Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the pathogen responsible for coronavirus disease 2019(COVID-19),continues to evolve,giving rise to more variants and global reinfections.Previous research ha...Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the pathogen responsible for coronavirus disease 2019(COVID-19),continues to evolve,giving rise to more variants and global reinfections.Previous research has demonstrated that barcode segments can effectively and cost-efficiently identify specific species within closely related populations.In this study,we designed and tested RNA barcode segments based on genetic evolutionary relationships to facilitate the efficient and accurate identification of SARS-CoV-2 from extensive virus samples,including human coronaviruses(HCoVs)and SARSr-CoV-2 lineages.Nucleotide sequences sourced from NCBI and GISAID were meticulously selected and curated to construct training sets,encompassing 1733 complete genome sequences of HCoVs and SARSr-CoV-2 lineages.Through genetic-level species testing,we validated the accuracy and reliability of the barcode segments for identifying SARS-CoV-2.Subsequently,75 main and subordinate species-specific barcode segments for SARS-CoV-2,located in ORF1ab,S,E,ORF7a,and N coding sequences,were intercepted and screened based on single-nucleotide polymorphism sites and weighted scores.Post-testing,these segments exhibited high recall rates(nearly 100%),specificity(almost 30%at the nucleotide level),and precision(100%)performance on identification.They were eventually visualized using one and two-dimensional combined barcodes and deposited in an online database(http://virusbarcodedatabase.top/).The successful integration of barcoding technology in SARS-CoV-2 identification provides valuable insights for future studies involving complete genome sequence polymorphism analysis.Moreover,this cost-effective and efficient identification approach also provides valuable reference for future research endeavors related to virus surveillance.展开更多
Virus-encoding RNA-dependent RNA polymerase(RdRp)is essential for genome replication and gene transcription of human coronaviruses(HCoVs),including severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).We previo...Virus-encoding RNA-dependent RNA polymerase(RdRp)is essential for genome replication and gene transcription of human coronaviruses(HCoVs),including severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).We previously identified the interaction between the catalytic subunit NSP12 of SARS-CoV-2 RdRp and the host protein CREB-regulated transcription coactivator 3(CRTC3),a member of the CRTC family that regulates cyclic AMP response element-binding protein(CREB)-mediated transcriptional activation.Currently,the implication of CRTC3 in the pathogenesis of HCoVs is poorly understood.Herein,we demonstrated that CRTC3 attenuates RdRp activity and SARS-CoV-2 genome replication,therefore reducing the production of progeny viruses.The interaction of CRTC3 with NSP12 contributes to its inhibitory effect on RdRp activity.Furthermore,we expanded the suppressive effects of two other CRTC family members(CRTC1 and CRTC2)on the RdRp activities of lethal HCoVs,including SARS-CoV-2 and Middle East respiratory syndrome coronavirus(MERS-CoV),along with the CREB antagonization.Overall,our research suggests that CRTCs restrict the replication of HCoVs and are antagonized by CREB,which not only provides new insights into the replication regulation of HCoVs,but also offers important information for the development of anti-HCoV interventions.展开更多
This study explores the antiviral properties of high-voltage low-frequency electric field exposure on the replication of human viruses, including Herpes Simplex Virus type 1 (HSV-1), Human Coronavirus OC43 (HCoV OC43)...This study explores the antiviral properties of high-voltage low-frequency electric field exposure on the replication of human viruses, including Herpes Simplex Virus type 1 (HSV-1), Human Coronavirus OC43 (HCoV OC43), and Influenza A virus (A H1N1). Using the HealectricsTM device (model S02), which operates by applying high-voltage direct current (30 - 50 kV) with a polarity change frequency of ~0.2 Hz, we investigated the impact on viral infectivity and host cell viability. Virus cultures were exposed to electric fields during different stages: virion adsorption (0 - 1 hour), intracellular replication (1 - 8 hours), and both stages. Viral infectivity was assessed through titration, and cytotoxic effects were evaluated using MTT assays. Electric field exposure significantly reduced viral infectivity, particularly during the combined sorption and replication stages, with up to a 90% decrease in viral activity. Among the viruses tested, HCoV OC43 showed the least sensitivity, with a reduction in viral activity by a factor of 5. Comparisons revealed statistically significant reductions for influenza and herpes viruses, and a trend towards significance for HCoV OC43. The electric field treatment did not significantly affect the viability of Vero and MDCK cells, indicating the method’s safety. Our findings suggest that high-voltage low-frequency electric fields can effectively reduce viral infectivity and may serve as a potential therapeutic and preventive measure against a wide range of membrane-bound viruses, including SARS-CoV-2.展开更多
