Antigenomic RNA Research Articles

Analysis of Replication, Cell Division-Mediated Spread, and HBV Envelope Protein-Dependent Pseudotyping of Three Mammalian Delta-like Agents.

The human hepatitis delta virus (HDV) is a satellite RNA virus that depends on hepatitis B virus (HBV) surface proteins (HBsAg) to assemble into infectious virions targeting the same organ (liver) as HBV. Until recently, the evolutionary origin of HDV remained largely unknown. The application of bioinformatics on whole sequence databases lead to discoveries of HDV-like agents (DLA) and shed light on HDV's evolution, expanding our understanding of HDV biology. DLA were identified in heterogeneous groups of vertebrates and invertebrates, highlighting that the evolution of HDV, represented by eight distinct genotypes, is broader and more complex than previously foreseen. In this study, we focused on the characterization of three mammalian DLA discovered in woodchuck (Marmota monax), white-tailed deer (Odocoileus virginianus), and lesser dog-like bat (Peropteryx macrotis) in terms of replication, cell-type permissiveness, and spreading pathways. We generated replication-competent constructs expressing 1.1-fold over-length antigenomic RNA of each DLA. Replication was initiated by transfecting the cDNAs into human (HuH7, HeLa, HEK293T, A549) and non-human (Vero E6, CHO, PaKi, LMH) cell lines. Upon transfection and replication establishment, none of the DLA expressed a large delta antigen. A cell division-mediated viral amplification assay demonstrated the capability of non-human DLA to replicate and propagate in hepatic and non-hepatic tissues, without the requirement of envelope proteins from a helper virus. Remarkably L-HDAg but not S-HDAg from HDV can artificially mediate envelopment of WoDV and DeDV ribonucleoproteins (RNPs) by HBsAg to form infectious particles, as demonstrated by co-transfection of HuH7 cells with the respective DLA expression constructs and a plasmid encoding HBV envelope proteins. These chimeric viruses are sensitive to HDV entry inhibitors and allow synchronized infections for comparative replication studies. Our results provide a more detailed understanding of the molecular biology, evolution, and virus-host interaction of this unique group of animal viroid-like agents in relation to HDV.

Analysis of Negative-Strand RNA Viruses by RT-qPCR: Rift Valley Fever Virus and Toscana Virus.

RT-qPCR allows the detection of viruses and the monitoring of viral replication. This technique was extensively employed during the SARS-CoV-2 pandemic, where it demonstrated its efficiency and robustness. Here we describe the analysis of Rift Valley fever and Toscana virus infections over time, achieved through the RT-qPCR quantification of the viral genome. We further elaborate on the method to discriminate between genomic and antigenomic viral RNAs by using primers specific for each strand during the reverse transcription step.

Autochthonous Dengue Fever in 2 Patients, Rome, Italy.

Since August 2023, outbreaks of dengue virus (DENV) infection have occurred in Italy. We report 2 autochthonous case-patients and their extended follow-up. Despite persistent DENV detected in blood by PCR, results for antigenomic DENV RNA were negative after day 5, suggesting that a 5-day isolation period is adequate to avoid secondary cases.

Structures of the promoter-bound respiratory syncytial virus polymerase

The respiratory syncytial virus (RSV) polymerase is a multifunctional RNA-dependent RNA polymerase composed of the large (L) protein and the phosphoprotein (P). It transcribes the RNA genome into ten viral mRNAs and replicates full-length viral genomic and antigenomic RNAs1. The RSV polymerase initiates RNA synthesis by binding to the conserved 3′-terminal RNA promoters of the genome or antigenome2. However, the lack of a structure of the RSV polymerase bound to the RNA promoter has impeded the mechanistic understanding of RSV RNA synthesis. Here we report cryogenic electron microscopy structures of the RSV polymerase bound to its genomic and antigenomic viral RNA promoters, representing two of the first structures of an RNA-dependent RNA polymerase in complex with its RNA promoters in non-segmented negative-sense RNA viruses. The overall structures of the promoter-bound RSV polymerases are similar to that of the unbound (apo) polymerase. Our structures illustrate the interactions between the RSV polymerase and the RNA promoters and provide the structural basis for the initiation of RNA synthesis at positions 1 and 3 of the RSV promoters. These structures offer a deeper understanding of the pre-initiation state of the RSV polymerase and could aid in antiviral research against RSV.

