RESUMO
A method based on Melting Temperature analysis of Hypervariable regions (HVR) of S1 gene within a RT-qPCR was developed to detect different genotypes of avian infectious bronchitis virus (IBV) and identify the Mass genotype. The method was able to rapidly identify the Mass genotype among IBV field isolates, vaccine attenuated strains and reference M41 strain in allantoic liquid and also directly in tissues. The RT-qPCR developed detected the virus in both tracheal and pulmonary samples from M41-infected or H120-infected birds, in a larger post-infection period compared to detection by standard method of virus isolation. RT-qPCR method tested provided a sensitivity and rapid approach for screening on IBV detection and Mass genotyping from IBV isolates.
Assuntos
Infecções por Coronavirus/veterinária , Vírus da Bronquite Infecciosa/genética , Doenças das Aves Domésticas/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Benzotiazóis , Galinhas/virologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Diaminas , Genótipo , Pulmão/virologia , Desnaturação de Ácido Nucleico , Compostos Orgânicos/uso terapêutico , Doenças das Aves Domésticas/virologia , Quinolinas , Traqueia/virologiaRESUMO
ABSTRACT Objective. The aim of this study was identify the different genotypes of infectious bronchitis virus (IVB) present in commercial poultry farms from different localities of the Tolima Department, Colombia. Materials and methods. 105 samples of tracheal swabs of poultry of 21 farms were collected. Poultry had been vaccinated against IVB. An screen to identify positive samples and posteriorly the sequencing of the partial region of the S1 subunit and phylogenetic analysis of the isolates with the reference strains, including the vaccine currently used in the country was performed. Results . Poultry all farms had respiratory signs, but only four farms was confirmed the disease. Positive samples of the IBV (HT6, HT9, HT10 and HT11) were pathogenic for embryos 9-days-old. The HT6 sample was grouped in the same cluster that the Massachusetts strains. The HT9 and HT11 samples showed 99% similarity and were grouped genetically distant from the reference strains and other isolated. The HT10 sample showed low similarities with the isolates and reference strains, grouping alone in another cluster. Conclusions . New genotypes are circulating in the Tolima Department, where there is a risk of genetic recombination. Is believed that vaccines used were not providing cross-protection against the new genotypes.
RESUMEN Objetivo. El objetivo de este estudio fue identificar los diferentes genotipos del virus de bronquitis infecciosa (VBI) presente en granjas avícolas comerciales de diferentes localidades del departamento de Tolima, Colombia. Materiales y métodos . Se recolectaron 105 muestras de hisopados traqueales provenientes de aves de 21 granjas. Las aves habían sido vacunadas contra el VBI. Se realizó un "screen" para identificar muestras positivas y posteriormente la secuenciación de la región parcial de la subunidad S1 y el análisis filogenético de los aislamientos con las cepas de referencia, incluidas las vacunas utilizadas actualmente en el país. Resultados. Aves de todas las granjas tenían signos respiratorios, pero sólo cuatro granjas confirmaron la enfermedad. Las muestras positivas de VBI (HT6, HT9, HT10 y HT11) fueron patogénicas para embriones de 9 días de edad. La muestra HT6 se agrupó en el mismo clúster que las cepas de la vacuna Massachusetts. Las muestras HT9 y HT11 mostraron 99% de similitud y agrupan genéticamente distantes de las cepas de referencia y de los aislados. El HT10 mostró baja similitud con aislamientos y cepas de referencia, agrupándose separadamente en otro clúster. Conclusiones. Nuevos genotipos circulan en el departamento del Tolima, donde hay un riesgo de recombinación genética. Se estima que las vacunas utilizadas no están ofreciendo una protección cruzada contra los nuevos genotipos.
RESUMO
Bovine coronavirus (BCoV) causes severe diarrhea in newborn calves, is associated with winter dysentery in adult cattle and respiratory infections in calves and feedlot cattle. The BCoV S protein plays a fundamental role in viral attachment and entry into the host cell, and is cleaved into two subunits termed S1 (amino terminal) and S2 (carboxy terminal). The present study describes a strategy for the sequencing of the BCoV S1 gene directly from fecal diarrheic specimens that were previously identified as BCoV positive by RT-PCR assay for N gene detection. A consensus sequence of 2681 nucleotides was obtained through direct sequencing of seven overlapping PCR fragments of the S gene. The samples did not undergo cell culture passage prior to PCR amplification and sequencing. The structural analysis was based on the genomic differences between Brazilian strains and other known BCoV from different geographical regions. The phylogenetic analysis of the entire S1 gene showed that the BCoV Brazilian strains were more distant from the Mebus strain (97.8 percent identity for nucleotides and 96.8 percent identity for amino acids) and more similar to the BCoV-ENT strain (98.7 percent for nucleotides and 98.7 percent for amino acids). Based on the phylogenetic analysis of the hypervariable region of the S1 subunit, these strains clustered with the American (BCoV-ENT, 182NS) and Canadian (BCQ20, BCQ2070, BCQ9, BCQ571, BCQ1523) calf diarrhea and the Canadian winter dysentery (BCQ7373, BCQ2590) strains, but clustered on a separate branch of the Korean and respiratory BCoV strains. The BCoV strains of the present study were not clustered in the same branch of previously published Brazilian strains (AY606193, AY606194). These data agree with the genealogical construction and suggest that at least two different BCoV strains are circulating in Brazil.