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T-Cell tropism of simian varicella virus during primary infection.

Ouwendijk WJ, Mahalingam R, de Swart RL, Haagmans BL, van Amerongen G, Getu S, Gilden D, Osterhaus AD, Verjans GM - PLoS Pathog. (2013)

Bottom Line: Except for pneumonitis, pathology produced by SVV-EGFP was less compared to SVV-wt.In ganglia, SVV was found primarily in neurons and occasionally in memory T-cells adjacent to neurons.In conclusion, the data suggest the role of memory T-cells in disseminating SVV to its target organs during primary infection of its natural and immunocompetent host.

View Article: PubMed Central - PubMed

Affiliation: Department of Viroscience, Erasmus MC, Rotterdam, the Netherlands.

ABSTRACT
Varicella-zoster virus (VZV) causes varicella, establishes a life-long latent infection of ganglia and reactivates to cause herpes zoster. The cell types that transport VZV from the respiratory tract to skin and ganglia during primary infection are unknown. Clinical, pathological, virological and immunological features of simian varicella virus (SVV) infection of non-human primates parallel those of primary VZV infection in humans. To identify the host cell types involved in virus dissemination and pathology, we infected African green monkeys intratracheally with recombinant SVV expressing enhanced green fluorescent protein (SVV-EGFP) and with wild-type SVV (SVV-wt) as a control. The SVV-infected cell types and virus kinetics were determined by flow cytometry and immunohistochemistry, and virus culture and SVV-specific real-time PCR, respectively. All monkeys developed fever and skin rash. Except for pneumonitis, pathology produced by SVV-EGFP was less compared to SVV-wt. In lungs, SVV infected alveolar myeloid cells and T-cells. During viremia the virus preferentially infected memory T-cells, initially central memory T-cells and subsequently effector memory T-cells. In early non-vesicular stages of varicella, SVV was seen mainly in perivascular skin infiltrates composed of macrophages, dendritic cells, dendrocytes and memory T-cells, implicating hematogenous spread. In ganglia, SVV was found primarily in neurons and occasionally in memory T-cells adjacent to neurons. In conclusion, the data suggest the role of memory T-cells in disseminating SVV to its target organs during primary infection of its natural and immunocompetent host.

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Macroscopic and microscopic detection of SVV-infected cells in lungs of infected African green monkeys.(A) Macroscopic appearance of consolidated dark-red lesions (black arrow) in the lung of an SVV-wt−infected monkey at 13 dpi. (B) Macroscopic detection of EGFP fluorescence in affected area of lung (white arrow) of an SVV-EGFP-infected monkey at 9 dpi. (C) Magnification of the affected area in panel B shows EGFP fluorescence. (D–G) Serial lung sections obtained from an SVV-EGFP−infected monkey at 9 dpi analyzed by immunohistochemistry (IHC) for SVV antigens (D) or by immunofluorescence (IF) for EGFP (E), with two sections analyzed by IHC (F) or IF (G) using normal rabbit serum (NRS) and isotype control antibodies, respectively. Lung sections obtained from an SVV-wt−infected monkey at 9 dpi were analyzed using dual IF for SVV (green) and: cytokeratin (red) (H), CD3 (red) (I), CD68 (red) (J), and CD11c (red) (K) antigens. Arrows indicate double-positive cells. Asterisks indicate autofluorescent erythrocytes. Dashed lines indicate alveolar septa. Br: bronchus. Nuclei were counterstained with DAPI. D–G: 100× magnification; H, J: 400× magnification; I, K: 400× magnification and 2× digital zoom.
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ppat-1003368-g002: Macroscopic and microscopic detection of SVV-infected cells in lungs of infected African green monkeys.(A) Macroscopic appearance of consolidated dark-red lesions (black arrow) in the lung of an SVV-wt−infected monkey at 13 dpi. (B) Macroscopic detection of EGFP fluorescence in affected area of lung (white arrow) of an SVV-EGFP-infected monkey at 9 dpi. (C) Magnification of the affected area in panel B shows EGFP fluorescence. (D–G) Serial lung sections obtained from an SVV-EGFP−infected monkey at 9 dpi analyzed by immunohistochemistry (IHC) for SVV antigens (D) or by immunofluorescence (IF) for EGFP (E), with two sections analyzed by IHC (F) or IF (G) using normal rabbit serum (NRS) and isotype control antibodies, respectively. Lung sections obtained from an SVV-wt−infected monkey at 9 dpi were analyzed using dual IF for SVV (green) and: cytokeratin (red) (H), CD3 (red) (I), CD68 (red) (J), and CD11c (red) (K) antigens. Arrows indicate double-positive cells. Asterisks indicate autofluorescent erythrocytes. Dashed lines indicate alveolar septa. Br: bronchus. Nuclei were counterstained with DAPI. D–G: 100× magnification; H, J: 400× magnification; I, K: 400× magnification and 2× digital zoom.

