Utility of RT-PCR-based Dot-blot Hybridization for Detecting and Genotyping Echoviruses

We attempted to detect and identify virus types quickly by improving an RT-PCR-based dot-blot hybridization test for echoviruses, important human pathogens mainly causing aseptic meningitis. This test was applied to reference viruses of seven echovirus serotypes prevalent in Korea (E6, 7, 9, 11, 13, 25, and 30) and seventy isolates of echovirus isolated in Korea between 2002 and 2004. The primers for target DNA and hybridization probes (25mer, 50mer, and 70mer) were designed within the VP1 region of the echovirus. In RT-PCR, a nonradioactive digoxigenin-DNA labeling mix was added instead of dNTP to initiate PCR. The PCR product was then hybridized against 25mer, 50mer, and 70mer probe DNA spotted on nylon membranes and the reaction was observed. To investigate the optimal conditions for hybridization, various concentrations of target DNA (0.1, 1, 10, and 100 ng/micron l), size of probe DNA (25mer, 50mer, and 70mer), concentrations of probe DNA (10~50 pM), and reaction time were included. In the test zone, the optimal condition in terms of time and cost was a reaction time of 1 h with 10 ng/micron l target DNA concentration and 10 pM of a 50mer probe. We found 100% diagnosis of the serotypes for seven reference echoviruses and 90% (63/70) sensitivity for clinical isolates. Also, tests with this probe for reactivity with seven reference echoviruses by using DNA chips showed that diagnostic identification was possible without other serotype cross-reactivity. Therefore, efficiency analysis of probe and target DNA on clinical specimens by using dot-blot analysis indicated that this system can be applied to the prestages of the DNA chip and that the dot blot analysis itself can be used in applications to develop a tool for diagnosing specific viral serotypes.

A Quantitative Comparison of Vaccinia Virus Shedding from Conventional Dressing Sites and Vaccination Lesions after Smallpox Vaccination

BACKGROUND: We compared vaccinia virus shedding from the vaccine inoculation site (vaccination lesion) and two sites of a dressing covering the vaccination site; the outer surface of the semipermeable dressing (outer surface) and the inner surface of the semipermeable dressing, that is, the surface of a folded gauze under the semipermeable membrane (gauze surface) MATERIAL AND METHODS: Subjects were recruited from the volunteers who participated in a clinical trial of the efficacy of a 1:10 dilution of Lancy-Vaxina? (Berna Biotech, Switzerland), and were seen every 2-3 days (days 6, 8, 10, 13, and 15 after smallpox vaccination) for scheduled dressing changes. Swab specimens were obtained from the vaccination lesion, the outer surface, and the gauze surface. Quantitative viral culture assays for these specimens were done. RESULTS: Vaccinia virus was recovered from 126 (81%) of the 156 vaccination lesion samples collected from the 40 participants. A high virus titer was recovered from the vaccination lesion (geometric mean titer (log10)=3.91 on day 8). Of the 39 swab samples obtained from the gauze surface of the gauze, 16 (41%) were positive for virus. An intermediate titer was recovered from the gauze surface (geometric mean titer (log10)=0.91 on day 8). Of the 133 swab samples obtained from the outer surface, only one (0.8%) was positive for vaccinia. No virus was recovered from the outer surface on day 8. CONCLUSION: Our findings suggest that the addition of a semipermeable dressing to the folded gauze further reduces viral shedding and therefore increases protection.

A Quantitative Comparison of Vaccinia Virus Shedding from Conventional Dressing Sites and Vaccination Lesions after Smallpox Vaccination

BACKGROUND: We compared vaccinia virus shedding from the vaccine inoculation site (vaccination lesion) and two sites of a dressing covering the vaccination site; the outer surface of the semipermeable dressing (outer surface) and the inner surface of the semipermeable dressing, that is, the surface of a folded gauze under the semipermeable membrane (gauze surface) MATERIAL AND METHODS: Subjects were recruited from the volunteers who participated in a clinical trial of the efficacy of a 1:10 dilution of Lancy-Vaxina? (Berna Biotech, Switzerland), and were seen every 2-3 days (days 6, 8, 10, 13, and 15 after smallpox vaccination) for scheduled dressing changes. Swab specimens were obtained from the vaccination lesion, the outer surface, and the gauze surface. Quantitative viral culture assays for these specimens were done. RESULTS: Vaccinia virus was recovered from 126 (81%) of the 156 vaccination lesion samples collected from the 40 participants. A high virus titer was recovered from the vaccination lesion (geometric mean titer (log10)=3.91 on day 8). Of the 39 swab samples obtained from the gauze surface of the gauze, 16 (41%) were positive for virus. An intermediate titer was recovered from the gauze surface (geometric mean titer (log10)=0.91 on day 8). Of the 133 swab samples obtained from the outer surface, only one (0.8%) was positive for vaccinia. No virus was recovered from the outer surface on day 8. CONCLUSION: Our findings suggest that the addition of a semipermeable dressing to the folded gauze further reduces viral shedding and therefore increases protection.

