Characterization of the viral genomes present in commercial batches of horse serum obtained by high-throughput sequencing.

Horses are often used as blood donors for commercial horse serum (HS) production and to manufacture biologicals. HS is an alternative for fetal bovine serum (FBS) used as a supplement for cell culture and vaccine production. Furthermore, HS is also frequently obtained in order to produce antisera toxins and pathogens. The advent of high-throughput sequencing (HTS) has promoted changes in virus detection, since previous knowledge of targets is not required. Thus, the present study aimed to describe the virome of five different batches of commercial HS from New Zealand (three batches) and Brazil and the United States (one batch each) using HTS. Each HS pool were processed and sequenced using an Illumina MiSeq platform. Sequences-related to viruses belonging to the Flaviviridae, Herpesviridae, and Parvoviridae families were detected. Particularly, equine hepacivirus (EqHV), equine pegivirus (EPgV), and Theiler's disease-associated virus (TDAV) were more frequent found in the batches analyzed. The presence of viral genomes in cell culture sera illustrates that these commercial sera can contain a mixture of different viruses and, therefore, can be regarded as potentially infectious for susceptible hosts. Moreover, the innocuity of commercial HS is important for the efficiency and security of diagnostics and the production of biological products.

Comprehensive evolutionary and phylogenetic analysis of Hepacivirus N (HNV).

Hepaciviruses (HVs) have been detected in several domestic and wild animals and present high genetic diversity. The actual classification divides the genus Hepacivirus into 14 species (A-N), according to their phylogenetic relationships, including the bovine hepacivirus [Hepacivirus N (HNV)]. In this study, we confirmed HNV circulation in Brazil and sequenced the whole genome of two strains. Based on the current classification of HCV, which is divided into genotypes and subtypes, we analysed all available bovine hepacivirus sequences in the GenBank database and proposed an HNV classification. All of the sequences were grouped into a single genotype, putatively named 'genotype 1'. This genotype can be clearly divided into four subtypes: A and D containing sequences from Germany and Brazil, respectively, and B and C containing Ghanaian sequences. In addition, the NS3-coding region was used to estimate the time to the most recent common ancestor (TMRCA) of each subtype, using a Bayesian approach and a relaxed molecular clock model. The analyses indicated a common origin of the virus circulating in Germany and Brazil. Ghanaian sequences seemed to have an older TMRCA, indicating a long time of circulation of these viruses in the African continent.

New polyomavirus species identified in nutria, Myocastor coypus polyomavirus 1.

A novel polyomavirus (PyVs) comprising 5,422 bp was identified by high-throughput sequencing (HTS) in pooled organs of nutria (Myocastor coypus). The new genome displays the archetypal organization of PyVs, which includes open reading frames for the regulatory proteins small T antigen (sTAg) and large T antigen (LTAg), as well as for the capsid proteins VP1, VP2 and VP3. Based on the International Committee on Taxonomy of Viruses (ICTV) Polyomaviridae Study Group criteria, this genome comprises a new PyVs species for the Alphapolyomavirus genus and is putatively named "Myocastor coypus Polyomavirus 1" . The complete genome sequence of this Myocastor coypus Polyomavirus 1 (McPyV1) isolate is publically available under the GenBank accession no. MH182627.

Natural Outbreak of BVDV-1d-Induced Mucosal Disease Lacking Intestinal Lesions.

