High numbers of viral RNA copies in the central nervous system of mice during persistent infection with Theiler's virus

Abstract

The low-neurovirulence Theiler's murine encephalomyelitis viruses (TMEV), such as BeAn virus, cause a persistent infection of the central nervous system (CNS) in susceptible mouse strains that results in inflammatory demyelination. The ability of TMEV to persist in the mouse CNS has traditionally been demonstrated by recovering infectious virus from the spinal cord. Results of infectivity assays led to the notion that TMEV persists at low levels. In the present study, we analyzed the copy number of TMEV genomes, plus- to minus-strand ratios, and full-length species in the spinal cords of infected mice and infected tissue culture cells by using Northern hybridization. Considering the low levels of infectious virus in the spinal cord, a surprisingly large number of viral genomes (mean of 3.0 x 10(9)) was detected in persistently infected mice. In the transition from the acute (approximately postinfection [p.i.] day 7) to the persistent (beginning on p.i. day 28) phase of infection, viral RNA copy numbers steadily increased, indicating that TMEV persistence involves active viral RNA replication. Further, BeAn viral genomes were full-length in size; i.e., no subgenomic species were detected and the ratio of BeAn virus plus- to minus-strand RNA indicated that viral RNA replication is unperturbed in the mouse spinal cord. Analysis of cultured macrophages and oligodendrocytes suggests that either of these cell types can potentially synthesize high numbers of viral RNA copies if infected in the spinal cord and therefore account for the heavy viral load. A scheme is presented for the direct isolation of both cell types directly from infected spinal cords for further viral analyses.

FIG. 1

Method for quantitation of BeAn genomes in individual mouse spinal cords. Total RNA was extracted from spinal cords, blotted onto nitrocellulose filters, probed with an [α-³²P]dCTP-labeled BeAn virus sequence, and quantified by phosphorimaging. (A) Northern dot blot of serial fivefold dilutions of a standard in vitro-transcribed BeAn virus RNA and representative spinal cord total RNAs from infected mice killed on the indicated days p.i. (B) Standard curve derived from Northern hybridization of known amounts of in vitro-transcribed BeAn virus RNA shown in panel A. The sensitivity of this technique was 9 × 10⁶ viral genomes.

FIG. 2

(Top) Temporal detection of BeAn virus RNA copies in the spinal cords of infected mice using an [α-³²P]dCTP-labeled BeAn sequence as a probe for Northern hybridization (left ordinate). The use of similar amounts of RNA in specimens was judged from 18S and 28S rRNAs following agarose gel electrophoresis. There was a rise in the number of viral RNA copies between p.i. days 7 and 28, as shown (the dashed line between p.i. days 0 and 7 is simulated), suggesting that active RNA viral replication is required for viral persistence, in contrast with viral infectivity (shown below). Samples with virus copy numbers below the level of detection (9 × 10⁶ viral genomes) are not shown (two samples on day 7, three on day 21, two on day 29, one on day 36, and one on day 70). (Bottom) Temporal profile of viral infectivity (number of PFU per cord) of clarified spinal cord homogenates showing a decline in virus titers after p.i. day 14 (right ordinate).

FIG. 3

Detection of full-length BeAn virus RNA genomes during persistent infection of mice. Total RNA was prepared from infected cells in culture or from mouse spinal cords. RNA was electrophoresed on a denaturing agarose gel, transferred to nylon filters, and probed with an [α-³²P]dCTP-labeled BeAn virus sequence. A, 100 ng of in vitro-transcribed BeAn RNA; B, 1 μg of uninfected BHK-21 cell total RNA; C, 1 μg of BeAn virus-infected BHK-21 cell total RNA; D, 1 μg of uninfected N20 cell total RNA; E, 1 μg of BeAn virus-infected N20 cell total RNA; F, 1 μg of BeAn virus-infected M1-D cell total RNA; G, 10 μg of spinal cord total RNA at p.i. day 46; H, 10 μg of spinal cord total RNA at p.i. day 70. The arrow indicates full-length BeAn RNA.

FIG. 4

Method used to isolate oligodendrocytes and macrophages from spinal cords of persistently infected SJL mice. The viral RNA copy number determined by Northern hybridization is indicated at each step. (A) During the persistent phase of infection, the mean number (± SD) of viral genomes in whole spinal cords was 3.0 × 10⁹ ± 0.6 × 10⁹ (n = 30; data from Fig. 2). (B) The mean number (± SD) of viral genomes in single-cell suspensions after collagenase treatment of minced spinal cords, including cells in efflux from PBS-forced flushing of spinal canals, was 2.8 × 10⁹ ± 2.4 × 10⁹ (n = 3). (C) Representative experiment for isolation of oligodendrocytes by centrifugation through 0.9 M sucrose (most of the macrophages were eliminated from this gradient by passage of cells through a 38-μm screen). (D) Isolation of macrophages in Percoll gradients at the 30 to 70% interface (since only larger-pore-size screens were used to dissociate the cells, all of the cell types were present in this gradient). (E) Distribution of the viral genome load based on the combined data in panels C and D, i.e., 18% in oligodendrocytes (Oligos; 4.2 × 10⁸/2.38 × 10⁹), 8% in macrophages (Macs; 2.0 × 10⁸/2.38 × 10⁹), and 74% in the myelin fraction at the top of Percoll gradient (2.1 × 10⁹/2.38 × 10⁹ to 4.2 × 10⁸/2.38 × 10⁹). RBCs, red blood cells; assoc., associated.

FIG. 5

Flow cytometric analysis of cells in the myelin fraction at the top of the Percoll gradient in Fig. 4D. (A) Combined fluorescence and light photomicrograph of an aliquot of the myelin fraction allowed to settle onto a slide. Three nucleated cells stained with 7-AAD (anti-DNA) are shown amid many unstained myelin profiles. Aggregation of myelin profiles is, in part, due to settling and superimposition of some profiles. Magnification, ×400. (B) Forward- and side-scatter characteristics of the two components (cells and myelin profiles) in the myelin fraction indicative of size and granularity. (C) Forward- and side-scatter characteristics of 7-AAD-gated cells. Not shown is separation of the two components on the 7-AAD forward-scatter dot plot that enabled nucleated cells to be gated. (D) Dot plot of 7-AAD-gated cells in panel C stained with MOMA-2 (cytoplasmic staining) and goat anti-rat immunoglobulin G-FITC. Quadrants are set on cells stained only with the secondary antibody.