Background
Hepatitis C virus (HCV) is a small enveloped, positive-stranded RNA virus classified within the family
Flaviviridae, genus
Hepacivirus. HCV affects an estimated 170 million people worldwide and is a global health problem. Unlike most RNA viruses which usually cause acute diseases, HCV establishes life-long, persistent, intrahepatic infections in a majority of infected individuals, leading frequently to the development of cirrhosis and hepatocellular carcinoma [
1,
2]. Because the current, interferon-based treatment regimens eradicate HCV in only about 50% of patients, prevention of HCV infection is pivotal for controlling this viral pathogen.
HCV is transmitted primarily via percutaneous exposure to infectious blood. Prior to the introduction of anti-HCV screening tests in the early 1990s, receiving blood and blood products or organ transplants was a major risk factor for acquiring HCV infection. Currently, injection of illicit drugs represents a major risk, while other routes of infection, including occupational exposure (such as needle stick), sex, and mother-to-infant transmission (with the exception of HIV-coinfected mother), seem infrequent [
3]. Interestingly, it was shown recently in the chimpanzee model that HCV in infectious plasma could survive drying and environmental exposure to room temperature for at least 16 h. This finding has raised the possibility of person-to-person transmission of HCV via blood-contaminated objects and medical devices [
4]. Clearly, it is fundamental to quantitatively determine the stability of HCV under environmental conditions and evaluate reliable procedures for inactivating this virus. However, such efforts have been hampered by the lack of an efficient cell culture system and convenient, small animal models for HCV. Although HCV RNA and antigens have been used as indicators for the presence or absence of virus particles, such detection methods do not distinguish between the infectious and inactivated viruses [
4‐
6]. To circumvent this, several related viruses in the family
Flaviviridae that can be readily cultured in vitro, e.g., bovine viral diarrhoea virus (BVDV, genus
Pestivirus), have been used as surrogates for HCV to study the inactivation process [
7,
8]. Although these model viruses show similarity in virion and genome structure to HCV, more relevant systems are still needed to assess the reliable procedures for inactivating HCV.
The recent establishment of an HCV cell culture system based on a particular molecular clone, JFH-1, offers the opportunity of evaluating the inactivation methods for HCV directly [
9‐
12]. Using the Huh7-25-CD81 cell line that is highly susceptible to HCVcc infection [
13], the stability of HCVcc (JFH-1 strain) at different environmental temperatures (37°C, room temperature, and 4°C) was assessed in this study. In addition, the efficacy of several commonly used viral inactivation methods, including heat treatment, UVC light irradiation, aldehyde-mediated fixation, and detergent treatments in eliminating HCVcc infectivity were evaluated. The results revealed that all of these methods were able to inactivate HCVcc, provided proper conditions are met.
Discussion
In this study, a detailed analysis was conducted on the stability of HCVcc at various environmental temperatures. Also evaluated was the efficacy of several conventional viral inactivation procedures in eliminating HCVcc infectivity.
It has been shown previously that genotype 1a HCV in infectious plasma could survive drying and environmental exposure to RT for at least 16 h [
4]. The results of the current study have demonstrated that JFH-1 virus (genotype 2a) grown in cell culture can survive 37°C and RT for 2 and 16 days, respectively (Figure
1A and
1B). Of note, the stability of JFH1 HCVcc spiked in human serum did not differ much from those in cell culture medium when incubated at RT (Fig.
1D). When stored at 4°C, JFH-1 virus was found to be relatively stable, without drastic loss of titer during the 6-week observation period (Figure
1C). The latter result is in agreement with a previous report dealing with the J6/JFH1 chimeric virus [
10]. The ability of HCVcc to survive various environmental temperatures warrants precautions in handling and disposing objects and materials that may have been contaminated with HCV, to minimize the risk of HCV transmission.
