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Comparative Study
. 2005 Oct;167(4):1043-9.
doi: 10.1016/S0002-9440(10)61193-5.

Apoptosis of hepatocytes caused by Punta Toro virus (Bunyaviridae: Phlebovirus) and its implication for Phlebovirus pathogenesis

Xiaohua Ding  1 , Fangling Xu, Hongli Chen, Robert B Tesh, Shu-Yuan Xiao
Affiliations
Comparative Study

Apoptosis of hepatocytes caused by Punta Toro virus (Bunyaviridae: Phlebovirus) and its implication for Phlebovirus pathogenesis

Xiaohua Ding et al. Am J Pathol. 2005 Oct.

Abstract

Experimental infection of hamsters with Punta Toro virus (PTV) produces a disease with clinical and pathological similarities to the severe human hemorrhagic fever caused by Rift Valley fever virus (RVFV), thus providing an animal model for RVFV pathogenesis. In this model, hepatocytic apoptosis is the main pathological component of liver injuries that are responsible for severe hemorrhagic manifestations. To further elucidate whether viral replication in hepatocytes directly causes apoptosis, we studied the morphological and biochemical changes of apoptosis in HepG2 cells at different time points after PTV infection. Cellular viability began to decrease 12 hours after infection compared with controls. Caspases 3/7 were activated significantly at 48 and 72 hours after infection, and phosphatidylserine translocation and DNA fragmentation were also detected at 48 and 72 hours. Cell cycle analysis by flow cytometry showed that infected HepG2 cells were arrested at G(0)/G(1) phase. Furthermore, virus titer increased with apoptosis progression, suggesting that viral replication is necessary for the apoptotic process. These results indicate that PTV infection alone, without a secondary inflammatory cellular reaction, induces hepatocytic apoptosis and suggest that future therapeutics for RVFV hemorrhagic disease might target inhibition of cellular apoptotic pathways during the acute infection.

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Figures

Figure 1
Figure 1
Growth curves of the Adames and Balliet strains of PTV in HepG2 cells (means of duplicate).
Figure 2
Figure 2
CPE associated with replication of PTV in HepG2 cells. A: Morphological appearance for HepG2 cells with PTV infection at 0, 24, and 72 hours p.i. (40× objective). Many infected cells have rounded up and detached from the wall of plate. B: Viability of HepG2 cells as determined by MTT assay (mean ± SD).
Figure 3
Figure 3
Activities of caspases. A: Casapase 3/7-positive staining cells (40× objective). Bright blue fluorescence of nuclei corresponds to caspase 3/7-positive staining cells. B: Number of positive HepG2 cells at different time points in in situ analysis (mean ± SD).
Figure 4
Figure 4
PS translocation assay. Number of apoptotic cells stained with annexin V-FLUOS in PTV-infected and noninfected (control) HepG2 cells (mean ± SD).
Figure 5
Figure 5
DNA fragmentation analysis. A: TUNEL analysis of Punta Toro virus-infected HepG2 cells (40× objective). Green nuclear fluorescence correspond to positive staining. B: Number of apoptotic cells stained with fluorescein isothiocyanate and propidium iodide in control and PTV-infected HepG2 cells (mean ± SD).
Figure 6
Figure 6
Percentage (ratio ± SD%) of HepG2 in various cell cycle phases at different time points. DNA content was detected by flow cytometry after propidium iodide staining. A: Percentage of total HepG2 at G0/G1 phases. B: Percentage of total HepG2 at S phases. C: Percentage of total HepG2 at G2/M phases. D: Percentage of total HepG2 at sub-G1 phases.
Figure 7
Figure 7
Analysis of gene expression of Balliet strain-infected and noninfected HepG2 cells (mean ± SD). A: Bcl-2 gene. B: Bax gene.
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