Type I interferon induction is correlated with attenuation of a South American eastern equine encephalitis virus strain in mice

Abstract

North American eastern equine encephalitis virus (NA-EEEV) strains cause high mortality in humans, whereas South American strains (SA-EEEV) are typically avirulent. To clarify mechanisms of SA-EEEV attenuation, we compared mouse-attenuated BeAr436087 SA-EEEV, considered an EEEV vaccine candidate, with mouse-virulent NA-EEEV strain, FL93-939. Although attenuated, BeAr436087 initially replicated more efficiently than FL93-939 in lymphoid and other tissues, inducing systemic IFN-alpha/beta release, whereas FL93-939 induced little. BeAr436087 was more virulent than FL93-939 in IFN-alpha/beta-deficient mice, confirming that type I IFN responses determined attenuation, but the viruses were similarly sensitive to IFN-alpha/beta priming in vitro. Infection with BeAr436087 protected against FL93-939 disease/death, even when given 8 h afterward, suggesting that the environment produced by BeAr436087 infection attenuated FL93-939. We conclude that avoidance of IFN-alpha/beta induction is a major virulence factor for FL93-939. Furthermore, BeAr436087 could be used for vaccination and therapeutic treatment in the event of exposure to NA-EEEV during a bioterrorism attack.

Figure 1. Age-dependence of BeAr436087 and FL93-939 virulence in mice

Morbidity and mortality data from CD1 mice infected subcutaneously with 10³ pfu of BeAr436087 or FL93-939. Age-dependence of mortality (A) and AST (B) in mice inoculated subcutaneously in the ventral thorax with BeAr436087 (hatched bar) or FL93-939 (black bar). Percentage survival (C) and average daily weight change (D) of 6 week-old mice infected in the hind footpad with BeAr436087 (open circle) or FL93-939 (closed circle). Error bars represent standard deviations.

Figure 2. Comparison of BeAr436087 and FL93-939 replication and dissemination

6-week-old CD1 mice were infected with 10³ pfu of BeAr436087 (open circle) or FL93-939 (closed circle) in each hind footpad. Mice were perfused with PBS-1% DCS prior to tissue harvesting, homogenization and mechanical disruption. Virus titers in serum and tissue homogenates were determined by BHK-21 plaque titration: A) popliteal draining lymph node; B) serum; C) spleen; D) knee joint (femur epiphyses and metaphyses); E) bone aspirate (femur); F) brain. LD = limit of detection of the plaque assay and error bars represent standard deviations.

Figure 3. Effect of type I IFN responses on BeAr436087 replication and dissemination

BHK cell plaque titration of virus replication in various tissues and serum of 6-8 week-old IFN-deficient and control mice infected with 10³ pfu of BeAr436087 in the hind footpad. Mice were perfused with PBS-1% DCS prior to tissue harvesting, homogenization and mechanical disruption: A) 129 Sv/Ev (black bars), IFNAR1^−/− (hatched bars), IFNAGR^−/− (white bars) and STAT1^−/− (crosshatched bars) mice were infected with BeAr436087 and samples were collected at 24 h p.i. LN = popliteal lymph node. Joint = knee joint. B) Mice were infected with BeAr436087 (hatched bars) or FL93-939 (black bars) and samples were collected at 48 h p.i. Error bars represent standard deviations.

Figure 4. Comparative replication of BeAr436087 and FL93-939 in relevant cell-types in vitro

Primary cultures of CD1-derived osteoblasts (A) and cortical neurons (B) were infected with BeAr436087 (open circle) or FL93-939 (closed circle) and progeny virions were titered. Replication of BeAr436087 (C) and FL93-939 (D) in bone marrow-derived cDCs generated from control 129 Sv/Ev (closed square) and IFNAR1^−/− (open square) mice. All cells were infected at an moi of 0.1. LD = limit of detection for the plaque assay and error bars represent standard deviations.

Figure 5. Relative sensitivity of BeAr436087 and FL93-939 to IFN-α/β priming of primary osteoblasts

Triplicate wells of primary osteoblasts generated from CD1 mice were exposed to a range of IFN-α/β concentrations for 24 h prior to infection with SINV (open square), FL93-939 (open circle), FL93-939 (closed circle) or VEEV (closed square) at an moi of 1 for each virus. Data are presented as a percentage of cells surviving at 24 h p.i. versus the dose (IU/mL) of IFN-α/β used to prime.

Figure 6. Induction of systemic IFN-α/β in BeAr436087-infected versus FL93-939-infected mice

Results of biological assay for detection of IFN-α/β in the serum of 6 week-old CD1 mice of infected with 10³ pfu of FL93-939 (black bars) or BeAr436087 (white bars) in the hind footpad. LD = limit of detection of the IFN-α/β assay and error bars represent standard deviations.

Figure 7. STAT1 phosphorylation in brains of BeAr436087-infected and FL93-939-infected mice

Western blot detection of the abundance of STAT1 and its phosphorylation state at 24 or 48 h p.i. in lysates from whole brains of 6 week-old CD-1 mice infected subcutaneously in the hind footpad with 10³ pfu of BeAr436087 (BeAr) or FL93-939 (FL93). (+) = positive control in which 100,000 IU of IFN-α/β were injected directly into the brains of CD1 mice and whole brain lysates were harvested 30 min later.

Figure 8. Rapid BeAr436087-mediated protection against FL93-939-induced mortality in CD1 mice

6 week-old mice were untreated or inoculated with 10³ pfu of BeAr436087 (“treatment” in the figure) either 24, 8 or 0 h prior to, or 8 or 24 h after, inoculation in the left hind footpad with 10³ pfu of FL93-939. BeAr436087 inoculations were either the same (ipsilateral) or opposite (contralateral) footpad as FL93-939. Data are presented as the percentage of mice surviving the FL93-939 infection at 21 d p.i.