title: Development of a reverse genetics system for respiratory syncytial virus long strain and an immunogenicity study of the recombinant virus.
abstract: BACKGROUND
  Respiratory Syncytial Virus (RSV) is an important human respiratory pathogen, particularly of infants and older adults, and despite several decades of research and development, no licensed vaccine is available. Studies have confirmed that enhancement of RSV disease does not occur after inoculation with RSV live-attenuated vaccine candidates, making such vaccines preferable. In this paper, reverse genetics was used to construct two recombinant viruses, a recombinant Long strain (rLong) and rLong-∆G-EGFP; rLong-∆G-EGFP is a recombinant mutant in which G was replaced with the EGFP gene, based on the Long strain of RSV.
  
  
  RESULTS
  Both rLong and rLong-∆G-EGFP were constructed successfully and recovered in Hep-2 cells, and autofluorescence was observed in rLong-∆G-EGFP-infected cells during consecutive passages. Titers of rLong and rLong-∆G-EGFP were ~100-fold lower than the parental strain. Although virulence was attenuated, high titers of neutralizing antibodies were induced in BALB/c mice after being inoculated with recombinant viruses in a three-dose schedule. Unexpectedly, the neutralizing antibody titer in rLong-∆G-EGFP-immunized recipients did not decline significantly compared with the rLong strain. Protective efficacy of recombinant viruses in lung tissue was up to 100%, and the serum neutralizing antibody levels could stabilize at 21 days with no significant fall post-challenge. Enzyme-linked immunospot (ELISPOT) assays showed that both recombinant viruses were capable of inducing CD8+ T cell immune responses, which are crucial for virus clearance, and that rLong stimulated a higher level of IFN-γ production by comparison. In terms of inducing a balanced immune response, rLong-∆G-EGFP elicited slightly higher levels of IgG2a antibodies and lower levels of IgG1/IgG2a than the rLong virus.
  
  
  CONCLUSIONS
  This study suggested that immunization with rLong and rLong-∆G-EGFP were immunogenic and protected against RSV infection in the lower respiratory tract of BALB/c mice better than in the nose. Because of a relative low IgG1/IgG2a ratio, rLong-∆G-EGFP was more inclined to make CD4+ T cells, shifting toward a Th1-type response, indicating that the generation of a more balanced Th1/Th2 response was desirable. This explorative study on the recombinant Long viruses also contributed to obtaining more RSV attenuated candidates by a reverse genetics approach.
authors.affiliation: Institute of Infectious Disease Control and Prevention, Hubei Provincial Center for Disease Control and Prevention, No,6 North Zhuodaoquan Road, Wuhan City, Hubei province 430079, China. hubing007hb@163.com.
authors.forename: Bing
authors.initials: B
authors.lastname: Hu
authors.forename: Jiawei
authors.initials: J
authors.lastname: Jiang
authors.forename: Jianbo
authors.initials: J
authors.lastname: Zhan
authors.forename: Guoming
authors.initials: G
authors.lastname: Li
authors.forename: Yongzhong
authors.initials: Y
authors.lastname: Jiang
authors.forename: Xuhua
authors.initials: X
authors.lastname: Guan
authors.forename: Yuanding
authors.initials: Y
authors.lastname: Chen
authors.forename: Zhizheng
authors.initials: Z
authors.lastname: Fang
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D004195:Disease Models, Animal; D015870:Gene Expression; D017930:Genes, Reporter; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics; D056945:Hep G2 Cells; D006801:Humans; D007111:Immunity, Cellular; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D018357:Respiratory Syncytial Virus Infections / Q000276:immunology* / Q000821:virology*; D022261:Respiratory Syncytial Virus Vaccines; D018113:Respiratory Syncytial Virus, Human / Q000235:genetics* / Q000254:growth & development / Q000276:immunology*; D059386:Reverse Genetics / Q000379:methods*; D016176:T-Lymphocyte Subsets / Q000276:immunology; D010948:Viral Plaque Assay; D014779:Virus Replication
chemical_list: D000914:Antibodies, Viral; D007074:Immunoglobulin G; D022261:Respiratory Syncytial Virus Vaccines
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