Heat treatment is a widely used viral inactivation method that is effective against both enveloped and nonenveloped viruses [
14]. The mechanisms of heat-mediated inactivation include denaturation of viral proteins, as well as disassembly of virus particles into noninfectious viral subunits and single proteins [
15]. Viruses other than HCV in the family
Flaviviridae have been shown to be sensitive to heat treatment. Yellow fever virus is routinely inactivated at 56°C for 30 min. At 60°C, BVDV and yellow fever virus have been reported to be inactivated effectively in 30 and 5 min, respectively [
8,
16]. In the current study, similar kinetics of viral inactivation following heat treatment was observed for both the HCVcc in culture medium and those in human serum. While 10 min at 60°C or 4 min at 65°C was sufficient to eliminate the infectivity of HCVcc, incubation for 40 min was required to achieve complete viral inactivation at 56°C (Figure
2). Therefore, pretreatment of HCV positive sera for 30 min at 56°C may not be absolutely reliable in eliminating their infectivity. However, because the efficiency of heat treatment could be affected by a variety of factors, such as the initial viral titer, protein concentration in virus suspension, as well as the existence of viral aggregates [
8,
17] the exact temperature and time required for reliable HCV inactivation should be evaluated under each specific condition.
UV light irradiation is another commonly used physical method for viral inactivation. UVC with a wavelength range of 200-280 nm prevents viral replication by inducing formation of pyrimidine dimers in the viral genome [
18]. A recent study reported that BVDV, when suspended in PBS, could be inactivated completely by 1.6 J/cm
2 UVC light, while viral suspension containing 5% FBS required a higher radiation dose [
7]. The current study demonstrated that HCVcc in culture medium (2.5 × 10
4 FFU/ml, volume depth of 0.2 cm) could be inactivated completely by UVC irradiation at a dose of 2.7 × 10
-2 J/cm
2 within 1 min (Figure
3A), while those spiked in human serum (1.0 × 10
5 FFU/ml) required an irradiation dose of 5.4 × 10
-2 J/cm
2 for full inactivation (Figure
3B). Therefore, UVC light irradiation represents a highly effective means for inactivating HCVcc, the efficiency of which is not affected by human serum components that may interact with HCV virons in vivo. However, the irradiation dose required for each specific occasion may depend on the sample volume and its initial viral titer.
As a chemical cross-linking reagent, formaldehyde inactivates viruses primarily by denaturing viral proteins, as well as the nucleic acids [
19,
20]. Because the immunogenicity of the viral particles can be retained during inactivation, formalin (37% formaldehyde) treatment is the most used technique for preparing inactivated virus vaccines. For tissue fixation for histology or immunohistochemistry, 10% formalin (or 4% paraformaldehyde) is routinely used. Glutaraldehyde is another effective protein cross-linking reagent, mostly used for fixation of tissues for electron microscopy. Although the detailed mechanisms are not entirely clear yet, successful inactivation of many viruses with glutaraldehyde, including hepatitis B virus, human immunodeficiency virus (HIV), and SARS coronavirus, has been reported [
18,
21,
22]. We demonstrated here that at RT, 3 h of exposure to formaldehyde (0.037%) or 20 min of exposure to glutaraldehyde (0.01%), respectively, could reduce HCVcc infectivity from 4.1 × 10
4 FFU/ml to undetectable levels (Table
1). At these concentrations both aldehydes were also effective in inactivating HCVcc in the presence of human serum (Table
2). The slightly longer times required (4 h for formaldehyde treated samples and 40 min for glutaraldehyde treated samples, respectively) were most likely attributed to the 2.5-fold higher initial titer of the HCVcc stock tested (1.0 × 10
5 FFU/ml). However, a limitation of the current study is that, because of the cytotoxic effect of the aldehydes, the infectivity of viral samples could be analyzed at only the 100-fold dilution, which may somehow have reduced the sensitivity of the assay. It should be noted, however, the routinely used concentrations of aldehydes for fixation purposes (4% for formaldehyde and 2.5% for glutaraldehyde) are far in excess of the ones examined in the current study and, therefore, should be highly efficient in achieving HCV inactivation.
