RAPID AND SENSITIVE MULTIPLEX REAL-TIME PCR FOR DIAGNOSIS OF BOVINE VIRAL DIARRHEA, BOVINE RESPIRATORY SYNCYTIAL VIRUS, AND BOVINE PARAINFLU

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0207686
• Project Start Date: Jun 1, 2006
• Project End Date: Sep 30, 2008
• Program Code: [(N/A)]- (N/A)

Recipient Organization
OREGON STATE UNIVERSITY
(N/A)
CORVALLIS,OR 97331

Performing Department
COLLEGE OF VETERINARY MEDICINE

Non Technical Summary
Currently, respiratory diseases are the main health problem in cattle. Bovine viral diarrhea (BVD), bovine respiratory syncytial virus (BRSV), and bovine parainfluenza 3 (BPIV-3) are among the most important pathogens worldwide, which contribute to causation of bovine respiratory disease complex and currently, there are no sensitive diagnostic techniques available to detect all the 3 pathogens in a single tube. The purpose of this project is to develop a rapid and sensitive multiplex real-time PCR for the diagnosis of diseases caused by BRSV, BPIV3, and BVD.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3999	1101	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3999 - Animal research, general;

Field Of Science
1101 - Virology;

Keywords

bovine viral diarrhea (bvd)

bovine respiratory syncytial virus (brsv) and bovine parainfluenza 3

(bpiv-3)

Goals / Objectives
Currently, respiratory diseases are the main health problem in cattle. Bovine viral diarrhea (BVD), bovine respiratory syncytial virus (BRSV), and bovine parainfluenza 3 (BPIV-3) are among the most important pathogens worldwide, which contribute to causation of bovine respiratory disease complex. As these respiratory viral pathogens cause very similar clinical symptoms, differential diagnosis of the pathogens is required in one sample. Current diagnostic techniques detect one pathogen at a time but not all three viruses in a single tube. Therefore, the objectives of this project are to develop a rapid and sensitive multiplex real-time PCR for the diagnosis of diseases caused by BRSV, BPIV3, and BVD. Specific objectives are : 1) Primer and probe design for multiplex real-time PCR. 2) Detection and differentiation of pathogens in cell culture. 3) Detection and differentiation of pathogens in Clinical samples.

Project Methods
APPROACH: The following approaches are undertaken to accomplish the 3 objectives: 1) Primer and probe design for multiplex real-time PCR. 2) Detection and differentiation of pathogens in cell culture. 3) Detection and differentiation of pathogens in Clinical samples. 1) Primer and probe design for multiplex real-time PCR. Primer and Taqman probe sequences were selected from an alignment of nucleotide sequences of the BRSV, BVD and BPIV3 from GenBank (Table 1). The alignment was performed to select a highly conserved region for each virus. The PCR primers and probes were optimized with Primer Designer version 2.0, to perform with an annealing temperature of 55C. 2) Detection and differentiation of pathogens in cell culture. Virus stocks: BRSV and BVD will be cultured on MDBK cells, and BPIV3 will be cultured on Vero cells. About 5 ml of each virus will be prepared, and the stocks will be stored in 0.5-ml aliquots. The limit of sensitivity of the multiplex real-time RNA PCR will be determined by testing 10-fold dilutions of the 50% tissue culture infectious dose (TCID50) for each virus type. RNA extraction: Ambion MagMAX magnetic bead system (Ambion) or TRIzol reagent (Invitrogen) for tissues will be used for RNA extraction in the development of the assay. All samples will be extracted according to the manufacturers instructions. Viral RNA will be extracted from 0.2 ml of each dilution and tested by the multiplex real-time PCR under the conditions described below. The detection limit of the assay will be determined as the highest dilution that resulted in a positive reaction. 3) Detection and differentiation of pathogens in Clinical samples. Clinical samples: From December 2004 to December 2005, a number of respiratory samples (nasal swabs, nasopharyngeal aspirates, and bronchoalveolar lavage samples) and serum samples were received in the veterinary diagnostic laboratory for routine culture and diagnosis of viruses. From each sample, an aliquot was stored at -70C for PCR analysis. Real-time PCR conditions: The assays will be optimized first in a monospecific PCR; subsequently, one multiplex PCR will be performed with BRSV, BPIV3 and BVD viruses. Real-time PCR will be performed according to Qiagen one-step RT-PCR kit.

