Electron Beam Susceptibility of Enteric Viruses and Surrogate Organisms on Fruit, Seed and Spice Matrices.

The objective of this study was to use high-energy electron beam (HEEB) treatments to find surrogate microorganisms for enteric viruses and to use the selected surrogates as proof of concept to investigate low-energy electron beam (LEEB) treatments for enteric virus inactivation at industrial scale on frozen blueberries. Six food matrices inoculated with HAV (hepatitis A virus), MNV S99 (murine norovirus), bacteriophages MS2 and Qß, and Geobacillus stearothermophilus spores were treated with HEEB at 10 MeV using 4, 8 and 16 kGy doses. G. stearothermophilus spores showed the highest inactivation on all matrices except on raisins, with a dose-dependent effect. HAV reached the maximum measurable log10 reduction (> 3.2 log10) when treated at 16 kGy on raisins. MNV showed the highest resistance of all tested microorganisms, independent of the dose, except on frozen blueberries. On frozen blueberries, freeze-dried raspberries, sesame seeds and black peppercorns, HAV showed a mean inactivation level in between those of MS2 and G. stearothermophilus. Based on this, we selected both surrogate organisms as first approximation to estimate HAV inactivation on frozen blueberries during LEEB treatment at 250 keV using 16 kGy. Reductions of 3.1 and 1.3 log10 were measured for G. stearothermophilus spores and MS2, respectively, suggesting that a minimum reduction of 1.4 log10 can be expected for HAV under the same conditions.

Inactivation of hepatitis A virus and murine norovirus on surfaces of plastic, steel and raspberries using steam-ultrasound treatment.

The leading causes of foodborne viral disease outbreaks are human norovirus and hepatitis A virus (HAV). Their environmental persistence enables contamination of kitchen surfaces and crops often consumed raw, such as berries. Many decontamination procedures are inefficient and unsuitable for surfaces of industrial kitchen environments and soft fruits. In this study, we investigated the efficiency of a novel surface decontamination technology, combining steam and ultrasound (steam-ultrasound). Plastic, steel or raspberry surfaces were spiked with the norovirus surrogate, murine norovirus (MNV), and HAV, and steam-ultrasound treated at 85, 90 and 95 °C for 0-5 s. Post treatment viruses were titrated for survival by plaque assay and for genome stability by real-time quantitative PCR (RT-qPCR) of nucleic acid extracts. Survival of viruses were estimated in a log-linear model and the treatment time requirements for each decimal reduction (D value) in viral survival were calculated. The estimated D values of MNV or HAV were 0.4-0.2 or 1.1-0.8 s on plastic, 0.9-0.7 or 1.4-0.8 s on steel and 1.6-1.7 or 3.2-4.7 s on raspberries. No clear trend of genome reduction was observed with tested treatment parameters. Raspberries treated up to 4 s retained its natural texture and visual appeal similar to untreated controls whilst monitored for 7 days. In conclusion, steam-ultrasound treatment can within seconds reduce the titre of foodborne viruses on surfaces of plastic, steel and raspberries. This may particularly benefit industrial scale production of soft fruits for raw consumption and for swift non-hazardous decontamination of industrial kitchen surfaces.

Ultraviolet C light efficiently inactivates nonenveloped hepatitis A virus and feline calicivirus in platelet concentrates.

BACKGROUND: Nonenveloped transfusion-transmissible viruses such as hepatitis A virus (HAV) and hepatitis E virus (HEV) are resistant to many of the common virus inactivation procedures for blood products. This study investigated the pathogen inactivation (PI) efficacy of the THERAFLEX UV-Platelets system against two nonenveloped viruses: HAV and feline calicivirus (FCV), in platelet concentrates (PCs). STUDY DESIGN AND METHODS: PCs in additive solution were spiked with high titers of cell culture-derived HAV and FCV, and treated with ultraviolet C at various doses. Pre- and posttreatment samples were taken and the level of viral infectivity determined at each dose. For some samples, large-volume plating was performed to improve the detection limit of the virus assay. RESULTS: THERAFLEX UV-Platelets reduced HAV titers in PCs to the limit of detection, resulting in a virus reduction factor of greater than 4.2 log steps, and reduced FCV infectivity in PCs by 3.0 ± 0.2 log steps. CONCLUSIONS: THERAFLEX UV-Platelets effectively inactivates HAV and FCV in platelet units.

