T-Cell Tropism of Simian Varicella Virus during Primary Infection

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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.

Author Summary

Varicella-zoster virus (VZV) causes varicella, establishes life-long latent infection in ganglia and reactivates later in life to cause zoster. VZV is acquired via the respiratory route, with skin rash occurring up to 3 weeks after exposure. The cell types that transport VZV to skin and ganglia during primary infection are unknown. Simian varicella virus (SVV) infection of non-human primates mimics clinical, pathological and immunological features of human VZV infection. African green monkeys were infected with recombinant SVV expressing enhanced green fluorescent protein (SVV-EGFP) or wild-type SVV (SVV-wt) as a control. By visualizing SVV-EGFP−infected cells in the living animal and in tissue samples, we identified the virus-infected cell types in blood, lungs, skin and ganglia during primary infection. Our data demonstrate that during viremia, SVV predominantly infects peripheral blood memory T-cells. Detection of SVV-infected memory T-cells in lungs, in early varicella skin lesions and also, albeit to a lesser extent, in ganglia suggests a role for memory T-cells in transporting virus to these organs. Our study provides novel insights into the cell types involved in virus dissemination and the overall pathology of varicella in a non-human primate model.

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THE NATURE OF HERPES ZOSTER: A LONG-TERM STUDY AND A NEW HYPOTHESIS.

Paul R Hope (1964)

Dr Hope-Simpson presents a study of all cases of herpes zoster occurring in his general practice during a sixteen-year period. The rate was 3.4 per thousand per annum, rising with age, and the distribution of lesions reflected that of the varicella rash.It was found that severity increased with age, but that the condition did not occur in epidemics, and that there was no characteristic seasonal variation. A low prevalence of varicella was usually associated with a high incidence of zoster.Dr Hope-Simpson suggests that herpes zoster is a spontaneous manifestation of varicella infection. Following the primary infection (chickenpox), virus becomes latent in the sensory ganglia, where it can be reactivated from time to time (herpes zoster). Herpes zoster then represents an adaptation enabling varicella virus to survive for long periods, even without a continuous supply of persons susceptible to chickenpox.

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Predominant Infection of CD150+ Lymphocytes and Dendritic Cells during Measles Virus Infection of Macaques

Rik L de Swart, Martin Ludlow, Lot de Witte … (2007)

