Proteome demonstration of alpha-1-acid glycoprotein and alpha-1-antichymotrypsin candidate biomarkers for diagnosis of enterovirus 71 infection.

BACKGROUND: Human enterovirus 71 (EV71) is the major causative agents of hand-foot-and-mouth disease and frequently associated with severe complications such as encephalitis and death. Understanding the host response following enteroviral infection may facilitate the development of biomarkers for EV71 infections. METHODS: We implemented two-dimensional gel electrophoresis technology on proteins prepared from serum obtained from 4 mild and 4 severe cases of EV71 infections and 4 healthy control children, to investigate the differentially expressed proteins. The differential expressed proteins were further identified with liquid chromatography-mass spectrometry/mass spectrometry analysis and western blotting validation. RESULTS: A total of 27 differentially expressed proteins were picked and identified with liquid chromatography-mass spectrometry/mass spectrometry. Of the 27 identified proteins, 6 proteins were up-regulated in the mild-infected and severe EV71-infected patients in comparison to the healthy control group. Two proteins, alpha-1-acid-glycoprotein (AGP1) and alpha-antichymotrypsin (AACT), were not detected in the EV71-infected patients, but appeared in the control patient. Western blotting analysis demonstrated that AGP1 and AACT proteins were negatively associated with the clinical severity of EV71 infection. Similarly, both of the proteins were not detected in the secretion medium from the EV71-infected neuroblastoma cells, but detected in the mock-infected cells, suggesting that differentially expressed AGP1/AACT protein levels are in response to EV71 infections. CONCLUSIONS: Two candidate proteins AGP1 and AACT, whose expression levels were reduced under the EV71 infection pathological condition, provide useful source of information for potential diagnostic biomarkers of EV71 infection in children.

Genetic diversity and evolution of satellite RNAs associated with the bamboo mosaic virus.

Satellite RNAs (satRNAs) are subviral agents that depend on cognate helper viruses for genome replication and encapsidation. Their negative impacts on helper viruses have been exploited to control plant viral diseases. SatBaMV is a commonly found satRNA associated with Bamboo mosaic virus (BaMV) that infects diverse bamboo species in the field. To investigate the genetic diversity and evolution of satRNAs, we examined seven satBaMV populations derived from five bamboo species and cultivars from Taiwan, China, and India and one from the greenhouse. We found 3 distinct clades among the seven populations. Clade I is consisted of all satBaMV isolates, except for those from Dendrocalamus latiflorus in Taiwan and Bambusa vulgaris in India, which belong to Clades II and III, respectively. Interestingly, nucleotide diversity was lower for Clade I than II and III. However, the nucleotide diversity did not seem to depend on bamboo species or geographic location. Our population genetic analyses revealed the presence of excessive low-frequency polymorphic sites, which suggests that the satBaMV population was under purifying selection and/or population expansion. Further analysis of P20, the only satBaMV gene that encodes a non-structural protein involved in the long-distance movement of satBaMV, showed evidence of purifying selection. Taken together, our results suggest that purifying selection against defective P20 protein is responsible at least in part for the evolution of the satBaMV genome.

Heat shock protein-90-beta facilitates enterovirus 71 viral particles assembly.

Molecular chaperones are reported to be crucial for virus propagation, but are not yet addressed in Human Enterovirus 71 (EV71). Here we describe the specific association of heat shock protein-90-beta (Hsp90ß), but not alpha form (Hsp90α), with EV71 viral particles by the co-purification with virions using sucrose density gradient ultracentrifugation, and by the colocalization with viral particles, as assessed by immunogold electron microscopy. The reduction of the Hsp90ß protein using RNA interference decreased the correct assembly of viral particles, without affecting EV71 replication levels. Tracking ectopically expressed Hsp90ß protein associated with EV71 virions revealed that Hsp90ß protein was transmitted to new host cells through its direct association with infectious viral particles. Our findings suggest a new antiviral strategy in which extracellular Hsp90ß protein is targeted to decrease the infectivity of EV71 and other enteroviruses, without affecting the broader functions of this constitutively expressed molecular chaperone.