Journal of Virology最新文献

{"title":"Adaptive selection of quasispecies during <i>in vivo</i> passaging in chickens, mice, and ferrets results in host-specific strains for the H9N2 avian influenza virus.","authors":"Yiliang Li, Xi Quan, Rujian Chen, Xiao Wang, Yiting Chen, Yingde Gan, David M Irwin, Yongyi Shen","doi":"10.1128/jvi.00151-25","DOIUrl":"10.1128/jvi.00151-25","url":null,"abstract":"<p><p>Sporadic human infections of avian influenza virus (AIV) raise significant public health concerns. A critical factor limiting the transmission of AIVs is the shift in receptor-binding preference from Siaα2,3 to Siaα2,6. To reveal the adaptive selection dynamics during the host adaptation process of AIVs, this study generated a viral library with random mutations in the HA gene of the H9N2 strain. Upon passaging the viral library in chickens and mice, the predominantly selected variants exhibited a preference for Siaα2,3 receptors. Notably, the wild-type strain remained dominant in both inoculated and direct-contact chickens, while variants with the ΔL226/R229I substitutions were preferentially selected in mice. Ferrets have a predominance of Siaα2,6 in their respiratory tract. As expected, the variant harboring the N289D mutation, which prefers Siaα2,6 binding, was enriched during <i>in vivo</i> passaging in ferrets. The mice-adapted variant with the ΔL226/R229I mutations causes reduced levels of TNF-α in the early days post-infection in mice, which correlated with an increase in its viral titers. Conversely, elevated levels of IL-6 and IL-1β at five dpi may contribute to the development of the cytokine release syndrome, potentially elucidating the higher fatality rate observed. In conclusion, based on the mutant spectra of the HA gene, this study elucidates the distinct quasispecies dynamics during the adaptation of H9N2 to different hosts, with receptor availability serving as one of the driving factors. Furthermore, a series of critical substitutions that influence the interspecific transmission potential of H9N2 AIVs were identified.IMPORTANCEThe mutation of viruses creates a quasispecies reservoir. In this study, we aimed to investigate the dynamics of quasispecies during the host adaptation of AIVs. We generated a viral library with random mutations in the HA gene of H9N2 and conducted serial passaging in chickens, mice, and ferrets for five generations, respectively. The wild-type strain was dominant in chickens, while mice selected viruses with the ΔL226/R229I substitutions. Both variants showed a preference for binding to Siaα2,3, which aligned with the abundance of Siaα2,3 found in the respiratory tract epithelial cells of chickens and mice. In ferrets, where Siaα2,6 is more prevalent, the variant with the N289D mutation, which prefers Siaα2,6, was found to be enriched. In summary, this study revealed the adaptive selection of H9N2 quasispecies in various hosts, contributing to our understanding of AIV host adaptation.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0015125"},"PeriodicalIF":4.0,"publicationDate":"2025-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12172485/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144000798","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A human cytomegalovirus prefusion-like glycoprotein B subunit vaccine elicits humoral immunity similar to that of postfusion gB in mice.","authors":"Krithika P Karthigeyan, Megan Connors, Christian R Binuya, Mackensie Gross, Adelaide S Fuller, Chelsea M Crooks, Hsuan-Yuan Wang, Madeline R Sponholtz, Patrick O Byrne, Savannah Herbek, Caroline Andy, Linda M Gerber, John D Campbell, Caitlin A Williams, Elizabeth Mitchell, Lara van der Maas, Itzayana Miller, Dong Yu, Matthew J Bottomley, Jason S McLellan, Sallie R Permar","doi":"10.1128/jvi.02178-24","DOIUrl":"10.1128/jvi.02178-24","url":null,"abstract":"<p><p>Human cytomegalovirus (HCMV) is the leading infectious cause of birth defects. Despite the global disease burden, there is no Food and Drug Administration (FDA)-approved HCMV vaccine. The most efficacious HCMV vaccine candidates to date have used glycoprotein B (gB), a class III viral fusion protein, in its postfusion form. While some viral fusion proteins have been shown to elicit stronger neutralizing responses in their prefusion conformation, HCMV prefusion-like and postfusion gB were recently shown to elicit antibodies with similar fibroblast neutralization titers in mice. We aimed to define and compare the specificity and functionality of plasma IgG elicited