Interferon-Induced Transmembrane Protein 1 Restricts Replication of Viruses That Enter Cells via the Plasma Membrane.

The acute antiviral response is mediated by a family of interferon-stimulated genes (ISGs), providing cell-intrinsic immunity. Mutations in genes encoding these proteins are often associated with increased susceptibility to viral infections. One family of ISGs with antiviral function is the interferon-inducible transmembrane proteins (IFITMs), of which IFITM3 has been studied extensively. In contrast, IFITM1 has not been studied in detail. Since IFITM1 can localize to the plasma membrane, we investigated its function with a range of enveloped viruses thought to infect cells by fusion with the plasma membrane. Overexpression of IFITM1 prevented infection by a number of Paramyxoviridae and Pneumoviridae, including respiratory syncytial virus (RSV), mumps virus, and human metapneumovirus (HMPV). IFITM1 also restricted infection with an enveloped DNA virus that can enter via the plasma membrane, herpes simplex virus 1 (HSV-1). To test the importance of plasma membrane localization for IFITM1 function, we identified blocks of amino acids in the conserved intracellular loop (CIL) domain that altered the subcellular localization of the protein and reduced antiviral activity. By screening reported data sets, 12 rare nonsynonymous single nucleotide polymorphisms (SNPs) were identified in human IFITM1, some of which are in the CIL domain. Using an Ifitm1-/- mouse, we show that RSV infection was more severe, thereby extending the range of viruses restricted in vivo by IFITM proteins and suggesting overall that IFITM1 is broadly antiviral and that this antiviral function is associated with cell surface localization.IMPORTANCE Host susceptibility to viral infection is multifactorial, but early control of viruses not previously encountered is predominantly mediated by the interferon-stimulated gene (ISG) family. There are upwards of 300 of these genes, the majority of which do not have a clearly defined function or mechanism of action. The cellular location of these proteins may have an important effect on their function. One ISG located at the plasma membrane is interferon-inducible transmembrane protein 1 (IFITM1). Here we demonstrate that IFITM1 can inhibit infection with a range of viruses that enter via the plasma membrane. Mutant IFITM1 proteins that were unable to localize to the plasma membrane did not restrict viral infection. We also observed for the first time that IFITM1 plays a role in vivo, and Ifitm1-/- mice were more susceptible to viral lung infection. These data contribute to our understanding of how ISGs prevent viral infections.

Recombinant canine distemper virus strain Snyder Hill expressing green or red fluorescent proteins causes meningoencephalitis in the ferret.

The propensity of canine distemper virus (CDV) to spread to the central nervous system is one of the primary features of distemper. Therefore, we developed a reverse genetics system based on the neurovirulent Snyder Hill (SH) strain of CDV (CDV(SH)) and show that this virus rapidly circumvents the blood-brain and blood-cerebrospinal fluid (CSF) barriers to spread into the subarachnoid space to induce dramatic viral meningoencephalitis. The use of recombinant CDV(SH) (rCDV(SH)) expressing enhanced green fluorescent protein (EGFP) or red fluorescent protein (dTomato) facilitated the sensitive pathological assessment of routes of virus spread in vivo. Infection of ferrets with these viruses led to the full spectrum of clinical signs typically associated with distemper in dogs during a rapid, fatal disease course of approximately 2 weeks. Comparison with the ferret-adapted CDV(5804P) and the prototypic wild-type CDV(R252) showed that hematogenous infection of the choroid plexus is not a significant route of virus spread into the CSF. Instead, viral spread into the subarachnoid space in rCDV(SH)-infected animals was triggered by infection of vascular endothelial cells and the hematogenous spread of virus-infected leukocytes from meningeal blood vessels into the subarachnoid space. This resulted in widespread infection of cells of the pia and arachnoid mater of the leptomeninges over large areas of the cerebral hemispheres. The ability to sensitively assess the in vivo spread of a neurovirulent strain of CDV provides a novel model system to study the mechanisms of virus spread into the CSF and the pathogenesis of acute viral meningitis.

Human Respiratory Syncytial Virus Subgroup A and B Infections in Nasal, Bronchial, Small-Airway, and Organoid-Derived Respiratory Cultures.

