Rabies Virus-Neutralizing Antibodies in Free-Ranging Invasive Wild Boars (Sus scrofa) from Brazil.

Rabies, one of the most lethal global zoonoses, affects all mammals. It remains circulating worldwide in sylvatic cycles through terrestrial and airborne reservoirs, and in Brazil, bats are currently the main reservoirs and source of transmission. Wild boars, an important invasive alien species in Brazil, are a proven food source for hematophagous bats and may participate in the Brazilian sylvatic cycle of rabies. We evaluated the presence of this pathogen in hunted wild boars from the São Paulo state using histopathology, the direct fluorescent antibody test (DFA), viral isolation in cell culture (VICC), the rapid fluorescent focus inhibition test (RFFIT), and quantitative reverse transcription polymerase chain reaction (RT-qPCR). The results of histopathological, DFA, VICC, and RT-qPCR analysis were negative for all samples; seven serum samples tested positive in the RFFIT, and titers ranged from 0.13 IU/mL to 0.5 IU/mL. The presence of rabies virus-neutralizing antibodies in the studied wild boars suggests the circulation of the virus in these animals. Educative actions directed at hunters should include information on the prevention of this important zoonosis.

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.

Evaluation of the effect of sample suspension concentration and viral load on the outcome of the rabies tissue culture infection test.

In this study, we examined various brain suspension concentrations and viral loads in Neuro-2a cell cultures using 20 rabies-positive bovine samples. The reproducibility of results varied: 65% showed consistent outcomes across all concentrations, while 35% disagreed in at least one. Viral titers ranged from less than 25 × 101 to 25 × 103.50 TCID50/mL, with 20% below 25 × 101 TCID50/mL. Concentrations between 5% and 20% yielded over 90% agreement in positive results, but at 30%, agreement dropped from 85% to 50%. Cell confluence was successfully maintained at 5%, 10%, and 20%, while concentrations of 30% and above led to confluence loss. Low viral loads also negatively impacted reproducibility. These results suggest that sample concentration has a direct influence on preservation of cell confluence and that low viral loads may influence the reproducibility of the rabies tissue culture infection test (RTCIT).

Survey of euthanasia methods used in neotropical chiropterans in São Paulo State, Brazil: critical analyses and proposal for standardization of bat euthanasia methods / evantamento dos métodos de eutanásia utilizados em quirópteros neotropicais no estado de São Paulo, Brasil: análise crítica e proposta de padronização de métodos de eutanásia em morcegos

Zoonotic Surveillance Divisions (ZSDs) rescue and euthanize bats in contact with humans. The euthanasia procedure should be conducted safely for the animal and caretaker and minimal stress for the animal is important. The objectives of this study were: i. evaluate the methods of euthanasia used by ZSDs in the State of São Paulo; ii. compare the methods with national and international guidelines for animal euthanasia practices; iii. assess the methodologies considering taxonomy and eating habits of the main bat species, and iv. propose standardization of euthanasia procedures. Sixty-five ZSDs locations received an online questionnaire or were contacted by telephone and 33 ZSDs (50.8%) responded and are distributed in 11 mesoregions in the state to remain anonymous. The euthanasia methods were divided into chemical (injectable or inhalation), physical, or mixed methods. Bat specimens (n = 550) were identified and classified to evaluate the main genera found in the state. The location of the ZSD, species, eating habits, and the method of euthanasia used were analyzed. The specimens by bat families were Molossidae (n = 340), Phyllostomidae (n = 171), and Vespertilionidae (n = 39). Chemical methods were used in 25 ZSDs (75.75%), physical in 5 (15.15%), and mixed in 3 (9.09%). There is no uniformity or standardization in bat euthanasia methods used by ZSDs, although most are based on acceptable chemical methods. It was proposed an algorithm to assist the veterinarian in choosing the method of euthanasia for bats that will allow standardizing euthanasia procedures for this species, considering physiological differences, and respecting technical, bioethical, and animal welfare guidelines.(AU)

