Serodiagnosis of Primary Infections with Human Parvovirus 4, Finland

To determine the prevalence of parvovirus 4 infection and its clinical and sociodemographic correlations in Finland, we used virus-like particle–based serodiagnostic procedures (immunoglobulin [Ig] G, IgM, and IgG avidity) and PCR. We found 2 persons with parvovirus 4 primary infection who had mild or asymptomatic clinical features among hepatitis C virus–infected injection drug users.

Protein expression and virus-like particle purifi cation were conducted as for human bocavirus (HBoV) (9). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis identifi ed a 73-kDa protein (Figure 1, panel A), which was immunoreactive by Western blotting (10) with 5 known PARV4 IgG-positive serum samples (6) but not with negative serum samples ( Figure 1, panel B). Electron microscopy showed spherical particles ≈25 nm in diameter (Figure 1, panel C) that resembled those seen in vivo (11). The capsid protein region is 109 aa longer (N terminally) than that reported by Sharp et al. (6); both constructs assembled into capsids.
PARV4 IgG avidity was determined in all persistently (>1 year) IgG-positive persons in group 2 (n = 29). Twenty-eight persons showed high IgG avidity, and 1 showed borderline IgG avidity. All 4 IgG-positive persons had high-avidity IgG, which indicated previous immunity.
In group 3, a second sample from patient A, who showed seroconversion for IgG showed borderline IgG avidity. Patient B showed low IgG avidity in both samples (Table 1). Groups 2 and 3 were also analyzed for PARV4 DNA by qualitative PCR (13) as modifi ed (94°C for 10 min; 45 cycles at 94°C for 20s, 51°C or 56°C for 20s, and 72°C for 20s; and extension at 72°C for 7 min). Amplicons were subjected to electrophoresis and sequenced. In group 2, all 151 serum samples were PCR negative. In group 3, two patients (A and B) were PCR positive (Table 1).
PARV4 IgG-positive and IgG-negative IDUs (group 2) were compared for demographic and clinical characteristics. PARV4 IgG-positive persons reported more in-jection of drugs, persistent (>10 y) injection, and lending of injection equipment ( Table 2). They also had a more frequent history of imprisonment and unemployment and were less educated. No differences were seen between PARV4 IgG-positive and IgG-negative persons with any symptoms (fever, tiredness, nocturnal sweating, cough, diarrhea, shortness of breath, swallowing complaints, muscle weakness, dizziness, skin abscesses or herpetic lesions, loss of eyesight, or headache) during 6 months before being interviewed.

Conclusions
We developed IgG-, IgM-, and IgG-avidity-based PARV4 serodiagnostic procedures; studied high-prevalence cohorts by PCR; and analyzed HIV-infected IDUs for demographic and clinical correlations with PARV4  IgG positivity. Among healthy university students, none had PARV4 IgG, which is consistent with low baseline IgG prevalences of 0% and 2.8% for another EIA (6). The PARV4 IgG seroprevalence of 78% among HIV-infected IDUs represents a high incidence of PARV4, which refl ects the lengthy history of drug use among socially marginalized IDUs during an HIV outbreak in Finland (7). Two HCV-infected patients had PARV4 primary infections, as shown by increasing IgG levels, detectable IgM, low or borderline IgG avidity, and viral DNA in serum. These 4 fi ndings are presented as diagnostic criteria for PARV4 primary infection. As estimated by known kinetics of B19 virus diagnostics (14), these 2 PARV4 infections probably occurred in 2005. During that time, neither patient had contacted local healthcare providers. Conversely, these 2 patients used intravenous drugs daily, and might not have sought medical care unless they were severely ill.
Because PARV4 IgG seroprevalence in group 1 was 0% in this study, in contrast to prevalences of 60% for B19 (12) and 96% for HBoV (9) in the same students, serologic cross-reactivity between PARV4 and the other human parvoviruses appears highly unlikely. Amino acid sequence similarity is <30% between B19 and PARV4 and ≈40% between HBoV and PARV4.
PCR-negative results for group 2, including 4 patients who were IgM positive, are evidence against viremic primary, chronic, and recurrent PARV4 infections. However, because of the relatively low sensitivity of this PCR, the data do not rule out low levels of viral DNA in blood.
Analysis of HIV-infected IDUs supports the view that in northern Europe PARV4 is primarily a blood-borne virus. No differences were seen for factors related to sexual activity. However, our sample size was too small to make this conclusion. In a recent PCR study, PARV4 genotype