Progress 06/01/06 to 09/30/08

Outputs
OUTPUTS: As several respiratory viral pathogens cause very similar clinical symptoms, differential diagnosis of the pathogens is required in one sample. Therefore, the goal of this project that has been completed is development of a single tube fluorogenic multiplex real time-PCR-based TaqMan assay for simultaneous detection of bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus Type 3 (BPI3) and bovine viral diarrhoea virus(BVDV) from bovine respiratory samples. The objectives of the projects that have been completed are 1) Primer and probe design for multiplex real-time PCR. 2) Detection and differentiation of pathogens in cell culture. 3) Detection and differentiation of pathogens in Clinical samples. PARTICIPANTS: Rocky Baker Oregon State Veterinary Diagnostic Laboratory Corvallis, Oregon 97331 Brian Zulauf Graduate Student College of Veterinary Medicine Oregon State University TARGET AUDIENCES: Presented the data to Veterinary students and discussed the data with diagnostic lab personnel from different states during American Association of Veterinary Diagnostic laboratories meeting held in Oct 22-27, 2008. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We have developed a single tube fluorogenic multiplex real time-PCR-based TaqMan assay for simultaneous detection of BRSV and BPI3 from bovine respiratory samples. BVD primers interfered with BRSV and BPI3 primers and therefore was excluded in the multiplex PCR. However, monoplex PCR for BVD was specific and sensitive. TaqMan-PCR was optimized to quantify the viruses using the BioRad iCycler IQ real-time PCR detection system and dual-labeled fluorogenic probes. Primers and probes for our study were designed from a 100% conserved region of BRSV N gene and from BPI3 NP gene using GenBank sequences such that they can be used to differentiate BRSV and BPI3 in one-tube in clinical respiratory samples obtained from bovine species. Once the technique was optimized with validated reference strains and field isolates, the assay was applied to routine diagnostic samples. Respiratory samples collected from 100 cattle across the state of Oregon were screened for BRSV and BPI3 viruses by real time-PCR and virus isolation. The monoplex real-time PCR identified 19 samples positive for BRSV, 11 for BPI-3 and 14 for BVD. The multiplex real-time PCR identified 18 samples positive for BRSV and 4 samples positive for BPI3. Among positive samples, 2 samples showed positive for both BRSV and BPI3. In conclusion, the multiplex real-time TaqMan-PCR described here for detection and quantitation of BRSV and BPI3 viruses has been shown to be sensitive and specific. Rapid turnaround time, reproducibility and ease of use make this technique a valuable diagnostic tool for detection of BRSV and BPI3 in individual or pooled respiratory samples. We may have to design a different primers pairs and probe for BVD in order to incorporate into multiplex PCR to detect all the three viruses simultaneously in future.

Publications

• No publications reported this period

Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: As several respiratory viral pathogens cause very similar clinical symptoms, differential diagnosis of the pathogens is required in one sample. Therefore, the goal of this project is to develop a single tube fluorogenic multiplex real time-PCR-based TaqMan assay for simultaneous detection of bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus Type 3 (BPI3) and bovine viral diarrhoea virus(BVDV)from bovine respiratory samples. The objectives of the projects are 1) Primer and probe design for multiplex real-time PCR. 2) Detection and differentiation of pathogens in cell culture. 3) Detection and differentiation of pathogens in Clinical samples. PARTICIPANTS: Manoj K. Pastey DVM, MS, PhD Diplomate ACVM Assistant Professor Head, Molecular Diagnostic Laboratory 210 Dryden Hall Department of Biomedical Sciences College of Veterinary Medicine Oregon State University Corvallis, OR 97331 Phone: [phone] Fax: [phone] Email: [e-mail] Brian Zulauf Graduate Student 308 Dryden Hall Department of Biomedical Sciences College of Veterinary Medicine Oregon State University Corvallis, OR 97331