Introduction In spite of significant progress in global measles control programs, each year infections with measles virus (MV) cause almost half a million deaths in developing countries [1,2]. Measles is associated with a profound but transient immunosuppression, and as a result, opportunistic infections may cause pneumonia, gastroenteritis, and otitis media [3,4]. MV is a member of the family Paramyxoviridae, genus Morbillivirus, which are enveloped viruses with a single-stranded RNA genome of negative polarity. Morbilliviruses are among the most infectious viruses of mammals, and are predominantly transmitted via the respiratory route [3]. Surprisingly, little is known about the specific cells involved in virus transmission and dissemination throughout the body. Classical textbook descriptions of measles pathogenesis suggest that MV initially infects epithelial cells of the respiratory tract, subsequently spreads to the regional lymph nodes, and is finally disseminated during a viremic phase mediated by infected monocytes [5–7]. However, epithelial cells and unstimulated monocytes do not express signaling lymphocyte activation molecule (SLAM, CD150), the receptor used by wild-type MV [8,9], making this sequence of events unlikely. Moreover, wild-type MV does not readily infect monocytes or epithelial cell lines in vitro [10–12]. CD150 is expressed on subsets of thymocytes, macrophages, dendritic cells (DCs), and activated B- and T-lymphocytes [8,9]; therefore, these cells are the most likely primary target cells for MV infection. It has been described that wild-type MV strains replicate efficiently in lymphocytes in vivo [13] and in vitro [14], although it has taken until the early 1990s before lymphoid cells replaced Vero cells as the golden standard for isolation of wild-type MV from clinical samples [15]. A decade ago it was shown that human DCs could be infected with wild-type MV [16–18], which was shown to be mediated by CD150 [19]. Infected DCs produced infectious virus [17] and were able to transmit the virus upon in vivo inoculation [20]. Recent studies with an animal morbillivirus, canine distemper virus in ferrets, also showed a viral tropism that was compatible with CD150-expressing cells: the major infected cell populations were T-and B-lymphocytes and thymocytes [21,22]. In this model, infection of macrophages or DCs could not be demonstrated. However, since infection with this ferret-adapted virus causes mainly neurological symptoms and is almost always fatal, it is difficult to extrapolate these data to measles in humans. Humans are the only natural host for MV, although measles outbreaks have also occurred in captive non-human primates following contact with human patients [23]. The incubation time of measles is approximately 2 wk, which has made it difficult to investigate the early phases of MV infection in humans. Onset of the typical rash coincides with the appearance of virus-specific neutralizing antibodies and T-lymphocytes, which correlate with a rapid decrease in viral load [3]. Although several small laboratory animal models for MV infection have been developed, none of these closely mimic the pathogenesis of measles in humans [24]. Experimental infection of non-human primates, especially macaques, has proven crucial for studying the pathogenesis of MV infection. Both rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques are highly susceptible to MV infection [25,26], although clinical signs such as rash and conjunctivitis may be more prominent in rhesus than in cynomolgus macaques [27–29]. A full-length anti-genomic clone (MV-IC323) was previously generated from a Japanese wild-type MV strain, and the resulting recombinant virus was shown to be virulent in macaques [30]. The virus was shown to utilize CD150, but not CD46, as receptor [30,31]. Subsequently, the gene encoding enhanced green fluorescent protein (EGFP) was inserted in this clone within an additional transcription unit upstream of the MV nucleocapsid gene. The resulting recombinant virus, MV-IC323-EGFP, displayed similar in vitro replication characteristics as its parental IC323 strain, and infected cells produced high amounts of EGFP [31]. Morbillivirus genes are transcribed by a start-stop mechanism from a single promoter in the 3′ leader region [4], resulting in the highest number of mRNAs being transcribed from promoter-proximal transcription units. Thus, in MV-IC323-EGFP-infected cells EGFP is the maximal virally expressed protein, and its amount is directly related to the level of virus replication. We therefore reasoned that experimental infection of macaques with MV-IC323-EGFP would provide an ideal opportunity to study measles pathogenesis at the cellular, tissue, and whole organism level. Results MV-IC323-EGFP Causes Measles in Macaques Three rhesus (#R1–3) and three cynomolgus macaques (#C1–3) were infected intra-tracheally with 104 cell culture infectious dose-50 of MV-IC323-EGFP. Two animals of each species were euthanized on day 9, the expected time of peak virus replication, and one on day 15, at which time no residual infectious MV was expected to be present. To assess the virulence of the recombinant virus in vivo, we performed virus isolations from broncho-alveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMCs). MV-IC323-EGFP was isolated from BAL cells and PBMCs of all six animals (Figure 1A and 1B), and kinetics and peak levels of infected cells were similar to those described previously for wild-type MV infection in macaques [25,29,32,33]. MV was also detected by reverse transcriptase (RT)-PCR in throat and nose swabs, but only after onset of viremia (Figure 1C and 1D). MV-specific serum IgM and IgG did not appear until after day 9 (Figure 1E and 1F). Of the two animals that were euthanized at a later time point, one (#C2) developed typical measles skin rash (Figure 2). Collectively, these results indicate that MV-IC323-EGFP infection in macaques is a valid model to study the pathogenesis of measles and is comparable to MV infection in humans. Figure 1 MV Replication and Specific Antibody Responses in Macaques at Different Time Points after Infection Virus isolation from BAL cells (A) and PBMCs (B); virus detection by TaqMan RT-PCR in throat (C) and nose (D) swabs; MV fusion protein (F)–specific serum IgM (E) and IgG (F) responses as determined by FACS-measured immunofluorescence. Symbols indicate rhesus macaques #R1 (•), #R2 (▪), #R3 (▾), and cynomolgus macaques #C1 (○), #C2 (⋄), and #C3 (Δ). Figure 2 EGFP Fluorescence in Tissues of Macaques after Experimental Infection with MV-IC323-EGFP Cynomolgus macaque #C3 on day 9: MV infection in the skin (A), gingiva and buccal mucosa (B), tongue and tonsils (C), inguinal lymph nodes (D), lungs with tracheo-bronchial lymph nodes (E), stomach (left), spleen (upper left), and large intestine with gut-associated lymphoid tissue (F), spleen (right), and large intestine with gut-associated lymphoid tissue (G); cynomolgus macaque #C2 on day 13 after infection: skin rash shown in normal light (H) or by EGFP fluorescence (I). Macroscopic Detection of Fluorescence in Skin, Mouth, and Lymphoid Tissues In the living animals, we examined the skin and mouth for EGFP fluorescence on days 3, 6, and 9 after infection. No fluorescence was detected on day 3, while on day 6 a few fluorescent spots (hallmarks of MV replication) were detected in the buccal mucosa of one animal (#C3). On day 9 after infection, many fluorescent spots were detected in the skin of four of the six animals (Figure 2A): all three cynomolgus macaques and one of the rhesus macaques (#R1). In all six animals fluorescent spots were detected in the buccal mucosa, gingiva, and/or on the tongue on day 9 (Figure 2B and 2C). Upon necropsy on day 9 we examined the major organs for fluorescence. Lymphoid tissues were brightly fluorescent, including the tonsils, inguinal lymph nodes, tracheo-bronchial lymph nodes, spleen, and gut-associated lymphoid tissue (Figure 2C–2G). Furthermore, fluorescence was also detected in the trachea (unpublished data) and in the wall of the stomach (Figure 2F, left). No fluorescence was visible macroscopically in the thyroid gland, heart, liver, kidneys, adrenal glands, pancreas, urinary bladder, or brain of any of the six animals. Thus, lymphoid tissues were the major sites of MV replication. On day 11, skin rash was observed on the abdomen and legs of cynomolgus macaque #C2, which co-localized with fluorescence indicating the presence of MV-infected cells in the skin (Figure 2H and 2I). The rash peaked on day 13 and had disappeared on day 15. In rhesus macaque #R2 no fluorescence was detected in the skin at any time point, and this animal did not develop skin rash during the period between day 9 and day 15. The other four animals had been euthanized before the expected time of onset of rash. Upon necropsy on day 15, fluorescence was no longer macroscopically detectable in lymphoid tissues or any of the other internal organs. However, we still detected fluorescent spots on the skin, tongue, gingiva, and buccal mucosa of one of the two animals (#C2), suggesting delayed clearance in these peripheral tissues. The ability to macroscopically detect MV-infected tissues in the intact host and select these for detailed microscopic analysis provides a unique opportunity for pathogenesis studies. Massive MV Replication in B- and T-Lymphocytes Next, we determined the phenotype of MV-infected cells in peripheral blood and lymphoid tissues by FACS analysis. Between 0.1% and 10% of circulating B-lymphocytes, CD4+ T-lymphocytes, and CD8+ T-lymphocytes proved to be MV-infected, while MV replication was virtually absent in monocytes (Figures 3 and 4A), natural killer cells, or polymorphonuclear cells (unpublished data). In all PBMC subpopulations, EGFP fluorescence was almost exclusively detected in cells expressing the MV receptor CD150 (Figures 3 and S1). Considering the subpopulation distribution in PBMC (Table S1), the highest absolute numbers of circulating MV-infected cells were detected in the CD4+ T-lymphocyte population. Notably, memory T-lymphocytes were preferentially infected, as demonstrated by the two to ten times higher percentage of EGFP+ cells in the CD45RA− as compared to the CD45RA+ subpopulation of T-lymphocytes (Table S2). Figure 3 EGFP+ Cells in PBMC Subpopulations of Macaque #C2 at Different Time Points after Infection Freshly isolated PBMCs were stained with monoclonal antibodies and analyzed in a FACScalibur measuring approximately 500,000 events per sample to allow detection of low-frequent MV-infected cell populations. Results are shown as dot plots, with EGFP expression on the y-axis and CD150 (SLAM) expression on the x-axis. EGFP-expression in CD3+CD4+ T-lymphocytes is shown in red; CD3+CD8+ T-lymphocytes in green; MHC class-II+CD20+ B-lymphocytes in blue, and CD14+ monocytes in orange. Figure 4 EGFP+ Cells in PBMC Subpopulations or in Lymphoid Tissue–Derived B- and T-Lymphocytes Symbols are the same as in Figure 1. Cells were stained with monoclonal antibodies and analyzed in a FACScalibur measuring approximately 500,000 events to allow detection of low-frequency infected cell populations. (A) Percentages of EGFP-positive cells in the same PBMC subpopulations as shown in Figure 3, for all six animals over time. (B) Percentages of EGFP-positive B- and T-lymphocytes in single cell suspensions of lymphoid tissues of the four animals that were euthanized on day 9. Large numbers of infected lymphocytes were detected in single cell suspensions of lymphoid tissues collected on day 9 after infection, in which up to 10% of T-lymphocytes and up to 30% of B-lymphocytes were EGFP+ (Figure 4B). In absolute numbers (Table S3), B-lymphocytes were usually the major infected lymphocyte population in lymphoid tissues. In addition, not only the percentage of infected cells but also the virus replication level, as measured by the level of EGFP expression per cell, was consistently higher in B-lymphocytes than in T-lymphocytes (Figure S2). MV Infection in Skin and Mouth In skin samples collected on day 9, we detected MV-infected cells in the dermis but not in the epidermis. Most fluorescent cells were present in association with aggregates of inflammatory cells near hair follicles, and many of the cells had the phenotype of DCs with long cellular processes (Figure 5A and 5B). We placed dermis sections in culture to allow DCs and inflammatory cells to migrate from the tissue, similar as described previously [34]. Fluorescent migrated cells proved to be a mixture of small MHC class-II− and large MHC class-II+ cells, suggesting the presence of MV-infected T-lymphocytes and DCs (Figure 5C). In addition, we could isolate MV-IC323-EGFP from these cells in Vero cells expressing CD150. Figure 5 MV-Infected Cells in the Skin of Animal #C3 on Day 9 after Infection (A) and (B) are serial sections of the same tissue. (A) Hematoxylin and eosin staining, showing aggregate of inflammatory cells (between arrows) adjacent to a hair follicle (asterisk). (B) Confocal microscopy image (EGFP-fluorescence in green, TO-PRO counter staining in blue) showing presence of fluorescent cells in the aggregate of inflammatory cells; the inset shows an example of a fluorescent cell with long processes interpreted as a DC. (C) FACS analysis of cells migrated from the dermis after 2 d in cell culture. Half of the migrated cells were small MHC class II-negative (i.e., most likely T-lymphocytes), of which approximately 3% were EGFP-positive. 