Detergents are highly efficient at disrupting the lipid-enveloped viruses, and solvent/detergent (S/D) treatment is a standard method for inactivating viruses present in human blood products [
22]. The effects of both ionic (SDS) and nonionic (Triton X-100 and NP-40) detergents on HCVcc infectivity have been investigated here. All three detergents at the tested concentrations reduced HCVcc infectivity rapidly to undetectable levels (Table
3). Importantly, both intracellular HCVcc and those released into culture fluid could be inactivated by each of these detergents, regardless of the presence of human serum, indicating that components of culture medium, human serum or intracellular proteins did not interfere with the disruptive processes exerted by these detergents. Under current experimental conditions, effective HCVcc inactivation took place immediately after vortex-mixing, rendering it impossible to delineate the kinetics of viral infectivity reduction during the detergent treatment process. This finding is reminiscent of that reported for HIV in a previous study, which demonstrated that HIV-1 spiked in solution containing 1% Triton X-100 was inactivated completely within 1 min [
23]. As in the case of the aldehyde-inactivation experiments, the cytotoxic effect of detergents limited the sensitivity of the current assays. Interestingly, although 0.0005% Triton X-100 and 0.0005% NP-40 had no detectable effect on cell viability (Figure
4C), they still lowered HCVcc infectivity by 1.7- to 2.5-fold when the latter was compared with those determined in the presence of 0.001% SDS or without any detergent (Table
3). Most likely, the residual Triton X-100 or NP-40 still had some disruptive effect on virion integrity, which is important for viral infectivity. Alternatively, these detergents may have caused some cell surface alterations at this extremely low concentration that affect the process of HCVcc entry. However, to maintain the sensitivity of the assay, the detergent-treated samples were not diluted further for the infectivity test. Collectively, the robustness and immediate action of detergents in destroying HCVcc infectivity support the use of S/D treatment procedures in eliminating potential HCV contaminations in blood products.
Methods
Cell culture and virus stocks
The Huh7-25-CD81 cell line (a generous gift from Dr. Takaji Wakita), a Huh7 cell clone that stably expresses human CD81 [
13], was used throughout the experiments. This cell line was chosen because we found it was approximately 1.5- to 2-fold more sensitive for titration of HCVcc infectivity than was the Huh7.5.1 cell line (data not shown). Cells were maintained in DMEM supplemented with 10% fetal bovine serum (Invitrogen), 10 mM HEPES (Invitrogen), and 400 μg/ml G418 (Merck, Germany) at 37°C in 5% CO
2. To generate JFH-1 virus stocks, cell culture supernatant collected from full-length JFH-1 RNA-transfected Huh7 cells (kindly provided by Dr. Takaji Wakita) was used to infect Huh7-25-CD81 cells grown in T25 flasks at a multiplicity of infection (MOI) of 0.01. The infected cells were passaged at 3-day intervals with 1:3 to 1:4 split ratios into progressively larger culture vessels. At 12 days postinfection, the culture supernatants were harvested, clarified by centrifugation (5 min at 4000 rpm), and stored in aliquots at -70°C as the HCVcc stock. The infectious titer of the virus stock was determined by focus-forming unit (FFU) assay as described immediately below (the infectious titers of the un-concentrated virus stocks used were either 2.5 × 10
4 FFU/ml or 4.1 × 10
4 FFU/ml in the current study, as specified in each experiment).
To determine the stability of HCVcc and its susceptibility to individual inactivation methods in the presence of human serum, a condition which better mimics circulating HCV virions in vivo, HCVcc stock was first concentrated using the Amicon Ultra-15 device (100,000 NMWL membrane; Millipore) as described previously [
11]. The concentrated virus stock (1.1 × 10
6 FFU/ml) was then diluted 11-fold in normal human serum that had been heat-inactivated to achieve an infectious titer of 1.0 × 10
