Kuei-Hsiang Lin

Molecular epidemiology of enterovirus 71 in Taiwan.

Summary. Taiwan suffered a severe and widespread outbreak of enterovirus infection in 1998. More than 400 children were hospitalized, with seventy-eight fatalities due to central nerve system (CNS) involvement and cardiopulmonary collapse. Enterovirus 71 (EV71) was incriminated as the causative agent for the fatal cases. To understand the viral molecular epidemiology in this outbreak, fragments of 207-bp length of the VP4 region in 23 Taiwanese EV 71 isolates were sequenced. Pair-wise comparison revealed a 17.5–24.4% difference between the isolates and the prototype BrCr. However, all the changes in the VP4 region of the isolated strains were synonymous substitutions. Phylogenetic analysis was performed on these 23 isolates and 21 others deposited in GenBank. In this study, forty-four EV71 isolates from the world were separated into three distinct genotypes: A, B and C. The EV71 prototype strain, BrCr/70, is the only strain of genotype A. Group B included strains from the United States, Japan and Taiwan. Most strains in genotype B were isolated prior to 1990. Group C consisted of strains from Japan and Taiwan. Most strains of genotype C were isolated after 1990, they were further divided into 3 clusters: i.e. C-1, C-2 and C-3. In Taiwan, two genotypes, B and C-3, were co-circulating during the outbreak in 1998, although a minor group of genotype B may have appeared in Taiwan before 1986. The majority of the isolates clustered in genotype C-3. Genotype C showed a higher evolutionary rate than genotype B (3.9 × 10−3vs. 1.4 × 1010−3) in the VP4 region. There seems to be a worldwide trend with strains of genotype B appearing earlier than strains of genotype C which took over later in the dominance.

Respiratory Adenoviral Infections in Children: A Study of Hospitalized Cases in Southern Taiwan in 2001–2002

Adenoviruses account for 5-10 per cent of respiratory illnesses in children. To analyse the clinical features and the temporal frequency in acute adenoviral respiratory infections in hospitalized children in southern Taiwan, a total of 4333 children who were admitted to the Department of Pediatrics, Kaohsiung Municipal Hsiaokang (KMHK) Hospital, with clinical evidences of acute respiratory infections between January 2001 and December 2002 were studied. Adenoviruses were isolated from 317 patients with an isolation rate of 7.67 per cent. Serotype analysis was performed by polymerase chain reaction (PCR) and/or PCR-restriction fragment length polymorphism (PCR-RFLP) in 186 specimens. In 2001, adenovirus type 4 was found in the majority (57 per cent), followed by type 1.5.6 (15 per cent), type 2 (13 per cent), type 14 (8 per cent), type 3 (5 per cent), and type 7 (2 per cent). In 2002, type 3 became the major type (46 per cent), whereas the previously predominant type 4 decreased to 6 per cent, and type 7 increased from 2 to 19 per cent. The symptoms and signs included fever (98.7 percent), cough (77.6 per cent), abnormal breathing sounds (crackles and/or wheezing 23.3 per cent), abdominal pain (18.9 per cent), vomiting (21.8 per cent), and diarrhea (25.2 per cent). The mean duration of fever was 4.8 days (range 0-19 days). In the 186 cases in whom serotypes were analysed, pharyngitis and tonsillitis (47.8 per cent) were the most common presentation, followed by pneumonia (25.2 per cent), bronchitis (12.9 per cent), and pharyngoconjunctival fever (PCF) (7.6 per cent). Children between 4 and 8 years old were the most common group of patients with respiratory adenoviral infections. Our patients all had good prognosis. This adenoviruses molecular epidemiological study provides information that helps physicians in clinical differential diagnosis and treatment of respiratory adenoviral infection in children in southern Taiwan.

