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. 2006:104:346-65.

Human adenovirus type 37 and the BALB/c mouse: progress toward a restricted adenovirus keratitis model (an American Ophthalmological Society thesis)

James Chodosh  1
Affiliations

Human adenovirus type 37 and the BALB/c mouse: progress toward a restricted adenovirus keratitis model (an American Ophthalmological Society thesis)

James Chodosh. Trans Am Ophthalmol Soc. 2006.

Abstract

Purpose: To establish a mouse model of adenovirus keratitis in order to study innate immune mechanisms in the adenovirus-infected cornea.

Methods: Balb/c 3T3 fibroblasts were inoculated with human adenovirus (HAdV) serotypes 8, 19, or 37 and observed for cytopathic effect. Viral growth titers were performed, and apoptosis was measured by TUNEL assay. Viral and host cytokine gene expression was assessed by RT-PCR in cultured Balb/c 3T3 fibroblasts and in the corneas of virus-injected Balb/c mice. Western blot analysis was performed to detect cell signaling in the virus-infected cornea.

Results: Only HAdV37 induced cytopathic effect in mouse cells. Viral gene expression was limited, and viral replication was not detected. Apoptotic cell death in HAdV37-infected Balb/c cells was evident 48 and 72 hours postinfection (P < .01). MCP-1, IL-6, KC, and IP-10 mRNA levels were increased maximally by 8.4, 9.6, 10.5, and 20.0-fold, respectively, at 30 to 90 minutes after HAdV37 infection. Similar cytokine elevations were observed in the corneas of Balb/c mice 4 hours after stromal injection of HAdV37, when viral gene expression for the viral capsid protein IIIa was not detected. Western blot showed increased phosphorylation of ERK1/2 at 4 and 24 hours after corneal infection.

Conclusions: Despite limited viral gene expression, HAdV37 infection of Balb/c 3T3 fibroblasts results in increased proinflammatory gene expression. A similar pattern of cytokine expression in the corneas of HAdV37-infected Balb/c mice suggests the mouse adenoviral keratitis model may be useful for the study of early innate immune responses in the adenovirus-infected corneal stroma.

