Molecular detection of ovine gammaherpesvirus 2 in free ranging wild boars (Sus scrofa) from Southern Brazil.

Ovine gammaherpesvirus 2 (OvGHV2) is a member of Macavirus genus, subfamily Gammaherpesvirinae, family Herpesviridae, and causes sheep associated-malignant catarrhal fever (SA-MCF) in a wide range of ungulates. However, no descriptions of SA-MCF and/or infections due to OvGHV2 were identified in the wild boar (Sus scrofa). This study investigated the occurrence of OvGHV2 in the lungs (n = 44) of asymptomatic, free ranging wild boars captured in several regions of Paraná State, Southern Brazil. A PCR assay targeting the OvGHV2 tegument protein gene amplified OvGHV2 DNA in 4.55% (2/44) of the pulmonary tissues evaluated. Sequence analysis confirmed that the OvGHV2 strains herein identified have 98.4% deduced amino acid (aa) sequence identity with the prototype strain of OvGHV2 and 96.4-100% aa identity with similar strains of OvGHV2 detected in several animal species from diverse countries. These findings confirmed that these two wild boars were infected by OvGHV2, represent the first description of this infection in these animals, and add to the number of pathogens identified in this animal species. Furthermore, these findings contrast earlier descriptions of OvGHV2 in swine since in all previous reports the infected pigs demonstrated clinical manifestations of disease. Consequently, these wild boars from Southern Brazil were subclinically infected or suffered asymptomatic infections by OvGHV2.

A Single Amino Acid Substitution in Porcine Reproductive and Respiratory Syndrome Virus Glycoprotein 2 Significantly Impairs Its Infectivity in Macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a restricted tropism for macrophages and CD163 is a key receptor for infection. In this study, the PRRSV strain NCV1 was passaged on MARC-145 cells for 95 passages, and two plaque-clones (C1 and C2) were randomly selected for further analysis. The C1 virus nearly lost the ability to infect porcine alveolar macrophages (PAMs), as well as porcine kidney cells expressing porcine CD163 (PK15-pCD163), while the C2 virus replicates well in these two cell types. Pretreatment of MARC-145 cells with an anti-CD163 antibody nearly blocked C1 virus infection, indicating that the virus still required CD163 to infect cells. The C1 virus carried four unique amino acid substitutions: three in the nonstructural proteins and a K160I in GP2. The introduction of an I160K substitution in GP2 of the C1 virus restored its infectivity in PAMs and PK15-pCD163 cells, while the introduction of a K160I substitution in GP2 of the low-passaged, virulent PRRSV strain NCV13 significantly impaired its infectivity. Importantly, pigs inoculated with the rNCV13-K160I mutant exhibited lower viremia levels and lung lesions than those infected with the parental rNCV13. These results demonstrated that the K160 residue in GP2 is one of the key determinants of PRRSV tropism.

Porcine Circovirus 3a Field Strains in Free-Living Wild Boars in Paraná State, Brazil.

Porcine circovirus 3 (PCV-3) was identified in domestic pigs worldwide. Although PCV-3 has also been detected in wild boars, information regarding its circulation in this free-living animal species is scarce. To investigate PCV-3 occurrence in free-living wild boars in Brazil, 70 serum samples collected between January 2017 and June 2019 in Paraná state, Brazil were analyzed by PCR assay. Amplicons measuring 330 bp in length were amplified in seven (10.0%) of the serum samples and confirmed to be PCV3-specific by nucleotide (nt) sequencing. As the amplified products from the serum samples yielded only intermediate levels of viral DNA, lung samples from the seven PCR-positive wild boars were also evaluated by PCR. Of these samples, five lung samples were positive and provided high levels of viral DNA. The three lung samples that presented the highest levels of viral DNA were selected for amplification and sequencing of the whole PCV-3 genome. The three full-length sequences obtained were grouped in PCV-3 clade "a", and the sequences exhibited 100% nucleotide similarity among them. The PCV-3 field strains of this study showed nucleotide and amino acid similarities of 98.5-99.8% and 98.8-100%, respectively, with whole-genome PCV-3 sequences from around the world.