As Divisões de Vigilância de Zoonoses (DVZs) coletam e eutanasiam morcegos que tiveram contato com humanos. O procedimento de eutanásia precisa ocorrer de modo seguro para o animal e para o manipulador, sendo importante garantir o mínimo de estresse ao animal. Os objetivos deste estudo foram: i. avaliar os métodos de eutanásia praticados nas DVZ do estado de São Paulo; ii. comparar esses métodos com os guias nacionais e internacionais de práticas de eutanásia animal; iii. avaliar as metodologias aplicadas considerando a taxonomia e os hábitos alimentares das principais espécies de ocorrência de morcegos; e iv. propor a padronização dos procedimentos de eutanásia. Sessenta e cinco DVZs receberam o questionário online ou foram contatadas por telefone, dessas, trinta e três DVZs (50,8%) que responderam ao questionário foram incluídas no estudo e distribuídas em 11 mesorregiões para garantir anonimato. Os métodos de eutanásia reportados foram divididos em métodos químicos (injetáveis; inalatórios), físicos ou mistos. Os espécimes de morcegos (n = 550) foram identificados e classificados para avaliar os principais gêneros encontrados no estado. A localização da DVZ, as espécies, os hábitos alimentares e o método de eutanásia utilizado foram analisados. Os espécimes por família de espécies de morcegos foram Molossidae (n = 340), Phyllostomidae (n = 171) e Vespertilionidae (n = 39). Métodos químicos foram utilizados em 25 DVZs (75,75%), físicos em 5 (15,15%) e mistos em 3 (9,09%). Não há uniformidade ou padronização nos métodos de eutanásia de morcegos, embora a maioria seja baseada em métodos químicos aceitáveis. É proposto um algoritmo para auxiliar o veterinário na escolha do método de eutanásia, que permite a padronização desses procedimentos para os morcegos, considerando as diferenças fisiológicas e respeitando as diretrizes técnicas, bioéticas e de bem-estar animal.(AU)

Seroprevalence to Rabies Virus in Wildlife in Brazil.

Serum samples of 638 free-ranging wild mammals from São Paulo state, Brazil, were tested for neutralizing antibodies against rabies virus by the rapid fluorescent focus inhibition test. Overall seroprevalence was 1.7% among 24 species surveyed, with individuals of six species having positive results indicating exposure to rabies virus.

Serological Surveillance of Rabies in Free-Range and Captive Common Vampire Bats Desmodus rotundus.

The control of vampire bat rabies (VBR) in Brazil is based on the culling of Desmodus rotundus and the surveillance of outbreaks caused by D. rotundus in cattle and humans in addition to vaccination of susceptible livestock. The detection of anti-rabies antibodies in vampire bats indicates exposure to the rabies virus, and several studies have reported an increase of these antibodies following experimental infection. However, the dynamics of anti-rabies antibodies in natural populations of D. rotundus remains poorly understood. In this study, we took advantage of recent outbreaks of VBR among livestock in the Sao Paulo region of Brazil to test whether seroprevalence in D. rotundus reflects the incidence of rabies in nearby livestock populations. Sixty-four D. rotundus were captured during and after outbreaks from roost located in municipalities belonging to three regions with different incidences of rabies in herbivores. Sixteen seropositive bats were then kept in captivity for up to 120 days, and their antibodies and virus levels were quantified at different time points using the rapid fluorescent focus inhibition test (RFFIT). Antibody titers were associated with the occurrence of ongoing outbreak, with a higher proportion of bats showing titer >0.5 IU/ml in the region with a recent outbreak. However, low titers were still detected in bats from regions reporting the last outbreak of rabies at least 3 years prior to sampling. This study suggests that serological surveillance of rabies in vampire bats can be used as a tool to evaluate risk of outbreaks in at risk populations of cattle and human.

Orthohepadnavirus infection in a neotropical bat (Platyrrhinus lineatus).

Hepatitis B virus (HBV) is the prototype of the Orthohepadnavirus genus and represents an important cause of chronic hepatitis, liver cirrhosis, and hepatic cancer in humans worldwide. To verify the occurrence and genetic variability of orthohepadnavirus among neotropical bats, we tested 81 liver samples of New World bats from São Paulo State, Southeastern Brazil, collected during 2012. PCR, sequencing, and phylogenetic analysis of Surface/Polymerase and Core viral genes confirmed the occurrence of the first isolate of bat orthohepadnavirus detected in South America. These results may contribute to subsequent studies of the origin, variability, host species, and evolution of bat orthohepadnaviruses in South America.

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.

Evaluation of polyclonal anti-RNP IgG antibody for rabies diagnosis by indirect rapid immunohistochemistry test.

Rabies still represents a major public health threat and estimated to cause 60,000 human deaths annually, particularly in developing countries. Thus, adequate surveillance based on rapid and reliable rabies diagnosis for both humans and animals is essential. The WHO and OIE recommended gold standard diagnostic technique for rabies is the direct immunofluorescence assay (dFAT). However, dFAT is expensive and requires a high level of expertise. As an alternative, the rapid immunohistochemistry technique is a promise to be a simple and cost effective diagnostic tool for rabies, and can be performed on field conditions prevalent in developing countries. However, no validated commercial conjugate antibody for rabies is available to meet the laboratory demand. Here, we evaluated the polyclonal anti-rabies virus ribonucleoprotein (RNP) IgG antibody for Rabies lyssavirus (RABV) detection by indirect rapid immunohistochemistry test (iRIT). We tested polyclonal anti-RNP IgG antibody against a batch of 100 brain specimens representing a wide phylogenetic origin in the State of São Paulo, Brazil. The purified IgG obtained 100% of diagnostic specificity and sensibility for RABV antigen detection in iRIT compared with the gold standard dFAT. In conclusion, our results demonstrate that the polyclonal anti-RNP IgG antibody may be used as a diagnostic reagent for rabies using iRIT, with the expectation of increase in availability and cost reduction of the epidemiological surveillance for developing countries.

