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.

Correction: Molecular modelling of the HCMV IL-10 protein isoforms and analysis of their interaction with the human IL-10 receptor.

[This corrects the article DOI: 10.1371/journal.pone.0277953.].

Molecular modelling of the HCMV IL-10 protein isoforms and analysis of their interaction with the human IL-10 receptor.

The human cytomegalovirus (HCMV) UL111A gene encodes several homologs of the cellular interleukin 10 (cIL-10). Alternative splicing in the UL111A region produces two relatively well-characterized transcripts designated cmvIL-10 (isoform A) and LAcmvIL-10 (isoform B). The cmvIL-10 protein is the best characterized, both structurally and functionally, and has many immunosuppressive activities similar to cIL-10, while LAcmvIL-10 has more restricted biological activities. Alternative splicing also results in five less studied UL111A transcripts encoding additional proteins homologous to cIL-10 (isoforms C to G). These transcripts were identified during productive HCMV infection of MRC-5 cells with the high passage laboratory adapted AD169 strain, and the structure and properties of the corresponding proteins are largely unknown. Moreover, it is unclear whether these protein isoforms are able to bind the cellular IL-10 receptor and induce signalling. In the present study, we investigated the expression spectrum of UL111A transcripts in fully permissive MRC-5 cells and semi permissive U251 cells infected with the low passage HCMV strain TB40E. We identified a new spliced transcript (H) expressed during productive infection. Using computational methods, we carried out molecular modelling studies on the three-dimensional structures of the HCMV IL-10 proteins encoded by the transcripts detected in our work (cmvIL-10 (A), LAcmvIL-10 (B), E, F and H) and on their interaction with the human IL-10 receptor (IL-10R1). The modelling predicts clear differences between the isoform structures. Furthermore, the in silico simulations (molecular dynamics simulation and normal-mode analyses) allowed us to evaluate regions that contain potential receptor binding sites in each isoform. The analyses demonstrate that the complexes between the isoforms and IL-10R1 present different types of molecular interactions and consequently different affinities and stabilities. The knowledge about structure and expression of specific viral IL-10 isoforms has implications for understanding of their properties and role in HCMV immune evasion and pathogenesis.

Laboratory validation of confirmatory tests for rabies diagnosis: Approaches to reduce animal use and facilitate sample collection.

Rabies is an encephalitis caused by rabies virus, whose transmission occurs upon contact with infected animals' saliva. The diagnosis is usually performed post-mortem through a direct fluorescent antibody test (DFAT). If the DFAT results are negative, they must be confirmed with an isolation test, usually the mouse inoculation test (MIT), which implies the suffering and death of the animals, high costs and most importantly, up to 28 days to confirm a negative result. Another issue related to rabies diagnosis is the sample collection and storage, which is critical for the rabies virus' RNA genome. Thus, this study aimed to evaluate (i) reverse transcriptase polymerase chain reaction (RT-PCR) and Rabies Tissue Culture Infection Tests (RTCIT) in comparison to DFAT and MIT and (ii) FTA® cards as an alternative sample collection and preservation method. Eighty animal samples were evaluated through DFAT, RTCIT and RT-PCR; MIT was performed only in DFAT-negative samples. FTA® cards were evaluated with a subset of 64 samples, with sufficient material for imprinting. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV), agreement and Cohen's kappa were calculated for each test combination. RTCIT had higher sensitivity (92.5%) and RT-PCR had higher specificity (92.3%) compared to DFAT. The combination of tests enhanced sensitivity, NPV and Cohen's kappa (considering positive results by RTCIT or RT-PCR), and specificity and PPV (when both tests were concordant). The PCR based on FTA® cards as sample source was specific (84.6%-96.2%) but presented lower sensitivity (29.7%-73.0%), although it could detect as positive four DFAT-negative samples. RTCIT and RT-PCR may be used as confirmatory tests in DFAT-negative samples. Moreover, FTA® cards may be helpful for sample collection in field situations where a long time is needed until the sample undergoes laboratory testing.

Detection of rabies virus in cranial cavity lavage of naturally infected bats.

