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:...

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, ...

Phylogeographic dispersion and diversification of rabies virus lineages associated with dogs and crab-eating foxes (Cerdocyon thous) in Brazil

Genetic lineages of dog-associated RABV still circulate in some areas of the North and Northeast of Brazil. In parallel, another RABV lineage circulates among wild canids in the Northeast, particularly the crab-eating fox (Cerdocyon thous). Although previous studies and phylogenetic analyses have been carried out, the way in which these lineages are dispersed temporally and spatially remained to be elucidated. In this study, RABV N gene sequences isolated from canids in North and Northeast Brazil were analyzed by the Bayesian Markov Chain Monte Carlo Method, and the results were then used in a phylogeographic study. It was inferred from the findings that the most recent common ancestor became established at the end of the nineteenth century on the border of the Brazilian states of Paraíba and Pernambuco and diversified into the lineages associated with dogs and C. thous. Around 1910, the original C. thous lineage diversified into two main sublineages in the same area, one of which migrated to the south and the other to the north. The dog-associated lineage diversified around 1945 and moved toward the north and south. From the phylogeographic analysis it was possible to infer not only the movement of the virus lineages but also the probable location where dispersion and diversification occurred. The methodology used here enabled the phylogeographic history of RABV in the region to be reconstructed, and the dispersion pattern of the virus can be used to predict its movements, making it easier to stop the advance of a rabies epidemic.

In vitro and in vivo inhibition of rabies virus replication by RNA interference

Rabies is a zoonotic disease that affects all mammals and leads to more than 55,000 human deaths every year, caused by rabies virus (RABV) (Mononegavirales: Rhabdoviridae: Lyssavirus). Currently, human rabies treatment is based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. The aim of this study was to assess the decrease in the titer of rabies virus both in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs were used with antisense strands complementary to rabies virus nucleoprotein (N) mRNA. BHK-21 cells monolayers were infected with 1000 to 0.1 TCID50 of PV and after 2 hours the cells were transfected with each of tree RNAs in separate using Lipofectamine-2000. All three siRNAs reduced the titer of PV strain in a least 0.72 logTCID50/mL and no cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000. Swiss albino mice infected with 10.000 to 1 LD of PV strain by the intracerebral route were also transfected after two hours of infection with a pool 3 siRNAs with Lipofectamine-2000 by the intracerebral route, resulting in a survival rate of 30% in mice inoculated with 100 LD50, while the same dose led to 100% mortality in untreated animals. Lipofectamine-2000 showed no toxic effect in control mice. These results suggest that intracerebral administration of siRNAs might be an effective antiviral strategy for rabies.

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.

Desmodus rotundus and Artibeus spp. bats might present distinct rabies virus lineages

In Brazil, bats have been assigned an increasing importance in public health as they are important rabies reservoirs. Phylogenetic studies have shown that rabies virus (RABV) strains from frugivorous bats Artibeus spp. are closely associated to those from the vampire bat Desmodus rotundus, but little is known about the molecular diversity of RABV in Artibeus spp. The N and G genes of RABV isolated from Artibeus spp. and cattle infected by D. rotundus were sequenced, and phylogenetic trees were constructed. The N gene nucleotides tree showed three clusters: one for D. rotundus and two for Artibeus spp. Regarding putative N amino acid-trees, two clusters were formed, one for D. rotundus and another for Artibeus spp. RABV G gene phylogeny supported the distinction between D. rotundus and Artibeus spp. strains. These results show the intricate host relationship of RABV's evolutionary history, and are invaluable for the determination of RABV infection sources.

Produção de sondas genéticas não radioativas para o diagnóstico do virus rábico pela técnica de RT-PCR e imunoquimioluminescencia / Production of nonisotopically lab

Foram produzidas sondas genéticas não radioativas utilizadas para a detecção dos genes N e G do vírus rábico. A hibridação das sondas foram efetuadas nos amplificados dos genes, obtidos através de RT-PCR e, reveladas por imunoquimioluminescência. Para o controle das reações foi construído um plasmídeo, chamado pSH-G, contendo o inserto do gene G, clonado a partir da amostra vacinal PV. A detecção do gene N tem valor diagnóstico, pelo fato deste gene ser conservado entre as diferentes amostras do vírus rábico. A detecção do gene G tem valor em prospecções epidemiológicas, mas sem valor diagnóstico, por estar em uma região variável do genoma viral. Onze amostras foram testadas através de RT-PCR e hibridação com a sonda N e, em 100% das amostras os resultados foram positivos por ambas as técnicas. Somente 2 amostras, das 11 testadas, foram positivas através de RT-PCR e hibridação com a sonda G. Os resultados obtidos através de RT-PCR e hibridações tiveram concordância absoluta. O plasmídeo pSH-G mostrou ser um controle positivo confiável e um marcador comparável a outros pesos moleculares. Assim, esta técnica poderá ser utilizada futuramente em laboratórios de diagnóstico da raiva, por conferir sensibilidade e especificidade, além de ser exeqüível e de baixo custo financeiro, quando comparada às técnicas tradicionais de diagnóstico.