Analysis of human plasma products: polymerase chain reaction does not discriminate between live and inactivated viruses

BACKGROUND: The viral safety of human plasma products is based on the careful selection of donors and donations and the removal and inactivation of human pathogenic viruses that could potentially contaminate human plasma. For the analysis of the final products for potential virus contamination, the use of polymerase chain reaction (PCR) has been proposed. To test whether this method can discriminate between infectious and inactivated viruses, the following studies were performed. STUDY DESIGN AND METHODS: Infectious and virus-inactivated preparations were titrated with specific PCR, using viruses such as hepatitis B virus (HBV), hepatitis C virus, bovine viral diarrhea virus, and poliovirus. The inactivation method employed was pasteurization (10 hours, 60 degrees C) or solvent/detergent (SD) treatment; in the case of HBV, there was consecutive treatment by both methods. RESULTS: Pasteurization of HBV and hepatitis C virus as well as SD treatment of HBV or pasteurization of HBV followed by SD treatment did not affect the detectability of these viruses by PCR, whereas an infectivity study in chimpanzees demonstrated that infectious hepatitis C virus was inactivated by pasteurization. Pasteurization also had no effect on the PCR titers of stabilized bovine viral diarrhea virus or poliovirus preparations, but it destroyed the infectivity of these viruses completely after only 4 hours' heat treatment. CONCLUSION: Pasteurization or SD treatment destroys the infectivity of the viruses tested, but neither significantly affects their detectability by specific PCR. Therefore PCR is not a suitable measure for testing the viral safety of finished plasma products that have been subjected to virus inactivation.     

Resistance of porcine circovirus and chicken anemia virus to virus inactivation procedures used for blood products

BACKGROUND: Virus inactivation procedures are used to prevent contamination of plasma-derived blood products with viruses. Pasteurization or prolonged dry heat has proven effective against several enveloped and nonenveloped viruses and provides an additional layer of safety for plasma products. STUDY DESIGN AND METHODS: The resistance of porcine circovirus 2 (PCV2) and chicken anemia virus (CAV), two small, nonenveloped viruses, to standard (pasteurization, 10 hr at 60 degrees C; dry heating, 80 degrees C for 72 hr) and more extreme heat inactivation procedures (temperatures up to 120 degrees C) was determined. The ability of these procedures to inactivate PCV2 and CAV was measured by comparison of in vitro infectivity before and after treatment. RESULTS: Infectivity of PCV2 and CAV was reduced by approximately 1.6 and 1.4 log by pasteurization and by 0.75 and 1.25 log by dry-heat treatment, both substantially more resistant than other viruses previously investigated. PCV2 and CAV were additionally almost completely resistant to dry-heat treatment up to 120 degrees C for 30 minutes (mean log infectivity reductions, 1.25 and 0.6), although both were more effectively inactivated when the temperature of wet-heat treatment was increased to 80 degrees C (>3.2 and >3.6 log infectivity reduction). CONCLUSION: Although neither PCV2 nor CAV are known to infect humans, their inactivation properties may represent those of other small DNA viruses known to be present (e.g., TT virus, small anellovirus) or potentially present in human plasma. Findings of extreme thermal resistance demonstrate that recipients of plasma-derived therapeutics may potentially still be exposed to small DNA viruses, despite the implementation of viral inactivation steps.     

West Nile virus and the safety of plasma derivatives: verification of high safety margins,and the validity of predictions based on model virus data

BACKGROUND: During the 2002 West Nile virus (WNV) epidemic in the US, virus transmission through solid organ transplantation and transfusion of blood components was observed. This raised concerns about the safety of plasma derivatives. To verify the safety margins of these products, which were initially shown with a panel of model viruses including some very similar to WNV, the effectiveness of the virus inactivation procedures incorporated into their manufacturing processes was reinvestigated. STUDY DESIGN AND METHODS: An infectivity assay for 1999 New York isolate of WNV was established to investigate virus inactivation steps commonly used during the manufacture of plasma derivatives, such as pasteurization for human albumin, S/D treatment for IVIG and FVIII, vapor heating for FVIII inhibitor-bypassing activity, and incubation at low pH for IVIG. RESULTS: The results show that WNV behaves exactly as had been predicted based on available data for similar model viruses; that is, it is readily inactivated by all the commonly used virus inactivation procedures tested. CONCLUSION: Our investigation verifies the safety margins of plasma derivatives against a potential transmission of WNV and that the model virus concept is valid for predicting the behavior of closely related viruses.     

