In this study, we have demonstrated that the addition of VA RNAI
, a known IFN antagonist and inhibitor of dsRNA-mediated antiviral pathways, permitted the persistent growth of a plasma-derived HCV in a cell line that lacks IFN genes. Most of the current knowledge of HCV biology and pathogenesis has been derived from the use of the unique JFH-1 cell culture system, which now allows the study of the complete virus life cycle, including entry, assembly and release. The limitation of this model, however, is that robust viral growth is restricted only to hepatic-derived cell lines such as Huh7.5 and Huh7 cells 
and only by a genotype 2a replicon-derived virus. The establishment of an alternative model to characterize other HCV genotypes from infected individuals is still needed and is critical for the development of efficient viral therapies to control the disease.
By passaging genotype 1b virus-infected VeroE6 cells for 20 weeks in the presence of VA RNAI and more than 2 years without VA RNAI, we generated a persistently infected cell line that expresses HCV antigens at levels high enough to be detected by immunofluorescence and Western blot (, ). We found that the LB-piVe virus is highly cytotoxic, and is capable of inducing massive Huh7.5 cell death (); indicating that the virus produced in the persistently infected cells is infectious to hepatocytes. CPE could be blocked by antibodies to CD81 (), by anti-HCV-specific immunoglobulins () and by anti-E2 monoclonal antibodies (), confirming the link between cell death and viral infection. While neutralization was not as potent using the anti-E2 monoclonal antibodies, we believe that this may be due to the antibodies being raised against recombinant genotype 1a proteins. The genotype differences in the E2 proteins (including hypervariable domains) may be reflected in loss of epitope recognition, thus explaining the 0.6 log10 decrease in viral titer.
The LB-piVe virus-mediated CPE has the advantage that it can be assessed visually, and quantified easily and rapidly. This represents a significant improvement over the current genotype 2a HCVcc systems that utilize FFU assays, RT-PCR or reporter assays for quantitation, which are both laborious and time-consuming 
. In addition, we have demonstrated the utility of this system in virus neutralization studies () and in testing virus inhibition by well characterized HCV-specific antivirals ().
CPE was observed in VeroE6 cells and more-exaggerated CPE was found when filterable supernatants were used to infect Huh7.5 cells. While it was possible to enhance viral titer by panning the LB-piVe cells, and effectively increasing the number of virus-infected cells, the new culture could not survive after several passages. We suspect that the virus cannot be maintained in a culture that demonstrates massive CPE, such as that seen in Huh7.5 cells. This may be the reason that we were unable to obtain persistently infected Huh7.5 cell line, while VeroE6 cells can support persistent HCV infection due to a low-level display of CPE.
HCV- associated cell death has also been reported in Huh7.5.1 cells after infection with JFH-1 when HCV RNA levels reached a maximum 
. Gene expression profiling of HCV-infected Huh7.5 cells showed both the presence of activated caspase-3 and induction of cell death-related genes, suggesting an association of virus infection with cytopathic effects. Although not yet resolved, it has been postulated that HCV could mediate direct apoptosis by deregulating the cell cycle, which may contribute to liver injury in infected individuals 
. While still requiring further studies and more comparisons between human pathology and cell culture, we suggest that the LB-piVe system may very well mimic a natural HCV infection in humans and could represent a useful tool to study the intricate process of viral pathogenesis.
VeroE6 cells were also permissive for replication of genotype 2a J6/JFH-1 virus 
(). VA RNAI
boosted replication and spread in these cells, as shown by the increase in the HCV RNA yield (). This may be attributable to an increase in viral RNA stability and possibly reflects the type of interplay between host and virus. The presence of VA RNAI
allowed for broadened cell tropism by HCV to include non-hepatic cells ( and S1
), perhaps due to its ability to circumvent the IFN-induced antiviral response. The full-extent of the mechanisms employed by VA RNAI
towards overcoming the negative effects of IFN is currently unknown. VA RNAI
is important to adenovirus infection and confers virus stability in the presence of IFN and IFN-induced proteins. It has been suggested that VA RNAI
has an effect on HCV RNA stability by inhibition of the IFN-induced protein, ADAR1 
. When we compared the relative amount of HCV RNA in VA RNAI
-transfected cells versus -untransfected cells () that were infected with HCV-positive human plasma LB, we observed a 60-fold increase in VeroE6 cells. Interestingly, a precipitous decline in HCV RNA was observed in these cells in the absence of VA RNAI
(). Thus, VA RNAI
has an effect in the VeroE6 cells, at least during the first 8 days of infection. We have yet to evaluate possible defects in the RIG-I pathway observed previously in Huh7.5 cells and likely to play a role in early infection 
. The fact that we did not observe any increase in the relative amount of HCV RNA in Huh7.5 cells after VA RNAI
transfection followed by infection with the parental genotype 1b serum-derived virus (), was unexpected. We suspect that the relatively stable amount of viral RNA reflects extremely low viral replication of the LB virus in Huh7.5 cells. These findings are supported by the evidence that we could not establish a persistently infected cell-line with Huh7.5 cells, suggesting that VeroE6 cells were more permissive for persistent infection, perhaps due to the lack of IFN genes.
was not able to rescue the virus during 2′-C-Me-A treatment and does not stimulate replication nor does it protect the virus from an antiviral that targets the HCV polymerase. We used this RNA polymerase inhibitor to evaluate RNA stability in the absence of viral RNA replication. Since VA RNAI
only increased the HCV RNA in the presence of viral replication, we believe that it may perhaps inhibit cellular factors that are activated during viral replication (e.g.
