Sheng-Fan Wang

DC-SIGN mediates avian H5N1 influenza virus infection in cis and in trans

Abstract DC-SIGN, a C-type lectin receptor expressed in dendritic cells (DCs), has been identified as a receptor for human immunodeficiency virus type 1, hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, and the SARS coronavirus. We used H5N1 pseudotyped and reverse-genetics (RG) virus particles to study their ability to bind with DC-SIGN. Electronic microscopy and functional assay results indicate that pseudotyped viruses containing both HA and NA proteins express hemagglutination and are capable of infecting cells expressing α-2,3-linked sialic acid receptors. Results from a capture assay show that DC-SIGN-expressing cells (including B-THP-1/DC-SIGN and T-THP-1/DC-SIGN) and peripheral blood dendritic cells are capable of transferring H5N1 pseudotyped and RG virus particles to target cells; this action can be blocked by anti-DC-SIGN monoclonal antibodies. In summary, (a) DC-SIGN acts as a capture or attachment molecule for avian H5N1 virus, and (b) DC-SIGN mediates infections in cis and in trans.

Molecular epidemiology of HCV genotypes among injection drug users in Taiwan: Full-length sequences of two new subtype 6w strains and a recombinant form_2b6w.

Human immunodeficiency virus type 1 (HIV‐1) circulating recombinant form (CRF) 07_BC strain has caused serious outbreaks among injection drug users in Taiwan since 2004. The objective of this study was to conduct a molecular epidemiological study of HCV genotypes in intravenous drug users in Taiwan. Blood samples and questionnaires from 591 intravenous drug users infected with HIV‐1 were collected nationwide. In total, 180 samples were selected for HCV genotyping using multiplex PCR and phylogenetic analysis of the core, E1 and NS5B regions. The Inno‐Lipa assay was used to confirm multiple infections with different genotypes. Eighty percent had a single infection with subtype 1b being the most common subtype (24%), 12% had double infections and two had triple infections. In addition, three recombinant forms (RFs)‐2a1a, 3a1b, and 2b6w were identified. Phylogenetic analyses showed that the 3a, 6a, and 6n strains were clustered with strains present in Thailand and mainland China. Full‐length sequence analysis showed that two 6w strains shared 89.4–90.2% sequence homology with the 6(r) strain from the Guangdong Province, China. Bootscan analysis revealed that the recombination breakpoint of RF_2b6w was located at the NS2‐NS3 junction. In summary, the distribution of HCV genotypes among Taiwanese intravenous drug users was complex and more than 12% of the drug users were infected with more than one genotype of HCV. J. Med. Virol. 82:57–68, 2010.

Detection of swine-origin influenza A (H1N1) viruses using a localized surface plasmon coupled fluorescence fiber-optic biosensor.

Abstract Swine-origin influenza A (H1N1) virus (S-OIV) was identified as a new reassortant strain of influenza A virus in April 2009 and led to an influenza pandemic. Accurate and timely diagnoses are crucial for the control of influenza disease. We developed a localized surface plasmon coupled fluorescence fiber-optic biosensor (LSPCF-FOB) which combines a sandwich immunoassay with the LSP technique using antibodies against the hemagglutinin (HA) proteins of S-OIVs. The detection limit of the LSPCF-FOB for recombinant S-OIV H1 protein detection was estimated at 13.9pg/mL, which is 103-fold better than that of conventional capture ELISA when using the same capture antibodies. For clinical S-OIV isolates measurement, meanwhile, the detection limit of the LSPCF-FOB platform was calculated to be 8.25×104 copies/mL, compared with 2.06×106 copies/mL using conventional capture ELISA. Furthermore, in comparison with the influenza A/B rapid test, the detection limit of the LSPCF-FOB for S-OIV was almost 50-fold in PBS solution and 25-fold lower in mimic solution, which used nasal mucosa from healthy donors as the diluent. The findings of this study therefore indicate that the high detection sensitivity and specificity of the LSPCF-FOB make it a potentially effective diagnostic tool for clinical S-OIV infection and this technique has the potential to be applied to the development of other clinical microbe detection platforms.

