Infection of Lymphoid Tissues in the Macaque Upper Respiratory Tract Contributes to the Emergence of Transmissible Measles Virus

Journal of General Virology (2013), 94, 1933–1944 DOI 10.1099/vir.0.054650-0

Infection of lymphoid tissues in the macaque upper respiratory tract contributes to the emergence of transmissible measles virus

Martin Ludlow,13 Rory D. de Vries,23 Ken Lemon,3 Stephen McQuaid,3,4 Emma Millar,3 Geert van Amerongen,2 Selma Yu¨ksel,2 R. Joyce Verburgh,2 Albert D. M. E. Osterhaus,2 Rik L. de Swart2 and W. Paul Duprex1,3

Correspondence 1Department of Microbiology, Boston University School of Medicine, Boston, MA, USA Rik L. de Swart 2Department Viroscience, Erasmus MC, Rotterdam, The Netherlands [email protected] 3School of Medicine, Dentistry and Biomedical Sciences, Queen’s University of Belfast, Belfast, Northern Ireland, UK 4Tissue Pathology Laboratories, Belfast Health and Social Care Trust, Belfast, Northern Ireland, UK

Measles virus (MV), a member of the family Paramyxoviridae, remains a major cause of morbidity and mortality in the developing world. MV is spread by aerosols but the mechanism(s) responsible for the high transmissibility of MV are largely unknown. We previously infected macaques with enhanced green fluorescent protein-expressing recombinant MV and euthanized them at a range of time points. In this study a comprehensive pathological analysis has been performed of tissues from the respiratory tract around the peak of virus replication. Isolation of virus from nose and throat swab samples showed that high levels of both cell-associated and cell-free virus were present in the upper respiratory tract. Analysis of tissue sections from lung and primary bronchus revealed localized infection of epithelial cells, concomitant infiltration of MV-infected immune cells into the epithelium and localized shedding of cells or cell debris into the lumen. While high numbers of MV-infected cells were present in the tongue, these were largely encapsulated by intact keratinocyte cell layers that likely limit virus transmission. In contrast, the integrity of tonsillar and adenoidal epithelia was disrupted with high numbers of MV-infected epithelial cells and infiltrating immune cells present throughout epithelial cell layers. Disruption was associated with large numbers of MV-infected cells or cell debris ‘spilling’ from epithelia into the respiratory tract. The coughing and sneezing response induced by disruption of the ciliated epithelium, leading to Received 24 April 2013 the expulsion of MV-infected cells, cell debris and cell-free virus, contributes to the highly Accepted 17 June 2013 infectious nature of MV.

INTRODUCTION outbreaks of measles, the specific underlying pathological mechanisms responsible for the high transmissibility of Measles virus (MV), a morbillivirus in the family MV from infected to susceptible individuals are still Paramyxoviridae, is the most contagious of all currently unknown. circulating human viruses, with an estimated basic reproductive number (R0) of 12–18 (Moss & Griffin, The contagious nature of MV and a postulated respiratory 2006). An important consequence of such a high rate of route of transmission were first outlined by Peter Panum virus transmission is that vaccine coverage of greater than following investigation of an epidemic in the Faro Islands 95 % is required to eliminate endemic transmission in 1846 (Panum, 1939). This established the incubation (Anderson & May, 1982). This increases the complexity period of measles at approximately 14 days, at which point and difficulty of MV eradication, even in the developed the outbreak of the typical exanthem rash and catarrhal world where the numbers of measles cases have increased cough was associated with the time at which infected in recent years due to poor adherence to MV vaccination individuals were most infectious. Rapid resolution of the regimes (Carrillo-Santisteve & Lopalco, 2012). However, rash and clearance of MV after 4–5 days was later shown to despite a multitude of epidemiological investigations into be associated with a strong immune response, with cell- mediated rather than humoral immunity being predomin- 3These authors contributed equally to this paper. antly responsible for clearance of MV (de Vries et al.,

