Objective: Infection by HBV is the main risk factor for hepatocellular carcinoma (HCC) worldwide. HBV directly drives carcinogenesis through integrations in the human genome. This study aimed to precisely characterise HBV integrations, in relation with viral and host genomics and clinical features.Design: A novel pipeline was set up to perform viral capture on tumours and non-tumour liver tissues from a French cohort of 177 patients mainly of European and African origins. Clonality of each integration event was determined with the localisation, orientation and content of the integrated sequence. In three selected tumours, complex integrations were reconstructed using long-read sequencing or Bionano whole genome mapping.Results: Replicating HBV DNA was more frequently detected in non-tumour tissues and associated with a higher number of non-clonal integrations. In HCC, clonal selection of HBV integrations was related to two different mechanisms involved in carcinogenesis. First, integration of viral enhancer nearby a cancer-driver gene may lead to a strong overexpression of oncogenes. Second, we identified frequent chromosome rearrangements at HBV integration sites leading to cancer-driver genes (TERT, TP53, MYC) alterations at distance. Moreover, HBV integrations have direct clinical implications as HCC with a high number of insertions develop in young patients and have a poor prognosis.Conclusion: Deep characterisation of HBV integrations in liver tissues highlights new HBV-associated driver mechanisms involved in hepatocarcinogenesis. HBV integrations have multiple direct oncogenic consequences that remain an important challenge for the follow-up of HBV-infected patients.

Cyclins A2 and E1 regulate the cell cycle by promoting S phase entry and progression. Here, we identify a hepatocellular carcinoma (HCC) subgroup exhibiting cyclin activation through various mechanisms including hepatitis B virus (HBV) and adeno-associated virus type 2 (AAV2) insertions, enhancer hijacking and recurrent CCNA2 fusions. Cyclin A2 or E1 alterations define a homogenous entity of aggressive HCC, mostly developed in non-cirrhotic patients, characterized by a transcriptional activation of E2F and ATR pathways and a high frequency of RB1 and PTEN inactivation. Cyclin-driven HCC display a unique signature of structural rearrangements with hundreds of tandem duplications and templated insertions frequently activating TERT promoter. These rearrangements, strongly enriched in early-replicated active chromatin regions, are consistent with a break-induced replication mechanism. Pan-cancer analysis reveals a similar signature in BRCA1-mutated breast and ovarian cancers. Together, this analysis reveals a new poor prognosis HCC entity and a rearrangement signature related to replication stress.

Objective: Adeno-associated virus (AAV) is a defective mono-stranded DNA virus, endemic in human population (35%–80%). Recurrent clonal AAV2 insertions are associated with the pathogenesis of rare human hepatocellular carcinoma (HCC) developed on normal liver. This study aimed to characterise the natural history of AAV infection in the liver and its consequence in tumour development. Design: Viral DNA was quantified in tumour and non-tumour liver tissues of 1461 patients. Presence of episomal form and viral mRNA expression were analysed using a DNAse/TaqMan-based assay and quantitative RT-PCR. In silico analyses using viral capture data explored viral variants and new clonal insertions. Results: AAV DNA was detected in 21% of the patients, including 8% of the tumour tissues, equally distributed in two major viral subtypes: one similar to AAV2, the other hybrid between AAV2 and AAV13 sequences. Episomal viral forms were found in 4% of the non-tumour tissues, frequently associated with viral RNA expression and human herpesvirus type 6, the candidate natural AAV helper virus. In 30 HCC, clonal AAV insertions were recurrently identified in CCNA2 , CCNE1 , TERT , TNFSF10 , KMT2B and GLI1 / INHBE . AAV insertion triggered oncogenic overexpression through multiple mechanisms that differ according to the localisation of the integration site. Conclusion: We provided an integrated analysis of the wild-type AAV infection in the liver with the identification of viral genotypes, molecular forms, helper virus relationship and viral integrations. Clonal AAV insertions were positive selected during HCC development on non-cirrhotic liver challenging the notion of AAV as a non-pathogenic virus.

Significance: We previously demonstrated that induced pluripotent stem cell (iPSC)-derived cortical neurons from HSV-1 encephalitis patients with Toll-like receptor 3 (TLR3) pathway deficiencies are highly susceptible to HSV-1, due to impairment of cell autonomous TLR3-IFN immunity. In this study we present a protocol for efficient derivation/purification of trigeminal ganglion (TG) neurons from human iPSCs. The resulting TG neurons are of sensory identity and exhibit robust biological function. We also show that TG neurons and cortical neurons play distinct roles in host defense against HSV-1 in the central nervous system: unlike cortical neurons, TG neurons are vulnerable to HSV-1 because they require preemptive induction of TLR3-, IFN-α/β–mediated immunity. This is an important step to further our understanding of the HSV-1 encephalitis disease mechanism.

Virus-related liver carcinogenesis is one of the main contributors of cancer-related death worldwide mainly due to the impact of chronic hepatitis B and C infections. Three mechanisms have been proposed to explain the oncogenic properties of hepatitis B virus (HBV) infection: induction of chronic inflammation and cirrhosis, expression of HBV oncogenic proteins, and insertional mutagenesis into the genome of infected hepatocytes. Hepatitis B insertional mutagenesis modifies the function of cancer driver genes and could promote chromosomal instability. In contrast, hepatitis C virus promotes hepatocellular carcinoma (HCC) occurrence mainly through cirrhosis development whereas the direct oncogenic role of the virus in human remains debated. Finally, adeno associated virus type 2 (AAV2), a defective DNA virus, has been associated with occurrence of HCC harboring insertional mutagenesis of the virus. Since these tumors developed in a non-cirrhotic context and in the absence of a known etiological factor, AAV2 appears to be the direct cause of tumor development in these patients via a mechanism of insertional mutagenesis altering similar oncogenes and tumor suppressor genes targeted by HBV. A better understanding of virus-related oncogenesis will be helpful to develop new preventive strategies and therapies directed against specific alterations observed in virus-related HCC.