The rabies virus (RABV) has been isolated in several bats species in the world, and among them, hematophagous, frugivorous and insectivorous species. Bats found in Brazil are small, which can lead to situations in which there are limitations in the collection of the central nervous system (CNS) and the amount of material may be insufficient to carry out laboratory diagnostic techniques for rabies. The objective of this work was to evaluate an alternative sample collection for the diagnosis of rabies in bats. A total of 92 bat samples, 82 positives and 10 negatives were selected. The cranial cavity was scraped with the aid of sterile tips and a virus diluent was added to create a suspension. All samples were submitted to Rabies Tissue Culture Infection Test (RTCIT) and reverse transcription polymerase chain reaction (RT-PCR). The diagnostic sensitivity and specificity of the RTCIT and RT-PCR using the cranial cavity lavage were calculated in comparison with the results of the laboratory routine (DFAT and RTCIT) performed with the CNS (considered gold standard). The results of the RTCIT show that the cranial cavity lavage is not an adequate sample for viral isolation, since the diagnostic sensitivity was low (37.8 %) when compared with the tests with the CNS. However, the RT-PCR of the cranial cavity lavage may be a tool to assist in the diagnosis, since it presented a sensitivity of 76.8 %. The results of this study suggest that cranial cavity lavage is an interesting alternative to enable the diagnosis of rabies in bats and increases the possibility of diagnosis contributing to rabies surveillance and control.

In vitro efects of bufotenine against RNA and DNA viruses

Bufotenine, an alkaloid that can be found in plant extracts and skin secretions of amphibians, is reported to have potential antiviral activity. The present study evaluated the antiviral activity of bufotenine against different genetic lineages of rabies virus (RABV, a single-stranded, negative-sense RNA virus), canine coronavirus (CCoV, a positive-sense RNA virus) and two double-stranded DNA viruses (two strains of herpes simplex virus type 1/HSV-1 [KOS and the acyclovir-resistant HSV-1 strain 29R] and canine adenovirus 2, CAV-2). The maximal non-toxic bufotenine concentrations in Vero and BHK-21 cells were determined by MTT assays. The antiviral activity of bufotenine against each virus was assessed by examination of reductions in infectious virus titres and plaque assays. All experiments were performed with and without bufotenine, and the results were compared. Bufotenine demonstrated significant RABV inhibitory activity. No antiviral action was observed against CCoV, CAV-2 or HSV-1. These findings indicate that the antiviral activity of bufotenine is somewhat linked to the particular infectious dose used and the genetic lineage of the virus, although the mechanisms of its effects remain undetermined.

Rabies in Callithrix sp. in the urban area of Niterói, Rio de Janeiro, Brazil.

In Brazil, rabies occurs mainly within an urban cycle, in which dogs and bats are reservoirs. This paper aims to report the occurrence of rabies in Callithrix sp. in Niterói, Rio de Janeiro, Brazil. In June 2019 a hybrid specimen was referred for diagnosis. The Direct Fluorescent Antibody, Mouse Inoculation, and Polymerase Chain Reaction tests were positive. A phylogenetic analysis was compatible with antigenic variant 3, characteristic of Desmodus rotundus. New studies should be undertaken to elucidate the real role of callitrichids in the urban rabies cycle.

Rabies in Callithrix sp. in the urban area of Niterói, Rio de Janeiro, Brazil

Abstract In Brazil, rabies occurs mainly within an urban cycle, in which dogs and bats are reservoirs. This paper aims to report the occurrence of rabies in Callithrix sp. in Niterói, Rio de Janeiro, Brazil. In June 2019 a hybrid specimen was referred for diagnosis. The Direct Fluorescent Antibody, Mouse Inoculation, and Polymerase Chain Reaction tests were positive. A phylogenetic analysis was compatible with antigenic variant 3, characteristic of Desmodus rotundus. New studies should be undertaken to elucidate the real role of callitrichids in the urban rabies cycle.

