Human enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD), particularly in infants and children below 4 years of age. Shikonin is a bioactive compound with anti-inflammatory, antiviral, and antibacterial activities derived from the roots of the Chinese medicinal herb Lithospermum erythrorhizon. This study aimed to examine the antiviral activity of PMM-034, a shikonin ester derivative, against EV71 in rhabdomyosarcoma (RD) cells. Cytotoxicity of PMM-034 on RD cells was determined using WST-1 assay. Dose- and time-dependent effects of PMM-034 on EV71 replication in RD cells were determined using plaque reduction assay. mRNA expression levels of EV71/VP1 and pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α) were determined by real-time RT-PCR, and EV71/VP1 and phospho-p65 protein expressions were determined by western blot analysis. PMM-034 exhibited only weak cytotoxicity against RD cells. However, PMM-034 exhibited significant antiviral activity against EV71 in RD cells with 50% inhibitory concentration of 2.31 μg/mL. The VP1 mRNA and protein levels were significantly reduced in cells treated with PMM-034. Furthermore, relative mRNA expression levels of IL-1β, IL-6, IL-8, and TNF-α significantly decreased in the cells treated with PMM-034, while the phospho-p65 protein expression was also significantly lower in the treated cells. These results indicated that PMM-034 suppressed the expressions of pro-inflammatory cytokines in RD cells, exhibiting antiviral activity against EV71, as evidenced by the reduced VP1 mRNA and protein levels in PMM-034-treated cells. Thus, PMM-034 is a promising candidate for further development as an EV71 inhibitor.

Enterovirus 71 (EV71) immunomagnetic enrichment technique and routine detection methods were combined to detect swab environmental specimens to elucidate the role of environmental specimens in the spread of EV71. Immunomagnetic beads with specific enrichment of EV71 virus were prepared, then the beads were used to absorb the EV71 virus from environmental samples. Obtained immunomagnetic bead-virus complexes were detected by RT-PCR, RT-qPCR and cell culture. Isolated virus were subjected to VP1 full-length amplification and homology analysis was performed. A total of 4 µg of EV71 monoclonal antibody was mixed with 50 µl magnetic beads, and the highest coating efficiency was reached after incubating at room temperature for 2 h. Satisfactory enrichment effect was achieved by adding 50 µl immunomagnetic beads to 1.5 ml sample and shaking at room temperature for 2 h. The method of EV71 enrichment has high sensitivity and specificity. A total of 346 specimens after enrichment by immunomagnetic beads, the positive rates of RT-qPCR, RT-PCR and cell culture were 20.52, 5.78, and 9.25%, respectively, which were also significantly higher than those before enrichment (15.90, 3.47 and 4.05%; P<0.05). After enrichment with immunomagnetic beads, isolation rate of EV71 virus from case specimens and home environment specimens increased from 27.45 to 43.14% and from 0 to 5.29%, respectively. In home environment-positive specimens, positive rate of toys and stationery was high (52.00 and 24.00%, respectively). In kindergarten environmental samples, the positive rate of RT-qPCR was 6.12%, and EV71 virus was not isolated. Sequence analysis showed that the nucleotide homology of case isolates and home environment isolates was 98.0–100%.