foetal rhesus kidney-4 ( frhk-4 ) cell culture and hav - a continuous frhk-4 cell line was grown at 37c in 175 cm disposable falcon flasks containing 199 medium supplemented with 10% heat - inactivated foetal bovine serum ( fbs ) , 2 mm l - glutamine , 0.1 mm mem non - essential amino acids , 25 mm hepes , 0.18% sodium bicarbonate , 100 u / ml penicillin and 100 g / ml streptomycin ( sigma chemical co ) . flat - bottomed 96-well microtitre plates ( tissue culture grade , nunc inc naperville , il , usa ) were seeded with 100 l of a suspension of frhk-4 cells prepared in growth medium ( 10 cells / ml ) . after incubation at 37c in a 5% co2 atmosphere for 24 h until confluence was just reached , 100 l dilutions of virus sample prepared in 199 medium supplemented with 2% heat - inactivated fbs were added to test eight wells per dilution without removal of the growth medium . hav plate assay - three aliquots of hav ( haf-203 ) ( undiluted , 5-fold diluted and 10-fold diluted ) were prepared to inoculate the 96-well plate to compare the quantification of hav by the tissue culture infective dose ( tcid50 ) method and rt - pcr . two columns of uninfected wells were included on each plate as negative controls and one extra column was included to blank the plate reader . the reproducibility of both tests was evaluated by performing the assays in triplicate and on different days . elisa in situ - to quantify the infectivity of hav in cell culture , elisa in situ was performed as previously described by villar et al . after days 7 , 10 and 14 , the medium was removed and the cells were fixed using 100 l per well of a 1:1 mixture of acetone and methanol at 4c for 5 min . the fixative was removed and the monolayers were allowed to dry uncovered at room temperature for at least 2 h. the presence of virus - specific antigen was detected using the conjugate diluted 1:8000 with phosphate buffered saline ( pbs)/0.5% bovine albumin ( w / v ) . the conjugate ( 100 l ) was added to each test and uninfected well but not to the blank wells . after incubation of the cells at 37c for 1 h , the antibody solution was removed and the monolayers were washed four times with pbs plus 0.1% tween 20 ( w / v ) ( 300 l / well ) using an automated plate washer . immediately after washing , bound antibody was detected using hydrogen peroxide and tetramethylbenzidine . the colour change was monitored visually and stopped after 15 min by the addition of 100 l of 1 m sulphuric acid per well . the absorbance of the plates was then read at 450 nm and a positive / negative ratio was determined using uninfected wells as the negative value . the titre of infectious virus was determined according to the method of reed and muench ( 1938 ) , which is based on the tcid50 endpoint . extraction of viral rna from cell culture supernatants - a taqman rt - pcr assay was performed to quantify hav genomes as previously described by de paula et al . rna was extracted from cells in microplates using the sv total rna isolation system ( promega corporation , madison , wi ) , which specifically purifies viral rna from tissues and cultured cells . this method is based on a lysis - centrifugation process , followed by column filtration through a silica membrane in an rnase - free environment . the trypsinised cells were pelleted by centrifugation at 500 g for 5 min and the supernatant was discarded . the whole cell pellets were disrupted by adding 300 l of lysis buffer ( 10 cells / ml ) and viral rna was extracted from lysed whole cell samples . the positive strands of the 5 non - translated region ( 5ntr ) of the hav genome were amplified . the positive strands were reverse - transcribed using superscript iii reverse transcriptase ( invitrogen , carlsbad , ca ) . the reaction was conducted at 50c for 1 h , followed by incubation for 10 min at 65c in a 20 l volume containing 10 l of rna ( 10 pg-5 g ) , 10 pmol/l anti - sense primer ( hav5ncri - r ) ( table ) , 1 u/l rnasin ( promega , madison , wi ) , 125 mm of each deoxynucleoside triphosphate and 1 u/l superscript iii reverse transcriptase . after cdna synthesis , a master mix containing 1taqman universal pcr master mix ( applied biosystems , hammonton , nj ) and 1.25 l of the assay mixture ( 300 nm of each primer , 150 nm probe ) ( applied biosystems assay , foster city , ca ) was prepared on ice . five microlitres of cdna and standard curve points were added to 20 l of the pcr master mix . the thermal cycling conditions included an initial step at 50c for 2 min , 95c for 10 min , 40 cycles of 95c for 15 s and 60c for 1 min . the primers and probe used to quantify the 5ntr are described by de paula et al . tablereproducibility analysis of the ensyme immunoassay ( eia ) and real - time ( rt)-polymerase chain reaction assays and correlation between hepatitis a virus ( hav ) particles and hav genomesday 
