Fabrício Souza Campos

High prevalence of co-infections with bovine herpesvirus 1 and 5 found in cattle in southern Brazil.

Based on small scale studies or on little sensitive serological tests, bovines in the south of the state of Rio Grande do Sul, Brazil, are known to be infected with either bovine herpesvirus 1 (BoHV-1) or 5 (BoHV-5). However, whether the prevalence of each of these viruses is high or low is currently still unknown. In order to determine the extent of BoHV (-1 and/or -5) infections in bovines in this region of Brazil, 200 bovines were studied for the presence of BoHV DNA. To this end, first a quantitative PCR was developed that amplified BoHV-1 DNA as well as BoHV-5 DNA. Using this PCR the number of BoHV genomes normally present in latently infected ganglia of naturally infected bovines was estimated. The new PCR was sensitive enough to detect most BoHV DNA in infected ganglia. The results of this first PCR showed that at least 87% of the bovines in the south of Rio Grande do Sul were (latently) infected with either BoHV-1 or BoHV-5. To determine the prevalence of BoHV-1 and BoHV-5 separately, two type-specific PCRs - one for each virus - were developed that used the products of the first PCR as a template. The results of these type-specific PCRs showed that 82.8% of the BoHV positive population was (latently) infected with BoHV-1, 93.1% with BoHV-5 and 75.9% with both BoHV-1 and BoHV-5. This is the first time that such a high frequency of co-infection of BoHV-1 and BoHV-5 in bovines has been demonstrated.

Detection of bovine herpesvirus 1 and 5 in semen from Brazilian bulls

Bovine herpesvirus 1 (BoHV-1) and 5 (BoHV-5) are important pathogens of the respiratory and genital tract of cattle and may also affect the central nervous system and cause meningoencephalitis. Both virus types are estimated to be widely distributed in Southern Brazil. In the present study, BoHV-1 and/or BoHV-5 DNA were detected in bovine semen samples from two states of Brazil by two species-specific nested polymerase chain reactions (nPCRs). These nPCRs were used to assay 53 samples of fresh semen and 23 samples of frozen semen from breeding bulls. Viral DNA was detected in all 76 semen samples: all were positive for BoHV-5, whereas 34 of these were positive for BoHV-1 as well. Moreover, in five fresh and in 13 frozen semen samples-of a total number of 40 samples suitable for virus isolation-infectious BoHV-1 and/or BoHV-5 virus were detected. In conclusion, that both BoHV-1 and BoHV-5 were detected in bovine semen in Brazil highlighted the importance of examining bull semen in search for both agents to reduce the risk of transmitting these viruses.

Soroprevalência de herpesvírus bovinos tipos 1 e/ou 5 no Estado do Rio Grande do Sul

This study was carried out to estimate the prevalence of antibodies to bovine herpesviruses types 1(BoHV-1) and 5 (BoHV-5) in the state of Rio Grande do Sul (RS), Brazil, by testing serum samples against different BoHV-1 and BoHV-5 strains. The sera examined were obtained from a larger sample designed to estimate the prevalence of bovine brucellosis within the state. All sera were collected from cows 24 months or older, not vaccinated to bovine herpesviruses, from both dairy and beef herds. The number of samples to be tested was calculated based on an estimated prevalence of infection of 33%, with an average standard deviation of £1% and a 95% limit of agreement. Sera from 2.200 cattle from 390 farms distributed in 158 counties were tested by serum neutralization (SN) tests in search for antibodies to the following strains: BoHV-1.1 (strains EVI123/98 and Los Angeles), BoHV-5a (strain EVI88/95) and BoHV-5b (strain A663). The overall seroprevalence to BoHV-1 and BoHV-5 in the sampled herds was 29.2% (642/2.200); seropositive animals were detected in 225 (57.7%) of the sampled farms. Prevalence estimates varied according to the virus used for challenge in SN tests. The highest prevalence and sensitivity were attained when positive SN results against the four different strains were added together. The use of only one virus for challenge in SN tests would lead to a loss in sensitivity from 20.4% to 34.6% when compared to the combined SN-positive results. These findings provide evidence that antibodies to BoHV-1 and BoHV-5 are largely spread in dairy and beef herds in RS, although prevalence in distinct geographic regions is quite variable. The results were strongly affected by the virus strains used for challenge in SN testing. This must be taken into account when performing serologic tests to detect BoHV-1 and BoHV-5 antibodies. As SN test is not capable of discriminating between antibody responses to BoHV-1 and BoHV-5, type-specific prevalence remains unknown.