Farnesoid X receptor alpha ligands inhibit HDV in vitro replication and virion infectivity.

HDV, a satellite of HBV, is responsible for the most severe form of human viral hepatitis, for which curative therapy is still awaited. Both HBV and HDV use the hepatic transporter of bile acids (ie, Na+-taurocholate cotransporting polypeptide) to enter hepatocytes. We have previously shown that ligands of the farnesoid-X-receptor alpha (FXR), a master regulator of bile acids metabolism, inhibit HBV replication. Here we asked whether FXR ligands can also control HDV infection. In vitro HDV monoinfections or HDV/HBV coinfections and superinfections were performed in differentiated HepaRG cells (dHepaRG) and primary human hepatocytes. Following treatment with FXR ligands, HDV RNAs and antigens were analyzed by RT-qPCR, northern blot, immunofluorescence, and western blot. Virus secretion was studied by RNA quantification in supernatants, and the infectivity of secreted HDV particles was measured by reinfection of naive HuH7.5-Na+-taurocholate cotransporting polypeptide cells. In HDV/HBV superinfection models, a 10-day treatment with FXR ligand GW4064 decreased intracellular HDV RNAs by 60% and 40% in dHepaRG cells and primary human hepatocytes, respectively. Both HDV genomic and antigenomic RNAs were affected by treatment, which also reduced the amount of intracellular delta antigen. This antiviral effect was also observed in HDV monoinfected dHepaRG cells, abolished by FXR loss of function, and reproduced with other FXR ligands. In HBV/HDV coinfected dHepaRG cells, HDV secretion was decreased by 60% and virion-specific infectivity by >95%. FXR ligands both inhibit directly (ie, independently of anti-HBV activity) and indirectly (ie, dependently of anti-HBV activity) the replication, secretion, and infectivity of HDV. The overall anti-HDV activity was superior to that obtained with interferon-α, highlighting the therapeutic potential of FXR ligands in HDV-infected patients.

Nonclinical pharmacokinetics and biodistribution of VSV-GP using methods to decouple input drug disposition and viral replication

Viral replication places oncolytic viruses (OVs) in a unique niche in the field of drug pharmacokinetics (PK) as their self-amplification obscures exposure-response relationships. Moreover, standard bioanalytical techniques are unable to distinguish the input from replicated drug products. Here, we combine two novel approaches to characterize PK and biodistribution (BD) after systemic administration of vesicular stomatitis virus pseudotyped with lymphocytic choriomeningitis virus glycoprotein (VSV-GP) in healthy mice. First: to decouple input drug PK/BD versus replication PK/BD, we developed and fully characterized a replication-incompetent tool virus that retained all other critical attributes of the drug. We used this approach to quantify replication in blood and tissues and to determine its impact on PK and BD. Second: to discriminate the genomic and antigenomic viral RNA strands contributing to replication dynamics in tissues, we developed an in situ hybridization method using strand-specific probes and assessed their spatiotemporal distribution in tissues. This latter approach demonstrated that distribution, transcription, and replication localized to tissue-resident macrophages, indicating their role in PK and BD. Ultimately, our study results in a refined PK/BD profile for a replicating OV, new proposed PK parameters, and deeper understanding of OV PK/BD using unique approaches that could be applied to other replicating vectors.

The mutational spectrum of SARS-CoV-2 genomic and antigenomic RNA.

