Evolução do diagnóstico virológico de raiva humana no Instituto Pasteur de São Paulo, Brasil (1970-2020): análise de dados / Evolution of the virological diagnosis of human rabies at the Pasteur Institute of São Paulo, Brazil (1970-2020): data analysis

O diagnóstico laboratorial precoce de raiva humana deve ser realizado por testes apropriados, visto que a aplicação de protocolos de tratamento médico em indivíduos internados depende dos resultados laboratoriais. O presente estudo analisou os dados referentes aos 560 casos suspeitos de raiva humana submetidos ao diagnóstico virológico no IP-SP entre os anos de 1970 e 2020. Houve um avanço das metodologias laboratoriais, especialmente as moleculares, que passaram a ser essenciais, possibilitando o tratamento de indivíduos expostos, bem como a determinação da fonte de infecção dos casos, fato fundamental para a efetividade de ações de controle em regiões vulneráveis à disseminação da doença. Intervenções no ciclo urbano da raiva, por meio de vacinação de cães e gatos e encaminhamento de amostras para diagnóstico, diminuiram os casos transmitidos por cães, principalmente no Sudeste. Em contrapartida, no mesmo período foi observado um aumento exponencial de casos relacionados ao ciclo silvestre nas regiões Norte (32%) e Nordeste (53,3%), tendo os morcegos como principais transmissores (72%), seguidos dos primatas não humanos (6%) e dos canídeos silvestres (1%). Esses resultados demonstraram a importância do aprimoramento do diagnóstico laboratorial, que é parte essencial na condução de estratégias de controle, bem como de tratamento de indivíduos expostos.

Early laboratory diagnosis of human rabies should be performed by appropriate tests, since the application of medical treatment protocols in hospitalized individuals depends on laboratory results. The present study analyzed the data referring to the 560 suspected cases of human rabies submitted to virological diagnosis in the IP-SP from 1970 to 2020. There has been an advance in laboratory methodologies, especially molecular ones, which have become essential, enabling the treatment of exposed individuals, as well as allowing the determination of the source of infection of cases, a fundamental fact for the effectiveness of control actions in regions vulnerable to the spread of the disease. Interventions in the urban cycle of rabies, through vaccination of dogs and cats and referral of samples for diagnosis decreased the cases transmitted by dogs, especially in the Southeast, on the other hand, an exponential increase of cases was observed in the same period, in the North (32%) and Northeast (53.3%) regions, with cases related to the wild cycle, with bats as the main transmitters (72%), followed by non-human primates (6%), and wild canids (1%). Our results demonstrated the importance of improving laboratory diagnosis, which is an essential part of conducting control strategies as well as the treatment of exposed individuals.

In situ histopathological and immunohistochemical characterization of rabies in the brains of naturally infected equines and bovines / Caracterização histopatológica e imuno-histoquímica In situ do vírus da raiva em cérebros de equinos e bovinos naturalmente infectados

The present study sought to characterize the phenomena involved in the histopathology of rabies and to assess the presence and amount of viral antigen in situ in different brain regions of naturally infected equines and bovines. The histopathological examination showed several changes due to inflammation, being most often infected cells neurons. The neuronal degeneration involved 100% of cases, in addition to a diffuse lymphocytic Infiltration and gliosis, characterized by vasculitis and perivasculitis. The presence of Negri bodies was in most cases in discreet, and the fragments with higher concentrations of antigen by both techniques employed were the cerebellum and the brain stem. Immunohistochemistry test (IHC) demonstrated greater sensitivity when applied to samples of bovines. Our results showed that in 37.5% of the total number of fragments analyzed, viral inclusions were not observed, however, was the presence of inflammatory process. In relation to the species, the fragments from bovine's animals showed a slight increase when examined under this feature. These findings highlight the importance of submitting samples from suspected animals for laboratory diagnostic, even when there are no apparent abnormal histological findings. (AU)