Mentions: All SVV-infected animals became dyspneic at the time of skin rash. Macroscopic examination of lungs showed multifocal pulmonary consolidation and hemorrhage affecting at least one lobe in all animals euthanized 9 or 13 dpi (Fig. 2A). Diffuse EGFP fluorescence was detected in an SVV-EGFP−infected monkey at 9 dpi (Fig. 2B and C). Combined immunohistochemical (IHC) and immunofluorescence (IF) analyses for SVV antigens and EGFP on consecutive sections of lung showed that EGFP expression was restricted to SVV antigen-positive cells (Fig. 2D–G), demonstrating that EGFP is a valid marker to identify SVV-infected cells in the monkeys. To investigate SVV-infected cell types in situ, lung tissue sections were analyzed by dual-IF staining with SVV-specific antiserum and anti-keratin, -CD3, -CD68 and -CD11c mouse monoclonal antibodies (mAbs). SVV-infected cells were readily detected in lungs at 9 dpi, but not at later times (data not shown). At 9 dpi, abundant SVVposkeratinpos lung epithelial cells were observed (Fig. 2H), as well as SVVposCD3pos T-cells (Fig. 2I). In addition, SVV antigens were found in intra-alveolar cells that co-expressed CD68 and/or CD11c, consistent with alveolar macrophages (AM), some of which appeared to have phagocytosed SVV-infected cells (Fig. 2J). Occasionally, SVVposCD11cpos dendritic cell (DC)-like cells displaying multiple branched projections were observed adjacent to bronchi (Fig. 2K).


T-Cell tropism of simian varicella virus during primary infection.

Ouwendijk WJ, Mahalingam R, de Swart RL, Haagmans BL, van Amerongen G, Getu S, Gilden D, Osterhaus AD, Verjans GM - PLoS Pathog. (2013)

