Measles Vaccination of Nonhuman Primates Provides Partial Protection against Infection with Canine Distemper Virus

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Rory D. de Vries, Martin Ludlow, R. Joyce Verburgh, Geert van Amerongen, Selma Yüksel, D. Tien Nguyen, Stephen McQuaid, Albert D. M. E. Osterhaus, W. Paul Duprex and Rik L. de Swart J. Virol. 2014, 88(8):4423. DOI: 10.1128/JVI.03676-13. Published Ahead of Print 5 February 2014.

Measles Vaccination of Nonhuman Primates Provides Partial Protection against Infection with Canine Distemper Virus Department of Viroscience, Erasmus MC, Rotterdam, the Netherlandsa; Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts, USAb; Northern Ireland Molecular Pathology Laboratory and Belfast Health and Social Care Trust, The Queen’s University of Belfast, Northern Ireland, United Kingdomc

ABSTRACT

Measles virus (MV) is being considered for global eradication, which would likely reduce compliance with MV vaccination. As a result, children will grow up without MV-specific immunity, creating a potential niche for closely related animal morbilliviruses such as canine distemper virus (CDV). Natural CDV infection causing clinical signs has never been reported in humans, but recent outbreaks in captive macaques have shown that CDV can cause disease in primates. We studied the virulence and tropism of recombinant CDV expressing enhanced green fluorescent protein in naive and measles-vaccinated cynomolgus macaques. In naive animals CDV caused viremia and fever and predominantly infected CD150ⴙ lymphocytes and dendritic cells. Virus was reisolated from the upper and lower respiratory tracts, but infection of epithelial or neuronal cells was not detectable at the time points examined, and the infections were self-limiting. This demonstrates that CDV readily infects nonhuman primates but suggests that additional mutations are necessary to achieve full virulence in nonnatural hosts. Partial protection against CDV was observed in measles-vaccinated macaques, as demonstrated by accelerated control of virus replication and limited shedding from the upper respiratory tract. While neither CDV infection nor MV vaccination induced detectable cross-reactive neutralizing antibodies, MV-specific neutralizing antibody levels of MV-vaccinated macaques were boosted by CDV challenge infection, suggesting that cross-reactive VN epitopes exist. Rapid increases in white blood cell counts in MV-vaccinated macaques following CDV challenge suggested that cross-reactive cellular immune responses were also present. This study demonstrates that zoonotic morbillivirus infections can be controlled by measles vaccination. IMPORTANCE

Throughout history viral zoonoses have had a substantial impact on human health. Given the drive toward global eradication of measles, it is essential to understand the zoonotic potential of animal morbilliviruses. Morbilliviruses are thought to have evolved from a common ancestral virus that jumped species and adapted to new hosts. Recently, canine distemper virus (CDV), a morbillivirus normally restricted to carnivores, caused disease outbreaks in nonhuman primates. Here, we report that experimental CDV infection of monkeys resulted in fever and leukopenia. The virus replicated to high levels in lymphocytes but did not spread to epithelial cells or the central nervous system. Importantly, like measles virus in macaques, the infections were selflimiting. In measles-vaccinated macaques CDV was cleared more rapidly, resulting in limited virus shedding from the upper respiratory tract. These studies demonstrate that although CDV can readily infect primates, measles immunity is protective, and CDV infection is self-limiting.

C

anine distemper virus (CDV) is a member of the family Paramyxoviridae, genus Morbillivirus, and has been described as an important pathogen of dogs since the 17th century (1). Like the closely related measles virus (MV), CDV initially targets lymphoid tissues and causes profound lymphopenia and immune suppression (2, 3). Subsequently, the virus is disseminated to peripheral tissues, resulting in large-scale infection of epithelial cells. Infection of the central nervous system (CNS) is common, while this is a rare complication in measles. This difference, together with the severe immune suppression resulting in impaired CDVspecific immune responses, may explain the high case fatality rates observed in canine distemper compared to the low mortality rates reported for measles. CD150 or signaling lymphocyte activation molecule F1 (SLAM/F1) is the primary entry receptor for wild-type morbilliviruses on immune cells (4, 5). A recombinant CDV (rCDV) with reduced binding affinity to CD150 was severely attenuated in ferrets, demonstrating that infection of CD150⫹ lymphocytes and dendritic cells (DC) is vital for entry and virulence (6). Poliovirus

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receptor-related 4 (PVRL4) was recently described as a cellular receptor for MV on epithelial cells (7, 8). PVRL4 is expressed in the adherens junctions at the basolateral surface of differentiated epithelial cells, explaining why morbilliviruses do not infect epithelial cells apically. Canine PVRL4 was recently demonstrated to be an entry receptor for CDV in the late stages of the disease (9). When ferrets were infected with an rCDV unable to infect epithelial cells, the virus replicated in lymphocytes and caused lymphopenia, but the ferrets did not display fever or rash; CDV could not be isolated from the throat, and epithelial cell infection was

Received 11 December 2013 Accepted 28 January 2014 Published ahead of print 5 February 2014 Editor: D. S. Lyles Address correspondence to Rik L. de Swart, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.03676-13

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Rory D. de Vries,a Martin Ludlow,b R. Joyce Verburgh,a Geert van Amerongen,a Selma Yüksel,a D. Tien Nguyen,a Stephen McQuaid,c Albert D. M. E. Osterhaus,a W. Paul Duprex,b Rik L. de Swarta

de Vries et al.

MATERIALS AND METHODS Ethics statement. Animal experiments were conducted in compliance with European guidelines (EU directive on animal testing 86/609/EEC) and Dutch legislation (Experiments on Animals Act, 1997). The protocols were approved by the independent animal experimentation ethical review

