Jae-Hyun Cho, Gyu Min Lee, Seyoung Ko, Youngju Kim, Donghyuk Kim
Bovine mastitis, primarily caused by Staphylococcus aureus, significantly affects the dairy industry by reducing milk production and quality. The rise of antibiotic-resistant bacteria has prompted the need for alternative treatments. The three newly isolated bacteriophages, OPT-SA02, OPT-SC01, and OPT-SX11, were isolated from chicken fecal and sewage samples in South Korea. These bacteriophages were characterized via physiological and genomic analyses, identifying their therapeutic potential against S. aureus-induced mastitis. The bacteriophages were identified as members of the Herelleviridae family, exhibiting stability across broad pH (2-12) and temperature (37-70°C) ranges, as well as strong antibacterial activity at low multiplicity of infection (MOI) levels. Genomic analysis revealed that the conservation of lysis-related genes (holin and endolysin) is responsible for their lytic capabilities. Additionally, protein structural predictions revealed multi-domain structures in their endolysins, enhancing their lytic potential. These findings suggest that OPT-SA02, OPT-SC01, and OPT-SX11 show significant promise as alternative treatments for bovine mastitis.IMPORTANCEBovine mastitis, caused by pathogens such as Staphylococcus aureus and Staphylococcus xylosus, remains a major challenge in dairy farming, leading to significant economic losses and reduced milk quality. The increasing prevalence of antibiotic-resistant strains further complicates treatment, emphasizing the need for alternative strategies. This study identifies three newly isolated bacteriophages with effective antibacterial activity against these pathogens and provides comprehensive genomic and structural insights into their mechanisms. Genomic characterization revealed conserved lytic cassettes and genetic diversity within related bacteriophages, offering a deeper understanding of their evolutionary relationships and potential applications. Furthermore, protein structure analysis of the endolysin derived from these bacteriophages identified multi-domain architectures with preserved catalytic cores, underscoring their lytic efficacy against bacterial cell walls. These findings advance the understanding of the genetic and structural mechanisms of bacteriophage-mediated lysis and highlight their potential as sustainable tools for managing bovine mastitis and improving milk quality in dairy farming.
{"title":"Characterization and therapeutic potential of newly isolated bacteriophages against <i>Staphylococcus</i> species in bovine mastitis.","authors":"Jae-Hyun Cho, Gyu Min Lee, Seyoung Ko, Youngju Kim, Donghyuk Kim","doi":"10.1128/jvi.01901-24","DOIUrl":"https://doi.org/10.1128/jvi.01901-24","url":null,"abstract":"<p><p>Bovine mastitis, primarily caused by <i>Staphylococcus aureus</i>, significantly affects the dairy industry by reducing milk production and quality. The rise of antibiotic-resistant bacteria has prompted the need for alternative treatments. The three newly isolated bacteriophages, OPT-SA02, OPT-SC01, and OPT-SX11, were isolated from chicken fecal and sewage samples in South Korea. These bacteriophages were characterized via physiological and genomic analyses, identifying their therapeutic potential against <i>S. aureus</i>-induced mastitis. The bacteriophages were identified as members of the <i>Herelleviridae</i> family, exhibiting stability across broad pH (2-12) and temperature (37-70°C) ranges, as well as strong antibacterial activity at low multiplicity of infection (MOI) levels. Genomic analysis revealed that the conservation of lysis-related genes (holin and endolysin) is responsible for their lytic capabilities. Additionally, protein structural predictions revealed multi-domain structures in their endolysins, enhancing their lytic potential. These findings suggest that OPT-SA02, OPT-SC01, and OPT-SX11 show significant promise as alternative treatments for bovine mastitis.IMPORTANCEBovine mastitis, caused by pathogens such as <i>Staphylococcus aureus</i> and <i>Staphylococcus xylosus</i>, remains a major challenge in dairy farming, leading to significant economic losses and reduced milk quality. The increasing prevalence of antibiotic-resistant strains further complicates treatment, emphasizing the need for alternative strategies. This study identifies three newly isolated bacteriophages with effective antibacterial activity against these pathogens and provides comprehensive genomic and structural insights into their mechanisms. Genomic characterization revealed conserved lytic cassettes and genetic diversity within related bacteriophages, offering a deeper understanding of their evolutionary relationships and potential applications. Furthermore, protein structure analysis of the endolysin derived from these bacteriophages identified multi-domain architectures with preserved catalytic cores, underscoring their lytic efficacy against bacterial cell walls. These findings advance the understanding of the genetic and structural mechanisms of bacteriophage-mediated lysis and highlight their potential as sustainable tools for managing bovine mastitis and improving milk quality in dairy farming.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0190124"},"PeriodicalIF":4.0,"publicationDate":"2025-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143414596","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}
Influenza A viruses with fewer amino acids in the neuraminidase (NA) stalk domain are primarily isolated from chickens rather than wild ducks, indicating that a shortened NA stalk is considered an adaptation marker of avian influenza viruses (AIVs) to chickens. Experimental passages of an H7N7 nonpathogenic AIV (rgVac2-P0) in chickens resulted in a highly pathogenic variant (Vac2-P3L4) with a 34-amino-acid deletion in the NA stalk, encompassing five potential N-glycosylation sites. To investigate how amino acid truncation and deglycosylation in the NA stalk contribute to increased pathogenicity, a virus with glycosylation-deficient mutations at these sites (rgVac2-P3L4/P0NAΔGlyco) was constructed. Contrary to expectations, chickens inoculated with rgVac2-P3L4/P0NAΔGlyco exhibited variable clinical outcomes, attributed to the genetic instability of the virus. A single mutation stabilized the virus, and the mutant (rgVac2-P3L4/P0NAΔGlyco-Y65H) resulted in higher pathogenicity compared with a virus with restored glycosylation (rgVac2-P3L4/P0NA-Y65H). Glycan occupancy analysis revealed 3-4 glycans at the five potential sites. In functional analysis, glycosylation-deficient mutants, similar to the short-stalk NA virus, showed significantly reduced erythrocyte elution activity. Additionally, mutational analysis indicated variable contributions of N-glycans to elution activity across the sites. Moreover, the functionally most contributing sites of the five potential N-glycosylation motifs were consistently included in the amino acid deletions of the stalk-truncated NA in N7-subtyped field isolates, despite the varying truncation position or length. These findings suggest that the loss of glycosylation is functionally equivalent to a reduction in amino acids, and it plays a crucial role in enhancing pathogenicity in chickens and affecting NA function.IMPORTANCEAvian influenza poses significant economic challenges to the poultry industry and presents potential risks to human health. Understanding the molecular mechanisms that facilitate the emergence of chicken-adapted avian influenza viruses (AIVs) from non-pathogenic duck-origin influenza viruses is crucial for improving AIV monitoring systems in poultry and controlling this disease. Amino acid deletions in the neuraminidase (NA) stalk domain serve as one of the molecular markers for AIV adaptation to Galliformes. This study highlights the critical role of N-glycosylation in the NA stalk domain in the pathogenesis of high pathogenicity avian influenza viruses in chickens. The findings propose a novel theory that the loss of glycosylation at the NA stalk domain, rather than a reduction in stalk length, is responsible for both NA function and increased virus pathogenicity in chickens.
