We found a new flavivirus that is widespread in Culex pipiens and other Culex mosquitoes in Japan. The virus isolate, named Culex flavivirus (CxFV), multiplied only in mosquito cell lines producing a moderate cytopathic effect, but did not grow in mammalian cells. The CxFV genome is single-stranded RNA, 10,834 nt in length and containing a single open reading frame encoding a polyprotein of 3362 aa with 5' and 3' untranslated regions (UTRs) of 91 and 657 nt, respectively. Phylogenetic analyses revealed that CxFV is closely related to the insect flaviviruses associated with Aedes mosquitoes, Cell fusing agent (CFA) and Kamiti River virus (KRV). The 3' UTR of CxFV contains four tandem repeats, which have sequence similarities to the two direct repeats in the CFA and KRV 3' UTRs. These results suggest that CxFV may be a new group of insect flaviviruses.

We isolated a new flavivirus from Aedes albopictus mosquito and a related species in Japan. The virus, designated Aedes flavivirus (AEFV), only replicated in a mosquito cell line and produced a mild cytopathic effect. The AEFV genome was positive-sense, single-stranded RNA, 11,064 nucleotides in length and contained a single open reading frame encoding a polyprotein of 3341 amino acids with 5' and 3' untranslated regions (UTRs) of 96 and 945 nucleotides, respectively. Genetic and phylogenetic analyses classified AEFV with the insect flavivirus, but distinct from Cell fusing agent (CFA), Kamiti river virus and Culex flavivirus. Interestingly, a partial sequence of AEFV showed significant similarity to that of Cell silent agent (CSA), the insect flavivirus-related nucleotide sequence integrated in the genome of A. albopictus. These results suggest that AEFV is a new member of the insect flaviviruses, which are intimately associated with Aedes mosquitoes and may share a common origin with CSA.

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The salivary glands of female mosquitoes contain a variety of bioactive substances that assist their bloodfeeding behavior. Here, we report a salivary protein of the malarial vector mosquito, Anopheles stephensi, that inhibits activation of the plasma contact system. This factor, named hamadarin, is a 16-kDa protein and a major component of the saliva of this mosquito. Assays using human plasma showed that hamadarin dose-dependently inhibits activation of the plasma contact system and subsequent release of bradykinin, a primary mediator of inflammatory reactions. Reconstitution experiments showed that hamadarin inhibits activation of the plasma contact system by inhibition of the reciprocal activation of factor XII and kallikrein. Direct binding assays demonstrated that this inhibitory effect is due to hamadarin binding to both factor XII and high molecular weight kininogen and interference in their association with the activating surface. The assays also showed that hamadarin binding to these proteins depends on Zn 2؉ ions, suggesting that hamadarin binds to these contact factors by recognizing their conformational change induced by Zn 2؉ binding. We propose that hamadarin may attenuate the host's acute inflammatory responses to the mosquito's bites by inhibition of bradykinin release and thus enable mosquitoes to take a blood meal efficiently and safely.

Novel antithrombin molecules were identified from the ixodidae tick, Haemaphysalis longicornis. These molecules, named madanin 1 and 2, are 7-kDa proteins and show no significant similarities to any previously identified proteins. Assays using human plasma showed that madanin 1 and 2 dose-dependently prolonged both activated partial thromboplastin time and prothrombin time, indicating that they inhibit both the intrinsic and extrinsic pathways. Direct binding assay by surface plasmon resonance measurement demonstrated that madanin 1 and 2 specifically interacted with thrombin. Furthermore, it was clearly shown that madanin 1 and 2 inhibited conversion of fibrinogen into fibrin by thrombin, thrombin-catalyzed activation of factor V and factor VIII, and thrombin-induced aggregation of platelets without affecting thrombin amidolytic activity. These results suggest that madanin 1 and 2 bind to the anionbinding exosite 1 on the thrombin molecule, but not to the active cleft, and interfere with the association of fibrinogen, factor V, factor VIII and thrombin receptor on platelets with an anion-binding exosite 1. They appear to be exosite 1-directed competitive inhibitors.Keywords: anticoagulant; Haemaphysalis longicornis; salivary gland; thrombin inhibitor; tick.Thrombin has various physiological functions and plays important roles in hemostasis. For example, in the final step of blood clot formation, thrombin converts soluble fibrinogen into fibrin and subsequently triggers cross-linking between fibrin monomers by activating factor XIII [1]. It also amplifies its own generation by activating nonenzymatic cofactors V and VIII as well as factor XI [2,3]. Conversely, it suppresses its own generation by activating protein C [4], which inactivates factor Va and factor VIIIa together with protein S [5], when bound to the endothelial membrane receptor thrombomodulin. In addition, thrombin induces platelet aggregation via proteolytic activation of G-protein-coupled protease-activated receptors (PARs) [6,7]. Specific interactions of thrombin with these substrates, cofactors, and receptors involve not only the catalytic site and the primary binding pocket, but also secondary recognition sites, termed anion-binding exosite 1 and 2. Anion-binding exosite 1 interacts with negatively charged domains on fibrinogen [8], PARs [6,7,9], and thrombomodulin [10,11]. Anion-binding exosite 2 interacts with heparin [12], promoting inhibition of thrombin by antithrombin III [13] and heparin cofactor II [14]. Furthermore, both exosites are involved in the recognition of factor V and factor VIII by thrombin [15].The salivary glands of blood-sucking animals, such as leeches, insects, and ticks, contain various anticoagulants [16]. These substances inhibit the host hemostatic response so that the blood-sucking organism can feed smoothly on host blood. The best known anticoagulant identified from bloodsucking organisms is hirudin, a highly specific thrombin inhibitor, isolated from the medical leech, Hirudo medicinalis [17]. It interacts with tw...