 ( dpi)dilutionreal - time 
 ( copies / ml)a 
 cv% 
 ( rt)eia 
 ( od)b 
 cv% 
 ( rt)rc 
 p ( 95% ci)d 
 71/13.180  30.20.90.819  0.0283.50.969 < 0.0001 ( 85.6 - 99.3)1/5201.0  3.01.50.46 0.014.1 - -1/1089.0  1.01.10.33  0.1033.0 - -101/1111.333  2.309.45.22.29  0.041.90.8530.003 ( 43.6 - 96.8)1/10707.3 34.54.80.87  0.067.5 - -141/111.233.3  404.13.32.34  0.14.40.7840.012 ( 25.1 - 95.2)1/5205.6  13.66.61.80 0.137.5 - -1/1027  1.03.70.04  0.044.5 - - 
 a : mean  standard deviation ( sd ) of triplicate analyses ; b : media  sd of triplicate analyses ; c : correlation coefficient be tween rt and eia ; d : two - tailed p < 0.05 was considered statistically significant ; ci : confidence interval ; cv : coefficient of variation ; dpi : days post - infection ; od : optical density . a : mean  standard deviation ( sd ) of triplicate analyses ; b : media  sd of triplicate analyses ; c : correlation coefficient be tween rt and eia ; d : two - tailed p < 0.05 was considered statistically significant ; ci : confidence interval ; cv : coefficient of variation ; dpi : days post - infection ; od : optical density . 
 statistical analysis - all graphs were generated using graphpad prism software 5.0 . data are presented as the mean and standard deviation of triplicates for the rt and ensyme immunoassay ( eia ) values . the statistical significance of the linear ( pearson ) correlation between the methods was determined , with a p value < 0.05 considered significant . an analysis of variance was used to evaluate significant differences between the values of each group . correlation between the number of hav particles and the hav genome - to determine a relative correlation between the number of hav genomes and hav particles , which could be used as a replacement to traditional viral titration methods , we quantified the virus by the standard method of titration ( elisa in situ ) and a molecular test ( rt - pcr ) and used a statistical approach to test correlations . the number of hav ( haf-203 ) particles in the three different stocks was determined by elisa in situ and compared with the viral load measured by rt - pcr ( fig . , there was a significant increase in detection of hav genome and particles by rt and eia , respectively ( p < 0.001 ) . however , from day 10 - 14 , there was a significant decrease in genome detection ( p < 0.001 ) and a stabilization of hav particles ( fig . the maximum concentration of hav rna was reached in all stocks ( 10 copies / ml ) ( p < 0.001 ) , while the highest number of hav particles was observed at day 14 ( optical density = 2.4 , p < 0.001 ) . a linear correlation analysis showed a strong positive correlation between the number of hav particles and number of hav genomes ( r = 0.96 , p < 0.0001 ) at seven days post - inoculation ( table ) . after 10 and 14 days of incubation , the linear correlation analysis showed a weak positive correlation between hav particles and hav genomes ( r = 0.85 and 0.784 , respectively ) ( table ) . 1comparison between the numbers of viral particles analyzed by elisa in situ and the number of amplified copies of the genome of hepa titis a virus analyzed by real - time ( rt ) polymerase chain reaction according to the days of incubation . dpi : days post - infection ; eia : ensyme immunoassay ; od : optical density ; * : analysis of variance of rt values ; * * : elisa values . relationship between the titre of infectious hav and rna copies - the titre of infectious hav at seven , 10 and 14 days post - infection ( dpi ) was estimated by endpoint titration using tcid50 . the highest tcid50 value occurred on day 7 using serial one - fold dilutions ( 10 . the cycle threshold ( ct ) values for each day of infection were measured and correlated with tcid50 ( fig . this analysis demonstrated that there was a significant correlation between titre equivalent values , measured by rt - pcr and tcid50 titres . this correlation allowed the calculation of the number of rna copies corresponding with the tcid50 . 