Hui-Wen Chen

Influenza A penetrates host mucus by cleaving sialic acids with neuraminidase

BackgroundInfluenza A virus (IAV) neuraminidase (NA) cleaves sialic acids (Sias) from glycans. Inhibiting NA with oseltamivir suppresses both viral infection, and viral release from cultured human airway epithelial cells. The role of NA in viral exit is well established: it releases budding virions by cleaving Sias from glycoconjugates on infected cells and progeny virions. The role of NA in viral entry remains unclear. Host respiratory epithelia secrete a mucus layer rich in heavily sialylated glycoproteins; these could inhibit viral entry by mimicking sialylated receptors on the cell surface. It has been suggested that NA allows influenza to penetrate the mucus by cleaving these sialylated decoys, but the exact mechanism is not yet established.MethodsWe tested IAV interaction with secreted mucus using frozen human trachea/bronchus tissue sections, and bead-bound purified human salivary mucins (HSM) and purified porcine submaxillary mucins (PSM). The protective effect of mucus was analyzed using MDCK cells coated with purified HSM and PSM with known Sia content. Oseltamivir was used to inhibit NA activity, and the fluorescent reporter substrate, 4MU-Neu5Ac, was used to quantify NA activity.ResultsIAV binds to the secreted mucus layer of frozen human trachea/bronchus tissues in a Sia dependent manner. HSM inhibition of IAV infection is Sia dose-dependent, but PSM cannot inhibit infection of underlying cells. HSM competitively inhibits NA cleavage of 4MU-Neu5Ac, reporter substrate. Human IAV effectively cleaves Sias from HSM but not from PSM, and binds to HSM but not to PSM.ConclusionIAV interacts with human mucus on frozen tissue sections and mucus-coated beads. Inhibition of IAV infection by sialylated human mucus is dose-dependent, and enhanced when NA is inhibited with oseltamivir. Thus NA cleaves sialylated decoys during initial stages of infection. Understanding IAV interactions with host mucins is a promising new avenue for drug development.

The Roles of IRF-3 and IRF-7 in Innate Antiviral Immunity against Dengue Virus

We investigated the roles of IFN regulatory factor (IRF)-3 and IRF-7 in innate antiviral immunity against dengue virus (DENV). Double-deficient Irf-3−/−7−/− mice infected with the DENV2 strain S221 possessed 1,000–150,000 fold higher levels of viral RNA than wild-type and single-deficient mice 24 h postinfection (hpi); however, they remained resistant to lethal infection. IFN-α/β was induced similarly in wild-type and Irf-3−/− mice post–DENV infection, whereas in the Irf-7−/− and Irf-3−/−7−/− mice, significantly low levels of IFN-α/β expression was observed within 24 hpi. IFN-stimulated gene induction was also delayed in Irf-3−/−7−/− mice relative to wild-type and single-deficient mice. In particular, Cxcl10 and Ifnα2 were rapidly induced independently of both IRF-3 and IRF-7 in the Irf-3−/−7−/− mice with DENV infection. Higher levels of serum IFN-γ, IL-6, CXCL10, IL-8, IL-12 p70, and TNF were also observed in Irf-3−/−7−/− mice 24 hpi, at which time point viral titers peaked and started to be cleared. Ab-mediated blockade experiments revealed that IFN-γ, CXCL10, and CXCR3 function to restrict DENV replication in Irf-3−/−7−/− mice. Additionally, the IFN-stimulated genes Cxcl10, Ifit1, Ifit3, and Mx2 can be induced via an IRF-3– and IRF-7–independent pathway that does not involve IFN-γ signaling for protection against DENV. Collectively, these results demonstrate that IRF-3 and IRF-7 are redundant, albeit IRF-7 plays a more important role than IRF-3 in inducing the initial IFN-α/β response; only the combined actions of IRF-3 and IRF-7 are necessary for efficient control of early DENV infection; and the late, IRF-3– and IRF-7–independent pathway contributes to anti-DENV immunity.

