Variability of parvovirus B19 to inactivation by liquid heating in plasma products

BACKGROUND AND OBJECTIVES: Previously, we reported that although human parvovirus B19 in albumin and intravenous immunoglobulin preparations was rapidly inactivated during liquid heating, in contrast to other parvoviruses such as canine parvovirus, sensitivity to heat was highly dependent on the composition of the solution. In this study, we aimed to further elucidate the sensitivity to heat of B19 in haptoglobin and antithrombin (previously named antithrombin III) preparations during liquid heating. MATERIALS AND METHODS: Two different solutions collected immediately before heat treatment of haptoglobin and antithrombin preparations were spiked with B19 and subsequently treated at 60 degrees C for 10 h. B19 DNA-positive, anti-B19 IgG/IgM-negative plasma was used as a source of B19. The residual infectivity in each sample was measured using a B19 cell-based infectivity assay with an mRNA polymerase chain reaction. RESULTS: B19 in different plasma preparations showed different heat-sensitivity patterns during liquid heating: (i) slow inactivation in haptoglobin preparations, and (ii) only limited inactivation in antithrombin preparations. The kinetics of inactivation was greatly different from that in our previous studies in which the virus was shown to be rapidly inactivated in albumin and intravenous immunoglobulin preparations. CONCLUSION: B19 has unique properties in terms of heat sensitivity, depending on the composition of the solution during liquid heating. This finding may indicate the need for caution when interpreting the sensitivity of B19 to heat.     

Heat sensitivity of a SARS-associated coronavirus introduced into plasma products

BACKGROUND AND OBJECTIVES: Various measures to inactivate/remove viruses have been implemented for manufacturing plasma-derived products. Here, we examined the heat inactivation ability of an agent of the severe acute respiratory syndrome (SARS), SARS coronavirus (CoV). MATERIALS AND METHODS: The Frankfurt-1 strain of SARS-CoV was incorporated in manufacturing processes of several products by using samples collected immediately before liquid heat treatment at 60 degrees C. RESULTS: SARS-CoV was easily inactivated by this treatment for 60 min in all in-process samples. However, the different composition of the tested samples affected the heat sensitivity of the virus strain: the infectivity of the virus in Antithrombin III preparation still remained after heating for 30 min at 60 degrees C. CONCLUSION: If by rare chance SARS-CoV contaminates source plasma, there should be no or only minor risk of this virus infection, due to sufficient inactivation by the 60 degrees C 10 h liquid heating step, although we must pay attention to the composition used for blood product preparation.     

Viral safety of Nanogam, a new 15 nm-filtered liquid immunoglobulin product

BACKGROUND AND OBJECTIVES: Producers of plasma derivatives continuously improve the viral safety of their products by, for example, introducing additional virus-reducing steps into the manufacturing process. Here we present virus-elimination studies undertaken for a number of steps employed in a new manufacturing process for liquid intravenous immunoglobulin (Nanogam) that comprises two specific virus-reducing steps: a 15-nm filtration step combined with pepsin treatment at pH 4.4 (pH 4.4/15NF); and solvent-detergent (SD) treatment. The manufacturing process also includes precipitation of Cohn fraction III and viral neutralization, which contribute to the total virus-reducing capacity of the manufacturing process. In addition, the mechanism and robustness of the virus-reducing steps were studied. MATERIALS AND METHODS: Selected process steps were studied with spiking experiments using a range of lipid enveloped (LE) and non-lipid-enveloped (NLE) viruses. The LE viruses used were bovine viral diarrhoea virus (BVDV), human immunodeficiency virus (HIV) and pseudorabies virus (PRV); the NLE viruses used were parvovirus B19 (B19), canine parvovirus (CPV) and encephalomyocarditis virus (EMC). After spiking, samples were collected and tested for residual infectivity, and the reduction factors were calculated. For B19, however, removal of B19 DNA was measured, not residual infectivity. To reveal the contribution of viral neutralization, bovine parvovirus (BPV) and hepatitis A virus (HAV) were used. RESULTS: For the pH 4.4/15NF step, complete reduction (> 6 log(10)) was demonstrated for all viruses, including B19, but not for CPV (> 3.4 but < or = 4.2 log(10)). Robustness studies of the pH 4.4/15NF step with CPV showed that pH was the dominant process parameter. SD treatment for 10 min resulted in complete inactivation (> 6 log(10)) of all LE viruses tested. Precipitation of Cohn fraction III resulted in the significant removal (3-4 log(10)) of both LE and NLE viruses. Virus-neutralization assays of final product revealed significant reduction (> or = 3 log(10)) of both BPV and HAV. CONCLUSIONS: The manufacturing process of Nanogam comprises two effective steps for the reduction of LE viruses and one for NLE viruses. In addition, the precipitation of Cohn fraction III and the presence of neutralizing antibodies contribute to the total virus-reducing capacity of Nanogam. The overall virus-reducing capacity was > 15 log(10) for LE viruses. For the NLE viruses B19, CPV and EMC, the overall virus-reducing capacities were > 10, > 7 and > 9 log(10), respectively. Including the contribution of immune neutralization, the overall virus-reducing capacity for B19 and HAV is estimated to be > 10 log(10).     

