Francisco Esmaile de Sales Lima

Genomic characterization of novel circular ssDNA viruses from insectivorous bats in Southern Brazil.

Circoviruses are highly prevalent porcine and avian pathogens. In recent years, novel circular ssDNA genomes have recently been detected in a variety of fecal and environmental samples using deep sequencing approaches. In this study the identification of genomes of novel circoviruses and cycloviruses in feces of insectivorous bats is reported. Pan-reactive primers were used targeting the conserved rep region of circoviruses and cycloviruses to screen DNA bat fecal samples. Using this approach, partial rep sequences were detected which formed five phylogenetic groups distributed among the Circovirus and the recently proposed Cyclovirus genera of the Circoviridae. Further analysis using inverse PCR and Sanger sequencing led to the characterization of four new putative members of the family Circoviridae with genome size ranging from 1,608 to 1,790 nt, two inversely arranged ORFs, and canonical nonamer sequences atop a stem loop.

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.

Prevalence, risk factors, and biochemical markers in dogs with ultrasound-diagnosed biliary sludge

Regarded as an incidental finding, biliary sludge is often diagnosed in dogs on abdominal ultrasound. The aims of the present study were to assess the risk factors, biochemical markers and ultrasonographic findings and to estimate the prevalence and influence of different breeds, sexes, and ages on biliary sludge in dogs. Results demonstrate that the prevalence of biliary sludge is high, especially in senior dogs. The biochemical markers did not have a significant correlation with biliary sludge, and the type of diet was not considered to be the major risk factor. Hepatomegaly was frequently observed on the ultrasound scan of affected animals and of dogs on different systemic drugs and with cardiopathies, which have been referred to as risk groups for the development of inspissated bile.

Efficacy of an inactivated, recombinant bovine herpesvirus type 5 (BoHV-5) vaccine

Bovine herpesvirus type 5 (BoHV-5) is the causative agent of bovine herpetic encephalitis. In countries where BoHV-5 is prevalent, attempts to vaccinate cattle to prevent clinical signs from BoHV-5-induced disease have relied essentially on vaccination with BoHV-1 vaccines. However, such practice has been shown not to confer full protection to BoHV-5 challenge. In the present study, an inactivated, oil adjuvanted vaccine prepared with a recombinant BoHV-5 from which the genes coding for glycoprotein I (gI), glycoprotein E (gE) and membrane protein US9 were deleted (BoHV-5 gI/gE/US9(-)), was evaluated in cattle in a vaccination/challenge experiment. The vaccine was prepared from a virus suspension containing a pre-inactivation antigenic mass equivalent to 10(7.69) TCID(50)/dose. Three mL of the inactivated vaccine were administered subcutaneously to eight calves serologically negative for BoHV-5 (vaccinated group). Four other calves were mock-vaccinated with an equivalent preparation without viral antigens (control group). Both groups were boostered 28 days later. Neither clinical signs of disease nor adverse effects were observed during or after vaccination. A specific serological response, revealed by the development of neutralizing antibodies, was detected in all vaccinated animals after the first dose of vaccine, whereas control animals remained seronegative. Calves were subsequently challenged on day 77 post-vaccination (pv) with 10(9.25) TCID(50) of the wild-type BoHV-5 (parental strain EVI 88/95). After challenge, vaccinated cattle displayed mild signs of respiratory disease, whereas the control group developed respiratory disease and severe encephalitis, which led to culling of 2/4 calves. Searches for viral DNA in the central nervous system (CNS) of vaccinated calves indicated that wild-type BoHV-5 did not replicate, whereas in CNS tissues of calves on the control group, viral DNA was widely distributed. BoHV-5 shedding in nasal secretions was significantly lower in vaccinated calves than in the control group on days 2, 3, 4 and 6 post-challenge (pc). In addition, the duration of virus shedding was significantly shorter in the vaccinated (7 days) than in controls (12 days). Attempts to reactivate latent infection by administration of dexamethasone at 147 days pv led to recrudescence of mild signs of respiratory disease in both vaccinated and control groups. Infectious virus shedding in nasal secretions was detected at reactivation and was significantly lower in vaccinated cattle than in controls on days 11-13 post-reactivation (pr). It is concluded that the inactivated vaccine prepared with the BoHV-5 gI/gE/US9(-) recombinant was capable of conferring protection to encephalitis when vaccinated cattle were challenged with a large infectious dose of the parental wild type BoHV-5. However, it did not avoid the establishment of latency nor impeded dexamethasone-induced reactivation of the virus, despite a significant reduction in virus shedding after challenge and at reactivation on vaccinated calves.

