Pathogenesis and Immunity

Extraintestinal Spread and Replication of a Homologous EC Rotavirus Strain and a Heterologous Rhesus Rotavirus in BALB/c Mice

M. Fenaux, M. A. Cuadras, N. Feng, M. Jaimes, H. B. Greenberg
DOI: 10.1128/JVI.02664-05

Article Figures & Data

Figures

  • FIG. 1.
    FIG. 1.

    A 12-h rotavirus replication curve in MA104 cells inoculated with RRV at an MOI of 1. Bars: □, total (+)RNA copy numbers per ml; ▧, total (−)RNA copy numbers detected per ml. Asterisks mark the time points when a significant excess of total (+)RNA over (−)RNA was detected [(+)RNA/(−)RNA ratio of >1.58], indicating rotavirus replication. The triangles connected with the solid line indicate the RRV titer (in PFU/ml) determined by plaque assay. The x axis shows the hours postinoculation, and the y axis indicates the log10 of both (+)RNA and (−)RNA copies/ml and PFU/ml.

  • FIG. 2 and 3.
    FIG. 2 and 3.

    (−)RNA and excess (+)RNA copy numbers detected in the feces, small intestines (SI), MLN, blood, livers, lungs, and kidneys in suckling BALB/c mice inoculated with EC or RRV. The black diamond indicates the (−)RNA copy numbers in the inoculum. The solid black dots (•) indicate the total (−)RNA copies/μg of tissue or feces in an individual animal. The black bar indicates the geometric mean of the data set on each day. The asterisks indicate the time points when rotavirus replication [defined as a significant excess of (+)RNA] was detectable. The “‡” symbol indicates that the mean (−)RNA copy numbers are significantly (P < 0.05) higher compared to the same day and tissue in the other inoculation group (EC versus RRV). Groups were evaluated by a two-sample t test. Significance was established if the P value was <0.05. The x axes show the days postinoculation, and the y axes indicate the log10 of the (−)RNA copy numbers per microgram of tissue.

  • FIG. 4.
    FIG. 4.

    Whole blood drawn at 3 dpi from four 5-day-old mice inoculated with a 106 ID50 of EC rotavirus. Plasma was separated from the leukocytes, RNA was extracted from the plasma and PBMC fractions, and an ssQRT-PCR assay was used to determine the (−)RNA and excess (+)RNA rotavirus copy numbers in the plasma and leukocyte fractions. The results are reported in rotavirus (−)RNA copies/μg of whole blood. Significant excess (+)RNA was not detected.

  • FIG. 5.
    FIG. 5.

    Serial sections from MLN and lungs were collected from 104 ID50 EC- and 107 PFU RRV-infected and noninfected controls. Rotavirus replication was detected with biotinylated MAbs to rotavirus NSP4 (B4-2) (27) and to VP7 on assembled rotavirus triple-layered particles (MAb 159) (12) and developed with a secondary streptavidin-Texas Red antibody. The cellular nucleus was stained blue with TOTO-3. NSP4 stained EC-infected MLN (A1), RRV-infected MLN (A2), and uninfected MLN (A3). 159 stained EC-infected MLN (B1), RRV-infected MLN (B2), and uninfected MLN (B3). NSP4 stained EC-infected lung (C1), RRV-infected lung (C2), and uninfected control lung (C3). MAb 159 stained RRV-infected liver at ×40 (D1) and ×100 (D2) magnifications. (D3) MAb 159 stained uninfected liver.

  • FIG. 6.
    FIG. 6.

    Cytospin of mechanically disrupted MLN collected 3 dpi from mice inoculated with a 106 ID50 EC when 5 days old. (A1) Merged photograph of NSP4 (Texas Red) (A2) and monoclonal antibody to B220 (FITC) (A3), cell surface markers for B cells and plasmacytoid DC. (B1) Merged photograph of NSP4 (Texas Red) (B2) and monoclonal antibody to CD11b (FITC) (B3), cell surface markers for macrophages, granulocytes, NK cells, and a subset of DC. (C1) Merged photograph of NSP4 (Texas Red) (C2) and monoclonal antibody to CD11c (FITC) (C3), cell surface markers for DC. (D1) Merged photograph of NSP4 (Texas Red) (D2) and monoclonal antibody to CD3 (FITC) (D3), cell surface markers for T cells.

Tables

  • TABLE 1.

    Quantities of rotavirus (−)RNA and antigen in suckling mice inoculated with EC or RRV

    SampleInoculumRotavirus (−)RNARotavirus antigenIFA
    % Positive mice (no. positive/no. tested)Geometric mean (±SD)a% Positive mice (no. positive/no. tested)Geometric mean (±SD)b
    FecesEC98 (49/50)6.8 (±0.6)95 (17/18)6.2 (±0.9)NAc
    RRV86 (43/50)4.5 (±1.0)26 (5/19)1.3 (±2.3)NA
    Small Int.EC98 (49/50)5.0 (±1.0)83 (15/18)4.7 (±1.8)+
    RRV16 (8/50)0.3 (±0.8)54 (6/11)1.2 (±2.4)+
    MLNEC80 (40/50)3.9 (±1.7)35 (6/17)3.0 (±2.5)+
    RRV72 (32/44)3.0 (± 1.9)80 (4/5)2.4 (±3.3)+
    BloodEC31 (14/44)1.0 (±1.1)23 (6/26)1.6 (±2.2)NA
    RRV13 (6/44)0.4 (±0.8)7 (1/14)0.7 (±1.8)NA
    LiverEC64 (32/50)2.2 (±1.0)4 (1/26)0.3 (±1.0)−
    RRV10 (5/50)0.3 (±0.8)47 (10/21)2.1(±2.4)+
    LungEC28 (14/50)0.9 (±0.9)0 (0/22)0+
    RRV34 (17/50)1.6 (±2.0)14 (2/14)1.7 (±2.7)+
    KidneyEC45 (18/40)1.8 (±1.2)(0/6)NA−
    RRV3 (1/29)0(0/3)NA−

    • ↵a The log value of the geometric mean (−)RNA copy numbers/microgram at 3 to 7 dpi.

    • ↵b The log value of the geometric mean of PFU/ml equivalent values of the ELISA titer at 3 to 7 dpi.

    • ↵c NA, not available.

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