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. 2007 Feb;81(4):2056-64.
doi: 10.1128/JVI.01911-06. Epub 2006 Dec 6.

Synergistic attenuation of vesicular stomatitis virus by combination of specific G gene truncations and N gene translocations

David K Clarke  1 , Farooq Nasar, Margaret Lee, J Erik Johnson, Kevin Wright, Priscilla Calderon, Min Guo, Robert Natuk, David Cooper, R Michael Hendry, Stephen A Udem
Affiliations

Synergistic attenuation of vesicular stomatitis virus by combination of specific G gene truncations and N gene translocations

David K Clarke et al. J Virol. 2007 Feb.

Abstract

A variety of rational approaches to attenuate growth and virulence of vesicular stomatitis virus (VSV) have been described previously. These include gene shuffling, truncation of the cytoplasmic tail of the G protein, and generation of noncytopathic M gene mutants. When separately introduced into recombinant VSV (rVSV), these mutations gave rise to viruses distinguished from their "wild-type" progenitor by diminished reproductive capacity in cell culture and/or reduced cytopathology and decreased pathogenicity in vivo. However, histopathology data from an exploratory nonhuman primate neurovirulence study indicated that some of these attenuated viruses could still cause significant levels of neurological injury. In this study, additional attenuated rVSV variants were generated by combination of the above-named three distinct classes of mutation. The resulting combination mutants were characterized by plaque size and growth kinetics in cell culture, and virulence was assessed by determination of the intracranial (IC) 50% lethal dose (LD(50)) in mice. Compared to virus having only one type of attenuating mutation, all of the mutation combinations examined gave rise to virus with smaller plaque phenotypes, delayed growth kinetics, and 10- to 500-fold-lower peak titers in cell culture. A similar pattern of attenuation was also observed following IC inoculation of mice, where differences in LD(50) of many orders of magnitude between viruses containing one and two types of attenuating mutation were sometimes seen. The results show synergistic rather than cumulative increases in attenuation and demonstrate a new approach to the attenuation of VSV and possibly other viruses.

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Figures

FIG. 1.
FIG. 1.
Construction of rVSVIN mutant cDNA. The BsaAI, XbaI, MluI, and HpaI endonuclease sites used for construction of N gene shuffles and insertion of G genes containing CT truncations and MNCP mutations are indicated with arrows. Virus leader (Le), trailer (Tr), GT, and CT intergenic dinucleotides and transcriptional start signals (shaded boxes) at the beginning of each gene are shown. Synthesis of positive-sense genomic RNA was under control of the T7 RNA polymerase transcription promoter (T7-Prom) and was terminated by a T7 transcription terminator (T7 Term). Hepatitis delta virus ribozyme (HDV Ribozyme) was used to generate the precise viral 3′ end on the positive-sense genomic RNA transcript.
FIG. 2.
FIG. 2.
Genetic organization of rVSVIN mutants and plaque size comparison. (A) Mutants were named to reflect genomic organization and attenuating mutations. The N gene shuffle mutants N2, N3, and N4 were named according to the position of the N gene relative to that of wt VSVIN. The G protein CT truncation mutants CT1 and CT9 were named according to the number of amino acids retained in the cytoplasmic tail region of the G protein. Vectors containing noncytopathic M gene mutations (M33A and M51A [triangles]) were named MNCP mutants. Combination mutants were named N2CT1, N3CT1, N2CT9, N3CT9, and MNCPCT1 to reflect contributing mutations. An additional empty TU containing transcription start and stop signals but no additional gene was present in N4 and CT9 mutants. The HIV-1 gag gene was present in the fifth position of virus genomes as indicated. (B) Representative plaques produced by wt VSVIN and rVSVIN variants following plaque assay on replicate Vero cell monolayers at 37οC for 1 to 4 days.
FIG. 3.
FIG. 3.
Growth kinetics of rVSVIN mutants on Vero cell monolayers. Replicate Vero cell monolayers in 25 cm2 flasks were infected in duplicate at an MOI of 5 PFU/cell. Infected-cell supernatants were collected at intervals postinfection and titrated on Vero cell monolayers. All datum points represent the average titers of samples taken from duplicate infections. Growth curves are shown for mutants containing N gene shuffles (A), CT truncations (B), N gene shuffle-CT truncation combinations (C and D), and MNCP mutations (E).
FIG. 4.
FIG. 4.
Neurovirulence properties of rVSVIN mutants in mice following IC inoculation. In a series of experiments, 5-week-old Swiss Webster mice were inoculated IC with log10-fold dilutions of virus. Mice were monitored for 2 weeks for mortality and morbidity (paralysis). (A) The LD50 and PD50 values were determined by the method of Reed and Muench. (B) Time to death was recorded for mice in the group receiving the dose immediately above the determined LD50. Arrowheads indicate results in which LD50 and PD50 were not achieved.
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