Measles Virus-infected Cells Research Articles

Immunogenicity of a peptide-based vaccine for measles: a pilot evaluation in a mouse model

Although neutralizing antibody is an established correlate of protection for measles, T cell-mediated responses play at least two critical roles in immunity to measles: first, through provision of ‘help’ enabling robust humoral immune responses; and second, through clearance of measles virus-infected cells. Previously, we identified 13 measles-derived peptides that bound to human leukocyte antigen (HLA) molecules in Priess cells infected with measles virus. In this study, we evaluated the immunogenicity of these peptides in a transgenic mouse model. Our results demonstrated that these peptides induced Th1-biased immune responses at varying levels. Of the 13 peptides, the top four immunogenic peptides were further selected for a viral challenge study in mice. A vaccine based on a combination of these four peptides reduced morbidity and weight loss after viral challenge compared to placebo. Our results emphasize the potential of T cell-mediated, peptide-based vaccines against measles.

Specific intracellular signature of SARS-CoV-2 infection using confocal Raman microscopy

SARS-CoV-2 infection remains spread worldwide and requires a better understanding of virus-host interactions. Here, we analyzed biochemical modifications due to SARS-CoV-2 infection in cells by confocal Raman microscopy. Obtained results were compared with the infection with another RNA virus, the measles virus. Our results have demonstrated a virus-specific Raman molecular signature, reflecting intracellular modification during each infection. Advanced data analysis has been used to distinguish non-infected versus infected cells for two RNA viruses. Further, classification between non-infected and SARS-CoV-2 and measles virus-infected cells yielded an accuracy of 98.9 and 97.2 respectively, with a significant increase of the essential amino-acid tryptophan in SARS-CoV-2-infected cells. These results present proof of concept for the application of Raman spectroscopy to study virus-host interaction and to identify factors that contribute to the efficient SARS-CoV-2 infection and may thus provide novel insights on viral pathogenesis, targets of therapeutic intervention and development of new COVID-19 biomarkers.

IgA targeting on the α-molecular recognition element (α-MoRE) of viral phosphoprotein inhibits measles virus replication by interrupting formation and function of P-N complex intracellularly

Secretory IgA (SIgA) antibody is unique for its capability to transit through epithelial cells by transcytosis and thus has opportunities and probabilities to interact with all viral components during viral replication which may result in the inhibition of viral replication intracellularly. Here, we report a novel IgA mAb 1D11-IgA against phosphoprotein (P) of measles virus (MV), which is able to interact specifically with P in MV infected Vero-pIgR cells grown in a two-chamber transwell system. The binding epitope of 1D11-IgA involves a key residue proline 23 in P protein, which is among the α-molecular recognition element (α-MoRE) of P and critical for N0-P complex. The antibody appears to block P to interact with N in P-N complex and thus may inhibit the function of viral RdRp complex, which results in decreased synthesis of viral genome RNA and mRNA. Our data together demonstrate that IgA is able to interact with viral phosphoprotein intraepithelial cells and neutralize viral replication by interrupting formation of P-N complex and function of RdRp. The findings highlight that IgA has a unique anti-viral activity by targeting viral conserved components critical for viral replication, which serves as a proof-of-concept assessment of the druggability of mononegavirales P-N interfaces.

Upon Infection, Cellular WD Repeat-Containing Protein 5 (WDR5) Localizes to Cytoplasmic Inclusion Bodies and Enhances Measles Virus Replication.

Replication of negative-strand RNA viruses occurs in association with discrete cytoplasmic foci called inclusion bodies. Whereas inclusion bodies represent a prominent subcellular structure induced by viral infection, our knowledge of the cellular protein components involved in inclusion body formation and function is limited. Using measles virus-infected HeLa cells, we found that the WD repeat-containing protein 5 (WDR5), a subunit of histone H3 lysine 4 methyltransferases, was selectively recruited to virus-induced inclusion bodies. Furthermore, WDR5 was found in complexes containing viral proteins associated with RNA replication. WDR5 was not detected with mitochondria, stress granules, or other known secretory or endocytic compartments of infected cells. WDR5 deficiency decreased both viral protein production and infectious virus yields. Interferon production was modestly increased in WDR5-deficient cells. Thus, our study identifies WDR5 as a novel viral inclusion body-associated cellular protein and suggests a role for WDR5 in promoting viral replication.IMPORTANCE Measles virus is a human pathogen that remains a global concern, with more than 100,000 measles-related deaths annually despite the availability of an effective vaccine. As measles continues to cause significant morbidity and mortality, understanding the virus-host interactions at the molecular level that affect virus replication efficiency is important for development and optimization of treatment procedures. Measles virus is an RNA virus that encodes six genes and replicates in the cytoplasm of infected cells in discrete cytoplasmic replication bodies, though little is known of the biochemical nature of these structures. Here, we show that the cellular protein WDR5 is enriched in the cytoplasmic viral replication factories and enhances virus growth. WDR5-containing protein complex includes viral proteins responsible for viral RNA replication. Thus, we have identified WDR5 as a host factor that enhances the replication of measles virus.

