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PubMed1991

[Virus inactivation of plasma].

A procedure for the virus inactivation of single donor plasma is described. Virus inactivation is achieved by the combination of 1 mumol of phenothiazin dye, e.g. methylene blue and toluidine blue, and visible light. Using this method VSV, HSV and HIV, as well as other viruses, were inactivated. It could be shown that virus inactivation caused only a minimal reduction in the biological activities of coagulation factors and inhibitors. Furthermore, there was no indication for either neo-antigens or IgE-antibodies. In tolerance studies on dogs it was demonstrated that virus-inactivated autologous plasma caused no toxic effects.

Animals↗
PubMed2001

Virus inactivation and protein modifications by ethyleneimines.

Virus inactivation by ethyleneimines was first introduced more than 30 years ago. Selective targeting of nucleic acids was reported for oligomeric ethyleneimines. In this study, trimeric ethyleneimine (TEI) was used to inactivate minute virus of mice (MVM; Parvoviridae) and Semliki forest virus (SFV; Togaviridae). The pH-dependency of the inactivation kinetics observed with MVM was different compared to the kinetics reported for other viruses. The higher inactivation rate at higher pH favoured the idea of a mechanism involving protein modifications. Alteration of the isoelectric point and changes in mass could be observed after treatment of soluble proteins with TEI. The uptake of MVM by host cells was reduced or completely blocked by TEI treatment, as shown by monitoring viral internalisation of DNA into target cells. The observed loss of virus infectivity coincided with the inhibition of virus uptake. Thus, virus inactivation by TEI is most likely also a result of chemical modifications of viral surface proteins.

Aedes↗
PubMed2001

Construction of recombinant vaccinia viruses using PUV-inactivated virus as a helper.

Recombinant vaccinia viruses (VVs) are widely used as expression vectors in molecular biology and immunology and are now under evaluation for gene therapy. The current techniques for inserting foreign DNA into the large VV genome are based on either homologous recombination between transfer plasmids and VVgenomes or direct DNA ligation and packaging using replication-deficient poxviruses. Here, we describe efficient new versions of both methods that produce 90%-100% of the recombinant viruses. In the new homologous recombination method, VV DNA "arms" obtained by NotI digestion and intact transfer plasmids were used for co-transfection. In the direct DNA ligation method, foreign DNA was inserted into a unique NotI restriction site of the VVgenome. In both methods, the generation of recombinant viruses was carried out in cells infected with a non-replicating, psoralen-UV (PUV)-inactivated helper VV. The convenience of these new techniques is demonstrated by the construction of recombinant VVs that produce E. coli beta-galactosidase. An important feature of these strategies is that any VV strain can be used as a helper virus after PUV inactivation.

Blotting, Southern↗
PubMed1994

[Possibilities of virus inactivation of pooled fresh plasma with tri-n-butylphosphate (TNBP) detergents (SD procedure)].

Treatment with tri-n-butyl-phosphate and detergent (SD-treatment) leads to efficient inactivation of viruses having a lipid enveloped surface, like hepatitis B virus, hepatitis C virus and human immunodeficiency virus, that are presently the most transfusion relevant viruses in Germany. Other lipid enveloped viruses of the herpes group like cytomegalovirus and Epstein-Barr virus are inactivated as well. Non-enveloped viruses like parvovirus B19 and picornaviruses are not inactivated by SD-treatment. Future inactivation of blood components like plasma and blood products will be a combination of SD- and heat-treatment. Keeping single plasma units in quarantine for 6 months is one of the alternatives in elevating transfusion safety. For transfused blood the safety against infectious agent will continue to depend on the effectiveness of donor selection and the efficacy of testing.

Acquired Immunodeficiency Syndrome↗
PubMed1999

GB virus C/hepatitis G virus infection in HIV infected patients with haemophilia despite treatment with virus inactivated clotting factor concentrates.

