Detection of human parvovirus 4 viremia in the follow-up blood samples from seropositive individuals suggests the existence of persistent viral replication or reactivation of latent viral infection

The serological and PCR results from the longitudinal study are listed in Table 1. Notably, the immunoblotting results for the 10 study participants in this study did not change over time. Anti-PARV4 IgM persisted without weakening in all three of the IgM-positive participants and IgG reactivity also remained constant (participant 9 and 10 are shown in Figs. 1 and 2, respectively). The IgG reactivity of participant 9 was so weak that it could hardly be detected after scanning (Fig. 2). One blood sample collected from participant 9 at 32 months before the start of the study also had the same immunoblotting results (strip not shown). We cannot explain the persistence of IgM reactivity with weak IgG reactivity noted in participants 9 and 10 (both were PARV4 DNA-positive) but this pattern is not uncommon in our experience and these subjects were likely to be diagnosed as having current PARV4 infection without knowledge of their preceding immunoblotting results. Interestingly, the PARV4 DNA-positive participant (number 7) who was negative for anti-PARV4 IgM also had weak IgG reactivity (Fig. 2). PARV4 DNA was detected only in the first sample (one participant) or in the second sample (two participants). The number of positive PCR replicates was one (two samples) or 2 (one sample). The three nucleotide sequences of the PCR products are identical to the corresponding region of the GenBank sequence GU120197.1.

Both B19V and PARV4 viruses have two capsid proteins, VP1 and VP2 [18], and antibodies against linear B19V VP2 epitopes have been shown to disappear 6 months after acute B19V infection [19]. In the present study, the three PARV4-SUMO fusion proteins SUMOVP1, SUMOVP2 and SUMOVP3 represent the PARV4 VP1-unique sequence, the PARV4 VP1-VP2 overlapping region, and the carboxyl terminal region of PARV4 VP2, respectively. In contrast to B19V, the antibodies against the linear epitopes of PARV4 VP2 did not disappear over time (Figs. 1 and 2).

To date, studies on the anti-PARV4 IgM response have yielded inconsistent results. A monthly follow-up study of intravenous drug users found that five drug users had seroconversion to PARV4, but four of them were positive for anti-PARV4 IgM only in the blood sample collected at seroconversion; the anti-PARV4 IgM became undetectable in the blood sample collected after a month [10]. For the one exception, two sequential blood samples tested positive for anti-PARV4 IgM, and the duration of the IgM response was 32–93 days. All the IgM-positive blood samples collected at seroconversion had detectable PARV4 DNA, but viremia disappeared in the second IgM-positive blood sample collected after one month and the duration of viremia was estimated to have lasted between 32 and 98 days. This result suggested that anti-PARV4 IgM and PARV4 viremia were both short-lived and the existence of PARV4 DNA was expected in most of the IgM-positive blood samples. In contrast, a serological study conducted in Finland found that four (5.1 %) HIV-infected intravenous drug users tested positive for anti-PARV4 IgM without having PARV4 viremia [5]. Furthermore, the presence of high-avidity anti-PARV4 IgG indicated that the four drug users had previous immunity rather than primary infection. Additionally, the anti-PARV4 IgM-positive rate was 7 % in a Lithuanian study on patients with acute respiratory disease in contrast to 0.9 % in a Finnish study on university students [5, 6]. These IgM-positive Lithuanian patients were grouped into current (IgM+, IgG–) ongoing, or recent PARV4 infection groups (IgM+, IgG+); however, PARV4 DNA was not detectable in any of the IgM-positive blood samples. In our experience, IgM antibodies against PARV4 can remain detectable for 12 months or longer, and the high IgM-positive rates are explainable by a prolonged IgM response, but PARV4 DNA might not have been detected in IgM-positive blood samples without conducting PCR replicates.

In the present study, the possibility of low-level viral infections in some subjects is suggested by the infrequent positive results of the PCR replicates, but false positive results seem unlikely because not a single negative control was positive (a total of 12 negative control reactions were performed in this study). Furthermore, the PARV4 DNA-positive blood samples in our PARV4 studies to date have always shown PARV4 antibody positivity with only one exception; this uneven distribution conflicts with the false positive results. A low viral load in the presence of anti-PARV4 IgG without IgM was noted in one participant (number 7 in the table), this was considered to result from persistent viral replication in a B19V study of blood donors [14]. Our study does not demonstrate persistent viral replication directly because PARV4 viremia was not found at more than one time point. Nevertheless, the detection of PARV4 DNA in the second of the two blood samples from two participants who were positive for anti-PARV4 IgM and IgG strongly suggests the existence of persistent PARV4 replication for the following reasons. (1) Primary PARV4 infection can be excluded because of the pre-existing anti-PARV4 IgM and IgG, which suggests the possibility of persistent PARV4 replication, reactivation of latent PARV4 infection or re-infection. (2) Re-infection can boost the pre-existing IgG antibody response, but this was not observed in the two study participants. (3) The low (1/35) seropositive rate in siblings of PARV4-infected hemophiliac patients [4] and the low PARV4 seroprevalence in United Kingdom blood donors [7] suggest that PARV4 is not easily transmitted by person-to-person contact through non-parenteral routes hence the chance of re-infection is low. (4) It is possible that persistent low-level viral replication below the detection limit existed in the first blood samples, but reappearance of viremia due to reactivation of inactive virus in the tissues is also possible.