Seneca Valley virus induces immunodepressionin suckling piglets by selective apoptosis of B lymphocytes.

Seneca Valley virus (SVV) is the causative agent of an emerging infectious vesicular disease in swine that is clinically indistinguishable from other vesicular diseases of swine. This study utilized healthy suckling piglets (control) and SVV-naturally infected suckling piglets to determine the effects of SVV on lymphoid tissues and determined the SVV RNA load by quantitative RT-PCR (qRT-PCR). Furthermore, immunohistochemistry (IHC) analyses were performed to quantify the expression of T and B cell lymphocytes, natural killer cells, cleaved caspase 3, and ki-67. The main histopathologic finding in the infected group was severe lymphoid depletion. The highest average of SVV RNA load by qRT-PCR (Log10 genomic copies/g of tissue) occurred at the spleen (8.54 ± 0.8), followed by the tonsils (8.04 ± 1.42), and mesenteric lymph nodes (6.90 ± 1.42). The IHC analyses revealed that there was an increased in cellular apoptosis with concomitant reduction in the proliferation of B cells. The results from this study have demonstrated that SVV-infected piglets exhibited decreased lymphocyte density probably due to lymphoid apoptosis, affecting particularly B-cells lymphocytes.

Genome-Wide Variation in Betacoronaviruses.

The Severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 originated in bats and adapted to infect humans. Several SARS-CoV-2 strains have been identified. Genetic variation is fundamental to virus evolution and, in response to selection pressure, is manifested as the emergence of new strains and species adapted to different hosts or with novel pathogenicity. The combination of variation and selection forms a genetic footprint on the genome, consisting of the preferential accumulation of mutations in particular areas. Properties of betacoronaviruses contributing to variation and the emergence of new strains and species are beginning to be elucidated. To better understand their variation, we profiled the accumulation of mutations in all species in the genus Betacoronavirus, including SARS-CoV-2 and two other species that infect humans: SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Variation profiles identified both genetically stable and variable areas at homologous locations across species within the genus Betacoronavirus. The S glycoprotein is the most variable part of the genome and is structurally disordered. Other variable parts include proteins 3 and 7 and ORF8, which participate in replication and suppression of antiviral defense. In contrast, replication proteins in ORF1b are the least variable. Collectively, our results show that variation and structural disorder in the S glycoprotein is a general feature of all members of the genus Betacoronavirus, including SARS-CoV-2. These findings highlight the potential for the continual emergence of new species and strains with novel biological properties and indicate that the S glycoprotein has a critical role in host adaptation. IMPORTANCE Natural infection with SARS-CoV-2 and vaccines triggers the formation of antibodies against the S glycoprotein, which are detected by antibody-based diagnostic tests. Our analysis showed that variation in the S glycoprotein is a general feature of all species in the genus Betacoronavirus, including three species that infect humans: SARS-CoV, SARS-CoV-2, and MERS-CoV. The variable nature of the S glycoprotein provides an explanation for the emergence of SARS-CoV-2, the differentiation of SARS-CoV-2 into strains, and the probability of SARS-CoV-2 repeated infections in people. Variation of the S glycoprotein also has important implications for the reliability of SARS-CoV-2 antibody-based diagnostic tests and the design and deployment of vaccines and antiviral drugs. These findings indicate that adjustments to vaccine design and deployment and to antibody-based diagnostic tests are necessary to account for S glycoprotein variation.

Mycoplasma hyopneumoniae in free-living wild boars in Paraná, Brazil.