{"title":"Deglycosylation and truncation in the neuraminidase stalk are functionally equivalent in enhancing the pathogenicity of a high pathogenicity avian influenza virus in chickens.","authors":"Daiki Kobayashi, Takahiro Hiono, Hiromu Arakawa, Hiroyuki Kaji, Ayako Ohkawara, Takaya Ichikawa, Hinako Ban, Norikazu Isoda, Yoshihiro Sakoda","doi":"10.1128/jvi.01478-24","DOIUrl":"https://doi.org/10.1128/jvi.01478-24","url":null,"abstract":"<p><p>Influenza A viruses with fewer amino acids in the neuraminidase (NA) stalk domain are primarily isolated from chickens rather than wild ducks, indicating that a shortened NA stalk is considered an adaptation marker of avian influenza viruses (AIVs) to chickens. Experimental passages of an H7N7 nonpathogenic AIV (rgVac2-P0) in chickens resulted in a highly pathogenic variant (Vac2-P3L4) with a 34-amino-acid deletion in the NA stalk, encompassing five potential <i>N</i>-glycosylation sites. To investigate how amino acid truncation and deglycosylation in the NA stalk contribute to increased pathogenicity, a virus with glycosylation-deficient mutations at these sites (rgVac2-P3L4/P0NAΔGlyco) was constructed. Contrary to expectations, chickens inoculated with rgVac2-P3L4/P0NAΔGlyco exhibited variable clinical outcomes, attributed to the genetic instability of the virus. A single mutation stabilized the virus, and the mutant (rgVac2-P3L4/P0NAΔGlyco-Y65H) resulted in higher pathogenicity compared with a virus with restored glycosylation (rgVac2-P3L4/P0NA-Y65H). Glycan occupancy analysis revealed 3-4 glycans at the five potential sites. In functional analysis, glycosylation-deficient mutants, similar to the short-stalk NA virus, showed significantly reduced erythrocyte elution activity. Additionally, mutational analysis indicated variable contributions of <i>N</i>-glycans to elution activity across the sites. Moreover, the functionally most contributing sites of the five potential <i>N</i>-glycosylation motifs were consistently included in the amino acid deletions of the stalk-truncated NA in N7-subtyped field isolates, despite the varying truncation position or length. These findings suggest that the loss of glycosylation is functionally equivalent to a reduction in amino acids, and it plays a crucial role in enhancing pathogenicity in chickens and affecting NA function.IMPORTANCEAvian influenza poses significant economic challenges to the poultry industry and presents potential risks to human health. Understanding the molecular mechanisms that facilitate the emergence of chicken-adapted avian influenza viruses (AIVs) from non-pathogenic duck-origin influenza viruses is crucial for improving AIV monitoring systems in poultry and controlling this disease. Amino acid deletions in the neuraminidase (NA) stalk domain serve as one of the molecular markers for AIV adaptation to Galliformes. This study highlights the critical role of <i>N</i>-glycosylation in the NA stalk domain in the pathogenesis of high pathogenicity avian influenza viruses in chickens. The findings propose a novel theory that the loss of glycosylation at the NA stalk domain, rather than a reduction in stalk length, is responsible for both NA function and increased virus pathogenicity in chickens.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0147824"},"PeriodicalIF":4.0,"publicationDate":"2025-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143414597","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}
Timmy Richardo, Xiaokun Liu, Katinka Döhner, Tsung-Yu Chao, Anna Buch, Anne Binz, Anja Pohlmann, Madeleine de le Roi, Wolfgang Baumgärtner, Korbinian Brand, Rudolf Bauerfeind, Reinhold Förster, Beate Sodeik, Stephan Halle
Herpes simplex viruses (HSV) cause many skin diseases, particularly in immunocompromised patients. HSV-1 infection of murine skin recapitulates many aspects of human pathology. However, many protocols rely on mechanical or enzymatic skin disruption to induce lesions, although this can alter skin homeostasis and prime antiviral inflammation before inoculation. To investigate the initial events following HSV-1 primary skin infection before the onset of symptoms, we developed a novel murine ex vivo explant model using gentle depilation without further scarification and infected keratinocytes from the outside with minimal tissue damage. Two-photon microscopy showed that HSV-1 spread exclusively in the epidermis. The infection centers increased in number and size over time and contained hundreds of infected keratinocytes. We investigated the HSV-1 spread at the cellular level, using reporter strains with fluorescently tagged capsid protein VP26, and observed the formation of nuclear capsid assembly sites and nuclear capsid egress and the recruitment of the inner tegument protein pUL37GFP, the outer tegument protein VP11/12GFP, and the envelope protein gDGFP to cytoplasmic capsids. By using electron microscopy, the skin appeared intact, and keratinocytes contained many nuclear capsids, primary virions in the nuclear envelope, cytosolic membrane-associated capsids, and enveloped virions. Our protocol provides a robust and reproducible approach to investigate the very early events of HSV-1 spread in the skin, to characterize the phenotypes of HSV-1 mutants in terminally differentiated skin tissues, and to evaluate potentially antiviral small molecules in a preclinical ex vivo infection model.