UV-C inactivation of foodborne bacterial and viral pathogens and surrogates on fresh and frozen berries.

Outbreaks of foodborne illness associated with berries often involve contamination with hepatitis A virus (HAV) and norovirus but also bacteria such as Escherichia coli O157:H7 and parasites such as Cyclospora caytanensis. We evaluated the applicability of UV-C to the inactivation of pathogens on strawberries, raspberries and blueberries. Our three-step approach consisted of assessing the chemical safety of UV-C-irradiated berries, evaluating the sensory quality after UV-C treatment and finally studying the inactivation of the target microorganisms. Treatments lasting up to 9 min (4000 mJ cm-2) did not produce detectable levels of furan (<5 µg/kg), a known photolysis product of fructose with genotoxic activity and thus were assessed to be toxicologically safe. No effect on taste or appearance was observed, unless treatment was excessively long. 20 s of treatment (an average fluence of ~ 212 mJ cm-2) reduced active HAV titer by >1 log10 unit in 95% of cases except on frozen raspberries, while 120 s were required to inactivate murine norovirus to this extent on fresh blueberries. The mean inactivation of HAV and MNV was greater on blueberries (2-3 log10) than on strawberries and raspberries (<2 log10). MNV was more sensitive on fresh than on frozen berries, unlike HAV. Inactivation of Salmonella, E. coli O157:H7 and Listeria monocytogenes was poor on all three berries, no treatment reducing viable counts by >1 log10 unit. In most matrices, prolonging the treatment did not improve the result to any significant degree. The effect was near its plateau after 20 s of treatment. These results provide insight into the effectiveness of UV-C irradiation for inactivating bacterial and viral pathogens and surrogates on fresh and frozen berries having different surface types, under different physical conditions and at different levels of contamination. Overall they show that UV-C as single processing step is unsuitable to inactivate significant numbers of foodborne pathogens on berries.

Hepatitis A Virus Disinfection in Water by Solar Photo-Fenton Systems.

This study evaluates and compares the effectiveness of solar photo-Fenton systems for the inactivation of hepatitis A virus (HAV) in water. The effect of solar irradiance, dark- Fenton reaction and three different reactant concentrations (2.5/5, 5/10 and 10/20 mg/L of Fe2+/H2O2) on the photo-Fenton process were tested in glass bottle reactors (200 mL) during 6 h under natural sunlight. Disinfection kinetics were determined both by RT-qPCR and infectivity assays. Mean water temperatures ranged from 25 to 27.3 °C, with a maximum local noon UV irradiances of 22.36 W/m2. Photo-Fenton systems yielded increased viral reduction rates in comparison with the isolated effect under the Fenton reaction in darkness (negligible viral reduction) or the solar radiation (0.25 Log of RNA reduction). With the highest concentration employed (10-20 mg/L Fe2+-H2O2), an average RNA reduction rate of ~ 1.8 Log (initial concentration of 105 pfu/mL) and a reduction of 80% in the infectivity capacity were reached. Results showed a strong synergistic effect between Fe2+/H2O2 and sunlight, demonstrating that significant disinfection rates of HAV under photo-Fenton systems may occur with relatively higher efficiency at middle environmental temperatures and without the need for an energy-intensive light source.

Solar water disinfection (SODIS): Impact on hepatitis A virus and on a human Norovirus surrogate under natural solar conditions.

