Isolamento e caracterização molecular do vírus da cinomose canina, análise de antivirais e produção de uma proteína M recombinante

Tese (doutorado)—Universidade de Brasília, Programa em Rede Multi-Institucional do Pró-Centro-Oeste de Pós-Graduação em Biotecnologia e Biodiversidade, 2021.O canine distemper virus (CDV), gênero Morbillivirus, tem emergido e sido reconhecido como um patógeno altamente contagioso e letal de cães domésticos (Canis familiaris). CDV também infecta e ameaça uma grande variedade de animais selvagens, incluindo, por exemplo, as famílias Canidea (cães selvagens, raposa, lobo, cão-guaxinim), Mustelidae (furão, gambá, visom, lontra) e Procyonidae (guaxinim, quati). Estudos sobre o CDV são necessários por conta das diversas lacunas envolvendo o assunto, entre as quais estudos de prevalência e caracterização molecular, terapias antivirais e produção de insumos laboratoriais para o diagnóstico. Dessa forma, o presente estudo foi realizado e está constituído em seis capítulos. No primeiro capítulo foi realizada a revisão bibliográfica envolvendo a temática em questão. O segundo capítulo se refere à combinação dos dados obtidos do inquérito molecular, incluindo também os dados da literatura sobre dectecção molecular e sorológica do vírus nas diversas regiões do mundo. Embora este capítulo tenha sido publicado na forma de meta-análise, destaca-se a inserção dos nossos dados experimentais no estudo, permitindo a geração de dados mais robustos quanto à frequência de positividade viral, juntamente com a análise de diversas variáveis potencialmente envolvidas com o fator infecção viral. Ainda em relação ao segundo capítulo, frisa-se que o mesmo envolveu a coleta de amostras biológicas de cães com suspeita clínica de cinomose, procedentes do município de Jataí-Goiás. Em suma, houve 34% (48/141) de positividade para o RNA viral nas amostras. Essas amostras também serviram de base para a realização dos capítulos três e quatro. O terceiro capítulo envolveu o isolamento e sequenciamento completo do CDV. Para tanto, pela primeira vez foram desenvolvidos dezenas de primers que permitiram flanquear todo o material genético. Assim, houve sucesso na amplificação e sequenciamento completo de uma amostra (JA88/20, 15,624 nt, GenBank: MW460905). Interessantemente, a linhagem isolada se separou num ramo e formou um subgenótipo dentro do genótipo América do Sul-I/Europa. No quarto capítulo, as amostras laboratorialmente positivas para o CDV, foram testadas quanto à amplificação dos genes estruturais M (1008 nt), F (1989 nt) e H (1824 nt). Mediante a utilização de novos primers, foi obtido sucesso na amplificação de quatro amostras (4/48). A partir da caracterização molecular destes genes estruturais, foi observada a circulação na região de uma única linhagem durante o período de estudo. Surpreendentemente, os sequenciados revelaram assinaturas moleculares únicas dos isolados no local. O quinto capítulo está relacionado à dinâmica molecular das proteínas M e N, juntamente com o fornecimento de conhecimentos a respeito da geração de drogas antivirais. Os resultados obtidos de dinâmica molecular mostraram que os modelos gerados são de alta qualidade. A partir destes modelos, foi verificado que os resíduos aminoacídicos considerados chave, das proteínas M e N, estão em local acessível, representando assim excelente sítio para a ancoragem de moléculas com potenciais antivirais. Finalmente, no sexto capítulo a proteína M foi expressa em Escherichia coli (BL21) e purificada. Também houve sucesso no emprego da proteína recombinante como substrato no ELISA. Os dados, mostraram que houve acurácia laboratorial na distinção entre amostras IgG anti-CDV positivas e negativas. Em síntese, a proteína recombinante produzida confirma nossa hipótese referente ao seu emprego em ensaios de imunodetecção.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Fundação de Apoio à Pesquisa do Distrito Federal (FAP-DF) e Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).Canine distemper virus (CDV), genus Morbillivirus, has emerged and has been recognized as a highly contagious and lethal pathogen in domestic dogs (Canis familiaris). CDV also infects and threatens a wide variety of wild animals, including, for example, the Canidea (wild dogs, fox, wolf, raccoon dog), Mustelidae (ferret, opossum, mink, otter) and Procyonidae families (raccoon, coati). The studies on the CDV are necessary due to the several gaps involving the subject, including prevalence and molecular characterization studies, antiviral therapies and production of laboratory supplies for the diagnosis. Thus, the present study was carried out and consists of six chapters. In the first chapter, a bibliographic review involving the subject in question was carried out. The second chapter refers to the combination of data obtained from the molecular survey, also including data from the literature on molecular and serological detection of the virus in different regions of the world. Although this chapter was published in the form of a meta-analysis, the inclusion of our experimental data in the study stands out, allowing the generation of more robust data regarding the frequency of viral positivity, together with the analysis of several variables potentially involved with the viral infection factor. Still in relation to the second chapter, it is emphasized that it involved the collection of biological samples from dogs with clinical suspicion of distemper, coming from the municipality of Jataí-Goiás. In sum, CDV RNA was detected in 34% (48/141) of dogs suspected to have distemper. These collected samples served as the basis for the realization of chapters three and four. The third chapter involves the complete viral isolation and DNA sequencing. For this purpose, for the first time dozens of primers were developed that allowed the flanking of all genetic material. Thus, there was success in the amplification and complete sequencing of a sample (JA88/2020, 15,624 nt, GenBank: MW460905). Interestingly, the isolated strain separated into a branch and formed a subgenotype within the South America-I/Europe genotype. In the fourth chapter, the laboratory samples positive for CDV, were tested for the amplification of M (1008 nt), F (1989 nt) and H (1824 nt) structural protein genes. Through the use of novel primers, it was successful in amplifying four samples (4/48). From the molecular characterization of structural genes, circulation in the region of a single lineage was observed during the study period. Surprisingly, the sequences reveal unique molecular signatures of the isolates at the site. The fifth chapter is related to the molecular dynamics of M and N proteins, together with the generation of knowledge regarding the rational generation of drugs against CDV. The results obtained from molecular dynamics showed that the models generated are of high quality. From these models, it was verified that the amino acid residues considered key for M and N proteins are in an accessible location, thus representing an excellent site for the anchoring of molecules with potential antivirals. Finally, in the sixth chapter, M protein was expressed in Escherichia coli (BL21), purified. There was also success in the use of recombinant protein as a substrate in the ELISA. The data showed that there was laboratory accuracy in distinguishing between positive and negative anti-CDV IgG samples. In summary, the recombinant protein produced confirms our hypothesis regarding its use in immunodetection assays