Needle-free delivery of measles virus vaccine to the lower respiratory tract of non-human primates elicits optimal immunity and protection

Needle-free measles virus vaccination by aerosol inhalation has many potential benefits. The current standard route of vaccination is subcutaneous injection, whereas measles virus is an airborne pathogen. However, the target cells that support replication of live-attenuated measles virus vaccines in the respiratory tract are largely unknown. The aims of this study were to assess the in vivo tropism of live-attenuated measles virus and determine whether respiratory measles virus vaccination should target the upper or lower respiratory tract. Four groups of twelve cynomolgus macaques were immunized with 104 TCID50 of recombinant measles virus vaccine strain Edmonston-Zagreb expressing enhanced green fluorescent protein. The vaccine virus was grown in MRC-5 cells and formulated with identical stabilizers and excipients as used in the commercial MVEZ vaccine produced by the Serum Institute of India. Animals were immunized by hypodermic injection, intra-tracheal inoculation, intra-nasal instillation, or aerosol inhalation. In each group six animals were euthanized at early time points post-vaccination, whereas the other six were followed for 14 months to assess immunogenicity and protection from challenge infection with wild-type measles virus. At early time-points, enhanced green fluorescent protein-positive measles virus-infected cells were detected locally in the muscle, nasal tissues, lungs, and draining lymph nodes. Systemic vaccine virus replication and viremia were virtually absent. Infected macrophages, dendritic cells and tissue-resident lymphocytes predominated. Exclusive delivery of vaccine virus to the lower respiratory tract resulted in highest immunogenicity and protection. This study sheds light on the tropism of a live-attenuated measles virus vaccine and identifies the alveolar spaces as the optimal site for respiratory delivery of measles virus vaccine.

Measles Virus Matrix Protein Inhibits Host Cell Transcription.

Measles virus (MeV) is a highly contagious virus that still causes annual epidemics in developing countries despite the availability of a safe and effective vaccine. Additionally, importation from endemic countries causes frequent outbreaks in countries where it has been eliminated. The M protein of MeV plays a key role in virus assembly and cytopathogenesis; interestingly, M is localised in nucleus, cytoplasm and membranes of infected cells. We have used transient expression of M in transfected cells and in-cell transcription assays to show that only some MeV M localizes to the nucleus, in addition to cell membranes and the cytoplasm as previously described, and can inhibit cellular transcription via binding to nuclear factors. Additionally, MeV M was able to inhibit in vitro transcription in a dose-dependent manner. Importantly, a proportion of M is also localized to nucleus of MeV infected cells at early times in infection, correlating with inhibition of cellular transcription. Our data show, for the first time, that MeV M may play a role early in infection by inhibiting host cell transcription.

A chicken homologue of nectin-4 functions as a measles virus receptor

Measles virus (MV) vaccine strains use CD46, signaling lymphocyte activation molecule, and nectin-4 in human cells as receptors. Meanwhile, many of them are propagated in primary chicken embryonic fibroblasts (CEFs). Our data revealed that CEFs express a nectin-4 homologous molecule (CEF nectin-4) containing well-conserved motifs in the FG and BC loops, but not in the C′C″ loop. MV infected CHO cells expressing CEF nectin-4 and induced syncytia in these cells, confirming that CEF nectin-4 functions as an MV receptor and that the C′C″ loop is not critical for this function. Nectin-4-blind mutations in MV H protein reduced the infectivity of MV in CEF nectin-4-expressing cells. Infection of CEFs with the MV vaccine AIK-C strain was partially blocked by an anti-nectin-4 antibody, indicating that CEF nectin-4 plays a role for propagation of MV vaccines in CEFs.

