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).

Investigation of avian rotavirus infections in broiler chicks from commercial flocks with different performance efficiency indexes.

The aim of this study was to investigate and compare the frequency of occurrence of avian rotavirus (AvRV) in poultry flocks according to its Performance Efficiency Index (PEI) scores. A total of 256 individual intestinal content samples of small sized-chicks (runts) with clinical signs of Runting Stunting Syndrome (RSS) and 24 clinically healthy chicks (control) were collected from twelve flocks in southern Brazil with different PEI scores: good (n = 4, PEI mean = 365); moderate (n = 4, PEI mean = 342) or poor (n = 4, PEI mean = 319). Silver-stained polyacrylamide gel electrophoresis (ss-PAGE) was used to detect and identify the AvRV species followed by RT-PCR and sequencing of the partial VP6 gene for species confirmation. AvRV was detected in 83% (10/12) of the flocks and 23.4% (60/256) of the chicks. The electrophoretic migration patterns of viral dsRNA segments were compatible with AvRV species A (AvRV- A), D (AvRV-D) and F (AvRV-F) in 9 (15%), 18 (30%), and 33 (55%) of the positive chicks fecal samples, respectively. The AvRV species identified by ss-PAGE were confirmed by RT-PCR and partial sequence analysis of the VP6 gene. The AvRV detection rate was statistically higher (p = 0.007) in chicks from flocks with poor PEI when compared to those with good PEI. The occurrence of AvRV-D and AvRV-F was statistically higher in 7 to 9 days old chicks, while AvRV-A was detected only in 13 to 14 days old animals.

Detection and partial molecular characterization of Picobirnavirus in swine from the state of Minas Gerais, Brazil.

Picobirnavirus (PBV) is a small two-segmented double-stranded RNA (dsRNA) virus that has been identified in diarrheic feces of a large range of animal hosts, including humans. For this reason, PBV has been recognized as an opportunistic agent of gastrointestinal disease. Even under these circumstances, there is a lack of studies regarding this pathogen. Not outstanding, in Brazil, the single description of the PBV occurrence in pigs was provided in the 1980s. Hence, this study aimed to verify the PBV occurrence in Brazilian swine farms and to perform molecular characterization of the identified strains. High genetic variability was found in the analyzed sequences. Further studies comprehending the infection of swine by PBV in Brazilian herds should be performed to provide more accurate information on its epidemiology and to discuss the role of the virus in gastrointestinal diseases.

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.

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.

Electrophoretic RNA genomic profiles of Brazilian Picobirnavirus (PBV) strains and molecular characterization of a PBV isolated from diarrheic calf.

Picobirnavirus (PBV) belongs to the family Picobirnaviridae. PBV are a group of emerging non-enveloped viruses, with a bisegmented double-stranded RNA genome that can infect a wide range of hosts. This study reports the occurrence of PBV in fecal samples from five Brazilian dairy cattle herds. From the 289 stool samples of individual calves analyzed by silver-stained polyacrylamide gel electrophoresis (ss-PAGE) the PBV was detected in 8.3 % (24/289), of which 10.2% (18/176) had diarrheic consistency. Of the 24 positive samples in ss-PAGE, 5 (20.8%) of them showed a small electrophoretic profile and 19 (79.2%) samples had large profile. From the 24 positives samples by ss-PAGE, 15 (62.5%) were successfully amplified (201 bp) using GI specific primers targeting the RdRp gene of PBV. The analysis of nucleotide identity matrix revealed that the bovine PBV strain identified in this study, showed the highest nucleotide identity (81%) with PBV strain detected in turkey (MD-2010/HM803965). This is the first nucleotide sequence of a bovine PBV strain in the American continent and the first detection of small genome profile of PBV-like strains in bovine hosts.

An outbreak of winter dysentery caused by bovine coronavirus in a high-production dairy cattle herd from a tropical country

Bovine coronavirus (BCoV) is a known cause of winter dysentery (WD) in adult cattle. The morbidity of the disease is high, that results in a significant decrease in milk production and consequently, economic losses. In the present study, we report on a classical outbreak of WD that affected a high-production Holstein dairy herd raised in a tropical country. The lactating batch included 154 cows, and 138 (90 percent) presented diarrhea in a short (nine days) period of time. Three (2 percent) cows died. The other batches of animals did not become ill. The evolution of the disease in the herd, including the clinical signs and epidemiological features, strongly suggested a WD case. Semi-nested PCR and RFLP confirmed that BCoV was the cause of the infection. Samples tested negative for all other enteric pathogens. This case report highlights the importance of BCoV in WD even in tropical countries such as Brazil.