Identification of Taiwan and China-like recombinant avian infectious bronchitis viruses in Taiwan

Abstract Infectious bronchitis virus (IBV) infections in poultry cause great economic losses to the poultry industry worldwide. The emergence of viral variants complicates disease control. The IBV strains in Taiwan were clustered into two groups, Taiwan group I and Taiwan group II, based on the S1 gene. A variant was previously identified and showed a distinct S1 gene homology with other local strains. This study investigated the 3′ 7.3kb genome of eight Taiwan strains isolated from 1992 to 2007. The genes of interest were directly sequenced. Sequence analyses were performed to detect any recombination event among IBVs. The results demonstrated that all of the examined viruses maintained the typical IBV genome organization as 5′-S-3a-3b-E-M-5a-5b-N-UTR-3′. In the phylogenetic analyses, various genes from one strain were clustered into separate groups. Moreover, frequent recombination events were identified in the Simplot analyses among the Taiwan and China CK/CH/LDL/97I-type strains. Putative crossover sites were located in the S1, S2, 3b, M genes and the intergenic region between the M and 5a genes. All of the recombinants showed chimeric IBV genome arrangements originated from Taiwan and China-like parental strains. Field IBVs in Taiwan undergo genetic recombination and evolution.

Protective Role of Cross-Reactive CD8 T Cells Against Dengue Virus Infection.

Infection with one of the four dengue virus serotypes (DENV1-4) presumably leads to lifelong immunity against the infecting serotype but not against heterotypic reinfection, resulting in a greater risk of developing Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS) during secondary infection. Both antibodies and T cell responses have been implicated in DHF/DSS pathogenesis. According to the T cell-based hypothesis termed “original antigenic sin,” secondary DENV infection is dominated by non-protective, cross-reactive T cells that elicit an aberrant immune response. The goal of our study was to compare the roles of serotype-specific and cross-reactive T cells in protection vs. pathogenesis during DENV infection in vivo. Specifically, we utilized IFN-α/βR−/− HLA*B0702 transgenic mice in the context of peptide vaccination with relevant human CD8 T cell epitopes. IFN-α/βR−/− HLA*B0702 transgenic mice were immunized with DENV serotype 2 (DENV2)-specific epitopes or variants found in any of the other three serotypes (DENV1, DENV3 or DENV4), followed by challenge with DENV. Although cross-reactive T cell responses were lower than responses elicited by serotype-specific T cells, immunization with either serotype-specific or variant peptide epitopes enhanced viral clearance, demonstrating that both serotype-specific and cross-reactive T cells can contribute to protection in vivo against DENV infection.

Inhibitory and combinatorial effect of diphyllin, a v-ATPase blocker, on influenza viruses

Abstract An influenza pandemic poses a serious threat to humans and animals. Conventional treatments against influenza include two classes of pathogen-targeting antivirals: M2 ion channel blockers (such as amantadine) and neuraminidase inhibitors (such as oseltamivir). Examination of the mechanism of influenza viral infection has shown that endosomal acidification plays a major role in facilitating the fusion between viral and endosomal membranes. This pathway has led to investigations on vacuolar ATPase (v-ATPase) activity, whose role as a regulating factor on influenza virus replication has been verified in extensive genome-wide screenings. Blocking v-ATPase activity thus presents the opportunity to interfere with influenza viral infection by preventing the pH-dependent membrane fusion between endosomes and virions. This study aims to apply diphyllin, a natural compound shown to be as a novel v-ATPase inhibitor, as a potential antiviral for various influenza virus strains using cell-based assays. The results show that diphyllin alters cellular susceptibility to influenza viruses through the inhibition of endosomal acidification, thus interfering with downstream virus replication, including that of known drug-resistant strains. In addition, combinatorial treatment of the host-targeting diphyllin with pathogen-targeting therapeutics (oseltamivir and amantadine) demonstrates enhanced antiviral effects and cell protection in vitro.