Molecular and serological surveys of canine distemper virus : a meta-analysis of crosssectional studies

Background Canine morbillivirus (canine distemper virus, CDV) persists as a serious threat to the health of domestic dogs and wildlife. Although studies have been conducted on the frequency and risk factors associated with CDV infection, there are no comprehensive data on the current epidemiological magnitude in the domestic dog population at regional and national levels. Therefore, we conducted a cross-sectional study and included our results in a meta-analysis to summarize and combine available data on the frequency and potential risk factors associated with CDV infection. Methods For the cross-sectional study, biological samples from dogs suspected to have canine distemper (CD) were collected and screened for viral RNA. Briefly, the PRISMA protocol was used for the meta-analysis, and data analyses were performed using STATA IC 13.1 software. Results CDV RNA was detected in 34% (48/141) of dogs suspected to have CD. Following our meta-analysis, 53 studies were selected for a total of 11,527 dogs. Overall, the pooled frequency of CDV positivity based on molecular and serological results were 33% (95% CI: 23–43) and 46% (95% CI: 36–57), respectively. The pooled subgroup analyses of clinical signs, types of biological samples, diagnostic methods and dog lifestyle had a wide range of CDV positivity (range 8–75%). Free-ranging dogs (OR: 1.44, 95% CI: 1.05–1.97), dogs >24 months old (OR: 1.83, 95% CI: 1.1–3) and unvaccinated dogs (OR: 2.92, 95% CI: 1.26– 6.77) were found to be positively associated with CDV infection. In contrast, dogs <12 months old (OR: 0.36, 95% CI: 0.20–0.64) and dogs with a complete anti-CDV vaccination (OR: 0.18, 95% CI: 0.05–0.59) had a negative association. Conclusion Considering the high frequency of CDV positivity associated with almost all the variables analyzed in dogs, it is necessary to immediately and continuously plan mitigation strategies to reduce the CDV prevalence, especially in determined endemic localities

The Enteric Glial Network Acts in the Maintenance of Intestinal Homeostasis and in Intestinal Disorders

The enteric nervous system (ENS), also known as second brain, innervates our gastrointestinal tract controlling its functions, such as motility, fluid secretion, nutrient absorption, and even involvement in the control of immunity and inflammatory processes. In the gut, the gliocytes are known as enteric glial cells (EGCs). Enteric glial cells form a network that permeates the entire gut. Enteric glia express the cell surface hemichannel of connexin-43 (Cx43) necessary for the propagation of Ca2 + responses, necessary to maintain their functions. In this chapter, besides the development of ENS and its glial cells and the similarities with the astrocytes in the central nervous system, we approached the important role of the glial network in the control of gut homeostasis, in the interaction with the immune system, and its participation in pathological conditions. EGCs are even capable of replacing lost neurons. Thus the enteric glia is a multifunctional cell, which through its multiple interactions maintains the integrity of the ENS allowing it to be resistant to the different and constant aggressions suffered by the digestive system

