RESUMO
Cross-species infection with ovine herpesvirus 2 (OvHV-2) in cattle causes malignant catarrhal fever (MCF). MCF may involve the central nervous system (CNS) with necrotizing arteritis and/or vasculitis described to be unique to MCF and discriminatory compared to other viral CNS infections. However, a systematic histopathological characterization of the neural form of MCF in cattle is lacking. We examined medulla oblongata (n = 9) or the entire brain (n = 9) of 18 cattle in which OvHV-2 was identified by quantitative polymerase chain reaction (qPCR), in order to pinpoint potential variations in neuropathology. In 2/18 animals (11%) no lesions were identified, while 16/18 cattle (89%) had brain lesions of varying severity. Presence and quantities of OvHV-2 nucleic acid were determined by in situ hybridization and qPCR, respectively, and were related to the severity of lesions. Fifteen of 18 animals (83%) showed vasculitis, which was mainly of the lymphohistiocytic type, while pathognomonic necrotizing arteritis was only rarely present. Neuroparenchymal lesions included gliosis and/or neuronal changes in 7/16 brains with lesions (44%). The number of CD3+ lymphocytes was highest in animals with simultaneous vascular and neuroparenchymal lesions and high viral genome load. In one animal, OvHV-2 was exclusively observed in CD3+ lymphocytes but not in neurons or microglia. In conclusion, the neuropathological phenotype of bovine MCF in the brain was variable. In some cases, lesions mimicked neurotropic viral encephalitis, while pathognomonic necrotizing arteritis was not a consistent feature of neural MCF. Therefore, molecular detection of OvHV-2 is warranted in the presence of nonsuppurative encephalitis and in the absence of necrotizing arteritis.
Assuntos
Doenças dos Bovinos , Gammaherpesvirinae , Febre Catarral Maligna , Poliarterite Nodosa , Doenças dos Ovinos , Animais , Bovinos , Fenótipo , Poliarterite Nodosa/veterinária , OvinosRESUMO
We report the identification of a neurotropic astrovirus associated with encephalitis in a sheep. This virus is genetically almost identical to an astrovirus recently described in neurologically diseased cattle. The similarity indicates that astroviruses of the same genotype may cause encephalitis in different species.
Assuntos
Infecções por Astroviridae/veterinária , Astroviridae/genética , Doenças dos Bovinos/transmissão , Encefalite Viral/veterinária , Genoma Viral , Doenças dos Ovinos/transmissão , Animais , Astroviridae/classificação , Astroviridae/isolamento & purificação , Infecções por Astroviridae/epidemiologia , Infecções por Astroviridae/patologia , Infecções por Astroviridae/transmissão , Encéfalo/patologia , Encéfalo/virologia , Bovinos , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/patologia , Encefalite Viral/epidemiologia , Encefalite Viral/patologia , Encefalite Viral/transmissão , Especificidade de Hospedeiro , Humanos , Imuno-Histoquímica , Filogenia , Ovinos , Doenças dos Ovinos/epidemiologia , Doenças dos Ovinos/patologia , Suíça/epidemiologiaRESUMO
An 8-year-old alpaca was admitted to the emergency service of the Clinic for Ruminants in Bern due to a reduced general condition and progressive neurological signs. Despite supportive treatment, its condition deteriorated and the animal had to be euthanized. Histopathological analysis revealed a severe non-suppurative polioencephalomyelitis with neuronal necrosis, most likely of viral origin. We detected abundant neuronal labelling with antibodies directed against two different epitopes of Bovine Astrovirus CH13/NeuroS1 (BoAstV-CH13/NeuroS1), which is a common viral agent associated with non-suppurative encephalitis in Swiss cattle. These findings were further verified by detection of viral RNA by use of in-situ hybridization and real-time RT-PCR. Next generation sequencing revealed that the detected virus genome had a pairwise identity of 98.9% to the genome of BoAstV-CH13/NeuroS1. To our knowledge, this is the first report of an astrovirus-associated polioencephalomyelitis in an alpaca. These results point to the possibility of an interspecies transmission of BoAstV-CH13/NeuroS1.