2correlation of the amounts of hepatitis a virus particles and rna , estimated by tissue culture infective dose ( tcid50 ) and cycle threshold ( ct ) values of real - time polymerase chain reaction . analyses of assays accuracy - to test the reproducibility of the eia and rt - pcr assays , the assays were conducted in triplicate using different aliquots ( 1:1 , 1:5 and 1:10 ) of the same stock . in addition , the assays were performed three times as an inter - assay analysis . the low variations in the eia ( with a coefficient of variation ranging from 3 - 7.5% ) and rt - pcr ( with a coefficient of variation ranging from 0.9 - 6.6% ) demonstrated the reproducibility of the tests ( table ) . over the last few decades , several types of inactivated hav vaccines have been developed . the vaccines are whole - virus preparations produced by the growth of hav strains in cell culture , followed by inactivation of the virus . the viral load can be quantified by tcid50 determination or rt - pcr . in this study , a statistical approach was used to verify the correlation between the number of particles and number of hav rna copies . the number of hav particles was estimated by eia and this value was compared with the number of hav genomes determined by rt - pcr ( fig . the maximum concentration of hav rna was reached at 10 dpi and the highest number of hav particles was observed at 14 dpi . however , at seven days post - incubation , there was a strong significant positive correlation ( r = 0.969 , p < 0.001 ) between the number of particles and number of hav genomes ( table ) for all three stocks , which suggests that there is a statistical correlation between the number of hav genomes and the infectivity . a possible explanation for this result is that at seven days post - infection , the new hav genomes are predominantly encapsidated to generate mature virions instead of being used as templates for hav genome replication . this correlation enabled the calculation of the number of rna copies in a sample and the association with tcid50 , as described recently for enterovirus . for enterovirus , the rt data were correlated over a wide range of viral concentrations with the titre obtained by endpoint titration ( jonsson et al . ( 2004 ) , who reported that at 14 days of incubation , there was a higher optical density by elisa in situ , while the highest hav titre was observed at seven dpi . from days 7 - 10 , there was a significant increase in the detection of both the hav genome and particles by rt and eia ( p < 0.001 ) . this suggests that during this period , most of the new hav genomes synthesized are encapsidated to generate virions . linear correlation analysis showed that there was a weak positive correlation between the tcid50 and the number of hav genomes at 10 and 14 days post - infection . from days 10 - 14 , there was a significant decrease in genome detection ( p < 0.001 ) and a slight increase of the number of hav particles . while these results did not provide biochemical or virological evidence of the presence of defective particles , we speculate that during this period , a higher number of procapsids or empty particles are present in the viral stocks , likely due a reduced rate of genome replication and predominant use of the genome to produce viral protein as mrnas to produce viral proteins . to evaluate the reproducibility of the tests , all elisas and rt - pcr assays were conducted in triplicate . the coefficients of variation were lower than 10% , which demonstrates the reproducibility of the tests . in a previous study , the density and sedimentation analyses of extracts from cells infected with hav at 21 days revealed that the virion itself usually represented less than 50% of the total mass of the viral antigen . a major portion of the antigen was associated with non - infectious , empty particles ( ruchti et al . replication - defective mutant viruses are essential for viral genome replication or synthesis and the assembly of viral particles . these mutant viruses possess the advantages of both classical types of viral vaccines because these mutant viruses are as safe as inactivated viruses , but express viral antigens inside infected cells so that major histocompatibility complex class i and class ii presentation can occur efficiently . replication - defective viruses have served both as vaccines for the parent virus and as a vector for the expression of heterologous antigens . additionally , these replication - defective viruses have