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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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Detection of SVV-infected cells in ganglia of infected African green monkeys.(A) Virus DNA load was determined in ganglia at 9, 13 and 20 dpi by SVV-specific real-time qPCR. Filled and open squares represent pooled ganglia from the same level of the neuraxis from animals infected with SVV-wt and SVV-EGFP, respectively. Colors indicate level of the neuraxis: trigeminal (black), cervical (red), thoracic (blue), lumbar (green) and sacral (pink) ganglia. Horizontal bars represent mean viral DNA load per animal. (B) Immunohistochemical detection of SVV proteins (arrowheads) in a cervical ganglion at 9 dpi. Squares indicate corresponding tissue areas shown at higher magnification in (C) and (D). (E) Dual-immunofluorescence (IF) staining of a thoracic ganglion at 9 dpi for SVV proteins (green) and neural cell adhesion molecule (NCAM; red). Arrows indicate SVV-positive neurons. (F) Dual-IF staining of a thoracic ganglion at 9 dpi for SVV protein (green) and glial fibrillary acidic protein (GFAP; red). Arrow indicates neuron-adjacent SVV-positive cell. (G) Dual-IF staining of a thoracic ganglion from a monkey at 9 dpi for SVV protein (green) and CD3 (red). Arrows indicate SVV-positive T-cells. Asterisks indicate autofluorescent lipofuscin and the borders of the neuronal cell bodies are indicated with dashed lines. (H) Ganglion-derived single-cell suspensions were analyzed by flow cytometry and T-cells were categorized as naive, central memory (CM) and effector memory (EM) T-cells. E–G: nuclei were counterstained with DAPI (blue). ** p<0.01 by Mann-Whitney test. B: 200× magnification; C, D: 400× magnification, 2× digital zoom; E: 400× magnification; F, G: 400× magnification, 2× digital zoom.
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ppat-1003368-g007: Detection of SVV-infected cells in ganglia of infected African green monkeys.(A) Virus DNA load was determined in ganglia at 9, 13 and 20 dpi by SVV-specific real-time qPCR. Filled and open squares represent pooled ganglia from the same level of the neuraxis from animals infected with SVV-wt and SVV-EGFP, respectively. Colors indicate level of the neuraxis: trigeminal (black), cervical (red), thoracic (blue), lumbar (green) and sacral (pink) ganglia. Horizontal bars represent mean viral DNA load per animal. (B) Immunohistochemical detection of SVV proteins (arrowheads) in a cervical ganglion at 9 dpi. Squares indicate corresponding tissue areas shown at higher magnification in (C) and (D). (E) Dual-immunofluorescence (IF) staining of a thoracic ganglion at 9 dpi for SVV proteins (green) and neural cell adhesion molecule (NCAM; red). Arrows indicate SVV-positive neurons. (F) Dual-IF staining of a thoracic ganglion at 9 dpi for SVV protein (green) and glial fibrillary acidic protein (GFAP; red). Arrow indicates neuron-adjacent SVV-positive cell. (G) Dual-IF staining of a thoracic ganglion from a monkey at 9 dpi for SVV protein (green) and CD3 (red). Arrows indicate SVV-positive T-cells. Asterisks indicate autofluorescent lipofuscin and the borders of the neuronal cell bodies are indicated with dashed lines. (H) Ganglion-derived single-cell suspensions were analyzed by flow cytometry and T-cells were categorized as naive, central memory (CM) and effector memory (EM) T-cells. E–G: nuclei were counterstained with DAPI (blue). ** p<0.01 by Mann-Whitney test. B: 200× magnification; C, D: 400× magnification, 2× digital zoom; E: 400× magnification; F, G: 400× magnification, 2× digital zoom.