This is the first study conducted in Paraná, Brazil, to investigate Mycoplasma hyopneumoniae (Mhyo) infection in free-living wild boars. Eighty-eight wild boars were managed by authorized controllers between 2017 and 2019 in the state of Paraná in southern Brazil. Management georeferencing, sex, and weight were recorded for each animal. The presence of Mhyo antibodies in wild boar serum samples was evaluated using a commercial indirect ELISA kit. The presence of enzootic pneumonia-like gross lesions was evaluated, and the observed macroscopic lesions were subjected to immunohistochemistry (IHC). The Chi-square test and the intensity of the association with the odds ratio and 95% confidence interval were used to evaluate the differences in the qualitative variables between groups (sex and municipality). Juvenile wild boars exhibited a higher seroprevalence than older ones (p = 0.005). The Teixeira Soares municipality differed in Mhyo seroprevalence in comparison with Castro (p < 0.001), Ponta Grossa (p = 0.004), and Carambeí (p < 0.001). Females were 6.79 times more likely to present consolidation lesions than males (p = 0.004). Among the evaluated lung samples with injuries, 57.1% (8/14) and 53.8% (7/13) were Mhyo positive by IHC in Castro and Ponta Grossa, respectively, confirming that the identified macroscopic lesions were caused by Mhyo. This study demonstrates the circulation of Mhyo in free-living wild boars, which raises concerns regarding the epidemiological role of this animal species for the spread of the pathogen.

Porcine rotavirus B as primary causative agent of diarrhea outbreaks in newborn piglets.

Rotavirus (RV) is considered a major cause of acute viral gastroenteritis in young animals. RV is classified into nine species, five of which have been identified in pigs. Most studies worldwide have highlighted diarrhoea outbreaks caused by RVA, which is considered the most important RV species. In the present study, we described the detection and characterization of porcine RVB as a primary causative agent of diarrhoea outbreaks in pig herds in Brazil. The study showed a high frequency (64/90; 71.1%) of RVB diagnosis in newborn piglets associated with marked histopathological lesions in the small intestines. Phylogenetic analysis of the VP7 gene of wild-type RVB strains revealed a high diversity of G genotypes circulating in one geographic region of Brazil. Our findings suggest that RVB may be considered an important primary enteric pathogen in piglets and should be included in the routine differential diagnosis of enteric diseases in piglets.

Molecular characterization of Brazilian wild-type strains of bovine respiratory syncytial virus reveals genetic diversity and a putative new subgroup of the virus.

Background: Bovine orthopneumovirus, formerly known as bovine respiratory syncytial virus (BRSV), is frequently associated with bovine respiratory disease (BRD).Aim: To perform the molecular characterization of the G and F proteins of Brazilian wild-type BRSV strains derived from bovine respiratory infections in both beef and dairy cattle.Materials and Methods: Ten BRSV strains derived from a dairy heifer rearing unit (n = 3) in 2011 and steers of three other feedlots (n = 7) in 2014 and 2015 were analyzed. For the BRSV G and F partial gene amplifications, RT-nested-PCR assays were performed with sequencing in both directions with forward and reverse primers used.Results: The G gene-based analysis revealed that two strains were highly similar to the BRSV sequences representative of subgroup III, including the Bayovac vaccine strain. However, the remaining seven Brazilian BRSV strains were diverse when compared with strains representative of the BRSV I to VIII subgroups. The central hydrophobic region of the Brazilian BRSV G gene showed the replacement of conserved cysteines and other residues of importance to antibody reactivity. The deduced F gene amino acid sequences from the Brazilian BRSV strains showed changes that were absent in the representative sequences of the known subgroups. Viral isolation on the nasopharyngeal swab suspensions failed to isolate BRSV.Conclusion: Results suggest that these strains represent a putative new subgroup of BRSV with mutations observed in the immunodominant region of the G protein. However, further studies on these Brazilian BRSV strains should be performed to establish their pathogenic potential.

Detection of Equid gammaherpesvirus 2 and 5 DNA in the upper respiratory tract of asymptomatic horses from Southern Brazil.

Equid gammaherpesvirus 2 (EHV-2) and 5 (EHV-5) are members of the Herpesviridae family and have been reported in horse populations worldwide. This study aimed to evaluate the presence of herpesvirus DNA in the upper respiratory tract of horses. Twenty-six nasal swabs were collected from asymptomatic adult horses of two different horse farms (A, n = 18; B, n = 8), both located in Southern Brazil. The EHV-1, EHV-2, EHV-4, and EHV-5 DNA analyses were performed using nested PCR assays targeting the glycoprotein B gene. Four (15.3%) and 12 (46.1%) of the 26 nasal swab samples were positive for the EHV-2 and EHV-5, respectively. Four (15.3%) horses were detected with both viruses simultaneously. DNA of EHV-2 and EHV-5 in both single and mixed infections was identified in horses from both herds. All swab samples were negative for EHV-1 and EHV-4. This study reports the first detection of EHV-2 and EHV-5 in the upper respiratory tracts of horses in Brazil. The high detection rate of EHV-2 and EHV-5 in asymptomatic adult horses demonstrates that these gammaherpesviruses are circulating in Brazil.

