Washington University Polyomavirus Research Articles

Development of a Recombinant Protein-Based Immunoassay for Detection of Antibodies Against Karolinska Institute and Washington University Polyomaviruses.

To develop polyomavirus VP1 recombinant protein-based immunoassay, the expression of two polyomavirus (Karolinska Institute Polyomavirus; KIPyV, and Washington University Polyomavirus; WUPyV) VP1s in insect cells was investigated using an improved baculovirus system (BacMagic). The reliability of the purified VP1 to serve as antigens in serological tests was confirmed by the establishment of an enzyme-linked immunosorbent assay (ELISA). Two panels of serum samples were used, with Panel I comprising 60 sera (20 KIPyV-positive, 20 WUPyV-positive, and 20 negative) and Panel II consisting of 134 sera with unknown status. The seroprevalence of KIPyV and WUPyV in the study population was determined to be 62% and 50%, respectively. Antibody-negative sera exhibited low reactivities in both ELISAs, whereas antibody-positive sera displayed high reactivity with median optical density values of 1.37 and 1.47 in the KIPyV and WUPyV ELISAs, respectively. The differences in seroreactivities between antibody positive and negative for each virus were statistically significant (p < 0.0001; with 95% confidence interval). The study suggests that seroconversion for KIPyV and WUPyV occurs in childhood, with KIPyV seropositivity reaching 70% and WUPyV seropositivity reaching 60% after the age of 5 years. Adult seroprevalence for polyomaviruses was high, with more than 64% and 51% of the adult population being seropositive for KIPyV and WUPyV, respectively. The constant prevalence of KIPyV and WUPyV antibody in the age groups suggested that this antibody persists for life. The fact that antibody titers were generally stable over time revealed a persistent infection of polyomaviruses in the human population. The insect cell-derived recombinant VP1-based ELISA has been demonstrated to be valuable as a serological assay, offering a valid, reliable, fast, nonlaborious, and economical procedure.

KI and WU Polyomavirus in Respiratory Samples of SARS-CoV-2 Infected Patients.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been declared a global pandemic. Our goal was to determine whether co-infections with respiratory polyomaviruses, such as Karolinska Institutet polyomavirus (KIPyV) and Washington University polyomavirus (WUPyV) occur in SARS-CoV-2 infected patients. Oropharyngeal swabs from 150 individuals, 112 symptomatic COVID-19 patients and 38 healthcare workers not infected by SARS-CoV-2, were collected from March 2020 through May 2020 and tested for KIPyV and WUPyV DNA presence. Of the 112 SARS-CoV-2 positive patients, 27 (24.1%) were co-infected with KIPyV, 5 (4.5%) were positive for WUPyV, and 3 (2.7%) were infected simultaneously by KIPyV and WUPyV. Neither KIPyV nor WUPyV DNA was detected in samples of healthcare workers. Significant correlations were found in patients co-infected with SARS-CoV-2 and KIPyV (p < 0.05) and between SARS-CoV-2 cycle threshold values and KIPyV, WUPyV and KIPyV and WUPyV concurrently detected (p < 0.05). These results suggest that KIPyV and WUPyV may behave as opportunistic respiratory pathogens. Additional investigations are needed to understand the epidemiology and the prevalence of respiratory polyomavirus in COVID-19 patients and whether KIPyV and WUPyV could potentially drive viral interference or influence disease outcomes by upregulating SARS-CoV-2 replicative potential.

Study of Karolinska Institutet and Washington University polyomaviruses in tonsil, adenoid, throat swab and middle ear fluid samples.

To study prevalence of Karolinska Institutet (KI) and Washington University (WU) polyomavirus (PyV) in 100 tonsils, 100 adenoids, 146 throat swab and 15 middle ear fluid samples collected from 146 patients (120 children and 26 adults), to analyze the sequence of noncoding control region (NCCR) and complete WUPyV genomes. Viruses were detected by quantitative real-time PCR. The NCCRs and WUPyV genomes were sequenced and analyzed. The frequency of WUPyV and KIPyV DNA was 27 and 11% in adenoids, 4 and 3% in tonsils, 4.1 and 1.4% in throat swab samples, respectively. The WUPyV DNA was detected in one middle ear fluid sample as well. The WUPyV NCCRs showed mutations which may alter the putative transcription factor binding sites. Phylogenetic analysis revealed three clades of WUPyV. Tonsils and adenoids might be site of virus replication and/or persistence, and WUPyV may invade into the middle ear.

