B Cell-Based Vaccine Transduced With ESAT6-Expressing Vaccinia Virus and Presenting α-Galactosylceramide Is a Novel Vaccine Candidate Against ESAT6-Expressing Mycobacterial Diseases.

Early secretory antigenic target-6 (ESAT6) is a potent immunogenic antigen expressed in Mycobacterium tuberculosis as well as in some non-tuberculous mycobacteria (NTM), such as M. kansasii. M. kansasii is one of the most clinically relevant species of NTM that causes mycobacterial lung disease, which is clinically indistinguishable from tuberculosis. In the current study, we designed a novel cell-based vaccine using B cells that were transduced with vaccinia virus expressing ESAT6 (vacESAT6), and presenting α-galactosylceramide (αGC), a ligand of invariant NKT cells. We found that B cells loaded with αGC had increased levels of CD80 and CD86 after in vitro stimulation with NKT cells. Immunization of mice with B/αGC/vacESAT6 induced CD4+ T cells producing TNF-α and IFN-γ in response to heat-killed M. tuberculosis. Immunization of mice with B/αGC/vacESAT6 ameliorated severe lung inflammation caused by M. kansasii infection. We also confirmed that immunization with B/αGC/vacESAT6 reduced M. kansasii bacterial burden in the lungs. In addition, therapeutic administration of B/αGC/vacESAT6 increased IFN-γ+ CD4+ T cells and inhibited the progression of lung pathology caused by M. kansasii infection. Thus, B/αGC/vacESAT6 could be a potent vaccine candidate for the prevention and treatment of ESAT6-expressing mycobacterial infection caused by M. kansasii.

Rotavirus diarrhea in hospitalized children under 5 years of age in Vientiane, Lao PDR, 2009-2015.

BACKGROUND: Diarrhea is a leading cause of death in children <5 years worldwide, causing an estimated 215,000 deaths in 2013. This evaluation tracks the epidemiologic patterns and most common rotavirus genotypes among hospitalized children in this age group with acute gastroenteritis in Lao PDR. METHODS: Children <5 years in a central tertiary hospital in the capital city were prospectively enrolled into the surveillance platform during January 2009-December 2015. We collected information regarding clinical characteristics of enrolled children. Stool samples were obtained within 24 h of hospital admission and tested for rotavirus using rotavirus antigen detection enzyme immunoassay. Samples were sent to the regional reference laboratories in Australia and South Korea for genotyping. Bivariate analyses compared demographic and clinical characteristics between rotavirus positive and negative children using Chi-square statistical testing. Seasonality of rotavirus and annual genotype distribution are also described. RESULTS: We enrolled 1853 children <5 years with acute gastroenteritis during the surveillance period and collected 1772 fecal specimens, 982 (55%) of which tested positive for rotavirus. A higher proportion of rotavirus acute gastroenteritis was observed among children 12-23 months of age as compared to rotavirus negative children in the same age group, 41% vs 36%. Eighty-six percent of rotavirus positive children experienced vomiting, as compared to 65% of rotavirus negative children. Eighty-five percent (n = 830/982) of rotavirus positive specimens occurred during the dry season (January-April). The most common genotypes identified were G1, G2, G3 and P8 and P4. The most prevalent combined genotype differed annually during the surveillance period. CONCLUSION: Surveillance continues to be important in documenting the burden of rotavirus in children <5 years in Lao PDR as well as providing a baseline for determining the impact of rotavirus vaccine once it is introduced into Lao PDR's national immunization schedule.

High incidence of group A rotaviruses G4P[6] strains among children in Gyeonggi province of South Korea, from 2009 to 2012.

The genotype distribution of group A rotaviruses (RVAs) circulating in Gyeonggi province, South Korea between 2009 and 2012 was investigated. A total of 2619 stool specimens from sporadic acute gastroenteritis cases and 117 acute gastroenteritis outbreaks were analyzed. Among them, RVAs were detected from 263 (10.0%) sporadic cases and 3 (2.6%) outbreaks. The G4P[6] strains predominated (29.7%), followed by G1P[8] (19.4%), G2P[4] (15.6%), G3P[8] (13.3%) and G9P[8] (6.5%) strain. Especially 96.2% of the genotype G4P[6] strains were isolated from children<1year of age. Phylogenetic analysis revealed that genotype G4P[6] strains were members of sub-lineage Ie(G4) and Ia(P[6]). Intensified monitoring of RVAs, especially G4P[6] strains among young children, is essential to control RVA infections.

Detection of Rotavirus Genotypes in Korea 5 Years after the Introduction of Rotavirus Vaccines.