Raiva em colônias de morcegos insetívoros no litoral do Rio Grande do Sul / Rabies in insectivorous bat colonies on the coast of Rio Grande do Sul

Estudo da ocorrência de morcegos insetívoros positivos ao vírus da raiva (RABV) em cidades litorâneas do Rio Grande do Sul (RS). (AU)

Phylogeography of rabies isolated from cattle and horses transmitted by haematophagous bat Desmodus Rotundus in Brazil

Rabies is Public Health problem and is very important in Animal Health too. The illness demands continuous prophylactic care for herbivores with economic interest, such as cattle and horses. The main vector of rabies virus (RABV) for these animals are the hematophagous bats Desmodus rotundus. RABV is a RNA genome with low level of fidelity during replication cycle due to lack of repair of its polymerase. This causes the incorporation of mutations that increase the genotypic variation of the viral population. In the project, the nucleoprotein (N) gene of the RABV isolated mainly from cattle in different cities of State of São Paulo (SP), will be sequenced. In addition, genetic sequences deposited in GenBank will be also used. N is the most conserved gene of RABV for these reason is the most appropriated for phylogeographic studies. Because the RABV display evolutionary and ecological dynamics on the same time scale reliable phylogeographic inferences can be obtained from molecular data. As phylogeography expresses the contemporary pattern of geographic distribution of an organism according to gene genealogies the objective of this project is to determine the dispersion over time and space of the RABV transmitted by D. rotundus in SP. The phylogeography of RABV will be studied by phylogenetic analysis using Bayesian statistics using Monte Carlo methods via Markov Chains (MCMC), available on the Bayesian Evolutionary Analysis Sampling Trees (BEAST) plataform. In this way and after the test of different evolutionary models the data of the phylogenetic trees of substitution and more probable time will be converted into a KML file that allows the visualization of the spatial projection of the diffusion of the genetic lineages in the time and space using Google Earth. In this way, the final results can aid epidemiological surveillance and also strategic planning for the control of rabies.

Detecção de papilomavírus em morcegos no Brasil / Detection of papilomavirus in bats in Brazil

Objetivo do estudo é a identificação do PV em morcegos no Brasil. (AU)

Monitoramento do vírus da raiva em diferentes espécies de morcegos em Maringá, sul do Brasil

A raiva é uma enfermidade infectocontagiosa de caráter zoonótico, causada pelo vírus da raiva (RABV). Os quirópteros juntamente com os canídeos são os principais reservatórios do RABV, sendo responsáveis respectivamente pela manutenção dos ciclos aéreo e terrestre da doença. O presente trabalho teve por objetivo identificar interações entre o vírus e o reservatório no ciclo aéreo da raiva. Para isto, a presença do RABV foi investigada em amostras de saliva de morcegos de diferentes espécies. Foram realizadas trinta e seis capturas de morcegos, na Região de Maringá, Paraná, sul do Brasil, no período de abril a dezembro de 2013. Os morcegos foram capturados com auxílio de redes de nylon e acondicionados em sacos de algodão. Foram registrados os dados biométricos e coletada uma amostra de swab oral de cada exemplar. Para a identificação do RABV, foi realizada a técnica de Semi-Nested RT-PCR (“Reverse transcription polymerase chain reaction”) tendo como alvo o gene N que codifica a nucleoproteína do vírus. A análise de dados foi realizada por estatística descritiva. Ao longo do estudo, foram capturados 444 morcegos, pertencentes a quatro famílias e quinze espécies. O RABV não foi identificado em nenhuma das amostras analisadas. Estes resultados demonstram a ausência de excreção do RABV pela saliva de morcegos saudáveis na região alvo do estudo e salientam para a necessidade de mais estudos sobre a manutenção da raiva nas diferentes espécies de morcegos.

