Background: Until 2007, the polyomaviruses, BKPyV and JCPyV were the only known human polyomaviruses, and were shown to be ubiquitous in the population. First infection is generally asymptomatic, but these viruses may re-emergence leading to significant disease in immunocompromised subjects. Also, polyomaviruses produce T-antigens that have the ability to transform cells in vitro. Since 2007, eleven new polyomaviruses have been discovered, including Merkel cell polyomavirus which was shown to be integrated into the host genome of patients with Merkel cell carcinoma. It is imperative therefore that the biology, epidemiology and pathogenesis of the newly discovered human polyomaviruses be closely examined in order to gain a better understanding of their role in clinical disease. This study aimed at determining the nature and extent of infection of the new polyomaviruses WUPyV, KIPyV, MCPyV, HPyV6, 7, 9 12, TSPyV, MWPyV, STLPyV in children and adults.
Methods: Specimen cohorts: In this study, four separate sample cohorts were used to examine the epidemiology and pathogenesis of the newly described polyomaviruses. 1) The first cohort consisted of approximately 2000 hospital-based samples of convenience, including healthy controls. These specimens were used to determine the prevalence of HPyV 6, 7, 9, TSPyV and MWPyV in respiratory and faecal specimens, blood, urine, and cerebrospinal fluid. 2) Sample cohort 2 consisted of approximately 8000 samples longitudinally collected in a birth cohort from the respiratory and gastroenterial tract of infants. This cohort was used to determine the age of acquisition, infection dynamics and clinical association of the newly described polyomaviruses. 3) A third sample cohort of longitudinally collected blood, urine and respiratory swabs from 167 renal transplant recipients was examined to determine the prevalence and clinical burden of human polyomaviruses. 4) The last sample cohort consisted of carcinoma specimens; including two micropapillary urothelial bladder carcinomas from renal transplant recipents, 52 frozen breast carcinoma biopsies and 100 fresh frozen lung carcinoma tissues paired with matched histologically normal controls. Molecular methods: A range of molecular techniques were developed for this study, including real-time PCR assays to screen the specimen cohorts for human polyomaviruses, and sequencing procedures to generate whole genome sequence data. In addition a novel viral enrichment procedure was applied using targeted rolling circle PCR amplification, and two molecular-based techniques were designed to investigate polyomavirus genome integration in carcinoma tissues. Histology methods: Immunohistochemical staining was conducted using monoclonal antibodies specific for BKPyV, MCPyV, HPyV 6 and HPyV 7 antigens. This method was applied to confirm the molecular detection of polyomaviruses in carcinoma tissues.
Results: Specimen Cohort 1): Results demonstrated that the newly described polyomaviruses MCPyV, HPyV 6, 7 and TSPyV could be detected in respiratory swabs and faecal specimens, but not in blood, urine or cerebrospinal fluid. MWPyV was the most prevalent polyomavirus but was only detected in patients younger than 10 years. MWPyV was detected at a higher frequency in asymptomatic children than symptomatic patients suggesting this polyomavirus may not be associated with pathology.
Specimen Cohort 2): Results from the birth cohort demonstrated that WUPyV, KIPyV and MWPyV were first acquired around year 1, and were associated with mild respiratory symptoms in one-third of sole detections. Viral mRNA could be detected for WUPyV and KIPyV suggesting active replication in the respiratory tract.
Specimen Cohort 3): Most new polyomaviruses were commonly detected within the respiratory tract of renal transplant recipients at low viral load, in the absence of clinical symptoms. In contrast, only KIPyV, and no other virus, was detected at high viral load in sequential detections from two patients who displayed respiratory symptoms. This suggested KIPyV may be a significant pathogen in the immunocompromised.
Specimen Cohort 4): Examination of the carcinoma samples demonstrated that BKPyV was detected in bladder carcinoma tissues from two renal transplant recipients and, in one patient, was integrated within the coding sequence for the VP1 protein. This finding was confirmed by histological staining which demonstrated that BKPyV antigens were present in the nucleus of tumour cells but not surrounding normal tissue. MCPyV, HPyV 6 and 7 were detected within a subset of breast and lung carcinomas. In several cases, MCPyV was detected in tumour tissue only, and was confirmed by histological staining. In one patient a truncation within the VP2 coding sequence was detected and several patients had only partial MCPyV sequence. However, MCPyV integration into the tumour cell genome could not be demonstrated.
Conclusions: This study showed that the newly described human polyomaviruses were highly prevalent within the human respiratory tract, and were typically acquired early in life via the faecal-oral route, causing mild pathology in up to one-third of children. However, immunocompromised subjects were at greater risk of disease caused by polyomaviruses, particularly infections with KIPyV, and therefore should be seriously considered in the management of these patients. The most significant findings of this study were the clear evidence that the human polyomaviruses play a role in the development of human cancer, as demonstrated by the case of BKPyV-induced micropapillary urothelial bladder carcinoma, and the compelling data suggesting a potential role for MCPyV in breast and lung carcinomas.
It is important therefore that further studies be considered using appropriately controlled sample sets, to clearly delineate the role of the polyomaviruses in tumourgenesis. Once such a role is proven, appropriate vaccine development strategies may be devised, as successfully applied for the papillomaviruses, which genetically are closely related to the polyomaviruses.