054650 G 2013 SGM Printed in Great Britain 1933 M. Ludlow and others

2010b). Subsequent clinical studies demonstrated that MV and Waldeyer’s ring tonsillar tissues is a key determinant is first detected in respiratory secretions during the pre- governing virus transmission, leading to the release of both ceding 2–4 day prodromal period (Fulton & Middleton, cell-associated and cell-free virus into the lumen of the 1975; Tompkins & Macaulay, 1955), a feature of the disease respiratory tract. which complicates both the management and control of outbreaks (Moss & Griffin, 2012). Thus, MV-infected individuals are capable of transmitting virus to susceptible RESULTS contacts for a total of 7–9 days. Interestingly, it is has been reported that less than 20 % of cases are responsible MV infection of and shedding from the upper for more than 80 % of virus transmission (Lloyd-Smith respiratory tract of macaques et al., 2005), suggesting that differences in virus loads or Macroscopic visualization of enhanced green fluorescent the degree of tissue damage during infection and/or protein (EGFP) in MV-infected macaques at the peak of host genetic differences have prominent roles in virus infection [9 days post-infection (p.i.)] showed the presence transmission. of extensive foci of MV infection in the buccal mucosa, Historically, MV has been known as a respiratory pathogen gingiva, tongue and Waldeyer’s ring lymphoid tissues such which is also capable of spreading systemically in the as the tonsils (Fig. 1a–c). Between 9 and 11 days p.i. we blood, leading to a temporally well-characterized disease also observed widespread infection of epithelial tissues in course (Hektoen, 1905; Home, 1759; Panum, 1939). A the nose (Ludlow et al., 2013). In order to assess whether number of subsequent pathological studies, primarily virus infection in the upper respiratory tract (URT) of based on histological analysis of tissue samples from MV macaques was associated with virus shedding we examined patients, showed that giant cell formation in lymphoid the release of virus into the airways between 0 and 13 days tissue and lung epithelium are characteristic features of p.i. by virus isolation from nose and throat swabs. Virus measles (Enders et al., 1959; Schellmann & Sanson, 1965; was not isolated from throat or nose samples until 3 and Warthin, 1931). However, it is only in recent years that the 5 days p.i., respectively. Virus shedding attained a peak in underlying mechanisms governing the cellular tropism of the throat and nose at 7–9 days p.i. (Fig. 1d). It is MV have been elucidated. The identification of CD150 as important to note that the titres shown are those measured the primary MV cellular receptor on activated B- and T- in the virus transport medium, which is a significant lymphocytes, macrophages and mature dendritic cells dilution of the virus titres in the original mucosal tissues. (Tatsuo et al., 2000) explains the largely lymphotropic Although the exact magnitude of this dilution is difficult to nature of measles. The more recent discovery that PVRL4 assess, nose swabs always contained less fluid than the (Nectin 4), a component of the adherens junction, is also a throat brushes, resulting in a higher dilution factor. Thus, bona fide MV cellular receptor (Mu¨hlebach et al., 2011; peak MV titres in the macaque respiratory tract may reach 6 21 Noyce et al., 2011) explains the epitheliotropism observed as high as 10 TCID50 ml , suggesting that transmission of in the late stages of the disease (Ludlow et al., 2010, 2013). 0.01–1 ml of mucosal fluid could be enough for transmis- + sion of infection. Infectious virus was not isolated from Following entry into the body and infection of CD150 throat or nose swabs at time points beyond 11 days p.i. myeloid cells in the alveoli (Lemon et al., 2011), MV These results were confirmed by real-time reverse tran- spreads first to local lymph nodes, and then systemically via + scriptase PCR assays (data not shown). To determine infection of CD150 lymphocytes and dendritic cells in whether infectious virus was released from the airways in peripheral lymphoid tissues (de Swart et al., 2007). These the form of MV-infected cells, cellular debris or cell-free MV-infected immune cells contacting or infiltrating into + virus, we separated cell-associated and cell-free virus respiratory epithelium spread infection to PVRL4 obtained from nose and throat swabs by repeated epithelial cells in the late stages of the disease (Frenzke centrifugation. Isolation of virus from cell-associated or et al., 2013; Ludlow et al., 2013). Macaques provide an supernatant fractions showed equivalent levels of cell- excellent natural model in which to investigate the late associated and cell-free virus in throat and nose samples, stages of measles as a number of outbreaks have been indicating that either could be involved in transmission to reported in primate colonies with extensive chains of susceptible hosts (Fig. 1e). transmission between animals (Choi et al., 1999; MacArthur et al., 1979; Willy et al., 1999). MV infection in the adenoid and tonsil is In the present study we have examined the mechanisms associated with extensive damage to the through which MV crosses the epithelium to emerge into epithelium the respiratory tract at the peak of infection by utilizing fluorescent recombinant (r) MV to infect macaques. This We have previously shown that Waldeyer’s ring tissues, facilitated sensitive pathological assessment and analysis of including the adenoids (nasopharyngeal tonsils) and both respiratory tract and lymphoid tissues. We show that palatine tonsils, of macaques infected with pathogenic extensive disruption induced by immune cells infiltrating rMV contain large numbers of MV-infected immune cells into or residing in the epithelium of the respiratory tract approximately 1 week after MV infection (de Swart et al.,

1934 Journal of General Virology 94 Sources of transmissible measles virus in macaques

TN (a) (b)

(c)

(d) 105 105 Throat swabs Nose swabs

) 104 ) 104 –1 –1 ml ml

50 103 50 103

102 102

101 101 Virus load (TCID Virus load (TCID

100 100 0 123 4 5 6 7 89 11 13 0 123 4 5 6 7 8 9 11 13 Time (days p.i.) Time (days p.i.)

