title: Recombinant subgroup B human respiratory syncytial virus expressing enhanced green fluorescent protein efficiently replicates in primary human cells and is virulent in cotton rats.
abstract: Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which cocirculate during, or alternate between, yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSV(B05)) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSV(B05)EGFP(5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well-differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP(+) cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tracts. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies.
  
  
  OBJECTIVE
  Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with diminished virulence in animal models of disease. To address this, we determined the genome sequence of an unpassaged human respiratory syncytial virus from a sample obtained directly from an infected infant, assembled a molecular clone, and recovered a wild-type recombinant virus. Addition of a gene encoding enhanced green fluorescent protein allowed this wild-type virus to be tracked in primary human cells and living animals in the absence of significant cytopathic effects. Imaging of fluorescent cells proved to be a highly valuable tool for monitoring the spread of virus and may help improve assays for evaluating novel intervention strategies.
authors.affiliation: School of Medicine, Dentistry and Biomedical Sciences, The Queen's University of Belfast, Belfast, Northern Ireland, United Kingdom.
authors.forename: Ken
authors.initials: K
authors.lastname: Lemon
authors.affiliation: Department of Viroscience, Erasmus MC, Rotterdam, the Netherlands.
authors.forename: D Tien
authors.initials: DT
authors.lastname: Nguyen
authors.affiliation: Tissue Pathology, Belfast Health and Social Care Trust, The Queen's University of Belfast, Belfast, Northern Ireland, United Kingdom.
authors.forename: Martin
authors.initials: M
authors.lastname: Ludlow
authors.affiliation: Tissue Pathology, Belfast Health and Social Care Trust, The Queen's University of Belfast, Belfast, Northern Ireland, United Kingdom.
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Rennick
authors.affiliation: Department of Viroscience, Erasmus MC, Rotterdam, the Netherlands.
authors.forename: Selma
authors.initials: S
authors.lastname: Yüksel
authors.affiliation: Department of Viroscience, Erasmus MC, Rotterdam, the Netherlands.
authors.forename: Geert
authors.initials: G
authors.lastname: van Amerongen
authors.affiliation: Tissue Pathology, Belfast Health and Social Care Trust, The Queen's University of Belfast, Belfast, Northern Ireland, United Kingdom.
authors.forename: Stephen
authors.initials: S
authors.lastname: McQuaid
authors.affiliation: School of Medicine, Dentistry and Biomedical Sciences, The Queen's University of Belfast, Belfast, Northern Ireland, United Kingdom.
authors.forename: Bert K
authors.initials: BK
authors.lastname: Rima
authors.affiliation: Department of Viroscience, Erasmus MC, Rotterdam, the Netherlands.
authors.forename: Rik L
authors.initials: RL
authors.lastname: de Swart
authors.affiliation: Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts, USA pduprex@bu.edu.
authors.forename: W Paul
authors.initials: WP
authors.lastname: Duprex
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004195:Disease Models, Animal; D005260:Female; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D006801:Humans; D007223:Infant; D018357:Respiratory Syncytial Virus Infections / Q000473:pathology / Q000821:virology; D018113:Respiratory Syncytial Virus, Human / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D012137:Respiratory System / Q000821:virology; D016205:Sigmodontinae; D013194:Staining and Labeling; D014774:Virulence; D014779:Virus Replication
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins