Estudo metagenômico para monitoramento de vírus em amostras de morcegos do Programa da Raiva do Rio Grande do Sul

Morcegos são reservatórios naturais de uma grande variedade de vírus de interesse a saúde pública, incluindo vírus zoonóticos que podem causar patologias graves aos humanos. Dados epidemiológicos demonstram que o morcego é a espécie animal responsável pela maioria dos casos de raiva humana no Brasil nas últimas duas décadas. A mudança no perfil epidemiológico desta doença, que anteriormente era predominantemente transmitida por cães, gerou um aumento nas amostras de morcegos enviadas aos órgãos públicos para a vigilância do vírus rábico. No presente estudo, amostras obtidas de morcegos urbanos da espécie Tadarida brasiliensis monitorados no âmbito do Programa de Controle e Profilaxia da Raiva do Estado do Rio Grande do Sul foram analisadas por abordagem de metagenômica, com objetivo de identificar outros vírus, que representassem risco à saúde pública ou não, colaborando para a melhor compreensão da diversidade viral destes animais. Foram encontradas sequências relacionadas a 04 famílias virais que infectam uma ampla variedade de hospedeiros: Anelloviridae, Circoviridae, Papillomaviridae e Poxviridae. Estes achados auxiliam no melhor conhecimento sobre os vírus carreados por morcegos desta região e demonstram que o uso da metagenômica como instrumento de vigilância amplia a gama de vírus de interesse que podem ser monitorados nestas amostras.Bats are natural reservoirs for a wide variety of viruses of public health concern, including zoonotic viruses that can cause severe diseases in humans. Epidemiological data revealed that bats are the animal species responsible for most cases of human rabies in Brazil in the last two decades. This change in the rabies epidemiological profile, which was previously predominantly transmitted by dogs, generated an increase in the number of bats samples sent to public organs for surveillance of rabies virus. In the present study, samples obtained from urban bats from the species Brazilian free-tailed bat, monitored under the Rio Grande do Sul State Rabies Control and Prophylaxis Program were analyzed by a metagenomic approach with the objective of identifying other viruses, that pose as a risk to public health or not, providing a better understanding of bats viral diversity locally and in the general way. Sequences related to 04 viral families that infect a wide variety of hosts have been found: Anelloviridae, Circoviridae, Papillomaviridae e Poxviridae. These findings improves the knowledege about the viruses carried by these animals and demonstrate that the use of metagenomics as a surveillance tool expand the range of viruses of public health concern investigated in these samples

Fecal bacterial communities of wild black capuchin monkeys (Sapajus nigritus) from the Atlantic Forest biome in Southern Brazil are divergent from those of other non-human primates

Gut microbiota are influenced by factors such as diet, habitat, and social contact, which directly affect the host's health. Studies related to gut microbiota in non-human primates are increasing worldwide. However, little remains known about the gut bacterial composition in wild Brazilian monkeys. Therefore, we studied the fecal microbiota composition of wild black capuchin monkey (Sapajus nigritus) (n=10) populations from two different Atlantic Forest biome fragments (five individuals per fragment) in south Brazil. The bacterial community was identified via the high-throughput sequencing and partial amplification of the 16S rRNA gene (V4 region) using an Ion Personal Genome Machine (PGMTM) System. In contrast to other studies involving monkey microbiota, which have generally reported the phyla Firmicutes and Bacteroidetes as predominant, black capuchin monkeys showed a high relative abundance of Proteobacteria (X= 80.54%), followed by Firmicutes (X= 12.14%), Actinobacteria (X= 4.60%), and Bacteriodetes (X= 1.31%). This observed particularity may have been influenced by anthropogenic actions related to the wild habitat and/or diet specific to the Brazilian biome's characteristics and/or monkey foraging behavior. Comparisons of species richness (Chao1) and diversity indices (Simpson and InvSimpson) showed no significant differences between the two groups of monkeys. Interestingly, PICRUSt2 analysis revealed that metabolic pathways present in the bacterial communities were associated with xenobiotic biodegradation and the biosynthesis of secondary metabolites, which may suggest positive effects on monkey health and conservation in this anthropogenic habitat. Infectious disease-associated microorganisms were also observed in the samples. The present study provides information about the bacterial population and metabolic functions present in fecal microbiota, which may contribute to a better understanding of the ecology and biology of black capuchin monkeys living in forest fragments within the Atlantic Forest biome in southern Brazil. Additionally, the present study demonstrates that the fecal bacterial communities of wild black capuchin monkeys in this area are divergent from those of other wild non-human primates

