Journal of Virological Methods 140 (2007) 106–114 Preparation and evaluation of a recombinant Rift Valley fever virus N protein for the detection of IgG and IgM antibodies in humans and animals by indirect ELISA Petrus Jansen van Vuren a , Abraham C. Potgieter b , Janusz T. Paweska c,∗ , Alberdina A. van Dijk a a North-West University, Potchefstroom 2520, South Africa b Biochemistry Division, Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa c Special Pathogens Unit, National Institute for Communicable Diseases, Private Bag X4, Sandringham 2131, South Africa Received 14 September 2006; received in revised form 2 November 2006; accepted 8 November 2006 Available online 15 December 2006 Abstract This paper describes the cloning, sequencing and bacterial expression of the N protein of the Rift Valley fever virus (RVFV) ZIM688/78 isolate and its evaluation in indirect ELISAs (I-ELISA) for the detection of IgM and IgG antibodies in human and sheep sera. Sera used for the evaluation were from 106 laboratory workers immunised with an inactivated RVF vaccine, 16 RVF patients, 168 serial bleeds from 8 sheep experimentally infected with wild type RVFV and 210 serial bleeds from 10 sheep vaccinated with the live attenuated Smithburn RVFV strain. All human and animal sera that tested positive in the virus neutralisation test were also positive in the IgG I-ELISA. There was a high correlation (R 2 = 0.8571) between virus neutralising titres and IgG I-ELISA readings in human vaccinees. In experimentally infected sheep IgG antibodies were detected from day 4 to 5 post-infection onwards and IgM antibodies from day 3 to 4. The IgG I-ELISA was more sensitive than virus neutralisation and haemagglutination-inhibition tests in detecting the early immune response in experimentally infected sheep. The I-ELISAs demonstrated that the IgG and IgM response to the Smithburn vaccine strain was slower and the levels of antibodies induced markedly lower than to wild type RVFV infection. © 2006 Elsevier B.V. All rights reserved. Keywords: Rift Valley fever virus; Recombinant N protein; IgM and IgG indirect ELISA; Humans; Sheep 1. Introduction Rift Valley fever (RVF) is a mosquito-borne viral disease that is a significant global threat to humans and livestock. Historically, Rift Valley fever virus (RVFV) was restricted to sub-Saharan Africa (Swanepoel and Coetzer, 2004). However, in 1977 it first emerged in Egypt where it caused severe outbreaks in livestock and humans (Meegan, 1981). The recent RVF out- breaks in the Arabian Peninsula (CDC, 2000; Jupp et al., 2002; Shoemaker et al., 2002), the first outbreaks outside Africa, have the implication that it is likely that RVFV will now spread fur- ther into non-endemic RVF areas since it is capable of utilizing a wide range of mosquito vectors (Turrel et al., 1998). RVFV ∗ Corresponding author. Tel.: +27 11 3866382; fax: +27 11 8823741. E-mail address: januszp@nicd.ac.za (J.T. Paweska). infections in livestock are characterised by an acute hepatitis, abortion and high mortality rates, particularly in young animals. Humans infected with RVFV typically develop a mild self lim- ited febrile illness, but retinal degeneration, severe encephalitis, fatal hepatitis and hemorrhagic fever may also occur (Swanepoel and Coetzer, 2004). RVFV is a member of the Phlebovirus genus in the Bunyaviri- dae family (Bishop et al., 1980), a family of spherical enveloped viruses with a trisegmented, single stranded RNA genome of negative (L and M segments) or ambisense (S segment) polar- ity. The large (L) segment encodes the RNA dependent RNA polymerase. The medium (M) segment encodes the envelope glycoproteins G1 and G2 and non structural proteins 14K and 78K (Giorgi et al., 1991; Schmaljohn and Hooper, 2001). The small (S) segment encodes the N protein and a non structural (NSs) protein using an ambisense coding strategy (Ihara et al., 1984; Giorgi et al., 1991; Gauliard et al., 2006). The N protein 0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2006.11.005