Respiratory diseases have a large proportion among those various diseases. Among those, the main diseases that we are now dealing with are viruses which have no majority vaccine found: Human Rhinovirus 14 (HRV), Human...Respiratory diseases have a large proportion among those various diseases. Among those, the main diseases that we are now dealing with are viruses which have no majority vaccine found: Human Rhinovirus 14 (HRV), Human Coronavirus OC43 (HCoV), Respiratory Syncytial Virus (RSV), and Human Para influenza virus 1(HVJ). Even though the body can cure most of these viruses by itself, there are some incidents which end up with death. Starting an experiment with those reasons, we separated viruses by the basic symptoms and appearances, and by using data mining, we found similarities and differences of various sequences. As a result, having a high frequency, decision tree prove that each sequences are too different from each other, but still decision tree only shows the difference of the sequences. According to apriori algorithm, it could be able to find a remedy which can block amino acid L, Leucine.展开更多
The recent SARS-CoV-2 pandemic renewed interest in other previously discovered human coronaviruses—the non-severe acute respiratory syndrome human coronavirus (non-SARS hCoVs) and this study is a starting point for a...The recent SARS-CoV-2 pandemic renewed interest in other previously discovered human coronaviruses—the non-severe acute respiratory syndrome human coronavirus (non-SARS hCoVs) and this study is a starting point for a closer investigation of those. With the pandemic behind us we believe it to be important to also examine the current and past respiratory pathogen landscape in the patient population in our care. Therefore, 954 nasopharyngeal swabs of patients with respiratory diseases collected between October 2018 and March 2020 were tested against the pathogens Mycoplasma pneumoniae, Bordetella pertussis, Influenza A and virus, Human metapneumovirus, respiratory syncytial virus, Parainfluenza virus, human Adenovirus and Polyoma virus BK/JC. Swabs negative against these pathogens were further tested for OC43 and 229E by RT-qPCR. Human coronaviruses 229E and OC43 were proven as the causative agents in a considerable number of cases, confirmed by PCR. Overall, our results show that those two non-SARS hCoVs were responsible for 13.9% (11 of 79) of infections with flu-like symptoms of unknown etiology in the study area. In the subsequent seroprevalence study, it was shown that the seroprevalence rate of IgG antibodies against 229E and OC43 was over 50%, indicating that a big part of the population in our study area has been in contact with these non-SARS-CoVs and has built the specific humoral immune response accordingly.展开更多
Viral infectious clones(ICs)serve as robust platforms for studying viral biology and screening antiviral agents using reverse genetics.However,the molecular profiles and complex limitations of human coronaviruses(HCoV...Viral infectious clones(ICs)serve as robust platforms for studying viral biology and screening antiviral agents using reverse genetics.However,the molecular profiles and complex limitations of human coronaviruses(HCoVs)pose a challenge to ICs development.In this study,we report a novel platform to develop the ICs for HCoV-OC43-VR1558 using a one-step assembly method in yeast by transformation-associated recombination(TAR)technology.Recombinant HCoV-OC43-VR1558,named as rOC43(1558)-WT,was rapidly generated by TAR.In addition,recombinant HCoV-OC43-VR1558-expressing reporter genes,named as rOC43(1558)-ns2FusionRluc,was also generated based on TAR by inserting the ns2 region of the IC with Renilla luciferase(Rluc).We further characterized their replication through virus titration using 50%tissue culture infective dose(TCID50)and indirect immunofluorescence assay(IFA),luciferase reporter assay,and western blotting(WB)assay.The genetic stability of the recombinant HCoV-OC43 was assessed through viral genome sequencing following passaging in BHK-21 cells.These reporter viruses were validated as screening tools for inhibitorsin vitro by evaluating the antiviral activities of remdesivir and chloroquine.The phenotypes of HCoV-OC43-VR1558 and HCoV-OC43-VR759 were comparedin vitro andin vivo.The TAR-based one-step assembly of IC was successfully applied,facilitating the rapid generation of recombinant HCoV-OC43 and providing a useful platform for the investigation of biological mechanisms,development of vaccines and diagnostic tests,and screening inhibitors of HCoVs.展开更多