Human respiratory syncytial virus (HRSV) is the leading cause of bronchiolitis in infants. Two subgroups of HRSV (A and B) routinely cocirculate. Most research has been performed with HRSV-A strains because these are easier to culture than HRSV-B strains. In this study, we aimed to compare the replicative fitness and HRSV-induced innate cytokine responses of HRSV-A and HRSV-B strains in disease-relevant cell culture models. We used two recombinant (r) clinical isolate-based HRSV strains (A11 and B05) and one recombinant laboratory-adapted HRSV strain (A2) to infect commercially available nasal, bronchial, and small-airway cultures. Epithelial cells from all anatomical locations were susceptible to HRSV infection despite the induction of a dominant type III interferon response. Subgroup A viruses disseminated and replicated faster than the subgroup B virus. Additionally, we studied HRSV infection and innate responses in airway organoids (AOs) cultured at air-liquid interface (ALI). Results were similar to the commercially obtained bronchial cells. In summary, we show that HRSV replicates well in cells from both the upper and the lower airways, with a slight replicative advantage for subgroup A viruses. Lastly, we showed that AOs cultured at ALI are a valuable model for studying HRSV ex vivo and that they can be used in the future to study factors that influence HRSV disease severity.IMPORTANCE Human respiratory syncytial virus (HRSV) is the major cause of bronchiolitis and pneumonia in young infants and causes almost 200,000 deaths per year. Currently, there is no vaccine or treatment available, only a prophylactic monoclonal antibody (palivizumab). An important question in HRSV pathogenesis research is why only a fraction (1 to 3%) of infants develop severe disease. Model systems comprising disease-relevant HRSV isolates and accurate and reproducible cell culture models are indispensable to study infection, replication, and innate immune responses. Here, we used differentiated AOs cultured at ALI to model the human airways. Subgroup A viruses replicated better than subgroup B viruses, which we speculate fits with epidemiological findings that subgroup A viruses cause more severe disease in infants. By using AOs cultured at ALI, we present a highly relevant, robust, and reproducible model that allows for future studies into what drives severe HRSV disease.

Measles studies in the macaque model.

Much of our current understanding of measles has come from experiments in non-human primates. In 1911, Goldberger and Anderson showed that macaques inoculated with filtered secretions from measles patients developed measles, thus demonstrating that the causative agent of this disease was a virus. Since then, different monkey species have been used for experimental measles virus infections. Moreover, infection studies in macaques demonstrated that serial passage of the virus in vivo and in vitro resulted in virus attenuation, providing the basis for all current live-attenuated measles vaccines. This chapter will review the macaque model for measles, with a focus on vaccination and immunopathogenesis studies conducted over the last 15 years. In addition, recent data are highlighted demonstrating that the application of a recombinant measles virus strain expressing enhanced green fluorescent protein dramatically increased the sensitivity of virus detection, both in living and sacrificed animals, allowing new approaches to old questions on measles vaccination and pathogenesis.

Development of a semi-quantitative real-time RT-PCR for the detection of measles virus.

Real-time detection of polymerase chain reactions allows convenient detection and quantification of virus-derived nucleic acids in clinical specimens. We have developed a real-time RT-PCR assay for the detection of measles virus (MV) genomic RNA, and compared it to a well-established conventional RT-PCR assay. Based on a serial dilution of the live-attenuated MV Edmonston Zagreb vaccine, the detection limits were approximately 0.1 and 0.02 cell culture infectious dose 50% units (CCID50) per test for the conventional and TaqMan RT-PCR assays, respectively. Furthermore, tissue materials spiked with known quantities of MV were equally well detected in both assays. The TaqMan assay was linear within a range of 10(4.4) to 10(-0.6)CCID50/ml, with an intra-assay variability lower than 3% and an inter-assay variability ranging from 1.5% at 10(4.4)CCID50/ml to 8.7% at 10(-0.6)CCID50/ml. The TaqMan assay could detect representative wild-type viruses from the currently active MV clades, and could detect MV genome in clinical specimens obtained from measles patients. Finally, quantification of MV RNA in peripheral blood mononuclear cells or broncho-alveolar lavage cells from cynomolgus macaques collected at different time points after experimental infection showed a good correlation with virus isolation data. In conclusion, the TaqMan assay developed is specific, sensitive, rapid and reproducible, and can be of use for diagnostic purposes or for studies on the pathogenesis of measles.