O presente estudo buscou caracterizar os fenômenos envolvidos na histopatologia da raiva e avaliar a presença e quantidade de antígeno viral in situ nas diferentes regiões cerebrais de equinos e bovinos naturalmente infectados. O exame histopatológico demonstrou várias mudanças devido à inflamação, sendo mais frequentemente infectadas as células neuronais. A degeneração neuronal foi observada em 100% dos casos, além de uma infiltração linfocitária difusa e gliose, caracterizada por vasculite e perivasculite. A presença de corpúsculos de Negri foi observada na maioria dos casos de maneira discreta, e os fragmentos com maior concentração de antígeno, por ambos os testes empregadas foram o cerebelo e o tronco encefálico. O teste de Imuno-histoquímica (IHC) demonstrou maior sensibilidade quando aplicada em amostras de bovinos. Nossos resultados demostraram que em 37,5% do número total de fragmentos analisados, inclusões virais não foram observadas, no entanto, havia processo inflamatório. Em relação à espécie, os fragmentos de bovinos demonstraram um ligeiro aumento quando examinado sob este aspecto. Esses achados destacam a importância de submeter amostras de animais suspeitos para diagnóstico laboratorial, mesmo quando não houver nenhum achado histopatológico anormal.Palavras-chave: raiva, equinos, bovinos, imuno-histoquímica, IFD, alterações histopatológicas. (AU)

Non-human primates as a reservoir for rabies virus in Brazil.

Rabies virus (RABV) does not persist in the environment as it is a very fragile agent. The primary hosts are mammalian species in the orders Carnivora and Chiroptera. Since the late 1980s, RABV has been isolated from non-human primates, Callithrix jacchus (the white-tufted marmoset), in four coastal states (Rio Grande do Norte, Ceará, Piauí and Pernambuco) in north-eastern Brazil, where this species is indigenous. The original habitat of C. jacchus consisted of two Brazilian biomes, the Atlantic Forest and the Caatinga. However, these marmosets have since adapted to other ecosystems as a result of human activities. Between 1988 and 1989, RABV isolates were obtained from white-tufted marmosets in the state of Rio Grande do Norte, but antigenic and genetic identification studies were not conducted at that time. In the following years, three additional states reported cases (Ceará, Piauí and Pernambuco). In two of these states (Ceará and Piauí), human cases of rabies transmitted by marmosets were reported. According to Brazilian Health Ministry data, at least 19 human cases in which this species was the source of infection were registered in between 1990 and 2016. Recent findings in laboratory tests of 12 rabid samples from humans and marmosets and the regional transmission among these animals for over 20 years, together with the gradual increase in the affected geographic area, support the concept of the emergence of a new RABV reservoir. Regional tourism, the wild animal trade and the cultural practice of maintaining these animals as pets, particularly in coastal regions, appear to be major risk factors for the increase in human cases. Additional epidemiological and ecological studies are required to better understand local disease dynamics and to identify ideal opportunities for prevention and control of this fatal infection.

Non-human primates as a reservoir for rabies virus in Brazil

Rabies virus (RABV) does not persist in the environment as it is a very fragile agent. The primary hosts are mammalian species in the orders Carnivora and Chiroptera. Since the late 1980s, RABV has been isolated from non-human primates, Callithrix jacchus (the white-tufted marmoset), in four coastal states (Rio Grande do Norte, Ceará, Piauí and Pernambuco) in north-eastern Brazil, where this species is indigenous. The original habitat of C. jacchus consisted of two Brazilian biomes, the Atlantic Forest and the Caatinga. However, these marmosets have since adapted to other ecosystems as a result of human activities. Between 1988 and 1989, RABV isolates were obtained from white-tufted marmosets in the state of Rio Grande do Norte, but antigenic and genetic identification studies were not conducted at that time. In the following years, three additional states reported cases (Ceará, Piauí and Pernambuco). In two of these states (Ceará and Piauí), human cases of rabies transmitted by marmosets were reported. According to Brazilian Health Ministry data, at least 19 human cases in which this species was the source of infection were registered in between 1990 and 2016. Recent findings in laboratory tests of 12 rabid samples from humans and marmosets and the regional transmission among these animals for over 20 years, together with the gradual increase in the affected geographic area, support the concept of the emergence of a new RABV reservoir. Regional tourism, the wild animal trade and the cultural practice of maintaining these animals as pets, particularly in coastal regions, appear to be major risk factors for the increase in human cases. Additional epidemiological and ecological studies are required to better understand local disease dynamics and to identify ideal opportunities for prevention and control of this fatal infection.

