Plasmids Expressing shRNAs Specific to the Nucleocapsid Gene Inhibit the Replication of Porcine Deltacoronavirus In Vivo

Simple Summary Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen distributed globally, which causes substantial economic losses in the swine industry. The characterization of the receptor promiscuity may pose a risk of cross-species transmission. However, the options for pharmaceutical interventions are limited. In this study, the vectors expressing shRNAs targeting the nucleocapsid gene were generated to assess the inhibition effect of PDCoV reproduction. Our preliminary results demonstrate that a dual shRNA expression system is an effective strategy in combating PDCoV infection without cytotoxicity, which would facilitate the ongoing development of RNAi-based therapeutic drugs against viral diseases. Abstract Porcine deltacoronavirus (PDCoV) is a novel enteric coronavirus and is becoming one of the major causative agents of diarrhea in pig herds in recent years. To date, there are no commercial vaccines or antiviral pharmaceutical agents available to control PDCoV infection. Therefore, developing a reliable strategy against PDCoV is urgently needed. In this study, to observe the antiviral activity of RNA interference (RNAi), four short hairpin RNAs (shRNAs) specific to the nucleocapsid (N) gene of PDCoV were designed and tested in vitro. Of these, a double-shRNA-expression vector, designated as pSil-double-shRNA-N1, was the most effectively expressed, and the inhibition of PDCoV replication was then further evaluated in neonatal piglets. Our preliminary results reveal that plasmid-based double-shRNA-expression targeting the N gene of PDCoV can significantly protect LLC-PK1 cells and piglets from pathological lesions induced by PDCoV. Our study could benefit the investigation of the specific functions of viral genes related to PDCoV infection and offer a possible methodology of RNAi-based therapeutics for PDCoV infection.


Introduction
Based on antigenic relationships [1], coronaviruses, members of the Coronaviridae family, are conventionally composed of three genera, that is, Alphacoronavirus, Betacoronavirus, and Gammacoronavirus. Recently, a novel genus, Deltacoronavirus, has been discovered in varied host species, including birds and mammals [2,3]. In a survey conducted in Hong Kong, deltacoronaviruses were first identified in both avian and porcine specimens in 2012 [1]. Thereafter, a porcine deltacoronavirus (PDCoV) associated with diarrhea was first

shRNA Design and Selection
The N gene sequence of CH/JXNI/02/2015 was analyzed using the online siRNA target design tool to choose the candidate target sequences (https://www.thermofisher.com/ cn/zh/home/life-science/rnai/synthetic-rnai-analysis/stealth-rnai-technology.html (accessed on 10 April 2018). There were 4 potential target shRNAs designed, specific to different conserved nucleotide positions of the N gene of PDCoV ( Table 1). The specificity of the sequences was then examined by BLAST to ensure that they would not hit any sequences of the swine genome, and had 100% identity with the targeted gene.

Construction of shRNA Plasmids
The protocol for the construction of shRNA plasmids was described previously [27,28]. Briefly, sense and antisense DNA oligonucleotides dissolved in sterile ddH 2 O were annealed in 25 µL of reaction system containing 5 µL (100 µM) of sense (forward) oligonucleotide, 5 µL (100 µM) of antisense (reverse) oligonucleotide, and 15 µL of ddH 2 O. The mixture was heated to 95 • C for 5 min, then cooled down to 50 • C and kept for 30 s, and subsequently incubated at 4 • C for 30 min. There were 2 shRNA-expressing plasmids constructed, pSil-U6-mcherry and pSil-double-U6-mcherry, as mentioned before [29], and then transformed into Escherichia coli DH5a competent cells. The positive clones were selected and then validated by PCR amplification and DNA sequencing.

