Simultaneous detection of Rift Valley Fever, bluetongue, rinderpest, and Peste des petits ruminants viruses by a single-tube multiplex reverse transcriptase-PCR assay using a dual-priming oligonucleotide system.

The aim of this study was to develop a highly sensitive and specific one-step multiplex reverse transcriptase PCR assay for the simultaneous and differential detection of Rift Valley Fever virus (RVFV), bluetongue virus (BTV), rinderpest virus (RPV), and Peste des petits ruminants virus (PPRV). These viruses cause mucosal lesions in cattle, sheep, and goats, and they are difficult to differentiate from one another based solely on their clinical presentation in suspected disease cases. In this study, we developed a multiplex reverse transcriptase PCR to detect these viruses using a novel dual-priming oligonucleotide (DPO). The DPO contains two separate priming regions joined by a polydeoxyinosine linker, which blocks extension of nonspecifically primed templates and consistently allows high PCR specificity even under less-than-optimal PCR conditions. A total of 19 DPO primers were designed to detect and discriminate between RVFV, BTV, RPV, and PPRV by the generation of 205-, 440-, 115-, and 243-bp cDNA products, respectively. The multiplex reverse transcriptase PCR described here enables the early diagnosis of these four viruses and may also be useful as part of a testing regime for cattle, sheep, or goats exhibiting similar clinical signs, including mucosal lesions.

Specific detection of Rinderpest virus by real-time reverse transcription-PCR in preclinical and clinical samples from experimentally infected cattle.

A highly sensitive detection test for Rinderpest virus (RPV), based on a real-time reverse transcription-PCR (rRT-PCR)system, was developed. Five different RPV genomic targets were examined, and one was selected and optimized to detect viral RNA in infected tissue culture fluid with a level of detection ranging from 0.59 to 87.5 50% tissue culture infectious doses (TCID(50)) per reaction depending on the viral isolate. The strain sensitivity of the test was validated on 16 RPV strains belonging to all three phylogenetic branches described for RPV. No cross-reactivity was detected with closely related peste des petit ruminants or with symptomatically similar viruses, including all seven serotypes of foot-and-mouth disease virus, two serotypes of vesicular stomatitis virus, bluetongue virus, and bovine herpes virus type 2. In samples from experimentally infected cattle, our real-time RT-PCR test was significantly more sensitive than the gold standard test of virus isolation, allowing the detection of the disease 2 to 4 days prior to the appearance of clinical signs. The comparison of clinical samples with putative diagnostic value from live animals showed that conjunctival swabs and blood buffy coat were the samples of choice for epidemiological surveillance, while lymph nodes performed the best as postmortem specimens. This portable and rapid real-time RT-PCR has the capability of the preclinical detection of RPV and provides differential diagnosis from look-alike diseases of cattle. As RPV is declared globally eradicated, this test provides an important rapid virus detection tool that does not require the use of infectious virus and allows the processing of a large number of samples.

Recombinant Newcastle disease viruses with targets for PCR diagnostics for rinderpest and peste des petits ruminants.

Since February 1st 2011, rinderpest (RP) has been officially declared eradicated worldwide. National authorities have been requested to destroy all their RP related materials. Nonetheless, their national reference laboratories performing real time reverse transcription polymerase chain reaction assays (PCR diagnostics) need RP positive control samples, since some countries still prefer to maintain diagnostic capability for RP for several reasons. In the future, a similar situation will arise for peste des petits ruminants (PPR) as the ambition has been expressed to eradicate PPR. Anticipating on this, we intended to perform qualified PCR diagnostics without use of infectious RPV or PPRV. Therefore, Newcastle disease virus (NDV) with small RNA inserts based on RPV or PPRV sequences were generated and used as positive control material. Recombinant NDVs (recNDVs) were differentially detected by previously established PCR diagnostics for RPV or PPRV. Both recNDVs contain a second PCR target showing that additional targets in NDV are feasible and would increase the diagnostic sensitivity by use of two PCR assays. RecNDV with small PCR targets is not classified as RPV or PPRV containing material, and can be used to mimic RPV or PPRV. Using these recNDVs as virus positive material contributes to the ambition of worldwide eradication, while qualified PCR diagnostics for these OIE-listed diseases remains operational.

Observations on rinderpest and rinderpest-like diseases throughout West and Central African countries during rinderpest eradication projects.

