Haruhiko Isawa

First Molecular Evidence of Bartonella quintana in Pediculus humanus capitis (Phthiraptera: Pediculidae), Collected from Nepalese Children

Abstract Trench fever is a body louse-borne disease caused by Bartonella quintana Brenner. The recent status of louse infestation in Nepalese children is not well known. We collected head and body lice, Pediculus humanus capitis De Geer and Pediculus humanus humanus L., respectively, from 30 children, including 11 cases of double infestation with both head and body lice. Detection of B. quintana in both louse species identified was carried out by polymerase chain reaction (PCR). PCR products with B. quintana DNA sequences were detected in both head and body lice from two children as well as in body lice derived from two other children. These results demonstrate that head lice may also play a role in the transmission of trench fever.

Host-Feeding Habits of Culex pipiens and Aedes albopictus (Diptera: Culicidae) Collected at the Urban and Suburban Residential Areas of Japan

ABSTRACT To evaluate the vectorial capacity of mosquitoes for viruses in Japan, the host-feeding habits of the mosquitoes were analyzed by sequencing polymerase chain reaction-amplified fragments of the cytochrome b and 16S ribosomal RNA regions of the mitochondrial DNA of 516 mosquitoes of 15 species from seven genera that were collected from residential areas during 2003–2006. Culex pipiens L. and Aedes albopictus Skuse were the most commonly collected species in urban and suburban residential areas. Anautogenous Culex pipiens pallens Coquillett was distinguished from the autogenous Cx. pipiens form molestus Forskal using a polymerase chain reaction-based identification method. Both Cx. p. pollens and Cx. p. form molestus exhibited similar host-feeding habits, broadly preferring avian (50.0 and 42.5% of avian, respectively) and mammalian (38.6 and 45.0% of avian, respectively) hosts, such as tree sparrows, ducks, and humans. Conversely, Ae. albopictus exhibited a highly mammalophilic and anthropophilic feeding pattern, with 84.2% feeding on mammalian hosts and 68.5% of these on humans. We concluded that in Japan, Cx. pipiens might play a significant role in the avian-to-mammal transmission of viruses, such as West Nile virus, whereas Ae. albopictus might play a role in the human-human transmission of dengue and Chikungunya viruses.

Identification and molecular characterization of a new nonsegmented double-stranded RNA virus isolated from Culex mosquitoes in Japan.

Two infectious agents were isolated from Culex species mosquitoes in Japan and were identified as distinct strains of a new RNA virus by a method for sequence-independent amplification of viral nucleic acids. The virus designated Omono River virus (OMRV) replicated in mosquito cells in which it produced a severe cytopathic effect. Icosahedral virus particles of approximately 40 nm in diameter were detected in the cytoplasm of infected cells. The OMRV genome was observed to consist of a nonsegmented, 7.6-kb double-stranded RNA (dsRNA) and contain two overlapping open reading frames (ORFs), namely ORF1 and ORF2. ORF1 was found to encode a putative dsRNA-binding protein, a major capsid protein, and other putative proteins, which might be generated by co- and/or post-translational processing of the ORF1 polyprotein precursor, and ORF2 was found to encode a putative RNA-dependent RNA polymerase (RdRp), which could be translated as a fusion with the ORF1 product by a -1 ribosomal frameshift. Phylogenetic analysis based on RdRp revealed that OMRV is closely related to penaeid shrimp infectious myonecrosis virus and Drosophila totivirus, which are tentatively assigned to the family Totiviridae. These results indicated that OMRV is a new member of the family of nonsegmented dsRNA viruses infecting arthropod hosts, but not fungal or protozoan hosts.