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committee DCC in Driebergen, The Netherlands. This committee is not affiliated with the Erasmus MC, where the experiments were performed. Animals were housed in groups prior to CDV challenge, received standard primate feed and fresh fruit on a daily basis, and had access to water ad libitum. In addition, their cages contained several sources of environmental enrichment, for example, hiding places, hanging ropes, tires, and other toys. During the infection study animals were housed in HEPA-filtered, negatively pressurized biosafety level 3 (BSL-3) isolator cages. Animal welfare was observed on daily basis, and all animal handling was performed under light anesthesia using ketamine and medetomidine. After handling, atipamezole was administered to antagonize the effect of medetomidine. Viruses. CDV strains used in this study were Snyder-Hill (SH) and Ohio R252 (26). MV strains used were the vaccine strain EdmonstonZagreb (EZ) and wild-type strain Khartoum Sudan (KS). These viruses were selected because we had recombinant viruses expressing EGFP from an additional transcription unit that had been well characterized in vitro and in vivo. In the nomenclature used for the viruses, r refers to recombinant, the superscript symbol refers to the strain name, and the number in parentheses refers to the position of the additional transcription unit encoding EGFP in the viral genome: rCDVSHEGFP(6), rCDVR252EGFP(6), MVEZ, and rMVKSEGFP(3). Animal study design. Nine CDV- and MV-seronegative and six MVEZ-vaccinated cynomolgus macaques (Macaca fascicularis) were used for the infection studies. All animals were males between 3 and 5 years of age. A temperature probe was implanted intraperitoneally 2 weeks before the beginning of the experiments to monitor body temperature noninvasively. CDV- and MV-seronegative animals (n ⫽ 3 per group) were infected with a high dose of rCDVSHEGFP(6), low dose of rCDVSHEGFP(6), or a high dose of rCDVR252EGFP(6). MV-vaccinated animals had received an intratracheal (i.t.) infection with MVEZ (104 50% tissue culture infectious doses [TCID50]) 10 months before inclusion in the CDV study as part of another experiment. In the current study, they were infected with either a high (n ⫽ 3) or low (n ⫽ 3) dose of rCDVSHEGFP(6). Animals receiving a high viral dose were infected with 106 TCID50 of CDV, of which 50% of the inoculum was administered by the i.t route, 40% was administered by the intranasal (i.n.) route, and 10% was administered onto the eyes. Animals receiving a low dose were infected with 105 TCID50 of CDV exclusively via i.t. inoculation. Animals were euthanized at 6, 10, or 14 days postinfection (dpi) (n ⫽ 1/group/ time point). Necropsy. Animals were euthanized by exsanguination under deep ketamine/medetomidine anesthesia. Macroscopic detection of EGFP was performed as described previously (22–24). During necropsy, tissues from the upper and lower respiratory tract, including the nasal concha, nasal septum, trachea, primary bronchus, and lungs, were harvested and directly screened for EGFP expression. The lungs were inflated with 2% (wt/vol) low-melting-point agarose before being screened, as described previously (24, 27). After screening, tissues were transferred to buffered formalin (FA). Nonlymphoid tissues were collected directly in FA; lymphoid tissues were collected in either FA for immunohistochemistry or phosphate-buffered saline (PBS) for preparation of single-cell suspensions using cell strainers with a 100-␮m pore size (BD Biosciences) and direct use for flow cytometry. Blood samples. Small-volume blood samples were collected in Vacuette tubes (Greiner) containing K3EDTA as an anticoagulant at 3, 6, 8, 10, 12, and 14 dpi. Total white blood cell (WBC) counts were obtained using an automated counter (pocH-100iV; Sysmex). Plasma was separated by centrifugation, heat inactivated (30 min at 56°C), clarified and stored at ⫺20°C. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation, washed, and resuspended in complete RPMI 1640 medium (Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 2 mM L-glutamine, 10% (vol/vol) heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 ␮g/ml). Cells were counted using a hemocytometer and used directly for flow cytometry and virus isolation. Isolation of CDV was performed on Vero-

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not detected by immunohistochemistry (10). This suggested that epithelial cell infection is important for clinical disease and virus transmission, as was previously hypothesized for MV (11). Even though CDV was originally described as an infectious disease of dogs, it naturally infects a wide range of carnivores and has a relatively high propensity to cross species barriers. The virus has been reported to cause disease in members of the carnivore families of Ailuridae, Felidae, Hyaenidae, Mustelidae, Procyonidae, Ursidae, Viverridae, and Phocidae (12). In addition, CDV has also been reported to infect javelinas (13) and was recently detected in rodents (14). A natural outbreak with CDV in nonhuman primates was first reported in 1989 when 22 Japanese macaques (Macaca fuscata) housed in an animal research facility seroconverted to CDV (15). More recently, dramatic outbreaks in breeding colonies of rhesus macaques (Macaca mulatta) in China and cynomolgus macaques (Macaca fascicularis) in Japan were described (16, 17). Collectively these outbreaks included more than 10,000 animals and resulted in case fatality rates of 5 to 30%. The main cause of death was pneumonia, and a few animals displayed neurological signs. The frequent detection of secondary pathogens suggests that the high case fatality rates were probably related to opportunistic infections resulting from CDV-induced immune suppression. It remains unclear whether the viruses that caused these outbreaks had adapted to primates as experimental infection of macaques failed to recapitulate the dramatic clinical signs or lead to the death of the animals. The World Health Organization is considering measles as a target for global eradication. Although measles eradication would save many lives, it will eventually result in reduced compliance to MV vaccination across the world. In this scenario children would grow up without morbillivirus-specific immunity, possibly creating a niche for animal morbilliviruses to cross the species barrier. Morbillivirus infections induce partial cross-protection, as illustrated by the fact that live attenuated MV vaccines protect dogs from CDV (18, 19). Thus, cessation of MV vaccination could facilitate cross-species infection and adaptation of animal morbilliviruses to humans. Although it has been claimed that this risk is low (20), the large CDV outbreaks in nonhuman primates suggest that animal morbilliviruses represent more than a theoretical risk for primates lacking morbillivirus immunity. Furthermore, new morbilli-related viruses are still being discovered, emphasizing the need for surveillance (21). We have previously used recombinant MV (rMV) expressing enhanced green fluorescent protein (EGFP) in conjunction with macroscopic and microscopic imaging techniques to detect virusinfected cells and study measles pathogenesis in the macaque model (22–25). Here, we show that cynomolgus macaques are susceptible to infection with non-primate-adapted rCDV strains that express EGFP, resulting in virus replication in CD150⫹ lymphocytes and DC. Epithelial or neuronal cell infection was not detected at the time points examined. In addition, we show that MV-vaccinated macaques were partially cross-protected from CDV challenge and showed reduced virus shedding from the upper respiratory tract.

Experimental CDV Infection of Macaques

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amidino-2-phenylindole (DAPI) hard-set mounting medium (Vector). Fluorescently stained tissue sections were assessed, and digital fluorescent images were acquired with a Leica DFC350 FX digital camera and processed using Leica FW4000 software. Infection of primary normal macaque bronchial epithelial cells. Primary well-differentiated normal macaque bronchial epithelial (wd-NMBE) cells were obtained from bronchi of euthanized control macaques, as described previously for humans (31). Undifferentiated NMBE cells were grown in type 1 collagen and fibronectin-coated T75 flasks, trypsinized at 60 to 80% confluence, and seeded into 6.5-mm transwell inserts with a 0.4-␮m pore size at 5 ⫻ 104 cells per insert as described previously (32). After the NMBE cells had polarized and developed cilia, the monolayers on filters were wounded by scratching with the tip of a pipette (25) and subsequently infected apically with 2 ⫻ 105 TCID50 of either rMVKSEGFP(3), rCDVSHEGFP(6), or rCDVR252EGFP(6). The inoculum was removed 90 min after infection. Cells were screened for infection at 72 h postinfection (hpi), fixed, permeabilized, and counterstained for cilia with anti-human ␤-tubulin IV. Cells were analyzed by confocal laser scanning microscopy using an LSM700 system fitted on an Axio Observer Z1 inverted microscope (Zeiss). Images were generated using Zen software. Statistical analysis. The initial experiments in which animals were infected with two different CDV strains using high or low inocula were not designed to allow statistical analysis as we used three animals per group. Therefore, we have chosen to present the individual measurements of each animal. The symbols used per animal are identical between the figures, which allows cross-comparison of the data. The comparison of CDV infection in naive or MV-vaccinated animals was performed in 12 animals, of which 6 were vaccinated. The animals all received the same virus, but in each group three animals received a high dose, and three received a low dose. For statistical analysis we ignored the differences in doses. Animals were euthanized at 6, 10, or 14 dpi, prohibiting the use of the area under the curve for statistical analysis. Therefore, we compared the groups at each sampling point, using Student’s t test on log-transformed data. If the data were not normally distributed, we used a nonparametric test (Mann-Whitney U test).

