Wu-Tse Liu

Infection with enterovirus 71 or expression of its 2A protease induces apoptotic cell death.

Enterovirus 71 (EV71) is the causative agent of human diseases with distinct severity, from mild hand-foot-and-mouth disease to severe neurological syndromes, such as encephalitis and meningitis. Infection of several different cell lines with EV71 causes extensive cytopathic effect, leading to destruction of the entire monolayer and the death of infected cells. In this study, cell death processes during EV71 infection and the underlying mechanisms of them were investigated. The hallmarks of apoptosis, nuclear condensation and fragmentation, were observed 24 h after infection. Apoptosis in infected cells was also confirmed by detectable cleavage of cellular DNA and degradation of poly(ADP-ribose) polymerase. Transient expression of EV71 2A protease (2A(pro)) alone resulted in the induction of apoptotic change. Infection of EV71 or expression of EV71 2A(pro) leads to cleavage of the eukaryotic initiation factor 4GI, a key factor for host protein synthesis. This study added one more example to the growing list of human viruses that induce apoptosis by a virus-encoded protein.

Generating and characterizing monoclonal and polyclonal antibodies against avian H5N1 hemagglutinin protein.

Accurate and timely diagnoses are central to H5N1 infection control. Here we describe the cloning and expression of the HA1 protein of the A/Vietnam/1203/04 strain in a bacterial system to generate mono-/polyclonal antibodies. All of the eight generated monoclonal antibodies recognized the same linear epitope on the top globular region of the HA structure -- a highly conserved epitope among all circulating H5N1 clades identified by amino acid alignment. Results from immunofluorescence staining and Western blotting indicate that all monoclonal antibodies interacted with a denatured form of HA proteins, while the resultant polyclonal antibodies recognized both denatured and native HA proteins on H5N1 reverse-genetics (RG) viruses. Results from flow cytometry and microneutralization assays indicate that the polyclonal antibodies blocked viral binding and neutralized H5N1-RG viruses. Our results may prove useful to establishing future H5N1 mono-and polyclonal antibodies, and perhaps contribute to the development of an alternative H5N1 vaccine.

Rapid diagnosis and quantification of herpes simplex virus with a green fluorescent protein reporter system.

A genetically modified cell line (Vero-ICP10-EGFP) was constructed for detection of herpes simplex virus (HSV) by a simple, rapid and direct method. The cell line was developed by stable transfection of Vero cell with a plasmid encoding the green fluorescent protein (GFP) driven by the promoter of the HSV-2 ICP10 gene. As early as 6 h after infection with HSV, fluorescence-emitting cells can be observed under a fluorescence microscope. A single infected cell emitting fluorescence can be observed with soft agar overlay by inverted fluorescence microscopy. No induction of detectable fluorescence was seen following infections with human cytomegalovirus (HCMV), varicella zoster virus (VZV), coxsackievirus A16 and enterovirus 71. Analysis by flow cytometry also demonstrated that intensity of the triggered fluorescence is proportional to the titer of HSV inoculated. Taken together, this novel GFP reporter system could become a useful means for rapid detection and quantification of HSV in clinical specimens.

Influenza A virus in Taiwan, 1980–2006: Phylogenetic and antigenic characteristics of the hemagglutinin gene

Limited amount of information is available in Taiwan on the genetic or antigenic characteristics of influenza A virus prior to the establishment of a Taiwan surveillance network in 2000. Isolates of H1N1 and H3N2 viruses in Taiwan between 1980 and 2006 were studied, and part of the hemagglutinin gene was analyzed due to its importance in terms of viral infection and antibody neutralization. Results from a phylogenetic analysis indicate continuous evolutionary topology in H3N2 isolates, and two distinct H1N1 lineages. Many genetic relationships between vaccine strains and epidemic isolates appearing in Taiwan before other global locations were also observed and recorded in addition to a gradual increase in the number of N‐linked glycosylation sites on partial HA1 proteins since 1980. The results from pairwise comparisons of HA1 nucleotide and deduced amino acid sequences indicate shared identities within groups organized according to their bootstrap and P‐values of approximately 95.5–100% and 95.7–100% in H1N1 and 94.5–100% and 93.2–100% in H3N2 viruses, respectively. Comparisons of amino acid substitutions in the five antigenic regions reveal highly non‐synonymous changes occurring in the Sb region of H1N1 and in the B region of H3N2. The results of an antigenic analysis using a hemagglutinin inhibition (HI) test indicate the presence of some epidemic strains 1–2 years earlier in Taiwan than in other parts of the world, as well as higher vaccine mismatch rates. This information supports the need for continuous surveillance of emerging influenza viruses in Taiwan, which will be useful for making global vaccine decisions. J. Med. Virol. 81:1457–1470, 2009.