Generation of LLC-PK1 Cells Stably Expressing shRNA and Virus Infection
The LLC-PK1 cells were seeded (2 × 10 4 /well) into 6-well plates and incubated for 24 h at 37 • C in a 5% CO 2 atmosphere. When reaching 50-70% confluence, the cells were washed three times with sterilized 0.01 M pH7.4 phosphate buffered saline (PBS). Afterwards, the cells were transfected with 2.5 µg/well of shRNA-expressing plasmids using a Lipofectamine TM 3000 (Invitrogen, Waltham, MA, USA), according to the manufacturer's instructions. After 24 h of incubation, the growth media were substituted with maintenance media containing 2% FBS and 1000 µg/mL of Neomycin (G418). The survival cell clones were maintained in G418-containing media for 15 d with frequent media replacements until cell death could no longer be observed. Then, these monoclonal cells transfected with shRNA-expressing plasmids were screened by limiting dilution analysis (LDA), as previously described [29], and cultured in DMEM growth media containing G418 (500 µg/mL) in 6-well plates at 37 • C in a 5% CO 2 atmosphere. After reaching a confluence, these plasmid-transduced cells were inoculated with PDCoV at a multiplicity of infection (MOI) of 0.1. Non-transfected cells were set as a control. Cell transfection efficiency and cytopathic effect (CPE) images were taken under an inverted fluorescence/phase-contrast microscope (Nikon, Tokyo, Japan).

Cell Viability Determination (MTS Assay)
The constructed stable cell lines of LLC-PK1 with shRNA expression were seeded into 96-well plates at a density of 1 × 10 4 /well, and then treated as described above. The cells were infected with PDCoV at an MOI of 0.1. Cell viability was determined by CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) according to the manufacturer's protocol. Briefly, 20 µL/well of the MTS reagent [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfop henyl)-2H-tetrazolium] was added at 36 h post-infection (hpi) and the cells were incubated at 37 • C for 4 h in a 5% CO 2 atmosphere. The absorbance at 490 nm of each solution was read by a Varioskan Flash Spectral Scanning Multimode Reader (Thermo Fisher, Waltham, MA, USA). The cell viability was calculated as a percentage of the OD value of the treated cells versus the control cells. All experiments were conducted in triplicate.

Virus Titration (TCID 50 Determination)
The stable cell lines of LLC-PK1 with shRNA expression were harvested at 48 hpi and subjected to freeze-thaw three times. After clarification based on the standard methodology, the cell culture supernatants were collected and made into a 10-fold serial dilution (10 −1 to 10 −10 ), and then added into 96-well plates pre-seeded with LLC-PK1 cells to determine the virus titers expressed as TCID 50 , which was determined using the Reed-Muench method [30].

RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)
To estimate the impact of shRNA on viral replication, a qRT-PCR assay was used. For this purpose, total RNA was initially extracted from the LLC-PK1 cells at 36 hpi using the MiniBEST™ Viral RNA/DNA Extraction Kit Ver.5.0 (Takara, Kyoto, Japan), and then reversely transcribed into the first-strand cDNA using the GoScript TM Reverse Transcription System (Promega, Madison, WI, USA), following the manufacturer's instructions. The qRT-PCR of PDCoV genomic RNA was performed in a total volume of 20 µL reaction mixture comprising 10 µL of SYBR Premix Ex Taq II (Takara, Kyoto, Japan), 0.4 µL of forward primer, 0.4 µL of reverse primer, 2 µL of cDNA template, and 0.4 µL of Rox Reference Dye, and the β-actin gene was employed as an internal reference control ( Table 2). The qRT-PCR was performed on an ABI 7500 Real-Time PCR System (Thermo Fisher, Carlsbad, CA, USA) under the following parameters: initial denaturation at 95 • C for 30 s, and then 40 cycles of 95 • C for 5 s and 61 • C for 30 s; the melting curve stage comprised 95 • C for 1 min, 55 • C for 30 s, and 95 • C for 30 s. Each experiment was repeated three times. The relative expression level of the N gene of PDCoV in infected cells was estimated by the 2 − Ct method [28].

Western Blotting
The stable LLC-PK1 cell lines expressing shRNA were infected with PDCoV at an MOI of 0.1. The infected cells were collected at 36 hpi and washed three times with cold PBS (0.01 M, pH7.4), following the aforementioned procedures. The cell pellets were lysed in radio immunoprecipitation assay (RIPA) lysis buffer with a protease inhibitor phenylmethane sulfonyl fluoride (PMSF) (Beyotime, Shanghai, China). Each sample was denatured in 5 × loading buffer at 100 • C for 10 min. The total proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membranes were incubated with rabbit anti-PDCoV polyclonal antibodies prepared in our laboratory at 4 • C overnight, washed three times with 0.01 M pH 7.4 PBS containing 0.05% Tween-20 (PBST), and then incubated with HRP-conjugated goat anti-rabbit antibody (Transgen, Beijing, China) for 2 h at room temperature. The membranes were washed with PBST and visualized by enhanced chemiluminescence reagents and recorded on a Chemi-Doc imaging system (Bio-Rad, Hercules, CA, USA).