20% of the migrated cells were identified as large granular cells in scatter, approximately 40% of these were MHC class II-positive (i.e., most likely dendritic cells), of which approximately 10% were EGFP-positive. In the submucosa of tongue and buccal wall, fluorescence was also mainly detected in association with aggregates of inflammatory cells, which in these tissues were present near the mucous glands (Figure S3). Similar to those in the skin, the aggregates appeared to contain both infected lymphocytes and DCs. In addition, in these tissues fluorescent cells were also detected in the keratinized epithelium, in many cases associated with intercellular vacuolization, indicative for epithelial necrosis (Figure S3). In a biopsy of skin-exhibiting rash that was collected on day 13, we observed sero-purulent crusting. Infected cells were present in aggregates of inflammatory cells close to the hair follicles and sebaceous glands, but a substantial amount of fluorescence was localized to the keratin layer of the stratum corneum (unpublished data). MV Infection in Internal Organs Large numbers of MV-infected lymphocytes and DCs were detected in lymphoid tissues and in tissues of the respiratory and digestive tracts collected on day 9 after infection. MV-infected cells in the submucosa of the trachea were interconnected by long dendritic processes (Figure 6A and 6B), suggesting cell-to-cell transmission of the virus. We also detected MV-positive cells in the lumen of the trachea (Figure 6C), which could play a role in virus transmission if expelled during coughing. Figure 6 Assessment of the Distribution of MV-Infected Cells in Fixed Tissue Sections Collected 9 d after Infection MV infection was visualized by detection of EGFP fluorescence in paraformaldehyde-fixed vibratome-cut tissue sections (V, 100 μm) or through the use of anti-EGFP or anti-MV N antibodies in formalin-fixed microtome-cut tissue sections (FF, 6 μm) from infected rhesus (#R1) or cynomolgus (#C1) macaques. Propidium iodide (red) was used as a structural counterstain (A, B, D, G, I, N, P). The asterisk indicates the approximate location of the MV-infected cells shown in the inset in (B, D, K and N). (A) Animal #R1 (V). A single MV-infected ciliated epithelial cell (arrow) is visible in the mucociliary epithelium of the tracheal mucosa. A number of MV-infected cells of lymphoid origin (arrowheads) are present in subjacent lamina propria and submucosa. MCE, mucociliary epithelium. (B) Animal #R1 (V). Numerous MV-infected cells (green) with extended cellular processes (inset) are visible in different levels of the tracheal mucosa. (C) Animal #R1 (FF). MV-infected cells (arrow) are present in the tracheal lumen; the arrowhead indicates infected cells in the tracheal lamina propria / submucosa. (D) Animal #R1 (V). A number of MV-infected germinal centers (inset) are visible in a composite image of the spleen. (E) Animal #R1 (V). Co-localization of EGFP fluorescence (green) and CD20-expression (blue) in infected B-lymphocytes in the spleen. (F) Animal #R1 (V). Co-localization of EGFP fluorescence (green) and CD3-expression (blue) in infected T-lymphocytes in the spleen. (G) Animal #R1 (FF). Co-localization of EGFP fluorescence (green) and CD11c-expression (blue) in infected DCs in the spleen. (H) Animal #R1 (FF). No co-localization between MV infection (red) and expression of the macrophage marker Mac287 (green) in the tracheo-bronchial lymph node. (I) Animal #R1 (V). Multiple interconnected foci of MV infection in the tracheo-bronchial lymph node. (J) Animal #R1 (FF). Infection of cells with the morphological characteristics of DCs (arrows) in the tracheo-bronchial lymph node. (K) Animal #R1 (V). Numerous MV-infected cells (green) in a composite image of the ileum. EGFP fluorescence does not co-localize with cytokeratin expression (red) (inset). (L) Animal #R1 (FF). MV-infected cells in lymphoid tissue of the duodenum. (M) Animal #C1 (FF). MV-infected cells in lymphoid tissue of the lamina propria of the stomach. (N) Focus of MV-infected cells (green) in the ciliated bronchial epithelial cell layer of the bronchus adjacent to the bronchial lumen. Fluorescent cilia (inset) are readily identified at the periphery of the infected cells. CBE, ciliated bronchial epithelial cell layer; BL, bronchial lumen. (O) Animal #R1 (FF). MV-infected cells (green) in the bronchus express the epithelial cell marker cytokeratin (red). Cell nuclei were counterstained with DAPI (blue). (P) Animal #C1 (FF). Detection of multiple foci of MV-infected cells (green) in the lung. Confocal analysis of the spleen showed that red pulp areas contained almost no EGFP+ cells, while white pulp areas were strongly fluorescent (Figure 6D). Fluorescent cells were CD20+ (Figure 6E), CD3+ (Figure 6F), or CD11c+ (Figure 6G), but did not co-stain with the macrophage-specific marker Mac287 (Figure 6H, see also Figure S4). Multinucleated EGFP+ Warthin-Finkeldey cells (Figure 6I) and EGFP+ cells with the distinctive morphology of DCs (Figure 6J) were detected in the lymph nodes. In the walls of the stomach and the small and large intestines both large process-rich cells (DCs) and small round cells (lymphocytes) were concentrated in aggregates of lymphoid tissue (Figure 6K–6M). EGFP+ cells were not detected in the outer epithelial cell layer in these organs. However, EGFP+ ciliated epithelial cells were detected in the trachea and in the lungs (Figure 6N–6P). Cell-to-cell fusion of these cells was particularly common in these tissues. Fluorescent ciliated epithelial cells were also detected in BAL samples collected on days 6 and 9 after infection, but the majority of EGFP+ cells in the BAL were large MHC class-II+ CD11c+ cells (most likely alveolar macrophages) or small MHC class-II− CD11c− cells (most likely T-lymphocytes). Discussion Here, we have infected macaques with a recombinant MV strain expressing high levels of EGFP, enabling highly sensitive detection of infected cells. The recombinant virus proved to be virulent in macaques, allowing macroscopic and microscopic assessment of the course of measles. Lymphocytes expressing the MV receptor CD150 were found to be the major target cells for MV replication in vivo. In submucosal tissues we detected large numbers of MV-infected lymphocytes and DCs in aggregates of inflammatory cells. Finally, MV infection was also detected in the epithelia of mouth and trachea. We identified CD150+ B- and T-lymphocytes as major targets for MV infection in vivo. The preferential infection of lymphocytes expressing CD150, a T cell activation marker [35,36], suggests that MV targets activated rather than resting T-lymphocytes. Our in vivo data are in agreement with recently published results of ex vivo infection studies in human tonsillar tissue, demonstrating that wild-type MV strains predominantly infected human CD150+ B- and T-lymphocytes [37]. In previous studies monocytes were postulated to be the key MV-infected cell population in peripheral blood of humans, based on the detection of MV RNA by RT-PCR in monocyte-enriched PBMC samples obtained from measles patients [5]. However, the samples used in those studies were collected after onset of rash, and these results may have reflected binding of opsonized virus particles to Fc-receptors rather than active virus replication. Our data are in accordance with the observation that non-activated human monocytes do not express CD150 and are only inefficiently infected in vitro [10,12]. MV-infection of human and non-human primate lymphocytes has been described previously [27,38–40], but the data we collected in macaques are the first to show the kinetics and high frequency of MV-infected lymphocytes in peripheral blood and lymphoid tissues. Estimations of MV-infected cells in PBMCs by an infectious center assay (Figure 1B) or by EGFP detection (Figure 3) suggested that the latter method was one to two log values more sensitive. This clearly illustrates the strength of our model based on infection with an autofluorescent virus as compared to conventional pathogenesis studies. MHC class II+ CD11c+ DCs in the dermis of the skin and the submucosa of respiratory and digestive tracts were identified as a second major target cell population for MV infection. Since DCs are known to express CD150 and can be infected with MV in vitro, a role for this cell population in the pathogenesis of measles had been hypothesized [41,42]. Here we formally prove that this is indeed the case in macaques in vivo. Interestingly, infected DCs in our study were in many cases found in association with lymphoid tissue or aggregates of inflammatory cells near hair follicles and mucous glands. Theoretically DCs could also have become fluorescent by uptake of EGFP rather than by MV-infection. However, this is unlikely to result in high EGFP expression levels as those observed in our study (Figures 5 and 6). We are currently undertaking additional studies to further characterize the phenotype and functionality of these infected DCs. Our observation that high percentages of infected lymphocytes and DCs were detected in lymphoid tissues of infected macaques sheds new light on measles-associated immunosuppression. Measles causes lymphopenia [3], which has previously been attributed to apoptosis of uninfected cells [40]. Our data suggest that actual depletion of the many MV-infected lymphocytes may contribute significantly to the observed lymphopenia. In addition, MV was found to preferentially infect CD150+ lymphocytes with a memory phenotype (CD45RA−). This is in accordance with data recently published by Condack et al., describing the preferential MV-infection of human memory T-lymphocytes as compared to naive T-lymphocytes in ex vivo infection studies in human tonsillar tissue [37]. Overall, these data strongly suggest that depletion of memory T- and B-lymphocytes as a direct consequence of MV infection may play an important role in immunosuppression. In addition, MV infection of DCs may lead to immune modulation as previously demonstrated in ex vivo studies [41–43]. Levels of MV-infected cells varied substantially between individual animals. Whereas the viral load in PBMCs of animal #R3 resembled those detected in the other animals, the absolute percentages of EGFP-positive cells were much lower (Figure 3B). This animal also had the lowest percentages of EGFP-positive cells in lymphoid tissues (Figure 3C), and no EGFP expression was macroscopically detected in the skin. This variation in responses is likely related to the use of outbred non-SPF animals, and may reflect natural variations in the course of measles in humans, where children may develop either mild or more severe measles. The case-fatality rate of measles in humans is approximately 0.01% in industrialized countries, but may be up to 10% in developing countries [44]. The increased mortality may be explained by factors such as crowding, poor supportive care, and the high pressure of opportunistic infections [45], which are often considered to be the ultimate cause of measles-associated mortality [3]. However, opportunistic infections may also influence the pathogenesis of measles by another mechanism. Our data demonstrate that CD150+ lymphocytes are major targets for MV replication. Opportunistic pathogens may cause chronic immune activation [46], likely resulting in higher numbers of CD150+ lymphocytes in circulation and in lymphoid tissues. This suggests that chronic immune activation from high pathogen pressure may facilitate MV replication and spread, thus increasing the severity of MV infection. Apart from MV infection of lymphocytes and DCs, we also detected MV infection in the squamous stratified epithelium of tongue and buccal mucosa and in the ciliated epithelium of the trachea. In these tissues infection was associated with syncytium formation. Since epithelial cells do not express CD150, these observations suggest the possibility of non-CD150–mediated MV infection and spread. In previous in vitro studies it was shown that MV could infect human primary small airway epithelial cells or lung carcinoma cells via a CD150- and CD46-independent mechanism, resulting in the formation of syncytia [11,47]. It has been suggested that high local concentrations of virus would enable the virus to enter cells using low affinity receptors [9,31]. However, the existence of an additional high affinity MV receptor cannot be excluded [11,47]. On basis of our data we hypothesize that, upon natural MV infection, initial MV replication takes place in the tonsils. However, it remains unclear how the virus gains access to this organ. Given its high person-to-person transmissibility, the virus would need an easily accessible target cell in the upper respiratory or digestive tract that could “trap” the virus and subsequently transport it into lymphoid tissues. The observed substantial MV infection of DCs suggests that CD150+ DCs may fulfill this role, as has also been suggested for HIV-1 infection [48]. Notably, DC-SIGN was recently identified as a new attachment receptor for MV, which could enhance viral transmission to CD150+ lymphocytes [49]. Whether this process takes place at the respiratory mucosa, in the tonsillar crypts, or elsewhere will need to be determined in future studies focusing on earlier time points after MV infection. In conclusion, our data demonstrate that in non-human primates MV targets CD150+ lymphocytes and CD11c+ myeloid cells, and to a lesser extent epithelial cells. In contrast to previous studies in human measles patients, circulating monocytes in peripheral blood do not sustain productive MV infection in macaques. Although the experiments described here are based on a single recombinant MV strain, and important differences may exist between the course of MV infection in humans and macaques, these data warrant re-evaluation of the tropism of MV in humans. This non-human primate model provides new opportunities to address specific aspects of measles pathogenesis in vivo. Materials and Methods Study design. Three cynomolgus macaques (animals #C1–3) and three rhesus macaques (animals #R1–3) were infected by intra-tracheal inoculation with 104 cell culture infectious dose-50 of MV-IC323-EGFP diluted to a volume of 5 ml in phosphate buffered saline (PBS). The animals were juvenile (2–4 y), seronegative for measles as determined by virus neutralization, and were housed in negatively pressurized hepa-filtered BSL-3 isolator cages. The virus stock was grown in human B-lymphoblastic B-lymphocytes (BLCL) and tested negative for contamination with Mycoplasma species. The infections were performed in pairs, the first (#C1 and #R1) and third (#C3 and #R3) set of animals were euthanized on day 9 while the animals of the second set (#C2 and #R2) were euthanized on day 15. Necropsies were performed in a laminar flow biosafety cabinet. The study was approved by the animal ethics committee and performed according to Dutch guidelines for animal experimentation. Samples. Heparinized blood samples were collected at days 0, 3, 6, 9, 11, 13, and 15 after infection. Plasma was separated by centrifugation, heat inactivated (30 min 56 °C) and stored at −20 °C. PBMCs were isolated by density gradient centrifugation, resuspended in RPMI-1640 supplemented with antibiotics and heat-inactivated fetal bovine serum, counted, and used fresh for virus isolation (see below). BAL were collected on days 3, 6, 9, and 13 after infection, by intra-tracheal infusion of 10-ml PBS through a flexible catheter. Recovered BAL fluid was centrifuged, and BAL-cells were resuspended in culture medium with supplements as described above, counted and used fresh for virus isolation. MV detection and serology. MV was isolated in human BLCL using an infectious centre test as previously described [29,33]. Cytopathic changes were monitored by light and fluorescence microscopy after co-cultivation for 3–6 d, and results were expressed as numbers of infected cells per 106 total cells. In our hands, virus isolation from PBMC and BAL cells in BLCL proved to be slightly more sensitive than isolation in Vero cells expressing CD150 (unpublished data). Real-time RT-PCR was performed as described previously [50]. Fusion protein–specific serum IgM and IgG antibody levels were determined by FACS-measured immunofluorescence as described previously [51]. Macroscopic detection of EGFP fluorescence. For the purpose of detecting EGFP fluorescence inside a BSL-3 isolator cage or a laminar flow biosafety cabinet, a lamp was custom-made containing six 5-volt LEDs (Luxeon Lumileds, lambertian, cyan, peak emission 490–495 nm) mounted with D480/40 bandpass filters (Chroma) in a frame that allowed decontamination with 70% alcohol or fumigation with formaldehyde. Emitted fluorescence was visualized through the amber cover of a UV transilluminator (UVP) normally used for screening DNA gels. Photographs were made using a Nikon D80 digital SLR camera. Necropsies. Animals were euthanized by sedation with ketamine (20 mg/kg body weight) followed by exsanguination. Samples were collected both in 4% paraformaldehyde and in buffered formalin. A selection of samples was also collected in PBS for direct processing of fresh tissues or was snap-frozen in liquid nitrogen and stored at −80 °C. FACS analysis. Freshly isolated PBMCs were stained with four different combinations of subset-specific monoclonal antibodies cross-reactive with macaque cells. Staining 1: CD150PE (Pharmingen, clone A12) / CD3PerCP (Pharmingen, clone SP34–2) / CD8APC (DAKO, clone DK25); staining 2: CD150PE / HLA-DRPerCP (BD Biosciences, clone G46–6) / CD20APC (Beckman Coulter, clone B9E9); staining 3: CD150PE / CD14PerCP (Pharmingen, clone M5E2) / CD16AF647 (Pharmingen, clone 3G8); staining 4: CD45RAPE (Pharmingen, clone 5H9) / CD3PerCP / CD4APC (BD Biosciences, clone SK3). Fluorescence was detected in a FACSCalibur, obtaining approximately 500,000 events to allow detection of low-frequent EGFP+ subpopulations. Lymphoid tissues were minced, and single cell suspensions were prepared by using cell strainers with 100-μm pore size (BD Biosciences); these cells were directly used for FACS analysis. Skin cultures. Shaven skin sections were collected in PBS during necropsies and processed as previously described [34] with adaptations. Briefly, tissues were incubated overnight in trypsin (0.05%), after which dermis and epidermis were mechanically separated. The two tissues were subsequently cultured for 2 d in complete medium to allow DCs to migrate out of the tissue. The single cell suspension that remained in the medium was used for FACS analysis. Preparation of tissue samples for vibratome sectioning. 4% (w/v) paraformaldehyde-fixed tissue samples were immersed in 0.2 M sodium cacodylate buffer (pH 7.2) for 20 min and embedded in 5% (w/v) agarose (Type VII low gelling temperature, Sigma) in PBS. A vibratome (Leica Microsystems) was used to cut serial 100-μm sections into 0.2 M TRIS buffered saline. A number of tissue sections were counterstained by incubation for 60 s in propidium iodide. Sections were mounted in Eukitt mounting medium (Electron Microscopy Sciences) onto glass slides and cover slips. Immunocytochemical staining of 100-μm vibratome-cut tissue slices. Vibratome-cut tissue sections (100 μm) were permeabilized in PBS with 0.2% (v/v) Triton X-100 (TX-100) for 30 min at room temperature to facilitate the dissemination of primary and secondary antibodies through the tissue slice. Sections were incubated overnight at 4 °C in an appropriate primary antibody diluted in PBS with 0.1% (v/v) TX-100 and 1% (w/v) bovine serum albumin (BSA). Monoclonal antibodies to CD20 (1:100, DAKO) and CD3 (1:100, DAKO) were used to detect B- and T-lymphocytes, respectively. CAM 5.2 was used to detect cells of an epithelial origin. After incubation in primary antibodies, tissue sections were rinsed three times in PBS with 0.1% (v/v) TX-100. Sections were incubated for 2 h at room temperature in goat anti-mouse Alexa 568 or goat anti-mouse Alexa 647 (Molecular Probes) diluted in PBS with 0.1% TX-100 (v/v) and 1% (w/v) BSA. Sections were rinsed several times in PBS with 0.1% (v/v) TX-100 and mounted as described previously. Imaging of vibratome- and microtome-cut brain slices. Photomicrographs of vibratome-cut tissue slices were collected by confocal scanning laser microscopy. Tissue slices were viewed using an upright DM-IRBE fluorescence microscope (Leica) and appropriate fields selected. A Leica TCS/NT confocal microscope equipped with a krypton-argon laser as the source for the ion beam was used to detect EGFP and appropriate secondary antibodies as described previously [52]. Selected histological or immunocytochemically stained sections were digitally scanned using an Aperio Scanscope T3 with a ×40 objective. From these scans selected images could be viewed or displayed at a range of magnifications. Immunohistochemical and immunofluorescence analysis. All formalin-fixed sections were deparaffinized, and antigen retrieval was performed in a pressure cooker at full power for 2 min in 0.01M TRIS-EDTA buffer (pH 6.0). MV-infected cells were detected using a polyclonal antibody to EGFP (Invitrogen). Sections were incubated in primary antibody overnight at 4 °C, and specific antibody-antigen binding sites were detected using an Envision-Peroxidase system with DAB (DAKO) as substrate. Single and dual labeling immunofluorescence was performed using anti-EGFP and monoclonal antibodies to the DC marker CD11c (Novacastra), the macrophage cell marker Mac387 (Abcam), and the epithelial cell–specific marker CAM 5.2 (Becton-Dickinson). Antigen binding sites were detected with either goat anti-mouse or anti-rabbit Alexa 488 or 568 (Molecular Probes). In some instances, sections were counterstained with either propidium iodide (Sigma) or DAPI mounting medium (Vector). Supporting Information Figure S1 Expression of CD150 on T-Lymphocytes, B-Lymphocytes, and Monocytes PBMCs of animal #R1 collected 9 d after infection with MV-IC323-EGFP were stained with anti-CD150PE, anti-CD3PerCP, and anti-CD20APC. As an isotype control for the anti-CD150 monoclonal, a second sample was stained with mouse IgG1kPE, anti-CD3PerCP, and anti-CD20APC. Three PBMC subpopulations were gated (A): T-lymphocytes (CD3+CD20−, red), B-lymphocytes (CD3−CD20+, green), and non-T/non-B cells (blue). The percentages of the total PBMCs and the scatter plots (B) of these subpopulations suggested that the non-T/non-B cells mainly consisted of monocytes. The histograms showed that CD150 was expressed by T-lymphocytes (C) and B-lymphocytes (D), but not by the non-T/non-B cell population (E). Isotype controls are shown as dotted lines. (2.7 MB TIF) Click here for additional data file. Figure S2 Percentages of EGFP+ Cells in Lymphoid Tissue–Derived Lymphocytes EGFP+ lymphocyte subpopulations were detected in single cell suspensions of a mandibular lymph node (A–F) or gut-associated lymphoid tissue (G–L) of macaque #C3, collected 9 d after infection with MV-IC323-EGFP. EGFP-expression in CD3+CD8− T-lymphocytes is shown in green (D and J), after gating on region R1 (A and G), and R2 (B and H); EGFP-expression in CD3+CD8+ T-lymphocytes is shown in purple (E and K), after gating on region R1 (A and G), and R3 (B and H); EGFP-expression in MHC-class II+CD20+ B-lymphocytes is shown in blue (F and L), after gating on region R1 (A and G), and R4 (C and I). (2.9 MB TIF) Click here for additional data file. Figure S3 EGFP+ Cells in Tissues of the Oral Cavity Samples were collected from cynomolgus macaque #C3 on day 9 after infection with MV-IC323-EGFP. Subsequent panels represent serial sections of the same tissue, of which the first shows fluorescence (EGFP-fluorescence in green, TO-PRO counter staining in red or blue) and the second the corresponding hematoxylin and eosin staining. EGFP+ cells were detected in the lamina propria/submucosa of the tongue (A), seromucous glands of the tongue (C), and buccal wall (E) localized to aggregates of mononuclear cells in these tissues (B, D, and F). Fluorescent cells in the keratinized epithelium of the tongue (G) were detected in association with intercellular vacuolization, indicative for epithelial necrosis (H). (9.7 MB TIF) Click here for additional data file. Figure S4 Identification of MV-Infected Cells in Tissue Sections MV-infected cells in paraformaldehyde-fixed vibratome-cut tissue sections (A and B) or formalin-fixed microtome-cut tissue sections (C–E) from macaque #R1 on day 9 after infection with MV-IC323-EGFP. (A) Identification of MV-infected cells (green) in the spleen that express the B cell marker CD20 (blue). (B) Identification of MV-infected cells (green) in the spleen that express the T cell marker CD3 (blue). (C) Identification of MV-infected cells (green) in the spleen that express the DC marker CD11c (blue). Cell nuclei are counterstained with propidium iodide (red). (D) No co-localization between MV-infected cells (red) in the tracheo-bronchial lymph node and cells expressing the macrophage cell marker Mac287 (green). (E) MV-infected cells (green) in the bronchus expressing the epithelial cell marker cytokeratin (red). Cell nuclei are counterstained with DAPI (blue). (2.6 MB TIF) Click here for additional data file. Table S1 PBMC Lymphocyte Subpopulations Percentages of CD3+CD4+, CD3+CD8+, CD20+, and CD14+ cells in PBMCs collected on different sampling points and percentages of EGFP+ cells per PBMC subpopulation. (64 KB DOC) Click here for additional data file. Table S2 PBMC T Cell Subpopulations Percentages of CD3+CD4+CD45RA−, CD3+CD4+CD45RA+, CD3+CD8+CD45RA−, and CD3+CD8+CD45RA+ cells in PBMCs collected on different sampling points and percentages of EGFP+ cells per subpopulation. In addition, the ratio between the percentage of EGFP+ cells in CD45RA− versus CD45RA+ cells is shown, indicating preferential MV-infection of CD45RA− T cells (i.e., T cells with a memory phenotype). (66 KB DOC) Click here for additional data file. Table S3 Organ Suspension Lymphocyte Subpopulations Percentages of CD3+ and CD20+ cells in single cell suspensions prepared of different lymphoid tissues collected from animals R1, C1, R3, and C3 and the percentages of EGFP+ cells per subpopulation. (36 KB DOC) Click here for additional data file. Accession Numbers The GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for proteins discussed in this manuscript are CD150 (NP_003028) and EGFP (AAB02574).

