Qingwei Liu

Hand, foot, and mouth disease (HFMD) is an infectious disease that mainly affects infants and children, causing considerable morbidity and mortality worldwide. HFMD is commonly caused by enteroviru...

In recent years, enterovirus D68 (EVD68) has been reported increasingly to be associated with severe respiratory tract infections and acute flaccid myelitis (AFM) in children all over the world. Yet, no effective vaccines or antiviral drugs are currently available for EVD68. Although several experimental animal models have been developed, immunogenicity and protective efficacy of inactivated EVD68 vaccines has not been fully evaluated. To promote the development of vaccines, we established an Institute of Cancer Research (ICR) suckling mouse model of EVD68 infection in this study. The results showed that ICR neonatal mice up to about nine days of age were susceptible to infection with EVD68 clinical strain US/MO/14-18947 by intraperitoneal injection. The infected mice exhibited progressive limb paralysis prior to death and the mortality of mice was age- and virus dose-dependent. Tissue viral load analysis showed that limb muscle and spinal cord were the major sites of viral replication. Moreover, histopathologic examination revealed the severe necrosis of the limb and juxtaspinal muscles, suggesting that US/MO/14-18947 has a strong tropism toward muscle tissues. Additionally, β-propiolactone-inactivated EVD68 vaccine showed high purity and quality and induced robust EVD68-specific neutralizing antibody responses in adult mice. Importantly, results from both antisera transfer and maternal immunization experiments clearly showed that inactivated EVD68 vaccine was able to protect against lethal viral infection in the mouse model. In short, these results demonstrate the successful establishment of the mouse model of EVD68 infection for evaluating candidate vaccines against EVD68 and also provide important information for the development of inactivated virus-based EVD68 vaccines.

Noroviruses (NoVs) are the main pathogens responsible for sporadic and epidemic nonbacterial gastroenteritis, causing an estimated 219,000 deaths annually worldwide. There is no commercially available vaccine for NoVs, due partly to the difficulty in establishing NoV cell culture models. The histo-blood group antigen (HBGA) blocking assay is used extensively to assess the protective potential of candidate vaccine-elicited antibodies, but there is still no widely used cellular evaluation model. In this study, we have established a cell line-based NoV vaccine evaluation model through the construction of human α1,2-fucosyltransferase 2-overexpressing 293T (293T-FUT2) cell lines. The 293T-FUT2 cells stably expressed H type 2 and Lewis y antigens. Virus-like particles (VLPs) of the NoV prototype strain genogroup I.1 (GI.1) and the predominant strains GII.4 and GII.17 could attach to the cell line efficiently in a dose-dependent manner. Importantly, antisera against these NoV VLPs could inhibit the attachment of the VLPs, where the inhibitory effects measured by the attachment inhibition assay correlated significantly with the antibody levels determined by the HBGA blocking assay. Collectively, our attachment inhibition assay could serve as a surrogate neutralization assay for the evaluation of NoV vaccines at the cellular level.

Coxsackievirus A16 (CVA16) is one of the major pathogens responsible for hand, foot and mouth disease, which affects more than two million children in the Asian-Pacific region annually. Previous studies have shown that scavenger receptor B2 is a functional receptor for CVA16 that facilitates the uncoating process. However, it remains unclear whether other receptors are required for efficient CVA16 infection. In this study, by using a variety of assays we demonstrated that CVA16 utilizes surface heparan sulfate glycosaminoglycans as its attachment receptor. We further showed that five surface-exposed positively charged residues located in a cluster at the five-fold vertex of the virion are critical to heparan sulfate binding and cellular attachment of CVA16. Among the five residues, the arginine at position 166 (R166) of VP1 capsid protein appeared to be the most important for the interaction between CVA16 and heparan sulfate. Alanine substitution at this site (R166A) almost completely abolished heparan su...

