A tick-borne Langat virus mutant that is temperature sensitive and host range restricted in neuroblastoma cells and lacks neuroinvasiveness for immunodeficient mice

Abstract

Langat virus (LGT), the naturally attenuated member of the tick-borne encephalitis virus (TBEV) complex, was tested extensively in clinical trials as a live TBEV vaccine and was found to induce a protective, durable immune response; however, it retained a low residual neuroinvasiveness in mice and humans. In order to ablate or reduce this property, LGT mutants that produced a small plaque size or temperature-sensitive (ts) phenotype in Vero cells were generated using 5-fluorouracil. One of these ts mutants, clone E5-104, exhibited a more than 10(3)-fold reduction in replication at the permissive temperature in both mouse and human neuroblastoma cells and lacked detectable neuroinvasiveness for highly sensitive immunodeficient mice. The E5-104 mutant possessed five amino acid substitutions in the structural protein E and one change in each of the nonstructural proteins NS3 and NS5. Using reverse genetics, we demonstrated that a Lys(46)-->Glu substitution in NS3 as well as a single Lys(315)-->Glu change in E significantly impaired the growth of LGT in neuroblastoma cells and reduced its peripheral neurovirulence for SCID mice. This study and our previous experience with chimeric flaviviruses indicated that a decrease in viral replication in neuroblastoma cells might serve as a predictor of in vivo attenuation of the neurotropic flaviviruses. The combination of seven mutations identified in the nonneuroinvasive E5-104 mutant provided a useful foundation for further development of a live attenuated TBEV vaccine. An evaluation of the complete sequence of virus recovered from brain of SCID mice inoculated with LGT mutants identified sites in the LGT genome that promoted neurovirulence/neuroinvasiveness.

FIG. 1.

Plaque morphology and growth analysis of parental E5 virus and its E5-104 derivative in Vero cells and growth in neuroblastoma cells of human or murine origin. (A) Plaques of E5 and E5-104 on Vero cell monolayers were visualized using immunoperoxidase staining on day 4 postinfection. The mean plaque diameter of 11 randomly selected E5 plaques was 2.2 mm. Plaques of E5-104 (mean diameter of 0.18 ± 0.02 mm; n = 9) are indicated by arrows. (B) Growth of the wt LGT, E5, and E5-104 viruses in Vero and neuroblastoma cell cultures at temperatures ranging from 32°C to 39°C. Confluent cell monolayers in 24-well plates were infected with parental or mutant virus at an MOI of 0.01 and incubated at the indicated temperature for 4 days. Virus titers in cell culture medium were determined by plaque assay in Vero cells incubated at 32°C. The limit of detection was 0.7 log₁₀ PFU/ml. Bars represent the standard error of three to four separate experiments. (C) Replication kinetics of wt LGT, E5, and E5-104 viruses in Vero cells or in Neuro-2A or SH-SY5Y neuroblastoma cells following incubation at 32°C or 37°C. Virus was inoculated at an MOI of 0.01, and the virus titer in cell culture medium was determined in Vero cells cultivated at 32°C.

FIG. 2.

Plaque morphology and growth analysis of cDNA-derived E5-651 virus and its recombinant mutants in Vero and neuroblastoma cells. (A) Plaque size of the E5-651 parent and its mutants on Vero cells monolayers. Monolayers of confluent cells were infected with the indicated viruses and incubated at 37°C for 4 days, and resulting plaques were visualized by immunostaining. The value below each well corresponds to the mean plaque diameter in mm ± standard error (n = 10 to 20 plaques per virus). (B) Analysis of growth of parental E5-651 and its recombinant mutant derivatives in Vero cells and in Neuro-2A or SH-SY5Y neuroblastoma cells following incubation at 32°C. Cells were infected with the indicated virus at an MOI of 0.01, and the virus titers in cell culture medium were determined as described in Materials and Methods.

FIG. 3.

Proposed three-dimensional structure of the E protein of LGT E5, based on the structure of the TBEV E protein ectodomain (31). The three structural domains I, II, and III are shown in red, yellow, and blue, respectively. Mutated residues identified in the E protein of the E5-104 mutant (Table 1) are shown in the upper panel. Mutated residues present in virus isolates from brain of mice are shown in the lower panel. The mutant residues are shown in green, and their position numbers and amino acid substitutions are indicated. The structure of the E protein ectodomain is displayed using the Swiss-Pdb Viewer version 3.7 (http://swissmodel.expasy.org/spdbv).