Non-human primates as a reservoir for rabies virus in Brazil

Rabies virus (RABV) does not persist in the environment as it is a very fragile agent. The primary hosts are mammalian species in the orders Carnivora and Chiroptera. Since the late 1980s, RABV has been isolated from non-human primates, Callithrix jacchus (the white-tufted marmoset), in four coastal states (Rio Grande do Norte, Ceará, Piauí and Pernambuco) in north-eastern Brazil, where this species is indigenous. The original habitat of C. jacchus consisted of two Brazilian biomes, the Atlantic Forest and the Caatinga. However, these marmosets have since adapted to other ecosystems as a result of human activities. Between 1988 and 1989, RABV isolates were obtained from white-tufted marmosets in the state of Rio Grande do Norte, but antigenic and genetic identification studies were not conducted at that time. In the following years, three additional states reported cases (Ceará, Piauí and Pernambuco). In two of these states (Ceará and Piauí), human cases of rabies transmitted by marmosets were reported. According to Brazilian Health Ministry data, at least 19 human cases in which this species was the source of infection were registered in between 1990 and 2016. Recent findings in laboratory tests of 12 rabid samples from humans and marmosets and the regional transmission among these animals for over 20 years, together with the gradual increase in the affected geographic area, support the concept of the emergence of a new RABV reservoir. Regional tourism, the wild animal trade and the cultural practice of maintaining these animals as pets, particularly in coastal regions, appear to be major risk factors for the increase in human cases. Additional epidemiological and ecological studies are required to better understand local disease dynamics and to identify ideal opportunities for prevention and control of this fatal infection.

Analysis of rabies diagnosis in dogs and cats in the state of São Paulo, Brazil.

The genetic lineage of rabies virus (RABV) associated with dogs has not been found in the state of São Paulo since 1998, and all cases of rabies in domestic animals reported since then have involved the RABV lineage that circulates in bats. As there has been a change in the rabies transmission cycle in cats and dogs, we decided to analyze the tests used to diagnose rabies in these animals in the 15-year period from 2002 to 2016 in the state of São Paulo. During this period, 85,508 central nervous system (CNS) samples from dogs and cats were submitted to the Rabies Diagnosis Section at the Pasteur Institute of São Paulo for testing. All of the samples were tested by the fluorescent antibody test (FAT) and at least one of the following three tests: mouse inoculation test (MIT), rabies tissue culture infection test (RTCIT) and reverse transcription polymerase chain reaction (RT-PCR). Of all the samples tested, twenty were positive in at least one of these assays. Four other positive samples were identified at other institutions in the state of São Paulo. Of the twenty samples that tested positive at the Pasteur Institute of São Paulo, nine were tested by FAT, and the results were subsequently confirmed by other techniques; five gave inconclusive results, and therefore, other techniques had to be used as soon as possible in case the samples were positive; and six were negative by FAT and positive by one or more of the following tests: RTCIT, MIT and RT-PCR. Genetic typing of isolates from eighteen samples identified them as the lineage circulating in bats. In light of this finding, which indicates that genetic lineages associated with bats are circulating in domestic animals in the state of São Paulo, when the results of FAT carried out with samples from aggressive cats and dogs are inconclusive, complementary tests should be used. Decomposing samples and samples for which FAT was inconclusive should be tested using molecular techniques so that a definitive result can be obtained quickly and timely post-exposure prophylaxis can be administered to exposed individuals.