Metagenomic Survey of Viral Diversity Obtained from Feces of Subantarctic and South American Fur Seals.

The Brazilian South coast seasonally hosts numerous marine species, observed particularly during winter months. Some animals, including fur seals, are found dead or debilitated along the shore and may harbor potential pathogens within their microbiota. In the present study, a metagenomic approach was performed to evaluate the viral diversity in feces of fur seals found deceased along the coast of the state of Rio Grande do Sul. The fecal virome of two fur seal species was characterized: the South American fur seal (Arctocephalus australis) and the Subantarctic fur seal (Arctocephalus tropicalis). Fecal samples from 10 specimens (A. australis, n = 5; A. tropicalis, n = 5) were collected and viral particles were purified, extracted and amplified with a random PCR. The products were sequenced through Ion Torrent and Illumina platforms and assembled reads were submitted to BLASTx searches. Both viromes were dominated by bacteriophages and included a number of potentially novel virus genomes. Sequences of picobirnaviruses, picornaviruses and a hepevirus-like were identified in A. australis. A rotavirus related to group C, a novel member of the Sakobuvirus and a sapovirus very similar to California sea lion sapovirus 1 were found in A. tropicalis. Additionally, sequences of members of the Anelloviridae and Parvoviridae families were detected in both fur seal species. This is the first metagenomic study to screen the fecal virome of fur seals, contributing to a better understanding of the complexity of the viral community present in the intestinal microbiota of these animals.

Molecular detection and characterization of BK and JC polyomaviruses in urine samples of renal transplant patients in Southern Brazil

The human polyomaviruses JC (JCPyV) and BK (BKPyV) are widespread in the human population. Following the primary infection, virus reactivation may lead to nephropathy and graft rejection in renal transplant patients. This study was carried out to access the presence of BKPyV and JCPyV DNA in urine samples collected from renal transplant patients (n = 92) and healthy individuals (n = 88) in Porto Alegre, Rio Grande do Sul. The samples were submitted to a nested PCR. A significantly higher frequency (P < 0.001) of BKPyV was found in renal transplant patients (65.2%) in comparison to the control group (32.9%). JCPyV was detected equally in both groups. Phylogenetic analysis of both BKPyV and JCPyV amplicons demonstrates the presence of the BKPyV subtypes I and II, whereas for JCPyV, four different groups are found (1, 2, 3, and 4). J. Med. Virol. 87:522–528, 2015.

Prevalence of enterotoxin-encoding genes and antimicrobial resistance in coagulase-negative and coagulase-positive Staphylococcus isolates from black pudding

