Development of diagnostic methods and surveillance of Rift valley fever in Republic of Korea

Abstract

Rift Valley fever (RVF) is an acute mosquito-borne viral zoonotic disease that affects mainly domestic ruminants and humans. This disease causes abortions in pregnant animals, and it has a high mortality rate in newborn animals. RVF virus (RVFV) was first identified in Kenya in 1931 and was reported to be endemic in Africa but has recently spread to the Arabian Peninsula. With increasing climate change and global movement in animals and animal products, there is great concern that the disease might be spreaded worldwide to regions such as Europe, Asia and the Americas. Therefore, early detection and surveillance are important for preventing the introduction of RVF in non-endemic or disease free countries. Although RVF has not been reported in the Republic of Korea (ROK), the possibility of RVFV introduction will be increasing because transmissible mosquito vectors are present and direct flights to Africa were added in 2012. Thus diagnostic methods were developed and conducted a surveillance study to detect RVFV in mosquito vectors. In the first study, a new quantatitative real time RT-PCR (qRT-PCR) assay was developed that can safely and cost-effectively differentiate infected from vaccinated animals (DIVA) with possible application in RVF-free countries. The new qRT-PCR assay targeting the S segment (NSs and N gene) was tested with synthesized standard RNA and MP-12 strain viruses. The detection limit of the new qRT-PCR assay was 1 copy/µl of NSs and N, and was able to differentiate the Smithburn strain from the Clone 13 vaccine strain. No cross reactivity with other vector-borne viruses was observed, a factor which is especially important in the ROK, was observed. To examine the performance of the new qRT-PCR, intra-and inter-assay variability data were analyzed and showed high reproducibility. These results indicate that the new qRT-PCR can be used as a safe and cost-effective DIVA diagnostic test in RVF-free countries including ROK. In second study, a monoclonal antibody-based competitive ELISA was developed for the detection of antibodies to RVFV in goat and cattle. The recombinant N protein of RVFV was expressed in E. coli with six-histidine tag and the purified N protein was used for detecting antigen with competitive monoclonal antibody against RVFV antibodies. The competitive ELISA (C-ELISA) could detect antibodies at 9-11 days after inoculation in goat and cattle with the sensitivity of 94.7 % (virus neutralization titer >32) and specificity of 99.7%, respectively. In addition, C-ELISA did not show any cross-reactivity with positive sera against Arboviruses such as Akabane, Aino, Chuzan, Ibaraki, and Bovine ephemeral fever virus which are prevalent viral agents in ruminant animals throughout Southeast Asia. The results in present study indicate that C-ELISA is a simple, rapid and convenient sero-diagnostic method for RVFV in goat and cattle. In third study, a surveillance study was conducted to detect RVFV in mosquito vectors collected around the airport and harbor from 2012 to 2013. A total of 36,734 mosquitoes were collected and tested by real time RT-PCR. A total of 1,837 mosquito pools were used, and all were confirmed to be negative. This is the first report in the ROK concerning RVFV surveillance in mosquito vectors, and continuous surveillance should be conducted for the early warning of RVFV introduction. In final study, a serological surveillance was conducted to detect antibodies against RVFV. A total of 2,382 serum samples from goats and cattle were randomly collected from nine areas in ROK from 2011 to 2013. These samples were tested for antibodies against RVFV, using commercial ELISA kits. None of the goats and cattle was positive for antibodies against RVFV. This finding suggests that this disease is not present in ROK, and furthermore presents the evidence of the RVFV-free status of this country. Taken together, the results of this study have important meaning in animal and public health because RVF is transboundary disease and zoonosis. Furthermore, continuous surveillance and regulatory activities need to be undertaken to maintain this status.