(e) 105 105

104 104 ) )

–1 3 10 –1 103 ml ml

50 50 102 102 (TCID (TCID

101 101 Nose swab cell-free virus isolation Throat brush cell-free virus isolation

<1 101 102 103 104 105 <1 101 102 103 104 105 Throat brush cell-associated virus isolation Nose swab cell-associated virus isolation –1 –1 (TCID50 ml ) (TCID50 ml )

Fig. 1. Detection of macroscopic EGFP fluorescence and virus isolation from recombinant wild-type MV-infected macaques. (a– c) Macroscopic fluorescence images of rMV-infected (KS strain) cynomolgus macaques. At the peak of infection (9–11 days p.i.) extensive MV infection was present in (a) buccal mucosa and gingiva, (b) tongue and tonsils (TN), and (c) gingiva. (d) Isolation of MV from throat and nose swabs by assay on Vero-hCD150 cells. Median values are shown based on samples obtained from 38 wild-type MV-infected animals between 0 and 13 days p.i. (e) Isolation of cell-free and cell-associated MV from throat and nose

swabs. Data are presented as X–Y pairs of TCID50 values of cell-associated (X) and cell-free virus (Y) in a simple scatter plot based on samples obtained from 11 wild-type MV-infected animals. KS strain (a–c, e); KS and IC323 strains (d). http://vir.sgmjournals.org 1935 M. Ludlow and others

2007; de Vries et al., 2010a). Although the role of these commonly observed in the lumen of the respiratory tract. tissues in MV transmission has not been examined, Examination of serial H&E- and immunohistochemically disruption of adenoid or tonsillar epithelium would (IHC)-stained sections of the primary bronchus revealed facilitate the release of both MV-infected cells and cell- extensive infiltration of mononuclear cells into the epithelium, free virus into the throat and nasal cavity. To address this including locations which did not contain prominent MV hypothesis we examined sections of Waldeyer’s ring tissue infection, suggesting that both uninfected and MV-infected from MV-infected macaques. Prior to the peak of infection, immune cells infiltrate into the epithelium from underlying at 7 days p.i., large numbers of MV-infected immune cells aggregates of lymphoid cells (Fig. 3d). Epithelial disruption were observed beneath and within the squamous epithelium was more extensive than the observed level of epithelial cell of tonsillar crypts in the absence of detectable epithelial cell infection and thus cannot be attributed to epithelial cell infection (Fig. 2a, b). However, by 9 days p.i. epithelia infection alone. Epithelial cell disruption was much more showed extensive infection-associated lesions (Fig. 2c), which prominent in adenoid and tonsillar epithelium than in the were not observed in corresponding tissues of non-infected bronchi and lung. Interestingly, epithelial damage in the lungs macaques. The adenoid and tonsillar epithelial architecture was predominantly observed in close proximity to bronchus was disrupted and breakdown of the cytokeratin network was associated lymphoid tissue (BALT), supporting the role of observed, together with a reduction in the thickness of the high concentrations of both infected and non-infected epithelium and a concomitant infiltration of MV-infected lymphocytes and myeloid cells in the pathogenesis of these lesions. Breaks in the integrity of adenoid epithelium and uninfected inflammatory immune cells. The epithelial + facilitated the release of MV-infected CD20 B cells into the cell barrier was breached at multiple locations, with the + lumen exposed to the underlying immune cells. Extensive nasopharynx (Fig. 3e), and large numbers of CD11c myeloid disruption of the epithelium was also associated with high cells were present beneath and within disrupted tonsillar levels of MV infection of stratified squamous or pseudo- epithelium (Fig. 3f). stratified ciliated columnar epithelial cells in the palatine tonsil or adenoid, respectively (Fig. 2c, d). Characterization of MV-infected cells in the oral The availability of formalin-fixed samples of tonsillar cavity tissues collected from a large number of macaques enabled MV infection of oral epithelium was investigated by + a retrospective temporal histopathological analysis to be targeted histopathological analysis of EGFP foci taken performed on serial sections of tonsillar tissue from the late from the inner cheek, tongue, gingiva and buccal mucosa, stages of infection (Fig. 2d). MV-infected cells and which were readily identified macroscopically in the oral associated epithelial disruption were not evident in control cavity (Fig. 1). MV infection of the inner cheek was tonsillar tissues, but large numbers of MV-infected cells predominantly associated with infection of aggregates of were present in underlying lymphoid tissue and within the immune cells adjacent to minor salivary gland ducts (Fig. stratified squamous epithelium by 9–11 days p.i. This was 4a). Non-keratinized stratified squamous epithelial cells associated with epithelial disruption and the presence of lining the ducts were infected and this was associated with large amounts of cellular debris containing mononuclear exfoliation of infected cells into the lumen of the duct and cells in tonsillar crypts. MV-infected cells were no longer disruption of the epithelium (Fig. 4b). Foci of MV detected and some degree of epithelium recovery was infection in the buccal mucosa consisted of aggregates of evident at 15 days p.i., but analysis of haematoxylin and infected mononuclear cells in the oral mucosa, with spread eosin (H&E)-stained serial sections showed cellular debris to adjacent non-keratinized stratified squamous epithelial consisting predominantly of mononuclear cells (Fig. 2d, cells (Fig. 4c). Examination of intact foci of MV infection inset) still trapped within the lumen of the crypts. from the buccal mucosa by live cell confocal microscopy showed loosely connected aggregates of infected cells, with Epithelial disruption in the respiratory tract is focal areas of cell-to-cell fusion visible in areas containing associated with immune cell infiltration the highest density of MV-infected cells (Fig. 4d). Importantly, no disruption of the oral epithelium adjacent Extensive infiltration of immune cells into respiratory tract to the lumen of the oral cavity was apparent, suggesting epithelium was a prominent feature of MV infection at the that these MV-infected cells present within the parenchyma peak of infection at 9 days p.i. Extensive disruption of of the tissue were unlikely to contribute to transmission. alveolar epithelium was apparent in sections of lung tissue Similarly, analysis of serial histological and immunostained with cells in the submucosa observed ‘spilling’ into the sections showed that MV-infected cells in the tongue were alveolar lumen (Fig. 3a). In many locations, the normal surrounded by uninfected keratinocytes and the overlying architecture of the alveolar epithelium was disrupted due keratin layer, with little evidence of immune cell infiltra- to widespread formation of giant cells within the tion and no disruption of the integrity of the tongue epithelium (Fig. 3b). Dual labelling of bronchial epithelium epithelium (Fig. 4e). Dual labelling with cytokeratin with antibodies to EGFP or cytokeratin identified many showed that infected cells within tongue epithelium + exfoliated epithelial cells and EGFP cells which lacked consisted of non-keratinized stratified squamous epithelial + cytokeratin (Fig. 3c). Cytokeratin2/EGFP cells were cells (Fig. 4f).

