Pestivirus infections in ruminants result in significant economic losses worldwide. The aetiological agents are three species from the genus Pestivirus, family Flaviviridae, including bovine viral diarrhoea virus type 1 (BVDV-1), BVDV-2, border disease virus (BDV), and an atypical pestivirus named HoBi-like pestivirus. In this study, eighty-nine pestivirus isolates that were collected in Brazil between 1995 and 2014 and that originated from either cattle, fetal bovine serum (FBS) or as cell culture contaminants were genotyped based on a comparison of gene sequences from their 5' untranslated regions (5'UTR), N-terminal autoprotease (N ) and envelope glycoprotein 2 (E2). Of these isolates, 53.9% of the sequences were genotyped as BVDV-1, 33.7% as BVDV-2 and 12.4% as HoBi-like pestivirus. The prevalence of subgenotypes within the species was as follows: BVDV-1a (35.9%), BVDV-2b (31.4%), BVDV-1b (10.1%), BVDV-1d (6.7%), BVDV-2c (2.2%) and BVDV-1e (1.1%). BVDV-2c and BVDV-1e were detected for the first time in Brazil. This study revealed extensive genetic diversity among Brazilian pestivirus isolates, and the combination of pestiviruses that was detected is unique to Brazil. This information may serve as a foundation for designing and evaluating diagnostic tools and in the development of more effective vaccines; therefore, it may potentially contribute to pestivirus control and eradication.

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.

Summary 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.

Viruses from the genus Pestivirus of the family Flaviviridae have a non-segmented, single-stranded RNA genome and can cause diseases in animals from the order Artiodactyla. Homologous recombination is rarely reported in this virus family. To detect possible recombination events, all complete pestivirus genomes that are available in GenBank were screened using distinct algorithms to detect genetic conversions and incongruent phylogenies. Three putative recombinant viruses derived from recombination from different pestivirus subtypes/genogroups were detected: Bovine viral diarrhea virus 1 (BVDV-1) strain 3156, BVDV-2 strain JZ05-1 and Classical swine fever virus (CSFV) strain IND/UK/LAL-290. The present study demonstrated that the pestivirus classification cannot be based only on the analysis of one fragment of the genome because genetic conversions can lead to errors. The designation of the recombinant forms (RF) provides a more informative structure for the nomenclature of the genetic variant. The present work reinforces that homologous recombination occurs in pestivirus populations under natural replication and describes the first evidence of recombination in BVDV-2.

Passive monitoring for detection of influenza A viruses (IAVs) in pigs has been carried out in Brazil since 2009, detecting mostly the A(H1N1)pdm09 influenza virus. Since then, outbreaks of acute respiratory disease suggestive of influenza A virus infection have been observed frequently in Brazilian pig herds. During a 2010-2011 influenza monitoring, a novel H1N2 influenza virus was detected in nursery pigs showing respiratory signs. The pathologic changes were cranioventral acute necrotizing bronchiolitis to subacute proliferative and purulent bronchointerstitial pneumonia. Lung tissue samples were positive for both influenza A virus and A(H1N1)pdm09 influenza virus based on RT-qPCR of the matrix gene. Two IAVs were isolated in SPF chicken eggs. HI analysis of both swine H1N2 influenza viruses showed reactivity to the H1δ cluster. DNA sequencing was performed for all eight viral gene segments of two virus isolates. According to the phylogenetic analysis, the HA and NA genes clustered with influenza viruses of the human lineage (H1-δ cluster, N2), whereas the six internal gene segments clustered with the A(H1N1)pdm09 group. This is the first report of a reassortant human-like H1N2 influenza virus derived from pandemic H1N1 virus causing an outbreak of respiratory disease in pigs in Brazil. The emergence of a reassortant IAV demands the close monitoring of pigs through the full-genome sequencing of virus isolates in order to enhance genetic information about IAVs circulating in pigs.

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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.

The HoBi‐like pestivirus (HoBiPeV), currently classified as Pestivirus H species, is a pathogen associated with a broad spectrum of clinical manifestations in ruminants, particularly in cattle. Since HoBiPeV complete genome sequencing data is scarce, in the present study we described five nearly complete new Brazilian HoBiPeV genomes and further perform a more complete genetic and evolutionary characterization with all additional genome sequences available in the GenBank database. Entropy and selection pressure analysis showed the E2 gene, a surface glycoprotein, is the most variable gene, which also displays the greatest number of sites under positive selection. Phylogenetic and Bayesian inference based on complete genome and Npro gene, respectively, from all HoBiPeV sequences available so far, confirms the existence of three main clades (a, b, and c). The abovementioned analysis suggests that this pestivirus species probably emerged in Asia and spread to different regions including Brazil, where only strains belonging to specific genetic group ‘a’ have been found. The hypothesis of the HoBiPeV introduction in Brazil (between 1,890 and 1,962), formulated based on Bayesian inference, coincides with a period of intensive importation of water buffalo (Bubalus arnee) and indicine cattle (Bos taurus indicus) from Asia to Brazil, suggesting that this could be the origin of the current Brazilian HoBiPeV genetic group ‘a’.