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
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.
