RNA interference targeting UL25 gene as a gene therapy approach against BHV-1 virus

Background: In recent decades RNAi has become a novel and effective tool to silence gene expression. The expression of shRNAs against target genes has now become a standard and powerful strategy for antiviral therapeutic approaches. Infectious bovine rhinotracheitis (IBR) is a domestic and wild ruminant viral disease that caused by bovine herpesvirus-1. Occasionally, its entry into the herd may cause vigorous economic damage due to losing weight, reduced output and limitation on livestock international trade. Vaccines against the disease are not fully effective and the need for an effective therapeutic approach is highly felt. In the present study an RNAi- based gene therapy was designed and implemented. Due to the essential role of UL25 gene for BHV-1 replication. Method: the sequence of UL25 gene was purposed for designing shRNA molecules. The UL25 gene sequence was extracted from the NCBI database and suitable shRNA molecules were purposed by using online soft wares. Three recombinant lentiviral vectors expressing short hairpin RNAs (shRNAs) versus the UL25 gene of IBRV were constructed and co-transfected into HEK 293T cells. The effectiveness of designed shRNA molecules was assayed by calculating TCID50 titers and observation of BHV-1 cytopathic effects. Results: The nal calculations showed that all shRNAs had antiviral effects. In the TCID50 test, the shRNAs 1, 2 and 3 suciently decreased BHV1 output in comparison with the control groups (almost 98.22%, 99.63% and 99.54%, respectively than cells with IBRV inoculation and nearly 90.84%, 97.9% and 96.84%, respectively than cells with ORFV and scrambled vector inoculation). ShRNA-2 achieved maximal inhibition of viral replication. Conclusion: The results indicated that shRNAs targeting the UL25 gene showed considerable antiviral attributes and reduced IBRV multiplication in MDBK cells. In conclusion; RNA interference can be used as a gene therapy instrument against BHV-1 virus.


Introduction
Bovine herpesviruses-1 causes a viral disease of domestic and wild cows which is called bovine infectious rhinotracheitis (Turin et al., 1999). The major host of the virus is cattle, but other small ruminants may also be infected. After high fever, anorexia, sudden depression, the signs of respiratory infections, abortion in pregnant animals, conjunctival rhinorocyte and traumatic in ammation may occur.
Although BHV-1 infection isn't a common life-threatening pathogen, it is a risk factor for the expansion of additive bacterial infections and ultimately causes severe economic losses due to the reduction of healthy products and the limitation of international trade (Biswas et al., 2013).
Bovine herpesvirus-1 belongs to the genus Varicellovirus, subfamily Alphaherpesvirinae and the family Herpesviridae. The virus genome is a double-stranded DNA with the size of 135301 bp that is divided into 4 segment such as a unique long (UL) segment, a unique short (US) segment and two inverted repeat region that named inverted internal repeat (IR) and terminal repeat (TR) sequences. Two isomeric forms of the viral genome organized by UL, US and inverted repeat regions. 73 open reading frames (ORF) that encode for 70 proteins have been identi ed in BHV-1 genome (Bowman et al., 2006). The UL25 gene is one of the 40 nuclear genes of the virus and is conserved in the all members of herpesvirinae subfamily (Bowman et al., 2006). This gene is located at the position 60602-62398 of the BHV-1 genome and codes a 63 KDa tegument protein with 598 amino acids which is necessary in the accumulation and packaging of genomic DNA into the capsid structure (Desloges et al., 2001). BHV-1 virus has distributed into three subtypes such as BHV-1.1, BHV-1.2, and BHV-1.3. BHV-1.1 often is linked to the respiratory manifestation of the disease (IBR), BHV-1.2 causes reproductive infections and BHV-1.3/BHV-5 is a neuropathogenic agent that is associated with neurological disorders (Muylkens et al., 2007). It is worth noting that all subtypes are antigenically equivalent and indistinguishable with typical serological tests (Keuser et al., 2004).
Over the years, continuous researches have been conducted to discover effective anti-IBR vaccines or therapeutic agents. Although, currently, several vaccines are available for the disease; but none of them are completely speci c and effective. Therefore, the necessity of providing e cient and speci c anti BHV-1 preventive/ therapeutic methods is felt (Song et al., 2016).
The RNAi pathway is a process in which two strands RNA molecules with speci c length and secondary structure degrade the homologous RNA targets or suppress the expression of their complementary genes.
This pathway in the cell is naturally aimed to protect the genome against external genetic threats such as viral genes, transgenes and internal threats like transposons. Furthermore , it is responsible for regulate gene expression and development (Ma et al., 2007).
Nowadays the RNA interference pathway has become a mere biological phenomenon to a powerful therapeutic tool for treating a wide range of diseases, including viral infections. Ease of use, rapidity, excellent performance and remarkable speci city when exerted at various phases of virus-host interplay are some of the potential advantages of using the RNAi pathway as an antiviral approach over traditional methods such as antiviral drugs or vaccines (Fischer et al., 2004).
RNAi is supplied to the cell from short interfering RNA (siRNA) or short hairpin RNA (shRNA) to down regulate the expression of purposed genes. Many studies have shown shRNA offers advantages in silencing the target genes such as stability, cost-effectiveness and ease of delivery. The shRNA expression cassettes are consistently integrated into the host cell genome and suppress expression of the target gene by homologous mRNA degradation without change in other mRNAs. Thus, RNAi seems to be an appropriate option as a therapeutic method for protecting plant and animal species against various viruses (Rao et al., 2009;Narute et al., 2009).
In the present study, due to the essential task of the UL25 gene in BHV-1 multiplication [11], this gene sequence was chosen to design shRNA molecules against IBR disease. For a better e ciency, shRNA molecules must be designed to disrupt expression of conserved regions of the viral target gene. So by using NCBI database, the sequence of BHV-1-UL25 gene (Accession number: AJ004801.1) was obtained and was aligned with the sequence of this gene in the other strains of virus by CLUSTAL OMEGA online software. Therefore the most homologous regions were selected for further analyzes. Several shRNA molecules were designed using three online web servers such as WI siRNA selection program, BLOCK-IT RNAi designer and www.invivogen.com/siRNA-wizard. The sequences of proposed shRNA molecules were matched to the target gene sequence to nd those which target the conserved regions. Since it is important that shRNA molecules should easily access to the UL25 mRNA, CLC Genomic Work brench software was applied to predict the secondary structure RNA of UL25 gene and the position of shRNA molecules that are related to the conserved regions in this structure. Selected sequences were submitted to a BLAST search against the cattle genome sequence to ensure that the host genome was not targeted. After the subsequent investigation of optimal CG percentage and off-target effects, three shRNA sequences were nalized. The sequences of the cut-off site of the BamHI and EcoRI restriction enzymes were placed on both sides of shRNA sequences and TCAAGAG were placed as a loop sequence. Afterwards, the oligonucleotides were synthesized by Bioneer Co.

