Genetic characterization of the rare Bruconha virus (Bunyavirales: Orthobunyavirus) isolated in Vale do Ribeira (Atlantic Forest biome), Southeastern Brazil

Brazil is a great source of arbovirus diversity, mainly in the Amazon region. However, other biomes, especially the Atlantic Forest, may also be a hotspot for emerging viruses, including Bunyaviruses (Negarnaviricota: Bunyavirales). For instance, Vale do Ribeira, located in the Southeastern region, has been widely studied for virus surveillance, where Flavivirus, Alphavirus and Bunyaviruses were isolated during the last decades, including Bruconha virus (BRCV), a member of Orthobunyavirus genus Group C, in 1976. Recently, a new isolate of BRCV named Span321532 was obtained from an adult sentinel mouse placed in Iguape city in 2011, and a full-length genome was generated with nucleotide differences ranging between 1.5%, 5.3% and 5% (L, M and S segments, respectively) from the prototype isolated 35 years earlier. In addition, each segment placed BRCV into different clusters, showing the high variety within Bunyavirales. Although no evidence for reassortants was detected, this finding reiterates the need for new surveillance and genomic studies in the area considering the high mutation rates of arbovirus, and also to identify the hosts capable of supporting the continuous circulation of Orthobunyavirus

Ocorrência de leucoencefalomalácia (LEME) em equídeos no estado de São Paulo, Brasil: achados anatomopatológicos

O trabalho relata a ocorrência de leucoencefalomalácia em equídeos (LEME) com sintomatologia nervosa e com diagnóstico negativo para raiva, herpesvírus equino e encefalomielite equina durante o período de dois anos, no Estado de São Paulo, Brasil. Foram examinadas 67 amostras de sistema nervoso central e em 10,4% (cinco equinos, um pônei e um asinino) observaram-se lesões macroscópicas de LEME, confirmadas pela análise histopatológica. Os animais acometidos eram cinco machos e duas fêmeas, com idades que variavam de 11 meses a nove anos. Os sete casos ocorreram tanto no inverno como em outras estações do ano. As principais manifestações clínicas relatadas foram incoordenação, ataxia, paralisia dos membros posteriores, profunda depressão, levando ao óbito. Macroscopicamente, observaram-se congestão dos vasos meníngeos, áreas de malácia da substância branca, caracterizadas por coloração amarelada e/ou hemorrágica, com cavitação e amolecimento circundados por hiperemia. As lesões microscópicas observadas em todos os casos eram de necrose de liquefação da substância branca do cérebro, caracterizada por substância eosinofílica amorfa e homogênea, presença de edema axonal e perivascular, hemorragia e vacuolização do neurópilo adjacente e esferoides axonais. Em algumas áreas de malácia havia também células Gitter. Em apenas um animal observou-se manguito perivascular mononuclear. O presente trabalho confirma que o diagnóstico diferencial é importante na distinção da LEME com outras neuropatias encefálicas que acometem equídeos. A ocorrência da LEME relatada neste estudo demonstra que esta enfermidade é importante para a equideocultura do Estado de São Paulo.This article describes clinical and pathological findings of leukoencephalomalacia in equids with neurological signs which tested negative to rabies, equine herpesvirus and equine encephalomyelitis. This work was carried during the period of two years in São Paulo State, Brazil. A total of 67 brain samples were examined and in 10.4% (five equines, one poney, and one donkey) were observed gross lesions of ELEM, confirmed by histopathological analysis. The animals were five males and two females ranged from 11 months to nine years old. The seven cases happened in all seasons of the year. The most characteristic clinical signs were incoordination, ataxia, paralysis of the hind legs, profound depression and death. Necropsy was performed to collect brain samples for virological and histopathological diagnosis. Gross lesions included congestion of meningeal blood vessels, malacia of the white matter characterized by yellowish depressed areas sometimes hemorrhagic, with cavitations, and softening surrounded by hyperemic area. Microscopically, the lesions were liquefactive necrosis of the white matter brain, characterized by eosinophilic and amorphous material, axonal and perivascular edema, hemorrhage and vacuolization of the neuropil and axonal sferoids. Gitter cells were seen in some areas of malacia. Perivascular mononuclear cuffing was observed in only one case. The present study confirms that differential diagnosis is very important to distinguish equid neuropathies. The occurence of ELEM in the present study shows that the disease is important for the equideoculture in São Paulo State

Ocorrência de enfermidades virais em asininos (Equus asinus) no estado de São Paulo, Brasil

