Inactivation of viruses by chemically and photochemically generated singlet molecular oxygen.

Inactivation of viruses in blood plasma can be achieved by photodynamic procedures using methylene blue (MB) or other photoactive dyes. Singlet molecular oxygen (1O2) probably contributes to the virucidal effects of photosensitization. We report the inactivation of herpes simplex virus type 1 (HSV-1) and suid herpes virus type 1 (SHV-1) by chemically generated singlet oxygen, produced by thermal decomposition of the endoperoxide of 3,3'-(1,4-naphthylidene)dipropionate (NDPO2). We demonstrate that viruses can be inactivated by 1O2 generated by chemiexcitation in a reaction in the dark, even in the presence of human plasma. Virus inactivation in phosphate-buffered saline (PBS) was enhanced when water was replaced by deuterium oxide (D2O) and diminished when human plasma or quenchers (imidazole or histidine) were added. The singlet oxygen quenching activities of plasma, imidazole and histidine correlated with their inhibitory effects on virus inactivation. The production of 1O2 was assessed by an indicator reaction: the bleaching of p-nitrosodimethylaniline (RNO) with imidazole as 1O2 acceptor. Virus inactivation and singlet oxygen generation of NDPO2 were compared with those of MB/light-mediated photosensitization. Based on similar amounts of 1O2 generated by either procedure, virus inactivation by MB/light was more effective. Virus inactivation by MB/light was not affected by type I quenchers (e.g. mannitol), but was inhibited by human plasma or singlet oxygen quenchers. Furthermore, in D2O-based PBS, virus inactivation was more effective than that in H2O. These observations confirm that singlet oxygen is involved in virus inactivation by MB/light. Taken together, the results demonstrate that singlet oxygen produced by either procedure is virucidal. The enhanced effect of the photochemical procedure suggests that, in addition to type II, type I reactions and/or the binding affinity of the dye for the virus contribute to virus killing by MB/light.

Viral marker rates among unpaid blood donors in Europe decreased from 1990 to 1996.

Not for profit plasma fractionators in Europe believe that the standardised measurement and reporting of such epidemiological data is a key issue for public health. Accordingly, under the auspices of the European Plasma Fractionation Association (EPFA), t

Results of viral marker screening of unpaid blood donations and probability of window period donations in 1997. EPFA Working Group on Quality Assurance.

BACKGROUND AND OBJECTIVES: To monitor the safety of the blood supply and evaluate the potential benefits of additional measures, the likelihood of virus transmission must be assessed. The European Plasma Fractionation Association and its member organisations have therefore developed a surveillance system to monitor infection markers among unpaid blood and plasma donors. We report and analyse the results of this surveillance for 1997. MATERIALS AND METHODS: Data from the screening of unpaid donations for anti-HIV, anti-HCV and HBsAg during 1997 were collected retrospectively by EPFA member organisations. We identified seroconverters and estimate the probability of window period donations. RESULTS: Data included screening results from 11 million unpaid donations in Europe, the USA and Australia. Prevalence of viral markers varied, with marker rates from repeat donations in Europe and Australia being significantly lower than in the USA. For first-time donations, in contrast, prevalence of HBsAg in the USA was within the ranges of those measured in Europe and Australia. Screening data of about 5 million European and 0.5 million Australian repeat donations were used to identify seroconverters. From the seroconverters that were detected among the European organisations, we estimated that 1 in every 2,323,778 repeat donations (range: 707,090-20,922,520) was made during the window period of anti-HIV screening. One in 620,754 (201,216-2,316,805) and 1 in 398,499 (155,209 to >1,088,511) repeat donations were made during the anti-HCV or HBsAg window period, respectively. Probabilities of window period donations in Australia were within the ranges of those measured in Europe. CONCLUSIONS: The collated surveillance data of 1997 illustrate the high degree of safety in blood and plasma products from unpaid donors.

Infection cycle of herpes viruses after photodynamic treatment with methylene blue and light.

Herpes simplex virus type 1 was inactivated by illumination with red light in the presence of low concentrations (1 microM) of methylene blue (MB). In ultrastructural examinations after photodynamic treatment, no significant damage of the viral envelope was observed. The inactivated viruses were able to bind to host cells and to penetrate them. Viral DNA was released within the cells, but DNA replication was completely inhibited by photodynamic treatment. In Southern blots, a degradation of the viral DNA was detected with increasing illumination time. Taken together, these data show that MB/light treatment of herpes viruses gives rise to DNA damage and blocks DNA replication.

Hepatitis C and human immunodeficiency virus RNA degradation by methylene blue/light treatment of human plasma.

Treatment of human plasma with methylene blue in combination with visible light (MB/light) inactivates several bloodborne viruses such as retro viruses and herpes viruses. The viral nucleic acid is thought to be a critical target for the inactivation procedure. We investigated the effects of photodynamic treatment on the RNA of hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) using Amplicor reverse transcriptase polymerase chain reaction (RT-PCR), which detects and quantifies a small fragment of the viral RNA. The detectable HCV RNA load (5-nontranslated region) in infected human plasma declined by 94-97 % within 10 min of illumination in small-scale experiments (1-2 ml vol.). Since the same effect was observed in both anti-HCV positive and negative ("window") samples, it can be concluded that HCV antibodies do not influence virus inactivation by photodynamic treatment. The effect of treatment on RT-PCR signals of HIV-1, which is known to be inactivated rapidly by MB/light treatment, was examined. Plasma was infected with HIV-1 and subjected to RT-PCR, which detected a part of the gag gene. The extent and kinetics of PCR signal reduction induced by MB/light treatment were similar to those observed for HCV. Experiments at production scale where single plasma units (300 ml) were infected with HCV showed reduction rates of PCR signals consistent with those measured in the small-scale experiments. The data support the view that MB/light treatment affects the viral nucleic acids and suggest that HCV is susceptible to the procedure.

Sixth EPFA/NIBSC workshop on NAT in rome 1999: outcome of discussions.

The sixth international workshop on "Nucleic Acid Amplification Technology for the Detection of Blood Borne Viruses" was held on 5-6 May 1999 at the Istituto Superiore di Sanità in Rome, Italy. The purpose of the workshop was to bring together regulators, blood and plasma product manufacturers, national control agencies, test kits manufacturers and scientists to discuss recent experience as well as regulatory topics related to the use of NAT for the detection of blood borne viruses. Various papers were presented and discussed in 6 sessions.