The raw material for viral evolution is provided by intra-host mutations occurring during replication, transcription or post-transcription. Replication and transcription of Coronaviridae proceed through the synthesis of negative-sense 'antigenomes' acting as templates for positive-sense genomic and subgenomic RNA. Hence, mutations in the genomes of SARS-CoV-2 and other coronaviruses can occur during (and after) the synthesis of either negative-sense or positive-sense RNA, with potentially distinct patterns and consequences. We explored for the first time the mutational spectrum of SARS-CoV-2 (sub)genomic and anti(sub)genomic RNA. We use a high-quality deep sequencing dataset produced using a quantitative strand-aware sequencing method, controlled for artefacts and sequencing errors, and scrutinized for accurate detection of within-host diversity. The nucleotide differences between negative- and positive-sense strand consensus vary between patients and do not show dependence on age or sex. Similarities and differences in mutational patterns between within-host minor variants on the two RNA strands suggested strand-specific mutations or editing by host deaminases and oxidative damage. We observe generally neutral and slight negative selection on the negative strand, contrasting with purifying selection in ORF1a, ORF1b and S genes of the positive strand of the genome.

The hepatitis delta virus and chronic hepatitis D.

The hepatitis delta virus and chronic hepatitis D.

Rescue of the first alphanucleorhabdovirus entirely from cloned complementary DNA: An efficient vector for systemic expression of foreign genes in maize and insect vectors.

Recent reverse genetics technologies have enabled genetic manipulation of plant negative-strand RNA virus (NSR) genomes. Here, we report construction of an infectious clone for the maize-infecting Alphanucleorhabdovirus maydis, the first efficient NSR vector for maize. The full-length infectious clone was established using agrobacterium-mediated delivery of full-length maize mosaic virus (MMV) antigenomic RNA and the viral core proteins (nucleoprotein N, phosphoprotein P, and RNA-directed RNA polymerase L) required for viral transcription and replication into Nicotiana benthamiana. Insertion of intron 2 ST-LS1 into the viral L gene increased stability of the infectious clone in Escherichia coli and Agrobacterium tumefaciens. To monitor virus infection in vivo, a green fluorescent protein (GFP) gene was inserted in between the N and P gene junctions to generate recombinant MMV-GFP. Complementary DNA (cDNA) clones of MMV-wild type (WT) and MMV-GFP replicated in single cells of agroinfiltrated N.benthamiana. Uniform systemic infection and high GFP expression were observed in maize inoculated with extracts of the infiltrated N.benthamiana leaves. Insect vectors supported virus infection when inoculated via feeding on infected maize or microinjection. Both MMV-WT and MMV-GFP were efficiently transmitted to maize by planthopper vectors. The GFP reporter gene was stable in the virus genome and expression remained high over three cycles of transmission in plants and insects. The MMV infectious clone will be a versatile tool for expression of proteins of interest in maize and cross-kingdom studies of virus replication in plant and insect hosts.

Ebola virus infection induces a delayed type I IFN response in bystander cells and the shutdown of key liver genes in human iPSC-derived hepatocytes.

SummaryLiver damage and an exacerbated inflammatory response are hallmarks of Ebola virus (EBOV) infection. Little is known about the intrinsic response to infection in human hepatocytes and their contribution to inflammation. Here, we present an induced pluripotent stem cell (iPSC)-derived hepatocyte-like cell (HLC) platform to define the hepato-intrinsic response to EBOV infection. We used this platform to show robust EBOV infection, with characteristic ultrastructural changes and evidence for viral replication. Transcriptomics analysis revealed a delayed response with minimal early transcriptomic changes, followed by a general downregulation of hepatic function and upregulation of interferon signaling, providing a potential mechanism by which hepatocytes participate in disease severity and liver damage. Using RNA-fluorescence in situ hybridization (FISH), we showed that IFNB1 and CXCL10 were mainly expressed in non-infected bystander cells. We did not observe an inflammatory signature during infection. In conclusion, iPSC-HLCs are an immune competent platform to study responses to EBOV infection.

Detection of Japanese Encephalitis Virus RNA in Host-Questing Ticks in Japan, 2019-2020.