RESULTS

rCDVSHEGFP(6) and rCDVR252EGFP(6) efficiently replicate in macaques. Six morbillivirus-naive cynomolgus macaques were infected i.n., i.t., and onto the eyes with a high dose (106 TCID50) of rCDVSHEGFP(6) (n ⫽ 3) or rCDVR252EGFP(6) (n ⫽ 3). Three additional animals were infected with a 10-fold-lower dose of rCDVSHEGFP(6), delivered exclusively by i.t. inoculation. This route was chosen as it is a highly reproducible route of administration for experimental MV infection of macaques. Within each group, one animal was euthanized at 6, 10, or 14 dpi. Flow cytometry was used to detect EGFP directly in blood and BAL samples obtained at 3, 6, 8, 10, 12, and 14 dpi. EGFP-positive (EGFP⫹) cells were detected in the BAL fluid (Fig. 1A) and peripheral blood mononuclear cells (PBMC) (Fig. 1B) of all animals. In PBMC, rCDVSHEGFP(6) infected approximately 10 times more cells than rCDVR252EGFP(6) (Fig. 1B). In the low- and high-dose rCDVSHEGFP(6) infection groups, similar infection percentages were detected in BAL cells and PBMC although virus replication peaked approximately 1 or 2 days later in the low-dose group (Fig. 1A and B). Such a dose-dependent peak of replication was previously described for MV (33). Macroscopic detection of EGFP during the necropsies of the low- and high-dose rCDVSHEGFP(6)infected macaques at 6 dpi showed EGFP fluorescence in lymphoid tissues (Fig. 1C to G). rCDVSHEGFP(6) and rCDVR252EGFP(6) are virulent in the macaque and cause lymphopenia and fever. Virus isolations were performed from eye, nose, and throat swabs, from BAL cells,

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dogSLAM (VDS) cells (4) using an infectious center test as previously described (28). Virus isolations were monitored by UV microscopy for EGFP fluorescence after cocultivation with VDS for 3 to 6 days, and results were expressed as number of virus-infected cells/106 total cells. Bronchoalveolar lavage (BAL). A BAL was performed at 3, 6, 8, 10, 12 and 14 dpi by i.t. infusion of 10 ml of PBS through a flexible catheter. In animals that were sacrificed, a BAL was performed postmortem by direct infusion of PBS (10 ml) into the right-hand side of the lung. BAL cells were resuspended in culture medium with supplements as described above, counted, and used directly for flow cytometry and virus isolation. Virus isolation was performed on VDS cells as described for MV on VerohCD150 cells (22). Throat, nose, and eye swabs. Throat swabs (cytobrush plus; Medscand Medical) and nose and eye swabs (polyester-tipped minitip urethral swab; Copan) were collected at 3, 6, 8, 10, 12, and 14 dpi in transport medium (Eagle’s minimal essential medium [EMEM] with Hanks’ salts, supplemented with lactalbumin enzymatic hydrolysate, penicillin, streptomycin, polymyxin B sulfate, nystatin, gentamicin, and glycerol) and frozen at ⫺80°C. After being thawed, samples were vortexed, the swab was removed, and the remaining transport medium was used for virus isolation. Isolation of CDV was performed on VDS cells using an infectious center test as previously described (28). The isolations were screened for EGFP fluorescence at days 3 and 7 posttitration, and results are expressed as TCID50/ml. Serological analysis of macaque plasma and serum. Virus-neutralizing (VN) antibody responses were measured by an endpoint neutralization assay. Briefly, serial 2-log dilutions (starting at 2⫺2) of heat-inactivated plasma samples were incubated in triplicate with rCDVSHEGFP(6), rCDVR252EGFP(6), or the MV Edmonston strain (MVEd) for 1 h at 37°C in 96-well flat-bottom plates. Subsequently, trypsinized VDS (for CDV) or Vero (for MV) cells were added at a concentration of 1 ⫻ 104 cells/well. Plates were incubated for 5 to 7 days at 37°C and visually monitored for cytopathic effects. VN titers were calculated as the 50% endpoint of triplicate measurements using the method of Reed and Muench (29). The additional dilution caused by the addition of the virus was included in the titer calculation. MV-F and MV-H glycoprotein-specific serum IgG antibody levels were determined by flow cytometry as described previously (30). Flow cytometry. CDV-infected cells in PBMC were phenotyped by flow cytometry. PBMC were stained with CD3APC-Cy7 (where APC is allophycocyanin) (clone SP34-2; BD), CD4V450 (clone L200; BD), CD8AmCyan (clone SK1; BD), CD20PE-Cy7 (where PE is phycoerythrin) (clone L27; BD), CD14PerCP (where PerCP is peridinin chlorophyll protein) (clone M5E2; BD), and CD150PE (clone A12; BD). Lymphoid organs were stained with the same set of monoclonal antibodies, with the exception of CD14PerCP. Single-cell suspensions were stained with CD3APC-Cy7 (clone SP34-2; BD), CD20PE-Cy7 (clone L27; BD), HLA-DRPacificBlue (clone L243; Biolegend), and CD11cPE (clone BU15; AbD Serotec) to identify infected DC in lymphoid organs. The infection percentages within the populations were determined by detection of EGFP. All flow cytometry was performed on a FACSCanto II (BD). Histological and immunohistochemical analysis. Hematoxylin and eosin (H&E) staining was performed to evaluate histological changes. Immunohistochemical staining was performed using a fully automated BondMax immunostainer with a polymer-based peroxidase detection system. CDV-infected cells were detected using a polyclonal rabbit antibody to EGFP (Invitrogen). Dual-labeling indirect immunofluorescence was performed manually after pressure cooker pretreatment in 0.01 M citrate buffer, pH 9.0, using polyclonal rabbit anti-EGFP and monoclonal mouse antibodies to the macrophage/DC marker CD11c (clone 5D11; Novocastra), the T-lymphocyte marker CD3 (clone F7.2.38; Dako), the B-lymphocyte marker CD20 (clone L26; Dako), and the epithelial cell marker cytokeratin (clone AE1/AE3; Dako). 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 4=,6=-di-

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and from PBMC to confirm the pathogenicity of rCDVSHEGFP(6) and rCDVR252EGFP(6) in macaques and demonstrate that the EGFP⫹ cells produced infectious virus. Peak virus replication was reached at 6 to 8 dpi with both strains (Fig. 2A to E) although, in general, rCDVSHEGFP(6) replicated to higher levels than rCDVR252EGFP(6). Furthermore, all groups developed transient leukopenia, but white blood cell (WBC) levels returned to normal from day 8 onward, except in the low-dose rCDVSHEGFP(6) infection group (Fig. 2F). All animals developed fever, which peaked earlier in animals infected with rCDVSHEGFP(6) (Fig. 2G). CDV-specific virus-neutralizing (VN) antibodies against either CDV strain were detected from 8 dpi onward (Fig. 2H to I), but no MVEd-specific VN antibodies could be detected in these animals (Fig. 2J). Tropism of rCDV in lymphoid tissues. The phenotype of CDV-infected cells in PBMC and lymphoid tissues was assessed by flow cytometry and immunohistochemistry. Helper and cytotoxic T lymphocytes and B lymphocytes were the predominant CDVinfected cell types in PBMC, and infection percentages in these subpopulations exceeded those observed previously in experi-