Variability of parvovirus B19 genotype 2 in plasma products with different compositions in the inactivation sensitivity by liquid-heating

Background and Objectives Our previous report showed that parvovirus B19 genotype 1 in different solutions derived from plasma preparations showed different heat‐sensitivity patterns during liquid‐heating. In this study, we similarly examined B19 genotype 2. Materials and Methods Two plasma samples one containing B19 genotype 1 and the other genotype 2 DNA were used. Four process samples collected immediately before the heat treatment step in the manufacture of albumin, immunoglobulin, haptoglobin and antithrombin preparations were spiked with B19 and subsequently treated at 60°C for 10 h. A low pH immunoglobulin solution was also spiked with B19 and treated at room temperature for 14 days. Infectivity was then measured. Results B19 genotype 2, similar to genotype 1, showed three patterns of inactivation: (i) a rapid inactivation in the albumin and immunoglobulin preparations, (ii) a slow inactivation in the haptoglobin preparation and (iii) only limited inactivation in the antithrombin preparation. Its sensitivity in the low pH immunoglobulin solutions also resembled that of genotype 1. Conclusion Both genotypes 1 and 2 of B19 varied in sensitivity to liquid‐heating and low pH among different plasma preparations.     

Inactivation of parvovirus B19 by liquid heating incorporated in the manufacturing process of human intravenous immunoglobulin preparations

Several reports have suggested the possible transmission of human parvovirus B19 (B19) through the administration of plasma derivatives that had undergone virus inactivation by various types of heat treatment. However, none of the reports evaluated and discussed the inactivation of B19 by the heat treatment that is implemented in the individual manufacturing processes of such products. The present study evaluated the ability to inactivate B19 of liquid-heat treatment at 60 degrees C for 10 h that was incorporated in the manufacturing process of intravenous human immunoglobulin preparations. The results showed that B19 was rapidly inactivated under the conditions used for the liquid-heat treatment.     

Extent of hepatitis E virus elimination is affected by stabilizers present in plasma products and pore size of nanofilters

Background and Objective To investigate the physico‐chemical properties of hepatitis E virus (HEV) with regard to inactivation/removal, we have studied four isolates with respect to sensitivity to heat during liquid/dry‐heating as well as removal by nanofiltration. Materials and Methods Hepatitis E virus in an albumin solution or phosphate‐buffered saline (PBS) was liquid‐heated at 60°C for a preset time. HEV in a freeze‐dried fibrinogen containing stabilizers was also dry‐heated at 60 or 80°C for a preset time. In addition, to clarify the removal of HEV, the purified virus in PBS was filtered using several types of virus‐removal filter (nanofilters) that have different pore sizes. HEV infectivity or genome equivalents before and after the treatments were assayed by a semiquantitative cell‐based infectivity assay or quantitative polymerase chain reaction assay, respectively. Results Hepatitis E virus isolates in albumin solutions were inactivated slowly at 60°C for 5 h and the resultant log reduction factor (LRF) was from 1·0 to ≥ 2·2, whereas the virus in PBS was inactivated quickly to below the detection limit and the LRF was ≥ 2·4 to ≥ 3·7. The virus in a freeze dried fibrinogen containing trisodium citrate dihydrate and l ‐arginine hydrochloride as stabilizers was inactivated slowly and the LRF was 2·0 and 3·0, respectively, of the 72 h at 60°C, but inactivated to below the detection limit within 24 h at 80°C with an LRF of ≥ 4·0. The virus in PBS was also confirmed as to be approximately 35 nm in diameter by nanofiltration. These results are useful for evaluating viral safety against HEV contamination in blood products. Conclusion The sensitivity of HEV to heat was shown to vary greatly depending on the heating conditions. On the other hand, the HEV particles were completely removed using 20‐nm nanofilters. However, each inactivation/removal step should be carefully evaluated with respect to the HEV inactivation/removal capacity, which may be influenced by processing conditions such as the stabilizers used for blood products.     