Importance: This study describes a novel murine ex vivo skin explant model to investigate early events in HSV-1 infection without causing significant tissue damage. To infect from the outside, via the apical keratinocytes, this method relies on gentle depilation, which maintains skin integrity. HSV-1 spread exclusively within the epidermis, with infection centers increasing over time and involving hundreds of keratinocytes. Using advanced microscopy techniques, we tracked HSV-1 spread at the cellular level and intracellular assembly of all intermediate virus structures. This model offers a valuable tool for studying the initial stages of HSV-1 infection, assessing viral mutant phenotypes, and testing antiviral compounds in a more physiological context to provide critical insights into HSV-1 pathogenesis and therapeutic strategies.
{"title":"Herpes simplex virus assembly and spread in murine skin after infection from the outside.","authors":"Timmy Richardo, Xiaokun Liu, Katinka Döhner, Tsung-Yu Chao, Anna Buch, Anne Binz, Anja Pohlmann, Madeleine de le Roi, Wolfgang Baumgärtner, Korbinian Brand, Rudolf Bauerfeind, Reinhold Förster, Beate Sodeik, Stephan Halle","doi":"10.1128/jvi.01638-24","DOIUrl":"https://doi.org/10.1128/jvi.01638-24","url":null,"abstract":"<p><p>Herpes simplex viruses (HSV) cause many skin diseases, particularly in immunocompromised patients. HSV-1 infection of murine skin recapitulates many aspects of human pathology. However, many protocols rely on mechanical or enzymatic skin disruption to induce lesions, although this can alter skin homeostasis and prime antiviral inflammation before inoculation. To investigate the initial events following HSV-1 primary skin infection before the onset of symptoms, we developed a novel murine <i>ex vivo</i> explant model using gentle depilation without further scarification and infected keratinocytes from the outside with minimal tissue damage. Two-photon microscopy showed that HSV-1 spread exclusively in the epidermis. The infection centers increased in number and size over time and contained hundreds of infected keratinocytes. We investigated the HSV-1 spread at the cellular level, using reporter strains with fluorescently tagged capsid protein VP26, and observed the formation of nuclear capsid assembly sites and nuclear capsid egress and the recruitment of the inner tegument protein pUL37GFP, the outer tegument protein VP11/12GFP, and the envelope protein gDGFP to cytoplasmic capsids. By using electron microscopy, the skin appeared intact, and keratinocytes contained many nuclear capsids, primary virions in the nuclear envelope, cytosolic membrane-associated capsids, and enveloped virions. Our protocol provides a robust and reproducible approach to investigate the very early events of HSV-1 spread in the skin, to characterize the phenotypes of HSV-1 mutants in terminally differentiated skin tissues, and to evaluate potentially antiviral small molecules in a preclinical <i>ex vivo</i> infection model.</p><p><strong>Importance: </strong>This study describes a novel murine <i>ex vivo</i> skin explant model to investigate early events in HSV-1 infection without causing significant tissue damage. To infect from the outside, via the apical keratinocytes, this method relies on gentle depilation, which maintains skin integrity. HSV-1 spread exclusively within the epidermis, with infection centers increasing over time and involving hundreds of keratinocytes. Using advanced microscopy techniques, we tracked HSV-1 spread at the cellular level and intracellular assembly of all intermediate virus structures. This model offers a valuable tool for studying the initial stages of HSV-1 infection, assessing viral mutant phenotypes, and testing antiviral compounds in a more physiological context to provide critical insights into HSV-1 pathogenesis and therapeutic strategies.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0163824"},"PeriodicalIF":4.0,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143408646","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}
Gairu Li, Xinxin Li, Jie Chen, Phillipe Lemey, Bram Vrancken, Shuo Su, Simon Dellicour, Fabiana Gámbaro
Japanese encephalitis is a viral disease caused by the Japanese encephalitis virus (JEV), primarily affecting rural areas of Asia and western Pacific region. China remains one of the main epicenters, experiencing a significant burden of human and animal cases despite vaccination efforts. The ecology of this arbovirus is complex, involving Culex mosquitoes as primary vectors, wading birds as natural reservoirs, and pigs as amplifying hosts. Given the virus's epidemiological importance in China, combined with the country's expanding pig farming industry and diverse climates, investigating the virus spread and its environmental drivers is needed to address its persistent burden. In this study, we conducted phylogeographic analyses by combining publicly available JEV envelope gene sequences from China and other regions. Our reconstructions revealed multiple introduction events leading to various circulating JEV clades in China, with one predominant clade. Additionally, our analyses showed a diffusion capacity of JEV exceeding previous estimates for co-circulating arboviruses. These differences could be attributed to pig trade or bird migration, calling for further investigations into the drivers of JEV spread.
Importance: Japanese encephalitis virus (JEV) is the cause of Japanese encephalitis, a significant health concern in China. Despite being one of the most studied mosquito-borne viruses, no previous studies have combined genomic and geographic data to investigate the spatial epidemiology and dispersal capacity of the virus. In this study, we analyzed genomic, geographic, and environmental data to trace the dispersal history of JEV in China and explore the environmental factors influencing its distribution. Our findings show that JEV circulates predominantly in areas with higher temperatures, dense human and pig populations, and favorable conditions for Culex mosquitoes. Notably, our analyses showed a higher diffusion capacity of JEV compared to co-circulating viruses, possibly driven by factors like pig trade and bird migration. Our analysis calls for improved genomic surveillance and establishes a baseline for future studies on the effects of climate change, agricultural practices, and bird migration on JEV circulation.
