Abstract
Background: Border disease is believed to be one of the most important diseases in the animal husbandry industry, which has not yet been eradicated in Iran. The development of approaches based on the application of interfering RNA (RNAi) for antiviral therapy has attracted a great deal of attention over the recent years. The present research was conducted to design, construct, and apply shRNA against the NS3 gene of BDV to evaluate the prevention of BDV proliferation in the cell culture system. For this purpose, the suitable oligonucleotide sequence of NS3 gene coding was selected utilizing BDV- X818 strain. Afterwards, using shRNA design software, shRNA molecules were designed and synthesized. These shRNAs were cloned into the desired vectors and were finally transfected in HEK293T cells employing the third generation of lentiviral packaging system. Subsequently, these shRNA expressing lentiviruses were transduced to the MDBK cell line to challenge to border virus. In order to evaluate the efficacy of shRNAs, the viral infectious titer and RNA copy number were calculated with TCID50 and Real-time RT-PCR tests, respectively.Results: The results revealed that shRNAs 1, 2, and 3 decreased viral RNA by more than 90% compared to the control groups. BDV titer noticeably decreased after the challenge with shRNAs 1, 2, and 3 from ~88% up to 99% in comparison with the control groups.Conclusions: Overall, it could be concluded that RNAi may be considered as a strong treatment proposal against viruses, such as BDV.
Highlights
Border disease is a viral sickness of small ruminants
Afterwards, the HEK 293 T cells were cultured in 10-cm plates and in a confluency of approximately 70–80%; they were cotransfected by three lentiviral plasmids with the names psPAX— a packaging vector (21 μg), pMD2.G—a pseudo-type (VSV-G) envelope vector (10.5 μg), and pCDH containing anti-BDVNS3 short hairpin RNA (shRNA) or Border disease virus (BDV)-NS3 HELICc and binding site cassette as transfer vector (21 μg) using the calcium phosphate protocol according to the instructions of Bonbiotech company (Iran)
At 72 h following the infection of MDBK cells with the prepared lentiviruses, the GFP expression indicated that the lentiviral vectors were efficiently integrated into the cells
Summary
Border disease is a viral sickness of small ruminants. Infertile ewes, abortion, stillbirth, and the birth of tiny, faint lambs that may have a vibration, unusual body conformation, and hairy fleeces are the most prevalent manifestations of the disease. The disease is prevalent all over the world and leads to basic economic losses in animal farms due to certain complications, Inhibition of BDV With RNAi such as weak reproduction in herds and expensive follow-up tests [4]. The present research was conducted to design, construct, and apply shRNA against the NS3 gene of BDV to evaluate the prevention of BDV proliferation in the cell culture system. Afterwards, using shRNA design software, shRNA molecules were designed and synthesized These shRNAs were cloned into the desired vectors and were transfected in HEK293T cells employing the third generation of lentiviral packaging system. These shRNA expressing lentiviruses were transduced to the MDBK cell line to challenge to border virus. In order to evaluate the efficacy of shRNAs, the viral infectious titer and RNA copy number were calculated with TCID50 and Real-time RT-PCR tests, respectively
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