six samples and Mycoplasma hyopneumoniae in three of six. Actinobacillus pleuropneumoniae (1/6), Haemophilus parasuis (1/6) and PCV2 (1/6) were also detected. These findings indicate that subtypes A(H1N1)pdm09 and H1N2 were present in pigs in southern Brazil and were associated with PRDC outbreaks.
Assuntos
Surtos de Doenças/veterinária , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H1N2/isolamento & purificação , Infecções por Orthomyxoviridae/veterinária , Doenças dos Suínos/virologia , Criação de Animais Domésticos , Animais , Brasil/epidemiologia , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N2/genética , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , Suínos , Doenças dos Suínos/epidemiologiaRESUMO
Adenovirus (AdV), enterovirus (EV), genogroup A rotaviruses (GARV) and Torque teno virus (TTV) are non-enveloped viral agents excreted in feces and so may contaminate water bodies. In the present study, the molecular detection of these viruses was performed in samples of surface water collected from the Arroio Dilúvio, a waterstream that crosses the city of Porto Alegre, RS, Brazil, receiving great volumes of non-treated sewage from a large urban area. Sampling was performed during 2009, in three different occasions (January, April and September). The highest detection rate was observed for EV (64.28%), followed by TTV (28.57%) and AdV (21.43%). Rotaviruses were not detected. More than on kind of tested virus was detected in five (35. 71%) of 14 samples. January was the month with the highest viral detection rate, being all samples, collected in this month, positive for at least one group of tested virus. The correlation between the detection of these different viral agents and environmental factors is discussed. To the knowledge of the authors, this is the first description of viral genomes in water samples taken from the Arroio Dilúvio, Porto Alegre (Brazil).
Assuntos
Adenoviridae/isolamento & purificação , Enterovirus/isolamento & purificação , Rotavirus/isolamento & purificação , Torque teno virus/isolamento & purificação , Microbiologia da Água , Adenoviridae/genética , Brasil , DNA Viral/genética , Enterovirus/genética , Reação em Cadeia da Polimerase , Rios , Rotavirus/genética , Torque teno virus/genéticaRESUMO
In this study, a preparation of saponins (QB-90U) extracted from leaves of Quillaja brasiliensis collected in Uruguay was evaluated as a vaccine adjuvant by comparison with alum and the well known saponin-based adjuvant, Quil A. The haemolytic activity and cellular toxicity of the saponin preparations were also evaluated. QB-90U was only slightly haemolytic and showed a low cytotoxicity when compared to Quil A. The adjuvant properties of QB-90U were assayed by sub-cutaneous immunization of mice with a preparation of inactivated bovine herpesvirus 5 (BoHV-5) either with no adjuvant or adjuvanted with QB-90U, Quil A or alum. Serum levels of anti-BoHV-5 IgG, IgG1, IgG2a, IgG2b and also IgG3 were significantly increased by QB-90U and were of the same order as those elicited by Quil A. Furthermore, high titres of neutralizing antibodies were found to be present in the serum of immunized animals from both groups. The cellular response induced by QB-90U did also reproduce the one elicited by Quil A. In fact, a robust DTH response was observed in mice immunized with both saponin preparations; as well as increased splenocytes levels of Th1-type cytokines, namely IFN-γ and IL-2. Taken together, the above results confirm and extend our previous observation regarding the similarity of the responses elicited by Quil A and the saponin preparation from Q. brasiliensis (Fleck et al., 2006) and indicate that QB-90U is worth of further studies as a safe and potent vaccine adjuvant.
Assuntos
Adjuvantes Imunológicos/farmacologia , Infecções por Herpesviridae/prevenção & controle , Herpesvirus Bovino 5/patogenicidade , Quillaja/química , Saponinas/imunologia , Adjuvantes Imunológicos/toxicidade , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Chlorocebus aethiops , Citocinas/imunologia , Feminino , Hemólise , Infecções por Herpesviridae/imunologia , Imunidade Humoral , Camundongos , Saponinas de Quilaia , Saponinas/farmacologia , Saponinas/toxicidade , Baço/imunologia , Células VeroRESUMO
An immunoperoxidase inhibition assay (IIA) for detection of rabies antibodies in human sera is described. Diluted test sera are added to microplates with paraformaldehyde-fixed, CER cells infected with rabies virus. Antibodies in test sera compete with a rabies polyclonal rabbit antiserum which was added subsequently. Next, an anti-rabbit IgG-peroxidase conjugate is added and the reaction developed by the addition of the substrate 3-amino-9-ethylcarbazole (AEC). The performance of the assay was compared to that of the "simplified fluorescence inhibition microtest" (SFIMT), an established virus neutralization assay, by testing 422 human sera. The IIA displayed 97.6% sensitivity, 98% specificity and 97.6% accuracy (Kappa correlation coefficient=0.9). The IIA results can be read by standard light microscopy, where the clearly identifiable specific staining is visible in antibody-negative sera, in contrast to the absence of staining in antibody-positive samples. The assay does not require monoclonal antibodies or production of large amounts of virus; furthermore, protein purification steps or specialized equipment are not necessary for its performance. The IIA was shown to be suitable for detection of rabies antibodies in human sera, with sensitivity, specificity and accuracy comparable to that of a neutralization-based assay. This assay may be advantageous over other similar methods designed to detect rabies-specific binding antibodies, in that it can be easily introduced into laboratories, provided basic cell culture facilities are available.
