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. 2001 Apr;75(8):3706-18.
doi: 10.1128/JVI.75.8.3706-3718.2001.

Role of alpha/beta interferon in Venezuelan equine encephalitis virus pathogenesis: effect of an attenuating mutation in the 5' untranslated region

L J White  1 , J G Wang, N L Davis, R E Johnston
Affiliations

Role of alpha/beta interferon in Venezuelan equine encephalitis virus pathogenesis: effect of an attenuating mutation in the 5' untranslated region

L J White et al. J Virol. 2001 Apr.

Abstract

Venezuelan equine encephalitis virus (VEE) is an important equine and human pathogen of the Americas. In the adult mouse model, cDNA-derived, virulent V3000 inoculated subcutaneously (s.c.) causes high-titer peripheral replication followed by neuroinvasion and lethal encephalitis. A single change (G to A) at nucleotide 3 (nt 3) of the 5' untranslated region (UTR) of the V3000 genome resulted in a virus (V3043) that was avirulent in mice. The mechanism of attenuation by the V3043 mutation was studied in vivo and in vitro. Kinetic studies of virus spread in adult mice following s.c. inoculation showed that V3043 replication was reduced in peripheral organs compared to that of V3000, titers in serum also were lower, and V3043 was cleared more rapidly from the periphery than V3000. Because clearance of V3043 from serum began 1 to 2 days prior to clearance of V3000, we examined the involvement of alpha/beta interferon (IFN-alpha/beta) activity in VEE pathogenesis. In IFN-alpha/betaR(-/-) mice, the course of the wild-type disease was extremely rapid, with all animals dying within 48 h (average survival time of 30 h compared to 7.7 days in the wild-type mice). The mutant V3043 was as virulent as the wild type (100% mortality, average survival time of 30 h). Virus titers in serum, peripheral organs, and the brain were similar in V3000- and V3043-infected IFN-alpha/betaR(-/-) mice at all time points up until the death of the animals. Consistent with the in vivo data, the mutant virus exhibited reduced growth in vitro in several cell types except in cells that lacked a functional IFN-alpha/beta pathway. In cells derived from IFN-alpha/betaR(-/-) mice, the mutant virus showed no growth disadvantage compared to the wild-type virus, suggesting that IFN-alpha/beta plays a major role in the attenuation of V3043 compared to V3000. There were no differences in the induction of IFN-alpha/beta between V3000 and V3043, but the mutant virus was more sensitive than V3000 to the antiviral actions of IFN-alpha/beta in two separate in vitro assays, suggesting that the increased sensitivity to IFN-alpha/beta plays a major role in the in vivo attenuation of V3043.

