The Rift Valley fever virus (RVFV), a member of the genus Phlebovirus (family Bunyaviridae) is an enveloped negative-strand RNA virus with a tripartite genome. Until 2000, RVFV circulation was limited to the African continent, but the recent deadly outbreak in the Arabian Peninsula dramatically illustrated the need for rapid diagnostic methods, effective treatments, and prophylaxis. A method for quantifying the small RNA segment by a real-time detection reverse transcription (RT)-PCR using TaqMan technology and targeting the nonstructural protein-coding region was developed, and primers and a probe were designed. After optimization of the amplification reaction and establishment of a calibration curve with synthetic RNA transcribed in vitro from a plasmid containing the gene of interest, real-time RT-PCR was assessed with samples consisting of RVFV from infected Vero cells. The method was found to be specific for RVFV, and it was successfully applied to the detection of the RVFV genome in animal sera infected with RVFV as well as to the assessment of the efficiency of various drugs (ribavirin, alpha interferon, 6-azauridine, and glycyrrhizin) for antiviral activity. Altogether, the results indicated a strong correlation between the infectious virus titer and the amount of viral genome assayed by real time RT-PCR. This novel method could be of great interest for the rapid diagnosis and screening of new antiviral compounds, as it is sensitive and time saving and does not require manipulation of infectious material.The Rift Valley fever (RVF) virus (RVFV), a member of the genus Phlebovirus, belongs to the Bunyaviridae family and possesses a negative-stranded, tripartite RNA genome composed of a large, a medium, and a small (S) segment (for reviews, see references 9 and 37). Like other phleboviruses, the S segment utilizes an ambisense strategy to code for two proteins, the nucleocapsid protein and the nonstructural protein (NS s ), which are synthesized from subgenomic viral complementary and viral sense mRNA, respectively.RVF is a mosquito-borne zoonosis predominantly provoking the death of young animals and abortion (e.g., sheep and goats) (for reviews, see references 24, 39 and 41). The disease was first identified in sheep by Daubney et al. in Kenya in 1931, and it is endemic almost everywhere in subtropical Africa (6). Transmission to humans occurs primarily by contact with infected animal body fluids and by mosquito bites. Infection is usually asymptomatic or associated with a brief self-limited febrile illness. However, complications such as retinitis, encephalitis, or hemorrhagic fever occur in some patients with mortality rates of up to 10 to 12% (21, 28).The potential of RVF as a disease emerging in new areas was first documented in Egypt in 1977 (16), and since then, epidemics have occurred in Mauritania (1987Mauritania ( to 1988Mauritania ( and 1998, Madagascar (1990to 1991), Egypt (1993, and eastern Africa (in Kenya, Somalia, and Tanzania) (references 33 and 34 and references therein). Recently, the...

Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic, tick-borne member of the family Bunyaviridae and the genus Nairovirus. To better elucidate the pathogenesis of CCHFV, we analysed the host innate immune response induced in antigen-presenting cells (APCs) infected in vitro by CCHFV. Monocyte-derived dendritic cells (DCs) and macrophages (MPs) were both shown to be permissive for CCHFV and to replicate the virus, as monitored by genomic and antigenomic strand quantification. Virus replication was, however, controlled, corroborating an efficient alpha interferon-induced response. The upregulation of CD-83 and CD-86 indicated that CCHFV induced a partial maturation of DCs, which were also shown to activate the secretion of interleukin (IL)-6 and IL-8, but no tumour necrosis factor alpha (TNF-alpha). On the other hand, in MPs, CCHFV infection elicited a high IL-6 and TNF-alpha response and a moderate chemokine response. Nevertheless, when we compared these APC responses with those seen after infection with Dugbe virus (DUGV), a mildly pathogenic virus genetically close to CCHFV, we found that, in spite of some similarities, DUGV induced a higher cytokine/chemokine response in MPs. These results suggest that CCHFV is able to inhibit the activation of inflammatory mediators selectively in infection in vitro and that these differences could be relevant in pathogenesis.