Assuntos
Anticorpos Antivirais/sangue , Técnicas de Laboratório Clínico/métodos , Vírus da Raiva/imunologia , Raiva/diagnóstico , Virologia/métodos , Humanos , Técnicas Imunoenzimáticas/métodos , Sensibilidade e EspecificidadeRESUMO
Bovine herpesvirus 1 (BoHV-1) and 5 (BoHV-5) are important pathogens of the respiratory and genital tract of cattle and may also affect the central nervous system and cause meningoencephalitis. Both virus types are estimated to be widely distributed in Southern Brazil. In the present study, BoHV-1 and/or BoHV-5 DNA were detected in bovine semen samples from two states of Brazil by two species-specific nested polymerase chain reactions (nPCRs). These nPCRs were used to assay 53 samples of fresh semen and 23 samples of frozen semen from breeding bulls. Viral DNA was detected in all 76 semen samples: all were positive for BoHV-5, whereas 34 of these were positive for BoHV-1 as well. Moreover, in five fresh and in 13 frozen semen samples-of a total number of 40 samples suitable for virus isolation-infectious BoHV-1 and/or BoHV-5 virus were detected. In conclusion, that both BoHV-1 and BoHV-5 were detected in bovine semen in Brazil highlighted the importance of examining bull semen in search for both agents to reduce the risk of transmitting these viruses.
Assuntos
Doenças dos Bovinos/virologia , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 1/isolamento & purificação , Herpesvirus Bovino 5/isolamento & purificação , Sêmen/virologia , Animais , Brasil , Bovinos , Doenças dos Bovinos/epidemiologia , DNA Viral/química , Infecções por Herpesviridae/epidemiologia , MasculinoRESUMO
Bovine herpesvirus type 5 (BoHV-5) is the causative agent of bovine herpetic encephalitis. In countries where BoHV-5 is prevalent, attempts to vaccinate cattle to prevent clinical signs from BoHV-5-induced disease have relied essentially on vaccination with BoHV-1 vaccines. However, such practice has been shown not to confer full protection to BoHV-5 challenge. In the present study, an inactivated, oil adjuvanted vaccine prepared with a recombinant BoHV-5 from which the genes coding for glycoprotein I (gI), glycoprotein E (gE) and membrane protein US9 were deleted (BoHV-5 gI/gE/US9(-)), was evaluated in cattle in a vaccination/challenge experiment. The vaccine was prepared from a virus suspension containing a pre-inactivation antigenic mass equivalent to 10(7.69) TCID(50)/dose. Three mL of the inactivated vaccine were administered subcutaneously to eight calves serologically negative for BoHV-5 (vaccinated group). Four other calves were mock-vaccinated with an equivalent preparation without viral antigens (control group). Both groups were boostered 28 days later. Neither clinical signs of disease nor adverse effects were observed during or after vaccination. A specific serological response, revealed by the development of neutralizing antibodies, was detected in all vaccinated animals after the first dose of vaccine, whereas control animals remained seronegative. Calves were subsequently challenged on day 77 post-vaccination (pv) with 10(9.25) TCID(50) of the wild-type BoHV-5 (parental strain EVI 88/95). After challenge, vaccinated cattle displayed mild signs of respiratory disease, whereas the control group developed respiratory disease and severe encephalitis, which led to culling of 2/4 calves. Searches for viral DNA in the central nervous system (CNS) of vaccinated calves indicated that wild-type BoHV-5 did not replicate, whereas in CNS tissues of calves on the control group, viral DNA was widely distributed. BoHV-5 shedding in nasal secretions was significantly lower in vaccinated calves than in the control group on days 2, 3, 4 and 6 post-challenge (pc). In addition, the duration of virus shedding was significantly shorter in the vaccinated (7 days) than in controls (12 days). Attempts to reactivate latent infection by administration of dexamethasone at 147 days pv led to recrudescence of mild signs of respiratory disease in both vaccinated and control groups. Infectious virus shedding in nasal secretions was detected at reactivation and was significantly lower in vaccinated cattle than in controls on days 11-13 post-reactivation (pr). It is concluded that the inactivated vaccine prepared with the BoHV-5 gI/gE/US9(-) recombinant was capable of conferring protection to encephalitis when vaccinated cattle were challenged with a large infectious dose of the parental wild type BoHV-5. However, it did not avoid the establishment of latency nor impeded dexamethasone-induced reactivation of the virus, despite a significant reduction in virus shedding after challenge and at reactivation on vaccinated calves.