Genetic analysis of Asian measles virus strains--new endemic genotype in Nepal.

In many parts of Asia measles virus (MV) continues to be endemic. However, little is known about the genetic characteristics of viruses circulating on this continent. This study reports the molecular epidemiological analysis based on the entire nucleocapsid (N) and hemagglutinin (H) genes of the first isolates from Nepal and Taiwan, as well as of recent MV strains from India, Indonesia, and China. Four isolates collected in various regions in Nepal during 1999 belonged to a new genotype, tentatively called D8. Another Nepalese isolate and one from India belonged to genotype D4. The diversity of the Nepalese strains indicated that measles continues to be endemic in this country. The isolate from Taiwan grouped with D3 viruses and one Chinese strain isolated in The Netherlands was assigned to the previously described clade H, known to be endemic in Mainland China. Molecular characterization emerges as an important tool for monitoring virus endemicity and vaccination efforts.

Impaired immunity in harbour seals (Phoca vitulina) exposed to bioaccumulated environmental contaminants: review of a long-term feeding study.

Mass mortalities among seals and dolphins inhabiting contaminated marine regions have led to speculation about a possible involvement of immunosuppression associated with environmental pollution. To evaluate whether contaminants at ambient environmental levels can affect immune function of seals, we carried out an immunotoxicological study under semifield conditions. Two groups of 11 harbour seals (Phoca vitulina) originating from a relatively uncontaminated area were fed herring from either the highly polluted Baltic Sea or the relatively uncontaminated Atlantic Ocean. Changes in immune function were monitored over a 2 1/2-year period. The seals that were fed contaminated Baltic herring developed significantly higher body burdens of potentially immunotoxic organochlorines and displayed impaired immune responses as demonstrated by suppression of natural killer cell activity and specific T-cell responses. During a 2-week fasting experiment performed at the end of the feeding study, mobilization of organochlorines from the blubber did not lead to a strong increase of contaminant levels in the blood, and no enhancement of the existing immunosuppression was observed. These results demonstrate that chronic exposure to environmental contaminants accumulated through the food chain affects immune function in harbour seals, whereas short-term fasting periods, which are normal for seals, do not seem to pose an additional risk. The seals of this study were not exposed perinatally to high levels of environmental chemicals, and body burdens of organochlorines measured near the end of the study were lower than those generally observed in free-ranging seals inhabiting many contaminated regions. Therefore, it may be expected that environmental contaminants adversely affect immune function of free-ranging seals inhabiting contaminated regions at least as seriously as observed in these studies.

Contaminant-related suppression of delayed-type hypersensitivity and antibody responses in harbor seals fed herring from the Baltic Sea.

Recent mass mortalities among several marine mammal populations have led to speculation about increased susceptibility to viral infections as a result of contaminant-induced immunosuppression. In a 2.5-year study, we fed herring from either the relatively uncontaminated Atlantic Ocean or the contaminated Baltic Sea to two groups of captive harbor seals and monitored immune function in the seals. Seals fed the contaminated fish were less able to mount a specific immunological response to ovalbumin, as measured by in vivo delayed-type hypersensitivity (DTH) reactions and antibody responses. The skin reaction to this protein antigen was characterized by the appearance of mononuclear cells which peaked at 24 hr after intradermal administration, characteristic of DTH reactions in other animals studied. These DTH responses correlated well with in vitro tests of T-lymphocyte function, implicating this cell type in the reaction. Aryl-hydrocarbon (Ah) receptor-dependent toxic equivalent (TEQ) profiles in blubber biopsies taken from the seals implicated polychlorinated biphenyls rather than dioxins or furans in the observed immunosuppression. Marine mammal populations currently inhabiting polluted coastal environments in Europe and North America may therefore have an increased susceptibility to infections, and pollution may have played a role in recent virus-induced mass mortalities.

Evaluation of different measles IgG assays based on recombinant proteins using a panel of low-titre sera.

