Use of internal standard RNA molecules for the RT-PCR amplification of the faeces-borne RNA viruses.

The diagnostic system based on reverse transcription (RT)-PCR has been used widely for the detection of viral genomes of faecal-borne RNA viruses. However, faecal specimens often produce both false positive and false negative results. Therefore, there is a need for a diagnosis procedure that can control for 'false-results'. In this study, an internal standard RNA that can serve as a non-competitive positive template was developed and used directly to detect faecal-borne RNA viruses without noticeable competitive inhibition of the target viral genome. These results suggest that the internal standard RNA is a useful standard molecule when undertaking diagnostic qualitative RT-PCR procedures for enteroviruses and related faecal-borne RNA viruses.

Seroprevalence of sexually transmitted viruses in Korean populations including HIV-seropositive individuals.

In order to investigate the prevalence of sexually transmitted viruses (STVs) in Korean populations, the prevalence of specific antibodies to herpes simplex type 2 (HSV-2) and hepatitis C virus (HCV) and surface antigen to hepatitis B virus (HBsAg) was evaluated in blood donors (n = 200), voluntary visitors for STI testing in public health centre (n = 204), HIV seropositive individuals (n = 200) and commercial sex workers (CSWs) (n = 200). All blood samples were tested using commercial enzyme-linked immunosorbent assay (ELISA) kits that detect type-specific IgG to HSV-2 and HBsAg and anti-HCV. The prevalence of type-specific IgG to HSV-2 among the above four groups was 5.0%, 21.9%, 65.0% and 71.0%, respectively. The seroprevalence of HBsAg was 0% in blood donors, 7.0% of visitors for STI, 7.5% of HIV infected individuals and 1.2% in CSWs. That of anti-HCV was 0%, 2.5%, 5.0% and 10.3%, respectively. This study underlines the very high seroprevalence of STVs, especially HSV-2, in the group at high risk of STI. The prevalences of HCV in CSWs and HIV infected individuals were two to three times higher than STI patients. It means sexual transmission might be a possible route of transmission of HCV, because HIV infection is closely related with sexual behaviour in Korea. The spread of HSV-2 is dependent on sexual transmission and that the presence of antibody to HSV-2 may be suitable for use as a serological marker of the degree at risk of STI.

Isolation and molecular identification of echovirus 13 isolated from patients of aseptic meningitis in Korea, 2002.

During 2002, several epidemics of aseptic meningitis were attributed to echovirus 13 in Korea. The causative agents of these outbreaks were isolated and identified using rhabdosarcoma cells, HEp-2 and Buffalo green monkey kidney cells, and a neutralization test using monospecific antiserum. Fifty-four echovirus 13 isolates were isolated from patients with aseptic meningitis in the provinces, Seoul, Kyonggi, Gwangju, Jeonju, Busan, and Ulsan. Symptoms associated with aseptic meningitis infection in patients included the occurrence of headaches and mild fever. Molecular characterization of echovirus 13 samples was achieved by sequence and phylogenetic analyses on partial VP1 sequences from 20 Korean isolates and 10 foreign isolates listed in Genbank. Minor variation was observed among the Korean isolates, which formed a unique cluster with isolates of German and Japanese origin. The marked similarities between isolates could be attributed to a relatively recent arrival of the virus in Korea. This is the first such investigation of aseptic meningitis caused by echovirus 13 on the Korean peninsula.

Identification of enteroviruses by using monoclonal antibodies against a putative common epitope.

A common epitope region of enteroviruses was identified by sequence-independent single-primer amplification (SISPA), followed by immunoscreening of 11 cDNA libraries from two Korean enterovirus isolates (echoviruses 7 and 30) and a coxsackievirus B3 (ATCC-VR 30). The putative common epitope region was localized in the N terminus of VP1 when the displayed recombinant proteins from the phages were chased by the convalescent-phase sera. The genomic region encoding the common epitope region was amplified and then expressed by using the vector pGEX-5X-1. The antigenicity of the expressed recombinant protein was identified by Western blotting with guinea pig antisera for six different serotypes of enteroviruses. After successive immunization of mice with the recombinant common epitope protein, splenocytes were extracted and hybridized with P3X63-Ag8-653 cells. A total of 24 hybridomas that produced monoclonal antibodies (MAbs) against the putative common epitope of enteroviruses were selected. Four of these were immunoglobulin G1 isotypes with a kappa light chain. These MAbs recognized 15 Korean endemic serotypes and prototypes of enteroviruses in an indirect immunofluorescence assay. These results suggest that the expressed protein might be a useful antigen for producing group common antibodies and that the use of the MAbs against the putative common epitope of enteroviruses might be a valuable diagnostic tool for rapidly identifying a broad range of enteroviruses.