Three kinds of treatment and their combination against coronavirus PEDV

Background Porcine epidemic diarrhea virus (PEDV) of the family Coronaviridae has caused substantial economic losses in the swine husbandry industry. The currently available vaccines are only partially effective and there is no specific drug available for treatment of viral infection. Method In the current study, we use animal coronavirus PEDV as a model to study antiviral agents. Briefly, a fusion inhibitor tHR2, recombinant lentivirus-delivered shRNAs targeted to conserved M and N sequences, homoharringtonine (HHT), and hydroxychloroquine (HCQ) were surveyed for their antiviral effects. Results Treatment with HCQ at 50 mM reduced virus titer in TCID 50 by 30 fold, and the combination with HHT at 150 nM reduced virus titer in TCID 50 by 200 fold. Conclusion The combination of two drugs with low dose can more effectively inhibit PEDV infection compared to one alone. The present study offers information to contribute to the development of effective antiviral strategies against coronavirus infection, and demonstrates that the combination treatment, even at low concentrations, presents more potent antiviral activity than the separate components acting alone. 1 protease for 30 min at ice. The cell suspension was then fractionated by centrifugation at 6000 × g for 10 min at 4℃. Solubilized proteins were harvested, electrophoresed in denaturing polyacrylamide gels, electro blotted onto a polyvinylidene fluoride (PVDF) membrane, and reacted with the antibodies indicated overnight. Protein bands were detected with secondary antibody conjugated to horseradish peroxidase (HRP) for 45 min at room temperature, and actin was and two-way ANOVA with Dennett’s multiple-comparison test. Partial correlation analyses were evaluated by unpaired student's test. combination treatment HHT and tHR2 in additive protective effects by inhibiting fusion and viral replication. found that the combination of HHT and shRNA-MN improved effects they have the same target in the viral replication process. More details remain to be studied. The combination of a low dose of HHT host targeting antivirals tHR2 direct-acting

Introduction induce the body to produce an effective immune response [4]. M protein plays an essential role in viral assembly [5]. In RNA interference (RNAi), short interfering RNA molecules or short hairpin RNA (shRNA) targeting specific mRNA leads to degradation of mRNA and inhibition of gene expression [6][7]. RNAi targeting viral genes is an effective way to protect cells from virus infection,and has been shown to mediate genetic suppression of many human and animal pathogenic coronaviruses [8,9,10]. Class I enveloped viruses such as coronaviruses utilize a conserved membrane fusion mechanism, in which highly conserved heptad repeat (HR) regions interact to form six-helix bundle  structures, contributing to the fusion of virus and cell membranes. The HIV-1 fusion inhibitor T20, for example, and several modified peptides (e.g., T1249, T2635, T-20 s 138A ), which are derived from the HR region, have been clinically approved to suppress HIV-1 and T-20-resistant variants [11][12][13][14]. Coronavirus S glycoprotein shares a mechanism similar to other Class I virus glycoproteins for membrane fusion [15][16][17]. Our previous work has shown that the modified HR peptide efficiently blocks coronavirus attack [17,18].
Viruses are able to utilize cellular machineries for viral replication, and a growing understanding of cellular proteins and associated pathways in viral replication has supported the design of new strategies for developing novel antiviral agents. Small molecule chemicals that are able to target cellular machinery act as efficient antiviral agents. The natural compound homoharringtonine (HHT), for instance, is known to inhibit the first cycle of the elongation phase of eukaryotic translation. HHT antagonizes the phosphorylation level of endogenous and exogenous eIF4E (p-eIF4E), which may regulate the selective translation of specific mRNA [19,20]. In a previous study, we observed that HHT completely inhibited infection by PEDV at a concentration of 500 nM in cell cultures; treatment with HHT at doses of 0.05 mg/kg significantly reduced viral load and relieved severe symptoms in PEDV-infected animals [20]. One crucial cellular mechanismautophagy, relies on lysosomes for the clearance and recycling of abnormal proteins or organelles; the deregulation of autophagy is associated with the development of various diseases, including viral infection by coronavirus [21]. Hydroxychloroquine (HCQ), an autophagy inhibitor chloroquine hydroxy-derivative, acts as an antiinflammatory agent by attenuating tissue injury through downregulation of inflammatory activation [22]. It also triggers the host defense machinery by inducing innate immune activation [23]. HCQ shows efficiently antiviral activity against a broad spectrum of viruses, including Zika virus and Dengue virus [23,24].
In the present study, we constructed a truncated HR2 peptide and recombinant lentivirus-delivered shRNAs targeted to conserved M and N sequences, as well as small molecule chemicals HHT and HCQ, to evaluate their antiviral activity. We identified that a combination treatment more significantly improved the antiviral effects compared to the action of individual components. This paper may provide a foundation to leverage these agents in treating coronavirus infection in humans and animals.

Design of shRNA plasmids
The shRNA expression vector pGPU6/GFP/Neo was selected to be the parental plasmid. The shRNA sequences were as follows

Statistics
All results were expressed as means and standard deviations (SD). Statistical analyses were performed using Prism 5.01 (GraphPad Software). Significance was determined by one-way analysis of variance (ANOVA) and two-way ANOVA with Dennett's multiple-comparison test. Partial correlation analyses were evaluated by unpaired student's test.