Between 1998 and 2005, the Regional Reference Laboratory at Bingerville (Ivory-Coast) received samples for analysis from Western and Central African countries. From a total of 606 sera; 65 tissue samples and 75 swabs received, no rinderpest virus or specific gene products or antibodies against rinderpest were detected. Use of the PCR on the tissue and swabs (total of 140 samples) identified the genomic presence of BVD (4/140), MCF (2/140), IBR (1/140) and FMD (6/140) viruses. These cause diseases that produce similar clinical signs to rinderpest. The quality of many samples sent to the reference laboratory did not meet the laboratory requirements and this compromised analysis of some specimens.

Identification of T-helper and linear B epitope in the hypervariable region of nucleocapsid protein of PPRV and its use in the development of specific antibodies to detect viral antigen.

Peste des petits ruminants is a highly contagious viral disease of small ruminants making its diagnosis difficult from the similar symptoms of Rinderpest. Computer based prediction algorithms was applied to identify antigenic determinants on the nucleocapsid (N) protein of PPRV. Specificity and antigenicity of each peptide was evaluated by solid phase ELISA. Six specific peptide sequences were evaluated in multiple antigenic peptide (MAP) form and immune response was evaluated by supplementing universal T-helper epitope human IL-1beta peptide (VQGEESNDK, amino acids 163-171). Out of the six peptides 19mer sequence corresponding to 454-472 region of N protein of PPRV was found to be highly immunogenic and specific to PPRV. Evaluation of overlapping peptides differing in length for this 452-472 region, showed minimum length of 14 amino acid residues were required for the stable affinity binding of antigen-antibody. The results of immunization and indirect ELISA indicated the presence of T-helper epitope at the N-terminal end and linear B epitope at the C-terminal region of 454-472 19mer of nucleocapsid peptide of PPRV-nucleocapsid protein. The antipeptide antibodies developed against this region showed specificity to PPRV antigen differentiating it from RPV when used in indirect ELISA and western blot analysis.

One-step multiplex RT-PCR assay for the detection of peste des petits ruminants virus in clinical samples.

A single-tube one-step multiplex RT-PCR was standardized to amplify both 337 bp and 191 bp fragments of N and M genes of peste des petits ruminants virus (PPRV), respectively, and only a 337 bp fragment of N gene of Rinderpest virus (RPV). The RT-PCR using purified viral RNA was easily adopted for direct detection of PPRV in clinical field samples and its differentiation from RPV. The amplified N and M gene products were confirmed to be PPRV- and RPV-specific by their size in 1.5% agarose gel and restriction analysis. In the assay, the Qiagen one-step RT-PCR kit containing the Ominiscript and Sensiscript reverse transcriptases and Hot star Taq DNA polymerase was utilized. The sensitivity of the assay was found to be 100 fg of PPRV RNA. Compared with a two-step assay, the one-step assay is easier and time-saving as it requires just a single buffer for both reactions, reverse transcription (RT) and PCR. In experimentally infected goats, PPRV was detectable by the one-step RT-PCR in nasal and ocular swabs 7-17 days post infection (p.i.). and in oral swabs 7-15 days p.i. Out of 32 clinical field samples tested, 18 were positive by sandwich ELISA (S-ELISA), while 22 were positive by the one-step RT-PCR.

Enhanced immunosurveillance for animal morbilliviruses using vesicular stomatitis virus (VSV) pseudotypes.

The measurement of virus-specific neutralising antibodies represents the "gold-standard" for diagnostic serology. For animal morbilliviruses, such as peste des petits ruminants (PPRV) or rinderpest virus (RPV), live virus-based neutralisation tests require high-level biocontainment to prevent the accidental escape of the infectious agents. In this study, we describe the adaptation of a replication-defective vesicular stomatitis virus (VSVΔG) based pseudotyping system for the measurement of neutralising antibodies against animal morbilliviruses. By expressing the haemagglutinin (H) and fusion (F) proteins of PPRV on VSVΔG pseudotypes bearing a luciferase marker gene, neutralising antibody titres could be measured rapidly and with high sensitivity. Serological responses against the four distinct lineages of PPRV could be measured simultaneously and cross-neutralising responses against other morbilliviruses compared. Using this approach, we observed that titres of neutralising antibodies induced by vaccination with live attenuated PPRV were lower than those induced by wild type virus infection and the level of cross-lineage neutralisation varied between vaccinates. By comparing neutralising responses from animals infected with either PPRV or RPV, we found that responses were highest against the homologous virus, indicating that retrospective analyses of serum samples could be used to confirm the nature of the original pathogen to which an animal had been exposed. Accordingly, when screening sera from domestic livestock and wild ruminants in Tanzania, we detected evidence of cross-species infection with PPRV, canine distemper virus (CDV) and a RPV-related bovine morbillivirus, suggesting that exposure to animal morbilliviruses may be more widespread than indicated previously using existing diagnostic techniques.

