Shih Cheng Chang

Viral and host proteins involved in picornavirus life cycle

Picornaviruses cause several diseases, not only in humans but also in various animal hosts. For instance, human enteroviruses can cause hand-foot-and-mouth disease, herpangina, myocarditis, acute flaccid paralysis, acute hemorrhagic conjunctivitis, severe neurological complications, including brainstem encephalitis, meningitis and poliomyelitis, and even death. The interaction between the virus and the host is important for viral replication, virulence and pathogenicity. This article reviews studies of the functions of viral and host factors that are involved in the life cycle of picornavirus. The interactions of viral capsid proteins with host cell receptors is discussed first, and the mechanisms by which the viral and host cell factors are involved in viral replication, viral translation and the switch from translation to RNA replication are then addressed. Understanding how cellular proteins interact with viral RNA or viral proteins, as well as the roles of each in viral infection, will provide insights for the design of novel antiviral agents based on these interactions.

Development of antiviral agents for enteroviruses

Enteroviruses (EVs) are common human pathogens that are associated with numerous disease symptoms in many organ systems of the body. Although EV infections commonly cause mild or non-symptomatic illness, some of them are associated with severe diseases such as CNS complications. The current absence of effective vaccines for most viral infection and no available antiviral drugs for the treatment of EVs highlight the urgency and significance of developing antiviral agents. Several key steps in the viral life cycle are potential targets for blocking viral replication. This article reviews recent studies of antiviral developments for EVs based on various molecular targets that interrupt viral attachment, viral translation, polyprotein processing and RNA replication.

Diverse apoptotic pathways in enterovirus 71-infected cells

Mechanisms related to the neuropathogenesis of enterovirus 71 infection remain unclear. This investigation conducts a comprehensive study of the apoptotic pathways in neural and non-neural cells following enterovirus 71 infection. Infections with enterovirus 71 not only induce classical cytopathic effects in SF268 (human glioblastoma), SK-N-MC (human neuroblastoma), RD, and Vero cells, but also induce classic signs of apoptosis in all cells, including DNA fragmentation and phosphatidylserine translocation. Apoptosis has also been caused by the efflux of cytochrome c from mitochondria, and subsequently by cleavage of caspase 9 in all cells. Activation of caspase 8 followed by cleavage of the proapoptotic protein Bid only occurs in non-neural cells. Results of this study demonstrate that a mitochondrial pathway of apoptosis mediated by activation and cleavage of caspase 9 is a main pathway in enterovirus 71-induced apoptosis, especially for enterovirus 71-infected neural cells.

An investigation of epidemic enterovirus 71 infection in Taiwan, 2008: Clinical, virologic, and serologic features

Background: Enterovirus 71 (EV71) is causing life-threatening hand-foot-mouth disease in Asia. In Taiwan, EV71 epidemics with different predominant genotypes occurred in 1998 (C2), 2000–2001 (B4), and 2004–2005 (C4). This genotype replacement may have important implications for vaccine development and prediction of epidemics. A nationwide EV71 outbreak occurred again in 2008, which provided a unique opportunity to characterize clinical, virologic, and serologic features of this epidemic. Methods: We analyzed clinical and virologic data of 111 EV71 patients hospitalized in 2008 and prospectively conducted follow-ups of healthy children from June 2006 to December 2008. Results: Among the 111 EV71 inpatients, 21 (19%) developed complications. Among the 21 complicated cases, 15 had central nervous system complication only, 2 had acute heart failure, and 4 had central nervous system and pulmonary complications. In the prospective study, 11 symptomatic infections and 4 asymptomatic infections were detected. Twenty-two EV71 isolates were genotyped, and 21 of them belong to genotype B5, which is phylogenetically close to B5 viruses circulating in Southeast Asia. Serologic tests show that children infected with B5 viruses have lower geometric mean titers of neutralizing antibody against genotype C4 than those against genotype B5 (P = 0.004, t test). Conclusions: The 2008 nationwide EV71 epidemic was caused by genotype B5 that was likely introduced to Taiwan from Southeast Asia. Clinical features of the 2008 epidemic were not different from those observed before in Taiwan. Potential antigenic variations between genotype C4 and B5 viruses could be detected and its long-term epidemiologic significance needs further investigation to clarify.