A modified caprylic acid method for manufacturing immunoglobulin G from human plasma with high yield and efficient virus clearance

BACKGROUND AND OBJECTIVES: The increasing demand for intravenous immunoglobulin (IVIG) necessitates the development of improved plasma fractionation methods, providing higher immunoglobulin G (IgG) recovery. Here, we describe a new IVIG production process resulting in a high yield of IgG and effective reduction of physico-chemically resistant viruses. MATERIALS AND METHODS: IgG was purified from Cohn fraction II+III by caprylic acid treatment, polyethylene glycol precipitation, anion-exchange chromatography, nanofiltration and ultrafiltration. Stability of the purified IgG was studied in different formulations. Virus reduction was studied with two viruses: bovine viral diarrhoea virus, assessed by an infectivity assay; and human parvovirus B19, assessed by polymerase chain reaction. RESULTS: The combination of caprylic acid treatment with polyethylene glycol precipitation and a single anion-exchange chromatography yielded polymer-free, pure IgG. The purified IgG could be filtered through a small pore-size virus filter (Millipore V-NFP) with high throughput and excellent yield. The formulated product was stable as a 100 g/l IgG solution. Bovine viral diarrhoea virus was effectively inactivated by the caprylic acid treatment, and parvovirus B19 was effectively removed in the polyethylene glycol precipitation and nanofiltration stages, the total reduction of parvovirus being approximately 14 log10. CONCLUSIONS: The new process gives pure and stable IgG solution with an average yield of 4.8 g of IgG per kg of recovered plasma and has a very high capacity to remove even physico-chemically resistant viruses.     

Human parvovirus PARV4 in clotting factor VIII concentrates

BACKGROUND AND OBJECTIVES: Parvoviruses are small non-enveloped DNA viruses, relatively resistant to virus inactivation procedures. The recently identified human parvovirus PARV4, including a related genotype 2 virus (also termed PARV5), has been found to be a contaminant of pooled plasma used in the manufacture of plasma-derived products. This report describes an investigation to determine whether PARV4 is present in clotting factor concentrates. MATERIALS AND METHODS: Factor VIII concentrates manufactured in the past 30-35 years were screened for PARV4 and human parvovirus B19 (B19V) sequences. Viral loads in products testing positive for PARV4 were quantified using a consensus TaqMan assay designed to a highly conserved region. DNA sequence analysis was performed to confirm the genotypes present. RESULTS: From a total of 175 lots of factor VIII concentrate, 28 of these contained PARV4 sequences, and in two lots both genotypes 1 and 2 were found to be present. The highest viral loads observed exceeded 10(5) copies per ml. The majority of factor VIII concentrates testing positive for PARV4 were manufactured in the 1970s and 1980s. Human B19V was also a frequent contaminant of these products. CONCLUSIONS: PARV4 was detected in 16% of factor VIII concentrates, particularly in older batches from the 1970s and 1980s. The significance in terms of the viral safety and potential transmission to recipients of these products is not yet known.     

