Journal of Medical Virology 86:627–633 (2014) Assessing the Detection of Human Papillomavirus Late mRNA in Liquid Base Cytology Samples for Risk Stratification of Cervical Disease

Molecular human papillomavirus (HPV) testing is an important and developing tool for cervical disease management. However there is a re-quirement to develop new HPV tests that can differentiate between clinically significant and benign, clinically insignificant infection. Evi-dence would indicate that clinically significant infection is linked to an abortive HPV replication cycle. In particular the later stages of the replication cycle (i.e., production of late mes-senger (m) RNAs and proteins) appear compro-mised. Compared to current DNA-based tests which indicate only presence or absence of virus, detecting virus mRNAs by reverse tran-scriptase PCR (RT-PCR) may give a more refined insight into viral activity and by implica-tion, clinical relevance. A novel quantitative (q) RT-PCR assay was developed for the detection of mRNAs produced late in the viral replication cycle. Initially this was validated on HPV-con-taining cell lines before being applied to a panel of 223 clinical cervical samples represent-ing the cervical disease spectrum (normal to high grade). Samples were also tested by a commercial assay which detects expression of early HPV E6/E7 oncoprotein mRNAs. Late mRNAs were found in samples associated with no, low and high grade disease and did not risk-stratify HPV infection. The data reveal hid-den complexities within the virus replication cycle and associated lesion development. This suggests that future mRNA tests for cervical disease may require quantitative detection of specific novel viral mRNAs. J. Med. Virol. 86:627–633, 2014. # 2013 Wiley Periodicals, Inc. KEY WORDS: human papillomavirus; gene expression; cervical intrae-pithelial neoplasia; liquid based cytology; mRNA test