{"title":"Tracing more than two decades of Japanese encephalitis virus circulation in mainland China.","authors":"Gairu Li, Xinxin Li, Jie Chen, Phillipe Lemey, Bram Vrancken, Shuo Su, Simon Dellicour, Fabiana Gámbaro","doi":"10.1128/jvi.01575-24","DOIUrl":"https://doi.org/10.1128/jvi.01575-24","url":null,"abstract":"<p><p>Japanese encephalitis is a viral disease caused by the Japanese encephalitis virus (JEV), primarily affecting rural areas of Asia and western Pacific region. China remains one of the main epicenters, experiencing a significant burden of human and animal cases despite vaccination efforts. The ecology of this arbovirus is complex, involving <i>Culex</i> mosquitoes as primary vectors, wading birds as natural reservoirs, and pigs as amplifying hosts. Given the virus's epidemiological importance in China, combined with the country's expanding pig farming industry and diverse climates, investigating the virus spread and its environmental drivers is needed to address its persistent burden. In this study, we conducted phylogeographic analyses by combining publicly available JEV envelope gene sequences from China and other regions. Our reconstructions revealed multiple introduction events leading to various circulating JEV clades in China, with one predominant clade. Additionally, our analyses showed a diffusion capacity of JEV exceeding previous estimates for co-circulating arboviruses. These differences could be attributed to pig trade or bird migration, calling for further investigations into the drivers of JEV spread.</p><p><strong>Importance: </strong>Japanese encephalitis virus (JEV) is the cause of Japanese encephalitis, a significant health concern in China. Despite being one of the most studied mosquito-borne viruses, no previous studies have combined genomic and geographic data to investigate the spatial epidemiology and dispersal capacity of the virus. In this study, we analyzed genomic, geographic, and environmental data to trace the dispersal history of JEV in China and explore the environmental factors influencing its distribution. Our findings show that JEV circulates predominantly in areas with higher temperatures, dense human and pig populations, and favorable conditions for <i>Culex</i> mosquitoes. Notably, our analyses showed a higher diffusion capacity of JEV compared to co-circulating viruses, possibly driven by factors like pig trade and bird migration. Our analysis calls for improved genomic surveillance and establishes a baseline for future studies on the effects of climate change, agricultural practices, and bird migration on JEV circulation.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0157524"},"PeriodicalIF":4.0,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143408581","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}
Type I interferons exert their antiviral effects against SARS-CoV-2 by inducing the expression of interferon-stimulated genes (ISGs), including but not limited to LY6E, CH25H, IFITM2/3, and IFIH1. However, the antiviral effect and underlying mechanisms of action of most ISGs in SARS-CoV-2 infection are not yet fully understood. By screening 109 ISG-knockout cell lines, we identify that phospholipid scramblase 1 (PLSCR1), an interferon-inducible protein, acts as a crucial restriction factor against SARS-CoV-2 infection. Cells lacking PLSCR1 are highly susceptible to SARS-CoV-2 infection. Conversely, overexpression of PLSCR1 inhibits SARS-CoV-2 infection. Depletion of PLSCR1 enhances cellular entry of both pseudotyped and authentic SARS-CoV-2. Mechanistically, PLSCR1 inhibits SARS-CoV-2 entry by specifically downregulating plasma membrane expression of ACE2, the virus's receptor, without affecting the overall levels of ACE2 within the cell. As such, we unraveled previously unappreciated mechanisms by which PLSCR1 exerts its restrictive effect on SARS-CoV-2. These data provide new insights into the interplay between host innate antiviral immunity and SARS-CoV-2 and shed light on novel antiviral therapeutics.
Importance: Phospholipid scramblase 1 (PLSCR1) has been identified as a critical host restriction factor against SARS-CoV-2 infection. In this study, we demonstrated that PLSCR1 inhibited SARS-CoV-2 entry by downregulating the plasma membrane expression of ACE2, the primary receptor for viral entry. Our findings elucidate a novel host-pathogen interaction that not only deepens our understanding of the innate immune response to SARS-CoV-2 but offers potential strategies for therapeutic interventions against COVID-19.
{"title":"PLSCR1 suppresses SARS-CoV-2 infection by downregulating cell surface ACE2.","authors":"Ruiyi Ma, Xinyi Zhang, Ruonan Li, Xiaojing Dong, Wenjing Wang, Qi Jiang, Xia Xiao, Yujin Shi, Lan Chen, Tian Zheng, Zichun Xiang, Lili Ren, Zhuo Zhou, Xiaobo Lei, Jianwei Wang","doi":"10.1128/jvi.02085-24","DOIUrl":"https://doi.org/10.1128/jvi.02085-24","url":null,"abstract":"<p><p>Type I interferons exert their antiviral effects against SARS-CoV-2 by inducing the expression of interferon-stimulated genes (ISGs), including but not limited to LY6E, CH25H, IFITM2/3, and IFIH1. However, the antiviral effect and underlying mechanisms of action of most ISGs in SARS-CoV-2 infection are not yet fully understood. By screening 109 ISG-knockout cell lines, we identify that phospholipid scramblase 1 (PLSCR1), an interferon-inducible protein, acts as a crucial restriction factor against SARS-CoV-2 infection. Cells lacking PLSCR1 are highly susceptible to SARS-CoV-2 infection. Conversely, overexpression of PLSCR1 inhibits SARS-CoV-2 infection. Depletion of PLSCR1 enhances cellular entry of both pseudotyped and authentic SARS-CoV-2. Mechanistically, PLSCR1 inhibits SARS-CoV-2 entry by specifically downregulating plasma membrane expression of ACE2, the virus's receptor, without affecting the overall levels of ACE2 within the cell. As such, we unraveled previously unappreciated mechanisms by which PLSCR1 exerts its restrictive effect on SARS-CoV-2. These data provide new insights into the interplay between host innate antiviral immunity and SARS-CoV-2 and shed light on novel antiviral therapeutics.</p><p><strong>Importance: </strong>Phospholipid scramblase 1 (PLSCR1) has been identified as a critical host restriction factor against SARS-CoV-2 infection. In this study, we demonstrated that PLSCR1 inhibited SARS-CoV-2 entry by downregulating the plasma membrane expression of ACE2, the primary receptor for viral entry. Our findings elucidate a novel host-pathogen interaction that not only deepens our understanding of the innate immune response to SARS-CoV-2 but offers potential strategies for therapeutic interventions against COVID-19.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0208524"},"PeriodicalIF":4.0,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143408651","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}
Muhammad Fadli, Sakae Hisano, Guy Novoa, José R Castón, Hideki Kondo, Nobuhiro Suzuki
