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The simian immunodeficiency virus transmembrane protein is poorly immunogenic in inactivated virus vaccine.

The transmembrane proteins (TMP) of immunodeficiency lentiviruses are primary candidates for inclusion in AIDS vaccines, the design and testing of which is facilitated by the SIV-macaque infection model. Antibody responses to linear determinants in the SIVmac TMP were investigated in rhesus macaques either infected with the SIVmac J5 molecular clone or vaccinated with partially purified, formalin-inactivated SIVmac. Infected animals were shown to recognise predominantly four regions in the external domain and three regions in the internal domain of the TMP defined by a series of nominally 20mer overlapping peptides. In contrast SIV vaccinates had extremely restricted and weak antibody responses to the TMP, indicating a selective loss of immunogenicity of this component in the vaccine.

Amino Acid Sequence↗

Replacement therapy with virus-inactivated plasma concentrates in von Willebrand disease.

In von Willebrand disease, the goal of treatment is to correct the two laboratory hallmarks of abnormal hemostasis, i.e. the deficiency of factor VIII (FVIII) and the prolonged bleeding time (BT). Since desmopressin (DDAVP) is able to achieve both these goals in the majority of patients, it is the treatment of choice. Some patients, however, are not responsive or become refractory to DDAVP. In these, blood products maintain an important therapeutic role, and there is a need to assess the efficacy of the recently available virus-inactivated plasma concentrates, which contain both FVIII and von Willebrand factor and carry a low risk of transmitting blood-borne viruses. Our survey of the data reported in the literature indicates that all available concentrates are similarly effective in attaining high and sustained levels of FVIII after infusion. Although they often shorten or normalize the prolonged BT, that effect is less uniform. Since concentrates appear efficacious in the majority of clinical situations that require the use of blood products, they should be preferred, because of their greater safety, to cryoprecipitate produced by blood banks, which cannot be virus inactivated.

Bleeding Time↗

Respiratory synctial virus infection in BALB/c mice previously immunized with formalin-inactivated virus induces enhanced pulmonary inflammatory response with a predominant Th2-like cytokine pattern.

Vaccination with formalin-inactivated respiratory syncytial virus (FI-RSV) caused excessive disease in infants upon subsequent natural infection with RSV. Recent studies with BALB/c mice have suggested that T cells are important contributors to lung immunopathology during RSV infection. In this study, we investigated vaccine-induced enhanced disease by immunizing BALB/c mice with live RSV intranasally or with FI-RSV intramuscularly. The mice were challenged with RSV 6 weeks later, and the pulmonary inflammatory response was studied by analyzing cells obtained by bronchoalveolar lavage 4 and 8 days after challenge. FI-RSV-immunized mice had an increased number of total cells, granulocytes, eosinophils, and CD4+ cells but a decreased number of CD8+ cells. The immunized mice also had a marked increase in the expression of mRNA for the Th2-type cytokines interleukin-5 (IL-5) and IL-13 as well as some increase in the expression of IL-10 (a Th2-type cytokine) mRNA and some decrease in the expression of IL-12 (a Th1-type cytokine) mRNA. The clear difference in the pulmonary inflammatory response to RSV between FI-RSV- and live-RSV-immunized mice suggests that this model can be used to evaluate the disease-enhancing potential of candidate RSV vaccines and better understand enhanced disease.

Animals↗

Quinacrine enhances vesicular stomatitis virus inactivation and diminishes hemolysis of dimethylmethylene blue-phototreated red cells.

Several photodynamic methods for virus inactivation in red blood cell (RBC) suspensions have resulted in unwanted hemolysis during extended 1-6 degrees C storage. To explore the possibility that hemolysis may be mediated by a membrane-bound dye, a molecule similar in structure to yet different in light absorption properties from the photosensitizer was used as an inhibitor for RBC membrane binding in virus photoinactivation and photohemolysis studies. The addition of 500 pM quinacrine to oxygenated RBC before treatment with 3.6 microM dimethylmethylene blue (DMMB) and 219 mJ/cm2 red light resulted in an increased extracellular concentration of the sensitizer, increased extracelluar viral inactivation kinetics, and decreased hemolysis during 1-6 degrees C storage without alteration of quinacrine absorption properties. These results collectively suggest that despite its recognized affinity for viral nucleic acid, DMMB also binds to RBC membranes and that the bound dye is, in part, responsible for photoinduced hemolysis.

Blood Preservation↗

Comparison of four virus-inactivated plasma concentrates for treatment of severe von Willebrand disease: a cross-over randomized trial.

