Delwart, E.

Hedman, K.

Hedman, L.

Kantele, A.

Kivelä, P.

Kumar, A.

Lahtinen, A.

Lappalainen, M.

Liitsola, K.

Ristola, M.

Sharp, C.

Simmonds, P.

Söderlund-Venermo, M.

English

Edinburgh Research Explorer

     Edinburgh Research Explorer                                      Serodiagnosis of primary infections with human parvovirus 4,FinlandCitation for published version:Lahtinen, A, Kivelä, P, Hedman, L, Kumar, A, Kantele, A, Lappalainen, M, Liitsola, K, Ristola, M, Delwart, E,Sharp, C, Simmonds, P, Söderlund-Venermo, M & Hedman, K 2011, 'Serodiagnosis of primary infectionswith human parvovirus 4, Finland' Emerging Infectious Diseases, ???volume??? 17, ???journalNumber???1, ???pages??? 79-82.Link:Link to publication record in Edinburgh Research ExplorerDocument Version:Publisher final version (usually the publisher pdf)Published In:Emerging Infectious DiseasesGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 20. Feb. 2015Serodiagnosis of Primary Infections with Human Parvovirus 4, Finland Anne Lahtinen, Pia Kivelä, Lea Hedman, Arun Kumar, Anu Kantele, Maija Lappalainen, Kirsi Liitsola, Matti Ristola, Eric Delwart, Colin Sharp, Peter Simmonds, Maria Söderlund-Venermo, and Klaus HedmanTo determine the prevalence of parvovirus 4 infection and its clinical and sociodemographic correlations in Fin-land, we used virus-like particle–based serodiagnostic pro-cedures (immunoglobulin [Ig] G, IgM, and IgG avidity) and PCR. We found 2 persons with parvovirus 4 primary infec-tion who had mild or asymptomatic clinical features among hepatitis C virus–infected injection drug users.A new member of family Parvoviridae, human par-vovirus 4 (PARV4), was identifi ed in plasma of an injection drug user (IDU) with unexplained fatigue, head-aches, fever, night sweats, nausea, and diarrhea (1). In PCR studies of blood and postmortem tissues, virus was detected mainly in persons with histories of injection drug use (2–5). A recent PARV4 immunoglobulin (Ig) G study also showed higher prevalence of antibodies to PARV4 in IDUs and HIV-positive persons who had hemophilia than in HIV-positive men who have sex with men (6). The clini-cal role of this virus is unknown.We report virus-like particle–based comprehensive serodiagnosis for PARV4 and determine its occurrence in Finland in 3 diverse population groups. In the highest prev-alence group, we comparatively analyzed PARV4 IgG–positive and IgG–negative persons for sociodemographic and behavioral background factors and symptoms by using an HIV risk factor database (7).The StudyGroup 1 (low risk) comprised 115 university students (1 serum sample/student). Group 2 (high risk) comprised 78 HIV IgG–positive IDUs from the Helsinki Cohort Study (8) (151 plasma samples, 1–7 samples/person). Group 3 (high risk) comprised 200 hepatitis C virus (HCV) IgG–positive patients (1 sample/person). Informed consent was obtained from persons in groups 1 and 2 and from 2 patients with primary infections in group 3. The study was reviewed and approved by the Helsinki University Central Hospital Ethics Committee (#281/13/03/01/09 and #469/2001).Four genomic regions of open reading frame 2 of PARV4 genotype 1 (AY622943) were cloned for bac-ulovirus expression by using PARV4 PCR–positive plasma (1) as initial template. The clone with nt region 3137–5122 (AY622943) and infectivity was construct-ed with primers PARV4EcoRI_3137: 5′-TATGAATTCATGATTGAGCATGGGG-3′ and PARV4EagI_5122: 5′-TACGGCCGTTACAGCAAATGAGAATAA-3′.Protein expression and virus-like particle purifi cation were conducted as for human bocavirus (HBoV) (9). So-dium 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 nega-tive serum samples (Figure 1, panel B). Electron micros-copy showed spherical particles ≈25 nm in diameter (Fig-ure 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 enzyme immunoassay (EIA) was con-ducted as for HBoV (9). Specifi c results were obtained by subtracting antigen-free background levels. For IgM EIA, a μ-capture format was used (9). IgG and IgM cutoff values, obtained from group 1 absorbances (mean + 4 × SD), were 0.141 and 0.205, respectively. IgG-avidity EIA was con-ducted as for HBoV (method A) (12); cutoff values for high and low avidity were 25% and 15%, respectively.None of the 115 students (group 1) were PARV4 IgG positive, and 1 (0.9%) of 115 was weakly IgM positive (Figure 2, panel A). Sixty-one (78.2%) of 78 HIV-infected patients (group 2) were IgG positive, and 4 (5.1%) of 78 were IgM positive (Figure 2, panel B). Sixty-nine (34.5%) of 200 HCV-infected patients (group 3) were IgG positive, and 3 (1.5%) of 200 were IgM positive (Figure 2, panel C). Previous samples were available for 2 of the IgM-positive patients (A and B) in group 3. These samples showed sero-conversion for IgG and an increase in IgG (Table 1).PARV4 IgG avidity was determined in all persistently (>1 year) IgG-positive persons in group 2 (n = 29). Twen-ty-eight persons showed high IgG avidity, and 1 showed  Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 1, January 2011 79 Author affi liations: University of Helsinki, Helsinki, Finland (A. Lahtinen, L. Hedman, A. Kumar, A. Kantele, M. Söderlund-Ven-ermo, K. Hedman); Helsinki University Central Hospital, Helsinki (P. Kivelä, L. Hedman, A. Kantele, M. Lappalainen, M. Ristola, K. Hedman); National Institute for Health and Welfare, Helsinki (K. Liitsola); Blood System Research Institute, San Francisco, Califor-nia, USA (E. Delwart); University of California, San Francisco (E. Delwart); and University of Edinburgh, Summerhall, UK (C. Sharp, P. Simmonds)DOI: 10.3201/eid1701.100750borderline 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 charac-teristics. 