A Single Amino Acid Change in nsP1 Attenuates Neurovirulence of the Sindbis-Group Alphavirus S.A.AR86

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.

Eilat Virus Host Range Restriction Is Present at Multiple Levels of the Virus Life Cycle

“…Similar to infection of C. quinquefasciatus with SINV-eGFP and the SINV/EILV structural chimera, the majority of mosquitoes became infected with all doses of SINV/EILV 3= UTR. Infection rates ranged from 80% to 100%, and an ID 50 was not determined (Table 1). Surprisingly, infection of newborn mice produced minimal clinical disease and little lethality.…”

“…To further inves- tigate the host range restriction of SINV/EILV, infections of mosquitoes and newborn mice were performed. Adult female Culex quinquefasciatus, selected because Culex mosquitoes are the main SINV vectors and are susceptible to EILV, were injected via the intrathoracic route with SINV-eGFP and SINV/EILV at doses ranging from ϳ10 to 10 4 PFU/mosquito to determine the mosquito 50% infectious dose (ID 50 ), and at 7 dpi, whole mosquitoes were analyzed via eGFP expression and CPE assays for the presence of virus. C. quinquefasciatus infections with both viruses yielded similar results at all doses, with infection rates ranging from 73% to 100% even after the smallest doses; consequently, an ID 50 could not be determined (Table 1).…”

An Attenuating Mutation in nsP1 of the Sindbis-Group Virus S.A.AR86 Accelerates Nonstructural Protein Processing and Up-Regulates Viral 26S RNA Synthesis

“…The 5Ј untranslated region also contributes to neuroinvasion and the development of encephalitis (2,16). Recent studies with S.A.AR86 (9) and Semliki Forest virus (32) demonstrated a role for the viral nonstructural proteins in adult mouse neurovirulence. In S.A.AR86, a single amino acid change at nsP1 position 538 dramatically reduces adult mouse neurovirulence (9).…”

“…However, S.A.AR86, a Sindbis-group alphavirus isolated in South Africa, does cause lethal disease in adult mice following i.c. inoculation (9,28,34). This ability to cause lethal disease in adult immunocompetent mice makes S.A.AR86 a valuable tool for studying viral and host determinants that contribute to viral encephalitis.…”

Sindbis-Group Alphavirus Replication in Periosteum and Endosteum of Long Bones in Adult Mice

“…At the time of sacrifice, mice were perfused with 4% paraformaldehyde in PBS (pH 7.4) prior to decalcification and in situ hybridization. Paraffin-embedded limb sections were probed using either a riboprobe complementary for S.A.AR86 nucleotides 7371 to 7816 or a control riboprobe specific for the influenza virus strain PR/8 hemagglutinin (HA) as described previously (9). S.A.AR86-specific in situ signal was observed in the periosteum and tendons of the long bones adjacent to the joints in mice infected with either dose of virus.…”