A comprehensive molecular analysis of bovine coronavirus strains isolated from Brazil and comparison of a wild-type and cell culture-adapted strain associated with respiratory disease.

Bovine coronavirus (BCoV) has dual tropisms that can trigger enteric and respiratory diseases in cattle. Despite its global distribution, BCoV field strains from Brazil remain underexplored in studies investigating the virus's worldwide circulation. Another research gap involves the comparative analysis of S protein sequences in BCoV isolates from passages in cell lines versus direct sequencing from clinical samples. Therefore, one of the objectives of our study was to conduct a comprehensive phylogenetic analysis of BCoV strains identified from Brazil, including a respiratory strain obtained during this study, comparing them with global and ancestral BCoV strains. Additionally, we performed a comparative analysis between wild-type BCoV directly sequenced from the clinical sample (nasal secretion) and the cell culture-adapted strain, utilizing the Sanger method. The field strain and multiple cell passage in cell culture (HRT-18) adapted BCoV strain (BOV19 NS) detected in this study were characterized through molecular and phylogenetic analyses based on partial fragments of 1,448 nt covering the hypervariable region of the S gene. The analyses have demonstrated that different BCoV strains circulating in Brazil, and possibly Brazilian variants, constitute a new genotype (putative G15 genotype). Compared with the ancestral prototype (Mebus strain) of BCoV, 33 nt substitutions were identified of which 15 resulted in non-synonymous mutations (nine transitions and six transversions). Now, compared with the wild-type strain was identified only one nt substitution in nt 2,428 from the seventh passage onwards, which resulted in transversion, neutral-neutral charge, and one substitution of asparagine for tyrosine at aa residue 810 (N810Y).

Porcine lymphotropic herpesvirus DNA detection in multiple organs of pigs in Brazil.

We investigated the porcine lymphotropic herpesvirus (PLHV) DNA presence in multiple organs of pigs. Biological samples (n = 136) included tissue fragments of the central nervous system, heart, kidney, liver, lungs, spleen, urinary bladder, and urine. Sixty-eight (50%) organs were PLHV DNA-positive. None of the urine samples were detected with the virus genome. Although the presence of the PLHV DNA in the urinary bladder and kidney has been detected, it was not possible to show whether urine can be considered an effective route of virus shedding. This study warns to the risk of PLHV zoonotic transmission by xenotransplantation of tissues of porcine origin.

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.

Longitudinal study of rotavirus C VP6 genotype I6 in diarrheic piglets up to 1 week old.

The reports of rotavirus C (RVC) involvement in diarrhea outbreaks in newborn piglets have been increasing in recent years. This longitudinal study, conducted over a 37-day period, aimed to evaluate the frequency of RVC infection in piglets aged up to 7 days obtained from a pig herd with a previous diagnosis of RVC infection in this age group. Piglets from 50 different litters were monitored daily for the occurrence of diarrhea, and all litters were classified into the following categories: sow parity order (PO) 1 to 5; litter size (LS) ≤ 10 piglets and > 10 piglets; and piglet birth weight (BW) 1.2 to 1.3 kg and > 1.3 to 1.4 kg. Two hundred six diarrheic fecal samples were collected and classified according to the fecal consistency score (pasty, semiliquid, liquid). Ten fecal samples were collected from asymptomatic piglets (control group). Fecal samples were screened for rotavirus (RV) by silver stained-polyacrylamide gel electrophoresis (ss-PAGE), and samples with inconclusive and negative-ss-PAGE results were submitted to RVC VP6 gene amplification by RT-PCR. RVC was identified in 71 (34.5%) samples, in 1 (10%) sample of the control group, and in piglets from 33 (66%) litters. The electrophoretic profile of RV species A was identified in only two samples. Of the 72 RVC-positive samples, 51 (70.8%) presented semiliquid or liquid consistency. There was no significant difference in either group regarding the production parameters (PO, LS, BW) evaluated. An analysis of the whole VP6 gene of three RVC field strains collected on the first, fifteenth, and last day of the experiment enabled us to identify genotype I6. This report describes the first longitudinal study examining epidemiological aspects of RVC infection in newborn piglets.

Detection of canine parvovirus types 2b and 2c in canine faecal samples contaminating urban thoroughfares in Brazil.