Identification of intertypic recombinant infectious bronchitis viruses from slaughtered chickens

ABSTRACT Avian coronavirus infectious bronchitis virus (IBV) poses a major threat to the global poultry industry. New IBV geno- and serotypes are continually reported. However, information on IBV prevalence is not frequently addressed in these reports. This study reports on a viral surveillance program in Taiwan from 2005 to 2006 with sampling conducted in poultry slaughterhouses. The genetic features of the obtained field isolates were investigated using sequence analysis and SimPlot analysis. A 1-directional neutralization test was performed to examine the antigenic variations among the collected viruses. The selection pressures that may contribute to the evolution of Taiwan IBV during recent decades were assessed. The surveillance program revealed that 8 out of 47 flocks (17%) were IBV-infected, from which 13 IBV isolates were recovered. Based on the phylogenetic analysis of the S1 gene, 11 of 13 isolates (84.6%) clustered with Taiwan group I. One IBV isolate showed evidence of frequent recombination events with China-like IBV in the spike glycoprotein (S) gene. Another isolate demonstrated the incorporation of China-like and H120-like genome fragments within the S2 gene and the membrane protein (M) gene region, respectively. Some antigenic changes were found in the 1-directional neutralization test. However, no positive selection pressures were related to those variations in the S1 genes among Taiwan IBV. Based on our work, we suggest that sampling chickens in poultry slaughterhouses is an effective and valuable means of compiling viral prevalence data, particularly in situations where there is subclinical infection. Infectious bronchitis viruses from slaughtered chickens revealed intertypic genetic recombination and antigenic diversity.

A Multiplex Reverse Transcriptase–PCR Assay for the Genotyping of Avian Infectious Bronchitis Viruses

Abstract Infectious bronchitis viruses (IBVs) in Taiwan have been divided into two genogroups, Taiwan group I (TW-I) and Taiwan group II (TW-II). Heterologous Mass-type strains are widely used as vaccines in the field. This work reports on a rapid and reliable multiplex reverse transcriptase–polymerase chain reaction (mRT-PCR) assay for the genotyping of IBVs. Multiplex primer sets were designed to amplify the region covering hypervariable regions 1 and 2 of the S1 glycoprotein gene. Several local strains and commercially available vaccines were used for evaluating the viral genotyping assay, and a number of field isolates were examined for clinical application. The results showed that all of the examined IBVs were accurately genotyped by identifying the corresponding bands on agarose gels (TW-I: 322 bp, TW-II: 161 bp, Mass type: 256 bp) after the mRT-PCR, in agreement with the viral genome sequence data. The mRT-PCR assay was able to detect viral RNA copies as low as 103, 105, and 103 for the TW-I, TW-II, and Mass-type strains, respectively. The mRT-PCR assay accurately detected and discriminated vaccine viruses from wild-type strains in the field. This assay may be beneficial for virus identification and differentiation in routine disease surveillance.

A type-specific blocking ELISA for the detection of infectious bronchitis virus antibody.

Abstract Infectious bronchitis virus (IBV) infection has been a major threat to the poultry industry worldwide. Current commercially available ELISA kits detect group-specific antibodies; however, to understand the status of field infection, a monoclonal antibody (mAb) blocking ELISA (b-ELISA) against local IBVs was developed. The selected mAb showed specificity to Taiwan IBV strains but had no cross-reactivity with the vaccine strain H120. Using the hemagglutination inhibition (HI) test as a standard, the cut-off value, sensitivity, and specificity of a b-ELISA using this mAb were evaluated in 390 field samples. The type-specificity of detection was validated using a panel of chicken hyperimmune sera. The results showed that the b-ELISA demonstrated high sensitivity (98.0%) and specificity (97.2%) of detection. The agreement between the results of the b-ELISA and the HI test was statistically significant (Kappa=0.95), and there was no significant difference between these two methods (McNemar p =0.72). The b-ELISA specifically detected Taiwan IBV serotypes but not three non-Taiwan IBV serotypes nor sera against other avian pathogens. This b-ELISA provides type-specific antibody detection of local IBV strains. It has the potential to serve as a rapid and reliable diagnostic method for IBV clinical infections in the field in Taiwan.