1936 Journal of General Virology 94 Sources of transmissible measles virus in macaques

(a) DAPI/EGFP DAPI/CYK/EGFP (b) DAPI/EGFP DAPI/CYK/EGFP Day 7

H&E MV Cytokeratin (c) * * * Day 9

(d) Day 11 Day 15 Uninfected

Fig. 2. Disruption of tonsillar epithelium in MV-infected cynomolgus macaques due to infiltration of immune cells. (a, b) Immunocytochemical staining of formalin-fixed tissue sections taken from an rMV-infected macaque at 7 days p.i., prior to the peak of infection. (a) MV-infected immune cells (green) in simple squamous epithelium (red) of tonsillar crypts. Bar, 200 mm. (b) Higher magnification of MV-infected immune cells present in tonsillar epithelium in the absence of epithelial cell infection. Bar, 50 mm. (c, d) Serial H&E- (left) and IHC-stained sections (MV N, centre; cytokeratin, right) of tonsillar tissue from MV-infected macaques. (c) Extensive MV infection of epithelial cells and infiltration of infected and uninfected lymphoid/myeloid cells was associated with damage to the integrity of adenoidal epithelium at the peak of infection at 9 days p.i. Bar: top, 160 mm; bottom, 40 mm. (d) Progressive infection and disruption was observed in tonsillar crypt epithelium. No positive MV staining or pathology was observed in control tonsillar epithelium (uninfected), but extensive MV infection and disruption was evident in sections from infected macaques at 11 days p.i. MV-infected cells were not observed in the epithelium at 15 days p.i. but aggregates of cellular debris remained in the lumen of the crypt (arrow). (Inset) Higher magnification image of mononuclear cells indicated by the arrow in the main panel. Bar: uninfected, 160 mm; days 11–15, 80 mm. DAPI was used as a nuclei counterstain (blue) in (a, b). KS strain (a, b, d, bottom panel); IC323 strain (c, d, middle panel).

Cell-associated MV in the respiratory tract may tracheal epithelium contained MV antigen (Fig. 5a). In + + contribute to MV transmission addition to EGFP cells in epithelia, EGFP cellular debris Analysis of tracheal rings from MV-infected macaques by was readily detected on the surface of tracheal epithelium immunofluorescence staining using hyperimmune serum (Fig. 5b, c) and dual immunofluorescence staining showed from an subacute sclerosing panencephalitis (SSPE) patient that MV antigen was present within these structures (Fig. + + showed that large foci of EGFP cells present in the 5d). The observation that EGFP cellular debris was http://vir.sgmjournals.org 1937 M. Ludlow and others

(a) (b) DAPI/CYK/MV

(e) DAPI/CD20 DAPI/MV N DAPI/CD20/EGFP

(f) DAPI/CD11c DAPI/MV N DAPI/CD11c/MV N

Fig. 3. Characterization of pathology in epithelium of MV-infected macaques. (a) MV-infected cells in an IHC-stained section of a lung alveolus ‘spill’ from two breaks in the epithelium (arrow). Bar, 80 mm. (b) Giant cell formation (arrows) is visible in MV- infected (green) alveolar epithelium (red). Bar, 50 mm. (Inset) Clustering of nuclei in absence of cytokeratin stain. (c) Disruption + of bronchial epithelium architecture due to extensive immune cell infiltration. Cytokeratin epithelial cells (red) at the luminal surface contain EGFP (green) (asterisks) and in some instances are exfoliated into the lumen (arrowhead). A single MV-infected cell on the apical surface of the epithelium does not contain cytokeratin (arrow). Bar, 50 mm. (d) Extensive infiltration of mononuclear cells (arrows) and giant cell formation (arrowhead) in primary bronchus epithelium in an H&E-stained section of primary bronchus. Bar, 80 mm. (Inset) MV-infected cells in an IHC-stained serial section showing focal infection of the + epithelium. (e) Dual labelling of MV antigen (green) and CD20 B cells (red) in adenoid tissue showing B cells infiltrating through a break in the epithelium (arrows). Contiguous MV infection of pseudostratified ciliated columnar adenoidal epithelial cells is observed adjacent to this area (arrowheads). The approximate location of the demarcation boundary of the epithelium + from underlying tissue is indicated by a dashed line. (f) Dual labelling of MV-infected cells (green) and CD11c myeloid cells (red) beneath and within tonsillar epithelium. MV-infected simple squamous epithelial cells are present adjacent to ‘breaks’ in the integrity of the epithelium (arrows). Bar (e, f), 100 mm. DAPI was used to counterstain nuclei (blue). KS strain (a, b, e, f); IC323 strain (c, d).