Canine parvovirus type 2 (CPV-2) is a highly contagious virus that causes acute gastroenteritis in dogs all over the world. Because of its stability in the environment, CPV-2 can remain infective for a long time, especially if protected in organic matter. To demonstrate CPV-2's potential as an environmental hazard for nonimmunized susceptible hosts, we investigated 50 faecal samples collected from public areas in a municipality of Paraná state, Brazil. Seven samples tested positive for CPV by a PCR assay targeting the partial VP2 gene, with three strains being confirmed as CPV-2b variant and one as CPV-2c variant by sequence analysis. These findings were supported by phylogenetic analysis, and the species identity of faecal samples source was confirmed by canine mitochondrial DNA amplification and sequencing. Our results demonstrate the presence of CPV in canine faeces contaminating urban thoroughfares and reinforce the importance of environmental control to reduce the potential exposure risks to susceptible hosts.

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.

High detection rate and genetic diversity of picobirnavirus in a sheep flock in Brazil.

This study reports the detection by RT-PCR and molecular characterization of partial RdRp gene of picobirnavirus (PBV) dsRNA in fecal samples (n = 100) from a meat sheep flock in southern Brazil. The analysis of the results allowed the identification of two important characteristics of PBV infection. The first was the high frequency of infection in the sheep flock evaluated where 62% of the analyzed fecal samples were PBV-positive. The second was the high genetic variability found in field strains of ovine PBV genogroup I circulating in animals of the same sheep flock.

Unusual outbreak of post-weaning porcine diarrhea caused by single and mixed infections of rotavirus groups A, B, C, and H.

Rotaviruses (RVs) are a major cause of severe diarrhea in humans and animals. Five of the nine RV groups (RVA, RVB, RVC, RVE, and RVH) have been previously detected in pigs; however, in pig herds worldwide, most studies highlight diarrhea outbreaks caused by RVA. In the present study, we describe detection and characterization of RV groups A, B, C, and H in fecal samples from pigs with single and mixed infections during a post-weaning diarrhea outbreak. The outbreak occurred in a single pig herd routinely vaccinated with an inactivated commercial vaccine for neonatal diarrhea control that included the RVA OSU (G5P[7]) strain. RVC (78%) was the most prevalent group found in single (34%) and mixed (44%) infections, followed by RVA (46%), RVB (32%), and RVH (18%). Phylogenetic analysis of three RVA strains allowed the characterization of two distinct G/P genotypes represented by G5P[13] and G9P[23], different from G5P[7] found in vaccines. Regardless of the RV group, mixed infections (54%) were more prevalent than single infections. Detection of RVB or RVH was associated with the presence of other RV groups, suggesting a secondary action of these RV groups in the reported outbreak. The detection of RV groups B, C, and H in the same pig herd suggests that these RVs act as causative agents of diarrhea and should be included in the diagnostic tests of porcine enteric diseases. These data provide new epidemiological information on RV diversity that need to be addressed in future studies for a better understanding and prevention of RV infections.

Genetic heterogeneity of the VP6 gene and predominance of G6P[5] genotypes of Brazilian porcine rotavirus C field strains.

The aim of this study was to investigate the genetic diversity of the VP6, VP7, and VP4 genes of 15 Brazilian wild-type porcine RVC strains identified in diarrheic fecal samples. The VP6 gene analysis demonstrated heterogeneity between the 15 RVC strains, which clustered in three distinct genotypes (I1, I5, and I6). In the VP7 and VP4 gene analysis, the genotype combination G6P[4] was detected in only one strain (UEL-77), while G6P[5] was the most commonly (n = 14) detected in RVC strains identified in the Brazilian pig herds evaluated, indicating its probable predominance in this country, mainly in 2014.

VP6 gene diversity in 11 Brazilian strains of porcine group C rotavirus.

Porcine group C rotavirus (RVC) is recognised as an enteric pathogen in piglets worldwide. The VP6 gene of RVC is divided into seven I-genotypes. Genotypes I2 and I3 are found in human and bovine strains, respectively; the porcine strains are divided into the other five genotypes (I1, I4-I7). In this study, molecular analysis of nearly the full length of the VP6 gene was performed in 11 Brazilian wild-type porcine RVC strains identified in diarrhoeic faecal samples, which were collected from eight pig farms located in five Brazilian states from piglets of 1-4 weeks of age. The nucleotide sequences of the VP6 gene showed 82.9-100 % identity between the Brazilian strains, 84.9-93.1 % with the prototype Cowden strain, and 82.4-92.2 % with other porcine RVC strains. In the 11 diarrhoeic faecal samples analysed in this study, three distinct porcine RVC genotypes (I1, I5, and I6) were identified and none were predominant. The results presented in this study revealed a high nucleotide diversity of the VP6 gene in porcine RVC field strains circulating in Brazil, which highlights the importance of further epidemiological and molecular surveys worldwide.

