RESUMO
To understand the population genetics events during coronavirus host switches, the Beaudette strain of Avian coronavirus (AvCoV) adapted to BHK-21 cells was passaged 15 times in VERO cells, the virus load and the variants at each passage being determined by RT-qPCR and genome-length deep sequencing. From BHK-21 P2 to VERO P3, a trend for the extinction of variants was followed by stability up to VERO P11 and both the emergence and the rise in frequency in some variants, while the virus loads were stable up to VERO P12. At the spillover from BHK-21 to VERO cells, variants that both emerged, showed a rise in frequency or were extinguished were detected on the spike, while variants at the M gene showed the same pattern only at VERO passage 13. Furthermore, nsps 3-5, 9 and 15 variants were detected at lower passages compared to the consensus sequences, with those at nsp3 being detected in the spectra also at higher passages. This suggests that quasispecies coronavirus evolution in spillovers follows the virus life cycle, starting with the evolution of the receptor binding proteins, followed by the replicase and then proteins involved in virion assembly, keeping the general fitness of the mutant spectrum stable.
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Feline immunodeficiency virus (FIV) is an important pathogen of domestic and wild felids. Although serological tests suggest the presence of FIV in cats from Colombia, no molecular characterization has been reported. Here, we describe the near-complete genome of FIV subtype A from a Colombian domestic cat.
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Avian coronavirus (AvCoV) is ubiquitously present on poultry as a multitude of virus lineages. Studies on AvCoV phenotypic traits are dependent on the isolation of field strains in chicken embryonated eggs, but the mutant spectrum on each isolate is not considered. This manuscript reports the previously unknown HTS (high throughput sequencing)-based complete genome haplotyping of AvCoV isolates after passages of two field strains in chicken embryonated eggs. For the first and third passages of strain 23/2013, virus loads were 6.699 log copies/ µL and 6 log copies/ µL and, for 38/2013, 5.699 log copies/µL and 2.699 log copies/µL of reaction, respectively. The first passage of strain 23/2013 contained no variant haplotype, while, for the third passage, five putative variant haplotypes were found, with > 99.9% full genome identity with each other and with the dominant genome. Regarding strain 38/2013, five variant haplotypes were found for the first passage, with > 99.9% full genome identity with each other and with the dominant genome, and a single variant haplotype was found. Extinction and emergence of haplotypes with polymorphisms in genes involved in receptor binding and regulation of RNA synthesis were observed, suggesting that phenotypic traits of AvCoV isolates are a result of their mutant spectrum.
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Avian coronavirus, the causative agent of avian infectious bronchitis, occurs as multiple genotypes and lineages, and full genomes are not available for the majority of them. This paper reports the (previously unknown) complete genome sequence of strain D274 of this virus (27,599 nucleotides), isolated from chickens in The Netherlands in 1979.
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Rabies transmitted by the hematophagous bat Desmodus rotundus represents a public health concern and a burden for the Brazilian livestock industry. Current evidence suggests that rabies occurrence is related to landscape characteristics, topography, hydrography, animal production systems and land use...
A raiva transmitida por morcegos hematófagos da espécie Desmodus rotundus representa uma preocupação de saúde pública e causa de importantes prejuízos para a pecuária brasileira. A evidência atual sugere que a ocorrência de raiva está relacionada às características da paisagem, topografia, hidrografia, sistemas de produção animal e usos da terra...