Non-human primates as a reservoir for rabies virus in Brazil

Rabies virus (RABV) does not persist in the environment as it is a very fragile agent. The primary hosts are mammalian species in the orders Carnivora and Chiroptera. Since the late 1980s, RABV has been isolated from non­human primates, Callithrix jacchus (the white­tufted marmoset), in four coastal states (Rio Grande do Norte, Ceará, Piauí and Pernambuco) in north­eastern Brazil, where this species is indigenous. The original habitat of C. jacchus consisted of two Brazilian biomes, the Atlantic Forest and the Caatinga. However, these marmosets have since adapted to other ecosystems as a result of human activities. Between 1988 and 1989, RABV isolates were obtained from white­tufted marmosets in the state of Rio Grande do Norte, but antigenic and genetic identification studies were not conducted at that time. In the following years, three additional states reported cases (Ceará, Piauí and Pernambuco). In two of these states (Ceará and Piauí), human cases of rabies transmitted by marmosets were reported. According to Brazilian Health Ministry data, at least 19 human cases in which this species was the source of infection were registered in between 1990 and 2016. Recent findings in laboratory tests of 12 rabid samples from humans and marmosets and the regional transmission among these animals for over 20 years, together with the gradual increase in the affected geographic area, support the concept of the emergence of a new RABV reservoir. Regional tourism, the wild animal trade and the cultural practice of maintaining these animals as pets, particularly in coastal regions, appear to be major risk factors for the increase in human cases. Additional epidemiological and ecological studies are required to better understand local disease dynamics and to identify ideal opportunities for prevention and control of this fatal infection. (AU)

The epidemiological importance of bats in the transmission of rabies to dogs and cats in the State of São Paulo, Brazil, between 2005 and 2014

In Brazil, rabies control in dogs and cats was pioneered by the state of São Paulo with the adoption of the Pan American Health Organization recommendations for prophylaxis and control, which led to a reduction in rabies cases from 1994 onwards. As a result of these measures, the rabies virus (RABV) genetic lineage associated with dogs has not been found in the state since 1998, and all the cases in domestic animals reported since then have been caused by bat­associated lineages of RABV. In the light of this, this study sought to investigate rabies cases in dogs and cats in the state of São Paulo between 2005 and 2014 and identify the associated transmission cycles by characterizing the RABV lineages responsible for these cases. Nine samples from dogs (n = 5) and from cats (n = 4) were collected between 2005 and 2014. The tenth animal, a rabid cat, was analysed by a different laboratory. The N gene nucleotide sequences obtained were analysed with the neighbor­joining algorithm and Kimura 2­parameter model using the MEGA 6 program. Phylogenetic analysis revealed that the genetic lineages identified in all the samples were those circulating in Brazilian bats. The findings of this study demonstrate that bats play an important role in the transmission of rabies to domestic animals in São Paulo state and that emphasis should be placed on the implementation of public policies to support surveillance of chiropterans for rabies.(AU) i

Phylogenetic analysis of rabies virus isolated from canids in North and Northeast Brazil.

Cases of canine rabies continue to occur in North and Northeast Brazil, and the number of notifications of rabies cases in wild canids has increased as a result of the expansion of urban areas at the expense of areas with native vegetation. In light of this, we performed molecular characterization of rabies virus isolates from dogs and Cerdocyon thous from various states in North and Northeast Brazil. In all, 102 samples from dogs (n = 56) and Cerdocyon thous (n = 46) collected between 2006 and 2012 were used. The nucleotide sequences obtained for the N gene of rabies virus were analyzed, and phylogenetic analysis revealed the presence of two distinct genetic lineages, one associated with canids and one with bats, and, within the canid cluster, two distinct sublineages circulating among dogs and Cerdocyon thous. In addition, phylogenetic groups associated with geographic region and fourteen cases of interspecific infection were observed among the isolates from canids. Our findings show that analysis of rabies virus lineages isolated from reservoirs such as canids must be constantly evaluated because the mutation rate is high.

Genetic identification of species of bats that act as reservoirs or hosts for viral diseases

Introduction: Viruses have been identified as the main etiologic agents of both zoonoses and emerging infectious diseases (EIDs) and various species of wild fauna can be involved in the maintenance of these diseases. The very wide variety of bats, together with their ability to adapt to different environments and fly long distances, means that these animals are currently one of the main reservoirs for zoonoses and EIDs. For these reasons the correct identification of different bat species is essential.Aims: This paper describes the genetic identification of 56 samples isolated from different bat species.Methodology: Sequencing and phylogenetic analysis of the mitochondrial DNA cytochrome b (mtDNA cyt-b) gene. Results: Four families (Molossidae, Vespertilionidae, Noctilionidae and Phyllostomidae), twelve genera and nineteen different species of bats were identified, and the Basic Local Alignment Search Tool (BLAST) was used to confirm species identity. The phylogenetic tree constructed revealed two main clusters (1 and 2), both consist in two subclusters.Conclusions: Our results were concordant with those obtained by morphometric identification and genetic identification carried out by other authors, showing that the method described here can be used as an effective alternative to, or in combination with, morphometric identification of bats

