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.

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.

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.

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.

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.

Nationwide surveillance for pathogenic microorganisms in groundwater near carcass burials constructed in South Korea in 2010.

Widespread outbreaks of foot-and-mouth disease and avian influenza occurred in South Korea during 2010. In response to the culling of many animals to attenuate the spread of disease, South Korea used mass burial sites to dispose of the large number of carcasses; consequently, concerns about groundwater contamination by leachate from these burial sites are increasing. Groundwater is one of the main sources of drinking water, and its cleanliness is directly related to public health. Thus, this study aimed to evaluate the safety of groundwater around the burial sites (total of 600 sites). A total of 1,200 groundwater samples were collected though the country, and microbial analysis was conducted during two time periods: during the spring (n = 600; April to June 2012) and after rainfall (n = 600; August to October, 2012; fall). Fecal coliform and Escherichia coli were detected in 173 (14.4%) and 85 (7.1%) of the 1,200 samples, respectively. Salmonella spp. and Shigella spp. each were detected only once (0.083%). Clostridium perfringens was detected from 7 groundwater samples (0.583%), and E. coli O157:H7 was not detected. With respect to norovirus, only the GII type was detected from six groundwater samples (0.5%), and enterovirus was detected in 15 groundwater samples (1.25%). The frequency of E. coli that we detected was lower than that found in previous studies conducted in South Korea, but we detected higher frequency of fecal coliform than that observed in a previous report. The contamination frequencies of Salmonella spp. and Shigella spp. were very low, but C. perfringens, which could be an indicator of fecal pollution, was detected in seven regions. Overall, the results of the present study indicate a low possibility of contamination from burial sites. However, consistent monitoring is required to prevent microbial contamination of groundwater near the burial sites.

Mitochondria-targeted apoptosis in human cytomegalovirus-infected cells.

Mitochondria often play central roles in apoptotic pathways, and disruption of the mitochondrial transmembrane potential (ΔΨm) has been observed in various cells undergoing apoptosis. Human cytomegalovirus (HCMV) infection induces apoptosis in permissive cells; however, investigations of mitochondria-targeted apoptosis in HCMV-infected human foreskin fibroblast (HFF) cells have been limited. Here, we investigated the mitochondrial apoptosis pathway in HCMV-infected HFF cells. Flow cytometry analysis using JC-1 revealed that HCMV infection induces disruption of ΔΨm in HFF cells when administered 24 h postinfection (hpi), and this disruption was maximized at 48 hpi. Moreover, cytochrome c, normally a mitochondrial inner membrane protein, was detected in cytoplasmic extracts of HCMV-infected cells, but not mock-infected cells, by western blot analysis at 24 hpi. A caspase activity assay based on fluorescence spectrophotometry using a fluorogenic substrate revealed an increase in caspase-3 activity at 48 hpi in HCMV-infected cells. Caspase-8 activity was increased at 72 hpi in HCMV-infected cells. These results imply that HCMV infection induces mitochondria-mediated apoptosis in HFF cells.

Detection of human cytomegalovirus UL97 D605E mutation in Korean stem cell transplantation recipients and donors.

Ganciclovir resistance of human cytomegalovirus is associated with mutations in the viral UL97 gene and poses severe problems for immunocompromised patients. In this study, PCRbased restriction fragment length polymorphism and sequencing analyses detected the UL97 D605E mutation in all five clinical isolates from patients with ganciclovir-resistant human cytomegalovirus infection during prolonged ganciclovir therapy, whereas the M460V mutation was only present in 1 of 5 isolates. On the other hand, the detection rates of the D605E mutation in the stored available DNA samples from the donor and allogeneic stem cell transplantation recipients were 66.7% and 93.7%, respectively, suggesting that the presence of D605E mutation was not associated with the ganciclovir exposure. Although the D605E mutation may not be related to ganciclovir resistance, we suggest that this mutation could be an important molecular marker of human cytomegalovirus evolution in East Asian countries. Moreover, the restriction fragment length polymorphism method using the restriction enzyme HaeIII, which is generally used to detect the UL97 A591V mutation, could also detect the D605E mutation and may therefore be a useful tool for future research on the investigation of UL97 gene mutations.

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.

Full sequence analysis and characterization of a human astrovirus type 1 isolate from South Korea.

Human astroviruses are recognized as an important cause of infantile gastroenteritis around the world. In South Korea, sporadic cases of HAstV infection have been reported since 2002. However, hitherto, there have been no studies reporting the whole genome sequence of an HAstV isolate from South Korea. Hence, we sequenced and analyzed the entire genome of an HAstV-1 strain (lhar) that was isolated in Seoul, South Korea. The whole-genome sequence analysis revealed 3 open reading frames comprising the whole genome: ORF1a (2,763 bp), ORF1b (1,548 bp), and ORF2 (2,364 bp). The lhar strain showed amino acid identities with 8 other reference strains of 87.6-98.7%, 94.2-98.8%, and 62.6-99.0% in the ORF1a, ORF1b, and ORF2 regions, respectively. The amino acid sequence of the capsid region encoded by ORF2 was compared with a total of 19 HAstV-1 strains and 8 HAstVs reference strains isolated in various countries. This revealed 1 amino acid substitution, at aa412 (Pro → Arg) in ORF2. This study, the first to report the full-length sequence of an HAstV isolated in South Korea, is meaningful in that it can be used as a full-length HAstV sequence standard for future comparison studies. It may also prove useful to the field of public health field by facilitating the diagnosis and the prediction of new emerging variants.

Detection and genotyping of vancomycin-resistant Enterococcus spp. by multiplex polymerase chain reaction in Korean aquatic environmental samples.