Diarrhea outbreaks in suckling piglets due to rotavirus group C single and mixed (rotavirus groups A and B) infections / Surtos de diarreia em leitões lactentes por rotavírus grupo C em infecções singulares e mistas (rotavirus grupos A e B)

Porcine group A rotavirus (PoRVA) is a major cause of neonatal diarrhea in suckling and recently weaned piglets worldwide. The involvement of non-group A rotavirus in cases of neonatal diarrhea in piglets are sporadic. In Brazil there are no reports of the porcine rotavirus group C (PoRVC) as etiologic agent of the diarrhea outbreaks in piglets. The aim of this study was to describe the identification of rotavirus group C in single and in mixed infection with rotavirus groups A and B in three neonatal diarrhea outbreaks in suckling (<21-day-old) piglets, with 70 percent to 80 percent and 20 percent to 25 percent of morbidity and lethality rates, respectively, in three pig herds located in the state of Santa Catarina, Brazil. The diagnosis of PoRV in the diarrheic fecal samples was performed using polyacrylamide gel electrophoresis (PAGE) to identify the presence of porcine rotavirus groups A, B (PoRVB), and C, and by RT-PCR (PoRVA and PoRVC) and semi-nested (SN)-PCR (PoRVB) to partially amplify the VP4 (VP8*)-VP7, NSP2, and VP6 genes of PoRVA, PoRVB, and PoRVC, respectively. […] The PoRVB strains (first and second outbreaks) and the PoRVC strains (first, second, and third outbreaks) showed higher nt identity and clustered in the phylogenetic tree with PoRVB and PoRVC strains that belong to the N4 and I1 genotypes, respectively. This is the first description in Brazil of the involvement of PoRVC in the etiology of diarrhea outbreaks in suckling piglets. The results of this study demonstrated that PoRVC, in both single and mixed infections, is an important enteropathogen involved in neonatal diarrhea outbreaks in piglets and that the use of more sensitive diagnostic techniques allows the identification of mixed infections involving two or even three groups of PoRV, which may be more common than previously reported.(AU)

O rotavírus suíno grupo A (PoRVA) é uma das principais causas de diarreia neonatal em leitões lactentes e recém-desmamados em todo o mundo. As descrições do envolvimento de rotavírus não-grupo A em quadros de diarreia neonatal em leitões são esporádicas. No Brasil não há relatos do envolvimento do rotavírus suíno grupo C (PoRVC) na etiologia dos surtos de diarreia em leitões. O objetivo deste estudo foi descrever a identificação de rotavírus grupo C em infecções singulares e mistas com os rotavírus grupos A e B em três surtos de diarreia neonatal em leitões lactentes (<21 dias de idade), com taxas de morbidade de 70 por cento a 80 por cento e de letalidade de 20 por cento a 25 por cento, em três rebanhos suínos localizados no estado de Santa Catarina, Brasil. O diagnóstico de PoRV nas amostras de fezes diarreicas foi realizado por eletroforese em gel de poliacrilamida (PAGE) para identificar a presença dos grupos A, B (PoRVB), e C de rotavírus suíno e por RT-PCR (PoRVA e PoRVC) e semi-nested (SN)-PCR (PoRVB) com a amplificação parcial dos genes VP4 (VP8*)-VP7, NSP2 e VP6 de PoRVA, PoRVB e PoRVC, respectivamente. [...] As cepas de PoRVB (primeiro e segundo surtos) e as cepas de PoRVC (primeiro, segundo e terceiro surtos) mostraram maior identidade de nt com cepas de PoRVB e PoRVC que pertencem aos genotipos N4 e I1, respectivamente. Esta é a primeira descrição realizada no Brasil do envolvimento de PoRVC na etiologia de surtos de diarreia em leitões lactentes. Os resultados deste estudo demonstram que o PoRVC, tanto em infecções singulares quanto em infecções mistas, é um importante enteropatógeno envolvido em surtos de diarreia neonatal em leitões e que o uso de técnicas de diagnóstico mais sensíveis permite caracterizar que infecções mistas, com dois ou até mesmo com três grupos de PoRV, podem ser mais comuns do que anteriormente relatado.(AU)