Bovine viral diarrhea virus (BVDV) belongs to the Pestivirus genus, which is further divided into subgenotypes (1a-1u and 2a-c). When persistent infection occurs, the calf will be immunotolerant to BVDV and possibly develop mucosal disease. This study describes an outbreak of BVDV-1d-induced mucosal disease lacking intestinal lesions. Eleven calves presented with anorexia, sialorrhea, lameness, recumbency, and death. Three calves were necropsied, showing ulceration of the interdigital skin and the oral and nasal mucosa; linear ulcers in the tongue, esophagus, and rumen; and rounded ulcers in the abomasum. Microscopically, mucosa and skin had superficial necrosis, with single-cell necrosis and vacuolation in epithelial cells, and severe parakeratosis. Immunohistochemistry (IHC) showed BVDV antigen in the cytoplasm of epithelial cells in skin and mucosa. All 11 dead calves were positive upon reverse transcription-polymerase chain reaction (RT-PCR) for the detection of Pestivirus along with another 11 live calves from the herd, which were positive again by RT-PCR and IHC after a 4-week interval. Sequencing of the 5' untranslated region and N-terminal protease showed that viruses from these 22 calves were homologous and of subgenotype BVDV-1d. Cytopathic BVDV was isolated from 8 of 11 dead calves, but only noncytopathic BVDV was isolated from the 11 live animals. The findings indicate that this was an outbreak of mucosal disease caused by BVDV-1d, with high morbidity, and lesions restricted to the upper alimentary system and skin and absent from intestine. Thus, the epidemiological and pathological features in this form of mucosal disease may be similar to vesicular diseases, including foot and mouth disease.

Fibropapillomatosis in green turtles Chelonia mydas in Brazil: characteristics of tumors and virus.

Fibropapillomatosis (FP) is a benign neoplasia that affects physiological functions of sea turtles and may lead to death. High prevalence of FP in sea turtle populations has prompted several research groups to study the disease and the associated herpesvirus, chelonid herpesvirus 5 (ChHV5). The present study detected and quantified ChHV5 in 153 fibropapilloma samples collected from green turtles Chelonia mydas on the Brazilian coast between 2009 and 2010 to characterize the relationship between viral load and tumor characteristics. Of the tumor samples collected, 73 and 87% were positive for ChHV5 in conventional PCR and real-time PCR, respectively, and viral loads ranged between 1 and 118.62 copies cell⁻¹. Thirty-three percent of turtles were mildly, 28% were moderately and 39% were severely affected with FP. Skin samples were used as negative control. High viral loads correlated positively with increasing FP severity in turtles sampled on the Brazilian coast and with samples from turtles found dead in the states of São Paulo and Bahia. Six viral variants were detected in tumor samples, 4 of which were similar to the Atlantic phylogenetic group. Two variants were similar to the western Atlantic/eastern Caribbean phylogenetic group. Co-infection in turtles with more than one variant was observed in the states of São Paulo and Bahia.

Spleen and lung virome analysis of South American fur seals (Arctocephalus australis) collected on the southern Brazilian coast.

South American fur seals (Arctocephalus australis) are believed to reach the coast of Rio Grande do Sul (RS) through sea currents. They live in colonies and are frequently found resting on the beach. However, it is also common to find dead pinnipeds on beaches, sharing the environment with humans, domestic animals and other wild species on the coast and facilitating the transmission of pathogens. In the present study, a metagenomic approach was applied to evaluate the viral diversity in organs of fur seals found deceased along the coast of the state of RS, southern Brazil. The lungs and spleens of 29 animals were collected, macerated individually, pooled separately (one pool for lungs and another for spleens) and sequenced using the Illumina MiSeq platform. Sequences more closely related to members of the Anelloviridae and Circoviridae families were detected. Nine putative new species of anellovirus and one putative new genus, named Nitorquevirus, were described. Additionally, the circovirus sequences found in the lungs of A. australis have a common ancestor with PCV3, a proposed swine pathogen. Our study expanded the knowledge about viral communities in pinnipeds and could be useful for monitoring new viruses and potential viral sharing among wildlife, domestic animals, and humans.

Antimicrobial resistance and subtyping of Salmonella enterica subspecies enterica serovar Enteritidis isolated from human outbreaks and poultry in southern Brazil.