Rotavirus (RV) is one of the most important viral etiologic agents of acute gastroenteritis (AGE) in children. Although effective RV vaccines (RVVs) are now used worldwide, novel genotypes and outbreaks resulting from rare genotype combinations have emerged. This study documented RV genotypes in a Korean population of children with AGE 5 yr after the introduction of RVV and assessed potential genotype differences based on vaccination status or vaccine type. Children less than 5-yr-old diagnosed with AGE between October 2012 and September 2013 admitted to 9 medical institutions from 8 provinces in Korea were prospectively enrolled. Stool samples were tested for RV by enzyme immunoassay and genotyped by multiplex reverse-transcription polymerase chain reaction. In 346 patients, 114 (32.9%) were RV-positive. Among them, 87 (76.3%) patients were infected with RV alone. Eighty-six of 114 RV-positive stool samples were successfully genotyped, and their combinations of genotypes were G1P[8] (36, 41.9%), G2P[4] (12, 14.0%), and G3P[8] (6, 7.0%). RV was detected in 27.8% of patients in the vaccinated group and 39.8% in the unvaccinated group (P=0.035). Vaccination history was available for 67 of 86 cases with successfully genotyped RV-positive stool samples; RotaTeq (20, 29.9%), Rotarix (7, 10.4%), unvaccinated (40, 59.7%). The incidence of RV AGE is lower in the RV-vaccinated group compared to the unvaccinated group with no evidence of substitution with unusual genotype combinations.

A survey on the status of hepatitis e virus infection among slaughterhouse workers in South Korea.

OBJECTIVES: The seroprevalence of hepatitis E virus (HEV) among high-risk groups overseas is high, but studies in these groups are rare in South Korea. We conducted the present study from April to November 2012 to obtain data on the seroprevalence and associated risk factors for HEV among slaughterhouse workers in South Korea. METHODS: Slaughterhouse workers from 80 workplaces nationwide were surveyed in South Korea in 2012. The subjects comprised 1848 cases: 1434 slaughter workers and 414 residual products handlers. By visiting 80 slaughterhouses, which were mixed with 75 of which also performed residual products handling, we conducted a questionnaire survey for risk factors and obtained blood samples in order to determine the seropositivity and seroprevalence of HEV. Anti-HEV IgG and IgM were measured using HEV IgG and IgM enzyme-linked immunospecific assay kits and HEV antigen was measured by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The seropositivity of anti-HEV IgG was 33.5% (slaughter workers 32.8% and residual products handlers 36.2%), and among the seropositive individuals the seroprevalence of anti-HEV IgM was 0.5% (slaughter workers 0.5%, residual products handlers 0.7%). The response rate of HEV-antigen as measured by RT-PCR was 0.2%. Risk factors significantly related to anti-HEV IgG seropositivity were age, sex , and working duration (slaughter workers only). CONCLUSIONS: There were significant risk factors (sex, age, and working duration) for HEV identified in our study. All three positive cases for HEV-antigen by RT-PCR were related to pig slaughter but without statistical significance. To prevent HEV, an educational program and working guidelines may be needed for high risk groups.

Epidemics of Norovirus GII.4 Variant in Outbreak Cases in Korea, 2004-2012.

Norovirus GII.4 is recognized as a worldwide cause of nonbacterial outbreaks. In particular, the GII.4 variant occurs every 2-3 years according to antigenic variation. The aim of our study was to identify GII.4 variants in outbreaks in Korea during 2004-2012. Partial VP1 sequence of norovirus GII.4-related outbreaks during 2004-2012 was analyzed. The partial VP1 sequence was detected with reverse transcription-polymerase chain reaction, seminested polymerase chain reaction, and nucleotide sequence of 312-314 base pairs for phylogenetic comparison. Nine variants emerged in outbreaks, with the Sydney variant showing predominance recently. This predominance may persist for at least 3 years, although new variants may appear in Korea.

Updates on the genetic variations of norovirus in sporadic gastroenteritis in Chungnam Korea, 2009-2010.

Previously, we explored the epidemic pattern and molecular characterization of noroviruses (NoVs) isolated in Chungnam, Korea in 2008, and the present study extended these observations to 2009 and 2010. In Korea, NoVs showed the seasonal prevalence from late fall to spring, and widely detected in preschool children and peoples over 60 years of age. Epidemiological pattern of NoV was similar in 2008 and in 2010, but pattern in 2009 was affected by pandemic influenza A/H1N1 2009 virus. NoV-positive samples were subjected to sequence determination of the capsid gene region, which resolved the isolated NoVs into five GI (2, 6, 7, 9 and 10) and eleven GII genotypes (1, 2, 3, 4, 6, 7, 8, 12, 13, 16 and 17). The most prevalent genotype was GII.4 and occupied 130 out of 211 NoV isolates (61.6%). Comparison of NoV GII.4 of prevalent genotype in these periods with reference strains of the same genotype was conducted to genetic analysis by a phylogenetic tree. The NoV GII.4 strains were segregated into seven distinct genetic groups, which are supported by high bootstrap values and previously reported clusters. All Korean NoV GII.4 strains belonged to either VI cluster or VII cluster. The divergence of nucleotide sequences within VI and VII intra-clusters was > 3.9% and > 3.5%, respectively. The "Chungnam(06-117)/2010" strain which was isolated in June 2010 was a variant that did not belong to cluster VI or VII and showed 5.8-8.2%, 6.2-8.1% nucleotide divergence with cluster VI and VII, respectively.

