Search Results (72)

Hendra virus (HeV) is a highly pathogenic member of the Henipavirus genus (family Paramyxoviridae, order Mononegavirales), for which all basic replication processes are located in the cytoplasm. The HeV matrix (M) protein plays essential roles in viral assembly and budding at the plasma membrane, but also undergoes dynamic nuclear and nucleolar trafficking, accumulating in nucleoli early in infection, before relocalising to the plasma membrane. We previously showed that M targets sub-nucleolar compartments—the fibrillar centre (FC) and dense fibrillar component (DFC)—to modulate rRNA biogenesis by mimicking a process occurring during a nucleolar DNA-damage response (DDR). Here, we show that M protein sub-nucleolar localisation is regulated by ubiquitination, which controls its redistribution between the FC-DFC and granular component (GC). The mutagenesis of a conserved lysine (K258) reported to undergo ubiquitination, combined with the pharmacological modulation of ubiquitination, indicated that a positive charge at K258 is required for M localisation to the FC-DFC, while ubiquitination regulates subsequent egress from the FC-DFC to the GC. M proteins from multiple Henipaviruses exhibited similar ubiquitin-dependent sub-nucleolar trafficking, indicating a conserved mechanism. These findings reveal a novel mechanism regulating viral protein transport between phase-separated sub-nucleolar compartments and highlight ubiquitination as a key modulator of intra-nucleolar trafficking. Full article

Hepatitis E, caused by the hepatitis E virus (HEV), is a zoonotic disease that extends beyond hepatocellular necrosis to replicate in multiple organs. While most infections are self-limiting, HEV infection during pregnancy is associated with severe outcomes, including acute liver failure, preterm delivery, and miscarriage, with the mechanisms underlying this high pathogenicity remaining poorly understood. This study established a pregnant tree shrew model with a late-stage HEV infection and a cellular model using zoonotic HEV genotypes GT3 and GT4 to investigate the effects of estrogen on HEV replication. Results showed that negative-strand RNA detection revealed replicative intermediates in feces and tissues during the acute phase, with peak viral loads occurring within one week and the highest titers in bile. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels rose at 3 days post-inoculation (DPI), peaking at 7 DPI. Elevated estrogen levels post-miscarriage correlated with increased viral loads, a trend mirrored in cell culture models showing linear relationships between estrogen and viral replication. Histopathology demonstrated viral hepatitis lesions in liver tissues and abnormalities in the uterus, ovaries, and brain, including hydropic degeneration, neuronal disruption, and granulosa cell necrosis. This study developed a pregnant tree shrew model for HEV infection, providing a robust tool for exploring pathogenic mechanisms during pregnancy and genotype-specific differences in zoonotic HEV pathogenicity. These findings offer new insights into the role of estrogen in HEV replication and its contribution to adverse pregnancy outcomes. Full article

Hepatitis E virus (HEV) is a positive-sense, single-stranded RNA virus that poses a significant public health risk, yet its study is hindered by the complexity of conventional RNA-based reverse genetics systems. These systems require multiple steps, including genome cloning, in vitro transcription, and capping, making them labor-intensive and susceptible to RNA degradation. In this study, we developed a single-step, plasmid-based HEV expression system that enabled direct intracellular transcription of the full-length HEV genome under a cytomegalovirus immediate-early (CMV-IE) promoter. The viral genome was flanked by hammerhead (HH) and hepatitis delta virus (HDV) ribozymes to ensure precise self-cleavage and the generation of authentic 5′ and 3′ termini. This system successfully supported HEV genome replication, viral protein expression, and progeny virion production at levels comparable to those obtained using in vitro-transcribed, capped HEV RNA. Additionally, a genetic marker introduced into the plasmid construct was stably retained in progeny virions, demonstrating the feasibility of targeted genetic modifications. However, plasmid-derived HEV exhibited delayed replication kinetics, likely due to the absence of an immediate 5′ cap. Attempts to enhance capping efficiency through co-expression of the vaccinia virus capping enzyme failed to improve HEV replication, suggesting that alternative strategies, such as optimizing the promoter design for capping, may be required. This plasmid-based HEV reverse genetics system simplifies the study of HEV replication and pathogenesis and provides a versatile platform for the genetic engineering of the HEV genome. Full article

