METHODS: In this study, human rhabdomyosarcoma (RD) cells were infected with EV71 at different multiplicity of infection (MOI). After infection, the cytopathic effect (CPE) was monitored and recorded using a phase contrast microscope associated with a CCD camera at different time points post viral infection (0, 6, 12, 24 h post infection). Cell growth and viability were measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay in both EV71 infected and mock infected cells at each time point. EV71 replication kinetics in RD cells was determined by measuring the total intracellular viral RNA with real-time reverse-transcription polymerase chain reaction (qRT-PCR). Also, the intracellular and extracellular virion RNA was isolated and quantified at different time points to analyze the viral package and secretion. The expression of viral protein was determined by analyze the levels of viral structure protein VP1 with Western blotting.

RESULTS: EV71 infection induced a significant CPE as early as 6 h post infection (pi) in both RD cells infected with high ratio of virus (MOI 10) and low ratio of virus (MOI 1). In EV71 infected cells, the cell growth was inhibited and the number of viable cells was rapidly decreased in the later phase of infection. EV71 virions were uncoated immediately after entry. The intracellular viral RNA began to increase at as early as 3 h pi and the exponential increase was found between 3 h to 6 h pi in both infected groups. For viral structure protein synthesis, results from western-blot showed that intracellular viral protein VP1 could not be detected until 6 h pi in the cells infected at either MOI 1 or MOI 10; and reached the peak at 9 h pi in the cells infected with EV71 at both MOI 1 and MOI 10. Simultaneously, the viral package and secretion were also actively processed as the virus underwent rapid replication. The viral package kinetics was comparable for both MOI 1 and MOI 10 infected groups. It was observed that at 3 h pi, the intracellular virions obviously decreased, thereafter, the intracellular virions began to increase and enter into the exponential phase until 12 h pi The total amounts of intracellular virons were decreased from 12 to 24 h pi Consistent with this result, the increase of virus secretion occurred during 6 to 12 h pi

CONCLUSION: The viral kinetics of EV71 were established by analyzing viral replication, package and secretion in RD cells.