ID: 32: Roles of A-to-I editing by RNA adenosine deaminase ADAR1 in innate immunity RNA sensing

ADAR1, an interferon inducible adenosine deaminase acting on RNA, catalyzes the C6 deamination of adenosine (A) to produce inosine (I) in RNA with double-stranded (ds) character, leading to genetic recoding and altered RNA structure as I pairs as G instead of A. Adar1 expression occurs from multiple promoters and encodes an IFN-inducible p150 cytoplasmic protein and a constitutively expressed N-terminally truncated p110 nuclear protein. The roles of ADAR1 p150 and p110 in uninfected and virus-infected cells were examined using human cell clones stably deficient in ADAR1, mouse cells genetically deficient in ADAR1, and by complementation strategies. Exemplified by measles virus (MV) infected cells, ADAR1 suppressed activation of PKR and IRF3, suppressed IFN β induction, and protected cells from stress granule formation and cytopathic damage. RNA expression profiles and A-to-I editing were characterized by qPCR and RNA deep sequencing, with differences found between CKO mutant and parental MV infections, and between untreated and IFN-treated uninfected cells. Both non-self (viral) and self (cellular) RNAs with ds character were identified and their editing characterized. Results obtained with mouse cells genetically deficient in either adar1 or the related deaminase adar2, or stat1 or stat2, suggest a central role of ADAR1 in overall A-to-I editing and p150 in the IFN-induced editing and innate immunity RNA sensing. In summary, ADAR1 behaved as an anti-apoptotic host factor that suppressed innate immune activities and was pro-viral. Our findings furthermore implicate a balanced interplay between PKR and ADAR1, two IFN inducible dsRNA binding enzymes, in modulating IFN β protein production and stress responses.

In vivo ligands of MDA5 and RIG-I in measles virus-infected cells.

RIG-I-like receptors (RLRs: RIG-I, MDA5 and LGP2) play a major role in the innate immune response against viral infections and detect patterns on viral RNA molecules that are typically absent from host RNA. Upon RNA binding, RLRs trigger a complex downstream signaling cascade resulting in the expression of type I interferons and proinflammatory cytokines. In the past decade extensive efforts were made to elucidate the nature of putative RLR ligands. In vitro and transfection studies identified 5′-triphosphate containing blunt-ended double-strand RNAs as potent RIG-I inducers and these findings were confirmed by next-generation sequencing of RIG-I associated RNAs from virus-infected cells. The nature of RNA ligands of MDA5 is less clear. Several studies suggest that double-stranded RNAs are the preferred agonists for the protein. However, the exact nature of physiological MDA5 ligands from virus-infected cells needs to be elucidated. In this work, we combine a crosslinking technique with next-generation sequencing in order to shed light on MDA5-associated RNAs from human cells infected with measles virus. Our findings suggest that RIG-I and MDA5 associate with AU-rich RNA species originating from the mRNA of the measles virus L gene. Corresponding sequences are poorer activators of ATP-hydrolysis by MDA5 in vitro, suggesting that they result in more stable MDA5 filaments. These data provide a possible model of how AU-rich sequences could activate type I interferon signaling.

MeV-RII_HepG2, a persistently measles virus (MeV) infected human hepatoma cell line, displays resistance towards secondary infections with MeV virotherapeutics

MeV-RII_HepG2, a persistently measles virus (MeV) infected human hepatoma cell line, displays resistance towards secondary infections with MeV virotherapeutics

Systemically delivered measles virus-infected mesenchymal stem cells can evade host immunity to inhibit liver cancer growth

