Restricted accessResearch articleFirst published online 2026-4
Development and Evaluation of a Baculovirus-Expressed VP1-Based Immunoassay for BK Polyomavirus Serology and Preliminary Seroepidemiological Applications
BK polyomavirus (BKPyV) is highly prevalent and clinically relevant in immunocompromised populations, underscoring the need for reliable serological tools to support epidemiological monitoring and clinical follow-up. This study aimed to generate a recombinant VP1-based enzyme-linked immunosorbent assay (ELISA) using a baculovirus–Sf9 insect cell system and to explore its application in a human serum cohort. Recombinant VP1 was produced by baculovirus-mediated expression, purified by nickel–nitrilotriacetic acid affinity chromatography, and used to establish an indirect IgG ELISA. A plasmid-based transient expression approach in Sf9 cells was evaluated only as a feasibility experiment and did not yield sufficient VP1 for assay development. Analytical performance was assessed using a polymerase chain reaction (PCR)-defined serum panel (n = 30), and the assay was subsequently applied to 149 sera with unknown BKPyV status. Baculovirus-mediated expression generated high-purity VP1 with an average yield of approximately 1.55 mg/mL from a 10 mL culture. PCR-confirmed positive sera showed markedly higher optical density values than PCR-negative sera, with very good analytical discrimination in this small validation set (area under the curve = 0.996). In the cohort analysis, overall seroreactivity was 89.7%, antibody reactivity increased with age, and higher ELISA signals were observed in renal transplant and hemodialysis patients than in healthy individuals. Detectable reactivity in young children may reflect early exposure or passive maternal antibodies. These findings indicate that insect cell–derived BKPyV VP1 is a suitable antigen for serological assays and a practical platform for preliminary seroepidemiological applications, which require confirmation in larger independent validation cohorts.
Abedi KiasariB. Comparative evaluation of human papillomavirus type 16 L1 protein expressed in plasmid- and baculovirus-based systems in insect cells. Arch Razi Inst, 2020; 75(2):187–195.
2.
Ahlenstiel-GrunowT, PapeL. Diagnostics, treatment, and immune response in BK polyomavirus infection after pediatric kidney transplantation. Pediatr Nephrol, 2020; 35(3):375–382.
3.
AlMalakiKA, Asmaa’BA-O, GhaziHF. The potential role of BK and JC polyomaviruses in urothelial cancer. Iraqi J. Med. Sci, 2025; 23(1).
4.
BahrulolumH, TarrahimofradH, RouzbahaniFN, et al.Potential of CRISPR/Cas system as emerging tools in the detection of viral hepatitis infection. Virol. J., 2023; 20(1):91.
BlackardJT, DaviesSM, LaskinBL. BK polyomavirus diversity—why viral variation matters. Rev Med Virol, 2020; 30(4):e2102.
7.
Burger-CalderonR, Webster-CyriaqueJ. Human BK polyomavirus—the potential for head and neck malignancy and disease. Cancers (Basel), 2015; 7(3):1244–1270.
8.
ChenXS, StehleT, HarrisonSC. Interaction of polyomavirus internal protein VP2 with the major capsid protein VP1 and implications for participation of VP2 in viral entry. Embo J, 1998; 17(12):3233–3240.
9.
Contreras-GómezA, Sánchez-MirónA, García-CamachoF, et al.Protein production using the baculovirus-insect cell expression system. Biotechnol Prog, 2014; 30(1):1–18.
10.
CookL. (2016) Polyomaviruses. In: Diagnostic Microbiology of the Immunocompromised Host, Wiley, pp. 197–216.
11.
FurmagaJ, KowalczykM, ZapolskiT, et al.BK polyomavirus—biology, genomic variation and diagnosis. Viruses, 2021; 13(8):1502.
12.
GillockE, RottinghausS, ChangD, et al.Polyomavirus major capsid protein VP1 is capable of packaging cellular DNA when expressed in the baculovirus system. J Virol, 1997; 71(4):2857–2865.