Assuntos
Animais , Quirópteros/virologia , Vírus da Raiva/genéticaRESUMO
Rabies transmitted by the hematophagous bat Desmodus rotundus represents a public health concern and a burden for the Brazilian livestock industry. Current evidence suggests that rabies occurrence is related to landscape characteristics, topography, hydrography, animal production systems and land use. However, a few studies have analyzed the possible connections among geographic factors and the molecular diversity of the rabies virus, furthering the understanding of the spatial and temporal dynamics of outbreaks. A study reported that the latest rabies epizootics in herbivores reported in the eastern region of São Paulo (close to the Minas Gerais border) occurred in two epidemic waves; the first was before 1998, and the other occurred after 1999. Using this evidence, the aim of the present study was to analyze cases of rabies in herbivores in the southern region of Minas Gerais (2000-2009) and their possible relationship with the aforementioned epidemics, considering the geographic characteristics of the region. Partial sequences of glycoprotein (539 nt) and nucleoprotein genes (414 nt) were obtained from 31 rabies virus isolates from herbivores. A phylogenetic tree was proposed for each genomic region using the Neighbor joining method, fixing the Kimura 2-parameter evolution model with a bootstrap level of 1,000 replications. Genetic sublineages were plotted on maps, considering rabies risk areas for herbivores in São Paulo, as well as topographic characteristics and hydrographic basins, to visualize any apparent distribution pattern influenced by those features. The phylogenetic trees had concordant topologies, suggesting a possible common origin for rabies outbreaks in herbivores along the SP/MG border, surrounding the less elevated portions of the Serra da Mantiqueira and along the hydrographic basins of Piracicaba/Jaguarí, Paranaíba do Sul, Grande, Pardo and Mogi-Guaçu rivers.The co-circulation of several viral lineages was observed in some municipalities, possibly due to an overlapping of rabies outbreaks. Inferred protein sequences of both genes showed synonymous mutations, except among residues 20 to 200, corresponding to the external domain of the glycoprotein. This information prompted cooperation among the animal health services of both states to reinforce rabies control in the border area.(AU)
A raiva transmitida por morcegos hematófagos da espécie Desmodus rotundus representa uma preocupação de saúde pública e causa de importantes prejuízos para a pecuária brasileira. A evidência atual sugere que a ocorrência de raiva está relacionada às características da paisagem, topografia, hidrografia, sistemas de produção animal e usos da terra. Contudo, existem poucos estudos que analisem as possíveis conexões entre fatores geográficos e a diversidade molecular do vírus da raiva, permitindo a compreensão da dinâmica espacial e temporal dos focos de raiva. Um desses trabalhos estabeleceu que a última epizootia de raiva dos herbívoros registrada no leste do estado de São Paulo (na fronteira com Minas Gerais), aconteceu em duas ondas epidêmicas, sendo a primeira em 1998 e, em 1999, a segunda. Considerando esta evidência, o intuito do presente estudo foi analisar casos de raiva em herbívoros na região sudeste de Minas Gerais (2000-2009) e sua possível relação com a epidemia previamente mencionada, incluindo as características geográficas da região. Foram obtidas sequencias parciais dos genes da glicoproteína (539 nt) e da nucleoproteína (414 nt) a partir de 31 isolados de vírus da raiva procedentes de herbívoros. Foi proposta uma árvore filogenética para cada região genômica usando o método de Neighbor joining, fixando o modelo evolutivo Kimura 2 - parâmetros com um nível de bootstrap de 1000 replicações. As sublinhagens genéticas foram localizadas sobre mapas, considerando as áreas de risco para raiva dos herbívoros em São Paulo, assim como as características topográficas e bacias hidrográficas com o intuito de visualizar qualquer padrão aparente de distribuição segundo essas características. As duas árvores filogenéticas mostraram topologias concordantes, sugerindo uma possível origem comum para os surtos que aconteceram ao longo da fronteira SP/MG, ao redor das porções menos elevadas da Serra da Mantiqueira e acompanhando as bacias hidrográficas dos rios Piracicaba/Jaguarí, Paranaíba do Sul, Grande, Pardo e Mogi-Guaçu. Foi possível observar circulação de varias linhagens virais simultaneamente em alguns municípios, possivelmente por causa de sobreposição de surtos. As sequencias de proteína inferidas a partir dos dois genes mostraram mutações sinônimas, excetuando aquelas encontradas entre os resíduos 20 a 200, correspondentes ao domínio externo da glicoproteína. Esta informação salienta a importância da cooperação entre as autoridades sanitárias de ambos os estados para reforçar o programa de controle da doença nas áreas limítrofes.(AU)