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mental MV infection (22, 23, 34). Virtually no CD14⫹ monocytes or CD3⫺ CD8⫹ natural killer (NK) cells were infected by CDV (Fig. 3A). Five key lymphoid tissues were examined, and highest infection levels were detected in B lymphocytes, followed by helper and cytotoxic T lymphocytes. Analogous to the virus isolations (Fig. 2D and E), macaques infected with a high dose of rCDVSHEGFP(6) reached higher infection percentages on 6 dpi, and low-dose-infected macaques reached higher infection percentages at 10 dpi (Fig. 3B). Flow cytometric staining with CD150 indicated that the CDVinfected EGFP⫹ cells were CD150⫹ (Fig. 3C). Many EGFP⫹ cells with the phenotype of DC were detected in the lymphoid tissues (Fig. 3D, left panel), and dual indirect immunofluorescence using anti-EGFP and anti-CD11c monoclonal antibodies showed that EGFP⫹ CD11c⫹ cells were present in these tissues (Fig. 3D, right panel). This was confirmed using flow cytometry to examine single-cell suspensions prepared from lymphoid tissues which also contained CDV-infected DC. After lymphocytes were excluded on the basis of forward and side scatter (Fig. 3E, red plot, left panel), large cells were gated as noncluster-

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FIG 1 rCDVSHEGFP(6) and rCDVR252EGFP(6) replication in macaques. Nine morbillivirus-naive cynomolgus macaques were infected with either a low-dose rCDVSHEGFP(6) (red), a high-dose rCDVSHEGFP(6) (green), or a high-dose rCDVR252EGFP(6) (blue) virus. PBMC and BAL cells were obtained at 3, 6, 8, 10, 12, and 14 dpi and used for detection of EGFP⫹ cells by flow cytometry. Tissues were screened for macroscopic EGFP fluorescence during necropsy. (A and B) Detection of EGFP⫹ cells in BAL fluid or PBMC. Lines connect the geometric means per group; symbols show the data of individual animals. (C to G) Macroscopic detection of EGFP during the necropsies of the high- and low-dose rCDVSHEGFP(6)-infected macaques at 6 dpi showed EGFP fluorescence in the lungs and tracheobronchial lymph node (C), spleen (D), tonsils (E), mandibular lymph node (F), and the Peyer’s patches (G). Tr, trachea; Tg, tongue; il, ileum. FACS, fluorescence-activated cell sorting.

Downloaded from http://jvi.asm.org/ on August 13, 2014 by UNIVERSITY OF DAYTON LIBRARIES FIG 2 rCDVSHEGFP(6) and rCDVR252EGFP(6) viruses are virulent in macaques and cause lymphopenia, fever, and VN antibody responses. The animals and legends correspond to those of Fig. 1. (A to E) Virus was isolated in VDS cells, and results are expressed in TCID50/ml of transport medium (eye, nose, and throat swabs) or as numbers of infected cells/106 total cells (BAL cells and PBMC). (F and G) White blood cell (WBC) counts and body temperature are shown relative to the starting values of the individual animals. All groups showed leukopenia and fever. (H to J) VN antibody responses to CDV strains SH (H) and R252 (I) were detected from 8 dpi onward, but MVEd-specific VN antibodies were not detected (J). VI, virus isolation.

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and immunohistochemistry. (A) Percentages of EGFP⫹ cells in CD3⫹ CD4⫹ helper T lymphocytes, CD3⫹ CD8⫹ cytotoxic T lymphocytes, CD20⫹ B lymphocytes, CD14⫹ monocytes, or CD3⫺ CD8⫹ NK cells in PBMC. Legends and symbols correspond with those of Fig. 1. (B) Percentages of EGFP⫹ cells in CD3⫹ CD4⫹ helper T lymphocytes, CD3⫹ CD8⫹ cytotoxic T lymphocytes, and CD20⫹ B lymphocytes in five key lymphoid tissues. Bars indicate the mean percentages of EGFP⫹ cells of the five lymph nodes; symbols indicate the individual percentages per lymph node. (C) Staining with CD150 indicated that the CDV-infected EGFP⫹ cells were CD150⫹. (D) Many EGFP⫹ cells with the phenotype of DC were detected in the lymphoid tissues. Dual indirect immunofluorescence using anti-EGFP and anti-CD11c monoclonal antibodies showed that EGFP⫹ CD11c⫹ cells were present in these tissues. (E) Flow cytometry of single-cell suspensions prepared from lymphoid tissues showed CDV-infected DC. After lymphocytes were excluded on the basis of forward scatter (FSC) and side scatter (SSC) (red plot in left panel), large cells were gated as nonclustering cells that were CD3⫺ CD20⫺ HLA-DRhigh. CDV-infected DC were shown as CD11c⫹ EGFP⫹ cells within this population (right panel). PP, Peyer’s patches; TB-LN, tracheo-bronchial lymph node; Ax-LN, axillary lymph node.

ing cells that were CD3⫺ CD20⫺ with a high level of HLA-DR expression (HLA-DRhigh). CDV-infected DC were shown as CD11c⫹ EGFP⫹ cells within this population (Fig. 3E, right panel). rCDV-infected epithelial cells were not detected in macaques. During necropsy, the upper and lower respiratory tracts were sampled extensively; tissues were processed and paraffin embedded, and sections were analyzed for the presence of EGFP by indirect immunofluorescence and immunohistochemical staining. Submucosal infection of lymphoid and myeloid cells was detected in all tissues of the respiratory tract, including the adenoids, tonsil, nasal septum, trachea, primary bronchus, and lung (Fig. 4A to F). Notably, epithelial cell infection was not detected in any of the tissues at the time points examined. Although in several cases CDV-infected cells were detected within the epithelial layer, dual immunofluorescence consistently identified these as CD3⫹, CD11c⫹, or CD20⫹ (Fig. 4G to I, insets) and cytokeratin negative (data not shown). Limited spread of rCDV in well-differentiated normal macaque bronchial epithelial cells. Primary NMBE cells were

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obtained from the bronchus of a euthanized macaque and differentiated on the air-liquid interface (32). The monolayer of wd-NMBE cells was scratched and infected with cell-free rMVKS EGFP(3), rCDVSHEGFP(6), or rCDVR252EGFP(6) virus. rMVKS EGFP(3), which served as a positive control, caused foci of infection around the scratch (Fig. 5A). No MV infection was observed outside the scratched area of the wd-NMBE cell culture. rCDVSHEGFP(6) and rCDVR252EGFP(6) infected a minimal number of single cells in the wd-NMBE cell cultures around the scratch, but the infection did not spread or cause fusion (Fig. 5B and C, respectively). The top images of panels A, B, and C show a scan of a complete well; the images below are an enlargement of the dotted square box shown above. Staining of human ␤-tubulin IV clearly demonstrated the presence of ciliated cells in the wdNMBE cell cultures (Fig. 5D, upper and lower panel, respectively). MV vaccination induces partial protection against CDV challenge infection in macaques. Six macaques, vaccinated 10 months earlier with MVEZ, were challenged with either a low (n ⫽

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FIG 3 Tropism of rCDV in PBMC and lymphoid tissues. The phenotype of CDV-infected cells in PBMC and lymphoid tissues was assessed by flow cytometry