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Early Target Cells of Measles Virus after Aerosol Infection of Non-Human Primates

Ken Lemon, Rory D. de Vries, Annelies W Mesman … (2011)

Introduction Measles virus (MV) is one of the most contagious human viruses known, and is transmitted via aerosols or by direct contact with contaminated respiratory secretions. Clinical signs appear approximately two weeks after infection and include fever, rash, cough, coryza and conjunctivitis [1]. Measles is associated with a transient but profound immunosuppression, resulting in increased susceptibility to opportunistic infections. While significant progress has recently been made in global control programs, 164,000 deaths were still attributed to measles in 2008 [2]. It has been well established that MV infects cells via receptor-dependent fusion of the virion at the plasma membrane [3]. Two cellular receptors for MV have been identified. In 1993 the membrane cofactor protein CD46, expressed by virtually all nucleated human cells, was the first protein to be identified as MV receptor [4], [5]. However, it soon became evident that only vaccine and laboratory-adapted MV strains were able to utilize this molecule as an entry receptor [6]. Signaling lymphocyte activation molecule (SLAM or CD150), a membrane glycoprotein expressed on subsets of immune cells, was identified as the receptor for wild-type MV strains in 2000 [7], [8]. It is now generally accepted that pathogenic wild-type MV strains use CD150 as high affinity cellular receptor, whereas vaccine and laboratory-adapted strains can use either CD46 or CD150. Distribution of CD150 explains most, but not all aspects of measles pathogenesis and it may be possible that the utilization of additional low-affinity cellular receptors explains how wild-type viruses enter CD150− epithelial or neuronal cells [9]–[11]. Previously, we successfully infected macaques with a recombinant MV based on the pathogenic IC323 strain [12] that expresses enhanced green fluorescent protein (EGFP) from a promoter-proximal additional transcription unit (ATU); this wild-type recombinant virus (rMVIC323EGFP) uses CD150 but not CD46 as a cellular entry receptor in vitro [13]. In macaques, rMVIC323EGFP proved to be virulent and CD150-expressing lymphocytes and dendritic cells (DC) were identified as the predominant target cells for MV replication [14]. In a more recent study, macaques were infected with a rMVIC323 that inefficiently binds CD150, showing that this virus was attenuated and indicating that CD150-mediated entry is indeed essential for MV to be fully virulent in vivo [15]. In vitro studies have demonstrated that at a high multiplicity of infection (MOI) wild-type MV can infect cells that do not express CD150, although this process is inefficient and usually does not result in cell-to-cell spread or virus release [13]. However, a number of CD150− cell types of epithelial or neuronal origin have been identified in which wild-type MV infection at a low MOI results in cytopathic effects and virus release [9]–[11]. It is thought that entry into these CD150− cells is mediated by an unidentified cellular receptor for MV, which is often referred to as the epithelial cell receptor (epR). Even though the receptor has not been identified, epR-binding sites on the MV hemagglutinin protein have been mapped [9], [10], and the receptor appears to be a protein expressed on the basolateral side of polarized epithelial cells associated with tight junctions [9], [16], [17]. In human tissues, cells within the epithelium have historically been shown to be infected by wild-type MV. More recently, epithelial cell infection has been demonstrated with dual immunofluorescence, using the recombinant rMVIC323EGFP strain in non-human primates [14], [16]. However, the limited epithelial cell infection observed predominantly occurs in the presence of substantial infection of lymphoid and myeloid cells, which is consistent with the differential expression of CD150 on these cell types. It has been postulated that MV infection starts from the luminal side of the upper respiratory epithelium [1]. However, there is no direct evidence for initial MV infection and replication in epithelial cells. Furthermore, the absence of CD150 or epR on the apical side of these cells makes it highly unlikely that respiratory epithelial cells are an initial target for MV. However, the respiratory epithelium contains many other cell types besides epithelial cells. Several research groups have postulated new strategies for MV to enter a host, namely via direct initial infection of CD150+ immune cells present throughout the respiratory tract and interdigitated within the epithelium [14], [18]. In 2006, the C-type lectin DC-SIGN was identified as an attachment receptor for MV [19]. In vitro DC-SIGN expressing DC could efficiently capture and transmit MV to CD4+ and CD8+ T-lymphocytes expressing CD150. This suggests a potential role for the DC as an initial target cell in vivo, where DC capture the virus from the luminal side of the respiratory tract and, with or without productive infection, transport the virus to draining lymph nodes (LN) containing many CD150+ cells, thereby initiating the typical systemic infection [19], [20]. In the present study, we have generated a rMV based on a genotype B3 wild-type MV isolate from Khartoum, Sudan. The open reading frame (ORF) encoding EGFP was introduced into the virus genome in the promoter-proximal position within an ATU using a similar approach as previously described for rMVIC323EGFP [12]. In an attempt to identify the early target cells of MV in non-human primates, macaques were infected with rMVKSEGFP and sacrificed 2, 3, 4 or 5 days post-infection (d.p.i). Infections were performed by inhalation of a high dose of virus formulated as small-particle size nebulized aerosol, thus exposing the entire upper and lower respiratory tract to the virus. MV-infected EGFP+ cells were identified at all four time points, albeit at low levels 2 and 3 d.p.i. Infection is initiated in large mononuclear cells in the alveolar lumen, most likely either AM or DC. Results Generation and characterization of rMVKSEGFP MVi/Khartoum.SUD/34.97/2 (MVKS) was isolated from a measles case in Khartoum, Sudan in 1997 [21]–[23]. This virus was previously shown to be highly virulent in macaques [24]. A consensus sequence of the complete viral genome was derived de novo, including the 3′ and 5′ ends which were sequenced following rapid amplification of cDNA ends (RACE), and a full-length anti-genomic plasmid (pMVKS) was constructed (figure 1A). The plasmid was modified by the addition of an ATU encoding EGFP at the promoter proximal position (figure 1A) to generate pMVKSEGFP. Recombinant viruses rMVKS and rMVKSEGFP were recovered following transfection of Vero-SLAM cells, and were passaged exclusively on Epstein-Barr virus-transformed human B-lymphoblastic cells (B-LCL) (figure 1B). Presence of a silent point mutation in the MV nucleocapsid (N) ORF (T1245C) acts as a genetic tag and its presence was confirmed by RT-PCR and sequencing of the ORF (data not shown). Observation of rMVKSEGFP by fluorescence microscopy revealed a high level of EGFP expression associated with single infected cells and multinucleated syncytia. Growth analysis of MVKS, rMVKS, rMVKSEGFP and rMVIC323EGFP in B-LCL over a period of 4 days demonstrated that the viruses reached equivalent titers (figure 1C). 10.1371/journal.ppat.1001263.g001 Figure 1 Generation and growth of rMVKSEGFP. (A) Plasmids generated after RT-PCR, cloning and sequencing of MV RNA isolated from MVKS-infected PBMC. pMVKS is a full-length plasmid containing the complete antigenome of MVKS and pMVKSEGFP was modified by the insertion of an ATU at the promoter proximal position containing the ORF encoding EGFP. (B) rMVKS and rMVKSEGFP were rescued from Vero-SLAM cells and passaged in B-LCL. Fluorescence microscopy confirmed high levels of EGFP expression in rMVKSEGFP infected cells. (C) Growth curves of MVKS, rMVKS, rMVKSEGFP and rMVIC323EGFP in human B-LCL. Virus was harvested 24, 48, 72 and 96 hours post infection, CCID50 was determined in an endpoint titration test. Measurements shown are averages of triplicates ± SD. Key: h.p.i.: hours post infection. Early rMVKSEGFP replication in the respiratory tract Four groups of three cynomolgus macaques were infected with rMVKSEGFP via the aerosol route as described previously [25]. Throat and nose swabs were collected daily and virus isolations were performed to determine the MV load in these clinical samples. Necropsies were performed 2, 3, 4 and 5 d.p.i. BAL cells were collected for virus isolation and the entire respiratory tract was screened macroscopically and microscopically for fluorescence by live cell UV fluorescence and confocal scanning laser microscopy. At 2 and 3 d.p.i., no macroscopic fluorescence was detectable, probably because of low levels of viral replication. No virus could be isolated from the nose, whereas from throat swabs virus was only isolated at 4 (2/6 animals) and 5 (3/3 animals) d.p.i. (figure 2A). However, MV was isolated from BAL cells as early as 2 d.p.i. (2/3 animals) and by 3, 4 and 5 d.p.i. virus was isolated from BAL cells of all animals with virus loads increasing over time (figure 2A). Microscopic detection of MV replication in freshly collected tissues of the respiratory tract proved that as early as 2 d.p.i. the virus was consistently present in the lungs of all animals (figure 2B and supporting videos S1, S2 and S3). On 2 and 3 d.p.i. single infected mononuclear cells with the appearance, size and typical tissue distribution of AM and/or DC were detected attached to the alveolar wall or inside the alveolar lumen. At these time points no MV-infected epithelial cells were detected in the lungs of any animal, either phenotypically, following screening of lung slices for EGFP-positive cells or histologically, by dual staining of MV proteins and cytokeratin. MV-infected cells could not be detected in the nasal septum, nasal concha, nasal lining, trachea or primary bronchus 2 and 3 d.p.i. By 4 d.p.i. a fluorescent signal was detected in the nasal septum of a single animal, and by 5 d.p.i. the nose, trachea and primary bronchus were consistently positive (table 1). 10.1371/journal.ppat.1001263.g002 Figure 2 Early rMVKSEGFP replication in the respiratory tract. (A) Virus isolation performed from nose and throat swabs (left two panels), and from BAL cells (right panel). Each symbol represents an individual animal, bars indicate the geometric mean. Key: VI: virus isolation; d.p.i.: days post infection. (B) Live cell confocal microscopy performed on agarose-inflated lung slices from animals on 2 and 3 d.p.i. EGFP+ cells are shown in green, DAPI was used to counter stain nuclei (blue). Three images were collected, labeled i, ii and iii. Panels iia and iib show infected cells in one image from different orientations. Matching 3D-videos for (Bi, Bii and Biii) are available as supporting data. 10.1371/journal.ppat.1001263.t001 Table 1 Dissemination of MV in tissues during early stage of infection. Days post-infection (d.p.i.) Tissue 2 3 4 5 Lung 3 3 3 3 Tracheobronchial LN1 1 3 3 3 PBMC2 - 4 - 3 3 Adenoid 1 - 3 3 Retropharyngeal LN - - 1 3 Mandibular LN - - - 3 GALT3 - - 1 3 Spleen - - 1 2 Tonsil - - 1 1 Thymus - - 1 1 Nasal septum - - 1 1 Nasal concha - - - 2 Trachea - - - 2 Inguinal LN - - - 2 Axillary LN - - - 1 Mesenteric LN - - - 1 Numbers indicate the number of macaques with EGFP+ cells in this tissue (n = 3). 1 LN: lymph node. 2 PBMC: peripheral blood mononuclear cells. 3 GALT: gut-associated lymphoid tissue. 4 -: no EGFP+ cells detected. Systemic rMVKSEGFP replication Virus isolations were performed from peripheral blood mononuclear cells (PBMC) and single cell suspensions of four lymphoid organs (retropharyngeal LN, mandibular LN, tonsil and tracheo-bronchial LN). RNA isolations and virus detection by RT-PCR were performed on the axillary LN and tracheo-bronchial LN, which drain the arm and the lungs, respectively. Furthermore, PBMC and all lymphoid organs were analyzed directly by flow cytometry and UV microscopy for fluorescence. Viremia was detected in all animals on 4 and 5 d.p.i. but in none of the animals sampled 2 and 3 d.p.i. (figure 3A, left panel, and table 1). Virus was not isolated from any lymphoid organ 2 d.p.i. However, by 3 d.p.i. virus was isolated from the tracheo-bronchial LN of all animals (data not shown). RT-PCR and flow cytometry confirmed the early presence of MV in the tracheo-bronchial LN, but not in more distally located LN, for example the axillary and retropharyngeal LN (figure 3A, right panel and 3B). Flow cytometry confirmed that the number of EGFP+ cells increased over time. Virus was detected by almost all methods in multiple lymphoid organs 4 and 5 d.p.i. by which time the MV was spreading systemically (table 1). Macroscopic detection of EGFP proved possible only 4 and 5 d.p.i (figure 3C). The tonsil of a single animal was positive 4 d.p.i . By 5 d.p.i. MV was detected macroscopically at multiple locations (adenoids, tonsil, retropharyngeal LN, trachea, tongue, tracheo-bronchial LN) in all animals indicating widespread dissemination. Phenotyping of the MV-infected cells in PBMC or single cell suspensions of lymphoid tissues collected on 4 and 5 d.p.i. showed that these were predominantly T- or B-lymphocytes (data not shown). 