Identification of Different Species of Mammalians Involved in Zoonoses as Reservoirs or Hosts by Sequencing of the Mitochondrial DNA Cytochrome B Gene

Introduction: The identification of species that act as reservoirs or hosts of zoonotic agents is essential for control and epidemiological surveillance of the important illness in public health. Identification of the reservoirs for zoonoses can help to clarify how the pathogens are maintained in nature, leading to more effective disease control and avoiding indiscriminate extermination of wild animals.Aims: The objective of this study was to describe the genetic identification of 106 samples isolated from different mammalians species.Methodology: This study was conducted using 106 tissue samples from wild and domestic mammals sent to rabies diagnosis in Pasteur Institute, Brazil. Sequencing of the mitochondrial DNA b gene and Basic Local Alignment Search Tool (BLAST) was used to confirm species identity.Results and Conclusion: By sequencing the mtDNA cyt-b gene 10 orders, 20 families, 34 genera and 38 species of mammalians were identified. In conclusion, the method used at this work was efficient for identification of different species of mammalians. Animals identified at this work with same method, belong to high distance order, as marsupials, chiropters and primates.

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

Antigenic and genetic stability of rabies virus

Rabies virus (RABV) is a single stranded RNA genome virus that encodes five proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA dependent polymerase (L). However, RABV seem to be remarkably stable antigenic and genomic differences among isolates from different species have been recognized for many years.Analysis of RABV isolates from different natural reservoirs reveals antigenic variants and/or genetic lineages with specific characteristics, suggesting selection and adaptation of viruses to each of the particular species. Such selections and adaptations are so specific that they allow for the identification of the natural reservoir of origin of a determined isolate.This work was conducted to investigate the genomic and antigenic stability of four different genetic lineages of RABV,originated from different host species, following successive passages in mice. Four RABV isolates (IP4005/10, IP964/06, IP3629/11 and IP4871/11) were inoculated intracerebrally into 3-4 weeks-old mice. After each passage, the viruses were examined in their antigenic profile with a panel of monoclonal antibodies to rabies virus antigens. Viral RNA wasextracted from the 1st, 5th and 10th passages and submitted to reverse transcription (RT) followed by polymerase chain reaction (RT-PCR), sequencing and phylogenetic analyses. Antigenic profile of the isolates did not reveal any recognizable alteration throughout. No nucleotide substitutions were noticed in the final sequences in any of genes sequenced fromthe four RABV isolates, with the exception of one nonsynonymous substitution in the putative protein P in position of amino-acid 222 in the isolate of non-hematophagous bat origin. These findings highlight the high antigenic and genetic stability of RABV, as opposed to the alleged high genomic variability of viruses with RNA genomes. On the other hand, itseems that different isolates may present different degrees of genetic stability, ...

Virus isolation in cell culture for confirmatory diagnostic of rabies in bovine specimens (AU)

This study investigated the suitability of virus isolation (VI) in mouse neuroblastoma cells (N2A) and baby hamster kidney cells (BHK-21) as a confirmatory test for diagnosis of bovine rabies. Fourty-eight brain samples from cattle suspected of rabies were initially submitted to fluorescent antibody test (FAT) and mouse inoculation test (MIT) for routine diagnostic. Subsequently, these specimens were submitted to three protocols of VI in each cell line: a single 24h or 72h passage (T1, T2), or three 48h passages (T3). The FAT and MIT combined detected 32/48 positive samples, from which MIT detected 32 and FAT 31. The average time required for final MIT results was 12.3 days (8 - 21). VI in BHK-21 cells provided definitive, positive results in 100% of the samples in 72h (T2) and in 96.9% after three 48h passages (T3). VI in N2A cells yielded positive results in 100% in 72h (T2) and in 93.7% of samples after three 48h passages (T3). Sensitivity, specificity, positive and negative predictive values were 100% in T2 in N2A and BHK-21 cells, and the Kappa value was excellent in both cells (k=1). A single 24h passage (T1) in both cell lines performed poorly, detecting less than 40% of the positive samples. Taking together, these results indicate that VI in both cell lines, especially in BHK-21 cells that grow faster and are much easier to maintain, does represent an adequate alternative for MIT as a confirmatory test for rabies diagnostic in bovine specimens, yielding reliable results in reduced time.