Experimental CDV Infection of Macaques

3) or high (n ⫽ 3) dose of rCDVSHEGFP(6). Prior to challenge we confirmed that vaccinated macaques had MV-specific VN antibody levels that are considered protective against measles (mean, 2.4 IU/ml; range, 0.84 to 6.7 IU/ml). To compare CDV loads in vaccinated and unvaccinated macaques, virus was isolated from nose (Fig. 6A) and throat (Fig. 6B) swabs, from BAL cells (Fig. 6C), and from PBMC (Fig. 6D). All vaccinated macaques were productively infected by rCDVSHEGFP(6) independent of the dose. However, virus replication levels were significantly reduced, and the infection was more rapidly cleared in all samples tested from the vaccinated macaques (Fig. 6A to D). Interestingly, virus loads detected in the upper respiratory tract of vaccinated macaques were substantially reduced compared to those of unvaccinated animals, suggesting that MV vaccination might not offer sterile immunity but still reduces transmission of CDV between macaques during an outbreak, as observed in the breeding colony in China. Correlates of cross-protection in macaques. In agreement with the finding that experimental CDV infection of macaques failed to induce MV-specific VN antibodies (Fig. 2J), MV vaccination failed to induce CDV-specific VN antibodies (Fig. 7A to C, 0 dpi). However, CDV-specific VN antibodies appeared approximately 8 dpi in both vaccinated and unvaccinated macaques although antibody levels were higher in vaccinated than in unvaccinated animals (Fig. 7A and B). Interestingly, MV-specific VN antibody levels of vaccinated macaques were boosted by the CDV

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infection (Fig. 7C), suggesting the existence of cross-neutralizing B-cell epitopes. Serum IgG antibody levels to the MV-F or -H glycoproteins were measured to determine the specificity of the antibodies mediating this secondary immune response. Although both curves showed a slight increase, boosting of F-specific IgG antibodies was more pronounced (Fig. 7D). At 10 dpi all vaccinated animals showed a more than 4-fold increase in MV-F-specific IgG antibody levels, while for MV-H-specific IgG, an increase was noted for only two out of four animals (data not shown). Accelerated clearance of CDV in MV-vaccinated macaques may also have resulted from cross-specific cellular immune responses. Whereas CDV infection of unvaccinated macaques resulted in severe leukopenia, with lowest WBC counts measured at 8 dpi, leukopenia in MV-vaccinated animals was less pronounced and was followed by a rapid increase in WBC counts from 6 dpi onward (Fig. 7E). DISCUSSION

In the present study, we show that two recombinant CDV strains readily infected cynomolgus macaques and caused fever and leukopenia. The viruses predominantly replicated in CD150⫹ lymphocytes and DC, indicating that CDV was capable of using macaque CD150. Partial immunity in MV-vaccinated macaques from CDV challenge was observed, resulting in restricted virus shedding from the upper respiratory tract. In recent years, we have studied the tropism of MV in ma-

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FIG 4 rCDV-infected epithelial cells were not observed in macaques. During necropsy, the upper and lower respiratory tracts were sampled extensively; samples were fixed in buffered formalin and paraffin embedded, and sections were analyzed by EGFP staining (brown staining). (A to F) In all tissues, including the adenoid (A), tonsil (B), nasal septum (C), trachea (D), primary bronchus (E), and lung (F), submucosal infection of lymphoid and myeloid cells was detected. Notably, epithelial cell infection was not detected in any of these tissues at the time points examined. Even though at first glance positivity might be interpreted in the epithelial cells of the adenoid (A), this proved to be background staining. (G to I) CDV-infected cells (green) detected within the epithelial layer were identified as CD3⫹ (G, red), CD11c⫹ (H, red), or CD20⫹ (I, red) by dual immunofluorescence consistently. Nuclei were counterstained with DAPI (blue). BALT, bronchus-associated lymphoid tissue.

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caques (22–24). The predominant infection of CD150⫹ lymphocytes and DC in macaques we report here for CDV corresponds well with the reported tropism of MV in macaques (22). In addition to being lymphotropic, in its natural host species CDV readily infects epithelial and neuronal cells (2, 3, 26). The epitheliotropic nature has also been described for MV in nonhuman primates (22, 25, 35) although CDV infects higher percentages of epithelial cells in carnivores than MV does in primates. Morbillivirus entry into epithelial cells requires the cellular receptor PVRL4. This receptor is present in the adherens junction on the basolateral side of epithelial cells, and entry into these cells is postulated to be important for viral shedding (7, 36). In contrast to previously reported experimental infections with rMV or rCDV modified to prevent PVRL4 binding (10, 11), we isolated significant amounts of CDV from BAL cells, throat swabs, and nasopharyngeal swabs, demonstrating shedding of CDV in the absence of detectable infection of

epithelial cells. This observation suggests that recently proposed hypotheses explaining transmission of morbilliviruses (37) may not tell the complete story. Even though both CDV strains infected macaque lymphocytes and DC, at the time points examined, we were not able to detect CDV in either epithelial cells or the CNS. Moreover, in vitro infection studies in primary wd-NMBE cells showed that cell-free rCDVSHEGFP(6) and rCDVR252EGFP(6) could infect single cells when the monolayer was scratched to expose the basolaterally expressed PVRL4, but both CDV strains were unable to spread to the neighboring cells. In contrast, these cultures were readily infected with wild-type MV. In vivo large numbers of CDV-infected immune cells were seen within the respiratory epithelium or in the respiratory submucosa, demonstrating that epithelial cells were exposed to CDV. In a recent report Sakai et al. described experimental infections of macaques with the CDV strain isolated dur-

FIG 6 MV vaccination induces partial cross-protection against CDV. Comparison of virus isolation data from the six unvaccinated and the six MV-vaccinated rCDVSHEGFP(6)-infected macaques, expressed as geometric means ⫾ standard errors of the means. All macaques were productively infected by rCDVSHEGFP(6), but virus replication levels were significantly reduced in the nose (A), throat (B), BAL cells (C), and PBMC (D) of MV-vaccinated macaques. Statistically significant differences are indicated with an asterisk (P ⬍ 0.05). VI, virus isolation.

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FIG 5 Limited spread of rCDV in scratched well-differentiated normal macaque bronchial epithelial cells. (A) As shown in the upper panel, infection of wd-NMBE cells with cell-free rMVKSEGFP(3) (MV) caused several foci of infection around the scratch. The lower panel shows an enlargement of the boxed area. (B and C) Infection of scratched wd-NMBE cells with cell-free rCDVSHEGFP(6) or rCDVR252EGFP(6) resulted in single EGFP⫹ cells without cell-to-cell spread. (D) Staining with anti-human ␤-tubulin IV (red) identified the presence of ciliated epithelial cells in the wd-NMBE cell cultures infected with MV or CDV. CDVSH, rCDVSHEGFP(6); CDVR252, rCDVR252EGFP(6).

