Highlights from the 2023 International Meeting on the Molecular Biology of Hepatitis B virus.

Since its discovery in 1965, our understanding of the hepatitis B virus (HBV) replication cycle and host immune responses has increased markedly. In contrast, our knowledge of the molecular biology of hepatitis delta virus (HDV), which is associated with more severe liver disease, is less well understood. Despite the progress made, critical gaps remain in our knowledge of HBV and HDV replication and the mechanisms underlying viral persistence and evasion of host immunity. The International HBV Meeting is the leading annual scientific meeting for presenting the latest advances in HBV and HDV molecular virology, immunology, and epidemiology. In 2023, the annual scientific meeting was held in Kobe, Japan and this review summarises some of the advances presented at the Meeting and lists gaps in our knowledge that may facilitate the development of new therapies.

Correction: The Tudor Domain Protein Spindlin1 Is Involved in Intrinsic Antiviral Defense against Incoming Hepatitis B Virus and Herpes Simplex Virus Type 1.

[This corrects the article DOI: 10.1371/journal.ppat.1004343.].

Hepatitis B Virus Pregenomic RNA in Hepatocellular Carcinoma: A Nosological and Prognostic Determinant.

Hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC). However, very little is known about the replication of HBV in HCC tissues. We analyzed viral and cellular parameters in HCC (T) and nontumor liver (NT) samples from 99 hepatitis B surface antigen (HBsAg)-positive, virologically suppressed patients treated by tumor resection or liver transplantation. We examined total HBV DNA and RNA as well as covalently closed circular DNA (cccDNA) and pregenomic RNA (pgRNA), which are considered as markers of active HBV replication. Total HBV DNA and RNA were detected in both T and NT samples in a majority of cases, but only a subset of tumors harbored detectable levels of HBV cccDNA and pgRNA (39% and 67%) compared to NT livers (66% and 90%; P < 0.01). Further evidence for HBV replication in tumor tissues was provided by sequencing of the X gene derived from episomal forms, showing that HBV genotypes differed between T and matched NT samples in 11 cases. The detection of pgRNA and cccDNA in tumors was correlated to the absence of tumorous microvascular invasion and to better patient survival. Analysis of gene expression profiles by Agilent microarrays revealed that pgRNA-positive HCCs were characterized by low levels of cell cycle and DNA repair markers and expression of the HBV receptor, sodium taurocholate cotransporting polypeptide, indicating well-differentiated tumors. CONCLUSION: HCC replicating HBV represents a subtype of weakly invasive HCC with a transcriptomic signature. pgRNA originating from nonintegrated, complete HBV genomes is a sensitive marker for viral replication and prognosis. (Hepatology 2018;67:86-96).

Viral Load Affects the Immune Response to HBV in Mice With Humanized Immune System and Liver.

BACKGROUND & AIMS: Hepatitis B virus (HBV) infects hepatocytes, but the mechanisms of the immune response against the virus and how it affects disease progression are unclear. METHODS: We performed studies with BALB/c Rag2-/-Il2rg-/-SirpaNODAlb-uPAtg/tg mice, stably engrafted with human hepatocytes (HUHEP) with or without a human immune system (HIS). HUHEP and HIS-HUHEP mice were given an intraperitoneal injection of HBV. Mononuclear cells were isolated from spleen and liver for analysis by flow cytometry. Liver was analyzed by immunohistochemistry and mRNA levels were measured by quantitative reverse transcription polymerase chain reaction (PCR). Plasma levels of HBV DNA were quantified by PCR reaction, and antigen-specific antibodies were detected by immunocytochemistry of HBV-transfected BHK-21 cells. RESULTS: Following HBV infection, a complete viral life cycle, with production of HBV DNA, hepatitis B e (HBe), core (HBc) and surface (HBs) antigens, and covalently closed circular DNA, was observed in HUHEP and HIS-HUHEP mice. HBV replicated unrestricted in HUHEP mice resulting in high viral titers without pathologic effects. In contrast, HBV-infected HIS-HUHEP mice developed chronic hepatitis with 10-fold lower titers and antigen-specific IgGs, (anti-HBs, anti-HBc), consistent with partial immune control. HBV-infected HIS-HUHEP livers contained infiltrating Kupffer cells, mature activated natural killer cells (CD69+), and PD-1+ effector memory T cells (CD45RO+). Reducing the viral inoculum resulted in more efficient immune control. Plasma from HBV-infected HIS-HUHEP mice had increased levels of inflammatory and immune-suppressive cytokines (C-X-C motif chemokine ligand 10 and interleukin 10), which correlated with populations of intrahepatic CD4+ T cells (CD45RO+PD-1+). Mice with high levels of viremia had HBV-infected liver progenitor cells. Giving the mice the nucleoside analogue entecavir reduced viral loads and decreased liver inflammation. CONCLUSION: In HIS-HUHEP mice, HBV infection completes a full life cycle and recapitulates some of the immunopathology observed in patients with chronic infection. Inoculation with different viral loads led to different immune responses and levels of virus control. We found HBV to infect liver progenitor cells, which could be involved in hepatocellular carcinogenesis. This is an important new system to study anti-HBV immune responses and screen for combination therapies against hepatotropic viruses.

