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Cryptosporidium parvum is a highly prevalent zoonotic and anthroponotic protozoan parasite that causes a diarrheal syndrome in children and neonatal livestock, culminating in growth retardation and mortalities. Despite the high prevalence of C . parvum , there are no fully effective and safe drugs for treating infections, and there is no vaccine. We have previously reported that the bacterial-like C . parvum lactate dehydrogenase (CpLDH) enzyme is essential for survival, virulence and growth of C . parvum in vitro and in vivo . In the present study, we screened compound libraries and identified inhibitors against the enzymatic activity of recombinant CpLDH protein in vitro . We tested the inhibitors for anti- Cryptosporidium effect using in vitro infection assays of HCT-8 cells monolayers and identified compounds NSC158011 and NSC10447 that inhibited the proliferation of intracellular C . parvum in vitro , with IC 50 values of 14.88 and 72.65 μM, respectively. At doses tolerable in mice, we found that both NSC158011 and NSC10447 consistently significantly reduced the shedding of C . parvum oocysts in infected immunocompromised mice’s feces, and prevented intestinal villous atrophy as well as mucosal erosion due to C . parvum . Together, our findings have unveiled promising anti- Cryptosporidium drug candidates that can be explored further for the development of the much needed novel therapeutic agents against C . parvum infections.

Type I interferons (IFNs-α/β) play a key role for the antiviral state of host, and the porcine arterivirus; porcine reproductive and respiratory syndrome virus (PRRSV), has been shown to down-regulate the production of IFNs during infection. Non-structural protein (nsp) 1 of PRRSV has been identified as a viral IFN antagonist, and the nsp1α subunit of nsp1 has been shown to degrade the CREB-binding protein (CBP) and to inhibit the formation of enhanceosome thus resulting in the suppression of IFN production. The study was expanded to other member viruses in the family Arteriviridae: equine arteritis virus (EAV), murine lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV). While PRRSV-nsp1 and LDV-nsp1 were auto-cleaved to produce the nsp1α and nsp1β subunits, EAV-nsp1 remained uncleaved. SHFV-nsp1 was initially predicted to be cleaved to generate three subunits (nsp1α, nsp1β, and nsp1γ), but only two subunits were generated as SHFV-nsp1αβ and SHFV-nsp1γ. The papain-like cysteine protease (PLP) 1α motif in nsp1α remained inactive for SHFV, and only the PLP1β motif of nsp1β was functional to generate SHFV-nsp1γ subunit. All subunits of arterivirus nsp1 were localized in the both nucleus and cytoplasm, but PRRSV-nsp1β, LDV-nsp1β, EAV-nsp1, and SHFV-nsp1γ were predominantly found in the nucleus. All subunits of arterivirus nsp1 contained the IFN suppressive activity and inhibited both interferon regulatory factor 3 (IRF3) and NF-κB mediated IFN promoter activities. Similar to PRRSV-nsp1α, CBP degradation was evident in cells expressing LDV-nsp1α and SHFV-nsp1γ, but no such degradation was observed for EAV-nsp1. Regardless of CBP degradation, all subunits of arterivirus nsp1 suppressed the IFN-sensitive response element (ISRE)-promoter activities. Our data show that the nsp1-mediated IFN modulation is a common strategy for all arteriviruses but their mechanism of action may differ from each other.

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