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, di-arrhea, shortness of breath, swallowing complaints, muscle weakness, dizziness, skin abscesses or herpetic lesions, loss of eyesight, or headache) during 6 months before be-ing interviewed.ConclusionsWe developed IgG-, IgM-, and IgG-avidity–based PARV4 serodiagnostic procedures; studied high-preva-lence cohorts by PCR; and analyzed HIV-infected IDUs for demographic and clinical correlations with PARV4 DISPATCHES80 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 1, January 2011Figure 1. Parvovirus 4 (PARV4) virus-like particle (VLP) expression and immunoreactivity, Finland. A) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of PARV4-like particles in Spodoptera frugiperda armyworm (Sf)9 cells (lane 1), purifi ed VLPs (lane 2), and uninfected Sf9 cells (lane 3). B) Western blotting with PARV4 immunoglobulin (Ig) G–positive serum (lanes 1, 3, and 4) or PARV4 IgG–negative serum (lane 2). Lanes 1 and 2, purifi ed VLPs as antigen; lane 3, Sf9 cells expressing VLPs; lane 4, Sf9 cells expressing glutathione-S-transferase control antigen; lanes M, molecular mass marker. Arrows in panels A and B indicate the PARV4 capsid protein. C) Electron micrograph of purifi ed VLPs. Scale bar = 200 nm.Figure 2. Parvovirus 4 (PARV4) enzyme immunoassay (EIA) results, Finland. Red dots, immunoglobulin (Ig) M; ×, IgG. Upper dashed line indicates IgM cutoff value (0.205), and lower dashed line indicates IgG cutoff value (0.141). A) Group 1: 115 university students (1 serum sample/person); none positive for PARV4 IgG, and 1 positive for PARV4 IgM. B) Group 2: 78 HIV-infected injection drug users (151 serum samples [1–7 samples/person]). Prevalences of PARV4 IgG and IgM were 78.2% (61/78) and 5.1% (4/78), respectively. C) Group 3: 200 hepatitis C virus–infected patients (1 sample/person). Prevalences of PARV4 IgG and IgM were 34.5% (69/200) and 1.5% (3/200), respectively.Primary Infections with PARV4, FinlandIgG 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 marginal-ized IDUs during an HIV outbreak in Finland (7).Two HCV-infected patients had PARV4 primary infec-tions, 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 pa-tient 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 par-voviruses 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 pri-mary, 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 vi-rus. 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  Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 1, January 2011 81 Table 1. Virologic findings for PARV4 primary infections in 2 patients, Finland* Patient Date of sampling IgG EIA absorbance IgM EIA absorbance PCR IgG avidity, % A 2004 Sep 8 0.016 0.051 – ND 2006 Mar 14 2.873 0.218 + 17.7B 2006 Mar 13 0.950 1.461 + 8.92006 Apr 6 1.946 0.661 + 10.1*PARV4, parvovirus 4; Ig, immunoglobulin; EIA, enzyme inmmunoassay; –, negative; ND, not determined; +, positive. Table 2. Characteristics of PARV4 IgG–positive and IgG–negative HIV-infected injection drug users, Finland* Characteristic IgG positive, n = 61 IgG negative, n = 17 p value† Age, y, median (range) 35 (17–61) 31 (21–55) 0.069 Male sex 44/61 (72) 12/17 (71) 1.000 Main drug was amphetamine‡ 38/61 (62) 10/17 (59) 0.786 Duration of injection, y, median (range) 10 (0–36) 7 (0–30) 0.259 Duration of injection >10 y 45/59 (76) 7/17 (44) 0.029 History of imprisonment 49/61 (80) 9/17 (53) 0.031 Education <9 y 52/59 (88) 10/17 (59) 0.011 HCV antibody positive 59/60 (98) 14/15 (93) 0.362 HBsAg positive 3/61 (5) 3/17 (18) 0.114 HBc IgG positive 46/60 (77) 9/17 (56) 0.124 Used antiretroviral therapy§ 29/61 (48) 4/17 (24) 0.099 CD4 cell count/μL, median§ 303 323 0.168 Present situation  Unstable living conditions (no permanent address) 35/59 (60) 5/15 (33) 0.088  Employed‡ 0/53 (0) 3/16 (19) 0.009 Risk behavior  Loaned needles or syringes 49/59 (83) 9/16 (56) 0.040  Borrowed needles or syringes 56/58 (97) 15/16 (94) 0.524  Had sexually transmitted diseases 37/57 (65) 7/17 (41) 0.097  Had commercial sex 18/61 (30) 8/15 (53) 0.127 Risk behavior past 6 mo  Used drugs 48/61 (79) 8/17 (47) 0.016  Used injection drugs 46/61 (75) 8/17 (47) 0.037  Used condoms inconsistently 25/61 (41) 7/16 (44) 1.000  Had >2 sex partners 16/61 (26) 7/17 (41) 0.244 *Values are no. positive/no. tested (%) unless otherwise indicated. PARV4, parvovirus 4; Ig, immunoglobulin; HCV, hepatitis C virus; HBsAg, hepatitis B surface antigen; HBc, hepatitis B core antigen; †By Fisher exact test or Mann-Whitney U test. Values in boldface are significant. ‡At the time of interview. §Closest to the sampling for PARV4 tests. 3 was commonly found among infants in West Africa, and there was no evidence of parenteral exposure (15).Using comprehensive serodiagnosis, we showed that PARV4 is ubiquitously present in IDUs in Finland and de-tected primary infections in 2 patients who had a full spec-trum of diagnostic fi ndings. Neither of these had sought medical help, which suggested that their primary infections may have been clinically mild or asymptomatic.This study was supported by the Helsinki University Central Hospital Research and Education, and Research and Develop-ment funds, the Sigrid Jusélius Foundation, the Medical Society of Finland, and the Academy of Finland (project 1122539).Ms Lahtinen is a scientist at the Haartman Institute, Depart-ment of Virology of the University of Helsinki. Her research in-terests include clinical characteristics of parvoviruses.References  1.  Jones MS, Kapoor A, Lukashov VV, Simmonds P, Hecht F, Delwart E. New DNA viruses identifi ed in patients with acute viral infection syndrome. J Virol. 2005;79:8230–6. DOI: 10.1128/JVI.79.13.8230-8236.2005  2.  Simmonds P, Manning A, Kenneil R, Carnie FW, Bell JE. Parenteral transmission of the novel human parvovirus PARV4. Emerg Infect Dis. 2007;13:1386–8.  3.  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DOI: 10.3201/eid1607.100025Address for correspondence: Klaus Hedman, Department of Virology, Haartman Institute, University of Finland, Box 21, Haartmaninkatu 3, Helsinki FIN-00014, Finland; email: klaus.hedman@helsinki.fi DISPATCHES82 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 1, January 2011