Differentiation of rinderpest and peste des petits ruminants viruses using specific cDNA clones.

The morbilliviruses which infect ruminants, rinderpest (RPV) and peste des petits ruminants (PPRV), are difficult to distinguish serologically. They can be distinguished by differential neutralisation tests and by the migration of the major virus structural protein, the nucleocapsid protein, on polyacrylamide gels. Both these methods are time consuming and require the isolation of live virus for identification; they are not suitable for analysis of material directly from post-mortem specimens. We describe a rapid method for differential diagnosis of infections caused by RPV or PPRV, which uses specific cDNA probes, derived from the mRNAs for the nucleocapsid protein of each virus, which can be used to distinguish unequivocally the two virus types rapidly.

A rapid chromatographic strip test for the pen-side diagnosis of rinderpest virus.

Rinderpest is a contagious viral disease of cloven-hoofed domestic and wild animals. Eradication of the virus following outbreaks depends on rapid and accurate diagnosis of infection and the implementation of control measures. Reporting and confirmatory diagnosis precede the implementation of control measures. A number of techniques have been used for diagnosis such as agar gel immunodiffusion, enzyme-linked immunosorbent assay (ELISA), molecular biological techniques such as polymerase chain reaction (PCR) and virus isolation in tissue culture. Many of these methods are both time consuming and require skilled personnel. The development of a rapid pen-side test for the detection of rinderpest virus (RPV) antigen in lachrymal fluid of cattle is described using the Clearview chromatographic strip test technology (Unipath, Bedford). Optimum conditions for binding monoclonal antibody to nitrocellulose and latex microspheres were determined and a prototype device was developed. The device detected viral antigen in lachrymal fluids from experimentally and naturally infected cattle and showed no cross-reactivity with other related viruses. A field trial was carried out at the Landhi Cattle Colony (LCC), Pakistan, to assess the performance of the rinderpest test under field conditions. Ninety-seven animals, some of which were showing various clinical signs, at LCC and neighbouring colonies were sampled and tested at the pen-side by Clearview and later by immunocapture ELISA (IC-ELISA) at IAH, Pirbright. Nineteen animals were positive by Clearview and/or IC-ELISA. Seventeen out of 19 rinderpest positive animals were positive by Clearview and 15 out of 19 were positive by IC-ELISA. Reverse transcription polymerase chain reaction (RT-PCR) confirmed the 19 animals to be rinderpest positive. This simple, rapid, specific test allows for the first time, accurate pen-side diagnosis of rinderpest.

Dipstick enzyme immunoassay for rinderpest antibody in cattle.

A dipstick enzyme immunoassay (ELISA) has been standardized for the detection of rinderpest antibodies. One hundred and thirty bovine serum samples were analysed by the dipstick ELISA method and the results compared with the conventional plate ELISA method. The sensitivity was found to be similar in both methods. The dipstick ELISA does not require expensive micro-plates and an ELISA reader, and is recommended for use in field laboratories where the qualitative detection of rinderpest antibodies is required.

Recent developments in the diagnosis of rinderpest and peste des petits ruminants.

