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.

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.

Viral diversity in oral cavity from Sapajus nigritus by metagenomic analyses.

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

Rabies surveillance in wild mammals in South of Brazil.

The sylvatic cycle of rabies, caused by the Rabies lyssavirus (RABV), is maintained in the American Continent by aerial and terrestrial wild mammals. In this study, we combined passive surveillance of rescued wild animals with active serological surveillance in targeting areas at Rio Grande do Sul State and Santa Catarina State, south of Brazil, where bites of humans by wild animals have been reported. Circulation of RABV in Brazilian bats has been extensively demonstrated; however, the observation of such infections in unvaccinated terrestrial mammals is restricted to some regions of the Brazilian territory. The occurrence of rabies infection in unvaccinated animals has been identified by the detection of RABV antigens in brain tissues of dead animals or anti-rabies antibodies in live animals. Such strategies allow the surveillance of rabies and the assessment of spillover risks from infected animals to humans. Our aim included the identification of species of wild mammals that are involved in the sylvatic cycle of rabies virus in Southern Brazil and to assess the risk of rabies infection in patients bitten by wild animals in the state. To assess the anti-rabies seropositivity, sera were submitted to the Rapid Fluorescent Focus Inhibition Test (RFFIT). Among the 100 mammals tested, five animals were seropositive (5%) including three (one primate and two wild canids) with rabies virus neutralizing antibodies titres >0.5 IU/ml. Our results highlight the exposure to RABV of both primates and wild canids in Southern Brazil and suggest the occurrence of RABV exposure without the development of further symptoms. Further research should clarify the dynamics of rabies in wild canids and whether primates are accidental hosts or reservoirs for RABV at this region.

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.

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.

Phylogenetic analysis of partial RNA-polymerase blocks II and III of Rabies virus isolated from the main rabies reservoirs in Brazil.

This study describes the results of the sequencing and analysis of segments of Blocks II and III of the RNA polymerase L gene of Rabies virus isolates from different reservoir species of Brazil. The phylogenetic relations of the virus were determined and a variety of species-specific nucleotides were found in the analyzed areas, but the majority of these mutations were found to be synonymous. However, an analysis of the putative amino acid sequences were shown to have some characteristic mutations between some reservoir species of Brazil, indicating that there was positive selection in the RNA polymerase L gene of Rabies virus. On comparing the putative viral sequences obtained from the Brazilian isolates and other Lyssavirus, it was determined that amino acid mutations occurred in low-restriction areas. This study of the L gene of Rabies virus is the first to be conducted with samples of virus isolates from Brazil, and the results obtained will help in the determination of the phylogenetic relations of the virus.