Identification and characterization of a collagen-induced platelet aggregation inhibitor, triplatin, from salivary glands of the assassin bug, Triatoma infestans

To facilitate feeding, certain hematophagous invertebrates possess inhibitors of collagen‐induced platelet aggregation in their saliva. However, their mechanisms of action have not been fully elucidated. Here, we describe two major salivary proteins, triplatin‐1 and ‐2, from the assassin bug, Triatoma infestans, which inhibited platelet aggregation induced by collagen but not by other agents including ADP, arachidonic acid, U46619 and thrombin. Furthermore, these triplatins also inhibited platelet aggregation induced by collagen‐related peptide, a specific agonist of the major collagen‐signaling receptor glycoprotein (GP)VI. Moreover, triplatin‐1 inhibited Fc receptor γ‐chain phosphorylation induced by collagen, which is the first step of GPVI‐mediated signaling. These results strongly suggest that triplatins target GPVI and inhibit signal transduction necessary for platelet activation by collagen. This is the first report on the mechanism of action of collagen‐induced platelet aggregation inhibitors from hematophagus invertebrates.

Identification and characterization of the plasma kallikrein-kinin system inhibitor, haemaphysalin, from hard tick, Haemaphysalis longicornis

The plasma kallikrein-kinin system inhibitor, haemaphysalin, from the hard tick, Haemaphysalis longicornis, was identified. It was found that haemaphysalin inhibited activation of the plasma kallikrein-kinin system by interfering with reciprocal activation between factor XII and prekallikrein. It did not, however, inhibit amidolytic activities of factor XIIa and kallikrein. Direct binding assay indicated that factor XII/XIIa and high molecular weight kininogen (HK) are the target molecules of haemaphysalin, and that Zn2+ ions are involved in the interactions of haemaphysalin with these target molecules. This suggests that haemaphysalin interacts with target molecules by recognizing their conformational changes induced by Zn2+ ions. Furthermore, haemaphysalin interacted with the fibronectin type II domain and domain D5, the cell binding domains of factor XII and HK, respectively. This finding suggests that haemaphysalin interferes with the association of factor XII and the prekallikrein-HK complex with a biologic activating surface by binding to these cell-binding domains, leading to inhibition of the reciprocal activation between factor XII and prekallikrein.

Biting density and distribution of Aedes albopictus during the September 2014 outbreak of dengue fever in Yoyogi Park and the vicinity in Tokyo Metropolis, Japan.

A total of 160 autochthonous dengue cases transmitted by Aedes albopictus were reported between August and October of 2014 in Tokyo Metropolis, Japan. Ae. albopictus is a medically important vector of dengue virus, which has expanded its geographic distribution in temperate regions. Understanding the distribution and biting density of Ae. albopictus during the 2014 dengue outbreak in Tokyo is important to evaluate the epidemic risks of dengue fever in other highly populated cities in Europe and Asia. Of the 160 patients, 134 visited the same park (Yoyogi Park) located in central Tokyo. Mosquitoes infected with dengue virus were collected from this park, suggesting that it was the exclusive location for the transmission of dengue. This study aimed to collect referential data to estimate the transmission threshold of dengue virus in terms of biting density of Ae. albopictus and determined high transmission risk areas of dengue virus in Yoyogi Park and its vicinity. The overall mean density of biting Ae. albopictus (7.13/man/8 min) was sufficiently high for successful transmission of dengue virus, and areas with biting densities higher than the overall mean density were classified as high risk areas for the transmission of dengue virus in Yoyogi Park.

RNA Splicing in a New Rhabdovirus from Culex Mosquitoes

ABSTRACT Among members of the order Mononegavirales, RNA splicing events have been found only in the family Bornaviridae. Here, we report that a new rhabdovirus isolated from the mosquito Culex tritaeniorhynchus replicates in the nuclei of infected cells and requires RNA splicing for viral mRNA maturation. The virus, designated Culex tritaeniorhynchus rhabdovirus (CTRV), shares a similar genome organization with other rhabdoviruses, except for the presence of a putative intron in the coding region for the L protein. Molecular phylogenetic studies indicated that CTRV belongs to the family Rhabdoviridae, but it is yet to be assigned a genus. Electron microscopic analysis revealed that the CTRV virion is extremely elongated, unlike virions of rhabdoviruses, which are generally bullet shaped. Northern hybridization confirmed that a large transcript (approximately 6,500 nucleotides [nt]) from the CTRV L gene was present in the infected cells. Strand-specific reverse transcription-PCR (RT-PCR) analyses identified the intron-exon boundaries and the 76-nt intron sequence, which contains the typical motif for eukaryotic spliceosomal intron-splice donor/acceptor sites (GU-AG), a predicted branch point, and a polypyrimidine tract. In situ hybridization exhibited that viral RNAs are primarily localized in the nucleus of infected cells, indicating that CTRV replicates in the nucleus and is allowed to utilize the hosts nuclear splicing machinery. This is the first report of RNA splicing among the members of the family Rhabdoviridae.