Although attenuated measles virus (MV) has demonstrated potent oncolytic activities towards human cancers, it has not yet been widely adopted into clinical practice. One of the major hurdles is the presence of pre-existing anti-MV immunity in the recipients. In this study, we have evaluated the combination of the potent oncolytic activity of the attenuated MV with the unique immunoprivileged and tumor-tropic biological properties of human bone marrow-derived mesenchymal stem cells (BM-hMSCs) to combat human hepatocellular carcinoma (HCC), orthotopically implanted in SCID mice, passively immunized with human neutralizing antibodies against MV as a preclinical model. SCID mice were orthotopically implanted with patient-derived HCC tissues and established HCC cell lines. SCID mice were passively immunized with human neutralizing anti-measles antibodies. Bioluminescence and fluorescence imaging were employed to monitor the ability of systemically delivered MV-infected BM-hMSCs to infiltrate the implanted tumors and their effects on tumor growth. Systemically delivered MV-infected BM-hMSCs homed to the HCC tumors implanted orthotopically in the liver and it was evidenced that BM-hMSCs could transfer MV infectivity to HCC via heterofusion. Furthermore, therapy with MV-infected BM-hMSCs resulted in significant inhibition of tumor growth in both measles antibody-naïve and passively-immunized SCID mice. By contrast, when cell-free MV viruses were delivered systemically, antitumor activity was evident only in measles antibody-naïve SCID mice. MV-infected BM-hMSCs cell delivery system provides a feasible strategy to elude the presence of immunity against MV in most of the potential cancer patients to be treated with the oncolytic MV viruses.

Protein Kinase PKR Amplification of Interferon β Induction Occurs through Initiation Factor eIF-2α-mediated Translational Control

The protein kinase PKR is activated by RNA with double-stranded (ds) structure and subsequently impairs translation through phosphorylation of protein synthesis initiation factor eIF-2α. PKR also mediates activation of signal transduction pathways leading to interferon beta (IFN-β) gene induction following virus-infection or RNA transfection. We previously demonstrated in measles virus-infected cells that PKR is required for the maximal induction of IFN-β gene expression by the interferon promoter stimulator gene 1 (IPS-1) adaptor-dependent cytosolic RNA sensor pathway. While both IPS-1 and PKR are important mediators of IFN-β induction, with PKR contributing to an enhanced NF-κB activation, the mechanism by which PKR enhances NF-κB activity and amplifies IFN-β induction is unresolved. Herein we tested the possibility that PKR could activate signal transduction pathways indirectly through translational control responses. Following transfection with synthetic or natural dsRNAs or infection with measles virus, we observed increased mRNA but decreased protein levels for the inhibitor of NF-κB signaling, IκB-α, that correlated with PKR activation and eIF-2α phosphorylation. Importantly, knockdown of PKR increased IκB-α protein levels and impaired IFN-β induction. Additionally, inhibition of translation by cycloheximide treatment rescued IFN-β induction following PKR knockdown but not IPS-1 knockdown. Mutation of eIF-2α to prevent phosphorylation also impaired IFN-β induction in PKR-sufficient virus-infected cells. These results suggest that an eIF-2α-dependent translation inhibition mechanism is sufficient to explain the PKR-mediated amplification of IPS-1-dependent IFN-β induction by foreign RNA.

The Use of Oral Fluid Samples Spotted on Filter Paper for the Detection of Measles Virus Using Nested RT‐PCR

Measles is the leading cause of death in infants, although a vaccine is available for its prevention. At this stage of measles elimination and eradication, it is so important to confirm clinically diagnosed measles cases in the laboratory but, developing countries have troubles in collecting and maintaining the cold chain of the specimens while transporting them to the laboratories. Therefore, filter papers are good candidates for simplification of specimen collection and transportation. In this research, the effects of the temperature, at which the dried specimens were kept, and the time duration the dried specimens were kept before being tested, were studied. Since there were not enough patients' oral fluid samples available, a nested reverse transcriptase PCR (RT-PCR) that detected measles virus (MV) from dried filter papers was set up using MV infected cells diluted in sterile phosphate-buffered saline (PBS). Dried specimens were stored at -25°C, 4°C, and room temperature for 1 day, 1, 2, and 3 weeks before being tested. This method was then applied to filter paper oral fluids collected from nine clinically diagnosed measles patients in Iran in 2010 which were tested after being kept at room temperature for 1 day, 1 and 3 weeks after preparation. The results showed that dried oral fluids on filter papers are reliable specimens for the detection of MV RNA using nested RT-PCR, but the nested RT-PCR results of low titer viruses dried onto filter papers are not reproducible and reliable.