Antibodies play a critical role in immunity against enterovirus 71 (EV71). However, how EV71-specific antibodies neutralize infections remains poorly understood. Here we report the working mechanism for a group of three monoclonal antibodies (MAbs) that potently neutralize EV71. We found that these three MAbs (termed D5, H7, and C4, respectively) recognize the same conserved neutralizing epitope within the VP1 GH loop of EV71. Single MAbs in this group, exemplified by D5, could inhibit EV71 infection in cell cultures at both the pre- and postattachment stages in a cell type-independent manner. Specifically, MAb treatment resulted in the blockade of multiple steps of EV71 entry, including virus attachment, internalization, and subsequent uncoating and RNA release. Furthermore, we show that the D5 and C4 antibodies can interfere with EV71 binding to its key receptors, including heparan sulfate, SCARB2, and PSGL-1, thus providing a possible explanation for the observed multi-inhibitory function of the MAbs. Collectively, our study unravels the mechanism of neutralization by a unique group of anti-EV71 MAbs targeting the conserved VP1 GH loop. The findings should enhance our understanding of MAb-mediated immunity against enterovirus infections and accelerate the development of MAb-based anti-EV71 therapeutic drugs. IMPORTANCE Enterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease (HFMD), which has caused significant morbidities and mortalities in young children. Neither a vaccine nor an antiviral drug is available. Neutralizing antibodies are major protective components in EV71 immunity. Here, we unraveled an unusual mechanism of EV71 neutralization by a group of three neutralizing monoclonal antibodies (MAbs). All of these MAbs bound the same conserved epitope located at the VP1 GH loop of EV71. Interestingly, mechanistic studies showed that single antibodies in this MAb group could block EV71 attachment and internalization during the viral entry process and interfere with EV71 binding to heparan sulfate, SCARB2, and PSGL-1 molecules, which are key receptors involved in different steps of EV71 entry. Our findings greatly enhance the understanding of the interplays among EV71, neutralizing antibodies, and host receptors, which in turn should facilitate the development of an MAb-based anti-EV71 therapy.

Enterovirus 71 (EV71) is the main causative agent of hand, foot and mouth disease that occurs in young children. Neither antiviral agents nor vaccines are available for efficiently combating viral infection. Study of EV71–host interplay is important for understanding viral infection and developing strategies for prevention and therapy. Here the interactions of EV71 with human dendritic cells were analyzed. EV71 capture, endocytosis, infection, and degradation in monocyte-derived dendritic cells (MDDCs) were detected by Flow cytometry or real-time (RT-) PCR, and MDDCs-mediated EV71 trans-infection of RD cells was determined via coculture system. Cell morphology or viability was monitored with microscopy or flow cytometry. SiRNA interference was used to knock down gene expression. MDDCs can bind EV71, but these loaded-EV71 particles in MDDCs underwent a rapid degradation in the absence of efficient replication; once the captured EV71 encountered susceptible cells, MDDCs efficiently transferred surface-bound viruses to target cells. The molecule of DC-SIGN (DC-specific intercellular adhesion molecule-3 grabbing nonintegrin) mediated viral binding and transfer, because interference of DC-SIGN expression with specific siRNAs reduced EV71 binding and impaired MDDC-mediated viral trans-infection, and exogenous expression of DC-SIGN molecule on Raji cell initiated viral binding and subsequent transmission. MDDCs could bind efficiently EV71 viruses through viral binding to DC-SIGN molecule, and these captured-viruses could be transferred to susceptible cells for robust infection. The novel finding of DC-mediated EV71 dissemination might facilitate elucidation of EV71 primary infection and benefit searching for new clues for preventing viruses from initial infection.

To obtain recombinant VP0 protein of enterovirus 71, and generate the corresponding VP0-specific polyclonal antibodies, for molecular detection and characterization of EV71. The VP0 gene was amplified by PCR and cloned into vector pET26b to make pET-VP0 for the prokaryotic expression of VP0. The recombinant VP0 protein was expressed in E.coli BL21 harboring pET-VP0, purified from inclusion bodies, renatured, and subsequently used to immunize guinea pigs. The resultant antisera were evaluated for anti-VP0 titer, binding capacity and specificity by ELISA, immunofluorescence staining and Western blot assays. Recombinant protein VP0 was efficiently produced in E.coli. Immunization of guinea pigs with recombinant VP0 elicited high-titer (1:10(6)) VP0-specific antibodies. Western blot analysis showed the resultant anti-VP0 sera reacted with E.coli-expressed VP0 as well as EV71 propagated in Vero cells. Moreover, the antisera positively recognized EV71 infected cells by immunofluorescence staining. The recombinant VP0 and the corresponding polyclonal antibodies can be used to identify and characterize EV71, and therefore represent useful agents and tools for the development of new diagnostic methods and vaccines for EV71.