In order to clarify the pre-exist immunity background of different human coronaviruses(HCoV),this study investigated the positive rate of spike(S)protein antibodies of HCoV,including HCoV-severe acute respiratory synd...In order to clarify the pre-exist immunity background of different human coronaviruses(HCoV),this study investigated the positive rate of spike(S)protein antibodies of HCoV,including HCoV-severe acute respiratory syndrome(SARS)-associated coronavirus(SARS-CoV-1),severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),Middle East respiratory syndrome coronavirus(MERS-CoV),HCoV-229E,HCoV-NL63,HCoV-HKU1 and HCoV-OC43,before and after the Coronavirus Disease 2019(COVID-19)outbreak.We utilized pseu-dotyped virus-based neutralization assays(PBNA)or enzyme-linked immunosorbent assays(ELISA)to detect antibody levels against HCoV in serum samples collected in 2009-2010 and 2023.The PBNA results showed that neutralizing antibodies against SARS-CoV-1 and the MERS-CoV were negative.In the serum samples from 2009 to 2010,neutralizing antibodies against SARS-CoV-2(D614G)were negative,whereas in the serum sam-ples from 2023,73 samples(73%)showed neutralizing reactions with the SARS-CoV-2 D614G strain,96 sam-ples(96%)with the BA.5 strain,and 91 samples(91%)with the BF.7 strain.Among pre-COVID-19 samples,33%(33/100)showed neutralizing reactions with HCoV-229E and 63%(63/100)with HCoV-NL63.Among post-COVID-19 samples,50%(50/100)showed neutralizing reactions with HCoV-229E and 49%(49/100)with HCoV-NL63.Due to the different receptors of alpha coronavirus genus compared to other beta coron-avirus genus,neutralizing antibodies against HCoV-OC43 and HCoV-HKU1 virus cannot be detected by con-structing corresponding pseudotyped virus.Binding antibodies against HCoV-OC43 and HCoV-HKU1 virus were detected using ELISA.The results revealed that among pre-COVID-19 samples,83%(83/100)and 45%(45/100)had binding activity with HCoV-OC43 and HCoV-HKU1,respectively.Among post-COVID-19 samples,100%(100/100)and 81%(81/100)had binding activity with HCoV-0C43 and HCoV-HKU1,respectively.展开更多
基金supported by grants from Key Research&Development Project of Nanhua Biomedical Co.,Ltd.(No.H202191490139)National Natural Science Foundation of China(No.31872866)+1 种基金China Postdoctoral Science Foundation(Nos.2021M701160 and 2022M721101)Funds of Hunan university(521119400156).
文摘Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the pathogen responsible for coronavirus disease 2019(COVID-19),continues to evolve,giving rise to more variants and global reinfections.Previous research has demonstrated that barcode segments can effectively and cost-efficiently identify specific species within closely related populations.In this study,we designed and tested RNA barcode segments based on genetic evolutionary relationships to facilitate the efficient and accurate identification of SARS-CoV-2 from extensive virus samples,including human coronaviruses(HCoVs)and SARSr-CoV-2 lineages.Nucleotide sequences sourced from NCBI and GISAID were meticulously selected and curated to construct training sets,encompassing 1733 complete genome sequences of HCoVs and SARSr-CoV-2 lineages.Through genetic-level species testing,we validated the accuracy and reliability of the barcode segments for identifying SARS-CoV-2.Subsequently,75 main and subordinate species-specific barcode segments for SARS-CoV-2,located in ORF1ab,S,E,ORF7a,and N coding sequences,were intercepted and screened based on single-nucleotide polymorphism sites and weighted scores.Post-testing,these segments exhibited high recall rates(nearly 100%),specificity(almost 30%at the nucleotide level),and precision(100%)performance on identification.They were eventually visualized using one and two-dimensional combined barcodes and deposited in an online database(http://virusbarcodedatabase.top/).The successful integration of barcoding technology in SARS-CoV-2 identification provides valuable insights for future studies involving complete genome sequence polymorphism analysis.Moreover,this cost-effective and efficient identification approach also provides valuable reference for future research endeavors related to virus surveillance.
基金supported by grants from the National Natural Science Foundation of China(32071236)the National Science Fund for Distinguished Young Scholars(32225001)+6 种基金the 1.3.5 Project for Disciplines Excellence of West China Hospital,Sichuan University(ZYGD23018)Key Science and Technology Research Projects in Key Areas of the Corps(2023AB053)the National Key Research and Development Program of China(2022YFC2303700)the Joint Project of Pengzhou People's Hospital with Southwest Medical University(2024PZXNYD02)Project funded by China Postdoctoral Science Foundation(2020M683304)Sichuan Science and Technology Support Project(2021YJ0502)Post-Doctor Research Project,West China Hospital,Sichuan University(2020HXBH082).