Experimental CDV Infection of Macaques

MV-vaccinated and unvaccinated macaques before and after infection with rCDVSHEGFP(6). MV vaccination did not induce detectable CDV-specific VN antibodies, and CDV-specific VN antibodies appeared in both vaccinated and unvaccinated macaques at approximately 8 dpi (A and B). MV-specific VN antibody levels in sera from vaccinated macaques were boosted by the CDV infection (C). (D) Serum IgG antibody levels to the MV-F and -H glycoproteins were measured by a flow cytometric assay (30), demonstrating a boosting of antibodies specific to MV-F and MV-H. (E) WBC counts showed severe leukopenia in unvaccinated macaques, whereas leukopenia in MV-vaccinated animals was less pronounced and followed by a rapid increase of WBC counts from 6 dpi onward. All plots show geometric means ⫾ standard errors of the means. Statistically significant differences are indicated with an asterisk (P ⬍ 0.05).

ing an outbreak among cynomolgus macaques in China. The authors showed that this virus not only could efficiently use both macaque CD150 and PVRL4 in vitro but also infected both lymphocytes and epithelial cells in vivo (17). These observations suggest that the continuing chain of CDV transmissions in macaques has resulted in adaptation of the virus, enabling cell-to-cell spread in macaque PVRL4-positive (PVRL4⫹) epithelial cells. However, based on our data it is impossible to conclude whether the absence of viral spread in epithelial cells was based on poor binding affinity for macaque PVRL4 or on an intracellular block of virus replication. In previous experimental morbillivirus cross-species infection studies, viruses were typically less pathogenic in the new host than in their natural host. Experimental infection of pigs showed that they were susceptible to CDV infection although the virus replicated exclusively in lymphoid tissues, and there were no clinical signs of disease (38, 39). Experimental infection of cats resulted in subclinical infection, and again the virus did not infect epithelial or neuronal cells or transmit from cat to cat (38–40). However, large outbreaks among felids in Africa and North America caused by CDV were reported in the 1990s (41–43), and an outbreak among tigers in a Japanese zoo was recently reported (44). Strikingly similar to these observations, experimental infection of seals with CDVSH led to an exclusively lymphoid and not epithelial infection, which did not transmit to sentinel hosts (45). However, in 1987 and 2002 CDV caused massive lethal outbreaks in Baikal and Caspian seals (46–49). Taken together, these observations are

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reminiscent of what we observe as a self-limiting nonlethal experimental infection in macaques, contrasting with recently reported lethal CDV outbreaks in macaques in China and Japan (16, 17). Apparently, a morbillivirus that crosses the species barrier and is transmissible within the new host species can readily adapt to achieve full virulence. Immunological relationships between morbilliviruses have long been reported (50–52). Cross-reactivity of sera was shown in the 1980s using monoclonal antibodies. However, the cross-neutralizing capacity of specific antibodies is not fully understood. ¨ rvell et al. reported that convalescent-phase sera from CDVO infected dogs could not cross-neutralize MV (53). Furthermore, it was shown that MV-monospecific antibodies against the H and F glycoproteins failed to cross-neutralize CDV (54). These data are surprising since vaccination with attenuated morbilliviruses cross-protects from infection with heterologous morbilliviruses. In fact, live attenuated and recombinant MV vaccines have been used to immunize dogs against CDV in the presence of maternal antibodies (19, 55, 56), resulting in partial protection from CDV even though CDV-specific antibodies could not be detected. In macaques we found that MV vaccination did not lead to detectable CDV cross-neutralizing antibodies, but MV-specific antibodies were boosted upon CDV challenge, indicating that cross-reactivity on an antibody level is present. In addition, we observed a rapid proliferation of WBC in MV-vaccinated macaques upon CDV challenge, reminiscent of a secondary cellular immune re-

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FIG 7 Correlates of protection in MV-vaccinated macaques. (A to C) VN titers against rCDVSHEGFP(6) (A), rCDVR252EGFP(6) (B), and MVEd (C) in

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ACKNOWLEDGMENTS We thank Ken Lemon and Linda Rennick for their contributions to these studies and our colleagues of the Erasmus MC animal facility for taking care of the animals and preparing and cleaning the isolators. We are grateful to Yusuke Yanagi for providing the VDS cells. This work was supported by ZonMw (grant number 91208012), MRC (grant number G0801001), and FNIH (grant number DUPREX09GCGH0).

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REFERENCES 1. Blancou J. 2004. Dog distemper: imported into Europe from South America? Hist. Med. Vet. 29:35– 41. 2. 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. U. S. A. 101:14216 –14421. http://dx.doi.org/10.1073/pnas.0403597101. 3. Beineke A, Puff C, Seehusen F, Baumgartner W. 2009. Pathogenesis and immunopathology of systemic and nervous canine distemper. Vet. Immunol. Immunopathol. 127:1–18. http://dx.doi.org/10.1016/j.vetimm .2008.09.023. 4. Seki F, Ono N, Yamaguchi R, Yanagi Y. 2003. Efficient isolation of wild strains of canine distemper virus in Vero cells expressing canine SLAM (CD150) and their adaptability to marmoset B95a cells. J. Virol. 77:9943– 9950. http://dx.doi.org/10.1128/JVI.77.18.9943-9950.2003. 5. Tatsuo H, Ono N, Yanagi Y. 2001. Morbilliviruses use signaling lymphocyte activation molecules (CD150) as cellular receptors. J. Virol. 75:5842– 5850. http://dx.doi.org/10.1128/JVI.75.13.5842-5850.2001. 6. 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. http://dx.doi.org/10.1128/JVI.00357-06. 7. Mühlebach MD, Mateo M, Sinn PL, Prufer S, Uhlig KM, Leonard VH, Navaratnarajah CK, Frenzke M, Wong XX, Sawatsky B, Ramachandran S, McCray PB, Jr, Cichutek K, von Messling V, Lopez M, Cattaneo R. 2011. Adherens junction protein nectin-4 is the epithelial receptor for measles virus. Nature 480:530 –533. http://dx.doi.org/10.1038/nature 10639. 8. Noyce RS, Bondre DG, Ha MN, Lin LT, Sisson G, Tsao MS, Richardson CD. 2011. Tumor cell marker PVRL4 (nectin 4) is an epithelial cell receptor for measles virus. PLoS Pathog. 7:e1002240. http://dx.doi.org/10.1371 /journal.ppat.1002240. 9. Pratakpiriya W, Seki F, Otsuki N, Sakai K, Fukuhara H, Katamoto H, Hirai T, Maenaka K, Techangamsuwan S, Lan NT, Takeda M, Yamaguchi R. 2012. Nectin4 is an epithelial cell receptor for canine distemper virus and involved in the neurovirulence. J. Virol. 86:10207–10210. http: //dx.doi.org/10.1128/JVI.00824-12. 10. Sawatsky B, Wong XX, Hinkelmann S, Cattaneo R, von Messling V. 2012. Canine distemper virus epithelial cell infection is required for clin-

4432

jvi.asm.org

19. 20. 21.

22.

23.

24.

25.

26.

