The distinguishing features of human metapneumovirus and respiratory syncytial virus

Acute respiratory tract infections (RTIs) are a leading cause of morbidity and mortality worldwide. Human Metapneumovirus (hMPV) is a member of the Metapneumovirus genus within the Pneumovirinae subfamily of the Paramyxoviridae family. Though hMPV was only discovered in 2001, a large body of work has already shown that it is the aetiologic agent of a substantial proportion of upper and lower RTIs across all age groups in both healthy and immunocompromised hosts throughout the world. RSV, also a pneumovirus, is the human pathogen most closely related to hMPV. RSV is the leading cause of pneumonia and bronchiolitis in infants and young children, but can also cause respiratory tract disease in all age groups. In this paper, we will review the salient features of the virology, epidemiology, pathogenesis, host immune responses, clinical manifestations and diagnostic modalities of hMPV, using RSV as a comparison. In addition, we will show how immunoprophylactic and therapeutic strategies studied and used in clinical practice for RSV—some with great success, and others tragic failure—have led to promising areas of research for the prevention and treatment of the significant burden of disease caused by hMPV. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparison of the seroprevalence of human metapneumovirus and human respiratory syncytial virus

Human metapneumovirus (hMPV) is a virus that induces human respiratory syncytial virus (hRSV)-like illnesses, ranging from upper respiratory tract infection to severe bronchiolitis and pneumonia. The 100 serum samples from children aged 1 month to 5 years were tested for the presence of hMPV and hRSV antibodies using an indirect immunofluorescence assay and a neutralizing-antibody assay, respectively. The seroprevalence of hMPV was significantly lower than that of hRSV in children over 4-months-old (43% vs. 60%, P < 0.025), and the difference was particularly notable between the ages of 4 months and 1 year (11% vs. 48%, P = 0.006). The results suggest that primary infection with hMPV occurs somewhat later than that with hRSV.     

Cell-free and cell-bound antibody in nasal secretions from infants with respiratory syncytial virus infection.

Twenty-two infants under 9 months of age hospitalized with bronchiolitis or pneumonia due to respiratory syncytial virus (RSV) were serially sampled to determine the pattern of secretory antibody response. Using double labeling techniques, we found several types of immunoglobulin in secretions: cell-free antibody to RSV of the immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) classes; and immunoglobulins of all three classes bound to RSV-infected cells shed from the nasal epithelium (presumably cell-bound antibody to RSV). IgA attached to RSV-infected epithelial cells was almost always detected in the first available nasal sample (day 1 or 2 of hospitalization). In contrast, cell-free anti-RSV IgA first appeared an average of 3.5 days later at a time when virus antigen was disappearing from the secretion. IgG and IgM attached to RSV-infected cells appeared more irregularly. The titer of cell-free anti-RSV IgM was often higher than that of IgA early in the illness and declined as the infection resolved. Cell-free anti-RSV IgG was usually present earlier than IgA and rose during convalescence.     

Chemokines in nasal secretions of normal adults experimentally infected with respiratory syncytial virus

The goal of this study was to determine time courses of upregulation of several chemokines in nasal secretions after inoculation of human subjects with a low dose of live respiratory syncytial virus (RSV). Healthy, nonsmoking young adults were admitted to an inpatient clinical research unit. After baseline studies, subjects were nasally inoculated with approximately 10(3) plaque-forming units of RSV (strain A2), followed by daily nasal lavages. Nasal lavage fluid (NLF) was assayed for chemokines by specific ELISA. Of 10 subjects inoculated with RSV, 3 developed clinical symptoms of upper respiratory infection and also shed virus. Among infected subjects, there was a transient postinoculation increase in interleukin-8 (IL-8) in NLF to an average of 2.7-fold compared to baseline, followed by a prolonged increase (maximum mean 5.4-fold) during virus shedding. RANTES, MIP-1alpha, and MCP-1 all increased during virus shedding only (maximum mean increases of 5.3-fold, 13-fold, and 7.2-fold, respectively). Semiquantitative RT-PCR in brushed nasal epithelial cells on day 6 after inoculation suggested upregulation of RANTES, but not IL-8, mRNA during virus shedding. We conclude that chemokines IL-8, RANTES, MIP-1alpha, and MCP-1 are all increased in nasal secretions in human RSV infection at the time of virus shedding and symptomatic illness and that the epithelium lining the nasal turbinate contributes to the increase in RANTES.     