Effective implementation of control measures for rinderpest and peste des petits ruminants requires that a proper and rapid diagnosis of the disease is made. Peste de petits ruminants (PPR) can be confused clinically with other infections such as pasteurellosis or contagious ecthyma. Rinderpest, in its classical form, is easy to identify clinically; however, mass vaccination in many countries and also the emergence of mild strains of the virus have made clinical diagnosis more difficult. Clinical observations for both diseases should always be confirmed by a laboratory. Diagnostic techniques used in the past were virus neutralization, agar gel immunodiffusion and virus isolation in cell culture, followed sometimes by reproducing the disease in susceptible animals. All these techniques are either time-consuming, labour intensive, insensitive, or expensive to perform. With the advent of hybridoma and molecular biological techniques, new reagents to assist diagnosis have become available and have led to the development of specific and rapid tests for the diagnosis of each disease. The present article reviews the diagnostic techniques currently available. An indirect ELISA was used successfully to evaluate the status of cattle following the Pan African Rinderpest Campaign. More recently competitive or blocking ELISAs have been developed based on monoclonal antibodies specific for the N or H proteins of the viruses, and which enable differential diagnosis between rinderpest and PPR. This is particularly important in sheep and goats, which may be infected with either virus. In future, improved standardization and reduced costs may be expected with the introduction of ELISAs based on purified antigens expressed in gene vector systems such as baculovirus. ELISA may also be adapted to antigen detection. Nucleic acid technology has also been applied to virus detection procedures. Hybridization probes showed a disappointing sensitivity for diagnostic applications, but more recently the polymerase chain reaction method has shown great promise, providing the potential of high sensitivity combined with specificity.

Monoclonal antibody-based competitive ELISA for simultaneous detection of rinderpest virus and peste des petits ruminants virus antibodies.

An experimental competitive enzyme-linked immunosorbent assay (morbillivirus cELISA) using a recombinant N antigen (rRPV N) expressed in a baculovirus and a ruminant morbillivirus (RPV and PPRV)-specific monoclonal antibody (P-13A9) was developed for simultaneous detection of rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) antibodies and its diagnostic performance was evaluated. A set of known reference antisera against RPV and PPRV belonging to different lineages, experimental sera from cattle vaccinated for a RPV of Asian lineage, and field sera from cattle and sheep/goat populations known to be positive (West Africa) and negative (Korea) for RPV and PPRV were used for the evaluation. Morbillivirus cELISA results on the panel of experimental RPV and PPRV antisera showed high correlation (r=0.97) between the whole virus and the rRPV N antigens, suggesting that the rRPV N contains a ruminant morbillivirus-specific antigenic determinant recognized by the P-13A9 and it may be suitable as an ELISA antigen in place of the whole virus. Morbillivirus cELISA detected anti-RPV and anti-PPRV antibodies in all reference RPV and PPRV antisera containing VN titers >/=1:8, suggesting that the assay can simultaneously detect antibodies against RPV and PPRV. Anti-RPV antibody was detected by morbillivirus cELISA in vaccinated cattle as early as the VNT and continued to be detectable by both the cELISA and the VNT until termination of the study. When applied to field samples from Africa, morbillivirus cELISA showed good agreement with a RP cELISA kit (kappa value of 0.86) in bovine sera and with a peste des petits ruminant cELISA kit (kappa value of 0.81) in caprine/ovine sera. Usefulness of morbillivirus cELISA using the rRPV N protein was discussed.

Isolation of peste des petits ruminants virus from the Sudan.

Based on clinical signs, the presence of a rinderpest related antigen and the ability of rinderpest immune serum to prevent virus isolation, rinderpest was diagnosed in two outbreaks in goats in central Sudan during 1971 and 1972. Two viruses isolated from these goats have been re-examined both serologically and by the inoculation of experimental cattle, sheep and goats. Each is now considered to represent the virus of peste des petits ruminants, thereby extending the geographical range of this agent.

Detection of rinderpest virus antigens in vitro and in vivo by direct immunofluorescence.

Cell-culture attenuated and virulent strains of rinderpest virus (RV) were inoculated on to bovine kidney cell cultures. A direct immunofluorescent antibody test detected RV antigens in cell cultures within one to three days after inoculation whereas RV cytopathic effects usually took three to nine days to develop. Cells containing RV antigens were also detected in impression smears and frozen sections of tissues collected from RV infected animals at post mortem examination, and in smears of lymph node biopsies taken from cattle with clinical rinderpest. These techniques may offer additional methods for rapid diagnosis of rinderpest.

Immunohistochemical detection of rinderpest virus: effects of autolysis and period of fixation.

Samples of eyelid, tongue, soft palate and palatine tonsil were collected from calves infected experimentally with rinderpest virus. The tissues were fixed in 10 per cent neutral buffered formalin immediately, 24 or 48 hours post mortem. Then, after three days, 10 days, 28 days or three months in formalin, they were processed into paraffin blocks and examined immunohistochemically for rinderpest viral antigen. The tonsil was the best of the four tissues in providing a consistently positive immunohistochemical signal for the presence of virus, despite autolytic changes and/or prolonged fixation.