Identification and characterization of plasma kallikrein-kinin system inhibitors from salivary glands of the blood-sucking insect Triatoma infestans.

Two plasma kallikrein–kinin system inhibitors in the salivary glands of the kissing bug Triatoma infestans, designated triafestin‐1 and triafestin‐2, have been identified and characterized. Reconstitution experiments showed that triafestin‐1 and triafestin‐2 inhibit the activation of the kallikrein–kinin system by inhibiting the reciprocal activation of factor XII and prekallikrein, and subsequent release of bradykinin. Binding analyses showed that triafestin‐1 and triafestin‐2 specifically interact with factor XII and high molecular weight kininogen in a Zn2+‐dependent manner, suggesting that they specifically recognize Zn2+‐induced conformational changes in factor XII and high molecular weight kininogen. Triafestin‐1 and triafestin‐2 also inhibit factor XII and high molecular weight kininogen binding to negatively charged surfaces. Furthermore, they interact with both the N‐terminus of factor XII and domain D5 of high molecular weight kininogen, which are the binding domains for biological activating surfaces. These results suggest that triafestin‐1 and triafestin‐2 inhibit activation of the kallikrein–kinin system by interfering with the association of factor XII and high molecular weight kininogen with biological activating surfaces, resulting in the inhibition of bradykinin release in an animal host during insect blood‐feeding.

Characterization of Dak Nong virus, an insect nidovirus isolated from Culex mosquitoes in Vietnam.

In this study, we isolated and characterized an insect nidovirus from the mosquito Culex tritaeniorhynchus Giles (Diptera: Culicidae) in Vietnam, as an additional member of the new family Mesoniviridae in the order Nidovirales. The virus, designated “Dak Nong virus (DKNV),” shared many characteristics with Cavally virus and Nam Dinh virus, which have also been discovered recently in mosquitoes, and these viruses should be considered members of a single virus species, Alphamesonivirus 1. DKNV grew in cultured mosquito cells but could not replicate in the cultured vertebrate cells tested. N-terminal sequencing of the DKNV structural proteins revealed two posttranslational cleavage sites in the spike glycoprotein precursor. DKNV is assumed to be a new member of the species Alphamesonivirus 1, and the current study provides further understanding of viruses belonging to the new family Mesoniviridae.

Analysis of Mosquito-Borne Flavivirus Superinfection in Culex tritaeniorhynchus (Diptera: Culicidae) Cells Persistently Infected with Culex Flavivirus (Flaviviridae)

ABSTRACT Superinfection exclusion is generally defined as a phenomenon in which a pre-existing viral infection prevents a secondary viral infection; this has also been observed in infections with mosquito-borne viruses. In this study, we examined the superinfection exclusion of the vertebrate-infecting flaviviruses, Japanese encephalitis virus (JEV) and dengue virus (DENV), by stable and persistent infection with an insect-specific flavivirus, Culex flavivirus (CxFV), in a Culex tritaeniorhynchus Giles cell line (CTR cells). Our experimental system was designed based on the premise that wild Cx. tritaeniorhynchus mosquitoes naturally infected with CxFV are superinfected with JEV by feeding on JEV-infected animals. As a result, we found no evidence of the superinfection exclusion of both JEV and DENV by pre-existing CxFV infection at the cellular level. However, JEV superinfection induced severe cytopathic effects on persistently CxFV-infected CTR cells. These observations imply the possibility that JEV superinfection in CxFV-infected Cx. tritaeniorhynchus mosquitoes has an adverse effect on their fitness.