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Veterinary Surveillance of Rift Valley Fever (RVF)
Closed for proposals
Project Type
Project Code
D32023CRP
1363Approved Date
Status
Start Date
Expected End Date
Completed Date
28 September 2011Objectives
The aim of the CRP is to develop, evaluate, validate and harmonize nuclear and nuclear related serological and molecular diagnostic technologies to improve Member State capacities to effectively control rift Valley Fever (RVF) by supporting the build up of competence in the use of modern biotechnology.
Specific objectives
• Evaluation of recombinant antigens for use in indirect and competition ELISA’s.).
• Harmonization of Standard Operating Procedures (SOPs) and introduction of quality assurance procedures for RVF-ELISA and RVFV RT-PCR.
Setting up of a serological and molecular epidemiological database (based on antibody prevalence and virus isolate genetic variation
The Specific research objectives are to evaluate, validate and implement RT-PCR and PCR sequencing procedures for early and sensitive detection of the RVF virus and to apply molecular epidemiology using isotopic techniques to improve diagnostic sensitivity (via isotope incorporation into PCR amplicons) and to confirm diagnostic specificity (via hybridization of amplicons with isotope labelled probes). In laboratories equipped with real-time PCR capabilities, adapt the manual PCR procedures to include their use as part of the Standard Operating Procedures (SOPs). Manual isotope based slab PCR-sequencing procedures will be implemented (in laboratories equipped with automated sequencing equipment, these procedures will be adapted for use)
.• Evaluation, validation and use of different ELISA formats to detect virus-specific antibodies.
Impact
• A real-time reverse transcription-loop-mediated isothermal amplification assay (RT-LAMP) targeting the genomic large RNA segment of Rift Valley fever virus (RVFV) was developed and validated. A set of six designed RT-LAMP primers identified strains of RVFV isolated in geographically distinct areas over a period of 50 years; there was no cross-reactivity with other genetically related and unrelated arboviruses. When testing serial sera and plasma from sheep experimentally infected with wild-type RVFV, there was 100% agreement between results of the RT-LAMP, a TaqMan-based real-time PCR, and virus isolation. Similarly, the assay had very high levels of diagnostic sensitivity and specificity when testing various clinical specimens from humans and animals naturally infected with the virus during recent outbreaks of the disease in Africa. The detection of specific viral genome targets in positive clinical specimens was achieved in less than 60 minutes.
• A highly accurate, rapid, and very simple nucleic acid detection format, the RT-LAMP, was developed in a thermostable kit format that has been used in less-well-equipped laboratories in Africa (Mauretania RVF outbreak 2010) and a portable reader performing this test was developed together with some software to allow the rapid diagnosis during RVF outbreaks in remote areas.
• The RT-LAMP will be a valuable tool for the differential diagnosis of viral haemorrhagic fevers as well for humans.
• A quantitative real-time reverse transcription-PCR (qRT-PCR) was used in an outbreak in Kenya to see if it could be used in the field to rapidly identify viraemic RVF cases with risk of death. Levels of viraemia in fatal cases were significantly higher than those in nonfatal cases. A negative correlation between the levels of infectious virus particles and the qRT-PCR crossover threshold (CT) values allowed the use of qRT-PCR to assess prognosis. The correlation between high viraemia and fatality indicates that this qRT-PCR testing can be used to identify severe RVF cases in humans requesting specific treatment.
• A safe laboratory procedure, based on a sandwich ELISA (sAg-ELISA), was developed and evaluated for the detection of nucleocapsid protein (NP) of Rift Valley fever virus (RVFV) in specimens inactivated at 56 °C for 1 h in the presence of 0.5% Tween-20 (v/v) before testing. The assay was highly repeatable and specific; it detected strains of RVFV from the entire distributional range of the disease, isolated over a period of 53 years; no cross-reactivity with genetically related African viruses or other members of the family Bunyaviridae was observed. The sAg-ELISA detection limit ranged from log10102.2 to 103.2 TCID50/reaction volumes. The ELISA detected NP antigen in spiked bovine and sheep liver homogenates up to at least 8 days of incubation at 37 °C whereas infectious virus could not be detected at 48 h incubation in these adverse conditions. Compared to virus isolation from sera of RVF patients and sheep infected experimentally, the ELISA had 67.7% and 70% sensitivity, and 97.97% and 100% specificity, respectively. The assay was 100% accurate when testing tissues from buffalo foetuses infected naturally. The assay was able to detect NP antigen in infective culture supernatants 16–24 h before cytopathic effects were observed microscopically and as early as 8 h after inoculation with 105.8 TCID50/ml of RVFV. The assay will be valuable therefore for rapid identification of the virus when its primary isolation is attempted in vitro. As a highly specific, safe and simple assay format, the sAg-ELISA represents a valuable diagnostic tool for use in less equipped laboratories in Africa, and for routine differential diagnosis of viral hemorrhagic fevers.