Japanese encephalitis virus (JEV), a mosquito-borne virus, causes severe clinical symptoms in humans in the Asian-Pacific region, where it circulates in a primary transmission cycle among Culex tritaeniorhynchus mosquitoes, domestic swine (Sus scrofa domesticus), and wading birds. We report here an anomalous result that mosquito-borne JEV was detected in unfed host-questing ticks collected from the field in Japan. JEV genomic RNA was detected in four pools of Haemaphysalis flava nymphs collected in November and December 2019, and March 2020, when Cx. tritaeniorhynchus adults were not presumed to be active. Moreover, JEV antigenomic RNA was detected in some JEV-positive tick samples, suggesting virus replication in ticks. However, taken together with no infectious virus isolated, the possibility that the antigenomic RNA was derived from the undigested bloodmeal source in ticks cannot be ruled out. Thus, the role of the ticks as a natural reservoir for JEV remains to be confirmed.

Interaction of HnRNP F with the guanine-rich segments in viral antigenomic RNA enhances porcine reproductive and respiratory syndrome virus-2 replication

BackgroundHeterogeneous nuclear ribonucleoprotein (HnRNP) F is a member of HnRNP family proteins that participate in splicing of cellular newly synthesized mRNAs by specifically recognizing tandem guanine-tracts (G-tracts) RNA sequences. Whether HnRNP F could recognize viral-derived tandem G-tracts and affect virus replication remain poorly defined.MethodsThe effect of HnRNP F on porcine reproductive and respiratory syndrome virus (PRRSV) propagation was evaluated by real-time PCR, western blotting, and plaque-forming unit assay. The association between HnRNP F and PRRSV guanine-rich segments (GRS) were analyzed by RNA pulldown and RNA immunoprecipitation. The expression pattern of HnRNP F was investigated by western blotting and nuclear and cytoplasmic fractionation.ResultsKnockdown of endogenous HnRNP F effectively blocks the synthesis of viral RNA and nucleocapsid (N) protein. Conversely, overexpression of porcine HnRNP F has the opposite effect. Moreover, RNA pulldown and RNA immunoprecipitation assays reveal that the qRMM1 and qRRM2 domains of HnRNP F recognize the GRS in PRRSV antigenomic RNA. Finally, HnRNP F is redistributed into the cytoplasm and forms a complex with guanine-quadruplex (G4) helicase DHX36 during PRRSV infection.ConclusionsThese findings elucidate the potential functions of HnRNP F in regulating the proliferation of PRRSV and contribute to a better molecular understanding of host-PRRSV interactions.

Prevalence of Occult Hepatitis C Virus Infection in Egyptian Patients with Lymphoma: A New Vision.

Occult hepatitis C virus infection (OCI) is the absence of HCV RNA in serum and the presence of actively replicating HCV RNA in hepatocytes and peripheral blood mononuclear cells (PBMCs), as evidenced by the presence of antigenomic negative sense single-stranded RNA. This study aimed to determine the prevalence of OCI in Egyptian lymphoma patients and assess changes in biochemical parameters in patients with confirmed OCI. The current study was conducted on 100 apparently healthy subjects as control group and 100 patients with lymphoma as a case group. HCV RNA was extracted and detected in both plasma and PBMCs using qRT-PCR. Total protein, albumin, ALT, AST, and total and direct bilirubin were measured in serum. OCI was detected in 6% of the patient group. OCI patients had lower levels of total protein and serum albumin and higher ALT and AST compared with lymphoma patients without OCI. Our study revealed that six out of 100 patients with lymphoma disorders had occult HCV infection (6%). Therefore, the possibility of this infection should be considered in patients with lymphoma.

αVβ3 Integrin Expression Is Essential for Replication of Mosquito and Tick-Borne Flaviviruses in Murine Fibroblast Cells.

