Reverse genetic systems (RGS) have been widely used for fixed rabies virus (RABV) strains. However, RGS, for wild-type (wt) strains, have been seldom reported despite the value of this approach in defining the biological characteristics of these strains. In this work, we developed a wt RGS using a swine-origin RABV strain (GD-SH-01) for the first time. In order to have a better understanding of the contribution and function of individual gene on viral proliferation for wt RABV isolates, we constructed a full-length cDNA clone of GD-SH-01 and exchanged the single genes encoding RABV protein of a highly attenuated RABV strain HEP-Flury with those of the virulent strain. Analysis of the viral growth kinetics, cell-to-cell spread, and genomic RNA (gRNA) synthesis of the both the rescued and parental virus strains revealed that replacement of the HEP-Flury N or L genes with those from GD-SH-01 resulted in higher proliferative capacity of both chimeric rHEP-shN and rHEP-shL while the former seemed to have a better viral gRNA synthesis ability, the latter spread faster. Replacement of HEP-Flury P gene with GD-SH-01 P gene resulted in reduction of the virus titer in cell culture supernatants with a poor replicative and spreading ability. However, replacement of HEP-Flury M or G genes with those from GD-SH-01 seemed to impact less on viral proliferation. Taken together, we show that we have successfully rescued a wt RABV strain, and assessed the impact of each gene on viral proliferative capacity using a series of single-gene-substituted viruses.
Rabies virus Reverse genetic system Gene substitution Viral proliferation
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We thank the Guangdong Haid Institute of Animal Husbandry and Veterinary for the support of instruments. This study was partially supported by National Nature Science Foundation of China (No.31172322), Nature Science Foundation of Guangdong (No.2015A03031103) and the National Key Research and Development Program of China (No. 2016YF D0500400).
XFG, YFW, and QT conceived the study; QT, YFW, QZ, JL, and MZM performed the research; QT, YFW, QZ, and JL analyzed data; QT, YFW, XFG, and YWL wrote the manuscript.
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Conflicts of interest
The authors declare that they have no competing interests.
This article does not contain any studies with human participants or animals performed by any of the authors.
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