Although several veterinary coronavirus vaccines are currently available, their efficacy is variable. Among them, the infectious bronchitis virus vaccine is very effective for chickens , whereas the canine and porcine vaccines are only partially effective. Furthermore, there is currently no effective antiviral treatment against these virus infections ; therefore, rapid and potent anti-TGEV therapeutic agents are urgently needed. RNAi technology provides an important methodology for rational drug design and gene therapy for many viral diseases, which has proven to be a potent tool for host protection against viral infection, suppression of viral genome transcription, and blocking viral replication [25, 26]. RNAi can be introduced into the cells using two different approaches. The first is chemically synthesized siRNAs. Cells transfected with chemically synthesized siRNAs can achieve rapid and effective silencing of a target gene, but the effects are transient. Second, shRNAs, which can be cleaved by Dicer to produce siRNAs in the host cell, can circumvent the disadvantages of chemically synthesized siRNAs by using stably transfected plasmids or virus vectors [27–29]. The present study demonstrated the use of RNAi against TGEV via shRNA-expressing plasmid vector pGPU6-GFP, which significantly reduced viral genomic RNA replication and protected ST cells from TGEV destruction, by targeting ORF 7 gene.
RNAi is highly sequence-specific and requires 100% identity between the target and targeting sequences to achieve virus clearance from cell culture [30–32]. The application of shRNAs targeting the conserved region of the gene is one way to overcome the lack of knowledge of the target sequence. To ensure that shRNAs can be used for a wide range of virus strains, we evaluated the cross-inhibitory capabilities of these shRNAs by conducting multiple alignments of TGEV ORF 7 gene sequences, based on the available sequences in GenBank. Our results showed that pGPU6-GFP/207, pGPU6-GFP/241and and pGPU6-GFP/276 could cover 95% (19/20), 75% (15/20) and 75% (15/20) of TGEV strains, respectively (data not shown). Interestingly, we have also conducted multiple alignments of the sequences among TGEV, feline coronavirus virus and canine coronavirus, and have shown that they are also conservative. This conservative characteristic of the sequences between the three viruses means that they have greater potential application for the treatment of the diseases caused by these viruses, which also indicates that further studies are necessary to search for the cross-inhibitory effects in a range of TGEV strains and other coronaviruses.
In our study, two strategies were used to detect the inhibitory effect of shRNAs on both the ORF 7 gene and TGEV genome. The first approach was that cells were infected with TGEV after being transfected with shRNAs, in which the shRNAs were used as preventive substances. As shown in Figures 1 and 2, there was a marked decrease in both the ORF 7 and N genes, which could be amplified simultaneously from all the same regions within the TGEV genome RNA and sub-genomic mRNAs (except the shortest one). The reduction showed that the viral genome RNA and its transcripts were decreased significantly. A similar suppression pattern was observed for virus titers. In the second strategy, shRNAs of ORF 7 gene were used as therapeutic agents and transfected after the cells were infected with TGEV. Almost all the cells maintained their normal morphology before they were collected for real-time RT-PCR. The ORF 7 and N genes were reduced significantly when the shRNAs were transfected at 4 h after TGEV infection (Figures 1 and 2). More experiments were carried out at 8 and 12 h after TGEV infection, but the results were not as remarkable as those after 4 h (data not shown), which indicates that shRNAs affect the initial stage of TGEV infection.
In the study by Ortego et al., recombinant TGEV with ORF 7 gene deletion replicated in cell culture with similar efficiency to the wild-type virus, and stably maintained the modifications introduced into the genome . This observation seems to contrast with the results of the present study, which provides evidence that RNAi of the ORF 7 gene could lead to decreased virus replication. There are three possible reasons for this. First, the genome of parental TGEV could be degraded directly because it consists of a positive-sense ssRNA. Second, all the other sub-genomic mRNAs could be degraded as the ORF 7 gene is included in all the sub-genomic mRNAs. As shown in Figures 2 and 4, N gene was significant decreased in the shRNA expressing cells. Besides, the 3′ end of the TGEV genome gene, the downstream gene of ORF 7 gene, could be degraded with the ORF 7 gene according to the characteristics of RNAi, and its degradation could delay the viral replication, because it has been reported that it could interact with host cell proteins and have a great influence on the replication of TGEV . In the study by Cruz et al., it was reported that TGEV without ORF 7 enhanced virulence in infected piglets . We speculate that this phenomenon won’t happen on the RNAi treated pigs, because the RNAi method is different from the reverse genetics technique. By the RNAi used in this study, the other viral sub-genomic mRNAs were degraded (Figure 2 and 4). While the reverse genetics deletion will only affect the ORF 7 gene itself. Nevertheless, whether the technology could be used in vivo still needs more explored.
To elucidate the protection of shRNAs against TGEV in more detail, ST cells stably expressing the shRNAs were established. TGEV replication in these cells was observed through detection of virus titers and real-time quantitative RT-PCR. Cells expressing pGPU6-GFP/207 showed 99% inhibition in the viral genome, whereas pGPU6-GFP/241 showed up to 89% and pGPU6-GFP/276 85% inhibition (Figure 4). TGEV destroys ST cells in two ways, by apoptosis and necrosis [33, 34]. Therefore, CPE and apoptosis were analyzed. Examination of TGEV-induced CPE indicated that ST cells were very sensitive to TGEV infection. Cells expressing pGPU6-GFP/NC formed large bodies and were stained red by PI (Figure 7), whereas cells expressing pGPU6-GFP/207 were not stained red, indicating that the cell membrane was intact. Compared with the condensed, irregular nuclei of pGPU6-GFP/NC-expressing cells, cells expressing pGPU6-GFP/207 were completely protected from apoptosis and the nuclei showed normal morphology. The sequence-specific shRNAs, especially pGPU6-GFP/207, exhibited potent ability to protect ST cells from TGEV-induced destruction. Our present results indicate a close relationship between inhibition of TGEV replication and cell viability by shRNAs. Our data also suggest that siRNA targeting ORF 7 gene can elicit viral RNA from infected cells and potentially offers an efficient therapeutic option for TGEV infection.