Single-Nucleotide Resolution Mapping of Hepatitis B Virus Promoters in Infected Human Livers and Hepatocellular Carcinoma.

Hepatitis B virus (HBV) is a major cause of liver diseases, including hepatocellular carcinoma (HCC), and more than 650,000 people die annually due to HBV-associated liver failure. Extensive studies of individual promoters have revealed that heterogeneous RNA 5' ends contribute to the complexity of HBV transcriptome and proteome. Here, we provide a comprehensive map of HBV transcription start sites (TSSs) in human liver, HCC, and blood, as well as several experimental replication systems, at a single-nucleotide resolution. Using CAGE (cap analysis of gene expression) analysis of 16 HCC/nontumor liver pairs, we identify 17 robust TSSs, including a novel promoter for the X gene located in the middle of the gene body, which potentially produces a shorter X protein translated from the conserved second start codon, and two minor antisense transcripts that might represent viral noncoding RNAs. Interestingly, transcription profiles were similar in HCC and nontumor livers, although quantitative analysis revealed highly variable patterns of TSS usage among clinical samples, reflecting precise regulation of HBV transcription initiation at each promoter. Unlike the variety of TSSs found in liver and HCC, the vast majority of transcripts detected in HBV-positive blood samples are pregenomic RNA, most likely generated and released from liver. Our quantitative TSS mapping using the CAGE technology will allow better understanding of HBV transcriptional responses in further studies aimed at eradicating HBV in chronic carriers. IMPORTANCE: Despite the availability of a safe and effective vaccine, HBV infection remains a global health problem, and current antiviral protocols are not able to eliminate the virus in chronic carriers. Previous studies of the regulation of HBV transcription have described four major promoters and two enhancers, but little is known about their activity in human livers and HCC. We deeply sequenced the HBV RNA 5' ends in clinical human samples and experimental models by using a new, sensitive and quantitative method termed cap analysis of gene expression (CAGE). Our data provide the first comprehensive map of global TSS distribution over the entire HBV genome in the human liver, validating already known promoters and identifying novel locations. Better knowledge of HBV transcriptional activity in the clinical setting has critical implications in the evaluation of therapeutic approaches that target HBV replication.

The Tudor domain protein Spindlin1 is involved in intrinsic antiviral defense against incoming hepatitis B Virus and herpes simplex virus type 1.

Hepatitis B virus infection (HBV) is a major risk factor for the development of hepatocellular carcinoma. HBV replicates from a covalently closed circular DNA (cccDNA) that remains as an episome within the nucleus of infected cells and serves as a template for the transcription of HBV RNAs. The regulatory protein HBx has been shown to be essential for cccDNA transcription in the context of infection. Here we identified Spindlin1, a cellular Tudor-domain protein, as an HBx interacting partner. We further demonstrated that Spindlin1 is recruited to the cccDNA and inhibits its transcription in the context of infection. Spindlin1 knockdown induced an increase in HBV transcription and in histone H4K4 trimethylation at the cccDNA, suggesting that Spindlin1 impacts on epigenetic regulation. Spindlin1-induced transcriptional inhibition was greater for the HBV virus deficient for the expression of HBx than for the HBV WT virus, suggesting that HBx counteracts Spindlin1 repression. Importantly, we showed that the repressive role of Spindlin1 is not limited to HBV transcription but also extends to other DNA virus that replicate within the nucleus such as Herpes Simplex Virus type 1 (HSV-1). Taken together our results identify Spindlin1 as a critical component of the intrinsic antiviral defense and shed new light on the function of HBx in HBV infection.

