Incidence and molecular characterization of hepatitis A viruses in Korean surface water between 2007 and 2010.

Several bodies of surface water in Korea were surveyed for the presence of hepatitis A virus (HAV) between 2007 and 2010. Of 265 surface water samples, 9 (3.4%) were HAV-positive. HAVs were mainly detected in the summer (3/62, 4.8%) and spring (4/96, 4.2%) seasons. Comparing different water sources, the highest prevalence (6.6%) of positive samples was seen in lake water, four HAV-positive samples being from lakes. Comparing prevalence rates across the four representative Korean basin systems, no HAVs were found in the Han or Nakdong river basins. The highest HAV prevalence was found in samples from the Yeongsan river and other basins (6.3%); the Geum/Seom river was also found to have a high HAV prevalence (5.7%). HAVs from the nine positive samples were then sequenced and analyzed phylogenetically. Two of the HAVs belong to genotype IA and fall within the same cluster as HAVs 6-3(ASAN4) (EU049548), KANSAN-PS1 (EU049554), and ASAN-KM (EU049563), which were collected from the stools of patients with gastroenteritis in Korea. The seven other HAV nucleotide sequences belong to the genotype IB cluster. This is the first nationwide surveillance of HAV in major Korean water sources.

A 5-year survey (2007-2011) of enteric viruses in Korean aquatic environments and the use of coliforms as viral indicators.

Three hundred and thirty-nine water samples obtained from 90 locations in Korea from 2007 to 2011 were tested for the presence of enteric viruses (EV), total coliforms (TC), and fecal coliforms (FC). A total culturable virus assay revealed that 89 samples (26.3%) were positive for EVs, the average concentration being 5.8 most probable number (MPN)/100 L. The Han river basin exhibited the highest contamination by EVs (occurrence, 41.3%; average concentration, 24.0 MPN/100 L). EV contamination was found more frequently in river water (occurrence, 33.6%; concentration, 8.4 MPN/100 L) than in lake water or groundwater. The concentration of EVs was highest in spring (7.7 MPN/100 L), whereas it was found most frequently in winter (36.1%). The number of TCs ranged from 0 - 1.2 × 10(5) colony forming units (CFU)/100 mL and that of FCs from 0-6.2 × 10(3) CFU/100 mL per sample. Statistical analyses showed that the presence of EVs, TCs and FCs did not correlate significantly with temperature or turbidity. In addition, presence of TCs and FCs was not significantly correlated with presence of EVs. In conclusion, TCs and FCs may not be accurate microbial indicators of waterborne EVs in Korean aquatic environments.

Development and diagnostic application/evaluation of pandemic (H1N1) 2009 influenza virus-specific monoclonal antibodies.

We prepared mAb specific to the H1N1 2009 virus (H1N1 2009) to facilitate development of an RDT with enhanced sensitivity and specificity. Among these antibodies, we identified two clones--hybridomas 1H7E1 and 3A3H7-that specifically bound to H1N1 2009 (non-seasonal) and were very suitable for application to a diagnostic kit. The affinity constants (K(a)) of 1H7E1 and 3A3H7 were 1.10 × 10(10) and 2.35 × 10(10), respectively. To identify the antibodies, we performed ELISA and immunoblot analyses and found that 1H7E1 recognized a conformational epitope of HA while 3A3H7 recognized a linear epitope. In clinical evaluations using specimens from 215 patients, a lateral flow rapid testing kit comprising these mAb showed a sensitivity of 81.5% (75/92) and a specificity of 96.7% (119/123). Results using the RDT kit were well correlated with conventional RT-PCR methods as commonly and commercially used. Based on our findings, we believe that use of these mAb with a rapid evaluation kit could serve as a good diagnostic tool for H1N1 2009.

Detection and molecular characterization of adenoviruses in Korean children hospitalized with acute gastroenteritis.

