Chan-Hee Lee

A Single N-Linked Glycosylation Site in the Japanese Encephalitis Virus prM Protein Is Critical for Cell Type-Specific prM Protein Biogenesis, Virus Particle Release, and Pathogenicity in Mice

ABSTRACT The prM protein of Japanese encephalitis virus (JEV) contains a single potential N-linked glycosylation site, N15-X16-T17, which is highly conserved among JEV strains and closely related flaviviruses. To investigate the role of this site in JEV replication and pathogenesis, we manipulated the RNA genome by using infectious JEV cDNA to generate three prM mutants (N15A, T17A, and N15A/T17A) with alanine substiting for N15 and/or T17 and one mutant with silent point mutations introduced into the nucleotide sequences corresponding to all three residues in the glycosylation site. An analysis of these mutants in the presence or absence of endoglycosidases confirmed the addition of oligosaccharides to this potential glycosylation site. The loss of prM N glycosylation, without significantly altering the intracellular levels of viral RNA and proteins, led to an ≈20-fold reduction in the production of extracellular virions, which had protein compositions and infectivities nearly identical to those of wild-type virions; this reduction occurred at the stage of virus release, rather than assembly. This release defect was correlated with small-plaque morphology and an N-glycosylation-dependent delay in viral growth. A more conservative mutation, N15Q, had the same effect as N15A. One of the four prM mutants, N15A/T17A, showed an additional defect in virus growth in mosquito C6/36 cells but not human neuroblastoma SH-SY5Y or hamster BHK-21 cells. This cell type dependence was attributed to abnormal N-glycosylation-independent biogenesis of prM. In mice, the elimination of prM N glycosylation resulted in a drastic decrease in virulence after peripheral inoculation. Overall, our findings indicate that this highly conserved N-glycosylation motif in prM is crucial for multiple stages of JEV biology: prM biogenesis, virus release, and pathogenesis.

Molecular epidemiology of norovirus infections in children with acute gastroenteritis in South Korea in November 2005 through November 2006.

ABSTRACT Norovirus infections were detected in 114 of 762 children with acute gastroenteritis in South Korea from November 2005 to November 2006. Seasonality peaks in December, March, and October were also assessed in this study. We identified seven noroviral genotypes (GI-6, GII-2, GII-3, GII-4, GII-5, GII-6, and GII-8) and a C1-120 strain showing low identity (79.3%) with GII-13 and GII-17.

Immunogenicity of the E1E2 proteins of hepatitis C virus expressed by recombinant adenoviruses.

The E1 and E2 proteins of hepatitis C virus (HCV) are believed to be the viral envelope glycoproteins that are major candidate antigens for HCV vaccine development. We reported previously that the replication-competent recombinant adenovirus encoding core-E1-E2 genes of HCV (Ad/HCV) produces serologically reactive E1 and E2 proteins forming a heterodimer in substantial amounts. Here, we examined immunogenicity of the E1E2 proteins copurified from HeLa cells infected with Ad/HCV virus in mice. Furthermore, we constructed a replication-defective recombinant adenovirus encoding the core-E1-E2 genes of HCV (Ad.CMV.HCV) and examined immunogenicity of the virus in mice. The mice immunized intraperitoneally with the copurified E1E2 proteins induced mainly antibodies to E2, but not to E1 by Western blot analysis. The sera of mice immunized with the E1E2 inhibited the binding of E2 protein to the major extracellular loop of human CD81. E2-specific cytotoxic T cells (CTLs), but not antibodies to the E1E2 antigens were induced in the mice intramuscularly immunized with Ad.CMV.HCV virus. When immunized with both Ad.CMV.HCV virus and the E1E2, mice elicited E2-specific CTLs and antibodies to the E1E2 antigens. The results suggest that immunization of Ad.CMV.HCV virus combined with E2 protein is an effective modality to induce humoral as well as cellular immune response to E2 antigen.

A complex RNA motif defined by three discontinuous 5-nucleotide-long strands is essential for Flavivirus RNA replication.