The Flavivirus genus includes a number of important viruses that are pathogenic to humans and animals and are responsible for outbreaks across the globe. Integrins, a family of heterodimeric transmembrane molecules expressed in all nucleated cells mediate critical functions of cell physiology and cell cycle. Integrins were previously postulated to be involved in flavivirus entry and to modulate flavivirus replication efficiency. In the present study, mouse embryonic fibroblasts (MEF), lacking the expression of αVβ3 integrin (MEF-αVβ3−/−), were infected with four different flaviviruses, namely yellow fever virus (YFV), West Nile virus (WNV), Usutu virus (USUV) and Langat virus (LGTV). The effects of the αVβ3 integrin absence in double-knockout MEF-αVβ3−/− on flavivirus binding, internalization and replication were compared to the respective wild-type cells. Binding to the cell surface for all four flaviviruses was not affected by the ablation of αVβ3 integrin, whereas internalization of USUV and WNV was slightly affected by the loss of αVβ3 integrin expression. Most interestingly, the deletion of αVβ3 integrin strongly impaired replication of all flaviviruses with a reduction of up to 99% on virus yields and a strong reduction on flavivirus anti-genome RNA synthesis. In conclusion, our results demonstrate that αVβ3 integrin expression in flavivirus-susceptible cell lines enhances the flavivirus replication.

Inducers of the NF-κB pathways impair hepatitis delta virus replication and strongly decrease progeny infectivity in vitro

Background & AimsHDV superinfection of chronically HBV-infected patients is the most aggressive form of chronic viral hepatitis, with an accelerated progression towards fibrosis/cirrhosis and increased risk of liver failure, hepatocellular carcinoma, and death. While HDV infection is not susceptible to available direct anti-HBV drugs, suboptimal responses are obtained with interferon-α-based therapies, and the number of investigational drugs remains limited. We therefore analyzed the effect of several innate immune stimulators on HDV replication in infected hepatocytes.MethodsWe used in vitro models of HDV and HBV infection based on primary human hepatocytes (PHHs) and the non-transformed HepaRG cell line that are relevant to explore new innate immune therapies.ResultsWe describe here, for the first time, anti-HDV effects of Pam3CSK4 and BS1, agonists of Toll-like receptor (TLR)-1/2, and the lymphotoxin-β receptor (LTβR), respectively. Both types of agonists induced dose-dependent reductions of total intracellular HDV genome and antigenome RNA and of HDV protein levels, without toxicity in cells monoinfected with HDV or co/superinfected with HBV. Moreover, both molecules negatively affected HDV progeny release and strongly decreased their specific infectivity. The latter effect is particularly important since HDV is thought to persist in humans through constant propagation.ConclusionsImmune-modulators inducing NF-κB pathways in hepatocytes can inhibit HDV replication and should be further evaluated as a possible therapeutic approach in chronically HBV/HDV-infected patients.Lay summaryHepatitis delta virus causes the most severe form of viral hepatitis. Despite positive recent developments, effective treatments remain a major clinical need. Herein, we show that immune-modulators that trigger the NF-κB pathways could be effective for the treatment of hepatitis delta infections.

Hepatitis Delta Virus Genome RNA Synthesis Initiates at Position 1646 with a Nontemplated Guanosine.

Hepatitis delta virus (HDV) is a significant human pathogen that causes acute and chronic liver disease; there is no licensed therapy. HDV is a circular negative-sense single-stranded RNA (ssRNA) virus that produces three RNAs in infected cells, genome, antigenome, and mRNA; the latter encodes hepatitis delta antigen (HDAg), the viral protein. These RNAs are synthesized by host DNA-dependent RNA polymerase acting as an RNA-dependent RNA polymerase. Although HDV genome RNA accumulates to high levels in infected cells, the mechanism by which this process occurs remains poorly understood. For example, the nature of the 5' end of the genome, including the synthesis start site and its chemical composition, is not known. Analysis of this process has been challenging because the initiation site is part of an unstable precursor in the rolling-circle mechanism by which HDV genome RNA is synthesized. In this study, circular HDV antigenome RNAs synthesized in vitro were used to directly initiate HDV genome RNA synthesis in transfected cells, thus enabling the detection of the 5' end of the genome RNA. The 5' end of this RNA is capped, as expected for a polymerase II product. Initiation begins at position 1646 on the genome, which is located near the loop end proximal to the start site for HDAg mRNA synthesis. Unexpectedly, synthesis begins with a guanosine that is not conventionally templated by the HDV RNA. IMPORTANCE Hepatitis delta virus (HDV) is a unique virus that causes severe liver disease. It uses host RNA polymerase II to copy its circular RNA genome in a unique and poorly understood process. Although the virus RNA accumulates to high levels within infected cells, it is not known how the synthesis of the viral RNA begins or even where on the genome synthesis starts. Here, we identify the start site for the initiation of HDV genome RNA synthesis as position 1646, which is at one end of the closed-hairpin-like structure of the viral RNA. The 5' end of the RNA is capped, as expected for polymerase II products. However, RNA synthesis begins with a guanosine that is not present in the genome. Thus, although HDV uses polymerase II to synthesize the viral genome, some details of the initiation process are different. These differences could be important for successfully targeting virus replication.