Diagnóstico de cinomose canina por RT-PCR em amostras de cães do Estado de São Paulo enviadas para a vigilância laboratorial da raiva

A cinomose canina (CD) é uma das doenças infecciosas mais importantes dos cães domésticos. No Brasil é ainda a principal causa de mortalidade de cães em algumas populações urbanas. Embora sequências de diferentes genes do vírus sejam utilizadas como alvo para detecção do vírus da cinomose canina (CDV), o gene N parece ser o melhor para a amplificação de todas as suas linhagens. Utilizando-se a técnica de RT-PCR direcionada ao gene N do CDV, foram analisadas 190 amostras de sistema nervoso central (SNC) de cães do estado de São Paulo com quadros sugestivos de encefalite e que foram encaminhadas ao Instituto Pasteur para o diagnóstico da raiva, durante o ano de 2014. A positividade foi superior a 50% indicando que a cinomose continua a ser uma importante causa de mortalidade canina.

Eastern equine encephalitis cases among horses in Brazil between 2005 and 2009.

Eastern equine encephalitis is a viral zoonosis that exhibits complex distribution and epidemiology, and greater importance should be given to this disease by the public-health authorities. In Brazil, although eastern equine encephalitis virus (EEEV) has been identified in vectors and antibodies are sometimes detected in horses and humans, there have been no records of equine encephalitis in horses caused by this virus during the last 24 years. This study describes eighteen cases of eastern equine encephalomyelitis that occurred in six Brazilian states between 2005 and 2009. Viral RNA was identified using semi-nested RT-PCR to detect members of the genus Alphavirus, and by genetic sequencing. The gene encoding NSP1 was partially amplified, and after genetic sequencing, eighteen sequences were generated. All eighteen strains were classified as belonging to lineage III of American EEEV. These findings could be an indication of the importance of this virus in animal and human public health.

Método de diagnóstico antemortem da raiva humana por meio de técnicas de biologia molecular, utilizando saliva e biópsia de pele da região da nuca / An antemortem method for diagnosis of human rabies by molecular techniques using saliva and neck-skin biopsy

Por meio das técnicas de RT-PCR com primers direcionados para o gene da

glicoproteína e RT-PCR e hemi-nested RT-PCR com primers direcionados para o gene da nucleoproteína, o RNA do vírus da raiva foi identificado em 95,2% de 21 amostras, 18 de saliva e três de biópsia de pele da região da nuca, coletadas entre a hospitalização e a morte de um paciente com sinais clínicos da raiva. O tratamento administrado ao paciente incluiu a indução de coma e terapia antiviral. Cada técnica, isoladamente, detectou RNA viral em 90,5%; 57,1% e 85,7% das amostras, respectivamente. Nossos resultados sugerem que a amplificação em paralelo de diferentes regiões do genoma do vírus da raiva pode fornecer maior confiabilidade ao diagnóstico antemortem da doença, auxiliando a decisão médica quanto à aplicação do protocolo de tratamento com antivirais.

Phylogeography of rabies virus isolated from dogs in Brazil between 1985 and 2006.

To establish the phylogeographic relationships in rabies viruses in Brazil, we studied a dataset retrieved from GenBank consisting of 71 genetic sequences from the coding region of the N gene of rabies viruses isolated in dogs over a period of 22 years. The Bayesian Markov chain Monte Carlo method available in the BEAST package was used with the GTR+G+Г4 evolutionary model in conjunction with the relaxed uncorrelated lognormal molecular clock model and an exponential growth tree prior. A discrete phylogeographic diffusion model was also analyzed using a standard continuous-time Markov chain viewed with Google Earth to provide a spatial projection of the diffusion of genetic lineages based on their phylogeographic relationships. The topology of the time and substitution phylogenetic trees agreed with the spatial dispersal of the lineages. It was possible to infer that the lineages in the southeastern region of Brazil in the 1970s are the closest to the most common recent ancestor and that all the lineages in the midwestern, northern and northeastern regions are more distant. The importance of this study lies in the fact that it can help with the planning of rabies control measures, as dogs continue to be the main reservoir of rabies virus throughout the world.

