Rift Valley fever virus (RVFV) is responsible for large and recurrent outbreaks of acute febrile illness among humans and domesticated animals in Africa. It belongs to the family Bunyaviridae, genus Phlebovirus, and its negative-stranded RNA genome consists of three segments. Here, we report the establishment and characterization of two different systems to rescue the RVFV wild-type strain ZH548. The first system is based on the BHK-21 cell clone BSR-T7/5, which stably expresses T7 RNA polymerase (T7 pol). Rescue of wild-type RVFV was achieved with three T7 pol-driven cDNA plasmids representing the viral RNA segments in the antigenomic sense. The second system involves 293T cells transfected with three RNA pol I-driven plasmids for the viral segments and two RNA pol II-driven support plasmids to express the viral polymerase components L and N. It is known that the 59 triphosphate group of T7 pol transcripts strongly activates the antiviral interferon system via the intracellular RNA receptor RIG-I. Nonetheless, both the T7 pol and the pol I/II system were of similar efficiency. This was even true for the rescue of a RVFV mutant lacking the interferon antagonist nonstructural proteins. Further experiments demonstrated that the unresponsiveness of BHK-21 and BSR-T7/5 cells to T7 pol transcripts is most probably due to a deficiency in the RIG-I pathway. Our reverse genetics systems now enable us to manipulate the genome of RVFV and study its virulence mechanisms. Moreover, the finding that BHK-derived cell lines have a compromised RIG-I pathway may explain their suitability for propagating and rescuing a wide variety of viruses. INTRODUCTIONRift Valley fever virus (RVFV) is a serious pathogen affecting humans and livestock in sub-Saharan Africa, Egypt, Yemen and Saudi Arabia. Recurrent epidemics have killed thousands of animals, hundreds of humans, and caused significant economic losses (Balkhy & Memish, 2003). The severity of RVFV zoonosis as well as the capability to cause major epidemics have prompted authorities to list RVFV as a notifiable disease and a potential biological weapon (Borio et al., 2002).RVFV belongs to the genus Phlebovirus, family Bunyaviridae (Elliott, 1997). Bunyaviruses are enveloped and have a tri-segmented single-stranded RNA genome of negative or ambisense polarity, replicate in the cytoplasm, and bud into the Golgi apparatus. RVFV encodes five structural proteins: the viral polymerase on the large (L) segment, two glycoproteins (Gn and Gc) and the 78 kDa protein on the medium (M) segment, and the viral nucleocapsid protein (N) on the smallest (S) segment (Struthers et al., 1984). In addition, there are two nonstructural proteins, encoded on the M segment (termed NSm) and the S segment (termed NSs). These accessory proteins are dispensable for viral multiplication in cell culture (Gerrard et al., 2007;Vialat et al., 2000;Won et al., 2006), but play important roles for pathogenesis in vivo. In particular, the NSm and 78 kDa proteins were found to enhance intrahost viral spread , whereas NS...

Rift Valley fever virus (RVFV) is a highly pathogenic member of the family Bunyaviridae that needs to be handled under biosafety level (BSL) 3 conditions. Here, we describe reverse genetics systems to measure RVFV polymerase activity in mammalian cells and to generate virus-like particles (VLPs). Recombinant polymerase (L) and nucleocapsid protein (N), expressed together with a minireplicon RNA, formed transcriptionally active nucleocapsids. These could be packaged into VLPs by additional expression of viral glycoproteins. The VLPs resembled authentic virus particles and were able to infect new cells. After infection, VLP-associated nucleocapsids autonomously performed primary transcription, and co-expression of L and N in VLP-infected cells allowed subsequent replication and secondary transcription. Bunyaviruses are potently inhibited by a human interferon-induced protein, MxA. However, the affected step in the infection cycle is not entirely characterized. Using the VLP system, we demonstrate that MxA inhibits both primary and secondary transcriptions of RVFV. A set of infection assays distinguishing between virus attachment, entry, and subsequent RNA synthesis confirmed that MxA is able to target immediate early RNA synthesis of incoming RVFV particles. Thus, our reverse genetics systems are useful for dissecting individual steps of RVFV infection under non-BSL3 conditions.