O coronavirus bovino (BCoV) pode causar a diarreia de inverno (WD - Winter Dysentery) ao infectar bovinos adultos, particularmente em regiões de clima temperado ou frio. A morbidade da doença é alta, resultando em queda na produção de leite e, consequentemente, perdas econômicas. No presente estudo, é descrito um surto clássico de WD acometendo um rebanho de bovinos leiteiros da raça Holandesa PB, de alta produção, proveniente do estado do Paraná. O lote afetado era composto por 154 vacas em lactação, sendo que 138 (90 por cento) apresentaram diarreia em um curto (nove dias) período de tempo e 3 (2 por cento) vacas morreram em consequência da diarreia, desidratação e desequilíbrio eletrolítico. As outras categorias de animais do rebanho (bezerras, novilhas e vacas secas) não apresentaram sinal clínico. A evolução da doença clínica, assim como a epidemiologia da infecção sugeriu um quadro clássico de WD. O diagnóstico foi realizado por meio da identificação do BCoV, pela técnica de semi-nested PCR e confirmação por RFLP, em amostra fecal de uma vaca que veio a óbito. A presença de outros patógenos entéricos também foi avaliada e apresentou resultados negativos. O surto de WD descrito na região sul do Brasil alerta para a possibilidade da ocorrência dessa virose também em países situados em regiões de clima tropical.

Frequency of BCoV detection by a semi-nested PCR assay in faeces of calves from Brazilian cattle herds.

Bovine coronavirus (BCoV) is one of the main causes of neonatal calf diarrhoea. Several diagnostic assays have been employed to detect the presence of the virus in stool samples from calves. Despite this, the frequency of BCoV infection among Brazilian and even South American cattle herds has yet to be well characterised. This study describes the occurrence of BCoV infection among calves from dairy and beef herds in four Brazilian states. A total of 282 stool samples from 1 to 60-day-old calves were evaluated for the presence of BCoV by a semi-nested (SN) PCR assay. The animals were from herds (n = 23) located in three geographical regions in Brazil (south, southeast, and center-west). The specific BCoV amplicon was detected in 15.6% (44/282) of the faecal specimens examined, of which 95.4% (42/44) were from diarrhoeic and 4.6% (2/44) from asymptomatic calves. The specificity of the SN-PCR amplicons was evaluated by restriction fragment length polymorphism (RFLP) analysis. The results show that the BCoV is widespread, mainly among calves from 16 to 30-days-old (p = 0.0023), and verify the association between BCoV infection and clinical signs of diarrhoea (p = 0.005). These findings emphasise the importance of this virus in enteric infections of Brazilian cattle herds.

Identification of a mutation in the spike protein cleavage site in Brazilian strains of wild-type bovine coronavirus

The spike (S) protein of coronaviruses, a type I membrane glycoprotein, is primarily responsible for entry into susceptible cells by binding with specific receptors on cells and mediating subsequent virus-cell fusion. The bovine coronavirus (BCoV) S protein is cleaved into two subunits, the N-terminal S1 and the C-terminal S2. The proteolytic cleavage site of S protein is highly conserved among BCoV strains and is located between amino acids 763 and 768 (KRRSRR). This study describes a single mutation in the S protein cleavage site of three Brazilian strains of BCoV detected in diarrheic fecal samples from calves naturally infected. The sequenced PCR products revealed that amino acid sequence of the cleavage site of our strains was KRRSSR, indicating a mutation at amino acid position 767 (R ® S). This amino acid substitution occurred due to a single nucleotide substitution in the sequence of DNA corresponding to the proteolytic cleavage site, CGT to AGT. This is the first description of this nucleotide mutation (C to A), which resulted in the substitution of arginine to serine in the S cleavage site. In this study we speculated the probable effects of this mutation in the proteolytic cleavage site using the murine hepatitis coronavirus (MHV) as a comparative model.

