Rapid and sensitive detection of peste des petits ruminants virus by a polymerase chain reaction assay
Introduction
Peste des petits ruminants (PPR) is a severe disease of goats and sheep with high morbidity and sometimes high mortality. It continues to cause serious economic losses in these species in Africa, the Middle East, and Asia (Taylor, 1984, Shaila et al., 1989, Lefevre and Diallo, 1990, Abu-Elzein et al., 1990, Nanda et al., 1996, Govindarajan et al., 1997). The causative agent is a morbillivirus, peste des petits ruminants virus (PPRV). It is closely related to rinderpest virus (RPV), another member of the morbillivirus genus which can also cause a disease in small ruminants (Anderson et al., 1990, Couacy-Hymann et al., 1995). At present, rinderpest eradication projects are carried out in Africa, the Middle East and Asia. However, for the diagnosis of rinderpest in small ruminants, it is essential to differentiate it clearly from PPR. This is impossible clinically because the symptoms of both diseases are very similar. In the laboratory, this is possible by virus isolation followed by either the differential neutralisation test in cell culture or by the animal inoculation because PPRV causes inapparent infection in cattle whereas small ruminants can show clinical symptoms after RPV infection. These techniques are all time-consuming. Moreover, RPV infection of small ruminants is sometimes asymptomatic (Couacy-Hymann et al., 1995). In the last decade, molecular biology techniques have permitted the development of specific and sensitive tests for rapid diagnosis. The structure and biology of peste des petits ruminants is now well known. As with other Morbilliviruses, it has an unsegmented negative stranded RNA genome encoding six structural and two non-structural proteins (Haffar et al., 1999). Among them, the nucleocapsid protein (NP) is the major viral protein. It has been the target for developing diagnostic tests that can be used to distinguish rinderpest infection from PPR. Monoclonal antibodies, anti-NP, were used to develop ELISAs for both specific serological diagnosis in the competitive format and for antigen detection by immunocapture (Libeau et al., 1994, Libeau et al., 1995). For this latter test, the limit of detection has been estimated at 100.6 TCID50 of virus in 50 μl of sample. In 1989, nucleic acid technology was applied for the first time to the detection of RPV and PPRV by using, as probes, the cDNA corresponding to the nucleocapsid gene of each virus and labelled with 32P nucleotide (Diallo et al., 1989). Although sensitive, such a test is not suitable for routine use in most of the developing countries due the health hazard linked to the radioactive labels and the lack of adequate equipment. An alternative and very sensitive technique, the amplification of the viral nucleic acid by the reverse transcription-polymerase chain reaction (RT-PCR) is described for specific detection of PPRV. In this test, we use a simplified RNA extraction technique instead of the classical phenol–chloroform extraction method.
Section snippets
Virus
Nineteen PPRV isolates available in CIRAD (Montpellier, France) were grown in Vero cells: Guinea Bissau, Senegal (94), Burkina-Faso (86), Côte-d'Ivoire (89), Guinea, Dorcas (Sultanate of Oman), Ibri (Sultanate of Oman), Sinnar (Sudan), Ethiopia 94, Nigeria 75/1, Nigeria 75/2, Nigeria 75/3, Nigeria 76/1, Accra, Egypt, Ghana 78/1, Israel, Central Africa Republic, India 94/1, India Calcutta, India Pradesh. Rinderpest attenuated vaccine strain has also been used. Cell growth medium was Eagle's MEM
Rapid extraction of RNA
Attempts were made to simplify the RNA extraction method, a step critical for RT-PCR technique. The glassmilk was used as RNA binding matrix in different conditions: guanidine thiocyanate or sodium iodide solutions as lysis buffers and RNase inhibitors, different times of incubation as indicated in materials and methods. The RNAs that were extracted under different conditions from PPRV infected cells then dot-blotted and revealed by PPRV NP radioactive probe. The results obtained from this
Discussion
PPRV and RPV infections of small ruminants are clinically similar so the differential diagnosis has to be made between these two diseases when they coexist in an area. Such a diagnosis could be made now by competitive ELISA (antibody detection), or by immunocapture (antigen detection). Shaila et al. (1996) have reported the application of the RT-PCR to detect PPR RNA in pathological samples. To purify the RNA, they used the phenol–chloroform method described by Chomczynski and Sacchi (1987)
References (25)
- et al.
Observation on the pathogenicity for sheep and goats and the transmissibility of the strain of virus isolated during the rinderpest outbreak in Sri Lanka in 1987
Veterinary Microbiology
(1990) - et al.
Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction
Analytical Biochemistry
(1987) - et al.
Goats vaccinated with the attenuated peste des petits ruminants virus are protected against rinderpest disease
Research in Veterinary Science
(1995) - et al.
Differentiation of rinderpest and peste des petits ruminants viruses using specific cDNA clones
Journal of Virological Methods
(1989) - et al.
Development of a competitive ELISA for detecting antibodies to the peste des petits ruminants virus using a recombinant N protein
Research in Veterinary Science
(1995) - et al.
Application of two RNA extraction methods prior to amplification of hepatitis E virus nucleic acid by the polymerase chain reaction
Journal of Virological Methods
(1991) - et al.
The isolation of peste des petits ruminants virus from northern India
Veterinary Microbiology
(1996) - et al.
Geographic distribution and epidemiology of Peste des Petits Ruminants Viruses
Virus Research
(1996) The distribution and epidemiology of peste-des-petits-ruminants
Preventive in Veterinary Medecine
(1984)- et al.
Techniques for the evaluation of Nucleic Acid Amplification Technology performance with specimens containing interfering substances: efficacy of Boom methodology for the extraction of HIV-1 RNA
Journal of Virological Methods
(1999)
Increased sensitivity for detection of human cytomegalovirus in urine by removal of inhibitors for the polymerase chain reaction
Journal of Virological Methods
Isolation of peste des petits ruminants from goats in Saudi Arabia
Veterinary Record
Cited by (0)
- 1
Deceased.