Assuntos
Infecções por Astroviridae/veterinária , Astroviridae , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/virologia , Poliomielite/veterinária , Animais , Astroviridae/genética , Biópsia , Encéfalo/patologia , Encéfalo/virologia , Bovinos , Imuno-Histoquímica , Hibridização In Situ , RNA Viral , Reação em Cadeia da Polimerase em Tempo RealRESUMO
In mammals, the small, positive-sense single-stranded RNA astroviruses are known as being mostly enterotropic and host-specific. Over the past years, however, they were identified several times in central nervous system tissues of humans, minks, cattle, sheep, and pigs with nonsuppurative inflammatory disease of that organ system. We recently reported such neurotropic astroviruses, amongst which bovine astrovirus CH15 (BoAstV-CH15) in two cows, and ovine astrovirus CH16 (OvAstV-CH16) in a sheep, which were genetically almost identical to one another. In order to investigate the occurrence of this virus species in Switzerland over time, we selected formalin-fixed, paraffin-embedded (FFPE) brain tissues of small ruminants diagnosed with severe encephalitis between 1969 and 2012 and screened those by immunohistochemistry for the capsid protein of BoAstV-CH15/OvAstV-CH16. We found one sheep, which died in 1992, that displayed positive immunostaining in various brain regions, and observed that immunostained cells were generally co-localized with the strongest histopathological lesions. We confirmed the virus presence with a second immunohistochemical protocol and demonstrated its close genetic relationship to other BoAstV-CH15/ OvAstV-CH16 strains by next-generation sequencing of an RNA extract from FFPE brain material. Our findings demonstrate that astrovirus BoAstV-CH15/OvAstV-CH16 existed in Switzerland already more than 2 decades ago and underline again the close relationship of the bovine and ovine strains of this virus.
RESUMO
Astroviruses (AstV) are single-stranded, positive-sense RNA viruses, best known for causing diarrhea in humans and are also found in many other mammals; in those, the relevance in gastroenteritis remains unclear. Recently described neurotropic AstV showed associations with encephalitis in humans as well as in other mammals. In Switzerland, two different neurotropic AstV were identified in cattle, as well as one in a sheep. The high genetic similarity between the ovine and one of the bovine AstV strengthens the hypothesis of an interspecies transmission. In humans, AstV associated with encephalitis were found also in human stool samples, suggesting that in these patients the infection spreads from the gastrointestinal tract to the brain under certain conditions, such as immunosuppression. Whether a similar pathogenesis occurs in ruminants remains unknown. The aims of this study were (1) the investigation of the potential occurrence of neurotropic AstV in feces samples, (2) the discovery and analysis of so far unknown AstV in small ruminants and other ruminant species' fecal samples and (3) the examination of a potential interspecies transmission of AstV. To achieve these aims, RNA extraction out of 164 fecal samples from different ruminant species was performed and all samples were screened for known neurotropic AstV occurring in Switzerland, as well as for various AstV using RT-PCR. Positive tested samples were submitted to next generation sequencing. The generated sequences were compared to nucleotide- and amino acid databases, virus properties were identified, and phylogenetic analyses as well as recombination analysis were performed. The excretion of neurotropic AstV in small ruminants' feces could not be demonstrated, but this work suggests the first identification of AstV in goats as well as the discovery of multiple and highly diverse new genetic variants in small ruminants, which lead to a classification into novel genotype-species. Additionally, the prediction of multiple recombination events in four of five newly discovered full or almost full-length genome sequences suggests a plausible interspecies transmission. The findings point out the occurrence and fecal shedding of previously unknown AstV in sheep and goats and pave the way towards a better understanding of the diversity and transmission of AstV in small ruminants.
RESUMO
A novel bovine astrovirus genotype species (BoAstV-CH13/NeuroS1) was recently identified in brain tissues of cattle as a plausible cause of encephalitis. The purpose of the present study was to develop and validate real time RT-PCR assays for the detection of BoAstV-CH13/NeuroS1 in brain tissues of cattle. Three different primer-probe combinations were designed based on BoAstV-CH13/NeuroS1 full-genome sequences of 11 different strains identified in cattle, and established in three distinct one-step real time RT-PCR protocols. These protocols were compared regarding their diagnostic performance using brain tissues of cattle with and without astrovirus associated encephalitis. The limit of detection (LOD) of all three assays was between 1.34 × 101 and 1.34 × 102 RNA copies, leading to an analytical sensitivity two orders of magnitude superior compared to a conventional pan-astrovirus RT-PCR protocol (LOD 1.31 × 104 RNA copies). Amplification efficiency was in the range of 97.3% to 107.5% with linearity (R2) > 0.99. The diagnostic sensitivity and specificity of the assays was determined as 100%, and all three revealed good intra- and inter-test repeatability. In conclusion, the newly developed RT-qPCRs are sensitive, specific, and reliable test formats that will facilitate BoAstV-CH13/NeuroS1 detection in routine diagnostics as well as in research settings.