INTRODUCTION Staphylococcal species are pathogens that are responsible for outbreaks of foodborne diseases. The aim of this study was to investigate the prevalence of enterotoxin-genes and the antimicrobial resistance profile in staphylococcus coagulase-negative (CoNS) and coagulasepositive (CoPS) isolates from black pudding in southern Brazil. METHODS Two hundred typical and atypical colonies from Baird-Parker agar were inoculated on mannitol salt agar. Eighty-two mannitol-positive staphylococci were submitted to conventional biochemical tests and antimicrobial susceptibility profiling. The presence of coagulase (coa) and enterotoxin (se) genes was investigated by polymerase chain reaction. RESULTS The isolates were divided into 2 groups: 75.6% (62/82) were CoNS and 24.4% (20/82) were CoPS. The biochemical tests identified 9 species, of which Staphylococcus saprophyticus (37.8%) and Staphylococcus carnosus (15.9%) were the most prevalent. Antimicrobial susceptibility tests showed resistance phenotypes to antibiotics widely administered in humans, such as gentamicin, tetracycline, chloramphenicol, and erythromycin. The coa gene was detected in 19.5% (16/82) of the strains and 4 polymorphic DNA fragments were observed. Five CoNS isolates carrying the coa gene were submitted for 16S rRNA sequencing and 3 showed similarity with CoNS. Forty strains were positive for at least 1 enterotoxin-encoding gene, the genes most frequently detected were sea (28.6%) and seb (27.5%). CONCLUSIONS The presence of antimicrobial resistant and enterotoxin-encoding genes in staphylococci isolates from black pudding indicated that this fermented food may represent a potential health risk, since staphylococci present in food could cause foodborne diseases or be a possible route for the transfer of antimicrobial resistance to humans.

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.

A new marseillevirus isolated in Southern Brazil from Limnoperna fortunei

Members of the family Marseilleviridae are giant viruses that have the ability to infect amoebas. Such viruses were initially described in 2009. Since then, this family has grown, and diverse members have been found in different environments and geographic locations. Previous phylogenetic analyses suggested the existence of four marseillevirus lineages. A fourth lineage was described with the discovery of the Brazilian marseillevirus (BrMr), isolated from Pampulha Lake, Brazil. Here we describe the isolation and characterization of the Golden marseillevirus (GMar), a new marseillevirus isolated from golden mussels (Limnoperna fortunei) in South of Brazil. This new representative of Marseilleviridae has circular, double-stranded (dsDNA) that contains 360, 610 base pairs and encodes 483 open read frames (ORFs). The complete virus genome was sequenced and phylogenic analyses indicated clear differences between this virus and other marseilleviruses. In addition, this is the only marseillevirus so far that has been isolated from mussels, and this report expands the diversity of environments from which giant viruses could be recovered.

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.

Detection of bovine herpesvirus 2 and bovine herpesvirus 4 DNA in trigeminal ganglia of naturally infected cattle by polymerase chain reaction.

Establishment of latent infection within specific tissues in the host is a common biological feature of the herpesviruses. In the case of bovine herpesvirus 2 (BoHV-2), latency is established in neuronal tissues, while bovine herpesvirus 4 (BoHV-4) and ovine herpesvirus 2 (OvHV-2) latent virus targets on cells of the monocytic lineage. This study was conducted in quest of BoHV-2, BoHV-4 and OvHV-2 DNA in two hundred trigeminal ganglia (TG) specimens, derived from one hundred clinically healthy cattle, majority of them naturally infected with bovine herpesvirus 1 (BoHV-1) and bovine herpesvirus 5 (BoHV-5). Total DNA extracted from ganglia was analyzed by polymerase chain reaction (PCR) designed to amplify part of the genes coding for BoHV-2, and BoHV-4 glycoprotein B and, for OvHV-2, the gene coding for phosphoribosylformylglycinamidine synthase-like protein. BoHV-2 DNA was detected in TG samples of two (2%) and BoHV-4 DNA in nine (9%) of the animals, whereas OvHV-2 DNA could not be detected in any of the TG DNA. The two animals in which BoHV-2 DNA was identified were also co-infected with BoHV-1 and BoHV-5. Within the nine animals in which BoHV-4 DNA was detected, six were also co-infected with BoHV-1 and BoHV-5. This report provides for the first time evidence that viral DNA from BoHV-2 and BoHV-4 can be occasionally detected in TG of naturally infected cattle. Likewise, in this report we provided for the first time evidence that the co-infection of cattle with three distinct bovine herpesviruses might be a naturally occurring phenomenon.