Epidemiologia molecular de surto de raiva bovina na região central do Rio Grande do Sul, 2012 / Molecular epidemiology of an outbreak of bovine rabies in central Rio Grande do Sul, Brazil, 2012

A raiva é uma doença infecciosa do sistema nervoso central de mamíferos causada pelo vírus da raiva (RabV), geralmente, transmitido pela mordedura de animais infectados. No Brasil, os morcegos hematófagos Desmodus rotundus são as principais fontes de infecção do RabV para bovinos e equinos. Este artigo descreve uma investigação epidemiológica e molecular de surtos de raiva ocorridos na região central do Rio Grande do Sul, entre maio e agosto de 2012. Nesse período, 45 casos suspeitos de raiva foram relatados em 22 pequenos rebanhos, localizados dentro de um raio de 4,7km, no município de Pinhal Grande. Desses, 32 amostras foram submetidas para diagnóstico da raiva, sendo que o RabV e/ou antígenos virais foram identificados em 27 amostras. Em um segundo momento, 11 amostras foram submetidas à transcrição reversa/reação em cadeia da polimerase (RT-PCR) para o gene da nucleoproteína (N) do RabV, seguido de sequenciamento nucleotídico e análise filogenética. Sete das 11 amostras apresentaram sequências nucleotídicas idênticas e uma apresentou mutação sinônima, não-codificante, indicando uma provável origem comum dos vírus. Por outro lado, três amostras apresentaram mutações que resultaram em alterações de aminoácidos, sugerindo uma origem diferente do vírus. Esses resultados sugerem que RabV de diferentes origens/linhagens co-circulam na região e foram envolvidos nos surtos descritos. Investigações sobre a circulação de ambos os genótipos em morcegos na região estão em andamento.

Rabies is an infectious disease of the central nervous system of mammals caused by rabies virus (RabV), generally transmitted by the bite of rabid animals. In Brazil, vampire bats Desmodus rotundus are the main reservoirs of RabV for livestock. The present study describes a molecular and epidemiological investigation of outbreaks of bovine rabies occurring in the central region of Rio Grande do Sul state, Brazil, between May and August 2012. In this period, 45 cases suspected of rabies were reported in 22 small herds, located within a 4.7km range, in the county of Pinhal Grande. From these, 32 samples were submitted to rabies diagnosis and RabV and/or viral antigens were identified in 27 samples. Subsequently, 11 brain samples were submitted to reverse transcription/polymerase chain reaction (RT-PCR) for the nucleoprotein gene (N) followed by nucleotide sequencing and phylogenetic analysis. Seven out of 11 samples yielded identical sequences; one presented a synonymous, non-coding mutation, indicating a likely common origin of the virus. However, three other samples presented nucleotide mutations which resulted in amino acid changes, suggesting a different origin of the virus. In summary, these results suggest that RabV strains of different origin/lineages co-circulate in the region and were involved in the outbreaks. Investigations on the circulation of both genotypes in bats in the region are currently underway.

First detection of adenovirus in the vampire bat (Desmodus rotundus) in Brazil.

This paper describes the first detection of adenovirus in a Brazilian Desmodus rotundus bat, the common vampire bat. As part of a continuous rabies surveillance program, three bat specimens were captured in Southern Brazil. Total DNA was extracted from pooled organs and submitted to a nested PCR designed to amplify a 280 bp long portion of the DNA polymerase gene of adenoviruses. One positive sample was subjected to nucleotide sequencing, confirming that this DNA fragment belongs to a member of the genus Mastadenovirus. This sequence is approximately 25 % divergent at the nucleotide level from equine adenovirus 1 and two other recently characterized bat adenoviruses.

Detection of Alphacoronavirus in velvety free-tailed bats (Molossus molossus) and Brazilian free-tailed bats (Tadarida brasiliensis) from urban area of Southern Brazil.

A survey was carried out in search for bat coronaviruses in an urban maternity roost of about 500 specimens of two species of insectivorous bats, Molossus molossus and Tadarida brasiliensis, in Southern Brazil. Twenty-nine out of 150 pooled fecal samples tested positive by reverse transcription-PCR contained fragments of the RNA-dependent RNA polymerase gene of coronavirus-related viruses. The sequences clustered along with bat alphacoronaviruses, forming a subcluster within this group. Our findings point to the need for risk assessment and continued surveillance of coronavirus infections of bats in Brazil.