AIM: To determine the frequency of GB virus C (GBV-C)/hepatitis G virus (HGV) infection before and after switch to the use of virus inactivated concentrates in haemophiliac patients infected with human immunodeficiency virus (HIV). PATIENTS AND METHODS: Initial and follow up sera from 49 children with haemophilia were analysed for the presence of GBV-C/HGV RNA and antibodies to HGV (anti-HGV). All patients had been infected with HIV while receiving concentrates without virus inactivation before 1984 and were subsequently treated with virus inactivated concentrates. RESULTS: In the first available serum sample (1987 or later), two of 49 patients were GBV-C/HGV RNA positive and two further patients were anti-HGV positive. During follow up (mean, 6 years), 14 patients developed markers of GBV-C/HGV infection. Eleven of these had received no blood products except clotting factor concentrates that had been prepared with virus inactivation. CONCLUSIONS: Despite being treated with virus inactivated clotting factor concentrates, HIV positive patients with haemophilia are at an increased risk of manifesting GBV-C/HGV infection. We hypothesise that GBV-C/HGV is transmitted by these clotting factor concentrates. However, we cannot rule out the emergence of markers of GBV-C/HGV infection as a result of the progression of immune impairment in the course of HIV infection.

Adolescent↗
PubMed2003

Destruction of Cucumber green mottle mosaic virus by heat treatment and rapid detection of virus inactivation by RT-PCR.

Heat treatment is commonly used to control viral contamination of seeds. To study virus inactivation, virus was purified from seeds contaminated with Cucumber green mottle mosaic virus (CGMMV) after various heat treatments. CGMMV particles were observed to be physically disrupted by high temperature. Analysis of viral RNA revealed that the 5' and 3' termini of the genome were protected whereas regions between 2-2.5, 3.2-3.7 and 4-4.8 kb from the 5' terminus were not. Heat inactivation of virus on seeds was confirmed by RT-PCR using primers for a heat-sensitive region. The RT-PCR approach developed here may prove preferable to time- and labor-intensive bioassays for assessing virus heat inactivation.

Cucumis sativus↗
PubMed1985

Immune response of mice and sheep to bluetongue virus inactivated by gamma irradiation.

Gamma irradiation is being tested as a means of inactivating bluetongue virus (BTV) for use in vaccines. Exposure of BTV 17 to various levels of irradiation revealed that a dose of approximately 0.6 megarad was required to reduce the virus titer by one log10, or 90%. To test the immunogenicity of irradiated BTV, mouse brain passaged virus and concentrated cell culture passaged virus were inactivated by 6 megarads of gamma irradiation, and vaccines were prepared by emulsifying the virus preparations in equal volumes of a modified incomplete Freund's adjuvant. These vaccines stimulated the production of neutralizing antibodies in mice and sheep, a cell mediated immune response in mice, and a protective immune response in sheep. The results suggest that gamma irradiation would be an effective means of inactivating BTV for the preparation of vaccines.

Animals↗
PubMed2000

Pressure cycling technology: a novel approach to virus inactivation in plasma.

BACKGROUND: Hydrostatic-pressure virus inactivation is a novel approach to the inactivation of pathogens in plasma and blood-derived components, that retains the therapeutic properties of these products. STUDY DESIGN AND METHODS: A custom-built apparatus was used to pressurize human plasma samples spiked with lambda phage. Phage titer and plasma protein activities were monitored after pressure treatment. RESULTS: Pressure-mediated inactivation of lambda phage was found to be an effective means for virus inactivation, particularly when performed at near-zero (0 degrees C) temperatures, rather than at temperatures above 20 degrees C and below -40 degrees C. The efficiency of inactivation was improved by an increase in applied pressure and repeated cycling from atmospheric to high pressure. In contrast, activities of plasma proteins alkaline phosphatase and total amylase did not vary with temperature and remained within 29 percent and 6 percent, respectively, of starting values after the same pressure treatments. By combining cycling, near-zero temperatures, and high pressure, phage titers in serum were reduced approximately 6 log after 10 to 20 minutes of treatment. Activities of plasma proteins IgG, IgM, and factor X were at 104 percent, 89 percent, and 80 percent, respectively, of starting values after 20 minutes of the same temperature and pressure treatment. CONCLUSION: High-pressure procedures may be useful for the inactivation of viruses in blood and other protein-containing components.

Antibodies, Viral↗
PubMed1991

Protection against feline leukemia virus infection by use of an inactivated virus vaccine.

