Full genome sequencing of archived wild type and vaccine rinderpest virus isolates prior to their destruction.

When rinderpest virus (RPV) was declared eradicated in 2011, the only remaining samples of this once much-feared livestock virus were those held in various laboratories. In order to allow the destruction of our institute's stocks of RPV while maintaining the ability to recover the various viruses if ever required, we have determined the full genome sequence of all our distinct samples of RPV, including 51 wild type viruses and examples of three different types of vaccine strain. Examination of the sequences of these virus isolates has shown that the African isolates form a single disparate clade, rather than two separate clades, which is more in accord with the known history of the virus in Africa. We have also identified two groups of goat-passaged viruses which have acquired an extra 6 bases in the long untranslated region between the M and F protein coding sequences, and shown that, for more than half the genomes sequenced, translation of the F protein requires translational frameshift or non-standard translation initiation. Curiously, the clade containing the lapinised vaccine viruses that were developed originally in Korea appears to be more similar to the known African viruses than to any other Asian viruses.

Rinderpest eradication: appropriate technology and social innovations.

Rinderpest is only the second infectious disease to have been globally eradicated. In the final stages of eradication, the virus was entrenched in pastoral areas of the Greater Horn of Africa, a region with weak governance, poor security, and little infrastructure that presented profound challenges to conventional control methods. Although the eradication process was a development activity rather than scientific research, its success owed much to several seminal research efforts in vaccine development and epidemiology and showed what scientific decision-making and management could accomplish with limited resources. The keys to success were the development of a thermostable vaccine and the application of participatory epidemiological techniques that allowed veterinary personnel to interact at a grassroots level with cattle herders to more effectively target control measures.

The detection of rinderpest virus RNA extracted from a rapid chromatographic strip-test by RT-PCR.

The Global Rinderpest Eradication Program (GREP) aimed to eradicate rinderpest by 2010 and it is widely believed to have been successful. An integral part of the program was the submission of samples from suspect rinderpest positive animals to a local Reference Laboratory for final confirmation. Confirmation of rinderpest in field samples is often hampered because of poor quality of the sample upon receipt. As part of GREP a rapid diagnostic strip test for the detection of rinderpest virus (RPV) in the field was developed allowing a rapid response to suspect outbreaks. The feasibility of extracting viral RNA from the used rapid diagnostic rinderpest devices for final confirmation in the laboratory is described. Viral material contained within used rinderpest devices was stable enough after storage for one week at 21°C to extract RNA from five different RPV strains and amplify it by reverse transcriptase polymerase chain reaction (RT-PCR). Temperature did not affect adversely the extraction and amplification of the viral RNA but humidity impaired RNA extraction and amplification. Used rinderpest devices from field diagnosed rinderpest-positive animals could represent an ideal additional sample for submission to the Reference Laboratories for confirmation of preliminary diagnosis in the field.

Improvement and development of rapid chromatographic strip-tests for the diagnosis of rinderpest and peste des petits ruminants viruses.

This paper describes the improvement of a rapid diagnostic test for the detection of rinderpest virus (RPV) at pen-side and the development of a similar test for the detection of another Morbillivirus, peste de petits ruminants virus (PPRV). Using the Svanova Biotech format, prototype chromatographic strip test devices were developed for RPV and PPRV detection. For the RP device, the incorporation of a monoclonal antibody (Mab), which recognises additional RPV strains of RPV lineage 2, enhanced the range of reactivity of the rapid diagnostic test. The device detected antigen in animals infected experimentally with different RPV strains. It also showed detection levels similar to the RP Clearview™ device reported previously. In addition, RPV was also detected under field conditions in Pakistan. A PPRV specific Mab (C77) was used for the development of the PPR test. This Mab recognised a wide range of PPRV isolates and did not show any cross-reactivity with any other virus tested. In animal experiments the device was able to detect viral antigen in eye swabs taken from the animals. The PPRV test should be invaluable for future PPR control eradication programs.

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.

The M and N genes-based simplex and multiplex PCRs are better than the F or H gene-based simplex PCR for Peste-des-petits-ruminants virus.