Assuntos
Animais , Vírus da Raiva/genética , Quirópteros/virologiaRESUMO
Gastroenteritis is one of the leading causes of morbidity and mortality among young and newborn animals and is often caused by multiple intestinal infections, with rotavirus and bovine coronavirus (BCoV) being the main viral causes in cattle. Given that BCoV is better studied than equine coronaviruses and given the possibility of interspecies transmission of these viruses, this research was designed to compare the partial sequences of the spike glycoprotein (S), hemagglutinin-esterase protein (HE), and nucleoprotein (N) genes from coronaviruses from adult cattle with winter dysentery, calves with neonatal diarrhea, and horses. To achieve this, eleven fecal samples from dairy cows with winter dysentery, three from calves, and two from horses, all from Brazil, were analysed. It could be concluded that the enteric BCoV genealogy from newborn and adult cattle is directly associated with geographic distribution patterns, when S and HE genes are taken into account. A less-resolved genealogy exists for the HE and N genes in cattle, with a trend for an age-related segregation pattern. The coronavirus strains from horses revealed Betacoronavirus sequences indistinguishable from those found in cattle, a fact previously unknown.
Assuntos
Coronavirus Bovino/classificação , Coronavirus Bovino/genética , Genes Virais , Filogenia , Animais , Brasil , Bovinos , Geografia , CavalosRESUMO
Cryptosporidium spp. are cosmopolitan protozoa that infect fishes, reptiles, amphibians, birds and mammals. More than 20 species are recognized within this genus. Rodents are a group of abundant and ubiquitous organisms that have been considered reservoirs of Cryptosporidium for humans and livestock. The aim of this study was to design specific primers for the gene encoding 18S rRNA, potentially capable of amplifying any species or genotype of Cryptosporidium spp. and evaluate the diagnostic attributes of the nested-PCR based on such probes. The primers were designed to amplify the shortest segment as possible to maximize the sensitivity of the test, but preserving the discriminatory potential of the amplified sequences for phylogenetic inferences. The nested-PCR standardized in this study (nPCR-SH) was compared in terms of sensitivity with another similar assay (nPCR-XIAO) that has been largely used for the detection and identification of Cryptosporidium spp. worldwide. We also aimed to molecularly characterize samples of Cryptosporidum spp. isolated from synanthropic rodents using these probes. Forty-five rodents were captured in urban areas of the municipality of Umuarama, Paraná State, Brazil. Fecal samples were submitted to three molecular tests (nested-PCRs), two of them targeted to the 18S rDNA gene (nPCR-SH and nPCR-XIAO) and the third targeted to the gene encoding actin (nPCR-actin). The nPCR-SH was tested positive on samples of Cryptosporidum parvum, Cryptosporidum andersoni, Cryptosporidum meleagridis, Cryptosporidum hominis, Cryptosporidum canis, and Cryptosporidum serpentis. Sixteen samples of rodents were positive by nPCR-SH, six by nPCR-XIAO and five by nPCR-actin. Sequencing of amplified fragments allowed the identification of Cryptosporidum muris in three samples of Rattus rattus, and two genotypes of Cryptosporidium, the genotypes mouse II and III. Cryptosporidium genotype mouse II was found in one sample of Mus musculus and genotype mouse III, in twelve samples, being five from R. rattus and seven from M. musculus. The results of this study demonstrated that the primers designed for detection of Cryptosporidium spp. were more efficient than those used in the nPCR-XIAO. Genotypes or species of Cryptosporidium that can be usually transmitted for human beings and livestock were not found in synanthropic rodents, suggesting that the importance of these animals in zoonotic transmission of cryptosporidiosis should be revisited.