DDX3 is an ATP-dependent RNA helicase that is involved in multiple cellular activities, including RNA metabolism and innate immunity. DDX3 is known to assist the replication of some viruses while restricting others through its direct interaction with viral proteins. However, the role of DDX3 in the replication of the hepatitis E virus (HEV) is unknown. In this study, DDX3 was shown to interact with the HEV capsid protein and provide an important role in HEV replication. The DDX3 C-terminal domain was demonstrated to interact with the capsid protein. The depletion of DDX3 led to a significant reduction in HEV replication. Also, the ATPase motif of DDX3 was shown to be required in HEV replication as an ATPase-null mutant DDX3 failed to rescue the viral replication in the DDX3-depleted cells. These results demonstrate a pro-viral role of DDX3 in HEV replication, providing further insights on the virus–cell interactions. Full article

The emergence of RNA viruses driven by global population growth and international trade highlights the urgent need for effective antiviral agents that can inhibit viral replication. Nucleoside analogs, which mimic natural nucleotides, have shown promise in targeting RNA-dependent RNA polymerases (RdRps). Starting from protected 5-iodouridine, we report the synthesis of hitherto unknown C5-substituted-(1,3-diyne)-uridines nucleosides and their phosphoramidate prodrugs. The modifications at C5 include 4-(trifluoromethyl)benzene (a), 4-pentyl-benzene (b), 3,5-dimethoxy-benzene (c), 4-(trifluoromethoxy)benzene (d), 3-aniline (e), 4-pyridine (f), 3-thiophene (g), C₆H₁₃ (h), 2-pyrimidine (i), cyclopropyl (j), and phenyl (k) groups. These compounds were synthesized using Sonogashira palladium-catalyzed reactions and nickel–copper-catalyzed C-H activation between various alkynes, yielding between 25% and 67%. The antiviral activities of obtained compounds were measured through HTS against RNA viruses including influenza H1N1 and H3N2, human respiratory syncytial virus (RSV), SARS-CoV-2, Zika, hepatitis C virus (HCV), Hepatitis E virus (HEV), as well as against coronavirus (HCoV-229E). Unfortunately, none of them showed promising antiviral activity, with less than 85% inhibition observed in the cell viability screening of infected cells. Full article

Hepatitis E virus (HEV) exists in two distinct forms: a non-enveloped form (neHEV), which is present in feces and bile, and a quasi-enveloped form (eHEV), found in circulating blood and culture supernatants. This study aimed to elucidate the roles of Ras-associated binding 13 (Rab13) and protein kinase A (PKA) in the entry mechanisms of both eHEV and neHEV, utilizing small interfering RNA (siRNA) and chemical inhibitors. The results demonstrated that the entry of both viral forms is dependent on Rab13 and PKA. Further investigation into the involvement of tight junction (TJ) proteins revealed that the targeted knockdown of zonula occludens-1 (ZO-1) significantly impaired the entry of both eHEV and neHEV. In addition, in ZO-1 knockout (KO) cells inoculated with either viral form, HEV RNA levels in culture supernatants did not increase, even up to 16 days post-inoculation. Notably, the absence of ZO-1 did not affect the adsorption efficiency of eHEV or neHEV, nor did it influence HEV RNA replication. In cell-to-cell spread assays, ZO-1 KO cells inoculated with eHEV showed a lack of expression of HEV ORF2 and ORF3 proteins. In contrast, neHEV-infected ZO-1 KO cells showed markedly reduced ORF2 and ORF3 protein expression within virus-infected foci, compared to non-targeting knockout (NC KO) cells. These findings underscore the crucial role of ZO-1 in facilitating eHEV entry and mediating the cell-to-cell spread of neHEV in infected cells. Full article