Viral diversity in oral cavity from Sapajus nigritus by metagenomic analyses

Sapajus nigritus are non-human primates which are widespread in South America. They are omnivores and live in troops of up to 40 individuals. The oral cavity is one of the main entry routes for microorganisms, including viruses. Our study proposed the identification of viral sequences from oral swabs collected in a group of capuchin monkeys (n = 5) living in a public park in a fragment of Mata Atlantica in South Brazil. Samples were submitted to nucleic acid extraction and enrichment, which was followed by the construction of libraries. After high-throughput sequencing and contig assembly, we used a pipeline to identify 11 viral families, which are Herpesviridae, Parvoviridae, Papillomaviridae, Polyomaviridae, Caulimoviridae, Iridoviridae, Astroviridae, Poxviridae, and Baculoviridae, in addition to two complete viral genomes of Anelloviridae and Genomoviridae. Some of these viruses were closely related to known viruses, while other fragments are more distantly related, with 50% of identity or less to the currently available virus sequences in databases. In addition to host-related viruses, insect and small vertebrate-related viruses were also found, as well as plant-related viruses, bringing insights about their diet. In conclusion, this viral metagenomic analysis reveals, for the first time, the profile of viruses in the oral cavity of wild, free ranging capuchin monkeys

Diversity, resistance profiles and virulence of Enterococcus spp. from fecal samples of Tadarida brasiliensis urban bats (Brazilian free-tailed bats)

O objetivo deste trabalho foi avaliar o perfil dos enterococos em amostras de fezes de morcegos urbanos Tadarida brasiliensis coletadas no Rio Grande do Sul. Fezes de morcegos foram coletadas e submetidas à identificação das espécies de enterococos e teste de suscetibilidade aos antimicrobianos rifampicina, eritromicina, norfloxacino, ciprofloxacino, tetraciclina ampicilina, cloranfenicol, estreptomicina, gentamicina, linezolida, nitrofurantoína e vancomicina. A presença dos genes de resistência (ermA, ermB, ermC, msrC, vanA, vanB, vanC1, vanC2/3, tetM, tetM e tetS) e virulência (ace, agg, cylA, esp e gelE) foi determinada por PCR. Além disso, o DNA fecal foi extraído e submetido a qPCR e PCR para detectar as espécies E. casseliflavus, E.faecalis, E. faecium, E. gallinarum, E. hirae e E. mundtii e os genes de resistência, respectivamente. Foram isolados 73 enterococos, sendo E. faecalis, E. casseliflavus, E. gallinarum e E. mundtii identificados. Fenótipos de resistência foram observados para rifampicina (n=53), eritromicina (n=32), norfloxacino (n=7), ciprofloxacino (n=6) e tetraciclina (n=1). Dos genes de resistência testados nos isolados resistentes, somente os genes ermC e tetM estavam presentes. Seis E. faecalis suscetíveis à vancomicina foram positivos para vanC1 e vanC2/3. Os genes gelE, ace, agg, cylA e esp foram detectados nos isolados. Nas amostras de DNA fecal, todas as espécies analisadas e os genes ermC, tetM, vanA, vanB e vanC2/3 foram observados. Como conclusão, diferentes espécies de enterococos estão presentes nas fezes de morcegos urbanos de T. brasiliensis. A presença de enterococos resistentes nestes animais pode estar relacionada com ação antropogênica e/ou ligada ao resistoma.We aimed to evaluate the profile of enterococci from fecal samples of urban bats Tadarida brasiliensis collected at Rio Grande do Sul state, southern Brazil. Bat feces were collected and subjected to enterococci species identification and antimicrobial susceptibility tests for rifampicin, erythromycin, norfloxacin, ciprofloxacin, tetracycline, ampicillin, chloramphenicol, streptomycin, gentamicin, linezolid, nitrofurantoin, and vancomycin. The presence of resistance (ermA, ermC, ermB, msrC, vanA, vanB, vanC1, vanC2/3, tetM, tetM, and tetS) and virulence (ace, agg, cylA, esp, and gelE) genes was determined by PCR. In addition, fecal DNA was extracted and subjected to qPCR and PCR to detect the species E. casseliflavus, E. faecalis, E. faecium, E. gallinarum, E. hirae and E. mundtii, and resistance genes, respectively. A total 73 enterococci were isolated, of which E. faecalis, E. casseliflavus, E. gallinarum, and E. mundtii were identified. Resistance phenotypes were observed for rifampicin (n= 53), erythromycin (n= 32), norfloxacin (n= 7), ciprofloxacin (n= 6) and tetracycline (n=1). Of the resistance genes tested in resistant isolates, only ermC and tetM were present. Six vancomycin-susceptible E. faecalis were positive for vanC1 and vanC2/3. Genes gelE, ace, agg, cylA and esp were detected in the isolates. In fecal DNA samples, all analyzed species and the genes ermC, tetM, vanA, vanB and vanC2/3 were observed. We conclude that different species of enterococci are present in feces of T. brasiliensis urban bats. The presence of antibiotic-resistant enterococci in those animals may be related to anthropogenic action and/or linked to the resistome