Until recently, cryoprecipitate has been the treatment of choice in patients with severe von Willebrand disease (vWD) because it can transiently correct low plasma levels of factor VIII coagulant activity (FVIII:C) and shorten or normalize the prolonged bleeding time (BT), the two laboratory hallmarks of the disease. However, cryoprecipitate may still transmit blood-borne viruses, whereas the development of virucidal methods have rendered plasma concentrates containing FVIII:C and von Willebrand factor (vWF) safer. To establish their potential usefulness in the treatment of vWD, we compared the effect of four virus-inactivated concentrates on FVII:C and vWF plasma levels and the BT (template method) in 10 patients with severe vWD using a crossover randomized design. The concentrates were an intermediate-purity, pasteurized FVIII-vWF concentrate; an intermediate-purity, dry-heated FVIII-vWF concentrate; a solvent/detergent-treated vWF concentrate, containing little FVIII; and a high-purity solvent/detergent-treated FVIII-vWF concentrate. All concentrates were equally effective in attaining normal and sustained levels of FVIII:C postinfusion, although peak levels were more delayed after the vWF concentrate. The effect of concentrates on the BT, however, was less uniform and satisfactory. The pasteurized FVIII-vWF concentrate transiently corrected, completely or partially, the BT in 8 of 10 patients, the dry-heated and solvent/detergent FVIII/vWF concentrates in five, whereas in no patient did the vWF concentrate correct the BT according to the criteria used in this study. These effects on the BT were not related to the plasma levels of ristocetin cofactor activity-attained postinfusion (100 U/dL or more in the majority of patients) or to the multimeric structure of vWF in concentrates (defective in larger multimers in all cases). In conclusion, even though virus-inactivated concentrates can be used to increase FVIII:C levels in patients with severe vWD, none of the concentrates studied by us consistently normalizes the BT in a sustained fashion.

Bleeding Time↗

Cellular and antibody responses to equine herpesviruses 1 and 4 following vaccination of horses with modified-live and inactivated viruses.

The ability of monovalent and bivalent equine herpesvirus (EHV) vaccines to stimulate cellular and antibody responses to EHV-1 and EHV-4 was compared in healthy horses. Comparison of data from lymphocyte blastogenesis tests in which live viruses were used as antigens and that were conducted prior to vaccination and after 2 vaccinations revealed that horses given modified-live EHV-1 had significant increases in proliferative responses to EHV-1 (P = 0.03) and EHV-4 (P = 0.04). Responses to EHV-1 and EHV-4 in horses given the inactivated-virus bivalent vaccine were less; however, significant differences were not noticed when postvaccinal lymphocyte blastogenesis tests were compared between the groups of vaccinees. Interleukin-2 activity was not detected in leukocyte cultures from either group of vaccines following stimulation with live EHV-1 or EHV-4; however, interferon activity was found in similar cultures from both groups of vaccinees. For EHV-4, interferon activity in cultures from both groups of vaccinees was significantly (P < 0.05) greater than that in leukocyte cultures from unvaccinated controls. Both vaccines induced significant (P < 0.05) increases in serum antibodies that neutralized EHV-1 infectivity. The ELISA for EHV-1 and EHV-4 antibodies revealed that both vaccines induced significant (P < 0.05) increases (compared with preinoculation values) in antibodies reactive with these 2 types of EHV. Total serum antibody responses, as measured by ELISA, to EHV-1 and EHV-4 were significantly (P < 0.05) higher in horses that received the bivalent inactivated-virus vaccine, compared with that in horses that received monovalent vaccine. Evaluation of these data revealed that vaccination with modified-live EHV-1 can stimulate cellular and antibody responses that cross-react with EHV-4.

Analysis of Variance↗

The band III ligand dipyridamole protects human RBCs during photodynamic treatment while extracellular virus inactivation is not affected.

BACKGROUND: Recently, the potential usefulness of dipyridamole (DIP) in protecting RBCs against the harmful side effects of photodynamic sterilization was demonstrated. In the present study, the use of DIP for selective protection of RBCs was investigated under conditions more relevant for blood bank practice. STUDY DESIGN AND METHODS: WBC-reduced RBC suspensions (30% Hct) were treated with 1,9-dimethylmethylene blue and red light, and the influence of the inclusion of DIP on photohemolysis was assessed as a function of sensitizer concentration, light dose, and storage time. Furthermore, the possible interference of DIP with inactivation of extracellular virus by use of a panel of different viruses (HIV-1, pseudorabies virus [PRV], bovine viral diarrhea virus [BVDV], VSV, encephalomyocarditis, and canine parvovirus) was investigated. RESULTS: In WBC-reduced RBC suspensions (30% Hct), DIP exerted a clear protective effect against photohemolysis. Part of this protection was achieved with concentrations near the dissociation constant for band III binding. Importantly, efficiency of inactivation of extracellular HIV-1, PRV, BVDV, and VSV was not significantly impaired by the inclusion of DIP. Phototreatment conditions, resulting in a 4 to 5 log inactivation of extracellular HIV-1 and PRV, resulted in a high level of hemolysis after 28 days of storage. This long-term hemolysis could be decreased, but not completely prevented, by the inclusion of DIP. CONCLUSION: Photohemolysis in RBC concentrates can be reduced substantially by the application of DIP, while the efficacy of inactivation of HIV-1 and other viruses remains unchanged.

