Fatal disseminated mouse adenovirus type 1 infection in mice lacking B cells or Bruton's tyrosine kinase

Abstract

Mouse adenovirus type 1 (MAV-1) infection of B-cell-deficient and Bruton's tyrosine kinase (Btk)-deficient mice resulted in fatal disseminated disease resembling human adenovirus infections in immunocompromised patients. Mice lacking B cells or Btk were highly susceptible to acute MAV-1 infection, in contrast to controls and mice lacking T cells. To our knowledge, this is the first demonstration that mice with an X-linked immunodeficiency phenotype (Btk deficient) are susceptible to virus-induced disease. Mice lacking B cells or Btk on a C57BL/6 background succumbed with encephalomyelitis, hepatitis, and lymphoid necrosis. Mice lacking B cells on a BALB/c background succumbed with enteritis and hepatitis. Survival of acute MAV-1 infection correlated with early T-cell-independent neutralizing antibody and T-cell-independent antiviral immunoglobulin M. Treatment of MAV-1-infected Btk(-/-) mice 4 to 9 days postinfection with antiserum harvested 6 to 9 days postinfection from MAV-1-infected Btk(+/+) mice was therapeutic. Our findings implicate a critical role for B-cell function in preventing disseminated MAV-1 infection, particularly production of early T-cell-independent antiviral immunoglobulin M.

FIG. 1.

Survival of (A) B6 (n = 3) and RAG-1^−/− (n = 5), (B) B6 (n = 6), μMT (n = 6), and TCRβxδ^−/− (n = 6), (C) B6 (n = 6), BALB/c (n = 6), Btk^−/− (n = 9), and Jh (n = 6), and (D) B6 (n = 3) and Btk^−/− (n = 6) mice. Mice were injected intraperitoneally with (A) 100 PFU, (B) 700 PFU, (C) 700 PFU, and (D) 1 PFU.

FIG. 2.

Quantitation of virus from (A to C) B6 and μMT, (D) BALB/c and Jh, and (E and F) B6 and Btk^−/− mice; (E) three mice each were infected intraperitoneally or intravenously (symbols to the left and right, respectively, in each group). Each symbol represents an individual mouse. B6, □; μMT, ▵; BALB/c, □; Jh, ▿; Btk^−/−, ○. Solid symbols, mice that were moribund when euthanized. The short horizontal lines indicate means for three or more log-transformed titers; the dotted line at 2 × 10³ PFU/g indicates the limit of detection. *, P < 0.01; in panel F the asterisk relates to moribund Btk^−/− mice compared to B6 mice and to Btk^−/− mice not showing disease signs.

FIG. 3.

(A to L) Organs taken at 7 d.p.i. from B6, μMT, and Btk^−/− mice infected with 700 PFU. Tissue sections were stained with hematoxylin and eosin or processed for in situ hybridization. Insets (magnification, 400×) show endothelial cells of the same organ stained positively with a viral in situ hybridization probe. (M to T) Organs taken at 9 d.p.i. from BALB/c and Jh mice infected with 700 PFU. Tissue sections were stained with hematoxylin and eosin. Arrowheads in N and P show the boundary of germinal centers. The inset in panel T is a 400× view of the boxed area showing a viral inclusion body. e, perivascular edema; f, fibrin in the perivascular space; hn, hepatic necrosis; pp, Peyer's patch; v, blood vessel.

FIG. 4.

Representative hematoxylin- and eosin-stained tissue sections from organs taken at 12 d.p.i. from B6 and Btk^−/− mice that had been mock infected or infected with 1 PFU. Arrowheads in E show the germinal center boundary. *, lymphocytes adherent to endothelial cells. va, vasculitis; m, meningeal vascular necrosis; h, hepatitis.

FIG. 5.

Northern analysis of FasL polyadenylated RNA in BALB/c and Jh liver. RNA isolated 9 d.p.i. from the liver of BALB/c and Jh mice that had been mock infected (m) or infected (i) with 700 PFU. Each lane has RNA from an individual mouse. The level of FasL was normalized to the actin level and then to the value for the mock-infected BALB/c mouse, as indicated under the lanes. FasL levels were higher in infected Jh than BALB/c livers (P = 0.002).

FIG. 6.

Serum neutralizing antibody titers. Sera obtained (A) preinfection (open symbols) and at 6 d.p.i. (solid symbols) from B6, TCRα^−/−, and Btk^−/− mice that had been infected with 10⁴ PFU, (B) 7 d.p.i. from B6 and Btk^−/− mice that had been infected with 700 PFU, (C) 9 d.p.i. from B6 and A_β^b−/− mice that had been mock infected (open symbols) or infected (solid symbols) with 700 PFU, and (D) 12 d.p.i. from B6 and Btk^−/− mice that had been mock infected (open symbols) or infected (solid symbols) with 1 PFU of MAV-1. The dotted line represents the limit of detection. Each symbol represents serum from an individual mouse.

FIG. 7.

Anti-MAV-1 IgM and IgG. (A) Pooled sera preinfection (open symbols) and 6 d.p.i. (solid symbols) from B6 (□, n = 3), TCRα^−/− (⋄, n = 5), and Btk^−/− (○, n = 2) mice infected with 1 × 10⁴ PFU assayed for antiviral IgM. (B) Sera obtained preinfection (open symbols) and 6 d.p.i. (solid symbols) from five B6 and five TCRβxδ^−/− mice infected with 700 PFU assayed for antiviral IgM. Mean values ± standard deviation are shown. (C) Sera from mock-infected B6 (□, n = 1) or Btk^−/− (○, n = 1) mice and pooled sera from B6 (▪, n = 4) and Btk^−/− (•, n = 6) mice infected with 1 PFU were assayed for antiviral IgM (solid lines) and antiviral IgG (dashed lines) at 12 d.p.i. (D) Mean values for sera from five B6 (▪) mice and five CD4^−/− (★) mice, pooled sera from 5 A_β^b−/− (+) mice, and pooled sera from two TCRβxδ^−/− (×) mice assayed for antiviral IgG at 12 weeks p.i. with 700 PFU. Mean values ± standard deviation are shown for B6 and CD4^−/− samples.

FIG. 8.

(A) RAG-1^−/− mice infected with 100 PFU were treated intravenously daily on days 0 to 7 postinfection with 0.25 ml of PBS, pooled sera from naïve RAG-1^−/− mice, pooled sera from naïve B6 mice, or pooled MAV-1-immune sera diluted 1:4 in PBS (panels 1 to 4, respectively). MAV-1-immune sera were harvested 12 weeks postinfection from B6 mice infected with 700 PFU and had neutralizing antibody titers of >1:1,000, high levels of antiviral IgG, and no detectable antiviral IgM (data not shown and Fig. 5B). Virus levels were determined at 7 d.p.i. (B) Sera pooled from days 6 to 9 postinfection from MAV-1-infected Btk^+/+ (▪) or Btk^−/− (•) mice were assayed for antiviral IgM (solid line), IgG (dashed line), and neutralizing antibody (box). (C) Btk^−/− mice infected with 100 PFU were treated intraperitoneally daily from days 4 to 9 postinfection with 0.1 ml of the Btk^+/+ (•, n = 4) or Btk^−/− (○, n = 3) antiserum shown in panel B.