13.
GoldmannC, PetryH, FryeS, et al.Molecular cloning and expression of major structural protein VP1 of the human polyomavirus JC virus: Formation of virus-like particles useful for immunological and therapeutic studies. J Virol, 1999; 73(5):4465–4469.
14.
GossaiA, WaterboerT, NelsonHH, et al.Seroepidemiology of human polyomaviruses in a US population. Am J Epidemiol, 2016; 183(1):61–69.
15.
GovindS, HockleyJ, MorrisC, et al.; Collaborative Study Group. The development and establishment of the 1st WHO BKV International Standard for nucleic acid based techniques. Biologicals, 2019; 60:75–84.
16.
HelleF, BrochotE, HandalaL, et al.Biology of the BKPyV: An update. Viruses, 2017; 9(11):327.
17.
HirschH, VincentiF, FrimanS, et al.Polyomavirus BK replication in de novo kidney transplant patients receiving tacrolimus or cyclosporine: A prospective, randomized, multicenter study. Am. J. Transplant., 2013; 13(1):136–145.
18.
HitchmanRB, PosseeRD, KingLA. Baculovirus expression systems for recombinant protein production in insect cells. Recent Pat Biotechnol, 2009; 3(1):46–54.
19.
HöckerB, SchnebleL, MurerL, et al.Epidemiology of and risk factors for BK polyomavirus replication and nephropathy in pediatric renal transplant recipients: An international CERTAIN registry study. Transplantation, 2019; 103(6):1224–1233.
20.
HongM, LiT, XueW, et al.Genetic engineering of baculovirus-insect cell system to improve protein production. Front Bioeng Biotechnol, 2022; 10:994743.
21.
HurdissDL, MorganEL, ThompsonRF, et al.New structural insights into the genome and minor capsid proteins of BK polyomavirus using cryo-electron microscopy. Structure, 2016; 24(4):528–536.
22.
HusseinyMI, LaceySF. Development of infectious recombinant BK virus. Virus Res., 2011; 161(2):150–161.
KausarA, AmraizD, AliL, et al.A systematic review of BK and JC polyomaviruses in Asia: Molecular insights and prevalence. Rev Med Virol, 2025; 35(4):e70048.
25.
KeanJM, RaoS, WangM, et al.Seroepidemiology of human polyomaviruses. PLoS Pathog, 2009; 5(3):e1000363.
26.
KiasariBA, AlipourAH, HemmatiN, et al.Lymphotropic polyomavirus and Merkel cell polyomavirus in patients infected with HIV or hepatitis B or C virus. New Microbes New Infect, 2024; 62:101471.
27.
KottonCN, KamarN, WojciechowskiD, et al.; Transplantation Society International BK Polyomavirus Consensus Group. The second international consensus guidelines on the management of BK polyomavirus in kidney transplantation. Transplantation, 2024; 108(9):1834–1866.
28.
LampinenV, GröhnS, LehmlerN, et al.Production of norovirus-, rotavirus-, and enterovirus-like particles in insect cells is simplified by plasmid-based expression. Sci. Rep., 2024; 14(1):14874.
29.
LeavittA, RobertsT, GarceaR. Polyoma virus major capsid protein, VP1. Purification after high level expression in Escherichia coli. J Biol Chem, 1985; 260(23):12803–12809.
30.
LeeM-S, HseuY-C, LaiG-H, et al.High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development. Microb Cell Fact, 2011; 10(1):1–12.
31.
LevicanJ, AcevedoM, LeónO, et al.Role of BK human polyomavirus in cancer. Infect Agents Cancer, 2018; 13(1):1–8.
32.
LiT-C, TakedaN, KatoK, et al.Characterization of self-assembled virus-like particles of human polyomavirus BK generated by recombinant baculoviruses. Virology, 2003; 311(1):115–124.
33.
MoensU, Van GhelueM, EhlersB. Are human polyomaviruses co-factors for cancers induced by other oncoviruses? Rev Med Virol, 2014; 24(5):343–360.