Anion Exchange Protein 1, Erythrocyte↗

Virus inactivation by anilinonaphthalene sulfonate compounds and comparison with other ligands.

Bis-(8-anilinonaphthalene-1-sulfonate) (bis-ANS) causes inactivation of vesicular stomatitis virus (VSV) at micromolar concentrations while butyl-ANS and ANS are effective at concentrations one and two orders of magnitude higher, respectively. VSV fully inactivated by the combined effects of 10 microM bis-ANS and 2.5 kbar hydrostatic pressure elicited a high titer of neutralizing antibodies. Incubation of VSV with >/=2 M urea at atmospheric pressure caused very little virus inactivation, whereas at a pressure of 2.5 kbar, 1 M urea caused inactivation that exceeded by more than two orders of magnitude the sum of the inactivating effects produced by urea and pressure separately. Measurements of bis-ANS fluorescence showed that increasing the urea concentration reduces the pressure required to disrupt the structure. We conclude that anilinonaphthalene sulfonate compounds inactivate VSV by a mechanism similar to that produced by pressure. The most effective antiviral compound was bis-ANS which can be used for the preparation of safe viral vaccines or as an antiviral drug eventually.

Anilino Naphthalenesulfonates↗

Virus inactivation by nucleic acid extraction reagents.

Many assume that common methods to extract viral nucleic acids are able to render a sample non-infectious. It may be that inactivation of infectious virus is incomplete during viral nucleic acid extraction methods. Accordingly, two common viral nucleic acid extraction techniques were evaluated for the ability to inactivate high viral titer specimens. In particular, the potential for TRIzol LS Reagent (Invitrogen Corp., Carlsbad, CA) and AVL Buffer (Qiagen, Valencia, CA) were examined to render suspensions of alphaviruses, flaviviruses, filoviruses and a bunyavirus non-infectious to tissue culture assay. The dilution series for both extraction reagents consistently caused cell death through a 100-fold dilution. Except for the DEN subtype 4 positive control, all viruses had titers of at least 10(6)pfu/ml. No plaques were detected in any extraction reagent plus virus combination in this study, therefore, the extraction reagents appeared to inactivate completely each of the high-titer viruses used in this study. These results support the reliance upon either TRIzol LS Reagent or AVL Buffer to render clinical or environmental samples non-infectious, which has implications for the handling and processing of samples under austere field conditions and low level containment.

Alphavirus↗

[The possibilities and limits for virus inactivation in the household].

Viruses of importance brought into the household or excreted by its members vary in their resistance and are decimated or even completely inactivated by natural environmental influences as well as by washing and cleaning. Steps to disinfect the household should be limited to specific indications and performed with virucidal compounds harmless to the environment. Naturally antiviral influences such as heat, UV-rays, sunshine, acids, the effectiveness of washing and cleaning procedures in the household and some selected strongly virucidal, but non-pollutant disinfectants are discussed.

Disinfection↗

Development and biochemical characterization of a double-virus-inactivated factor VIII preparation.

This paper describes the development of a double-virus-inactivated preparation. The original goal was to inactivate and/or remove parvovirus from plasma-derived factor VIII. More recently, attention has also been focused on the controversial issue of transmission of the non-lipid-coated hepatitis A virus by preparations of factor VIII which have been solvent/detergent-treated and purified by ion-exchange chromatography.

Antiviral Agents↗

Virus inactivation in blood components by photoactive phenothiazine dyes.

The virucidal properties of photoactive phenothiazine dyes, such as methylene blue, have been known approximately 70 years. The mechanism of virus inactivation involves binding of the dye to nucleic acid, absorption of light, generation of reactive oxygen species, guanine oxidation in the viral genome. The first practical research involving phenothiazine photosensitizers in transfusion medicine was sparked by efforts to prevent transmission of hepatitis from pooled plasma administered during the Korean War. During the last decade, methylene blue was used by some European countries for virus photoinactivation in fresh frozen plasma, whereas other phenothiazine derivatives with greater affinities for nucleic acids and capabilities to inactivate intracellular virus have been investigated for photochemical decontamination of red cell suspensions. The toxicity of methylene blue is well characterized; the drug has been used for many years to treat methemoglobinemia using concentrations orders of magnitude greater than those used for virus photoinactivation, and several filters have been developed to remove the dye from plasma. Future widespread use of phenothiazines for virus photoinactivation in blood components may depend on whether the risk inherent in their use is less than the residual risk from transfusion-transmitted viruses.