This study evaluates the effectiveness of solar water disinfection (SODIS) in the reduction and inactivation of hepatitis A virus (HAV) and of the human Norovirus surrogate, murine Norovirus (MNV-1), under natural solar conditions. Experiments were performed in 330 ml polyethylene terephthalate (PET) bottles containing HAV or MNV-1 contaminated waters (10(3) PFU/ml) that were exposed to natural sunlight for 2 to 8 h. Parallel experiments under controlled temperature and/or in darkness conditions were also included. Samples were concentrated by electropositive charged filters and analysed by RT-real time PCR (RT-qPCR) and infectivity assays. Temperature reached in bottles throughout the exposure period ranged from 22 to 40ºC. After 8 h of solar exposure (cumulative UV dose of ~828 kJ/m2 and UV irradiance of ~20 kJ/l), the results showed significant (P<0.05) reductions from 4.0 (+/-0.56)x10(4) to 3.15 (+/-0.69)x10(3) RNA copies/100ml (92.1%, 1.1 log) for HAV and from 5.91 (+/-0.59)x10(4) to 9.24 (+/-3.91)x10(3) RNA copies/100 ml (84.4%, 0.81 log) for MNV-1. SODIS conditions induced a loss of infectivity between 33.4% and 83.4% after 4 to 8 h in HAV trials, and between 33.4% and 66.7% after 6 h to 8 h in MNV-1 trials. The results obtained indicated a greater importance of sunlight radiation over the temperature as the main factor for viral reduction.

Ultraviolet-C efficacy against a norovirus surrogate and hepatitis A virus on a stainless steel surface.

In this study, the effects of 10-300 mWs/cm(2) of ultraviolet radiation (UV-C) at 260 nm were investigated for the inactivation of two foodborne viruses: murine norovirus-1 (MNV-1; a human norovirus [NoV] surrogate) and hepatitis A virus (HAV). We used an experimentally contaminated stainless steel surface, a common food-contact surface, to examine the effects of low doses of UV-C radiation on MNV-1 and HAV titers. The modified Gompertz equation was used to generate non-linear survival curves and calculate dR-values as the UV-C dose of 90% reduction for MNV-1 (R(2)=0.95, RMSE=0.038) and HAV (R(2)=0.97, RMSE=0.016). Total MNV-1 and HAV titers significantly decreased (p<0.05) with higher doses of UV-C. MNV-1 and HAV were reduced to 0.0-4.4 and 0.0-2.6 log10PFU/ml, respectively, on the stainless steel surfaces by low-dose UV-C treatment. The dR-value, 33.3 mWs/cm(2) for MNV-1 was significantly (p<0.05) lower than 55.4 mWs/cm(2) of HAV. Therefore, the present study shows that HAV is more resistant to UV-C radiation than MNV-1. These data suggest that low doses of UV-C light on food contact surfaces could be effective to inactivate human NoV and HAV in restaurant, institutional, and industrial kitchens and facilities.

Solar water disinfection (SODIS): impact on hepatitis A virus and on a human Norovirus surrogate under natural solar conditions

This study evaluates the effectiveness of solar water disinfection (SODIS) in the reduction and inactivation of hepatitis A virus (HAV) and of the human Norovirus surrogate, murine Norovirus (MNV-1), under natural solar conditions. Experiments were performed in 330 ml polyethylene terephthalate (PET) bottles containing HAV or MNV-1 contaminated waters (103 PFU/ml) that were exposed to natural sunlight for 2 to 8 h. Parallel experiments under controlled temperature and/or in darkness conditions were also included. Samples were concentrated by electropositive charged filters and analysed by RT-real time PCR (RT-qPCR) and infectivity assays. Temperature reached in bottles throughout the exposure period ranged from 22 to 40ºC. After 8 h of solar exposure (cumulative UV dose of ~828 kJ/m2 and UV irradiance of ~20 kJ/l), the results showed significant (P < 0.05) reductions from 4.0 (±0.56) ×104 to 3.15 (±0.69) × 103 RNA copies/100 ml (92.1%, 1.1 log) for HAV and from 5.91 (±0.59) × 104 to 9.24 (±3.91) × 103 RNA copies/100 ml (84.4%, 0.81 log) for MNV-1. SODIS conditions induced a loss of infectivity between 33.4% and 83.4% after 4 to 8 h in HAV trials, and between 33.4% and 66.7% after 6 h to 8 h in MNV-1 trials. The results obtained indicated a greater importance of sunlight radiation over the temperature as the main factor for viral reduction (AU)

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Susceptibility of murine norovirus and hepatitis A virus to electron beam irradiation in oysters and quantifying the reduction in potential infection risks.

