Nannan Song

A Novel Enterovirus 71 (EV71) Virulence Determinant: The 69th Residue of 3C Protease Modulates Pathogenicity

Human enterovirus type 71 (EV71), the major causative agent of hand-foot-and-mouth disease, has been known to cause fatal neurological complications. Unfortunately, the reason for neurological complications that have been seen in fatal cases of the disease and the relationship between EV71 virulence and viral genetic sequences remains largely undefined. The 3C protease (3Cpro) of EV71 plays an irreplaceable role in segmenting the precursor polyprotein during viral replication, and intervening with host life activity during viral infection. In this study, for the first time, the 69th residue of 3C protease has been identified as a novel virulence determinant of EV71. The recombinant virus with single point variation, in the 69th of 3Cpro, exhibited obvious decline in replication, and virulence. We further determined the crystal structure of 3C N69D at 1.39 Ǻ resolution and found that conformation of 3C N69D demonstrated significant changes compared with a normal 3C protein, in the substrate-binding site and catalytic active site. Strikingly, one of the switch loops, essential in fixing substrates, adopts an open conformation in the 3C N69D-rupintrivir complex. Consistent with this apparent structural disruption, the catalytic activity of 3C N69D decreased sharply for host derived and viral derived substrates, detected for both in vitro and in vivo. Interestingly, in addition to EV71, Asp69 was also found in 3C proteases of other virus strains, such as CAV16, and was conserved in nearly all C type human rhinovirus. Overall, we identified a natural virulence determinant of 3C protease and revealed the mechanism of attenuated virulence is mediated by N69D substitution. Our data provides new insight into the enzymatic mechanism of a subdued 3C protease and suggests a theoretical basis for virulence determinantion of picornaviridae.

MA104 Cell line presents characteristics suitable for enterovirus A71 isolation and proliferation.

Enterovirus A71 (EV‐A71), one of the most important causative agents of hand, foot and mouth disease (HFMD) in children, can lead to severe clinical outcomes, even death. However, the infection spectrum of EV‐A71 in different cell lines remains unknown. Therefore, in this study, the biological characteristics of EV‐A71 Subgroup C4 in different cell lines were investigated. To this end, the infectivity of EV‐A71Jinan1002 isolated from children with severe HFMD was assessed in 18 different host cell lines. It was found that the MA104 cell line displayed biological characteristics suitable for EV‐A71 Subgroup C4 strain isolation and proliferation; indeed, it was found that a broad spectrum of cell lines can be infected by EV‐A71Jinan1002. Among the screened cells, four cell lines (HEK293, RD, MA104 and Marc145) produced high 50% tissue culture infective dose (TCID50) values calculated in viral proliferations (ranged from 107.6 to 107.8); the TCID50 being negatively associated with the time to appearance of CPE. Proliferation curves demonstrated that EV‐A71Jinan1002 amplifies more efficiently in MA104, Hep‐2 and RD cells. Remarkably, the virus isolation rate was much higher in MA104 cells than in RD cells. Thus this study, to our knowledge, is for the first to explore the infection spectrum of EV‐A71 subgroup C4 in such a large number of different cell lines. Our data provide useful reference data for facilitating further study of EV‐A71.

Genomic and immunologic factors associated with viral pathogenesis in a lethal EV71 infected neonatal mouse model

Hand, foot and mouth disease (HFMD) caused by enterovirus 71 (EV71) has emerged as a major health problem in China and worldwide. The present study aimed to understand the virological features of EV71 and host responses resulting from EV71 infection. Six different EV71 strains were isolated from HFMD patients with severe or mild clinical symptoms, and were analyzed for pathogenicity in vitro and in vivo. The results demonstrated that the six virus strains exhibited similar cytopathogenic effects on susceptible MA104 cells. However, marked differences in histological and immunopathological changes were observed when mice were inoculated with the different virus strains. Thus, the viruses studied were divided into two groups, highly or weakly pathogenic. Two representative virus strains, JN200804 and JN200803 (highly and weakly pathogenic, respectively) were studied further to investigate pathogenicity-associated factors, including genetic mutations and immunopathogenesis. The present study has demonstrated that highly pathogenic strains have stable genome and amino acid sequences. Notably, the present study demonstrated that a highly pathogenic strain induced a significant increase of the bulk CD4 T cell levels at 3 days post-inoculation. In conclusion, the current study demonstrates that genomic and immunologic factors may be responsible for the multiple tissue damage caused by highly pathogenic EV71 infection.