Tertiary or higher-order RNA motifs that regulate replication of positive-strand RNA viruses are as yet poorly understood. Using Japanese encephalitis virus (JEV), we now show that a key element in JEV RNA replication is a complex RNA motif that includes a string of three discontinuous complementary sequences (TDCS). The TDCS consists of three 5-nt-long strands, the left (L) strand upstream of the translation initiator AUG adjacent to the 5-end of the genome, and the middle (M) and right (R) strands corresponding to the base of the Flavivirus-conserved 3 stem-loop structure near the 3-end of the RNA. The three strands are arranged in an antiparallel configuration, with two sets of base-pairing interactions creating L-M and M-R duplexes. Disrupting either or both of these duplex regions of TDCS completely abolished RNA replication, whereas reconstructing both duplex regions, albeit with mutated sequences, fully restored RNA replication. Modeling of replication-competent genomes recovered from a large pool of pseudorevertants originating from six replication-incompetent TDCS mutants suggests that both duplex base-pairing potentials of TDCS are required for RNA replication. In all cases, acquisition of novel sequences within the 3M-R duplex facilitated a long-range RNA-RNA interaction of its 3M strand with either the authentic 5L strand or its alternative (invariably located upstream of the 5 initiator), thereby restoring replicability. We also found that a TDCS homolog is conserved in other flaviviruses. These data suggest that two duplex base-pairings defined by the TDCS play an essential regulatory role in a key step(s) of Flavivirus RNA replication.

Complete genome sequence and phylogenetic analysis of a recombinant Korean norovirus, CBNU1, recovered from a 2006 outbreak.

We have determined the complete nucleotide and deduced amino acid sequences of the RNA genome of CBNU1, a human norovirus (NoV) recovered from a 2006 outbreak in South Korea. The genome of 7547 nucleotides, excluding a 3-poly(A) tail of 11-105 nucleotides, encodes three overlapping open reading frames (ORFs): ORF1 (nucleotides 5-5104), ORF2 (nucleotides 5085-6731), and ORF3 (nucleotides 6731-7495). In a comparison to 108 other currently available completely sequenced NoVs representing all five genogroups (GI-GV) except GIV, the CBNU1 strain was highly similar to GII.3 NoVs. Multiple sequence alignments of the completely sequenced NoV genomes revealed five hypervariable regions throughout their genomes: two in ORF1, one in ORF2, and two in ORF3. At both the nucleotide and amino acid levels, genome-based phylogenetic analyses invariably showed that the CBNU1 strain was most closely related to three GII.3 NoVs: the American Texas/TCH04-577 and the two Japanese Saitama U18 and Saitama U201 strains; furthermore, these genome-based phylogenetic topologies corresponded most closely to those based on the ORF2 genes, as compared to those based on the ORF1 and ORF3 genes. Subsequent ORF2-based phylogenetic analyses of a selection of 126 other NoVs representing all 19 GII genotypes, in combination with genome-based Simplot analyses, showed that the CBNU1 strain was a recombinant GII.3 NoV with a breakpoint at the ORF1/ORF2 junction between two putative parent-like strains, Guangzhou/NVgz01 and Texas/TCH04-577. Overall, the CBNU1 strain represents the first Korean human NoV whose genome has been completely sequenced and for which its relationship with a large panel of genetically diverse NoVs has been extensively characterized.

Use of Time-Saving Flow Cytometry for Rapid Determination of Resistance of Human Cytomegalovirus to Ganciclovir

ABSTRACT There are two ways to assess the susceptibility of human cytomegalovirus (HCMV) to ganciclovir (GCV): one is a genotypic test that detects resistance-related mutations and the other is a phenotypic test that actually assesses susceptibility. The advantages of genotyping the UL97 gene are its rapidity and accuracy. However, to detect novel mutations or mutations affecting the UL54 DNA polymerase, a phenotypic test such as the plaque reduction assay (PRA) is also required. To avoid the shortcomings of PRA such as its time-consuming nature and labor-intensiveness, we developed a time-saving fluorescence-activated cell sorting (TS-FACS) technique. We obtained a GCV 50% inhibitory concentration (IC50) from five clinical isolates and an HCMV laboratory strain (AD169) and compared the results with those from the PRA. The laboratory strain and three clinical isolates were sensitive to GCV. Although there was a minor discrepancy in the case of one of the three isolates, the GCV IC50 values obtained by TS-FACS analysis correlated well with the results of the PRA. The remaining two isolates were resistant to GCV; one was GCV resistant due to the mutation M460V, and the GCV IC50 results obtained by TS-FACS analysis and by PRA were also comparable. The advantages of TS-FACS analysis are the shorter time required, the possibility of automation, and its comparability to PRA, considered the gold standard. Thus, TS-FACS analysis may be useful as an alternative to PRA in the clinic.

