hMPV Infection Research Articles

Activity and Regulation of Alpha Interferon in Respiratory Syncytial Virus and Human Metapneumovirus Experimental Infections

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) cause a similar spectrum of respiratory infections in humans. Classified within the Paramyxoviridae family, Pneumovirinae subfamily, RSV and hMPV present a significant degree of divergence in genome constellation, organization, and protein sequences. RSV has been reported to be a poor inducer of alpha/beta interferons (IFN-alpha/beta) and partially resistant to its antiviral activity. The nature of the innate immune response to hMPV is currently unknown. Herein, an experimental mouse model was used to investigate the interplay between RSV and hMPV infections and IFN-alpha in the airways. RSV-infected BALB/c mice treated intranasally with either poly-ICLC, a potent inducer of IFN-alpha, or directly with recombinant IFN-alpha showed significantly reduced lung viral titers, inflammation, and clinical disease than untreated controls. However, RSV was significantly less sensitive to the antiviral activity of IFN-alpha than hMPV. Similarly, when the ability to directly induce IFN-alpha production was assessed, RSV was clearly a weaker inducer of IFN-alpha than hMPV, as shown by both kinetics and the absolute amount of IFN-alpha secreted into the bronchoalveolar lavage. To further investigate the putative inhibitory effect of these viruses on IFN-alpha production, mice were infected for 48 h prior to treatment with poly-ICLC or a specific Toll-like receptor 9 ligand, CpG oligodeoxynucleotides. Strikingly, both poly-ICLC- and CpG-mediated IFN-alpha production was abrogated by either RSV or MPV infection. These results suggest that a complex interplay between virus-specific and host-mediated responses regulates IFN-alpha in the lung during infection by members of the Pneumovirinae family.

Human Metapneumovirus Infection in Lung Transplant Recipients: Clinical Presentation and Epidemiology

In immunocompromised patients, respiratory viruses are likely to lead to lower respiratory tract infections that cause severe morbidity and mortality. We conducted a prospective study from September 2003 to March 2004 to investigate the epidemiology and impact of human metapneumovirus (hMPV) on lung transplant recipients. We collected 265 nasopharyngeal aspirates and bronchoalveolar lavages: 51 samples originated from immunocompromised adults, 49 from lung transplant recipients, and 2 from a bone marrow recipient. Additionally, 209 samples from hospitalized non-immunocompromised children and 5 samples from immunocompromised children were analyzed for replicating hMPV by a combined cell culture and reverse transcriptase polymerase chain reaction method that includes DNA sequencing of selected isolates. Twelve samples from lung transplant recipients (25%), 29 from non-immunocompromised children (14%), and 2 from a child with a renal transplant were positive for hMPV. Most of the cases clustered within 2 outbreaks in October/November and March. In immunocompromised patients, hMPV was isolated throughout the entire observation period. The same viral strains circulated in hospitalized children and in lung transplant recipients. A different strain was isolated during the interepidemic period, suggesting that hMPV infections may be transmitted among lung transplant recipients independently from the community outbreak situation. Clinical signs and symptoms varied from no symptoms to severe pneumonia or acute graft rejection. Significantly, the only deaths occurred in the hMPV-positive group. Of interest, identification of replicating hMPV significantly correlated with rejection symptoms present at the time point of sample collection. Results of the study suggest that hMPV may be added to the list of pathogens that are possibly associated with episodes of allograft rejection.

Immune response and alteration of pulmonary function after primary human metapneumovirus (hMPV) infection of BALB/c mice

Human metapneumovirus (hMPV), a recently identified virus, causes upper and lower respiratory tract diseases. In this study, we show that BALB/c mice inoculated with hMPV exhibited significant morbidity on 1–2 days post-infection, when airway obstruction was found. Increased airway hyper-responsiveness to metacholine was found on day 4 concurrent with lung viral replication. Both IgG1 and IgG2a hMPV-specific antibodies were found in sera, while interferon-gamma (IFN-γ) and interleukin-4 (IL-4) were found in bronchoalveolar lavage. Lung histology showed parenchymal pneumonia and increased lymphocytic infiltration. We present here an animal model that may be helpful in studying hMPV pathogenesis and evaluating the effects of vaccines.

The Immune Response to Human Metapneumovirus Is Associated with Aberrant Immunity and Impaired Virus Clearance in BALB/c Mice

Human metapneumovirus (HMPV), recently identified in isolates from children hospitalized with acute respiratory tract illness, is associated with clinical diagnosis of pneumonia, asthma exacerbation, and acute bronchiolitis in young children. HMPV has been shown to cocirculate with respiratory syncytial virus (RSV) and mediate clinical disease features similarly to RSV. Little is known regarding the pathophysiology or immune response associated with HMPV infection; thus, animal models are needed to better understand the mechanisms of immunity and disease pathogenesis associated with infection. In this study, we examine features of the innate and adaptive immune response to HMPV infection in a BALB/c mouse model. Primary HMPV infection elicits weak innate and aberrant adaptive immune responses characterized by induction of a Th2-type cytokine response at later stages of infection that coincides with increased interleukin-10 expression and persistent virus replication in the lung. Examination of the cytotoxic T lymphocyte and antibody response to HMPV infection revealed a delayed response, but passive transfer of HMPV-specific antibodies provided considerable protection. These features are consistent with virus persistence and indicate that the immune response to HMPV is unique compared to the immune response to RSV.

