First detection of a phylogenetically distinct and highly prevalent human polyomavirus 6 in human bile samples

Abstract

Oncovirus associated malignancies are potentially preventable diseases with major public health significance. Human polyomaviruses (HPyVs) may be associated with oncogenesis or symptomatic illnesses in immunocompromised patients, but the site of viral shedding of most recently discovered HPyVs remains obscured. Bile samples were collected from patients with malignant biliary obstruction caused by cholangiocarcinoma, hepatocellular carcinoma (HCC), pancreatic carcinoma, or gallbladder carcinoma. Bile samples were also collected from patients with acute gallstone cholangitis as controls. A consensus polymerase chain reaction (PCR) assay targeting the large tumor antigen (LTAg) gene of 11 known HPyVs was designed and utilized to screen for HPyVs in 124 bile samples. Two of 124 (1.6%) bile samples were positive for HPyV6 deoxyribonucleic acid (DNA) in the screening. Subsequently, a total of 171 bile samples were screened with a specific conventional PCR assay targeting the viral capsid protein (VP1) gene of HPyV6, which detected HPyV6 DNA in 11/171 (6.4%) samples. To better understand the prevalence and the viral loads of HPyV6 in the bile samples, a more sensitive real-time PCR assay using specific primers targeting the HPyV6 LTAg gene was performed. The overall prevalence of HPyV6 was higher in the bile samples of patients with malignant biliary obstruction (18.8%) than those with acute gallstone cholangitis (5.5%). The prevalence and mean viral load of HPyV6 were the highest in the cholangiocarcinoma subgroup (27.6% and 2.41×104 copies/ml). These findings were confirmed with another real-time PCR assay using specific primers targeting the HPyV6 VP2 gene. Complete genome of 2 representative bile HPyV6 strains were sequenced, including one from a patient with cholangiocarcinoma (Bile-81) and another from a patient with acute gallstone cholangitis (Bile-72). These bile HPyV6 strains are phylogenetically distinct from other known HPyV6 strains and may represent a novel clade of HPyV6. They formed a distinct cluster from the other HPyV6s and exhibited >2% differences in amino acid sequences in their major proteins, including small T antigen (STAg) (1.1% to 2.2%), LTAg (1.1% to 2.3%), VP2 (1.2% to 2.1%), and VP3 (1.9% to 2.8%). Comparison of patients with and without HPyV6 in bile samples showed that the virus was unlikely the cause of the patients’ acute symptoms and liver dysfunction. HPyV6 may be associated with the pathogenesis of cholangiocarcinoma in patients without coinfection with other well established oncogenic pathogens in our locality, such as Clonorchis sinensis, Hepatitis B virus (HBV), and Hepatitis C virus (HCV). Further studies utilizing in situ hybridization for detection of HPyV6 in cholangiocarcinoma tissues should be conducted to further investigate on the possible association between HPyV6 and cholangiocarcinoma.