Development of monoclonal antibodies to Rift Valley Fever Virus and their application in antigen detection and indirect immunofluorescence.

Rift Valley fever virus is a mosquito-borne virus which is associated with acute hemorrhagic fever leading to large outbreaks among ruminants and humans in Africa and the Arabian Peninsula. RVFV circulates between mosquitoes, ruminants, camels and humans, which requires divergent amplification and maintenance strategies that have not been fully explored on the cellular and molecular level. We therefore assessed monoclonal antibodies for their applicability to monitor the expression pattern and kinetics of viral proteins in different RVFV infected cell species. Sequences of RVFV vaccine strain MP-12 were used in a bacterial expression system to produce recombinant non-structural proteins directed to NSs and NSm. After immunization of balb/c mice a set of monoclonal antibodies were generated and extensively characterized. The kinetics of RVFV proteins in vertebrate (Vero76) and mosquito-derived (C6/36) cells were evaluated with monoclonal antibodies against the nucleocapsid protein (NP) and the glycoproteins (Gn and Gc) as well as with the newly generated NSs and NSm derived monoclonal antibodies. Significant differences of viral protein distribution and accumulation in vertebrate compared to mosquito-derived cells could be demonstrated. Differences were observed for the nonstructural NSm and most intriguingly for the NSs protein indicating significant divergency of replication strategies of RVFV in Vero 76 cells and C6/36 cells. The described monoclonal antibodies are therefore powerful tools to elucidate the discrepancies of virus replication and interaction within the mammalian host compared to the mosquito vector.

Seroprevalence of Rift Valley fever virus in livestock during inter-epidemic period in Egypt, 2014/15.

BACKGROUND: Rift Valley fever virus (RVFV) caused several outbreaks throughout the African continent and the Arabian Peninsula posing significant threat to human and animal health. In Egypt the first and most important Rift Valley fever epidemic occurred during 1977/78 with a multitude of infected humans and huge economic losses in livestock. After this major outbreak, RVF epidemics re-occurred in irregular intervals between 1993 and 2003. Seroprevalence of anti-RVFV antibodies in livestock during inter-epidemic periods can be used for supporting the evaluation of the present risk exposure for animal and public health. A serosurvey was conducted during 2014/2015 in non-vaccinated livestock including camels, sheep, goats and buffalos in different areas of the Nile River Delta as well as the furthermost southeast of Egypt to investigate the presence of anti-RVFV antibodies for further evaluating of the risk exposure for animal and human health. All animals integrated in this study were born after the last Egyptian RVF epidemic in 2003 and sampled buffalos and small ruminants were not imported from other endemic countries. RESULTS: A total of 873 serum samples from apparently healthy animals from different host species (camels: n = 221; sheep: n = 438; goats: n = 26; buffalo: n = 188) were tested serologically using RVFV competition ELISA, virus neutralization test and/or an indirect immunofluorescence assay, depending on available serum volume. Sera were assessed positive when virus neutralization test alone or least two assays produced consistent positive results. The overall seroprevalence was 2.29% (95%CI: 1.51-3.07) ranging from 0% in goats, 0.46% in sheep (95%CI: 0.41-0.5), and 3.17% in camels (95%CI: 0.86-5.48) up to 5.85% in buffalos (95%CI: 2.75-8.95). CONCLUSION: Our findings assume currently low level of circulating virus in the investigated areas and suggest minor indication for a new RVF epidemic. Further the results may indicate that during long inter-epidemic periods, maintenance of the virus occur in vectors and also most probably in buffaloes within cryptic cycle where sporadic, small and local epidemics may occur. Therefore, comprehensive and well-designed surveillance activities are urgently needed to detect first evidence for transition from endemic to epidemic cycle.

A competitive ELISA for species-independent detection of Crimean-Congo hemorrhagic fever virus specific antibodies.

Crimean-Congo hemorrhagic fever virus (CCHFV) circulates in many countries of Asia, Africa, and Europe. CCHFV can cause a severe hemorrhagic fever in humans with case-fatality rates of up to 80%. CCHF is considered to be one of the major emerging diseases spreading to and within Europe. Ticks of the genus Hyalomma function as vector as well as natural reservoir of CCHFV. Ticks feed on various domestic animals (e.g. cattle, sheep, goats) and on wildlife (e.g. hares, hedgehogs). Those animal species play an important role in the life cycle of the ticks as well as in amplification of CCHFV. Here we present a competitive ELISA (cELISA) for the species-independent detection of CCHFV-specific antibodies. For this purpose nucleocapsid (N) protein specific monoclonal antibodies (mAbs) were generated against an Escherichia coli (E. coli) expressed CCHFV N-protein. Thirty-three mAbs reacted with homologous and heterologous recombinant CCHFV antigens in ELISA and Western blot test and 20 of those 33 mAbs reacted additionally in an immunofluorescence assay with eukaryotic cells expressing the N-protein. Ten mAbs were further characterized in a prototype of the cELISA and nine of them competed with positive control sera of bovine origin. The cELISA was established by using the mAb with the strongest competition. For the validation, 833 sera from 12 animal species and from humans were used. The diagnostic sensitivity and specificity of the cELISA was determined to be 95% and 99%, respectively, and 2% of the sera gave inconclusive results. This cELISA offers the possibility for future large-scale screening approaches in various animal species to evaluate their susceptibility to CCHFV infection and to identify and monitor the occurrence of CCHFV.