The Borna Disease Virus 2 (BoDV-2) Nucleoprotein Is a Conspecific Protein That Enhances BoDV-1 RNA-Dependent RNA Polymerase Activity.

An RNA virus-based episomal vector (REVec) based on Borna disease virus 1 (BoDV-1) is a promising viral vector that achieves stable and long-term gene expression in transduced cells. However, the onerous procedure of reverse genetics used to generate an REVec is one of the challenges that must be overcome to make REVec technologies practical for use. In this study, to resolve the problems posed by reverse genetics, we focused on BoDV-2, a conspecific virus of BoDV-1 in the Mammalian 1 orthobornavirus. We synthesized the BoDV-2 nucleoprotein (N) and phosphoprotein (P) according to the reference sequences and evaluated their effects on the RNA polymerase activity of the BoDV-1 large protein (L) and viral replication. In the minireplicon assay, we found that BoDV-2 N significantly enhanced BoDV-1 polymerase activity and that BoDV-2 P supported further enhancement of this activity by N. A single amino acid substitution assay identified serine at position 30 of BoDV-2 N and alanine at position 24 of BoDV-2 P as critical amino acid residues for the enhancement of BoDV-1 polymerase activity. In reverse genetics, conversely, BoDV-2 N alone was sufficient to increase the rescue efficiency of the REVec. We showed that the REVec can be rescued directly from transfected 293T cells by using BoDV-2 N as a helper plasmid without cocultivation with Vero cells and following several weeks of passage. In addition, a chimeric REVec harboring the BoDV-2 N produced much higher levels of transgene mRNA and genomic RNA than the wild-type REVec in transduced cells. Our results contribute to not only improvements to the REVec system but also to understanding of the molecular regulation of orthobornavirus polymerase activity. IMPORTANCE Borna disease virus 1 (BoDV-1), a prototype virus of the species Mammalian 1 orthobornavirus, is a nonsegmented negative-strand RNA virus that persists in the host nucleus. The nucleoprotein (N) of BoDV-1 encapsidates genomic and antigenomic viral RNA, playing important roles in viral transcription and replication. In this study, we demonstrated that the N of BoDV-2, another genotype in the species Mammalian 1 orthobornavirus, can participate in the viral ribonucleoprotein complex of BoDV-1 and enhance the activity of BoDV-1 polymerase (L) in both the BoDV-1 minireplicon assay and reverse genetics system. Chimeric recombinant BoDV-1 expressing BoDV-2 N but not BoDV-1 N showed higher transcription and replication levels, whereas the propagation and infectious particle production of the chimeric virus were comparable to those of wild-type BoDV-1, suggesting that the level of viral replication in the nucleus is not directly involved in the progeny virion production of BoDVs. Our results demonstrate a molecular mechanism of bornaviral polymerase activity, which will contribute to further development of vector systems using orthobornaviruses.

Dual Promoters Improve the Rescue of Recombinant Measles Virus in Human Cells.

