Patent Application: US-56488604-A

Abstract:
the present invention relates to hepatitis c virus . more particularly , the invention relates to the development of a tool suitable for the search , discovery and validation of novel hcv antiviral drugs and therapies . the invention further relates to methods for inducing hcv replication in vitro , and more particularly to a simple in vitro replication assay for hcv . in addition , the invention relates to the use of the methods of the present invention to prognose the resistance / sensitivity of a particular strain of hcv to a chosen anti - hcv agent . in one embodiment , the present invention relates to an adaptation of a therapeutic regimen for a patient infected with hcv which takes into account the resistance / sensitivity phenotype of the hcv strain which infects same . the invention more particularly , relates to a method for generating an established cell line which produces hepatitis c virus comprising transforming peripheral blood mononuclear cells which produce hcv with epstein barr virus . the invention also relates to an ebv established b - cell line capable of replicating complete and infectious hcv . as well , the invention relates to a cell - based in vitro replication system for hcv comprising an ebv - transformed b - cell capable of replicating complete and infectious hcv , and a second cell population having hcv tropism and in which robust hcv replication occurs , so that under appropriate culture conditions the second cell population can become infected by the infectious hcv produced by the ebv - transformed b - cell . the present invention also relates to kits for transforming a hcv - producing cell and to kits for diagnosing hcv in a patient .

Description:
the existence of extrahepatic reservoirs of hepatitis c virus ( hcv ) replication remains controversial . several groups have described the presence of hepatitis c virus ( hcv ) genomic sequences ( plus - strand ) and replicative . intermediate ( minus - strand ) in peripheral blood mononuclear cells ( pbmc ). the association of hcv rna with peripheral blood leukocytes has been documented since 1992 however , the specificity of the methods used in these studies has been questioned . more recent reports , which used an optimized negative strand - specific reverse - transcriptase polymerase chain reaction ( rt - pcr ) assay , detected negative - strand hcv only in pbmc taken from post - transplant or human immunodeficiency virus ( hiv )- coinfected hcv patients , and not in pbmc from typical patients with chronic hcv infection . of note , a number of studies have also reported that human b and t cell lines are capable of supporting a productive infection . however , the data supporting viral production was only based on rna detection ( proc . natl . acad . sci . usa , 1992 , 89 : 5477 ; j virol . 1993 , 67 : 1953 ; ibid , 1996 , 70 : 3325 - 9 ; ibid , 70 : 7219 - 23 ; hepatology 1996 , 23 : 205 ; antiviral research 2001 , 52 : 1 - 17 ). the validity of these data have been questioned ( laskus et al . 1998 , see below ). moreover , pbmc obtained from hcv negative donors were successfully infected using hcv - positive sera , demonstrating that pbmcs are permissive for hcv replication in vitro ( j . gen . virol . 1995 , 76 : 2485 - 2491 ). however , replication of the virus therein was really low . in addition , only rna was detected . thus , prior to the present invention , it remained unclear whether hcv could actively replicate to workable levels in pbmcs . using an immunodeficiency ( scid ) mouse model that allows long - term survival of human hematopoietic cells , bronowicki et al . ( 1998 ) presented strong evidence for persistence of hcv rna in pbmcs obtained from hcv positive donors ( hepatology 1998 , 28 : 211 - 218 ). the susceptibility of pbmc to hcv infection has been corroborated by in situ hybridization techniques showing both positive and negative polarity rna strands in circulating and / or bone marrow recruited mononuclear cells . recent reports have established that hcv is in fact associated to b cells . based on the model of epstein - barr virus another b - cell - tropic virus , that remains latent while the host cell is quiescent but is reactivated and enters a lytic replication phase once the host cell is activated ( j . virol . methods , 1988 , 21 : 223 - 227 ; annual rev . microbiol . 2000 , 54 : 19 - 48 ), boisvert et al ., ( 2001 ) examined the possibility that hcv could replicate in peripheral b cells , but under altered physiological conditions , such as immunosupression or cellular activation . the authors could not detect hcv replication in enriched b cells obtained from hcv positive donors upon cell stimulation with cd40l . considering the observations of laskus et al ., ( 1998 ) showing the presence of active hcv replication in lymphoid tissue in patients coinfected with hiv ( not in non - hiv infected patients ), suggesting that co - infection of hiv would be required in hcv cell - based assay , and those of boisvert et al ., ( 2001 ), we hypothesized that hcv replication in peripheral blood leukocytes ( pbml ) requires cell activation ( e . g . in the mixture of the t - and b - cell population ). until now , all studies of hcv replication have concentrated on documenting the presence of the replicative intermediate ( minus - strand ). rna . however , the validity of these reports has been criticized because the presence of viral proteins was not demonstrated . it stands to reason that in order for replication to occur , protein expression is required . therefore , in order to sustain the observations relating to activated pbmcs , it was important to show protein expression . thus , non - structural ( ns ) hcv proteins were chosen as an indicator of viral replication ( see fig3 , 5 - 9 , 13 - 16 , 21 , 24 - 27 , 31 - 36 , 40 , 42 - 43 and 53 ). the studies presented hereinbelow clearly demonstrate that pbmcs obtained from hcv seropositive donors are able to support at least one complete cycle of viral replication upon activation . for this , a simple method that actively induces virus replication within the infected cell was developed . most circulating leukocytes are . in a resting state , but remain responsive to mitogenic signal that can induce cell activation . lymphocyte activation in response to extrinsic signals results in either progression through the cell cycle , or activation of proapoptotic pathway ( s ) ( cell 1991 , 65 : 921 - 923 ; science 1996 , 274 : 1664 - 1672 ). lymphocyte activation correlates with a strong increase in translation rates and expression of translation initiation factors ( j . immunol . 