Clinical and genetic characterization of severe influenza B-associated diseases during an outbreak in Taiwan.

BACKGROUND Mismatches between circulating and vaccine strains of influenza virus had been observed in Taiwan. A comprehensive clinical and genetic analysis of influenza B viruses-associated important diseases was lacking. OBJECTIVES Clinical and phylogenetic analysis of influenza B viruses during an outbreak in Taiwan. STUDY DESIGNS Clinical manifestations of hospitalized, culture-confirmed patients were analyzed from July 2004 to June 2005. Partial genome sequence analysis of hemagglutinin (HA), neuraminidase (NA), and nonstructural (NS) genes were performed in 54 influenza B isolates during the study period, and nine srandomly chosen isolates during 2000 and 2003. RESULTS Three specific diseases were found in these patients, including 13 of encephalitis/encephalopathy, 28 of influenza-associated myositis (IAM), and one of acute respiratory distress syndrome (ARDS). Three phylogenetic groups were identified, including reassortant strains-group 1 (Victoria lineage of HA, Yamagata lineage of NA, clade A of NS), group 2 (Yamagata lineage of HA, Yamagata lineage of NA, clade A of NS), and group 3 (Yamagata lineage of HA, Yamagata lineage of NA, clade B of NS). CONCLUSIONS Severe influenza B-associated disease in children was not rare and might be fatal. We offered the evidence of co-circulation of the two HA lineages in the same outbreak.

Comparative Genomic Analysis of Coxsackievirus A6 Strains of Different Clinical Disease Entities

Background Studies regarding coxsackievirus A6 (CVA6) infection were limited. In Taiwan, outbreaks of CVA6 occurred in 2009 and 2010, respectively, but the clinical manifestations were markedly different. We conducted a study to compare the clinical features and genomic sequence between the two years. Methodology/Principal Findings In 2009 and 2010, 205 patients with coxsackievirus A6 (CVA6) infection were treated at Chang Gung Memorial Hospital. Detailed clinical features were obtained from 126 inpatients, 62 in 2009 and 64 in 2010. Between the inpatients in 2009 and 2010, no statistically significant difference was noted in terms of demographics, length of hospital stay and laboratory data. Significantly more patients in 2009 presented with herpangina (82%) while more patients in 2010 presented with hand-foot-mouth disease (HFMD; 67%) and skin rash beyond the typical sites for HFMD. Complete genomic sequences were determined and compared for three isolates from patients with herpangina in 2009 and three isolates from patients with HFMD in 2010. The complete sequences showed that 2009 and 2010 CVA6 isolates were indistinguishable by partial VP1 genes, but there were 5 unique nucleotide changes in 3′ UTR, and 23 out of 2201 (1%) amino acids were different. 2010 viruses underwent the largest number of amino acid changes in 3CD protein, which is the precursor of both 3C protease and 3D polymerase. Conclusions Since 2008 in Finland, outbreaks of HFMD due to CVA6 were noted internationally. CVA6 of different genetic background may cause different clinical manifestations such as herpangina and HFMD.

Genetic characterization of enterovirus 71 isolated from patients with severe disease by comparative analysis of complete genomes

Enterovirus 71 (EV71) which causes mild illness in children is also associated with severe neurological complications. This study analyzed the complete genomes of EV71 strains derived from mild and severe diseases in order to determine whether the differences of EV71 genomes were responsible for different clinical presentations. Compared to complete genomes of EV71 strains derived from mild cases (less virulent strains), nucleotide differences in EV71 strains isolated from severe cases (more virulent strains) were observed primarily in the internal ribosomal entry site (IRES) of the 5′‐untranslated region (UTR), which is vital for the cap‐independent translation of viral proteins. In the protein‐coding region, an E–Q substitution at amino acid position 145 of structural protein VP1 that occurred in more than one of more virulent strains was observed. This site is known to be related functionally to receptor binding and virulence in mice. Overall, strains (Group III) isolated from patients with fatal or severe sequelae outcomes had greater sequence substitutions in the 5′‐UTR and/or protein‐coding region and exhibited a relatively low‐average homology to less virulent strains across the entire genome, indicating the possibility of significant genomic diversity in the most virulent EV71 strains. Further studies of EV71 pathogenesis should examine the significance of genomic diversity and the effects of multiple mutations in a viral population. J. Med. Virol. 84:931–939, 2012.