NK cells are negatively regulated by sCD83 in experimental autoimmune uveitis

Natural killer (NK) cells represent a subset of lymphocytes that contribute to innate immunity and have been reported to play a role in autoimmune uveitis. However, the mechanisms regulating NK cellular function in this condition remain unclear. Herein, we investigated the status of NK cells in experimental autoimmune uveitis (EAU). We found that the number of CD83+CD3−NK1.1+ cells was increased in the inflamed eyes and spleens of the EAU mouse model. At the recovery stage of EAU, serum concentrations of soluble CD83 (sCD83) were increased. sCD83 treatment relieved retinal tissue damage and decreased the number of infiltrating NK cells in inflamed eyes. Further analysis of the effects of sCD83 treatment in EAU revealed that it reduced: 1) the expressions of CD11b and CD83 in NK cells, 2) the percent of CD11bhighCD27lowCD3−NK1.1+ cells and 3) the secretion of granzyme B, perforin and IFN-γ in NK cells as demonstrated both in vivo and in vitro. When sCD83 treated-NK cells were transferred into EAU mice, retinal tissue damage was relieved. These results demonstrate sCD83 down-regulate NK cellular function and thus provide important, new information regarding the means for the beneficial effects of this agent in the treatment of autoimmune uveitis.

Soluble CD83 Alleviates Experimental Autoimmune Uveitis by Inhibiting Filamentous Actin-Dependent Calcium Release in Dendritic Cells

Soluble CD83 (sCD83) is the extracellular domain of the membrane-bound CD83 molecule, and known for its immunoregulatory functions. Whether and how sCD83 participates in the pathogenesis of uveitis, a serious inflammatory disease of the eye that can cause visual disability and blindness, is unknown. By flow cytometry and imaging studies, we show that sCD83 alleviates experimental autoimmune uveitis (EAU) through a novel mechanism. During onset and recovery of EAU, the level of sCD83 rises in the serum and aqueous humor, and CD83+ leukocytes infiltrate the inflamed eye. Systemic or topical application of sCD83 exerts a protective effect by decreasing inflammatory cytokine expression, reducing ocular and splenic leukocyte including CD4+ T cells and dendritic cells (DCs). Mechanistically, sCD83 induces tolerogenic DCs by decreasing the synaptic expression of co-stimulatory molecules and hampering the calcium response in DCs. These changes are caused by a disruption of the cytoskeletal rearrangements at the DC–T cell contact zone, leading to altered localization of calcium microdomains and suppressed T-cell activation. Thus, the ability of sCD83 to modulate DC-mediated inflammation in the eye could be harnessed to develop new immunosuppressive therapeutics for autoimmune uveitis.

Salmonella STM1697 coordinates flagella biogenesis and virulence by restricting flagellar master protein FlhD4C2 from recruiting RNA polymerase

Abstract Salmonella reduces flagella biogenesis to avoid detection within host cells by a largely unknown mechanism. We identified an EAL-like protein STM1697 as required and sufficient for this process. STM1697 surges to a high level after Salmonella enters host cells and restrains the expression of flagellar genes by regulating the function of flagellar switch protein FlhD4C2, the transcription activator of all other flagellar genes. Unlike other anti-FlhD4C2 factors, STM1697 does not prevent FlhD4C2 from binding to target DNA. A 2.0 Å resolution STM1697–FlhD structure reveals that STM1697 binds the same region of FlhD as STM1344, but with weaker affinity. Further experiments show that STM1697 regulates flagella biogenesis by restricting FlhD4C2 from recruiting RNA polymerase and the regulatory effect of STM1697 on flagellar biogenesis and virulence are all achieved by interaction with FlhD. Finally, we describe a novel mechanism mediated by STM1697 in which Salmonella can inhibit the production of flagella antigen and escape from the host immune system.

Antiviral activity of a polysaccharide from Laminaria japonica against enterovirus 71

This in vitro study investigated the antiviral activity of an acidic polysaccharide from Laminaria japonica against enterovirus 71 (EV71) as well as its mechanism of action. The LJ04 polysaccharide was purified from Laminaria japonica by affinity chromatography. To investigate its antiviral activity, an MTT assay, q-PCR, immunofluorescent staining and western-blot analysis were performed. To define its mechanism of action, ELISA, q-PCR and flow cytometry were conducted. LJ04 had a low EC50, high CC50 and high SI. LJ04 inhibited not only JN200804, but also JN200803 in RD cells, and viral proliferation was strongly inhibited, whereas LJ04 suppressed viral-induced apoptosis as detected by flow cytometry. In conclusion, LJ04 was found to have robust antiviral activity by inhibiting apoptosis and inducing IFN-β expression. Our findings indicate that LJ04 is a good candidate for the treatment of EV71.