To study the pathogenesis and better development of therapy against HCV there is need of a cell culture based system which supports HCV replication. Previously we have used MDBK, HELA, HEK-293 T and Huh-7 cell lines for viral inoculation experiments and found that Huh-7 cell line supported the HCV replication the most [
23]. Huh-7 derived cell lines, are most widely used infectious cell culture system for liver associated diseases and fundamental studies for the development of antiviral agents against HCV [
19,
27,
28]. Several alternative strategies are being used to back up the viral component in the different models. Subgenomic replicons system is one of the most commonly used cell culture system to investigate HCV-RNA replication and different steps of viral life cycle [
29]. Despite of its effectiveness it can not exactly mimics the actual HCV replication cycle and shedding of viral particles to the medium. In spite of viral replication, the biologically relevant infectious viral particles cannot be demonstrated by such approach. Buck et al. and Molina et al. has successfully infected human primary hepatocytes with the serum of patients infected with HCV genotype 1, 2, 3 and 4 and found efficient HCV replication [
21,
22].
In the present study, we used serum of HCV genotype 3a infected patient, the most prevalent genotype in Pakistan [
3,
4] to infect Huh-7 cell line. Recently, it is demonstrated that both 5' and 3' untranslated regions of the viral HCV RNA genome play a pivotal role in translation of viral proteins via interaction with cellular factors including eukaryotic initiation factor 3 eIF3 [
30], 40S ribosomal subunit [
31], poly pyrimidine tract binding protein (PTB) [
32] and microRNA 122 [
33]. Besides, it has been shown that intra genetic viral interactions such as NS4a/NS5a are required for key pathways in HCV life cycle. The hypothesis of using HCV infected serum in the present study was, it would have full length HCV genotype 3a RNA genome ensuring the presence of all the necessary ingredients involved in viral replication and poly protein precursor to infect Huh-7 cells
in vitro. Our results showed the presence of HCV RNA in serum infected cells from day 1 to 40 post serum inoculation. The serum infected cells steadily showed the expression of viral core gene (Figure
1). In the media HCV RNA was not detected till 10
th day due to fact Huh-7 cells were not shedding viral particles in to culture medium (Figure
2). This may be due to lack of active exocytosis of viral particles showing absence of replication intermediate [
21]. It is interesting to find that Huh-7 showed continuous expression of core protein from 2
nd day of infection to 40
th day suggesting that replication of HCV is going on in serum infected Huh-7 cells (Figure
3A, B). This finding is in accordance with the earlier work done by El-Awady and his coworkers, who reported HCV genotype 4a serum infection in HepG2 cell line and found that viral proteins started to express themselves 1 week post infection [
21]. We presume that our
in vitro system is highly likely to mimic the
in vivo HCV replication. Our work is in agreement with the earlier reports of infection experiments [
19,
23,
24].
Furthermore, in this study we evaluated siRNA based HCV genome silencing in our persistent HCV infection Huh-7 cell model that efficiently supported HCV replication up to 40 days. In order to confirm that the HCV is self replicating or due to HCV carry over, we subjected our persistent HCV infection model in Huh-7 cells (post 40 days) to HCV RNA silencing through our previously reported core gene specific siRNA [
34]. Excitingly we found significant HCV genotype 3a replication inhibition (
P-value = 0.00) nearly up to 92% after 24 h (Day 1) post transfection but HCV titer started to increase after 48 h (Day 2) onward and showed continuous replication from 72th hours (Day 3) (Figure
4A), whereas the control siRNA (scramble) did not show any effect on inhibition of HCV replication. HCV replication in the Huh-7 cells was observed through semi quantitative RT-PCR by using core specific primers. Western blot results also showed significant down regulation of core protein at day one of post transfection (Figure
4B). These results validate our previous study in which we found significant inhibition in transiently infected Huh-7 cells. Our data is in disagreement with Zekri et al. [
26] who demonstrated that siRNAs against 5'UTR of HCV genotype-4 inhibited HCV replication in serum infected Huh-7 cells up to 7 days. Our results clearly depict that the most epic inhibitory effect of siRNA was seen 24 h post transfection. The difference may be due to the selection of two different HCV regions and genotype.