Inactivation of parvovirus B19 during pasteurization of human serum albumin

BACKGROUND: It has been shown that HSA may be contaminated with parvovirus B19 (B19) DNA. However, the presence of B19 DNA does not necessarily indicate infectious virus. HSA is pasteurized at 60 degrees C for 10 hours and it remains unclear whether this procedure inactivates B19. Studies with animal parvoviruses indicate considerable heat resistance at 60 degrees C. However, due to the lack of a suitable cell culture system, the pasteurization process has not been investigated in the past. STUDY DESIGN AND METHODS: The recently described cell clone KU812Ep6 was used to establish a system for investigation of B19 inactivation during pasteurization. Virus-infected cells were detected by immunofluorescent staining of viral capsid antigen and by RT-PCR assay of virus-specific capsid mRNA. RESULTS: B19 was inactivated after 10 minutes at 60 degrees C for > or = 4 log. In contrast, porcine parvovirus was widely resistant at 60 degrees C. Inactivation of B19 was independent of the analyzed albumin products (5, 20, and 25% albumin from three manufacturers) and from the specific virus source used for the inactivation experiments. Degradation of B19 DNA by deoxyribonuclease I treatment after pasteurization indicated that the virus capsid is destroyed during heat treatment. CONCLUSION: Heat resistance of B19 markedly differs from heat resistance of animal parvoviruses. While animal parvoviruses widely withstand pasteurization of albumin, B19 was rapidly inactivated. These results confirm the safety of pasteurized albumin and are in line with its good clinical safety record with respect to B19 infection. However, conclusions regarding the safety of other blood-derived medicinal products should not be derived from B19 inactivation in albumin, because different processes or different composition of product intermediates may significantly influence B19 stability during heat treatment.     

H5N1 influenza virus and the safety of plasma products

BACKGROUND: The ever-increasing number of human H5N1 influenza virus infections may enable these viruses to acquire the ability to spread effectively among humans and potentially to cause a pandemic. Recently, more systemic virus dissemination was reported during H5N1 virus infection of humans, resulting in significant virus concentrations also in the blood. The observation has raised concerns about the safety of labile blood products for transfusion and consequentially also for plasma derivatives. To confirm the safety margins of plasma products, dedicated virus inactivation processes used during their production were investigated for their effectiveness in inactivating this virus of recent concern. STUDY DESIGN AND METHODS: Virus inactivation by steps commonly used during the manufacture of plasma derivatives, such as pasteurization for human albumin, solvent/detergent treatment for intravenous immunoglobulin (IVIG), vapor heating for factor VIII inhibitor bypassing activity, and incubation at low pH for IVIG, were investigated with a reassortant strain of H5N1 influenza virus. RESULTS: The results show that H5N1 influenza behaves as expected for lipid-enveloped viruses; that is, the virus is effectively inactivated by all the commonly used virus inactivation procedures tested. CONCLUSION: The safety margins of plasma derivatives against the theoretical transmission of H5N1 influenza virus are very substantial.     

Inactivation of hepatitis A variants during heat treatment (pasteurization) of human serum albumin

BACKGROUND: Pasteurization of human serum albumin (HSA) is detailed in the US and European Pharmacopoeial monographs and therefore a process that allows for little variation in physiochemical variables. Nevertheless, differences of up to 3.9 log in hepatitis A virus (HAV) inactivation by pasteurization have been reported. Here, the hypothesis that the choice of HAV variant used in the pasteurization might contribute to this inactivation variability is evaluated experimentally. STUDY DESIGN AND METHODS: The identity of four widely used cytopathic variants of the original HAV HM175 strain was determined by partial sequencing. These variants were used in pasteurization studies conducted under the principles of good laboratory practice, for which HAV‐spiked HSA of 5 or 25% protein content was kept at 58 ± 1°C for 600 ± 10 minutes, and the virus inactivation was assessed. In addition, data from previous pasteurization studies were included in the analysis. RESULTS: The four HAV variants could be divided into two subgroups, with significantly different (p ≤ 0.0001) virus inactivation by pasteurization (4.7 and 4.8 log vs. 2.3 and 2.6 log, respectively). Also, the protein concentration of the HSA solution used for pasteurization had a significant effect on the achieved HAV inactivation, with reduction factors obtained in 5% HSA significantly lower than in 25% HSA (p < 0.002). CONCLUSION: HAV variant and protein concentration of the HSA solution affect the overall HAV inactivation that is achieved during pasteurization. As the HAV inactivation capacity should not be overestimated, an HAV variant more resistant to heat inactivation should be used for studies investigating the viral safety profiles of plasma derivatives.     

