Humans ; Parvoviridae Infections ; virology ; Parvovirus B19, Human ; genetics ; metabolism ; physiology

OBJECTIVETo establish a TaqMan-based Real-time PCR assay with internal control for the detection of parvovirus B19 DNA in human serum.

METHODSTwo DNA fragments in length of 113 bp each were artificially synthesized, cloned into T vector and used as standard DNA or internal control, respectively. One pair of primers and two probes were included in the Real-time PCR. The probes were labeled with different fluoresceins and could bind B19 DNA or internal control, respectively. Precision and specificity of the method were evaluated. Specimens of human serum were examined by this assay to find B19 DNA.

RESULTSThe standard curve was constructed using the quantified standard B19 DNA. The Real-time PCR method was established. It was stable according to precision evaluation by the intra- and inter-assay and specific without any evident cross-reaction with human hepatitis B virus (HBV). Among 160 samples of human serum, B19 DNA was detected in 2 with a concentration of 2.1 x 10(5) Geq/ml and 3.6 x 10(3) Geq/ml, respectively.

CONCLUSIONThe Real-time PCR for B19 DNA detection was developed successfully, in which the internal control was helpful to exclude false-negative results.

DNA, Viral ; blood ; Humans ; Parvovirus B19, Human ; genetics ; Polymerase Chain Reaction ; methods

Human parvovirus B19 (B19 virus) is one of the two parvoviruses that cause human diseases. As an important pathogen to humans, it causes infectious erythema in children, acute aplastic anemia, fetal edema and death. In this review, we focus on the recent advances in the molecular virology of B19V, such as viral genotypes, viral receptor, genomic features and viral replication, viral transcription and post-transcription regulation, viral nonstructural and structural protein features and functions, viral diagnosis and antiviral agents, to provide reference for further study of B19 pathogenesis mechanisms, treatment and diagnostic strategies.
Antiviral Agents ; DNA, Viral ; genetics ; Erythema Infectiosum ; diagnosis ; virology ; Genotype ; Humans ; Parvovirus B19, Human ; genetics ; Virology ; trends ; Virus Replication

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

OBJECTIVEHuman Parvovirus B19 (HPV B19) is a small (23 nm), non-enveloped DNA virus found in 1974. It has been proved that HPV B19 is associated with a variety of childhood diseases, such as erythema infectious, transient aplastic crisis, aplastic anemia, idiopathic thrombocytopenic purpura and arthropathy, etc. There have been no any effective vaccines to prevent HPV B19 infection so far. The HPV B19 genome is composed of 5.6 kb single strand DNA. This genome encodes a nonstructural protein NS1, two structural proteins VP1 and VP2. Most neutralizing linear epitopes of HPV B19 cluster in the VP1 unique and VP1-VP2 junction regions. Only proteins encoded by genes of the VP1 unique and VP1-VP2 junction regions can stimulate bodies to produce protective antibodies. Aim of the present study was to get the VP1 unique region gene of HPV B19 and to analyze the genetic diversity so as to further study its function and application.

METHODSThe VP1 unique region gene of HPV B19 was amplified from the serum of a child with idiopathic thrombocytopenic purpura by PCR. The purified PCR product was cloned into pGEM-T easy vector and transfected into the host strain E. coli (DH5 alpha). Positive clones were chosen and then the target gene was sequenced.

RESULTSThe target gene sequence of HPV B19 VP1 unique region was amplified and cloned successfully. It had 705 nucleotides. Compared with the relevant sequences published in Genbank, the sequencing results were revealed with two nucleotides changes in the HPV B19 VP1 unique region, but their coding amino acid were not changed.

CONCLUSIONIt is suggested that genetic diversity exists in the VP1 unique region of HPV B19. Construction of the recombinant plasmid of HPV B19 VP1 unique region gene might benefit to further study.

Capsid Proteins ; genetics ; Child ; DNA, Viral ; chemistry ; genetics ; Genetic Variation ; Humans ; Mutation ; Parvovirus B19, Human ; genetics ; Polymerase Chain Reaction ; Sequence Analysis, DNA

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: 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

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

Parvovirus B19 infection is known to cause chronic anemia in immunocompromised hosts, including organ transplant recipients. We report the first case of liver transplant recipient with parvovirus B19-induced pure red cell aplasia in Korea. A 57-yr-old female patient with hepatocellular carcinoma due to hepatitis C virus received a liver transplantation. Two months later, anemia developed and she received periodic red blood cell transfusions. However, chronic anemia persisted and bone marrow examination was performed 8 months after transplantation. Bone marrow aspiration smears showed markedly reduced erythroid precursors with atypical giant pronormoblasts and nuclear remnants with viral inclusions, and characteristic lantern cells were observed in biopsy sections. In addition, parvovirus B19 DNA PCR was positive. She was diagnosed as parvovirus B19-induced pure red cell aplasia and her anemia was improved following intravenous immunoglobulin therapy.
Blood Transfusion ; Bone Marrow/pathology ; Carcinoma, Hepatocellular/etiology/therapy ; DNA, Viral/analysis ; Female ; Hepatitis C/complications/diagnosis ; Humans ; Immunocompromised Host ; Immunoglobulins/therapeutic use ; Liver Neoplasms/etiology/therapy ; Liver Transplantation ; Middle Aged ; Parvoviridae Infections/complications/*diagnosis ; *Parvovirus B19, Human/genetics ; Red-Cell Aplasia, Pure/*diagnosis/therapy/virology