Mentions: The hallmark of primary SVV and VZV infection is the capacity of virus to infect and establish latency in ganglionic neurons along the entire neuraxis [1], [13], [39]–[42]. Virus may reach ganglia hematogenously or by retrograde axonal transport along axons innervating varicella lesions [12], [20], [21], [43], [44]. We determined the kinetics of virus infection and the cell types infected in ganglia during primary SVV infection. The SVV DNA load in ganglia was significantly higher in SVV-wt− compared to SVV-EGFP−infected monkeys (p<0.01; Mann-Whitney test) (Fig. 7A), peaking at 9 dpi and decreasing thereafter (Fig. 7A), as might be expected during the establishment of latency. Despite high SVV DNA loads, no virus-mediated cytopathology was seen in ganglia (data not shown). Virus antigen was more abundant at 9 dpi than at 13 and 20 dpi (data not shown). SVV-infected cells in ganglia were detected in situ by IHC using SVV-specific antiserum (Fig. 7B–D). Dual-IF staining for SVV and the neuron-specific marker NCAM (neural cell adhesion molecule) showed that most SVVpos cells were neurons (Fig. 7E). Occasionally, SVV antigens were seen at the neuronal cell surface or potentially within satellite glial cells (SGC) (Fig. 7D). SGC form a sheet that completely enwraps neuronal cell bodies, providing physical and metabolic support to the neurons and contributing to regulation of the immune response in the peripheral nervous system [45], [46]. Virus-infected cells located in vicinity to neurons did not express the SGC-specific marker glial fibrillary acidic protein (GFAP) [45], implicating that SGC were not infected with SVV at 9, 13 and 20 dpi (Fig. 7F and data not shown). To address the possibility of T-cell–mediated transfer of SVV to neurons, ganglia were examined using dual-IF staining for SVV antigens and CD3. In an SVV-wt−infected monkey euthanized at 9 dpi, SVV-infected T-cells were detected in close proximity to neurons (Fig. 7G). Notably, this animal also had the highest SVV DNA load in blood and ganglia. Flow cytometric analysis of ganglion-derived single-cell suspensions demonstrated that ganglion-resident T-cells were memory T-cells, predominantly EM T-cells (Fig. 7H).


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)

Detection of SVV-infected cells in ganglia of infected African green monkeys.(A) Virus DNA load was determined in ganglia at 9, 13 and 20 dpi by SVV-specific real-time qPCR. Filled and open squares represent pooled ganglia from the same level of the neuraxis from animals infected with SVV-wt and SVV-EGFP, respectively. Colors indicate level of the neuraxis: trigeminal (black), cervical (red), thoracic (blue), lumbar (green) and sacral (pink) ganglia. Horizontal bars represent mean viral DNA load per animal. (B) Immunohistochemical detection of SVV proteins (arrowheads) in a cervical ganglion at 9 dpi. Squares indicate corresponding tissue areas shown at higher magnification in (C) and (D). (E) Dual-immunofluorescence (IF) staining of a thoracic ganglion at 9 dpi for SVV proteins (green) and neural cell adhesion molecule (NCAM; red). Arrows indicate SVV-positive neurons. (F) Dual-IF staining of a thoracic ganglion at 9 dpi for SVV protein (green) and glial fibrillary acidic protein (GFAP; red). Arrow indicates neuron-adjacent SVV-positive cell. (G) Dual-IF staining of a thoracic ganglion from a monkey at 9 dpi for SVV protein (green) and CD3 (red). Arrows indicate SVV-positive T-cells. Asterisks indicate autofluorescent lipofuscin and the borders of the neuronal cell bodies are indicated with dashed lines. (H) Ganglion-derived single-cell suspensions were analyzed by flow cytometry and T-cells were categorized as naive, central memory (CM) and effector memory (EM) T-cells. E–G: nuclei were counterstained with DAPI (blue). ** p<0.01 by Mann-Whitney test. B: 200× magnification; C, D: 400× magnification, 2× digital zoom; E: 400× magnification; F, G: 400× magnification, 2× digital zoom.
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Related In: Results  -  Collection

