Background Several coronaviruses establish persistent infections in vitro and in vivo,however it is unknown whether persistence is a feature of the severe acute respiratory syndorme coronavirus(SARS-CoV)life cycle.Thi...Background Several coronaviruses establish persistent infections in vitro and in vivo,however it is unknown whether persistence is a feature of the severe acute respiratory syndorme coronavirus(SARS-CoV)life cycle.This study was conducted to investigate viral persistence.Methods We inoculated confluent monolayers of Vero cells with SARS-CoV at a multiplicity of infection of 0.1 TCID_(50)and passaged the remaining cells every 4 to 8 days for a total of 11 passages.Virus was titrated at each passage by limited dilution assay and nucleocapsid antigen was detected by Western blot and immunofluoresence assays.The presence of viral particles in passage 11 cells was assessed by electron microscopy.Changes in viral genomic sequences during persistent infection were examined by DNA sequencing.[KH*2/5D]Results Cytopathic effect was extensive after initial inoculation but diminished with serial passages.Infectious virus was detected after each passage and viral growth curves were identical for parental virus stock and virus obtained from passage 11 cells.Nucleocapsid antigen was detected in the majority of cells after initial inoculation but in only 10%-40%of cells at passages 2-11.Electron microscopy confirmed the presence of viral particles in passage 11 cells.Sequence analysis at passage 11 revealed fixed mutations in the spike(S)gene and ORFs 7a-8b but not in the nucleocapsid(N)gene.[KH*2/5D]Conclusions SARS-CoV can establish a persistent infection in vitro.The mechanism for viral persistence is consistent with the formation of a carrier culture whereby a limited number of cells are infected with each round of virus replication and release.Persistence is associated with selected mutations in the SARS-CoV genome.This model may provide insight into SARS-related lung pathology and mechanisms by which humans and animals can serve as reservoirs for infection.展开更多
Background:Critically ill patients in ICUs are highly vulnerable to infectious diseases.Early and accurate identification of pathogens is vital for initiating appropriate antimicrobial therapy.To evaluate the diagnost...Background:Critically ill patients in ICUs are highly vulnerable to infectious diseases.Early and accurate identification of pathogens is vital for initiating appropriate antimicrobial therapy.To evaluate the diagnostic effectiveness in patients with suspected infectious diseases;three different molecular technologies and conventional microbiological tests were used.Methods:A total of 97 individuals suspected of having infectious diseases were retrospectively enrolled from July 2023 to January 2024 at Shanghai General Hospital.Samples were collected for metagenomic next-generation sequencing(mNGS),droplet digital polymerase chain reaction(ddPCR),multiplex drop-off digital polymerase chain reaction(MDO-dPCR),and conventional microbiological tests(CMTs)for suspected pathogen detection.The diagnostic efficacies of the three molecular technologies and CMTs were compared,and the effects of their joint application on clinical outcomes were evaluated.Intergroup comparisons were performed using the Kruskal-Wallis test,with a P-value<0.05 considered statistically significant.Results:Joint detection exhibited a high negative predictive value.The sensitivity of MDO-dPCR,ddPCR,and mNGS was 52.6%,48.5%,and 96.6%,respectively;and the corresponding specificity was 72.5%,73.3%,and 50.0%.A positive correlation was observed between pathogen copies detected using MDO-dPCR and procalcitonin(Pearson’sρ=0.21,P=0.039),acute physiology and chronic health evaluation II(Pearson’sρ=0.24,P=0.018),and sequential organ failure assessment(Pearson’sρ=0.25,P=0.012).Therapeutic regimens were adjusted in 51.5%of the patients(50/97)based on the results of the combination tests.Conclusions:In the present study,we highlighted the significance of molecular technologies for the early diagnosis of patients with suspected infections.These technologies can serve as a complement to CMTs and should be implemented promptly to guide clinicians in providing timely and effective anti-infective treatments.Future studies should aim to confirm these findings in large-scale clinical trials to refine diagnostic protocols,while also incorporating cost-utility analyses.展开更多
基金This work was supported by grants from the National Institutes of Health(No.AI51292)the Ellison Medical Foundation(No.ID SS 0030).