Capsidless yadokariviruses (members of the order Yadokarivirales) with (+)RNA genomes divert the capsid of their partner icosahedral double-stranded RNA (dsRNA) viruses in different families of the order Ghabrivirales into the replication site. A yadokarivirus, AfSV2, has been reported from a German strain of the ascomycete fungus Aspergillus foetidus coinfected by two dsRNA viruses, a victorivirus (AfSV1, family Pseudototiviridae) and an alternavirus (AfFV, family Alternaviridae). Here, we identified AfSV1 as the partner of AfSV2 in a Japanese A. foetidus strain after showing the infectiousness of AfSV2 in three forms: virus particles (heterocapsid), transforming full-length complementary DNA (cDNA), and purified replicated form (RF) dsRNA that is believed to be inactive as a translational template. Virion transfection of virus-free A. foetidus protoplasts resulted in the generation of two strains infected either by AfSV1 alone or by both AfSV1 and AfSV2. Transformants with AfSV2 full-length cDNA launched AfSV2 infection only in the presence of AfSV1, but not those with AfSV2 RNA-directed RNA polymerase mutant cDNA. The purified fractions containing AfSV2 RF dsRNA also launched infection when transfected into protoplasts infected by AfSV1. Treatment with dsRNA-specific RNase III, but not with proteinase K, S1 nuclease, or DNase I, abolished the infectivity of AfSV2 RF dsRNA. Furthermore, we confirmed the infectiousness of gel-purified AfSV2 RF dsRNA in the presence of AfSV1. Taken together, our results show the unique infectious entity of AfSV2 and the expansion of yadokarivirus partners in the family Pseudototiviridae and provide interesting evolutionary insights.IMPORTANCEThe viral phylum Pisuviricota accommodates members with both double-stranded RNA (dsRNA) and (+)RNA genomes. Some members of the second group display peculiar virus lifestyles. These include (+)RNA yadkariviruses, which are capsidless and highjack the capsid of their partner dsRNA viruses in the order Ghabrivirales of a different phylum Duplornaviricota. We identified the partner dsRNA virus (AfSV1, a victorivirus) of a yadokarivirus (AfSV2) from the ascomycete Aspergillus foetidus. AfSV2 is infectious in the presence of AfSV1 in three forms: purified particles, transforming full-length complementary DNA, and, surprisingly, the purified replicative form dsRNA. These combined results expand yadokarivirus partner viruses to the family Pseudototiviridae and provide evidence for AfSV2 as a unique infectious entity as well as interesting evolutionary insights.
{"title":"A capsidless (+)RNA yadokarivirus hosted by a dsRNA virus is infectious as particles, cDNA, and dsRNA.","authors":"Muhammad Fadli, Sakae Hisano, Guy Novoa, José R Castón, Hideki Kondo, Nobuhiro Suzuki","doi":"10.1128/jvi.02166-24","DOIUrl":"https://doi.org/10.1128/jvi.02166-24","url":null,"abstract":"<p><p>Capsidless yadokariviruses (members of the order <i>Yadokarivirales</i>) with (+)RNA genomes divert the capsid of their partner icosahedral double-stranded RNA (dsRNA) viruses in different families of the order <i>Ghabrivirales</i> into the replication site. A yadokarivirus, AfSV2, has been reported from a German strain of the ascomycete fungus <i>Aspergillus foetidus</i> coinfected by two dsRNA viruses, a victorivirus (AfSV1<i>,</i> family <i>Pseudototiviridae</i>) and an alternavirus (AfFV, family <i>Alternaviridae</i>). Here, we identified AfSV1 as the partner of AfSV2 in a Japanese <i>A. foetidus</i> strain after showing the infectiousness of AfSV2 in three forms: virus particles (heterocapsid), transforming full-length complementary DNA (cDNA), and purified replicated form (RF) dsRNA that is believed to be inactive as a translational template. Virion transfection of virus-free <i>A. foetidus</i> protoplasts resulted in the generation of two strains infected either by AfSV1 alone or by both AfSV1 and AfSV2. Transformants with AfSV2 full-length cDNA launched AfSV2 infection only in the presence of AfSV1, but not those with AfSV2 RNA-directed RNA polymerase mutant cDNA. The purified fractions containing AfSV2 RF dsRNA also launched infection when transfected into protoplasts infected by AfSV1. Treatment with dsRNA-specific RNase III, but not with proteinase K, S1 nuclease, or DNase I, abolished the infectivity of AfSV2 RF dsRNA. Furthermore, we confirmed the infectiousness of gel-purified AfSV2 RF dsRNA in the presence of AfSV1. Taken together, our results show the unique infectious entity of AfSV2 and the expansion of yadokarivirus partners in the family <i>Pseudototiviridae</i> and provide interesting evolutionary insights.IMPORTANCEThe viral phylum <i>Pisuviricota</i> accommodates members with both double-stranded RNA (dsRNA) and (+)RNA genomes. Some members of the second group display peculiar virus lifestyles. These include (+)RNA yadkariviruses, which are capsidless and highjack the capsid of their partner dsRNA viruses in the order <i>Ghabrivirales</i> of a different phylum <i>Duplornaviricota</i>. We identified the partner dsRNA virus (AfSV1, a victorivirus) of a yadokarivirus (AfSV2) from the ascomycete <i>Aspergillus foetidus</i>. AfSV2 is infectious in the presence of AfSV1 in three forms: purified particles, transforming full-length complementary DNA, and, surprisingly, the purified replicative form dsRNA. These combined results expand yadokarivirus partner viruses to the family <i>Pseudototiviridae</i> and provide evidence for AfSV2 as a unique infectious entity as well as interesting evolutionary insights.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0216624"},"PeriodicalIF":4.0,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143408640","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}
Huixin Zhu, Jinxiu Lou, Zhen Yang, Juan Bai, Ping Jiang, Xianwei Wang, Xing Liu
Porcine epidemic diarrhea virus (PEDV), a highly pathogenic enteric coronavirus, has caused significant economic losses worldwide in recent years. The PEDV spike (S) protein has been reported to undergo extensive N-glycosylation, suggesting that glycosylation plays a crucial role in PEDV replication. In this study, we demonstrated that the N-glycosylation pathway promotes PEDV replication by facilitating the glycosylation of the S protein. First, we observed that pharmacological inhibition of host N-glycosylation using specific inhibitors significantly reduces viral replication. Furthermore, genetic ablation of STT3A or STT3B, the catalytically active subunits of the oligosaccharyltransferase (OST) complex, revealed that the STT3B-OST complex, but not STT3A, is preferentially required for PEDV replication. Notably, we showed that the N-glycosylation of the PEDV S protein depends on the oligosaccharyltransferase activity of STT3B. Together, the study demonstrated the critical role of the N-glycosylation pathway in PEDV replication by elucidating the relationship between the N-glycosylation of the PEDV S protein and STT3B, thereby presenting a potential new target for the prevention and control of PEDV.IMPORTANCEThe highly N-glycosylated spike protein of porcine epidemic diarrhea virus (PEDV) is a multifunctional protein that plays a crucial role in the viral replication cycle. In this study, using pharmacological inhibitors, we demonstrated the importance of the N-glycosylation pathway in PEDV replication. Genetic analysis revealed that STT3B, one of the catalytically active subunits of the oligosaccharyltransferase complex, promotes viral proliferation by regulating the N-glycosylation of the PEDV spike protein. Our findings enhance the understanding of the role of the N-glycosylation pathway in viral infection and identify STT3B as a potential therapeutic target for controlling PEDV infection.
