Early target cells of measles virus after aerosol infection of non-human primates.
ABSTRACT Measles virus (MV) is highly infectious, and has long been thought to enter the host by infecting epithelial cells of the respiratory tract. However, epithelial cells do not express signaling lymphocyte activation molecule (CD150), which is the high-affinity cellular receptor for wild-type MV strains. We have generated a new recombinant MV strain expressing enhanced green fluorescent protein (EGFP), based on a wild-type genotype B3 virus isolate from Khartoum, Sudan (KS). Cynomolgus macaques were infected with a high dose of rMV(KS)EGFP by aerosol inhalation to ensure that the virus could reach the full range of potential target cells throughout the entire respiratory tract. Animals were euthanized 2, 3, 4 or 5 days post-infection (d.p.i., n = 3 per time point) and infected (EGFP(+)) cells were identified at all four time points, albeit at low levels 2 and 3 d.p.i. At these earliest time points, MV-infected cells were exclusively detected in the lungs by fluorescence microscopy, histopathology and/or virus isolation from broncho-alveolar lavage cells. On 2 d.p.i., EGFP(+) cells were phenotypically typed as large mononuclear cells present in the alveolar lumen or lining the alveolar epithelium. One to two days later, larger clusters of MV-infected cells were detected in bronchus-associated lymphoid tissue (BALT) and in the tracheo-bronchial lymph nodes. From 4 d.p.i. onward, MV-infected cells were detected in peripheral blood and various lymphoid tissues. In spite of the possibility for the aerosolized virus to infect cells and lymphoid tissues of the upper respiratory tract, MV-infected cells were not detected in either the tonsils or the adenoids until after onset of viremia. These data strongly suggest that in our model MV entered the host at the alveolar level by infecting macrophages or dendritic cells, which traffic the virus to BALT or regional lymph nodes, resulting in local amplification and subsequent systemic dissemination by viremia.
- SourceAvailable from: ncbi.nlm.nih.gov[show abstract] [hide abstract]
ABSTRACT: The paramyxovirus family contains established human pathogens such as the measles virus and human respiratory syncytial virus, as well as emerging pathogens including the Hendra and Nipah viruses and the recently identified human metapneumovirus. Two major envelope glycoproteins, the attachment protein and the fusion protein, promote the processes of viral attachment and virus-cell membrane fusion required for entry. Although common mechanisms of fusion protein proteolytic activation and the mechanism of membrane fusion promotion have been shown in recent years, considerable diversity exists in the family relating to receptor binding and the potential mechanisms of fusion triggering.FEBS Journal 10/2009; 276(24):7217-27. · 4.25 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Measles virus normally causes disease in human beings, and the host range of this virus may be determined by a specific receptor on the surface of primate cells. Human-rodent somatic cell hybrids were tested for their ability to bind measles virus, and only cells that contained human chromosome 1 were capable of binding virus. A study of lymphocyte markers suggested that the complement regulator known either as membrane cofactor protein or CD46 was the measles virus receptor. We proved this hypothesis by demonstrating that hamster cell lines that expressed human CD46 could subsequently bind virus. Furthermore, infected CD46+ cells produced syncytia and viral proteins. Finally, polyclonal antisera against CD46 inhibited virus binding and infection. These results prove that human CD46 permits cells both to bind measles virus and to support infection.Cell 11/1993; · 31.96 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: A monoclonal antibody (MCI20.6) which inhibited measles virus (MV) binding to host cells was previously used to characterize a 57- to 67-kDa cell surface glycoprotein as a potential MV receptor. In the present work, this glycoprotein (gp57/67) was immunopurified, and N-terminal amino acid sequencing identified it as human membrane cofactor protein (CD46), a member of the regulators of complement activation gene cluster. Transfection of nonpermissive murine cells with a recombinant expression vector containing CD46 cDNA conferred three major properties expected of cells permissive to MV infection. First, expression of CD46 enabled MV to bind to murine cells. Second, the CD46-expressing murine cells were able to undergo cell-cell fusion when both MV hemagglutinin and MV fusion glycoproteins were expressed after infection with a vaccinia virus recombinant encoding both MV glycoproteins. Third, M12.CD46 murine B cells were able to support MV replication, as shown by production of infectious virus and by cell biosynthesis of viral hemagglutinin after metabolic labeling of infected cells with [35S]methionine. These results show that the human CD46 molecule serves as an MV receptor allowing virus-cell binding, fusion, and viral replication and open new perspectives in the study of MV pathogenesis.Journal of Virology 11/1993; 67(10):6025-32. · 5.08 Impact Factor
Early Target Cells of Measles Virus after Aerosol Infection
of Non-Human Primates
Ken Lemon1., Rory D. de Vries2., Annelies W. Mesman3, Stephen McQuaid4, Geert van Amerongen2,
Selma Yu ¨ksel2, Martin Ludlow1,2, Linda J. Rennick1, Thijs Kuiken2, Bertus K. Rima1, Teunis B. H.