To investigate antimicrobial resistance, 96 Salmonella enterica subspecies enterica serovar Enteritidis strains isolated from salmonellosis outbreaks and poultry-related products obtained in southern Brazil were analyzed. Macrorestriction patterns, obtained by pulsed-field gel electrophoresis and phage types, were assessed. Although 43.75% of samples were sensitive to all drugs tested, resistance to sulfonamide (34.37%), trimethoprim-sulfamethoxazole (25.00%), nalidixic acid (14.58%), streptomycin (2.08%), gentamicin, and tetracycline (1.04%) was identified. Furthermore, 89.60% of strains belonged to phage type 4, and a predominant pulsed-field gel electrophoresis genotype represented by 82.29% of the strains was identified, suggesting that a clonal group was distributed in poultry, food, and human isolates. Although it was not possible to associate strains from different sources, the occurrence of antimicrobial-resistant Salmonella Enteritidis strains supports the need to establish monitoring programs to identify the emergence of potential resistance patterns and to direct policies for use of these drugs in food-producing animals.

Insights on the genetic features of endometrial pathogenic Escherichia coli strains from pyometra in companion animals: Improving the knowledge about pathogenesis.

Endometrial pathogenic E. coli (EnPEC) isolates are involved in endometrial infections in animals and humans. Besides the high aggressiveness of the endometrial infections, the EnPEC virulence profile and pathogenesis are still little known. In this study, we have sequenced and analyzed an EnPEC strain from canine pyometra (E. coli_LBV005/17), following a molecular characterization of the virulence profile and phylogenetic evolution of an EnPEC collection from canines and felines (45 strains). Most of the strains belonged to phylo-group B2, and display a high virulence profile. In particular we highlight the classification of the E. coli_LBV005/17 as sequence type 131 (ST131), in addition to other five strains, as observed by gyrB phylogenetic analysis. Also, the phylogenetic position of EnPEC strains from pyometra in companion animals suggests that their origins are from both extraintestinal and commensal E. coli strains. Accordingly to Principal Coordinates Analysis (PCoA) and phylogenetic analysis we can propose that EnPEC strains have neither the same genetic profile, nor a unique common ancestral. In summary, the present work characterize an EnPEC genome from bitch pyometra and the genetic profile of 45 EnPEC strains from companion animals pyometra, being the commonest virulence pattern: fimA, papC, hlyA, hlyE, cnf1, entB, iroN, irp1, bssS, bssR, and hmsP. These data improving the background knowledge of this E. coli pathotype related to pyometra in companion animals and may support new methods to prevent the disease evolution.

Liver virome of healthy pigs reveals diverse small ssDNA viral genomes.

Brazil is a major exporter of pork meat worldwide. Swine liver is a common ingredient in food consumed by humans, thus emphasizing the importance of evaluating the presence of associated pathogens in swine liver. To obtain knowledge, this study aimed to provide insights into the viral communities of livers collected from slaughtered pigs from southern Brazil. The 46 livers were processed and submitted for high-throughput sequencing (HTS). The sequences were most closely related to Anelloviridae, Circoviridae and Parvoviridae families. The present work also describes the first Brazilian PCV1 and the first PPV6 and PPV7 from South America. Virus frequencies revelead 63% of samples positive for TTSuV1, 71% for TTSuVk2, 10.8% for PCV, 13% for PPV and 6% for PBov. This report addresses the diversity of the liver virome of healthy pigs and expands the number of viruses detected, further characterizing their genomes to assist future studies.

Virome of crab-eating (Cerdocyon thous) and pampas foxes (Lycalopex gymnocercus) from southern Brazil and Uruguay.

Crab-eating (Cerdocyon thous) and Pampas foxes (Lycalopex gymnocercus) are wild canids distributed in South America. Domestic dogs (Canis lupus familiaris) and wild canids may share viral pathogens, including rabies virus (RABV), canine distemper virus (CDV), and canine parvovirus 2 (CPV-2). To characterize the virome of these wild canid species, the present work evaluated the spleen and mesenteric lymph node virome of 17 crab-eating and five Pampas foxes using high-throughput sequencing (HTS). Organ samples were pooled and sequenced using an Illumina MiSeq platform. Additional PCR analyses were performed to identify the frequencies and host origin for each virus detected by HTS. Sequences more closely related to the Paramyxoviridae, Parvoviridae and Anelloviridae families were detected, as well as circular Rep-encoding single-stranded (CRESS) DNA viruses. CDV was found only in crab-eating foxes, whereas CPV-2 was found in both canid species; both viruses were closely related to sequences reported in domestic dogs from southern Brazil. Moreover, the present work reported the detection of canine bocavirus (CBoV) strains that were genetically divergent from CBoV-1 and 2 lineages. Finally, we also characterized CRESS DNA viruses and anelloviruses with marked diversity. The results of this study contribute to the body of knowledge regarding wild canid viruses that can potentially be shared with domestic canids or other species.

