Real-time RT-PCR Detection of Bovine Viral Diarrhoea Virus in Whole Blood Using an External RNA Reference

Overview

Journal J Virol Methods

Specialty Microbiology

Date 2006 Oct 13

PMID 17030066

Citations 17

Authors

N J Young

C J Thomas

M E Collins

J Brownlie

Affiliations

Soon will be listed here.

Abstract

A novel two-step real-time RT-PCR assay using SYBR Green I was developed for the detection of acute Bovine Viral Diarrhoea virus (BVDV) infection in whole blood from cattle. During infection animals experience a characteristic transient leucopenia and the number of cells per volume of blood changes over time; so quantitation of viral load by reference to a cellular housekeeping gene is not ideal as this may hide significant animal to animal variation. Therefore, to facilitate comparison of different samples, an external RNA reference was used for normalisation whereby each sample was spiked with the RNA virus, Canine Enteric Coronavirus (CECov), prior to RNA extraction, for comparative purposes. Real-time RT-PCR was carried out with two primer sets designed to amplify either a 156 bp region of the BVDV 5'-UTR or a 280 bp region of the CECov nucleocapsid protein gene. Linearity and efficiency of the assay was established and the method assessed using samples from BVDV-challenged calves. Viral RNA was quantified on days 6 and 14 post-challenge by real-time RT-PCR. Infectious virus isolation by traditional cell culture was negative after day 7. This study demonstrates encouraging results for rapid, sensitive and reliable detection of acute BVDV infection and provides an alternative real-time RT-PCR method for use on whole blood samples or samples where suitable housekeeping genes are not available.

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References

Bustin S . Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol. 2000; 25(2):169-93. DOI: 10.1677/jme.0.0250169. View

Bae H, Nitsche A, Teichmann A, Biel S, Niedrig M . Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay. J Virol Methods. 2003; 110(2):185-91. DOI: 10.1016/s0166-0934(03)00129-0. View

Brownlie J . The pathways for bovine virus diarrhoea virus biotypes in the pathogenesis of disease. Arch Virol Suppl. 1991; 3:79-96. DOI: 10.1007/978-3-7091-9153-8_10. View

Baker J . The clinical manifestations of bovine viral diarrhea infection. Vet Clin North Am Food Anim Pract. 1995; 11(3):425-45. DOI: 10.1016/s0749-0720(15)30460-6. View

Paton D, Sharp G, Ibata G . Foetal cross-protection experiments between type 1 and type 2 bovine viral diarrhoea virus in pregnant ewes. Vet Microbiol. 1999; 64(2-3):185-96. DOI: 10.1016/s0378-1135(98)00269-7. View

Pfaffl M . A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001; 29(9):e45. PMC: 55695. DOI: 10.1093/nar/29.9.e45. View

Collett M, Larson R, Belzer S, Retzel E . Proteins encoded by bovine viral diarrhea virus: the genomic organization of a pestivirus. Virology. 1988; 165(1):200-8. DOI: 10.1016/0042-6822(88)90673-3. View

Niesters H . Quantitation of viral load using real-time amplification techniques. Methods. 2002; 25(4):419-29. DOI: 10.1006/meth.2001.1264. View

Bhudevi B, Weinstock D . Detection of bovine viral diarrhea virus in formalin fixed paraffin embedded tissue sections by real time RT-PCR (Taqman). J Virol Methods. 2003; 109(1):25-30. DOI: 10.1016/s0166-0934(03)00040-5. View

10.

Whelan J, Russell N, Whelan M . A method for the absolute quantification of cDNA using real-time PCR. J Immunol Methods. 2003; 278(1-2):261-9. DOI: 10.1016/s0022-1759(03)00223-0. View

11.

Johansson M, Pisa E, Tormanen V, Arstrand K, Kagedal B . Quantitative analysis of tyrosinase transcripts in blood. Clin Chem. 2000; 46(7):921-7. View

12.

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A . Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002; 3(7):RESEARCH0034. PMC: 126239. DOI: 10.1186/gb-2002-3-7-research0034. View

13.

Cleland A, Nettleton P, Jarvis L, Simmonds P . Use of bovine viral diarrhoea virus as an internal control for amplification of hepatitis C virus. Vox Sang. 1999; 76(3):170-4. DOI: 10.1159/000031044. View

14.

Bhudevi B, Weinstock D . Fluorogenic RT-PCR assay (TaqMan) for detection and classification of bovine viral diarrhea virus. Vet Microbiol. 2001; 83(1):1-10. PMC: 7117215. DOI: 10.1016/s0378-1135(01)00390-x. View

15.

Nobiron I, Thompson I, Brownlie J, Collins M . DNA vaccination against bovine viral diarrhoea virus induces humoral and cellular responses in cattle with evidence for protection against viral challenge. Vaccine. 2003; 21(17-18):2082-92. DOI: 10.1016/s0264-410x(02)00745-4. View

16.

Castelain S, Descamps V, Thibault V, Francois C, Bonte D, Morel V . TaqMan amplification system with an internal positive control for HCV RNA quantitation. J Clin Virol. 2004; 31(3):227-34. DOI: 10.1016/j.jcv.2004.03.009. View

17.

Polak M, Zmudzinski J . Experimental inoculation of calves with laboratory strains of bovine viral diarrhea virus. Comp Immunol Microbiol Infect Dis. 2000; 23(3):141-51. DOI: 10.1016/s0147-9571(99)00060-0. View

18.

Dent A, Shaffer A, Yu X, Allman D, Staudt L . Control of inflammation, cytokine expression, and germinal center formation by BCL-6. Science. 1997; 276(5312):589-92. DOI: 10.1126/science.276.5312.589. View

19.

Letellier C, Kerkhofs P . Real-time PCR for simultaneous detection and genotyping of bovine viral diarrhea virus. J Virol Methods. 2003; 114(1):21-7. DOI: 10.1016/j.jviromet.2003.08.004. View

20.

Sandvik T . Selection and use of laboratory diagnostic assays in BVD control programmes. Prev Vet Med. 2005; 72(1-2):3-16. DOI: 10.1016/j.prevetmed.2005.08.015. View