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Jianming Qiu - One of the best experts on this subject based on the ideXlab platform.
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parvoVirus B19 infection of human primary erythroid progenitor cells triggers atr chk1 signaling which promotes B19 Virus replication
Journal of Virology, 2011Co-Authors: Yong Luo, Steve Kleiboeker, Sai Lou, Xuefeng Deng, Zhengwen Liu, Jianming QiuAbstract:Human parvoVirus B19 (B19V) infection is restricted to erythroid progenitor cells of the human bone marrow. Although the mechanism by which the B19V genome replicates in these cells has not been studied in great detail, accumulating evidence has implicated involvement of the cellular DNA damage machinery in this process. Here, we report that, in ex vivo-expanded human erythroid progenitor cells, B19V infection induces a broad range of DNA damage responses by triggering phosphorylation of all the upstream kinases of each of three repair pathways: ATM (ataxia-telangiectasi mutated), ATR (ATM and Rad3 related), and DNA-PKcs (DNA-dependent protein kinase catalytic subunit). We found that phosphorylated ATM, ATR, and DNA-PKcs, and also their downstream substrates and components (Chk2, Chk1, and Ku70/Ku80 complex, respectively), localized within the B19V replication center. Notably, inhibition of kinase phosphorylation (through treatment with either kinase-specific inhibitors or kinase-specific shRNAs) revealed requirements for signaling of ATR and DNA-PKcs, but not ATM, in Virus replication. Inhibition of the ATR substrate Chk1 led to similar levels of decreased Virus replication, indicating that signaling via the ATR-Chk1 pathway is critical to B19V replication. Notably, the cell cycle arrest characteristic of B19V infection was not rescued by interference with the activity of any of the three repair pathway kinases.
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the small 11kda nonstructural protein of human parvoVirus B19 plays a key role in inducing apoptosis during B19 Virus infection of primary erythroid progenitor cells
Blood, 2010Co-Authors: Aaron Yun Chen, Wuxiang Guan, Fang Cheng, Steve Kleiboeker, Elizabeth Yan Zhang, Thomas M Yankee, Jianming QiuAbstract:Human parvoVirus B19 (B19V) infection shows a strong erythroid tropism and drastically destroys erythroid progenitor cells, thus leading to most of the disease outcomes associated with B19V infection. In this study, we systematically examined the 3 B19V nonstructural proteins, 7.5kDa, 11kDa, and NS1, for their function in inducing apoptosis in transfection of primary ex vivo–expanded erythroid progenitor cells, in comparison with apoptosis induced during B19V infection. Our results show that 11kDa is a more significant inducer of apoptosis than NS1, whereas 7.5kDa does not induce apoptosis. Furthermore, we determined that caspase-10, an initiator caspase in death receptor signaling, is the most active caspase in apoptotic erythroid progenitors induced by 11kDa and NS1 as well as during B19V infection. More importantly, cytoplasm-localized 11kDa is expressed at least 100 times more than nucleus-localized NS1 at the protein level in primary erythroid progenitor cells infected with B19V; and inhibition of 11kDa expression using antisense oligos targeting specifically to the 11kDa-encoding mRNAs reduces apoptosis significantly during B19V infection of erythroid progenitor cells. Taken together, these results demonstrate that the 11kDa protein contributes to erythroid progenitor cell death during B19V infection.