2.3.Preparation of anti-BHV1-UL25 shRNAs
The proposed shRNA molecules were synthesized as single-strand oligoes by Bioneer Co. and were annealed with each other. In order to annealing, a mixture of 1µL of sense oligo (200 µM), 1µL of antisense oligo (200 µM), and 2 µl of sterile deionized water were mixed, then the mixture was placed in a boiling water at 95 ° C for 4 minutes to remove all possible secondary structures. Finally, it was placed at room temperature for 10 minutes to form the desired double strands.
2.4.2. Production of Lentiviral transfer Plasmaid: To construct lentiviral plasmids carrying shRNA sequences, pCDH-CMV-MCS-EF1-CGFP-T2A-Puro lentiviral plasmid was double-digested with BamHI (Roche, Germany,) and EcoRI (Roche, Germany). The Ligation reaction was carried out with T4 10X ligation buffer (Fermentas, Germany, Cat. No.: B69), T4 DNA ligase (10U) (Fermentas, Germany), synthetized shRNA oligos and lentiviral plasmid and were transformed in the E.coli DH5α. Then, colony PCR reaction was performed using the general primers of pCDH-CMV-MCS-EF1-cGFP-T2A-Puro lentiviral plasmid. The sequences of primers were as follows: CMV-F: AATGGGCGGTAGGCGTGTA -3'and EF1-R: 5'-GGACTGTGGGCGATGTG -3'. The PCR thermal cycle program was consisted of denaturation at 95 º C for 5 min followed by 30 cycles at 95 º C for 30 s, 55 º C for 40 s and 72 º C for 60 s, followed by a nal extension at 72 º C for 10 minutes. Finally the recombinant lentiviral plasmids were sequenced by Bioneer Co. Subsequently, transduced MDBK cells were infected by BHV-1 and monitored for the development of cytopathic effects. Finally TCID50 titers were calculated for the wells that infected with the BHV-1 and shRNA expressing lentivectors and positive (cells infected with only BHV-1 or BHV-1 and scrambled lentivector) and negative controls (cells infected with scrambled lentivector or without viral infection).