Pharmacological action of tick saliva upon haemostasis and the neutralization ability of sera from repeatedly infested hosts.

Ticks are blood-feeding arthropods widely distributed in the world and vectors of several diseases. As haematophagy demands evasion strategies and repeatedly infested hosts develop protective immune responses, we investigated the mechanisms of the Rhipicephalus (Boophilus) microplus saliva anti-haemostatic activity and the possible relationship between the acquired natural anti-tick host resistance and anti-haemostatic action. For this purpose, we studied the effects of R. microplus saliva on different pathways of haemostasis and tested whether repeated infested bovine sera (RIBS) are able to abolish salivary anti-haemostatic activities. R. microplus saliva (i) displays inhibitory activity upon collagen-induced platelet aggregation; (ii) inhibits the induction of endothelial pro-coagulant state; and (iii) reduces thrombogenesis in vivo. RIBS were shown to be able to partially block the delay of coagulation and the anti-thrombotic effect of saliva, and to totally abolish the modulation of endothelium activation. Conversely, RIBS has no effect on the inhibition of platelet aggregation. These results show, for the first time, the neutralization ability of sera from acquired resistance hosts against tick anti-haemostatics. Moreover, this is the first report of a haematophagous parasite able to modulate endothelial cell pro-coagulant state, and addresses the presence of anti-platelet and anti-thrombotic activity in R. microplus saliva.

In vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains using the PrimeFlow RNA assay.

Bovine viral diarrhea viruses (BVDV), segregated in BVDV-1 and BVDV-2 species, lead to substantial economic losses to the cattle industry worldwide. It has been hypothesized that there could be differences in level of replication, pathogenesis and tissue tropism between BVDV-1 and BVDV-2 strains. Thus, this study developed an in vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains. To this end the competitive dynamics of BVDV-1a, BVDV-1b, and BVDV-2a strains in cell cultures was evaluated by a PrimeFlow RNA assay. Similar results were observed in this study, as was observed in an earlier in vivo transmission study. Competitive exclusion was observed as the BVDV-2a strains dominated and excluded the BVDV-1a and BVDV-1b strains. The in vitro model developed can be used to identify viral variations that result in differences in frequency of subgenotypes detected in the field, vaccine failure, pathogenesis, and strain dependent variation in immune responses.

Molecular diagnosis of Salmonella species in captive psittacine birds.

Cloacal swabs were collected from 280 captive psittacine birds belonging to 13 species. Samples of dna were tested by PCR using a pair of primers that amplify a 284 base pair fragment of the Salmonella genus invA gene, and the PCR-positive samples were tested by standard microbiological techniques. Thirteen per cent of the samples were positive by PCR, but negative by microbiological techniques. The infection rates were significantly different among the 13 species, the most commonly infected being Amazona amazonica (28 per cent) and Amazona pretrei (20 per cent). Specific tests for Salmonella Typhimurium Salmonella Enteritidis, Salmonella Pullorum and Salmonella Gallinarum did not produce positive results.

Serological survey for antibodies against pestiviruses in Wyoming domestic sheep.