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block to the production of full length B19 Virus transcripts by internal polyadenylation is overcome by replication of the viral genome
Journal of Virology, 2008Co-Authors: Wuxiang Guan, Fang Cheng, Yuko Yoto, Steve Kleiboeker, Susan Wong, Ning Zhi, David J Pintel, Jianming QiuAbstract:The pre-mRNA processing strategy of the B19 Virus is unique among parvoViruses. B19 Virus-generated pre-mRNAs are transcribed from a single promoter and are extensively processed by alternative splicing and alternative polyadenylation to generate 12 transcripts. Blockage of the production of full-length B19 Virus transcripts at the internal polyadenylation site [(pA)p] was previously reported to be a limiting step in B19 Virus permissiveness. We show here that in the absence of genome replication, internal polyadenylation of B19 Virus RNAs at (pA)p is favored in cells which are both permissive and nonpermissive for B19 viral replication. Replication of the B19 Virus genome, however, introduced either by viral infection or by transfection of an infectious clone into permissive cells or forced by heterologous replication systems in nonpermissive cells, enhanced readthrough of (pA)p and the polyadenylation of B19 Virus transcripts at the distal site [(pA)d]. Therefore, replication of the genome facilitates the generation of sufficient full-length transcripts that encode the viral capsid proteins and the essential 11-kDa nonstructural protein. Furthermore, we show that polyadenylation of B19 viral RNA at (pA)p likely competes with splicing at the second intron. Thus, we conclude that replication of the B19 Virus genome is the primary limiting step governing B19 Virus tropism.
Giorgio Gallinella - One of the best experts on this subject based on the ideXlab platform.
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hepg2 hepatocellular carcinoma cells are a non permissive system for B19 Virus infection
Journal of General Virology, 2008Co-Authors: Francesca Bonvicini, Claudia Filippone, Elisabetta Manaresi, Marialuisa Zerbini, Monica Musiani, Giorgio GallinellaAbstract:ParvoVirus B19 has been associated with liver dysfunction and has been considered a potential aetiological agent of fulminant hepatitis and hepatitis-associated aplastic anaemia. The possible effects of B19 Virus infection on the liver have been investigated using HepG2 hepatocellular carcinoma cells as a model system, but the reported results are inconsistent. To investigate this relationship further, this study followed the course of B19 Virus infection of HepG2 cells in terms of viral DNA, RNA and protein production by quantitative PCR, RT-PCR and immunofluorescence assays. The data showed that B19 Virus is able to bind and possibly enter HepG2 cells, but that viral genome replication or transcription is not supported and that viral proteins are not produced. As far as HepG2 cells can be considered a representative model system, any possible pathogenic role of B19 Virus on the liver cannot be ascribed to infection or to a direct cytopathic effect on hepatocytes.
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prevention of iatrogenic transmission of B19 infection different approaches to detect remove or inactivate Virus contamination
Clinical Laboratory, 2006Co-Authors: Francesca Bonvicini, Monica Musiani, Giorgio Gallinella, Simone Ambretti, Giovanna Angela Gentilomi, Marialuisa ZerbiniAbstract: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.
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molecular testing for detection of in vitro infectivity of plasma pools contaminated with B19 Virus
Journal of Medical Virology, 2004Co-Authors: Francesca Bonvicini, Monica Musiani, Giorgio Gallinella, Monica Cricca, Simone Ambretti, Stefania Delbarba, Marialuisa ZerbiniAbstract:B19 Virus can be transmitted by contaminated blood or blood products. Recent observations, in healthy volunteers, suggest that active B19 infection can follow the administration of plasma pools with a concentration ≥107 genome equivalents/ml (geq/ml) of B19 DNA. However, patients receiving batches with levels of Virus DNA lower than 104 geq/ml do not show any evidence of transmission of the Virus. The aim of the study was to show, by in vitro assays, a threshold of viral load in B19 contaminated plasma pools over which the infection can be transmitted. Twenty plasma pools, each containing 960 single donations, were tested to correlate the viral load and the level of antibodies anti-B19 with the in vitro infectivity and expression of B19 Virus. All the plasma pools, titrated for B19 viral load by competitive PCR, were inoculated into KU812Ep6 erythroid human cell line. Five of the nine contaminated plasma pools, with a B19 DNA concentration ≥3.60 × 106 geq/ml, were able to infect KU812Ep6 cells. In vitro infectivity was shown in KU812Ep6 cells at 24 h post-infection by in situ hybridisation and amplification assays for viral DNA and RNAs. Plasma pools with a viral load in the range of 6.00 × 103–8.96 × 104 geq/ml did not show infectivity when inoculated into KU812Ep6 cells. Medium-high titres of IgG antibodies anti-B19 were detectable in all the plasma pools and the neutralising activity associated with specific IgG anti-B19 may explain the lack of infectivity of plasma pools contaminated with a low viral load. In conclusion, in situ hybridisation and amplification assays for viral DNA and RNAs in KU812Ep6 cells inoculated with plasma pools can be valid assays to test for the presence of infectous Virus in the production of B19-safe material. J. Med. Virol. 74:272–276, 2004. © 2004 Wiley-Liss, Inc.