Viral safety of solvent/detergent-treated plasma

BACKGROUND: Pooling of plasma donations increases the risk for blood-borne infections. In solvent/detergent (SD)-treated plasma, lipid-enveloped viruses are efficiently inactivated. This method, however, does not affect non-lipid-enveloped viruses. The current study investigated the viral safety of SD-treated plasma (Octaplas) and paid particular attention to the transmission of non-lipid-enveloped viruses. STUDY DESIGN AND METHODS: The study comprised 343 adults undergoing cardiac surgery. Follow-up was performed 6 to 12 months and 2 years after operation. The sera were tested for hepatitis B surface antigen and specific antibodies against hepatitis A, B, and C; cyto-megalovirus; HIV, human T-lymphotropic virus types I and II; and human parvovirus B19 (B19). A total of 25 batches of SD-treated plasma prepared from Norwegian plasma were used. All batches were tested for hepatitis A virus and B19 by nucleic acid amplification testing and investigated for neutralizing antibodies directed against these viruses. RESULTS: In patients who received SD-treated plasma, B19 seroconversion occurred at a rate similar to that in nontransfused patients. No other seroconversions could be ascribed to the transfusion of SD-treated plasma. All 25 SD-treated plasma batches contained neutralizing antibodies against hepatitis A virus and B19. In nucleic amplification testing, all SD-treated plasma batches tested positive for B19, while five demonstrated borderline reactions for hepatitis A virus. CONCLUSION: Transfusion of SD-treated plasma was found to be safe with regard to lipid-enveloped viruses. Immune antibodies neutralize viral particles in plasma and are of importance in avoiding clinical disease with the non-lipid-enveloped hepatitis A virus and B19.     

Duck hepatitis B photoinactivation bydimethylmethylene blue in RBC suspensions

BACKGROUND: Dimethylmethylene blue (DMMB) has been used to photoinactivate a number of model viruses, including VSV, in RBC suspensions under conditions that preserve in vitro RBC properties during storage. The relative sensitivity of duck HBV (DHBV) and VSV to photoinactivation by DMMB was investigated by performing an indirect immunofluorescence assay (IFA) using primary duck hepatocyte (PDH) cultures or a standard plaque assay for the respective viruses. STUDY DESIGN AND METHODS: DMMB was added to 45-percent Hct, WBC-reduced, oxygenated AS-3 RBCs at 10-, 1-, and 0.1-microM concentrations. Samples (1-mm thick) were illuminated with 5.4-mW per cm(2) of red light for 2 or 9 seconds. Unilluminated samples without DMMB or with 10 microM DMMB served as control. RESULTS: DHBV and VSV were rapidly photoinactivated by DMMB in a concentration and light-dose-dependent fashion. Neither virus was substantially inactivated by incubation with DMMB in the dark. For a given light exposure, DHBV required a concentration of DMMB one-one hundredth that of VSV to achieve approximately the same level of inactivation. CONCLUSION: DHBV appears to be considerably more sensitive than VSV to DMMB photoinactivation. Photoinactivation in 45-percent Hct RBCs can be achieved in seconds by using micromolar quantities of dye.     