The protective immunity induced by 3 experimental FeLV vaccines were evaluated: Prototype inactivated FeLV vaccine developed from a molecularly cloned FeLV isolate (FeLV-FAIDS-61E-A); a mixture of immunodominant synthetic peptides corresponding to regions of the FeLV-Gardner-Arnstein-B (FeLV-GA-B) envelope proteins; and an adjuvant-disrupted but non-activated virus prepared from a non-cloned FeLV field isolate comprised of subgroup A and B viruses (FeLV-05821-AB). Included as controls were parallel groups of cats inoculated with adjuvants alone or with an established commercial FeLV vaccine. After each inoculation and after virulent virus challenge exposure, sera from all cats were assayed for ELISA-reactive antibody against purified FeLV, FeLV neutralizing (VN) antibody, and FeLV antigenemia/viremia--viral p27 antigen in serum and within circulating leukocytes. Immunity was challenged by oral/nasal exposure of vaccinated and control cats with FeLV-FAIDS-61E-A or FeLV-05821-AB, an infective, noncloned, tissue-origin, FeLV field isolate containing subgroup-A and -B viruses. Vaccine-induced immunity was assessed by comparing the postchallenge-exposure incidence of persistent viremia and the pre- and postchallenge exposure titers of VN and ELISA antibody in cats of the control and vaccine groups. The percentage of cats, that resisted development of persistent viremia after FeLV challenge exposure and the preventable fraction (PF) for the vaccine groups (which adjusts for the severity of the challenge and the degree of innate resistance in the controls) were as follows: adjuvant controls, 26%; FeLV-FAIDS-61E-A inactivated virus vaccine, 95% (PF = 93.2%); FeLV-GA-B peptide vaccine, 5% (-28.4%); FeLV-05821-AB noninactivated vaccine, 67% (55.4%); and commercial FeLV vaccine, 35% (12.2%). The prechallenge exposure mean VN antibody titer for each group was: less than 1:8 in the adjuvant controls; 1:43 in the FeLV-FAIDS-61E-A-vaccinated cats; less than 1:8 in the peptide-vaccinated cats; 1:38 in the noninactivated virus-vaccinated cats group; and 1:12 in the cats vaccinated with the commercial vaccine. Thus, induction of VN antibody in the vaccinated cats, although modest, appeared to be correlated with induction of protective immunity as defined by resistance to FeLV challenge exposure. Results of these studies indicate that inoculation of cats with an experimental inactivated virus vaccine prepared from a molecularly cloned FeLV isolate was most effective in stimulating protective immunity against heterologous and homologous FeLV challenge exposure.

Adjuvants, Immunologic↗
PubMed1986

Antibody response of mice to lactate dehydrogenase-elevating virus during infection and immunization with inactivated virus.

BALB/c and Swiss mice were infected with lactate dehydrogenase-elevating virus (LDV) or immunized with glutaraldehyde-inactivated or ether-extracted virus and their plasma was monitored for anti-LDV IgG and IgM levels by ELISA and indirect fluorescent antibody staining, for neutralizing antibodies, for sensitized antibody-virus complexes, for immune complexes, and for total plasma IgG and IgM. In infected mice, anti-LDV IgM was transiently formed during the first 2 weeks post infection (p.i.) but only at a low level. Anti-LDV IgG was produced in a biphasic manner with an initial peak at about 10 days p.i. and a secondary rise reaching a maximum level 30-80 days p.i. which was retained throughout the persistent phase of infection. The concomitant appearance of comparable levels of low molecular weight immune complexes suggests that most anti-LDV IgG was complexed with LDV proteins. Also, as early as 10 days p.i., infectious antibody-LDV complexes developed, which were neutralizable by rabbit anti-mouse IgG, whereas antibodies that neutralize the infectivity of exogenously added LDV appeared only 1-2 months p.i. Throughout infection, most of the anti-LDV IgG was directed to VP-3, the envelope glycoprotein of LDV, which was found to exist in at least 10 distinct forms ranging in molecular weight from 24 to 42 kDa. Anti-LDV IgG levels as high as those observed in infected mice developed in mice immunized with inactivated LDV. Antibodies to glutaraldehyde-inactivated LDV were also mainly directed to VP-3, but exhibited no neutralizing activity. The polyclonal B cell activation associated with a persistent LDV infection and the formation of immune complexes were not observed in mice immunized with inactivated virus.