Severe Dengue Fever Outbreak in Taiwan

Dengue fever (DF) is a vector-borne disease caused by dengue viruses (DENVs). Epidemic dengue occurs intermittently in Taiwan. In 2014, Taiwan experienced its largest DF outbreak. There were 15,732 DF cases reported. There were a total of 136 dengue hemorrhagic fever (DHF) cases, of which 20 resulted in death. Most DF cases were reported in southern Taiwan. A total of 15,043 (96%) cases were from Kaohsiung, a modern city in southern Taiwan. This report reviews DF epidemics in Taiwan during 2005-2014. The correlation between DF and DHF along with temperature and precipitation were conjointly examined. We conclude that most dengue epidemics in Taiwan resulted from imported DF cases. Results indicate three main factors that may have been associated with this DF outbreak in Kaohsiung: an underground pipeline explosion combined with subsequent rainfall and higher temperature. These factors may have enhanced mosquito breeding activity, facilitating DENV transmission.

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.

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.

Consecutive large dengue outbreaks in Taiwan in 2014-2015.

Dear Editor, The World Health Organization (WHO) has recently raised a warning that many countries have dengue epidemics or outbreaks. Major dengue fever (DF) cases were reported in Southeast Asia.1 DF is a vector-borne disease caused by dengue virus that belongs to the genus Flavivirus in the family Flaviviridae. Dengue viruses are geographically distributed by two competent vectors, Aedes aegypti and Aedes albopictus. The Aedes aegyptimosquitoes are predominant in southern Taiwan and highly correlated with dengue epidemics or outbreaks.2 Previous reports have indicated that dengue epidemics or outbreaks have mainly occurred in the south of Taiwan, such as Tainan and Kaohsiung,3 which are neighboring cities. Dengue activity has mainly been found in Kaohsiung city.3 The co-circulation of two or even four dengue serotypes (DENV-1–4) has been reported in Kaohsiung city.4 A severe DF outbreak occurred in Taiwan in 2014. A total of 15 732 DENV laboratory confirmed cases were reported by the Centers for Disease Control, Taiwan (Taiwan CDC)4 (Supplementary Figure S1), including 15 492 indigenous and 240 imported cases (Supplementary Table S1). A total of 15 043 cases (96%) were reported in Kaohsiung city.4 We previously reported a correlation between the underground pipeline leaking gas explosion event, which was followed by continuous heavy rains, with the 2014 DF outbreak in Kaohsiung (Po0.0001; r= 0.87).5 The cavities created by the explosion might have led to an increase in stagnant waters suitable for breeding of the Aedes mosquitoes. We found that the Breteau index for measuring the density of mosquitoes increased from 10%–19% to 35%–49% from July to September in Kaohsiung. Serotyping using real-time reverse transcription polymerase chain reaction (real-time RT-PCR) for the NS5 gene and detection with type-specific primers and probes6 indicated DENV-1 as the predominant strain (Figure 1A; Supplementary Table S1). This 2014 DF outbreak was initiated by imported DF cases, and their viral envelope sequences were similar to the 2013 Indonesia isolates (Figure 1A; Supplementary Table S1). However, a consecutive larger DF outbreak occurred in Taiwan in 2015, and a total of 43 784 DF cases were reported by the Taiwan CDC4 (Supplementary Figure S1). Among them, 362 were imported cases (Supplementary Table S1). Most of the DF cases were distributed in Tainan (22 777; 52%) and Kaohsiung (19 784; 45%).4 Serotyping data indicated DENV-2 as the predominant strain in the 2015 DF outbreak (Figure 1A; Supplementary Table S1). DENV-2 infected cases were reported initially in May, and then the number surged to its highest peak in September but gradually decreased after October in Tainan city. In addition, the Taiwan CDC reported that DENV-2 was disseminated to the neighboring city, Kaohsiung, after July.4 We collaborated with the Kaohsiung Medical University Hospital (KMUH) and obtained 2000 DENV-positive serum samples in which the subjects were infected in 2015 as confirmed by the laboratory of Taiwan CDC.4 Written informed consent was obtained from each participant. All study protocols were approved by the institutional review board of the Kaohsiung Medical University. The serotype of 940 samples was determined using virus isolation and real-time RT-PCR.6,7 Most cases identified before July were DENV-1 infections. From August to September, 20% were DENV-1, and 80% were DENV-2. Samples collected after October were all DENV-2. Phylogenetic analysis (a neighbor-joining tree with p-distance inferred with 1000 bootstrap replicates in MEGA program version 5 for a 1485-nt fragment spanning the full gene of the envelope glycoprotein)8 proved that the DENV-2 isolated in Kaohsiung after August clustered with the 2015 outbreak-associated DENV-2 Tainan strain (bootstrap value was 100). However, it did not cluster with previous Taiwanese epidemic DENV-2 isolates (Figure 1A, left). Notably, the Taiwanese 2015 DENV-2 outbreak strains phylogenetically clustered with the 2014 China and 2015 Indonesia isolates. We suggest that this DF outbreak may have been caused by the imported DENV-2 infected cases from China or Indonesia. A similar phenomenon was found in the 2014 DENV-1 outbreak in Kaohsiung, which may have been caused by the imported cases infected with the 2013 Indonesia strain (Figure 1B, right) (Supplementary Table S1). We also