Finally, we found that both isoforms of chicken Mx protein appear to lack GTPase activity, which might explain the observed lack of antiviral activity.

From infection studies with cultured chicken cells and experimental mammalian hosts, it is well known that influenza viruses use the nonstructural protein 1 (NS1) to suppress the synthesis of interferon (IFN). However, our current knowledge regarding the in vivo role of virus-encoded NS1 in chickens is much more limited. Here, we report that highly pathogenic avian influenza viruses of subtypes H5N1 and H7N7 lacking fully functional NS1 genes were attenuated in 5-week-old chickens. Surprisingly, in diseased birds infected with NS1 mutants, the IFN levels were not higher than in diseased birds infected with wild-type virus, suggesting that NS1 cannot suppress IFN gene expression in at least one cell population of infected chickens that produces large amounts of the cytokine in vivo. To address the question of why influenza viruses are highly pathogenic in chickens although they strongly activate the innate immune system, we determined whether recombinant chicken alpha interferon (IFN-␣) can inhibit the growth of highly pathogenic avian influenza viruses in cultured chicken cells and whether it can ameliorate virus-induced disease in 5-week-old birds. We found that IFN treatment failed to confer substantial protection against challenge with highly pathogenic viruses, although it was effective against viruses with low pathogenic potential. Taken together, our data demonstrate that preventing the synthesis of IFN is not the primary role of the viral NS1 protein during infection of chickens. Our results further suggest that virus-induced IFN does not contribute substantially to resistance of chickens against highly pathogenic influenza viruses.The nonstructural protein 1 (NS1) of influenza A virus serves multiple functions in the viral life cycle. It regulates viral polymerase activity, and it modulates cellular signaling pathways (for a review, see reference 14). It is well established that NS1 can suppress innate immune responses in infected cells by blocking intracellular signaling pathways that lead to the synthesis of type I and type III interferon (IFN). Specifically, NS1 inhibits ubiquitinylation of RIG-I by TRIM25, thereby preventing efficient induction of IFN in infected cells (9,12,28,35). The NS1 proteins of most influenza viruses block global posttranscriptional processing of cellular mRNAs, including mRNAs encoding 27,33,34). NS1 further inhibits specific antiviral effector proteins, such as 2Ј-5Ј oligoadenylate synthetase (OAS) (29) and double-stranded RNA (dsRNA)-activated protein kinase (PKR) (23,30). In addition, NS1 was shown to modulate host cell apoptosis (39, 55), stimulate phosphoinositol 3-kinase signaling (13), and regulate viral polymerase activity (2, 30).Current knowledge regarding the in vivo functions of NS1 is mainly based on studies in mice with targeted deletions of several key components of the IFN system. Viruses expressing defective NS1 proteins are generally strongly attenuated in hosts with an intact IFN system but retain a high degree of virulence in hosts with defective IFN syst...

BackgroundStudies of the host response to infection often require quantitative measurement of the antiviral type I interferons (IFN-α/β) in biological samples. The amount of IFN is either determined via its ability to suppress a sensitive indicator virus, by an IFN-responding reporter cell line, or by ELISA. These assays however are either time-consuming and lack convenient readouts, or they are rather insensitive and restricted to IFN from a particular host species.ResultsAn IFN-sensitive, Renilla luciferase-expressing Rift Valley fever virus (RVFV-Ren) was generated using reverse genetics. Human, murine and avian cells were tested for their susceptibility to RVFV-Ren after treatment with species-specific IFNs. RVFV-Ren was able to infect cells of all three species, and IFN-mediated inhibition of viral reporter activity occurred in a dose-dependent manner. The sensitivity limit was found to be 1 U/ml IFN, and comparison with a standard curve allowed to determine the activity of an unknown sample.ConclusionsRVFV-Ren replicates in cells of several species and is highly sensitive to pre-treatment with IFN. These properties allowed the development of a rapid, sensitive, and species-independent antiviral assay with a convenient luciferase-based readout.