Elimination of both cell-free and cell-associated HIV infectivity in plasma by a filtration/methylene blue photoinactivation system

BACKGROUND: Methylene blue phototreatment effectively inactivates cell-free viruses in plasma while maintaining coagulation activities. However, this treatment is considered to be less effective for cell-associated virus inactivation. This report describes a new virus elimination system designed to eliminate cell-associated viruses with a cell-removal filter followed by methylene blue photoinactivation of cell-free viruses in plasma. STUDY DESIGN AND METHODS: Fresh plasma was inoculated with HIV or HIV-infected Molt4 cells (Molt4(IIIB)). The plasma was transferred to a bag containing methylene blue by passing it through a cell-removal filter and was irradiated with white fluorescent light. HIV infectivity was detected by indirect fluorescence assay. In parallel studies, coagulation activities in identically treated plasma were measured during 1 year of storage at -80 degrees C. RESULTS: Initial cell-free HIV titer of 10(6.2) TCID(50) per 0.1 mL dropped to 10(-0. 3) and <10(-0.5) TCID(50) per 0.1 mL after 10 or 20 J per cm(2) radiation, respectively. Cellular components were not detectable in plasma after filtration. The cell-free state of the plasma was ascertained from the observation that the DNase-resistant beta-globin gene, as a marker of intact WBCs, was not detected in the filtrates by PCR. The infectivity of Molt4(IIIB) was reduced to below the detection limit after filtration and radiation, and proviral HIV DNA was not detected in the filtrates by PCR. Coagulation activities including factor VIII in the treated plasma were maintained at more than 76 percent compared with the percentage in untreated plasma after 1 year of storage. CONCLUSION: The filtration/methylene blue photoinactivation system eliminated both cell-free and cell-associated HIV infectivities from plasma while maintaining coagulation activities for 1 year at -80 degrees C storage.     

巴斯德消毒法可否影响血红蛋白理化性质及生物学功能？

背景：巴斯德消毒法对白蛋白的病毒灭活条件已很完善,可不要求进行病毒灭活验证,但将其应用于血红蛋白类血液代用品病毒灭活的研究在国内外尚未见有系统报道。目的：探讨巴斯德消毒法对血红蛋白类血液代用品理化性质及生物学功能的影响。方法：取适量脐血,经离心、洗血、破膜、添加稳定剂处理后,对照组于55℃水浴加热,待血红蛋白溶液温度达到（55±1）℃开始计时,2 h后加热处理完成;巴氏消毒组于60℃水浴加热,待血红蛋白溶液温度达到（60±1）℃开始计时,10 h后加热处理完成,该过程持续通氮保护。然后置冰浴冷却到4℃以下,低温高速离心,微孔滤膜过滤,得到纯化及病毒灭活的脐血血红蛋白。结果与结论：巴氏消毒组与对照组制品外观都为红色澄明液体;在得率、高铁血红蛋白含量、氧结合量方面两组差异无显著性意义;两组的纯度都在98%以上;两种纯化方法并没有对血红蛋白携氧功能造成影响。因此,可以用巴斯德病毒灭活的方法来代替一直在使用的55℃,2 h的热敏法纯化方式。这样不仅能保证血红蛋白的理化性质、生物学性质,还能同时达到病毒灭活的目的。     

Virus reduction in the preparation of intravenous immune globulin: in vitro experiments

BACKGROUND: While immune globulins for intravenous administration (IGIV) have an excellent record with respect to virus safety, concern regarding these preparations has been raised by reports of transmission of hepatitis C virus (HCV) to patients treated with IGIV and the presence of genetic material for HCV in IGIV preparations. STUDY DESIGN AND METHODS: This in vitro study evaluated the effectiveness of several manufacturing steps, including ethanol precipitation and pasteurization, in reducing HIV and model viruses including encephalomyocarditis (EMC) virus, pseudorabies virus (PRV), bovine viral diarrhea virus (BVDV), Sindbis virus, vaccinia virus, and vesicular stomatitis virus (VSV), as well as HCV RNA, in IGIV. RESULTS: Ethanol precipitation carried out after pasteurization resulted in virus reductions (log10) of >3.97 for HIV, 1.95 for EMC virus, >5.39 for PRV, and 3.52 for BVDV. Pasteurization inactivated EMC virus by 4.52 log10 and resulted in a log10 reduction of >6.54 for HIV, >5.39 for PRV, >6.64 for BVDV, >7.78 for Sindbis virus, >5.84 for vaccinia virus, and >6.99 for VSV. All viruses except EMC virus were reduced below the limit of detection within 6 hours of the beginning of pasteurization. Cohn processing of Fraction II + III paste and the 4.5-percent alcohol precipitation step prior to pasteurization provided additional virus removal. Studies using the polymerase chain reaction technique found that HCV RNA was detectable in the starting fraction of Cohn Fraction II paste, but not in the final IGIV preparation. CONCLUSION: These findings strongly support the viral safety of IGIV prepared by this method and show a significant added measure of virus safety associated with pasteurization of this preparation.     