Consumption of raw oysters is an exposure route for human norovirus (NoV) and hepatitis A virus (HAV). Therefore, efficient postharvest oyster treatment technology is needed to reduce public health risks. This study evaluated the inactivation of HAV and the NoV research surrogate, murine norovirus-1 (MNV-1), in oysters (Crassostrea virginica) by electron beam (E-beam) irradiation. The reduction of potential infection risks was quantified for E-beam irradiation technology employed on raw oysters at various virus contamination levels. The E-beam dose required to reduce the MNV and HAV titer by 90% (D(10) value) in whole oysters was 4.05 (standard deviations [SD], ±0.63) and 4.83 (SD, ±0.08) kGy, respectively. Microbial risk assessment suggests that if a typical serving of 12 raw oysters was contaminated with 10(5) PFU, a 5-kGy treatment would achieve a 12% reduction (from 4.49 out of 10 persons to 3.95 out of 10 persons) in NoV infection and a 16% reduction (from 9.21 out of 10 persons to 7.76 out of 10 persons) in HAV infections. If the serving size contained only 10(2) PFU of viruses, a 5-kGy treatment would achieve a 26% reduction (2.74 out of 10 persons to 2.03 out of 10 persons) of NoV and 91% reduction (2.1 out of 10 persons to 1.93 out of 100 persons) of HAV infection risks. This study shows that although E-beam processing cannot completely eliminate the risk of viral illness, infection risks can be reduced.

The efficacy of simulated solar disinfection (SODIS) against coxsackievirus, poliovirus and hepatitis A virus.

The antimicrobial activity of simulated solar disinfection (SODIS) against enteric waterborne viruses including coxsackievirus-B5, poliovirus-2 and hepatitis A virus was investigated in this study. Assays were conducted in transparent 12-well polystyrene microtitre plates containing the appropriate viral test suspension. Plates were exposed to simulated sunlight at an optical irradiance of 550 Wm(-2) (watts per square metre) delivered from a SUNTEST™ CPS+ solar simulator for 6 hours. Aliquots of the viral test suspensions were taken at set time points and the level of inactivation of the viruses was determined by either culture on a HeLa cell monolayer for coxsackievirus-B5 and poliovirus-2 or by utilising a chromogenic antibody-based approach for hepatitis A virus. With coxsackievirus-B5, poliovirus-2 and hepatitis A virus, exposure to SODIS at an optical irradiance of 550 Wm(-2) for 1-2 hours resulted in complete inactivation of each virus. The findings from this study suggest that under appropriate conditions SODIS may be an effective technique for the inactivation of enteric viruses in drinking water. However, further verification studies need to be performed using natural sunlight in the region where the SODIS technology is to be employed to validate our results.

Stability of human enteric viruses in seawater samples from mollusc depuration tanks coupled with ultraviolet irradiation.

AIMS: To evaluate the stability in seawater of human adenovirus (HAdV2), murine norovirus (MNV-1) and hepatitis A virus (HAV) in a shellfish depuration system with and without ultraviolet (UV) treatment. METHODS AND RESULTS: Seawater was seeded with viruses and disinfected using a 36 W lamp. Samples were collected at 24, 48, 72, 96 and 120 h; viruses were concentrated and the viral decay was evaluated using molecular and cell culture methods. Based on the molecular results, at 120 h of disinfection, there was a reduction of more than 3 log(10) for HAdV2 and HAV; MNV-1, a 4.5 log(10) reduction was observed at 72 h. Infectious MNV-1 was not detected after 72 h of treatment; while HAdV2 remained infectious. Seawater not treated demonstrated a progressive viral reduction for the three viruses tested. CONCLUSIONS: The UV reduced the number of viral particles, and the results indicate there is natural and gradual decrease of viral load and viability in seawater. SIGNIFICANCE AND IMPACT OF THE STUDY: UV irradiation is the method of choice for shellfish depuration in many countries; this work showed useful information about the viral stability in seawater and application of UV to water disinfection to be used in shellfish depuration tanks.

Temperature and density dependent induction of a cytopathic effect following infection with non-cytopathic HAV strains.