1938 Journal of General Virology 94 Sources of transmissible measles virus in macaques

(a) (b) DAPI/CYK/EGFP Inner cheek

(e) (f) DAPI/CYK/EGFP Tongue

Fig. 4. MV infection of oral epithelium. (a) Serial H&E- (top) and IHC-stained (bottom) sections of the inner cheek. MV infection of lymphoid tissue was observed adjacent to epithelial cell-lined minor salivary gland ducts (arrows). Bar, 320 mm. (b) Dual labelling of EGFP (green) and cytokeratin (red) showing MV-infected epithelial cells (arrow) lining a duct located in the inner cheek. Disruption of the normal cytokeratin architecture and exfoliation of MV-infected cells is visible at the base of the duct (arrowhead). Bar, 100 mm. (c) Serial H&E- (top) and IHC-stained (bottom) sections of a focus of infection present in the buccal mucosa. MV infection was predominantly observed in infiltrates of immune cells but a few foci of infected epithelial cells are present (arrows). Bar, 160 mm. (d) Three-dimensional reconstruction of a focus of infection in the buccal mucosa obtained from a50mm vibratome-cut section showing interconnected MV-infected immune cells (green). Bar, 60 mm. (e) Serial H&E- (top) and IHC-stained (bottom) sections of tongue. No infiltration of immune cells or disruption of epithelium was observed. Foci of MV-infected stratified squamous epithelial cells are visible in tongue epithelium. Bar, 320 mm. (f) Indirect dual immunofluorescence on a transverse tongue section. Large foci of MV-infected (EGFP, green) cells dual labelled with the epithelial cell marker cytokeratin (red). Bar, 200 mm. (Inset) Little disruption of the stratified squamous (red) is apparent. DAPI was used as a nuclei counterstain (blue) in (b, f). KS strain (a–f).

present above epithelial cells in the nasal concha confirmed structures at higher magnification showed that they are that this debris was present in the lumen and thus did not approximately 4 mm in diameter and extend 4–5 mmwhen represent subcellular structures within infected cells (Fig. 5e). viewed in cross-section (Fig. 5f, insets). Transport towards the Further dual immunofluorescence staining showed instances oral and nasal cavity of cellular debris and infectious virus + of specific staining for MV antigen in EGFP subcellular released from MV-infected cells by the mucociliary escalator debris in the tracheal lumen in close proximity to large foci of would represent an efficient mechanism through which MV- infected tracheal epithelial cells (Fig. 5f). Analysis of these infected cells/debris are removed from the body. http://vir.sgmjournals.org 1939 M. Ludlow and others

(a) DAPI/EGFP DAPI/MV DAPI/EGFP/MV

(b) (c) (d) DAPI/EGFP/MV

(e) DAPI/CYK DAPI/EGFP DAPI/CYK/EGFP

(f) DAPI/EGFP DAPI/MV DAPI/EGFP/MV

Fig. 5. Visualization of cell-associated virus in the lumen of the respiratory tract of MV-infected cynomolgus macaques. (a) Large + foci of EGFP cells (red) in the tracheal epithelium show positive staining for MV antigen (green). Bar, 50 mm. (b) Detection of + EGFP subcellular debris from MV-infected cells on the surface of trachea epithelium. Bar, 160 mm. (c) Higher magnification + image of boxed area in (b). Bar, 20 mm. (d) Detection of MV antigen (green) in EGFP subcellular structures (red) in the + + tracheal lumen. Bar, 12 mm. (e) EGFP subcellular debris (green) is present adjacent to cytokeratin nasal concha epithelial cells (red). Bar, 25 mm. (f) Dual labelling of MV antigen (green) and EGFP (red) shows MV-infected cells in the tracheal epithelium (arrowheads) and MV antigen in subcellular structures in the tracheal lumen (arrows). Bar, 20 mm. (Insets) Higher + magnification image of MV antigen subcellular debris shows that these structures are approximately 4 mm in diameter and extend 4–5 mm. Bar: left, 3 mm; right, 2 mm. MV antigen was detected with SSPE serum in (a, c, f). DAPI was used as a nuclear counterstain in (a, d–f). The lumen of the tracheal epithelium (d, f) and nasal concha (e) is indicated with a dashed line. KS strain (a–e).

