Anti-Nucleocapsid Protein Immune Responses Counteract Pathogenic Effects of Rift Valley Fever Virus Infection in Mice Petrus Jansen van Vuren1,2, Caroline T. Tiemessen2,3, Janusz T. Paweska1,2* 1 Special Pathogens Unit, National Institute for Communicable Diseases of the National Health Laboratory Service, Sandringham, South Africa, 2 Division Virology and Communicable Diseases Surveillance, School of Pathology, University of the Witwatersrand, Johannesburg, South Africa, 3 Cell Biology/AIDS Virus Research Unit, National Institute for Communicable Diseases of the National Health Laboratory Service, Sandringham, South Africa Abstract The known virulence factor of Rift Valley fever virus (RVFV), the NSs protein, counteracts the antiviral effects of the type I interferon response. In this study we evaluated the expression of several genes in the liver and spleen involved in innate and adaptive immunity of mice immunized with a RVFV recombinant nucleocapsid protein (recNP) combined with Alhydrogel adjuvant and control animals after challenge with wild type RVFV. Mice immunized with recNP elicited an earlier IFNb response after challenge compared to non-immunized controls. In the acute phase of liver infection in non-immunized mice there was a massive upregulation of type I and II interferon, accompanied by high viral titers, and the up- and downregulation of several genes involved in the activation of B- and T-cells, indicating that both humoral and cellular immunity is modulated during RVFV infection. Various genes involved in pro-inflammatory responses and with pro- apoptotic effects were strongly upregulated and anti-apoptotic genes were downregulated in liver of non-immunized mice. Expression of many genes involved in B- and T-cell immunity were downregulated in spleen of non-immunized mice but normal in immunized mice. A strong bias towards apoptosis and inflammation in non-immunized mice at an acute stage of liver infection associated with suppression of several genes involved in activation of humoral and cellular immunity in spleen, suggests that RVFV evades the host immune response in more ways than only by inhibition of type I interferon, and that immunopathology of the liver plays a crucial role in RVF disease progression. Citation: Jansen van Vuren P, Tiemessen CT, Paweska JT (2011) Anti-Nucleocapsid Protein Immune Responses Counteract Pathogenic Effects of Rift Valley Fever Virus Infection in Mice. PLoS ONE 6(9): e25027. doi:10.1371/journal.pone.0025027 Editor: Patricia V. Aguilar, University of Texas Medical Branch, United States of America Received March 3, 2011; Accepted August 25, 2011; Published September 16, 2011 Copyright: 2011 Jansen van Vuren et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Poliomyelitis Research Foundation (PRF Grant number 08/14) (www.prf.ac.za). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: januszp@nicd.ac.za Introduction Rift Valley fever virus (RVFV), a mosquito borne Phlebovirus of the Bunyaviridae family, causes irregular but large outbreaks characterized by high fatality rates in young domestic ruminants and abortion storms in pregnant animals [1–3]. The RVFV RNA genome consists of large (L) and medium (M) segments which are in the negative sense, and a small (S) segment utilizing an ambisense coding strategy. The L segment encodes a viral RNA- dependent RNA polymerase, the M segment encodes two structural glycoproteins, two non-structural proteins, and the S segment encodes the nucleocapsid protein (NP) and a non- structural protein (NSs) [4,5]. The NP is highly conserved and constitutes the most immunodominant viral protein in the Bunyaviridae family [6–11]. The NP associates with the viral RNA-dependent RNA polymerase and viral RNA to form the ribonucleoproteins (RNP) necessary for transcription and replica- tion. The known virulence factor of RVFV, the NSs protein, counteracts the antiviral effects of the type I interferon response. Not much is known, however, about other molecular aspects of RVFV pathogenicity. Although the nucleocapsid protein (NP) of the virus does not induce production of neutralizing anti- bodies, anti-NP antibodies have been shown to be protective in mice [12–17]. Humoral responses against the RVFV glycoproteins effectively neutralize the virus and are believed to solely protect against challenge [14,16,18–20]. RVFV is sensitive to the actions of type I interferons [21–24] but it has developed several counteracting mechanisms. The NSs protein of RVFV interacts with a repressor complex (Sin3A/ NCoR/HDAC) to inhibit transcriptional activation of the IFNb gene [25,26]. A more general shutdown of cellular gene expression is caused by interaction of NSs with the p44 subunit of the basal transcription factor II H (TFIIH), resulting in reduced transcrip- tional activity in RVFV infected cells [27]. The NSs has also been shown to act on a post-translational level by degrading Protein Kinase R (PKR) [28]. Interferon gamma (IFNc) is a strong immunoregulator and has direct antiviral properties, but its role in the protection of the host against RVFV infection is debatable [29]. A study in rhesus monkeys showed that prophylactic treatment with recombinant human IFNc before RVFV infection protected monkeys from clinical disease and decreased viremia significantly [30]. However, there was no marked difference in pathogenicity of RVFV MP-12 or Clone-13 in wild type mice compared to mice deficient in IFNc receptor (IFNGR2/2), suggesting that NSs does not have the same inhibitory effect on the type II interferon response (IFNc) as it has on the type I PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e25027