Seneca Valley virus RNA detection in pig feed and feed ingredients in Brazil.

We investigated Seneca Valley virus (SVV) contamination in pig feed and feed ingredients. Twenty-seven samples were collected from two Brazilian feed mills and subjected to conventional RT-nested-PCR and qRT-PCR assays. Seven samples were SVV-positive with viral loads of 3.94-4.33 log10 genomic copies/g of feed. The study reveals SVV feed and feed ingredient contamination under natural conditions in Brazil.

Molecular analysis of the full-length F gene of Brazilian strains of canine distemper virus shows lineage co-circulation and variability between field and vaccine strains.

Canine distemper is a highly contagious systemic viral disease, with worldwide distribution that affects a wide variety of terrestrial carnivores. This study characterized full-length fusion (F) genes from 15 Brazilian wild-type canine distemper virus (CDV) strains collected between 2003-2004 (n = 6) and 2013-2016 (n = 9). Using deduced amino acid (aa) sequence analysis, 14 strains were classified into Europe 1/South America 1 (EU1/SA1) lineage, with a temporal clustering into past (2003-2004) and contemporary (2013-2016) strains. One strain clustered to Rockborn-like lineage, showing high similarity (98.5%) with the Rockborn vaccine strain. In analyzed strains, the fusion protein signal-peptide (Fsp) coding region was highly variable at the aa level (67.4%-96.2%). The Brazilian strains were more Fsp-divergent from the North America 1 (NA1) strains (24.5%-36.3%) than from the Rockborn (11.2%-14.9%) vaccine strain. Seventeen cysteine residues in the full-length F gene and four non-conserved glycosylation sites in the Fsp region were detected. The results reveal that past and contemporary CDV strains are currently co-circulating. This first analysis of full-length F genes from Brazilian wild-type CDV strains contributes to knowledge of molecular epidemiology of CDV viral infection and evolution.

Sanitary program to reduce embryonic mortality associated with infectious diseases in cattle.

Among reproductive disorders in dairy and beef cattle worldwide, embryonic mortalities stand out as one of the most frequent. Because of the multifactorial etiology, the clinical and laboratory diagnoses of embryonic mortality causes in cattle are quite complex. Often, infectious causes may account for up to 50% of bovine embryonic mortality rates after 30 days of conception. This review will address the main causes of early and late embryonic mortality, with emphasis on infectious causes and, particularly, those more frequent in the Brazilian cattle herds. In addition, we will discuss ways of controlling and prophylaxis including those related to reproductive and sanitary management, with emphasis on immunoprophylaxis of the three most frequent reproductive infectious diseases in Brazilian dairy and beef cattle herds.

Pathologic and molecular findings associated with atypical porcine pestivirus infection in newborn piglets.

Atypical porcine pestivirus (APPV) has been associated with congenital tremor (CT) type A-II in newborn piglets. Although the number of APPV-based studies is increasing, the associated pathologic findings in infected piglets are underreported. This study describes the histopathologic features of spontaneous APPV infection in CT-affected piglets and complements a previous report by our group. Four two-day-old piglets with CT were evaluated by histopathology, immunohistochemistry (IHC), and molecular assay. The main histopathologic findings at the brain and spinal cord included neuronal necrosis, gliosis, neuronophagia, satellitosis, demyelination, Wallerian degeneration, and Purkinje cell necrosis. An IHC assay designed to detect the proliferation of glial fibrillary acidic protein (GFAP) in affected areas of the brain and spinal cord revealed that the proliferation of GFAP + cells and fibers was predominant in APPV-infected piglets relative to asymptomatic piglets of the same age group. The RT-nested-PCR assays identified APPV RNA in the cerebrum, cerebellum, and brainstem of all piglets; other viruses known to produce similar manifestations were not detected. These results suggest that the APPV-induced histopathologic findings are predominantly degenerative and necrotic and correlate with our previous findings. Consequently, it is proposed that neuronal necrosis, gliosis, neuronophagia, and satellitosis should be considered as important histologic features of APPV-induced infection in symptomatic CT piglets.