Design of shRNAs:
Several shRNA molecules were designed using online soft wares and three of them were selected based on de ned parameters and secondary structure status. The sequences of three anti-BHV1-UL25 shRNA oligonucleotides have been listed in the table 1. Secondary structure of viral mRNA for UL25 gene was predicted by CLC software and the interaction sites with the designed shRNAs are shown in gure 1. <Figure 1 near here> ShRNA expressing lentivector production: After co-transfection of transfer and mock lentiviral plasmids with psPAX2 and pMD2G, the expression of the EGFP reporter protein was evaluated by uorescent microscopy and since the shRNA sequence was at the upstream of EGFP gene, the production of lentivectors that expressed shRNAs was con rmed (Fig.  2).

<Figure 2 near here>
Challenge of BHV-1 and lentivector expressing shRNAs: After lentiviral vectors infection, 48 h and 72 h pi, MDBK cells were observed using a uorescent microscope and EGFP expression was monitored for con rmation of shRNA-expression (Fig. 3).
The development of cytopathic effects in MDBK cells was monitored after inoculation of BHV-1. The cytopathic effects observed in the cells were co-infected with BHV-1 and shRNA expressing lentivectors and compared with positive controls (the cells were infected with only BHV-1 and those were infected by BHV-1 and scrambled lentivector) and normal cells as negative control. The results showed that cells infected with anti-BHV-1 shRNAs visibly reduced CPE 72h after the infection, compared to cells of positive control group (Fig. 4). <Figure 3 near here> <Figure 4 near here> TCID50 assay: The TCID50 assay was performed to evaluate the e ciency of shRNAs to inhibit IBRV replication. The nal calculations showed that in the TCID50 test, the shRNAs 1, 2 and 3 also significantly reduced virus yield compared to the control groups (approximately 98.22%, 99.63% and 99.54%, respectively compared to cells infected with ORFV and approximately 90.84%, 97.9% and 96.84%, respectively compared to cells infected with ORFV and scrambled vector) ( Figure 5).

Discussion
In spite of many attempts have been made to discover and apply effective vaccines or antiviral agents, the disease remain prevalent and the effectiveness of common available vaccines isn't ideal (Romera rt al., 2014;Song et al., 2016)..
It is established that treatment based on altered target gene expression and employing a natural intracellular pathway has fewer side effects than other therapies. Therefore, RNAi has been considered as a powerful strategy for the treatment of many viral diseases in recent years (Perrimon et al., 2010).
Despite the advantages of using siRNA molecules for therapeutic applications against acute viral infections, the use of shRNA molecules that triggers a more stable gene expression and less off-target effects seems to be more appropriate. On the other hand, so far, some features of lentiviral vectors include extensive tissue tropism, persistent gene expression and low carcinogenic risk has been reported in the in vivo conditions (Kafri et al., 2000). Considering the advantages of lentivectors and shRNA molecules, in the present study lentivectorbased shRNAs were selected to suppress gene expression.
UL25 gene is one of the 40 core IBRV genes that is conserved among all members of the alpha-, beta-, and gamma herpesvirinae subfamilies. Its role in DNA packaging and attainment of the tegument layer by the virion is very important and therefore in the present study it UL25 gene was chosen.
In recent years many studies have been conducted for applying RNAi pathway to suppress gene expression in order to determine the role of unknown genes or providing antiviral strategies against many After performing TCID50 assay, the results indicated that silencing viral gD gene expression was successful (Song et al., 2016). In the present study three shRNAs against IBRV-UL25 gene were constructed and expressed via lentiviral vectors. We demonstrated the designed shRNAs could successfully inhibit IBRV multiplication in vitro. As demonstrated by TCID50 assay, shRNA2, shRNA3 and shRNA1 downregulated the IBRV replication in the amount of 97.9, 96.84 and 90 percent, respectively.
Finally, it should be stated although the results of the present study veri ed the feasibility of a suitable treatment against IBRV infection via RNAi pass way in the cell culture system, the use of this method in the in vivo system requires further investigation and studies.

Declarations
Declarations of interest: none Submission declaration and veri cation: This work has not been published previously and it is not under consideration for publication elsewhere. Its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out. If the manuscript accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright-holder.
Use of inclusive language: Authors ensure that writing is free from bias Authors' contributions: EA and SS designed shRNAs and performed the cloning and cell culture tests. AM and BS calculated and interpreted the results. AM performed the nal interpretation and wrote the manuscript.
Role of the funding source: This work was supported by grants from Shahrekord University (97GRN1M1801).