DISCUSSION (Mu¨hlebach et al., 2011; Noyce et al., 2011) has Measles is characterized by a number of unique patho- reinvigorated the study of MV infection and spread within logical and epidemiological features, which slow progress polarized epithelial cells. Apical budding from these towards controlling and eradicating the disease. These epithelial cells is assumed to be the primary contributor include a relatively long incubation period prior to the to infectious transmissible virus in the airways of infected onset of clinical symptoms, the fact that individuals in the individuals (Racaniello, 2011). However, in vitro epithelial prodromal stage are highly infectious and the requirement cell culture models fail to fully recapitulate the complex of vaccine coverage of greater than 95 % in order to prevent architecture of respiratory epithelium in vivo, which also endemic transmission. The discovery of PVRL4 as a cellular encompasses both dendritic cells and macrophages and is receptor for MV on the basolateral surface of epithelial cells often infiltrated by underlying immune cells during

1940 Journal of General Virology 94 Sources of transmissible measles virus in macaques inflammatory responses. Thus, investigations into the patients at the prodromal stage (Warthin, 1931). In this mechanism(s) by which MV spreads through squamous study, multi-nucleated giant cells of assumed lymphocyte or columnar epithelium into the respiratory tract must be origin were observed beneath and within tonsillar and based on a thorough understanding of the pathology adenoid epithelium, with elongated syncytia observed to observed in vivo following natural MV infection of either extend between epithelial cells towards the surface of the humans or macaques. tonsils. The pathology associated with the spread of uninfected and infected immune cells into tonsillar Virus present in aerosol droplets expelled from MV- epithelium may be responsible for the ‘spots’ on the infected individuals by coughing is suspected of being the surface of the tonsil that have been reported during the primary route of virus transmission to susceptible hosts prodromal stage of measles (Herrman, 1915). In more (Griffin, 2007). However, MV can also spread through serious cases, alterations to the tonsillar epithelium have fomites and/or close contact with bodily secretions. The been associated with rupture and haemorrhaging during high transmissibility of MV has been extensively docu- the prodromal stage (John et al., 1988; Redman & Chaney, mented with infected individuals in schools, sports 1959). stadiums, aeroplanes and offices responsible for triggering outbreaks (Bloch et al., 1985; Coleman & Markey, 2010; The high density of MV-infected cells observed in the oral Edelson, 2012; Ehresmann et al., 1995; Paunio et al., 1998). cavity of infected macaques at necrospy suggested addi- In many of these cases, aerosol transmission of MV was tional routes through which MV could be released into the suspected due to the absence of direct contact between environment. However, detailed pathological assessment of many of the secondary cases with the index case. The tissues collected from this area revealed differential extreme infectiousness of some MV-infected individuals pathology and patterns of virus infections in comparison has resulted in the use of the term ‘superspreader’. In a to tonsillar lymphoid tissue. While exfoliation of MV- well-documented case from April 1951, a large measles infected epithelial cells and epithelium disruption in minor outbreak was initiated in Qaqortoq (formerly Julianeha˚b), salivary gland ducts in the inner cheek was readily Greenland, when an asymptomatic sailor infected approxi- observed, MV-infected immune and epithelial cells in the mately 250 people in a dancehall in a single evening buccal mucosa and tongue were present beneath uninfect- (Christensen et al., 1953). The fact that this event occurred ed squamous epithelium, not in contact with the when the index case was still in the prodromal stage of environment, and thus presumably have a minimal measles, 3 days before the onset of rash, demonstrates the contribution towards MV transmission. Although discrete difficulty in controlling outbreaks of measles. This provides foci of MV-infected cells in the buccal mucosa were only a prime example of the complexity and heterogeneity of detected upon illumination under fluorescent light, such virus transmission as modelling has shown that approxi- foci may be analogous to the ‘Koplik spots’ observed in the mately 20 % of measles cases are responsible for 80 % of buccal mucosa of human measles patients (Koplik, 1896; virus transmission (Lloyd-Smith et al., 2005), thus Suringa et al., 1970). However, the absence of ulceration, adhering to the general ‘20/80’ rule governing individual necrosis or exudates suggests that in the macaque such variation in virus transmission (Woolhouse et al., 1997). It lesions are less prominent with a reduced inflammatory remains to be determined if such variation is linked to response. either differences in the extent of epithelial disruption or Our studies into mechanisms of MV cell-to-cell spread led the virus burden in the epithelium of the URT. Although in us to hypothesize that cell-associated virus may also vitro studies have shown that epithelial cells can be infected contribute to host-to-host transmission, especially via by MV and produce new virus particles by budding from close contact. This is supported by the fact that the release the apical membrane (Sinn et al., 2002; Tahara et al., 2008; of giant cells into the lumen of the respiratory tract is a Takeuchi et al., 2003), MV cell-to-cell spread is relatively consistent pathological feature of measles (Mcquillin et al., slow and results in the release of low virus titres (Leonard 1976; Scheifele & Forbes, 1972; Tompkins & Macaulay, et al., 2008; Ludlow et al., 2010; Mu¨hlebach et al., 2011). In 1955). Such cells obtained from nasopharyngeal or contrast, the MV replication cycle in lymphocytes is usually conjunctival swabs can be detected during the prodromal more rapid and results in higher levels of viral shedding (de stage and have proven invaluable as an additional Vries et al., 2010a; Lemon et al., 2011). Therefore, the high diagnostic feature of measles in resource-limited areas MV infection levels in the lymphoid tissues of the URT (Lightwood et al., 1970). Similarly, we also detected MV- should be considered as an important potential source of infected cells and associated subcellular debris in the lumen transmissible MV, especially when taking into account the of the respiratory tract together with cell-associated virus in high degree of epithelial disruption we observed in these nose and throat swabs taken from infected macaques. The tissues. + infectious nature of subcellular EGFP structures in the The extensive pathology we observed in the tonsillar lumen of the respiratory tract is suggested by the presence epithelium of infected macaques, such as immune cell of MV antigen within the spheroid structures. With a infiltration, giant cell formation and epithelium dissolu- diameter of 4 mm, these structures are approximately 15 tion, mirrors findings reported previously in a compre- times larger than single virus particles, which are typically hensive analysis of tonsils obtained from human measles 