Geijtenbeek3, Albert D. M. E. Osterhaus2, W. Paul Duprex1,5*, Rik L. de Swart2
1Centre for Infection and Immunity, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University of Belfast, Belfast, United Kingdom, 2Department of
Virology, Erasmus MC, Rotterdam, The Netherlands, 3Centre for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands, 4Tissue
Pathology, Belfast Health and Social Care Trust, Queen’s University of Belfast, Belfast, United Kingdom, 5Department of Microbiology, Boston University School of
Medicine, Boston, Massachusetts, United States of America
Measles virus (MV) is highly infectious, and has long been thought to enter the host by infecting epithelial cells of the
respiratory tract. However, epithelial cells do not express signaling lymphocyte activation molecule (CD150), which is the
high-affinity cellular receptor for wild-type MV strains. We have generated a new recombinant MV strain expressing
enhanced green fluorescent protein (EGFP), based on a wild-type genotype B3 virus isolate from Khartoum, Sudan (KS).
Cynomolgus macaques were infected with a high dose of rMVKSEGFP by aerosol inhalation to ensure that the virus could
reach the full range of potential target cells throughout the entire respiratory tract. Animals were euthanized 2, 3, 4 or 5
days post-infection (d.p.i., n=3 per time point) and infected (EGFP+) cells were identified at all four time points, albeit at low
levels 2 and 3 d.p.i. At these earliest time points, MV-infected cells were exclusively detected in the lungs by fluorescence
microscopy, histopathology and/or virus isolation from broncho-alveolar lavage cells. On 2 d.p.i., EGFP+cells were
phenotypically typed as large mononuclear cells present in the alveolar lumen or lining the alveolar epithelium. One to two
days later, larger clusters of MV-infected cells were detected in bronchus-associated lymphoid tissue (BALT) and in the
tracheo-bronchial lymph nodes. From 4 d.p.i. onward, MV-infected cells were detected in peripheral blood and various
lymphoid tissues. In spite of the possibility for the aerosolized virus to infect cells and lymphoid tissues of the upper
respiratory tract, MV-infected cells were not detected in either the tonsils or the adenoids until after onset of viremia. These
data strongly suggest that in our model MV entered the host at the alveolar level by infecting macrophages or dendritic
cells, which traffic the virus to BALT or regional lymph nodes, resulting in local amplification and subsequent systemic
dissemination by viremia.
Citation: Lemon K, de Vries RD, Mesman AW, McQuaid S, van Amerongen G, et al. (2011) Early Target Cells of Measles Virus after Aerosol Infection of Non-Human
Primates. PLoS Pathog 7(1): e1001263. doi:10.1371/journal.ppat.1001263
Editor: Christopher Richardson, Dalhousie University, Canada
Received September 10, 2010; Accepted December 23, 2010; Published January 27, 2011
Copyright: ? 2011 Lemon et al. 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.
Funding: This work was supported by ZonMw (grant# 91208012), MRC (grant# G0801001) and the VIRGO consortium, an innovative cluster approved by the
Netherlands Genomics Initiative and partially funded by the Dutch Government (grant# BSIK03012). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: A.D.M.E. Osterhaus wishes to declare, for the avoidance of any misunderstanding on competing interests, that he founded and is chief
scientific officer of Viroclinics, a company set up in collaboration with Erasmus University. 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.
* E-mail: email@example.com
. These authors contributed equally to this work.
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 approx-
imately two weeks after infection and include fever, rash, cough,
coryza and conjunctivitis . Measles is associated with a transient
but profound immunosuppression, resulting in increased suscep-
tibility to opportunistic infections. While significant progress has
recently been made in global control programs, 164,000 deaths
were still attributed to measles in 2008 .
