Expression and Serological Characterization of Polyomavirus WUPyV and KIPyV Structural Proteins Kalle Kantola, 1 Mohammadreza Sadeghi, 1 Moritz J. Ewald, 6 Benedikt Weissbrich, 6 Tobias Allander, 3,4 Cecilia Lindau, 3,4 Kalle Andreasson, 7 Anne Lahtinen, 1 Arun Kumar, 1 Pa ¨ ivi Norja, 1 Tuomas Jartti, 5 Pasi Lehtinen, 5 Eeva Auvinen, 1,2 Olli Ruuskanen, 5 Maria So ¨ derlund-Venermo, 1 and Klaus Hedman 1,2 Abstract The polyomaviruses WUPyV and KIPyV were recently discovered. We expressed their structural proteins VP1, VP2, and VP3, and the corresponding proteins of BKV and JCV, for immunoblotting of IgG antibodies from 115 wheezing young children and 25 asymptomatic adults. Furthermore, nasopharyngeal aspirates (NPA) and sera from the children were examined by PCR for viral DNA. The overlapping minor proteins VP2 and VP3 of WUPyV and KIPyV were more reactive in immunoblots than the major protein VP1; of 100 NPA PCR-negative wheezing children aged 4 y, 31 (31%) and 31 (31%) were positive for WUPyV and KIPyV VP2/VP3, compared to only 3 (3%) and 5 (5%) for VP1, respectively. For comparison, the respective WUPyV and KIPyV IgG seroprevalences as determined by immunofluorescence assay (IFA) with nondenatured VP1 were 80% and 54%, respectively, among 50 NPA PCR-negative children aged 2 y. This difference shows the importance of conformational VP1 antige- nicity. Of the 25 adults, 52% and 68% were IgG-positive in immunoblots for VP2/VP3 of WUPyV and KIPyV, and 8% and 12% were for VP1, respectively. Of the 192 NPA samples studied by PCR, 7 (3.6%) were positive for WUPyV, and 3 (1.5%) were positive for KIPyV DNA. Unlike the NPA samples, none of the corresponding 443 sera contained WUPyV or KIPyV DNA. Together with the high VP2/VP3 IgG prevalence, this points to a paucity or brevity of KIPyV and WUPyV viremias among immunocompetent children. Our results indicate the significance of protein conformation in immunoreactivity of VP1, and show the antigenic importance of the WUPyV and KIPyV minor proteins VP2 and VP3. The high and rapidly increasing IgG prevalence rates observed in this study for WUPyV and KIPyV support the notion that these novel polyomaviruses are widespread and are acquired early in childhood. Introduction H uman polyomaviruses BKV and JCV are globally widespread, with adult seroprevalences of 80–90% and 35–60%, respectively (10,16,18). Primary infection by BKV typically occurs in early childhood, whereas that by JCV oc- curs a few years later (27). The primary encounters are usually asymptomatic; however, under immunosuppression both of these viruses may be reactivated and cause disease. BKV causes nephropathy in renal transplant recipients (13), whereas JCV gives rise to progressive multifocal leukoence- phalopathy (17). Two new members of the Polyomaviridae family, provi- sionally named KI polyomavirus (KIPyV) and WU poly- omavirus (WUPyV), were recently discovered in Sweden and in the U.S. by screening of human respiratory samples with large-scale sequencing and bioinformatics (3,11). In genome organization KIPyV and WUPyV are typical poly- omaviruses, yet in amino acid sequence their structural proteins are only 15–29% identical to those of BKV and JCV. The ‘‘late’’ regions of KIPyV and WUPyV genomes show 64–71% amino acid sequence identity. KIPyV and WUPyV, like other polyomaviruses, encode three structural proteins, VP1, VP2, and VP3, and the early 1 Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland. 2 Helsinki University Central Hospital Laboratory Division, Helsinki, Finland. 3 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden. 4 Department of Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden. 5 Department of Pediatrics, Turku University Hospital, Turku, Finland. 6 Institute of Virology and Immunobiology, University of Wu ¨ rzburg, Wu ¨ rzburg, Germany. 7 Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden. VIRAL IMMUNOLOGY Volume 23, Number 4, 2010 ª Mary Ann Liebert, Inc. Pp. 385–393 DOI: 10.1089/vim.2009.0083 385