Volume and surface changes in vero cell and its nucleus after infection with measles virus: a stereological study

Measles virus has no or indistinctive cytopathic effects (CPE) in cell couture system. Employment of some detecting methods like plaque assay or stereologic experiments, as a method of detecting of viral infection in the cells would be applicable. The aim of this study was investigating the early changes in quantitative parameters of measles virus infected Vero cells. Stereological methods using invariator, were applied for the first time to estimate cell and nucleus volume and cell surface of the infected Vero cell line with the measles virus.This method can be applied on other cultured cells.Vero cells grown in tissue culture plates for 48 hours at 36˚C were infected with 100TCID50 of AiK strain of measles virus. Volume and surface of the infected Vero cells were studied at 4, 9 and 25 hours post infection along with uninfected control cells. The mean cell volume and surface of the cells infected with measles virus, increased ~87% and ~50%, respectively, 4 hours post-infection, as compared with the uninfected control. The nuclei did not show any differences. The mean parameters of infected cells in other time intervals showed no significant difference comparing with the control cells. Although there are other specific methods, stereology may be used as an integrated protocol to detect cytophatic changes of the measles virus infected cells early in the permissive cell culture system.

VSV Oncolysis in Combination With the BCL-2 Inhibitor Obatoclax Overcomes Apoptosis Resistance in Chronic Lymphocytic Leukemia

In chronic lymphocytic leukemia (CLL), overexpression of antiapoptotic B-cell leukemia/lymphoma 2 (BCL-2) family members contributes to leukemogenesis by interfering with apoptosis; BCL-2 expression also impairs vesicular stomatitis virus (VSV)-mediated oncolysis of primary CLL cells. In the effort to reverse resistance to VSV-mediated oncolysis, we combined VSV with obatoclax (GX15-070)—a small-molecule BCL-2 inhibitor currently in phase 2 clinical trials—and examined the molecular mechanisms governing the in vitro and in vivo antitumor efficiency of combining the two agents. In combination with VSV, obatoclax synergistically induced cell death in primary CLL samples and reduced tumor growth in severe combined immunodeficient (SCID) mice-bearing A20 lymphoma tumors. Mechanistically, the combination stimulated the mitochondrial apoptotic pathway, as reflected by caspase-3 and -9 cleavage, cytochrome c release and BAX translocation. Combination treatment triggered the release of BAX from BCL-2 and myeloid cell leukemia-1 (MCL-1) from BAK, whereas VSV infection induced NOXA expression and increased the formation of a novel BAX-NOXA heterodimer. Finally, NOXA was identified as an important inducer of VSV-obatoclax driven apoptosis via knockdown and overexpression of NOXA. These studies offer insight into the synergy between small-molecule BCL-2 inhibitors such as obatoclax and VSV as a combination strategy to overcome apoptosis resistance in CLL.

Systemic Therapy of Disseminated Myeloma in Passively Immunized Mice Using Measles Virus-infected Cell Carriers

Multiple myeloma (MM) is bone marrow plasma cell malignancy. A clinical trial utilizing intravenous administration of oncolytic measles virus (MV) encoding the human sodium-iodide symporter (MV-NIS) is ongoing in myeloma patients. However, intravenously administered MV-NIS is rapidly neutralized by antiviral antibodies. Because myeloma cell lines retain bone marrow tropism, they may be ideal as carriers for delivery of MV-NIS to myeloma deposits. A disseminated human myeloma (KAS 6/1) model was established. Biodistribution of MM1, a myeloma cell line, was determined after intravenous infusion. MM1 cells were found in the spine, femurs, and mandibles of tumor-bearing mice. Lethally irradiated MM1 cells remained susceptible to measles infection and transferred MV to KAS 6/1 cells in the presence of measles immune sera. Mice-bearing disseminated myeloma and passively immunized with measles immune serum were given MV-NIS or lethally irradiated MV-NIS-infected MM1 carriers. The antitumor activity of MV-NIS was evident only in measles naive mice and not in passively immunized mice. In contrast, survivals of both measles naive and immune mice were extended using MV-NIS-infected MM1 cell carriers. Hence, we demonstrate for the first time that systemically administered cells can serve as MV carriers and prolonged survival of mice with pre-existing antimeasles antibodies.