It was recently shown that infection of ISE6 tick cells by a recombinant Semliki Forest virus (SFV) expressing a heterologous gene induced small interfering RNAs (siRNAs) and silencing of the gene. To gain information on RNA interference (RNAi) in ticks, three known viral inhibitors that act in different ways, the NS1 protein of Influenza virus, NSs of Tospovirus Tomato spotted wilt virus and HC-Pro of Zucchini yellow mosaic virus were expressed and investigated to determine if they antagonize induced RNAi. Using the recombinant SFV replicon expressing firefly luciferase, silencing was induced and the suppressor activity of these inhibitors during or after initiation of siRNA synthesis was tested, to determine which step of the RNAi pathway is impaired. It was found that these proteins, identified in mammalian or plant systems, also display activity in tick cells. These data suggest that ticks utilize a mechanism similar to the one found in other eukaryotes.RNA silencing or RNA interference (RNAi) is a highly conserved process in which the introduction or the production of double-stranded RNA (dsRNA) into cells triggers the degradation of mRNAs containing homologous sequences by sequence-specific cleavage of the mRNAs (for review, see e.g. Lecellier & Voinnet, 2004;Voinnet, 2005). The degradation is mediated by 21-24 nt long small interfering RNAs (siRNA), which are produced by the RNase III-like enzyme Dicer and incorporated into the RNA-induced silencing complex (RISC) (Elbashir et al., 2001;Hamilton & Baulcombe, 1999). The RNAi pathway is an ancient evolutionary conserved mechanism, which has been retained in plants, worms, insects and mammals and appears to represent an early form of innate immunity.Among the different systems in which RNA silencing has been studied, plants, mammalian cells and insects (particularly Drosophila and mosquitoes) represent a significant field, but less is known about ticks and tick cells. Only a few reports described RNAi in ticks, in vitro or in vivo, and these studies describe silencing of specific genes but do not explore the mechanisms (Aljamali et al., 2003; Karim et al., 2004;Narasimhan et al., 2004). Ticks are obligate ectoparasites distributed worldwide and transmit human pathogens, such as the Lyme disease agent, the agent of Ehrlichiosis and arboviruses, some of which are serious human pathogens (i.e. the Crimean-Congo hemorrhagic fever virus). Recently, we showed that the tick cell line ISE6, established from Ixodes scapularis (Munderloh et al., 1994), was able to support the replication of the mosquito-borne Semliki Forest virus (SFV) and recombinant replicon (rSFV) and we found that infection of ISE6 cells with Hazara virus, a tick-borne arbovirus of the family Bunyaviridae (genus Nairovirus) was silenced through RNAi by a rSFV expressing nairovirus sequences (Garcia et al., 2005). The induction of RNAi in tick cells by SFV infection was clearly associated with the synthesis of siRNAs and a Dicer-like activity.The discovery that plant viruses encode suppressors of g...

We report the successful infection of the cell line ISE6 derived from Ixodes scapularis tick embryos by the tick-borne Hazara virus (HAZV), a nairovirus in the family Bunyaviridae. Using a recombinant Semliki Forest alphavirus replicon that replicates in these cells, we were able to inhibit replication of HAZV, and we showed that this blockage is mediated by the replication of the Semliki Forest alphavirus replicon; the vector containing the HAZV nucleoprotein gene in sense or antisense orientation efficiently inhibited HAZV replication. Moreover, expression of a distantly related nucleoprotein gene from Crimean-Congo hemorrhagic fever nairovirus failed to induce HAZV silencing, indicating that the inhibition is sequence specific. The resistance of these cells to replicate HAZV correlated with the detection of specific RNase activity and 21-to 24-nucleotide-long small interfering RNAs. Altogether, these results strongly suggest that pathogen-derived resistance can be established in the tick cells via a mechanism of RNA interference.