Inactivation of hepatitis A virus in plasma products by vapor heating

BACKGROUND: The transmission of hepatitis A virus (HAV) has been associated with the use of a number of solvent/detergent-treated factor VIII concentrates and possibly a factor IX concentrate. These reports have emphasized the necessity of using virus-inactivation methods for plasma products that are capable of inactivating nonenveloped viruses such as HAV. STUDY DESIGN AND METHODS: A simple, highly accurate titration procedure for HAV, which allows extensive kinetic investigations of virus-inactivation procedures, has been developed. This system has now been used to evaluate the efficacy of vapor heating in inactivating HAV after the addition of the virus to a range of human plasma products. RESULTS: It was demonstrated that HAV was significantly more thermostable than other picornaviruses, which reinforced the fact that such viruses cannot be used as model viruses for HAV-inactivation studies. A one-step vapor-heating procedure was demonstrated to inactivate between 5.9 and > 6.3 log10 of HAV in different products. A two-step vapor-heating procedure had the capacity to inactivate between > 8.7 and > 10.4 log10 of HAV. Both procedures were more effective in inactivating HAV than was the pasteurization procedure used for virus inactivation in human albumin solutions. CONCLUSION: These data demonstrate the efficacy of vapor heating in inactivating high-titer HAV after the spiking of plasma products with virus. This study confirms and explains the results of controlled clinical trials and long-term clinical usage with respect to the lack of HAV transmission by such vapor-heated products.     

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.     

四种病毒灭活方法用于登革病毒灭活效果的比较

目的评价常用病毒灭活方法对血液制品中登革病毒(DENV)的灭活效果。方法将单采新鲜冰冻血浆(FFP),人凝血因子Ⅷ(FⅧ)和静脉注射免疫球蛋白(IVIG)3种血浆及其制品中加入高滴度(8.00-9.25)的登革病毒液,分别采用亚甲蓝(MB)光化学法灭活FFP、有机溶剂/去污剂(S/D)法灭活FⅧ、低pH常温孵化法和巴氏消毒法灭活IVIG;以1×10~6/mL A549细胞接种于T25的培养瓶中作为病毒传代及滴度滴定指示细胞,将灭活前后的血浆及制品接种于A549细胞并检测其DENV滴度,并通过qRT-PCR对DENV RNA做定量检测;评估不同的灭活方法对DENV的灭活效果。结果 DENV滴度下降:MB光化学法灭活FFP下降滴度≥5.92 log,S/D法灭活FⅧ下降滴度≥5.17 log、巴氏法和低pH法灭活IVIG均下降滴度≥5.92 log,其中MB光化学法灭活FFP、SD灭活FⅧ及巴氏法灭活IVIG后DENV RNA(cp/mL)降低1.25 log-2.25 log,低pH法灭活IVIG后DENV RNA(cp/mL)降低0.17 log。所有血浆和血液制品样品经灭活后,在DENV宿主细胞上3代盲传后均未检测到DENV RNA。结论 4种常用病毒灭活方法均能有效灭活血浆及血液制品中的DENV。     

