Lentivirus expressing shRNAs inhibit the replication of contagious ecthyma virus by targeting DNA polymerase gene

Leila Asadi Samani,Ehsan Arefian,Behnaz Saffar,Azam Mokhtari

doi:10.1186/s12896-020-00611-4

Leila Asadi Samani, Ehsan Arefian + Show 2 more

Open Access

https://doi.org/10.1186/s12896-020-00611-4

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Journal: BMC Biotechnology	Publication Date: Mar 23, 2020
Citations: 5	License type: open-access

Affiliation: University of Tehran, Shahrekord University

Abstract

BackgroundContagious ecthyma or Orf is known as a zoonotic disease remains prevalently worldwide despite the application of some control strategies against it. RNAi particularly shRNA provides us with the chance to tackle this obstacle by an encouraging new approach. The current study indicates the design and experiment of third-generation lentivirus packaging systems delivering shRNAs to inhibit Orf virus (ORFV) replication and infection. Given the importance of DNA-pol gene in virus replication, in this study, three shRNAs against this gene were designed and cloned into lentiviral vectors to stabilize the expression of shRNAs. After producing lentivectors expressing ORFV-DNA– pol in HEK293T cells, the synthesized shRNAs were applied to downregulate viral replication and gene expression. The reduction in viral titer and RNA was evaluated by TCID50 test as well as real-time RT-PCR. The results were then analyzed in comparison with the control group.ResultsDesigned shRNAs significantly reduced virus yield approximately 90 to 97% and 96.8 to 99.4%, respectively compared to the control groups (cells infected with ORFV and infected with ORFV and scrambled vector) by TCID50 test. Real-time RT-PCR revealed a dramatic reduction in the expression of viral RNA approximately 99% compared to cells infected with ORFV and from 92.6 to 99%, respectively compared to cells infected with ORFV and scrambled vector.ConclusionsTherefore, it can be stated that RNAi is capable of being used as a potent therapeutically option against viruses like ORFV.

Highlights

Contagious ecthyma or Orf is known as a zoonotic disease remains prevalently worldwide despite the application of some control strategies against it
The survival rate of the cells infected with ORFV-Short hairpin RNA (shRNA) was higher than the infected cells with the scrambled lentivector, which indicates the lower development rate of the cytopathic effects of Orf virus in these cells (Fig. 1c)
The inhibition of ORFV replication using shRNAs was determined by Enhanced Green fluorescent protein (GFP) (EGFP) expression in transduced cells with lentiviral vector and observed for Cytopathic effects (CPE) induced by ORFV

Summary

Introduction

Contagious ecthyma or Orf is known as a zoonotic disease remains prevalently worldwide despite the application of some control strategies against it. The current study indicates the design and experiment of thirdgeneration lentivirus packaging systems delivering shRNAs to inhibit Orf virus (ORFV) replication and infection. The virus affects the skin and mucosal membrane of ruminants and as a zoonotic pathogen can infect humans, especially those working with animals [2, 3]. While recent advances in research on Orf virus provide a new understanding of the basic mechanisms of the virus pathogenesis, none of the existing vaccines induces complete and long-term immunity; vaccinated animals can be re-infected. There is no effective vaccine against Orf disease in humans, sheep or goats [5]

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