100–300 nm in size (Griffin, 2007). Such structures are http://vir.sgmjournals.org 1941 M. Ludlow and others probably derived from vesicles of cellular debris extruded Animals were housed and experiments were conducted in compliance from MV-infected cells and may be readily expelled into with European guidelines (EU directive on animal testing 86/609/ the air by coughing triggered directly by stimulation of EEC) and Dutch legislation (Experiments on Animals Act 1997). The protocols were approved by the independent animal experimentation cough receptors in damaged epithelium below the level of ethical review committee (DCC) in Driebergen, The Netherlands. the larynx in the trachea or lungs (Eccles, 2005; Animal welfare was observed on a daily basis; animal handling was Widdicombe, 1995). Alternatively, the generalized inflam- performed under light anaesthesia using ketamine and medetomi- matory response initiated by the high level of MV infection dine. After handling, atipamezole was administered to antagonize the in tonsillar tissue in the URT may also trigger a cough effect of medetomidine. response in epithelium below the larynx. Although there is a paucity of research into aerosol transmission of MV, it Virus detection in nose and throat swabs. Nose and throat swabs were collected using a polyester minitip urethral swab (Copan) or a has been reported that cell-free MV survives for longer in cytobrush plus cell collector (Medsc and Medical), respectively. The the air than influenza virus (De Jong & Winkler, 1964), but swabs were collected in virus transport medium (2 ml), consisting of shows increased rates of virus decay at humidity levels of Eagle’s minimal essential medium with Hanks’ salts, supplemented 50–70 %. It could be postulated that MV released into the with lactalbumin enzymatic hydrolysate (0.5 g l21), penicillin, environment in a cell-associated form would be better streptomycin, polymyxin B sulfate, nystatin, gentamicin and glycerol protected against environmental hazards such as humidity, (10 % v/v). Swabs were frozen at 280 uC within 2 h after collection. temperature and UV radiation, and may be more infectious Samples collected at different days p.i. were thawed, vortexed, 200 ml than cell-free virions due to the greater efficacy and was collected in lysis buffer for RNA isolation and the remainder was used for serial twofold titration (in eight rows) on Vero-hCD150 cells. speed of cell-to-cell fusion in comparison to virus-to-cell Virus loads in nose and throat swabs are expressed as TCID50. In two fusion. experiments, including 11 macaques, procedures were adapted to In summary, we propose that MV infection of adenoid and discriminate between cell-free and cell-associated virus. To this end, nose and throat swabs were collected in RPMI 1640 medium tonsillar lymphoid tissue with subsequent epithelial supplemented with 10 % (v/v) FBS, and processed immediately after damage in the URT contributes to the emergence of collection. The samples were briefly vortexed and subsequently transmissible virus in the respiratory tract, augmenting centrifuged for 10 min at 300 g. The supernatant was transferred to a previously proposed sources such as MV-infected epithelial new centrifuge tube and the pellet was resuspended in an equivalent cells in the trachea (Mu¨hlebach et al., 2011) and nose volume of RPMI+10 % (v/v) FBS to assess the level of cell-associated (Ludlow et al., 2013). Future studies focused on aerosolized virus. The supernatant was centrifuged a second time for 15 min at transmission of fluorescent morbilliviruses in the ferret, a 1000 g and subsequently the supernatant was used to assess the level of cell-free virus. model used extensively for virus transmission studies (Herfst et al., 2012; Imai et al., 2012), would help to Necropsies. Animals were euthanized by exsanguination under illuminate natural routes of virus transmission and provide ketamine/medetomidine anaesthesia, macroscopic foci containing the basis for more challenging studies into natural EGFP were visualized and imaged, and samples collected for transmission of MV in the macaque. histological, immunohistochemical or immunocytochemical analysis as described previously (de Swart et al., 2007; de Vries et al., 2010a; Ludlow et al., 2013). Representative areas from lung, trachea, primary bronchus, tonsil, adenoid, nasal concha, tongue, buccal mucosa and METHODS inner cheek were sampled. Tonsillar and adenoidal lymphoid tissues Cells and viruses. Two recombinant fluorescent and pathogenic were bisected and embedded to provide full cross-sections. rMV strains, rMVIC323EGFP and rMVKSEGFP, which have been used previously to study the pathogenesis of MV in the macaque (de Swart Immunohistochemical and immunofluorescence analysis of et al., 2007; de Vries et al., 2010a, 2012; Lemon et al., 2011) were used formalin-fixed tissues. All formalin-fixed sections were deparaffin- for in vivo experiments. rMVKSEGFP was rescued from a molecular ized, antigen retrieval performed and MV-infected cells detected as clone based on a wild-type genotype B3 virus isolated in 1997 from described previously (de Swart et al., 2007) using polyclonal rabbit PBMCs taken from a measles case in Khartoum, Sudan (el Mubarak antibodies to EGFP (Invitrogen) or anti-measles N protein (Novus et al., 2000; Lemon et al., 2011) while rMVIC323EGFP was based on Biologicals). An Envision-Peroxidase system with 3,39-diaminobenzi- the wild-type IC (Ichinose)-B strain of MV isolated from a throat dine (DAB) (DAKO) as substrate was used to detect antigen binding swab taken from a patient in Japan in 1984 (Hashimoto et al., 2002; sites. Following assessment of histology and virus immunopathology Kobune et al., 1990). We did not observe strain- or host species- in respiratory tract and lymphoid tissues, blocks were selected for specific differences in the studies described in this paper. However, it dual-labelling immunofluorescence performed using polyclonal is important to emphasize that the experiments were not designed to rabbit antibodies to EGFP (Invitrogen) and measles N protein or identify such differences. Virus stocks were generated following human hyperimmune serum obtained from a patient with SSPE infection of a human B-lymphoblastic cell line (B-LCL) and were together with monoclonal mouse antibodies to the myeloid cell tested to ensure the absence of Mycoplasma species prior to use in marker CD11c (Novocastra; clone 5D11), B-lymphocyte marker animal infections. Virus titres were obtained by end-point titration in CD20 (DAKO; clone L26) and an epithelial cytokeratin cell marker Vero-human (h) CD150 cells and were expressed as 50 % tissue (DAKO; clone AE1/AE3). Antigen binding sites were detected using 21 culture infectious doses (TCID50 ml ). the antibodies anti-rabbit Alexa 488 and anti-mouse/human Alexa 568 (Invitrogen). Following washes to remove unbound antibody, Animal study design. Cells and tissues were collected from MV- sections were mounted on glass slides using DAPI hardset mounting seronegative cynomolgus (n535) and rhesus macaques (n55) that medium (Vector). Selected H&E-, IHC- or immunofluorescence- were infected with fluorescent rMV by intra-tracheal inoculation or stained sections were imaged as described previously (Ludlow et al., by aerosol inhalation as reported previously (de Vries et al., 2012). 2013).