1998 , 160 : 3269 - 3273 ). the change in the cellular environment associated with immune activation could induce hcv protein synthesis and initiate a cascade of events leading to an impaired cell cycle and an enhanced viral replication . in accordance with the present invention , the activation of pbmcs ( or pbls ) is achieved using at least one mitogenic ( or activating agent ). in one particular embodiment , the activating agent is a mixture of antigen - nonspecific t and / or b cell activators ( anti - cd3 antibody , phytohemagglutinin ( pha ), cd40l , staphylococcus aureus crown i ( sac ), il2 and il4 ). of course , it will be realized that other t and b cell activating agents exist and are well - known in the art . such agents could be used in the methods and culture systems of the present invention . the result of activation upon hcv viral replication can be seen in fig6 - 9 , 13 - 14 and 16 - 22 . in one particular embodiment , ag - specific t and / or b cell activating agents could also be used . it will also be understood that the present invention provides assays which can be used to identify further activating agents , mixtures thereof or other nutrients which can further activate the hcv - producing cells of the present invention and / or promote a longer survival thereof in culture . for example , having shown that pbmcs or pbls can be activated to replicate hcv , other inducers and mixtures thereof can be tested , and the hcv production or replication cycle monitored to identify other inducers or combination thereof ( cheaper , more efficient , more adapted to specific strains or the like ). in accordance with one embodiment , hcv non - structural proteins ( ns3 and ns5 ) were chosen and detected by western blot analysis . virus - like particles could be detected within the infected cells by electron microscopy demonstrating that viral proteins are assembling . viral particles could be isolated from the pbmcs supernatant . the presence of virus was evidenced from western blot ( anti - core ) analysis and genomic rna detection by real time rt - pcr , this observation shows that upon assembly , viral particles were actively being liberated to the supernatant . moreover , using a co - culture method ( see for example fig3 , 15 and 27 ) it was demonstrated that the hcv particles produced in pbmc could infect other cells ( fig3 ). non - limiting examples thereof include liver cells such as huh - 7 ( fig3 and 17 - 18 ), daudi ( b - cell ) ( fig2 ), mt4 ( t - cell ) cell lines ( fig1 ), naïve pbls and thus b and t cell lines as well as primary lymphocytes . thus , not only is it shown that hcv can replicate , and assemble in the tissue culture system of the present invention , but it is also shown that it can also infect other cells . infection was monitored by detection of viral rna ( real time rt - pcr ). the results generated by these experiments has a significant impact on the testing of anti - hcv agents . of course , it also serves as a proof of principle that pbmc are able to sustain hcv infection and generate infective hcv . moreover these data strongly suggest that both the serum and pbmcs obtained from hcv positive donors can be used as a source of infectious virus to infect naïve cells such as monocyte and / or monocyte - derived dendritic cells ( dcs ). therefore , the instant invention , which enables the infection of cells with hcv , is by itself a significant achievement . isolated nucleic acid molecules of the present invention are meant to include those that result from any known method , such as chemically synthesized . similarly , an oligomer which corresponds generically to a hcv nucleic acid molecule , or to a strain specific hcv nucleic acid , can be synthesized . such synthetic oligonucleotides can be prepared , for example , by the triester method of matteucci et al , j . am . chem . soc . 103 : 3185 - 3191 ( 1981 ) or by using an automated dna synthesizer . an oligonucleotide can be derived synthetically or by cloning . if necessary , the 5 ′- ends of the oligomers can be phosphorylated using t4 polynucleotide kinase . kinasing of single strands prior to annealing or for labeling can be achieved using an excess of the enzyme . if kinasing is for the labeling of probe , the atp can contain high specific activity radioisotopes . then , the dna oligomer can be subjected to annealing and ligation with t4 ligase or the like . the present invention relates in one embodiment , to a nucleic acid for the specific detection , in a sample , of the presence of hcv nucleic acid sequences . in one preferred embodiment , the present invention relates to oligomers which specifically target and enable amplification ( i . e . primers ) of hcv rna sequences associated with infection . in one embodiment , the amplified product can be detected following hybridizing with a probe which consists of an isolated nucleic acid consisting of 10 to 1000 nucleotides ( preferably , 10 to 500 , 10 to 100 , 10 to 50 , 10 to 35 , 20 to 1000 , 20 to 500 , 20 to 100 , 20 to 50 , or 20 to 35 ) which hybridizes preferentially to an amplified product which originated from hcv rna , wherein said nucleic acid probe is or is complementary to a nucleotide sequence consisting of at least 10 consecutive nucleotides ( preferably , 15 , 18 , 20 , 25 , or 30 ) from the known hcv polynucleotide sequence . amplification of chosen hcv sequences is exemplified herein . of course , other sequences , primers and probes could be used in accordance with the present invention . primer in accordance with the present invention can be designed as commonly known in the art based on the sequences of hcv available publicly or of the sequences of hcv shown herein . of course , as will be understood by the person of ordinary skill , a multitude of additional probes or primers can be designed from the same or other region of hcv . the hybridization probes of the present invention can be labeled by standard labeling techniques such as with a radiolabel , enzyme label , fluorescent label , biotin - avidin label , chemiluminescence , and the like . after hybridization , the probes can be visualized using known methods . the nucleic acid probes of the present invention include rna , as well as dna probes , such probes being generated using techniques known in the art . in one embodiment of the above described method , a nucleic acid probe is immobilized on a solid support . examples of such solid supports include , but are not limited to , plastics such as polycarbonate , complex carbohydrates such as agarose and sepharose , and acrylic resins , such as polyacrylamide and latex beads . techniques for coupling nucleic acid probes to such solid supports are well known in the art . the test samples suitable for nucleic acid probing methods of the present invention include , for example , cells or nucleic acid extracts of cells , or biological fluids . the sample used in the above - described methods will vary based on the assay format , the detection method and the nature of the tissues , cells or extracts to be assayed . methods for preparing nucleic acid extracts from cells , purifying cells and the like are well known in the art and can be readily adapted in order to obtain a sample which is compatible with the method utilized . iii . a method of detecting the presence of hcv in a sample in another embodiment , the present invention relates to a method of detecting the presence of hcv nucleic acid in a sample comprising a ) co - culturing a cell sample with a target cell under hcv - activating conditions , and b ) detecting the presence of the hcv nucleic acid using an amplification method . one skilled in the art would select the nucleic acid primers according to techniques known in the art as described above . in another embodiment , hcv protein is detected using a hcv - specific ligand , such as an antibody iv . a kit for detecting the presence of hcv in a sample in another embodiment , the present invention relates to a kit for detecting the presence of hcv in a sample comprising at least one container means having disposed therein at least one primer pair . in a preferred embodiment , the kit further comprises at least one further container comprising one or more of the following : amplification reagents , probes , wash reagents and reagents capable of detecting the presence of bound nucleic acid probe . examples of detection reagents include , but are not limited to radiolabelled probes , enzymatic labeled probes ( horse radish peroxidase , alkaline phosphatase ), and affinity labeled probes ( biotin , avidin , or steptavidin ). in detail , a compartmentalized kit includes any kit in which reagents are contained in separate containers . such containers include small glass containers , plastic containers or strips of plastic or paper . such containers allow the efficient transfer of reagents from . one compartment to another compartment such that the samples