Nucleocapsid (N), matrix (M) and hemagglutinin (H) genes-based simplex PCRs and an N and M genes-based multiplex PCR were developed for detection of Peste-des-petits-ruminants virus (PPRV). The M gene PCR was the most sensitive, followed by N, H and an already described fusion (F) gene PCRs, as they could detect the virus in samples with titers of 101, 102, 104and 105 TCID50/ml, respectively. The multiplex PCR was as sensitive as the M gene PCR, but it had the advantage of differentiating PPRV from Rinderpest virus (RPV).

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.

Re-infection of wildlife populations with rinderpest virus on the periphery of the Somali ecosystem in East Africa.

We report surveillance for rinderpest virus in wildlife populations in three major ecosystems of East Africa: Great Rift Valley, Somali and Tsavo from 1994 to 2003. Three hundred and eighty wild animals were sampled for detection of rinderpest virus, antigen or genome and 1133 sampled for antibody in sera from Kenya, Uganda, Ethiopia and Tanzania from 20 species. This was done modifying for wildlife the internationally recommended standards for rinderpest investigation and diagnosis in livestock. The animals were selected according to susceptibility and preference given to gregarious species, and populations were selected according to abundance, availability and association with livestock. Rinderpest virus, antigen and/or genome were detected in Kenya; within Tsavo, Nairobi and Meru National Parks. Serological results from 864 animals (of which 65% were buffalo) from the region were selected as unequivocal; showing the temporal and spatial aspects of past epidemics. Recent infection has been only in or peripheral to the Somali ecosystem (in Kenya). Our evidence supports the hypothesis that wildlife is not important in the long-term maintenance of rinderpest and that wildlife are infected sporadically most likely from a cattle source, although this needs to be proven in the Somali ecosystem. Wildlife will continue to be a key to monitoring the remaining virus circulation in Africa.

Surveillance of wildlife as a tool for monitoring rinderpest and peste des petits ruminants in West Africa.

The authors provide a report on the surveillance of rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) in the wildlife population in Côte d'Ivoire. For this purpose, 266 animals from nine different species, selected according to susceptibility and abundance, were captured and sampled from Comoé, Marahoué and Lamto Parks. Two hundred and forty seven sera and 214 nasal swabs were collected and analysed by competitive enzyme-linked immunosorbent assay (cELISA) and reverse-transcriptase polymerase chain reaction (RT-PCR) techniques, respectively. Serological data demonstrated that RPV was not circulating within the national Parks and estimated the PPR seroprevalence to be less than 1%. The analysis of the nasal swabs revealed no cases of RPV infection, but PPRV infection was detected in four species, including buffalo. To minimise the cost of the study without affecting the sensitivity of the test, samples were pooled into different groups and submitted to RT-PCR using nucleoprotein gene specific primers. The RT-PCR used in this study, which was derived from the method developed by Couacy-Hymann et al. in 2002, was followed by a hybridisation step using internal specific probes to confirm the identity of the deoxyribonucleic acid product. When used in conjunction with a cELISA this method accurately demonstrated the absence of rinderpest viral persistence in Côte-d'Ivoire.

Detection of rinderpest virus using N-protein monoclonal antibodies.

A panel of monoclonal antibodies (mAbs) was generated against the RBOK strain of rinderpest virus (RPV). All of them bound to the N protein of RPV. The antigen capture ELISA using the mAbs could detect the virus in crude viral preparations. The mAb 12BF8.1.1 showed higher reactivity with cell-associated (CA) virus, whereas the mAbs 12AD10.1.1, 12BD7.1.1 and 12DG7.1.1 showed higher reactivity with extracellular virus (hereafter referred to as cell-free (CF) virus). The mAbs 12BF8.1.1 and 12AD10.1.1 could detect the virus in infected Vero cell culture supernatants (CCS) as early as 24 h post-cytopathic effect (CPE) initiation. Detergent treatment (Triton X-100) of RPV preparations enhanced the binding of the mAbs to the virus. All the seven mAbs showed specific fluorescence in virus-infected cell cultures. The immunofluorescence (IFA) using mAbs was found to be more sensitive and reliable than the immunoperoxidase test (IPT) for detection of rinderpest.