Identification of genes involved in the host response to enterovirus 71 infection

Enterovirus 71 (EV71) infection may be asymptomatic or may cause diarrhea, rashes, and hand, foot, and mouth disease (HFMD). However, EV71 also has the potential to cause severe neurological disease. To date, little is known about the molecular mechanisms of host response to EV71 infection. In this report, we utilized cDNA microarray to profile the kinetics and patterns of host gene expression in EV71-infected human neural SF268 cells. We have identified 157 genes with significant changes in mRNA expression and performed hierarchical clustering to classify these genes into five different groups based on their kinetics of expression. EV71 infection led to increases in the level of mRNAs encoding chemokines, proteins involved in protein degradation, complement proteins, and proapoptotis proteins. cDNA microarray expression comparisons of EV71- and mock-infected cells also revealed the down-regulation of several genes encoding proteins involved in host RNA synthesis. Expression of interferon-regulated proteins was increased early in the infection and then decreased. Expression of proteins involved in cellular development and differentiation, some oncogenes, and transcription and translation regulators were suppressed and then stimulated late in the infection. Our findings illustrate the overall host response to EV71 infection, and will aid in understanding the host response to this virus.

Pyrosequencing reveals an oseltamivir- resistant marker in the quasispecies of avian influenza A (H7N9) virus

Prompt diagnosis of an oseltamivir-resistant marker is important for patient management, in particular to prevent the spread of resistant strains in the recent human H7N9 outbreak. We tailored a pyrosequencing assay to reveal neuraminidase R292K, a resistant marker found in one isolate from China, and demonstrated its performance in both sensitivity and specificity. In addition, a semi-nested polymerase chain reaction was applied, which enhanced the detection rate by at least 10-fold. We validated this assay by examining the marker in Taiwans first imported human case and found R and K in quasispecies.

Amino Acids Transitioning of 2009 H1N1pdm in Taiwan from 2009 to 2011

A swine-origin influenza A was detected in April 2009 and soon became the 2009 H1N1 pandemic strain (H1N1pdm). The current study revealed the genetic diversity of H1N1pdm, based on 77 and 70 isolates which we collected, respectively, during the 2009/2010 and 2010/2011 influenza seasons in Taiwan. We focused on tracking the amino acid transitioning of hemagglutinin (HA) and neuraminidase (NA) genes in the early diversification of the virus and compared them with H1N1pdm strains reported worldwide. We identified newly emerged mutation markers based on A/California/04/2009, described how these markers shifted from the first H1N1pdm season to the one that immediately followed, and discussed how these observations may relate to antigenicity, receptor-binding, and drug susceptibility. It was found that the amino acid mutation rates of H1N1pdm were elevated, from 9.29×10−3 substitutions per site in the first season to 1.46×10−2 in the second season in HA, and from 5.23×10−3 to 1.10×10−2 in NA. Many mutation markers were newly detected in the second season, including 11 in HA and 8 in NA, and some were found having statistical correlation to disease severity. There were five noticeable HA mutations made to antigenic sites. No significant titer changes, however, were detected based on hemagglutination inhibition tests. Only one isolate with H275Y mutation known to reduce susceptibility to NA inhibitors was detected. As limited Taiwanese H1N1pdm viruses were isolated after our sampling period, we gathered 8,876 HA and 6,017 NA H1N1pdm sequences up to April 2012 from NCBI to follow up the dynamics of mentioned HA mutations. While some mutations described in this study seemed to either settle in or die out in the 2011–2012 season, a number of them still showed signs of transitioning, prompting the importance of continuous monitoring of this virus for more seasons to come.