Diversidade, perfis de resistência e virulência de Enterococcus spp. em fezes de morcegos urbanos Tadarida brasiliensis (Brazilian free-tailed bats)

 We aimed to evaluate the profile of enterococci from fecal samples of urban bats Tadarida brasiliensis collected at Rio Grande do Sul state, southern Brazil. Bat feces were collected and subjected to enterococci species identification and antimicrobial susceptibility tests for rifampicin, erythromycin, norfloxacin, ciprofloxacin, tetracycline, ampicillin, chloramphenicol, streptomycin, gentamicin, linezolid, nitrofurantoin, and vancomycin. The presence of resistance (ermA, ermC, ermB, msrC, vanA, vanB, vanC1, vanC2/3, tetM, tetM, and tetS) and virulence (ace, agg, cylA, esp, and gelE) genes was determined by PCR. In addition, fecal DNA was extracted and subjected to qPCR and PCR to detect the species E. casseliflavus, E. faecalis, E. faecium, E. gallinarum, E. hirae and E. mundtii, and resistance genes, respectively. A total 73 enterococci were isolated, of which E. faecalis, E. casseliflavus, E. gallinarum, and E. mundtii were identified. Resistance phenotypes were observed for rifampicin (n= 53), erythromycin (n= 32), norfloxacin (n= 7), ciprofloxacin (n= 6) and tetracycline (n=1). Of the resistance genes tested in resistant isolates, only ermC and tetM were present. Six vancomycin-susceptible E. faecalis were positive for vanC1 and vanC2/3. Genes gelE, ace, agg, cylA and esp were detected in the isolates. In fecal DNA samples, all analyzed species and the genes ermC, tetM, vanA, vanB and vanC2/3 were observed. We conclude that different species of enterococci are present in feces of T. brasiliensis urban bats. The presence of antibiotic-resistant enterococci in those animals may be related to anthropogenic action and/or linked to the resistome. O objetivo deste trabalho foi avaliar o perfil dos enterococos em amostras de fezes de morcegos urbanos Tadarida brasiliensis coletadas no Rio Grande do Sul. Fezes de morcegos foram coletadas e submetidas à identificação das espécies de enterococos e teste de suscetibilidade aos antimicrobianos rifampicina, eritromicina, norfloxacino, ciprofloxacino, tetraciclina ampicilina, cloranfenicol, estreptomicina, gentamicina, linezolida, nitrofurantoína e vancomicina. A presença dos genes de resistência (ermA, ermB, ermC, msrC, vanA, vanB, vanC1, vanC2/3, tetM, tetM e tetS) e virulência (ace, agg, cylA, esp e gelE) foi determinada por PCR. Além disso, o DNA fecal foi extraído e submetido a qPCR e PCR para detectar as espécies E. casseliflavus, E.faecalis, E. faecium, E. gallinarum, E. hirae e E. mundtii e os genes de resistência, respectivamente. Foram isolados 73 enterococos, sendo E. faecalis, E. casseliflavus, E. gallinarum e E. mundtii identificados. Fenótipos de resistência foram observados para rifampicina (n=53), eritromicina (n=32), norfloxacino (n=7), ciprofloxacino (n=6) e tetraciclina (n=1). Dos genes de resistência testados nos isolados resistentes, somente os genes ermC e tetM estavam presentes. Seis E. faecalis suscetíveis à vancomicina foram positivos para vanC1 e vanC2/3. Os genes gelE, ace, agg, cylA e esp foram detectados nos isolados. Nas amostras de DNA fecal, todas as espécies analisadas e os genes ermC, tetM, vanA, vanB e vanC2/3 foram observados. Como conclusão, diferentes espécies de enterococos estão presentes nas fezes de morcegos urbanos de T. brasiliensis. A presença de enterococos resistentes nestes animais pode estar relacionada com ação antropogênica e/ou ligada ao resistoma

