The first aim of this study was to characterize B19 DNA persisting in human synovial tissue (I) and skin (II).
Samples
Synovial tissue: Samples of synovial tissue were obtained during arthroscopy from 30 constitutionally healthy adults (age span 18-53 years, mean 21) with joint trauma or exertion (I).
Skin: Biopsies of skin were obtained from 34 subjects; 20 had inflammatory B19-nonrelated dermatological lesions (Group 1) and 14 were healthy members of hospital staff (Group 2). The range of birth year of these subjects was 1913-1991 (mean 1957).
Sera: Sera were obtained from all tissue donors for antibody testing. B19-IgG antibodies were measured (I) by a commercial EIA (Dako, Glostrup, Denmark) or (II) by in-house EIAs, and B19-IgM antibodies by the EIA of Biotrin (Dublin, Ireland). For further evidence of the time of B19 primary infection, the sera of subjects examined in depth for persisting B19 DNA were studied for epitope-type-specificity (ETS) of VP2-IgG (Soderlund et al., 1995b) as described recently (Kaikkonen et al., 1999).
DNA extraction
DNA was purified by proteinase K digestion (0.45% NP-40, 0.45% Tween, 2.5 mM MgCl2, 50 mM KCl, 1% gelatine in 10 mM Tris, pH 8.3) over night at 55°C followed by 10 min at 95°C to inactivate the enzyme (group 1 skin samples), or by proteinase K digestion followed by phenol/chloroform extraction and ethanol precipitation (synovial tissue samples and group 2 skin samples).
PCRs
All the synovial tissue and skin samples were first screened for B19 DNA with a nested PCR (VP1-PCR) amplifying a 1066 bp region in the middle of the genome. This method is described in Soderlund et al. (1997b). Of all positive samples, the persisting B19 DNA was further characterized with two additional nested PCRs, NS1- and VP2-PCR, amplifying the NS1 gene and the VP2 gene, respectively. All the three PCRs (primer sequences are shown in Table 2) and the corresponding hybridization probes were
designed according to the sequence of the Au isolate (Shade et al., 1986). The second round products of all three PCRs were detected by agarose gel electrophoresis and ethidium bromide staining. In addition, the products were transferred to a nylon membrane and hybridized with a digoxigenin-labelled probe specific for the corresponding genomic region. Optimal reaction conditions for each primer set were determined using the control DNA, extracted from the viremic serum NAN, as template.
Semi-quantification
To assess whether B19 DNA persists as an intact molecule or is fragmented, we examined, by semi-quantification, whether or not all three genomic regions could be detected in equal amounts. The sensitivities of the NS1 and VP2-PCR methods were equalized to the level of the VP1 PCR, which was shown to detect one target molecule / reaction (Soderlund et al., 1997b). The detection sensitivities of all three PCRs were within one logarithmic unit. For semi-quantification of the different B19 genomic regions, DNA suspensions were diluted serially in 10-fold steps, and each dilution was studied by the nested VP1-PCR. The last dilution giving a positive signal by ethidium bromide staining (in this thesis referred to as end point titer) was then used as a template in the NS1- and VP2-PCRs.
Duplex-PCR
To support our theory for the genomic intactness, of synovial DNA preparations diluted to end point, both the VP2 and NS protein-coding sequences were amplified simultaneously in one tube (I). The first PCR round utilized the outer primer pairs of the VP2 and NS reactions. The product was purified with High Pure PCR Product Purification Kit (Boehringer Mannheim, Mannheim, Germany) and transferred to the second reaction tube.
Primers for the nested reaction were NSifwd, NSirev, rt1 and VP2irev (table 2). The resulting amplicons were 439 and 639 nucleotides in size, respectively, and they were separated electrophoretically on a 1 % TAE-agarose gel and were Southern blotted.
Hybridization was done simultaneously with NS1 and VP2 probes.