and reagents are not cross - contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another . such containers will include a container which will accept the test sample , a container which contains the probe or primers used in the assay , containers which contain wash reagents ( such as phosphate buffered saline , tris - buffers , and the like ), and containers which contain the reagents used to detect the hybridized probe , bound antibody , amplified product , or the like . one skilled in the art will readily recognize that the nucleic acid probes described in the present invention can readily be incorporated into one of the established kit formats which are well known in the art . of course , the kit can also be based on a detection of hcv protein as opposed to hcv nucleic acid . in one embodiment , a first container would contain an antibody specific to a hcv protein . in a particular embodiment , the kit is adapted to be used on hcv expressing cells obtained by the co - culturing system of the present invention , or by the immortalizing system of the present invention . it is to be understood that although the following discussion is specifically directed to human patients , the teachings are also applicable to any animal that expresses hcv . according to the invention , presymptomatic and post - symptomatic screening of an individual is now possible . this is especially valuable for the identification of non - symptomatic carriers of hcv . early diagnosis is also desired to maximize appropriate timely intervention . probes that detect a chosen hcv sequence may be labeled with any of a variety of labels that can , directly or indirectly , result in a signal when the probe is hybridized to the amplified target sequence . for example , a label may be any moiety that produces a luminescent , fluorescent , radioactive , or enzymatic signal that can be detected by using methods well known in the art . a probe need not be labeled with a label moiety if binding of the probe specifically to the amplified nucleic acid containing the exon - exon junction results in a detectable signal , such as , for example a detectable electrical impulse . adoptive transfer of donor - derived virus - specific t cells generated in cultures with antigen - bearing autologous monocyte - derived dendritic cells ( dcs ) has attracted considerable attention as a promising tool to generate a strong immune response ( int . j . cancer . 2001 , 94 : 459 - 73 ; exp . hematol 2001 , 29 : 1247 - 55 ; trends mol . med . 2001 , 7 : 388 - 94 ). this technique has not only proved useful as an alternative anti - cancer strategy but also as a novel anti - virus therapy . for example , when dcs were pulsed with human cytomegalovirus virus ( hcmv ) antigen and cocultured with autologous peripheral blood lymphocytes from hcmv - seropositive individuals , there was an increase in the numbers of cytolytic t cells . this technique was used to enhance immunity in hcmv - seropositive transplant patients ( blood . 2000 , 97 : 994 - 1000 ). now having developed a technology to infect cells with hcv , it becomes possible to adapt the dendritic cells ( dcs ) technology mentioned above , to generate t - cell responses to hcv . advantages for using dcs for this purpose include the fact that : i ) they are considered the most potent of the antigen - presenting cells ( apcs ) ( blood . 1997 , 90 : 32453287 ; nature . 1998 , 392 : 245 - 252 ); ii ) their role in resistance against experimental malignancies and infections is well documented ( j . immunol . 1998 , 161 : 2094 - 2098 , j . virol . 1998 , 72 : 3812 - 3818 ); iii ) dcs can be easily generated from bone marrow , cord blood , and peripheral blood ; iv ) dcs have the unique ability to process exogenously supplied antigen efficiently and present peptides on both class 1 and class 2 hla molecules along with an array of costimulatory molecules ( nature . 1998 , 392 : 245 - 252 ; nature . 1999 , 398 : 77 - 80 ); v ) the presentation of both helper and ctl - defined epitopes suggests that both cd4 + and cd8 + hcv - specific t cells will be generated . this will allow both the generation of cytolytic effector function and the potential for re - establishment of longer - term immune memory , which may be important in preventing subsequent viral reactivation ; and vi ) the lack of an absolute knowledge of the presented peptides means that this technique can be used for patients of any hla type and will trigger t - cell reactivity to undefined immunogenic determinants , thereby allowing a greater potential for augmentation of a broader t - cell response . it is thus expected that this will reduce the possibility that selective pressure will be applied to hcv in vivo . based on the foregoing , it is predicted that the approach described herein ( together with possible adaptations by a person of ordinary skill using the knowledge in the art ) will contribute significantly to the design of a vaccine therapy towards hcv infection . vii . robust hepatitis c virus replication in peripheral blood lymphocytes from infected donors there is considerable evidence that hepatitis c virus ( hcv ) resides in an extrahepatic reservoir . although peripheral blood lymphocytes ( pbls ) have been suspected of harboring hcv , virus production was not achieved in these cells despite many attempts . here , we show that pbls from hcv positive , injection drug users , harbor the virus and support viral replication . hcv replication was activated by ex vivo cell stimulation , with the use of a mixture of t and b cell activators . the presence of viral positive and negative rna strands and hcv proteins is documented herein . virus particles were isolated from cell supernatant and analyzed by density gradients centrifugation . virus structural proteins and viral rna could be readily detected in the supernatant of activated pbls by western blotting and real time rt - pcr , respectively . virus particles contain de novo synthesized genomic rna and structural proteins as shown by metabolic labeling with 32 p - orthophosphate and 35 s - labeled aminoacids . finally , hcv particles , released from cells , are infectious as demonstrated by co - culturing . studies using this novel hcv replication system should contribute to the understanding of the virus life cycle , host - virus relationship , pathogenesis and importantly , to the discovery and validation of new anti - hcv agents . to examine hcvs extrahepatic replication , we used pbls from seventy - eight hcv positive , hiv - negative , injection drug users ( idus ; all obtained with written consent ; table s1 detailing the available information on the participants is included in the on - line below ). pbls from the idus were treated with a mixture of t and b cell activators to show replication of hcv and infectivity of the de novo produced virus . the rationale behind the selection of idus as a source of pbls is addressed below . viii . hcv (+) and (−) strand rna and viral proteins are produced de novo in activated pbls . viral rna was detected in non - stimulated and stimulated pbls from a hcv positive donor by nested rt - pcr ( fig2 a ). viral rna was not detected in hcv negative donors or in negative controls ( fig2 a ; note that nested rt - pcr is neither strand specific nor quantitative ). these results confirm early evidence showing that pbls harbor hcv rna ( 12 - 16 ). to obtain quantitative results , total rna extracted from activated cells was subjected to a strand specific real time rt - pcr analysis to demonstrate the presence of hcv (−) rna strand ( fig2 b ). the kinetics of hcv rna induction was similar in activated pbls from two carriers , mll - 038 and mll - 039 ( fig2 b ). the amount of (−) strand rna increases slightly , but significantly , early ( 1 day ) upon cell