Among the diseases that affect equines, viral diseases play an important role from a health and economic point of view, especially influenza, viral arteritis, herpes infections and vesicular stomatitis. In the Brazilian literature, there is little or no account of the occurrence of infectious diseases in donkeys. Given the importance of donkeys in different activities and the lack of information on infections that may occur in these animals, the aim of this study was to determine the frequency of anti-equine herpesvirus (EHV), anti-equine arteritis virus (EAV), anti-vesicular stomatitis, and anti-equine influenza (H3N8) antibodies in the serum of 85 donkeys bred in some regions of the state of Sao Paulo. We found the following antibody frequencies: 50.6% (43/85) antibodies against influenza virus subtype H3N8, 47% (40/85) anti-EHV, and 20% (17/85) anti-EAV. The donkeys were not seropositive for vesicular stomatitis. The results suggested that the agents EHV, EAV, and equine influenza subtype H3N8 circulate among donkeys in some regions of the state of Sao Paulo, Brazil, reinforcing the importance of establishing a routine diagnosis and epidemiological study of this species.Dentre as doenças que acometem os equídeos, as enfermidades virais assumem um papel importante do ponto de vista sanitário e econômico, especialmente a influenza, arterite viral, as infecções herpéticas e a estomatite vesicular. Na literatura nacional, existe pouco ou nenhum relato sobre a ocorrência de enfermidades infecciosas nos asininos. Tendo em vista a importância dos asininos para diferentes atividades e a falta de informações sobre as doenças que acometem esses animais, este trabalho teve como objetivo estudar a frequência de anticorpos anti-EHV, antivírus da arterite equina, anti-estomatite vesicular e anti-influenza equina (H3N8) em 85 soros de jumentos criados no estado de São Paulo. Estimou-se que 50,6% apresentavam anticorpos contra o subtipo H3N8 do vírus da influenza; 47% (40/85) apresentavam anticorpos contra o EHV e 20% apresentavam anticorpos contra o vírus da arterite. Os jumentos não foram soro reagentes contra a estomatite vesicular. Os resultados obtidos sugerem que os agentes EHV, vírus da arterite equina e influenza equina subtipo H3N8, circulam entre os jumentos do estado de São Paulo, caracterizando a importância do estabelecimento de uma rotina diagnostica e estudos epidemiológicos na espécie

Inquérito sorológico da infecção por herpesvírus equino no Estado de Minas Gerais

Os herpesvírus equinos tipo 1 (HVE-1) e 4 (HVE-4) são agentes causadores de diferentes formas de doença em cavalos, das quais as mais comuns são a rinopneumonite, o abortamento, a mortalidade perinatal e a mieloencefalopatia herpética equinas, que causam grandes perdas econômicas. Tem sido descrita mundialmente, havendo poucos estudos no Brasil. O objetivo do presente trabalho foi estudar a ocorrência e a distribuição da infecção por herpesvírus equinos (HVE) em equídeos criados em dez Delegacias Regionais do Estado de Minas Gerais: Almenara, Bambuí, Curvelo, Governador Valadares, Montes Claros, Oliveira, São Gonçalo do Sapucaí, Teófilo Otoni, Unaí e Viçosa. Foi utilizada a técnica de soroneutralização em microplacas com o intuito de detectar anticorpos soro neutralizantes. Das amostras analisadas, 17,6% (145/826) foram soropositivas para o HVE, sendo 18,7% (140/749) cavalos soropositivos, 6,8% (5/73) muares soropositivos e nenhum asinino soropositivo (0/4). Conclui-se que o HVE-1 encontra-se amplamente disseminado no Estado de Minas Gerais, pois todas as regiões estudadas apresentaram animais sororreagentes ao HVE-1. Observou-se maior ocorrência de anticorpos contra o HVE em animais adultos, indicando assim o potencial desses animais como fonte de infecção para os potros.Equine herpesvirus type 1 (EHV-1) and 4 (EHV-4) are major pathogens affecting horses, and cause respiratory disease, abortion, perinatal mortality and neurological disease, bringing economical losses. This infection has been reported worldwide, but there are only a few studies in Brazil. The present study was undertaken in order to evaluate the occurrence and distribution of equine herpesviruses (EHV) infection in equids from ten regions of Minas Gerais State: Almenara, Bambuí, Curvelo, Governador Valadares, Montes Claros, Oliveira, São Gonçalo do Sapucaí, Teófilo Otoni, Unaí and Viçosa. To detect antibodies against EHV virus neutralization test in microplates was used. We found 17.6% (145/826) positive animals for EHV. 18.7% (140/749) positive horses, 6.8% (5/73) positive mules and none positive (0/4) donkeys. All ten regions studied showed animals reagents to EHV. The results suggest that EHV is widespread in equids of Minas Gerais State. It was observed a higher occurrence of antibodies against EHV in adult animals, indicating the potential of these animals as source of infection for foals

Validation and use of reverse transcription followed by real-time polymerase chain reaction (RT-qPCR) for the surveillance and diagnosis of flavivirus transmitted by mosquitoes in Brazil