Inactivation of West Nile virus, vaccinia virus and viral surrogates for relevant and emergent viral pathogens in plasma-derived products

BACKGROUND AND OBJECTIVES: Human plasma is the source of a wide variety of therapeutic proteins, yet it is also a potential source of viral contamination. Recent outbreaks of emergent viral pathogens, such as West Nile virus, and the use of live vaccinia virus as a vaccine have prompted a reassessment of the viral safety of plasma-derived products. The purpose of this study was to evaluate the efficacy of current viral inactivation methods for West Nile and vaccinia viruses and to reassess the use of model viruses to predict inactivation of similar viral pathogens. MATERIALS AND METHODS: Virus-spiked product intermediates were processed using a downscaled representation of various manufacturing procedures. Virus infectivity was measured before and after processing to determine virus inactivation. RESULTS: The results demonstrated effective inactivation of West Nile virus, vaccinia virus and a model virus, bovine viral diarrhoea virus, during pasteurization, solvent/detergent treatment and caprylate treatment. Caprylate provided rapid and effective inactivation of West Nile virus, vaccinia virus, duck hepatitis B virus and Sindbis virus. Inactivation of West Nile virus was similar to that of bovine viral diarrhoea virus. CONCLUSIONS: This study demonstrates that procedures used to inactivate enveloped viruses in manufacturing processes can achieve inactivation of West Nile virus and vaccinia virus. In addition, the data support the use of model viruses to predict the inactivation of similar emergent viral pathogens.     

Parvovirus B19 may have a role in the pathogenesis of juvenile idiopathic arthritis

OBJECTIVE: To determine the prevalence of human parvovirus B19 infection in patients with juvenile idiopathic arthritis (JIA) by detection of specific IgM, IgG, and viral DNA. METHODS: Serum samples of 50 patients with diagnosis of JIA and 39 healthy controls were analyzed by ELISA to detect IgG and IgM anti-B19-specific antibodies. The parvovirus B19 genome was detected by nested polymerase chain reaction (PCR). The average age of the patients was 9.6 years (2-14 yrs); 30 were female (60%) and 20 male (40%). The definitive diagnoses of these patients corresponded to 19 systemic forms (38%), 11 to the oligoarticular variety (22%) and 20 to the polyarticular (40%). The average age of the control group was 7.8 years (2-16 yrs); the distribution by sex was 25 females (64%) and 14 males (36%). RESULTS: IgM against parvovirus B19 was detected in 20% of the cases (10 patients) and B19 DNA genome by PCR in 48% (24 patients); in 10% of the cases (5 patients), both markers were detected. IgG was found in 32% (16 patients). In the control group neither IgM nor the viral genome was detected. However, 43.5% of the controls (17/39) had IgG against parvovirus B19, indicating past infection by the virus. CONCLUSION: Our study confirms recent observations regarding a high prevalence of viral DNA in JIA patients and a possible role of this viral infection in JIA pathogenesis.     

Effect of manufacturing process parameters on virus inactivation by dry heat treatment at 80 degrees C in factor VIII

BACKGROUND AND OBJECTIVES: Dry heat treatment at 80 degrees C for 72 h is used as a virus inactivation step for some coagulation factor concentrates such as Bio Products Laboratory's (BPL) factor VIII 8Y. In the current study, the effect of this process has been tested on a range of viruses. In addition the effect of various manufacturing process parameters on virus inactivation has been investigated. MATERIALS AND METHODS: Samples of product intermediate were obtained from manufacturing, spiked with virus and subjected to freeze drying and dry heat treatment. Virus inactivation was determined by infectivity assay. RESULTS: Freeze drying followed by dry heat treatment was effective for inactivating a wide range of enveloped and nonenveloped viruses. Sucrose or protein concentration had no effect on virus inactivation. Product presentation or the interruption of heat treatment also had no effect. The inactivation of some of the viruses was greater at higher residual water content but under such conditions the stability of the product was reduced. CONCLUSION: This virus inactivation step was effective for a wide range of viruses and over the range of process conditions encountered in manufacturing. This demonstrates the robustness of this process step.     