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ppat-1003368-g007: Detection of SVV-infected cells in ganglia of infected African green monkeys.(A) Virus DNA load was determined in ganglia at 9, 13 and 20 dpi by SVV-specific real-time qPCR. Filled and open squares represent pooled ganglia from the same level of the neuraxis from animals infected with SVV-wt and SVV-EGFP, respectively. Colors indicate level of the neuraxis: trigeminal (black), cervical (red), thoracic (blue), lumbar (green) and sacral (pink) ganglia. Horizontal bars represent mean viral DNA load per animal. (B) Immunohistochemical detection of SVV proteins (arrowheads) in a cervical ganglion at 9 dpi. Squares indicate corresponding tissue areas shown at higher magnification in (C) and (D). (E) Dual-immunofluorescence (IF) staining of a thoracic ganglion at 9 dpi for SVV proteins (green) and neural cell adhesion molecule (NCAM; red). Arrows indicate SVV-positive neurons. (F) Dual-IF staining of a thoracic ganglion at 9 dpi for SVV protein (green) and glial fibrillary acidic protein (GFAP; red). Arrow indicates neuron-adjacent SVV-positive cell. (G) Dual-IF staining of a thoracic ganglion from a monkey at 9 dpi for SVV protein (green) and CD3 (red). Arrows indicate SVV-positive T-cells. Asterisks indicate autofluorescent lipofuscin and the borders of the neuronal cell bodies are indicated with dashed lines. (H) Ganglion-derived single-cell suspensions were analyzed by flow cytometry and T-cells were categorized as naive, central memory (CM) and effector memory (EM) T-cells. E–G: nuclei were counterstained with DAPI (blue). ** p<0.01 by Mann-Whitney test. B: 200× magnification; C, D: 400× magnification, 2× digital zoom; E: 400× magnification; F, G: 400× magnification, 2× digital zoom.
Mentions: The hallmark of primary SVV and VZV infection is the capacity of virus to infect and establish latency in ganglionic neurons along the entire neuraxis [1], [13], [39]–[42]. Virus may reach ganglia hematogenously or by retrograde axonal transport along axons innervating varicella lesions [12], [20], [21], [43], [44]. We determined the kinetics of virus infection and the cell types infected in ganglia during primary SVV infection. The SVV DNA load in ganglia was significantly higher in SVV-wt− compared to SVV-EGFP−infected monkeys (p<0.01; Mann-Whitney test) (Fig. 7A), peaking at 9 dpi and decreasing thereafter (Fig. 7A), as might be expected during the establishment of latency. Despite high SVV DNA loads, no virus-mediated cytopathology was seen in ganglia (data not shown). Virus antigen was more abundant at 9 dpi than at 13 and 20 dpi (data not shown). SVV-infected cells in ganglia were detected in situ by IHC using SVV-specific antiserum (Fig. 7B–D). Dual-IF staining for SVV and the neuron-specific marker NCAM (neural cell adhesion molecule) showed that most SVVpos cells were neurons (Fig. 7E). Occasionally, SVV antigens were seen at the neuronal cell surface or potentially within satellite glial cells (SGC) (Fig. 7D). SGC form a sheet that completely enwraps neuronal cell bodies, providing physical and metabolic support to the neurons and contributing to regulation of the immune response in the peripheral nervous system [45], [46]. Virus-infected cells located in vicinity to neurons did not express the SGC-specific marker glial fibrillary acidic protein (GFAP) [45], implicating that SGC were not infected with SVV at 9, 13 and 20 dpi (Fig. 7F and data not shown). To address the possibility of T-cell–mediated transfer of SVV to neurons, ganglia were examined using dual-IF staining for SVV antigens and CD3. In an SVV-wt−infected monkey euthanized at 9 dpi, SVV-infected T-cells were detected in close proximity to neurons (Fig. 7G). Notably, this animal also had the highest SVV DNA load in blood and ganglia. Flow cytometric analysis of ganglion-derived single-cell suspensions demonstrated that ganglion-resident T-cells were memory T-cells, predominantly EM T-cells (Fig. 7H).

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