Pestiviruses including Bovine viral diarrhea virus type 1 (BVDV-1), BVDV-2 and Border disease virus (BDV) have been reported in both sheep and cattle populations, together with the HoBi-like, an emerging group of pestiviruses. Pestivirus control programs in the United States have focused on the control of BVDV-1 and 2. The incidence of pestivirus infection in sheep in the United States and the risk of transmission between cattle and sheep populations are unknown. The aim of this study was to perform serological surveillance for pestivirus exposure in sheep from an important sheep producing state in the Unites States, Wyoming. For this, sera from 500 sheep, collected across the state of Wyoming (US) in 2015-2016, were examined by comparative virus neutralization assay against four species/proposed species of pestiviruses: BVDV-1, BVDV-2, BDV and HoBi-like virus. Rates of exposure varied between geographic regions within the state. The overall pestivirus prevalence of antibodies was 5.6%. Antibodies were most frequently detected against BVDV-1 (4%), and the highest antibody titers were also against BVDV-1. Data from this study highlights understanding of the dynamics of sheep pestivirus exposure, consideration of reference strains used for VN assays, transmission patterns, and potential vaccination history should be taken into account in implementation of control measures against pestiviruses in sheep and for successful BVDV control programs in cattle.

Papillomaviruses in ruminants: An update.

Papillomaviruses (PVs) are complex viruses which infect the skin or mucosae of a broad range of amniotes worldwide. They cause benign or malignant lesions depending on environmental factors, virus oncogenicity and the location of infection. Bovine papillomaviruses (BPVs) are the second most studied PVs beyond human PVs. In the past few years, genetic characterization of animal PVs has increased due to the availability of new techniques, which simplified the sequencing of entire genomes. Therefore, this review aims to provide an update of the current epidemiology, classification and genome features of ruminant PVs (mainly BPVs) affecting animals worldwide. The review also aimed to clarify the key differences between the high-risk Delta papillomaviruses and the seemingly low-risk Xi, Epsilon, Dyoxi and Dyokappapillomavirus as well as the recently described PVs BPV18, 19, 21 and PpuPV1 that belongs to an unclassified genus.

HoBi-like is the most prevalent ruminant pestivirus in Northeastern Brazil.

The ruminant pestiviral species BVDV-1, BVDV-2 and BDV, along with the putative species HoBi-like, may cause substantial economic losses in cattle, sheep and goats. Brazil's large size, variable biomes and wide range of ruminant animal production within different geographic regions suggest that the presence and prevalence of ruminant pestivirus may differ by regions within Brazil. This study investigated the genetic diversity of ruminant pestiviruses and determined the frequency of active infections within two states of the Northeast Region of Brazil, Maranhão and Rio Grande do Norte. Serum samples from 16,621 cattle and 2,672 small ruminants from 569 different herds residing in this region were tested by RT-PCR followed by DNA sequencing. Seventeen positive cattle were detected (0.1%) from fifteen different herds (2.64%). All isolates were classified as HoBi-like pestiviruses based on phylogenetic analysis. All small ruminant samples tested negative. The findings presented herein suggest that the Northeast Region of Brazil has a uniquely high prevalence of HoBi-like viruses. The increasing reports of HoBi-like viruses detected in cattle in the field suggest that natural infection with these viruses may be more widespread than previously thought. The identification of HoBi-like viruses as the most prevalent type of ruminant pestivirus circulating in the Northeast Region of Brazil indicates the need for both continued monitoring and determination of the extent of economic losses associated with HoBi-like virus infections. In addition, it must be taken into account in the choice of diagnostic tests and in vaccine formulations.

Presence of atypical porcine pestivirus (APPV) in Brazilian pigs.

Recently, a putative new pestivirus species, provisionally named as Atypical Porcine Pestivirus (APPV), was associated with the congenital tremor in piglets in North America and consequently in Europe and Asia. The present research aimed to describe the detection and characterization of APPV employing NS5B gene partial sequencing, gross pathology and histologic examination of piglets displaying congenital tremor from two different farms of Southern Brazil. No gross lesions were observed, and the histological findings revealed moderate vacuolization of the white matter of the cerebellum. RT-PCR followed by DNA sequencing and a phylogenetic analysis confirmed the presence of APPV in samples from the two farms, which the samples were distinct in nature. Phylogenetic reconstruction reinforced the high genetic variability within the APPVs previously reported. This is the first report of APPV in South America suggesting that this new group of viruses may be widespread in swine herds in other countries as it is in Brazil.

Backyard pigs are a reservoir of zoonotic hepatitis E virus in southern Brazil.

