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Research interests of Prof. Jen-Ren WangViral pathogenesis, virus-host cell interaction, diagnostic virology, enterovirus 71, epidemiology of enteroviruses and influenza viruses, virulence gene analysis of enterovirus 71 and influenza viruses. My research focuses on clinical diagnostic virology, viral surveillance, molecular epidemiology of re-emergent virus, analysis of virulence gene and pathogenesis of enterovirus and influenza virus 1. Clinical Virology1.1 Echovirus 30 (Wang et al, J Clin Microbiol 40: 4439-4444, 2002) For clinical diagnostic virology, we have collaborated with the physicians at the National Cheng Kung University Medical Center, the regional hospitals, and primary care clinics to establish virus watch program in southern Taiwan. For example, we found a large outbreak of aseptic meningitis occurred from April to November in Taiwan in 2001. Of the 1,130 enterovirus-infected patients, echovirus 30 (E30) infection was diagnosed in 188 (16.6%). The virus isolates were initially identified as echovirus 4 (E4) on the basis of immunofluorescence staining with anti-E4 and anti-E30 (Bastianni prototype) monoclonal antibodies. However, upon performance of the neutralization test, E30-specific reverse transcription-PCR, and sequencing of the VP1 gene, the results identified these isolates as E30, not E4. The findings indicate that the reagent used to type E30, which is produced with the Bastianni strain as the immunogen, is inadequate for the identification of recent E30 isolates in Taiwan. Phylogenetic analyses of the VP1 genes of these isolates showed that their sequences differed from those of E30 isolates from the GenBank database, suggesting that this outbreak was caused by a new variant strain of E30 introduced into Taiwan in 2000 that resulted in the widespread aseptic meningitis epidemic in 2001. 1.2 Echovirus 18 (Tasi et al, J Med Microbiol 60(9):1360-1365, 2011. figure on cover)An outbreak of aseptic meningitis occurred in Taiwan from April to August 2006. We found that major isolates from the 2006 outbreak of aseptic meningitis in Taiwan were echovirus 18 (27.4%, 93 cases) and the case fatality rate among children was 4%. Phylogenetic analyses of the partial VP1 gene showed there is 3.7-23.8% variation in their sequences compared from the GenBank database, and two notable position changes were substitution in V152S or V152Q and deletion in Q158. These mutations resulted in an antigenic drift in the loop of VP 1 protein in three-dimensional structure. In conclusion, a new variant E18 was circulating and caused an outbreak of aseptic meningitis. 2. Enterovirus 71 (Huang, et al. J Clin Microbiol. 2009. 47(11):3653-62.)In recent years, enterovirus 71 (EV71) has been a cause of numerous outbreaks of hand-foot-and-mouth disease, with severe neurological complications in the Asia-Pacific region. We found that the reemergence of EV71 genotype B5 in 2008 resulted in the largest outbreak of EV71 in Taiwan in the past years. Phylogenetic analyses indicated that dominant genotype changes from B to C or C to B occurred at least three times between 1986 and 2008. Furthermore, antigenic cartography of EV71 by using neutralization tests revealed that the reemerging EV71 genotype B5 strains formed a separate cluster which was antigenically distinct from the B4 and C genotypes. Moreover, analyses of full-length genomic sequences of EV71 circulating in Taiwan during this period showed the occurrence of intra- and interserotypic recombination. Therefore, continuous surveillance of EV71 including the monitoring of genetic evolution and antigenic changes is recommended and may contribute to the development of a vaccine for EV71. 3. Pathogenesis of enterovirus 713.1 Introduction of a strong temperature-sensitive phenotype into enterovirus 71 by altering an amino acid of virus 3D polymerase (Kung, et al. Virology. 2010. 396:1-9.) In 1998, an enterovirus 71 (EV71) epidemic in Taiwan resulted in 78 deaths; however, the molecular basis ofEV71 pathogenicity remains poorly understood. Comparison of the deduced amino acid sequences in 3Dpolymerases of EV71clinical isolates showed the T251V or T251I substitution from 1986 to 1998 outbreaks. An EV71 replicon system showed that introducing an I251T mutation did not affect luciferase activities at35 °C when compared with wild type; however, lower luciferase activities were observed when they were incubated at 39.5 °C. In addition, the I251T mutation in the EV71 infectious clone not only reduced viral replication at 39.5 °C in vitro but also decreased the virulence of the mouse adaptive strain MP4 in neonatal mice in an i.p. infection model. Therefore, these results suggested that the threonine at position 251 results in a temperature sensitivity phenotype of EV71 which may contribute to the attenuation of circulating strains in 1986. 3.2 A single nucleotide in stem loop II of 5'-untranslated region contributes to virulence of enterovirus 71 in mice (Yeh, et al. Plos One. 2011. 6(11): e27082.)In this report, we investigated increased virulence of unadapted EV71 clinical isolate 237 as compared with isolate 4643 in mice. A fragment 12 nucleotides in length in stem loop (SL) II of 237 5'-untranslated region (UTR) visibly reduced survival time and rate in mice was identified by constructing a series of infectious clones harboring chimeric 5'-UTR. In cells transfected with bicistronic plasmids, and replicon RNAs, the 12-nt fragment of isolate 237 enhanced translational activities and accelerated replication of subgenomic EV71. Finally, single nucleotide change from cytosine to uridine at base 158 in this short fragment of 5'-UTR was proven to reduce viral translation and EV71 virulence in mice. Results collectively indicated a pivotal role of novel virulence determinant C158 on virus translation in vitro and EV71 virulence in vivo. These results presented the first reported virulence determinant in EV71 5'-UTR and first position discovered from unadapted isolates. 