10.1371/journal.ppat.1001263.g003 Figure 3 Systemic rMVKSEGFP replication. (A) MV load in PBMC and LN. The left panel shows virus isolations performed from PBMC, each symbol represents an individual animal, bars indicate the geometric means. The right panel shows the presence of MV genome in the axillary LN (crosshairs, geometric mean in blue) and in the tracheobronchial LN (triangles, geometric mean in red). Key: VI: virus isolation; RT-PCR: real-time reverse transcriptase PCR; d.p.i.: days post infection. (B) Detection of EGFP+ cells by flow cytometry from the retropharyngeal LN (left) and the tracheobronchial LN (right) on 2, 3, 4 and 5 d.p.i. Data are shown as dot plots of FL-1 (EGFP) versus FL-2 (empty channel), generated with BD FACSDiva software. In these plots autofluorescent cells usually appear on a diagonal line as they cause comparable signals in both channels. The EGFP-positive events were gated as indicated by the curvilinear line. Data of a representative animal are shown on each time point. Numbers of EGFP+ cells per million total cells are shown in each plot. (C) Representative example of macroscopic EGFP detection at 5 d.p.i. Arrow indicates the infected tonsillar tissue expressing EGFP. Key: Tg: tongue; Tn: tonsil; L: larynx. Phenotyping of early MV-infected cells in the lungs Early after infection MV was consistently present in the lungs. In order to characterize the early target cells, live agarose-inflated lung slices containing EGFP+ cells were formalin-fixed and paraffin-embedded. Serial sections were cut and used for immunohistochemistry and indirect immunofluorescence to determine the precise location of MV infection, identify the phenotype of the infected cells and gain an understanding of how such “seeding” of MV infection in the lungs might lead to the establishment of systemic infection. At 3 d.p.i., two foci of infection were identified in paraffin-embedded lung sections of one of the three infected animals, interestingly both in BALT (figure 4A and supporting online, annotated immunohistochemical and H&E pathological scans figure S3). These BALT structures were lined by a cytokeratin-positive epithelial cell layer and contained numerous immune cells (figure 4B), that stained positive with CD11c for AM or DC, Mac387 for macrophages, CD20 for B-lymphocytes and/or CD3 for T-lymphocytes. Blood vessels, identified using the endothelial cell-specific marker CD31, were always present in BALT structures irrespective of the presence or absence of MV-infected cells. Since there were a limited number of foci of infection identified this early after infection, we were unable to quantify the levels of infection in different cell types. However, MV-infected B-lymphocytes, T-lymphocytes and DC could readily be detected by specific dual labeling at higher magnifications (figure 4C and Figure S2). Multiple foci of MV-infected cells were detected 4 and 5 d.p.i. in the alveolar lumina and walls of all animals and the majority of these infected cells were CD11c+ (Figure S1), consistent with what had been observed previously in macaques euthanized 7 d.p.i. [25]. 10.1371/journal.ppat.1001263.g004 Figure 4 Characterization of MV infection in BALT structures. (A) H&E staining on lung slice from an animal euthanized on 3 d.p.i.. The number of EGFP+ foci was extremely low, the boxed area (Ai) is a BALT which was the only area on the section where EGFP+ cells were present. (Aii) shows a serial section stained with anti-GFP (black) to detect the presence of virus (see also Figure S3, annotated immunohistochemical and H&E annotated pathology scans). (B) Indirect dual immunofluorescence of the infected BALT structure, showing the presence of T-lymphocytes (CD3), DC or macrophages (CD11c, mac387) and B-lymphocytes (CD20) within the BALT. The BALT is lined by a layer of cytokeratin-positive epithelial cells, and has a blood vessel with CD31-positive endothelium running through it transversely. (C) Higher magnifications of dual immunofluorescence within the BALT indicates the presence of MV-infected T-lymphocytes (CD3), DC or macrophages (CD11c) and B-lymphocytes (CD20), Double positive cells are indicated by arrows. In panel (B) and (C), EGFP+ cells are shown in green, cell-type specific staining is shown in red. DAPI was used to counter stain nuclei in blue. (D) Dual immunofluorescence performed on uninfected BALT region. Dual labelling with cytokeratin (green) and CD3, CD11c or CD20 (red) showed that T-lymphocytes, B-lymphocytes and DC or macrophages are present in very close proximity or in direct contact with the alveolar or bronchiolar lumen (asterisks). Single colour images for (C) are available as supporting data (figure S2). Analysis of BALT structures in the lungs of non-infected macaques indicated that even though cytokeratin-positive epithelial cells lined these structures, cells of lymphoid and myeloid origin were present both within the epithelium and in direct contact with the adjacent lumen. Indirect immunofluorescence identified CD11c+, CD3+ and CD20+ cells in direct contact with the lumen of alveoli, bronchioles or bronchi (figure 4D, asterisks). This was also confirmed in virus-negative BALT structures from uninfected animals (supporting online, annotated immunohistochemical and H&E pathological scans S3). Early MV infection in lymphoid tissue is frequently associated with the presence of blood vessels Within the infected BALT structures, MV-infected cells were readily detected in direct contact with or in close proximity to the endothelial wall of blood vessels. A similar distribution was observed in the tracheo-bronchial LN, tonsils and adenoids on 4 and 5 d.p.i., in which MV-infected cells were mostly detected in close proximity to venules (figure 5). On rare occasions, MV-infected cells with the morphology of dendritic cells could be seen migrating through the endothelium (figure 5, right panel and figure S2). 10.1371/journal.ppat.1001263.g005 Figure 5 Dissemination of MV into the lymphoid organs via blood vessels. (A, B) H&E staining (left panel) and EGFP staining (right panel) on serial sections of tonsils at 4 d.p.i. (A) and 5 d.p.i. (B). Asterisk denote the proximity of venules to MV-infected cells. (C) Dual labeling of EGFP (green) and the endothelial marker CD31 (red) performed on the tonsils from animals euthanized 5 d.p.i. The left panel shows MV-infected cells in close proximity to CD31+ endothelial cells of venules (arrows), the right panel shows an MV-infected cell migrating through the wall of the venule (arrow). DAPI was used to counter stain nuclei in blue. Single color images for (C) are available as supporting data (figure S2). Discussion In the present study, we have generated and utilized a virulent rMV strain expressing EGFP, based on a wild-type genotype B3 MV isolate from Khartoum, Sudan. In growth curves in human B-LCL recombinant strains rMVKS and rMVKSEGFP reached equivalent titers, which were slightly higher than those reached by rMVIC323EGFP. These data suggest that the addition of EGFP into the genome had no detectable effect on virus fitness as determined in vitro. Pathogenesis studies performed with molecular clones of wild-type MV have thus far exclusively been based on the Japanese strain IC323 [12]. Development of a second recombinant wild-type MV serves to complement ongoing studies of MV pathogenesis and ensures that observations are not strain-specific. Expression of EGFP from a promoter-proximal ATU leads to significant amounts of EGFP and, interestingly in the case of MV, has no or only a limited effect on the virulence in vivo. This was not the case for other morbilliviruses, for example canine distemper virus [26]. The new recombinant virus rMVKSEGFP described here also proved to be virulent in cotton rats [Lemon K, manuscript in preparation], and allowed sensitive microscopic detection of the virus in vitro, ex vivo and in vivo. Macaques were infected with a high dose of rMVKSEGFP via the aerosol route and early necropsies were performed to identify the initial target organs, tissues and cells. The nebulizer used was similar to the type that is used in ongoing clinical trials of measles aerosol vaccination, organized in India by the World Health Organization. The nebulizer produced a volume median diameter (VMD) of 4–6 µm, allowing the inoculum to deposit both in the upper respiratory and lower respiratory tract, and reach the alveolar lumina. A high infectious dose (106 CCID50) was nebulized to ensure that all potential early target cells for MV infection in the respiratory tract were exposed to the virus. Such an approach is important in any study which aims to identify key cells targeted by a respiratory virus as it ensures the pathogen can access the broadest range of cell types and associated tissues throughout the respiratory tract. However, it is important to acknowledge our limited understanding of how MV is transmitted from human to human: in our current study we have infected animals with cell-free virus but transmission between humans could also involve excretion of cell-associated virus. Studies which examine the pathological consequences of MV infection in animals at later time points would greatly facilitate our understanding of virus transmission, both for MV and other respiratory viruses. The techniques and bank of tissues collected in this and other studies could be used to shed light on person to person transmission. Our data strongly suggest the following sequence of events. At early time points (2 and 3 d.p.i.), MV infected large mononuclear cells with the phenotype and location of AM or DC. Targeting of these cells was followed by the establishment of localized MV replication in close proximity, lymphoid aggregates in the lungs (BALT). These BALT structures contained a large number of B-cells and memory CD4+ T-cells [27], both cell types previously described as preferential targets for MV in lymphoid tissue at later time points [14]. Seeding and amplification of the infection in these microenvironments, which are well suited to a lymphotropic virus such as MV, is likely to be critical in the establishment of the infection. From the lungs, MV was transported by infected cells to the draining tracheo-bronchial LN. After localized replication in the lungs and increased replication in the tracheo-bronchial LN, MV spread systemically through viremia to the majority of lymphoid organs by 4 or 5 d.p.i. MV-infected cells were always detected in close proximity to venules within lymphoid organs, suggesting that these were involved in spreading the virus. It has been stated that MV initially targets the epithelium of the upper respiratory tract to establish infection [1]. However, all known wild-type MV receptors are absent on the luminal side of respiratory epithelial cells, making their initial infection by MV highly unlikely. Other potential entry strategies by which MV might enter a susceptible host have been described in the literature. For example, the Trojan horse strategy that has been described for HIV-1 has also been considered for MV [20]. DC could capture MV from the respiratory tract using dendrites protruding through the epithelium and transmit virus to CD4+ and CD8+ T-lymphocytes, leading to infection. In vivo in the macaque model, infection of DC has indeed been described in submucosal tissues [14]. Furthermore, in the present study we demonstrate that MV was detectable in the lungs 2 d.p.i., since MV-infected cells could be both isolated from BAL and imaged in situ by live cell confocal scanning laser microscopy. These data confirm that large mononuclear cells present in the alveolar lumen or lining the alveolar epithelium, most likely AM and/or DC, are among the earliest cells infected by MV in the macaque model. Even though the IfnarKO-SLAMGe mouse model does not recapitulate the whole spectrum of measles pathogenesis, initial infection of AM and DC was also shown in this model by flow cytometry [28]. We show here that, in the respiratory tract, BALT structures were the only MV-infected tissues at 3 d.p.i. BALT is normally lined by a continuous epithelial layer, making direct entry of MV unlikely. However, the epithelium of the BALT has previously been described to be a flattened respiratory epithelium, with common influx and efflux of lymphocytes, AM and DC [29]. Furthermore, the epithelium of BALT of many mammalian species contains M-cells [30], cells that are specialized for antigen uptake. In mouse models, it has been shown that BALT plays a role in the uptake of multiple bacteria (Pseudomonas aeruginosa [31], Mycobacterium tuberculosis [32]). Reoviruses have also been described to be taken up by M-cells, with subsequent spread to the regional lymph nodes [33]. In this study we did not observe antigen uptake by M-cells. Instead, we observed infected cells resembling AM or DC at 2 d.p.i. and suggest that they transported MV through the BALT epithelium into the underlying lymphoid tissue. An alternative route for MV to enter a susceptible host would be via direct infection of CD150+ cells in Waldeyer's tonsillar ring, consisting of tonsils and