HBx relieves chromatin-mediated transcriptional repression of hepatitis B viral cccDNA involving SETDB1 histone methyltransferase.

BACKGROUND & AIMS: Maintenance of the covalently closed circular HBV DNA (cccDNA) that serves as a template for HBV transcription is responsible for the failure of antiviral therapies. While studies in chronic hepatitis patients have shown that high viremia correlates with hyperacetylation of cccDNA-associated histones, the molecular mechanisms controlling cccDNA stability and transcriptional regulation are still poorly understood. This study aimed to decipher the role of chromatin and chromatin modifier proteins on HBV transcription. METHODS: We analyzed the chromatin structure of actively transcribed or silenced cccDNA by infecting primary human hepatocytes and differentiated HepaRG cells with wild-type virus or virus deficient (HBVX-) for the expression of hepatitis B virus X protein (HBx), that is required for HBV expression. RESULTS: In the absence of HBx, HBV cccDNA was transcriptionally silenced with the concomitant decrease of histone 3 (H3) acetylation and H3K4me3, increase of H3 di- and tri-methylation (H3K9me) and the recruitment of heterochromatin protein 1 factors (HP1) that correlate with condensed chromatin. SETDB1 was found to be the main histone methyltransferase responsible for the deposition of H3K9me3 and HBV repression. Finally, full transcriptional reactivation of HBVX- upon HBx re-expression correlated with an increase of histone acetylation and H3K4me3, and a concomitant decrease of HP1 binding and of H3K9me3 on the cccDNA. CONCLUSION: Upon HBV infection, cellular mechanisms involving SETDB1-mediated H3K9me3 and HP1 induce silencing of HBV cccDNA transcription through modulation of chromatin structure. HBx is able to relieve this repression and allow the establishment of active chromatin.

The four and a half LIM-only protein 2 (FHL2) activates transforming growth factor ß (TGF-ß) signaling by regulating ubiquitination of the E3 ligase Arkadia.

Arkadia is a RING-based ubiquitin ligase that positively regulates TGF-ß signaling by targeting several pathway components for ubiquitination and degradation. However, little is known about the mechanisms controlling Arkadia activity. Here we show that the LIM-only protein FHL2 binds and synergistically cooperates with Arkadia to activate Smad3/Smad4-dependent transcription. Knockdown of FHL2 by RNA interference decreases Arkadia level and restricts the amplitude of Arkadia-induced TGF-ß target gene responses. We found that Arkadia is ubiquitinated via K63- and K27-linked polyubiquitination. A single mutation at the RING domain that abolishes the E3 activity diminishes Arkadia ubiquitination, indicating that this modification partly involves autocatalytic process. Mutation of seven lysines at the C-terminal region of Arkadia severely impairs ubiquitination through the K27 but not the K63 linkage and slows down the turnover of Arkadia, suggesting that K27-linked polyubiquitination might promote proteolysis-dependent regulation of Arkadia. We show that FHL2 increases the half-life of Arkadia through inhibition of ubiquitin chain assembly on the protein, which provides a molecular basis for functional cooperation between Arkadia and FHL2 in enhancing TGF-ß signaling. Our study uncovers a novel regulatory mechanism of Arkadia by ubiquitination and identifies FHL2 as important regulator of Arkadia ubiquitination and TGF-ß signal transduction.

Methyltransferase PRMT1 is a binding partner of HBx and a negative regulator of hepatitis B virus transcription.

The hepatitis B virus X protein (HBx) is essential for virus replication and has been implicated in the development of liver cancer. HBx is recruited to viral and cellular promoters and activates transcription by interacting with transcription factors and coactivators. Here, we purified HBx-associated factors in nuclear extracts from HepG2 hepatoma cells and identified protein arginine methyltransferase 1 (PRMT1) as a novel HBx-interacting protein. We showed that PRMT1 overexpression reduced the transcription of hepatitis B virus (HBV), and this inhibition was dependent on the methyltransferase function of PRMT1. Conversely, depletion of PRMT1 correlated with increased HBV transcription. Using a quantitative chromatin immunoprecipitation assay, we found that PRMT1 is recruited to HBV DNA, suggesting a direct effect of PRMT1 on the regulation of HBV transcription. Finally, we showed that HBx expression inhibited PRMT1-mediated protein methylation. Downregulation of PRMT1 activity was further observed in HBV-replicating cells in an in vivo animal model. Altogether, our results support the notion that the binding of HBx to PRMT1 might benefit viral replication by relieving the inhibitory activity of PRMT1 on HBV transcription.