Quantitative analysis of senecavirus A in tissue samples from naturally infected newborn piglets.

In this study, we determined the distribution of senecavirus A (SVA) and viral RNA load in different organs and tissues of naturally infected piglets. A TaqMan-based qRT-PCR assay was performed using RNA extracted from brainstem, cerebellum, cerebrum, heart, kidney, liver, lungs, small intestine, spleen, urinary bladder, and tonsils of seven newborn piglets. SVA was detected in 57 out of 70 tissue samples (81.4%). Viral loads ranged from 4.07 to 10.38 log10 genomic copies per g of tissue. The results show that SVA has tropism for various organs in naturally infected newborn piglets, especially for tonsils, spleen, lungs, and liver. Lymphoid organs had the highest viral loads and may be important sites for SVA replication.

A TaqMan-based qRT-PCR assay for Senecavirus A detection in tissue samples of neonatal piglets.

This study describes a sensitive (1.3 × 101 genomic copies/µL) and specific TaqMan-based qRT-PCR assay able to detect and quantify SVA RNA in porcine biological samples. The technique represents an efficient tool for the virus diagnosis and assessment of SVA load in tissues of infected animals and for epidemiological studies.

Age distribution of porcine sapovirus asymptomatic infection and molecular evidence of genogroups GIII and GIX? circulation in distinct Brazilian pig production systems.

This study aimed to evaluate the natural infection by SaV in pigs of different categories of production cycle in an important Brazilian pig-producing region. Faecal samples (n = 169) of suckling, post-weaning, finisher and breeder pig categories were analysed. Animals were from five farrow-to-weaning and nine grower-to-finish commercial pig farms. The RT-PCR assay was performed targeting the partial RNA-dependent RNA polymerase (RdRp) gene of porcine SaV genome. The virus was detected in 23.7% (40/169) of faecal samples and in 10/14 (5/5 farrow-to-weaning; 5/9 grower-to-finish) of pig farms evaluated. Porcine SaV was most frequently (p < 0.05) detected in pigs at post-weaning than in grower-to-finish and breeder categories. The phylogenetic analysis revealed that the porcine SaV strains belong to the GIII and GIX? genogroups. This study showed that the porcine SaV GIII genogroup has spread in the pig herds and provides the first evidence of GIX? genogroup circulation in South America.

Longitudinal study of Salmonella spp., diarrheagenic Escherichia coli, Rotavirus, and Coronavirus isolated from healthy and diarrheic calves in a Brazilian dairy herd.

This prospective longitudinal study investigated the epidemiology of enteric disease associated with infections in calves aging up to 70 days. A total of 850 fecal samples were collected from 67 calves. Seventeen isolates of Salmonella spp. were recovered from feces of 11 calves (16.4%), and statistical analysis revealed no association between the presence of Salmonella spp. and clinical signs of diarrhea or age. Virulence factors of Escherichia coli were identified in 103 strains: eae (7), K99/STa (7), Stx1 (7), Stx1/eae (36), Stx1/Stx2/eae (2), Stx2 (43), and Stx2/eae (1). There was statistical association between diarrheic animals carrying E. coli Stx1/eae (+) in their feces at 2 and 4 weeks of age (P = 0.003) and E. coli Stx2 (+) at 5 weeks of age (P = 0.03). Rotavirus was detected in 49 (5.76%) fecal samples collected from 33 calves (49.2%). The presence of rotavirus was correlated with diarrheic feces (P < 0.0001) rather than feces with normal consistency. There was a significant relationship between age group and diarrhea (P = 0.001). Bovine coronavirus (BCoV) was detected in 93 fecal samples collected from 46 calves (68.6%). There was an association (P < 0.0001) between diarrheic animals positive for BCoV and age groups. The results demonstrate the importance of the pathogens studied in the etiology of diarrhea in calves.