Background: Hepatitis E virus (HEV) is the causative agent of acute hepatitis worldwide. There is no seroprevalence study in backyard farms, which are characterized by suboptimal hygienic conditions in Brazil. We aimed to determine the seroprevalence and genetic diversity of HEV in backyard pigs in Brazil. Methods: Swine serum samples collected in 2012 (n=731) and 2014 (n=713) were analysed. The presence of anti-HEV immunoglobulin G in pig serum was evaluated by indirect enzyme-linked immunosorbent assay. Reverse transcription polymerase chain reaction was performed and phylogenetic analyses were carried out based on the partial ORF1 and ORF2 coding regions. Results: Anti-HEV antibodies were detected in 77.6% (567/731; 95% confidence interval [CI] 74.5 to 90.6%) of serum samples in 2012 and 65.5% (467/713; 95% CI 62.0 to 69.0%) in 2014. The herd seroprevalence was 91.7% (187/204; 95% CI 91% to 99%) in 2012 and 83.7% (164/196; 95% CI 78% to 89%) in 2014. Further, HEV RNA was detected in 0.8% (6/713) of samples from 2014. Phylogenetic analysis showed three different genotype 3 subtypes with high similarity to human HEV strains. Conclusions: This study showed that backyard pigs are a reservoir of HEV and alerts us to the need to control infection and spillover from backyard farms. GenBank accession numbers: MF438128-MF438135.

Characterization of dog serum virome from Northeastern Brazil.

Domestic dogs share habitats with human, a fact that makes them a potential source of zoonotic viruses. Moreover, knowledge regarding possible bloodborne pathogens is important due to the increasing application of blood transfusion in dogs. In the present study, we evaluated the serum virome of 520 dogs using throughput sequencing (HTS). The serum samples were pooled and sequenced using an Illumina MiSeq platform. Our unbiased method identified prevalent canine pathogens as canine protoparvovirus 1 (canine parvovirus 2), undersearched agents as canine bocaparvovirus 1 (minute virus of canines) and canine circovirus, circular viruses closely related to viruses recently found in human samples, and new parvovirus and anelloviruses. The dog virome described in the present work furthers the knowledge concerning the viral population in domestic animals. The present data includes information regarding viral agents that are potentially transmitted through blood transfusion among dogs.

Antigenic diversity of Brazilian isolates of HoBi-like pestiviruses.

Hobi-like viruses comprise an unclassified group of bovine pestiviruses related to bovine viral diarrhea virus 1 (BVDV-1) and 2 (BVDV-2). These viruses were originally identified in fetal bovine serum from Brazilian origin and, subsequently, isolated from diseased animals in several countries. Herein we performed an antigenic characterization of eight Brazilian HoBi-like viruses isolated from persistently infected (PI) animals and from gastroenteric disease (2007-2015). Phylogenetic analysis based on the 5' unstranslated region (UTR) clustered these viruses with other HoBi-like viruses from European and Asiatic origin. Monoclonal antibody (MAb) binding indicated variability in the Hobi-like virus glycoprotein E2 and significant differences from the homologous BVDV-1 and BVDV-2 glycoprotein. Analysis of antigenic relatedness based on virus-neutralizing titers using virus-specific antisera revealed that HoBi-like viruses are antigenically very different from BVDV-1 and, to a lesser extent, from BVDV-2. Cross-neutralizing assays between pairs of HoBi-like viruses and their respective antisera indicated the existence of antigenic variability among these viruses, even for viruses isolated from the same herd in different occasions. Moreover, the identification of a HoBi-like isolate with low antigenic similarity with the other isolates indicates the potential existence of antigenic subgroups among HoBi-like virus isolates. Finally, sera of lambs immunized with commercial BVDV vaccines showed low or undetectable neutralizing activity against HoBi-like isolates. These results indicate significant antigenic differences between BVDV genotypes and Brazilian HoBi-like viruses and the existence of antigenic variability within this atypical group of pestiviruses. These findings extend the knowledge about the antigenic diversity of HoBi-like viruses and reinforce the need for their inclusion in current BVDV vaccines.