It has been well established that MV infects cells via receptor-
dependent fusion of the virion at the plasma membrane . 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 MVstrains were ableto utilize this molecule
as an entry receptor . 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,
whereasvaccineand laboratory-adapted strainscan useeitherCD46
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 CD1502epithelial or neuronal cells [9–11].
Previously, we successfully infected macaques with a recombi-
nant MV based on the pathogenic IC323 strain  that expresses
PLoS Pathogens | www.plospathogens.org1January 2011 | Volume 7 | Issue 1 | e1001263
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 . 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 . In a more recent
study, macaques were infected with a rMVIC323that 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 .
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 . However, a
number of CD1502cell 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 CD1502cells 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 . 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 . 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 . 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
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 . A
consensus sequence of the complete viral genome was derived de
novo, including the 39 and 59 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 rMVKSand 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
Early rMVKSEGFP replication in the respiratory tract
Four groups of three cynomolgus macaques were infected with
rMVKSEGFP via the aerosol route as described previously .
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
Measles remains an important vaccine-preventable cause
of morbidity and mortality in developing countries. The
causative agent, measles virus (MV), is one of the most
contagious viruses known. Measles has an incubation time
of approximately two weeks, and surprisingly little is
known about the early events after MV infection. Epithelial
cells in the upper respiratory tract have long been
considered as early target cells, but more recently alveolar
macrophages (AM) and dendritic cells (DC) have been
proposed as alternatives. We have infected cynomolgus
macaques with a high dose of a recombinant EGFP-
expressing MV strain via aerosol inhalation, to ensure that
the virus had access to the entire respiratory tract. At 2
days post-infection, MV-infected mononuclear cells were
detectable in the alveolar lumen but not in the upper
respiratory tract. These infected cells migrated through the
bronchus-associated lymphoid tissue to the draining
tracheo-bronchial lymph node at 3 days post-infection.
Systemic infection was initiated from this point, as
observed in macaques euthanized 4 or 5 days post-
infection. Thus, even though the aerosolized virus had
access to epithelial cells and lymphoid tissues along the
entire respiratory tract, AM and DC in the lungs were the
first cells that sustained MV replication.
Early Target Cells of Measles Virus in Macaques
PLoS Pathogens | www.plospathogens.org2January 2011 | Volume 7 | Issue 1 | e1001263
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
Figure 1. Generation and growth of rMVKSEGFP. (A) Plasmids generated after RT-PCR, cloning and sequencing of MV RNA isolated from MVKS-
infected PBMC. pMVKSis a full-length plasmid containing the complete antigenome of MVKSand pMVKSEGFP was modified by the insertion of an ATU
at the promoter proximal position containing the ORF encoding EGFP. (B) rMVKSand 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, CCID50was determined in an endpoint titration test.
Measurements shown are averages of triplicates 6 SD. Key: h.p.i.: hours post infection.
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.
Early Target Cells of Measles Virus in Macaques
PLoS Pathogens | www.plospathogens.org3January 2011 | Volume 7 | Issue 1 | e1001263
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).
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 retropha-
ryngeal 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).
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 de-
termine 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. .
Analysis of BALT structures in the lungs of non-infected
macaques indicated that even though cytokeratin-positive epithe-
lial 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 immunohisto-
chemical 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
Table 1. Dissemination of MV in tissues during early stage of
Days post-infection (d.p.i.)
Numbers indicate the number of macaques with EGFP+cells in this tissue
1LN: lymph node.
2PBMC: peripheral blood mononuclear cells.
3GALT: gut-associated lymphoid tissue.
4-: no EGFP+cells detected.
Early Target Cells of Measles Virus in Macaques
PLoS Pathogens | www.plospathogens.org4January 2011 | Volume 7 | Issue 1 | e1001263
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
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 rMVKSand 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 . Development of a second recombi-
nant 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 . 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 mm, allowing the inoculum to deposit both in the
upper respiratory and lower respiratory tract, and reach the
alveolar lumina. A high infectious dose (106CCID50) 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
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.
Early Target Cells of Measles Virus in Macaques
PLoS Pathogens | www.plospathogens.org5 January 2011 | Volume 7 | Issue 1 | e1001263
Early Target Cells of Measles Virus in Macaques
PLoS Pathogens | www.plospathogens.org6January 2011 | Volume 7 | Issue 1 | e1001263
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 , both cell types previously
described as preferential targets for MV in lymphoid tissue at later
time points . 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 . 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 . 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 . 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 .
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 .
