Isolation and initial characterization of new betanodaviruses in shellfish.

Betanodaviruses cause the disease viral nervous necrosis (VNN) in finfish. Using a novel approach with two consecutive PCRs, detection semi-nested two-step RT-PCR (DSN-2 RT-PCR) and discriminative multiplex two-step RT-PCR (DMT-2 RT-PCR), we have identified the presence of a new type of betanodavirus in shellfish and called it Korean shellfish nervous necrosis virus (KSNNV). Partial nucleotide sequences of the T4 region in RNA2 fragment of KSNNVs were 73%-75% homologous to those of other reported genotypes and formed a new cluster of betanodavirus in phylogenetic tree analysis. Successful isolation of KSNNV was achieved in two of six shellfish samples containing high concentrations of virus using the blind passage method, and the typical shapes of betanodavirus were confirmed in KSNNV-KOR1 by electron microscopy. In the experimental infection test, seven of 14 fish species showed susceptibility to KSNNV-KOR1 isolate but without clinical signs or death. Although the range of susceptible host species was not significantly different from the RGNNV type, the concentration of KSNNV in the brain of infected fish (102 -105 copies/mg brain) was much lower compared to that found in sevenband grouper (Epinephelus septemfasciatus Thunberg) sampled in the moribund stage with RGNNV infection (106 -107 copies/mg brain). However, histopathological analyses showed the presence of multiple vacuoles in brains of all KSNNV-infected fish at 14 days postinjection. In detection test, as a single or multiple type with the other genotype(s) (RGNNV or BFNNV), the prevalence of KSNNV was 8.4% and 8.7% in domestic (62 of 741 samples) and Chinese samples (12 of 138 samples), respectively, but not in finfish. We propose that KSNNVs obtained from shellfish be classified into a separate and new genotype of betanodavirus.

High prevalence of betanodavirus barfin flounder nervous necrosis virus as well as red-spotted grouper nervous necrosis virus genotype in shellfish.

Using two serially executed PCRs, the discriminative multiplex two-step RT-PCR (DMT-2 RT-PCR) following the detection seminested two-step RT-PCR (DSN-2 RT-PCR), we found a high frequency presence of BFNNV genotype as well as RGNNV in various domestic and imported shellfish. This was definitely different from the previous reports of outbreaks and asymptomatic infection only by the RGNNV genotype in cultured finfish in Korea. Cultivation of NNV entrapped in shellfish was performed successfully by a blind passage. Thus, in an attempt to elucidate the epidemiology of betanodavirus, experiments conducted on 969 shellfish samples concluded that (i) distribution of NNV genotype, especially BFNNV, in shellfish is clearly different from that found in finfish of the world; (ii) unlike RGNNV, which showed a high rate in summer, BFNNV showed no seasonal variation and this result suggests BFNNVs in the marine environment remain fairly constant throughout the year; and (iii) the entrapped virus in shellfish was alive and culturable in vitro. These results are the first report of high level prevalence of in vitro culturable NNV in shellfish, for both BFNNV and RGNNV, which may present a potential risk in transmitting nodaviruses to host species in a marine environment.

A case of Behçet's disease with superior and inferior vena caval occlusion.

Behçet's disease is a chronic multisystemic disorder involving many organs and characterized by recurrent oral and genital ulcers and relapsing iritis. A case of BD with large vein thrombosis involving superior and inferior vena cava is presented. Large vein thrombosis in BD is not commonly developed and most commonly observed in the inferior or superior vena cava. A review of the literature emphasizes the rarity of the combined superior and inferior vena caval occlusion. Existence of extensive large vein occlusion in BD is associated with limited therapy and poor prognosis.

Progress in studies on the overwintering of the mosquito Culex tritaeniorhynchus.

A detailed review is presented of half a century's research in Japan, Korea and the temperate zone of China on the overwintering of Culex tritaeniorhynchus, the main vector of Japanese encephalitis. The evidence indicates that in the temperate zone, two types of overwintering strategy can be distinguished in Cx. tritaeniorhynchus, ie survival by adults in situ in a dormant state, or migration by individuals which are in reproductive diapause. The authors contend that the former strategy is not very effective, and individuals which overwinter in this way do not constitute the main source of the next season's mosquito populations. On the contrary, new mosquito populations seem to be established by groups of adults which appear suddenly, early in the year, and which are probably windborne migrants from the south. It is concluded that further research into the mode of overwintering of Cx. tritaeniorhynchus should focus on the effective source of the populations which appear in temperate areas in early spring, and that efforts should be made to increase our understanding of the migration patterns of this species.

[Comparative studies on diapause responses between Culex tritaeniorhynchus and Culex pipiens pallens].

By comparing the diapause responses between locally hibernating species, C. pipiens pallens, and C. tritaeniorhynchus the overwintering way of which is yet unknown, we found the relative identities of these 2 species in their ovarian diapause criteria, change of the mid-gut and the behaviour of refusing blood feeding. The increases in body weight and fatty contents in the dispersed tritaeniorhynchus were much less than those in diapause pipiens species. Moreover, their ability to resist low temperature was also found significantly weaker. In Shanghai area, the date of the critical photoperiod for C. tritaeniorhynchus was found around the 25th of September with a daylight exposure length (including twilight) of about 13 hours and 43 minutes, being 20 days earlier than that of the locally hibernating species C. pipiens pallens. The gonotrophic dissociation during critical photoperiod occurred more frequently in tritaeniorhynchus than in pipiens mosquitoes. In contrast to pipiens species, it is extremely difficult to obtain diapause tritaeniorhynchus in nature post critical photoperiod. The possibility of a seasonal migratory behaviour of C. tritaeniorhynchus was discussed.