Dispersion and diversification of Lyssavirus rabies transmitted from haematophagous bats Desmodus rotundus: a phylogeographical study.

Rabies is worldwide zoonosis caused by Lyssavirus rabies (RABV) a RNA negative sense virus with low level of fidelity during replication cycle. Nucleoprotein of RABV is the most conserved between all five proteins of the virus and is the most used gene for phylogenetic and phylogeographic studies. Despite of rabies been very important in Public Health concern, it demands continuous prophylactic care for herbivores with economic interest, such as cattle and horses. The main transmitter of RABV for these animals in Brazil is the hematophagous bats Desmodus rotundus. The aim of this study was to determine the dispersion over time and space of RABV transmitted by D. rotundus. Samples of RABV from the State of São Paulo (SP), Southeast Brazil isolated from the central nervous system (CNS) of cattle, were submitted to RNA extraction, RT-PCR, sequencing and phylogeographic analyzes with BEAST (Bayesian Evolutionary Analysis Sampling Trees) v 2.5 software. Was possible to identify high rate of diversification in starts sublineages of RABV what are correlated with a behavior of D. rotundus, the main transmitter of rabies to cattle. This study also highlights the importance of continuous monitoring of genetic lineages of RABV in Brazil.

No molecular evidence for influenza A virus and coronavirus in bats belonging to the families Phyllostomidae, Vespertilionidae, and Molossidae in the state of São Paulo, Brazil.

This study aimed to evaluate, by molecular methods, the presence of influenza A virus (IAV) and coronavirus in non-hematophagous bats collected in the state of São Paulo, Brazil. Samples of lung tissue and small intestine from 105 bats belonging to three families (Phyllostomidae, Vespertilionidae, and Molossidae) were collected in 22 municipalities in the state of São Paulo. Genetic identification of bats species was performed by amplification and sequencing of a fragment of 710 bp of the mitochondrial COI gene. In the detection of IAV, genomes were performed by RT-PCR, aiming at the amplification of a 245-bp fragment of the IAV matrix (M) protein gene. For coronaviruses, two fragments of 602 and 440 bp corresponding to segments along the gene encoding the RNA-dependent RNA polymerase (RdRp) were targeted. The detection limit for each of the PCRs was also determined. All samples analyzed here were negative for both viruses, and the lower limit of detection of the PCRs for the amplification of influenza virus A and coronavirus was estimated at 3.5 × 103 and 4.59 genomic copies per microliter, respectively. Although bats have been shown to harbor a large number of pathogens, the results of the present study support the theory that virus circulation in bats in the wild often occurs at low viral loads and that our understanding of the complex infectious dynamics of these viruses in wild conditions is still limited.

In vitro effects 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.

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.

Human embryonic kidney (HEK-293) cell line: An alternative for rabies virus diagnosis and research.

Rabies is a serious public health problem in developing countries and is caused by Rabies lyssavirus (RABV), a neurotropic RNA virus. The gold standard test for rabies diagnosis is the direct fluorescent antibody test (DFAT). Nevertheless, a confirmatory method is recommended, such as rabies tissue culture infection test (RTCIT). Several cell lines have been tested for RTCIT, and the murine neuroblastoma (Neuro-2a) cell line has been shown to be the most permissive for infection. The human embryonic kidney (HEK-293) cell line was recently thought as an option, due to neuronal protein expression and easy maintenance. In the present work, we evaluated the susceptibility of HEK-293 cell line to RTCIT compared to Neuro-2a. We used a total of 93 brain samples, 48 negatives and 45 positives for RABV previously tested by DFAT or RT-PCR and by RTCIT in Neuro-2a. Of the positive samples, 43 were positive in the traditional RTCIT using Neuro-2a. Two protocols of HEK-293 cell line to RTCIT were tested (with and without virus adsorption) with different incubations times: 24, 48 and 72 h. The highest positive rate in HEK-293 (41 positive samples) resulted from the adsorption protocol with 72 h incubation period, in contrast to 43 positive samples with the traditional RTCIT with Neuro-2a. No satisfactory results were observed using the protocol without adsorption, regardless of the incubation time. Despite the slightly higher sensitivity of Neuro-2a cells, the use of the HEK-293 cells still offers positive aspects, such as, more rapid results, with the advantage of fast and easy growth over Neuro-2a cell line. Therefore, our findings confirm that HEK-293 cells are susceptible to RABV and can be an alternative for RTCIT.

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.

Detection of adenovirus, papillomavirus and parvovirus in Brazilian bats of the species Artibeus lituratus and Sturnira lilium.

Bats play a significant role in maintaining their ecosystems through pollination, dispersal of seeds, and control of insect populations, but they are also known to host many microorganisms and have been described as natural reservoirs for viruses with zoonotic potential. The diversity of viruses in these animals remains largely unknown, however, because studies are limited by species, location, virus target, or sample type. Therefore, the aim of this study was to detect fragments of viral genomes in bat samples. We performed high-throughput sequencing analysis and specific PCR and RT-PCR on pools of anal and oropharyngeal swabs from Artibeus lituratus and Sturnira lilium collected in southern Brazil. As a result, a member of the family Adenoviridae related to human adenovirus C was detected in anal swabs from S. lilium. In addition, we detected a papillomavirus in an anal swab from A. lituratus. Our analyses also allowed the detection of adenoviruses and parvoviruses in oropharyngeal swabs collected from A. lituratus. These results increase our knowledge about viral diversity and illustrate the importance of conducting virus surveillance in bats.

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)