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Figures

FIGURE 1
FIGURE 1
General schematic of adenovirus internalization cascade, based on studies performed with HAdV2 and SW480 cells., After binding of the HAdV2 penton fiber knob to one of several possible primary receptors, a secondary interaction between the viral penton base and cellular integrins mediates activation of phosphoinositide 3-kinase (PI3K) and Rho GTPases. Subsequent dynamin-dependent actin polymerization induces clathrin-mediated endocytosis of viral particles.
FIGURE 2
FIGURE 2
Model of cell signaling and downstream effects in HAdV19-infected human corneal fibroblasts.–,, Following primary and secondary binding to CD46 or sialic acid, and then αvβ3 integrin, respectively, the subgroup D adenovirus HAdV19 is internalized by the activity of Src kinase, leading to multiple downstream signaling events, and culminating in enhanced cell survival and proinflammatory gene expression.
FIGURE 3
FIGURE 3
Cytopathic effect in Balb/c 3T3 fibroblasts observed at 72 hours after HAdV37 but not mock infection. Cells were infected at a multiplicity of infection of 50 TCID/cell and photographed with a Zeiss Axiovert inverted microscope (original magnification, ×200).
FIGURE 4
FIGURE 4
Growth curve for HAdV37 in Balb/c 3T3 fibroblasts. Cell monolayers at 95% confluence were infected in triplicate with HAdV37 at a multiplicity of infection of 50 TCID/cell, and cells and supernatants harvested daily for 6 days postinfection for titering. Error bars show the standard deviation of the mean titer at each time point.
FIGURE 5
FIGURE 5
Expression of specific HAdV37 mRNAs by reverse transcriptase polymerase chain reaction (RT-PCR) in Balb/c 3T3 cells (○) vs A549 cells (▪). Cell monolayers at 95% confluence were infected in triplicate with HAdV37 at a multiplicity of infection (MOI) of 50 TCID/cell, and RNA harvested as described in the “Methods” section, and subjected to RT-PCR with primers listed in Table 1. The time of earliest detection of each adenoviral transcript in at least three independent experiments is shown for each cell type. Those genes with an asterisk (*) were found to be expressed only in the human A549 cells, not in the mouse cells.
FIGURE 6
FIGURE 6
TUNEL-positive Balb/c 3T3 cells per high-powered field (hpf) at indicated times postinfection. Balb/c 3T3 fibroblasts were infected with buffer or HAdV37 at a multiplicity of infection of 50 TCID/cell. Significantly increased numbers of apoptotic cells were noted in HAdV37-infected cell cultures at 48 and 72 hours after infection, as compared to cells mock-infected with buffer (* P < .01). The experiment shown is representative of three independent experiments. Error bars show the standard deviation of the mean number of apoptotic cells at each time point.
FIGURE 7
FIGURE 7
Real-time polymerase chain reaction (PCR) analysis of cytokine mRNA change induced in vitro by HAdV37 infection of Balb/c 3T3 fibroblasts. Cell monolayers were infected at a multiplicity of infection of 50 TCID/cell with HAdV37 or mock-infected with buffer and the RNA harvested at 30, 60, and 90 minutes postinfection. The height of each bar indicates the fold-increase in host cell cytokine gene expression induced at the indicated time after HAdV37 infection, as compared to mock-infected cells. Fold-increases in KC and MCP-1 mRNA expression appear maximal at 30 minutes postinfection, whereas fold-increases in IL-6 and IP-10 mRNA expression appear to be highest at 90 minutes postinfection. This experiment was repeated twice with similar results.
FIGURE 8
FIGURE 8
Real-time PCR analysis of change in host cytokine gene expression in Balb/c mice corneas at 4 hours after HAdV37 injection as compared to untouched and buffer-injected control corneas. The RNA from three corneas was pooled for analysis of each cytokine at each time point. The level of mRNA for each cytokine in buffer and HAdV37-injected corneas was expressed as the fold-increase over that of untouched corneas, set arbitrarily at a value of 1.0. mRNA levels for IL-6, KC, and IP-10 were all increased at 4 hours postinfection A similar pattern of increased expression for all three cytokines was seen in two replicates of this experiment.
FIGURE 9
FIGURE 9
Analysis of KC protein expression in corneas of Balb/c mice at 0, 4, and 8 hours postinfection. Right corneas were injected with 108 TCID/ml of HAdV37 or buffer in 1 μL, and the left corneas used as untouched controls. Corneas were harvested at the specified times postinfection and dissected into chilled 1% SDS lysis buffer with protease inhibitors, lysed, and homogenized. Supernatants were collected after centrifugation, and KC protein quantity in each sample was analyzed by the Bio-Plex cytokine assay system and reported as the quantity of KC protein per cornea. KC levels were highest in HAdV37-injected corneas at 8 hours postinfection. This experiment was performed three times with equivalent results.
FIGURE 10
FIGURE 10
Real-time polymerase chain reaction (PCR) analysis for expression of adenoviral gene transcripts in corneas of Balb/c mice 4 hours after intrastromal injection with HAdV37. An increase in threshold cycle (CT) units reflects an increase in the level of viral gene expression as compared to CT levels obtained immediately after injection (before any viral gene expression has occurred). Viral mRNAs for E1A and E1B 19k transcripts were present in HAdV37-injected mouse corneas at 4 hours postinfection, but the IIIa viral capsid gene was not detected (n.d.) in the virus-infected cornea in any of three separate experiments at this time postinfection.
FIGURE 11
FIGURE 11
Immunoblot analysis for phosphorylation of ERK1/2 at 4 and 24 hours after HAdV37 injection into the corneas of Balb/c mice. HAdV37 or buffer was injected intrastromally at the indicated time points, and proteins from three corneas for each condition and time point were harvested and pooled for Western blot analysis. Each phospho-ERK blot (upper of each paired blot) was stripped and reprobed for total ERK (lower of each paired blot). Phosphorylation of ERK1/2 was increased at both 4 and 24 hours after infection with HAdV37 as compared to buffer-injected controls. No differences in total ERK1/2 levels were seen at either time point. This experiment was repeated twice with similar results.
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