Nasal commensal Staphylococcus epidermidis counteracts influenza virus

Several microbes, including Staphylococcus epidermidis (S. epidermidis), a Gram-positive bacterium, live inside the human nasal cavity as commensals. The role of these nasal commensals in host innate immunity is largely unknown, although bacterial interference in the nasal microbiome may promote ecological competition between commensal bacteria and pathogenic species. We demonstrate here that S. epidermidis culture supernatants significantly suppressed the infectivity of various influenza viruses. Using high-performance liquid chromatography together with mass spectrometry, we identified a giant extracellular matrix-binding protein (Embp) as the major component involved in the anti-influenza effect of S. epidermidis. This anti-influenza activity was abrogated when Embp was mutated, confirming that Embp is essential for S. epidermidis activity against viral infection. We also showed that both S. epidermidis bacterial particles and Embp can directly bind to influenza virus. Furthermore, the injection of a recombinant Embp fragment containing a fibronectin-binding domain into embryonated eggs increased the survival rate of virus-infected chicken embryos. For an in vivo challenge study, prior Embp intranasal inoculation in chickens suppressed the viral titres and induced the expression of antiviral cytokines in the nasal tissues. These results suggest that S. epidermidis in the nasal cavity may serve as a defence mechanism against influenza virus infection.

Detection of Anti-Reticuloendotheliosis Virus Antibody by Blocking Enzyme-Linked Immunosorbent Assay with Expression Envelope Protein

SUMMARY. The current reticuloendotheliosis virus (REV) antibody detection kit that uses enzyme-linked immunosorbent assay (ELISA) needs concentrated virus, which is difficult to obtain due to its poor propagation in cells. In addition, this kit detects only chicken antibody but not other species. To overcome these disadvantages, we cloned and expressed REV env gene to develop monoclonal antibodies (mAbs), which we used for antibody detection in ELISA. Three mAbs were prepared from mice. These three mAbs could recognize REVs from ducks and geese by immunodot assay. In addition, the epitopes that the three mAbs recognized were determined by using three different env protein fragments by western blotting. One mAb was used to develop a blocking ELISA (bELISA) coated with expressed env protein to detect anti-REV antibody in chicken serum. This assay had a 98.8% (79/80) agreement with a commercial ELISA kit. Another 146 chicken sera with known neutralization antibodies were used as positive controls to evaluate this bELISA. The sensitivity and specificity this bELISA were 88.9% (40/45) and 94.8% (91/96), respectively. Thus, this bELISA could be used for anti-REV antibody detection in birds. RESUMEN. Detección de anticuerpos contra el virus de la reticuloendoteliosis mediante el ensayo de inmunoabsorción con enzimas ligadas en formato de bloqueo con la expresión de la proteína de de la envoltura. El estuche actual para la detección de anticuerpos contra la reticuloendoteliosis (REV) que utiliza un ensayo de inmunoabsorción con enzimas ligadas (ELISA) necesita virus concentrado, que es difícil de obtener debido a su baja propagación en células. Además, este estuche detecta sólo los anticuerpos de pollo, y no de otras especies. Para superar estas desventajas, se clonó y se expresó el gene env del virus de la reticuloendoteliosis para desarrollar anticuerpos monoclonales, que se utilizaron para la detección de anticuerpos por ELISA. Se prepararon tres anticuerpos monoclonales a partir de ratones. Estos tres anticuerpos pudieron reconocer virus de reticuloendoteliosis de patos y gansos por un ensayo inmunodot . Además, se determinaron los epítopos que los tres anticuerpos monoclonares reconocieron mediante el uso de tres diferentes fragmentos de la proteína env por inmunotransferencia Western. Un anticuerpo monoclonal fue usado para desarrollar un ELISA de bloqueo (bELISA) recubierto con una proteína env expresada y se utilizó para detectar anticuerpos anticuerpos contra el virus de la reticuloendoteliosis en el suero de pollos. Este ensayo mostró una concordancia del 98.8% (79/80) con un estuche comercial de ELISA. Otros 146 sueros de pollos reconocidos por contener anticuerpos neutralizantes fueron utilizados como controles positivos para evaluar este método de ELISA de bloqueo. La sensibilidad y la especificidad del método de ELISA de bloqueo fueron del 88.9% (40/45) y del 94.8% (91/96), respectivamente. Por lo tanto, este método de ELISA de bloqueo podría ser utilizado para la detección de anticuerpos contra el virus de la reticuloendoteliosis en aves.