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.

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.

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.

Highly divergent cattle hepacivirus N in Southern Brazil.

The genus Hepacivirus includes 14 species (Hepacivirus A-N). In this study, we determined a partial genome sequence of a highly divergent bovine hepacivirus (hepacivirus N, HNV) isolate from cattle in Southern Brazil. Previously described HNV isolates have shared 80-99.7% nucleotide sequence identity in the NS3 coding region. However, the sequence determined in this study had 72.6% to 73.8% nucleotide sequence identity to known HNV NS3 sequences. This high divergence could be seen in a phylogenetic tree, suggesting that it represents a new genotype of HNV. These data expand our knowledge concerning the genetic variability and evolution of hepaciviruses.

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.

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.

Clinical Presentation Resembling Mucosal Disease Associated with 'HoBi'-like Pestivirus in a Field Outbreak.

The genus Pestivirus of the family Flaviviridae consists of four recognized species: Bovine viral diarrhoea virus 1 (BVDV-1), Bovine viral diarrhoea virus 2 (BVDV-2), Classical swine fever virus (CSFV) and Border disease virus (BDV). Recently, atypical pestiviruses ('HoBi'-like pestiviruses) were identified in batches of contaminated foetal calf serum and in naturally infected cattle with and without clinical symptoms. Here, we describe the first report of a mucosal disease-like clinical presentation (MD) associated with a 'HoBi'-like pestivirus occurring in a cattle herd. The outbreak was investigated using immunohistochemistry, antibody detection, viral isolation and RT-PCR. The sequence and phylogenetic analysis of 5'NCR, N(pro) and E2 regions of the RT-PCR positive samples showed that four different 'HoBi'-like strains were circulating in the herd. The main clinical signs and lesions were observed in the respiratory and digestive systems, but skin lesions and corneal opacity were also observed. MD characteristic lesions and a pestivirus with cytopathic biotype were detected in one calf. The present study is the first report of a MD like presentation associated with natural infection with 'HoBi'-like pestivirus. This report describes the clinical signs and provides a pathologic framework of an outbreak associated with at least two different 'HoBi'-like strains. Based on these observations, it appears that these atypical pestiviruses are most likely underdiagnosed in Brazilian cattle.

High frequency of bovine viral diarrhea virus type 2 in Southern Brazil.

Ruminant pestiviruses can infect cattle populations worldwide and cause significant economic losses due to their impact on productivity and health. Knowledge of pestivirus diversity is important for control programs and vaccine development and for determining probable sources of infection. In this paper, we describe a search for ruminant pestiviruses with RT-PCR in sera of 9078 calves from 6 to 12 months of age. The calves were first analyzed in pools and then analyzed individually. Thirty-three RT-PCR positive animals were detected (0.36%) from 6.9% (24) of the 346 herds. The sequencing analysis of the 5' non-coding region and N terminal autoprotease showed the presence of BVDV-1a (15 isolates), -1b (3), -1d (1) and -2b (14), with a higher frequency (42.4%) of BVDV-2 in comparison with other countries. The presence of sheep was significantly associated with BVDV infection. Our results also suggested that a BVDV control program based only on the investigation of cattle would not be successful, especially in regions with farms harboring multiple animal species. This study may also serve as a reference for future control programs in Southern Brazil because it reports the prevalence of cattle with active infections and the genetic background of the circulating strains.

Evaluation of prenucleic acid extraction for increasing sensitivity of detection of virus in bovine follicular fluid pools.

Bovine viral diarrhea virus (BVDV), bovine herpesvirus type 1 (BoHV-1), and bovine herpesvirus type 5 (BoHV-5) are major cattle pathogens that can be present in biological materials used in assisted reproduction biotechnologies. The aim of the present study was to increase the sensitivity of the polymerase chain reaction (PCR) for detection of BVDV, BoHV-1, and BoHV-5 in bovine follicular fluid (FF) collected during oocyte retrieval for in vitro embryo production. Ovaries were collected immediately after slaughter at a commercial abattoir, aspirated, and the 7336 samples of FF were pooled in 84 samples. Before testing the FF field samples, sensitivity of the protocol was determined using a prenucleic acid extraction procedure that was directly compared with standard RNA or DNA extraction protocols. The prenucleic acid extraction procedure increased sensitivity of reverse transcription (RT)-PCR for BVDV and nested PCR for BoHV-1 and BoHV-5 by 100 and 10 times, respectively. The 84 FF pools were assayed for BVDV, BoHV-1, and BoHV-5 using virus isolation and RT-PCR or nested PCR. Fourteen (16.7%) FF pools were positive for BVDV RNA, and one (1.2%) was positive for BoHV-1 DNA. Two of the BVDV RT-PCR positive samples and the one BoHV-1 PCR positive sample were also positive in cell culture, demonstrating that FF contained infectious viruses. In this study, the prenucleic acid extraction procedure increased the sensitivity of RT-PCR and PCR detection. This study highlighted the importance of assuring biosecurity by detecting the presence of viral pathogens in biological materials used during in vitro embryo production.