by distinct prefusion-like and postfusion gB constructs. Prefusion-like and postfusion gB elicited comparable IgG responses that predominantly mapped to the AD-5 antigenic domain known to elicit neutralizing antibodies. Interestingly, postfusion gB elicited significantly higher plasma IgG binding to cell-associated gB and antibody-dependent cellular phagocytosis than that of prefusion-like gB. The vaccines elicited comparable neutralization titers of heterologous HCMV strain AD169r in fibroblasts; however, neither elicited neutralizing titers against the vaccine-matched strain Towne in fibroblasts. Our data indicate that gB in this prefusion-like conformation elicits similar specificity and functional humoral immunity to that of postfusion gB, unlike certain class I viral fusion proteins that have been used as vaccine antigens. These findings deepen our understanding of the immune response elicited by class III fusion proteins and may inform further design and testing of conformationally dependent herpesvirus glycoprotein vaccine candidates.IMPORTANCEVaccines against human cytomegalovirus (HCMV) still remain elusive in spite of the high disease burden of the virus, especially in pre-term infants and immunocompromised individuals. While vaccine efforts have focused on vaccine-induced antibodies to neutralize the virus, studies have increasingly shown the importance of other antibody functions in protection against cytomegalovirus (CMV) transmission. In this study, we comprehensively evaluated immune responses elicited by the prefusion state of an important HCMV protein called glycoprotein B (gB) in mice. Our results indicate that prefusion gB elicits immune responses similar to that of postfusion gB in mice and reveals areas for further redesign and testing for prefusion vaccine antigens against CMV and other herpesviruses, which could help in furthering vaccine development against HCMV.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0217824"},"PeriodicalIF":4.0,"publicationDate":"2025-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12172439/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144008767","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Autographa californica multiple nucleopolyhedrovirus Ac51 interacts with Ac66 and facilitates its nuclear localization to promote the nuclear egress of nucleocapsids.","authors":"Jianxiang Qiu, Guo-Sheng Zhu, Jiaxin Liu, Longkuan Feng, Zefen Pang, Dandong Zhu, Kanghong Chen, Xiukui Yan, Ao Li, Chuming You, Zhixin Fang","doi":"10.1128/jvi.01969-24","DOIUrl":"10.1128/jvi.01969-24","url":null,"abstract":"<p><p>Autographa californica multiple nucleopolyhedrovirus is a large DNA virus that replicates within host nuclei. The mechanism underlying the nuclear egress of viral nucleocapsids is not fully understood. We previously identified Ac51 as a key player in facilitating efficient nuclear egress of nucleocapsids for the release of budded viruses (BVs). This protein joins Ac66 and EXON0 as the third identified protein with a similar function. In this study, we evaluated the functional importance of conserved domains of Ac51 during virus replication and found that the coiled-coil (CC) motif of Ac51 was crucial for the protein's accumulation and BV production. Our study further revealed a strong interaction and colocalization between Ac51 and Ac66. Deletion of <i>ac51</i> significantly impaired the nuclear import of Ac66, leading to its aggregation in the cytoplasm. Deletion of the CC motif also significantly reduced the nuclear localization of Ac66 and their colocalization, while deletion of the DnaJ domain of Ac51 seemed to impair the interaction between Ac51 and Ac66 <i>in vitro</i>. Molecular docking analysis provided a potential interaction mechanism between Ac51 and Ac66. Intriguingly, our study also identified two other viral nucleocapsid proteins, ME53 and Ac132, which interacted and colocalized with both Ac51 and Ac66. Molecular docking analysis suggested distinct binding regions of ME53 and Ac132 within the Ac51-Ac66 complex. In summary, these results demonstrated that Ac51 and Ac66 cooperate to facilitate the nuclear egress of nucleocapsids and uncovered a functional correlation between ME53/Ac132 and the Ac51-Ac66 complex in nucleocapsid assembly or transport.IMPORTANCEThe nuclear egress of AcMNPV nucleocapsids is a crucial step for producing high levels of budded viruses, but its underlying mechanism is not fully understood. Previous studies have highlighted the significant roles of three