Phylogenetic analysis of a coxsackievirus A24 variant: The most recent worldwide pandemic was caused by progenies of a virus prevalent around 1981

Nucleotide substitutions in the viral-encoded proteinase 3C (3Cpro) region (549 nucleotides) of the RNA genome of a coxsackievirus A24 variant (CA24v), one of the agents causing acute hemorrhagic conjunctivitis (AHC), were studied using 32 isolates collected from the Eastern hemisphere in 1970-1989. Based on regression analysis of nucleotide differences among isolates, the nucleotide substitution rate of CA24v 3Cpro was estimated to be 3.7 x 10(-3)/nucleotide/year. A phylogenetic tree constructed by the modified unweighted pair group method using arithmetic averages (UPGMA) indicated that CA24v had evolved from a common ancestor which appeared in one focal place in November 1963 +/- 21 months, about 7 years before the first isolation of CA24v in Singapore. The tree also revealed that all the recent epidemic isolates in 1985-1989 including Asian and Ghanian strains diverged from each other after 1981. This finding is consistent with the evidence that AHC due to CA24v had been confined to Southeast Asia and the Indian subcontinent until 1985, then suddenly and explosively spread to other areas where no CA24v isolations had been reported.

TLR7 and TLR8 Gene Variations and Susceptibility to Hepatitis C Virus Infection

Toll-like receptors (TLRs) play pivotal roles in the innate immune system and control inflammatory responses and adaptive immunity. We previously evaluated associations between TLR7 and TLR8 gene SNPs and susceptibility to hepatitis C virus (HCV) infection. Our results suggested that TLR7IVS2-151G and TLR8-129G alleles were present at higher frequency in males of an HCV-infected group as compared to a control group (24.1% vs. 14.4%, p = 0.028; 17.6% vs. 6.8%, p = 0.004, respectively). Based upon their recognition of single stranded viral RNA, this suggested that TLR7 and TLR8 played a significant role in anti-HCV immune responses. Here, we studied the functional effects of these polymorphisms by analyzing the mRNA expressions of TLR7 and TLR8 and cytokine production induced ex vivo by TLR7- and TLR8-specific agonists using whole blood of subjects with different genotypes. The percentage of CD14+ cells from those with an AG haplotype that expressed TLR7 and TLR8 was significantly lower, but higher in intensity compared to cells from those with GG and AC haplotypes. Cells from those with an AG haplotype produced more IFN-α and less amounts of pro-inflammatory cytokines upon stimulation. This suggests that variations in TLR7 and TLR8 genes might impair immune responses during HCV infection.

The change of etiological agents and clinical signs of epidemic viral conjunctivitis over an 18-year period in southern Taiwan

BackgroundEpidemic viral conjunctivitis is a highly contagious eye disease that occurs worldwide and is caused mainly by adenoviruses and enteroviruses. An 18-year analysis of the changes of pathogens and clinical signs in a subtropical and densely populated island presents certain special features.MethodsWe retrospectively analyzed the clinical information and laboratory records of the conjunctivitis patients with positive conjunctival swabs from 1980 to 1997.ResultsThe positive rate of laboratory diagnosis of epidemic conjunctivitis was 50.0% (1,233/2,467). From 1980 to 1994, the predominant causative agent of adenoviral keratoconjunctivitis was adenovirus type 8 (Ad8), with six genotypes being evolved. Three of the new Ad8 genotypes each caused a new epidemic. After 1995 the predominant adenoviral pathogens shifted to Ad37 and Ad19, and no more Ad8 was isolated. Enterovirus type 70 (EV70) was isolated from four outbreaks of acute hemorrhagic conjunctivitis (AHC) from 1980 to 1984, but rarely in later years. Coxsackievirus A type 24 variant (CA24v), which first appeared in 1985, appeared later as the causes of four major epidemics of AHC from 1985 to 1994. The overall clinical symptoms of viral conjunctivitis were more severe in the 1990s than in the 1980s.ConclusionIn southern Taiwan, outbreaks of adenoviral keratoconjunctivitis caused by new genomic variants could be associated with the long-term endemic co-circulation of Ad8, Ad19, and Ad37, while epidemics of CA24v AHC were caused mainly by introduction of new viral strains from neighboring countries. The aggravation of host symptoms in the 1990s needs further investigation and close follow-up.