Differential response of dendritic cells to human metapneumovirus and respiratory syncytial virus

Dendritic cells (DCs) play a pivotal role in shaping antiviral immune responses in the respiratory tract. Human metapneumovirus (hMPV) is a recently identified pathogen and like its better known relative, respiratory syncytial virus (RSV), has been increasingly recognized as a major cause of respiratory morbidity in infants and in elderly persons. In the present study, we examined susceptibility as well as cellular responses of human DCs to hMPV compared with RSV. Monocyte-derived DCs (moDCs) were susceptible to infection by both viruses, but only RSV was able to induce a productive infection with release of viral progeny. Despite the fact that viral infection resulted in phenotypic maturation of moDCs, as shown by the upregulation of cell surface markers and antigen-presenting molecules (MHC I and II, CD80, CD83, CD86, CD38), RSV-infected moDCs showed a severely impaired capacity to stimulate CD4+ T cell proliferation. Compared with hMPV, RSV was a more potent inducer of inflammatory and immunomodulatory cytokines, including TNF-alpha, IL-6, IL-1beta, IL-10, and IL-12p70 in both moDCs and plasmacytoid dendritic cells (pDCs). On the other hand, hMPV, but not RSV, was able to trigger production of IFN-alpha by moDCs, while both viruses strongly induced IFN-alpha in pDCs. Finally, both viruses strikingly suppressed IFN-alpha production by moDCs or pDCs stimulated with synthetic dsRNA and CpG-ODN, respectively. The findings provide novel evidence that RSV and hMPV differentially activate human DCs and may use distinct mechanisms to interfere with the host innate and adaptive immune responses.     

Use of egg-yolk antibody for detection of respiratory syncytial virus in nasal secretions by ELISA

Summary Egg-yolk immunoglobulins extracted from the eggs of hens immunized with respiratory syncytial virus (RSV) have been used as a reagent in double sandwich ELISA for detecting RSV in nasal secretions. The sensitivity of virus detection was the same in indirect ELISA, using rabbit anti chicken globulin conjugate, as when biotinylated yolk globulin and labeled avidin were used for detection. The specificity of ELISA for detecting RSV using yolk antibody was similar to that achieved by indirect immunofluorescence using commercial reagents of mammalian origin. Purified immunoglobulin was easily extracted from egg yolk; the amount of globulin present in a single preparation obtained from a batch of ten eggs was sufficient to carry out 10⁶ ELISA tests for RSV detection.With 3 FiguresTelephone number of corresponding author (M. H. V. van Regenmortel) 33/88/610202.     

Identification of potential human respiratory syncytial virus and metapneumovirus T cell epitopes using computational prediction and MHC binding assays

Human respiratory syncytial virus (RSV) and human metapneumovirus (MPV) are two of the most common causes of serious viral lower respiratory tract illness in humans. CD8+ T cells have been shown to be important in animal models and human clinical studies for the clearance of viral infection, and they may contribute in part to protection against severe disease during reinfections. Precise enumeration and accurate phenotyping of RSV- or MPV-specific CD8+ T cells in humans is currently limited by the relatively small number of T cell epitopes that have been mapped with accompanying identification of MHC restriction patterns. We sought to expand the number of potential RSV and MPV epitopes for use in clinical and translational studies by identifying an expanded set of MHC-binding peptides based on RSV and MPV wild-type virus strain protein sequences. We interrogated the full protein sequences of all 9 or 11 proteins of MPV or RSV respectively using four established epitope prediction algorithms for human HLA A*0101, A*0201, or B*0702 binding and attempted to synthesize the top-scoring 150-152 peptides for each of the two viruses. Synthesis resulted in 442 synthesized and soluble peptides of the 452 predicted epitopes for MPV or RSV. We then determined the binding of the synthetic peptides to recombinant human HLA A*0101, A*0201 or B*0702 molecules with the predicted restriction using a commercially available plate-based assay, iTopia. A total of 230 of the 442 peptides tested exhibited binding to the appropriate MHC molecule. The binding results suggested that existing algorithms for prediction of MHC A*0201 binding are particularly robust. The binding results also provided a large benchmarking data collection for comparison of new prediction algorithms.     