Human rabies transmitted by vampire bats: antigenic and genetic characterization of rabies virus isolates from the Amazon region (Brazil and Ecuador).

Since 2004, the main transmitter of human rabies in Latin America has been the vampire bat (Desmodus rotundus). Based on the nucleoprotein of the rabies virus (RV), we analyzed antigenic and genetic profiles of isolates from 29 samples taken from humans living in different areas of the Amazon region. Two isolates were from Ecuador and 27 from the Northern and Northeastern regions of Brazil, which were obtained during outbreaks in various municipalities in the states of Pará and Maranhão in the years 2004 and 2005. The partial N gene (nt 104-1477) of the 29 isolates was sequenced, and the sequences were used to build a neighbor-joining tree with the Kimura-2 parameter model. All 29 human RV isolates were identified as belonging to antigenic variant 3 (AgV3) and were genetically grouped into the D. rotundus cluster, which was divided into two subclusters (A and B), subcluster A in turn being divided into four genetic groups (A1, A2, A3 and A4). Genetic and molecular markers characterizing these genetic lineages were also identified. The results of this study show that the isolates belong to the same rabies cycle as that of the vampire bat D. rotundus. However, the division of clusters within the lineage associated with D. rotundus shows that different genetic sublineages of the virus were circulating in the Amazon region during the study period. Our findings suggest that there are phylogeographic differences between isolates obtained over a short period.

Rabies virus in insectivorous bats: implications of the diversity of the nucleoprotein and glycoprotein genes for molecular epidemiology.

Insectivorous bats are the main reservoirs of rabies virus (RABV) in various regions of the world. The aims of this study were to (a) establish genealogies for RABV strains from different species of Brazilian insectivorous bats based on the nucleoprotein (N) and glycoprotein (G) genes, (b) investigate specific RABV lineages associated with certain genera of bats and (c) identify molecular markers that can distinguish between these lineages. The genealogic analysis of N and G from 57 RABV strains revealed seven genus-specific clusters related to the insectivorous bats Myotis, Eptesicus, Nyctinomops, Molossus, Tadarida, Histiotus and Lasiurus. Molecular markers in the amino acid sequences were identified which were specific to the seven clusters. These results, which constitute a novel finding for this pathogen, show that there are at least seven independent epidemiological rabies cycles maintained by seven genera of insectivorous bats in Brazil.

Genetic characterization of rabies virus isolated from bovines and equines between 2007 and 2008, in the States of São Paulo and Minas Gerais.

INTRODUCTION: Rabies is an acute disease of the central nervous system and is responsible for the deaths of thousands of humans, wild animals and livestock, particularly cattle, as well as causing major economic losses. This study describes the genetic characterization of rabies virus variants that circulate in Desmodus rotundus populations and are transmitted to herbivores. METHODS: Fifty rabies virus isolates from bovines and equines in the States of São Paulo and Minas Gerais, Brazil, were genetically characterized and compared with sequences retrieved from GenBank. RESULTS: Two clusters (I and II) with mean nucleotide identities of 99.1 and 97.6% were found. The first of these contained nearly all the samples analyzed. Lineages from other Brazilian states grouped in cluster II. CONCLUSIONS: Analysis of the amino acid sequences of the N proteins revealed the existence of genetic markers that may indicate possible variations between geographic regions, although the biologically active regions are conserved within the species over space and time.

Comparative analysis of rabies virus isolates from Brazilian canids and bats based on the G gene and G-L intergenic region.

Rabies virus (RABV) isolates from two species of canids and three species of bats were analyzed by comparing the C-terminal region of the G gene and the G-L intergenic region of the virus genome. Intercluster identities for the genetic sequences of the isolates showed both regions to be poorly conserved. Phylogenetic trees were generated by the neighbor-joining and maximum parsimony methods, and the results were found to agree between the two methods for both regions. Putative amino acid sequences obtained from the G gene were also analyzed, and genetic markers were identified. Our results suggest that different genetic lineages of RABV are adapted to different animal species in Brazil.