An infectious clone of enterovirus 71(EV71) that is capable of infecting neonatal immune competent mice without adaptive mutations.

Enterovirus 71 (EV71) is a major pathogen that causes hand, foot and mouth disease (HFMD), which is a life threatening disease in certain children. The pathogenesis of EV71-caused HFMD is poorly defined due to the lack of simple and robust animal models with severe phenotypes that recapitulate symptoms observed in humans. Here, we generated the infectious clone of a clinical isolate from a severe HFMD patient. Virus rescued from the cDNA clone was infectious in cell lines. When administrated intraperitoneally to neonatal ICR, BALB/c and C57 immune competent mice at a dosage of1.4 × 104 pfu per mouse, the virus caused weight loss, paralysis and death in the infected mice after 4-5 days of infection. In the infected mice, detectable viral replication was detected in various tissues such as heart, liver, brain, lung, kidney, small intestine, leg skeletal muscle and medulla oblongata. The histology of the infected mice included massive myolysis, glomerular atrophy, villous blunting in small intestine, widened alveolar septum, diminished alveolar spaces and lymphocytes infiltration into the lung. By using the UV-inactivated virus as a control, we elucidated that the virus first amplified in the leg skeletal muscle tissue and the muscle tissue served as a primary viral replication site. In summary, we generated a stable EV71 infectious clone that is capable of infecting neonatal immune competent mice without adaptive mutations and provide a simple, valuable animal model for the studies of EV71pathogenesis and therapy.

The Establishment of Infectious Clone and Single Round Infectious Particles for Coxsackievirus A10.

Coxsackievirus A10 (CVA10) is one of the major etiological agents of hand, foot, and mouth disease. There are no vaccine and antiviral drugs for controlling CVA10 infection. Reverse genetic tools for CVA10 will benefit its mechanistic study and development of vaccines and antivirals. Here, two infectious clones for the prototype and a Myc-tagged CVA10 were constructed. Viable CVA10 viruses were harvested by transfecting the viral mRNA into human rhabdomyosarcoma (RD) cells. Rescued CVA10 was further confirmed by next generation sequencing and characterized experimentally. We also constructed the vectors for CVA10 subgenomic replicon with luciferase reporter and viral capsid with EGFP reporter, respectively. Co-transfection of the viral replicon RNA and capsid expresser in human embryonic kidney 293T (HEK293T) cells led to the production of single round infectious particles (SRIPs). Based on CVA10 replicon RNA, SRIPs with either the enterovirus A71 (EVA71) capsid or the CVA10 capsid were generated. Infection by EVA71 SRIPs required SCARB2, while CVA10 SRIPs did not. Finally, we showed great improvement of the replicon activity and SRIPs production by insertion of a cis-active hammerhead ribozyme (HHRib) before the 5'-untranslated region (UTR). In summary, reverse genetic tools for prototype strain of CVA10, including both the infectious clone and the SRIPs system, were successfully established. These tools will facilitate the basic and translational study of CVA10.

Genome-wide linear B-cell epitopes of enterovirus 71 in a hand, foot and mouth disease (HFMD) population.

BACKGROUND: Enteroviruses cause hand, foot and mouth disease (HFMD). The host B-cells recognize the viral proteins and provoke humoral responses. Deciphering the B-cell responses to the viral epitopes helps diagnosis and vaccine development. OBJECTIVES: The objective of the present study was to investigate for the first time the landscape of genome-wide linear B-cell epitopes of enterovirus 71 in HFMD population. STUDY DESIGN: The peptides encompassing the entire coding region of EV71 were chemically synthesized and displayed on a microarray. The peptide microarray was used to screen serum samples from an HFMD population, including EV71-, CAV10-, CAV16- and CAV6-infected patients. We identified the dominant epitope-containing-peptides (DECPs) that react with the sera of more than 20% of the HFMD population and the common DECPs that cross-react with the sera from other enteroviruses-infected population. RESULTS: Ten DECPs reacting with IgM and 9 DECPs reacting with IgG antibodies were identified, of which, 6 IgM and 5 IgG common DECPs cross-reacted with the sera from other enteroviruses. Some DECPs preferentially reacted with IgG or IgM antibodies and some epitope-antibody interactions correlated with the severity of HFMD. CONCLUSIONS: We uncovered the DECPs and the common DECPs among a group of enteroviruses in HFMD population and found that some epitope-antibody reactions were associated with the outcome of HFMD. These data may guide developing vaccines against the enteroviruses and help the diagnosis and prognosis of HFMD.