Detection of respiratory syncytial virus in nasopharyngeal secretions by DNA-RNA hybridization.

We have developed an RNA-cDNA hybridization assay for the detection of respiratory syncytial virus (RSV) RNA in nasopharyngeal samples. We chose to use as probe a cDNA complementary to the nucleocapsid protein gene of RSV, integrated into the plasmid vector pBR322. The lower limit of sensitivity of the assay is 8.2 X 10(2) PFU of the Long strain of RSV. In throat washes with added cell-free virus, the assay can detect 3.3 X 10(3) PFU of RSV. Respiratory secretions were collected from a group of 104 infants in New Orleans, and 73 of the samples were tested for RSV by immunofluorescence (IF). All were then frozen at -70 degrees C for later testing by hybridization, and 67 were tested for RSV antigens by enzyme immunoassay (EIA). A second set of respiratory secretions from 48 infants in Denver were cultured for virus, assayed for RSV antigen by EIA, and then frozen for later testing by hybridization. For those samples on which IF was performed, hybridization, compared with IF, had a sensitivity of 49% and a specificity of 66%. For samples tested by EIA, hybridization had a sensitivity of 60% and a specificity of 81% compared with EIA. Compared with virus isolation, hybridization assay had a sensitivity of 73% and a specificity of 92%. With clinical samples, the sensitivity and specificity of the assay were improved with the addition of a control blot, which was hybridized to the plasmid vector (pBR322). The performance of the hybridization assay can be expected to improve when the assay is used with fresh clinical material rather than frozen samples.     

Rapid detection of respiratory syncytial virus antigens in nasopharyngeal secretions

The combined use of fluorescein-labelled monoclonal antibody and a cytocentrifuge for preparation of cell spots greatly reduced the time for rapid diagnosis, and improved the sensitivity and ease of detection of respiratory syncytial (RS) virus antigen in specimens of nasopharyngeal secretions.     

A reverse passive haemagglutination test for the detection of respiratory syncytial virus in nasal secretions from infants

A reverse passive haemagglutination (RPH) test has been developed for the detection of respiratory syncytial (RS) virus in nasal secretions, taken from infants with acute respiratory illness. In the final form of the procedure, RS virus was detected in 24 of 25 samples positive for RS virus by tissue culture and/or fluorescence antibody staining and in two samples negative for RS virus by these techniques. The simplicity of the technique and the rapidity with which it may be performed together with its apparently high degree of sensitivity should make RPH useful in the rapid diagnosis of RS virus.     

Detection of respiratory syncytial virus in nasopharyngeal secretions by shell vial technique.

A shell vial technique was used to recover respiratory syncytial virus (RSV) from frozen nasopharyngeal specimens previously tested by rapid diagnostic methods. With specimens determined to be positive by direct fluorescence assay (DFA), the shell vial technique was at least as sensitive as conventional tissue culture (92 versus 90%). The majority of RSV isolates were detected within 16 h postinoculation, versus an average of 4.5 days by conventional techniques. Also, the shell vial method recovered RSV from 16 of 17 specimens (94%) which had previously tested positive by enzyme immunoassay (EIA). In addition, the shell vial method detected RSV in 4 and 11% of specimens previously determined to be negative by DFA and EIA, respectively. Therefore, we recommend the use of the shell vial technique for specimens testing negative by the rapid methods of DFA or EIA.     

Rapid immunoperoxidase assay for detection of respiratory syncytial virus in nasopharyngeal secretions.