Animal Challenge Experiments
There were 12 healthy colostrum-deprived (CD) piglets randomly divided into 6 groups (2 piglets for each group; Table 3). Each piglet was injected via the pre-cava with 3 mL of pSil-double-shRNA-N1-mcherry or pSil-shRNA-NC-mcherry at a dose of 3 mg/pig and an equal volume of phosphate buffered saline (PBS), respectively. At 48 h post-injection, all piglets in groups P + V, N + V, and C + V were inoculated with 10 5 TCID 50 PDCoV orally, while groups P, N, and C were non-infection controls. The clinical signs of the piglets were monitored every 4 h before 12 h of PDCoV infection, including appetite, body weight, diarrhea, and vomiting. Fecal samples were collected by rectal swabs for qRT-PCR analysis at the same time points. All piglets were then euthanized and necropsied to examine the macroscopic lesions, and the small intestines were harvested at 48 hpi. The paraffin-embedded duodenum, jejunum, and ileum tissues were processed for histopathological analysis with hematoxylin and eosin (H&E) staining. The length of the villi and crypts of each small intestine segment were measured respectively and the villous height-to-crypt depth (V/C) ratios were further calculated.

Statistical Analysis
The data were statistically analyzed in GraphPad Prism Software version 5.01 (Graph-Pad Software, San Diego, CA, USA). The data are presented as the mean ± SD for all experiments. A two-tailed t-test was used to assess the significance of the differences between the means. A p-value of <0.05 (*) was considered significantly different and a p-value of <0.001 (***) was considered highly significant.

Anti-PDCoV Activity of pSil-Double-shRNA-N1-Mcherry in Infected LLC-PK1
To examine if there was enhanced inhibition potential of double-shRNA plasmids during PDCoV infection, the LLC-PK1 cell lines expressing pSil-dou N1-mcherry were developed. After inoculation with PDCoV for 36 h, the typi observed microscopically in infected control cells, whereas no apparent CPE in cells expressing double shRNA-N1 sequences ( Figure 4). Meanwhile, PD amounts and N protein synthesis in the samples from pSil-double-shRNAgroup were dramatically reduced as compared with the samples from the sin N1 plasmid group ( Figure 5 and Supplementary Figure S1). The virus titers PK1 cell lines with double shRNAs co-expressing were significantly lower pared with the infected control, which corresponded to approaching over a 215 ( Figure 6). To examine if there was enhanced inhibition potential of double-shRNA-expressing plasmids during PDCoV infection, the LLC-PK1 cell lines expressing pSil-double-shRNA-N1-mcherry were developed. After inoculation with PDCoV for 36 h, the typical CPE was observed microscopically in infected control cells, whereas no apparent CPE was found in cells expressing double shRNA-N1 sequences ( Figure 4). Meanwhile, PDCoV RNA amounts and N protein synthesis in the samples from pSil-double-shRNA-N1-mcherry group were dramatically reduced as compared with the samples from the single shRNA-N1 plasmid group ( Figure 5 and Supplementary Figure S1). The virus titers in the LLC-PK1 cell lines with double shRNAs co-expressing were significantly lower when compared with the infected control, which corresponded to approaching over a 2154-fold drop ( Figure 6).      To further access the cell viability under the influence of shRN fection, an MTS assay was performed on the LLC-PK1 cell lines co mids. As shown in Figure 7, pSil-double-shRNA-N1-mcherry was h tecting the LLC-PK1 cells against PDCoV compared with the infec viability (%) of 81.624 ± 0.024%. However, pSil-shRNA-NC-mCherr ilar impact, only keeping 21.525 ± 0.010% cells alive. These data un ble-shRNA-N1-mcherry exhibited strong potency against PDCoV i when compared with the mock control, the cell viability contribute mCherry and pSil-double-shRNA-N1-mcherry was 96.68 ± 0.025% respectively, which was not significantly different from the mock c the introduction of shRNA into LLC-PK1 cells did not induce obvio To further access the cell viability under the influence of shRNAs during PDCoV infection, an MTS assay was performed on the LLC-PK1 cell lines containing shRNA plasmids. As shown in Figure 7, pSil-double-shRNA-N1-mcherry was highly effective in protecting the LLC-PK1 cells against PDCoV compared with the infected control, with a cell viability (%) of 81.624 ± 0.024%. However, pSil-shRNA-NC-mCherry could not exert similar impact, only keeping 21.525 ± 0.010% cells alive. These data uncovered that pSil-double-shRNA-N1-mcherry exhibited strong potency against PDCoV infection. Remarkably, when compared with the mock control, the cell viability contributed by pSil-shRNA-NC-mCherry and pSil-double-shRNA-N1-mcherry was 96.68 ± 0.025% and 88.099 ± 0.031%, respectively, which was not significantly different from the mock control, indicating that the introduction of shRNA into LLC-PK1 cells did not induce obvious cytotoxicity.
ble-shRNA-N1-mcherry exhibited strong potency against PDCoV infection. when compared with the mock control, the cell viability contributed by pSil-mCherry and pSil-double-shRNA-N1-mcherry was 96.68 ± 0.025% and 88.0 respectively, which was not significantly different from the mock control, in the introduction of shRNA into LLC-PK1 cells did not induce obvious cytoto