Hepatitis A virus infection and growth in cultured cells is protracted, cell-type restricted, and generally not accompanied by the appearance of a cytopathic effect, with the exception of some culture-adapted strains. We demonstrate that the non-cytopathic HAV strain HM175/clone 1 can be induced to exhibit a cytopathic phenotype in both persistently or acutely infected cells under co-dependent conditions of lower incubation temperature (<34°C) and reduced cell density in both monkey (FRhK-4) and human (A549) cells. This phenotype is not virus-strain restricted, as it was also observed in cells infected with HAV strains, HAS-15 and LSH/S. Cytopathic effect was accompanied by rRNA cleavage, indicating activation of the RNase L pathway, viral negative strand synthesis, caspase-3 activation, and apoptosis. The results indicate that a cytopathic phenotype may be present in some HAV strains that can be induced under appropriate conditions, suggesting the potential for development of a plaque assay for this virus.

The depuration dynamics of oysters (Crassostrea gigas) artificially contaminated with hepatitis A virus and human adenovirus.

Within the country of Brazil, Santa Catarina is a major shellfish producer. Detection of viral contamination is an important step to ensure production quality and consumer safety during this process. In this study, we used a depuration system and ultraviolet (UV) disinfection to eliminate viral pathogens from artificially infected oysters and analysed the results. Specifically, the oysters were contaminated with hepatitis A virus (HAV) or human adenovirus type 5 (HAdV5). After viral infection, the oysters were placed into a depuration tank and harvested after 48, 72 and 96 h. After sampling, various oyster tissues were dissected and homogenised and the viruses were eluted with alkaline conditions and precipitated with polyethylene glycol. The oyster samples were evaluated by cell culture methods, as well as polymerase chain reaction (PCR) and quantitative-PCR. Moreover, at the end of the depuration period, the disinfected seawater was collected and analysed by PCR. The molecular assays showed that the HAdV5 genome was present in all of the depuration time samples, while the HAV genome was undetectable after 72 h of depuration. However, viral viability tests (integrated cell culture-PCR and immunofluorescence assay) indicated that both viruses were inactivated with 96 h of seawater recirculation. In conclusion, after 96 h of UV treatment, the depuration system studied in this work purified oysters that were artificially contaminated with HAdV5 and HAV.

The depuration dynamics of oysters (Crassostrea gigas) artificially contaminated with hepatitis A virus and human adenovirus

Within the country of Brazil, Santa Catarina is a major shellfish producer. Detection of viral contamination is an important step to ensure production quality and consumer safety during this process. In this study, we used a depuration system and ultraviolet (UV) disinfection to eliminate viral pathogens from artificially infected oysters and analysed the results. Specifically, the oysters were contaminated with hepatitis A virus (HAV) or human adenovirus type 5 (HAdV5). After viral infection, the oysters were placed into a depuration tank and harvested after 48, 72 and 96 h. After sampling, various oyster tissues were dissected and homogenised and the viruses were eluted with alkaline conditions and precipitated with polyethylene glycol. The oyster samples were evaluated by cell culture methods, as well as polymerase chain reaction (PCR) and quantitative-PCR. Moreover, at the end of the depuration period, the disinfected seawater was collected and analysed by PCR. The molecular assays showed that the HAdV5 genome was present in all of the depuration time samples, while the HAV genome was undetectable after 72 h of depuration. However, viral viability tests (integrated cell culture-PCR and immunofluorescence assay) indicated that both viruses were inactivated with 96 h of seawater recirculation. In conclusion, after 96 h of UV treatment, the depuration system studied in this work purified oysters that were artificially contaminated with HAdV5 and HAV.

Inactivation of hepatitis A virus and norovirus surrogate in suspension and on food-contact surfaces using pulsed UV light (pulsed light inactivation of food-borne viruses).

This study was conducted to evaluate the inactivation of murine norovirus (MNV-1) and hepatitis A virus (HAV) by pulsed ultraviolet (UV) light. MNV-1 was used as a model for human norovirus. Viral suspensions of about 10(6) PFU/ml were exposed to pulses of UV light for different times and at different distances in a Xenon Steripulse device (model RS-3000C). Inactivation studies were also carried out on 1-cm(2) stainless steel and polyvinyl chloride disks with 10(5) PFU/ml. Inactivation of MNV-1 and HAV at 10.5 cm from the UV source was greater on inert surfaces than in suspension. The presence of organic matter (fetal bovine serum) reduced the effectiveness of pulsed light both in suspension and on surfaces. However, 2-s treatment in the absence of FBS completely inactivated (5 log reduction) the viral load at different distances tested, whether in suspension (MNV-1) or on disks (MNV-1 and HAV). The same treatment in the presence of fetal bovine serum (5%) allowed a reduction of about 3 log. This study showed that short duration pulses represent an excellent alternative for inactivation of food-borne viruses. This technology could be used to inactivate viruses in drinking water or on food-handling surfaces.

