Towards of this compound should provide insights about its

Towards development of potent antivirals against HEV Several laboratories have been focusing on identifying suitable drug targets and developing antivirals against HEV (48). Summarized below is the outcome of recent efforts in identifying potent antivirals against HEV.

(a) Antiviral effect of inhibitors of nucleotide synthesis pathway: IMPDH is an essential enzyme in the purine biosynthesis pathway. Several inosine monophosphate dehydrogenase inhibitors such as Mycophenolic acid (MPA), ribavirin and 5-ethynyl-1-?-D-ribofuranosylimidazole-4-carboxamide (EICAR) inhibit HEV replication (32, 49). The combination of MPA and ribavirin acts more effectively to inhibit HEV replication than MPA or ribavirin alone (49).

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Further, Mycophenolate mofetil, a prodrug of MPA, exhibited frequent HEV clearance in heart transplant patients, providing protection from chronification (49). Dihydroorotate dehydrgenase (DHODH) and orotidine-5′-monophosphate decarboxylase (ODCase) are essential enzymes in the pyrimidine biosynthesis pathway. DHODH inhibitors such as brequinar, leflunomide and ODCase inhibitor 6-azauracil also inhibit HEV replication in mammalian cell culture model (50).

These compounds deserve further validation as antivirals against HEV. (b) Antiviral effect of nucleoside analogues: 2′-C-methylcytidine (CMC) is a nucleoside analogue that efficiently inhibits HEV replication in the cell culture system (51). It was also shown that CMC retained anti-HEV activity even after long-term exposure with the virus, implying its potential use to combat drug resistance development (51).

However, CMC showed an antagonistic effect when tested in combination therapy with ribavirin (51).  Further in vivo evaluation of this compound should provide insights about its anti-HEV effects. Sofosbuvir, a prodrug of a uridine nucleoside analogue that acts as a direct acting antiviral against HCV RdRp in its active form, was reported by Thi et al. to inhibit HEV genotype-3 replication in vitro and showed additive effect when combined with ribavirin (52).

However, those data were not fully reproducible by Wang et al (53) and moreover, sofosbuvir treatment failed to clear HEV viremia in an immunosuppressed patient with chronic HCV and HEV without ribavirin (54). Therefore, usage of sofosbuvir as an anti-HEV therapeutic needs further validation (further discussed in RdRp section). (c) Antiviral effect of peptide-conjugated morpholino oligomers (PPMO): Zhang laboratory developed HEV specific PPMOs and evaluated their efficacy in inhibiting viral replication.

Out of the four PPMOs tested, PPMO HP1 was most effective in reducing viral replication in mammalian cell culture (55).  PPMO HP1 specifically inhibits viral translation by targeting a highly conserved sequence in the start region of ORF1 of genotype-1 and genotype-3 HEV. Treatment of cells with 2, 4, and 8 ?M of PPMO HP1 reduced luciferase expression by 53.

4%, 94.4%, and 99.7%, respectively, in a luciferase reporter based HEV replicon system (55). The antiviral activity of PPMO HP1 was specific, dose-responsive and potent. Hence, its further validation as a potential HEV-specific antiviral is warranted. (d) 1-(9-ethylcarbazol-3-yl)-3-(2-methyl-4-nitrophenyl) urea 66E2:  66E2 1-(9-ethylcarbazol-3-yl)-3-(2-methyl-4-nitrophenyl) urea has been identified as an inhibitor of HEV replication in hepatocytes (56).

66E2 inhibits genotype-3 HEV replication by ~50%, without showing any cytotoxicity. Interestingly, 66E2 also inhibits hepatitis C virus (HCV) and Dengue virus replication (56). The mechanism by which 66E2 inhibits viral replication remains to be explored. (e) MG132: Karpe et al reported significant inhibition of HEV replication-related luciferase activity in cells treated with MG132 (57). However, subsequently it was shown that MG132 also reduced the cellular RNA and protein levels, indicating its effect to be non-specific (58). (f) Zinc: A recent report by Kaushik et al has demonstrated the antiviral activity of zinc against HEV (59). Zinc is an essential micronutrient, which plays crucial role in multiple cellular processes.

It also acts as a broad spectrum antimicrobial against several pathogens (60, 61). Zinc salts blocked the replication of both gentype-1 and genotype-3 HEV by inhibiting the activity of viral RdRp in cultured human hepatoma cells (59). Further, zinc salts did not affect virus entry into the host cell. Zinc also displayed moderate cooperativity with ribavirin in inhibiting viral replication. These data indicate the possible therapeutic usage of zinc in controlling HEV infection. However, considering the complexities involved in serum/plasma and intracellular zinc homeostasis (62), the efficacy of zinc in inhibiting HEV replication in vivo remains to be evaluated.

Moreover, the detailed mechanism(s) underlying the inhibitory action of zinc on HEV replication needs to be investigated.