also served as important probes of the host immune response by helping define the importance of the first round of infected cells in the host immune response , the mechanisms of activation of the innate immune response and the role of the complement pathway in humoural immune responses to viruses ( dudek & knipe 2006 ) . in this study , we demonstrated that the number of defective hav particles in cell culture at 14 days should be considered . the linear correlation analysis suggested that varying amounts of defective particles can modulate the results of hav quantification by elisa or rt - pcr . on the first day of infection , the replication cycle occurred normally and progeny virions were produced . however , these virions were non - infectious and thus , the infection did not spread to a second round of cells . the released , non - infectious virions may provide inert antigen that can spread beyond the infected cells ; nevertheless , the blockage of spreading may not be complete for all viruses ( loudon et al . for this reason , stocks of rna viruses can contain different mixtures of infectious and defective particles that are indistinguishable by elisa in situ because the surfaces of both particles share the same immunoreactivity ( handke et al . generally , only the number of infectious virions in the reference stocks is of interest and the quantity of defective particles is neglected . the relative number of defective particles can be at least five - fold higher in the stock with the lowest titre compared with the stock with the highest titre ( handke et al . 2009 ) . to further determine whether the viral titre was correlated with the calculated ct value from rt - pcr , titres of hav - containing stocks were estimated by endpoint titration . the rt data were correlated over a wide range of viral concentrations with the hav titre obtained by tcid50 ( fig . this correlation enabled the calculation of the number of rna copies in a sample and the association with tcid50 , as described recently for enterovirus ( jonsson et al . the need for a virus quantification assay is particularly important to determine viral quantities , follow hav growth in cell culture and establish the quantity of antigen for diagnostic assays and inactivation . the results reported here indicate that the duration of hav growth in cell culture must be taken into account when correlating the quantification methods . at seven days post - infection , a correlation between the quantity of hav genomes determined by rt - pcr and the number of hav particles determined by elisa in situ was observed . it was also demonstrated here that titre equivalents generated by rt - pcr correlated with the titres obtained by the endpoint dilution method , which implies that rt - pcr may be a feasible , faster alternative assay to estimate hav titres at approximately 10 days post - infection . the results for 14 days post - infection imply that defective particles in viral stocks should be considered when performing virus quantification assays .
elisa in situ can be used to titrate hepatitis a virus ( hav ) particles and real - time polymerase chain reaction ( rt - pcr ) has been shown to be a fast method to quantify the hav genome . 
 precise quantification of viral concentration is necessary to distinguish between infectious and non - infectious particles . 
 the purpose of this study was to compare cell culture and rt - pcr quantification results and determine whether hav genome quantification can be correlated with infectivity . 
 for this purpose , three stocks of undiluted , five - fold diluted and 10-fold diluted hav were prepared to inoculate cells in a 96-well plate . 
 monolayers were then incubated for seven , 10 and 14 days and the correlation between the elisa in situ and rt - pcr results was evaluated . at 10 days post - incubation , 
 the highest viral load was observed in all stocks of hav via rt - pcr ( 105 copies / ml ) ( p = 0.0002 ) , while elisa revealed the highest quantity of particles after 14 days ( optical density = 0.24 , p < 0.001 ) . at seven days post - infection 
 , there was a significant statistical correlation between the results of the two methods , indicating equivalents titres of particles and hav genome during this period of infection . 
 the results reported here indicate that the duration of growth of hav in cell culture must be taken into account to correlate genome quantification with infectivity .