ical disease but not for immunosuppression. J. Virol. 86:3658 –3666. http: //dx.doi.org/10.1128/JVI.06414-11. Leonard VHJ, Sinn PL, Hodge G, Miest T, Devaux P, Oezguen N, Braun W, McCray PB, McChesney MB, Cattaneo R. 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. http://dx.doi.org/10.1172/JCI35454. Ludlow M, Rennick LJ, Nambulli S, De Swart RL, Duprex WP. 2014. Using the ferret model to study morbillivirus entry, spread, transmission and cross-protection. Curr. Opin. Virol. 4:15–23. http://dx.doi.org/10 .1016/j.coviro.2013.11.001. Appel MJ, Reggiardo C, Summers BA, Pearce-Kelling S, Mare CJ, Noon ¨ rvell C. 1991. Canine distemper virus infecTH, Reed RE, Shively JN, O tion in javelinas (collared peccaries). Arch. Virol. 119:147–152. http://dx .doi.org/10.1007/BF01314331. Origgi FC, Sattler U, Pilo P, Waldvogel AS. 2013. Fatal combined infection with canine distemper virus and orthopoxvirus in a group of Asian marmots (Marmota caudata). Vet. Pathol. 50:914 –920. http://dx .doi.org/10.1177/0300985813476060. Yoshikawa Y, Ochikubo F, Matsubara Y, Tsuruoka H, Ishii M, Shirota K, Nomura Y, Sugiyama M, Yamanouchi K. 1989. Natural infection with canine distemper virus in a Japanese monkey (Macaca fuscata). Vet. Microbiol. 20:193–205. http://dx.doi.org/10.1016/0378-1135(89)90043-6. Qiu W, Zheng Y, Zhang S, Fan Q, Liu H, Zhang F, Wang W, Liao G, Hu R. 2011. Canine distemper outbreak in rhesus monkeys, China. Emerg. Infect. Dis. 17:1541–1543. http://dx.doi.org/10.3201/eid1708 .101153. Sakai K, Nagata N, Ami Y, Seki F, Suzaki Y, Iwata-Yoshikawa N, Suzuki T, Fukushi S, Mizutani T, Yoshikawa T, Otsuki N, Kurane I, Komase K, Yamaguchi R, Hasegawa H, Saijo M, Takeda M, Morikawa S. 2013. Lethal canine distemper virus outbreak in cynomolgus monkeys in Japan in 2008. J. Virol. 87:1105–1114. http://dx.doi.org/10.1128/JVI.02419-12. Barrett T. 1999. Morbillivirus infections, with special emphasis on morbilliviruses of carnivores. Vet. Microbiol. 69:3–13. http://dx.doi.org/10 .1016/S0378-1135(99)00080-2. Strating A. 1975. Measles vaccine in dogs: efficacy against aerosol challenge with virulent canine distemper virus. J. Am. Vet. Med. Assoc. 167: 59 – 62. Sanders R, Dabbagh A, Featherstone D. 2011. Risk analysis for measles reintroduction after global certification of eradication. J. Infect. Dis. 204(Suppl 1):S71–S77. http://dx.doi.org/10.1093/infdis/jir133. Drexler JF, Corman VM, Muller MA, Maganga GD, Vallo P, Binger T, Gloza-Rausch F, Rasche A, Yordanov S, Seebens A, Oppong S, Sarkodie YA, Pongombo C, Lukashev AN, Schmidt-Chanasit J, Stocker A, Carneiro AJ, Erbar S, Maisner A, Fronhoffs F, Buettner R, Kalko EK, Kruppa T, Franke CR, Kallies R, Yandoko ER, Herrler G, Reusken C, Hassanin A, Kruger DH, Matthee S, Ulrich RG, Leroy EM, Drosten C. 2012. Bats host major mammalian paramyxoviruses. Nat. Commun. 3:796. http://dx.doi.org/10.1038/ncomms1796. de Swart RL, Ludlow M, De Witte L, Yanagi Y, Van Amerongen G, McQuaid S, Yüksel S, Geijtenbeek TB, Duprex WP, Osterhaus AD. 2007. Predominant infection of CD150⫹ lymphocytes and dendritic cells during measles virus infection of macaques. PLoS Pathog. 3:e178. http: //dx.doi.org/10.1371/journal.ppat.0030178. de Vries RD, Lemon K, Ludlow M, McQuaid S, Yüksel S, Van Amerongen G, Rennick LJ, Rima BK, Osterhaus AD, De Swart RL, Duprex WP. 2010. In vivo tropism of attenuated and pathogenic measles virus expressing green fluorescent protein in macaques. J. Virol. 84:4714 – 4724. http://dx.doi.org/10.1128/JVI.02633-09. Lemon K, de Vries RD, Mesman AW, McQuaid S, Van Amerongen G, Yüksel S, Ludlow M, Rennick LJ, Kuiken T, Rima BK, Geijtenbeek TB, Osterhaus AD, Duprex WP, de Swart RL. 2011. Early target cells of measles virus after aerosol infection of non-human primates. PLoS Pathog. 7:e1001263. http://dx.doi.org/10.1371/journal.ppat.1001263. Ludlow M, Rennick L, Sarlang S, Skibinski G, McQuaid S, Moore T, De Swart RL, Duprex WP. 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. 91:971–979. http: //dx.doi.org/10.1099/vir.0.016428-0. Ludlow M, Nguyen DT, Silin D, Lyubomska O, De Vries RD, von Messling V, McQuaid S, De Swart RL, Duprex WP. 2012. Recombinant canine distemper virus strain Snyder Hill expressing green or red fluores-

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sponse. Combined, these data suggest that MV vaccination can induce CDV cross-immunity on both humoral and cellular levels. In conclusion, we have demonstrated that nonhuman primates are susceptible to infection with two different recombinant CDV strains and that measles immunity provides partial protection and limits viral shedding. Thus, morbillivirus cross-specific immunity of the human population restricts the possibilities of CDV or other animal morbilliviruses replicating in or adapting to humans. However, if measles eradication is followed by significant drops in MV vaccination coverage, such adaptation cannot be excluded. Although this should not be used to argue against measles eradication, it reinforces the general idea that sustained MV vaccination and serological and virological surveillance of morbillivirus infections will remain essential. Potentially, the use of rMV strains as vectors for vaccination against heterologous pathogens could provide the necessary incentive to sustain compliance to MV vaccination, retain morbillivirus seropositivity, and take advantage of what is one of the safest and efficacious live attenuated virus vaccines.

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27.

28.

30.

31. 32.

33.

34.

35.

36. 37. 38. 39. 40.

41.

