In 2005, we isolated a new species of virus from mosquitoes in the Philippines. The virion was elliptical in shape and had a short single projection. The virus was named Tanay virus (TANAV) after the locality in which it was found. TANAV genomic RNA was a 9562 nt+poly-A positive strand, and polycistronic. The longest ORF contained putative RNA-dependent RNA polymerase (RdRP); however, conserved short motifs in the RdRP were permuted. TANAV was phylogenetically close to Negevirus, a recently proposed taxon of viruses isolated from haemophagic insects, and to some plant viruses, such as citrus leprosis virus C, hibiscus green spot virus and blueberry necrotic ring blotch virus. In this paper, we describe TANAV and the permuted structure of its RdRP, and discuss its phylogeny together with those of plant viruses and negevirus.In 2005, as part of our tropical arbovirus surveillance, we attempted to isolate mosquito-borne viruses in the Philippines. Mosquitoes were collected from a pig farm in Tanay, Rizal province, Luzon Island. Collected mosquitoes were Culex spp. and Armigeres spp. Mosquito pools were homogenized and centrifuged. The supernatants were passed through 0.22 mm filter and inoculated onto monolayer cultures of Aedes albopictus C6/36 cells, which were then incubated at 28 u C. Four samples (11-2 from Culex quinquefasciatus, 11-3 from Culex spp., 11-4 and 11-5 from Armigeres spp.) caused cytopathic effect to C6/36 cells. We attempted to amplify the viral genome by PCR using consensus primers of Flavivirus, Alphavirus and Bunyavirus, but were unsuccessful (data not shown).To observe the virion, we conducted plaque purification three times, negatively stained the concentrated virus samples with 1.5 % uranyl acetate, and then examined them at 80 kV using a JEM-1230 transmission electron microscope (JEOL). We found elliptical particles (~50 nm) with a short single projection (Fig. 1a). Some particles seemed to be attached to each other through their projection.

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Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral disease that is endemic in China, Korea and Japan. No effective vaccine or specific treatment for SFTS is currently available. Here, we used a mouse model to examine the effects of ribavirin, site-1 protease inhibitor PF-429242, steroids, and combination of minocycline and ciprofloxacin (MC) on SFTS infection. The antiserum from a patient who recovered from SFTS was also examined for its effect on mice. Administration of antiserum completely protected mice against lethal infection with SFTSV. It could also protect mice from showing clinical signs of the disease due to non-lethal infection. MC-treatment resulted in prolonged survival times during lethal infection. Although other agents had no significant protective effects, they did not provide detrimental effects that could lead to progression of the disease in mice. Our results suggest that antiserum treatment may be clinically useful for post-exposure prophylaxis against SFTSV infection.

Ixodid ticks transmit several important viral pathogens. We isolated a new virus (Tofla virus: TFLV) from Heamaphysalis flava and Heamaphysalis formsensis in Japan. The full-genome sequences revealed that TFLV belonged to the genus Nairovirus, family Bunyaviridae. Phylogenetic analyses and neutralization tests suggested that TFLV is closely related to the Hazara virus and that it is classified into the Crimean-Congo hemorrhagic fever group. TFLV caused lethal infection in IFNAR KO mice. The TFLV-infected mice exhibited a gastrointestinal disorder, and positron emission tomography-computed tomography images showed a significant uptake of 18F-fluorodeoxyglucose in the intestinal tract. TFLV was able to infect and propagate in cultured cells of African green monkey-derived Vero E6 cells and human-derived SK-N-SH, T98-G and HEK-293 cells. Although TFLV infections in humans and animals are currently unknown, our findings may provide clues to understand the potential infectivity and to develop of pre-emptive countermeasures against this new tick-borne Nairovirus.

Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units.