Discussion
In the current study, we found that the combination of HHT and HCQ at low concentrations reduced TCID 50 by 200 fold (Fig. 4). In our previous study, we found that HHT exerted broad-spectrum antiviral action and completely inhibited infection by PEDV at a concentration of 500 nM in Vero cells [20].
Treatment with HHT at doses of 0.05 mg/kg significantly reduced viral load and relieved severe symptoms in PEDV-infected animals, but treatment with 0.2 mg/kg of HHT led to severe side effects, fatal to about 50% of the animals [20]. The inhibitory effect of HCQ is associated with host defense machinery and immune regulation; HCQ at high concentration also exhibits tissue toxicity [21-24].
The study of preparations therefore provides new ideas, but the damage of host cells resulting from increasing doses is a problem that cannot be ignored.
Recent research has shown that combination therapy can be more effective than monotherapy. The combination of ribavirin with interferon-β, for instance, inhibits SARS-CoV replication very strongly, compared to either single treatment [25]. A combination of RNAi and a virus receptor trap exerts additive antiviral activity in coxsackievirus B3-induced myocarditis in mice [26]. Likewise, antibody administered with Sunitinib significantly reduces vascular leakage and synergizes to provide superior protection from lethal DENV infection compared with either agent alone [27]. Combination therapies targeting viruses at different stages of their lifecycle are a common strategy to improve the efficiency of the antiviral treatment, and the combination of multi-target preparations is expected to be less cytotoxic [28].
The RNAi-based strategy has different mechanisms of action than antibodies, small molecules, and other protein drugs. It is worth mentioning that the RNAi drug Onpattro (patisiran) was the first FDAapproved for adult patients with peripheral neuropathy (polyneuropathy) caused by hereditary transthyretin amyloidosis (hATTR). Methods to express multiple shRNAs from a single vector have been explored, including expression from multiple cassettes using the same promoter [29], or from a single vector containing multiple promoters [30]. The inhibition of viruses by means of RNAi is an approach to antivirals that has gained importance in recent years [10,[31][32][33][34], but the use of multiple shRNAs in the elimination of viral infection has been less successful. Our study demonstrates that dual shRNA-expressing plasmids effectively inhibit the replication of PEDV, better than individual shRNA (Fig. 1). We have established a concept for developing transfection vectors that may have wide applications in gene antiviral strategies, including the delivery of siRNA to combat viral infection. This approach has the potential to be utilized for the prevention and control of coronavirus PEDV.
However, the inhibitory effect of double shRNA needs to be further improved. In future work, the human U6 promoter in the vector pGPU6/GFP/Neo will be replaced with the porcine U6 promoter to increase the transfection efficiency of shRNA and enhance the inhibitory effect.
The membrane fusion process has been considered as a significant antiviral target and has received wide attention; the recombinant HR2 region of fusion glycoprotein is the ideal fusion inhibitor [11].
Based on a LearnCoil-VMF prediction result, amino acid L1252 to L1286 (35Aa) from the HR2 domain of S glycoprotein was selected to design a fusion inhibitor that efficiently blocks MERS-CoV entry [16][17]. The predicted HR2 domain (65Aa) from S glycoprotein of PEDV, however, is longer than that in MERS-CoV and other coronaviruses [16][17][18]35], and synthetic HR2 peptide at the concentration of 40 µM statistically inhibits PEDV infection [36]. Reducing the length of the active peptide may be an important consideration to reduce the cost of peptide agents. Here we truncated the tHR2 domain to produce the peptide tHR2 (39Aa), with the aim of identifying a minimal domain capable of disrupting the formation of the six-helix bundle required for viral cell entry. Our results suggest that tHR2, but not scrambled peptide, has the ability to inhibit PEDV infection (Figs. 3E, 3F). Furthermore, the antiviral effect of tHR2 is not as strong as expected, perhaps due to the short length or disrupted core structure. It is noteworthy that by 2013 the FDA had already approved the listing of more than 60 peptide drugs [37]. Peptides will be widely used in medicine and biotechnology in the near further.
In this study, we explored how to maximize the antiviral effect of HHT while reducing host cell toxicity. We found that tHR2 peptide designed based on PEDV S genes effectively inhibits the membrane fusion of PEDV, and the site of action is clear and does not affect the cell structure. The combination treatment with HHT and tHR2 results in additive protective effects by inhibiting membrane fusion and viral replication. We also found that the combination of HHT and shRNA-MN did not exhibit improved effects (not shown here), perhaps because they have the same target in the viral replication process. More details remain to be studied. The combination of a low dose of HHT and/or HCQ as host targeting antivirals (HTA) with tHR2 as direct-acting antiviral (DAA) has the potential to improve safety and increase the antiviral effect. These results provide a reference for the

Consent to publication
Not applicable.

Availability of data and materials
All data generated or analyzed during this study are included in this published article. financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.