Chikungunya virus and the safety of plasma products

BACKGROUND: Chikungunya virus (CHIKV) outbreaks were previously restricted to parts of Africa, Indian Ocean Islands, South Asia, and Southeast Asia. In 2007, however, the first autochthonous CHIKV transmission was reported in Europe. High‐level viremia, a mosquito vector that is also present in large urban areas of Europe and America, and uncertainty around the resistance of this Alphavirus toward physiochemical inactivation processes raised concerns about the safety of plasma derivatives. To verify the safety margins of plasma products with respect to CHIKV, commonly used virus inactivation steps were investigated for their effectiveness to inactivate this newly emerging virus. STUDY DESIGN AND METHODS: Pasteurization for human serum albumin (HSA), vapor heating for Factor VIII inhibitor bypassing activity, solvent/detergent (S/D) treatment for intravenous immunoglobulin (IVIG), and incubation at low pH for IVIG were investigated for their capacity to inactivate CHIKV and the closely related Sindbis virus (SINV). The obtained results were compared to previous studies with West Nile virus and the commonly used model virus bovine viral diarrhea virus. RESULTS: The data generated demonstrate the effective inactivation of CHIKV as well as SINV by the inactivation steps investigated and thereby support results from earlier validation studies in which model viruses were used. CONCLUSION: High inactivation capacities with respect to CHIKV were demonstrated. This provides solid reassurance for the safety of plasma products and the results verify that the use of model viruses is appropriate to predict the inactivation characteristics of newly emerging viruses when their physicochemical properties are well characterized.     

Prevention of iatrogenic transmission of B19 infection: different approaches to detect, remove or inactivate virus contamination

Parvovirus B19 is a frequent contaminant of human blood and plasma derivatives and iatrogenic transmission of B19 infection has been shown to occur through the administration of contaminated products. Manufacturing procedures, generally used for removal or inactivation of enveloped viruses (HIV, HCV and HBV) are not always effective in the elimination of B19 virus. A certain risk of contamination remains for some plasma derivatives due to the high-titer viral load in the starting blood donations and the extreme heat resistance and small size of the virus. This review provides an update on the different approaches currently available to detect, remove or inactivate B19 virus in order to enhance the safety margins of plasma products. Nucleic acid amplification techniques are the methods of choice for the detection of viruses, due to their high specificity and sensitivity. NAT assays are beneficial tools for the identification of contaminated mini-pools or plasma pools and the quantification of B19 contamination. They may also be valuable for testing the removal of B19 virus during manufacturing: since the virus may not be completely inactivated or removed by chemical or physical treatments, residual B19 contamination should always be checked. Solvent-detergent treatments fail to destroy B19 capsids because of the absence of a lipid-envelope, and heat treatments (pasteurization and dry-heat methods) cannot guarantee a complete viral inactivation because of the variable heat sensitivity of the virus.     

Removal of neutralized model parvoviruses and enteroviruses in human IgG solutions by nanofiltration

BACKGROUND: Nanofiltration has proven to be an effective technology for virus removal. The small nonenveloped viruses, such as HAV and parvovirus B19 (B19), are evidently more difficult to remove than the larger enveloped viruses. However, since antibodies against these viruses, HAV and B19, are constantly present in large plasma pools, it is conceivable that antibody binding will lead to an increase in size of the viruses due to a corona of antibodies on the virus surfaces. These enlarged viruses should be easier to remove by nanofiltration than naked viruses. In this study, antibody-coated viruses and free virions were subjected to nanofiltration to determine whether the antibody-mediated increase in size contributes to the elimination of viruses which are otherwise too small to be retained by filters of a given nominal pore diameter. STUDY DESIGN AND METHODS: One-percent IgG or 1-percent albumin solutions were spiked with bovine enterovirus (BEV) and filtered through 50-nm filters. This virus cross-reacts with antibodies in pooled human IgG solutions but persists as free virions in albumin solutions. Similarly, two parvoviruses, bovine parvovirus (BPV) and minute virus of mice (MVM), in 1-percent IgG solutions were filtered through 20-nm filters. BPV cross-reacts with antibodies in human IgG, whereas MVM remains unaffected. Virus removal was assessed by in vitro infectivity assays. Two methods were developed to displace the antibodies from BEV and BPV and thereby render them accessible for titration. RESULTS: Antibody-coated BEV was eliminated to below detection limit by the 50-nm filters (logarithmic reduction factors [LRF] > or = 5), while free BEV virions passed without restraint through the same filters. Similarly, antibody-coated BPV was completely retained by the 20-nm filters (LRF > or = 5). MVM virions were strongly retained by the 20-nm filters; however, the virus was still detectable in the filtrate. CONCLUSION: These results indicate that viruses with bound antibodies are efficiently eliminated by nanofiltration by use of filters having nominal pore sizes larger than the diameter of the respective free virions.     