Updates on Enterovirus Surveillance in Korea

PURPOSE: We identified the causative viruses from patients with aseptic meningitis, acute hemorrhagic conjunctivitis and other enterovirus-related diseases to understand the epidemiological patterns and prevailing strains of enterovirus infections each year. MATERIALS AND METHODS: During 1999-2003, we examined 3,260 specimens from 2,939 patients with aseptic meningitis or other clinical manifestations for the presence of enteroviruses by using both cell culture/ neutralisation test and reverse transcription-polymerse chain reaction-sequencing. To investigate the etiological agents which caused an epidemic of acute haemorrhagic conjunctivitis, conjunctival swab samples from acute haemorrhagic conjunctivitis patients showing cytopathic effects in HEp2 cells were tested by enteroviral specific PCR. RESULTS: We identified 603 isolates of enteroviruses (20.5%) among 2,939 cases and 22 serotypes of human enteroviruses were isolated during this 5 year period. Echovirus 13 and coxsackievirus A24 in 2002 and coxsackievirus A9 in 2003 were the first enterovirus to be indentified in Korea since we began the enterovirus surveillance in 1993. While an epidemic of echovirus 13 infection in Korea began in Gwangju and Jeolla province in 2002 and spread to Seoul, Gyunggi, Busan, Ulsan and other regions, echovirus 6 isolates in 2002 were mainly detected in Busan specimens and some Gwangju samples. From the nucleotide sequencing of enteroviral PCR products of conjunctival swab specimens, we found 85% nucleotide homology to coxsackievirus A24 (D90457). CONCLUSIONS: We isolated 603 enteroviral isolates among 2939 cases during 1999-2003. Echovirus 13 and coxsackievirus A24 were the first enterovirus to be identified in Korea and caused nationwide epidemics in 2002.

Updates on Enterovirus Surveillance in Korea

PURPOSE: We identified the causative viruses from patients with aseptic meningitis, acute hemorrhagic conjunctivitis and other enterovirus-related diseases to understand the epidemiological patterns and prevailing strains of enterovirus infections each year. MATERIALS AND METHODS: During 1999-2003, we examined 3,260 specimens from 2,939 patients with aseptic meningitis or other clinical manifestations for the presence of enteroviruses by using both cell culture/ neutralisation test and reverse transcription-polymerse chain reaction-sequencing. To investigate the etiological agents which caused an epidemic of acute haemorrhagic conjunctivitis, conjunctival swab samples from acute haemorrhagic conjunctivitis patients showing cytopathic effects in HEp2 cells were tested by enteroviral specific PCR. RESULTS: We identified 603 isolates of enteroviruses (20.5%) among 2,939 cases and 22 serotypes of human enteroviruses were isolated during this 5 year period. Echovirus 13 and coxsackievirus A24 in 2002 and coxsackievirus A9 in 2003 were the first enterovirus to be indentified in Korea since we began the enterovirus surveillance in 1993. While an epidemic of echovirus 13 infection in Korea began in Gwangju and Jeolla province in 2002 and spread to Seoul, Gyunggi, Busan, Ulsan and other regions, echovirus 6 isolates in 2002 were mainly detected in Busan specimens and some Gwangju samples. From the nucleotide sequencing of enteroviral PCR products of conjunctival swab specimens, we found 85% nucleotide homology to coxsackievirus A24 (D90457). CONCLUSIONS: We isolated 603 enteroviral isolates among 2939 cases during 1999-2003. Echovirus 13 and coxsackievirus A24 were the first enterovirus to be identified in Korea and caused nationwide epidemics in 2002.