Localization of antigenic sites at the amino-terminus of rinderpest virus N protein using deleted N mutants and monoclonal antibody.

The nucleocapsid (N) protein of rinderpest virus (RPV) is highly conserved, immunogenic, and abundantly expressed during infection. Six antigenic sites (sites A, B, C, D, E and F), defined previously by a competitive binding assay using corresponding monoclonal antibodies (Mabs), have been further localized by immunoassays using deleted N mutants. Five different forms of RPV N protein, containing residues aa 1-79, aa 1-149, aa 1-421, aa 414-525 and aa 1-525, were expressed as glutathione S transferase (GST) fusion proteins (designated as GST-N1-79, GST-N1-149, GST-N1-421, GST-N414-525, and GST-N1-525, respectively) in E.coli BL21 cells. In ELISA using deleted N mutants, Mabs recognizing sites A, B, C, D and E reacted with 3 GST fusion proteins (GST-N1-149, GST-N1-421 and GST-N1-525), indicating that they are located at aa 80-149. Mab recognizing site F reacted with 4 GST fusion proteins (GST-N1-79, GST-N1-149, GST-N1-421 and GST-N1-525), indicating that site F is located at aa 1-79. Identification of the amino-terminal antigenic sites of the N protein would provide antigen basis for developing sensitive and specific diagnostic reagents for RPV, although it remains to be further investigated antigenic sites at the carboxyl-terminus.

Use of participatory epidemiology in studies of the persistence of lineage 2 rinderpest virus in East Africa.

In 1994, rinderpest virus of African lineage 2 was detected in East Africa after an apparent absence of more than 30 years. In 1996, a disease search, based on participatory epidemiological techniques supplemented by serological and virological analyses, was undertaken in southern Somalia and north-eastern Kenya to collate past and current epidemiological information about rinderpest-compatible disease events, and to test the hypothesis that African lineage 2 rinderpest virus persists in populations of transhumant cattle in the Somali ethnic areas. The findings in Afmadu in Lower Juba led the search for rinderpest to the communities in the Bardera area and then on to the Kenya/Somalia border areas between Mandera and El Wak. The herders had a specific knowledge of the clinical signs of rinderpest and provided detailed and accurate descriptions of cases. They differentiated between classical acute rinderpest and a milder syndrome characterised by an ocular discharge and diarrhoea, few oral lesions, corneal opacity and occasional mortality. The studies provided evidence for the endemic occurrence of rinderpest back to at least 1981, with a periodicity of five years in the incidence of the disease. After a period of high mortality in 1992 to 1993, around Afmadu, herders reported a mild disease, with occasional increases in mortality, from other areas of Lower Juba and the Gedo Region. Reports by herders of a rinderpest-compatible disease in the El Wak area were pursued until active cases were located and rinderpest was confirmed.

Competitive enzyme-linked immunosorbent assay based on monoclonal antibody and recombinant hemagglutinin for serosurveillance of rinderpest virus.

A competitive enzyme-linked immunosorbent assay (C-ELISA) which detects antibodies unique to rinderpest virus (RPV) has been developed. This test can differentiate antibodies against RPV and those against peste des petits ruminants virus. The recombinant RPV hemagglutinin (H)-protein C-ELISA (recH C-ELISA) is based on the ability of a well-characterized monoclonal antibody (MAb) produced with the soluble, secreted form of the H protein (Sec H protein) of RPV made in a baculovirus expression system to compete with the binding of RPV antibodies in the serum of vaccinated or infected, recovered animals to the Sec H protein. The B-cell epitope recognized by the MAb corresponds to amino acids 575 to 583 on the H protein, which is not present on the antigenically closely related peste des petits ruminants virus hemagglutinin-neuraminidase protein. Initially, a positive-negative threshold cutoff value for percent inhibition of 34 was established with 500 known RPV-negative serum samples. The recH C-ELISA was developed with the enzyme immunoassay software of a commercial RPV C-ELISA kit. Comparative analysis of the test results for 700 serum samples obtained with the commercial kit gave a sensitivity of 112.4% and a specificity of 72.4%. Variations in percent inhibition values were observed for the two assay systems. These variations may have been due to the undefined amount of antigen present in the commercial kit as well as the use of a different MAb. The recH C-ELISA detected more positive serum samples compared to the number detected by the commercial kit, with the results confirmed by a virus neutralization test. Thus, recH C-ELISA is a sensitive tool for RPV serosurveillance in disease eradication programs.