1942 Journal of General Virology 94 Sources of transmissible measles virus in macaques

ACKNOWLEDGEMENTS el Mubarak, H. S., Van De Bildt, M. W., Mustafa, O. A., Vos, H. W., Mukhtar, M. M., Groen, J., el Hassan, A. M., Niesters, H. G., Ibrahim, We would like to thank the staff of the Tissue Core Technology Unit S. A. & other authors (2000). Serological and virological character- and the Bioimaging Unit, Queen’s University of Belfast for expert ization of clinically diagnosed cases of measles in suburban technical assistance, Yusuke Yanagi for providing the Vero-hCD150 Khartoum. J Clin Microbiol 38, 987–991. cell line, and Thijs Kuiken and Bert Rima for helpful discussions. In Enders, J. F., McCarthy, K., Mitus, A. & Cheatham, W. J. (1959). addition, we thank Robert Dias D’Ullois, Tien Nguyen and Linda Isolation of measles virus at autopsy in cases of giant-cell pneumonia Rennick for their contributions to these studies. This work was without rash. N Engl J Med 261, 875–881. funded by the Medical Research Council (grant no. G0801001) and Frenzke, M., Sawatsky, B., Wong, X. X., Delpeut, S., Mateo, M., ZonMW (grant no. 91208012). The funders had no role in study Cattaneo, R. & von Messling, V. (2013). design, data collection and analysis, decision to publish or Nectin-4-dependent measles preparation of the manuscript. virus spread to the cynomolgus monkey tracheal epithelium: role of infected immune cells infiltrating the lamina propria. J Virol 87, 2526–2534. REFERENCES Fulton, R. E. & Middleton, P. J. (1975). Immunofluorescence in diagnosis of measles infections in children. J Pediatr 86, 17–22. Anderson, R. M. & May, R. M. (1982). Directly transmitted infections Griffin, D. E. (2007). Measles virus. In Fields Virology, 5th edn, pp. diseases: control by vaccination. Science 215, 1053–1060. 1551–1585. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Bloch, A. B., Orenstein, W. A., Ewing, W. M., Spain, W. H., Mallison, Lippincott Williams & Wilkins. G. F., Herrmann, K. L. & Hinman, A. R. (1985). 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