5 FFU/ml. This human serum containing HCVcc was stored at -70°C in aliquots until use.
HCV infectivity assay
The infectious titers of virus stocks and treated viral samples were determined by FFU assay, as described previously [
24], using an indirect immunofluorescence assay (IFA) for HCV NS3. In brief, 100 μl of 10-fold serially diluted samples (the dilution factors generally ranged from 1:1 to1:1000) were inoculated onto naïve Huh7-25-CD81 cells seeded in 96-well plates 1 day before infection (7000 cells/well). After 6 h of incubation at 37°C, cells were refed with 100 μl fresh medium. Following an additional 72 h, cells were fixed in 4% paraformaldehyde for 30 min at room temperature (RT), blocked for 60 min in a blocking buffer (3% BSA, 0.3% Triton X-100, 10% FBS in PBS), followed by incubation with a polyclonal antibody against HCV NS3 (kindly provided by Dr. Takaji Wakita) at 1:500 dilution. After 2 h incubation at RT, cells were washed extensively with PBS and then incubated with an FITC-conjugated goat anti-rabbit IgG (Beijing Zhongshanjinqiao, China) at 1:100 dilution for 1 h. Following PBS washes, the numbers of fluorescent foci (a focus is defined as a cluster of infected cells immunostained positive for NS3 antigen) per well at appropriate dilutions (generally containing 5 to 100 foci per well) were counted. The infectious titers, expressed as FFU/ml, were calculated from the average foci number of triplicate or duplicate (for samples derived from human serum spiked with HCVcc) wells. The detection limit of the FFU assay was 10 FFU/ml. For samples with infectious titers below the detection limit of the assay, the potential residual infectivity was examined as follows. Naïve Huh7-25-CD81 cells seeded in 96-well plates were inoculated with samples to be tested (100 μl/well). Inoculated cells were passaged at 3-day intervals from one well into three wells at each passage (with a 1:3 split ratio) to allow growth of residual infectious virus. IFA for NS3 were performed on each cell passage. If the IFA results remained negative for three successive cell passages (up to 9 days postinoculation), the tested sample was considered to be inactivated completely.
Viral stability assays
An HCVcc stock with a titer of 2.5 × 104 FFU/ml was dispensed into 300-μl aliquots in tightly capped, 1.5-ml microcentrifuge tubes and then incubated at 37°C, RT (25 ± 2°C), and 4°C, respectively, and protected from light. Aliquots incubated at 37°C were removed every 8 h, while those incubated at RT or 4°C were removed every 2 days or every 2 weeks, respectively, and stored at -70°C until virus titration on Huh7-25-CD81 cells. For viral stability assays for HCVcc spiked in normal human serum (1.0 × 105 FFU/ml), aliquots were incubated at RT and removed every 7 days for titration. All the time points selected in the experiments were designed based on the results of several pilot experiments.
Heat treatment
An HCVcc stock in culture medium (2.5 × 104 FFU/ml) or concentrated HCVcc stock diluted in human serum (1.0 × 105 FFU/ml) was dispensed into 100-μl aliquots in tightly capped, 1.5-ml microcentrifuge tubes and then incubated in water baths with temperatures of 56°C, 60°C, and 65°C, respectively. At designated time points, aliquots were removed, transferred immediately into ice-water bath to stop the effect of heat, and then subjected to FFU assay for virus titration.
UVC light irradiation
Two hundred-microliter aliquots of an HCVcc stock (2.5 × 104 FFU/ml) or HCVcc diluted in normal human serum (1.0 × 105 FFU/ml) were placed in 48-well plates to give a volume depth of about 0.2 cm and then exposed to continuous UVC light 30 cm beneath the longitudinal midpoint of a UVC lamp (model: ZSZ20D, wavelength = 253.7 nm, Beijing Haidian Konghou High Temperature Composite Material Factory, China). At the distance of 30 cm, the radiant intensity of the UVC lamp was 450 μW/cm2 (where μW = 10-6 J/sec), as specified by the manufacturer. After varying lengths of exposure, samples (200 μl) were removed, and their residual infectivity was titrated on Huh7-25-CD81 cells immediately. Control samples were set up in parallel and incubated for the same time period but protected from UVC light.
MTT cytotoxicity assay
MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] cytotoxicity assay was carried out to determine the concentrations of aldehydes or detergents at which they were no longer cytotoxic to Huh7-25-CD81 cell. Solutions of aldehydes (37% formaldehyde and 50% glutaraldehyde) or detergents [sodium dodecyl sulfate (SDS, 0.1%), Triton X-100 (0.2%) or nonidet P-40 (NP-40, 0.2%)] were diluted serially in cell culture medium, respectively (the range of concentrations for each reagent was designed based on the results of pilot experiments). The diluted reagents were then added to Huh7-25-CD81 cells seeded in 96-well plates (7000 cells/well) 1 day before. After 6 h of incubation at 37°C, the treated cells were refed with 100 μl of fresh culture medium to keep the exposure time to the individual reagents the same as that in HCV infectivity assay. Following an additional 72 h, 20 μl of MTT solution (5 mg/ml, Sigma-Aldrich) was added to each well. After a 4-h incubation at 37°C, the MTT solution was removed and replaced with 200 μl of dimethyl sulfoxide (DMSO, Sigma-Aldrich) per well. After the formazan crystals were dissolved by agitation (10 min at RT), the absorbance of solution in each well was measured at 490 nm using an enzyme-linked immunosorbent assay plate reader (Bio-Rad). The percentage of cell viability was calculated as the ratio of absorbance in treated cells compared with that in untreated controls. All experiments were performed in triplicate and repeated twice.
