Consensus siRNA for inhibition of HCV genotype-4 replication.
BACKGROUND
HCV is circulating as a heterogeneous group of quasispecies. It has been addressed that siRNA can inhibit HCV replication in-vitro using HCV clone and/or replicon which have only one genotype. The current study was conducted to assess whether siRNA can inhibit different HCV genotypes with many quasispecies and to assess whether consensus siRNA have the same effect as regular siRNA.


METHODS
We generated two chemically synthesized consensus siRNAs (Z3 and Z5) which cover most known HCV genotype sequences and quasispecies using Ambium system. Highly positive HCV patient's serum with nine quasispecies was transfected in-vitro to Huh-7 cell line which supports HCV genotype-4 replication. siRNA (Z3&Z5) were transfected according to Qiagen Porta-lipid technique and subsequently cultured for eight days. HCV replication was monitored by RT-PCR for detection of plus and minus strands. Real-time PCR was used for quantification of HCV, whereas detection of the viral core protein was performed by western blot.


RESULTS
HCV RNA levels decreased 18-fold (P = 0.001) and 25-fold (P = 0.0005) in cells transfected with Z3 and Z5, respectively, on Day 2 post transfection and continued for Day 3 by Z3 and Day 7 by Z5. Reduction of core protein expression was reported at Day 2 post Z3 siRNA transfection and at Day 1 post Z5 siRNA, which was persistent for Day 4 for the former and for Day 6 for the latter.


CONCLUSIONS
Consensus siRNA could be used as a new molecular target therapy to effectively inhibit HCV replication in the presence of more than one HCV quasispecies.
Zekri
Abdel Rahman N
AR
Virology and Immunology Unit, Cancer Biology Department, National Cancer Institute, Cairo University, 1st Kasr El-Aini st, Cairo, Egypt. ncizekri@yahoo.com
Bahnassy
Abeer A
AA
El-Din
Hanaa M Alam
HM
Salama
Hosny M
HM
D045744:Cell Line, Tumor; D015967:Gene Expression Regulation, Viral; D020868:Gene Silencing; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism*; D013997:Time Factors; D014758:Viral Core Proteins / Q000378:metabolism; D014779:Virus Replication
D034741:RNA, Small Interfering; D014758:Viral Core Proteins