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B19 Virus genome diversity epidemiological and clinical correlations
Journal of Clinical Virology, 2003Co-Authors: Giorgio Gallinella, Elisabetta Manaresi, Simona Venturoli, M Musiani, M ZerbiniAbstract:Genetic analysis of parvoVirus B19 has been carried out mainly to establish a framework to track molecular epidemiology of the Virus and to correlate sequence variability with different pathological and clinical manifestations of the Virus. A good amount of information regarding B19 Virus sequence variability is available, and presently there are about 400 sequence records deposited in the nucleotide database of NCBI. A few are almost complete genomic sequences, and these allow the construction of a global alignment framework. Many others are partial genomic sequences, limited to selected regions, and these allow comparison of a higher number of isolates from well-defined epidemiological settings and/or pathological conditions. Most studies showed that the genetic variability of B19 Virus is low, that molecular epidemiology is possible only on a limited geographical and temporal setting, and that no clear correlations are present between genome sequence and distinctive pathological and clinical manifestations. More recently, several viral isolates have been identified that show remarkable sequence diversity with respect to reference sequences. The identification of variant isolates added to the knowledge of genetic diversity in this Virus group and allowed the identification of three divergent genetic clusters, about 10% divergent from each other and still quite distinct from other parvoViruses, that can be thought of as different genotypes within the human erythroVirus group and that show clearly resolved phylogenetic relationship. These variant isolates pose interesting questions regarding the real extent of genetic variability in the human erythroViruses, the relevance of these Viruses in terms of epidemiology and their possible implication in the pathogenesis of erythroVirus-related diseases.
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analysis of B19 Virus contamination in plasma pools for manufacturing by using a competitive polymerase chain reaction assay
Vox Sanguinis, 2002Co-Authors: Giorgio Gallinella, Francesca Bonvicini, Monica Musiani, E Moretti, G Nardi, Elisa Zuffi, E Bucci, M ZerbiniAbstract:Background and Objectives The presence of B19 Virus in blood poses a risk of transmission of the Virus via blood or blood products. Screening processes for manufacturing should be aimed at achieving production plasma pools with B19 Virus contamination levels below 104 genome equivalents/ml (geq/ml) in order to prevent transmission of infection through plasma derivatives. Materials and Methods The suitability of a competitor plasmid as an internal analytical standard for the detection of B19 Virus in plasma pools was assessed by using a competitive polymerase chain reaction (PCR) assay. Seventy-five plasma pools, each consisting of 960 single donations, were analysed for B19 Virus contamination following a lysis treatment. Results The amount of competitor plasmid in the competitive PCR assay established, with good accuracy, a threshold value for discrimination of the viral load in plasma pools. Analysis of samples from plasma pools showed that 12% of pools were contaminated with B19 Virus at levels above the set threshold value. Conclusions The competitive PCR assay developed proved to be effective for discrimination of the B19 Virus contamination level in screening of plasma pools for manufacturing.
Kazuo Sugamura - One of the best experts on this subject based on the ideXlab platform.
