Characterization of Parvovirus B19 genotype 2 in KU812Ep6 cells

Abstract

An infectious parvovirus B19 (B19V) genotype 2 variant was identified as a high-titer contaminant in a human plasma donation. Genome analysis revealed a 138-bp insertion within the p6 promoter. The inserted sequence was represented by an additional 30 bp from the end of the inverted terminal repeat adjacent to a 108-bp element found also, in inverted orientation, at the extreme right end of the unique sequence of the genome. However, despite the profound variations in the promoter region, the pattern of gene expression and DNA replication did not differ between genotype 1 and genotype 2 in permissive erythroid KU812Ep6 cells. Capsid proteins of both genotypes differ in their amino acid sequences. However, equivalent kinetics of virus inactivation at 56 degrees C or pH 4 indicated a comparable physicochemical stability of virus capsids. Sera from six individuals infected by B19V genotype 1 were investigated on cross-neutralization of B19V genotype 2 in vitro. Similar neutralization of both B19V genotypes was observed in sera from three individuals, while the sera from three other individuals showed weaker cross-neutralization for genotype 2. In conclusion, the in vitro replication characteristics and physical stability of B19V capsids are very similar between human parvovirus B19 genotypes 1 and 2, and cross-neutralization indicates a close antigenic relation of genotypes 1 and 2.

FIG. 1.

Characterization of B19V from various plasma samples. DNA obtained from five B19V-containing plasma samples was digested with XmnI and subjected to Southern analysis using a mixture of digoxigenin (DIG)-labeled DNA from a plasmid containing the B19V genotype 1 genome and DIG-labeled IM-81 viral DNA. (A) DNA sizes are given in kilobase pairs (kb). Samples IM-71, IM-72, IM-73, and IM-80 contained genotype 1 B19V, while sample IM-81 contained the variant genotype 2. (B) The theoretical pattern and length of the restriction fragments is sketched. P6 indicates the location of the p6 promoter. Additionally, genome concentrations were determined by quantitative PCR (1), and the infectivity was quantified by titration on Ku812Ep6 cells (C).

FIG. 2.

Sequence alignment in the p6 promoter region (nucleotides 103 to 368 from the B19V Au sequence; GenBank accession no. M13178). Major potential transcription factor binding sites, as described previously, are indicated by boxes (13, 29). Compared to the genotype 1 Au sequence, the genotype 2 LaLi sequence (GenBank accession no. AY044266), the genotype 2 A6 sequence (GenBank accession no. AY064475), and the genotype 3 V9 sequence (GenBank accession no. AX003421) are given. In the IM-81 sequence (GenBank accession no. AY903437), there is a 108-bp sequence insertion flanked by two 30-bp direct repeats which are indicated by arrows. The vertical arrow shows the position of the nick site as established for the opposite strand of the B19 Au genome. The end of the ITR is indicated by a vertical dotted arrow.

FIG. 3.

Kinetics of NS-1 mRNA (A) and VP mRNAs (B) in KU812Ep6 cells. KU812Ep6 cells were infected with B19V genotype 1 (strain S-9) or genotype 2 (strain IM-81) at a multiplicity of infection of 1,000 genomes per cell. Samples of approximately 2 × 10⁶ cells were taken at various time points after infection, and mRNA extraction and quantification by TaqMan real-time PCR was performed as described in Materials and Methods. Amounts of mRNA are expressed in relative units.

FIG. 4.

Kinetics of VP2 capsid protein expression. KU812Ep6 cells were infected with B19V genotype 1 (strain S-9) or genotype 2 (strain IM-81) at a multiplicity of infection of 1,000 genomes per cell. Samples were taken at various time points after infection, and protein lysates were subjected to SDS-gel electrophoresis in 8% Tris-glycine polyacrylamide gels. Proteins were transferred onto nitrocellulose membranes by Western blotting, and B19V capsid proteins were detected using a mouse monoclonal antibody (Chemicon International, Temecula, Calif.).

FIG. 5.

Kinetics of DNA replication. KU818Ep6 cells were infected with B19V genotype 1 (strain S-9) or genotype 2 (strain IM-81) at a multiplicity of infection of 1,000 genomes per cell. Samples were taken at various time points after infection, and DNA was prepared in agarose plugs as described in Materials and Methods. Alternatively, DNA was also prepared by conventional extraction (QIAmp Blood kit; QIAGEN, Hilden, Germany), and B19V genome copy numbers were determined by TaqMan PCR. Agarose-embedded DNA was separated by 0.7% agarose gel electrophoresis and blotted onto a nylon membrane. B19V DNA was detected using DIG-labeled B19V plasmid DNA.

FIG. 6.

Inactivation of B19V genotypes at high temperature (A) or low pH (B). Human albumin heated to 56°C (A) or at pH 4 (B) was spiked 1:11 with B19V genotype 1 (strain S-1) or genotype 2 (strain IM-81). During incubation at 56°C or at low pH, samples were withdrawn and titrated immediately for infectious B19V concentration by quantification of the mRNA-inducing virus dose (indicated as mRNA₅₀ per ml). (C) The difference in the amino acid sequence of the viral capsid proteins of genotype 1 (strain S-1) and genotype 2 (strain IM-81) is shown. Additionally, the location of variant amino acid positions (indicated as dots) with respect to the VP1 and VP2 open reading frames (indicated as bars) is sketched.

FIG. 7.

Neutralization of genotype 1 and genotype 2 by sera from individuals after infection with genotype 1. Neutralization reactions were set up by mixing 50 μl of dilutions from genotype 1 strain S-1 or genotype 2 strain IM-81 at a concentration of 9.6 log₁₀ genomes per ml with 50 μl serum from a twofold dilution series. The mixture was incubated for 2 h at 37°C for neutralization. Thereafter, the samples were inoculated onto KU812Ep6 cells for titration of infectious virus by quantitative determination of induced viral mRNA. (A to F) Results from sera from six individuals who had been infected with B19V genotype 1 virus 6 to 18 months prior to sampling. (A) Serum at 11 months postinfection (p.i.); (B) serum B at 6 months p.i.; (C) serum C at 16 months p.i.; (D) serum D at 10 months p.i.; (E) serum E at 9 months p.i.; (F) serum F at 18 months p.i. B19V-specific antibody titer was determined by a calibrated commercial B19V ELISA containing VP2 antigen. Antibody titers are expressed in infectious units per milliliter as defined by international standard NIBSC 93/724.