Os flavivírus são considerados uma séria ameaça à saúde pública em diversas partes do mundo, pois muitos são agentes altamente patogênicos a seres humanos e animais, tais como os vírus da febre amarela, vírus do Oeste do Nilo, vírus da encefalite japonesa e vírus da dengue, capazes de causar encefalites ou febres hemorrágicas em seus hospedeiros. Muitos deles têm avançado a diferentes regiões geográficas onde sua circulação não havia sido detectada previamente, causando novos surtos. O diagnóstico clínico destas infecções é, muitas vezes, difícil, devido ao grande número de sintomas apresentados, que podem se confundir com outras enfermidades de diferentes causas etiológicas. Os principais métodos diretos utilizados atualmente no Brasil para detecção destes vírus são a inoculação intracerebral em camundongos neonatos, inoculação em culturas de células e RTPCR específica. O presente trabalho tem como objetivos avaliar a sensibilidade e validar a detecção dos vírus pertencentes ao gênero Flavivirus circulantes no Brasil por meio de uma reação single de RT-PCR em tempo real e implementá-la, tanto na rotina diagnóstica de casos com suspeita de arbovirose como na pesquisa de amostras de campo para monitoramento viral. Amostras dos flavivírus padrões da Febre Amarela, Bussuquara, Iguape, Ilheus, Encefalite de Saint Louis, Cacipacore e Zika foram quantificados por titulação em unidades formadoras de placa (UFP) ou TCID50 para se avaliar os limites de detecção para cada um deles por RT-qPCR que detecta o gênero Flavivirus. Os limites encontrados variaram de 0,01 UFP, para o vírus Ilheus, a 1 UFP, para os vírus da Febre Amarela e Iguape, e 1x101,6 TCID50/100µL para o vírus Bussuquara. Além disso, o presente trabalho foi capaz de identificar, após sequenciamento de cDNA gerado, os vírus Zika, isolado de um paciente febril, e os vírus Ilheus e Iguape, isolados a partir de diferentes espécies de Culicídeos, após uma única reação de RT-qPCR, e um possível novo flavivírus específico de insetos, isolado de mosquitos Aedes coletados em Guapiaçu, São Paulo. Não houve sinal de amplificação para os Alphavirus Mayaro e Chikungunya. O presente protocolo mostrou-se com alta sensibilidade e especificidade, podendo dessa forma ser utilizado para o diagnóstico diferencial dos diferentes flavivírus que ocorrem no Brasil, bem como para estudos de monitoramento viral em animais sentinelas e vetores, colaborando dessa forma com a saúde pública. Pode-se, ainda, detectar possíveis novos vírus específicos de artrópodesFlaviviruses are considered a serious threat to public health in many parts of the world, as many are highly pathogenic to humans and animals, such as Yellow Fever virus, West Nile virus, Japanese encephalitis virus and dengue virus, which are capable of causing encephalitis or hemorrhagic fever in their hosts. Many of them have spread to different geographic regions where their circulation had not been detected previously, causing new outbreaks. Diagnosis of these infections is often difficult, due to the large number of symptoms presented, which can be confused with other diseases of different etiological causes. The main direct methods currently used in Brazil for detecting these viruses are intracerebral inoculation in neonatal mice, inoculation in cell cultures and specific RT-PCR. The present work aims to evaluate the sensitivity and validate the detection of viruses belonging to the genus Flavivirus circulating in Brazil through a single real-time RT-PCR reaction and to implement it, both in the diagnostic routine of cases with arbovirus suspicions and in field samples for viral monitoring. Samples of the standard flaviviruses Yellow Fever, Bussuquara, Iguape, Ilheus, Saint Louis Encephalitis, Cacipacore and Zika were quantified by titration by plaque forming units (UFP) or TCID50 to evaluate the detection limits for each of them by RT- qPCR that detects genus Flavivirus. The limits found ranged from 0.01 PFU for Ilheus virus to 1 PFU for Yellow Fever and Iguape viruses and 1x101.6 TCID50 / 100L for the Bussuquara virus. In addition, the present work was able to identify, after cDNA sequencing Zika virus, isolated from a febrile patient, and both Ilheus and Iguape viruses, isolated from different species of Culicidae, and a possible new insect-specific flavivirus, isolated from Aedes mosquitoes collected in Guapiaçu, São Paulo. The Alphaviruses Mayaro and Chikungunya were not amplified. The present protocol shoed high sensitivity and specificity, and therefore it may may be used for the differential diagnosis of the different flaviviruses that occur in Brazil, as well as for viral monitoring studies in sentinel animals and vectors, thus collaborating with public health. It is also possible to detect new flavivirus that are arthopode-specific