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Figures

FIG. 1
FIG. 1
Virus titers of V3000 and V3043 in 129Sv/Ev mouse tissues. Six- to 8-week-old 129Sv/Ev mice were inoculated s.c. in the LRFP with 103 PFU of V3000 (solid bars) or V3043 (hatched bars). At the indicated times p.i., three mice per group were sacrificed and the following tissues were harvested: DLN (A), spleen (B), serum (C), and brain (D). Virus titers were determined by plaque assay of BHK-21 cells. The values are the geometric mean titers (log10 PFU/milliliter or gram) of three mice. Error bars represent standard deviations. The lower limit of detection is indicated by a broken line. ∗, all samples had virus titers below the limit of detection; ∗∗, one out of three samples had virus liters above the limit of detection.
FIG. 2
FIG. 2
Survival of 129Sv/Ev (closed symbols) and IFN-α/βR−/− (open symbols) mice infected with V3000 or V3043. Six- to 8-week-old mice, 11 mice per group, were inoculated in the LRFP with 103 PFU of V3000 or V3043. The mice were observed every 6 h for the first 48 h and every 24 h for 12 days. The survival curves of V3000 and V3043 in the IFN-α/βR−/− mice are superimposable.
FIG. 3
FIG. 3
Virus titers of V3000 and V3043 in IFN-α/βR−/− mouse tissues. Six- to 8-week-old IFN-α/βR−/− mice were inoculated s.c. in the LRFP with 103 PFU of V3000 (solid bars) or V3043 (hatched bars). At the indicated times p.i., three mice per group were sacrificed, tissues were harvested, and virus titers were determined as indicated in Fig. 1. The following tissues were harvested: DLN (A), spleen (B), serum (C), and brain (D). The values are the geometric mean titers (log10 PFU/milliliter or gram) of three mice. Error bars represent the standard deviations. The lower limit of detection is indicated as a broken line. ∗, samples with virus titers below the limit of detection.
FIG. 4
FIG. 4
In vitro growth of V3000 and V3043 in L929 cells (A), Swiss 3T3 cells (B), BHK-21 cells (C), and primary BMMΦ (D) from 129Sv/Ev (closed symbols) and IFN-α/βR−/− (open symbols) mice. Monolayers grown in 60-mm-diameter dishes were infected in triplicate with V3000 (solid line) or V3043 (broken line) at an MOI of 5 to 12. After 1 h of adsorption at 37°C, the monolayers were washed three times and complete media were added. The first aliquot was collected at this time. Aliquots of culture media were collected at various times p.i., and equal volumes of fresh media were replaced every time. Virus titers were determined by a plaque assay on BHK-21 cells.
FIG. 5
FIG. 5
In vivo (A) and in vitro (B) induction of IFN-α/β in 129Sv/Ev and IFN-α/βR−/− mice or cells derived from them. (A) Six- to 8-week-old 129Sv/Ev and IFN-α/βR−/− mice were inoculated s.c. in the LRFP with 105 IU of wild-type R5005-3000 or mutant R5505-3000 replicons expressing GFP. At 6, 12, and 24 hpi, three mice per experimental group were euthanatized for blood collection. Aliquots of serum were frozen at −70°C and were thawed only once for IFN-α/β detection by using a bioassay on L929 cells and EMCV as indicator virus. Values are the averages of data from three mice, and the error bars represent the standard deviations. The lower limit of detection is indicated as a broken line. ∗, samples with virus titers below the limit of detection (dotted line). The levels of IFN-α/β in the mock-infected mice were below the limit of detection. (B) Monolayers of BMMΦ from IFN-α/βR−/− mice prepared as described for Fig. 4 were infected with R5005-3000 or R5505-3000 GFP-expressing replicons at an MOI of 20. At 18 and 24 hpi, the media were collected and clarified by centrifugation and aliquots were frozen until used in IFN-α/β assays as described for panel A.
FIG. 6
FIG. 6
IFN-α/β sensitivity assay of L929 (A) and 3T3 (B) cells. Monolayers of L929 cells in 96-well plates were treated with twofold dilutions of murine IFN-α/β (concentrations ranging from 506 to 0 IU/ml) for 24 h. Mice were infected with either V3000 or V3043 at an MOI of 10 to 20 PFU/cell in the presence of 20,000 IU of anti-IFN-α/β antibodies/ml to minimize any possible autocrine effects of IFN-α/β induction. The percentage of CPE was measured at 72 hpi by the MTT colorimetric assay.
FIG. 7
FIG. 7
Mutant virus is more sensitive to IFN-α/β in L929 cells early in infection at the RNA synthesis level. L929 cells pretreated for 24 h with 0, 21, or 500 IU/ml of murine IFN-α/β were infected with V3000 or V3043 at an MOI of 4 or were mock infected. Total cytoplasmic RNAs were isolated at 4 hpi, and the levels of VEE plus-strand genomic and subgenomic RNA and of the mRNA for the housekeeping gene GAPDH were determined by RPA. Lane 1, undigested probes; lanes 2 to 4, 10, 100, and 1,000 ng, respectively, of plus-strand VEE RNA transcripts used as controls and subjected to the RPA. The presence (+) or absence (−) of IFN-α/β pretreatment is indicated. The concentrations of IFN-α/β (+) (in IU/milliliter) used are indicated.
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