During the WHO campaign to eradicate measles, accurate discrimination between immune and non-immune individuals will become increasingly important. Due to waning immunity in vaccinated populations, the performance of a measles IgG assay depends mainly on its ability to detect reliably seronegative individuals among many vaccinees with low antibody levels. New serological tests based on recombinant proteins detect only a fraction of the total measles virus (MV) specific antibodies. Therefore, several assays based on recombinant MV-haemagglutinin (ELISA and flow cytometry) or MV-fusion protein (flow cytometry) as well as neutralisatlon and haemagglutination test have been evaluated using a large panel of low-titre and negative sera. Since such an evaluation is highly dependent on threshold values for positivity, the receiver operating characteristic curve analysis was applied. The H-FACS and the H-ELISA showed the best performing characteristics (specificity: 97.4 and 96.1%, respectively; sensitivity: 88.1 and 89.6%, respectively) and may be an alternative to the neutralisation assay. The number of undefined/grey zone sera was significantly lower compared to a commercial whole virus-based ELISA and therefore fewer individuals would be vaccinated unnecessarily.

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.

A FACS-measured immunofluorescence assay was developed for the detection of antibodies directed against the hemagglutinin (H) and fusion (F) glycoproteins of measles virus (MV). Human melanoma cell lines transfected with either the MV H or F genes, which showed a high surface expression of the respective proteins in their native conformation, were used as target cells. The cells were incubated with diluted plasma samples, and stained subsequently with FITC-conjugated secondary antibodies. The FACS-measured fluorescence signals correlated directly with the amount of specific immunoglobulins over a wide concentration range. The use of different conjugates enabled the separate detection of MV-specific IgG, IgM, IgA and IgG subclasses, with relatively low backgrounds. Hemagglutinin-specific IgG, IgM and IgA fluorescence signals were shown to correlate well with MV-specific IgG ELISA titers and MV-specific IgM or IgA capture ELISA OD450-values, respectively. The polyclonal conjugates with specificity for human immunoglobulins offered sufficient cross-reactivity to detect MV-specific IgG, IgM and IgA in plasma samples of cynomolgus macaques, making this technique a useful tool for studying serological responses in vaccination and challenge experiments in non-human primate models.

Morbillivirus infections of aquatic mammals: newly identified members of the genus.

Several disease outbreaks, which have caused the deaths of many thousands of seals and dolphins during the last decade, have now been attributed to infections with newly identified Morbilliviruses. Outbreaks in the late eighties amongst harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus) in northwestern Europe and amongst baikal seals (Phoca sibirica) in Siberia were caused by the newly discovered phocine distemper virus and by a strain of canine distemper virus, respectively. Although closely related these two viruses were not identical. They were more distantly related to the viruses which caused mass mortality amongst striped dolphins (Stenella coeruleoalba) in the Mediterranean sea in the early nineties. This dolphin morbillivirus was shown to be closely related to a virus that was found in harbour porpoises (Phocoena phocoena) which had stranded at the coasts of northwestern Europe in the late eighties: porpoise morbillivirus. The present knowledge of the genetic and antigenic relationships of these apparently new members of the genus Morbillivirus with the established members of the genus is presented. In addition, the origin and epizootiological aspects of these newly discovered viruses are discussed. Finally experimental evidence that environmental pollution may have contributed to the severity and extent of these infections in recent years is presented.

Relative immunocompetence of the newborn harbour seal, Phoca vitulina.

The immune system of many mammalian species is not fully developed at birth, with newborns obtaining temporary immunological protection from maternal antibodies. Little is known of the immune system of the harbour seal, and developmental aspects of its immune system have not been systematically studied. We collected blood and milk samples from nine free-ranging mother-pup pairs throughout the lactation period on Sable Island, Canada, in an effort to characterise developmental aspects of the immune system of this newborn pinniped. Pup lymphocytes responded stronger to the mitogens concanavalin A, phytohaemagglutinin, and pokeweed mitogen than the lymphocytes of their mothers. In contrast to newborn cats and dogs, newborn seal pups developed high specific antibody responses after immunisation with an inactivated rabies vaccine. Circulating levels of total IgG in newborn pups were low (3% of maternal levels), but increased rapidly after colostrum intake (to 65% of maternal levels after 15 days). A similar pattern of increase in pup serum was observed for phocine distemper virus specific antibodies which had been detected in the serum and milk of mothers, suggesting that the transfer of colostral antibodies is an important feature of temporary protection for the pup. We speculate that the relative immunocompetence of the harbour seal at birth reflects an adaptation to its relatively short nursing period and limited maternal care.