adenoids. Tonsils and adenoids are lined by CD150− epithelial cells, but at sites of damage or in tonsillar crypts direct infection of CD150+ cells at the luminal surface might be possible. In our model, tonsils and adenoids were directly exposed to a high dose of nebulized virus, but a consistent level of infection was only detected 4 and 5 d.p.i., when the infection already was systemic. Only one out of six animals had MV-infected cells in the adenoid 2 d.p.i. and no infection was observed in the tonsils 2 and 3 d.p.i. These data suggest that MV cannot easily penetrate the epithelial layer to initiate MV infection of CD150+ cells in tonsillar tissue of the Waldeyer's ring. Following the initial infection of cells in the lung, the draining TB-LN was the first lymphoid organ being consistently MV-positive 3 d.p.i. Since this LN drains the lungs, it is most likely that MV-infected cells are transported through lymphatic vessels to reach the TB-LN. In the BALT and TB-LN, MV-infected cells were often detected in close proximity of venules. We hypothesize that MV-infected cells are transported through these venules into the bloodstream, from where they reach the spleen and other lymphoid organs, initiating the systemic infection as observed 4 and 5 d.p.i. The proximity of MV-infected cells to venules in the tonsils and adenoids 4 and 5 d.p.i. substantiates this hypothesis. In conclusion, aerosol exposure of the entire respiratory tract of macaques to a high dose of infectious MV leads to initial infection of mononuclear cells in the alveoli (2 d.p.i.), followed by MV replication in BALT (3 d.p.i.). Phenotypically and based on location it is likely that the initial target cells in the alveoli are AM or DC. In BALT, T-lymphocytes, B-lymphocytes and DCs are all productively infected. CD11c+ cells are the major target cell population in the lungs 4 and 5 d.p.i. indicating an important role for AM and/or DC early in establishing the infection. Materials and Methods Ethics statement Animals were housed and experiments were conducted in strict compliance with European guidelines (EU directive on animal testing 86/609/EEC) and Dutch legislation (Experiments on Animals Act, 1997). The protocol was approved by the independent animal experimentation ethical review committee DCC in Driebergen, the Netherlands (Erasmus MC permit number EUR1664). Animal welfare was observed on a daily basis, and all animal handling was performed under light anesthesia using a mixture of ketamine and medetomidine to minimize animal suffering. After handling atipamezole was administered to antagonize the effect of medetomidine. Generation of wild-type recombinant MV expressing EGFP rMVKSEGFP is based on a wild-type genotype B3 virus isolated from PBMC collected in 1997 from a severe measles case in Khartoum, Sudan [21]. The clinical isolate (MVKS) was passaged exclusively in CD150+ human B-LCL and was previously shown to be highly pathogenic in macaques [24]. Total RNA was isolated from B-LCL infected with MVKS and the complete consensus genomic sequence determined following RT-PCR (GenBank accession number HM439386). The sequences of the genomic termini were confirmed by 5′ RACE. A full-length cDNA which expressed the MVKS anti-genome (pMVKS) was constructed based on a modified pBluescript vector [34]. A single silent mutation was introduced into the N ORF (T1245C) to act as a genetic tag to distinguish recombinant virus from the clinical isolate. The full-length plasmid was modified further by the introduction of an ATU expressing EGFP at the promoter proximal position to generate pMVKSEGFP. Plasmid sequences are available on request. Recombinant viruses were recovered from MVA-T7-infected Vero-SLAM cells transfected with the full-length plasmids along with plasmids expressing MV N, P and L. Virus stocks were grown in B-LCL and tested negative for contamination with Mycoplasma species. Virus titers were determined by endpoint titration in Vero-SLAM cells, and expressed in 50% cell culture infectious dose (CCID50). Virus fitness of MVKS, rMVKS, rMVKSEGFP and rMVIC323EGFP was compared in a growth curve. Human B-LCL were infected in triplicate with MVKS, rMVKS, rMVKSEGFP or rMVIC323EGFP in 24-wells plates at MOI 0.1. At 24, 48, 72 and 96 hours post infection plates were freeze-thawed at −80°C, and cells and supernatant fluids were harvested. After sonification and clarification, the amounts of cell-free virus at different time-points were determined by endpoint titration in Vero-CD150 cells using ten-fold dilutions and testing eight wells per dilution, and expressed in CCID50. Early target cell animal study Twelve juvenile, MV-seronegative cynomolgus macaques (Macaca fascicularis) were housed in negatively pressurized, HEPA-filtered BSL-3 isolator cages. Animals were infected with rMVKSEGFP by aerosol inhalation using a pediatric face mask (ComfortSeal silicone mask assembly, small, Monaghan Medical Corp., Plattsburgh NY). Aerosol was generated using the Aerogen Aeroneb Lab nebulizer with an OnQ aerosol generator (kind gift of Dr. J. Fink, Aerogen) as previously described [25]. This nebulizer generates a small particle size aerosol (VMD 4–6 µm), which is deposited on epithelia throughout the entire respiratory tract upon inhalation [35]. A total dose of 106 CCID50 was nebulized, but we previously found that a substantial part of nebulized virus is lost due to inactivation during nebulization, condensation in the nebulizer tubing or face mask, deposition on the skin of the animals or deposition in the mouth followed by swallowing. We therefore estimated that the delivered dose was approximately 105 CCID50, of which based on previous studies approximately 10% is expected to reach epithelia in the lungs [35]. Animals were euthanized on 2, 3, 4 or 5 d.p.i. (n = 3 per time point). Necropsy Animals were euthanized by sedation with ketamine (20 mg/kg body weight) followed by exsanguination. Macroscopic detection of EGFP was performed at necropsy as described previously [14]. Briefly, fluorescence was detected with a custom-made lamp containing 6 LEDs (peak emission 490–495nm); emitted fluorescence was detected through an amber cover of a UV transilluminator used for screening DNA gels. Photographs were made using a Nikon D80 SLR camera. Organs were collected in PBS, directly processed and screened for presence of EGFP by UV microscopy. From here, EGFP+ samples were transferred to 4% (w/v) paraformaldehyde in PBS (to preserve EGFP autofluorescence) or to 10% neutral buffered formalin. The left lung lobe was inflated as described previously [Lemon K, manuscript in preparation] using a solution of 4% (w/v) agarose in PBS mixed 1∶1 with DMEM/Ham's F12 medium supplemented with L-glutamine (2 mM), 10% (v/v) heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 µg/ml). The inflated lung was allowed to solidify on ice, and ∼1 mm slices were cut by hand. Slices were permeabilized with 0,1% (v/v/) Triton-X100, counterstained with DAPI and directly analyzed for EGFP fluorescence by confocal laser scanning microscopy with a LSM700 system fitted on an Axio Observer Z1 inverted microscope (Zeiss). Images and videos were generated using Zen software. Blood samples Small volume blood samples were collected in Vacuette tubes containing K3EDTA as an anticoagulant daily after infection. White blood cells (WBC) were obtained by treatment of EDTA blood with red blood cell lysis buffer (Roche diagnostics, Penzberg, Germany) and used directly for detection of EGFP by flow cytometry. During necropsy blood was collected in heparin to prevent coagulation, PBMC were isolated by density gradient centrifugation, washed, resuspended in complete RPMI-1640 medium (Gibco Invitrogen, Carlsbad, CA, USA) supplemented with L-glutamine (2 mM), 10% (v/v) heat-inactivated FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml), counted using a haemocytometer and used directly for flow cytometry and virus isolation. Isolation of MV was performed on human B-LCL using an infectious center test as previously described [24]. Virus isolations were monitored by UV microscopy for EGFP fluorescence after co-cultivation with B-LCL for 3–6 days and results were expressed as number of virus-infected cells per 106 total cells. Broncho-alveolar lavage A BAL was performed post-mortem by direct infusion of 10 ml PBS into the right lung lobe. BAL cells were resuspended in culture medium with supplements as described above, counted and used directly virus isolation. Virus isolation was performed on B-LCL as described above. The remaining BAL cells were examined for EGFP expression by UV microscopy. Throat and nose swabs Throat and nose swabs were collected daily in transport medium (EMEM with Hanks' salts, supplemented with lactalbumine enzymatic hydrolysate, penicillin, streptomycin, polymyxine B sulphate, nystatin, gentamicin and glycerol) and frozen at −80°C. After thawing samples were vortexed, the swab was removed and the remaining transport medium was used for virus isolation [36]. Isolation of MV was performed on Vero-SLAM cells using an infectious center test as previously described [24]. The isolations were screened for EGFP fluorescence at day 3 and 7 post titration and results are expressed as the number of EGFP+ wells per 96 total wells. Lymphoid organs Lymphoid organs were collected during necropsy in PBS for direct preparation of single cell suspensions using cell strainers with a 100 µm pore size (BD Biosciences). Single cell suspensions were used directly for detection of EGFP by flow cytometry. From a selection of lymphoid organs (retropharyngeal LN, mandibular LN, tonsil and tracheobronchial LN) single cell suspensions were also used for virus isolation on Vero-SLAM cells as described above. The isolations were screened for EGFP fluorescence at day 3 and 7 post titration. The axillary and tracheobronchial LN were also collected in RNA later (Ambion) during necropsy for virus detection by real-time RT-PCR. Flow cytometry Freshly isolated WBC, PBMC and single cell suspensions prepared from lymphoid organs were analyzed unstained for EGFP expression by flow cytometry. EGFP was detected in the FITC channel on a FACS Canto II, approximately 106 events were obtained per sample to allow detection of low frequent EGFP+ populations. Immunohistochemical and immunofluorescence analysis of formalin-fixed tissues Only lung slices which were scored positive on live UV fluorescent screening were processed to paraffin. At days 2 and 3, 8/49 and 16/95 slices were scored positive, respectively. Sections (7 µm) were cut and deparaffinized, antigen retrieval was performed in a pressure cooker at full power for 3 min in 0.01 M TRIS-EDTA buffer (pH 9.0). MV-infected cells were detected using a polyclonal rabbit antibody to EGFP (Invitrogen). Sections were incubated in primary antibody overnight at 4°C, and specific antibody-antigen binding sites were detected using an Envision-Peroxidase system with DAB (DAKO) as substrate. Dual labeling indirect immunofluorescence was performed using polyclonal rabbit anti-EGFP and monoclonal mouse antibodies to the macrophage/DC marker CD11c (Novocastra, clone 5D11), the T-lymphocyte marker CD3 (DAKO, clone F7.2.38), the B-lymphocyte marker CD20 (DAKO, clone L26), the epithelial cell marker cytokeratin (DAKO, clone AE1/AE3), the endothelial cell marker CD31 (DAKO, clone JC70A) and the macrophage marker Mac387 (Abcam). Further dual labeling to assess the organization of epithelia and different cell types within BALT were carried out with a polyclonal antibody to epithelial cytokeratin (DAKO, Cat. No. Z0622) in combination with the above monoclonal antibodies to CD3, CD20 or CD11c. In all cases antigen binding sites were detected with a mixture of anti-mouse Alexa 568 and anti-rabbit Alexa 488 (Invitrogen). Sections were counterstained with DAPI hardset mounting medium (Vector). All fluorescently stained slides were assessed and digital fluorescent images acquired with a Leica DFC350 FX digital camera and processed using Leica FW4000 software. Supporting Information Figure S1 CD11c+ DC and macrophages targeted in the lung at 4 and 5 d.p.i. At 4 and 5 d.p.i. the CD11c+ DC or macrophage population was the major cell type in the lung in which MV replicates. Dual labelling for EGFP (green) and CD11c (red), DAPI was used to counter stain nuclei in blue. Left panels show EGFP alone (green), centre panels show CD11c alone (red), right panels show overlay of EGFP and CD11c. The two rows are two representative examples of double positive cells as indicated by arrows. (2.21 MB TIF) Click here for additional data file. Figure S2 Single color images for figure 4C and 5C. (9.75 MB TIF) Click here for additional data file. Figure S3 On-line immunohistochemical and H&E pathology scans. Four pathology slides were scanned and digitized at a high resolution and annotated. (0.06 MB PDF) Click here for additional data file. Video S1 Z-stack as rendered 3D-movie corresponding to figure 2, panel Bi. (1.91 MB MOV) Click here for additional data file. Video S2 Z-stack as rendered 3D-movie corresponding to figure 2, panel Bii. (2.16 MB MOV) Click here for additional data file. Video S3 Z-stack as rendered 3D-movie corresponding to figure 2, panel Biii. (1.51 MB MOV) Click here for additional data file.