Macroscopic and microscopic detection of SVV-infected cells in lungs of infected African green monkeys.(A) Macroscopic appearance of consolidated dark-red lesions (black arrow) in the lung of an SVV-wt−infected monkey at 13 dpi. (B) Macroscopic detection of EGFP fluorescence in affected area of lung (white arrow) of an SVV-EGFP-infected monkey at 9 dpi. (C) Magnification of the affected area in panel B shows EGFP fluorescence. (D–G) Serial lung sections obtained from an SVV-EGFP−infected monkey at 9 dpi analyzed by immunohistochemistry (IHC) for SVV antigens (D) or by immunofluorescence (IF) for EGFP (E), with two sections analyzed by IHC (F) or IF (G) using normal rabbit serum (NRS) and isotype control antibodies, respectively. Lung sections obtained from an SVV-wt−infected monkey at 9 dpi were analyzed using dual IF for SVV (green) and: cytokeratin (red) (H), CD3 (red) (I), CD68 (red) (J), and CD11c (red) (K) antigens. Arrows indicate double-positive cells. Asterisks indicate autofluorescent erythrocytes. Dashed lines indicate alveolar septa. Br: bronchus. Nuclei were counterstained with DAPI. D–G: 100× magnification; H, J: 400× magnification; I, K: 400× magnification and 2× digital zoom.
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ppat-1003368-g002: Macroscopic and microscopic detection of SVV-infected cells in lungs of infected African green monkeys.(A) Macroscopic appearance of consolidated dark-red lesions (black arrow) in the lung of an SVV-wt−infected monkey at 13 dpi. (B) Macroscopic detection of EGFP fluorescence in affected area of lung (white arrow) of an SVV-EGFP-infected monkey at 9 dpi. (C) Magnification of the affected area in panel B shows EGFP fluorescence. (D–G) Serial lung sections obtained from an SVV-EGFP−infected monkey at 9 dpi analyzed by immunohistochemistry (IHC) for SVV antigens (D) or by immunofluorescence (IF) for EGFP (E), with two sections analyzed by IHC (F) or IF (G) using normal rabbit serum (NRS) and isotype control antibodies, respectively. Lung sections obtained from an SVV-wt−infected monkey at 9 dpi were analyzed using dual IF for SVV (green) and: cytokeratin (red) (H), CD3 (red) (I), CD68 (red) (J), and CD11c (red) (K) antigens. Arrows indicate double-positive cells. Asterisks indicate autofluorescent erythrocytes. Dashed lines indicate alveolar septa. Br: bronchus. Nuclei were counterstained with DAPI. D–G: 100× magnification; H, J: 400× magnification; I, K: 400× magnification and 2× digital zoom.
Mentions: All SVV-infected animals became dyspneic at the time of skin rash. Macroscopic examination of lungs showed multifocal pulmonary consolidation and hemorrhage affecting at least one lobe in all animals euthanized 9 or 13 dpi (Fig. 2A). Diffuse EGFP fluorescence was detected in an SVV-EGFP−infected monkey at 9 dpi (Fig. 2B and C). Combined immunohistochemical (IHC) and immunofluorescence (IF) analyses for SVV antigens and EGFP on consecutive sections of lung showed that EGFP expression was restricted to SVV antigen-positive cells (Fig. 2D–G), demonstrating that EGFP is a valid marker to identify SVV-infected cells in the monkeys. To investigate SVV-infected cell types in situ, lung tissue sections were analyzed by dual-IF staining with SVV-specific antiserum and anti-keratin, -CD3, -CD68 and -CD11c mouse monoclonal antibodies (mAbs). SVV-infected cells were readily detected in lungs at 9 dpi, but not at later times (data not shown). At 9 dpi, abundant SVVposkeratinpos lung epithelial cells were observed (Fig. 2H), as well as SVVposCD3pos T-cells (Fig. 2I). In addition, SVV antigens were found in intra-alveolar cells that co-expressed CD68 and/or CD11c, consistent with alveolar macrophages (AM), some of which appeared to have phagocytosed SVV-infected cells (Fig. 2J). Occasionally, SVVposCD11cpos dendritic cell (DC)-like cells displaying multiple branched projections were observed adjacent to bronchi (Fig. 2K).

Bottom Line: Except for pneumonitis, pathology produced by SVV-EGFP was less compared to SVV-wt.In ganglia, SVV was found primarily in neurons and occasionally in memory T-cells adjacent to neurons.In conclusion, the data suggest the role of memory T-cells in disseminating SVV to its target organs during primary infection of its natural and immunocompetent host.

View Article: PubMed Central - PubMed

Affiliation: Department of Viroscience, Erasmus MC, Rotterdam, the Netherlands.

ABSTRACT
Varicella-zoster virus (VZV) causes varicella, establishes a life-long latent infection of ganglia and reactivates to cause herpes zoster. The cell types that transport VZV from the respiratory tract to skin and ganglia during primary infection are unknown. Clinical, pathological, virological and immunological features of simian varicella virus (SVV) infection of non-human primates parallel those of primary VZV infection in humans. To identify the host cell types involved in virus dissemination and pathology, we infected African green monkeys intratracheally with recombinant SVV expressing enhanced green fluorescent protein (SVV-EGFP) and with wild-type SVV (SVV-wt) as a control. The SVV-infected cell types and virus kinetics were determined by flow cytometry and immunohistochemistry, and virus culture and SVV-specific real-time PCR, respectively. All monkeys developed fever and skin rash. Except for pneumonitis, pathology produced by SVV-EGFP was less compared to SVV-wt. In lungs, SVV infected alveolar myeloid cells and T-cells. During viremia the virus preferentially infected memory T-cells, initially central memory T-cells and subsequently effector memory T-cells. In early non-vesicular stages of varicella, SVV was seen mainly in perivascular skin infiltrates composed of macrophages, dendritic cells, dendrocytes and memory T-cells, implicating hematogenous spread. In ganglia, SVV was found primarily in neurons and occasionally in memory T-cells adjacent to neurons. In conclusion, the data suggest the role of memory T-cells in disseminating SVV to its target organs during primary infection of its natural and immunocompetent host.

Show MeSH
Related in: MedlinePlus