Low-dose irradiation improves microbial quality and shelf life of fresh mint (Mentha piperita L.) without compromising visual quality.

UNLABELLED: The effects of low-dose irradiation (0.25 to 2 kGy) and postirradiation storage (at 4 degrees C) on microbial and visual quality, color values (L*, a*, b*, chroma, and hue [ degrees ]), and chlorophyll content (Chl a, Chl b, and total Chl) of fresh mint were evaluated. Samples inoculated with E. coli O157:H7, Salmonella, and MS2 bacteriophage were irradiated and evaluated. E. coli O157:H7 and Salmonella populations were reduced by 2 to 2.4, 3.5, and 5.8 log CFU/g, respectively, 1 d after treatment with 0.25, 0.60, and 1 kGy, respectively, and were completely eliminated at 2 kGy. None of the irradiation doses (P < 0.0001) reduced MS2 bacteriophage populations by more than 0.60 log PFU/g. Irradiation doses did not affect visual quality and samples remained of excellent to good quality (score 7.75 to 9) for up to 9 d of storage. Irradiation at 0.60, 1, and 2 kGy increased (P < 0.0001) Chl a, Chl b, and total Chl. Both total Chl and Chl a decreased significantly after 3 d of storage. Significant decreases in Chl b were not observed until day 12 of storage. Color values (L*, b*, and chroma) were not significantly different until day 6 of storage and hue ( degrees ) remained unchanged (179 degrees ) for the entire storage period of 12 d. Overall, irradiation did not change L*, a*, b*, or chroma. These results demonstrate that irradiation of fresh mint at 2 kGy has the potential to improve its microbial quality and extend its shelf life without compromising its visual quality and color. PRACTICAL APPLICATION: Mints and other raw fresh herbs are widely used for flavoring as well as garnish in a variety of dishes without further cooking. However, mint is one considered as one of the high-risk herbs when it comes to microbial contamination. We have evaluated the use of gamma irradiation treatment at very low doses ranging from 0 to 2 kGy to eliminate seeded Salmonella spp, E. coli O157:H7, and MS2 bacteriophage, a surrogate of hepatitis A virus. We found that low-dose irradiation (1.0 to 2.0 kGy) appears to be a promising method for improving the microbiological quality of fresh mint without compromising its visual and color attributes. This method may be applied to many popular fresh culinary herbs that are commonly used as garnishes in Asian cuisine.

UV light inactivation of hepatitis A virus, Aichi virus, and feline calicivirus on strawberries, green onions, and lettuce.

A majority of illnesses caused by foodborne viruses are associated with fresh produce. Fruits and vegetables may be considered high-risk foods, as they are often consumed raw without a specific inactivation step. Therefore, there is a need to evaluate nonthermal treatments for the inactivation of foodborne pathogens. This study investigates the UV inactivation of three viruses: feline calicivirus (a surrogate for norovirus), and two picornaviruses, hepatitis A virus and Aichi virus. Three produce types were selected for their different surface topographies and association with outbreaks. Green onions, lettuce, and strawberries were individually spot inoculated with 10(7) to 10(9) 50% tissue culture infective doses (TCID50) of each virus per ml and exposed to UV light at various doses (< or = 240 mW s/cm2), and viruses were eluted using an optimized recovery strategy. Virus infection was quantified by TCID50 in mammalian cell culture and compared with untreated recovered virus. UV light applied to contaminated lettuce resulted in inactivation of 4.5 to 4.6 log TCID50/ml; for contaminated green onions, inactivation ranged from 2.5 to 5.6 log TCID50/ml; and for contaminated strawberries, inactivation ranged from 1.9 to 2.6 log TCID50/ml for the three viruses tested. UV light inactivation on the surface of lettuce is more effective than inactivation on the other two produce items. Consistently, the lowest results were observed in the inactivation of viruses on strawberries. No significant differences (P > 0.05) for virus inactivation were observed among the three doses applied (40, 120, and 240 mW s/cm2) on the produce, with the exception of hepatitis A virus and Aichi virus inactivation on green onions, where inactivation continued at 120 mW s/cm2 (P < 0.05).

