Preventing the expression of BDV-NS2-3 using the design specific shRNA as a potential therapeutic method

Border disease first was reported in 1959 from United Kingdom.Patients between England and Wales border had been infected, so the disease calledborder disease [1].Border disease virus (BDV) belongs to the genus Pestivirus, which also include bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV). The genera Pestivirus and Flavivirus and the hepatitis C virus group are included in thefamily Flaviviridae [2]. BDV is the causative agent of an important congenital disease of sheep and is probably distributed worldwide. Intrauterine infections during early pregnancy can cause fetal death and abortions. Alternatively, intrauterine infections during the first half of gestation can result in lambs which show tremor, ataxia, hairy fleece, brain malformations, and poor growth. Affected lambs, termed hairy shakers, are persistently infected (PI) and play an important role in virus transmission [3]. In addition, a syndrome similar to mucosal disease of cattle has been described for sheep infected with ruminant pestiviruses [4].Furthermore, several studies have shown that pestiviruses are not strictly host specific but may infect many species within the Artiodactyla [3].Two biotypes of BDVare distinguished by their effects in cell cultures: the cytopathic biotype (cp) and the noncytopathic one (ncp). Most of the field isolates of BDV are non cytopathic whereas some isolates include the two virus biotypes.The non-cytopathic (NCP) biotype of the virus can cross the placenta and establish a persistent infection (PI) in the developing fetus. This biotype should be regarded as the normal biotype and the cytopathic (CP) biotype is an abnormal virus that is usually isolated only from PI animals dying from mucosal disease [5].Pestiviruses are enveloped particles, spherical in shape and approximately 40–60 nm in diameter. The genome is a positive singlestranded RNA molecule with a length of 12.3 kb which encodes a polyprotein of about 4000 amino acids [6].Generally, 11 to 13 pestiviral proteins can be found as products of polyprotein processing that is mediated by viral and host cell proteases. In the polyprotein, the mature viral proteins are arranged in the following order (from the N to C terminus): Npro, C, Erns, E1, E2, p7, NS2-3, (NS2), (NS3), NS4A, NS4B, NS5A, NS5B. The first protein of the polyprotein, a nonstructural autoprotease (Npro), is followed by the structural proteins C, Erns, E1, and E2.(Fig. 1) The remaining proteins are presumably nonstructural (NS). Processing at the cleavage sites NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B has been shown to be mediated by the NS3 serine protease.In addition, NS3 possesses multiple enzymatic activities, namely NTPase and helicase activity [7].Recent molecular evidence points to CP viruses arising from their NCP counterparts by recombination in regions encoding NS2-3 that include the insertion of host RNA and/or the duplication of viral RNA sequences [5].It has been proposed that recombination results from a template switch of the viral RNA polymerase during genome replication [8].For the BDV, expression of NS3 (p80) is also restricted to cpviruses, while NS2-3 (p125) is expressed from both non-cp and cp isolates.The most conserved pestiviral proteins are represented by NS3 and NS4A, while E2, p7, NS2, and NS5A appear to be least conserved [9].A few decades ago, drugs which were used to treat different diseases were only limited tochemical drugs, peptides, monoclonal antibodies and recombinant proteins [10].RNA interference (RNAi) is now a popular method for silencing gene expression in a variety of systems. RNAi methods use double-stranded RNAs (dsRNAs) to target complementary RNAs for destruction. In mammalian systems, very short dsRNAs (22–25 bp) such as short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) are used to avoid endogenous nonspecific antiviral responses that target longer dsRNAs. siRNAs elicit a transient silencing response, while shRNAs can be expressed continuously to establish stable gene silencing. shRNAs can be introduced into cells and animals using a variety of standard vectors as well as retroviral or lentiviral expression systems [11].Materials Methods:In this study, proper sequences of gene encoding for the BDV-X818 NS2-3 selected using NCBI gene bank (GenBank Accession AF092448). Several shRNAs were designated to silence NS2-3 gene expression using online softwares for siRNA designing (Ambion, Invitrogen, SiDirect, Genescript).The most appropriated suggested siRNAs were 19nt in length . These chosen siRNAs were checked for secondary structure formation using CLC and mfold softwares, as well as low level of off-targets. Then, in order to design hairpin, the proposed vector and loop sequences submitted, so the most effective shRNAs with desired restriction enzymesites were designed.Results Discussion:Four potentially effective shRNA molecules were designed. Their sequences and start positions in the target gene in the table 1.The results showed that there are potentially effective shRNA molecules against NS2-3 gene of BDV that can suppress its proliferation.However, in addition to great success of RNAi strategies in the treatment and prevention of human viruses, the use of this concept against animal viruses should be performed to better understand the effectiveness or ineffectiveness of this method and such studies helps in suppressing viral disease. Our understanding and application of RNAi has dramaticallyadvanced. Despite limitations in developingeffective delivery vehicles and concerns regarding potential off-targetactivity, clinical development of RNAi strategies has been initiated. As the science of thisfledgling technology advances, it is evident that issues such as targetselection, effectors potency, delivery vehicle design, and off-targeteffects will continue to be addressed and resolved. Bi-functional RNAiproducts are evolving components in this transition to clinicallyeffective and safer therapeutics. The interaction of these molecules in the treatment of viral diseases is frequently studied and some research groups tried to inhibit the proliferation of infections caused by viruses such as Flaviviridae. In one of these studies,the suppression of BVDV NS4B and NS5A proteins withsiRNA molecules was evaluated by Lambeth et al (2007). Using these molecules as siRNA targets has been examined by Chisari et al (2003) too.They designed7 siRNA molecules to inhibit hepatitis virus C to test and demonstrate the efficiency of these molecules in reducing viral replication. Adrish Sen et al (2003) designed specific siRNAs against NS5A of HCV genotype 1a and successfully inhibited RNA and protein expression of NS5A in human liver cell lines. They showed that siRNA molecules can inhibit the expression of HCV proteins. BDV establishes persistent infections in sheep populationsworldwide, often resulting in significant economic impacts.Although the control measures of eradication, selective test and slaughter and vaccination was widely used, BDV remainsprevalent due to diversified antigenicity. The efficiency and specificity of RNAi areattractive therapeutic characteristics that may prove useful forthe development of antiviral therapies.In studies done on RNAi against Flaviviridae most attention has been to NS2-3 due to being conserved and its specific functional roles. So in this study shRNAswere designed for NS2-3.