文摘Virus-encoding RNA-dependent RNA polymerase(RdRp)is essential for genome replication and gene transcription of human coronaviruses(HCoVs),including severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).We previously identified the interaction between the catalytic subunit NSP12 of SARS-CoV-2 RdRp and the host protein CREB-regulated transcription coactivator 3(CRTC3),a member of the CRTC family that regulates cyclic AMP response element-binding protein(CREB)-mediated transcriptional activation.Currently,the implication of CRTC3 in the pathogenesis of HCoVs is poorly understood.Herein,we demonstrated that CRTC3 attenuates RdRp activity and SARS-CoV-2 genome replication,therefore reducing the production of progeny viruses.The interaction of CRTC3 with NSP12 contributes to its inhibitory effect on RdRp activity.Furthermore,we expanded the suppressive effects of two other CRTC family members(CRTC1 and CRTC2)on the RdRp activities of lethal HCoVs,including SARS-CoV-2 and Middle East respiratory syndrome coronavirus(MERS-CoV),along with the CREB antagonization.Overall,our research suggests that CRTCs restrict the replication of HCoVs and are antagonized by CREB,which not only provides new insights into the replication regulation of HCoVs,but also offers important information for the development of anti-HCoV interventions.
文摘This study explores the antiviral properties of high-voltage low-frequency electric field exposure on the replication of human viruses, including Herpes Simplex Virus type 1 (HSV-1), Human Coronavirus OC43 (HCoV OC43), and Influenza A virus (A H1N1). Using the HealectricsTM device (model S02), which operates by applying high-voltage direct current (30 - 50 kV) with a polarity change frequency of ~0.2 Hz, we investigated the impact on viral infectivity and host cell viability. Virus cultures were exposed to electric fields during different stages: virion adsorption (0 - 1 hour), intracellular replication (1 - 8 hours), and both stages. Viral infectivity was assessed through titration, and cytotoxic effects were evaluated using MTT assays. Electric field exposure significantly reduced viral infectivity, particularly during the combined sorption and replication stages, with up to a 90% decrease in viral activity. Among the viruses tested, HCoV OC43 showed the least sensitivity, with a reduction in viral activity by a factor of 5. Comparisons revealed statistically significant reductions for influenza and herpes viruses, and a trend towards significance for HCoV OC43. The electric field treatment did not significantly affect the viability of Vero and MDCK cells, indicating the method’s safety. Our findings suggest that high-voltage low-frequency electric fields can effectively reduce viral infectivity and may serve as a potential therapeutic and preventive measure against a wide range of membrane-bound viruses, including SARS-CoV-2.
文摘Respiratory diseases have a large proportion among those various diseases. Among those, the main diseases that we are now dealing with are viruses which have no majority vaccine found: Human Rhinovirus 14 (HRV), Human Coronavirus OC43 (HCoV), Respiratory Syncytial Virus (RSV), and Human Para influenza virus 1(HVJ). Even though the body can cure most of these viruses by itself, there are some incidents which end up with death. Starting an experiment with those reasons, we separated viruses by the basic symptoms and appearances, and by using data mining, we found similarities and differences of various sequences. As a result, having a high frequency, decision tree prove that each sequences are too different from each other, but still decision tree only shows the difference of the sequences. According to apriori algorithm, it could be able to find a remedy which can block amino acid L, Leucine.
文摘The recent SARS-CoV-2 pandemic renewed interest in other previously discovered human coronaviruses—the non-severe acute respiratory syndrome human coronavirus (non-SARS hCoVs) and this study is a starting point for a closer investigation of those. With the pandemic behind us we believe it to be important to also examine the current and past respiratory pathogen landscape in the patient population in our care. Therefore, 954 nasopharyngeal swabs of patients with respiratory diseases collected between October 2018 and March 2020 were tested against the pathogens Mycoplasma pneumoniae, Bordetella pertussis, Influenza A and virus, Human metapneumovirus, respiratory syncytial virus, Parainfluenza virus, human Adenovirus and Polyoma virus BK/JC. Swabs negative against these pathogens were further tested for OC43 and 229E by RT-qPCR. Human coronaviruses 229E and OC43 were proven as the causative agents in a considerable number of cases, confirmed by PCR. Overall, our results show that those two non-SARS hCoVs were responsible for 13.9% (11 of 79) of infections with flu-like symptoms of unknown etiology in the study area. In the subsequent seroprevalence study, it was shown that the seroprevalence rate of IgG antibodies against 229E and OC43 was over 50%, indicating that a big part of the population in our study area has been in contact with these non-SARS-CoVs and has built the specific humoral immune response accordingly.