Mitogen and antigen induced B and T cell responses of peripheral blood mononuclear cells from the harbour seal (Phoca vitulina).

In vitro assays were developed for studies concerning the functioning of the immune system of the harbour seal (Phoca vitulina). Proliferative responses of peripheral blood mononuclear cells (PBMC) were measured after stimulation with different concentrations of the mitogens concanavalin A (Con A), pokeweed mitogen (PWM), phytohaemagglutinin (PHA) or lipopolysaccharide from Salmonella typhimurium (LPS). Con A and PWM induced strong proliferative responses, while PHA and LPS induced comparatively low proliferative responses. Responses of mitogen stimulated PBMC to recombinant human interleukin-2 (rhIL-2) and in vitro immunoglobulin production by mitogen stimulated PBMC were measured to discriminate between stimulation of T cells and B cells. It was found that Con A and PHA stimulate phocine T cells, PWM stimulates both T cells and B cells and LPS predominantly stimulates phocine B cells. Antigen-specific immune responses were measured after immunization of seals with an inactivated rabies vaccine and/or with tetanus toxoid. Antigen-specific proliferation of PBMC and the presence of antigen-specific antibody forming cells were demonstrated for both antigens in the PBMC of immunized animals. The responses measured in vitro correlated well with the development of specific serum antibody titers to these antigens.

Administration of an insulin powder to the lungs of cynomolgus monkeys using a Penn Century insufflator.

A powder formulation of live-attenuated measles vaccine is being developed for administration to the lungs. The safety and efficacy of the powder will be assessed by insufflation into cynomolgus monkeys. A Penn Century insufflator has been evaluated for powder dosing to the monkeys using an insulin formulation having similar physicochemical characteristics to the vaccine powder. Insulin pharmacokinetics were compared following dosing by powder insufflation, solution instillation into the trachea and subcutaneous injection. The insulin dosed to the lungs and trachea was more rapidly absorbed than that administered subcutaneously. Insulin bioavailability was greater from the inhaled powder than from the instilled solution. The findings confirm that the Penn Century device is suitable for vaccine powder dosing to the deep lung.

Short term fasting does not aggravate immunosuppression in harbour seals (Phoca vitulina) with high body burdens of organochlorines.

Two groups of 11 harbour seals (Phoca vitulina) with different body burdens of organochlorines were subjected to an experimental 15-day fasting period, during which they lost an average 16.5% of their body weights. Blood levels of the most persistent organochlorines showed an approximate twofold increase, while levels of aryl hydrocarbon receptor-binding organochlorines remained largely unaffected. Few differences in immunological parameters were observed between the two dietary groups. Numbers of circulating lymphocytes dropped to about 65% of the initial values and NK cell activity showed a slight increase in both groups. Mitogen- and antigen-induced lymphoproliferative responses of the Baltic group of seals remained within normal ranges. These results suggest that relatively short-term fasting periods do not present an additional immunotoxicological risk to seals with high body burdens of organochlorines.

Impaired immunity in harbour seals [Phoca vitulina] fed environmentally contaminated herring.

In recent years, mass mortalities among seals and dolphins have been attributed to infections with different morbilliviruses. In all cases, these marine top predators were exposed to high levels of persistent lipophilic environmental contaminants accumulated through the food chain. This observation led to the hypothesis that a contaminant-related suppression of the immune system might have contributed to the severity of the virus outbreaks. We conducted a semi-field feeding experiment, in which we fed two groups of harbour seals (Phoca vitulina) fish with different levels of contaminants. During a period of 2 1/2 years, blood samples were taken at regular intervals, and the functioning of different compartments of the immune system was monitored and compared. We found impaired natural killer (NK) and specific T cell responses in the seals fed contaminated fish. This is the first demonstration of immunosuppression in mammals following chronic exposure to environmental contaminants at ambient environmental levels.

Major immunogenic proteins of phocid herpes-viruses and their relationships to proteins of canine and feline herpesviruses.