A review of three pathology-based techniques for retrospective diagnosis of rinderpest, with comparison to virus isolation.

Base of tongue, eyelid, and retropharyngeal lymph node were collected from three animals experimentally infected with rinderpest and utilised in a study comparing virus isolation with histopathology, immunohistochemistry, and in situ hybridisation to determine the usefulness of the latter three techniques as retrospective diagnostic aids for this disease. Virus isolation was positive for all nine samples. Histopathology was suggestive in all the tissues and definitive in some. Immunohistochemistry and in situ hybridisation highlighted the presence of rinderpest antigen of rinderpest nucleic acid in all of the sections. However, in situ hybridisation was more specific than immunohistochemistry.

Recommended standards for epidemiological surveillance systems for rinderpest. Office International des Epizooties.

A process involving time-bound steps used to verify the transition from the status of freedom from rinderpest to that of freedom from infection with the rinderpest virus is described. The procedure, informally but widely known as the 'OIE Pathway' (OIE: Office International des Epizooties), originated with the report of the 1989 Expert Consultation on Rinderpest Surveillance Systems. The OIE Foot and Mouth Disease and Other Epizootics Commission, with the assistance of experts on the disease, proposed a revision of the recommended standards in the reference document presented at the 66th General Session of the OIE (66 SG/12/CS3 B, Appendix III). During that General Session, the standards were amended and adopted by the OIE International Committee (Resolution No. IX dated 28 May 1998).

Peste des petits ruminants.

The peste des petits ruminants (PPR) is proving to be a disease which has an increasingly significant economic impact on a number of countries in Africa and the Middle East, and possibly also on the Indian sub-continent. The antigenic relationships which exist between the PPR and rinderpest viruses pose problems for diagnosis which complicates rinderpest control and eradication programmes. Progress has recently been made in regard to diagnosis (specific nucleic probes and monoclonal antibodies), as well as control (homologous vaccine). International legislation remains to be established and epidemiological surveys should be conducted in order to determine the exact geographical distribution of the disease.

Prevalence of bovine viral diarrhoea virus antibodies in India.

A study was undertaken regarding the prevalence of bovine viral diarrhoea virus (BVDV) antibodies in bovine sera which tested negative for rinderpest and peste des petits ruminants virus antibodies. The samples were collected between January 1996 and December 1997. A total of 439 samples (327 cattle and 112 buffalo from 17 states of India) were tested using a commercial enzyme-linked immunosorbent assay (ELISA) kit. The mean prevalence of BVDV antibodies in cattle was 15.29% (50/327) in 16 states compared to 23.21% (26/112) in buffalo in 9 states, with an overall prevalence of 17.31% (76/439) in 17 states. The serological evidence that BVDV infection is widespread in India is of utmost practical importance because of the clinical resemblance to rinderpest. A differential diagnosis between these two diseases is critical in view of the declaration by India of provisional freedom from rinderpest disease; active sero-surveillance is to begin in 2000 to achieve certification of freedom from rinderpest infection by the Office International des Epizooties.

Field diagnostic kits: a solution for developing countries?

An exact assessment of the animal health situation in a country is an essential element in formulating eradication and control programmes, and in regulating international trade in animals and animal products from that country. Due to a lack of human and technical resources, Veterinary Services in developing countries often lack precise knowledge on disease occurrence. Since the collection and transmission of reliable information on animal diseases in developing countries are major concerns of the Office International des Epizooties (OIE), a project aimed at improving this situation was implemented with international financial support. This project involved the development by the Centre for the Application of Methodology for the Diagnosis of Animal Diseases (CAMDA) of field kits for the diagnosis of the main diseases present in tropical Africa: rinderpest, peste des petits ruminants (PPR), contagious bovine pleuropneumonia (CBPP) and contagious caprine pleuropneumonia (CCPP). Several tests already exist, such as complement deoxyribonucleic acid (cDNA)-specific probes and polymerase chain reaction (PCR) for rinderpest and PPR, DNA probes and PCR for CBPP, capture enzyme-linked immunosorbent assay, the agglutination test and the immunobinding peroxidase test for CCPP, etc. With specific reference to these examples, the various problems faced by the OIE and CAMDA are reviewed.