Real Time RT-PCR for the detection and quantitation of bovine respiratory syncytial virus

https://doi.org/10.1016/j.jviromet.2005.01.008Get rights and content

Abstract

A quantitative Real Time RT-PCR assay was developed to detect and quantify bovine respiratory syncytial virus (BRSV) in the respiratory tract of infected animals. A pair of primers and a TaqMan probe targeting conserved regions of the nucleoprotein gene of BRSV were designed. The detection limit of the assay was shown to be 103 RNA copies and standard curve demonstrated a linear range from 103 to 108 copies as well as an excellent reproducibility. The efficiency of the BRSV Real Time RT-PCR was then assessed by detecting BRSV in lungs, tracheas and bronchoalveaolar fluids (BAL) samples of experimentally infected calves. The assay was shown to be 100 times more sensitive than conventional RT-PCR and was more efficient for BRSV diagnosis. Finally, the Real Time RT-PCR was used to quantify BRSV load in BAL fluids of four experimentally infected calves for 14 days. The high sensitivity, rapidity and reproducibility of the BRSV Real Time RT-PCR make this method suitable for diagnostic and for the evaluation of the efficiency of new vaccines.

Introduction

Bovine respiratory syncytial virus (BRSV) and human respiratory syncytial virus (HRSV) belong to the Pneumovirus genus of the Paramyxoviridae family (Stott and Taylor, 1985). These two negative single-stranded RNA viruses share common genomic, antigenic, epidemiological and pathological characteristics (Van Der Poel et al., 1994) and are the major cause of lower respiratory tract diseases in cattle and young infants, respectively (Stott and Taylor, 1985).

Seventy percent of calves exhibit a positive serological response against BRSV at the age of 12 months. Infection of cattle remains asymptomatic, can lead to mild respiratory symptoms or even cause severe mortalities up to 20% (Wellemans, 1990). Based on antigenic properties, BRSV isolates have been divided in three subgroups (A, A/B and B) (Furze et al., 1994, Furze et al., 1997, Schrijver et al., 1996). More recently, analysis of nucleotidic sequences of BRSV have revealed that subgroups are the result of a continuous evolution of the BRSV F, N and G proteins, occurring in isolates since 1967 (Prozzi et al., 1997, Valarcher et al., 2000).

Inactivated and attenuated vaccines have been available for long time. However, several problems associated with vaccination of calves have been described as short time protection, inefficacy on young calves under colostral protection (Kimman et al., 1989b) and amplified illness after infection (Kimman et al., 1989c, Schreiber et al., 2000).

Development of new vaccines against BRSV is needed and in such experiments, the study of virus shedding and replication is of first importance. However, classical methods of diagnosis based on BRSV antigen detection or virus isolation from lung samples, nasal swabs or bronchoalveaolar lavage (BAL) fluids demonstrated poor sensitivity due to low viral titers shed and lability of the BRSV (Kimman et al., 1986, Kimman et al., 1989a). To overcome these problems, reverse transcription-polymerase chain reaction (RT-PCR), directed against the fusion and glycoprotein genes (Larsen et al., 1999, Valarcher et al., 2001) and nested RT-PCR, directed against the nucleoprotein gene (Valarcher et al., 1999) and fusion gene (Valentova et al., 2003), have been developed for the detection of BRSV in naturally and experimentally infected calves and have been shown to be more sensitive than antigen detection and virus isolation. Nevertheless, these techniques do not allow a rapid and quantitative method for the detection of the BRSV.

Recently, the Real Time PCR technology has been developed. This technique, based on thermal annealing and real time fluorescence measurement, is a rapid, sensitive and quantitative method for the detection of nucleic acids (Holland et al., 1991, Livak et al., 1995).

In this report, a Real Time RT-PCR assay targeting the nucleoprotein gene of BRSV is described. This technique was used for the detection and quantitation of the virus in the respiratory tract of experimentally infected calves.

Section snippets

Viruses isolates

Nine strains of BRSV were used to validate the Real Time PCR assay (Table 1). Strains RB94 and MRV533, were isolated in Belgium and described by Wellemans and Leunen (1975). BovX strain was isolated in Switzerland and described by Paccaud and Jacquier (1970). The Lelystad strain was isolated in The Netherlands and was described by Kimman et al. (1986). NMK7 was isolated in Japan by Inaba et al. (1970). The 85–1330 was isolated in 1985 in The USA. FS-1 was isolated in The USA by Smith et al.

Detectability and linearity of the BRSV Real Time PCR

Serial log 10 dilutions ranging from 101 to 108 copies of control RNA were reverse transcribed. cDNAs were then used to determine the detectability and the linearity of the assay (Fig. 1). Ct values were measured in triplicate and were plot against the log of the input RNA copies number. The detection limit of the assay was shown to be 102 RNA copies. Standard curves demonstrated a strong linear correlation between 103 and 108 copies (R2 = 0.997) and a slope of −3.67.

Reproducibility

Reproducibility of the assay

Discussion

Real Time PCR has become one of the most important tools in medical and veterinary diagnosis and research. This molecular technique provides a rapid, specific and sensitive method for the diagnosis and the quantitation of pathogens. Here, Real Time PCR was used for the detection and quantitation of bovine respiratory syncytial virus.

The nucleoprotein gene of BRSV has been shown to be highly conserved due to strong selective structural constrains that limit its evolution (Samal et al., 1991,

Acknowledgements

We are grateful to K. Lentz for excellent technical assistance and to G. Meyer (Ecole Nationale Vétérinaire de Toulouse, France) for the challenge inoculum. This work is supported by the Federal Public Service public health, chain food safety and environment (convention no. S-6117).

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