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human parvoVirus B19 nonstructural protein ns1 induces cell cycle arrest at g1 phase
Journal of Virology, 2003Co-Authors: Eiji Morita, Hironobu Asao, Hiroyuki Sato, Akitoshi Nakashima, Kazuo SugamuraAbstract:Human parvoVirus B19 is known to cause several diseases, such as aplastic crisis in patients with chronic hemolytic anemia, erythema infectiosum, persistent infections manifesting as chronic anemia in immunocompromised patients, nonimmune hydrops fetalis leading to intrauterine fetal death, and arthralgia/arthritis (6). The mechanistic basis for these B19 Virus-associated diseases is poorly understood; however, most symptoms appear to be related to the unique tissue tropism of B19 Virus. Cells permissive for B19 Virus replication are proliferating erythroid precursors in human bone marrow and fetal liver tissues, and B19 Virus infection to the target cells directly induces cell death (6, 16). B19 Virus is a single-stranded DNA Virus that lacks an envelope and has a genome length of 5.4 kb with hairpin structures at each extremity (12). Two major open reading frames (ORFs) extend through almost the entire genome of B19 Virus (23). A nonstructural protein (NS1) on the left side of the genome with a molecular mass of 70 to 77 kDa appears as two bands on Westernblot analysis (17). NS1 has been found to be essential for replication of viral DNA, and for the regulation of its own viral promoters (6). NS1 contains a consensus sequence for purine nucleotide binding in the middle part (3). This consensus is predicted to be an ATP- or GTP-binding site, which is associated with ATPase and DNA helicase activities (3). NS1 is also known to be cytotoxic for erythroid cells and is possibly related to the pathogenesis of B19 Virus infection (19). Our previous studies demonstrated that NS1 expression induced apoptosis in erythroid linage cells both in vivo and in vitro (7, 15, 30); however, the molecular mechanism by which NS1 mediates apoptosis has not been clarified. We previously demonstrated that B19 Virus infection induces growth inhibition of erythroid cells immediately after infection, with almost all of B19 Virus-infected cells arresting at the G2 phase of the cell cycle (17). This was accompanied by an accumulation of mitotic cyclins such as cyclin A and cyclin B (17). In minute Virus of mice (MVM), a rodent parvoVirus, however, Virus-infected cells were shown to fall into G1- and S-phase arrest, as well as G2 arrest, and these multistep cell cycle arrests were induced by expression of NS1 of the MVM (11). In the present study, we show that B19 Virus-infected cells also falls into not only G2 arrest but also G1 arrest, and this G1 arrest is mediated by NS1.
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human parvoVirus B19 induces cell cycle arrest at g2 phase with accumulation of mitotic cyclins
Journal of Virology, 2001Co-Authors: Eiji Morita, K Tada, Hiroshi Chisaka, Hironobu Asao, Hiroyuki Sato, Nobuo Yaegashi, Kazuo SugamuraAbstract:Human parvoVirus B19 infects specifically erythroid progenitor cells, which causes transient aplastic crises and hemolytic anemias. Here, we demonstrate that erythroblastoid UT7/Epo cells infected with B19 Virus fall into growth arrest with 4N DNA, indicating G(2)/M arrest. These B19 Virus-infected cells displayed accumulation of cyclin A, cyclin B1, and phosphorylated cdc2 and were accompanied by an up-regulation in the kinase activity of the cdc2-cyclin B1 complex, similar to that in cells treated with the mitotic inhibitor. However, degradation of nuclear lamina and phosphorylation of histone H3 and H1 were not seen in B19 Virus-infected cells, indicating that the infected cells do not enter the M phase. Accumulation of cyclin B1 was persistently localized in the cytoplasm, but not in the nucleus, suggesting that B19 Virus infection of erythroid cells raises suppression of nuclear import of cyclin B1, resulting in cell cycle arrest at the G(2) phase. The B19 Virus-induced G(2)/M arrest may be the critical event in the damage of erythroid progenitor cells seen in patients with B19 Virus infection.
M Zerbini - One of the best experts on this subject based on the ideXlab platform.
