Genotype ; Hepatitis B, Chronic ; complications ; Hepatitis C, Chronic ; complications ; Humans ; Parvoviridae Infections ; complications ; virology ; Parvovirus ; classification ; genetics ; isolation & purification ; Parvovirus B19, Human ; isolation & purification

A new nested-polymerase chain reaction (nested-PCR) assay was developed to detect human parvovirus B19 DNA corresponding to the nonstructural protein in clinical specimens in a routine diagnostic laboratory. The sensitivity of this highly specific assay was up to 0.005 fg of B19 DNA. Parvovirus B19 was identified in sera of 20 pregnant women with abnormal pregnant outcome. Among these 20 cases, intrauterine parvovirus infection did exist in 7 pregnant women because parvovirus B19 DNA was detected in the pregnant tissues of them such as placenta tissues, chorionic villi, amniotic fluid, fetal spleen, liver and abdominal fluids.
DNA, Viral/*analysis ; Parvoviridae Infections/*virology ; Parvovirus B19, Human/genetics ; Parvovirus B19, Human/*isolation & purification ; Placenta/virology ; Pregnancy Complications, Infectious/diagnosis ; Pregnancy Complications, Infectious/*virology ; Viral Nonstructural Proteins/*analysis

BACKGROUND: The safety of plasma derivatives has been reinforced since 1980s by variable pathogen inactivation or elimination techniques. Nucleic acid amplification test (NAT) for the source plasma has also been implemented worldwide. Recently nanofiltration has been used in some country for ensuring safety of plasma derivatives to eliminate non-enveloped viruses such as parvovirus B19 (B19V) and hepatitis A virus (HAV). We evaluated the efficacy of nanofiltration for the elimination of B19V and HAV. METHODS: To verify the efficacy of nanofiltration, we adopted a 20 nm Viresolve NFP (Millipore, USA) in the scaling down (1:1,370) model of the antithrombin III production. As virus stock solutions, we used B19V reactive plasma and porcine parvovirus (PPV) and HAV obtained from cell culture. And 50% tissue culture infectious dose was consumed as infectious dose. The methods used to evaluate the virus-elimination efficacy were reverse-transcriptase polymerase chain reaction for B19V and the cytopathic effect calculation after filtration for PPV and HAV. RESULTS: B19V was not detected by RT-PCR in the filtered antithrombin III solutions with initial viral load of 6.42x10(5) IU/mL and 1.42x10(5) IU/mL before filtration. The virus-elimination efficacy of nanofiltration for PPV and HAV were > or =10(3.32) and > or =10(3.31), respectively. CONCLUSIONS: Nanofiltration would be an effective method for the elimination of B19V and HAV. It may be used as a substitute for NAT screening of these viruses in source plasma to ensure safety of plasma derivatives in Korea.
Antithrombin III/isolation & purification ; DNA, Viral/analysis ; Filtration/*methods ; Hepatitis A virus/genetics/*isolation & purification ; Humans ; Nanotechnology/*methods ; Parvovirus B19, Human/genetics/*isolation & purification ; RNA, Viral/analysis ; Reverse Transcriptase Polymerase Chain Reaction

The baculovirus expression system was employed to prepare the virus-like particles (VLPs) of human parvovirus B19. The synthesized VP2 gene of B19 was inserted into the multi-cloning site (MCS) of pFastBac1 vector; the resulting plasmid was transferred to the Escherichia coli DH10Bac competent cells, which contain a baculovirus shuttle vector (Bacmid), to generate Bacmid-VP2 by site-specific transposition. Recombinant baculovirus carrying VP2 gene (rBac-VP2) was then rescued from Bacmid-VP2-transfected Sf9 cells. Indirect immunofluorescence and Western blotting were used to identify the VP2 protein in rBac-VP2-infected Sf9 cells, and the VLPs were observed under transmission electron microscope after being enriched by ultracentrifugation. The B19 VLPs were successfully produced in insect cells with baculovirus expression system, which will facilitate the development of diagnostic reagents to detect the antibody against B19 virus in human serum.
Animals ; Antibodies, Viral ; blood ; Baculoviridae ; genetics ; metabolism ; Capsid Proteins ; biosynthesis ; genetics ; Cell Line ; Cloning, Molecular ; Genetic Vectors ; genetics ; Parvovirus B19, Human ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Virion ; genetics ; metabolism

BACKGROUND: To ensure the safety of plasma derivatives, some countries have been screening for the human parvovirus B19 (B19V) antigen or DNA in blood donors. We investigated the prevalence of B19V DNA and anti-B19V antibodies in Korean plasmapheresis donors to evaluate the necessity of B19V DNA screening test. METHODS: Plasma samples were collected between March and July 2008 from 10,032 plasmapheresis donors. The B19V DNA test was performed using the LightCycler 2.0 (Roche, Germany) with quantification kits. Anti-B19V IgM and IgG were tested in 928 randomly selected samples from the 10,032 donors using recomWell Parvovirus B19 ELISA IgM, IgG assay (Mikrogen, Germany). RecomLine Parvovirus B19 LIA IgG, IgM assay (Mikrogen, Germany) was used to analyze the epitopes of antibodies in donors showing positive results for B19V DNA and anti-B19V antibodies. DNA sequencing was performed to identify the genotypes. RESULTS: The prevalence of B19V DNA was 0.1% (10/10,032). Virus titers in B19V DNA positive donors were less than 10(5) IU/mL (range: 2.7x10(1)-3.2x10(4) IU/mL) except for 1 donor (1.33x10(8) IU/mL). All the isolated B19V DNAs from 6 donors were identified as genotype I. Nine out of 10 B19V DNA positive donors also possessed anti-B19V IgG only or IgG and IgM. The prevalence of anti-B19V IgG was 60.1% (558/928). CONCLUSIONS: The prevalence of B19V DNA in Korean blood donors was not high and most donors also possessed neutralizing anti-B19V antibodies. Thus, the implementation of a B19V screening test for Korean blood donors does not appear to be imperative.
Adolescent ; Adult ; Aged ; Antibodies, Viral/blood ; *Blood Donors ; DNA, Viral/*blood ; Enzyme-Linked Immunosorbent Assay/methods ; Female ; Follow-Up Studies ; Genotype ; Humans ; Immunoglobulin G/blood ; Immunoglobulin M/blood ; Male ; Middle Aged ; Parvoviridae Infections/epidemiology ; Parvovirus B19, Human/genetics/immunology/*isolation & purification ; *Plasmapheresis ; Polymerase Chain Reaction/methods ; Prevalence ; Republic of Korea/epidemiology ; Retrospective Studies