Nonstructural protein 2A modulates replication and virulence of enterovirus 71.

Enterovirus 71 (EV71) can cause hand, foot, and mouth disease in children, and severe infections can induce neurological complications and even death. However, the pathogenesis of EV71 remains unknown. The 2A proteinase (2Apro) of EV71 plays an important role in segmenting the precursor polyprotein during viral replication, inhibiting host protein synthesis, and evading innate immunity. This study was to determine the function of EV71 2Apro in replication and virulence. A chimeric strain (SDLY 107-2A-1) was recombined by replacing 2Apro of a severe strain (SDLY107) with that of a mild strain (SDLY1) based on an infectious cDNA clone. The replication kinetics of the chimeric strain in vitro and in vivo were determined by qRT-PCR, which showed that the chimeric strain replicated slower and generated less viral RNA than the severe strain. The pathological change and viral load of chimeric strain infected mice were intermediate between severe strain infected mice and mild strain infected mice. Cellular cytotoxicity assays revealed that 2Apro was associated with the neurotoxicity of EV71. Histopathological and immunohistochemical assays detected tissue pathological damage in the lungs, muscles, brain, and intestinal tissues. Together, these results suggest that 2Apro modulates replication and virulence of EV71. This provides a theoretical basis for virulence determination of EV71.

Contribution of 3CD Region to the Virulence of Enterovirus 71.

Enterovirus 71 is a neuroinvasive virus that is associated with severe neurological complications. We had earlier suggested that the replication capacity of a severe strain was higher than that of a mild strain. The recombinant 3CRV and 3CDRV virus strains were successfully rescued in our previous study. In the present study, we found no difference in virulence between 3CRV and severe strains. However, the capacity of replication and to cause cell injury of 3CDRV strain decreased in vitro, especially at 39.5 °C. Replacement of 3CD region in the severe strain led to milder symptoms, less body weight loss, and lower viral load in ICR mice. Histopathological findings indicated less severe injury in mice infected with 3CDRV strain. This study suggests that the 3CD region contributes to the attenuation of the severe strain, including its replication capacity and temperature sensitivity.

Association of viral replication capacity with the pathogenicity of enterovirus 71.

Enterovirus 71 (EV71) is a major cause of hand-foot-and-mouth disease, which is associated with fatal neurological disease. The mechanism of EV71 pathogenesis remains obscure. We compared the replication capacity of the severe and mild enterovirus 71 isolates. The replication kinetics of EV71 in RD cells and ICR mice was determined by qRT-PCR. The lung, muscular, brain, intestine tissues were used for histopathological and immunohistochemical assays. The growth curves of EV71 strains in RD cells showed that the severe EV71 strains (SDLY107 and SDLY52) replicated faster and generated more viral RNA than the mild EV71 strains (SDLY11 and SDLY1). The mice infected by the severe EV71 strains (SDLY107) showed more severe clinical symptoms, pathological changes and higher viral load than the mice infected by the mild EV71 strains (SDLY11). These results suggest that there was a difference in replication capacity between the severe and mild EV71 strains, which was possibly associated with EV71 pathogenesis.

Complete genome sequencing and analysis of six enterovirus 71 strains with different clinical phenotypes.

BACKGROUND: Hand, foot and mouth diseases (HFMD) caused by enterovirus 71(EV71) presents a broad spectrum of clinical manifestations ranging from mild febrile disease to fatal neurolocal disease. However, the mechanism of virulence is unknown. METHODS: We isolated 6 strains of EV71 from HFMD patients with or without neurological symptoms, and sequenced the whole genomes of the viruses to reveal the virulence factors of EV71. RESULTS: Phylogenetic tree based on VP1 region showed that all six strains clustered into C4a of C4 sub-genotype. In the complete polypeptide, 298 positions were found to be variable in all strains, and three of these positions (Val(P814)/Ile(P814) in VP1, Val(P1148)/Ile(P1148) in 3A and Ala(P1728)/Cys)/Val(P1728) in 3C) were conserved among the strains with neurovirulence, but variable in strains without neurovirulence. In the 5'-UTR region, it showed that the first 10 nucleotides were mostly conserved, however from the 11th nucleotide, nucleotide insertions and deletions were quite common. The secondary structure prediction of 5'-UTR sequences showed that two of three strains without neurovirulence (SDLY11 and SDLY48) were almost the same, and all strains with neurovirulence (SDLY96, SDLY107 and SDLY153) were different from each other. SDLY107 (a fatal strain) was found different from other strains on four positions (C(P241)/T(P241), A(P571)/T(P571), C(P579)/T(P579) in 5'-UTR and T(P7335)/C(P7335) in 3'-UTR). CONCLUSIONS: The three positions (Val(P814)/Ile(P814) in VP1, Val(P1148)/Ile(P1148) in 3A and Ala(P1728)/Cys(P1728)/Val(P1728) in 3C), were different between two phenotypes. These suggested that the three positions might be potential virulent positions. And the three varied positions were also found to be conserved in strains with neurovirulence, and variable in strains without neurovirulence. These might reveal that the conservation of two of the three positions or the three together were specific for the strains with neurovirulence. Varation of secondary structure of 5'-UTR, might be correlated to the changes of viral virulence. SDLY107 (a fatal strain) was found different from other strains on four positions, these positions might be related with death.