Rinderpest virus lineage differentiation using RT-PCR and SNAP-ELISA.

An RT-PCR/ELISA system has been developed that detects and differentiates Rinderpest virus (RPV) from the other closely related morbillivirus of ruminants, Peste des petits Ruminants virus (PPRV). In addition, using lineage specific probes, it is possible to determine whether the virus sample is wild-type or vaccine, and the likely origin of the outbreak if it is wild-type. It involves carrying out a RT-PCR with one digoxygenin (Dig)-labelled primer followed by a hybridisation step with a virus-specific, biotin-labelled, probe. The hybridisation step is carried out in an ELISA format on a streptavidin-coated plate. The DIG-labelled products are detected using a specific anti-DIG monoclonal antibody and an anti-mouse horseradish peroxidase conjugate. The hybridisation step replaces nucleotide sequencing or nested PCR for confirmation of the identity of DNA product. The assay is fast and easy to carry out and can give semi-quantitative estimates of the virus content of samples.

The use of antigen-capture enzyme-linked immunosorbent assay (ELISA) for the diagnosis of rinderpest and peste des petits ruminants in ethiopia.

Rinderpest had been reported in most parts of Ethiopia when the Pan African Rinderpest Campaign (PARC) was launched. As a result of intensive disease investigation and strategic vaccination, most parts of the country are now considered provisionally free, and widespread vaccination has been replaced by clinical and serological surveillance. Details of any episodes of disease are recorded and followed up after laboratory confirmation of suspected cass using antigen-capture ELISA. This paper is based on observations on the performance of the antigen detection ELISA compared to the agar gel immunodiffusion (AGID) test, which also differentiates rinderpest from peste des petits ruminants (PPR). The stability of the specific viral antigen was monitored for 4 days, and rinderpest and PPR antigens were still detected, depending on the type of specimen. Antigen capture ELISA is more rapid, sensitive and virus specific than the AGID. Even if the cold chain of the specimen is compromised for a day or two during sample collection and submission, the specimen may still be suitable for testing by ELISA.

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.

Rinderpest virus isolates of different virulence vary in their capacity to infect bovine monocytes and macrophages.

Three isolates of rinderpest virus (RPV) with different in vivo virulence were able to infect and productively replicate in bovine monocytic cells. They differed in their kinetics of replication and the morphological changes induced in infected cultures. The highly virulent RPV-Saudi infected > 80% of cells within 6 days p.i. (m.o.i. = 0.1 TCID50 per cell). Under identical conditions, > 50% of cells were infected by the 'mild' (causes minimal mortality in vivo) isolate RPV-Egypt, whereas only 25% were infected by the avirulent RPV-RBOK. Infection by all three viruses produced infectious progeny, induced the formation of syncytia and stellate cells with long processes, and down-regulated MHC class II expression; there was no apparent effect on MHC class I nor LFA-1. RPV-Saudi was the most efficient at generating progeny virus and producing syncytia. While RPV-RBOK was the least efficient at inducing syncytia, RPV-Egypt was the least efficient for progeny virus production. In contrast, RPV-Egypt was particularly efficient at inducing stellate cell formation and down-regulating MHC class II expression. These results indicate a relationship between in vivo virulence and the characteristics of replication and induced morphological changes in monocytes/macrophages. The down-regulation of MHC class II expression would offer a means by which the virus could evade immune recognition. This would be particularly useful for the more cell-associated, but less efficient at maturing, RPV-Egypt.

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.