Inhibition Effects of pSil-Double-shRNA-N1-Mcherry Plasmids on PDCoV Replication in Neonatal Piglets
To observe the anti-PDCoV effects of the double-shRNAs-expressing plasmids in vivo, the piglets were given paravertebral injections with pSil-double-shRNA-N1-mcherry plasmids, and then challenged with PDCoV. At 36 hpi, the challenged neonatal piglets in groups N + V and C + V started showing depression, anorexia, watery diarrhea, dehydration, and weight loss, and the characteristic gross lesions were discovered in the small intestines (duodenum, jejunum, and ilium), including transparent and thin intestinal walls, mesenteric congestion, and hemorrhage ( Figure 8). In contrast, no clinical symptoms were observed in the piglets from the P + V and non-infection groups; the only pathological changes encountered in postmortem examination at 48 hpi was an intermediate level of yellow fluid accumulation in the small intestine lumen. Histopathological analysis demonstrated moderate villous atrophy in the PDCoV-infected piglets after pSil-double-shRNA-N1-mcherry administration, in which the villus-to-crypt (V/C) ratios of each intestine segment were significantly higher than those of the infection control (Table 4 and Supplementary Table  S1). Notably, the average V/C ratios of the double-shRNA-N1 plasmid group and the shRNA-NC plasmid groups were relatively lower when compared with those of the mock control, indicating that shRNA-expressing plasmids might not completely protect the infection caused by PDCoV, or perhaps had mild cytotoxicity toward the piglets. However, the histological lesions in the P + V group were not more pronounced than those of the positive control group (Figure 9). Therefore, our results evidence that double expression shRNAs specific to the N gene could alleviate the intestinal injury caused by PDCoV infection. Most importantly, as displayed in Figure 10

Discussion
Widely used for gene regulation in mammalian and human cells [31][32][33], RNAi is a conserved biological response to small dsRNA that initiates sequence-specific gene suppression/silencing at the post-transcriptional level [17]. RNAi can be delivered into the cells via two different ways, that is, either through exogenously synthetic siRNAs or plasmid/viral vectors encompassing shRNA−encoding fragments. Although synthesized siR-NAs can effectively induce the degradation of targeted RNA in transfected cells, the effects are transient. The plasmids or virus vectors-based shRNAs are much more stable than RNAs in certain environments and can be processed by a host cellular mechanism to continuously produce siRNAs in the host cells, which can overcome the disadvantages of chemically synthesized siRNAs [21]. Specifically, viral vectors, including lentiviruses, adenovirus, and other self-replicating RNA viruses, could trigger long-term inhibition activity [34].