Parvovirus B19 DNA in plasma pools and plasma derivatives

BACKGROUND AND OBJECTIVES: Human parvovirus B19 (B19) has been transmitted by various plasma-derived medicinal products. The aim of this study was to determine the frequency and the level of B19 DNA contamination in plasma pools destined for fractionation and in a broad range of plasma derivatives. In addition, removal of B19 DNA by the manufacturing process was investigated in cases where corresponding samples from plasma pool and product were available. MATERIALS AND METHODS: Plasma pool samples and blood products were tested for B19 DNA by nested polymerase chain reaction (PCR), and the viral DNA content was determined by TaqMan quantitative PCR. RESULTS: Two-hundred and twenty two of 372 plasma pools for fractionation contained B19 DNA at concentrations of 10(2)-10(8) genome equivalents/ml (geq/ml). While approximately 65% of the DNA-positive plasma pools were only moderately contaminated (< 10(5) geq/ml), 35% contained > 10(6) geq/ml. High frequencies of contamination were detected in Factor VIII (79 of 91), prothrombin complex concentrates (38 of 43) and Factor IX (41 of 62), where the concentration of B19 DNA ranged between 102 and 107 geq/ml. A lower level of B19 DNA contamination was found in antithrombin III (five of 26 samples), in anti-D immunoglobulins (three of 37 samples) and in albumin (four of 51 samples), with levels ranging between 10(2) and 10(3) geq/ml. Furthermore, investigation of plasma pools for solvent/detergent plasma (S/D plasma), from two manufacturers, revealed B19 DNA in 15 of 66 batches at concentrations of 10(2)-10(8) geq/ml. Similar concentrations were detected in the corresponding final S/D plasma products. Anti-B19 immunoglobulin G (IgG) was found in plasma pools and S/D plasma at concentrations of approximately 40 IU/ml. CONCLUSION: Although positive PCR results do not necessarily reflect infectivity, these data show that B19 is a common contaminant in plasma pools and in plasma-derived medicinal products. Considering the resistance of animal parvoviruses to inactivation by heat and chemical agents, and the absence of specific information for B19, the risk of B19 transmission by plasma products should be considered. Physicians should be aware of this problem when treating patients of B19-related risk groups. The plasma fractionation industry should continue their efforts to avoid B19 contamination of plasma derivatives and develop methods which are effective in removing/inactivating parvovirus B19.     

Hepatitis B virus, hepatitis non-A, non-B virus and hepatitis delta virus in lyophilized antihemophilic factor: relative sensitivity to heat

Lyophilized plasma derivatives are more stable to heat than when they are in the liquid state. Commercial Factor VIII (antihemophilic factor) was seeded with a measured quantity of hepatitis B virus. The contaminated material was then lyophilized and subjected to heat of 60 degrees C for 30 hr. Chimpanzees were inoculated with the heat-treated antihemophilic factor or sham-treated antihemophilic factor that had been held at 4 degrees C. Surprisingly, hepatitis B virus survived the heating procedure with no apparent loss in titer: the incubation period to appearance of HBsAg was that expected for the challenge dose of virus. Even more surprising, one chimpanzee (the recipient of the unheated antihemophilic factor) also developed non-A, non-B hepatitis and two chimpanzees (recipients of the heated antihemophilic factor) also developed delta hepatitis. Neither of these agents was a contaminant of the hepatitis B virus challenge pool, since the purity of this hepatitis B virus pool was established previously in chimpanzees. Thus, both a non-A, non-B agent and the delta agent apparently contaminated the commercial antihemophilic factor. This is the first direct evidence for contamination of antihemophilic factor with the delta agent and confirms previous seroepidemiologic evidence for its presence in pooled plasma derivatives. Subsequent inactivation studies were performed with antihemophilic factor experimentally contaminated with the Hutchinson strain of non-A, non-B hepatitis virus. In these studies, heating at 60 degrees C for 30 hr in the dry state rendered antihemophilic factor free of detectable non-A, non-B hepatitis virus.     

Evaluation of an automated high-volume extraction method for viral nucleic acids in comparison to a manual procedure with preceding enrichment

BACKGROUND AND OBJECTIVES: Nucleic acid extraction still harbours the potential for improvements in automation and sensitivity of nucleic acid amplification technology (NAT) testing. This study evaluates the feasibility of a novel automated high-volume extraction protocol for NAT minipool testing in a blood bank setting. MATERIALS AND METHODS: The chemagic Viral DNA/RNA Kit special for automated purification of viral nucleic acids from 9.6 ml of plasma by using the chemagic Magnetic Separation Module I was investigated. Analytical sensitivity for hepatitis C virus (HCV), human immunodeficiency virus-1 (HIV-1), hepatitis B virus (HBV), hepatitis A virus (HAV) and parvovirus B19 (B19) was compared to our present manual procedure that involves virus enrichment by centrifugation. RESULTS: Chemagic technology allows automation of the viral DNA/RNA extraction process. Viral nucleic acids were bound directly to magnetic beads from 9.6-ml minipools. By combining the automated magnetic beads-based extraction technology with our in-house TaqMan polymerase chain reaction (PCR) assays, 95% detection limits were 280 IU/ml for HCV, 4955 IU/ml for HIV-1, 249 IU/ml for HBV, 462 IU/ml for HAV and 460 IU/ml for B19, calculated for an individual donation in a pool of 96 donors. The detection limits of our present method were 460 IU/ml for HCV, 879 IU/ml for HIV-1, 90 IU/ml for HBV, 203 IU/ml for HAV and 314 IU/ml for B19. CONCLUSIONS: The 95% detection limits obtained by using the chemagic method were within the regulatory requirements for blood donor screening. The sensitivities detected for HCV, HBV, HAV and B19 were found to be in a range similar to that of the manual purification method. Sensitivity for HIV-1, however, was found to be inferior for the chemagic method in this study.     