Reverse genetics is a technology that allows the production of a virus from its complementary DNA (cDNA). It is a powerful tool for analyzing viral genes, the development of novel vaccines, and gene delivery vectors. The standard reverse genetics protocols are laborious, time-consuming, and inefficient for negative-strand RNA viruses. A new reverse genetics platform was established, which increases the recovery efficiency of the measles virus (MV) in human 293-3-46 cells. The novel features compared with the standard system involving 293-3-46 cells comprise (a) dual promoters containing the RNA polymerase II promoter (CMV) and the bacteriophage T7 promoter placed in uni-direction on the same plasmid to enhance RNA transcription; (b) three G nucleotides added just after the T7 promoter to increase the T7 RNA polymerase activity; and (c) two ribozymes, the hairpin hammerhead ribozyme (HHRz), and the hepatitis delta virus ribozyme (HDVrz), were used to cleavage the exact termini of the antigenome RNA. Full-length antigenome cDNA of MV of the wild type IC323 strain or the vaccine AIK-C strain was inserted into the plasmid backbone. Both virus strains were easily rescued from their respective cloned cDNA. The rescue efficiency increased up to 80% compared with the use of the standard T7 rescue system. We assume that this system might be helpful in the rescue of other human mononegavirales.

Significantly Improved Recovery of Recombinant Sonchus Yellow Net Rhabdovirus by Expressing the Negative-Strand Genomic RNA

Generation of recombinant negative-stranded RNA viruses (NSVs) from plasmids involves in vivo reconstitution of biologically active nucleocapsids and faces a unique antisense problem where the negative-sense viral genomic RNAs can hybridize to viral messenger RNAs. To overcome this problem, a positive-sense RNA approach has been devised through expression of viral antigenomic (ag)RNA and core proteins for assembly of antigenomic nucleocapsids. Although this detour strategy works for many NSVs, the process is still inefficient. Using Sonchus yellow net rhabdovirus (SYNV) as a model; here, we develop a negative-sense genomic RNA-based approach that increased rescue efficiency by two orders of magnitude compared to the conventional agRNA approach. The system relied on suppression of double-stranded RNA induced antiviral responses by co-expression of plant viruses-encoded RNA silencing suppressors or animal viruses-encoded double-stranded RNA antagonists. With the improved approach, we were able to recover a highly attenuated SYNV mutant with a deletion in the matrix protein gene which otherwise could not be rescued via the agRNA approach. Reverse genetics analyses of the generated mutant virus provided insights into SYNV virion assembly and morphogenesis. This approach may potentially be applicable to other NSVs of plants or animals.

Murine hepatocytes do not support persistence of Hepatitis D virus mono-infection in vivo.

As a result of the limited availability of in vivo models for hepatitis D virus (HDV), treatment options for HDV chronically infected patients are still scant. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as HDV entry receptor has enabled the development of new infection models. To comparatively assess the efficacy and persistence of HDV mono-infection in murine and human hepatocytes in vivo. Mice with humanized NTCP (hNTCPed84-87 mice) were generated by editing amino acid residues 84-87 of murine NTCP in C57BL/6J mice. HDV infection was assessed in hNTCPed84-87 mice and in immune deficient uPA/SCID/beige (USB) mice, whose livers were reconstituted with human or murine (hNTCPed84-87 ) hepatocytes. Livers were analysed between 5 and 42days post-HDV inoculation by qRT-PCR, immunofluorescence and RNA in situ hybridization (ISH). hNTCPed84-87 mice could be infected with HDV genotype 1 or 3. ISH analysis demonstrated the presence of antigenomic HDV RNA positive murine hepatocytes with both genotypes, proving initiation of HDV replication. Strikingly, murine hepatocytes cleared HDV within 21days both in immunocompetent hNTCPed84-87 mice and in immunodeficient USB mice xenografted with murine hepatocytes. In contrast, HDV infection remained stable for at least 42days in human hepatocytes. Intrinsic innate responses were not enhanced in any of the HDV mono-infected cells and livers. These findings suggest that in addition to NTCP, further species-specific factors limit HDV infection efficacy and persistence in murine hepatocytes. Identifying such species barriers may be crucial to develop novel potential therapeutic targets of HDV.