Blood↗

Studies on host-virus interactions in the chick embryo-influenza virus system. VI. Evidence for multiplicity reactivation of inactivated virus.

Evidence has been presented that influenza viruses both of type A and B partially inactivated by ultraviolet irradiation may regain their capacity to propagate in the allantoic membrane of the chick embryo. In using such irradiated preparations as inocula for growth curve experiments it could be shown that the development of hemagglutinins as well as of infectivity preceded at rates resembling those noted with more than 10 times the amount of infective virus actually found in the irradiated seed. Partial inactivation of the inocula by heating to 56 degrees C. gave similar results. The phenomenon was observed only with seed irradiated for short periods of time so that the virus particles sustained only few hits of radiation. On prolonged exposure resulting in numerous hits per virus particle the capacity of reactivation was lost. Likewise, an irradiated preparation capable of reactivation in the allantoic membrane, could not be diluted more than about 30-fold and still clearly produce this phenomenon. This indicated that reactivation is obtained only when one host cell adsorbs more than one non-infective virus particle but not upon adsorption of a single particle. These data are in striking agreement with the phenomenon of "multiplicity reactivation" observed in the bacteriophage-E. coli system by Luria and Dulbecco.

Escherichia coli↗

Complete deoxygenation from a hemoglobin solution by an electrochemical method and heat treatment for virus inactivation.

Hemoglobin (Hb) has been widely studied as a raw material for various types of oxygen carriers. In the purification of Hb from red blood cells including virus inactivation and denaturation of other proteins and the long-term storage of Hb vesicles (HbV), a deoxygenation process is one of the important processes because of the high stability of deoxygenated Hb to heating and metHb formation. Though an oxygenated Hb solution can be deoxygenated with an artificial lung, it is difficult to reduce the oxygen partial pressure of the Hb solution to less than 10 Torr. We developed an electrochemical system for complete deoxygenation of the Hb solution at the cathode compartment using hydrogen containing nitrogen gas at the anode compartment. Oxygen in the Hb solution was reduced to OH(-) at the cathode compartment within several minutes at a potential value of -1.67 V and was finally converted to water by neutralization with H(+) from the anode in the whole system. The resulting completely deoxygenated Hb could tolerate heat treatment at 62 degrees C for 10 h with no denaturation of deoxygenated Hb. The metHb formation rate of reoxygenated Hb at 37 degrees C was not changed after heat treatment. Furthermore, vesicular stomatitis virus (VSV) could be inactivated at an inactivation degree of more than 5.96 log by heat treatment.

Animals↗

Use of several second generation serological assays to determine the true prevalence of hepatitis C virus infection in haemophiliacs treated with non-virus inactivated factor VIII and IX concentrates.

To investigate the prevalence of hepatitis C virus infection in two risk groups, stored serum samples from treated haemophiliacs and intravenous drug users were tested for anti-HCV by both anti-C-100 based and second generation ELISAs (Abbott and Ortho) followed by testing in two confirmatory immunoblot assays that incorporate core as well as other non-structural antigens (Innogenetics LIA and Chiron RIBA-HCV test). Clear evidence of HCV infection was found in all but one of 78 haemophiliacs treated with non-virus inactivated clotting factor concentrates, but in none exposed only to super dry heat-treated concentrates. Only four samples gave rise to conflicting serological results between the four tests, two of these occurred in patients with advanced HIV related disease and almost certainly reflected loss of humoral immunity associated with disease progression, and the others occurred in the only two patients tested who were chronic carriers of hepatitis B infection and may reflect an interaction between the two viruses. Comparison of anti-C-100 versus second generation tests in immunocompetent drug users revealed a false negative rate of 20% using C-100 alone, indicating the advantage of using second generation assays for detection of past or current HCV infection. Of all of the antigens used in the confirmatory assay, positive sera showed strongest and most frequent reactivity with the C22 and C33c proteins (Ortho RIBA).

Drug Contamination↗

Specific sorbent to remove solvent-detergent mixtures from virus-inactivated biological fluids.

A method involving a sorbent designed to capture specifically virus-inactivating solvent-detergent mixtures is described. Its specificity allows the adsorption of these undesirable chemicals with a high capacity in order to treat large amounts of inactivated biological fluids in small-sized columns. Typically, the volume of sorbent that can be used repeatedly is between one quarter and one tenth of the sample volume to be treated. Cleaning and sanitization can be done by classical methods while strong oxidizing agents can also be used to sterilize the sorbent.

Body Fluids↗