34.
MoensU, Van GhelueM, SongX, et al.Serological cross-reactivity between human polyomaviruses. Rev Med Virol, 2013; 23(4):250–264.
35.
NagshabandiMK. (2020) Polyomavirus Infection in Renal Transplant Recipients. PhD thesis, The University of Manchester, United Kingdom.
36.
NotebornMHM, VerschuerenCAJ, KochG, et al.Simultaneous expression of recombinant baculovirus-encoded chicken anaemia virus (CAV) proteins VP1 and VP2 is required for formation of the CAV-specific neutralizing epitope. J Gen Virol, 1998; 79 (Pt 12)(12):3073–3077.
37.
NourieN, BoueriC, Tran MinhH, et al.BK polyomavirus infection in kidney transplantation: A comprehensive review of current challenges and future directions. Int J Mol Sci, 2024; 25(23):12801.
38.
SalunkeD, CasparD, GarceaR. Polymorphism in the assembly of polyomavirus capsid protein VP1. Biophys J, 1989; 56(5):887–900.
39.
ShishkovaK, SirakovI, ShishkovS, et al.Detection of viruses with oncogenic and oncomodulatory potential in head and neck tumors—external auricle. Biomedicines, 2025; 13(10):2339.
40.
SookhooJR, SchiffmanZ, AmbagalaA, et al.Protein expression platforms and the challenges of viral antigen production. Vaccines (Basel), 2024; 12(12):1344.
41.
SrivastavaV, NandKN, AhmadA, et al.Yeast-based virus-like particles as an emerging platform for vaccine development and delivery. Vaccines (Basel), 2023; 11(2):479.
42.
TognonM, CoralliniA, MartiniF, et al.Oncogenic transformation by BK virus and association with human tumors. Oncogene, 2003; 22(33):5192–5200.
43.
TouzéA, BousarghinL, SterC, et al.Gene transfer using human polyomavirus BK virus-like particles expressed in insect cells. J Gen Virol, 2001; 82(Pt 12):3005–3009.
44.
Van OersMM. Opportunities and challenges for the baculovirus expression system. J. Invertebr. Pathol, 2011; 107 Suppl:S3–S15.
45.
VassylyevaMN, KlyuyevS, VassylyevAD, et al.Efficient, ultra-high-affinity chromatography in a one-step purification of complex proteins. Proc Natl Acad Sci U S A, 2017; 114(26):E5138–E5147.
46.
ViscidiRP, ClaymanB. (2006) Serological cross reactivity between polyomavirus capsids. In: Polyomaviruses and Human Diseases, Springer, pp. 73–84.
47.
ViscidiRP, RollisonDE, ViscidiE, et al.Serological cross-reactivities between antibodies to simian virus 40, BK virus, and JC virus assessed by virus-like-particle-based enzyme immunoassays. Clin Diagn Lab Immunol, 2003; 10(2):278–285.
48.
WangY, ZhangS, LiF, et al.Therapeutic target database 2020: Enriched resource for facilitating research and early development of targeted therapeutics. Nucleic Acids Res, 2020; 48(D1):D1031–D1041.
49.
ZhaoY, KolliopoulouA, RenF, et al.Transcriptional response of immune-related genes after endogenous expression of VP1 and exogenous exposure to VP1-based VLPs and CPV virions in lepidopteran cell lines. Mol Genet Genomics, 2019; 294(4):887–899.
50.
ZhouX, NakashimaK, ItoM, et al.Prevalence and viral loads of polyomaviruses BKPyV, JCPyV, MCPyV, TSPyV and NJPyV and hepatitis viruses HBV, HCV and HEV in HIV-infected patients in China. Sci Rep, 2020; 10(1):17066.
51.
ZhouX, ZhuC, LiH. BK polyomavirus: Latency, reactivation, diseases and tumorigenesis. Front Cell Infect Microbiol, 2023; 13:1263983.
