**4. NA-based antivirals against CVB3 infection**

#### **4.1 Anti-CVB3 ASONs**

CVB3, one of the most frequently used model systems for study of viral replication and pathogenesis, is also widely employed for evaluation of NA-based antiviral agents. The early investigations mainly focused on the application of the second and third generations of the ASONs. McManus and coworkers are one of the pioneer groups to study the potential possibility to inhibit CVB3 replication using ASONs. Their earliest work using regular ASONs to target the different sites of 5'UTR of CVB3 genome successfully mapped the IRES by *in vitro* translation inhibition assay (Yang et al., 1997). This study provided useful information for the design of ASON for inhibiting CVB3 replication *in vitro* and in mouse models. Later, they used PS-ASONs targeting the 5' and 3'UTRs as well as the start codon region and found that the oligomers targeting the 5' and 3' proximate ends of the CVB3 genome are the most effective candidates to inhibit viral replication in HeLa cells. Each of these two ASONs resulted in ~80% reduction of viral particle production, which is followed by the candidates targeting the IRES and the initiation codon region (A. Wang et al., 2001). The importance of these sites for ASON binding was further confirmed by *in vivo* evaluation using a murine myocarditis model, although the antiviral efficiency is not as high as that obtained from *in vitro* evaluation (Yuan et al., 2004).

To improve the stability of the oligomers, our group designed eight phosphorodiamidate morpholino oligomers (PMO) targeting both the sense and antisense strands of the CVB3 replication intermediate. To increase the efficiency of drug internalization, the PMO were conjugated to a cell-penetrating arginine-rich peptide. These modified ASONs were evaluated in HeLa cells and HL-1 cardiomyocytes in culture and in a murine myocarditis model (Yuan et al., 2006). One of the oligomers, designed to target a sequence in the 3' portion of the CVB3 IRES, was found to be especially potent against CVB3. Treatment of cells with this oligomer prior to CVB3 infection produced an approximately 3-log10 decrease in viral titer and largely protected cells from a virus-induced cytopathic effect. A similar antiviral effect was observed when this oligomer treatment began shortly after the virus infection period. A/J mice receiving intravenous administration of this oligomer once prior to and once after CVB3 infection showed an ~2-log10-decreased viral titer in the myocardium at 7 days post infection and a significantly decreased level of cardiac tissue damage, compared to the controls (Yuan et al., 2006).

In addition to the many ASON reports, another strategy using CpG containing oligodeoxynucleotide to activate antiviral immunity has been reported (Cong et al., 2007). The mechanism is that the C-type of CpG oligomer can induce anti-CVB3 activity in human peripheral blood mononuclear cells (PBMCs) through the induction of synthesis of natural mixed interferons.
