A recent study published in Gut provides a portrait of wild-type adeno-associated virus (AAV) infection in the liver with an identification of molecular forms, viral genotypes, viral integrations, and helper virus relationship and with AAV insertions positive selected during human hepatocellular carcinoma (HCC) development on noncirrhotic liver, which challenges the concept of AAV as nonpathogenic.
AAV is a defective, nonenveloped DNA virus that requires a helper virus to become an active infection, and it is endemic (30%-80%) in human populations. Because recurrent clonal AAV2 was recently found to be involved in the pathogenesis of HCC in normal livers without the presence of classic HCC risk factors such as hepatitis C virus infection, this study was designed to characterize the natural history of wild-type AAV and its relationship with tumor development in a large patient cohort with malignant or benign liver tumors. A DNase/TaqMan-based assay and quantitative RT-PCR (qRT-PCR) were used to analyze episomal form presence and viral mRNA expression.
Viral DVA was quantified in tumor and nontumor liver tissues of 1461 patients. Two major genotypes of AAV were ultimately identified by screening frozen liver tissue samples of 1319 participants with AAV DNA identified in 18% (n=233) nontumor liver tissues. One of the major genotypes is similar to AAV2, and the other is a hybrid between AAV2 and AAV13 sequences, named AAV2/13. In 27.5% (64 of 233) of the tissues that tested positive for AAV DNA, all AAV genomic regions were amplified, suggesting the presence of the entire viral genome. The DNase/TaqMan-based assay detected episomal viral forms in 26% of AAV-positive samples and 4.6% of all participants.
After potential AAV helper viruses were screened in all 1319 liver tissue samples, at least one of the following viruses was detected by qRT-PCR in 570 (43%) of participants: human herpesviruses (HHV) (most frequently HHV type 6 at 39%, followed by HHV type 4 at 4%) and human adenoviruses (although rare). In a multivariate analysis of the overall cohort, the factors independently associated with AAV positivity were coinfection with HHV6 (odds ratio [OR], 1.15; P =.031), age (OR, 1.42; P =.044), female sex (OR, 1.83; P <.001), and noncirrhotic liver (OR, 1.96; P <.001). When 30 independent clonal AAV insertions were reanalyzed, viral insertions occurred in both directions with AAV2 and AAV2/13 subtypes being equally represented (55% vs 45% of interpretable cases). Insertions were recurrently identified in CCNA2, CCNE1, TERT, TNFSF10, KMT2B, and GLI1/INHBE and in almost all cases triggered oncogenic overexpression by a promoter and/or enhancer cis mechanism.
Study investigators conclude, “We provided a portrait of AAV infection in the liver with a description of viral genotypes, molecular forms and helper virus paving the way for a renovated interest in wildtype AAV biology. New highlights on the understanding of the oncogenic consequences of AAV integration in HCC tumours emerged from this work. However, further studies are necessary to clarify the impact of AAV infection in additional cohort of patients and the frequency of insertional mutagenesis across different countries.”
La Bella T, Imbeaud S, Peneau C, et al. Adeno-associated virus in the liver: natural history and consequences in tumour development [published online August 2, 2019]. Gut. doi: 10.1136/gutjnl-2019-318281