Constructing artificial tertiary lymphoid structures (TLSs) opens new avenues for advancing cancer immunotherapy and personalized medicine by creating controllable immune niches. Mesenchymal stromal cells (MSCs) offer an ideal stromal source for such constructs, given their potent immunomodulatory abilities and accessibility. In this study, we explored the potential of adipose-derived MSCs to adopt TLS-supportive phenotypes and facilitate lymphocyte organization. Single-cell RNA sequencing revealed a distinct subpopulation of MSCs expressing key fibroblastic reticular cell (FRC)-associated markers, including IL-7, PDPN, and IL-15, though lacking follicular dendritic cell (FDC) markers. TNF-α stimulation, but not LTα2β1, further enhanced FRC marker expression (IL-7, PDPN, and ICAM1). Notably, in 3D spheroid co-culture with lymphocytes, MSCs upregulated additional FRC markers, specifically CCL21. Upon implantation into adipose tissue, MSC-lymphocyte organoids maintained structural integrity and showed extensive T-cell infiltration and partial vascularization after 15 days in vivo, although organized B-cell follicles and FDC markers were still lacking. These findings highlight MSCs’ intrinsic ability to adopt an FRC-like phenotype that supports T-cell and HEV organization, suggesting that further optimization, including genetic modification, may be needed to achieve an FDC phenotype and replicate the full architectural and functional complexity of TLSs. Full article

Since Mongolian gerbils are broadly susceptible to hepatitis E virus (HEV), including genotypes 1, 4, 5, and 8 (HEV-1, HEV-5, HEV-5, and HEV-8) and rat HEV, they are a useful small animal model for HEV. However, we have observed that the subtypes HEV-3k and HEV-3ra in genotype 3 HEV (HEV-3) were not infected efficiently in the gerbils. A small-animal model for HEV-3 is also needed since HEV-3 is responsible for major zoonotic HEV infections. To investigate whether gerbils can be used as animal models for other subtypes of HEV-3, we injected gerbils with five HEV-3 subtypes (HEV-3b, -3e, -3f, -3k, and -3ra) and compared the infectivity of the subtypes. We detected viral RNA in the gerbils’ feces. High titers of anti-HEV IgG antibodies in serum were induced in all HEV-3b/ch-, HEV-3f-, and HEV-3e-injected gerbils. Especially, the HEV-3e-injected animals released high levels of viruses into their feces for an extended period. The virus replication was limited in the HEV-3b/wb-injected and HEV-3k-injected groups. Although viral RNA was detected in HEV-3ra-injected gerbils, the copy numbers in fecal specimens were low; no antibodies were detected in the sera. These results indicate that although HEV-3′s infectivity in gerbils depends on the subtype and strain, Mongolian gerbils have potential as a small-animal model for HEV-3. A further comparison of HEV-3e with different genotype strains (HEV-4i and HEV-5) and different genera (rat HEV) revealed different ALT elevations among the strains, and liver damage occurred in HEV-4i- and HEV-5-infected but not HEV-3e- or rat HEV-infected gerbils, demonstrating variable pathogenicity across HEVs from different genera and genotypes in Mongolian gerbils. HEV-4i- and HEV-5-infected Mongolian gerbils might be candidate animal models to examine HEV’s pathogenicity. Full article

Hepatitis E virus (HEV) poses a significant global health threat, with an estimated 20 million infections occurring annually. Despite being a self-limiting illness, in most cases, HEV infection can lead to severe outcomes, particularly in pregnant women and individuals with pre-existing liver disease. In the absence of specific antiviral treatments, the exploration of RNAi interference (RNAi) as a targeted strategy provides valuable insights for urgently needed therapeutic interventions against Hepatitis E. We designed small interfering RNAs (siRNAs) against HEV, which target the helicase domain and the open reading frame 3 (ORF3). These target regions will reduce the risk of viral escape through mutations, as they belong to the most conserved regions in the HEV genome. The siRNAs targeting the ORF3 efficiently inhibited viral replication in A549 cells after HEV infection. Importantly, the siRNA was also highly effective at inhibiting HEV in the persistently infected A549 cell line, which provides a suitable model for chronic infection in patients. Furthermore, we showed that a 5′ triphosphate modification on the siRNA sense strand activates the RIG-I receptor, a cytoplasmic pattern recognition receptor that recognizes viral RNA. Upon activation, RIG-I triggers a signaling cascade, effectively suppressing HEV replication. This dual-action strategy, combining the activation of the adaptive immune response and the inherent RNAi pathway, inhibits HEV replication successfully and may lead to the development of new therapies. Full article