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B19 Virus genome diversity epidemiological and clinical correlations
Journal of Clinical Virology, 2003Co-Authors: Giorgio Gallinella, Elisabetta Manaresi, Simona Venturoli, M Musiani, M ZerbiniAbstract:Genetic analysis of parvoVirus B19 has been carried out mainly to establish a framework to track molecular epidemiology of the Virus and to correlate sequence variability with different pathological and clinical manifestations of the Virus. A good amount of information regarding B19 Virus sequence variability is available, and presently there are about 400 sequence records deposited in the nucleotide database of NCBI. A few are almost complete genomic sequences, and these allow the construction of a global alignment framework. Many others are partial genomic sequences, limited to selected regions, and these allow comparison of a higher number of isolates from well-defined epidemiological settings and/or pathological conditions. Most studies showed that the genetic variability of B19 Virus is low, that molecular epidemiology is possible only on a limited geographical and temporal setting, and that no clear correlations are present between genome sequence and distinctive pathological and clinical manifestations. More recently, several viral isolates have been identified that show remarkable sequence diversity with respect to reference sequences. The identification of variant isolates added to the knowledge of genetic diversity in this Virus group and allowed the identification of three divergent genetic clusters, about 10% divergent from each other and still quite distinct from other parvoViruses, that can be thought of as different genotypes within the human erythroVirus group and that show clearly resolved phylogenetic relationship. These variant isolates pose interesting questions regarding the real extent of genetic variability in the human erythroViruses, the relevance of these Viruses in terms of epidemiology and their possible implication in the pathogenesis of erythroVirus-related diseases.
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analysis of B19 Virus contamination in plasma pools for manufacturing by using a competitive polymerase chain reaction assay
Vox Sanguinis, 2002Co-Authors: Giorgio Gallinella, Francesca Bonvicini, Monica Musiani, E Moretti, G Nardi, Elisa Zuffi, E Bucci, M ZerbiniAbstract:Background and Objectives The presence of B19 Virus in blood poses a risk of transmission of the Virus via blood or blood products. Screening processes for manufacturing should be aimed at achieving production plasma pools with B19 Virus contamination levels below 104 genome equivalents/ml (geq/ml) in order to prevent transmission of infection through plasma derivatives. Materials and Methods The suitability of a competitor plasmid as an internal analytical standard for the detection of B19 Virus in plasma pools was assessed by using a competitive polymerase chain reaction (PCR) assay. Seventy-five plasma pools, each consisting of 960 single donations, were analysed for B19 Virus contamination following a lysis treatment. Results The amount of competitor plasmid in the competitive PCR assay established, with good accuracy, a threshold value for discrimination of the viral load in plasma pools. Analysis of samples from plasma pools showed that 12% of pools were contaminated with B19 Virus at levels above the set threshold value. Conclusions The competitive PCR assay developed proved to be effective for discrimination of the B19 Virus contamination level in screening of plasma pools for manufacturing.
Francesca Bonvicini - One of the best experts on this subject based on the ideXlab platform.
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hepg2 hepatocellular carcinoma cells are a non permissive system for B19 Virus infection
Journal of General Virology, 2008Co-Authors: Francesca Bonvicini, Claudia Filippone, Elisabetta Manaresi, Marialuisa Zerbini, Monica Musiani, Giorgio GallinellaAbstract:ParvoVirus B19 has been associated with liver dysfunction and has been considered a potential aetiological agent of fulminant hepatitis and hepatitis-associated aplastic anaemia. The possible effects of B19 Virus infection on the liver have been investigated using HepG2 hepatocellular carcinoma cells as a model system, but the reported results are inconsistent. To investigate this relationship further, this study followed the course of B19 Virus infection of HepG2 cells in terms of viral DNA, RNA and protein production by quantitative PCR, RT-PCR and immunofluorescence assays. The data showed that B19 Virus is able to bind and possibly enter HepG2 cells, but that viral genome replication or transcription is not supported and that viral proteins are not produced. As far as HepG2 cells can be considered a representative model system, any possible pathogenic role of B19 Virus on the liver cannot be ascribed to infection or to a direct cytopathic effect on hepatocytes.