nucleocapsid proteins, including Ac51, Ac66, and EXON0, in facilitating efficient nuclear egress of nucleocapsids. Here, we demonstrated a strong interaction and colocalization between Ac51 and Ac66, but not between Ac51 and EXON0. Furthermore, Ac51 is required for efficient nuclear localization of Ac66, indicating that Ac51 exerts a role in Ac66 to promote nucleocapsid egress. Our study also identified two other nucleocapsid proteins, ME53 and Ac132, which interacted and colocalized with both Ac51 and Ac66, suggesting their functional correlation in nucleocapsid assembly or transport. These discoveries enhanced our understanding of the functional mechanism of Ac51 and nucleocapsid assembly and transport and provided insights for further investigation of the nucleocapsid transport process.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0196924"},"PeriodicalIF":4.0,"publicationDate":"2025-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12172431/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144159674","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The role of cysteine-rich protein in enhancing mandarivirus infectivity and pathogenicity.","authors":"Jiaxing Wu, Xiaofei Liang, Die Li, Xuedong Liu, Zongtao Sun, Changyong Zhou, Xuefeng Wang, Mengji Cao","doi":"10.1128/jvi.02237-24","DOIUrl":"10.1128/jvi.02237-24","url":null,"abstract":"<p><p>Mandariviruses pose a significant threat to the citrus industry due to their diverse transmission modes and high pathogenicity. However, the pathogenicity mechanisms of mandariviruses remain largely unknown, especially as there is a complete lack of understanding regarding the function of the cysteine-rich proteins (CRPs) encoded by mandariviruses during infection. In this study, ectopic expression of two mandarivirus CRPs from citrus yellow mottle-associated virus (CiYMaV) and citrus yellow vein clearing virus (CYVCV) using a potato virus X vector resulted in severe symptoms and increased viral accumulation in <i>Nicotiana benthamiana</i>. CiYMaV CRP induced cell death in <i>N. benthamiana</i> leaves, with the zinc finger motif identified as the critical region responsible for this induction. CYVCV CRP functioned as a suppressor to inhibit local gene silencing induced by single-stranded <i>GFP</i>, but not double-stranded <i>GFP</i>. Furthermore, mutational analysis of the infectious clones of CiYMaV and CYVCV revealed that their respective CRPs are essential for disease symptom development and viral accumulation in citrus plants. In summary, our findings indicate that CiYMaV and CYVCV CRPs act as pathogenicity determinants, thereby enhancing our understanding of the functional repertoire within the mandariviruses proteome and providing a target for citrus hosts to defend against mandariviruses.</p><p><strong>Importance: </strong>Mandariviruses, infecting a wide range of citrus varieties, cause serious epidemics in Pakistan, India, Turkey, China, Iran, Italy, and America. However, little information is available about pathogenicity mechanisms of mandariviruses. Here, we confirmed the importance of two mandarivirus CRPs of citrus yellow mottle-associated virus (CiYMaV) and citrus yellow vein clearing virus (CYVCV) in disease symptom development and viral accumulation in citrus plants. Our study first provides evidence that CiYMaV and CYVCV CRPs, nonstructural proteins, act as pathogenicity determinants with multiple functions. This offers a broad understanding of functional repertoire within the mandariviruses proteome. Further investigation of the underlying mechanisms of how CRP, as a virulence factor, modulates plant immunity may suggest a possible new strategy for combating mandarivirus infection in the field.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0223724"},"PeriodicalIF":4.0,"publicationDate":"2025-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12172455/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094182","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>In vitro</i> and <i>in vivo</i> characterization of a bat merbecovirus with ACE2- and DPP4-independent cell entry.","authors":"Hiromichi Matsugo, Tomoya Kitamura, Naohiro Takahashi, James Chambers, Ayano Ichikawa, Misa Katayama, Kaixin Li, Wataru Sekine, Kosuke Ohira, Hiroho Ishida, Akiko Takenaka-Uema, Kazuyuki Uchida, Masayuki Shimojima, Taisuke Horimoto, Shin Murakami","doi":"10.1128/jvi.00727-25","DOIUrl":"https://doi.org/10.1128/jvi.00727-25","url":null,"abstract":"<p><p>Betacoronaviruses, which have caused three