Targeting human telomerase reverse transcriptase with recombinant lentivector is highly effective to stimulate antitumor CD8 T-cell immunity in vivo.

The success of active immunotherapy is based on the vaccine's ability to overcome immune tolerance through recalibrating the immune system so that it is able to recognize tumor antigens as foreign rather than self. In this study, we used a lentiviral vector system to target human telomerase reverse transcriptase (lv-hTERT), a widely expressed tumor antigen. Immunization of HLA-A*0201 transgenic HHD mice with recombinant lv-hTERT vector induces potent and diversified cytotoxic T lymphocyte responses that recognize in vitro murine tumor cells, which overexpress telomerase. Compared with peptide-based vaccinations, the lv-hTERT vector triggers better and more sustained CD8(+) T-cell response against self/TERT epitope in vivo. The study found that the additional use of a heterologous boosted vaccination drastically improves self/TERT-specific CD8 responses in lv-hTERT primed mice. Both primary and long-lasting self/TERT-specific CD8(+) T-cell responses induced with Iv-hTERT vector required the presence of CD4 T cells in vivo. This lv-hTERT-based active immunotherapy efficiently inhibits the growth of telomerase expressing tumors (B16/HLA-A2.1 murine melanoma) in HHD mice. These data show that targeting hTERT with lentivector is highly effective in stimulating a broad range of CD8 T-cell immunity that can be exploited for cancer immunotherapy.

Impact of cellular autophagy on viruses: Insights from hepatitis B virus and human retroviruses.

Autophagy is a protein degradative process important for normal cellular metabolism. It is apparently used also by cells to eliminate invading pathogens. Interestingly, many pathogens have learned to subvert the cell's autophagic process. Here, we review the interactions between viruses and cells in regards to cellular autophagy. Using findings from hepatitis B virus and human retroviruses, HIV-1 and HTLV-1, we discuss mechanisms used by viruses to usurp cellular autophagy in ways that benefit viral replication.

Crosstalk between Hepatitis B Virus and the 3D Genome Structure.

Viruses that transcribe their DNA within the nucleus have to adapt to the existing cellular mechanisms that govern transcriptional regulation. Recent technological breakthroughs have highlighted the highly hierarchical organization of the cellular genome and its role in the regulation of gene expression. This review provides an updated overview on the current knowledge on how the hepatitis B virus interacts with the cellular 3D genome and its consequences on viral and cellular gene expression. We also briefly discuss the strategies developed by other DNA viruses to co-opt and sometimes subvert cellular genome spatial organization.

Diversity of the nucleic acid forms of circulating HBV in chronically infected patients and its impact on viral cycle.

BACKGROUND: Besides the prototypical hepatitis B virus (HBV) infectious particle, which contains a full-length double-stranded DNA (flDNA), additional circulating virus-like particles, which carry pregenomic RNA (pgRNA), spliced1RNA (sp1RNA) or spliced-derived DNA (defDNA) forms have been described. We aimed to determine the level of these four circulating forms in patients and to evaluate their impact on viral lifecycle. METHODS: Chronic HBV untreated patients (n = 162), included in the HEPATHER cohort, were investigated. Pangenomic qPCRs were set up to quantify the four circulating forms of HBV nucleic acids (HBVnaf). In vitro infection assays were performed to address the impact of HBVnaf. RESULTS: Hierarchical clustering individualized two clusters of HBVnaf diversity among patients: (1) cluster 1 (C1) showing a predominance of flDNA; (2) cluster 2 (C2) showing various proportions of the different forms. HBeAg-positive chronic hepatitis phase and higher viral load (7.0 ± 6.4 vs 6.6 ± 6.2 Log10 copies/ml; p < 0.001) characterized C2 compared to C1 patients. Among the different HBVnaf, pgRNA was more prevalent in C1 patients with high vs low HBV viral load (22.1% ± 2.5% vs 4.1% ± 1.8% of HBVnaf, p < 0.0001) but remained highly prevalent in C2 patients, whatever the level of replication. C2 patients samples used in infection assays showed that: (1) HBVnaf secretion was independent of the viral strain; (2) the viral cycle efficiency differed according to the proportion of HBVnaf in the inoculum, independently of cccDNA formation. Inoculum enrichment before infection suggests that pgRNA-containing particles drive this impact on viral replication. CONCLUSION: Besides the critical role of HBV replication in circulating HBVnaf diversity, our data highlight an impact of this diversity on the dynamics of viral cycle. CLINICAL TRIAL REGISTRATION: Patients were included from a prospective multicenter French national cohort (ANRS CO22 HEPATHER, NCT01953458).