, dsRNA-binding proteins) and cause instability of the virus. It's possible that VA RNAI
interacted with, and therefore blocked ADAR1 and PKR pathways. This would be consistent with our previous findings showing that the HCV replicon was stimulated by knock-down or inhibition of ADAR1 or PKR 
. Additionally, the inhibition of RNA replication (including loss of negative strand RNA) should inhibit the formation of dsRNA intermediates, thus avoiding the activation of dsRNA-activated proteins that can lead to viral instability 
. Again, we cannot exclude the possibility that VA RNAI
enhanced viral replication in the absence of the polymerase inhibitor. Taken together, these data suggest that VA RNAI
may possibly contribute to establishing a persistent infection in VeroE6 cells; however, the presence of VA RNAI
alone is not enough to overcome the cellular antiviral response in Huh7.5 cells. IFN-deficient VeroE6 cells probably provide a more ideal environment for a virus that is, usually, IFN responsive. We suspect that this may be due to the decreased expression of IFN-induced proteins which may actively inhibit HCV replication 
. Both Huh7.5 cells and VeroE6 cells express PKR and ADAR1, but only the VeroE6 cells lack the IFN genes that induce these proteins. We found that even the persistent virus was stimulated by the presence of VA RNAI
, suggesting that some of the dsRNA-activated proteins were still expressed and were inhibitory to the virus.
In patients, in general, genotype 2 and 3 viruses are more sensitive to current antiviral therapy than the genotype 1 viruses 
. Genotype 1b is thought to be the most IFN resistant and the most prevalent in North America, Europe and Japan. However, the HCV replicons (genotype 1b) and J6/JFH1 virus are sensitive to IFN in cell culture. It is not clear why viruses respond to IFN differently in vivo
versus in vitro
. Since HCV grows well in VeroE6 cells, especially when assisted by VA RNAI
, we suggest that endogenous IFNs may limit HCV replication in cell culture.
We suspect that the LB-piVe virus, like the parental LB from which it was derived, was relatively resistant to IFN (), a property that has not yet been reported in infectious cell culture (). While it warrants further investigation, it may be possible that we were able to obtain this virus because VA RNAI
was present in the early stages of infection and inhibited the antiviral response generated by viral RNA replication. Our results on the enhancement of virus replication by VA RNAI
are clearly consistent with evasion of the antiviral response, and correlate with the observation that susceptibility of human primary hepatocytes to HCV infection could be improved by impairing expression of other IFN signaling factors such us interferon regulatory factor-7 (IRF-7) 
. We suggest that in the early stages of cell culture infection, before viral proteins are in sufficient quantity, the innate immune pathways are active and control infection (RIG-I, PKR, ADAR1, RNaseL, etc.). However, once the virus is given a chance to accumulate, it can overcome these mechanisms of host control, either through the E2, NS5A or NS3 proteins 
Our findings have raised some interesting questions. Future studies with IFN-sensitive viruses, complemented with known IFN-resistant HCV proteins (such as NS5A and E2) using sequences from the LB-piVe virus, are planned. Additionally, the LB-piVe virus will be ideal for evaluating the genes responsible for conferring IFN resistance. We plan to construct an infectious clone and a replicon based on this virus with the aim of evaluating individual genes. At the same time, alignments with the IFN-resistant parental strain of LB with IFN-sensitive genotype 1b replicons may enable the identification of important amino acids that determine IFN resistance. Transient transfection experiments complementing the IFN-sensitive replicons will be among the experiments that will provide insights into the identification of the features that may confer IFN resistance by this genotype 1b virus.
In summary, here we demonstrate that wild-type HCV genotype 1b viruses from human plasma can replicate in African green monkey kidney cells, VeroE6, and that replication of viral genotypes 1a and 2a can be stimulated by the presence of VA RNAI. This is a new approach to culturing HCV and the first report of a cell culture system that represents a convenient assay for studying genotype 1b. This is an improvement in terms of utility for research, as the virus can be titrated without employing error-prone, quantitative RT-PCR methods nor arduous immunocytochemistry-based focus forming assays. The availability of the LB-piVe virus raises an exciting possibility; potentially opening a new era of HCV research through the use of a new model system. Moreover, a persistently infected cell line that exhibits CPE provides a novel assay that may be conducive to high throughput development and screening of new antivirals.