human outbreaks within the last two decades, are thought to originate from bats, raising the concern that bat coronaviruses could cause a novel human outbreak in the future. To determine whether the bat merbecovirus EjCoV-3 strain, previously detected in <i>Eptesicus japonensis</i> in Japan, has the potential to infect humans, we analyzed its cellular entry mechanism. Cellular entry of EjCoV-3 via the spike protein requires protease treatment and is mediated by an unknown receptor, other than DPP4 or ACE2. We generated cultivable recombinant EjCoV-3 using bacterial artificial chromosome-based reverse genetics and found that it efficiently replicated in human respiratory and intestinal cell cultures as well as nasal ciliated epithelium in hamsters. These findings suggest that bat merbecovirus with ACE2- and DPP4-independent cell entry has the potential to cause human infections, highlighting the importance of extensive bat surveillance for pandemic preparedness.</p><p><strong>Importance: </strong>Betacoronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2, have caused three significant outbreaks in the past two decades and are believed to have originated from bats. To investigate the potential for future outbreaks, we generated a Japanese bat-derived MERS-related coronavirus, designated EjCoV-3, using reverse genetics. Our results showed that EjCoV-3 does not utilize ACE2 and DPP4, cell entry receptors for SARS-CoV and MERS-CoV, as a means of infection. However, we found that EjCoV-3 is the first bat merbecovirus capable of efficiently replicating in human respiratory cells and the respiratory tract of hamsters. These findings provide new insight into the potential for MERS-related coronaviruses that do not use ACE2 and DPP4 to infect the human respiratory tract, highlighting the importance of preparedness for outbreaks caused by these viruses.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0072725"},"PeriodicalIF":4.0,"publicationDate":"2025-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144317295","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Zika virus-induced fetal demise is triggered by strain- and dose-specific RLR-driven activation of the interferon response in the decidua, placenta, and fetus in <i>Ifnar1</i>^-/- mice.","authors":"Ellie K Bohm, David Castañeda, Qun Lu, Michael D Cameron, Matthew T Aliota","doi":"10.1128/jvi.00666-25","DOIUrl":"10.1128/jvi.00666-25","url":null,"abstract":"<p><p>Congenital Zika syndrome (CZS), the set of fetal and neonatal complications associated with Zika virus (ZIKV) infection in pregnancy, was first noted during the outbreak in the Americas in 2015-2016. However, there was an unequal distribution of ZIKV cases and severe outcomes in all areas where ZIKV emerged in the Americas, demonstrating that the risk of CZS varied over space and time. Recently, we demonstrated that phenotypic heterogeneity existed between closely related ZIKV strains. All ZIKV strains tested infected the placenta but varied in their capacity to cause overt fetal harm. Here, we further characterized the relative contributions of virus genotype and infecting dose of two phenotypically distinct ZIKV strains across multiple timepoints in gestation in pregnant mice that lack type-I interferon receptor function (<i>Ifnar1</i>^-/-). To better understand the underlying causes of adverse fetal outcomes, we used RNA sequencing to compare ZIKV-infected and uninfected tissues. We found that ZIKV infection triggers retinoic acid-inducible gene I (RIG-I)-like receptor-mediated activation of the interferon response at the maternal-fetal interface. However, modest chemical inhibition of RIG-I activation in the decidua and placenta did not protect against fetal demise. Instead, the fetal interferon response was significantly associated with fetal demise. Together, these findings suggest that the response to ZIKV at the maternal-fetal interface can vary, depending on the infecting ZIKV genotype and dose, and that the fetal immune response is an important mediator of fetal harm.