Efficient rescue of recombinant Lassa virus reveals the influence of S segment noncoding regions on virus replication and virulence

Abstract

Lassa virus (LASV), is a significant cause of severe, often fatal, hemorrhagic fever in humans throughout western Africa, with an estimated 100,000 infections each year. No vaccines are commercially available. We report the development of an efficient reverse genetics system to rescue recombinant LASV and to investigate the contributions of the long 5' and 3' noncoding regions (NCRs) of the S genomic segment to in vitro growth and in vivo virulence. This work demonstrates that deletions of large portions of these NCRs confer an attenuated phenotype and are a first step toward further insights into the high virulence of LASV.

Fig. 1.

(A) Schematic of plasmids used to generate fully infectious LASV. (B) Recombinant (rec.) viruses were generated in BSRT7/5 cells and passaged onto VeroE6 cells as described before (1). Infected cells were fixed at 4 dpi, and LASV proteins were detected with anti-LASV rabbit serum and anti-rabbit Alexa Fluor 488. Fluorescent photomicrographs were taken using a specific wavelength filter. (C) Minor changes in the sequence of the L RNA segment were maintained as selective markers to differentiate the recombinant from the wild-type virus.

Fig. 2.

(A) RNA secondary structure of the full-length LASV S RNA segment predicted by Mfold (29). The box on right depicts a magnification of the 5′-3′-terminal region and the predicted panhandle structure. (B) The lengths of the 5′ and 3′ NCRs of S RNAs from representative LASV strains are shown at the top; below are the available L RNA NCR lengths. The lengths of NCRs of S RNAs from LCMV strains are shown in the middle, and those from three representative phleboviruses are shown at the bottom.

Fig. 3.

(A) Schematic of plasmids used to generate recombinant LASV mutants carrying single or double deletions in the 5′ and/or 3′ NCR. (B) Recombinant viruses were generated in BSRT7/5 cells and passaged twice on VeroE6 cells. Viral titers (right) were determined by standard TCID₅₀ assay.

Fig. 4.

(A) Growth curves generated by infecting VeroE6 cells with a multiplicity of infection of 0.01 and collecting supernatants at 24-h intervals. Virus titers were determined in a TCID₅₀ assay. Peak titers reached at 3 dpi are shown on the right. (B). Replication phenotype in the mouse model. Four groups of 14-day-old mice were infected intracranially with 500 TCID₅₀ of authentic rLASV or a single or double deletion mutant virus. Brains and spleens were collected at 7 dpi, and LASV RNA was detected with a quantitative RT-PCR assay. The number of positive (pos) detections and the average C_T (threshold cycle) value are shown. LASV RNA was normalized between specimens using a multiplexed primer-probe set for glyceraldehyde 3-phosphate dehydrogenase (GADPH) mRNA (ABI).