Transition from acute to persistent Theiler's virus infection requires active viral replication that drives proinflammatory cytokine expression and chronic demyelinating disease

Abstract

The dynamics of Theiler's murine encephalomyelitis virus (TMEV) RNA replication in the central nervous systems of susceptible and resistant strains of mice were examined by quantitative real-time reverse transcription-PCR and were found to correlate with host immune responses. During the acute phase of infection in both susceptible and resistant mice, levels of viral replication were high in the brain and brain stem, while levels of viral genome equivalents were 10- to 100-fold lower in the spinal cord. In the brain, viral RNA replication decreased after a peak at 5 days postinfection (p.i.), in parallel with the appearance of virus-specific antibody responses; however, by 15 days p.i., viral RNA levels began to increase in the spinal cords of susceptible mice. During the transition to and the persistent phase of infection, the numbers of viral genome equivalents in the spinal cord varied substantially for individual mice, but high levels were consistently associated with high levels of proinflammatory Th1 cytokine and chemokine mRNAs. Moreover, a large number of viral genome equivalents and high proinflammatory cytokine mRNA levels in spinal cords were only observed for susceptible SJL/J mice who developed demyelinating disease. These results suggest that TMEV persistence requires active viral replication beginning about day 11 p.i. and that active viral replication with high viral genome loads leads to increased levels of Th1 cytokines that drive disease progression in infected mice.

FIG. 1.

Kinetics of BeAn viral RNA replication in cerebrums (A and D), brain stems (B and E), and spinal cords (C and F) of susceptible SJL/J (A, B, and C) and resistant B6 (D, E, and F) mice. Data are plotted as numbers of viral RNA copy equivalents per microgram of total RNA, with each open circle representing one mouse. Linear regression of the increase in viral RNA copies between days 11 and 33 p.i. in SJL/J mice is shown with dark lines (note the logarithmic scale of the y axis). Insets in the lower left corner of some panels show arithmetic plots of the acute kinetics of viral RNA growth (10⁶ for A and E; 10⁵ for B and E). The detection limit of the assay was >100 viral RNA copy equivalents.

FIG. 2.

Virus-specific antibody titers in sera from SJL/J mice. Titers were assayed by ELISAs for the same SJL/J mice that were used to generate the data shown in Fig. 1. Anti-TMEV antibody titers in mouse sera at each time (n = 4) are plotted as means ± standard deviations (SD).

FIG. 3.

Proinflammatory cytokine and chemokine mRNA expression levels in brains and spinal cords of BeAn-virus infected SJL/J mice at the indicated times. RNase protection assays revealed an acute increased expression of cytokine and chemokine mRNAs in brains and spinal cords. Both cytokine and chemokine mRNA expression levels decreased in the brain thereafter. While the expression of selective cytokine mRNAs increased, chemokine expression decreased in spinal cords during the transition phase.

FIG. 4.

IFN-γ and TNF-α mRNA expression in spinal cords of BeAn virus-infected SJL/J mice with high and low viral genome levels. A threshold of 2.6 × 10⁵ viral RNA copy equivalents per μg of total RNA was determined by visual inspection of RNase protection results compared to viral genome copy levels for each sample (see the text). Percentages of IFN-γ (A) and TNF-α (B) expression relative to that of GAPDH on the indicated days p.i. are shown. Mouse spinal cords contained viral genome loads that were higher (○) or lower (•) than a threshold value of 2.6 × 10⁵ genome copy equivalents per μg of total RNA. Each circle represents one mouse.

FIG. 5.

Viral genome equivalents (copy numbers) in spinal cords of BeAn virus-infected SJL/J mice that were healthy (n = 9) or had developed clinical demyelinating disease (n = 5) when sacrificed on day 107 p.i. All diseased mice had viral RNA copy numbers of >2.6 × 10⁵ (threshold level; horizontal line), and with one exception, all healthy mice had copy numbers of <2.6 × 10⁵. The detection limit for this assay was 100 genome copy numbers per μg of total spinal cord RNA.

FIG. 6.

Comparison of SJL/J and SJL/H mice for susceptibility to TMEV-induced demyelinating disease. Mice from Jackson Laboratory (SJL/J; n = 27) (A) and from Harlan Laboratories (SJL/H; n = 18) (B) were inoculated i.c. with 2 × 10⁶ PFU of BeAn virus, and clinical disease was assessed weekly until day 100 p.i. The incidence of clinical disease (left axis [□]) was plotted as the percentages of mice with signs of clinical demyelinating disease. The incidence of disease severity (right axis [•]) was plotted as mean clinical scores for all animals on each day p.i.

FIG. 7.

Comparison of viral genome copy equivalents per microgram of total RNA in the brains and spinal cords of SJL/J and SJL/H mice, as determined by quantitative real-time RT-PCR. The time points shown represent the approximate end of the acute phase of virus infection (day 11 p.i.) and the early persistent phase (day 33 p.i.), when viral genome loads are elevated in spinal cords. Data shown are means ± SD.