The research progress of WU/KI polyomaviruses

Washington University(WU)Karolinska Institute(KI)polyomaviruses were first found from the respiratory tract secretion in 2007, successively have detected positive reports from all over the world.ZheJiang, ShenZhen and other areas from our country have also detected these two viruses recent years.WUPyv/KIPyv belong to the polyomavirus genus and infections always occurred in early childhood, the seasonal distribution across different areas, clinical features present for kinds of respiratory symptoms such as fever, cough, wheezing and so on, without specificity, and they often come with other respiratory viruses.The infection rate of WUPyv/KIPyv is higher in immunocompromised patients, patients with HIV infection may lead to severe disease.However, the pathogenesis, and pathogenic characteristics of WUPyv/KIPyv are not clear, so whether they are respiratory pathogenic agents is still controversal. Key words: WU polyomavirus; KI polyomavirus; Respiratory infection; Chidren

A Naturally Transmitted Epitheliotropic Polyomavirus Pathogenic in Immunodeficient Rats: Characterization, Transmission, and Preliminary Epidemiologic Studies.

We report the identification, pathogenesis, and transmission of a novel polyomavirus in severe combined immunodeficient F344 rats with null Prkdc and interleukin 2 receptor gamma genes. Infected rats experienced weight loss, decreased fecundity, and mortality. Large basophilic intranuclear inclusions were observed in epithelium of the respiratory tract, salivary and lacrimal glands, uterus, and prostate gland. Unbiased viral metagenomic sequencing of lesioned tissues identified a novel polyomavirus, provisionally named Rattus norvegicus polyomavirus 2 (RatPyV2), which clustered with Washington University (WU) polyomavirus in the Wuki clade of the Betapolyomavirus genus. In situ hybridization analyses and quantitative polymerase chain reaction (PCR) results demonstrated viral nucleic acids in epithelium of respiratory, glandular, and reproductive tissues. Polyomaviral disease was reproduced in Foxn1rnu nude rats cohoused with infected rats or experimentally inoculated with virus. After development of RatPyV2-specific diagnostic assays, a survey of immune-competent rats from North American research institutions revealed detection of RatPyV2 in 7 of 1,000 fecal samples by PCR and anti-RatPyV2 antibodies in 480 of 1,500 serum samples. These findings suggest widespread infection in laboratory rat populations, which may have profound implications for established models of respiratory injury. Additionally, RatPyV2 infection studies may provide an important system to investigate the pathogenesis of WU polyomavirus diseases of man.

Molecular epidemiology of WU polyomavirus in hospitalized children with acute respiratory tract infection in China.

To explore the molecular epidemiology and clinical characteristics of Washington University polyomavirus (WUPyV) infection in pediatric patients with acute respiratory tract infections in China. A laboratory surveillance was performed to recruit pediatric patients with acute respiratory tract infections. WUPyV was detected using real-time PCR and complete genome was sequenced for randomly selected positive nasopharyngeal aspirate. Altogether 122 (7.5%) of 1617 children found to be infected with WUPyV and 88 (72.1%) were coinfected with other viruses during 2012-2015. The phylogenetic analysis showed that 14 strains from our study formed two new clusters (Id and IIIc) within the Branch I and Branch III, respectively. WUPyV is persistently circulating in China. Surveillance on WUPyV infection in wider areas and long persistence is warranted.

Human Washington University Polyomavirus in Patients with Respiratory Tract Infection in Kuwait

Objective: To determine Washington University (WU) polyomavirus strains circulating among hospitalized patients with respiratory tract infections (RTI) in Kuwait. Materials and Methods: Samples from 459 hospitalized children and adult RTI patients were screened for respiratory viruses by polymerase chain reaction from April 2013 to April 2016. The VP2 gene from WU virus (WUV)-positive samples was sequenced and subjected to phylogenetic analysis. Results: Of the 459 hospitalized RTI patients, 18 (3.9%) patients were positive for WUV infection. WUV infection was common among children aged ≤11 years (9 patients, 50%). Among the 18 WUV-infected hospitalized patients, viral co-infection was detected in 9 patients (50%). The most common viruses associated with mixed infection were respiratory syncytial virus and human rhinovirus (2 patients, 11.1% each). Of the 18 WUV-infected patients, 4 were sequenced and subjected to phylogenetic analysis. The circulating strains belong to type Ia and IIIb. Conclusion: This study enabled us to detect WUV among hospitalized RTI patients. Co-infection with other respiratory viruses was notable. Two circulating WUV genotypes (Ia and IIIb) were identified among hospitalized RTI patients in Kuwait.

Production of recombinant VP1-derived virus-like particles from novel human polyomaviruses in yeast.