activation then decreases at later times ( 1 - 3 days ), but increases again afterwards ( 5 - 7 days ) ( fig2 b ). although these kinetics are not readily explained , the presence of hcv (−) rna strand supports the notion of virus replication in pbls . the hcv life cycle is cytoplasmic ( 5 ), therefore , to show that rna synthesis occurs in the cytoplasm , bromo - substituted uridine ( bru ) together with actinomycin d ( actd ) was added to stimulated pbls ( 19 ). incorporated bru was detected by immunofluorescence using antibodies to 5 ′- bromodeoxyuridine ( 19 ). cytoplasmic rna synthesis was detected in activated hcv positive pbls from two hcv positive donors ( fig2 c and 28 ). in contrast , no incorporation of bru was detected in actd treated pbls from a hcv negative donor ( fig2 d ). in the absence of actd , strong incorporation of bru in newly synthesized rna was detected in the nucleus ( fig2 c and d ). taken together , these data clearly show that hcv rna synthesis occurs in activated pbls from idus . next , we wished to document hcv - directed translation in pbls . upon mitogen stimulation of hcv positive pbls , ns3 and ns5b proteins were readily detected by western blotting using several different antibodies ( fig2 a - c ). the quantity and kinetics of ns3 appearance was dependent on the particular procedure of stimulation ( fig2 d and e ) and the hcv carrier ( fig2 f - h ). this suggests that the kinetics of hcv protein production in stimulated pbls is modulated by host factors . to show that the appearance of the proteins , which interact with the ns3 and ns5b antibodies , is dependent on hcv replication , we used sirna against the core protein coding sequence ( fig2 i - k ). ns3 and ns5b levels decreased drastically following electroporation of the core - sirna in a dose - dependent manner when compared a to a non - specific unrelated rna ( inverted 4e - t - sirna ; see materials and methods , below ) ( fig2 i ). sirna silencing resulted from a decrease of hcv rna , as compared a to a non - specific rna , as demonstrated by real - time pcr quantification ( fig2 j , k ). the presence of core protein in the cytoplasm of activated hcv positive pbls was further confirmed by indirect immunofluorescence ( fig2 ). based on surveying 10 fields , we estimate that 1 to 3 % of the cells expressed high levels of hcv core protein . taken together , the data demonstrate that translation of the hcv (+) strand rna ( fig2 and 25 ) and transcription of the (−) strand rna ( fig2 ) occur in activated pbls . to examine whether hcv particles are produced and released into the culture medium , the supernatant from pbls was harvested and sedimented by centrifugation through a 20 % sucrose cushion . the presence of hcv particles was demonstrated by western blotting with an anti - core monoclonal antibody , mab225p ( fig2 a ). similar results were obtained when other anti - core antibodies ( monoclonal 515s ( 20 ) and polyclonal rr8 ) were used ( data not shown ). viral rna co - sedimented with the hcv core protein as demonstrated by nested rt - pcr ( fig2 b ). pbls were stimulated by methods b , p and ps ( detailed in example 2 ) and genomic rna isolated from the cell supernatant was quantified by real time rt - pcr ( fig2 c ). consistent with the protein data shown above , the amount of viral rna in the cell supernatant varied among the different stimulation procedures ( fig2 c ). to further support the evidence for virus production , particles were examined following metabolic labeling with 35s - methionine / cysteine ( fig2 d - g ). particles were sedimented through a 20 % sucrose cushion , resuspended and floated on optiprep ™ density gradients ( 21 ) ( fig2 d ). the sedimentation range of the labeled particles ( 1 . 13 - 1 . 215 g / ml ) was similar to that reported by others ( 22 - 28 ). hcv - e2 protein was present in the particles as determined by western blotting using monoclonal anti - e2 1864 ( fig2 e ). the absolute quantity of hcv (+) strand rna present in each faction was determined by real - time rt - pcr ( fig2 f ). the hcv genomic rna and e2 co - sedimented through the density gradient ( fig2 f ). interestingly , western blotting revealed that the hcv core protein sedimented throughout the gradient ( data not shown ). to further examine this behaviour , fractions 1 - 4 and 5 - 11 from the gradient were pooled , and the presence of hcv e2 and core proteins was determined . the high ( h ) density complexes ( 1 . 111 to 1 . 215 g / ml ) contained e2 and core protein and are likely to represent viral particles , while the low ( l ) density complexes ( 1 . 006 to 1 . 1 g / ml ) contained only core ( fig2 g ). the biological significance of this observation is not immediately clear . however , it was suggested earlier that different types of particles are found in serum from chronically infected individuals ( 23 , 29 ), and in the supernatant of cells expressing the full length hcv rna ( 21 ). rna and proteins were isolated following metabolic labeling with 35 s - methionine / cysteine or 32 p - orthophosphate ( the latter in the presence of actd ) to determine whether the viral proteins and genomic rna isolated from the different fractions was synthesized de novo . supernatant was collected after labeling ( fig2 h ). significantly , labeled rna and proteins co - sedimented through the density gradient ( fig2 h ). thus , the results show that virus particles containing de novo synthesized proteins and genomic rna were released to the supernatant . it was highly pertinent to examine whether the hcv particles released from stimulated pbls are infectious . as it is impossible to estimate the real ratio of infectious to non - infectious virus particles produced by activated pbls , a co - culture strategy , in which two different cell types in two chambers are separated by a 0 . 45 μm polyethylene terephthalate track - etched membrane , was used ( fig2 a and fig4 ). the htlv - 1 transformed t cell line , mt4 was chosen as the target cell of infection ( 30 - 33 ). total rna was extracted from infected cells and the quantity of hcv rna was determined . strikingly , viral rna ( average of 1600 copies / μg of total rna ; as determined by real - time rt - pcr , data not shown ) and ns3 protein were detected in mt4 , upon co - culture with activated pbls ( fig2 b ), demonstrating that the released viral particles are infectious and that cell - to - cell contact is not required for infection . no viral proteins were detected in mt - 4 cells when co - cultured with pbls from two hcv negative donors ( fig2 b ). in conclusion , we demonstrated that hcv replication occurs in pbls . without being limited to a particular theory , our success in showing replication , while earlier studies failed , can be attributed to two important factors : activation of the pbls and the use of idu donors . idus were selected because they experience a long - term altered immune response ( 34 - 36 ) and hcv replication in pbls has been associated with induced immunodeficiencies ( 37 - 39 ). drugs have a variety of effects on the immune system including suppressed cell - mediated immunity ( 34 - 36 ). this is reflected in a depressed level of t - dependent antibody production by b lymphocytes and in an alteration of t lymphocyte function . the clinical consequences of this suppression include an increase in the incidence of viral infections such as hiv and hcv ( 40 - 42 ). thus , our observations support the notion that immunosuppression in combination with cell activation act as “ cofactors ” in hcv pathogenesis . studies including hcv infected individuals who are not idus and non - idu immuno - suppressed individuals are required to support this hypothesis . it is most probable that hcv enters lymphocytes during the primary infection