Human adenoviruses (HAdVs) are an important cause of acute gastroenteritis in children. However, few studies on the epidemiology or types of HAdVs associated with acute gastroenteritis have been conducted in Korea. Therefore, in the present study, the incidence of HAdV in 2064 stool samples from Korean children hospitalized with acute gastroenteritis (2004-2006) was assessed and the types of viruses present determined. Polymerase chain reaction, sequencing, and phylogenic analyses revealed that 113 samples (5.5%) were HAdV-positive. While HAdVs were mainly detected during July to October, no seasonal difference between the enteric and non-enteric viruses in the incidence of HAdV was observed. HAdV-41 and HAdV-40 were found in 54 (47.8%) and 3 (2.6%) HAdV-positive samples, respectively. HAdV-3, HAdV-7, HAdV-2, HAdV-31, HAdV-4, and HAdV-37 were detected in 11 (9.7%), 5 (4.4%), 2 (1.7%), 2 (1.7%), 1 (0.8%), and 1 (0.8%) of sample(s), respectively. Thus, not only enteric, but also non-enteric, HAdVs may play an important role in acute gastroenteritis in Korean children.

Complete genomic sequence analysis of norovirus isolated from South Korea.

The complete nucleotide and deduced amino acid sequences of the RNA genome of a recently isolated norovirus (NoV) from Korea, designated Hu/GII-4/CBNU2/2007/KR (CBNU2), were determined and characterized by phylogenetic comparison with several genetically diverse NoV sequences. The RNA genome of CBNU2 is 7,560 nucleotides in length, excluding the 3' poly (A) tract. It includes three open reading frames (ORFs): ORF1, which encodes the nonstructural polyprotein (5-5,104); ORF2, which encodes VP1 (5,085-6,707); and ORF3, which encodes VP2 (6,707-7,513). ORF2-based phylogenetic analysis revealed that CBNU2 belonged to the GII.4 genotype, the most prevalent genotype, and formed a cluster with NoVs isolated from Asian regions, between 2006 and 2008. Comparative analysis with the consensus sequence of 207 completely sequenced NoV genomes showed 47 mismatched nucleotides: 26 in ORF1, 14 in ORF2, and 7 in ORF3, resulting in 8 amino acid changes: 3 in ORF1, 2 in ORF2, and 3 in ORF3. Phylogenetic analysis with full genome ORF1, ORF2, and ORF3 nucleotide sequences obtained from CBNU2 and each of the other representative NoV genomes suggested that CBNU2 had not undergone recombination with any of the other NoVs. A SimPlot analysis further supported this finding.

Nationwide groundwater surveillance of noroviruses in South Korea, 2008.

To inspect the norovirus contamination of groundwater in South Korea, a nationwide study was performed in the summer (June to August) and winter (October to December) of 2008. Three-hundred sites designated by the government ministry were inspected. Water samples were collected for analysis of water quality, microorganism content, and viral content. Water quality was assessed by temperature, pH, turbidity, residual chlorine, and nitrite nitrogen content. Microorganism contents were analyzed bacteria, total coliforms, Escherichia coli, and bacteriophage. Virus analyses included panenterovirus and norovirus. Two primer sets were used for the detection of norovirus genotypes GI and GII, respectively. Of 300 samples, 65 (21.7%) were norovirus positive in the summer and in 52 (17.3%) were norovirus positive in the winter. The genogroup GI noroviruses that were identified were GI-1, GI-2, GI-3, GI-4, GI-5, GI-6, and GI-8 genotypes; those in the GII genogroup were GII-4 and GII-Yuri genotypes. The analytic data showed correlative relationships between the norovirus detection rate and the following parameters: water temperature and turbidity in physical-chemical parameters and somatic phage in microbial parameters. It is necessary to periodically monitor waterborne viruses that frequently cause epidemic food poisoning in South Korea for better public health and sanitary conditions.

Selective detection of viable Helicobacter pylori using ethidium monoazide or propidium monoazide in combination with real-time polymerase chain reaction.

Because Helicobacter pylori has a role in the pathogenesis of gastric cancer, chronic gastritis and peptic ulcer disease, detection of its viable form is very important. The objective of this study was to optimize a PCR method using ethidium monoazide (EMA) or propidium monoazide (PMA) for selective detection of viable H. pylori cells in mixed samples of viable and dead bacteria. Before conducting the real-time PCR using SodB primers of H. pylori, EMA or PMA was added to suspensions of viable and/or dead H. pylori cells at concentrations between 1 and 100 µM. PMA at a concentration of 50 µM induced the highest DNA loss in dead cells with little loss of genomic DNA in viable cells. In addition, selective detection of viable cells in the mixtures of viable and dead cells at various ratios was possible with the combined use of PMA and real-time PCR. In contrast, EMA penetrated the membranes of both viable and dead cells and induced degradation of their genomic DNA. The findings of this study suggest that PMA, but not EMA, can be used effectively to differentiate viable H. pylori from its dead form.

