The proteolytic activities present in midguts of both fed and unfed Haemaphysalis longicornis were assessed by using the gelatin-substrate gel electrophoresis and inhibitor sensitivity analyses. Three predominant (116, 48 and 40 kDa) and two weak (55 and 60 kDa) proteinase bands were commonly expressed in both unfed and fed ticks, while a weak 80 kDa band was only present in fed ticks. Consistent with observations on other tick species, proteolytic activity against the gelatin substrate was observed only under acidic conditions. Inhibition studies against the gelatin substrate using a panel of inhibitors showed that the predominant proteolytic enzymes of 40 and 48 kDa molecular mass are cysteine proteinases. These results are discussed in the context of host vaccination as an alternative tick control method to the current use of chemical acaricides

There are few copies of Borna disease virus (BDV) genome in peripheral blood mononuclear cells and no reliable standard reverse transcriptase-polymerase chain reaction (RT-PCR) method for the detection of BDV RNA, which is both highly sensitive and free of contamination. Single-step RT-PCR, in which both reverse transcription and amplification by Taq DNA polymerase work efficiently in a single buffer, was applied to detect the p24 region of BDV RNA in vitro and in vivo. Using in vitro synthesized RNA, it was demonstrated that at least 100 copies of BDV RNA could be detected and the sensitivity and specificity were nearly equal to those obtained by RT-nested PCR. We could detect BDV RNA from more than 1 pg of cellular RNA obtained from BDV-persistently infected MDCK cells. Furthermore, this method was successfully performed on brain specimens obtained from a BDV-infected rat at 11 weeks post-inoculation. This single-step RT-PCR method will be convenient for detecting limited amounts of BDV RNA in various cells and tissue samples