Molecular detection and characterization of human enteroviruses in Korean surface water.

In this study, the genetic epidemiology of enteroviruses (EVs) in Korean surface water was evaluated by conducting phylogenetic analyses of the nucleotide sequences of the 5′ non-coding region (5′ NCR), which was determined by RT-PCR analysis of total culturable virus assay-positive samples. The results showed that the nucleotide sequences of the EVs could be classified into 4 genetic clusters, and that the predominant presence of Korea EVs were very similar to echoviruses type 30. Interestingly, two nucleotide sequences were very similar to those of coxsackievirus type B1 isolated from aseptic meningitis patients in Seoul, Korea, implying the possibility of a common source for the viruses circulated in water systems and humans. In addition, 3 nucleotide sequences clustered strongly with the nucleotide sequences from China or Japan, and one fell into the same cluster as echovirus type 11 from Taiwan, which suggests that EVs in Asia may have evolved in a region-specific manner. Taken together, the results of this study revealed that EVs from Korea surface waters could be genetically classified as coxsackieviruses or echoviruses, and that they evolved in Asia in a region-specific manner.

Detection and molecular characterization of enteroviruses in Korean surface water by using integrated cell culture multiplex RT-PCR.

OBJECTIVEnTo identify waterborne enteric viruses in Korean surface water.nnnMETHODSnIntegrated cell culture (ICC)-multiplex reverse transcription-polymerase chain reaction (RT-PCR) was simultaneously designed to detect coxsackieviruses (CV), polioviruses (PV), and reoviruses (RV). ICC-multiplex RT-PCR and phylogenetic analysis were conducted using 21 total culturable virus assay (TCVA)-positive sample-inoculated cell cultures.nnnRESULTSnCV and RV were detected in 9 samples each, and 3 samples were positive for both CV and RV. PV was not detected in any sample. Molecular phylogenetic analysis of the VP1 gene sequences revealed that CV types B2 and B4 predominated in Korean surface water, and the nucleotide sequences of CV type B2 were clustered with those of CVs isolated from China and Japan. The results suggested that the evolution of these viruses occurred in a region-specific manner.nnnCONCLUSIONnCV and RV are detectable in Korean surface water, with a predominance of CV type B2, and the evolution of CV type B2 occur in a region-specific manner.

Timing and evolution of the most recent common ancestor of the Korean clade HIV subtype B based on Nef and Vif sequences

Molecular phylogenetic studies of the HIV-1 isolated from Koreans have suggested the presence of the so-called “Korean clade”, which can be defined as a cluster free of foreign isolates. The Korean clade accounts for more than 60% of Korean isolates and exerts characteristic amino acid sequences. Thus, it is merited to estimate when this Korean clade first emerged in order to understand the evolutionary pattern of the Korean clade. We analyzed and reconstructed the most recent common ancestor (MRCA) sequences from nef (n=229) and vif (n=179) Korean clade sequences. Linear regression analyses of sequence divergence estimates were plotted against sampling years to infer the year in which there was zero divergence from the MRCA sequences. MRCA sequences suggested the Korean clade was first emerged around 1984, before the first detection of HIV-1 in Korea in 1985. Further studies on synonymous and nonsynonymous substitution rates suggested positive selection event for the Korean clade, while other subtype B had undergone negative to neutral evolution.

Modeling Spatial-Temporal Epidemics Using STBL Model

The Space Time Bilinear (STBL) model is a special form of a multiple bilinear time series which can be used to model time series which exhibit bilinear behavior on a spatial neighborhood structure. The STBL model and its identification have been proposed and discussed by Dai and Billard (1998). In this paper, we compare the STBL model with STARMA and single ARMA model. All problems are addressed by setting up the model in state space form and applying the Kalman filter. An application of the STBL model to epidemic surveillance data is given and the results com pared with those from other models.