and remains latent in resting cells . viral latency is well documented for epstein - barr virus ( ebv ), which remains dormant in quiescent host b - cells , but enters a lytic replication phase once the cell is activated ( 43 , 44 ). interestingly , ebv can also infect t cells ( 45 , 46 ). therefore , a number of intriguing parallels can be drawn between the hcv and ebv life cycles . it is conceivable that like in ebv infection , t cell immunity plays a critical role in limiting the number of hcv infected pbls and that during a sustained immunodeficiency state , such as that manifested in idus , clonal proliferation of virus infected cells will be favored . most importantly , in this report we describe a simple cell - based system that supports robust hcv replication . the implications of these findings are paramount for several reasons . first , they clearly implicate pbls in hcv pathogenesis . second , they provide a model that should be useful in the quest to gain understanding of the hcv life cycle , host - virus relationship , viral infectivity and in the discovery and validation of novel anti - hcv agents . for the latter purpose we have established ebv - transformed b - cell lines from hcv - infected donors which should facilitate the discovery of anti - hcv drugs ( see below ). we also provide methods to establish b - cell lines which fully replicate hcv ( see below ). a number of antibodies can be used in accordance with the present invention . non limiting examples thereof include ns3 polyclonal antibody , monoclonal anti - ns5b and monoclonal anti - ns3 . more specifically , monoclonal anti - ns3 antibody , 1g3d2 and polyclonal anti - ns3 , k135 which were from dr . d . lamarre ( boehringer ingelheim canada ltd ). ns3 rabbit anti - serum - rb provided by dr . r . bartenschlager , department of molecular virology , institute of hygiene , university of heidelberg , germany and monoclonal anti - ns5b , 5b - 3b1 from dr . d . moradpour , department of medicine ii , university of freiburg , germany , were also used . monoclonal anti - e2 1864 ( 450 - 470aa ), monoclonal anti - 5b 10 ( ifa ), monoclonal anti - core 515s ( 2040aa ), and core rabbit anti - serum rr8 were developed in the tokyo metropolitan institute of medical science . monoclonal anti - core ( cat . no . : mab255p ; lot : hcv - core - 2 - 4 ) was purchased from maine biotechnology services , inc . monoclonal anti - human f - actin ( ab205 ) was purchased from abcam limited . monoclonal anti - human β - actin ( clone ac - 15 ) was purchased from sigma - aldrich co . anti - bromodeoxyuridine monoclonal antibody - alexa fluor 488 conjugated , and goat anti - rabbit alexa fluor 594 conjugated were purchased from molecular probes , inc . of course , other antibodies ( or combination of antibodies ), whether polyclonal or monoclonal can also be used . participants were recruited through the drug addiction unit of the saint - luc hospital of the centre hospitalier de l &# 39 ; université de montral ( chum ) and the saint - luc cohort study . characteristics of the donors can be found at table s1 . donors provided a written informed consent approved by the chum review board before having their blood drawn . individuals from both sexes ( 87 % males ) were enrolled in this study between 2001 and 2003 . their mean age was 42 . 1 years ( sd ± 8 . 8 ) and the average time since their first injection was 16 . 5 years ( sd ± 9 . 6 ). 80 % of the donors reported injecting drugs during the 6 month period before blood was withdrawn for this study . cocaine and opiates were the most frequently used drugs , with 77 % and 34 . 6 % use , respectively . all hcv positive donors tested positive in a serological screen for hcv antibodies performed in the laboratory of microbiology at saint - luc hospital of the chum using two enzyme linked immunosorbent assays ( elisa , axsym and cobas ). presence of hcv was confirmed by hcv - rna detection when elisa data were discordant . all participants recruited for this study were hiv - 1 and hiv - 2 negative . serological screening for hiv antibodies was performed in the microbiology laboratory at saint - luc hospital , chum , with an enzyme - linked immunosorbent assay ( elisa ). similar procedures were used to verify the hcv negative donors . hcv negative donors ( six ) were recruited from the different participating laboratories as well as from the support staff responsible for the st . luc cohort . peripheral blood ( 20 ml ) was collected from hcv positive idu or hcv negative donors into edta - containing vacutainer tubes ( becton dickinson ). polymorphonuclear leukocytes and red blood cells were separated by centrifugation over a density gradient ( lymphocyte separation medium , cellgro ®). monocytes were then removed by plastic adherence under serum free conditions as described in the current protocols of immunology textbook . when required , cells were frozen in 10 % dmso containing fcs and stored at − 80 ° c . prior to monocyte separation . total pbls were cultured in 24 - well plates at 1 × 10 6 cells per ml in rpmi 1640 supplemented with 10 % heat - inactivated fcs and antibiotics . mitogens were added to the media ( rpmi 1640 , 10 % fbs , and antibiotics ) upon starting the culture and maintained throughout the experiment . the protocols used for pbcls stimulation were as follows : method a , pbls were grown in the presence of irradiated l4 . 5 cells ( murine fibroblasts expressing the cd40 ligand , cd154 ) as described ( 49 ). method b , 1 μg / ml of anti - cd3 and 200 u / ml of il - 2 ( sigma - aldrich co ) were added . method p , 3 μg / ml phytohemagglutinin ( pha , sigma - aldrich co ), and 200 u / mi il - 2 were used . method ps , 1 : 10 4 vol / vol of staphylococcus aureus cowan fixed cells ( sac , calbiochem ) in combination with phytohemagglutinin and 200 u / ml il - 2 were added to the media . method s , 1 : 10 4 vol / vol of sac and 200 u / ml of il - 4 ( sigma - aldrich co ) were used . cell activation was verified by flow cytometry . cells were rinsed twice with 1 ml cold phosphate buffered saline ( pbs : 137 mm nacl , 2 . 7 mm kcl , 4 . 3 mm na 2 hpo 4 , 1 . 4 mm kh 2 po 4 , ph 7 . 4 ) and fixed in 80 % ethanol / pbs for 30 min at 4 ° c . pbs ( 2 volumes ) was added and cells were pelleted by centrifugation . cells were rinsed twice with 2 ml pbs and then resuspended in 0 . 5 ml pbs containing 0 . 2 μg / ml rnase a and incubated for 40 min at 37 ° c . propidium iodide was added to a final concentration of 1 . 2 μg / ml and samples were analyzed by flow cytometry using a single laser facs instrument ( becton - dickinson ) combined with the cellquest ™ software . total rna was extracted from cells using trizol ™ ( invitrogen ) according to the manufacturer &# 39 ; s protocol . yeast trna ( 1 mg / ml ) was added as a carrier . rna was resuspended in nuclease - free water ( sigma - aldrich co ). total rna was quantified by phosphoimagerm ( storm system , molecular dynamics ) using the ribogreen ™ rna quantification kit ( molecular probes , inc ). hcv - rna was detected in cells by a reverse transcription - polymerase - chain reaction ( one step rt - pcr reaction , 45 cycles , qiagen ) against the highly conserved 5 ′ untranslated region ( sense primer from nucleotide 13 to 38 and the anti - sense primer from nucleotide 383 to 359 ) of the hcv genome ( strain h77 pcv - h77c , embl : af011751 , medline : 97385173 ) followed by a second round of amplification , nested pcr ( 45 cycles , sense primer from nucleotide 59 to 82 and the anti - sense primer from nucleotide 307 to 285 , strain h77 pcv - h77c ) using taq dna polymerase ( mbi fermentas ). β - actin was amplified ( 30 cycles ) using the sense primer 5 ′- gtggggcgccccaggcacca - 3 ′ and antisense primer 5 ′- gtccttaatgtcacgcacgatttc - 3 ′. method i : reverse transcription was carried out at 50 ° c . for 20 minutes in a one - tube two - step rt - pcr reaction with thermoscript ™ reverse transcriptase ( invitrogen ), 10 μm of hcv - tagged strand - specific rt primer and 100 μm of anti - sense gapdh primer ( table s2 ). the reverse transcriptase was inactivated by heating for 5 minutes at 95 ° c . and pcr ( 22 cycles ) with platinum taq dna polymerase was performed in a trio - thermocycler ™ ( biometra ): at 94 ° c . for 45 s , 60 ° c . for 60 s , 68 ° c . for 2 min . the first round pcr products were then amplified for 40 cycles in the roche lightcycle ™ instrument : denaturation at 95 ° c . for 60 s , and amplification and quantification at 95 ° c . for 15 s , 60 ° c . for 10 s with a single fluorescence measurement , 72 ° c . for 15 s . real - time quantification of rna copy numbers for hcv and the human gapdh gene was based on a set of eight log 10 external standards covering 10 8 to 10 1 plasmid copies of a pcrii vector containing the 5 ′ hcv leader ( genotype 1a ) and the gapdh normalization pcr amplicons which were run in parallel with the test samples . rna extracted from pbls of a hcv negative donor was used as control . as a reaction control for the strand - specific signal , the rt step of the rt - pcr was carried out without a hcv - tagged primer . the presence of hcv non - structural proteins in the cell samples used for rna preparation was confirmed by western blotting ( data not shown ). method ii : real - time rt - pcr was performed on the abi prism 7700 sequence detection system using the taqman ez rt - pcr kit ( applied biosystems ). rna sample ( 5 μl ), combined with 45 μl of reagent mix , was used for the real - time rt - pcr reaction . in vitro transcribed replicon rna was used as a standard to determine hcv copy numbers ( 1 μg of replicon rna equals 2 . 15 × 10 11 hcv copies ). the rna copy number was normalized ( ribogreen ™ rna quantification , molecular probes inc .) and expressed as genome equivalents per ml of total supernatant . bromo - uridine ( bru , 5 - bromouridine 5 ′- triphosphate , sigma - aldrich co ) was incorporated into pbls using a modified version of the procedure of haukenes et al . ( 50 ). bru ( 10 mm ) was incubated with an equal volume of lipofectamine ™ 2000 transfection reagent ( invitrogen ) for 30 min at room temperature and added to 250 μl of cells resuspended in optimem ™ medium ( invitrogen ) in a 1 : 1 ( vol / vol ) ratio . the bru / lipofectamine ™ 2000 mixture was added to cells 6 h after activation . cells were incubated for 5 h , washed and resuspended in mitogen ( method p ) containing culture medium . cells were collected after a 12 h incubation period at 37 ° c . in a 5 % co 2 environment . when actinomycin d ( actd , sigma - aldrich co ) was used , cells were incubated with the drug ( 5 μg / ml ) starting 30 min prior to the addition of bru . actd was maintained throughout the experiment . immunofluorescence was performed on 5 × 10 4 cells . following cytospin for 7 min at 1100 rpm in a cytosin 2 ( shandon ), cells were dried for 30 min at room temperature and fixed for 30 min at − 20 ° c . in a mixture of acetone and methanol ( 1 : 1 vol / vol ). cells were blocked for 30 min at room temperature in 10 mm tris - hcl ph 8 . 0 containing 1 % bsa . slides were washed 3 times with pbs and incubated at room temperature for 2 h with the polyclonal anti - core rr8 antibody ( 1 / 50 ) or overnight at 4 ° c . with the anti - bromo - deoxyuridine alexa fluor 488 conjugate antibody ( 2 μg / ml ) in a humidified box . slides were washed 3 times with pbs . for core detection , slides were incubated 1 h at room temperature with an alexa - 594 conjugated antibody ( dilution 1 / 250 ). dapi staining was performed for 7 min at room temperature ( 1 μg / ml final concentration ). mounted slides ( permount mounting medium , fisher scientific ) were stored overnight at 40 ° c . prior to analysis . conventional epifluorescence micrographs were obtained on a zeiss cell observer ™ system equipped with an axiovert ™ 200 m microscope using the 100 × oil lens . images were digitally deconvoluted with the axiovision 3 . 1 ™ software using the nearest neighbor ™ deconvolution method that uses the agard &# 39 ; s formula . proteins extracts were prepared by sonification in ripa buffer ( 150 mm nacl , 1 % np - 40 , 0 . 5 % doc , 0 . 1 % sds , 50 mm tris - hcl ph 7 . 5 ) and quantified ( bsa assay , biorad ). proteins ( 10 ag of extracts from pbls or 5 μg of extract from huh7 cells stably expressing the hcv replicon ( 47 )) were resolved on sds - 10 % polyacrylamide gels ( page ) and transferred to 0 . 2 pm protran ™ nitrocellulose membrane ( schleider and schuell ) for 1 h at 100v . the membrane was blocked with pbs containing 0 . 5 % tween - 20 ( pbs - t ) and 5 % nonfat dry milk . blots were then incubated with the primary antibody for 2 h at room temperature , washed 3 times with pbs - t and incubated for 1 h with a horse radish peroxidase ( hrp ) conjugated secondary antibody . blots were visualized using an enhanced luminol reagent ( ecl ; perkinelmer life sciences inc ). a total of 1 × 10 6 activated pbls were first preincubated in methionine - or phosphate - free rpmi for 30 min , and then incubated for 12 h in the same media supplemented with [ 35s ] protein labeling mix ( 1175 ci / mmol ) or carrier - free inorganic 32 p ( 500 μci / ml , h 3 po 4 , icn biomedicals , inc ), the latter in presence of actd ( 5 μg / ml ). supernatant was collected , cells and cellular debris was removed by low - speed centrifugation at 1600 × g for 15 min at 40 ° c ., followed by filtration with 0 . 45 μm pore size filter ( fisherbrand , fisher scientific ). particles were partially purified by ultracentrifugation through a 20 % sucrose cushion for a minimum of 6 h at 4 ° c . ( in beckman l8 - 55 ultracentrifuge ) at 35 , 000 rpm in a sw - 41 rotor . sediments were resuspended in serum free rpmi and lodixanol ( optiprep ™, invitrogen ) was added to a final concentration of 40 % w / v ( p = 1 . 216 ). the sample was laid over a 60 % wt / vol optiprep ™ solution ( p = 1 . 320 g / ml ) and then overlaid with a linear iodixanol gradient ( p = 1 . 038 to 1 . 205 g / ml ) prepared in rpmi and spun for 20 h at 4 ° c . in beckman l8 - 55 ultracentrifuge at 30 , 000 rpm using a sw - 41 rotor . fractions were collected from the top of the tube and rna was prepared as described above . half of the final rna volume was mixed with liquid scintillation cocktail ( ecolite ™, icn biomedicals ) and 32 p radioactivity was counted in a beckman ls 6500 scintillation counter . proteins were extracted by directly adding 10 × ripa buffer to a final concentration of 1 × ripa . 1 / 100th of the protein extract was mixed with liquid scintillation cocktail and 35s radioactivity was determined using a beckman ls 6500 scintillation counter . 1 / 10 of the protein extract was directly mixed with concentrated laemmli sample buffer , resolved on a sds 15 %- page , and transferred to 0 . 2 μm protran nitrocellulose membrane over night at 30v . the membrane was dried and exposed against kodak biomax ™ mr film . the remaining protein extract was concentrated by tca precipitation ( 15 % final ). proteins were washed twice with ether , dried and dissolved in a solution containing 3 m urea , 26 mm edta ( ph 8 ), and 0 . 5 μg / ml of rnase a . samples were mixed with concentrated laemmli sample buffer , resolved on a sds 10 % page and transferred to 0 . 