Photoinactivation of hepatitis A virus by synthetic porphyrins.

Porphyrins are photosensitizers and may be applicable in situations where viral inactivation is required, as for in vitro inactivation of nonenveloped viruses in blood components or in other aqueous media. No study has examined the efficacy of porphyrin inactivation on human pathogens such as hepatitis A virus (HAV) in plasma or other liquids. Experiments were conducted to evaluate the effect of synthetic porphyrins on HAV in porphyrin-containing human plasma and phosphate-buffered saline exposed to long-wavelength (365 nm) UV light. Inactivation of bacteriophage MS2 (MS2) also was determined in some trials. Solutions containing cationic, anionic or amphiphilic porphyrins irradiated with an average light dose of 4.3 J/cm(2) for 90 min resulted in >3 log(10) (>99.9%) to >4 log(10) (>99.99%) inactivation of both HAV and MS2. Viral inactivation may have been greater than observed because the limits of detection of the assay had been reached. Under ambient lighting conditions, none of the porphyrins was mutagenic in the Ames assay and only the congener with the longest chain-length, tetrakis (N-[n-hexadecyl]-4-pyridiniumyl) porphyrin, was appreciably toxic to mammalian cells. Disinfection by photoactivated synthetic porphyrins therefore can offer an effective and relatively safe approach to removal of nonenveloped viruses from aqueous media.

Use of reverse transcription and PCR to discriminate between infectious and non-infectious hepatitis A virus.

Hepatitis A virus (HAV) is a major cause of infectious hepatitis worldwide. Detection of HAV in contaminated food or water is a priority research area in laboratories worldwide. Our laboratory has reported previously the development of reverse transcription-polymerase chain reaction (RT-PCR) based detection and typing methods for HAV in contaminated shellfish and produce. It is commonly held that RT-PCR can detect viral genome, but cannot distinguish between infectious and inactivated virus. Therefore, signals obtained after PCR should be considered as false positives unless it can be shown that the sample contains virus capable of infecting a suitable host cell line in culture. We present data to show that this general assumption is not valid. Evidence is provided that demonstrate that signals generated after RT-PCR amplification of viral genome correlated well with the presence of infectious virus in the sample. Viral samples inactivated by heat or UV treatment produced significantly lower signal strength that paralleled infectivity of the sample in cultured cells. The loss of signal strength is most likely the result of damage to the viral RNA that renders it unsuitable for RT-PCR. The correlation between PCR signal and infectivity was better following UV inactivation than heat treatment. The procedure may be adapted to other viruses and inactivating agents.

Effect of gamma irradiation on human cortical bone transplants contaminated with enveloped and non-enveloped viruses.

In the production of bone grafts intended for transplantation, basic safety measures to avoid the transmission of pathogens are selection and serological screening of donors for markers of virus infections. As an additional safety tool we investigated the effect of gamma irradiation on the sterility of human bone diaphysis transplants and evaluated its impact on the virus safety of transplants. Model viruses were included in the study to determine the dose necessary to achieve a reduction factor for the infectivity titres of at least 4 log(10) at a temperature of -30+/-5 degrees C. The following viruses were used: human immunodeficiency virus type 2 (HIV-2), hepatitis A virus (HAV), and poliovirus (PV-1), and the following model viruses: pseudorabies virus (PRV) as a model for human herpesviruses, bovine viral diarrhoea virus (BVDV) for HCV, and bovine parvovirus (BPV) for parvovirus B19. A first approach was to determine the D(10) values (kGy) for the different viruses (virus inactivation kinetics: BPV 7.3; PV-1 7.1; HIV-2 7.1; HAV 5.3; PRV 5.3; BVDV <3.0 kGy). Based on these results, inactivation of these viruses was studied in experimentally contaminated human bone transplants (femoral diaphyses). For BPV, the most resistant one of the viruses studied, a dose of approximately 34 kGy was necessary to achieve a reduction of infectivity titres of 4 log(10). We therefore recommend a dose of 34 kGy for the sterilisation of frozen bone transplants.