BackgroundEleven new human polyomaviruses (HPyVs) have been identified in the last decade. Serological studies show that these novel HPyVs sub-clinically infect humans at an early age. The routes of infection, entry pathways, and cell tropism of new HPyVs remain unknown. VP1 proteins of polyomaviruses can assembly into virus-like particles (VLPs). As cell culturing systems for HPyV are currently not available, VP1-derived VLPs may be useful tools in basic research and biotechnological applications.ResultsRecombinant VP1-derived VLPs from 11 newly identified HPyVs were efficiently expressed in yeast. VP1 proteins derived from Merkel cell polyomavirus (MCPyV), trichodysplasia spinulosa-associated polyomavirus (TSPyV), and New Jersey polyomavirus (NJPyV) self-assembled into homogeneous similarly-sized VLPs. Karolinska Institutet polyomavirus (KIPyV), HPyV7, HPyV9, HPyV10, and St. Louis polyomavirus (STLPyV) VP1 proteins formed VLPs that varied in size with diameters ranging from 20 to 60 nm. Smaller-sized VLPs (25–35 nm in diameter) predominated in preparations from Washington University polyomavirus (WUPyV) and HPyV6. Attempts to express recombinant HPyV12 VP1-derived VLPs in yeast indicate that translation of VP1 might start at the second of two potential translation initiation sites in the VP1-encoding open reading frame (ORF). This translation resulted in a 364-amino acid-long VP1 protein, which efficiently self-assembled into typical PyV VLPs. MCPyV-, KIPyV-, TSPyV-, HPyV9-, HPyV10-, and HPyV12-derived VLPs showed hemagglutination (HA) assay activity in guinea pig erythrocytes, whereas WUPyV-, HPyV6-, HPyV7-, STLPyV- and NJPyV-derived VP1 VLPs did not.ConclusionsThe yeast expression system was successfully utilized for high-throughput production of recombinant VP1-derived VLPs from 11 newly identified HPyVs. HPyV12 VP1-derived VLPs were generated from the second of two potential translation initiation sites in the VP1-encoding ORF. Recombinant VLPs produced in yeast originated from different HPyVs demonstrated distinct HA activities and may be useful in virus diagnostics, capsid structure studies, or investigation of entry pathways and cell tropism of HPyVs until cell culture systems for new HPyVs are developed.

The Novel KI, WU and MC Polyomaviruses and Human Diseases

From 2007 to 2012, the Polyomaviridae family expanded with the discovery of several novel human polyomaviruses. Infection by polyomaviruses is widespread in the human population, but only immunocompromised patients are at risk for disease. Currently, four polyomaviruses have been specifically associated with human diseases: BK polyomavirus, JC polyomavirus, Merkel cell polyomavirus and trichodysplasia spinulosa-associated polyomavirus. The pathogenic potential of the other human polyomaviruses is unknown. This review focuses on the novel Karolinska Institute polyomavirus, Washington University polyomavirus and Merkel cell polyomavirus. Karolinska Institute polyomavirus and Washington University polyomavirus were uncovered in respiratory specimens of children with acute respiratory symptoms. Merkel cell polyomavirus was found in an aggressive skin tumor named Merkel cell carcinoma. The scientific literature concerning the three human polyomaviruses is summarized and discussed including seroepidemiology, detection methods (real-time PCR), histopathological findings and association with clinical manifestations.

KI, WU, and Merkel Cell Polyomavirus DNA was not Detected in Guthrie Cards of Children who Later Developed Acute Lymphoblastic Leukemia

Neonatal dried blood spots (Guthrie cards) have been used to demonstrate a prenatal origin of clonal leukemia-specific genetic aberrations in several subgroups of childhood acute lymphoblastic leukemia (ALL). One hypothesis suggests that an infectious agent could initiate genetic transformation already in utero. In search for a possible viral agent, Guthrie cards were analyzed for the presence of 3 newly discovered polyomavirus Karolinska Institutet polymavirus (KIPyV), Washington University polyomavirus (WUPyV), and Merkel cell polyomavirus (MCPyV). Guthrie cards from 50 children who later developed ALL and 100 matched controls were collected and analyzed by standard or real-time polymerase chain reaction for the presence of the VP1 region of KIPyV, WUPyV, and MCPyV, and the LT region for MCPyV. DNA from KIPyV, WUPyV, and MCPyV was not detected in neonatal blood samples from children with ALL or controls. Prenatal infections with these viruses are not likely to be etiological drivers for childhood leukemogenesis.

Investigation on the presence of polyomavirus, herpesvirus, and papillomavirus sequences in colorectal neoplasms and their association with cancer

Investigation on the presence of polyomavirus, herpesvirus, and papillomavirus sequences in colorectal neoplasms and their association with cancer