Removal of small non-enveloped viruses by nanofiltration

BACKGROUND AND OBJECTIVES: Nanofiltration is one of the most effective virus reduction methods in the manufacturing process of plasma products. However, it is difficult to remove small viruses from high molecular weight protein preparations like immunoglobulin G or factor VIII complex by nanofiltration, because the size of the protein is similar to that of viruses. In order to separate the viruses from these proteins by nanofiltration, it is necessary to change the size of either one. In this study, we report that such non-enveloped viruses as human parvovirus B19 (B19), human encephalomyocarditis virus (EMC) or porcine parvovirus (PPV) aggregate in the presence of certain kinds of amino acids and could be easily removed by nanofiltration. MATERIALS AND METHODS: 0.3 M Glycine (or other amino acid) solution spiked with viruses was subjected to dead-end single filtration with a 35-nm pore-size filter. Virus removal by nanofiltration was either evaluated by PCR or by infectivity assay. RESULTS: B19 in a 0.3 M glycine solution was reduced to 1:10(7.5) (7.5-log) by nanofiltration with a 35-nm pore-sized filter, whereas in PBS it was not reduced. Similarly, B19 was also reduced when suspended in other amino acids solutions. This effect was also confirmed with the other small non-enveloped viruses EMC or PPV. When 5% globulin or 5% albumin was added to a 0.3 M glycine solution, the removal rate was decreased. CONCLUSIONS: These data suggest that viruses in the presence of certain kinds of amino acids could be aggregated and effectively removed by a filter that has a pore size larger than the size of the viruses.     

Quantification of viral inactivation by photochemical treatment with amotosalen and UV A light, using a novel polymerase chain reaction inhibition method with preamplification

BACKGROUND: In evaluating a photochemical treatment process for inactivating parvovirus B19, there lacked simple culture methods to measure infectivity. The recently developed enzyme-linked immunospot (ELISpot) infectivity assay uses late-stage erythropoietic progenitor cells and is labor intensive and time consuming. We evaluated a novel, efficient polymerase chain reaction (PCR) inhibition assay and examined correlations with reductions in infectivity. METHODS: Contaminated plasma was treated with 150 micromol/L amotosalen and 3 J/cm(2) ultraviolet A light and then tested for DNA modification using conventional PCR inhibition and a novel preamplification approach. The novel assay subjected the samples to preamplification cycles using long-template PCR, followed by quantitative PCR (QPCR) inhibition detection. Both approaches were tested for correlations with reductions in viral infectivity by comparing ELISpot assay results of identical samples. RESULTS: The B19 preamplification inhibition assay showed detection ranges of 2-2.5 log and demonstrated quantitative correlation with up to a 5.8-log reduction in viral infectivity in ELISpot results. Conventional PCR detected a >5 log reduction in amplification, correlated with a 4.4-log reduction in viral infectivity. A range of 4-log inhibition of hepatitis B virus DNA amplification was also achieved. CONCLUSIONS: The results demonstrated that a novel preamplification QPCR assay is a useful tool for predicting reductions in infectivity after photochemical treatment. This assay was extended to show utility in circumstances where practical in vitro assays are unavailable for the determination of the efficacy of pathogen inactivation.     

