Serologic Evidence of WU and KI Polyomaviruses | CDC EID

Volume 15, Number 8–August 2009
Research
Serologic Evidence of Frequent Human Infection with WU and KI Polyomaviruses
Nang L. Nguyen, Binh-Minh Le, and David Wang
Author affiliation: Washington University in St. Louis, St. Louis, Missouri, USA

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Abstract
WU polyomavirus (WUPyV) and KI polyomavirus (KIPyV) are novel human polyomaviruses. They were originally identified in human respiratory secretions, but the extent of human infection caused by these viruses has not been described to date. To determine the seroepidemiology of WUPyV and KIpyIV, we used an ELISA to screen serum samples from 419 patients at the St. Louis Children's Hospital and Barnes-Jewish Hospital during 2007–2008. The age-stratified deidentified samples were examined for antibodies to the major capsid proteins of WUPyV and KIPyV. Seropositivity for each virus was similar; antibody levels were high in the youngest age group (<6 months), decreased to a nadir in the next age group (6 to <12 months), and then steadily increased with subsequent age groups, eventually reaching a plateau of ≈80% for WUPyV and ≈70% for KIPyV. These results demonstrate that both KIPyV and WUPyV cause widespread infection in the human population.

WU polyomavirus (WUPyV) (1) and KI polyomavirus (KIPyV) (2) are newly described human polyomaviruses most closely related to JC virus (JCV) and BK virus (BKV). JCV and BKV are human pathogens that commonly infect the population. In the United States, seropositivity rates of 44%–75% for JCV and 63%–80% for BKV have been reported (3,4). Current models suggest that initial infection by BKV and JCV occurs asymptomatically during childhood; latency may establish in the kidneys and may reactivate during immune suppression. JCV causes a fatal demyelinating disease of progressive multifocal leukoencephalopathy in immunocompromised persons (5). BKV is associated with a number of renal and urinary tract infections including tubular nephritis, which can lead to allograft failure in renal transplant recipients (6), and hemorrhagic cystitis in hematopoietic stem cell transplant recipients (7). Another human polyomavirus, Merkel cell polyomavirus, was recently discovered and has tentatively been linked to Merkel cell carcinoma (8).

For KIPyV and WUPyV, neither disease association nor extent of infection in the human population has been established. Both viruses were originally identified in specimens from patients with respiratory illnesses of unknown etiology. Subsequent studies found WUPyV and KIPyV in the respiratory tract of patients with and without respiratory signs and symptoms (9–11), in fecal samples (12,13), and in lymphoid tissue from immunocompromised persons (14). Reported prevalence rates are 1%–9% for WUPyV and 0.5%–3% for KIPyV (1,2,12,13). The severity of diseases caused by BKV and JCV (5,6,15) raises the question of whether WUPyV and KIPyV can cause human disease. As a step toward determining the potential pathogenicity of these viruses, we developed serologic assays to assess the extent of infection by WUPyV and KIPyV in humans.

Materials and Methods
Plasmid Constructs, Protein Expression, and Purification
Genes encoding the major capsid proteins, KIPyV viral protein 1 (VP1) and WUPyV VP1, were cloned into the Gateway vector pENTR/SD/D-TOPO (Invitrogen, Carlsbad, CA, USA) by PCR from clinical samples. The primers were as follows: 5´-CACCATGAGCTGCACCCCGT-3´ (forward) and 5´-ATACATTCACTTTGAATTTTGTTGAG-3´ (reverse) for the KIPyV VP1 PCR and 5´-CACCATGGCCTGCACAGCAAAGCCAGCC-3´ (forward) and 5´-TTATCCTTGTGTGTTTAGTATTGG-3´ (reverse) for the WUPyV VP1 PCR. Sequencing analysis showed that the KIPyV VP1 gene cloned was identical to that of the Brisbane 002 strain (GenBank accession no. ABR68682), except for 2 silent nucleotide mutations at positions 537 and 1005. For WUPyV, the gene encoding VP1 was identical to that of the B0 strain (GenBank accession no. ABQ09289). Positive clones containing the inserts were then transferred into the p-DEST15 plasmid (Invitrogen) by LR-homologous recombination to generate N-terminal–tagged glutathione S-transferase (GST)–WUPyV VP1 (plasmid NN003) and GST-KIPyV VP1 (NN006) constructs. N-terminal–tagged GST-VP1s from BKV, JCV, and simian virus 40 (SV40) were generously provided by Michael Pawlita (16), and GST-tagged microneme (Mic) protein encoded by Toxoplasma gondii was provided by David Sibley. VP1 was expressed in BL21(DE3)pLysS bacterial cells and affinity purified under native conditions by using the BugBuster GST-Bind Purification Kit (Novagen, Darmstadt, Germany) according to the manufacturer's suggested protocol.

Polyacrylamide Gel Electrophoresis and Western Blot Analysis
Proteins were separated by electrophoresis in 4%–15% polyacrylamide gradient gels (no. 161-1122; BioRad, Hercules, CA , USA) by using Tris/glycine/sodium dodecyl sulfate (SDS) buffer (no. 161–0732; BioRad). The proteins were then either stained with Coomassie brilliant blue or transferred to a polyvinylidene difluoride membrane (no. LC2002; Invitrogen) for Western blot immunoassay. Membranes were blocked with 5% nonfat milk in phosphate-buffered saline with Tween 20 (PBS-T) for 1 h, then incubated with the primary antibody followed by peroxidase-conjugated Protein A/G (no. 32490; Pierce Biotechnology, Rockford, IL, USA). The proteins were visualized by using a SuperSignal West Pico kit (no. 34077; Thermo Scientific, Rockford, IL , USA). Membranes that were probed >1× were stripped with Restore Western Blot Stripping Buffer (no. 21059; Thermo Scientific) and reblocked with 5% nonfat milk in PBS-T between immunoassays.

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