Assuntos
Infecções por Astroviridae , Astroviridae/genética , Encéfalo/virologia , Doenças dos Bovinos , Encefalite Viral , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Infecções por Astroviridae/diagnóstico , Infecções por Astroviridae/genética , Infecções por Astroviridae/veterinária , Infecções por Astroviridae/virologia , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/genética , Doenças dos Bovinos/virologia , Encefalite Viral/diagnóstico , Encefalite Viral/genética , Encefalite Viral/veterinária , Encefalite Viral/virologia , Sensibilidade e EspecificidadeRESUMO
Astroviruses are best known as being one of the leading causes of diarrhea in infants and were first described in this context in 1975. In its first years, astrovirus research was mainly restricted to electron microscopy and serology studies. The ability to culture some of these viruses in vitro allowed a first consequent step forward, especially at the molecular level. Since the emergence of more powerful genetic methods, though, the face of this research field has dramatically changed and evolved. From the exponential number of discoveries of new astrovirus strains in the most varied of animal species to their association with atypical diseases, these viruses revealed a lot of surprises, and many more are probably still waiting to be uncovered. This chapter summarizes the most important knowledge about astroviruses and discusses the implication of the latest findings in this area of research.
Assuntos
Infecções por Astroviridae/veterinária , Infecções por Astroviridae/virologia , Mamastrovirus/classificação , Mamastrovirus/isolamento & purificação , Animais , HumanosRESUMO
Members of the Astroviridae family are best known to cause diarrhea in different mammalian species. Lately, some strains have been associated with encephalitis in humans, minks and cattle. In this study, we developed an immunohistochemistry (IHC) procedure for the detection of a neurotropic bovine astrovirus (BoAstV-CH13/NeuroS1), which is associated with non-suppurative encephalitis in cattle. We expressed five recombinant antigens corresponding to different putative viral proteins of BoAstV-CH13/NeuroS1. Antigens were then used for the production of hyperimmune sera in rabbits. Out of the five hyperimmune sera, the one directed against the conserved N-terminus of the viral capsid protein, termed ORF2-con, clearly surpassed the others in the detection of viral antigens in IHC in terms of strong signal intensity and low background staining. The accuracy of the ORF2-con IHC protocol was then evaluated using different sets of brain tissue samples: 30 samples from 9 animals with confirmed BoAstV-CH13/NeuroS1 infection, 30 samples from 8 animals with non-suppurative encephalitis of another etiology and 30 samples from apparently healthy slaughtered animals. The IHC was positive only with tissue samples from animals with a known positive BoAstV-CH13/NeuroS1 status, but not with those from negative ones, indicating a good diagnostic sensitivity and specificity of the assay. The ORF2-con IHC procedure is therefore an adequate tool for the detection of BoAstV-CH13/NeuroS1 infections in cattle.
