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 and ). 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. ). 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 ). 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
]. 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 ). 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
] 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/cm2
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 × 104
FFU/ml, volume depth of 0.2 cm) could be inactivated completely by UVC irradiation at a dose of 2.7 × 10-2
within 1 min (Figure ), while those spiked in human serum (1.0 × 105
FFU/ml) required an irradiation dose of 5.4 × 10-2
for full inactivation (Figure ). 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
]. 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
]. 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 × 104
FFU/ml to undetectable levels (Table ). At these concentrations both aldehydes were also effective in inactivating HCVcc in the presence of human serum (Table ). 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 × 105
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 ). 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 ), 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 ). 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.