Furthermore, the epithelium of BALT of many mammalian
species contains M-cells , 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 ,
Mycobacterium tuberculosis ). Reoviruses have also been described
to be taken up by M-cells, with subsequent spread to the regional
lymph nodes . 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,
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
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).
Early Target Cells of Measles Virus in Macaques
PLoS Pathogens | www.plospathogens.org7 January 2011 | Volume 7 | Issue 1 | e1001263
consisting of tonsils and adenoids. Tonsils and adenoids are lined
by CD1502epithelial 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
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
rMVKSEGFP is based on a wild-type genotype B3 virus isolated
from PBMC collected in 1997 from a severe measles case in
Khartoum, Sudan . The clinical isolate (MVKS) was passaged
exclusively in CD150+human B-LCL and was previously shown
to be highly pathogenic in macaques . Total RNA was isolated
from B-LCL infected with MVKSand the complete consensus
genomic sequence determined following RT-PCR (GenBank
accession number HM439386). The sequences of the genomic
termini were confirmed by 59 RACE. A full-length cDNA which
expressed the MVKSanti-genome (pMVKS) was constructed based
on a modified pBluescript vector . 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 rMVI-
C323EGFP 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 280uC,
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
(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 . This
nebulizer generates a small particle size aerosol (VMD 4–6 mm),
which is deposited on epithelia throughout the entire respiratory
tract upon inhalation . A total dose of 106CCID50 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 105CCID50, of which based on previous studies
approximately 10% is expected to reach epithelia in the lungs .
Animals were euthanized on 2, 3, 4 or 5 d.p.i. (n=3 per time
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 .
Briefly, fluorescence was detected with a custom-made lamp
containing 6 LEDs (peak emission 490–495nm); emitted fluores-
cence was detected through an amber cover of a UV trans-
illuminator 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 autofluores-
cence) 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 mg/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
Early Target Cells of Measles Virus in Macaques
PLoS Pathogens | www.plospathogens.org8 January 2011 | Volume 7 | Issue 1 | e1001263
microscope (Zeiss). Images and videos were generated using Zen
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 mg/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 . Virus
isolations were monitored by UV microscopy for EGFP fluores-
cence after co-cultivation with B-LCL for 3–6 days and results were
expressed as number of virus-infected cells per 106total cells.
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 lactalbu-
mine enzymatic hydrolysate, penicillin, streptomycin, polymyxine
B sulphate, nystatin, gentamicin and glycerol) and frozen at
280uC. After thawing samples were vortexed, the swab was
removed and the remaining transport medium was used for virus
isolation . Isolation of MV was performed on Vero-SLAM
cells using an infectious center test as previously described .
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 were collected during necropsy in PBS for
direct preparation of single cell suspensions using cell strainers
with a 100 mm 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.
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 106events
were obtained per sample to allow detection of low frequent
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 mm) 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 4uC,
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
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
Found at: doi:10.1371/journal.ppat.1001263.s001 (2.21 MB TIF)
CD11c+ DC and macrophages targeted in the lung at
Found at: doi:10.1371/journal.ppat.1001263.s002 (9.75 MB TIF)
Single color images for figure 4C and 5C.
scans. Four pathology slides were scanned and digitized at a high
resolution and annotated.
Found at: doi:10.1371/journal.ppat.1001263.s003 (0.06 MB PDF)
On-line immunohistochemical and H&E pathology
figure 2, panel Bi.
Found at: doi:10.1371/journal.ppat.1001263.s004 (1.91 MB
Z-stack as rendered 3D-movie corresponding to
figure 2, panel Bii.
Found at: doi:10.1371/journal.ppat.1001263.s005 (2.16 MB
Z-stack as rendered 3D-movie corresponding to
figure 2, panel Biii.
Found at: doi:10.1371/journal.ppat.1001263.s006 (1.51 MB
Z-stack as rendered 3D-movie corresponding to
Early Target Cells of Measles Virus in Macaques
PLoS Pathogens | www.plospathogens.org9January 2011 | Volume 7 | Issue 1 | e1001263
The authors would like to thank Latoya Sarijoen, Tien Nguyen, Joyce
Verburgh, Monique van Velzen and Robert Dias D’Ullois for practical
assistance and are grateful to Yusuke Yanagi for providing the Vero-SLAM
cell line. We would also like to thank the staff of the Tissue Core
Technology Unit, QUB for their histological expertise. Furthermore, we
are grateful to Zeiss for the ‘‘on loan use’’ of the LSM700 system fitted on
an Axio Observer Z1 inverted microscope.