Formaldehyde (37%) or glutaraldehyde (50%) solutions (Beijing Chemical Reagents Company, China) were diluted in PBS at 1:10 (formaldehyde) or 1:50 (glutaraldehyde), respectively, then added to 500-μl viral samples [HCVcc stock in cell culture medium (4.1 × 104 FFU/ml) or HCVcc-containing human serum (1.0 × 105 FFU/ml)] to achieve a final concentration of 0.037% (formaldehyde) or 0.01% (glutaraldehyde), respectively. After different time periods at RT, treated samples were diluted 100-fold in culture medium immediately to stop the inactivation reaction, as well as to eliminate the cytotoxic effect of aldehydes in subsequent FFU assays (according to the results of the MTT assay, the presence of 0.00037% formaldehyde or 0.0001% glutaraldehyde had no appreciable effect on the viability of Huh7-25-CD81 cells). Immediately after the dilution, HCV infectivity in samples was titrated by FFU assay in Huh7-25-CD81 cells. Samples showing negative results in FFU assay were subjected to the residual infectivity test, as described in "HCV infectivity assay" As control, PBS was substituted for the aldehydes to treat the virus stocks, which were then diluted 100-fold to infect cells in the presence of either 0.00037% formaldehyde or 0.0001% glutaraldehyde, respectively.
Detergent treatments
Solutions of 0.5% SDS (w/v), 1% Triton X-100 (v/v), or 1% NP-40 (v/v) (all prepared in PBS) were added to 500-μl aliquots of viral samples [HCVcc stock in cell culture medium (4.1 × 104 FFU/ml) or normal human serum containing HCVcc (1.0 × 105 FFU/ml)] to achieve a final concentration of either 0.1% (SDS) or 0.2% (Triton X-100 and NP-40). After a gentle mixing (within 1 min), treated samples were diluted 100-fold (SDS-treated samples) or 400-fold (Triton X-100- or NP-40-treated samples) immediately in culture medium to negate the cytotoxic effect of detergents (based on the MTT assay results, the presence of 0.001% SDS, 0.0005% Triton X-100, or 0.0005% NP-40 had no demonstrable effect on the viability of Huh7-25-CD81 cells), then subjected to FFU assay. Samples with negative FFU assay results were examined for residual infectivity. As control, PBS was used in place of the detergents to treat the virus stocks, which were subsequently diluted either 100- or 400-fold to infect the Huh7-25-CD81 cells in the presence of 0.001% SDS, 0.0005% Triton X-100, or 0.0005% NP-40, respectively.
To assess the ability of detergents to disrupt intracellular HCV, JFH-1 infected Huh7-25-CD81 cell monolayers grown in 24-well plates (approximately 100% of cells stained positive for NS3 at the time of cell lysis as examined by IFA) were detached by trypsin/EDTA and washed extensively with PBS, and cell pellets were resuspended in 50 μl PBS containing 0.1% SDS, or 0.2% Triton X-100, or 0.2% NP-40, respectively (each detergent was disruptive to cells at these concentrations as visualized by microscopy). After centrifugation, the supernatants of cell lysates were diluted 100-fold (SDS-lysed samples) or 400-fold (Triton X-100- or NP-40-lysed samples) in culture medium for infectivity assays. IFA of HCV NS3 was performed on inoculated cells for three consecutive cell passages. As control, infected cells washed extensively with PBS were pelleted and resuspended in 50 μl PBS and lysed by three cycles of freezing and thawing (-70°C to 37°C), and the infectivity of supernatants was titrated on Huh7-25-CD81 cells.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
HZ and KL conceived the study and designed the experiments. HSS, JL, SS and LY carried out the experimental work. HSS, HZ and KL wrote the paper. All Authors have read and approved the final manuscript.