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. 2000 May;74(9):4207-13.
doi: 10.1128/jvi.74.9.4207-4213.2000.

A single amino acid change in nsP1 attenuates neurovirulence of the Sindbis-group alphavirus S.A.AR86

M T Heise  1 , D A Simpson, R E Johnston
Affiliations

A single amino acid change in nsP1 attenuates neurovirulence of the Sindbis-group alphavirus S.A.AR86

M T Heise et al. J Virol. 2000 May.

Abstract

S.A.AR86, a member of the Sindbis group of alphaviruses, is neurovirulent in adult mice and has a unique threonine at position 538 of nsP1; nonneurovirulent members of this group of alphaviruses encode isoleucine. Isoleucine was introduced at position 538 in the wild-type S.A.AR86 infectious clone, ps55, and virus derived from this mutant clone, ps51, was significantly attenuated for neurovirulence compared to that derived from ps55. Intracranial (i.c. ) s55 infection resulted in severe disease, including hind limb paresis, conjunctivitis, weight loss, and death in 89% of animals. In contrast, s51 caused fewer clinical signs and no mortality. Nevertheless, comparison of the virus derived from the mutant (ps51) and wild-type (ps55) S.A.AR86 molecular clones demonstrated that s51 grew as well as or better than the wild-type s55 virus in tissue culture and that viral titers in the brain following i.c. infection with s51 were equivalent to those of wild-type s55 virus. Analysis of viral replication within the brain by in situ hybridization revealed that both viruses established infection in similar regions of the brain at early times postinfection (12 to 72 h). However, at late times postinfection, the wild-type s55 virus had spread throughout large areas of the brain, while the s51 mutant exhibited a restricted pattern of replication. This suggests that s51 is either defective in spreading throughout the brain at late times postinfection or is cleared more rapidly than s55. Further evidence for the contribution of nsP1 Thr 538 to S.A.AR86 neurovirulence was provided by experiments in which a threonine residue was introduced at nsP1 position 538 of Sindbis virus strain TR339, which is nonneurovirulent in weanling mice. The resulting virus, 39ns1, demonstrated significantly increased neurovirulence and morbidity, including weight loss and hind limb paresis. These results demonstrate a role for alphavirus nonstructural protein genes in adult mouse neurovirulence.

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Figures

FIG. 1
FIG. 1
Virus derived from S.A.AR86 clone ps51 causes less morbidity and mortality than virus derived from the wild-type clone ps55. Six-week-old female CD-1 mice were infected i.c. with 500 PFU of the wild-type s55 or the mutant s51. (A) Mice were monitored for weight loss as an indicator of morbidity. Mouse weight is plotted as percentage of starting body weight over time. Data represent the mean percentage from five animals per group (s55 and s51) or two animals (mock). ○, s51-infected mice; ▴, s55-infected mice; +, mock-infected mice. Error bars represent the standard error. The data shown represent results from one of three comparable experiments. (B) Percentage of mice surviving over time after s51 or s55 infection. There were 7 mock-infected mice, 15 s51-infected mice, and 19 s55-infected mice. The data shown are pooled from three separate experiments.
FIG. 2
FIG. 2
s51 and s55 grow at equivalent rates in vitro. BHK-21 cells were infected in triplicate with the viruses s51 (○) and s55 (▴) at an MOI of 5.0. After 1 h of infection at 37°C, cells were washed three times with room temperature PBS (1% DCS), and 1 ml of growth medium was placed on the cells. One hundred microliters of supernatant was removed for titration by plaque assay at the indicated times. At the time of harvest, the sample volume removed was replaced with fresh media. Samples were titrated by plaque assay on BHK-21 cells. The data shown are from one of three representative experiments. Each data point represents the mean titer ± standard error.
FIG. 3
FIG. 3
s51 and s55 grow with equivalent kinetics in the brains of infected mice. Six-week-old female CD-1 mice were infected i.c. with 103 PFU of s51 or s55. Mice were sacrificed by exsanguination at 12, 24, 48, 72, 96, 120, and 144 h postinfection and perfused with PBS (pH 7.4). The left hemisphere of each brain was removed and placed in three volumes of PBS (1% DCS, Ca2+-Mg2+). Samples were frozen and thawed before homogenization and titration by plaque assay on BHK-21 cells. Each data point represents the mean titer ± the standard error. ○, s51; ▴, s55. n = 6 to 12 animals for each time point from three pooled experiments.
FIG. 4
FIG. 4
In situ hybridization to localize sites of infection by s55 or s51. Six-week-old female CD-1 mice were infected i.c. with 103 PFU of s51 (B, D, and F) or s55 (A, C, and E). At 12, 24, 48, 72, 96, 120, and 144 h postinfection, mice were exsanguinated and perfused with 4% paraformaldehyde in PBS (pH 7.4). Brains were divided sagitally down the midline, embedded in paraffin, and sectioned at 5 μm/section. Sections were subjected to in situ hybridization with riboprobes specific for S.A.AR86. Nonspecific binding controls consisted of sections from mock-infected mice probed with the S.A.AR86-specific riboprobe or sections from infected mice probed with a riboprobe specific for influenza virus strain PR/8 hemagglutinin. No specific signal was observed with either of these control groups. Shown are representative sections at 24 (A and B), 72 (C and D), and 144 (E and F) h. There were three mice per group per time point. The data shown are from one of two comparable experiments.
FIG. 5
FIG. 5
In vitro growth kinetics of the TR339 and 39ns1 viruses. BHK-21 cells were infected in triplicate at an MOI of 5.0 with either TR339 or 39ns1. Infection was performed as in Fig. 2. Supernatant was sampled at the indicated times postinfection, and virus levels were titrated on BHK-21 cells by plaque assay. ○, TR339; ▴, 39ns1. The data shown are from one of three comparable experiments.
FIG. 6
FIG. 6
39ns1 but not TR339 infection causes morbidity as defined by weight loss in 18-day-old mice. Eighteen-day-old mixed groups of male and female CD-1 mice were infected i.c. with 103 PFU of either TR339 or 39ns1. Mice were weighed daily as an indicator of virus-induced morbidity. Mouse weight is plotted as a percentage of starting body weight over time. The data represent the mean percentage from five (TR339 and 39ns1) or four (mock) animals per group. ○, TR339-infected mice; ▴, 39ns1-infected mice; +, mock-infected mice. Error bars represent the standard error. The data shown represent one of four comparable experiments with 18- to 21-day-old mice.
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