Early Steps of Hepatitis B Life Cycle: From Capsid Nuclear Import to cccDNA Formation.

Hepatitis B virus (HBV) remains a major public health concern, with more than 250 million chronically infected people who are at high risk of developing liver diseases, including cirrhosis and hepatocellular carcinoma. Although antiviral treatments efficiently control virus replication and improve liver function, they cannot cure HBV infection. Viral persistence is due to the maintenance of the viral circular episomal DNA, called covalently closed circular DNA (cccDNA), in the nuclei of infected cells. cccDNA not only resists antiviral therapies, but also escapes innate antiviral surveillance. This viral DNA intermediate plays a central role in HBV replication, as cccDNA is the template for the transcription of all viral RNAs, including pregenomic RNA (pgRNA), which in turn feeds the formation of cccDNA through a step of reverse transcription. The establishment and/or expression of cccDNA is thus a prime target for the eradication of HBV. In this review, we provide an update on the current knowledge on the initial steps of HBV infection, from the nuclear import of the nucleocapsid to the formation of the cccDNA.

Molecular basis of the interaction of the human tyrosine phosphatase PTPN3 with the hepatitis B virus core protein.

Interactions between the hepatitis B virus core protein (HBc) and host cell proteins are poorly understood, although they may be essential for the propagation of the virus and its pathogenicity. HBc has a C-terminal PDZ (PSD-95, Dlg1, ZO-1)-binding motif (PBM) that is responsible for interactions with host PDZ domain-containing proteins. In this work, we focused on the human protein tyrosine phosphatase non-receptor type 3 (PTPN3) and its interaction with HBc. We solved the crystal structure of the PDZ domain of PTPN3 in complex with the PBM of HBc, revealing a network of interactions specific to class I PDZ domains despite the presence of a C-terminal cysteine in this atypical PBM. We further showed that PTPN3 binds the HBc protein within capsids or as a homodimer. We demonstrate that overexpression of PTPN3 significantly affects HBV infection in HepG2 NTCP cells. Finally, we performed proteomics studies on both sides by pull-down assays and screening of a human PDZ domain library. We identified a pool of human PBM-containing proteins that might interact with PTPN3 in cells and that could be in competition with the HBc PBM during infection, and we also identified potential cellular partners of HBc through PDZ-PBM interactions. This study opens up many avenues of future investigations into the pathophysiology of HBV.

Mechanisms of HBV-related hepatocarcinogenesis.

The hepatitis B virus (HBV) is a small enveloped DNA virus, which primarily infects hepatocytes and causes acute and persistent liver disease. Epidemiological studies have provided overwhelming evidence for a causal role of chronic HBV infection in the development of hepatocellular carcinoma, but the molecular mechanisms underlying virally-induced tumourigenesis remain largely debated. In the absence of a dominant oncogene encoded by the HBV genome, indirect roles have been proposed, including insertional activation of cellular cancer-related genes by HBV DNA integration, induction of genetic instability by viral integration or by the regulatory protein HBx, and long-term effects of viral proteins in enhancing immune-mediated liver disease. Recent genetic studies indicate that HBV-related tumours display a distinctive profile with a high rate of chromosomal alterations and low frequency of beta-catenin mutations. This review will discuss the evidence implicating chronic HBV infection as a causal risk factor of primary liver cancer. It will also discuss the molecular mechanisms that are critical for the tumourigenic process due to long lasting infection with HBV.

Tbx3 is a downstream target of the Wnt/beta-catenin pathway and a critical mediator of beta-catenin survival functions in liver cancer.