Assuntos
Doenças dos Bovinos/prevenção & controle , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 5/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Formação de Anticorpos , Bovinos/imunologia , Doenças dos Bovinos/imunologia , Linhagem Celular , Encefalite Viral/imunologia , Encefalite Viral/prevenção & controle , Encefalite Viral/veterinária , Feminino , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/prevenção & controle , Herpesvirus Bovino 5/fisiologia , Masculino , Meningoencefalite/imunologia , Meningoencefalite/prevenção & controle , Meningoencefalite/veterinária , Testes de Neutralização , Vacinação/veterinária , Vacinas de Produtos Inativados/imunologia , Ativação Viral , Latência Viral , Eliminação de Partículas ViraisRESUMO
Verificou-se a incidência de herpesvírus bovinos (BoHVs) em encéfalos de bovinos submetidos ao diagnóstico de raiva no estado do Rio Grande do Sul. Para tanto, amostras coletadas durante dois anos (n=70) foram submetidas ao isolamento viral em cultivos celulares. Os BoHVs foram isolados em dois (2,9 por cento) encéfalos. Após serem submetidas à caracterização antigênica e molecular, as amostras foram subtipadas como BoHV-1.1 e BoHV-1.2b. A BoHV-1.1 foi isolada de um encéfalo que foi também positivo para raiva. O vírus da raiva foi identificado em 11 amostras (15,7 por cento). Estes achados revelam que a incidência de BoHVs em forma infecciosa em bovinos com encefalite foi baixa, embora represente 16,7 por cento (2/12) dos encéfalos nos quais um agente viral foi identificado. Tal fato confirma a já reportada associação entre BoHV-1 e encefalites. Esse é o primeiro relato da ocorrência de BoHV-1.2b, um subtipo considerado menos patogênico, em um caso de doença neurológica em bovinos.(AU)
The incidence of bovine herpesviruses (BoHVs) was determined in brains of cattle submitted to rabies diagnosis in the State of Rio Grande do Sul, Brazil. Brain samples collected in a two-year interval (n=70) were submitted to virus isolation in cell culture. The BoHVs were isolated from two (2.9 percent) of the brains. After the antigenic and molecular characterization, the isolated strains were subtyped as BoHV-1.1 and BoHV-1.2b. The BoHV-1.1 isolate was recovered from a brain sample that was also positive for rabies. Rabies virus was identified in 11 (15.7 percent) samples. These findings reveal that the incidence of infectious BoHVs in brains of cattle with encephalitis was low, although these represented 16.7 percent (2/12) of samples from which at least one viral agent was identified. This confirms the previously reported link between BoHV-1 and bovine encephalitis. However, it is the first report on the association of BoHV-1.2b, a putatively less pathogenic BoHV subtype, with neurological disease in cattle.(AU)
Assuntos
Animais , Bovinos , Herpesvirus Bovino 1/isolamento & purificação , Infecções por Herpesviridae/veterinária , Encéfalo , Bovinos , Raiva/veterináriaRESUMO
A sandwich ELISA (S-ELISA) was developed to detect antibodies to rabies virus in seraof different species. The test was performed as follows: ELISA plates were coated withpolyclonal mouse/anti-rabies antibodies for 2 hours at 37ºC. After adsorption, plateswere washed and non-specific binding blocked with 2% powdered milk. In a separateplate, serial threefold dilutions of test sera were incubated with inactivated rabies virusantigen. The mixtures were then placed on the rabies antibody-coated plates andincubated. These were then washed and incubated with polyclonal rabbit/anti-rabiesantibodies. Subsequently, a rabbit/IgG-peroxidase conjugate was added and platesincubated. After washing, the chromogen (ABTS with 0.15% H2O2) was added to platesand after incubation for 30 min were read in a spectrophotometer (OD405). To validatethe S-ELISA, 128 serum samples including humans, cattle, hematophagous and nonhaematophagous bats, mice, marmosets, ocelots - Leopardus pardalis, raccoons -Procyon lotor, jaguarondi - Herpailurus yaguarondi, fox - Cerdocyon thous and coati -Nasua nasua, were tested and compared to a standard fluorescent antibody virusneutralization test (FAVN). In comparison to FAVN, the S-ELISA showed highsensitivity (82.98%) and specificity (100%), with an accuracy of 87.5%. Subsequently,738 serum samples from different species were tested in the S-ELISA. Antibodies torabies were detected by S-ELISA in all species tested, with the exception of the threeserum samples from raccoons. The S-ELISA was shown to be a serological test of lowcost that can be easily implemented in diagnostic laboratories. In addition, no liveanimals, infectious virus, cell culture or fluorescence microscopy are required forperformance of the test. This is an additional advantage of the S-ELISA over othermethods of rabies antibody detection.