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prevention of iatrogenic transmission of B19 infection different approaches to detect remove or inactivate Virus contamination
Clinical Laboratory, 2006Co-Authors: Francesca Bonvicini, Monica Musiani, Giorgio Gallinella, Simone Ambretti, Giovanna Angela Gentilomi, Marialuisa ZerbiniAbstract: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.
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molecular testing for detection of in vitro infectivity of plasma pools contaminated with B19 Virus
Journal of Medical Virology, 2004Co-Authors: Francesca Bonvicini, Monica Musiani, Giorgio Gallinella, Monica Cricca, Simone Ambretti, Stefania Delbarba, Marialuisa ZerbiniAbstract:B19 Virus can be transmitted by contaminated blood or blood products. Recent observations, in healthy volunteers, suggest that active B19 infection can follow the administration of plasma pools with a concentration ≥107 genome equivalents/ml (geq/ml) of B19 DNA. However, patients receiving batches with levels of Virus DNA lower than 104 geq/ml do not show any evidence of transmission of the Virus. The aim of the study was to show, by in vitro assays, a threshold of viral load in B19 contaminated plasma pools over which the infection can be transmitted. Twenty plasma pools, each containing 960 single donations, were tested to correlate the viral load and the level of antibodies anti-B19 with the in vitro infectivity and expression of B19 Virus. All the plasma pools, titrated for B19 viral load by competitive PCR, were inoculated into KU812Ep6 erythroid human cell line. Five of the nine contaminated plasma pools, with a B19 DNA concentration ≥3.60 × 106 geq/ml, were able to infect KU812Ep6 cells. In vitro infectivity was shown in KU812Ep6 cells at 24 h post-infection by in situ hybridisation and amplification assays for viral DNA and RNAs. Plasma pools with a viral load in the range of 6.00 × 103–8.96 × 104 geq/ml did not show infectivity when inoculated into KU812Ep6 cells. Medium-high titres of IgG antibodies anti-B19 were detectable in all the plasma pools and the neutralising activity associated with specific IgG anti-B19 may explain the lack of infectivity of plasma pools contaminated with a low viral load. In conclusion, in situ hybridisation and amplification assays for viral DNA and RNAs in KU812Ep6 cells inoculated with plasma pools can be valid assays to test for the presence of infectous Virus in the production of B19-safe material. J. Med. Virol. 74:272–276, 2004. © 2004 Wiley-Liss, Inc.
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analysis of B19 Virus contamination in plasma pools for manufacturing by using a competitive polymerase chain reaction assay
Vox Sanguinis, 2002Co-Authors: Giorgio Gallinella, Francesca Bonvicini, Monica Musiani, E Moretti, G Nardi, Elisa Zuffi, E Bucci, M ZerbiniAbstract:Background and Objectives The presence of B19 Virus in blood poses a risk of transmission of the Virus via blood or blood products. Screening processes for manufacturing should be aimed at achieving production plasma pools with B19 Virus contamination levels below 104 genome equivalents/ml (geq/ml) in order to prevent transmission of infection through plasma derivatives. Materials and Methods The suitability of a competitor plasmid as an internal analytical standard for the detection of B19 Virus in plasma pools was assessed by using a competitive polymerase chain reaction (PCR) assay. Seventy-five plasma pools, each consisting of 960 single donations, were analysed for B19 Virus contamination following a lysis treatment. Results The amount of competitor plasmid in the competitive PCR assay established, with good accuracy, a threshold value for discrimination of the viral load in plasma pools. Analysis of samples from plasma pools showed that 12% of pools were contaminated with B19 Virus at levels above the set threshold value. Conclusions The competitive PCR assay developed proved to be effective for discrimination of the B19 Virus contamination level in screening of plasma pools for manufacturing.