Human-Leukocyte Antigen Class I Cw 1502 and Class II DR 0301 Genotypes Are Associated with Resistance to Severe Acute Respiratory Syndrome (SARS) Infection

One-hundred and thirty confirmed cases of severe acute respiratory syndrome (SARS) were recruited to evaluate their anti-SARS-coronavirus (CoV) antibody status and human leukocyte antigen (HLA) types in September 2006, 3 y after the SARS outbreaks in Taiwan. Western blot assay showed that 6.9% of participants still had anti-spike and anti-nucleocapside antibodies. A case-control study of the association of HLA with SARS revealed that the HLA-Cw1502 and DR0301 alleles conferred resistance against SARS infection (p<0.05).

Epidemiology of human herpesvirus type 8 and parvovirus B19 infections and their association with HIV-1 among men who have sex with men and injection drug users in Taiwan

BACKGROUND/PURPOSE Human herpesvirus 8 (HHV-8), the causal agent of Kaposis sarcoma (KS), is transmitted sexually among men who have sex with men (MSM), but little is known of its transmission among injection drug users (IDUs). By contrast, human parvovirus B19 (B19), a causative agent for anemia, is most frequently detected in IDUs. The aim of this study was to investigate the associations between HHV-8 infection and human immunodeficiency virus type 1 (HIV-1), and between B-19 and HIV-1 among MSM and IDUs patients. METHODS Serum samples from 553 IDUs and 231 MSM were analyzed for anti-HHV-8 lytic and anti-B19 viral structural capsid protein 2 (VP-2) antibodies using enzyme immunoassay, indirect immunofluorescence, and immunoblot assays. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to evaluate the associations between different viral infections. RESULTS HIV-1-seropositive MSM had significantly higher rates of HHV-8 infection than seronegative MSM (32.3% and 15.4%, respectively; OR = 2.62, 95% CI = 1.37-5.02). Among HIV-1/AIDS patient groups, MSM had significantly higher HHV-8 seropositive rates (32.3% vs. 6.6%, p < 0.0001) and lower B19 infection rates (35.4% vs. 78.8%, p < 0.001) than IDUs. In addition, HIV-1-infected MSM were 5.95 times (95% CI = 3.38-10.46) more likely to be infected with HHV-8 than male HIV-1-infected IDUs. By contrast, male IDUs were 6.74 times odds (95% CI = 4.28-10.61) more likely to contract B19 infection than MSM. CONCLUSION In Taiwan, MSM have a significantly higher prevalence for HHV-8 than IDUs. The contrasting risks of HHV-8 and B19 infections between different HIV-1/AIDS groups suggest that the efficiency of viral infection is affected by their distinct transmission routes.

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.