Assuntos
Anticorpos Antivirais , Estudos Soroepidemiológicos , Raiva , Vírus da RaivaRESUMO
Verificou-se a incidência de herpesvírus bovinos (BoHVs) em encéfalos de bovinos submetidos ao diagnóstico de raiva no estado do Rio Grande do Sul. Para tanto, amostras coletadas durante dois anos (n=70) foram submetidas ao isolamento viral em cultivos celulares. Os BoHVs foram isolados em dois (2,9 por cento) encéfalos. Após serem submetidas à caracterização antigênica e molecular, as amostras foram subtipadas como BoHV-1.1 e BoHV-1.2b. A BoHV-1.1 foi isolada de um encéfalo que foi também positivo para raiva. O vírus da raiva foi identificado em 11 amostras (15,7 por cento). Estes achados revelam que a incidência de BoHVs em forma infecciosa em bovinos com encefalite foi baixa, embora represente 16,7 por cento (2/12) dos encéfalos nos quais um agente viral foi identificado. Tal fato confirma a já reportada associação entre BoHV-1 e encefalites. Esse é o primeiro relato da ocorrência de BoHV-1.2b, um subtipo considerado menos patogênico, em um caso de doença neurológica em bovinos.
The incidence of bovine herpesviruses (BoHVs) was determined in brains of cattle submitted to rabies diagnosis in the State of Rio Grande do Sul, Brazil. Brain samples collected in a two-year interval (n=70) were submitted to virus isolation in cell culture. The BoHVs were isolated from two (2.9 percent) of the brains. After the antigenic and molecular characterization, the isolated strains were subtyped as BoHV-1.1 and BoHV-1.2b. The BoHV-1.1 isolate was recovered from a brain sample that was also positive for rabies. Rabies virus was identified in 11 (15.7 percent) samples. These findings reveal that the incidence of infectious BoHVs in brains of cattle with encephalitis was low, although these represented 16.7 percent (2/12) of samples from which at least one viral agent was identified. This confirms the previously reported link between BoHV-1 and bovine encephalitis. However, it is the first report on the association of BoHV-1.2b, a putatively less pathogenic BoHV subtype, with neurological disease in cattle.
Assuntos
Animais , Bovinos , Herpesvirus Bovino 1/isolamento & purificação , Infecções por Herpesviridae/veterinária , Encéfalo , Bovinos , Raiva/veterináriaRESUMO
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 (BoHV-1) and 5 (BoHV-5) as test challenge viruses. Bovine sera collected from a randomized sample (n=287) were tested in a 24h incubation SN against three type 1 viruses (BoHV-1.1 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.
Assuntos
Anticorpos Neutralizantes/imunologia , Doenças dos Bovinos/diagnóstico , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 1/imunologia , Herpesvirus Bovino 5/imunologia , Testes de Neutralização/veterinária , Animais , Bovinos , Doenças dos Bovinos/virologia , Infecções por Herpesviridae/diagnóstico , Infecções por Herpesviridae/virologia , Sensibilidade e EspecificidadeRESUMO
Based on small scale studies or on little sensitive serological tests, bovines in the south of the state of Rio Grande do Sul, Brazil, are known to be infected with either bovine herpesvirus 1 (BoHV-1) or 5 (BoHV-5). However, whether the prevalence of each of these viruses is high or low is currently still unknown. In order to determine the extent of BoHV (-1 and/or -5) infections in bovines in this region of Brazil, 200 bovines were studied for the presence of BoHV DNA. To this end, first a quantitative PCR was developed that amplified BoHV-1 DNA as well as BoHV-5 DNA. Using this PCR the number of BoHV genomes normally present in latently infected ganglia of naturally infected bovines was estimated. The new PCR was sensitive enough to detect most BoHV DNA in infected ganglia. The results of this first PCR showed that at least 87% of the bovines in the south of Rio Grande do Sul were (latently) infected with either BoHV-1 or BoHV-5. To determine the prevalence of BoHV-1 and BoHV-5 separately, two type-specific PCRs - one for each virus - were developed that used the products of the first PCR as a template. The results of these type-specific PCRs showed that 82.8% of the BoHV positive population was (latently) infected with BoHV-1, 93.1% with BoHV-5 and 75.9% with both BoHV-1 and BoHV-5. This is the first time that such a high frequency of co-infection of BoHV-1 and BoHV-5 in bovines has been demonstrated.