Samples of nasopharyngeal secretions obtained from 70 infants and young children with acute respiratory disease were examined for the presence of respiratory syncytial virus by immunoperoxidase assay (IPA). The IPA was compared with the immunofluorescence assay and with cell culture isolation. Respiratory syncytial virus antigen-positive cells were detected by both IPA and immunofluorescence assay in 28 specimens; 25 samples were positive in cell culture. The agreement between virus isolation and IPA and IFA was 89%. The applicability of IPA to rapid viral diagnosis of respiratory syncytial virus infection is discussed.     

Detection of human coronavirus NL63, human metapneumovirus and respiratory syncytial virus in children with respiratory tract infections in south-west Sweden

Two recently detected viruses, human metapneumovirus (hMPV) and coronavirus NL63 (HCoV-NL63), have been associated with acute respiratory tract infections, particularly in young children. This study investigated the frequency of hMPV and HCoV-NL63 infections in Swedish children by screening 221 nasopharyngeal aspirates, collected between November 2003 and May 2005, from 212 children attending the paediatric department of a county hospital in Sweden or submitted from local general practitioners. The samples were originally submitted to be tested for respiratory syncytial virus (RSV), and were examined retrospectively for hMPV and HCoV-NL63 by RT-PCR. Of the 212 patients, 101 were positive for RSV (48%), 22 (10%) were positive for hMPV, and 12 (6%) were positive for HCoV-NL63. The frequency of HCoV-NL63 infection increased from 1% in 2003-2004 to 10% in 2004-2005. Sequence analysis of parts of the coronavirus genomes showed considerable similarity to the HCoV-NL63 prototype sequence. The study demonstrated that HCoV-NL63 and hMPV occur in south-west Sweden with essentially the same frequency, seasonal distribution and clinical characteristics as have been reported in other countries.     

The pathogenesis of respiratory syncytial virus infection in infant ferrets.

The infant ferret is susceptible to respiratory syncytial virus infection in both the upper and lower respiratory tracts. In the nose, viral replication is restricted to the surface respiratory epithelium in the nasal passages and turbinates. In the lungs, viral replication is of a lower order of magnitude and is localized in the alveolar cells. The pattern of viral replication in nasal tissues is independent of the age of the animal at infection, whereas the pattern in lung tissues shows a striking age dependence, with viral replication progressively decreasing as a function of age. Thes age dependence appears to be due to an intrinsic age-related mechanism yet to be defined. We feel that the infant ferret is an acceptable model for the study of respiratory syncytial virus disease and that the study of age dependence observed in ferrets may allow elucidation of the mechanisms involved in the age dependence seen in humans.     

Infection and maturation of monocyte-derived human dendritic cells by human respiratory syncytial virus, human metapneumovirus, and human parainfluenza virus type 3

Human respiratory syncytial virus (HRSV), human metapneumovirus (HMPV), and human parainfluenza virus type 3 (HPIV3) are common, important respiratory pathogens, but HRSV has a substantially greater impact with regard to acute disease, long-term effects on airway function, and frequency of re-infection. It has been reported to strongly interfere with the functioning of dendritic cells (DC). We compared HRSV to HMPV and HPIV3 with regard to their effects on human monocyte-derived immature DC (IDC). Side-by-side analysis distinguished between common effects versus those specific to individual viruses. The use of GFP-expressing viruses yielded clear identification of robustly infected cells and provided the means to distinguish between direct effects of robust viral gene expression versus bystander effects. All three viruses infected inefficiently based on GFP expression, with considerable donor-to donor-variability. The GFP-negative cells exhibited low, abortive levels of viral RNA synthesis. The three viruses induced low-to-moderate levels of DC maturation and cytokine/chemokine responses, increasing slightly in the order HRSV, HMPV, and HPIV3. Infection at the individual cell level was relatively benign, such that in general GFP-positive cells were neither more nor less able to mature compared to GFP-negative bystanders, and cells were responsive to a secondary treatment with lipopolysaccharide, indicating that the ability to mature was not impaired. However, there was a single exception, namely that HPIV3 down-regulated CD38 expression at the RNA level. Maturation by these viruses was anti-apoptotic. Inefficient infection of IDC and sub-optimal maturation might result in reduced immune responses, but these effects would be common to all three viruses rather than specific to HRSV.