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Development of a reverse genetics system for respiratory syncytial virus long strain and an immunogenicity study of the recombinant virus

Bing Hu1*, Jiawei Jiang1, Jianbo Zhan1, Guoming Li1, Yongzhong Jiang1, Xuhua Guan1, Yuanding Chen2 and Zhizheng Fang3

Author Affiliations

1 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

2 Kunming Institute of Medical Biology, Chinese Academy of Medical Sciences, No.379 Jiaoling Road, Kunming City, Yunnan province 650118, China

3 Wuhan Institute of Biological Products, China Pharmaceutical Group Corporation, No.9 Linjiang Road, Wuhan City, Hubei province 430060, China

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Virology Journal 2014, 11:142  doi:10.1186/1743-422X-11-142

Published: 8 August 2014



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.


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.


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.

Respiratory syncytial virus (RSV) Long strain; Reverse genetics; Attenuated vaccine candidates; Immunogenicity; Protection