{"title":"STT3B promotes porcine epidemic diarrhea virus replication by regulating N-glycosylation of PEDV S protein.","authors":"Huixin Zhu, Jinxiu Lou, Zhen Yang, Juan Bai, Ping Jiang, Xianwei Wang, Xing Liu","doi":"10.1128/jvi.00018-25","DOIUrl":"https://doi.org/10.1128/jvi.00018-25","url":null,"abstract":"<p><p>Porcine epidemic diarrhea virus (PEDV), a highly pathogenic enteric coronavirus, has caused significant economic losses worldwide in recent years. The PEDV spike (S) protein has been reported to undergo extensive N-glycosylation, suggesting that glycosylation plays a crucial role in PEDV replication. In this study, we demonstrated that the N-glycosylation pathway promotes PEDV replication by facilitating the glycosylation of the S protein. First, we observed that pharmacological inhibition of host N-glycosylation using specific inhibitors significantly reduces viral replication. Furthermore, genetic ablation of STT3A or STT3B, the catalytically active subunits of the oligosaccharyltransferase (OST) complex, revealed that the STT3B-OST complex, but not STT3A, is preferentially required for PEDV replication. Notably, we showed that the N-glycosylation of the PEDV S protein depends on the oligosaccharyltransferase activity of STT3B. Together, the study demonstrated the critical role of the N-glycosylation pathway in PEDV replication by elucidating the relationship between the N-glycosylation of the PEDV S protein and STT3B, thereby presenting a potential new target for the prevention and control of PEDV.IMPORTANCEThe highly N-glycosylated spike protein of porcine epidemic diarrhea virus (PEDV) is a multifunctional protein that plays a crucial role in the viral replication cycle. In this study, using pharmacological inhibitors, we demonstrated the importance of the N-glycosylation pathway in PEDV replication. Genetic analysis revealed that STT3B, one of the catalytically active subunits of the oligosaccharyltransferase complex, promotes viral proliferation by regulating the N-glycosylation of the PEDV spike protein. Our findings enhance the understanding of the role of the N-glycosylation pathway in viral infection and identify STT3B as a potential therapeutic target for controlling PEDV infection.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0001825"},"PeriodicalIF":4.0,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143408578","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}
A successful strategy employed by RNA viruses to achieve replication is to evade host cell RNase degradation. However, the mechanisms through which plus-strand RNA viruses effectively shield viral RNA from cellular ribonuclease degradation remain unclear. In this study, we identified the phenomenon whereby plus-strand RNA viruses, including avian leukosis virus subgroup J (ALV-J), reticuloendotheliosis virus (REV), chicken astrovirus (CAstV), and porcine epidemic diarrhea virus (PEDV), hijacked host cellular Musashi homolog 1 (MSI1). These viruses upregulated MSI1 expression and facilitated its translocation from the cytoplasmic periphery to a position proximal to and within the nucleus, thereby protecting viral RNA from degradation. Mechanistic analyses revealed that these viruses use distinct regions, the unique (U3) region or three prime untranslated region (3'UTR), to engage with MSI1, consequently shielding their viral RNA from cytoplasmic ribonuclease degradation. These results offer significant implications for understanding the replication tactics used by plus-strand RNA viruses, thereby advancing our understanding of their biological behaviors.IMPORTANCEThe intricate interplay between RNA viruses and host cell RNA regulation encompasses viral mechanisms designed to circumvent RNase-mediated degradation. However, the specific strategies employed by plus-strand RNA viruses to shield their RNA from host ribonucleases remain inadequately characterized. In this study, Musashi homolog 1 (MSI1) is predominantly localized in the cytoplasm of normal cells, distinct from the nucleus. Following infection by plus-strand RNA viruses such as avian leukosis virus subgroup J (ALV-J), reticuloendotheliosis virus (REV), chicken astrovirus (CAstV), and porcine epidemic diarrhea virus (PEDV), these viruses hijack MSI1 to relocate near and within the nucleus. This hijacking is facilitated by specific regions, including unique or three prime untranslated regions, thereby preventing viral RNA from degradation by cytoplasmic ribonucleases. These findings have significant implications for elucidating the replication strategies of plus-strand RNA viruses, thereby advancing our understanding of their biological mechanisms.
{"title":"Plus-strand RNA viruses hijack Musashi homolog 1 to shield viral RNA from cytoplasmic ribonuclease degradation.","authors":"Defang Zhou, Menglu Xu, Qingjie Liu, Ruixue Xin, Gege Cui, Longying Ding, Xiaoyang Liu, Xinyue Zhang, Tianxing Yan, Jing Zhou, Shuhai He, Liangyu Yang, Bin Xiang, Ziqiang Cheng","doi":"10.1128/jvi.00023-25","DOIUrl":"https://doi.org/10.1128/jvi.00023-25","url":null,"abstract":"<p><p>A successful strategy employed by RNA viruses to achieve replication is to evade host cell RNase degradation. However, the mechanisms through which plus-strand RNA viruses effectively shield viral RNA from cellular ribonuclease degradation remain unclear. In this study, we identified the phenomenon whereby plus-strand RNA viruses, including avian leukosis virus subgroup J (ALV-J), reticuloendotheliosis virus (REV), chicken astrovirus (CAstV), and porcine epidemic diarrhea virus (PEDV), hijacked host cellular Musashi homolog 1 (MSI1). These viruses upregulated MSI1 expression and facilitated its translocation from the cytoplasmic periphery to a position proximal to and within the nucleus, thereby protecting viral RNA from degradation. Mechanistic analyses revealed that these viruses use distinct regions, the unique (U3) region or three prime untranslated region (3'UTR), to engage with MSI1, consequently shielding their viral RNA from cytoplasmic ribonuclease degradation. These results offer significant implications for understanding the replication tactics used by plus-strand RNA viruses, thereby advancing our understanding of their biological behaviors.IMPORTANCEThe intricate interplay between RNA viruses and host cell RNA regulation encompasses viral mechanisms designed to circumvent RNase-mediated degradation. However, the specific strategies employed by plus-strand RNA viruses to shield their RNA from host ribonucleases remain inadequately characterized. In this study, Musashi homolog 1 (MSI1) is predominantly localized in the cytoplasm of normal cells, distinct from the nucleus. Following infection by plus-strand RNA viruses such as avian leukosis virus subgroup J (ALV-J), reticuloendotheliosis virus (REV), chicken astrovirus (CAstV), and porcine epidemic diarrhea virus (PEDV), these viruses hijack MSI1 to relocate near and within the nucleus. This hijacking is facilitated by specific regions, including unique or three prime untranslated regions, thereby preventing viral RNA from degradation by cytoplasmic ribonucleases. These findings have significant implications for elucidating the replication strategies of plus-strand RNA viruses, thereby advancing our understanding of their biological mechanisms.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0002325"},"PeriodicalIF":4.0,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143399545","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}
Evan P Williams, Yi Xue, Peter Vogel, Dong Yang, Alejandro Ponce-Flores, Xiaoyu Li, Tyler J Ogorek, Manisha Saini, Jorge Iulek, Francesc Xavier Ruiz, Eddy Arnold, Jennifer E Golden, Bernd Meibohm, Colleen B Jonsson
Venezuelan, western, and eastern equine encephalitis virus (VEEV, WEEV, and EEEV) cause a febrile illness that may result in fatal neurological disease in humans and equines. Human infections are typically from mosquito bites, although cases from respiratory exposure in laboratory accidents have been documented. In addition to natural mosquito-borne infection, the potential biothreat inherent in the ability to disseminate these viruses via the respiratory route has driven the development of antiviral drugs for this route of exposure. To address this gap, we tested the prophylactic administration of a novel brain-penetrant, antiviral, BDGR-49, against a lethal intranasal challenge of VEEV, WEEV, or EEEV in BALB/c mouse model. BDGR-49 conferred 100% protection with 6 mg kg-1 twice per day for 6 days for VEEV, but not EEEV or WEEV. By 8 days post-infection (dpi), infectious virus, viral RNA, and viral antigen in the brain of BDGR-49-treated mice were significantly reduced. Brains of VEEV TrD-infected, BDGR-49-treated mice showed a significant reduction in the expression of genes associated with inflammation (IFNB1, TNF, IL6, and CCL5) and cell death (CASP4, GSDMD, PYCARD, and ZBP1). At dpi 14, histopathology showed that neuronal lesions and inflammatory cell infiltrates were essentially absent, and viral antigen was not detected in the brains of VEEV TrD-infected, BDGR-49-treated mice. In summary, although BDGR-49 treatment showed significant promise for the treatment of mice exposed intranasally to VEEV, the more rapid and efficient entry of EEEV and WEEV by this route into the central nervous system will require additional optimization of the dosing regimen.IMPORTANCEProphylactic and therapeutic treatment of viruses that cause encephalitis requires fast-acting drugs that rapidly penetrate the blood-brain barrier. Currently, clinicians have only a limited set of antivirals for the treatment of neurotropic infections such as herpesviruses or HIV-1, and none for alphaviruses, and treatment outcomes remain poor. New medical countermeasures will address the gap in treatment of viral encephalitis such as those caused by the neurotropic alphaviruses and others.