Assuntos
Criptosporidiose/veterinária , Cryptosporidium/isolamento & purificação , Primers do DNA , Camundongos/parasitologia , Ratos/parasitologia , Doenças dos Roedores/parasitologia , Actinas/genética , Animais , Sequência de Bases , Brasil , Criptosporidiose/parasitologia , Criptosporidiose/transmissão , Cryptosporidium/classificação , Cryptosporidium/genética , Primers do DNA/normas , DNA de Protozoário/isolamento & purificação , DNA Ribossômico , Reservatórios de Doenças/parasitologia , Fezes/parasitologia , Dados de Sequência Molecular , Oocistos/classificação , Filogenia , Reação em Cadeia da Polimerase/normas , RNA Ribossômico 18S/genética , Doenças dos Roedores/transmissão , Alinhamento de SequênciaRESUMO
Bovine coronavirus (BCoV) is a member of the group 2 of the Coronavirus (Nidovirales: Coronaviridae) and the causative agent of enteritis in both calves and adult bovine, as well as respiratory disease in calves. The present study aimed to develop a semi-nested RT-PCR for the detection of BCoV based on representative up-to-date sequences of the nucleocapsid gene, a conserved region of coronavirus genome. Three primers were designed, the first round with a 463bp and the second (semi-nested) with a 306bp predicted fragment. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) in DEPC treated ultra-pure water, in fetal bovine serum (FBS) and in a BCoV-free fecal suspension, when positive results were found up to the 10-2, 10-3 and 10-7 dilutions, respectively, which suggests that the total amount of RNA in the sample influence the precipitation of pellets by the method of extraction used. When fecal samples was used, a large quantity of total RNA serves as carrier of BCoV RNA, demonstrating a high analytical sensitivity and lack of possible substances inhibiting the PCR. The final semi-nested RT-PCR protocol was applied to 25 fecal samples from adult cows, previously tested by a nested RT-PCR RdRp used as a reference test, resulting in 20 and 17 positives for the first and second tests, respectively, and a substantial agreement was found by kappa statistics (0.694). The high sensitivity and specificity of the new proposed method and the fact that primers were designed based on current BCoV sequences give basis to a more accurate diagnosis of BCoV-caused diseases, as well as to further insights on protocols for the detection of other Coronavirus representatives of both Animal and Public Health importance.
O Coronavírus bovino (BCoV) pertence ao grupo 2 do gênero Coronavirus (Nidovirales: Coronaviridae) e é agente causador de enterites tanto em bezerros como em bovinos adultos, bem como de doença respiratória em bezerros. O presente estudo teve por objetivo desenvolver uma semi-nested RT-PCR para a detecção do BCoV com base em seqüências representativas e recentes do gene do nucleocapsídeo, região conservada do genoma dos coronavírus. Três primers foram desenhados, a primeira amplificação com um fragmento esperado de 463pb e a segunda (semi-nested) com um fragmento esperado de 306pb. A sensibilidade analítica foi determinada pela diluição do BCoV cepa Kakegawa (título HA: 256) na base de 10 em água ultra-pura tratada com DEPC, em soro fetal bovino (SFB) e em uma suspensão fecal negativa para o BCoV, onde foram encontrados resultados positivos até a diluição de 10-2, 10-3 e 10-7, respectivamente. Este resultado sugere que a quantidade total de RNA na amostra influencia na precipitação dos pellets pelo método de extração utilizado. Quando se utiliza amostra fecal, a grande quantidade de RNA total funciona como carreadora do RNA do BCoV, demonstrando elevada sensibilidade analítica e ausência de possíveis substâncias inibidoras da PCR. O protocolo final da semi-nested RT-PCR foi aplicado a 25 amostras fecais de vacas adultas, previamente avaliadas por uma nested RT-PCR RdRp utilizada como teste de referência, resultando em 20 e 17 amostras positivas para o primeiro e segundo teste, respectivamente. Os resultados dos dois sistema de diagnóstico apresentaram concordância substancial (kappa: 0,694). A elevada sensibilidade e especificidade do novo método proposto e o fato de que os primers foram desenhados baseados em sequências atuais do BCoV, oferecem bases para o diagnóstico mais acurado de infecções causadas pelo BCoV, assim como para novas perspectivas em protocolos de detecção de outros Coronavírus de importância tanto em saninade animal ...
