Identification of adult mouse neurovirulence determinants of the Sindbis virus strain AR86 - PubMed (original) (raw)

Identification of adult mouse neurovirulence determinants of the Sindbis virus strain AR86

Mehul S Suthar et al. J Virol. 2005 Apr.

Abstract

Sindbis virus infection of mice has provided valuable insight into viral and host factors that contribute to virus-induced neurologic disease. In an effort to further define the viral genetic elements that contribute to adult mouse neurovirulence, the neurovirulent Sindbis virus strain AR86 was compared to the closely related (22 single amino acid coding changes and the presence or absence of an 18-amino-acid sequence in nsP3 [positions 386 to 403]) but avirulent Girdwood strain. Initial studies using chimeric viruses demonstrated that genetic elements within the nonstructural and structural coding regions contributed to AR86 neurovirulence. Detailed mapping studies identified three major determinants in the nonstructural region, at nsP1 538 (Ile to Thr; avirulent to virulent), an 18-amino-acid deletion in nsP3 (positions 386 to 403), and nsP3 537 (opal to Cys; avirulent to virulent), as well as a single determinant in the structural genes at E2 243 (Leu to Ser; avirulent to virulent), which were essential for AR86 adult mouse neurovirulence. Replacing these codons in AR86 with those found in Girdwood resulted in the attenuation of AR86, while the four corresponding AR86 changes in the Girdwood genetic background increased virulence to the level of wild-type AR86. The attenuating mutations did not adversely affect viral replication in vitro, and the attenuated viruses established infection in the brain and spinal cord as efficiently as the virulent viruses. However, the virus containing the four virulence determinants grew to higher levels in the spinal cord at late times postinfection, suggesting that the virus containing the four attenuating determinants either failed to spread or was cleared more efficiently than the wild-type virus.

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Figures

FIG. 1.

FIG. 1.

Both the nonstructural and structural genes of AR86 contain neurovirulence determinants. A diagram of the Alphavirus genome organization is located at the top. On the left are the names of the cDNAs encoding full-length virus, beginning with the parental strains pS300 (wild-type AR86) and pG100 (wild-type Girdwood) and followed by the chimeric viruses. The cDNA constructs are diagrammed to show the sequences derived from pS300 (shaded box) and pG100 (open box). The chimeric clone pS350 contains AR86 nucleotide sequences between 1 and 6411, and the chimeric pG106 clone contains AR86 nucleotide sequences from 6411 to 11343. Groups (n, number of mice per group) of either 4-week-old (A) or 6-week-old (B) female CD-1 mice were inoculated i.c. with 103 PFU of each virus and observed daily for clinical signs. Virulence was assessed by morbidity (average clinical score [CS] on day 5), mortality, and average survival time (AST; number of days ± standard deviation [SD]).

FIG. 2.

FIG. 2.

Mutational analysis of neurovirulence determinants within the nonstructural genes of AR86. The cDNA constructs are diagrammed to show sequences derived from AR86 (pS300) along with amino acids located in nsP2 at positions 648 and 651 and nsP3 positions 344, 386 to 403, and 537. Groups (n) of 6-week-old female CD-1 mice were inoculated i.c. with 103 PFU of either wild-type AR86 (S300) or each mutant virus and observed daily for clinical signs. Virulence was assessed by morbidity (average CS on day 5), mortality, and AST (number of days ± SD). The mortalities for the mutant viruses S343 and S344 were statistically significant when compared to S300 (P < 0.05).

FIG. 3.

FIG. 3.

AR86 neurovirulence determinants within the nonstructural genes. The cDNA constructs are diagrammed to show the sequences derived from pS300 (shaded box) and pG100 (open box) along with amino acids located in nsP1 at position 538 (Thr in pS300 and Ile in pG100), nsP3 between positions 386 and 403 (del 386-403 in pS300), and nsP3 at position 537 (Cys in pS300 and Opal in pG100). All of the chimeric clones contain AR86 nucleotide sequences between 6411 and 11343. Groups (n) of 6-week-old female CD-1 mice were inoculated i.c. with 103 PFU of each virus and observed daily for clinical signs. Virulence was measured by morbidity (average CS on day 5), mortality, and AST (average number of days ± SD). The mortalities for the chimeric viruses S354, S355, and S356 were statistically significant when compared to the parental chimeric virus S350 (P < 0.05).

FIG. 4.

FIG. 4.

AR86 neurovirulence determinants within the structural genes. The cDNA constructs are diagrammed to show the sequences derived from pS300 (shaded box) and pG100 (open box) along with the amino acid located in E2 at position 243 (Ser in pS300 and Leu in pG100). All of the chimeric clones contain AR86 nucleotide sequences between 1 and 6411. Groups (n) of 6-week-old female CD-1 mice were inoculated i.c. with 103 PFU of virus and observed daily for clinical signs. Virulence was assessed by morbidity (average CS on day 5), mortality, and AST (average number of days ± SD). The mortalities for the chimeric viruses G107 and G117 were statistically significant when compared to the parental chimeric virus G106 (P < 0.05).

FIG. 5.

FIG. 5.

Identification of the major determinants of neurovirulence within AR86. The cDNA constructs are diagrammed to show the sequences derived from pS300 (shaded box) and pG100 (open box) along with amino acids located in nsP1 at position 538 (Thr in pS300 and Ile in pG100), nsP3 between positions 386 and 403 (del 386-403 in pS300), nsP3 at position 537 (Cys in pS300 and Opal in pG100), and E2 at position 243 (Ser in pS300 and Leu in pG100). Groups (n) of 6-week-old female CD-1 mice were inoculated i.c. with 103 PFU of virus and observed daily for clinical signs. Virulence was measured by morbidity (average CS on day 5), mortality, and AST (average number of days ± SD).

FIG. 6.

FIG. 6.

In vitro and in vivo growth analysis of virulent and attenuated viruses. (A) A single-step in vitro growth curve was performed on BHK-21 cells infected with S300 (solid line, filled circle), S363 (broken line, open circle), G100 (solid line, open square), or G163 (broken line, filled square) at an MOI of 5.0. Shown are data from a representative experiment where each point represents the average of results from three independent samples ± SD. (B) Six-week-old female CD-1 mice were infected i.c. with 103 PFU of S300 (solid line, filled circle), S363 (broken line, open circle), G100 (solid line, open square), and G163 (broken line, filled square). Mice were sacrificed by exsanguination at 6, 12, 24, 48, 72, 96, and 120 h postinfection and perfused with PBS (pH 7.4). The brain was harvested and evaluated for viral load by plaque assay on BHK-21 cells. The data shown represent results from one of three experiments for the brain. (C and D) Six-week-old CD-1 mice were infected with S300 (solid line, filled circle) or S363 (broken line, open circle) as in panel B. Mice were sacrificed at 48, 96, or 144 h postinfection and perfused with PBS, and viral titers in the thoracic or lumbar spinal cord were determined by plaque assay. (C) Viral titers in the thoracic spinal cord (n = 3 mice per time point; data shown represent results from one of two identical experiments). (D) Viral loads in the lumbar spinal cord. Data were pooled from two experiments and six mice per time point. Differences in viral loads in the thoracic and lumbar spinal cord at 96 h postinfection are statistically significant (P < 0.05) as measured by two-tailed Student's t test.

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