</p><p><strong>Importance: </strong>Congenital Zika syndrome is a constellation of fetal abnormalities ranging from fetal demise and microcephaly to infants that are born apparently healthy only to develop neurocognitive impacts later. ZIKV is now endemic in many regions worldwide, but how ZIKV harms the developing fetus remains an outstanding question. Previously, we used a mouse model of ZIKV infection during pregnancy to assess the pathogenic potential to the fetus of a panel of five low-passage ZIKV strains representing the viral genetic diversity in the Americas. We found that phenotypic heterogeneity existed between these closely related ZIKV strains. Here, we show that this heterogeneity is driven by RIG-I-like receptor-mediated activation of the interferon response at the maternal-fetal interface. We used chemical inhibition of the RIG-I pathway and measured the transcriptional activity of interferon-stimulated genes in fetuses to demonstrate that the fetal immune response may contribute to fetal demise.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0066625"},"PeriodicalIF":4.0,"publicationDate":"2025-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12172451/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144119925","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Contrasting interferon-mediated antiviral responses in human lung adenocarcinoma cells.","authors":"Matthew Esparza, Sara S El Zahed, Umut Karakus, Hanspeter Niederstrasser, Boning Gao, Kimberly Batten, Jerry W Shay, Bruce Posner, Fred R Hirsch, Luc Girard, Lily Jun-Shen Huang, John Minna, Adolfo García-Sastre, Beatriz M A Fontoura","doi":"10.1128/jvi.00469-25","DOIUrl":"10.1128/jvi.00469-25","url":null,"abstract":"<p><p>Lung cancers develop from lung epithelial cells after a series of genetic and epigenetic changes, and these cells are major sites of influenza virus infection. Thus, we explored how changes found in patient-derived lung cancer cell lines impacted influenza virus replication and identified two lines with opposite responses to influenza A viral infection. We show that the NCI-H820 lung adenocarcinoma (LUAD) is resistant to influenza A virus and VSV infection, while LUAD line NCI-H322 is highly susceptible to infection by both viruses. H322 cells have a homozygous deletion in a region of chromosome 9 encoding IFNαgenes, IFNβ1, IFNω1, and IFNε genes, leading to downregulation of immune response and high infection rates. In contrast, the resistant H820 cell line has three copies of these same interferon genes and shows increased expression of interferon-regulated genes. We found that the resistance of H820 cells to influenza infection is likely linked to impaired viral entry-due to high basal levels of interferon-induced proteins known to inhibit endocytosis (IFITM1/2/3, NCOA7, and CH25H)-and to increased expression of mRNAs that encode other antiviral factors. In contrast, H322 cells show the absence or low levels of interferon-regulated genes involved in the inhibition of viral entry. These results suggest that the opposite phenotypes on viral entry of H322 and H820 cells may be at least in part associated with impaired or enhanced interferon response, respectively. Since most lung cancer patients have genomic characterization of their tumors, individualized differences in interferon responses may have therapeutic and patient management implications.</p><p><strong>Importance: </strong>Lung cancers develop from genetic and epigenetic changes that can dramatically influence patients' susceptibility to viral infection and replication. This study evaluates the responses to influenza virus infection of two patient-derived lung cancer cell lines. Interestingly, the cell lines investigated are of the same cancer type, lung adenocarcinomas, yet one cell line is highly susceptible, while the other cell line is highly resistant to viral infection. This is in part due to contrasting genetic alterations that lead to changes in the interferon response pathways, which differentially impact viral entry. Thus, identifying these risk factors can inform the prognosis of patients infected with influenza virus and guide their personalized treatment plans.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0046925"},"PeriodicalIF":4.0,"publicationDate":"2025-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12172473/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144159685","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"BTP2 restricts Tulane virus and human norovirus replication independent of store-operated calcium entry.","authors":"Francesca J Scribano, J Thomas Gebert, Kristen A Engevik, Nicole M Hayes, Jorge Villanueva, Son Pham, Soni Kaundal, Janam J Dave, B V Venkataram Prasad, Mary K Estes, Sasirekha Ramani, Joseph M Hyser","doi":"10.1128/jvi.00444-25","DOIUrl":"10.1128/jvi.00444-25","url":null,"abstract":"<p><p>Human norovirus is the leading cause of viral gastroenteritis across all age groups. While there is a need