Community-based molecular epidemiology of HTLV type I in Taiwan and Kinmen: implication of the origin of the cosmopolitan subtype in northeast Asia.

To understand the possible origin and dissemination of HTLV-I infection in northeast Asia, community-based molecular epidemiological studies were conducted on the Kinmen Islands (off the coast of Fukien Province, China) and in Taiwan. A total of 3831 Taiwanese from 3 townships (Pu-Li, Chu-Dung, and Pu-Tze) and 993 aborigines from 4 tribes in Taiwan participated in this study. The prevalence rates of HTLV-I infection in adult residents from Pu-Li, Chu-Dung, and Pu-Tze were 0.82, 1.72, and 1.63%, respectively. None of the aborigines had HTLV-I infection. Previously, 0.73% of the adult population of Kin-Hu, Kinmen were found to have HTLV-I infection. Peripheral blood mononuclear cells were collected from HTLV-I carriers identified both in Taiwan and Kinmen and the HTLV-I LTR sequences were PCR amplified, subcloned, and sequenced for phylogenetic tree analysis. The results showed that all 6 HTLV-I isolates from Kinmen and 13 of 18 (72.2%) isolates from Taiwan were group a (transcontinental) of Cosmopolitan subtype, while 5 of 18 (27.8%) isolates from Taiwan were group b (Japanese) of Cosmopolitan subtype. Since all of the HTLV-I-infected persons were descendants of immigrants from mainland China, the origin of the Cosmopolitan subtype in Taiwan and Kinmen may not have been Japan, as previously theorized, but China, possibly the result of the migration of an infected population in the past several centuries.

A quantitative assay for measuring human foamy virus using an established indicator cell line.

In order to improve the accuracy for detecting human foamy virus (HFV), an indicator cell line was established by co-transfecting baby hamster kidney-21 cells with two plasmids: one containing a G418 antibiotic resistance marker and the other including the luc gene which was placed downstream of the inducible HFV long terminal repeat promoter (from -533 to +20). Among 11 independent subclones, IdB14 was found to be stable with a low basal level of luciferase activity. Although the changes in luciferase activity in infected clones showed time-dependency and peaked at day 8, it is possible to differentiate infected and uninfected cells on day 2. The sensitivity of the foamy virus activated luciferase (FAL) assay was 400 times higher than the end-point syncytium formation by TCID(50). The HFV LTR promoter in the IdB14 cell line was specific for this virus. Moreover, a linear relationship was found between the MOI and the activated intensity of luciferase expression. These findings suggest that the FAL assay using the IdB14 indicator cell line is a simple and useful technique for rapid diagnosis and quantitation of active HFV infection.

An indicator cell assay for detection of human cytomegalovirus based on enhanced green fluorescent protein.

An indicator cell line (ML-UL54-EGFP) for the detection of human cytomegalovirus (HCMV) by a simple and direct method was developed. The stable line was constructed by introducing into mink lung cells an expression cassette that contains the enhanced green fluorescent protein (EGFP) reporter gene under the control of an HCMV-inducible promoter. The promoter was from the upstream region of the HCMV UL54 (pol) gene, an early gene promoter that is activated in the early phase of HCMV infection. Following infection with HCMV for 48 h, the stable line expressed well detectable level of the EGFP as observed under a fluorescence microscope. The sensitivity of the indicator cell assay is at least comparable with that of a plaque assay as assessed with a panel of HCMV strains. There were no detectable fluorescent cells after inoculations with several viruses other than HCMV, indicating high specificity. Analysis with flow cytometry revealed that the induced fluorescence from the infected cells was proportional to the titer of HCMV inoculated, making it possible to quantify HCMV infectious particles. In summary, the EGFP-based indicator cell line is of potential use for rapid detection and quantification of HCMV in clinical specimens.