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Journal ID (nlm-ta): PLoS Pathog

Journal ID (iso-abbrev): PLoS Pathog

Journal ID (publisher-id): plos

Journal ID (pmc): plospath

Title: PLoS Pathogens

Publisher: Public Library of Science (San Francisco, USA )

ISSN (Print): 1553-7366

ISSN (Electronic): 1553-7374

Publication date Collection: May 2013

Publication date (Print): May 2013

Publication date (Electronic): 9 May 2013

Volume: 9

Issue: 5

Electronic Location Identifier: e1003368

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[1 ]Department of Viroscience, Erasmus MC, Rotterdam, the Netherlands

[2 ]Department of Neurology, University of Colorado School of Medicine, Aurora, Colorado, United States of America

[3 ]Department of Microbiology, University of Colorado School of Medicine, Aurora, Colorado, United States of America

University of Pittsburgh, United States of America

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* E-mail: g.verjans@ 123456erasmusmc.nl

Co-author ADMEO wishes to declare, for the avoidance of any misunderstanding on competing interests, that he co-founded and is chief scientific officer of Viroclinics Biosciences, a company set up in collaboration with Erasmus MC. However, for clarification, no materials or support were received from the company, and no agreements were in place concerning the execution or publication of this work. This does not alter our adherence to all PLoS Pathogens policies on sharing data and materials. The authors have no additional financial interests.

Conceived and designed the experiments: WJDO RM BLH ADMEO GMGMV. Performed the experiments: WJDO GvA SG. Analyzed the data: WJDO RLdS BLH SG GMGMV. Wrote the paper: WJDO RM RLdS BLH DG ADMEO GMGMV.

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Publisher ID: PPATHOGENS-D-13-00223

DOI: 10.1371/journal.ppat.1003368

PMC ID: 3649965

PubMed ID: 23675304

SO-VID: 3b8812e1-797c-43aa-8aff-dace81c0714d

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This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Date received : 24 January 2013

Date accepted : 2 April 2013

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This work was supported in part by Public Health Service grant AG032958 from the National Institutes of Health (WJDO, RM, DG, and GMGMV). RLdS was supported by the VIRGO Consortium, an innovative cluster approved by the Netherlands Genomics Initiative and partially funded by the Dutch Government (BSIK 031012). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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