Frequent infection with a viral pathogen,parvovirus B19, in rheumatic diseases of childhood

OBJECTIVE: To find further evidence of the association of parvovirus B19 infection with juvenile rheumatic diseases, and to get new insights into the immunopathogenesis of these diseases. METHODS: Paired serum and synovial fluid samples from 74 children with rheumatic disease were analyzed with respect to their content of viral DNA and antibodies directed against the B19 viral proteins VP1, VP2, and NS1. Control sera from 124 children with noninflammatory bone diseases or growth retardation were also analyzed. The sequence of the viral DNA, amplified by polymerase chain reaction (PCR), was determined. IgG-complexed virus was isolated from sera and synovial fluid by adsorption to protein A beads. The amount of free virus versus immunocomplexed virus particles was determined by quantification of the viral genomes by quantitative PCR. RESULTS: Twenty-six of the 74 patients (35%) had detectable amounts of parvovirus B19 DNA in the serum (n = 22 [30%]) and/or the synovial fluid (n = 16 [22%]), whereas only 9 of the 124 control sera (7%) were positive for the viral DNA (P < 0.0001). Forty-six patients (62%) had serum IgG against the structural proteins, indicating past infection with B19. NS1-specific antibodies were detected in sera from 29 patients (39%) and 27 controls (22%) (P < 0.001). In addition, 3 patients (4%) showed VP2-specific IgM. In 15 patients, viral DNA could be repeatedly detected in followup samples of serum and synovial fluid. Sequencing revealed low-degree nucleotide variations that are in the range of genotype 1 of parvovirus B19. Immunocomplexed virus was present in varying amounts, both in the sera and in the synovial fluid samples. CONCLUSION: Parvovirus B19 is frequently found in serum or synovial fluid of children with rheumatism. The rate of persistent B19 infection in these patients is significantly higher than in age-matched controls.     

Parvovirus transmission by blood products – a cause for concern?

The introduction of dual viral inactivation of clotting factor concentrates has practically eliminated infections by viruses associated with significant pathogenicity over the last 20 years. Despite this, theoretical concerns about transmission of infection have remained, as it is known that currently available viral inactivation methods are unable to eliminate parvovirus B19 or prions from these products. Recently, concern has been raised following the identification of the new parvoviruses, human parvovirus 4 (PARV4) and new genotypes of parvovirus B19, in blood products. Parvoviruses do not cause chronic pathogenicity similar to human immunodeficiency virus or hepatitis C virus, but nevertheless may cause clinical manifestations, especially in immunosuppressed patients. Manufacturers should institute measures, such as minipool polymerase chain reaction testing, to ensure that their products contain no known viruses. So far, human bocavirus, another new genus of parvovirus, has not been detected in fractionated blood products, and unless their presence can be demonstrated, routine testing during manufacture is not essential. Continued surveillance of the patients and of the safety of blood products remains an important ongoing issue.     

Inactivation of hepatitis A virus by heat and high hydrostatic pressure: variation among laboratory strains

Background and Objectives  Hepatitis A virus (HAV) transmission via contaminated blood products has been reported. Cell-adapted HAV strains are generally used to confirm virus inactivation in manufacturing blood products, but the strains may differ in their sensitivity to inactivation treatment. To select an appropriate cell-adapted HAV strain for virus validation, we compared the inactivation efficiency among four strains under two different physical inactivation treatments: heat and high hydrostatic pressure.
Materials and Methods  The cell-adapted HAV strains used here were KRM238, KRM003 (subgenotype IIIB), KRM031 (IA), and TKM005 (IB). The strains were treated at 60°C for up to 10 h or under high hydrostatic pressure (up to 420 MPa). The reduction in HAV infectivity was measured by an immunofocus-staining method.
Results  The heat treatment at 60°C for 10 h reduced HAV infectivity in the range of 3 to 5 log₁₀ among the strains; KRM238 and TKM005 were harder to inactivate than the other two. The high hydrostatic pressure treatment at 420 MPa also reduced infectivity in the range of 3 to 5 log₁₀ among the strains, and KRM031 was easier to inactivate than the other strains.
Conclusion  Heat treatment and high hydrostatic pressure treatment revealed differences in inactivation efficiencies among cell-adapted HAV strains, and each strain reacted differently depending on the treatment. KRM238 may be the best candidate for virus validation to ensure the safety of blood products against viral contamination, as it is harder to inactivate and it replicates better in cell culture than the other strains.