Conceived and designed the experiments: RDdV WPD RLdS. Performed
the experiments: KL RDdV AWM SM GvA SY ML LJR WPD RLdS.
Analyzed the data: KL RDdV AWM SM TK BKR TBHG ADMEO
WPD RLdS. Wrote the paper: KL RDdV TK WPD RLdS.
1. Griffin DE (2007) Measles virus. In: Knipe DM, Howley PM, eds. Fields
Virology. Philadelphia: Lippincott Williams & Wilkins. pp 1551–1585.
2. WHO (2009) Global reductions in measles mortality 2000–2008 and the risk of
measles resurgence. Wkly Epidemiol Rec 84: 509–516.
3. Smith EC, Popa A, Chang A, Masante C, Dutch RE (2009) Viral entry
mechanisms: the increasing diversity of paramyxovirus entry. FEBS J 276:
4. Do ¨rig RE, Marcil A, Chopra A, Richardson CD (1993) The human CD46
molecule is a receptor for measles virus (Edmonston strain). Cell 75: 295–305.
5. Naniche D, Varior-Krishnan G, Cervoni F, Wild TF, Rossi B, et al. (1993)
Human membrane cofactor protein (CD46) acts as a cellular receptor for
measles virus. J Virol 67: 6025–6032.
6. Buckland R, Wild TF (1997) Is CD46 the cellular receptor for measles virus?
Virus Res 48: 1–9.
7. Tatsuo H, Ono N, Tanaka K, Yanagi Y (2000) SLAM (CDw150) is a cellular
receptor for measles virus. Nature 406: 893–897.
8. Yanagi Y, Takeda M, Ohno S, Seki F (2006) Measles virus receptors and
tropism. Jpn J Infect Dis 59: 1–5.
9. Leonard VHJ, Sinn PL, Hodge G, Miest T, Devaux P, et al. (2008) Measles virus
blind to its epithelial cell receptor remains virulent in rhesus monkeys but cannot
cross the airway epithelium and is not shed. J Clin Invest 118: 2448–2458.
10. Tahara M, Takeda M, Shirogane Y, Hashiguchi T, Ohno S, et al. (2008)
Measles virus infects both polarized epithelial and immune cells using distinctive
receptor-binding sites on its hemagglutinin. J Virol 82: 4630–4637.
11. Takeda M, Tahara M, Hashiguchi T, Sato TA, Jinnouchi F, et al. (2007) A
human lung carcinoma cell line supports efficient measles virus growth and
syncytium formation via SLAM- and CD46-independent mechanism. J Virol 81:
12. Takeda M, Takeuchi K, Miyajima N, Kobune F, Ami Y, et al. (2000) Recovery
of pathogenic measles virus from cloned cDNA. J Virol 74: 6643–6647.
13. Hashimoto K, Ono N, Tatsuo H, Minagawa H, Takeda M, et al. (2002) SLAM
(CD150)-independent measles virus entry as revealed by recombinant virus
expressing green fluorescent protein. J Virol 76: 6743–6749.
14. De Swart RL, Ludlow M, De Witte L, Yanagi Y, Van Amerongen G, et al.
(2007) Predominant infection of CD150+ lymphocytes and dendritic cells during
measles virus infection of macaques. PLoS Pathog 3: e178.
15. Leonard VH, Hodge G, Reyes-del VJ, McChesney MB, Cattaneo R (2010)
Measles virus selectively blind to signaling lymphocytic activation molecule
(SLAM; CD150) is attenuated and induces strong adaptive immune responses in
rhesus monkeys. J Virol 84: 3413–3420.
16. Ludlow M, Rennick L, Sarlang S, Skibinski G, McQuaid S, et al. (2010) Wild-
type measles virus infection of primary epithelial cells occurs via the basolateral
surface without syncytium formation or release of infectious virus. J Gen Virol
17. Shirogane Y, Takeda M, Tahara M, Ikegame S, Nakamura T, et al. (2010)
Epithelial-mesenchymal transition abolishes the susceptibility of polarized
epithelial cell lines to measles virus. J Biol Chem 285: 20882–20890.
18. Von Messling V, Svitek N, Cattaneo R (2006) Receptor (SLAM [CD150])
recognition and the V protein sustain swift lymphocyte-based invasion of
mucosal tissue and lymphatic organs by a morbillivirus. J Virol 80: 6084–6092.