Assuntos
Animais , Bovinos , Coronavirus Bovino/genética , Infecções por Coronavirus/genética , Reação em Cadeia da Polimerase/métodos , Técnicas e Procedimentos Diagnósticos/veterinária , Fezes/virologia , Nucleocapsídeo/análise , Nucleocapsídeo/genéticaRESUMO
Bovine coronavirus (BCoV) is a member of the group 2 of the Coronavirus (Nidovirales: Coronaviridae) and the causative agent of enteritis in both calves and adult bovine, as well as respiratory disease in calves. The present study aimed to develop a semi-nested RT-PCR for the detection of BCoV based on representative up-to-date sequences of the nucleocapsid gene, a conserved region of coronavirus genome. Three primers were designed, the first round with a 463bp and the second (semi-nested) with a 306bp predicted fragment. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) in DEPC treated ultra-pure water, in fetal bovine serum (FBS) and in a BCoV-free fecal suspension, when positive results were found up to the 10-2, 10-3 and 10-7 dilutions, respectively, which suggests that the total amount of RNA in the sample influence the precipitation of pellets by the method of extraction used. When fecal samples was used, a large quantity of total RNA serves as carrier of BCoV RNA, demonstrating a high analytical sensitivity and lack of possible substances inhibiting the PCR. The final semi-nested RT-PCR protocol was applied to 25 fecal samples from adult cows, previously tested by a nested RT-PCR RdRp used as a reference test, resulting in 20 and 17 positives for the first and second tests, respectively, and a substantial agreement was found by kappa statistics (0.694). The high sensitivity and specificity of the new proposed method and the fact that primers were designed based on current BCoV sequences give basis to a more accurate diagnosis of BCoV-caused diseases, as well as to further insights on protocols for the detection of other Coronavirus representatives of both Animal and Public Health importance.(AU)
O Coronavírus bovino (BCoV) pertence ao grupo 2 do gênero Coronavirus (Nidovirales: Coronaviridae) e é agente causador de enterites tanto em bezerros como em bovinos adultos, bem como de doença respiratória em bezerros. O presente estudo teve por objetivo desenvolver uma semi-nested RT-PCR para a detecção do BCoV com base em seqüências representativas e recentes do gene do nucleocapsídeo, região conservada do genoma dos coronavírus. Três primers foram desenhados, a primeira amplificação com um fragmento esperado de 463pb e a segunda (semi-nested) com um fragmento esperado de 306pb. A sensibilidade analítica foi determinada pela diluição do BCoV cepa Kakegawa (título HA: 256) na base de 10 em água ultra-pura tratada com DEPC, em soro fetal bovino (SFB) e em uma suspensão fecal negativa para o BCoV, onde foram encontrados resultados positivos até a diluição de 10-2, 10-3 e 10-7, respectivamente. Este resultado sugere que a quantidade total de RNA na amostra influencia na precipitação dos pellets pelo método de extração utilizado. Quando se utiliza amostra fecal, a grande quantidade de RNA total funciona como carreadora do RNA do BCoV, demonstrando elevada sensibilidade analítica e ausência de possíveis substâncias inibidoras da PCR. O protocolo final da semi-nested RT-PCR foi aplicado a 25 amostras fecais de vacas adultas, previamente avaliadas por uma nested RT-PCR RdRp utilizada como teste de referência, resultando em 20 e 17 amostras positivas para o primeiro e segundo teste, respectivamente. Os resultados dos dois sistema de diagnóstico apresentaram concordância substancial (kappa: 0,694). A elevada sensibilidade e especificidade do novo método proposto e o fato de que os primers foram desenhados baseados em sequências atuais do BCoV, oferecem bases para o diagnóstico mais acurado de infecções causadas pelo BCoV, assim como para novas perspectivas em protocolos de detecção de outros Coronavírus de importância tanto em saninade animal ...(AU)