The distribution characteristics of Enterococcus spp., which are indicators of fecal pollution, were investigated at 33 sites within the 3 major water systems of Korea. Enterococci were detected at concentrations ranging from 1 to 37 CFU/100mL in 41 of 132 samples (31.1%) from the 3 major water systems. The overall average detected concentration was 1.2 CFU/100mL, while the average concentration for all detection sites was 5.3 CFU/100mL. After optimized multiplex polymerase chain reaction (PCR) was performed with newly developed VanA, VanB, VanC-1, and VanC-2/3 primers, concentrations of vancomycin-resistant Enterococcus spp. (VRE) ranging from 1 to 23 CFU/100mL were detected in 17 of 132 samples (12.9%). Of 216 individual enterococcal colonies, 64 (29.6%) displayed the VanC genotype. The results of a susceptibility test to vancomycin showed that the range of the minimal inhibitory concentration (MIC), an indicator of bacterial resistance, was 4 to 24µg/mL, with the average MIC at 9.2±4.5µg/mL. Of the bacterial isolates, 1 colony with the VanC-1 genotype was identified as E. gallinarum by 16S rDNA sequencing, whereas the other 63 colonies had the VanC-2/3 genotype and were identified as E. casseliflavus. Although these results imply that the major head bays of Korea are not contaminated with the highly vancomycin-resistant VanA- or VanB-type VREs, the misuse of antibiotics should be prohibited to minimize the presence of VREs and to maintain a safe water supply for protecting the health of the general population. Based on the study results, we also recommend the implementation of a continuous, broad-spectrum inspection program for Enterococcus spp. and VRE contamination in the major head bays. Furthermore, the multiplex PCR method described in this study can be used effectively for this purpose.

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.

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.

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.

Detection and molecular characterization of human noroviruses in Korean groundwater between 2008 and 2010.

RT-PCR, nucleotide sequencing, and phylogenetic analysis were performed for genotyping and molecular characterization of noroviruses isolated from Korean groundwater. Among 160 samples collected from 80 sites between 2008 and 2010, 14 samples (8.7 %) from 12 sites were positive for noroviruses (NoVs). The percentages of NoV-positive samples in 2008, 2009, and 2010 were 22.2, 3.2, and 0 %, respectively, representing a yearly decrease. GII-positive samples (n = 9, 5.6 %) outnumbered GI-positive samples (n = 5, 3.1 %). The genotypes of the GI NoVs were GI.2, GI.5, and GI.6, and the genotypes of the GII NoVs were all GII.4. One sample, HM623465, was very similar to CUK-3 and CBNU2 and two GII.4 sequences isolated from the stool of Korean gastroenteritis patients. A BLASTN search revealed several nucleotide sequences highly similar to those of NoVs isolated in this study. The original isolation sources for these similar NoVs were mostly stool (n = 731, 80.0 %) and groundwater (n = 135, 14.8 %), and all the countries from which they were isolated were almost in Asia (96.0 %); specifically, China (n = 192, 21.0 %), Japan (n = 383, 41.9 %), Korea (n = 296, 32.4 %), and other Asian countries (n = 6, 0.7 %). These results suggest that Korean groundwater might be contaminated with NoVs from the stool of infected patients and that these NoVs in turn cause new cases of gastroenteritis through a typical fecal-oral route with region-specific circulation. Therefore, it is important to properly treat sewage, which may include waterborne viruses and manage point sources in groundwater for national health and sanitation. In addition, continuous molecular surveillance remains important for understanding circulating NoVs.

Development and evaluation of a rapid diagnostic test for Plasmodium falciparum, P. vivax, and mixed-species malaria antigens.

Plasmodium falciparum and P. vivax malaria are endemic to many parts of the world and humans can be co-infected with both species. Because each Plasmodium species has different biological and clinical characteristics, accurate differentiation of the infecting species is essential for effective treatment. Therefore, we produced three monoclonal antibodies that recognize the lactate dehydrogenase of P. falciparum, P. vivax, or both to develop the first P. falciparum, P. vivax, and mixed-species infections malaria antigen detection kit. The detection limits of this kit were 150 and 250 parasites/µL for P. falciparum and P. vivax, respectively, and the kit was able to detect mixed-species infections. The sensitivity and specificity of this kit was assessed with 722 clinical specimens. Our results showed that its sensitivities for P. falciparum, P. vivax, and mixed-species infection were 96.5%, 95.3%, and 85.7%, respectively. In addition, its specificity was high (99.4%).

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.

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.

Full genome sequencing and analysis of human cytomegalovirus strain JHC isolated from a Korean patient.

Human cytomegalovirus (HCMV) is a ubiquitous human pathogen and contains double stranded DNA genome with approximately 230 kbp. Because of its huge size, comparative genomic studies of HCMV genome have been limited. In this study it was attempted to obtain and analyze the full genome sequence from clinical isolate from Korea. The strain JHC was isolated from Korean patient undergoing bone marrow transplantation who exhibited resistance to ganciclovir treatment (Lee et al., 2005). The virus was plaque-purified, and the full genome sequence was determined by pyrosequencing technique. The JHC genome was found to contain 235,476 bp and 165 open reading frames (ORFs). Comparison with the full genome nucleotide sequences of 11 other HCMV strains suggest that JHC is not closely related with any other strains at genome level. As expected, JHC lacked IRL sequences found in lab-adapted AD169-varUK strain and this region was replaced by ORFs UL133-UL150 as in other clinical isolates. Two ORFs (UL1 and UL119) of the strain JHC were found to be truncated due to early stop codons, and RL6 contains an unusual start codon TTG. The strain JHC contains all the genetic information for micro RNAs known to be present in HCMV.