BMDCs and BMMΦ, as well as MEFs from wt or STAT1^−/− mice, and RAW 264.7 cells were infected with a MOI of 0.05.
(A) MNV-1 causes CPE in permissive cells. MNV-1- or mock-infected cells were observed by light microscopy 2 d postinfection. The boxed area is magnified further to show the border of the plaque.
(B) Infected cell lysates were analyzed in two to four independent experiments by plaque assay at various timepoints postinfection to calculate standard deviations. For wt BMMΦ, MNV-1 growth was detected in two out of four experiments.

Cells were infected with MNV-1.CW1 (P3) (MOI of 2.0) (D–L) or mock-infected (A–C) and processed for electron microscopy 12 (D–F), 18 (G–I), or 24 (A–C; J–L) h.p.i. MNV-1 particles are indicated by arrows and confronting membranes by arrowheads. VA, vesiculated areas; Nuc, nucleus; rER, rough endoplasmic reticulum. Scale bars, 200 nm for (A), (D), (G), and (J); 500 nm for (B), (E), (H), and (K); 2 μm for (C), (F), (I), and (L).

(A) Serial passage of MNV-1.CW1 in cell culture causes attenuation. STAT1^−/− mice were infected orally with the indicated virus dose. The number of mice analyzed is indicated in parentheses.
(B) Summary of sequence analysis of MNV-1 over several passages. The nucleotide and amino acid differences between the indicated viruses are shown (for detail see Table 1).

Immunohistochemistry was performed on liver (A and B) and spleen (C and D) sections from STAT1^−/− mice 2 d after oral infection. MNV-1-specific staining was seen in Kupffer cells of infected livers when probed with MNV-1 immune (A) but not preimmune (B) serum. A selected Kupffer cell lining the sinusoid is indicated by an arrowhead. MNV-1-specific staining consistent with MΦ was seen in red pulp (C) and marginal zone (D) in the spleen. The arrow indicates a cell with MΦ morphology. No staining was observed in tissues from mice infected for 1 d, in infected tissues incubated with preimmune serum, or in mock-infected tissues incubated with immune serum. RP, red pulp; WP, white pulp.

(A–C) MNV-1.CW1 purified on CsCl density gradients was visualized by (A) negative staining electron microscopy, (C) Coomassie staining, and (B) Western blot analysis with a polyclonal anti-MNV-1-capsid antibody. Molecular weight markers are indicated in kiloDaltons.
(D) Specific binding of mAb A6.2 to two different concentrations of CsCl-purified MNV-1 particles in an enzyme-linked immunosorbent assay.
(E) Neutralization of MNV-1 from brain homogenate and MNV-1.CW1 by mAb A6.2 but not the isotype control (10H2) mAb in a plaque neutralization assay. The assay was repeated three times to calculate standard deviations. The limit of detection is indicated by the dashed line.
(F) Timecourse of viral RNA synthesis in RAW 264.7 cells. Northern blot analysis of viral RNA from cells infected with MNV-1.CW1 (MOI of 2.0) or mock-infected cells. The size of RNA markers in kilobases is shown on the left. The positions of subgenomic- and genomic-length RNA are indicated on the right. This timecourse is a representative of two independent experiments.
(G) Timecourse of viral protein synthesis in infected RAW 264.7 cells. MNV-1-specific proteins were precipitated from radiolabeled cell lysates of MNV-1.CW1-infected RAW 264.7 cells (MOI of 2.0) at indicated times after infection. The size of the proteins in kiloDaltons is indicated.

(A) MNV-1.CW1 has no defect in viral growth in vitro. Growth curves (MOI of 0.05) were performed two or three times with MNV-1.CW1 (P3) on indicated cells to calculate standard deviations.
(B) MNV-1 growth in MΦ is controlled by STAT-1 and Type I IFNs. BMMΦ of the indicated genotype were infected with MNV-1.CW1 (P3) at the indicated MOI. The experiment was performed twice to calculate standard deviations. The p-values for PKR versus wt infection at MOI 0.05 and 2.0, 0.8867 and 0.1616, respectively, are not significant. Statistical analysis was performed using the paired t-test (GraphPad Prism, version 3.03).