Presence, molecular characteristics and geosmin producing ability of actinomycetes isolated from South Korean terrestrial and aquatic environments.

The unpleasant odor of drinking water is one of the major problems in many water utilities in the world. Actinomycetes have long been associated with odorous compounds. Considering the paucity of research on Actinomycetes producing odorous compounds in South Korea, presence of Actinomycetes, their molecular characteristics and ability to produce odorous compounds were investigated in this study. Findings confirmed the presence of Actinomycetes in surface soil, sediment, and water samples from four sites: two artificial lakes [Paldang and Cheongpyeong (CP)], and two streams [Gyeongan (GA) and Yangpyeong]. Surface soil and sediment from CP area had the greatest concentration of Actinomycetes (8.2 x 10(7) and 6.8 x 10(6) colony forming units (CFUs)/gram, dry weight, respectively). When water samples are considered, samples from GA had the highest concentration (1.9 x 10(2) CFU/mL). 16S rRNA sequencing and molecular phylogenetic analysis showed that Streptomyces was the dominant genus (64.1%). In addition, the isolated Actinomycetes synthesized 5.4 ng/L geosmin as demonstrated by thermal desorption unit-gas chromatograph/mass spectrometry analysis.

Evaluation of a rapid diagnostic test, NanoSign® Influenza A/B Antigen, for detection of the 2009 pandemic influenza A/H1N1 viruses.

BACKGROUND: This study evaluated the clinical accuracy and analytical sensitivity of the NanoSign® Influenza A/B antigen kit in detecting 2009 pandemic influenza A/H1N1 viruses. The kit is one of the most popular rapid diagnostic tests for detecting influenza in Republic of Korea. RESULTS: The NanoSign® Influenza A/B kit resulted in 79.4% sensitivity and 97.2% specificity compared to RT-PCR in the detection of the viruses from 1,023 specimens. In addition, the kit was able to detect two strains of novel influenza viruses, Influenza A/California/12/2009(H1N1) and clinically isolated wild-type novel influenza A/H1N1, both of which are spreading epidemically throughout the world. In addition, the correlation between NanoSign® Influenza A/B test and conventional RT-PCR was approximately 94%, indicating a high concordance rate. Analytical sensitivity of the kit was approximately 73 ± 3.65 ng/mL of the purified viral proteins and 1.13 ± 0.11 hemagglutination units for the cultured virus. CONCLUSIONS: As the NanoSign® Influenza A/B kit showed relatively high sensitivity and specificity and the good correlation with RT-PCR, it will be very useful in the early control of influenza infection and in helping physicians in making early treatment decisions.

Molecular characterization of partial-open reading frames 1a and 2 of the human astroviruses in South Korea.

Human astroviruses (HAstVs) are among the major causes of gastroenteritis in South Korea. In this study, the partial regions of the open reading frame (ORF) 1a and ORF2 genes of HAstVs from gastroenteritis patients in nine hospitals were sequenced, and the molecular characterization of the viruses was revealed. 89 partial nucleotide sequences of ORF1a and 88 partial nucleotide sequences of ORF2 were amplified from 120 stool specimens. Phylogenetic analysis showed that most of the nucleotide sequences of ORF1a and ORF2 were grouped with HAstV type 1 but had evolutionary genetic distance compared with the reference sequences, such as the HAstV-1 prototype, Dresden strain, and Oxford strain. According to the phylogenetic analysis, some nucleotide sequences including SE0506041, SE0506043, and SE0506058, showed the discrepancy of the genotypes, but there was no proof of recombination among the HAstV types. In conclusion, this study showed that the dominant HAstV isolated from the Seoul metropolitan area in 2004-2005 was HAstV type 1, and that Korean HAstV-1 had the genetic distance in evolution compared with the reference sequences of HAstVs. Lots of nucleotide sequences of the ORF1a and ORF2 genes of HAstV will be useful for studying for the control and prevention of HAstV gastroenteritis in South Korea.

