| HSV-1 (herpes simplex virus 1) is a member of Herpesviridae and normally cause gingivostomatitis, keratoconjunctivitis and herpes genitalis. In immune-compromised individuals, the recurrent infection can cause severe symptoms such as herpes simplex encephalitis and corneal blindness. With the emergence of drug resistant strains in clinical practice, it is necessary to develop new antiviral complexes and new antiviral technologies.RNase P is a ribonucleoprotein complex which facilitates the maturation of tRNA by catalyzing a hydrolysis reaction to remove the leader sequence of precursor tRNA (pre-tRNA). It has been shown by previous studies that RNase P recognizes pre-tRNAs with their tertiary molecular structures. An external guide sequence (EGS) is a RNA sequence which can interact with target mRNA to form a tertiary structure like a pre-tRNA and recruit RNase P to degrade target mRNA. EGSs derived from natural pre-tRNA sequences had been applied to inhibit gene expression in both bacterial and mammalian cells. Furthermore, EGSs were shown to be effective in inhibiting gene expression and replication of several human viruses including human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), Kaposi sarcoma-associated herpesvirus (KSHV), and hepatitis B virus (HBV) in cultured cells.In this study, several EGSs were constructed to target the mRNA encoding HSV-1 ICP4 protein. ICP4, a viral immediate early (IE) protein functioning as a viral major transcription activator, is essential for the expression of viral (3 (early) and y (late) genes and viral replication. We determined the activity of the constructed EGSs in guiding RNase P to cleave the target ICP4 mRNA sequence with an in vitro reaction of extracted human RNase P, ICP4 mRNA substrate and designed EGSs. C468-A, an EGS variant, showed 60 times more efficient in guiding RNase P to cleave ICP4 mRNA sequence than SER-A derived from a natural pre-tRNA sequence. We infected HeLa and human primary oral keratinocyte (HOK) cell lines expressing EGSs with HSV-1 and detected the ICP4 mRNA and protein expression levels in these cell lines. By Northern and Western blot, we found cells expressing C468-A and SER-A showed 97% and 75% decrease in ICP4 gene expression comparing with parental cells expressing no EGSs. Meanwhile, the expressions of early and late genes of HSV such as TK, gC, ICP35 are also detected and found to be inhibited by 75-97%. Eventually, we determined the growth curve of HSV-1 in HeLa and HOK cell lines expressing different EGSs and found virus titers in cell lines expressing C468-A and SER-A showed a decrease of more than 7000- and 500 folds. Thus, we conclude that designed EGSs in this study can guide RNase P to specificly cleave ICP4 mRNA in cultured cells thus effectively inhibit HSV-1 gene expression and growth; designed EGSs in this study are promising to be reformed into an anti-HSV-1 drug.Hand, foot and mouth disease (HFMD) is a popular children's common disease in worldwide, and normally causes fever, mouth ulcers and rash or herpes on hands, feet and other body parts. Symptoms in severe patients include encephalitis, acute flaccid paralysis, cerebral edema, myocarditis serious complications and even death. The most common strains that cause HFMD are coxsackievirus A16 and entero-virus 71. Besides, Coxasckievirus A4, A5, A7, A9, A14, B2, B5, B13 and Echoviruses have been directly implicated to HFMD. Chinese Center for Disease Control and Prevention (China CDC) records millions of HFMD cases every year, thus HFMD has become a serious threat to the health of newborns and children in China.We have analysized 358 HFMD samples collected from Shawo, Ezhou, Hubei in 2012, 2013 and 2015 and investigated local environment and patient's families. We determined the serotype of viruses in these samples and found 51 samples are EV71 positive,66 are CA16 positive and 18 are both EV71 and CA16 positve. Then, we analysized the age-case distribution, gender-case distribution, time-case distribution, geological distribution and symptom-age relation features of HFMD outbreaks in different years. We found that the percentage of EV71 and CA16 infection are both higher among female than male HFMD patients; EV71 and CA16 can cause HFMD alternatively; enteroviruses other than EV71 and CA16 may play an important role in HFMD oubreaks. In addition, we isolated a CA16 strain, identified it as a B2b subtype by phylogenetic analysis. Moreover, we found 45 samples are enterovirus-positive among daily used water samples from 51 HFMD patients'families thus assumed that water source may be a new transmission route of HFMD. Our study concluded the epidemic pattern of HFMD outbreaks in Shawo in 2012-2015; speculated the source of prevailing HFMD viruses. Our findings of new transmission route shall provide reference to the control and prevention of HFMD. |
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