Detection of Human Metapneumovirus Antigens in Nasopharyngeal Secretions by an Immunofluorescent-Antibody Test

Human metapneumovirus (hMPV) is a recently discovered pathogen associated with respiratory tract infections, primarily in young children, immunocompromised individuals, and elderly individuals. Reverse transcription-PCR (RT-PCR) has been reported to be a more sensitive method for the diagnosis of hMPV infections than virus isolation by culture and serological study. However, there has been no report on rapid methods, such as an immunofluorescent-antibody test or an enzyme-linked immunosorbent assay, for the detection of hMPV antigens in nasopharyngeal secretions. In this study, we compared an indirect immunofluorescent-antibody test (IFA) with a monoclonal antibody with RT-PCR for detection of hMPV in nasal secretions from 48 hospitalized children with respiratory tract infections. Fifteen of the 48 children were positive for hMPV by RT-PCR. IFA results were positive for 11 of the 15 RT-PCR-positive children (sensitivity, 73.3%) and 1 of the 33 RT-PCR-negative children (specificity, 97.0%). Although the sensitivity of IFA is lower than that of RT-PCR, IFA is a rapid and useful test for the diagnosis of hMPV infections in children.

Development of a cotton rat–human metapneumovirus (hMPV) model for identifying and evaluating potential hMPV antivirals and vaccines

Hispid cotton rats were inoculated with two different human metapneumovirus (hMPV) subtype A strains and one subtype B hMPV. Although no overt disease was seen in any virus-inoculated animal, following an eclipse phase, significant pulmonary virus titers were observed in every hMPV-inoculated animal through day 7 post virus inoculation (p.i.) and in most through day 10. Peak virus titers occurred four days p.i., while virus-induced histopathology was most evident in lung sections obtained from animals 7 to 10 days p.i. The latter consisted primarily of desquamating and hypertrophic columnar epithelial cells lining the bronchi and bronchioles and the presence of large numbers of leukocytes in and around the bronchi and bronchioles. In fluorescent antibody studies, virus antigen-specific fluorescence was most evident in the desquamating tall columnar epithelial cells lining bronchi and bronchioles, in pneumocytes lining alveoli and in single or small groups of free cells, most probably leukocytes, present in the lumen of alveoli, bronchi and bronchioles. Virus was generally not detected in inoculated animals >10 days p.i. Although the pattern of virus replication in cotton rats was similar for all the three virus stains, the B subtype consistently grew to lower levels than the two A strains. Regardless, these findings indicate that hMPV replicates in cotton rats and that these animals may be used as a small animal model of hMPV infection and to facilitate the identification and development of vaccines and antivirals for preventing and/or ameliorating infections caused by this virus.

Development and Validation of an Enzyme-Linked Immunosorbent Assay for Human Metapneumovirus Serology Based on a Recombinant Viral Protein

The human metapneumovirus (hMPV) is a newly reported respiratory virus belonging to the Paramyxoviridae family that has been associated with bronchiolitis and pneumonia in young children. We developed a simple enzyme-linked immunosorbent assay (ELISA) for hMPV serological testing using the nucleoprotein (N) from group A or B (N-A or N-B) as the antigen, and we evaluated it in both children and adults. The N proteins were first used in a Western immunoblot assay to identify hMPV-negative sera, which were then used to determine the cutoff value of the ELISA test. Subsequent evaluation of the ELISA-N test revealed that the mean reciprocal antibody titer of 20 randomly selected seropositive children was 143, compared to 69 for 20 seropositive adults. In a prospective evaluation of 71 adults with acute exacerbations of chronic obstructive pulmonary disease, 58 (81.6%) had prior hMPV antibodies and 3 (4.2%) had evidence of recent hMPV infection. In testing paired sera from adults (n = 4) with recent hMPV group A infection confirmed by reverse transcriptase PCR (RT-PCR), ELISAs using the N-A or N-B proteins were able to detect hMPV seroconversion. Moreover, testing of paired sera from three adults with a recent infection by the human respiratory syncytial virus confirmed by RT-PCR and serology did not reveal any increase in hMPV antibodies over time. The ELISA-N is a simple, objective, and specific serological test useful for detecting anti-hMPV antibodies following group A or B viral infections, which should permit a better understanding of the epidemiology of this virus.