基金supported by the National Key Research and Development Program of China(2022YFC2304100 and 2021YFA1201003).
文摘Viral infectious clones(ICs)serve as robust platforms for studying viral biology and screening antiviral agents using reverse genetics.However,the molecular profiles and complex limitations of human coronaviruses(HCoVs)pose a challenge to ICs development.In this study,we report a novel platform to develop the ICs for HCoV-OC43-VR1558 using a one-step assembly method in yeast by transformation-associated recombination(TAR)technology.Recombinant HCoV-OC43-VR1558,named as rOC43(1558)-WT,was rapidly generated by TAR.In addition,recombinant HCoV-OC43-VR1558-expressing reporter genes,named as rOC43(1558)-ns2FusionRluc,was also generated based on TAR by inserting the ns2 region of the IC with Renilla luciferase(Rluc).We further characterized their replication through virus titration using 50%tissue culture infective dose(TCID50)and indirect immunofluorescence assay(IFA),luciferase reporter assay,and western blotting(WB)assay.The genetic stability of the recombinant HCoV-OC43 was assessed through viral genome sequencing following passaging in BHK-21 cells.These reporter viruses were validated as screening tools for inhibitorsin vitro by evaluating the antiviral activities of remdesivir and chloroquine.The phenotypes of HCoV-OC43-VR1558 and HCoV-OC43-VR759 were comparedin vitro andin vivo.The TAR-based one-step assembly of IC was successfully applied,facilitating the rapid generation of recombinant HCoV-OC43 and providing a useful platform for the investigation of biological mechanisms,development of vaccines and diagnostic tests,and screening inhibitors of HCoVs.
基金supported by National Key Research and Development Program of China:Analysis of Omicron Variants and Research of Prevention and Control(No.2023YFC3041500)National Natural Science Foundation of China:Research on in vivo and in vitro Efficacy Evaluation Technology for Novel Coronavirus Vaccine Standardization(No.82073621)State Key Laboratory of Drug Regulatory Science:Establishment of mouse infection model by influenza and SARS-CoV-2 pseudovirus(No.2023SKDLS0112).
文摘In order to clarify the pre-exist immunity background of different human coronaviruses(HCoV),this study investigated the positive rate of spike(S)protein antibodies of HCoV,including HCoV-severe acute respiratory syndrome(SARS)-associated coronavirus(SARS-CoV-1),severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),Middle East respiratory syndrome coronavirus(MERS-CoV),HCoV-229E,HCoV-NL63,HCoV-HKU1 and HCoV-OC43,before and after the Coronavirus Disease 2019(COVID-19)outbreak.We utilized pseu-dotyped virus-based neutralization assays(PBNA)or enzyme-linked immunosorbent assays(ELISA)to detect antibody levels against HCoV in serum samples collected in 2009-2010 and 2023.The PBNA results showed that neutralizing antibodies against SARS-CoV-1 and the MERS-CoV were negative.In the serum samples from 2009 to 2010,neutralizing antibodies against SARS-CoV-2(D614G)were negative,whereas in the serum sam-ples from 2023,73 samples(73%)showed neutralizing reactions with the SARS-CoV-2 D614G strain,96 sam-ples(96%)with the BA.5 strain,and 91 samples(91%)with the BF.7 strain.Among pre-COVID-19 samples,33%(33/100)showed neutralizing reactions with HCoV-229E and 63%(63/100)with HCoV-NL63.Among post-COVID-19 samples,50%(50/100)showed neutralizing reactions with HCoV-229E and 49%(49/100)with HCoV-NL63.Due to the different receptors of alpha coronavirus genus compared to other beta coron-avirus genus,neutralizing antibodies against HCoV-OC43 and HCoV-HKU1 virus cannot be detected by con-structing corresponding pseudotyped virus.Binding antibodies against HCoV-OC43 and HCoV-HKU1 virus were detected using ELISA.The results revealed that among pre-COVID-19 samples,83%(83/100)and 45%(45/100)had binding activity with HCoV-OC43 and HCoV-HKU1,respectively.Among post-COVID-19 samples,100%(100/100)and 81%(81/100)had binding activity with HCoV-0C43 and HCoV-HKU1,respectively.