Sequencing and phylogenetic analysis
Synovial isolates (I): In order to assess whether the synovial B19 is of a unique genotype or involves persistence-specific mutations that could be causally related to tissue tropism, the protein-coding region (constituting ~97 % of the whole B19 genome) was amplified and sequenced. These experiments were first conducted with synovial tissue of four individuals (I); two subjects represented acute-phase infection and two subjects represented long-term carriership. B19 DNA purified from these samples was amplified to five partly overlapping amplicons of ~1000 bp, which together covered the whole protein
coding region of the genome (I). Sequencing was done at the Institute of Biotechnology, University of Helsinki. The sequences of B19 DNA isolated from synovia recently or remotely after infection were compared with each other and with blood-derived B19 sequences from GenBank. Phylogenetic analyses were performed of the four synovial tissue-derived B19 sequences together with others from GenBank by using a 346-nt region corresponding to viral nt. 2246 through 2789. Sequence alignments were done by using CLUSTAL W version 1.75, and subsequent analyses were done by using the PHYLIP package (Thompson et al., 1994).
Dermal isolates (II): While determining the molecular characteristics of the skin-derived B19 DNA (II), we observed that most of the amplicons from the NS1- and VP2-PCRs did not hybridize or hybridized only weakly. These amplicons were therefore (partly) sequenced from several samples, either directly or after cloning (TA-cloning, Novagen Inc., Darmstadt, Germany). In addition to the three B19 primer sets (VP1, NS1 and VP2 PCRs), others (Table 2), prepared according to the blood-derived reference sequence Au or the current dermal B19 DNA, were used for amplification and sequencing to gain a near full-length sequence from one dermal isolate, LaLi. This was from a subject representing long-term carriership according to serological data. DNA sequences were obtained with ABI PRISM (Perkin-Elmer, Foster City, CA) at the sequencing core facility of the Haartman Institute, University of Helsinki, Finland. Again, sequences of the new variant, LaLi, were compared with the prototypic dermal and synovial sequences, as well with others found in GenBank. A phylogenetic analysis was performed by using the full-length protein-coding sequence of the dermal isolate LaLi together with 13 B19 sequences from GenBank (including 4 B19 DNA isolates persisting in synovial membranes and a Simian parvovirus sequence).
Table 2. Sequence data for primers used in this study.
PCR test Primer Sequence
VP1 PCR p6 GGAGAATCATTTGTCGGAAG
p3 CTTCTGCAGAATTAACTGAAGTC p8 TGTGCTTACCTGTCTGGATTG p5 AGG CTT GTG TAA GTC TTC AC p1 ATGAGTAAAGAAAGTGGCAAATGG
NS1 PCR NSofwd ATGGAGCTATTTAGAGGGGTG NSorev TTTGCAATCCAGACAGGTAAGC NSifwd ACTGGTTGTGTGAAAACAGAGTG NSirev TTTTCCTGCTACATCATTAAATGG NSpfwd AGAGGATAAGTGGAAACTAGTTG NSprev TACTGGAACACTTTTAGCAATGG
VP2 PCR VP2ofwd TGACTTCAGTTAATTCTGCAGAAG VP2orev TGG GTG CAC ACG GCT TTT GG VP2ifwd CTAGAATATCCTTACGCCCTGG VP2irev GTGGCTGATGCAAACCCCATC VP2pfwd CCATTTCTCATGGTCAGACCAC VP2prev CCATACAGAACCCACCATTAGG
Sequencing rt1 AATTTAGAGGGCTGCAGTCAAC NSs fwd GCAATGGCCATTGCTAAAAGTG
p9 CTTTAGGTATAGCCAACTGG
rts rev ATGTGTCAGGAACCCCTAAGC
K71 PCR ofwd TTTACTGAAGACAAATGGAAGT orev CACTGGGACAGTTTTGGCAATA ifwd AGTGGATTTCAATCAATATACA irev TCATAATTTTGGCATAATAATAG
VP1/2 cloning KK1 CTCAGAAAACGGGATCCCGATGACTTCAGTTAATTCTG KK2 CGGGATCCCGTTACAATGGGTGCACACGGCT
KK3 GTGAAGATCTTCATGAGTAAAGAAAGTGGTAAATG KK4 GGAATTCCTTACAATGGGTGCACACGGCTTT
Promoter cloning NheIfwd TTTGCTAGCTAGTTGCACGTCAACCCC B19RNArev TGCATAAGGCGGGAAAAAGCCC Prom5fwd TGTCCGCCATCTTGAACCG
Xhorev TTTCTCGAGGTTAGTAAGTTAAAAGTTAGTATAAATACCTGTTAG XhoD91.1rev TTTCTCGAGGTTAGTAAGTTAAAAGTTAGTATAAATACCTGTTAG