Impacts
TaqMan-PCR was optimized to quantify the viruses using the BioRad iCycler IQ real-time PCR detection system and dual-labeled fluorogenic probes. Primers and probes for our study were designed from a 100% conserved region of BRSV N gene, BPIV3 NP gene and BVDV noncoding 3' gene using GenBank sequences such that they can be used to differentiate BRSV,BPIV3 and BVDV in one-tube in clinical respiratory samples obtained from bovine species. Specificity of the assay was evaluated by testing different BRSV BVDV and BPI3 virus strains and other bovine viruses. Sensitivity of the single tube TaqMan assay was compared with two-tube TaqMan assay and enzyme linked immunosorbent assay (ELISA) used by our diagnostic lab. Single tube TaqMan assay was 10-100-fold more sensitive than the two-tube TaqMan assay and ELISA. The fluorogenic probes simultaneously identify BRSV, BVDV and BPI3 viruses from bovine samples. Quantitation of cRNA from BRSV, BVDV, and BPI3, viruses by TaqMan-PCR was accomplished by a standard curve plotting cycle threshold values (CT) verses copy number. Ambion MagMAX magnetic bead system and TRIzol reagent (Invitrogen) for tissues were used for RNA extraction in the development of the assay. Tissue culture propagated viruses and validated field samples were used to prove the specificity of the TaqMan-PCR assay. The specific probes designed in this study correctly identified all reference samples. Once the technique was optimized with validated reference strains and field isolates, the assay will be applied to routine diagnostic samples.

Publications

• No publications reported this period

Progress 01/01/06 to 12/31/06

Outputs
As several respiratory viral pathogens cause very similar clinical symptoms, differential diagnosis of the pathogens is required in one sample. Therefore, a single tube fluorogenic multiplex real time-PCR-based TaqMan assay was developed for simultaneous detection of bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus Type 3 (BPI3)and bovine viral diarrhea virus (BVD)from bovine respiratory samples. TaqMan-PCR was optimized to quantify the viruses using the BioRad iCycler IQ real-time PCR detection system and dual-labeled fluorogenic probes. Primers and probes for our study were designed from a 100% conserved region of BRSV N gene, from BPIV3 NP gene and from BVD 3'noncoding region using GenBank sequences such that they can be used to differentiate BRSV, BPIV3 and BVD in one-tube in clinical respiratory samples obtained from bovine species. Specificity of the assay was evaluated by testing different BRSV, BPI3 and BVD virus strains and other bovine viruses grown in cell culture. Sensitivity of the single tube TaqMan assay was compared with two-tube TaqMan assay and enzyme linked immunosorbent assay (ELISA)used by our diagnostic lab. Single tube TaqMan assay was 10-100-fold more sensitive than the two-tube TaqMan assay and ELISA. The fluorogenic probes simultaneously identified BRSV, BPI3 and BVD viruses from cell culture samples. Quantitation of cRNA from BRSV, BPI3, and BVD viruses by TaqMan-PCR was accomplished by a standard curve plotting cycle threshold values (CT) verses copy number. Ambion MagMAX magnetic bead system and TRIzol reagent (Invitrogen) for tissues were used for RNA extraction in the development of the assay. The specific probes designed in this study correctly identified all reference samples. Once the technique is optimized with validated reference strains and field isolates, the assay will be applied to routine diagnostic samples.

Impacts
Respiratory infections caused by respiratory syncytial virus, parainfluenza virus type 3 and bovine viral diarrhea are major causes of upper and lower respiratory tract diseases in young calves causing bronchiolitis, and pneumonia. Annually, US dairy and meat industry suffer from enormous economic losses due to respiratory diseases. Most of the losses arise from reduced milk yield, reduced fertility, and reduced meat production. Currently available laboratory diagnostics are not rapid diagnostic tests, less sensitive and specific and therefore, their clinical value is limited. Bovine respiratory syncytial virus, Bovine parainfluenza Type-3 virus and Bovine viral diarrhea virus cause very similar clinical respiratory symptoms, differential diagnosis of the pathogens is required in one sample. Developing multiplex real-time PCR assays for clinical diagnosis has a significant advantage, as it permits simultaneous amplification of several viruses in a single reaction mixture, facilitating cost-effective diagnosis of respiratory viral diseases in veterinary diagnostic laboratories. Therefore, the clinical utility of multiplex real-time assay shall show a sufficient net benefit weighing the total potential diagnostic benefits it produces, including direct health benefits to an animal and benefits to veterinary practitioners and animal industry.

Publications

• No publications reported this period