A wide range of virus-like particles (VLPs) is extensively employed as carriers to display various antigens for vaccine development to fight against different infections. The plant-produced truncated variant of the hepatitis E virus (HEV) coat protein is capable of forming VLPs. In this study, we demonstrated that recombinant fusion proteins comprising truncated HEV coat protein with green fluorescent protein (GFP) or four tandem copies of the extracellular domain of matrix protein 2 (M2e) of influenza A virus inserted at the Tyr485 position could be efficiently expressed in Nicotiana benthamiana plants using self-replicating vector based on the potato virus X genome. The plant-produced fusion proteins in vivo formed VLPs displaying GFP and 4M2e. Therefore, HEV coat protein can be used as a VLP carrier platform for the presentation of relatively large antigens comprising dozens to hundreds of amino acids. Furthermore, plant-produced HEV particles could be useful research tools for the development of recombinant vaccines against influenza. Full article

Hepatitis E virus (HEV) poses significant health concerns worldwide, particularly among people living with HIV (PLWHIV), due to an increased risk of chronic infection and progression to cirrhosis in individuals with low CD4 cell counts. This study aimed to investigate the prevalence, chronicity potential, and risk factors of HEV infection among PLWHIV in Greece, where data are currently absent. A synchronic multicentric study encompassing five major Greek university hospitals was executed over 24 months, recruiting 696 PLWHIV participants. The prevalence of HEV IgG antibodies was 16.5%, with 8.6% showing evidence of acute HEV infection (HEV IgM). Active viral replication (HEV RNA) was present in 2.3% of the study population. Longitudinal analysis revealed that of the 25 initially anti-HEV IgM-positive individuals, only 3 seroconverted to IgG positivity, and among those with prior HEV RNA positivity (16), none showed evidence of active replication in subsequent tests. Comparative subgroup analysis highlighted the lack of significant differences in HIV-related parameters between HEV seropositive and seronegative individuals. Laboratory evaluations generally showed no significant disparities across most parameters; however, a higher seropositivity for Hepatitis A was observed in the HEV-positive subgroup. Our findings highlight a considerable prevalence of HEV among PLWHIV in Greece, with no observed cases of chronicity. Full article

Hepatitis E virus (HEV) infection is typically a self-limiting, acute illness that spreads through the gastrointestinal tract but replicates in the liver. However, chronic infections are possible in immunocompromised individuals. The HEV virion has two shapes: exosome-like membrane-associated quasi-enveloped virions (eHEV) found in circulating blood or in the supernatant of infected cell cultures and non-enveloped virions (“naked”) found in infected hosts’ feces and bile to mediate inter-host transmission. Although HEV is mainly spread via enteric routes, it is unclear how it penetrates the gut wall to reach the portal bloodstream. Both virion types are infectious, but they infect cells in different ways. To develop personalized treatment/prevention strategies and reduce HEV impact on public health, it is necessary to decipher the entry mechanism for both virion types using robust cell culture and animal models. The contemporary knowledge of the cell entry mechanism for these two HEV virions as possible therapeutic target candidates is summarized in this narrative review. Full article