The immunogenic proteins of cells infected with the alpha- or the gamma-herpesvirus of seals, phocid herpesvirus-1 and -2 (PhHV-1, -2), were examined in radioimmunoprecipitation assays as a further step towards the development of a PhHV-1 vaccine. With sera obtained from convalescent seals of different species or murine monoclonal antibodies (Mabs), at least seven virus-induced glycoproteins were detected in lysates of PhHV-1-infected CrFK cells. A presumably disulphide-linked complex composed of glycoproteins of 59, 67 and 113/120 kDa, expressed on the surface of infected cells, was characterized as a major immunogenic infected cell protein of PhHV-1. This glycoprotein complex has previously been identified as the proteolytically cleavable glycoprotein B homologue of PhHV-1 (14). At least three distinct neutralization-relevant epitopes were operationally mapped, by using Mabs, on the glycoprotein B of PhHV-1. Among the infected cell proteins of the antigenically closely related feline and canine herpesvirus, the glycoprotein B equivalent proved to be the most highly conserved glycoprotein. Sera obtained from different seal species from Arctic, Antarctic, and European habitats did not precipitate uniform patterns of infected cell proteins from PhHV-1-infected cell lysates although similar titres of neutralizing antibodies were displayed. Thus, antigenic differences among the alphaherpesvirus species prevalent in the different pinniped populations cannot be excluded. PhHV-2 displayed a different pattern of infected cell proteins and only limited cross-reactivity to PhHV-1 at the protein level was detected, which is in line with its previous classification as a distinct species, based on nucleotide sequence analysis, of the gammaherpesvirus linenge. A Mab raised against PhHV-2 and specific for a major glycoprotein of 117 kDa, cross reacted with the glycoprotein B of PhHV-1. The 117-kDa glycoprotein could represent the uncleaved PhHV-2 glycoprotein B homologue.

Age-related disease in recurrent outbreak of phocid herpesvirus type-1 infections in a seal rehabilitation centre: evaluation of diagnostic methods.

The prevalence and clinical signs of phocid herpesvirus type-1 (PhHV-1) infections among harbour seals (Phoca vitulina) in a seal rehabilitation centre in the Netherlands were monitored between June and September 1993 and 1994 when 34 and 36 seals, respectively, were rehabilitated. In both years PhHV-1-related disease outbreaks occurred in the pupping season. PhHV-1 infections were diagnosed by the demonstration of a more than four-fold increase in virus neutralising serum antibodies in paired serum samples, by the isolation of the virus from swab samples in primary seal kidney cells, and by the detection of PhHV-1 DNA with a polymerase chain reaction (PCR) assay in swab samples. This assay targets a 290 bp fragment of the glycoprotein D (gD) gene equivalent of PhHV-1. The PCR assay when combined with Southern blotting (PCR-SB) was approximately 1000 times more sensitive than virus isolation when tested with serially diluted samples from PhHV-1-infected cell cultures. In contrast with virus isolation, the PCR-SB scored as positive all the animals with serological evidence of PhHV-1 infection. The majority of seals present in the centre during the outbreaks contracted the infection and developed benign upper respiratory disease. However, the severity of PhHV-1-related disease was inversely correlated with age and fatal generalised infections occurred only in neonates.

Streptococcus pneumoniae exposure is associated with human metapneumovirus seroconversion and increased susceptibility to in vitro HMPV infection.

It remains largely unknown which factors determine the clinical outcome of human metapneumovirus (HMPV) infections. The aim of the present study was to analyse whether exposure to bacterial pathogens can influence HMPV infections. From 57 children, serum samples and colonization data for Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and Streptococcus pneumoniae were collected at 1.5, 6, 14 and 24 months of age. Seroconversion rates to HMPV were determined and related to bacterial carriage. Frequent nasopharyngeal carriage (≥2 times in the first 2 years of life) of S. pneumoniae, but not of the other three pathogens, was associated with increased seroconversion rates of infants to HMPV at the age of 2 years (frequently vs. less exposed, 93% vs. 59%; p <0.05). Subsequently, the susceptibility of well-differentiated normal human bronchial epithelial cells (wd-NHBE) pre-incubated with bacterial pathogens to in vitro HMPV infection was evaluated. Pre-incubation of wd-NHBE with S. pneumoniae resulted in increased susceptibility to infection with HMPV-enhanced green fluorescent protein (EGFP), as determined by enumeration of EGFP-positive cells. This was not the case for cells pre-incubated with H. influenzae, M. catarrhalis on S. aureus. We conclude that exposure to S. pneumoniae can modulate HMPV infection.