Early and Late Pathogenic Events of Newborn Mice Encephalitis Experimentally Induced by Itacaiunas and Curionópolis Bracorhabdoviruses Infection

In previous reports we proposed a new genus for Rhabdoviridae and described neurotropic preference and gross neuropathology in newborn albino Swiss mice after Curionopolis and Itacaiunas infections. In the present report a time-course study of experimental encephalitis induced by Itacaiunas and Curionopolis virus was conducted both in vivo and in vitro to investigate cellular targets and the sequence of neuroinvasion. We also investigate, after intranasal inoculation, clinical signs, histopathology and apoptosis in correlation with viral immunolabeling at different time points. Curionopolis and Itacaiunas viral antigens were first detected in the parenchyma of olfactory pathways at 2 and 3 days post-inoculation (dpi) and the first clinical signs were observed at 4 and 8 dpi, respectively. After Curionopolis infection, the mortality rate was 100% between 5 and 6 dpi, and 35% between 8 and 15 dpi after Itacaiunas infection. We identified CNS mice cell types both in vivo and in vitro and the temporal sequence of neuroanatomical olfactory areas infected by Itacaiunas and Curionopolis virus. Distinct virulences were reflected in the neuropathological changes including TUNEL immunolabeling and cytopathic effects, more intense and precocious after intracerebral or in vitro inoculations of Curionopolis than after Itacaiunas virus. In vitro studies revealed neuronal but not astrocyte or microglial cytopathic effects at 2 dpi, with monolayer destruction occurring at 5 and 7 dpi with Curionopolis and Itacaiunas virus, respectively. Ultrastructural changes included virus budding associated with interstitial and perivascular edema, endothelial hypertrophy, a reduced and/or collapsed small vessel luminal area, thickening of the capillary basement membrane, and presence of phagocytosed apoptotic bodies. Glial cells with viral budding similar to oligodendrocytes were infected with Itacaiunas virus but not with Curionopolis virus. Thus, Curionopolis and Itacaiunas viruses share many pathological and clinical features present in other rhabdoviruses but distinct virulence and glial targets in newborn albino Swiss mice brain

Seropositivity diagnosis for hantavirus in Jataí, Goiás State, Brazil

Abstract INTRODUCTION: Emerging diseases are of great interest, especially those associated with high mortality rates such as hantaviruses. We aimed to conduct a seroepidemiological survey to determine the levels of hantavirus infection. METHODS: In-house enzyme-linked immunosorbent assay (ELISA) was used to detect specific antibodies. RESULTS: Of the 429 samples collected, seropositivity of 3.9% to anti-hantavirus immunoglobulin G (IgG) was observed (CI 95%: 2.3-5.7). Moreover, in three cases, immunoglobulin M (IgM) was detected, of which two were diagnosed as hantavirus cardiopulmonary syndrome (HCPS). CONCLUSIONS: Our data indicate the considerable occurrence of previous hantavirus infections, highlighting occurrences from sub-clinical cases to HCPS

Estudo transversal da letalidade da hantavirose no estado de Goiás, 2007-2013

Resumo OBJETIVO: descrever o perfil epidemiológico e clínico da hantavirose e analisar fatores associados à letalidade em Goiás, Brasil. MÉTODOS: foi conduzido um estudo transversal com dados do Sistema de Informação de Agravos de Notificação (Sinan), referentes ao período de 2007 a 2013, no estado de Goiás. Regressão logística foi empregada para estimar razões de chances (OR) com intervalos de confiança de 95% (IC95%). RESULTADOS: foram notificados 1.171 casos suspeitos de hantavirose e 73 (6,2%) confirmados. Entre os confirmados, observou-se maior frequência entre homens (n=50), febre como sintoma mais frequente (n=70) e elevada proporção de hospitalizações (n=68). A taxa de letalidade foi de 57,5%. Foram fatores associados ao óbito: insuficiência respiratória aguda (IRpA) (OR=3,6; IC95%1,2;10,6), hemoconcentração (OR=3,3; IC95%1,1;7,9) e não uso do respirador mecânico (OR=3,4; IC95%1,3;9,1). Após ajuste, a IRpA permaneceu associada ao óbito (OR=3,4; IC95%1,0;11,6). CONCLUSÃO: foi identificada alta taxa de letalidade, associada principalmente com insuficiência respiratória