Wangheng Hou

Construction and characterization of an infectious clone of coxsackievirus A6 that showed high virulence in neonatal mice

Atypical hand, foot, and mouth disease (aHFMD) outbreaks have been frequently reported worldwide in recent years. It is believed that coxsackievirus A6 (CA6) is the major pathogen for aHFMD. Studies regarding CA6 infection are limited and the genetic mechanism for the high pathogenicity of some new CA6 variants is still unclear. Infectious clones are powerful tools for studying the genetic mechanisms of RNA viruses. In this study, we describe the construction of a full-length cDNA clone of CA6 strain TW-2007-00141. The whole genome of CA6 was amplified in a single step and ligated into a plasmid vector through an efficient cloning method, Gibson assembly. The whole genome sequence of CA6 strain TW-2007-00141 was determined and phylogenetic analysis indicated that it shared a high degree of similarity (≥94%) with the CA6 strains found in Taiwan in 2009. The infectious clone of CA6 viruses were recovered by transfection into 293FT cells and showed similar biological properties to the parental virus. Viral particles were purified by CsCl isopycnic centrifugation, and two types of viral particles were observed under transmission electron microscopy. The rescued virus showed high virulence in one-day-old suckling mice. This clone may be useful for establishing animal models for the evaluation of CA6 vaccine efficiency in future.

Atomic structures of Coxsackievirus A6 and its complex with a neutralizing antibody

Coxsackievirus A6 (CVA6) has recently emerged as a major cause of hand, foot and mouth disease in children worldwide but no vaccine is available against CVA6 infections. Here, we demonstrate the isolation of two forms of stable CVA6 particles-procapsid and A-particle-with excellent biochemical stability and natural antigenicity to serve as vaccine candidates. Despite the presence (in A-particle) or absence (in procapsid) of capsid-RNA interactions, the two CVA6 particles have essentially identical atomic capsid structures resembling the uncoating intermediates of other enteroviruses. Our near-atomic resolution structure of CVA6 A-particle complexed with a neutralizing antibody maps an immune-dominant neutralizing epitope to the surface loops of VP1. The structure-guided cell-based inhibition studies further demonstrate that these loops could serve as excellent targets for designing anti-CVA6 vaccines.Coxsackievirus A6 (CVA6) causes hand, foot and mouth disease in children. Here the authors present the CVA6 procapsid and A-particle cryo-EM structures and identify an immune-dominant neutralizing epitope, which can be exploited for vaccine development.

The enhancement of RNAi against HIV in vitro and in vivo using H-2K(k) protein as a sorting method

Gene therapy offers a potentially an effective treatment for many human diseases, including HIV/AIDS. One of the most studied gene delivery systems is the use of lentivirus based vectors, which can deliver genes into both dividing and nondividing cells. However, low infection efficiency represents an obstacle for proper evaluation of their biological function. In this study, a recombinant lentiviral vector which expressed short hairpin RNAs (shRNAs) targeted against the HIV-1 vif/pol was transduced into various cells. An MHC class I molecule, H-2K(k), was used as a marker to accumulate the virally transduced cells through immunomagnetic sorting. In vitro testing of transduced cells showed 85% suppression of HIV in post-sorted PBMCs compared to 30% in pre-sorted PBMCs. In additional, using a mouse xenotransplantation model with the same treatment protocol for cell enrichment, a >95% decrease in HIV activity in post-sorted cells was achieved, as compared to nearly none in the pre-sorted cells. These studies offer a practical method to accumulate virally transduced cells, which can be applied to evaluate the performance of various shRNAs constructs.

Development of a coxsackievirus A16 neutralization test based on the enzyme-linked immunospot assay

Coxsackievirus A16 (CA16) is one of the major pathogens responsible for hand, foot and mouth disease (HFMD). The assessment of the humoral immunity response is indispensable in the development of vaccines against enteroviruses. The neutralization test based on the inhibition of cytopathic effects (Nt-CPE) is a common method for measuring neutralizing antibodies against CA16. However, an efficient neutralization test needs to be developed for seroepidemiological surveys and clinical trials of CA16 vaccines because Nt-CPE is time-consuming and labor-intensive. In this study, a high-throughput neutralization test for CA16 based on the enzyme-linked immunospot assay (Nt-ELISPOT) was developed. The monoclonal antibody 7D10, which reacted with the viral protein VP1, was used to detect the cells infected with CA16. The neutralizing titers of sera were proven to be unchanged over an infectious dose range from 10 to 10,000TCID50 per well. The Nt-ELISPOT results correlated well with the Nt-CPE results (R(2) = 0.9250), and the detection period was shortened from five days to approximately 30h. Overall, the Nt-ELISPOT is a reliable and efficient method for measuring neutralizing antibodies against CA16.