Torque teno sus virus 1 (TTSuV1) and 2 (TTSuV2) viral loads in serum of postweaning multisystemic wasting syndrome (PMWS)-affected and healthy pigs in Brazil.

Associations between Torque teno sus viruses (TTSuVs) and the occurrence of postweaning multisystemic wasting syndrome (PMWS) have been reported with controversial results. Currently, no studies have been performed comparing simultaneously viral loads of TTSuVs and PCV2. To examine the role for TTSuVs in PMWS-affected animals, a SYBR Green-based quantitative PCR (qPCR) was designed to detect and quantify TTSuV1, TTSuV2 and PCV2 genomes in swine sera. TTSuV1 genome loads were significantly higher in healthy adults than in young and SPF animals (p<0.05) suggesting that the prevalence of TTSuV1 infection increases with age and bears no association with PMWS. Regarding TTSuV2, no significant variation was detected in viral loads within any of the groups. As expected, PCV2 genome loads were higher in PMWS-affected swine than in healthy or SPF animals (p<0.001). These findings provide clear evidence to indicate that neither TTSuV1 nor TTSuV2 viral loads have any correlation with the occurrence of PMWS.

Diverse gammacoronaviruses detected in wild birds from Madagascar

To date, infectious bronchitis virus (IBV) is potentially found in wild birds of different species. This work reports the survey of coronaviruses in wild birds from Madagascar based on the targeting of a conserved genome sequence among different groups of CoVs. Phylogenetic analyses revealed the presence of gammacoronaviruses in different species of Gruiformes, Passeriformes, Ciconiiformes, Anseriformes, and Charadriiformes. Furthermore, some sequences were related to various IBV strains. Aquatic and migratory birds may play an important role in the maintenance and spread of coronaviruses in nature, highlighting their possible contribution in the emergence of new coronavirus diseases in wild and domestic birds.

Molecular Detection of Circovirus and Adenovirus in Feces of Fur Seals ( Arctocephalus spp.)

In some regions, little is known about exposure to viruses in coastal marine mammals. The present study aimed to detect viral RNA or DNA in 23 free-ranging fur seals on the northern coastline of Rio Grande do Sul State, Brazil. Polymerase chain reaction was used to detect nucleic acids of circoviruses, adenoviruses, morbilliviruses, vesiviruses, and coronaviruses in the feces from twenty-one South American fur seals (Arctocephalus australis) and two Subantarctic fur seals (A. tropicalis). Adenovirus DNA fragments were detected in two South American fur seals; nucleotide sequences of these fragments revealed a high degree of similarity to human adenovirus type C. Circovirus DNA fragments were detected in six animals of the same species. Two were phylogenetically similar to the Circovirus genus, whereas the other four nucleotide fragments showed no similarity to any of the known genera within the family Circoviridae. RNA fragments indicating the presence of coronavirus, vesivirus, and morbillivirus were not detected. These findings suggest that adenoviruses and circoviruses are circulating in fur seal populations found along the coast of Rio Grande do Sul State, Brazil.

Pathogenic Leptospira spp. in bats: Molecular investigation in Southern Brazil.

Abstract The present study aimed to investigate the frequency of pathogenic Leptospira spp. in Brazilian bats and to determine possible risk factors associated to it. Ninety two bats of 12 species were evaluated. Whole genomic DNA from kidneys was extracted and real-time PCR specific to pathogenic Leptospira spp. was applied. Association between the frequency of specimens positive for Leptospira spp. and sex, age, bat species or family, season of collection, geographic localization and feeding habits was evaluated. The results showed that 39.13% of analyzed bats were found positive for Leptospira spp. Nine bat species had at least one positive result. There was no association among the evaluated variables and frequency of pathogenic Leptospira spp. Although the limitations due to lack of Leptospira spp. isolation, leptospiral carriage was demonstrated in bats of different species from southern Brazil, which reinforces the need for surveillance of infectious agents in wild animals.

The constitutive expression of the V gene of Parainfluenza virus 5 affects the growth properties of bovine herpesvirus 5

This study aimed to analyze the effect of the expression of Parainfluenza virus 5 (PIV5) V protein in bovine cells on the replication of Bovine herpesvirus 5 (BoHV-5). Growth properties of BoHV-5 were evaluated in parental and PIV5 transfected cells. In one-step growth experiments, the BoHV-5 reached higher titers at earlier time points in the transfected cells when compared to the parental cells. The mean plaque size produced by the BoHV-5 in transfected cells was larger than the parental cells. This indicated that the expression of the PIV5 V gene facilitated the release and cell-to-cell spread of BoHV-5 in bovine cells.