2 μm protran nitrocellulose membrane for 1 h at 100v . proteins were detected by western blotting as described above . the target sequence for the sirna was chosen using the ambion ™ web - based criteria . the selected rna oligonucleotides , core ( from nucleotide 371 to nucleotide 391 , strain h77 pcv - h77c , embl : af011751 , medline : 97385173 ) and the unrelated non - specific rna ( inverted sequence for 4e - t from nucleotide 986 to nucleotide 1008 ; ddbj / embl / genbank ™ database , accession no . af240775 ), were synthesized by dharmacon research ( lafayette , colo .) and handled according to the manufacturer &# 39 ; s instructions . varying amounts ( 3 μ ; or 5 μl of a 20 μm solution ) of rna duplexes were electroporated using a gene pulser ® ii electroporator ( biorad ), into 1 × 10 6 pbls in 0 . 5 ml of serum free rpmi . cells were treated with a pulse of 975 μf and 300 v . then 0 . 5 ml of rpmi containing 20 % fcs was added and the cells were seeded in a 24 - well cell culture dish . protein and rna extracts were harvested 48 h after electroporation . immunoblots were performed as described above using an ns3 rabbit antiserum - rb and monoclonal anti - ns5b , 5b - 3b1 . hcv rna levels were quantified by real - time rt - pcr using method i . table s1 characteristics of the idu donors , enrolled between march 2001 and april 2003 : opioids excl idu under idu methdone cocaine age duration methadone ( past 6 ( past 6 ( past 6 participant ( years ) sex ( years ) treatment months ) months ) months ) sb - 1 41 male 22 yes no no no sb - 2 42 female 20 yes yes yes yes sb - 4 35 male 11 yes yes yes yes sb - 5 21 female 3 yes yes yes no sb - 6 32 male 1 yes yes yes yes sb - 7 45 male 18 yes no no no mll 001 48 male 31 no yes yes yes mll 002 39 male 3 no yes no yes mll 003 38 male 10 no yes yes yes mll 004 47 male 32 no yes yes yes mll 005 38 male 21 no yes no no mll 006 49 male 37 yes yes yes no mll 007 61 male 36 no yes no no mll 008 39 male 13 no no no yes mll 009 23 male 5 no yes no no mll 010 40 male 21 no no no yes mll 011 45 male 6 no yes no yes mll 012 48 male 14 no yes yes yes mll 013 49 male 24 no no yes no mll 014 41 male 18 no yes no yes mll 015 38 male 6 no yes yes yes mll 016 34 male 11 no no no no mll 018 42 male 13 no yes no yes mll 019 51 male 10 no yes no yes mll 020 38 male 13 no yes no yes mll 021 35 female 5 no no no no mll 022 43 male 29 no yes no yes mll 023 52 male 20 no yes no yes mll 024 37 male 13 no yes no yes mll 025 36 male 18 yes yes yes yes mll 026 29 female 13 yes yes yes yes mll 027 52 male 11 no yes yes yes mll 028 45 male 6 no yes yes yes mll 029 42 male 6 no yes yes yes mll 030 43 male 10 no yes no yes mll 031 36 male 19 yes yes no yes mll 032 22 male 11 yes yes yes no mll 033 24 male 7 yes yes yes yes mll 034 52 male 26 no yes no yes mll 035 61 male 36 no yes no no mll 036 49 male 31 no yes no yes mll 037 57 male 36 no no no no mll 038 27 male 11 no yes yes yes mll 039 42 female 17 yes yes yes no mll 040 53 male 40 no yes yes yes mll 041 34 male 11 no no no yes mll 042 47 male 7 no yes no yes mll 043 42 female 23 no no no no mll 044 30 male 11 no no no yes mll 045 41 male 22 no yes no yes mll 046 43 male 21 no yes yes yes mll 047 41 male 18 no yes no yes mll 048 47 male 22 no yes no yes mll 049 52 male 11 no no no yes mll 050 33 male 10 no yes no yes mll 051 45 male 30 yes yes no yes mll 052 33 male 8 no yes no yes mll 053 43 female 12 no yes no yes mll 054 46 male 22 no yes no yes mll 055 36 female 21 yes yes no yes mll 056 40 male 14 no no no yes mll 057 37 male 9 no yes yes yes mll 058 45 male 30 yes yes no yes mll 059 50 male 30 no yes yes yes mll 060 35 male 12 yes yes yes no mll 061 46 male 7 no no no yes mll 062 48 male 11 yes yes yes yes mll 063 66 female 35 no yes no yes mll 064 38 male 3 no yes no yes mll 065 33 male 10 no yes no yes mll 066 48 male 11 yes no no no mll 067 46 male 11 no yes no yes mll 068 42 male 6 no yes no yes mll 069 42 male 23 no yes yes yes mll 070 44 male 11 no yes no yes mll 071 47 female 22 no yes no yes mll 072 61 male 16 no yes no yes mll 073 37 male 9 yes no no no table s2 probes and primers used in real time rt - pcr method i . name orientation used in : target nucleotide position in target h250 sense 5 ′ external hcv 64 - 84 hc110 antisense 3 ′ external rt and hcv 456 - 475 1 st round pcr g . 24 sense 5 ′ external gapdh 15 - 32 g . 589 antisense 3 ′ external gapdh 581 - 597 h190 sense 5 ′ internal hcv 142 - 161 c40 antisense 3 ′ internal real - time pcr hcv 385 - 405 g . 174 sense 5 ′ internal gapdh 166 - 182 g . 511 antisense 3 ′ internal gapdh 502 - 520 297 . p1 sense fl1 probe hcv 274 - 297 300 . p2 sense fl2 probe hybridization hcv 300 - 324 probes g . p1 antisense fl1 probe gapdh 187 - 212 g . p2 antisense fl2 probe gapdh 214 - 238 xxi . established ebv - transformed cell line enabling robust hepatitis c virus replication in pbls from hcv infected donors it is shown herein that hcv can naturally infect blood cell and can replicate therein ( fig2 - 41 ). in order to assess whether the produced hcv was infectious the protocol of fig4 was followed . we show that hcv replicating in naturally infected pbls was indeed infectious . we further went on to generate an hcv expressing cell line . in an embodiment , we developed an ebv - cell line that is able to replicate hcv . b - cells from infected donors were identified as the cells that harbored hcv virus . these cells were immortalized by ebv infection . interestingly , when grown under normal conditions , the ebv - immortalized b cells from infected donors , do not produce detectable amounts of hcv proteins . however , when stimulated ( independent from the stimulation procedure p , s or ps ) virus proteins ( ns3 and ns5 ) become detectable ( fig4 ). peripheral blood lymphocytes ( pbls ) obtained from an hcv negative donor can be infected by co - culturing with stimulated ebv - transformed b - cells from an hcv positive donor ( fig4 ). this implies : a ) pbls are infectable , thus hcv has tropism for these cells , b ) hcv produced by the ebv - transformed b - cells from an hcv positive donor is infectious . a ) ebv - transformed b - cells grow in culture . therefore , a cell based replication system for hcv has been developed . b ) ebv - transformed b - cells proliferate under normal culture conditions ( rpmi 1640 , antibiotics and 10 % serum ), but produce the virus only when stimulated . c ) the released virus is infectious . therefore , this system can be used for hcv receptor identification . d ) this system should prove useful in the discovery and validation of new anti - hcv agents at all levels of the virus life cycle ( entry , protein synthesis , rna replication , assembly and release ). xxii . established ebv - transformed single cell clones enabling robust hepatitis c virus replication and characterization of the hcv harbored within thus , it has been demonstrated that hcv can be grown using a co - culture system assay . it has also been shown that hcv can be actively and fully expressed in immortalized cell lines . furthermore methods of actively producing hcv in vitro have been taught ( see fig5 , for an overview ). using established b - cell lines of the present invention , further selection of single cell clones and the characterization of the virus harbored within were carried out . such a characterization demonstrates the power and versatility of the present invention . it also demonstrates how the structure - function relationship of hcv can be scrutinized . reverse transcription was carried out at 50 ° c . for 20 minutes in a one - tube two - step rt - pcr reaction with thermoscript reverse transcriptase ( invitrogen ), 10 μm of hcv - tagged strand - specific rt primer and 100 μm of anti - sense gapdh primer . the reverse transcriptase was inactivated by heating for 5 minutes at 95 ° c . and pcr ( 22 cycles ) with platinum ™ taq dna polymerase was performed in a trio - thermocycler ™ ( biometra ): at 94 ° c . for 45 s , 60 ° c . for 60 s , 68 ° c . for 2 min . the first round pcr products were then amplified for 40 cycles in the roche lightcycler ™ instrument : denaturation at 950c for 60 s , and amplification and quantification at 95 ° c . for 15 s , 60 ° c . for 10 s with a single fluorescence measurement , 72 ° c . for 15 s . real - time quantification of rna copy numbers for hcv and the human gapdh gene was based on a set of eight log 10 external standards covering 10 8 to 10 1 plasmid copies of a pcrii vector containing the 5 ′ hcv leader ( genotype 1a ) and the gapdh normalization pcr amplicons which were run in parallel with the test samples . rna extracted from pbls of a hcv negative donor was used as control . as a reaction control for the strand - specific signal , the rt step of the rt - pcr was carried out without a hcv - tagged primer . immunofluorescence was performed on 5 × 10 4 cells . following cytospin ™ for 7 min at 1100 rpm in a cytosin ™ 2 ( shandon ), cells were dried for 30 min at room temperature and fixed for 30 min at − 20 ° c . in a mixture of acetone and methanol ( 1 : 1 vol / vol ). cells were blocked for 30 min at room temperature in 10 mm tris - hcl ph 8 . 