Lentivirus expressing shRNAs inhibit the replication of contagious ecthyma virus by targeting DNA polymerase gene.
DNA polymerase
Lentiviral plasmid
ORFV
RNAi
Real-time PCR
TCID50
Journal
BMC biotechnology
ISSN: 1472-6750
Titre abrégé: BMC Biotechnol
Pays: England
ID NLM: 101088663
Informations de publication
Date de publication:
23 03 2020
23 03 2020
Historique:
received:
24
11
2019
accepted:
06
03
2020
entrez:
16
4
2020
pubmed:
16
4
2020
medline:
16
6
2021
Statut:
epublish
Résumé
Contagious 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. Designed 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. Therefore, it can be stated that RNAi is capable of being used as a potent therapeutically option against viruses like ORFV.
Sections du résumé
BACKGROUND
Contagious 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.
RESULTS
Designed 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.
CONCLUSIONS
Therefore, it can be stated that RNAi is capable of being used as a potent therapeutically option against viruses like ORFV.
Identifiants
pubmed: 32293394
doi: 10.1186/s12896-020-00611-4
pii: 10.1186/s12896-020-00611-4
pmc: PMC7092477
doi:
Substances chimiques
DNA, Viral
0
RNA, Small Interfering
0
DNA-Directed DNA Polymerase
EC 2.7.7.7
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
18Subventions
Organisme : Iran National Science Foundation
ID : 95849043
Pays : International
Organisme : Shahrekord University
ID : 95GRN1M730
Pays : International
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