Laboratory safe detection of nucleocapsid protein of Rift Valley fever virus in human and animal specimens by a sandwich ELISA

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Abstract

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. Polyclonal capture and detection immune sera were generated respectively in sheep and rabbits immunized with recombinant NP antigen. 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 phleboviruses or other members of the family Bunyaviridae was observed. In specimens spiked with RVFV, including human and animal sera, homogenates of liver and spleen tissues of domestic ruminants, and Anopheles mosquito homogenates, the sAg-ELISA detection limit ranged from log10102.2 to 103.2 TCID50/reaction volume. 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 from 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 of various organs from mice infected experimentally and 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. This ability renders the assay 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.

Introduction

Rift Valley fever virus (RVFV), a member of the Phlebovirus genus of the Bunyaviridae family (Bishop et al., 1980), is the causative agent of Rift Valley fever (RVF), a mosquito-borne, viral, zoonotic disease that poses a significant health threat to domestic ruminants and humans in Africa. Infection with RVFV in livestock is characterized by an acute hepatitis, abortion and high mortality rates in new born animals. Humans infected with RVFV typically develop a mild self-limited febrile illness, but retinal degeneration, severe encephalitis, fatal hepatitis and hemorrhagic manifestations occur in a small proportion of patients (Swanepoel and Coetzer, 2004). Historically, RVF was restricted to sub-Saharan Africa but in 1977 spread to northern Africa (Meegan, 1979), in 1987 to West Africa (Jouan et al., 1988), and in 2000 to the Arabian Peninsula (Shoemaker et al., 2002). The ability of RVFV to utilise a wide range of mosquito vectors (Turrel et al., 2008) and cause extensive outbreaks with severe socio-economic losses, as well as global climate change that facilitates spread of vector-borne diseases outside their traditional geographical boundaries, are of great international concern. There is an increasing demand for diagnostic tools for rapid, accurate and safe laboratory diagnosis of RVF. This demand is challenged by the fact that work with RVFV requires high biocontainment facilities and also that the virus is regarded as a potential bioweapon agent. For these reasons, the current capacity for laboratory diagnosis of RVF is restricted to a limited number of reference laboratories worldwide. Diagnosis of RVF is achieved by different methods, including virus isolation (Shope and Sather, 1979, Anderson et al., 1989), nucleic acid techniques (Ibrahim et al., 1997, Garcia et al., 2001, Drosten et al., 2002) and detection of specific antibodies (Swanepoel et al., 1986, Paweska et al., 2003a). Virus isolation is lengthy, expensive and involves propagation of live virus. Nucleic acid techniques require highly specialized laboratory equipment, sophisticated reagents, and well-trained laboratory personnel, the availability of which could be problematic when outbreaks occur in remote regions and rapid diagnosis is necessary. Highly sensitive and specific ELISAs based on inactivated whole RVFV antigen (Paweska et al., 2003a, Paweska et al., 2003b, Paweska et al., 2005a, Paweska et al., 2005b) and recombinant nucleocapsid antigen (Jansen van Vuren et al., 2007, Paweska et al., 2007, Paweska et al., 2008b) have been validated extensively and shown to be highly accurate for detection of anti-RVFV antibody in humans and animals. An optical fiber immunosensor based on a sandwich ELISA has recently been reported for the detection of IgG antibody to RVFV in humans (Sobarzo et al., 2007). ELISAs for the detection of RVF viral antigen have been reported (Niklasson et al., 1983, Meegan et al., 1989, Zaki et al., 2006) but most of these assays are based on reagents that are difficult and expensive to produce, pose a biohazard risk to laboratory personnel, and were not validated according to current recommendations.

This paper describes the first development and evaluation of a sandwich ELISA for antigen detection (sAg-ELISA) based on an anti-nucleocapsid antibody capture and detection system and the use of thermo-chemically inactivated specimens to ensure safety of laboratory staff involved in the diagnosis of RVF.

Section snippets

Human and sheep sera

A total of 130 human sera submitted to the Special Pathogens Unit of the National Institute for Communicable Diseases, Sandringham, South Africa (SPU-NICD) for routine testing were used; 70 specimens were from suspected RVF cases sampled during the 2006–2008 disease outbreaks in Southern Africa.

A total of 105 sheep sera were used of which 20 were from sheep inoculated subcutaneously with the SPU22/118 KEN 07 strain of RVFV, isolated from a RVF human case during the 2007 Kenyan epidemic (CDC,

Internal quality control and repeatability

The sAg-ELISA generated minimal background noise and discriminated clearly between all the internal controls used. During routine runs of the assay there was no evidence for excessive variations within the upper and lower control limits (Fig. 1). CV values for intra- and inter-plate runs were below 5% (Table 1) demonstrating high repeatability of the assay.

Biological safety of inactivated specimens

Mice inoculated with inactivated samples of sera and tissue culture homogenates, which were spiked with a high concentration of RVFV (log1010

Discussion

The recent epidemics of RVF in the Arabian Peninsula have demonstrated the potential for RVFV to extend its traditional boundaries and cause severe outbreaks in naïve populations of humans and livestock. There is an increased need internationally for rapid diagnostic tools to detect RVFV in humans and animals both in endemic regions and potentially receptive regions.

A sandwich ELISA reported by Niklasson et al. (1983), using hyperimmune mouse and rabbit anti-sera, had a sensitivity of 105

Acknowledgements

We would like to thank Dr. Lizette Koekemoer from the Vector Control Reference Unit, National Institute for Communicable Diseases, South Africa for supplying Anopheles mosquito pools and the Poliomyelitis Research Foundation for financial support of the study.

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