Rabies virus, a member of the genus Lyssavirus of the family Rhabdoviridae, causes severe neurological disease and death in mammals, including humans. The genome is an unsegmented negative-sense RNA of about 12,000 bases that encodes five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and large protein (L). The G and M proteins form the viral envelope together with a lipid bilayer derived from the host cell membrane. The N, P and L proteins and the viral genomic RNA compose a ribonucleoprotein complex that is coated with the viral envelope in the virion. The N protein is responsible for encapsidation of the genomic and antigenomic RNAs, while the L protein, in cooperation with the P protein, functions as an RNAdependent RNA polymerase in infected cells.Schnell et al. (15) first established a system for recovering virus from cloned cDNA (reverse genetics system) of Mononegavirale using rabies virus. Later, various viruses belonging to the order Mononegavirales, including vesicular stomatitis virus (9, 17), human respiratory syncytial virus (2) and Sendai virus (3, 8), were recovered from cloned cDNA using almost the same principle as that used for rabies virus. This system made discretional manipulation of these viral genomes possible and, as a result, facilitated functional analysis of the genomes. Recently, we have also established a reverse genetics system of rabies virus, the attenuated RC-HL strain, and shown that the G gene is associated with the pathogenicity of rabies virus (7).In all of the systems described above, recovery of the virus is promoted by transfection of a full-length genome plasmid and the helper plasmids, which supply artificial viral genome RNA and N, P and L proteins under control of the T7 promoter to the cells after infection with the T7 RNA polymerase-expressing vaccinia virus. However, the systems have some disadvantages caused by the vaccinia virus. Firstly, effective propagation of the recombinant virus recovered from cDNA cannot be expected in transfected cells because of the strong cytopathic effect (CPE) caused by the vaccinia virus. This problem reduces the efficiency of recovering the virus from cloned cDNA. Secondly, homologous Agriculture, Gifu University, Gifu, Gifu 501-1193, Japan Received April 9, 2003 in revised form, May 19, 2003. Accepted May 23, 2003 Abstract: To improve efficiency of recovery of rabies virus from cloned cDNA, we established a BHK cell clone that stably expresses T7 RNA polymerase, which we named BHK/T7-9. We also constructed new helper plasmids for expression of nucleoprotein and RNA polymerase of the RC-HL strain using the pTM1 plasmid vector, which makes the T7 RNA polymerase-transcripts from the plasmid cap-independent for translation. After co-transfection of these helper plasmids and the previously constructed full-length genome plasmid of the RC-HL strain to BHK/T7-9 cells, recombinant rabies virus was efficiently recovered from the cloned cDNA.

Following virus infection of the central nervous system, microglia, the ontogenetic and functional equivalents of macrophages in somatic tissues, act as sources of chemokines, thereby recruiting peripheral leukocytes into the brain parenchyma. In the present study, we have systemically examined the growth characteristics of rabies virus (RV) in microglia and the activation of cellular signaling pathways leading to chemokine expression upon RV infection. In RV-inoculated microglia, the synthesis of the viral genome and the production of virus progenies were significantly impaired, while the expression of viral proteins was observed. Transcriptional analyses of the expression profiles of chemokine genes revealed that RV infection, but not exposure to inactivated virions, strongly induces the expression of CXC chemokine ligand 10 (CXCL10) and CC chemokine ligand 5 (CCL5) in microglia. RV infection triggered the activation of signaling pathways mediated by mitogen-activated protein kinases, including p38, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and c-Jun N-terminal kinase, and nuclear factor B (NF-B). RV-induced expression of CXCL10 and CCL5 was achieved by the activation of p38 and NF-B pathways. In contrast, the activation of ERK1/2 was found to down-regulate CCL5 expression in RV-infected microglia, despite the fact that it was involved in partial induction of CXCL10 expression. Furthermore, NF-B signaling upon RV infection was augmented via a p38-mediated mechanism. Taken together, these results indicate that the strong induction of CXCL10 and CCL5 expression in microglia is precisely regulated by the activation of multiple signaling pathways through the recognition of RV infection.

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Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne infectious disease caused by SFTS virus (SFTSV), which is a novel bunyavirus. SFTSV was first isolated from patients who presented with fever, thrombocytopenia, leukocytopenia, and multiorgan dysfunction in China. Subsequently, it was found to be widely distributed in Southeast Asia (Korea, Japan, and Vietnam). SFTSV can be transmitted not only from ticks but also from domestic animals, companion animals, and humans. Because the case fatality rate of SFTS is high (6-30%), development of specific and effective treatment for SFTS is required. Studies of potential antiviral drugs for SFTS-specific therapy have been conducted on existing or newly discovered agents in vitro and in vivo, with ribavirin and favipiravir being the most promising candidates. While animal experiments and retrospective studies have demonstrated the limited efficacy of ribavirin, it was also speculated that ribavirin would be effective in patients with a viral load <1 × 10 6 copies/mL. Favipiravir showed higher efficacy than ribavirin against SFTSV in in vitro assays and greater efficacy in animal models, even administrated 3 days after the virus inoculation. Although clinical trials evaluating the efficacy of favipiravir in SFTS patients in Japan are underway, this has yet to be confirmed. Other drugs, including hexachlorophene, calcium channel blockers, 2 ′ -fluoro-2 ′ -deoxycytidine, caffeic acid, amodiaquine, and interferons, have also been evaluated for their inhibitory efficacy against SFTSV. Among them, calcium channel blockers are promising because in addition to their efficacy in vitro and in vivo, retrospective clinical data have indicated that nifedipine, one of the calcium channel blockers, reduced the case fatality rate by >5-fold. Although further research is necessary to develop SFTS-specific therapy, considerable progress has been achieved in this area. Here we summarize and discuss recent advances in antiviral drugs against SFTSV.Keywords: severe fever with thrombocytopenia syndrome, severe fever with thrombocytopenia syndrome virus, antiviral, ribavirin, favipiravir Frontiers in Microbiology | www.frontiersin.org

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