{"title":"The antiviral BDGR-49 provides protection from lethal, neurotropic Venezuelan equine encephalitis virus intranasal infection in mice.","authors":"Evan P Williams, Yi Xue, Peter Vogel, Dong Yang, Alejandro Ponce-Flores, Xiaoyu Li, Tyler J Ogorek, Manisha Saini, Jorge Iulek, Francesc Xavier Ruiz, Eddy Arnold, Jennifer E Golden, Bernd Meibohm, Colleen B Jonsson","doi":"10.1128/jvi.01679-24","DOIUrl":"https://doi.org/10.1128/jvi.01679-24","url":null,"abstract":"<p><p>Venezuelan, western, and eastern equine encephalitis virus (VEEV, WEEV, and EEEV) cause a febrile illness that may result in fatal neurological disease in humans and equines. Human infections are typically from mosquito bites, although cases from respiratory exposure in laboratory accidents have been documented. In addition to natural mosquito-borne infection, the potential biothreat inherent in the ability to disseminate these viruses via the respiratory route has driven the development of antiviral drugs for this route of exposure. To address this gap, we tested the prophylactic administration of a novel brain-penetrant, antiviral, BDGR-49, against a lethal intranasal challenge of VEEV, WEEV, or EEEV in BALB/c mouse model. BDGR-49 conferred 100% protection with 6 mg kg<sup>-1</sup> twice per day for 6 days for VEEV, but not EEEV or WEEV. By 8 days post-infection (dpi), infectious virus, viral RNA, and viral antigen in the brain of BDGR-49-treated mice were significantly reduced. Brains of VEEV TrD-infected, BDGR-49-treated mice showed a significant reduction in the expression of genes associated with inflammation (<i>IFNB1</i>, <i>TNF</i>, <i>IL6</i>, and <i>CCL5</i>) and cell death (<i>CASP4</i>, <i>GSDMD</i>, <i>PYCARD</i>, and <i>ZBP1</i>). At dpi 14, histopathology showed that neuronal lesions and inflammatory cell infiltrates were essentially absent, and viral antigen was not detected in the brains of VEEV TrD-infected, BDGR-49-treated mice. In summary, although BDGR-49 treatment showed significant promise for the treatment of mice exposed intranasally to VEEV, the more rapid and efficient entry of EEEV and WEEV by this route into the central nervous system will require additional optimization of the dosing regimen.IMPORTANCEProphylactic and therapeutic treatment of viruses that cause encephalitis requires fast-acting drugs that rapidly penetrate the blood-brain barrier. Currently, clinicians have only a limited set of antivirals for the treatment of neurotropic infections such as herpesviruses or HIV-1, and none for alphaviruses, and treatment outcomes remain poor. New medical countermeasures will address the gap in treatment of viral encephalitis such as those caused by the neurotropic alphaviruses and others.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0167924"},"PeriodicalIF":4.0,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143399563","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}
In general, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates well at 37°C, which is the temperature of the human lower respiratory tract, but it poorly at 30°C‒32°C, which is the temperature of the human upper respiratory tract. The replication efficiency of SARS-CoV-2 in the upper respiratory tract may directly affect its transmissibility. In this study, an XBB.1.5 isolate showed superior replicative ability at 32°C and 30°C, whereas most other Omicron sub-variant isolates showed limited growth. Deep sequencing analysis demonstrated that the frequencies of viruses possessing the NSP6-S163P and NSP13-P238S substitutions increased to more than 97% during propagation of the XBB.1.5 isolate at 32°C but did not reach 55% at 37°C. Reverse genetics revealed that these substitutions contributed to superior virus growth in vitro at these low temperatures by improving virus genome replication. Mutant virus possessing both substitutions showed slightly higher virus titers in the upper respiratory tract of hamsters compared to the parental virus; however, transmissibility between hamsters was similar for the mutant and parental viruses. Taken together, our findings indicate that NSP6-S163P and NSP13-P238S contribute to superior virus growth at low temperatures in vitro and in the upper respiratory tract of hamsters.
Importance: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates efficiently at 37°C. However, the temperature of the human upper airway is 30°C-32°C. Therefore, the replicative ability of SARS-CoV-2 at low temperatures could influence virus replication in the upper airway and transmissibility. In this study, we assessed the growth of Omicron sub-variants at low temperatures and found that an XBB.1.5 isolate showed increased replicative ability. By deep sequencing analysis and reverse genetics, we found that amino acid changes in NSP6 and NSP13 contribute to the low-temperature growth; these changes improved RNA polymerase activity at low temperatures and enhanced virus replication in the upper airway of hamsters. Although these substitutions alone did not drastically affect virus transmissibility, in combination with other substitutions, they could affect virus replication in humans. Furthermore, since these substitutions enhance virus replication in cultured cells, they could be used to improve the production of inactivated or live attenuated vaccine virus.
{"title":"Amino acid substitutions in NSP6 and NSP13 of SARS-CoV-2 contribute to superior virus growth at low temperatures.","authors":"Yuri Furusawa, Maki Kiso, Ryuta Uraki, Yuko Sakai-Tagawa, Hiroyuki Nagai, Michiko Koga, Yukie Kashima, Masayuki Hojo, Noriko Iwamoto, Kiyoko Iwatsuki-Horimoto, Norio Ohmagari, Yutaka Suzuki, Hiroshi Yotsuyanagi, Peter J Halfmann, Wataru Kamitani, Seiya Yamayoshi, Yoshihiro Kawaoka","doi":"10.1128/jvi.02217-24","DOIUrl":"https://doi.org/10.1128/jvi.02217-24","url":null,"abstract":"<p><p>In general, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates well at 37°C, which is the temperature of the human lower respiratory tract, but it poorly at 30°C‒32°C, which is the temperature of the human upper respiratory tract. The replication efficiency of SARS-CoV-2 in the upper respiratory tract may directly affect its transmissibility. In this study, an XBB.1.5 isolate showed superior replicative ability at 32°C and 30°C, whereas most other Omicron sub-variant isolates showed limited growth. Deep sequencing analysis demonstrated that the frequencies of viruses possessing the NSP6-S163P and NSP13-P238S substitutions increased to more than 97% during propagation of the XBB.1.5 isolate at 32°C but did not reach 55% at 37°C. Reverse genetics revealed that these substitutions contributed to superior virus growth <i>in vitro</i> at these low temperatures by improving virus genome replication. Mutant virus possessing both substitutions showed slightly higher virus titers in the upper respiratory tract of hamsters compared to the parental virus; however, transmissibility between hamsters was similar for the mutant and parental viruses. Taken together, our findings indicate that NSP6-S163P and NSP13-P238S contribute to superior virus growth at low temperatures <i>in vitro</i> and in the upper respiratory tract of hamsters.</p><p><strong>Importance: </strong>Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates efficiently at 37°C. However, the temperature of the human upper airway is 30°C-32°C. Therefore, the replicative ability of SARS-CoV-2 at low temperatures could influence virus replication in the upper airway and transmissibility. In this study, we assessed the growth of Omicron sub-variants at low temperatures and found that an XBB.1.5 isolate showed increased replicative ability. By deep sequencing analysis and reverse genetics, we found that amino acid changes in NSP6 and NSP13 contribute to the low-temperature growth; these changes improved RNA polymerase activity at low temperatures and enhanced virus replication in the upper airway of hamsters. Although these substitutions alone did not drastically affect virus transmissibility, in combination with other substitutions, they could affect virus replication in humans. Furthermore, since these substitutions enhance virus replication in cultured cells, they could be used to improve the production of inactivated or live attenuated vaccine virus.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0221724"},"PeriodicalIF":4.0,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143399495","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}