Animals↗
PubMed1998

[How to prove complete virus inactivation in rabies vaccines. A comparison of an in vivo to an in vitro method]

At present, the complete inactivation of rabies virus in rabies vaccines ad us. vet. is proven by an animal experiment which causes severe suffering, the intracerebral injection of mice. This animal experiment yet is not validated. We have quantified the sensitivity of the mouse test and examined whether the animal experiment may be replaced by the immunofluorescence assay (IFT) as an in vitro method. Detection limits of both assays were determined depending on the examined product, i.e. prior to and after the addition of adjuvans and preservative, respectively. Furthermore, symptoms of the rabies desease were recorded and their severity was classified on a range of 1-5. Symptoms of rabies-infected mice were clear and highly specific. Symptoms classified as >/= 2 in context with a loss of >/= 15% of the initial weight were defined as humane endpoints of the desease. The quantitative detection of active virus was not inhibited in the presence of even high concentrations of inactivated virus. The detection limit of the mouse test was 10 viruses ml-1 independent of the examined product. The detection limit of the IFT prior to the addition of adjuvans and preservative was 10 viruses ml-1 as well. After the addition of these substances the detection limit rose to 103 viruses ml-1. Advantages and disadvantages of the mouse test and IFT are discussed.

Journal Article↗
PubMed1988

Analysis of the mechanism of influenza B virus inactivation by guinea pig serum.

Normal guinea pig serum lacking detectable antiviral antibody inactivated influenza B virus via the classical complement pathway. This virus inactivation appeared to result from the steric hindrance of HA activity by the association with the virus of serum proteins presumed to be complement components. Trypsin digestion of the associated proteins fully restored the HA activity but not infectivity. It was found that the virus underwent minor disruption of the envelope and degradation of M1 protein and genomic RNA.

Animals↗
PubMed2002

Pretreatment to avoid positive RT-PCR results with inactivated viruses.

Enteric viruses that are important causes of human disease must often be detected by reverse transcription-polymerase chain reaction (RT-PCR), a method that commonly yields positive results with samples that contain only inactivated virus. This study was intended to develop a pretreatment for samples, so that inactivated viruses would not be detected by the RT-PCR procedure. Model viruses were human hepatitis A virus, vaccine poliovirus 1 and feline calicivirus as a surrogate for the Norwalk-like viruses. Each virus was inactivated (from an initial titer of approximately 10(3) PFU/ml) by ultraviolet light, hypochlorite or heating at 72 degrees C. Inactivated viruses, that were treated with proteinase K and ribonuclease for 30 min at 37 degrees C before RT-PCR, gave a negative result, which is to say that no amplicon was detected after the reaction was completed. This antecedent to the RT-PCR method may be applicable to other types of viruses, to viruses inactivated in other ways and to other molecular methods of virus detection.

Animals↗
PubMed1995

Mammalian genotoxicity assessment of methylene blue in plasma: implications for virus inactivation.

BACKGROUND: The risk of adverse consequences of virus-inactivation procedures for plasma and cellular blood components must be less than the risk of transfusion-associated viral disease. Previous studies demonstrated that methylene blue, which is currently used in Europe for virus inactivation in fresh-frozen plasma, can elicit mutations in bacterial test systems. This study investigates the potential for methylene blue genotoxicity in two mammalian test systems. STUDY DESIGN AND METHODS: Different concentrations of methylene blue were prepared in plasma (heat-treated at 56 degrees C for 1 hour to reduce cytotoxicity) and used, without illumination, in an in vitro mouse lymphoma cell assay designed to detect forward mutations in the gene encoding thymidine kinase. The assay was performed in the presence or absence of rat liver S9 microsomal fraction. Similarly prepared samples of methylene blue in heat-treated plasma were used in an in vivo mouse micronucleus assay. Each system included a negative vehicle control (heat-treated plasma without methylene blue) and a positive control consisting of a known genotoxic agent. RESULTS: Intravenous administration to mice of 62 mg per kg of methylene blue did not increase the frequency of micronuclei in polychromatic red cells harvested from bone marrow. However, methylene blue concentrations of 10 micrograms per mL (with S9 activation) and 30 micrograms per mL (without S9 activation) significantly increased the thymidine kinase mutation frequency of mouse lymphoma cells to approximately 110 x 10(-6), from a spontaneous frequency of 28 x 10(-6). CONCLUSION: Methylene blue is mutagenic in cultured mammalian cells. In contrast, results from the mouse micronucleus assay suggest that the genotoxicity is not expressed in vivo. Considerably more investigation will be required to assess the genotoxic potential of intravenously administered methylene blue used in virus-inactivation procedures, because of the likelihood of the formation of methylene blue photoproducts or the impact of metabolic conversion of methylene blue to leukomethylene blue in vivo.

Animals↗