Evaluation of polyclonal anti-RNP IgG antibody for rabies diagnosis by indirect rapid immunohistochemistry test

Rabies still represents a major public health threat and estimated to cause 60,000 human deaths annually, particularly in developing countries. Thus, adequate surveillance based on rapid and reliable rabies diagnosis for both humans and animals is essential. The WHO and OIE recommended gold standard diagnostic technique for rabies is the direct immunofluorescence assay (dFAT). However, dFAT is expensive and requires a high level of expertise. As an alternative, the rapid immunohistochemistry technique is a promise to be a simple and cost effective diagnostic tool for rabies, and can be performed on field conditions prevalent in developing countries. However, no validated commercial conjugate antibody for rabies is available to meet the laboratory demand. Here, we evaluated the polyclonal anti-rabies virus ribonucleoprotein (RNP) IgG antibody for Rabies lyssavirus (RABV) detection by indirect rapid immunohistochemistry test (iRIT). We tested polyclonal anti-RNP IgG antibody against a batch of 100 brain specimens representing a wide phylogenetic origin in the State of São Paulo, Brazil. The purified IgG obtained 100% of diagnostic specificity and sensibility for RABV antigen detection in iRIT compared with the gold standard dFAT. In conclusion, our results demonstrate that the polyclonal anti-RNP IgG antibody may be used as a diagnostic reagent for rabies using iRIT, with the expectation of increase in availability and cost reduction of the epidemiological surveillance for developing countries.

Evaluation of polyclonal anti-RNP IgG antibody for rabies diagnosis by indirect rapid immunohistochemistry test

Rabies still represents a major public health threat and estimated to cause 60,000 human deaths annually, particularly in developing countries. Thus, adequate surveillance based on rapid and reliable rabies diagnosis for both humans and animals is essential. The WHO and OIE recommended gold standard diagnostic technique for rabies is the direct immunofluorescence assay (dFAT). However, dFAT is expensive and requires a high level of expertise. As an alternative, the rapid immunohistochemistry technique is a promise to be a simple and cost effective diagnostic tool for rabies, and can be performed on field conditions prevalent in developing countries. However, no validated commercial conjugate antibody for rabies is available to meet the laboratory demand. Here, we evaluated the polyclonal anti-rabies virus ribonucleoprotein (RNP) IgG antibody for Rabies lyssavirus (RABV) detection by indirect rapid immunohistochemistry test (iRIT). We tested polyclonal anti-RNP IgG antibody against a batch of 100 brain specimens representing a wide phylogenetic origin in the State of São Paulo, Brazil. The purified IgG obtained 100% of diagnostic specificity and sensibility for RABV antigen detection in iRIT compared with the gold standard dFAT. In conclusion, our results demonstrate that the polyclonal anti-RNP IgG antibody may be used as a diagnostic reagent for rabies using iRIT, with the expectation of increase in availability and cost reduction of the epidemiological surveillance for developing countries.

A rabies virus vampire bat variant shows increased neuroinvasiveness in mice when compared to a carnivore variant

Rabies is one of the most important zoonotic diseases and is caused by several rabies virus (RABV) variants. These variants can exhibit differences in neurovirulence, and few studies have attempted to evaluate the neuroinvasiveness of variants derived from vampire bats and wild carnivores. The aim of this study was to evaluate the neuropathogenesis of infection with two Brazilian RABV street variants (variant 3 and crab-eating fox) in mice. BALB/c mice were inoculated with RABV through the footpad, with the 50% mouse lethal dose (LD50) determined by intracranial inoculation. The morbidity of rabies in mice infected with variant 3 and the crab-eating fox strain was 100% and 50%, respectively, with an incubation period of 7 and 6 days post-inoculation (dpi), respectively. The clinical disease in mice was similar with both strains, and it was characterized initially by weight loss, ruffled fur, hunched posture, and hind limb paralysis progressing to quadriplegia and recumbency at 9 to 12 dpi. Histological lesions within the central nervous system (CNS) characterized by nonsuppurative encephalomyelitis with neuronal degeneration and necrosis were observed in mice infected with variant 3 and those infected with the crab-eating fox variant. However, lesions and the presence of RABV antigen, were more widespread within the CNS of variant-3-infected mice, whereas in crab-eating fox-variant-infected mice, RABV antigens were more restricted to caudal areas of the CNS, such as the spinal cord and brainstem. In conclusion, the results shown here demonstrate that the RABV vampire bat strain (variant 3) has a higher potential for neuroinvasiveness than the carnivore variant. (AU) i

A rabies virus vampire bat variant shows increased neuroinvasiveness in mice when compared to a carnivore variant.