Epidemic Keratoconjunctivitis Caused by a New Genotype of Adenovirus Type 8 (Ad8)—A Chronological Review of Ad8 in Southern Taiwan

PURPOSE To understand the pathogenic evolution of viral keratoconjunctivitis in Kaohsiung, Taiwan, a retrospective molecular and clinical analysis was conducted. METHODS From January 1990 to December 1994, conjunctival swab samples from patients suspected of having viral conjunctivitis were collected for viral culture isolation, neutralization test (NT), and endonuclease cleavage analysis. Six restriction endonucleases, comprising HindIII, BamHI, SalI, SstI, SmaI, and PstI, were used for cleavage. Clinical examinations of patients were performed by two senior ophthalmologists. RESULTS Twenty-one cases of a new genotype of adenovirus (Ad) type 8, designated as Ad8H, were discovered in the 27 detected adenovirus cases. The Ad8H has a distinct cleavage pattern, especially by HindIII and SalI. The Ad8H keratoconjunctivitis induced more subconjunctival hemorrhage (33.3%), keratitis (33.3%), and lymphadenopathy (85.7%) than other genotypes of Ad8 previously isolated in Kaohsiung, Taiwan. CONCLUSION We have discovered a new genotype, Ad8H, which was prevalent as the main pathogen of the adenoviral keratoconjunctivitis in Kaohsiung, Taiwan from 1990 to 1994. Adenovirus type 8 is evolving into more genotypes with a trend towards more severe symptoms, including subconjunctival hemorrhage, keratitis, and lymphadenopathy.

Characterization of newly emerging Newcastle disease viruses isolated during 2002–2008 in Taiwan

To elucidate the epidemiological relationships between ND outbreaks and genetic lineages, a portion of the F gene (535 bp) and the full-length HN gene (1922 bp) of recent Taiwanese NDVs isolated in 2002-2008 was amplified by reverse transcription (RT)-polymerase chain reaction (PCR) and sequenced. Only a portion of above amplified PCR products of the F and HN genes (374 and 1713 bp) and their deduced amino acid residues were compared with the other 60 NDVs retrieved from GenBank. Most (29/30) of the recent Taiwanese isolates were clustered in subgenotype VIIe while only one isolate was classified as subgenotype VIIc. All the 29 isolates of subgenotype VIIe were further subclassified and termed provisionally as sub-subgenotypes VIIe2 (13 isolates), VIIe3 (5 isolates), and VIIe4 (11 isolates). The sub-subgenotype VIIe2 isolates possessing the motif (112)R-R-Q-K-R-F(117) and amino acid residue substitutions at positions 23 (L to F) and 90 (T to A) were collected during 2002-2005. The sub-subgenotype VIIe3 isolates possessing the motif (112)R-R-K-K-R-F(117) and amino acid residue substitutions at positions 74 (E to G) and 75 (A to G) within epitopes and 114 (Q to K) within cleavage site of F protein were collected during 2003-2006. The sub-subgenotype VIIe4 isolates possessing the motif (112)R-R-Q-K-R-F(117) and amino acid residue substitutions at positions 23 (L to F), 26 (I to T), and 90 (T to A) were collected during 2007-2008. All the NDV isolates in this study exhibited a high intra-cerebral pathogenicity index (ICPI), they were all classified as velogenic type of NDVs. The sub-subgenotype VIIe2 and VIIe4 viruses are now dominant and have been implicated in most of the recent ND outbreaks in Taiwan. Phylogenetic analysis of these isolates revealed that they may have evolved from previously reported local strains (VIIe1). This finding is essential for improving the disease control strategies and development of vaccines for ND.

