Karen Miyuki Asano

Multiplex semi-nested RT-PCR with exogenous internal control for simultaneous detection of bovine coronavirus and group A rotavirus

Abstract Neonatal calf diarrhea is a multi-etiology syndrome of cattle and direct detection of the two major agents of the syndrome, group A rotavirus and Bovine coronavirus (BCoV) is hampered by their fastidious growth in cell culture. This study aimed at developing a multiplex semi-nested RT-PCR for simultaneous detection of BCoV (N gene) and group A rotavirus (VP1 gene) with the addition of an internal control (mRNA ND5). The assay was tested in 75 bovine feces samples tested previously for rotavirus using PAGE and for BCoV using nested RT-PCR targeted to RdRp gene. Agreement with reference tests was optimal for BCoV (kappa=0.833) and substantial for rotavirus detection (kappa=0.648). the internal control, ND5 mRNA, was detected successfully in all reactions. Results demonstrated that this multiplex semi-nested RT-PCR was effective in the detection of BCoV and rotavirus, with high sensitivity and specificity for simultaneous detection of both viruses at a lower cost, providing an important tool for studies on the etiology of diarrhea in cattle.

First detection and molecular diversity of Brazilian bovine torovirus (BToV) strains from young and adult cattle

Abstract Bovine torovirus (BToV) is an established enteric pathogen of cattle, but its occurrence in Brazilian cattle had not been reported until now. This article describes a survey on BToV in Brazil carried out on 80 fecal samples from diarrheic young and adult cattle, using a nested-RT-PCR targeting the nucleocapsid (N) gene. BToV was detected in 6.25% (5/80) of stool samples from three different geographic regions. Sequences analysis showed that Brazilian BToVs have a high degree of identity with European and Japanese BToVs and a lower degree of identity with North American Breda 1 strain. These results show that, albeit its low frequency and the scarce number of research on the field, BToV is still present amongst cattle populations.

Delayed progression of rabies transmitted by a vampire bat.

Here, we compared the growth kinetics, cell-to-cell spread, and virus internalization kinetics in N2a cells of RABV variants isolated from vampire bats (V-3), domestic dogs (V-2) and marmosets (V-M) as well as the clinical symptoms and mortality caused by these variants. The replication rate of V-3 was significantly higher than those of V-2 and V-M. However, the uptake and spread of these RABV variants into N2a cells were inversely proportional. Nevertheless, V-3 had longer incubation and evolution periods. Our results provide evidence that the clinical manifestations of infection with bat RABV variant occur at a later time when compared to what was observed with canine and marmoset rabies virus variants.

Intrahost Diversity of Feline Coronavirus: A Consensus between the Circulating Virulent/Avirulent Strains and the Internal Mutation Hypotheses?

To evaluate the most controversial issue concerning current feline coronavirus (FCoV) virology, the coexisting hypotheses of the intrahost and interhost origins of feline infectious peritonitis virus (FIPV) in regard to the pathogenesis of feline infectious peritonitis (FIP), this study aimed to assess the molecular diversity of the membrane gene FCoVs in 190 samples from 10 cats with signs of FIP and in 5 faecal samples from cats without signs of FIP. All samples from the non-FIP cats and 25.26% of the samples from the FIP cats were positive for the FCoV membrane (M) gene. Mutations in this gene consisted of SNP changes randomly scattered among the sequences; few mutations resulted in amino acid changes. No geographic pattern was observed. Of the cats without FIP that harboured FECoV, the amino acid sequence identities for the M gene were 100% among cats (Cats 1–3) from the same cattery, and the overall sequence identity for the M gene was ≥91%. In one cat, two different lineages of FCoV, one enteric and one systemic, were found that segregated apart in the M gene tree. In conclusion, the in vivo mutation transition hypothesis and the circulating high virulent-low virulent FCoV hypothesis have been found to be plausible according to the results obtained from sequencing the M gene.