19. De Witte L, Abt M, Schneider-Schaulies S, van Kooyk Y, Geijtenbeek TBH
(2006) Measles virus targets DC-SIGN to enhance dendritic cell infection. J Virol
20. De Witte L, De Vries RD, Van der Vlist M, Yu ¨ksel S, Litjens M, et al. (2008)
DC-SIGN and CD150 have distinct roles in transmission of measles virus from
dendritic cells to T-lymphocytes. PLoS Pathog 4: e1000049.
21. El Mubarak HS, Van de Bildt MWG, Mustafa OA, Vos HW, Mukhtar MM,
et al. (2000) Serological and virological characterization of clinically diagnosed
cases of measles in suburban Khartoum. J Clin Microbiol 38: 987–991.
22. El Mubarak HS, Van de Bildt MWG, Mustafa OA, Vos HW, Mukhtar MM,
et al. (2002) Genetic characterisation of wild type measles viruses circulating in
suburban Khartoum, 1997–2000. J Gen Virol 83: 1437–1443.
23. Ibrahim SA, Mustafa OM, Mukhtar MM, Saleh IA, El Mubarak HS, et al.
(2002) Measles in suburban Khartoum: an epidemiological and clinical study.
Trop Med Int Health 7: 442–449.
24. El Mubarak HS, Yu ¨ksel S, Van Amerongen G, Mulder PGH, Mukhtar MM,
et al. (2007) Infection of cynomolgus macaques (Macaca fascicularis) and rhesus
macaques (Macaca mulatta) with different wild-type measles viruses. J Gen Virol
25. De Vries RD, Lemon K, Ludlow M, McQuaid S, Yuksel S, et al. (2010) In vivo
tropism of attenuated and pathogenic measles virus expressing green fluorescent
protein in macaques. J Virol 84: 4714–4724.
26. Von Messling V, Milosevic D, Cattaneo R (2004) Tropism illuminated:
lymphocyte-based pathways blazed by lethal morbillivirus through the host
immune system. Proc Natl Acad Sci USA 101: 14216–14421.
27. Kawamata N, Xu B, Nishijima H, Aoyama K, Kusumoto M, et al. (2009)
Expression of endothelia and lymphocyte adhesion molecules in bronchus-
associated lymphoid tissue (BALT) in adult human lung. Respir Res 10: 97.
28. Ferreira CS, Frenzke M, Leonard VH, Welstead GG, Richardson CD, et al.
(2010) Measles virus infection of alveolar macrophages and dendritic cells
precedes spread to lymphatic organs in transgenic mice expressing human
signaling lymphocytic activation molecule (SLAM, CD150). J Virol 84:
29. Sternberg S (1997) Histology for pathologists. Philadelphia: Lippincott & Raven
30. Pabst R, Tschernig T (2010) Bronchus-associated lymphoid tissue: an entry site
for antigens for successful mucosal vaccinations? Am J Respir Cell Mol Biol 43:
31. Toyoshima M, Chida K, Sato A (2000) Antigen uptake and subsequent cell
kinetics in bronchus-associated lymphoid tissue. Respirology 5: 141–145.
32. Teitelbaum R, Schubert W, Gunther L, Kress Y, Macaluso F, et al. (1999) The
M cell as a portal of entry to the lung for the bacterial pathogen Mycobacterium
tuberculosis. Immunity 10: 641–650.
33. Morin MJ, Warner A, Fields BN (1994) A pathway for entry of reoviruses into
the host through M cells of the respiratory tract. J Exp Med 180: 1523–1527.
34. Lemon K, Rima BK, McQuaid S, Allen IV, Duprex WP (2007) The F gene of
rodent brain-adapted mumps virus is a major determinant of neurovirulence.
J Virol 81: 8293–8302.
35. Dubus JC, Vecellio L, De Monte M, Fink JB, Grimbert D, et al. (2005) Aerosol
deposition in neonatal ventilation. Pediatr Res 58: 10–14.
36. El Mubarak HS, De Swart RL, Osterhaus ADME, Schutten M (2005)
Development of a semi-quantitative real-time RT-PCR for the detection of
measles virus. J Clin Virol 32: 313–317.
Early Target Cells of Measles Virus in Macaques
PLoS Pathogens | www.plospathogens.org10 January 2011 | Volume 7 | Issue 1 | e1001263