Assuntos
Antígenos Virais/análise , Infecções por Astroviridae/veterinária , Doenças dos Bovinos/diagnóstico , Encefalite Viral/veterinária , Imuno-Histoquímica/métodos , Mamastrovirus/imunologia , Mamastrovirus/isolamento & purificação , Animais , Antígenos Virais/imunologia , Infecções por Astroviridae/diagnóstico , Infecções por Astroviridae/imunologia , Infecções por Astroviridae/virologia , Proteínas do Capsídeo/imunologia , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/virologia , Encefalite Viral/virologia , Mamastrovirus/genética , Sensibilidade e EspecificidadeRESUMO
Next-generation sequencing (NGS) has opened up the possibility of detecting new viruses in unresolved diseases. Recently, astrovirus brain infections have been identified in neurologically diseased humans and animals by NGS, among them bovine astrovirus (BoAstV) CH13/NeuroS1, which has been found in brain tissues of cattle with non-suppurative encephalitis. Only a few studies are available on neurotropic astroviruses and a causal relationship between BoAstV CH13/NeuroS1 infections and neurological disease has been postulated, but remains unproven. Aiming at making a step forward towards assessing the causality, we collected brain samples of 97 cases of cattle diagnosed with unresolved non-suppurative encephalitis, and analyzed them by in situ hybridization and immunohistochemistry, to determine the frequency and neuropathological distribution of the BoAstV CH13/NeuroS1 and its topographical correlation to the pathology. We detected BoAstV CH13/NeuroS1 RNA or proteins in neurons throughout all parts of the central nervous system (CNS) in 34% of all cases, but none were detected in cattle of the control group. In general, brain lesions had a high correlation with the presence of the virus. These findings show that a substantial proportion of cattle with non-suppurative encephalitis are infected with BoAstV CH13/NeuroS1 and further substantiate the causal relationship between neurological disease and astrovirus infections.
Assuntos
Infecções por Astroviridae/veterinária , Astroviridae/isolamento & purificação , Doenças dos Bovinos/patologia , Doenças dos Bovinos/virologia , Encefalite Viral/veterinária , Animais , Infecções por Astroviridae/patologia , Bovinos , Sistema Nervoso Central/patologia , Sistema Nervoso Central/virologia , Encefalite Viral/patologia , Imuno-Histoquímica , Hibridização In SituRESUMO
Neuroinfectious diseases in livestock represent a severe threat to animal health, but their prevalence is not well documented and the etiology of disease often remains unidentified. The aims of this study were to generate baseline data on the prevalence of neuroinfectious diseases in cattle in Switzerland by neuropathological survey, and to identify disease-associated pathogens. The survey was performed over a 1-year period using a representative number of brainstem samples (n=1816) from fallen cattle. In total, 4% (n=73) of the animals had significant lesions, the most frequent types of which were indicative of viral (n=27) and bacterial (n=31) etiologies. Follow-up diagnostics by immunohistochemistry, PCR protocols and next-generation sequencing identified infection with Listeria monocytogenes (n=6), ovine herpesvirus 2 (n=7), bovine astrovirus CH13 (n=2), bovine herpesvirus 6 (n=6), bovine retrovirus CH15 (n=2), posavirus 1 (n=2), and porcine astroviruses (n=2). A retrospective questionnaire-based investigation indicated that animals' owners observed clinical signs of neurological disease in about one-third of cases with lesions, which was estimated to correspond to approximately 85 cases per year in the adult fallen cattle population in Switzerland. This estimate stands in sharp contrast to the number of cases reported to the authorities and reveals a gap in disease surveillance. Systematic neuropathological examination and follow-up molecular testing of neurologically diseased cattle could significantly enhance the efficiency of disease detection for the purposes of estimating the prevalence of endemic diseases, identifying new or re-emerging pathogens, and providing "early warnings" of disease outbreaks.
Assuntos
Doenças dos Bovinos/microbiologia , Encefalite/veterinária , Viroses/veterinária , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/patologia , Encefalite/epidemiologia , Encefalite/microbiologia , Encefalite/virologia , Estudos Retrospectivos , Suíça/epidemiologia , Viroses/epidemiologiaRESUMO
Non-suppurative encephalitis is one of the most frequent pathological diagnosis in cattle with neurological disease, but there is a gap in the knowledge on disease-associated pathogens. In order to identify viruses that are associated with non-suppurative encephalitis in cattle, we used a viral metagenomics approach on a sample set of 16 neurologically-diseased cows. We detected six virus candidates: parainfluenza virus 5 (PIV-5), bovine astrovirus CH13/NeuroS1 (BoAstV-CH13/NeuroS1), bovine polyomavirus 2 (BPyV-2 SF), ovine herpesvirus 2 (OvHV-2), bovine herpesvirus 6 (BHV-6) and a novel bovine betaretrovirus termed BoRV-CH15. In a case-control study using PCR, BoAstV-CH13 (p=0.046), BoPV-2 SF (p=0.005) and BoHV-6 (p=4.3E-05) were statistically associated with the disease. These data expand our knowledge on encephalitis-associated pathogens in cattle and point to the value of NGS in resolving complex infection scenarios in a clinical disease setting.