A neonatal mouse model of coxsackievirus A10 infection for anti-viral evaluation

Abstract Epidemiological data indicate that coxsackievirus A10 (CVA10) has become one of the main causative agents of hand, foot and mouth disease (HFMD) and in recent years has often been found to co‐circulate with other enteroviruses, which poses a challenge for the prevention and control of HFMD. Although most CVA10‐associated HFMD cases present mild symptoms, severe manifestations and even death can also occur. However, the study of the pathogenesis and the development of drugs and vaccines for CVA10 infection are still far from complete. In this study, we established a neonatal mouse model for anti‐viral evaluation and characterized the pathology of CVA10 infection. To develop the mouse model, both inbred and outbred mouse strains were used to compare their sensitivity to CVA10 infection; then, one‐day‐old BALB/c mice were selected and inoculated intraperitoneally with a CVA10 clinical strain, CVA10‐FJ‐01. Clinical symptoms, such as wasting, hind‐limb paralysis and even death were observed in the CVA10‐infected mice. Moreover, pathological examination and immunohistochemistry staining showed that severe myonecrosis with inflammatory infiltration was observed in CVA10‐infected mice, indicating that CVA10 exhibited strong tropism to muscle tissue. Using real‐time PCR, we also found that the viral load in the blood and muscle was higher than that in other organs/tissues at different time points post‐infection, suggesting that CVA10 had a strong tropism to mice muscle and that viremic spread may also contribute to the death of the CVA10‐infected mice. Additionally, to evaluate the neonatal mouse model of CVA10 infection, female mice were immunized with formalin‐inactivated CVA10 and then allowed to mate after the third immunization. The results showed that maternal antibodies could protect mice against CVA10 infection. In summary, the results demonstrated that the neonatal mice model was a useful tool for evaluating the protective effects of CVA10 vaccines and anti‐viral reagents. HighlightsA neonatal mouse model of coxsackievirus A10 (CVA10) infection was successfully developed.This was first study to characterize the pathology of CVA10 infection, finding that CVA10 had a strong tropism to muscles.The infection model was successfully applied to evaluate a formalin‐inactivated CVA10 vaccine.

Structure of a Novel Shoulder-to-Shoulder p24 Dimer in Complex with the Broad-Spectrum Antibody A10F9 and Its Implication in Capsid Assembly

Mature HIV-1 viral particles assemble as a fullerene configuration comprising p24 capsid hexamers, pentamers and dimers. In this paper, we report the X-ray crystal structures of the p24 protein from natural HIV-1 strain (BMJ4) in complex with Fab A10F9, which recognizes a conserved epitope in the C-terminal domain of the BMJ4 p24 protein. Our structures reveal a novel shoulder-to-shoulder p24 dimerization mode that is mediated by an S-S bridge at C177. Consistent with these structures, the shoulder-to-shoulder dimer that was obtained from the BMJ4 strain was also observed in p24 proteins from other strains by the introduction of a cysteine residue at position 177. The potential biological significance was further validated by the introduction of a C177A mutation in the BMJ4 strain, which then displays a low infectivity. Our data suggest that this novel shoulder-to-shoulder dimer interface trapped by this unique S-S bridge could represent a physiologically relevant mode of HIV-1 capsid assembly during virus maturation, although Cys residue itself may not be critical for HIV-I replication.

A Chimeric Humanized Mouse Model by Engrafting the Human Induced Pluripotent Stem Cell-Derived Hepatocyte-Like Cell for the Chronic Hepatitis B Virus Infection

Humanized mouse model generated by grafting primary human hepatocytes (PHHs) to immunodeficient mouse has contributed invaluably to understanding the pathogenesis of hepatitis B virus (HBV). However, the source of PHHs is limited, which necessitates the search for alternatives. Recently, hepatocyte-like cells (HLCs) generated from human induced pluripotent stem cells (hiPSCs) have been used for in vitro HBV infection. Herein, we developed a robust human liver chimeric animal model to study in vivo HBV infection by engrafting the hiPSC-HLCs to Fah-/-Rag2-/-IL-2Rγc-/- SCID (FRGS) mice. After being optimized by a small molecule, XMU-MP-1, the hiPSC-HLCs engrafted FRGS (hHLC-FRGS) mice displayed approximately 40% liver chimerism at week 6 after engraftment and maintained at this level for at least 14 weeks. Viremia and HBV infection markers include antigens, RNA, DNA, and covalently closed circular DNA were detectable in HBV infected hHLC-FRGS mice. Furthermore, hiPSC-HLCs and hHLC-FRGS mice were successfully used to evaluate different antivirals. Therefore, we established a humanized mouse model for not only investigating HBV pathogenesis but also testing the effects of the anti-HBV drugs. Highlights: (1) The implanted hiPSC-HLCs established a long-term chimerism in FRGS mice liver. (2) hHLC-FRGS mice are adequate to support chronic HBV infection with a full viral life cycle. (3) hiPSC-HLCs and hHLC-FRGS mice are useful tools for evaluation of antivirals against HBV infection in vitro and in vivo. Research in Context To overcome the disadvantages of using primary human hepatocytes, we induced human pluripotent stem cells to hepatocyte-like cells (hiPSC-HLCs) that developed the capability to express important liver functional markers and critical host factors for HBV infection. The hiPSC-HLCs were permissive for the HBV infection and supported a full HBV replication. The hiPSC-HLCs were then engrafted to immunodeficient mouse to establish a chimeric liver mouse model, which was capable of supporting HBV infection in vivo and evaluating the effects of antiviral drugs. Our results shed light into improving the cellular and animal models for studying HBV and other hepatotropic viruses.