Discussion
Widely used for gene regulation in mammalian and human cells [31][32][33], RNAi is a conserved biological response to small dsRNA that initiates sequence-specific gene suppression/silencing at the post-transcriptional level [17]. RNAi can be delivered into the cells via two different ways, that is, either through exogenously synthetic siRNAs or plasmid/viral vectors encompassing shRNA−encoding fragments. Although synthesized siRNAs can effectively induce the degradation of targeted RNA in transfected cells, the effects are transient. The plasmids or virus vectors-based shRNAs are much more stable than RNAs in certain environments and can be processed by a host cellular mechanism to continuously produce siRNAs in the host cells, which can overcome the disadvantages of chemically synthesized siRNAs [21]. Specifically, viral vectors, including lentiviruses, adenovirus, and other self-replicating RNA viruses, could trigger long-term inhibition activity [34].
At present, the threat posed by viral infections remains a serious challenge due to the emergence of novel viral pathogens and reemergence of variant strains of known virus species. For fighting viral diseases with RNAi, considerable attempts have been made to date. Previous reports demonstrated that the plasmid vector constructs containing shRNAs targeting CSFV [25], FMDV [26], TGEV [27], and PEDV [28] could effectively inhibit viral replications [35][36][37][38]. RNAi-based technology is becoming a potent therapeutic strategy to exert antiviral activity [39].
PDCoV is a newly emerged enteric pathogen causing severe diarrheal disease and mortality in neonatal piglets, which has resulted in huge economic losses to the global pig industry. PDCoV has access to a variety of host cells, which implies that it may have a propensity for cross-species transmissibility [14]. To date, it remains a serious problem to efficiently prevent the spread of PDCoV infection. As known, the N protein of coronaviruses is a major structural protein that is involved in virus assembly, and plays a critical role in the entire life cycle of a coronavirus [18][19][20]. Therefore, the N gene is an ideal target for designing shRNA to inhibit PDCoV replication. In this study, we constructed four plasmids expressing shRNA targeting the N gene and evaluated whether shRNA-mediated RNAi could inhibit PDCoV infection in vitro and in vivo. Surprisingly, stably expressed shRNAs almost completely blocked PDCoV reproduction in vitro, including shRNA-N1, N2, and N3; while the amount of viral genomic RNAs in the shRNA-N4 group decreased relatively less, which suggests a limited antiviral effect. For this phenomenon, one of the possible reasons is that the efficacy of shRNA-N4 was influenced by the secondary structure on target sites [17]. However, the regarding information has rarely been clarified. To enhance the antiviral effectiveness of the shRNA-N1 plasmid against PDCoV, a double-shRNA-expressing plasmid was further generated in this study. As assessed, pSil-double-shRNA-N1-mcherry plasmids significantly inhibited PDCoV replication both in vitro and in vivo, indicating that PDCoV infection could be suppressed by the combinatorial RNAi (co-RNAi) method [40].
Recombinant viral vector and shRNA-expressing plasmids are the currently common strategies for RNAi delivery into cells. The viral vector could be used to internalize specific shRNA into cells with high efficiency and obtain long-term inhibition. As non-viral vectors, shRNA-expressing plasmids cannot readily cross the membrane into cells and exist stably in vivo. Further studies are needed to mitigate this problem. Because of the rich synthesis of shRNA and the efficient RNAi induced by the human U6 (hU6) promoter, the pSil-double-shRNA-N1 plasmids bearing dual hU6 promoter/shRNA cassettes were established for the transcription of shRNA-N1. Nevertheless, excessive shRNA expression may cause cytotoxicity through competitively inhibiting endogenous miRNAs in cells [41]. In our work, there was no significant cytotoxicity in stably transfected LLC-PK1 cells. However, in the histopathological examination of the different sections of the small intestines of the piglets challenged with PDCoV, both the villus heights and crypt depths decreased in the piglets after shRNA administration alone, compared with the mock control. Lymphocyte proliferation could also be found in the lamina propria of the small intestines. These phenomena may indicate that shRNA-expressing plasmids yielded toxicity effects to some extent in the piglets. Hence, more attention should be paid to eliminate the adverse reaction of exogenous shRNA expression.

Conclusions
The present study evidenced that the double-shRNA-expressing plasmid synergistically targeting the N gene of PDCoV could effectively inhibit PDCoV replication and protect piglets from PDCoV-induced intestinal injury. Our preliminary results demon-