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42. Harder TC, Kenter M, Appel MJ, Roelke-Parker ME, Barrett T, Osterhaus AD. 1995. Phylogenetic evidence of canine distemper virus in Serengeti’s lions. Vaccine 13:521–523. http://dx.doi.org/10.1016/0264 -410X(95)00024-U. 43. Roelke-Parker ME, Munson L, Packer C, Kock R, Cleaveland S, Carpenter M, O’Brien SJ, Pospischil A, Hofmann-Lehmann R, Lutz H, Mwamengele GL, Mgasa MN, Machange GA, Summers BA, Appel MJ. 1996. A canine distemper virus epidemic in Serengeti lions (Panthera leo). Nature 379:441– 445. http://dx.doi.org/10.1038/379441a0. 44. Nagao Y, Nishio Y, Shiomoda H, Tamaru S, Shimojima M, Goto M, Une Y, Sato A, Ikebe Y, Maeda K. 2012. An outbreak of canine distemper virus in tigers (Panthera tigris): possible transmission from wild animals to zoo animals. J. Vet. Med. Sci. 74:699 –705. http://dx.doi.org/10.1292 /jvms.11-0509. 45. Svansson V, Blixenkrone-Möller M, Skirnisson K, Have P, Heje NI, Nielsen J, Lund E. 1993. Infection studies with canine distemper virus in harbour seals. Arch. Virol. 131:349 –359. http://dx.doi.org/10.1007 /BF01378637. 46. Osterhaus AD. 1989. A morbillivirus causing mass mortality in seals. Vaccine 7:483– 484. http://dx.doi.org/10.1016/0264-410X(89)90265-X. 47. Grachev MA, Kumarev VP, Mamaev LV, Zorin VL, Baranova LV, Denikjna NN, Belikov SI, Petrov EA, Kolesnik VS, Kolesnik RS, Dorofeev VM, Beim AM, Kudelin VN, Nagieva FG, Sidorov VN. 1989. Distemper virus in Baikal seals. Nature 338:209. http://dx.doi.org/10.1038 /338209b0. 48. Kennedy S, Kuiken T, Jepson PD, Deaville R, Forsyth M, Barrett T, Van de Bildt MW, Osterhaus AD, Eybatov T, Duck C, Kydyrmanov A, Mitrofanov I, Wilson S. 2000. Mass die-Off of Caspian seals caused by canine distemper virus. Emerg. Infect. Dis. 6:637– 639. http://dx.doi.org /10.3201/eid0606.000613. 49. Kuiken T, Kennedy S, Barrett T, Van de Bildt MWG, Borgsteede FH, Brew SD, Codd GA, Duck C, Deaville R, Eybatov T, Forsyth MA, Foster G, Jepson PD, Kydyrmanov A, Mitrofanov I, Ward CJ, Wilson S, Osterhaus AD. 2006. The 2000 canine distemper epidemic in Caspian seals (Phoca caspica): pathology and analysis of contributory factors. Vet. Pathol. 43:321–338. http://dx.doi.org/10.1354/vp.43-3-321. 50. DeLay PD, Stone SS, Karzon DT, Katz S, Enders J. 1965. Clinical and immune response of alien hosts to inoculation with measles, rinderpest, and canine distemper viruses. Am. J. Vet. Res. 26:1359 –1373. 51. Imagawa DT, Goret P, Adams JM. 1960. Immunological relationships of measles, distemper, and rinderpest viruses. Proc. Natl. Acad. Sci. U. S. A. 46:1119 –1123. http://dx.doi.org/10.1073/pnas.46.8.1119. ¨ rvell 52. Sheshberadaran H, Norrby E, McCullough KC, Carpenter WC, O C. 1986. The antigenic relationship between measles, canine distemper and rinderpest viruses studied with monoclonal antibodies. J. Gen. Virol. 67:1381–1392. http://dx.doi.org/10.1099/0022-1317-67-7-1381. ¨ rvell C, Norrby E. 1974. Further studies on the immunological relation53. O ships among measles, distemper, and rinderpest viruses. J. Immunol. 113: 1850 –1858. 54. Gould EA, Armstrong M, Shirodaria PV, De’Ornellas D. 1981. Examination of the immunological relationship between measles virus and canine distemper virus using monospecific measles antisera. J. Med. Virol. 7:51– 60. http://dx.doi.org/10.1002/jmv.1890070106. 55. Moura RA, Warren J. 1961. Subclinical infection of dogs by canineadapted measles virus evidenced by their subsequent immunity to canine distemper virus. J. Bacteriol. 82:702–705. 56. Taylor J, Pincus S, Tartaglia J, Richardson C, Alkhatib G, Briedis D, Appel MJ, Norton E, Paoletti E. 1991. Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge. J. Virol. 65:4263– 4274.

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cent proteins causes meningoencephalitis in the ferret. J. Virol. 86:7508 – 7519. http://dx.doi.org/10.1128/JVI.06725-11. Nguyen DT, De Vries RD, Ludlow M, van den Hoogen BG, Lemon K, Van Amerongen G, Osterhaus AD, De Swart RL, Duprex WP. 2013. Paramyxovirus infections in ex vivo lung slice cultures of different host species. J. Virol. Methods 193:159 –165. http://dx.doi.org/10.1016/j .jviromet.2013.06.016. El Mubarak HS, Yüksel S, Van Amerongen G, Mulder PG, Mukhtar MM, Osterhaus AD, De Swart RL. 2007. Infection of cynomolgus macaques (Macaca fascicularis) and rhesus macaques (Macaca mulatta) with different wild-type measles viruses. J. Gen. Virol. 88:2028 –2034. http://dx .doi.org/10.1099/vir.0.82804-0. Reed LJ, Muench H. 1938. A simple method of estimating fifty percent endpoints. Am. J. Hygiene. 27:493– 497. de Swart RL, Vos HW, UytdeHaag FG, Osterhaus AD, Van Binnendijk RS. 1998. Measles virus fusion protein- and hemagglutinin-transfected cell lines are a sensitive tool for the detection of specific antibodies by a FACS-measured immunofluorescence assay. J. Virol. Methods 71:35– 44. http://dx.doi.org/10.1016/S0166-0934(97)00188-2. Fulcher ML, Gabriel S, Burns KA, Yankaskas JR, Randell SH. 2005. Well-differentiated human airway epithelial cell cultures. Methods Mol. Med. 107:183–206. http://dx.doi.org/10.1385/1-59259-861-7:183. Verkaik NJ, Nguyen DT, de Vogel CP, Moll HA, Verbrugh HA, Jaddoe VW, Hofman A, Van Wamel WJ, van den Hoogen BG, Buijs-Offerman RM, Ludlow M, de Witte L, Osterhaus AD, van Belkum A, de Swart RL. 2011. Streptococcus pneumoniae exposure is associated with human metapneumovirus seroconversion and increased susceptibility to in vitro HMPV infection. Clin. Microbiol. Infect. 17:1840 –1844. http://dx.doi.org /10.1111/j.1469-0691.2011.03480.x. van Binnendijk RS, van der Heijden RW, Van Amerongen G, UytdeHaag FG, Osterhaus AD. 1994. Viral replication and development of specific immunity in macaques after infection with different measles virus strains. J. Infect. Dis. 170:443– 448. http://dx.doi.org/10.1093/infdis/170.2 .443. de Vries RD, McQuaid S, Van Amerongen G, Yüksel S, Verburgh RJ, Osterhaus AD, Duprex WP, De Swart RL. 2012. Measles immune suppression: lessons from the macaque model. PLoS Pathog. 8:e1002885. http://dx.doi.org/10.1371/journal.ppat.1002885. Ludlow M, Lemon K, De Vries RD, McQuaid S, Millar E, Van Amerongen G, Yüksel S, Verburgh RJ, Osterhaus AD, De Swart RL, Duprex WP. 2013. Measles virus infection of epithelial cells in the macaque upper respiratory tract is mediated by sub-epithelial immune cells. J. Virol. 87: 4033– 4042. http://dx.doi.org/10.1128/JVI.03258-12. Noyce RS, Richardson CD. 2012. Nectin 4 is the epithelial cell receptor for measles virus. Trends Microbiol. 20:429 – 439. http://dx.doi.org/10 .1016/j.tim.2012.05.006. Racaniello V. 2011. An exit strategy for measles virus. Science 334:1650 – 1651. http://dx.doi.org/10.1126/science.1217378. Gaskin JM. 1974. Canine distemper virus in domesticated cats and pigs. Adv. Enzymol. Relat. Areas Mol. Biol. 40:803– 806. Appel MJ, Summers BA. 1995. Pathogenicity of morbilliviruses for terrestrial carnivores. Vet. Microbiol. 44:187–191. http://dx.doi.org/10.1016 /0378-1135(95)00011-X. Harder TC, Kenter M, Vos H, Siebelink K, Huisman W, Van Ameron¨ rvell C, Barrett T, Appel MJ, Osterhaus AD. 1996. Canine gen G, O distemper virus from large diseased felids: biological properties and phylogenetic relationships. J. Gen. Virol. 77:397– 405. http://dx.doi.org/10 .1099/0022-1317-77-3-397. Appel MJ, Yates RA, Foley GL, Bernstein JJ, Santinelli S, Spelman LH, Miller LD, Arp LH, Anderson M, Barr M, Pearce-Kelling S, Summers BA. 1994. Canine distemper epizootic in lions, tigers, and leopards in North America. J. Vet. Diagn. Invest. 6:277–288. http://dx.doi.org/10 .1177/104063879400600301.