for human norovirus antivirals, therapeutic development has been hindered by a lack of cell culture systems and animal models of infection. Surrogate viruses, such as Tulane virus (TV), have provided tractable systems to screen potential antiviral compounds. Our previous work demonstrated that TV encodes a viral ion channel, which dysregulates cytosolic calcium signaling. We set out to investigate whether host pathways triggered by viral ion channel activity, including store-operated calcium entry (SOCE), play a role in virus replication. Using pharmacologic inhibitors and genetically engineered cell lines, we establish that the SOCE inhibitor, BTP2, reduces TV replication in an SOCE-independent manner. We observed a significant reduction in TV replication, protein expression, and RNA synthesis in cells with both pre- and post-infection BTP2 treatment. By serial passage and plaque isolation, we demonstrate that TV quasi-species have mixed susceptibility and resistance to BTP2. Sequence comparison of the quasi-species revealed that amino acid changes in the structural proteins were associated with drug resistance. We utilized reverse genetics to generate TV with the resistance-associated VP1 and VP2 amino acid changes and found that amino acid changes in both proteins conferred BTP2 resistance. Together, this supports that TV structural proteins are the targets of BTP2. Finally, using human intestinal organoids, we demonstrate that BTP2 significantly reduces human norovirus replication.IMPORTANCEOur work identifies BTP2 as a potential human norovirus antiviral pharmacophore and highlights the utility of targeting calicivirus structural proteins to restrict viral replication. Furthermore, we establish a system whereby Tulane virus (TV) can be used to screen novel antiviral candidates and establish their mechanism of action. Together, this will facilitate rapid preclinical validation of other novel human norovirus therapeutics.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0044425"},"PeriodicalIF":4.0,"publicationDate":"2025-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12172428/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144174034","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Schmallenberg virus non-structural proteins NSs and NSm are not essential for experimental infection of <i>Culicoides sonorensis</i> biting midges.","authors":"Kerstin Wernike, Ana Vasic, Susanne Amler, Franziska Sick, Cristian Răileanu, Oliver Dähn, Helge Kampen, Cornelia Silaghi, Martin Beer","doi":"10.1128/jvi.00343-25","DOIUrl":"10.1128/jvi.00343-25","url":null,"abstract":"<p><p>The teratogenic orthobunyavirus Schmallenberg virus (SBV) is transmitted between its mammalian hosts by <i>Culicoides</i> biting midges. The genome of circulating SBV, i.e., variants present in viremic ruminants or insect vectors, is very stable, while variants found in malformed ruminant fetuses display a high genetic variability. It was suggested that fetal infection provides an environment that favors viral mutations that enable immune escape in the unborn but at the cost of limiting the ability of the virus to spread further. To investigate infection and dissemination rates of different SBV variants in the insect vectors, we fed laboratory-reared <i>Culicoides sonorensis</i> with blood containing the prototype strain BH80/11-4 from a viremic cow or strain D281/12, which was isolated from the brain of a sheep fetus and harbors multiple mutations in all three genome segments. Furthermore, virus variants lacking NSs, NSm, or both non-structural proteins were included. Six days after feeding, virus replication was found in about 2% of the midges exposed to wild-type strain BH80/11-4. The absence of the non-structural proteins had no obvious effect on the oral susceptibility to virus infection, as after 6 days, 2.78% of the midges fed with the NSs-deletion mutant displayed viral loads higher than the respective day-0 group, 1.92% of the midges exposed to the NSm-deletion mutant, and 1.55% of midges exposed to the NSs/NSm-deletion mutant. In contrast, strain D281/12 did not replicate at all in the midges, supporting the assumption that SBV variants arising in infected fetuses are unable to enter the normal insect-mammalian host cycle.IMPORTANCEBiting midges are responsible for the transmission of Schmallenberg virus (SBV), a pathogen of veterinary importance that primarily infects ruminants. Although SBV has been extensively studied in the mammalian host, the virus-intrinsic factors