Tbx3 encodes a transcriptional repressor that is important for diverse patterning events during development, and Tbx3 mutation in humans causes the ulnar-mammary syndrome. Here, we describe the identification of Tbx3 in array-based search for genes downstream Wnt/beta-catenin that are implicated in liver tumorigenesis. Overexpression of Tbx3 is closely associated with the mutational status of beta-catenin in murine liver tumors induced by Myc as well as in human hepatocellular carcinomas and hepatoblastomas. Moreover, Tbx3 transcription is activated by ectopic expression of beta-catenin in mouse liver and in human tumor cell lines. Evidence that Tbx3 transcription is directly regulated by beta-catenin is provided by chromatin immunoprecipitation and reporter assays. Although HepG2 cells stably transfected with Tbx3 display moderately enhanced growth rate, the dominant negative mutant Tbx3-Y149S drastically inhibits hepatoma cell growth in vitro and in vivo. Moreover, small interfering RNAs (siRNA) directed against Tbx3 inhibit anchorage-independent growth of liver and colon carcinoma cells. We further show that inhibition of Tbx3 expression by specific siRNAs blocks beta-catenin-mediated cell survival and renders cells sensitive to doxorubicin-induced apoptosis. Conversely, ectopic expression of Tbx3 inhibits apoptosis induced by beta-catenin depletion. Marked overexpression of Tbx3 in a subset of hepatoblastomas is associated with chemotherapy-resistant phenotype and unfavorable patient outcome. These results reveal an unsuspected role of Tbx3 as a mediator of beta-catenin activities on cell proliferation and survival and as an important player in liver tumorigenesis.

Hepatitis B virus replicating in hepatocellular carcinoma encodes HBx variants with preserved ability to antagonize restriction by Smc5/6.

Hepatitis B virus infection is a major cause of liver diseases including hepatocellular carcinoma (HCC). The viral regulatory protein HBx is essential for viral replication and has been involved in the development of HCC. Recently, we characterized a subset of HCCs that replicate HBV. Our aim was to characterize HBx encoded by the full-length HBV DNA (cccDNA) in HCC and non-HCC liver. HBx genes were amplified and sequenced from eight paired HCC and non-HCC tissues in which HBV cccDNA and pgRNA were both present. Sequence analyses identified twelve amino acid positions mutated between HCC and non-HCC liver, and detected in at least three cases. We next assessed the impact of these mutations on HBx function by testing their transcriptional activity. We examined their ability to rescue the transcription of HBV virus deficient for HBx in differentiated HepaRG cells and to induce Smc5/6 degradation, which is mandatory for viral replication. We assessed their capacity to activate a CREB-dependent reporter. Finally we analyzed their growth suppressive activity using colony formation assays. Our results showed that most HBx variants isolated from HCC retain their ability to support HBV cccDNA transcription and to degrade Smc5/6. Strikingly, HCC specific HBx variants are impaired in their antiproliferative activity, which may be detrimental for tumor growth. In conclusion, in contrast to previous observations that tumor HBx variants lack transcriptional activity, we showed here that HBx variants have retained their ability to counteract Smc5/6 and thus to activate cccDNA transcription although they tend to lose antiproliferative activity.

A potential robust antiviral defense state in the common vampire bat: Expression, induction and molecular characterization of the three interferon-stimulated genes -OAS1, ADAR1 and PKR.

Bats are known to harbor many zoonotic viruses, some of which are pathogenic to other mammals while they seem to be harmless in bats. As the interferon (IFN) response represents the first line of defense against viral infections in mammals, it is hypothesized that activation of the IFN system is one of the mechanisms enabling bats to co-exist with viruses. We have previously reported induction of type I IFN in a cell line from the common vampire bat, Desmodus rotundus, upon polyinosinic:polycytidylic acid (poly(I:C)) stimulation. To deepen our knowledge on D. rotundus' IFN-I antiviral response, we molecularly characterized three interferon-stimulated genes (ISGs), OAS1, PKR and ADAR1, closely implicated in the IFN-I antiviral response, and tested their functionality in our cellular model. We first found that D. rotundus encoded two OAS1 paralogs, OAS1a and OAS1b, and that the functional domains of the four ISGs characterized were highly conserved with those of other mammals. Despite their significant transcription level in the absence of stimulation, the transcription of the four ISGs characterized was enhanced by poly(I:C). In addition, the transcription of OAS1a and OAS1b appears to be differentially regulated. These findings demonstrate an active ISG antiviral response in D. rotundus in which OAS1b may play an important role.