Tetracysteine as a Reporter for Gene Therapy

OBJECTIVE To study the feasibility of using tetracysteine (TC) reporter in gene therapy. METHODS Effects of TC reporter and conventional reporter genes encoding green fluorescence protein (GFP) and luciferase (Luc) on expression and function of the therapeutic gene MGMT(P140K) were compared. Cytotoxicity and drug resistance were studied by Western blot. TC reporter used in therapy was analyzed by flow cytometry (FCM). RESULTS The TC reporter had no toxicity to cells and neither affected the expression or activity of therapeutic gene as compared to GFP and Luc. TC could be used in blood sample detection. CONCLUSION TC is a new kind of reporter gene for lentiviral vector in future gene therapy.

Atomic structures of enterovirus D68 in complex with two monoclonal antibodies define distinct mechanisms of viral neutralization

Enterovirus D68 (EV-D68) undergoes structural transformation between mature, cell-entry intermediate (A-particle) and empty forms throughout its life cycle. Structural information for the various forms and antibody-bound capsids will facilitate the development of effective vaccines and therapeutics against EV-D68 infection, which causes childhood respiratory and paralytic diseases worldwide. Here, we report the structures of three EV-D68 capsid states representing the virus at major phases. We further describe two original monoclonal antibodies (15C5 and 11G1) with distinct structurally defined mechanisms for virus neutralization. 15C5 and 11G1 engage the capsid loci at icosahedral three-fold and five-fold axes, respectively. To block viral attachment, 15C5 binds three forms of capsids, and triggers mature virions to transform into A-particles, mimicking engagement by the functional receptor ICAM-5, whereas 11G1 exclusively recognizes the A-particle. Our data provide a structural and molecular explanation for the transition of picornavirus capsid conformations and demonstrate distinct mechanisms for antibody-mediated neutralization.Structures of three enterovirus D68 capsid states and two monoclonal antibodies provide a molecular explanation for the transition of picornavirus capsid conformations and reveal distinct mechanisms for viral neutralization.

Optimized HepaRG is a suitable cell source to generate the human liver chimeric mouse model for the chronic hepatitis B virus infection

The human liver chimeric mouse with primary human hepatocytes (PHHs) engraftment has been demonstrated to be a useful animal model to study hepatitis B virus (HBV) pathogenesis and evaluate anti-HBV drugs. However, the disadvantages of using PHHs include the inability for cellular expansion in vitro, limited donor availability, individual differences, and ethical issues, necessitating the development of alternatives. To obtain in vitro expandable hepatocytes, we optimized the hepatic differentiation procedure of the human liver progenitor cell line, HepaRG, using four functional small molecules (4SM) and enriched the precursor hepatocyte-like cells (HLCs). HepaRG cells of different hepatic differentiation states were engrafted to immunodeficient mice (FRGS) with weekly 4SM treatment. The HepaRG-engrafted mice were challenged with HBV and/or treated with several antivirals to evaluate their effects. We demonstrated that the 4SM treatment enhanced hepatic differentiation and promoted cell proliferation capacity both in vitro and in vivo. Mice engrafted with enriched HepaRG of prehepatic differentiation and treated with 4SM displayed approximately 10% liver chimerism at week 8 after engraftment and were maintained at this level for another 16 weeks. Therefore, we developed a HepaRG-based human liver chimeric mouse model: HepaRG-FRGS. Our experimental results showed that the liver chimerism of the mice was adequate to support chronic HBV infection for 24 weeks and to evaluate antivirals. We also demonstrated that HBV infection in HepaRG cells was dependent on their hepatic differentiation state and liver chimerism in vivo. Overall, HepaRG-FRGS mice provide a novel human liver chimeric mouse model to study chronic HBV infection and evaluate anti-HBV drugs.