Occurrence of antibodies against Leptospira spp. in donkeys of São Paulo state

ABSTRACT: Among the diseases that affect equines, bacterial diseases play an important role from a health and economic point of view, especially leptospirosis and brucellosis. The study aimed to provide information on the occurrence of anti-Leptospira spp. and anti-Brucella abortus antibodies in donkeys of São Paulo state. We found a frequency of 62.4% (53/85) antibodies against Leptospira spp. The donkeys were not seropositive for Brucella spp

Simple protocol for population (Sanger) sequencing for Zika virus genomic regions

<div><p> BACKGROUND A number of Zika virus (ZIKV) sequences were obtained using Next-generation sequencing (NGS), a methodology widely applied in genetic diversity studies and virome discovery. However Sanger method is still a robust, affordable, rapid and specific tool to obtain valuable sequences. OBJECTIVE The aim of this study was to develop a simple and robust Sanger sequencing protocol targeting ZIKV relevant genetic regions, as envelope protein and nonstructural protein 5 (NS5). In addition, phylogenetic analysis of the ZIKV strains obtained using the present protocol and their comparison with previously published NGS sequences were also carried out. METHODS Six Vero cells isolates from serum and one urine sample were available to develop the procedure. Primer sets were designed in order to conduct a nested RT-PCR and a Sanger sequencing protocols. Bayesian analysis was used to infer phylogenetic relationships. FINDINGS Seven complete ZIKV envelope protein (1,571 kb) and six partial NS5 (0,798 Kb) were obtained using the protocol, with no amplification of NS5 gene from urine sample. Two NS5 sequences presented ambiguities at positions 495 and 196. Nucleotide analysis of a Sanger sequence and consensus sequence of previously NGS study revealed 100% identity. ZIKV strains described here clustered within the Asian lineage. MAIN CONCLUSIONS The present study provided a simple and low-cost Sanger protocol to sequence relevant genes of the ZIKV genome. The identity of Sanger generated sequences with published consensus NGS support the use of Sanger method for ZIKV population studies. The regions evaluated were able to provide robust phylogenetic signals and may be used to conduct molecular epidemiological studies and monitor viral evolution.</p></div

Detection of Zika and dengue viruses in wild-caught mosquitoes collected during field surveillance in an environmental protection area in São Paulo, Brazil.

Species of the genus Flavivirus are widespread in Brazil and are a major public health concern. The country's largest city, São Paulo, is in a highly urbanized area with a few forest fragments which are commonly used for recreation. These can be considered to present a potential risk of flavivirus transmission to humans as they are home simultaneously to vertebrate hosts and mosquitoes that are potential flavivirus vectors. The aim of this study was to conduct flavivirus surveillance in field-collected mosquitoes in the Capivari-Monos Environmental Protection Area (EPA) and identify the flavivirus species by sequence analysis in flavivirus IFA-positive pools. Monthly mosquito collections were carried out from March 2016 to April 2017 with CO2-baited CDC light traps. Specimens were identified morphologically and grouped in pools of up to 10 individuals according to their taxonomic category. A total of 260 pools of non-engorged females were inoculated into C6/36 cell culture, and the cell suspensions were analyzed by indirect immunofluorescence assay (IFA) after the incubation period. IFA-positive pools were tested by qRT-PCR with genus-specific primers targeting the flavivirus NS5 gene to confirm IFA-positive results and sequenced to identify the species. Anopheles cruzii (19.5%) and Wyeomyia confusa (15.3%) were the most frequent vector species collected. IFA was positive for flaviviruses in 2.3% (6/260) of the sample pools. This was confirmed by qRT-PCR in five pools (83.3%). All five flavivirus-positive pools were successfully sequenced and the species identified. DENV serotype 2 (DENV-2) was detected in Culex spp. and Culex vaxus pools, while ZIKV was identified in An. cruzii, Limatus durhamii and Wy. confusa pools. To the best of our knowledge, detection of flavivirus species of medical importance has never previously been reported in these species of wild-caught mosquitoes. The finding of DENV-2 and ZIKV circulating in wild mosquitoes suggests the existence of an enzootic cycle in the area. In-depth studies of DENV-2 and ZIKV, including investigation of mosquito infection, vector competence and infection in sylvatic hosts, are needed to shed light on the transmission dynamics of these important viruses and the potential risk of future outbreaks of DENV-2 and ZIKV infections in the region

Outbreak of Yellow Fever among Nonhuman Primates, Espirito Santo, Brazil, 2017

In January 2017, a yellow fever outbreak occurred in Espirito Santo, Brazil, where human immunization coverage is low. Histologic, immunohistologic, and PCR examinations were performed for 22 deceased nonhuman New World primates; typical yellow fever features were found in 21. Diagnosis in nonhuman primates prompted early public health response