0 containing 1 % bsa . slides were washed 3 times with pbs and incubated at room temperature for 2 h with the polyclonal anti - core rr8 antibody ( 1 / 50 ) in a humidified box . slides were washed 3 times with pbs . for core detection , slides were incubated 1 h at room temperature with an alexa - 594 conjugated antibody ( dilution 1 / 250 ). dapi staining was performed for 7 min at room temperature ( 1 μg / ml final concentration ). mounted slides ( permount ™ mounting medium , fisher scientific ) were stored overnight at 4 ° c . prior to analysis . conventional epifluorescence micrographs were obtained on a zeiss cell observer ™ system equipped with an axiovert 200 m ™ microscope using the 100 × oil lens . images were digitally deconvoluted with the axiovision 3 . 1 ™ software using the nearest neighbor ™ deconvolution method that uses the agard &# 39 ; s formula . mitogens were added to the media ( rpmi 1640 , 10 % fbs , 50 iu / ml penicillin , 50 mg / ml streptomycin ) upon starting the culture and maintained throughout the experiment . the protocols used for pbls stimulation were as follows : method a , pbls were grown in the presence of irradiated l4 . 5 cells ( murine fibroblasts expressing the cd40 ligand , cd154 ) as described [ loembe , 2001 # 1962 ]. method b , 1 μg / ml of anti - cd3 and 200 u / ml of il - 2 ( sigma - aldrich co ). method p , 3 μg / ml phytohemagglutinin ( pha , sigma - aldrich co ), and 200 u / ml il - 2 . method ps , 1 : 10 4 vol / vol of staphylococcus aureus cowan fixed cells ( sac , calbiochem ) in combination with phytohemagglutinin and 200 u / ml il - 2 . method s , 1 : 104 vol / vol of sac and 200 u / ml of il - 4 ( sigma - aldrich co ). cell activation was verified by flow cytometry . cells were rinsed twice with 1 ml cold phosphate buffered saline ( pbs : 137 mm nacl , 2 . 7 mm kcl , 4 . 3 mm na 2 hpo 4 , 1 . 4 mm kh 2 po 4 , ph 7 . 4 ) and fixed in 80 % ethanol / pbs for 30 min at 4 ° c . pbs ( 2 volumes ) was added and cells were pelleted by centrifugation . cells were rinsed twice with 2 ml pbs and then resuspended in 0 . 5 ml pbs containing 0 . 2 μg / ml rnase a and incubated for 40 min at 37 ° c . propidium iodide was added to a final concentration of 1 . 2 μg / ml and samples were analyzed by flow cytometry using a single laser facs instrument ( becton - dickinson ) combined with the cellquest ™ software . b cells from hcv positive donors were transformed by infection with ebv from b95 - 8 marmoset cell line supernatant [ miller , 1973 # 1983 ]. briefly , 5 × 10 6 pbls were infected by incubation in 1 ml of b95 - 8 supernatant for 2 hr at 37 ° c . in a 5 % co 2 atmosphere . pbls in 10 ml of media ( rpmi 1640 , 20 % fcs , 2mm l - glutamine , 50 iu / ml penicillin , 50 mg / ml streptomycin and 50 μm 2 - mercaptoethanol ( sigma ), 1 μg / ml of cyclosporin a ( sandimmun , novartis pharmaceuticals canada inc ., dorval qc , canada ), transferred to 25 cm 2 flasks and incubated for 2 to 3 weeks before expansion . established ebv - transformed cell lines were confirmed to be b cells ( cd19 (+), cd3 (−), cd16 (−), cd56 (−)). ebv transformed b cell lines were typed for major histocompatibility complex ( mhc ) class i antigen expression by the amplification refractory mutation system — polymerase chain reaction ( arms - pcr ) using 95 primer sets amplifying defined mhc class i alleles ( abc ssp unitray , pel - freez clinical systems , brown deer , wis .) [ bunce , 1995 # 1984 ]. genomic dna for molecular hla - typing was prepared from ebv transformed b cell lines using the qiaamp ™ dna blood kit ( qiagen inc ., mississauga , on ). total rna was extracted from cells using trizol ™ ( invitrogen ) according to the manufacturer &# 39 ; s protocol . yeast trna ( 1 mg / ml ) was added as a carrier . rna was resuspended in nuclease - free water ( sigma - aldrich co ). total rna was quantified by phosphoimager ™ ( storm system , molecular dynamics ) using the ribogreen ™ rna quantification kit ( molecular probes , inc ). xxiv . use of the cell lines of the invention to identify , validate or improve the antiviral activity of compounds non - limiting examples of candidate anti - hcv compounds ( pool thereof , librairies of compounds , pool thereof . . . ) to be used in screening using the assays and cells of the present invention are presented herein . in addition , the present invention provides the means to assess the resistance / phenotype profile of patients &# 39 ; strains of hcv toward a particular anti - hcv compound or candidate or pool thereof . non - limiting examples of compounds that could be used in such phenotype determination are listed in tables 1 and 2 . ( 1 ) hcv has pbmc tropism ; ( 2 ) hcv can naturally infect blood cells ; ( 3 ) hcv can replicate in pbmcs and pbmls ; ( 4 ) hcv replicating in naturally infected pbmcs is infectious ; ( 5 ) hcv can replicate in extrahepatic tissue ; and ( 6 ) hcv has a latent phase during pbmc infection , which can be ended by activation . it is interesting to note that hcv replication is activated upon immune response . thus , a person of ordinary skill in the art will be able to provide other methods of activation than those disclosed herein ( or complementary thereto ) to activate hcv replication in pbmcs or pblcs , without undue experimentation . the present invention provides the tools to study hepatitis c virus replication in a simple cell culture based system . this simple culturing tool is suitable for the search and validation of novel hcv antiviral drugs and therapies ( vaccine ). the assays and methods of the present invention enable the performance of screening assays to identify antiviral agents . of course , the assays can be highthroughput . compound libraries can now be used to identify candidate anti - hcv agents . these assays can thus be used to generate lead compounds for pharmaceutical anti - hcv formulations . the novel replication system of the present invention , in one embodiment , based on pbmcs ( or pbmls ) is simple , does not require facilities other than those normally used for hiv research , and allows experiments with the complete hcv . thus , novel drugs and therapies can be screened to target all the different stages of virus replication such as virus entry , cytoplasmic replication ( viral (−) and (+) strand synthesis ), viral protein synthesis , virus assembly , virus trafficking , and virus release . although the present invention has been described hereinabove by way of preferred embodiments thereof , it can be modified without departing from the spirit and nature of the subject invention as defined in the appended claims . 3 . j . g . mchutchison , m . w . fried , clin liver dis 7 , 149 - 61 . 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