Detection of replicating negative-sense RNAs in CaCo-2 cells infected with human astrovirus.

Human astrovirus (HAstV) is the second most important cause of viral diarrhea and acute gastroenteritis in infants under five. However, determination of the infectivity of clinical isolates is difficult, and the replication cycle of HAstV is not yet fully understood. In this study, it was attempted to detect negative-sense (-)RNAs generated during the replication of RNA viruses. We used clinical isolates of HAstV to infect CaCo-2 cells. Reverse transcription using only a sense primer followed by PCR using both sense and antisense primers showed that (-)RNAs were first detected in CaCo-2 cells between 9 and 12 h postinfection (p.i.). However, these (-)RNAs were not detected when cells were treated with the protein synthesis inhibitor cycloheximide during HAstV infection. Next, RT with only an antisense primer followed by PCR was performed to detect (+)RNA of HAstVs after production of (-)RNAs during replication. RT-PCR results using the antisense primer revealed that the amount of (+)RNA began to increase starting 9 h p.i., indicating an accumulation of the newly synthesized (+)RNA genome. Cycloheximide was observed to abrogate the increase of newly made (+)RNA during HAstV infection. In conclusion, the use of sense or antisense primers during the RT reaction together with cycloheximide enabled us to quantitatively detect (-)RNAs, and this proved to be an useful tool in understanding the replication cycle of HAstV.

Giardia duodenalis: improved detection of viable cysts by reverse transcription-PCR of heat shock-inducible hsp70 gene.

Giardia duodenalis is a waterborne protozoan parasite that causes the diarrhoeal disease, giardiasis. Its durable and thick cell wall allows the parasite to exhibit resistance to environmental stresses. Because G. duodenalis exists in a water system at low levels, it is necessary to develop a sensitive method to detect its viability in aquatic environments. In the present study, specific primers for the heat shock protein (hsp) 70 gene were designed on the basis of G. duodenalis genome sequence and bioinformatic analysis. Viable G. duodenalis cysts were successfully distinguished by reverse transcription-PCR (RT-PCR) analysis using these primers. The amplicon of hsp70 was obtained from one cyst of G. duodenalis/100 microl, and this detection sensitivity significantly increased by 10(3)-fold when the cysts were given heat shock treatment. These findings prove that viable G. duodenalis cysts were successfully detected with a high degree of sensitivity by RT-PCR analysis targeting the hsp70 gene of G. duodenalis, thereby suggesting its practical potential for detecting viable G. duodenalis in environmental samples.

Stimulation of interferon-beta gene expression by human cytomegalovirus via nuclear factor kappa B and phosphatidylinositol 3-kinase pathway.

Infection of human foreskin fibroblast (HFF) cells with human cytomegalovirus (HCMV) induces the secretion of soluble factors including interferon (IFN)-beta that stimulates human leukocyte antigen (HLA) class I expression. In this study, the mechanism of IFN-beta induction by HCMV was investigated. In HCMV-infected HFF cells, IFN-beta secretion increased at 6h post infection (h.p.i.). Reverse transcription polymerase chain reaction (RT-PCR) analysis using ultra violet (UV)-inactivated HCMV indicated that viral gene expression is not necessary for the stimulation of IFN-beta. Stimulation of IFN-beta by HCMV infection was not blocked by cycloheximide, an inhibitor of protein synthesis, further suggesting that the expression of HCMV genes is not required for the stimulation of IFN-beta gene transcription. IFN-beta may be produced from virus-infected cells as an inflammatory response and nuclear factor kappa B (NF-kappaB) plays a central role in inflammatory response. HCMV failed to induce the IFN-beta expression, when the virus-infected cells were treated with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, or LY294002 and wortmannin, inhibitors of phosphatidylinositol 3-kinase (PI3-K). The result suggests that PI3-K and/or NF-kappaB may be related with the induction pathway of IFN-beta by HCMV.

A Comparison between Low- and High-Passage Strains of Human Cytomegalovirus.