allowing infection of and replication in biting midges are largely unknown. Therefore, we infected laboratory-reared <i>Culicoides sonorensis</i> midges with SBV variants by feeding them virus-containing blood. The SBV variants differed in their genome composition, as we used the prototype wild-type strain, a strain with multiple mutations that was isolated from the brain of a malformed fetus, and recombinants lacking either NSs or NSm or both of these non-structural proteins. While the non-structural proteins had no obvious effect, the variant from the malformed fetus did not replicate at all, indicating that virus variants with characteristic genomic mutations present in fetuses lose their ability to infect the insect vector and will be excluded from the natural transmission cycle.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0034325"},"PeriodicalIF":4.0,"publicationDate":"2025-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12172452/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144032893","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Reconstitution of interferon regulatory factor 7 expression restores interferon beta induction in Huh7 cells.","authors":"Andreas Betz, Hao-En Huang, Zuguang Gu, Ombretta Colasanti, Teng-Feng Li, Jasper Hesebeck-Brinckmann, Nadine Gillich, Gnimah Eva Gnouamozi, Matthias Schlesner, Florian W R Vondran, Stephan Urban, Ralf Bartenschlager, Marco Binder, Volker Lohmann","doi":"10.1128/jvi.00703-25","DOIUrl":"10.1128/jvi.00703-25","url":null,"abstract":"<p><p>Abundance of essential components of double-stranded RNA (dsRNA) recognition and the subsequent interferon (IFN) response vary widely between primary human hepatocytes (PHHs) and commonly used cell culture models based on derivatives of Huh7 cells, supporting replication of all hepatitis viruses. We used RNA sequencing to compare the innate immune response in hepatoma cells with primary cells and non-neoplastic immortalized hepatocytes (PH5CH). Stimulation with the dsRNA analog poly(I:C) in Huh7 and Huh7.5, either by supernatant feeding or by transfection, resulted in an induction of interferon-stimulated genes widely comparable to that of PHH and PH5CH, but Huh7 and Huh7.5 lacked efficient production of IFN-β. We identified interferon regulatory factor 7 (IRF7) as a critical component missing in Huh7-derived cells. Upon reconstitution of IRF7 expression, <i>IFNB</i> induction was restored to the levels observed in PHH upon stimulation with poly(I:C). In contrast to PH5CH cells, for which IRF3 was sufficient for full IFN induction, the lack of IRF7 could not be compensated by increased IRF3 expression in Huh7-derived cells. We further found significant <i>IFNB</i> induction upon Sendai virus and hepatitis delta virus infections in Huh7.5 cells reconstituted with IRF7, but not in hepatitis A or hepatitis C virus-infected cells, widely representing the characteristics of the IFN response observed in other models and <i>in vivo</i>. Our data suggest that the reconstitution of IRF7 expression in Huh7 cells can aid a more physiological analysis of cell intrinsic immune responses to hepatotropic viruses in future studies.IMPORTANCECurrent <i>in vitro</i> studies often rely on Huh7-based hepatoma cells, which, although permissive for hepatitis viruses, lack critical components for double-stranded RNA recognition. We used RNA sequencing to compare the cell intrinsic innate immune responses of hepatoma cells with more authentic cellular models. We discovered that Huh7-derived cells, which are known to show very limited induction of <i>IFNB</i> upon pathogen recognition receptor stimulation, lack expression of interferon regulatory factor 7 (IRF7), an essential component for robust type I interferon induction. By reconstituting IRF7, we were able to restore the interferon response to levels observed in primary human hepatocytes. Our study not only identifies a key missing link in the Huh7/Huh7.5 innate immune response but also offers a way to enhance the physiological relevance of these cells in future studies. Our findings pave the way for more accurate modeling of the human hepatic response to viral infections, potentially improving the understanding and management of hepatitis.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0070325"},"PeriodicalIF":4.0,"publicationDate":"2025-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12172482/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128028","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}