3.3 Mutations in VP2 and VP1 capsid proteins increase cytotoxicity and mouse lethality of enterovirus 71 via virus binding and RNA accumulation enhancement. (Huang, et al. Virology. 2012. 422:132-143)EV71 infection occasionally associates with severe neurological sequelae such as brainstem encephalitis or poliovirus-like paralysis, but cytotoxicity and mouse lethality determinants remain unclear. We demonstrated how mouse-adapted virulent strain can induce more apoptosis of neuron cells and proved more virulent for neonatal mice than the non-virulent strain. Results pointed to EV71capsid region determining viral cytotoxicity, virus growth, and mouse lethality. In addition, mutant virus with lysine to methionine substitution at VP2149 (VP2149M) or glutamine to glutamic acid substitution at VP1145 (VP1145E) showed increased apoptosis and viral titers in Neuro-2a cells. Synergistic effect of VP2149M and VP1145E double mutations enhanced viral binding and RNA accumulation in infected Neuro-2a cells. These dual substitution mutants significantly reduced value of 50% lethal dose in neonatal mice infection, indicating they raised mouse lethality in vivo. In sum, VP2149Mand VP1145E mutations cooperatively promote viral binding and RNA accumulation of EV71, contributing to cytotoxicity and viral production in vitro and mouse lethality in vivo. 3.4 EV71 work accomplished and studies in progress by the PI:We continued to investigate the genetic evolution of EV71 for more than a decade long. We collaborated with Dr. Derek Smith from the University of Cambridge and established the first antigenic map of EV71. This cartography provided an all new sight for antigenic property change of EV71. In addition, we determined the viral genome recombination and performed a dynamic genetic evolution study of EV71. In the past of our studies, we primarily focused on molecular epidemiology, virulence determinants and pathogenesis of EV71. Through these studies, we have established various platforms, including in vitro culture in various cell lines, EV71 replicons, infectious clones, and in vivo animal model. 4. Influenza virus4.1 Genetic analysis of influenza virus (Tasi et al J Clin Microbiol 44: 2705-2713, 2006) As another re-emergent virus, we also have analyzed the genetic and antigenic evolution of influenza B virus field strains isolated in Taiwan from 1998 to 2005. All influenza B viruses isolated between 1998 and 2000 belonged to the B/Yamagata/16/88 lineage. The B/Victoria/2/87 lineage, which was cocirculated with the Yamagata lineage, was identified in Taiwan in March 2001. Since 2002, genetic reassortants of influenza B virus with the Victoria lineage of hemagglutinin and the Yamagata lineage of neuraminidase have been found at a rate of 46%. Therefore, in 2002, at least three sublineages of influenza B virus strains, the B/Shanghai/361/2002-like strain (Yamagata lineage), the B/Hong Kong/330/01-like strain (Victoria lineage), and the B/Hong Kong/1351/02-like strain (B reassortant lineage), were identified in Taiwan. The results showed that genetically distinct lineages can cocirculate in the population and that the reassortment among these strains plays a role in generating the genetic diversity of influenza B viruses. Interestingly, from January to April 2005, B reassortant viruses became dominant (73%) in Taiwan, which indicated that a mismatch had occurred between the influenza B vaccine strain recommended for the 2004-2005 season in the Northern hemisphere by the World Health Organization and the epidemic strain. 4.2 Humoral response against 2009 pH1N1 (Chi et al. Clin Vaccine Immunol 17(12):1958-1962, 2010.)Due to the important role of the antibody-mediated immunity in the protection of influenza infection, we investigated the cross-reactive neutralizing antibodies against 2009 pH1N1 of 229 stored sera from donors born between 1917 and 2008 in Taiwan using enzyme-linked immunosorbent assay-based microneutralization test. The peak of cumulative geometric mean titer was in donors aged over 90 years and declined sharply afterward. Sixteen of 27 subjects (59%) aged over 80 years had cross-reactive antibody titer of 160 or more against the 2009 pH1N1, whereas none of the donors from age 9 to 49 had an antibody titer of 160 or more. Interestingly, 2 of 51 children (4%) ranging in 6 months to 9 years old had an antibody titer of 40. We further tested the antibody response in 9 of the 51 pediatric sera to three endemic seasonal influenza viruses isolated in 2006 and 2008 in Taiwan, and the results showed only the 2 sera from children with antibody responses to 2009 pH1N1 had high neutralizing antibody titer against recent seasonal influenza strains. Our study shows the presence of some level of cross-reactive antibody in Taiwanese persons 50 years or older, and the elderly subjects who may already be exposed to the 1918 virus had high neutralizing antibody titer to the 2009 pH1N1. Our data also indicate that natural infection with the Taiwan 2006 and 2008 seasonal H1N1 viruses may induce cross-reactive antibody response to 2009 pH1N1. Currently, the EV71 and influenza virus investigations at National Cheng Kung University have been organized into two study groups. Many PIs were involved in these groups, including infectious disease specialists, pediatricians, epidemiologist, immunologists, virologists, and pathologists. Professional Experience
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