A proteína da espícula (S), uma glicoproteína de membrana do tipo I, é primariamente responsável pela entrada do vírus em células susceptíveis por meio da interação inicial com receptores celulares específicos e subseqüente mediação da fusão vírus-célula. A proteína S do coronavírus bovino (BCoV) é clivada em duas subunidades: a S1, na região N-terminal e a S2, na região C-terminal. O sítio de clivagem proteolítica da proteína S é altamente conservado entre as estirpes de BCoV e está situado entre os aminoácidos 763-768 (KRRSRR). Este estudo descreve uma mutação no sítio de clivagem da proteína S de três estirpes do BCoV detectadas em amostras fecais diarréicas de bezerros naturalmente infectados no Brasil. O seqüenciamento dos produtos de PCR identificou a seqüência de aminoácidos KRRSSR no sítio de clivagem de nossas amostras, indicando uma mutação na posição 767 (R->S). Esta mutação ocorreu devido a uma única substituição de nucleotídeo no sítio de clivagem proteolítica, alterando o códon CGT para AGT. Esta é a primeira descrição desta mutação de nucleotídeo (C para A), que resultou na substituição do aminoácido arginina por serina no sítio de clivagem da proteína S. Neste estudo também são sugeridos os prováveis efeitos desta mutação no sitio de clivagem proteolítica utilizando o coronavírus da hepatite dos camundongos (MHV) como um modelo comparativo.

Improved detection of bovine coronavirus N gene in faeces of calves infected naturally by a semi-nested PCR assay and an internal control.

Bovine coronavirus (BCoV), a positive sense single-stranded RNA virus, is an important causative agent of neonatal diarrhoea in calves from beef and dairy cattle worldwide. The routine detection and diagnosis of BCoV have been mainly dependent on assays with low sensitivity. The aim of the present study was to develop and evaluate a semi-nested PCR (SN-PCR) to amplify a 251bp fragment of BCoV N gene from fresh (n=25) and frozen (n=25) diarrhoeic faecal samples of naturally infected calves. To improve detection of BCoV in faecal samples by the SN-PCR an internal control was developed, and the results were compared with a conventional RT-PCR assay. The rates of positive samples by SN-PCR and RT-PCR were 24% (12/50) and 8% (4/50), respectively (K=0.43). Only fresh samples were positive in RT-PCR while the SN-PCR detected BCoV in both fresh and frozen faecal samples. The sensitivity of SN-PCR was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) that was detected until 10(-7) dilution. The specificity of the amplicons was assessed by restriction fragment length polymorphism and sequence analysis. The inclusion of an internal control provides a way to detect assay inhibition in faecal samples and failure of nucleic acid extraction that allow reduction of the number of false-negative results.

Avaliação da capacidade adjuvante do cloreto de dimetildioctadecilamônio associado ao hidróxido de alumínio na indução da resposta imune humoral de bovinos vacinados com o vírus da diarréia viral bovina / Evaluation of the adjuvant activity of dimethyl dioctadecyl ammonium chloride associated with the ammonium hydroxide to induction of humoral immune response of cattle vaccinated with bovine viral diarrhea virus

A resposta imunológica humoral de bovinos vacinados com o vírus da diarréia viral bovina (BVDV) inativado, tendo como adjuvante o cloreto de dimetildioctadecilamônio (DDA cloreto) associado ao hidróxido de alumínio (vacina B), foi comparada com uma vacina contendo o mesmo antígeno adsorvido apenas com hidróxido de alumínio (vacina A). Duas semanas após a segunda dose foi avaliado o título de anticorpos neutralizantes dos animais que receberam as duas preparações de antígenos. Os animais que receberam a vacina B apresentaram melhor resposta imune humoral quando comparados com os animais vacinados com a vacina A. O título médio de anticorpos neutralizantes, expresso em Log2, dos animais que receberam a vacina B foi superior (P<0,05) ao observado no grupo vacinado com a vacina A. Esse resultado demonstra que, em bovinos vacinados com o BVDV inativado, a inclusão do DDA cloreto em formulações de vacinas adsorvidas com hidróxido de alumínio potencializa a resposta imune humoral.