Genetic characterization of the hemagglutinin of two strains of influenza B virus co-circulated in Taiwan

Two isolates of influenza B virus were obtained in the spring of 1997. One strain, B/Taiwan/21706/97, was isolated from a patient who had acute tonsillitis. The other, B/Taiwan/3143/97, was isolated from a patient who was diagnosed with meningoencephalitis. This implies that the influenza B viruses not only cause respiratory symptoms but may also cause inflammation of the nervous system. Sequence analysis of the hemagglutinin (HA) gene, HA1 domain, indicated that there were remarkable amino acid changes in the strain B/Taiwan/3143/97 compared to B/Victoria/2/87, B/Yamagata/16/88, and B/Taiwan/7/88. The changes in the positions 116, 200, 238, 242, and 271 were correlated with receptor binding. Furthermore, a potential glycosylation site at position 233 was lost. In total, 30 amino acid changes were noted at positions ranging from 116 to 295. These changes may affect the antigenicity of the virus. Phylogenetic analyses also showed that the B/Taiwan/3143/97 was located in an independent lineage, when compared to the reference strains belonging to B/Victoria/2/87 and B/Yamagata/16/88 lineages. This supports the hypothesis that influenza B viruses with distinct genetic characteristic were co‐circulated in Taiwan. J. Med. Virol. 59:208–214, 1999.

Analysis of codon usage preference in hemagglutinin genes of the swine-origin influenza A (H1N1) virus

BACKGROUND The swine-origin influenza A (H1N1) virus (S-OIV) has come to the forefront since 2009 and was identified as a new reassortant strain. The hemagglutinin (HA) glycoprotein mediates virus binding, contains antigenic regions recognized by neutralizing antibodies, and is associated with viral cross-species infection and adaption. The comparison study of codon usage preferences in influenza viral genomes was less extensive. In this study, we used codon usage pattern analyses to validate the adaption and origins of S-OIV. METHODS Codon usage pattern was used to estimate the host adaption of S-OIVs. Phylogenetic analysis of the HA gene was conducted to understand the phylogeny of H1N1 viruses isolated from different hosts. Amino acid signature pattern on antigenic sites of HA was analyzed to understand the antigenic characteristics. RESULTS Results of phylogenetic analyses of HA gene indicate that S-OIVs group in identical clusters. The synonymous codon usage pattern analyses indicate that the effective number of codons versus GC content at the third codon position in the HA1 gene slightly differ from those in swine H1N1 and gradually adapted to human. Our data indicate that S-OIV evolution occurred according to positive selection within these antigenic regions. A comparison of antigenic site amino acids reveals similar signature patterns between S-OIV and 1918 human influenza strains. CONCLUSION This study proposes a new and effective way to gain a better understanding of the features of the S-OIV genome and evolutionary processes based on the codon usage pattern. It is useful to trace influenza viral origins and cross-species virus transmission.

Identifying conserved DR1501-restricted CD4+ T-cell epitopes in avian H5N1 hemagglutinin proteins.

Highly pathogenic avian influenza H5N1 viruses are capable of causing poultry epidemics and human mortality. Vaccines that induce protective neutralizing antibodies can prevent outbreaks and decrease the potential for influenza A pandemics. Identifying unique H5N1 virus-specific HLA class II-restricted epitopes is essential for monitoring cellular strain-specific immunity. Our results indicate that 80% of the 30 study participants who were inoculated with an H5N1 vaccine produced neutralizing antibodies. We used intracellular cytokine staining (ICS) to screen and identify six DR1501-restricted H5N1 virus epitopes: H5HA(148-162), H5HA(155-169), H5HA(253-267), H5HA(260-274), H5HA(267-281) and H5HA(309-323.) Tetramer staining results confirmed that two immunodominant epitopes were DR1501-restricted: H5HA(155-169) and H5HA(267-281). Both are located at the HA surface and are highly conserved in currently circulating H5N1 clades. These results suggest that a combination of ICS and tetramer staining can be used as a T-cell epitope-mapping platform, and the identified epitopes may serve as markers for monitoring vaccine efficacy.