Functional polymorphisms of TLR8 are associated with hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is a worldwide threat to public health. Toll‐like receptor 8 (TLR8) is critical for eliminating RNA viruses, and variation within the TLR8 gene may alter the function of TLR8 in response to HCV infection. Our previous study demonstrated that the TLR8‐129G>C (rs3764879) and TLR8+1G>A (rs3764880) variants were in complete linkage disequilibrium, and that the frequency of TLR8‐129C/+1A was significantly higher in male patients with HCV infection compared with the healthy controls. In the present study, we found that the promoter activity of TLR8‐129G was higher than that of TLR8‐129C in THP‐1 cells. Moreover, TLR8‐129G mRNA stability and competitive DNA‐binding ability were significantly lower than that of TLR8‐129C. To investigate the functional effects of TLR8 polymorphisms, we compared the nuclear factor‐κB (NF‐κB)‐driven luciferase activity in HEK293 cells transfected with the TLR8 variants. TLR8+1A plasmids induced less NF‐κB signalling than did those transfected with TLR8+1G after 20 μm CL075 (P = 0·011) stimulation. We also analysed the mRNA expression and cytokine production in whole blood and monocytes from people of various genotypes stimulated ex vivo by the interferon‐γ and TLR7/8 agonist CL075, R848. TLR8 expression in CD14+ cells derived from volunteers with TLR8‐129G/+1G was significantly higher than that derived from TLR8‐129C/+1A, and interleukin‐12p40 production was higher in volunteers with TLR8‐129G/+1G after stimulation. The data indicate that variations in TLR8 genes may modulate immune responses during HCV infection.

Interleukin-1α Released from Epithelial Cells after Adenovirus Type 37 Infection Activates Intercellular Adhesion Molecule 1 Expression on Human Vascular Endothelial Cells

ABSTRACT A key event in virus-induced inflammation (leukocyte extravasation through the endothelium) is the local activation of endothelial cells, as indicated by the expression of adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin. In order to identify triggers of inflammation in adenovirus infection, we inoculated respiratory and ocular epithelial cells with adenovirus type 37 (Ad37), a human pathogen associated with keratoconjunctivitis as well as urogenital and respiratory infections. Fluids from virus-infected epithelial cells activated ICAM-1 (and to a lesser extent, VCAM-1) expression on cultured human umbilical vein endothelial cells. Blocking studies with anticytokine antibodies implicated interleukin-1α (IL-1α) as the epithelial cell-derived factor which activated endothelial cell ICAM-1 expression. The results thus identify epithelial cell-derived IL-1α as a potentially important activator of endothelial cells in Ad37-induced inflammation.

The nucleotide sequence of 3C proteinase region of the coxsackievirus A24 variant: comparison of the isolates in Taiwan in 1985-1988.

Acute hemorrhagic conjunctivitis caused by coxsackievirus A24 variant (CA24v) first appeared in Taiwan in October 1985, followed by two other sequential epidemics in 1986 and 1988. In order to know the evolutionary relationship of the CA24v strains isolated in Taiwan, we first determined the nucleotide sequence of the 3C proteinase (3Cpro) region of the prototype strain (EH24/70), isolated in Singapore in 1970, by molecular cloning. The nucleotide sequence of the 3Cpro region thus sequenced showed striking homology with polioviruses and coxsackievirus A21.Viral RNA of eight isolates obtained from the three epidemics was reverse transcribed, amplified by the polymerase chain reaction, and cloned into M13 phage for the production of ssDNA for nucleotide sequencing by the dideoxy chain termination method. When the number of nucleotide difference was taken as a genetic distance between isolates, all isolates showed a very similar distance from the EH24/70, the earliest isolate of CA24v, indicating that they evolved at a constant evolutionary rate. Phylogenetic analysis by the unweighted pairwise grouping method of arithmetic average (UPGMA) indicated that the six isolates collected in 1985 and 1986 were closely related, while two 1988 isolates were more distant from them. The branching time between these two groups was estimated to be May 1984, 18 months before the first recognition of the CA24v epidemic in Taiwan.This is the first report of the nucleotide sequence of CA24v genome RNA and of an evolutionary analysis of the virus using the nucleotide sequence.