Rapid detection of bovine coronavirus by a semi-nested RT-PCR

O Coronavirus bovino (BCoV) pertence ao grupo 2 do genero Coronavirus (Nidovirales: Coronaviridae) e e agente causador de enterites tanto em bezerros como em bovinos adultos, bem como de doenca respiratoria em bezerros. O presente estudo teve por objetivo desenvolver uma semi-nested RT-PCR para a deteccao do BCoV com base em sequencias representativas e recentes do gene do nucleocapsideo, regiao conservada do genoma dos coronavirus. Tres primers foram desenhados, a primeira amplificacao com um fragmento esperado de 463pb e a segunda (semi-nested) com um fragmento esperado de 306pb. A sensibilidade analitica foi determinada pela diluicao do BCoV cepa Kakegawa (titulo HA: 256) na base de 10 em agua ultra-pura tratada com DEPC, em soro fetal bovino (SFB) e em uma suspensao fecal negativa para o BCoV, onde foram encontrados resultados positivos ate a diluicao de 10-2, 10-3 e 10-7, respectivamente. Este resultado sugere que a quantidade total de RNA na amostra influencia na precipitacao dos pellets pelo metodo de extracao utilizado. Quando se utiliza amostra fecal, a grande quantidade de RNA total funciona como carreadora do RNA do BCoV, demonstrando elevada sensibilidade analitica e ausencia de possiveis substâncias inibidoras da PCR. O protocolo final da semi-nested RT-PCR foi aplicado a 25 amostras fecais de vacas adultas, previamente avaliadas por uma nested RT-PCR RdRp utilizada como teste de referencia, resultando em 20 e 17 amostras positivas para o primeiro e segundo teste, respectivamente. Os resultados dos dois sistema de diagnostico apresentaram concordância substancial (kappa: 0,694). A elevada sensibilidade e especificidade do novo metodo proposto e o fato de que os primers foram desenhados baseados em sequencias atuais do BCoV, oferecem bases para o diagnostico mais acurado de infeccoes causadas pelo BCoV, assim como para novas perspectivas em protocolos de deteccao de outros Coronavirus de importância tanto em saninade animal quanto em saude publica.

Alphacoronavirus in urban Molossidae and Phyllostomidae bats, Brazil.

BackgroundBats have been implicated as the main reservoir of coronavirus (CoV). Thus the role of these hosts on the evolution and spread of CoVs currently deserve the attention of emerging diseases surveillance programs. On the view of the interest on and importance of CoVs in bats the occurrence and molecular characterization of CoV were conducted in bats from Brazil.FindingsThree hundred five enteric contents of 29 bat species were tested using a panCoV nested RT-PCR. Nine specimens were positive and eight was suitable for RdRp gene sequencing. RdRp gene phylogeny showed that all CoVs strains from this study cluster in Alphacoronavirus genus, with one Molossidae and one Phlyllostomidae-CoV specific groups. Phylogenetic analyses of two S gene sequences showed a large diversity within the Alphacoronavirus genus.ConclusionsThis study indicated a CoV-to-host specificity and draws attention for CoV detection in Cynomops sp, a potential new reservoir. The phylogenetic analyses indicate that diversity of CoV in bats is higher than previously known.

A Multigene Approach for Comparing Genealogy of Betacoronavirus from Cattle and Horses

Gastroenteritis is one of the leading causes of morbidity and mortality among young and newborn animals and is often caused by multiple intestinal infections, with rotavirus and bovine coronavirus (BCoV) being the main viral causes in cattle. Given that BCoV is better studied than equine coronaviruses and given the possibility of interspecies transmission of these viruses, this research was designed to compare the partial sequences of the spike glycoprotein (S), hemagglutinin-esterase protein (HE), and nucleoprotein (N) genes from coronaviruses from adult cattle with winter dysentery, calves with neonatal diarrhea, and horses. To achieve this, eleven fecal samples from dairy cows with winter dysentery, three from calves, and two from horses, all from Brazil, were analysed. It could be concluded that the enteric BCoV genealogy from newborn and adult cattle is directly associated with geographic distribution patterns, when S and HE genes are taken into account. A less-resolved genealogy exists for the HE and N genes in cattle, with a trend for an age-related segregation pattern. The coronavirus strains from horses revealed Betacoronavirus sequences indistinguishable from those found in cattle, a fact previously unknown.

Erratum to: Alphacoronavirus in urban Molossidae and Phyllostomidae bats, Brazil

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Differentiation of Bovine coronavirus (BCoV) genotypes by a restriction enzyme assay

This article reports the use of the GsuI restriction enzyme to differentiate genotypes of Bovine Coronavirus (BCoV), based on an 18-nucleotide deletion of S1-coding region found in one of the two genotypes. It was concluded that this assay can be used as a rapid tool for BCoV genotypes differentiation.This article reports the use of the GsuI restriction enzyme to differentiate genotypes of Bovine Coronavirus (BCoV), based on an 18-nucleotide deletion of S1-coding region found in one of the two genotypes. It was concluded that this assay can be used as a rapid tool for BCoV genotypes differentiation.