文摘Background Several coronaviruses establish persistent infections in vitro and in vivo,however it is unknown whether persistence is a feature of the severe acute respiratory syndorme coronavirus(SARS-CoV)life cycle.This study was conducted to investigate viral persistence.Methods We inoculated confluent monolayers of Vero cells with SARS-CoV at a multiplicity of infection of 0.1 TCID_(50)and passaged the remaining cells every 4 to 8 days for a total of 11 passages.Virus was titrated at each passage by limited dilution assay and nucleocapsid antigen was detected by Western blot and immunofluoresence assays.The presence of viral particles in passage 11 cells was assessed by electron microscopy.Changes in viral genomic sequences during persistent infection were examined by DNA sequencing.[KH*2/5D]Results Cytopathic effect was extensive after initial inoculation but diminished with serial passages.Infectious virus was detected after each passage and viral growth curves were identical for parental virus stock and virus obtained from passage 11 cells.Nucleocapsid antigen was detected in the majority of cells after initial inoculation but in only 10%-40%of cells at passages 2-11.Electron microscopy confirmed the presence of viral particles in passage 11 cells.Sequence analysis at passage 11 revealed fixed mutations in the spike(S)gene and ORFs 7a-8b but not in the nucleocapsid(N)gene.[KH*2/5D]Conclusions SARS-CoV can establish a persistent infection in vitro.The mechanism for viral persistence is consistent with the formation of a carrier culture whereby a limited number of cells are infected with each round of virus replication and release.Persistence is associated with selected mutations in the SARS-CoV genome.This model may provide insight into SARS-related lung pathology and mechanisms by which humans and animals can serve as reservoirs for infection.
基金This study was supported by the National Natural Science Foundation of China(grant number 82472218)the National Key Clinical Specialist Construction Project(grant number Z155080000004)+3 种基金National Key Research and Development Program of China(grant number 2024YFC3044400)Noncommunicable Chronic Diseases-National Science and Technology Major Project(grant number 2023ZD0506502)the Science and Technology of Shanghai Committee(grant numbers 23Y31900100 and 20Z11901105)the Key Supporting Discipline of Shanghai Healthcare System(grant number 2023ZDFC0102).
文摘Background:Critically ill patients in ICUs are highly vulnerable to infectious diseases.Early and accurate identification of pathogens is vital for initiating appropriate antimicrobial therapy.To evaluate the diagnostic effectiveness in patients with suspected infectious diseases;three different molecular technologies and conventional microbiological tests were used.Methods:A total of 97 individuals suspected of having infectious diseases were retrospectively enrolled from July 2023 to January 2024 at Shanghai General Hospital.Samples were collected for metagenomic next-generation sequencing(mNGS),droplet digital polymerase chain reaction(ddPCR),multiplex drop-off digital polymerase chain reaction(MDO-dPCR),and conventional microbiological tests(CMTs)for suspected pathogen detection.The diagnostic efficacies of the three molecular technologies and CMTs were compared,and the effects of their joint application on clinical outcomes were evaluated.Intergroup comparisons were performed using the Kruskal-Wallis test,with a P-value<0.05 considered statistically significant.Results:Joint detection exhibited a high negative predictive value.The sensitivity of MDO-dPCR,ddPCR,and mNGS was 52.6%,48.5%,and 96.6%,respectively;and the corresponding specificity was 72.5%,73.3%,and 50.0%.A positive correlation was observed between pathogen copies detected using MDO-dPCR and procalcitonin(Pearson’sρ=0.21,P=0.039),acute physiology and chronic health evaluation II(Pearson’sρ=0.24,P=0.018),and sequential organ failure assessment(Pearson’sρ=0.25,P=0.012).Therapeutic regimens were adjusted in 51.5%of the patients(50/97)based on the results of the combination tests.Conclusions:In the present study,we highlighted the significance of molecular technologies for the early diagnosis of patients with suspected infections.These technologies can serve as a complement to CMTs and should be implemented promptly to guide clinicians in providing timely and effective anti-infective treatments.Future studies should aim to confirm these findings in large-scale clinical trials to refine diagnostic protocols,while also incorporating cost-utility analyses.