Paslahepevirus balayani and Rocahepevirus ratti are genetically diverse species of hepatitis E virus [HEV]. Previously, only members of the Paslahepevirus genus were known to infect humans but recently some Rocahepevirus members have been found to be infectious to both immunocompromised and immunocompetent humans. Paslahepevirus balayani genotypes (gt) 1, 2, and 4 are known for their detrimental effects during pregnancy, causing pregnancy-related disorders. Recent findings have demonstrated the ability of Paslahepevirus balayani gt3 to replicate within placental cell lines, suggesting a direct effect on the placenta and fetus. To study whether zoonotic rat HEV strains possess a similar human-host placental tropism, we utilized JEG-3 cells to understand the replicative ability of an infectious clone of a recently reported strain of Rocahepevirus ratti, the LCK-3110 strain. Infectious cDNA clones of Pasla-, Avi-, and Rocahepevirus were transcribed and then, transduced into JEG-3 cells. Cells were harvested, and cell lysates were used for testing infectivity. Five days post-transfection or after inoculation onto naive HepG2/C3A cells, the cells were analyzed for infection. Replication in transduced JEG-3 cells and the infection potential in HepG2/C3A cells were assessed via an indirect immunofluorescence assay and a flow-cytometry assay. We found that the Rocahepevirus ratti LCK-3110 strain did not have efficient replication in JEG-3 cell cultures. Full article

The administration of vaccines using a combination approach ensures better coverage and reduces the number of injections and cost. The present study assessed liposome-complexed DNA-corresponding proteins of hepatitis E and B viruses (HEV and HBV) as combined vaccine candidates in rhesus monkeys. The HEV and HBV components consisted of 450 bps, neutralizing the epitope/s (NE) region, and 685 bps small (S) envelope gene-corresponding proteins, respectively. Three groups (n = 2 monkeys/group) were intramuscularly immunized with a total of three doses of NE Protein (Lipo-NE-P), NE DNA + Protein (Lipo-NE-DP), and each of NE and S DNA + Protein (Lipo-NES-DP), respectively, given one month apart. All immunized monkeys were challenged with 10,000 fifty percent monkey infectious dose of homologous HEV strain. Post-immunization anti-HEV antibody levels in monkeys were 59.4 and 148.4 IU/mL (Lipo-NE-P), 177.0 and 240.8 IU/mL (Lipo-NE-DP), and 240.7 and 164.9 IU/mL (Lipo-NES-DP). Anti-HBV antibody levels in Lipo-NES-DP immunized monkeys were 58,786 and 6213 mIU/mL. None of the challenged monkeys showed viremia and elevation in serum alanine amino transferase levels. Monkeys immunized with Lipo-NE-DP and Lipo-NES-DP exhibited a sterilizing immunity, indicating complete protection, whereas monkeys immunized with Lipo-NE-P showed limited viral replication. In conclusion, the liposome-complexed DNA-corresponding proteins of HEV and HBV induced protective humoral immune responses to both components in monkeys and are worth exploring further. Full article

Hepatitis is an inflammatory liver disease primarily caused by hepatitis A (HAV), B (HBV), C (HCV), D (HDV), and E (HEV) viruses. The chronic forms of hepatitis resulting from HBV and HCV infections can progress to cirrhosis or hepatocellular carcinoma (HCC), while acute hepatitis can lead to acute liver failure, sometimes resulting in fatality. Viral hepatitis was responsible for over 1 million reported deaths annually. The treatment of hepatitis caused by viral infections currently involves the use of interferon-α (IFN-α), nucleoside inhibitors, and reverse transcriptase inhibitors (for HBV). However, these methods do not always lead to a complete cure for viral infections, and chronic forms of the disease pose significant treatment challenges. These facts underscore the urgent need to explore novel drug developments for the treatment of viral hepatitis. The discovery of the CRISPR/Cas9 system and the subsequent development of various modifications of this system have represented a groundbreaking advance in the quest for innovative strategies in the treatment of viral infections. This technology enables the targeted disruption of specific regions of the genome of infectious agents or the direct manipulation of cellular factors involved in viral replication by introducing a double-strand DNA break, which is targeted by guide RNA (spacer). This review provides a comprehensive summary of our current knowledge regarding the application of the CRISPR/Cas system in the regulation of viral infections caused by HAV, HBV, and HCV. It also highlights new strategies for drug development aimed at addressing both acute and chronic forms of viral hepatitis. Full article