Diarrhea outbreaks in suckling piglets due to rotavirus group C single and mixed (rotavirus groups A and B) infections / Surtos de diarreia em leitões lactentes por rotavírus grupo C em infecções singulares e mistas (rotavirus grupos A e B)

Porcine group A rotavirus (PoRVA) is a major cause of neonatal diarrhea in suckling and recently weaned piglets worldwide. The involvement of non-group A rotavirus in cases of neonatal diarrhea in piglets are sporadic. In Brazil there are no reports of the porcine rotavirus group C (PoRVC) as etiologic agent of the diarrhea outbreaks in piglets. The aim of this study was to describe the identification of rotavirus group C in single and in mixed infection with rotavirus groups A and B in three neonatal diarrhea outbreaks in suckling (<21-day-old) piglets, with 70 percent to 80 percent and 20 percent to 25 percent of morbidity and lethality rates, respectively, in three pig herds located in the state of Santa Catarina, Brazil. The diagnosis of PoRV in the diarrheic fecal samples was performed using polyacrylamide gel electrophoresis (PAGE) to identify the presence of porcine rotavirus groups A, B (PoRVB), and C, and by RT-PCR (PoRVA and PoRVC) and semi-nested (SN)-PCR (PoRVB) to partially amplify the VP4 (VP8*)-VP7, NSP2, and VP6 genes of PoRVA, PoRVB, and PoRVC, respectively. […] The PoRVB strains (first and second outbreaks) and the PoRVC strains (first, second, and third outbreaks) showed higher nt identity and clustered in the phylogenetic tree with PoRVB and PoRVC strains that belong to the N4 and I1 genotypes, respectively. This is the first description in Brazil of the involvement of PoRVC in the etiology of diarrhea outbreaks in suckling piglets. The results of this study demonstrated that PoRVC, in both single and mixed infections, is an important enteropathogen involved in neonatal diarrhea outbreaks in piglets and that the use of more sensitive diagnostic techniques allows the identification of mixed infections involving two or even three groups of PoRV, which may be more common than previously reported.

O rotavírus suíno grupo A (PoRVA) é uma das principais causas de diarreia neonatal em leitões lactentes e recém-desmamados em todo o mundo. As descrições do envolvimento de rotavírus não-grupo A em quadros de diarreia neonatal em leitões são esporádicas. No Brasil não há relatos do envolvimento do rotavírus suíno grupo C (PoRVC) na etiologia dos surtos de diarreia em leitões. O objetivo deste estudo foi descrever a identificação de rotavírus grupo C em infecções singulares e mistas com os rotavírus grupos A e B em três surtos de diarreia neonatal em leitões lactentes (<21 dias de idade), com taxas de morbidade de 70 por cento a 80 por cento e de letalidade de 20 por cento a 25 por cento, em três rebanhos suínos localizados no estado de Santa Catarina, Brasil. O diagnóstico de PoRV nas amostras de fezes diarreicas foi realizado por eletroforese em gel de poliacrilamida (PAGE) para identificar a presença dos grupos A, B (PoRVB), e C de rotavírus suíno e por RT-PCR (PoRVA e PoRVC) e semi-nested (SN)-PCR (PoRVB) com a amplificação parcial dos genes VP4 (VP8*)-VP7, NSP2 e VP6 de PoRVA, PoRVB e PoRVC, respectivamente. [...] As cepas de PoRVB (primeiro e segundo surtos) e as cepas de PoRVC (primeiro, segundo e terceiro surtos) mostraram maior identidade de nt com cepas de PoRVB e PoRVC que pertencem aos genotipos N4 e I1, respectivamente. Esta é a primeira descrição realizada no Brasil do envolvimento de PoRVC na etiologia de surtos de diarreia em leitões lactentes. Os resultados deste estudo demonstram que o PoRVC, tanto em infecções singulares quanto em infecções mistas, é um importante enteropatógeno envolvido em surtos de diarreia neonatal em leitões e que o uso de técnicas de diagnóstico mais sensíveis permite caracterizar que infecções mistas, com dois ou até mesmo com três grupos de PoRV, podem ser mais comuns do que anteriormente relatado.