Rift Valley fever virus inhibits a pro-inflammatory response in experimentally infected human monocyte derived macrophages and a pro-inflammatory cytokine response may be associated with patient survival during natural infection

Abstract

Rift Valley fever virus (RVFV) causes significant morbidity and mortality in humans and livestock throughout Africa and the Middle East. The clinical disease ranges from mild febrile illness, to hepatitis, retinitis, encephalitis and fatal hemorrhagic fever. RVFV NSs protein has previously been shown to interfere in vitro with the interferon response, and RVFV lacking the NSs protein is attenuated in several animal models. Monocytes and macrophages are key players in the innate immune response via expression of various cytokines and chemokines. Here we demonstrate that wild-type RVFV infection of human monocyte-derived macrophages leads to a productive infection and inhibition of the innate immune response via decreased expression of IFN-α2, IFN-β and TNF-α. Using a recombinant virus lacking the NSs protein, we show that this effect is mediated by the viral NSs protein. Finally, analysis of RVF patient samples demonstrated an association between a pro-inflammatory cytokine response and patient survival.

Fig. 1.

RVFV productively infects monocyte derived human macrophages. MDM were infected with WT or ΔNSs RVFV. Supernatants were collected at various times post infection. Supernatants were titered on Vero E6 cells. WT virus (black bars) grew to higher titers than the ΔNSs virus (white bars) on cells from the same donor. Data are presented as PFU/mL at 24 h post infection. 4 different donors are represented in the figure.

Fig. 2.

WT virus causes marked CPE in infected cells while ΔNSs virus does not. MDM were mock-infected, or infected with WT, or ΔNSs RVFV. At 12 hpi cells were photographed under white light using the 20× objective to demonstrate the CPE caused by WT virus.

Fig. 3.

ΔNSs RVFV replicates to lower levels than wild-type RVFV in MDM. MDM were infected with WT or ΔNSs RVFV. RNA was purified from cells at various times post infection and analyzed by real time PCR. WT virus (black squares with solid lines) replicated to higher levels than the ΔNSs virus (white squares with dotted lines) on cells from the same donor. Data are presented as inverse Ct value at various times post infection. 3 different donors are represented in the figure. RNA from the 4th donor was not available for testing.

Fig. 4.

IFN-α2, IFN-β and TNF-α are expressed by MDM upon infection with ΔNSs virus but not WT RVFV virus. MDM were mock-infected, or infected with WT, ΔNSs, γ-WT or γ-ΔNSs RVFV. Supernatants were collected at various times post infection, and levels of cytokines were measured. 4 different donors are represented in the figure (A–D). Data for IFN-α2 (A), IFN-β (B) and TNF-α (C) at the 24 hpi time point are shown.

Fig. 5.

A pro-inflammatory response is associated with survival in human serum samples from the Saudi 2000–2001 outbreak. Serum samples from patients with known clinical outcome were analyzed in duplicate for various cytokines. The 5 cytokines that demonstrated statistical significance between fatal and non-fatal cases are shown. Two pro-inflammatory cytokines were elevated in non-fatal cases (white bars) and two immunosuppressive cytokines were elevated in fatal cases (black bars). The pro-inflammatory cytokine IL-1α is shown separately. Confidence interval is indicated by error bars and p values are noted.