Detection of Bovine Respiratory Syncytial Virus in Cell Cultures by nRT-PCR and Use of the Method for Virus Identification in Clinical Samples

Abstract

One-step RT-PCR and nested PCR were used to detect bovine respiratory syncytial virus (BRSV) in infected cell cultures. Specific 984 bp and 383 bp products, selected from gene encoding the F protein using suitable primers, were amplified and, if appropriate, reamplified. The method was further used for examination of nasal swabs and blood samples collected from animals showing signs of a respiratory disease. Lactating cows and 6-8-month-old bulls from two herds were investigated.The infection by BRSV was confirmed by a) demonstration of specific fragments of BRSV genome in all animals showing signs of an acute disease, and b) indirectly by serological methods in convalescent animals.Viral RNA was detected from nasal swabs and leukocytes of affected animals. The positive PCR product obtained by nested RT-PCR from RNA isolated from leukocytes was sequenced and nucleotide and amino acid homology were 97-100% and 94.5-100% respectively, when compared with sequences from the GenBank.Considering the difficulties associated with the demonstration of BRSV in cell cultures, the amplification techniques provide an effective tool for the identification of the causative agent of bovine respiratory diseases. Nested PCR; BRSV, F protein; diagnosticsBovine respiratory syncytial virus is a pneumovirus of the family Paramyxoviridae causing an acute respiratory disease in cattle. BRSV mostly attacks calves, in which the infection is manifested by pyrexia, anorexia, increased respiratory rate, and dyspnoea. Infections in lactating cows, accompanied by considerable decrease in milk yield by up to 60%, have also been described (Elvander 1996;Pritchard et al. 1997). Morbidity is high and death rate can reach up to 20% (Kovafiãík 1997). BRSV is regarded as the major causative agent of the bovine respiratory syndrome in West European states and the USA. The agent was also demonstrated in the local cattle herds (Pospí‰il et al. 1978; Kovafiãík 1999), but its prevalence in the Czech Republic has not yet been studied.The diagnosis is based on virus demonstration in cell cultures and serological methods for the demonstration of viral antigen and seroconversion. The methods used most frequently for antigen demonstration include the immunofluorescence test and the enzymatic immunosorbent assay (EIA) (Kimman et al. 1986;Krilov et al. 1988;Valarcher et al. 1999). Virus isolation is complicated by a high sensitivity of the agent and the fact that several passages are necessary before CPE develops. Tests on two blood samples collected at three-week intervals are necessary for the demonstration of seroconversion. Moreover, the interpretation of results is impeded by interaction with maternal antibodies present in blood sera of calves. Therefore, the serological methods are used mostly for the demonstration of past infections.

Molecular and Phylogenic Analysis of Bovine Respiratory Syncytial Virus in Nineveh province, Iraq

“…Diagnosis of BRSV isolated from nasal samples was determined using nested RT-PCR technique through a couple of primers target against (G) gene in nasal samples which indicate the accuracy and efficacy of the PCR technique for identification of BRSV in naturally infected cattle. This method can be used as a powerful technique in epidemiological studies, as reported in previous studies (Vilcek et al,1994;Valentova et al, 2003;Almeida et al, 2006). The result obtained in this study demonstrated that the G gene is very conservative and can be utilized as a target sequence for the identification of BRSV through RT-PCR.…”

Characterization of two BHV-4 strains isolated in the Czech Republic

“…We have isolated BHV-4 from nasal swabs obtained from calves (CH isolate) and dairy cows (Ni isolates) with respiratory distress. Duplicate serum samples from calves revealed elevated antibody titre against BRSV, although the virus could not be detected in clinical samples by specific nested RT-PCR (Valentova et al, 2003). Serum samples from cows were not available.…”

Antigenic and Molecular Identification of Some Viruses Causing Respiratory Disorder in Calves

“…Virus isolation was not attempted, since it is not considered to be a practical diagnostic method of choice (Vilcek, et al, 1994) as the viral isolation is complicated by the high sensitivity of the agent and the fact that several passages are necessary before CPE develops (Valentova and kovarcik, 2003). Also the isolation is a laborious procedure with unpredictable results because animal that develop the disease are not the best of choice as in most cases of BRSV or BPI-3 is obtained during isolation procedures for other viral pathogens rather than procedure specifically carried out for those viruses (Arns, et al, 2003).…”

“…Several RT-PCR for RSV detection based on the fusion (F) gene (Vilček, et al, 1994;Van der poel, et al, 1997 andValentova, et al, 2003) have been developed and evaluated in studies involving numerous field specimens. Therefore RT-PCR is potentially useful for improving the sensitivity of RSV detection.…”