• A multi-disciplinary approach is now imperative with regard to understanding and dealing with the geographical spread of vector-borne diseases, and groups need to collaborate in an integrated manner that includes vector control, vaccination programmes, improved therapy strategies, use of rapid, sensitive and specific diagnostic tools to improve monitoring and surveillance, increased public awareness, capacity building and improvement of infrastructure in endemic regions. An international network for capacity building, known as ARBO-ZOONET has been created that will promote: -
- identification of risk areas for RVF and updating risk maps;
- collection of viral isolates to enable improvement of diagnostic tests and vaccines design;
- establishment of surveillance networks for RVF;
- establishment of working groups on vector control, vaccines and therapy;
- promote the transfer of knowledge and technology to relevant countries;
• All Research Contract Holders installed ELISA tests for detection of IgG and IgM antibodies to RVFV in their laboratories for routine detection of RVF in livestock; in addition emphasis was placed on ensuring that competence in the use of these assays was maintained by a continuous schedule of training and, re-training where necessary, of staff involved in the programme. ELISA kits that had been developed by Onderstepoort Veterinary Institute and distributed by BDSL were tested and found fit for the given purpose. Since an alternative producer is distributing RVF ELISA kits these are now being validated making use of the serum banks developed during this project.
• RCHs established serum banks
• The laboratories developed their capacity for implementation of PCR for the detection of RVFV genome.
• Most of RCHs were able to carry out proficiency trials of ELISA tests developed by OVI, one an indirect ELISA for the detection of IgG antibodies, and the other an inhibition ELISA for the detection of RVF antibodies in humans, domesticated livestock and wild ruminants.
• The virus like particle vaccine (VLP) against RVF was tested in sheep at 1 RCH laboratory and induction of immunity could be shown.
• Production of RVF-VLP’s could be transferred to plants and initial results indicating successful oral immunization are under investigation.
• Epidemiological data on the distribution of the virus were gathered during the CRP. In all participating countries RVFV activity could be shown. In most cases this was low (below 5% sero-positivity). Specific disease indicators were developed like apparently higher abortion rate coincides with a minimum of 15 % sero-prevalence.
• The accumulation of so many results from the CRP led to several invitations to international meetings aiming at improving the forecasting of RVF events and intervention strategies.
Relevance
Rift Valley fever virus (RVFV) is one of a number of arthropod-borne Arboviruses, which include West Nile fever virus (WNFV), a mosquito-borne virus, and Crimean-Congo haemorrhagic fever virus (CCHFV), a tick-borne virus. These arthropod-borne viruses can cause disease in different domestic and wild animals and in humans, posing a threat to public health because of their epidemic and zoonotic potential. In 2000, RVF was reported for the first time outside the African continent, with cases being confirmed in Saudi Arabia and Yemen. This spread was probably caused by ruminant trade and highlights the threat of expansion of the virus into other parts of Asia and even Europe especially since RVFV can be spread by a wide range of mosquito vectors. In the light of global warming and globalization of trade and travel, public awareness of the spread of emerging zoonotic diseases has increased. Although transmission is mainly by mosquitoes, it can also occur via contact with infected animals (in the case of veterinarians or abattoir workers), infected blood or tissue samples (laboratory workers) and patients (family, physicians or nursing aides). The threat to Africa remains: in 2010 RVFV infection affected sheep, goats, cattle and wildlife on farms in a number of provinces in South Africa and Mauretania resulting in extensive livestock deaths as well as many human cases, including some fatalities. Most of these cases were in farmers, veterinarians and farm workers who were in direct contact with RVFV-infected livestock. Heavy rain at the start of 2011 in South Africa has presaged yet another outbreak of RVF in both livestock and humans.