488. Optimization of Measles Virus Infected Mesenchymal Stem Cell Carriers for Clinical Testing in Patients with Recurrent Ovarian Cancer

488. Optimization of Measles Virus Infected Mesenchymal Stem Cell Carriers for Clinical Testing in Patients with Recurrent Ovarian Cancer

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)

Recent studies of primate models suggest that wild-type measles virus (MV) infects immune cells located in the airways before spreading systemically, but the identity of these cells is unknown. To identify cells supporting primary MV infection, we took advantage of mice expressing the MV receptor human signaling lymphocyte activation molecule (SLAM, CD150) with human-like tissue specificity. We infected these mice intranasally (IN) with a wild-type MV expressing green fluorescent protein. One, two, or three days after inoculation, nasal-associated lymphoid tissue (NALT), the lungs, several lymph nodes (LNs), the spleen, and the thymus were collected and analyzed by microscopy and flow cytometry, and virus isolation was attempted. One day after inoculation, MV replication was documented only in the airways, in about 2.5% of alveolar macrophages (AM) and 0.5% of dendritic cells (DC). These cells expressed human SLAM, and it was observed that MV infection temporarily enhanced SLAM expression. Later, MV infected other immune cell types, including B and T lymphocytes. Virus was isolated from lymphatic tissue as early as 2 days post-IN inoculation; the mediastinal lymph node was an early site of replication and supported high levels of infection. Three days after intraperitoneal inoculation, 1 to 8% of the mediastinal LN cells were infected. Thus, MV infection of alveolar macrophages and subepithelial dendritic cells in the airways precedes infection of lymphocytes in lymphatic organs of mice expressing human SLAM with human-like tissue specificity.

Both RIG-I and MDA5 RNA Helicases Contribute to the Induction of Alpha/Beta Interferon in Measles Virus-Infected Human Cells

Measles virus (MV), a member of the family Paramyxoviridae, is a nonsegmented negative-strand RNA virus. The RNA helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are differentially involved in the detection of cytoplasmic viral RNAs and induction of alpha/beta interferon (IFN-alpha/beta). RIG-I is generally believed to play a major role in the recognition of paramyxoviruses, whereas many viruses of this family produce V proteins that can inhibit MDA5. To determine the individual roles of MDA5 and RIG-I in IFN induction after MV infection, small interfering RNA-mediated knockdown of MDA5 or RIG-I was performed in the human epithelial cell line H358, which is susceptible to wild-type MV isolates. The production of IFN-beta mRNA in response to MV infection was greatly reduced in RIG-I knockdown clones compared to that in H358 cells, confirming the importance of RIG-I in the detection of MV. The IFN-beta mRNA levels were also moderately reduced in MDA5 knockdown clones, even though these clones retained fully functional RIG-I. A V protein-deficient recombinant MV (MVDeltaV) induced higher amounts of IFN-beta mRNA at the early stage of infection in H358 cells compared to the parental virus. The reductions in the IFN-beta mRNA levels in RIG-I knockdown clones were less pronounced after infection with MVDeltaV than after infection with the parental virus. Taken together, the present results indicate that RIG-I and MDA5 both contribute to the recognition of MV and that the V protein promotes MV growth at least partly by inhibiting the MDA5-mediated IFN responses.

Measles virus blind to its epithelial cell receptor remains virulent in rhesus monkeys but cannot cross the airway epithelium and is not shed

The current model of measles virus (MV) pathogenesis implies that apical infection of airway epithelial cells precedes systemic spread. An alternative model suggests that primarily infected lymphatic cells carry MV to the basolateral surface of epithelial cells, supporting MV shedding into the airway lumen and contagion. This model predicts that a mutant MV, unable to enter cells through the unidentified epithelial cell receptor (EpR), would remain virulent but not be shed. To test this model, we identified residues of the MV attachment protein sustaining EpR-mediated cell fusion. These nonpolar or uncharged polar residues defined an area located near the binding site of the signaling lymphocytic activation molecule (SLAM), the receptor for MV on lymphatic cells. We then generated an EpR-blind virus maintaining SLAM-dependent cell entry and inoculated rhesus monkeys intranasally. Hosts infected with the selectively EpR-blind MV developed rash and anorexia while averaging slightly lower viremia than hosts infected with wild-type MV but did not shed virus in the airways. The mechanism restricting shedding was characterized using primary well-differentiated human airway epithelial cells. Wild-type MV infected columnar epithelial cells bearing tight junctions only when applied basolaterally, while the EpR-blind virus did not infect these cells. Thus, EpR is probably a basolateral protein, and infection of the airway epithelium is not essential for systemic spread and virulence of MV.