To understand how human cytomegalovirus (HCMV) might change and evolve after reactivation, it is very important to understand how the nucleotide sequence of cultured HCMV changes after in vitro passaging in cell culture, and how these changes affect the genome of HCMV and the consequent variation in amino acid sequence. Strain JHC of HCMV was propagated in vitro for more than 40 passages and its biological and genetic changes were monitored. For each passage, real-time PCR was performed in order to determine the genome copy number, and a plaque assay was employed to get virus infection titers. The infectious virus titers gradually increased with passaging in cell culture, whereas the number of virus genome copies remained relatively unchanged. A linear correlation was observed between the passage number and the log10 infectious virus titer per virus genome copy number. To understand the genetic basis underlying the increase in HCMV infectivity with increasing passage, the whole-genome DNA sequence of the high-passage strain was determined and compared with the genome sequence of the low-passage strain. Out of 100 mutations found in the high-passage strain, only two were located in an open reading frame. A G-T substitution in the RL13 gene resulted in a nonsense mutation and caused an early stop. A G-A substitution in the UL122 gene generated an S-F nonsynonymous mutation. The mutations in the RL13 and UL122 genes might be related to the increase in virus infectivity, although the role of the mutations found in noncoding regions could not be excluded.

Human cytomegalovirus induces apoptosis in promonocyte THP-1 cells but not in promyeloid HL-60 cells.

The effect of human cytomegalovirus (HCMV) infection on the viability of the cells in the monocyte/myeloid lineage was investigated. Two cell lines at different stages in the differentiation pathway, the less differentiated promyeloid HL-60 and the more differentiated promonocyte THP-1 cells, were used in this study. While the viability of THP-1 cells was significantly impaired by HCMV infection, the viability of HL-60 cells was not affected. The decrease in the viability of THP-1 cells appears to result from the increase in apoptosis following HCMV infection. Interestingly, HL-60 cells were more sensitive than THP-1 cells to the apoptotic effect of other apoptogenic agents such as ultraviolet irradiation and hydrogen peroxide. When HL-60 cells were induced to differentiate by treating cells with 12-O-tetradecanoyl-phorbol 13-acetate (TPA), HCMV infection induced an increase in apoptosis of the differentiated HL-60 cells by TPA. Therefore, HCMV-induced apoptosis in the cells of the myeloid/monocyte lineage appears to depend on the degree of cell differentiation.

Prevalence of human herpesvirus-6B in Korean hematopoietic stem cell transplantation recipients.

Human herpesvirus-6 (HHV-6) is a major pathogen associated with diseases of recipients of hematopoietic stem cell transplants (HSCT). We have isolated HHV-6 in Korean HSCT recipients and carried out a prospective investigation of its prevalence. We obtained peripheral blood from HSCT recipients who had signs of HHV-6 infection. Cord blood mononuclear cells (CBMC) and Sup-T1 cells were used to culture the HHV-6. Indirect immunofluorescence assays (IFA), and the polymerase chain reaction (PCR) were employed to detect HHV-6. The prevalence of HHV-6 infection in HSCT recipients was calculated on the basis of the PCR results. HHV-6 was isolated from four clinical samples. After culturing the HHV-6 in CBMC, the standard strain and the four clinical isolates were propagated in Sup-T1 cells. The infected cells became grossly enlarged and multinucleate after 7-21 days. The virus was identified primarily on the basis of the morphological changes of the cultured cells, and confirmed by specific IFA with monoclonal antibody to HHV-6. HHV-6 was detected in each sample by PCR with primers specific for the major immediate early gene. Sequencing of the standard strain and PCR products confirmed identification of the HHV-6B variant. By PCR we detected 415 instances of HHV-6 in 3966 samples (14.6% of peripheral blood mononuclear cells and 6.3% of sera), and HHV-6 DNAemia was most frequent from the second to the fourth week after HSCT.

Detection of coliforms in drinking water using skin patches: a rapid, reliable method that does not require an external energy source.

The detection of coliforms requires incubation in a laboratory, generally powered using electricity. In many parts of the developing world, however, external energy sources such as electricity are not readily available. To develop a fast, reliable method for detecting coliforms in water without an external energy source, we assessed the efficacy of six test kits for the identification of coliforms in water samples. To assess the possibility of using body temperature as the sole source of heat for incubation, bacterial samples were then mixed with the enzymatic test kit reagent and attached to the human body surface using a patch system. The patches were attached to the bodies of volunteers for 24 hours and the practicality and accuracy of the patches were assessed. Coliforms were detected within 24 hours in all patches. This innovation will facilitate the testing of water quality by researchers and by economically disadvantaged people without electricity.