Cost-effectiveness of transfusing virus-inactivatedplasma instead of standard plasma

BACKGROUND: Virus inactivation of plasma intended for transfusion avoids the transmission of hepatitis B virus, hepatitis C virus, and HIV. However, because most plasma recipients also receive other blood components concomitantly, the procedure reduces but cannot eliminate the risk of transfusion-transmitted infection. As virus-inactivated plasma has just been licensed in the United States and other countries, a cost-effectiveness analysis is pertinent. STUDY DESIGN AND METHODS: A Monte Carlo simulation of a Markov model representing the possible outcomes of plasma recipients was used to derive costs and utilities of transfusing virus-inactivated plasma instead of standard plasma. Probability distributions for patients' age and sex and for the number of blood components transfused per case were determined in 924 plasma recipients in a tertiary-care hospital. Other values were obtained from the medical literature. Results of the baseline and sensitivity analyses are the mean (+/- SD) of 10 simulations with 10(7) patients per simulation. RESULTS: In the baseline analysis, transfusing virus-inactivated plasma instead of standard plasma prolonged the quality-adjusted survival by 1 hour and 11 minutes per patient, at a cost-effectiveness ratio of $2,156,398 +/- $257,587 per quality-adjusted life year gained. Cost-effectiveness was most sensitive to the patients' mean age, the incremental cost per unit of virus-inactivated plasma, the HIV and hepatitis C virus transmission rates, and the short-term mortality of plasma recipients due to their underlying diseases. CONCLUSIONS: Compared to most accepted medical procedures, the transfusion of virus-inactivated plasma produces little health benefit at a very high cost. This poor cost-effectiveness ratio is due to the low current risk of infection with transfusion-transmitted viruses and to the greater age and poor short-term prognosis of most plasma recipients.     

Expansion of tropism of a feline parvovirus to target a human tumor cell line by display of an alpha(v) integrin binding peptide on the capsid

The autonomous parvoviruses are small, non-enveloped, single strand DNA viruses. They occur in many species and they have oncolytic properties. We are modifying the capsid of feline panleukopenia virus (FPV), a parvovirus which normally infects feline cells, with the goal of targeting human tumor cells for potential cancer therapy. Using recombinant viruses transducing a luciferase reporter, we show that insertion of a cyclically constrained, integrin-binding peptide at an exposed position on the FPV capsid enables transduction of an alpha(v) integrin-expressing human rhabdomyosarcoma cell line (Rh18A). These cells were not transduced by virus with the unmodified FPV capsid. Transduction of Rh18A was specifically inhibited by an alpha(v) integrin blocking antibody. However, other human tumor lines expressing alpha(v) integrins were not transduced by virus with either the modified or unmodified capsid. We conclude that modification of the FPV capsid to bind alpha(v) integrins can contribute to, but is not generally sufficient for, redirecting infection to human tumor cells. The permissiveness of Rh18A cells presumably involves additional factors unique to this line among various human cell lines tested.     

Inactivation of lipid-enveloped and non-lipid-enveloped model viruses in normal human plasma by crosslinked starch-iodine

BACKGROUND: The purpose of this study was to evaluate an iodine-based method of activating potentially harmful viruses that might be found in normal human plasma. STUDY DESIGN AND METHODS: A procedure has been developed for inactivating lipid-enveloped and non-lipid-enveloped model viruses in normal human plasma by using iodine complexed to crosslinked potato starch. The established conditions are an iodine concentration of 1.05 mg per mL and 60 minutes' incubation. RESULTS: Under these conditions, inactivation of more than 9 log10 of vesicular stomatitis virus, a lipid-enveloped virus, and more than 7 log10 of encephalomyocarditis virus, a non-lipid-enveloped virus, was achieved, with minimal losses of biologic activity of selected plasma proteins. Under these conditions, 70 percent of factor VIII activity, 77 percent of factor IX activity, and 100 percent of protein C activity in the plasma were retained. CONCLUSION: Crosslinked starch-iodine may be useful in the inactivation of viruses in single-donor plasma units and in pooled human plasma before fractionation.