Epidemiological investigation reveals local transmission of SARS-CoV-2 lineage P.1 in Southern Brazil

Since its detection in December of 2020, the SARS-CoV2 lineage P.1, descendent of B.1.1.28 lineage, has been identified in several places in Brazil and abroad. This Variant of Concern was considered highly prevalent in Northern Brazil and now is rapidly widening its geographical range. Here, we present epidemiological and genomic information of the first case of P1 lineage in Rio Grande do Sul state, in a patient with no reported travel history and a tracked transmission chain. These findings occurred in a tourist destination representing an important hub receiving tourists from diverse places

Abordagens na prevenção e vigilância da raiva : inquérito sorológico em animais silvestres e sequenciamento genômico

As ações de vigilância e controle da raiva no Brasil, e no Rio Grande do Sul, permanecem as mesmas há décadas. As diretrizes programáticas que orientam as ações dos órgãos públicos, e que são ensinadas e repassadas aos profissionais de saúde, foram instituídas quando o predomínio dos casos de raiva humana era relacionado à raiva canina. Este modelo foi exitoso e levou ao declínio acentuado dos casos de raiva humana no Brasil e nas Américas. Porém, desde o início dos anos 2000, predomina no Brasil a raiva humana relacionada a animais silvestres, principalmente a morcegos, o que demanda reavaliar as abordagens de vigilância e a propor ações que contemplem o ciclo silvestre da raiva. Paralelo à mudança do perfil epidemiológico, novas metodologias passaram a ser utilizadas na vigilância, caso da vigilância genômica, e outras se tornaram rotina, caso das técnicas moleculares no diagnóstico. No presente estudo novas abordagens de vigilância são discutidas, ao mesmo tempo que se avalia a qualidade da profilaxia antirrábica prestada no Rio Grande do Sul e o conhecimento dos profissionais sobre as condutas preconizadas. O uso da abordagem de pesquisa de anticorpos antirrábicos em espécies de animais silvestres de maior prevalência nos atendimentos antirrábicos, permitiu identificar que primatas e canídeos silvestres são expostos ao vírus rábico no RS, apesar de não haver casos diagnosticados nestas espécies no estado. A análise da profilaxia antirrábica do estado demonstrou que houveram avanços nas condutas prescritas pelos profissionais de saúde, mas ainda são necessárias ações para que se aperfeiçoe o entendimento do novo padrão epidemiológico da raiva, com especial atenção ao ciclo silvestre, e para que se faça o uso racional de imunobiológicos. Adicionalmente, informações relevantes ao entendimento da dinâmica do vírus da raiva estão sendo obtidas através da análise molecular de genomas de amostras positivas do estado, gerando conhecimento que auxiliará na compreensão sobre a disseminação e a manutenção do RABV no estado, e no direcionamento dos esforços de controle.Rabies surveillance and control actions in Brazil, and in Rio Grande do Sul, have remained the same for decades. The Rabies Control program guidelines that drive the actions of public agencies, and that are taught to and used by health professionals, were created when most cases of human rabies were related to canine rabies. This model was successful and led to a sharp decline in human rabies cases in Brazil and the Americas. However, since the beginning of the 2000s, human rabies related to wild animals, especially bats, has predominated in Brazil, which requires reassessing surveillance approaches and proposing actions that address the sylvatic cycle of rabies. Consecutively to the change in the epidemiological profile, new methodologies were incorporated in surveillance, such as genomic surveillance, and others became routine, such as molecular diagnostic techniques. In the present study, new surveillance approaches are discussed, and the quality of rabies prophylaxis provided in Rio Grande do Sul is assessed, as well as the professionals' knowledge about the recommended protocols for different potential rabies exposure. The use of the anti-rabies antibody serological surveillance approach in wild animal species with the highest prevalence in anti-rabies care allowed the understanding that wild primates and canids are exposed to the rabies virus in RS, although there are no cases diagnosed in these species in the State. The analysis of rabies prophylaxis in the State demonstrated that there were advances in the prescription recommended by health professionals, but it is still important to improve the understanding of the new epidemiological profile of rabies, with special attention to the sylvatic cycle, and for the rational use of immunobiological. Additionally, relevant information to fill some gaps about the dynamics of the rabies virus is being obtained through molecular analysis of genomes obtained from positive samples in our State, generating knowledge that will help in understanding the dissemination and maintenance of the RABV in the state, and in directing efforts to control