Occurrence and molecular characterization of noroviruses in Korean surface water between 2007 and 2010.

The occurrence of human norovirus (NoV) genogroup I (GI) and genogroup II (GII) strains was investigated in Korea. Between 2007 and 2010, 265 samples were collected from 89 Korean water source locations. NoV GI was detected in 4.5% and NoV GII in 1.5%. Samples collected in winter had the highest occurrence; 9.4% for NoV GI and 6.3% for NoV GII. NoV GI detection was highest in groundwater, with the next highest in river water and the lowest in lake water (5.9%, 5.4%, and 1.6%, respectively), and NoV GII was found only in river water. When three representative Korean basin systems (Han (H)-, Geum/Seom (G/S)-, and Nakdong (N)-river basins) were compared, both NoV genogroups were high in the G/S-, but absent in the H- river basin. The most prevalent genotypes within the GI and GII groups were GI.5 and GII.4, respectively. The NoVs found in surface water were identical to those found in patients and those found in groundwater. The NoVs appeared to be transmitted from the patient to the surface water, and then to the groundwater, suggesting a fecal-oral route of transmission. This is the first nationwide surveillance of NoV in major Korean water sources.

Development and clinical evaluation of a rapid diagnostic kit for feline leukemia virus infection.

Feline leukemia virus (FeLV) causes a range of neoplastic and degenerative diseases in cats. To obtain a more sensitive and convenient diagnosis of the disease, we prepared monoclonal antibodies specific for the FeLV p27 to develop a rapid diagnostic test with enhanced sensitivity and specificity. Among these antibodies, we identified two clones (hybridomas 8F8B5 and 8G7D1) that specifically bound to FeLV and were very suitable for a diagnostic kit. The affinity constants for 8F8B5 and 8G7D1 were 0.35 × 108 and 0.86 × 108, respectively. To investigate the diagnostic abilities of the rapid kit using these antibodies, we performed several clinical studies. Assessment of analytical sensitivity revealed that the detection threshold of the rapid diagnostic test was 2 ng/mL for recombinant p27 and 12.5 × 104 IU/mL for FeLV. When evaluating 252 cat sera samples, the kit was found to have a kappa value of 0.88 compared to polymerase chain reaction (PCR), indicating a significant correlation between data from the rapid diagnostic test and PCR. Sensitivity and specificity of the kit were 95.2% (20/21) and 98.5% (257/261), respectively. Our results demonstrated that the rapid diagnostic test would be a suitable diagnostic tool for the rapid detection of FeLV infection in cats.

Development of a new cell culture-based method and optimized protocol for the detection of enteric viruses.

The development of rapid and effective methods to detect water- and food-borne enteric viruses is important for the prevention and control of mass infection. This study represents an attempt to develop a reliable cell culture-based detection system and optimize an effective and rapid protocol for the assaying of environmental samples for the presence of infectious enteric viruses. Six enteric viruses were used in this study: poliovirus, Coxsackie virus A9, Coxsackie virus B5, human rotavirus G1, hepatitis A virus, and adenovirus type 41. Among the cell lines from humans (A549, HeLa, HEK293, and HFF) and other primates (Vero, BS-C-1, FRhK-4, BGMK, and MA104), a cytopathic effect (CPE) analysis indicated that the MA104 cell line was the most optimal for use in the detection of infectious enteric viruses. Both the sensitivity and specificity of virus detection in MA104 cells were similar to or higher than those in standard BGMK cells. Next, a method was developed for the determination of the infectiousness of enteric viruses using the colorimetric thiazolyl blue (MTT) assay. This assay utilizes 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide to yield % values based on colorimetric results. These results were compared with those from a conventional CPE-based TCID(50) assay, revealing no statistically significant difference between the two methods. The MTT% values in MA104 cells were comparable to those in BGMK cells. This MA104 cell-based MTT assay could substitute for the classical BGMK cell-based CPE assay for infectious enteric viruses.