Genetic analysis of hepatitis A virus strains that induced epidemics in Korea during 2007-2009.

Hepatitis A virus is one of the most prominent causes of fecally transmitted acute hepatitis worldwide. In order to characterize the viral agents causing an outbreak in Korea (comprising North and South Korea) from June 2007 to May 2009, we collected specimens and performed genotyping of the VP1/P2A and VP3/VP1 regions of hepatitis A virus. We then used a multiple-alignment algorithm to compare the nucleotide sequences of the 2 regions with those of reference strains. Hepatitis A virus antibodies were detected in 64 patients from 5 reported outbreaks (North Korea, June 2007 [n = 11]; Jeonnam, April 2008 [n = 15]; Daegu, May 2008 [n = 13]; Seoul, May 2009 [n = 22]; and Incheon, May 2009 [n = 3]). We found 100% homology between strains isolated from the Kaesong Industrial Region and Jeonnam. While those strains were classified as genotype IA strains, strains from Seoul and Incheon were identified as genotype IIIA strains and showed 98.9 to 100% homology. Genotype IIIA was also dominant in Daegu, where strains were 95.7 to 100% homologous. All hepatitis A virus strains isolated from the Kaesong Industrial Region, Jeonnam, Seoul, and Incheon belonged to a single cluster. However, strains from Daegu could be classified into 2 clusters, suggesting that the outbreak had multiple sources. This study indicates that hepatitis A virus strains of 2 different genotypes are currently cocirculating in Korea. Moreover, it documents an increasing prevalence of genotype IIIA strains in the country.

Infection status of hospitalized diarrheal patients with gastrointestinal protozoa, bacteria, and viruses in the Republic of Korea.

To understand protozoan, viral, and bacterial infections in diarrheal patients, we analyzed positivity and mixed-infection status with 3 protozoans, 4 viruses, and 10 bacteria in hospitalized diarrheal patients during 2004-2006 in the Republic of Korea. A total of 76,652 stool samples were collected from 96 hospitals across the nation. The positivity for protozoa, viruses, and bacteria was 129, 1,759, and 1,797 per 10,000 persons, respectively. Especially, Cryptosporidium parvum was highly mixed-infected with rotavirus among pediatric diarrheal patients (29.5 per 100 C. parvum positive cases), and Entamoeba histolytica was mixed-infected with Clostridium perfringens (10.3 per 100 E. histolytica positive cases) in protozoan-diarrheal patients. Those infected with rotavirus and C. perfringens constituted relatively high proportions among mixed infection cases from January to April. The positivity for rotavirus among viral infection for those aged < or = 5 years was significantly higher, while C. perfringens among bacterial infection was higher for > or = 50 years. The information for association of viral and bacterial infections with enteropathogenic protozoa in diarrheal patients may contribute to improvement of care for diarrhea as well as development of control strategies for diarrheal diseases in Korea.

Murine norovirus-1 3Dpol exhibits RNA-dependent RNA polymerase activity and nucleotidylylates on Tyr of the VPg.

We investigated the roles and biochemical properties of recombinant murine norovirus-1 (MNV-1) 3D(pol) in RNA synthesis and virus genome-linked protein (VPg) nucleotidylylation. We therefore expressed VPg and 3D(pol) of MNV-1 in Escherichia coli. MNV-1 3D(pol) exhibited RNA-dependent RNA polymerase (RdRp) activity in vitro with poly(A) RNA as a template and MnCl(2) as a cofactor. MNV-1 3D(pol) demonstrated optimum RNA-synthesis activity at pH 7.4 and 37 degrees C in the absence of a primer. Further, VPg was guanylylated by MNV-1 3D(pol) in the presence of MnCl(2) in a template-independent manner. The guanylylation reaction conducted with VPg substitution mutants (Y26F, Y40F, Y45F and Y117F) and a deletion mutant (Delta117-124) indicated that Tyr(117) was the probable target site of guanylylation. Homopolymeric RNAs did not enhance VPg guanylylation, whereas in vitro-transcribed (-) subgenomic (SG) and (+)SG RNA enhanced VPg guanylylation by 9.2 and 3.2 times, respectively. Within (-)SG RNA, the (-)ORF3 region played a critical role in enhancing VPg guanylylation, suggesting that the MNV-1 ORF3 region of negative-strand RNA contains a cis-acting element that stimulates 3D(pol)-mediated VPg guanylylation.