Rabies is one of the most important zoonotic diseases and is caused by several rabies virus (RABV) variants. These variants can exhibit differences in neurovirulence, and few studies have attempted to evaluate the neuroinvasiveness of variants derived from vampire bats and wild carnivores. The aim of this study was to evaluate the neuropathogenesis of infection with two Brazilian RABV street variants (variant 3 and crab-eating fox) in mice. BALB/c mice were inoculated with RABV through the footpad, with the 50% mouse lethal dose (LD50) determined by intracranial inoculation. The morbidity of rabies in mice infected with variant 3 and the crab-eating fox strain was 100% and 50%, respectively, with an incubation period of 7 and 6 days post-inoculation (dpi), respectively. The clinical disease in mice was similar with both strains, and it was characterized initially by weight loss, ruffled fur, hunched posture, and hind limb paralysis progressing to quadriplegia and recumbency at 9 to 12 dpi. Histological lesions within the central nervous system (CNS) characterized by nonsuppurative encephalomyelitis with neuronal degeneration and necrosis were observed in mice infected with variant 3 and those infected with the crab-eating fox variant. However, lesions and the presence of RABV antigen, were more widespread within the CNS of variant-3-infected mice, whereas in crab-eating fox-variant-infected mice, RABV antigens were more restricted to caudal areas of the CNS, such as the spinal cord and brainstem. In conclusion, the results shown here demonstrate that the RABV vampire bat strain (variant 3) has a higher potential for neuroinvasiveness than the carnivore variant.

Large protein as a potential target for use in rabies diagnostics

Rabies is a zoonotic viral disease that remains a serious threat to public health worldwide. The rabies lyssavirus (RABV) genome encodes five structural proteins, multifunctional and significant for pathogenicity. The large protein (L) presents well-conserved genomic regions, which may be a good alternative to generate informative datasets for development of new methods for rabies diagnosis. This paper describes the development of a technique for the identification of L protein in several RABV strains from different hosts, demonstrating that MS-based proteomics is a potential method for antigen identification and a good alternative for rabies diagnosis.(AU) i

Group A rotavirus in Brazilian bats: description of novel T15 and H15 genotypes.

This study aimed to survey for group A rotaviruses (RVA) in bats from Brazil and to perform phylogenetic inferences for VP4, VP7, NSP3, NSP4 and NSP5 genes. RVA was found in 9.18 % (28/305) of tested samples. The partial genotype constellation of a Molossus molossus RVA strain was G3-P[3]-Ix-Rx-Cx-Mx-Ax-Nx-T3-E3-H6, and that of a Glossophaga soricina RVA strain was G20-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-T15-Ex-H15. These findings demonstrate an important role of bats in RVA epidemiology and provide evidence of participation of bat RVA strains in interspecies transmission and reassortment events.

Alphacoronavirus in urban Molossidae and Phyllostomidae bats, Brazil.

BACKGROUND: Bats have been implicated as the main reservoir of coronavirus (CoV). Thus the role of these hosts on the evolution and spread of CoVs currently deserve the attention of emerging diseases surveillance programs. On the view of the interest on and importance of CoVs in bats the occurrence and molecular characterization of CoV were conducted in bats from Brazil. FINDINGS: Three hundred five enteric contents of 29 bat species were tested using a panCoV nested RT-PCR. Nine specimens were positive and eight was suitable for RdRp gene sequencing. RdRp gene phylogeny showed that all CoVs strains from this study cluster in Alphacoronavirus genus, with one Molossidae and one Phlyllostomidae-CoV specific groups. Phylogenetic analyses of two S gene sequences showed a large diversity within the Alphacoronavirus genus. CONCLUSIONS: This study indicated a CoV-to-host specificity and draws attention for CoV detection in Cynomops sp, a potential new reservoir. The phylogenetic analyses indicate that diversity of CoV in bats is higher than previously known.

Delayed progression of rabies transmitted by a vampire bat.

Here, we compared the growth kinetics, cell-to-cell spread, and virus internalization kinetics in N2a cells of RABV variants isolated from vampire bats (V-3), domestic dogs (V-2) and marmosets (V-M) as well as the clinical symptoms and mortality caused by these variants. The replication rate of V-3 was significantly higher than those of V-2 and V-M. However, the uptake and spread of these RABV variants into N2a cells were inversely proportional. Nevertheless, V-3 had longer incubation and evolution periods. Our results provide evidence that the clinical manifestations of infection with bat RABV variant occur at a later time when compared to what was observed with canine and marmoset rabies virus variants.

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