Clinical assessment and improved diagnosis of bocavirus-induced wheezing in children, Finland. Emerg Infect Dis.

Comparison of tissue distribution, persistence, and molecular epidemiology of parvovirus B19 and novel human parvoviruses PARV4 and human bocavirus.

Dating of human bocavirus infection with protein-denaturing IgG-avidity assays-secondary immune activations are ubiquitous in immunocompetent adults.

Frequent detection of the parvoviruses, PARV4 and PARV5, in plasma from blood donors and symptomatic individuals.

High frequencies of exposure to the novel human parvovirus PARV4 in hemophiliacs and injection drug users, as detected by a serological assay for PARV4 antibodies. J Infect Dis.

High prevalence of unprotected sex among Finnish HIV-positive and HIV-negative injecting drug users. Scand J Public Health.

HIV outbreak among injecting drug users in the Helsinki region: social and geographical pockets.

Human parvovirus B19 infection during pregnancy–value of modern molecular and serological diagnostics.

New DNA viruses identiﬁ ed in patients with acute viral infection syndrome.

Novel human parvovirus 4 genotype 3 in infants,

Parenteral transmission of the novel human parvovirus PARV4. Emerg Infect Dis.

Parvovirus 4 (PARV4) in serum of intravenous drug users and blood donors. Infection.

Parvovirus PARV4 visualization and detection.

Serodiagnosis of human bocavirus infection. Clin Infect Dis.

Serodiagnosis of primary infections with human parvovirus 4, Finland

Serodiagnosis of primary infections with human parvovirus 4, Finland

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