Immunofluorescence Assay for Detection of Human Metapneumovirus-Specific Antibodies by Use of Baculovirus-Expressed Fusion Protein

Human metapneumovirus (hMPV) has recently been identified as an etiological agent of acute respiratory infections. The hMPV fusion (F) protein has been indicated to be a major antigenic determinant that mediates effective neutralization and protection against hMPV infection. We developed a new immunofluorescence assay (IFA) using Trichoplusia ni (Tn5) insect cells infected with a recombinant baculovirus-expressing hMPV F protein (Bac-F IFA). A total of 200 serum samples from Japanese people 1 month to 41 years old were tested for immunoglobulin G antibodies to hMPV F protein by Bac-F IFA. The results were compared with those of the conventional IFA based on hMPV-infected LLC-MK2 cells (hMPV IFA). The titers obtained by the two IFAs correlated well (correlation coefficient of 0.88), and the concordance of seroreactivities between the two IFAs was 91% (kappa=0.76). For 192 of the 200 serum samples, the titers obtained by the Bac-F IFA were equal to or higher than those obtained by the hMPV IFA. These results indicated that the Bac-F IFA was more sensitive than the hMPV IFA and that the majority of the antibodies detected by the hMPV IFA reacted with the hMPV F protein. The Bac-F IFA is a more reliable, sensitive, and specific method for the detection of hMPV antibodies than is the hMPV IFA.

Expression of Recombinant Small Hydrophobic Protein for Serospecific Detection of Avian Pneumovirus Subgroup C

The small hydrophobic (SH) gene of the avian pneumovirus (APV) Colorado isolate (CO), which belongs to subgroup C (APV/C), was expressed with a baculovirus vector. The recombinant SH protein was evaluated as a potential subgroup-specific diagnostic reagent in order to differentiate infections resulting from APV/C from those induced by APV/A, APV/B, and human metapneumovirus (hMPV). When the recombinant baculovirus was used to infect insect cells, a 31- to 38-kDa glycosylated form of the SH protein was produced and subsequently tested for reactivity with antibodies specific for APV/A, APV/B, APV/C, and hMPV. Western blot analysis showed that the expressed recombinant SH protein could only be recognized by APV/C-specific antibodies. This result was consistent with sequence analysis of the APV/C SH protein, which had very low (24%) amino acid identity with the corresponding protein of hMPV and no discernible identity with the SH protein of APV/A or APV/B. A recombinant SH protein-based enzyme-linked immunosorbent assay (ELISA) was developed, and it further confirmed the lack of reactivity of this protein with antisera raised to APV/A, APV/B, and hMPV and supported its designation as a subgroup-specific antigen. This finding indicated that the recombinant SH protein was a suitable antigen for ELISA-based detection of subgroup-specific antibodies in turkeys and could be used for serologically based differential diagnosis of APV and hMPV infections.

Seasonality and clinical features of human metapneumovirus infection in children in Northern Alberta

Human metapneumovirus (hMPV) causes respiratory tract infections in all age groups. The characteristics of pediatric hMPV infection in Northern Alberta have not been studied. The objectives of this study were to determine the seasonality of pediatric hMPV infections over a 13-month period, the genetic relationship of hMPV isolates to hMPV detected in other parts of Canada, and the burden of illness and possible risk factors for pediatric hMPV hospitalization. Detection of hMPV by polymerase chain reaction was performed on nasopharyngeal specimens collected from outpatients and inpatients at the Stollery Children's Hospital in Edmonton, Alberta, November 12, 2002-December 31, 2003. Forty-two of 1,079 specimens were positive for hMPV (3.9%) from 41 patients (14 outpatients and 27 inpatients), with a peak incidence during January-April, but isolates were detected 10 months of the year. Co-infection was not detected in 39 specimens from which RSV had been detected. Two hMPV genetic clusters were detected, and the isolates were homologous to those of previous Canadian isolates. Four of the 14 outpatients had reactive airways disease. Possible risk factors in the 27 inpatients included prematurity (n = 8), congenital heart disease (n = 6), gastroesophageal reflux disease or aspiration (n = 6), global developmental delay (n = 5), and multiple congenital anomalies (n = 4). Risk factors for hospitalization appear to be similar to risk factors for respiratory syncytial virus hospitalization.

Human Metapneumovirus Persists in BALB/c Mice despite the Presence of Neutralizing Antibodies

Human metapneumovirus (HMPV) has emerged as an important human respiratory pathogen causing upper and lower respiratory tract infections in young children and older adults. Recent epidemiological evidence indicates that HMPV may cocirculate with respiratory syncytial virus, and HMPV infection has been associated with other respiratory diseases. In this study, we show that BALB/c mice are susceptible to HMPV infection, the virus replicates in the lungs with biphasic growth kinetics in which peak titers occur at days 7 and 14 postinfection (p.i.), and infectious HMPV can be recovered from lungs up to day 60 p.i. In addition, we show that genomic HMPV RNA can be detected in the lungs for >/=180 days p.i. by reverse transcription-PCR; however, neither HMPV RNA nor infectious virus can be detected in serum, spleen, kidneys, heart, trachea, and brain tissue. Lung histopathology revealed prevalent mononuclear cell infiltration in the interstitium beginning at day 2 p.i. and peaking at day 4 p.i. which decreased by day 14 p.i. and was associated with airway remodeling. Increased mucus production evident at day 2 p.i. was concordant with increased bronchial and bronchiolar inflammation. HMPV-specific antibodies were detected by day 14 p.i., neutralizing antibody titers reached >/=6.46 log(2) end-point titers by day 28 p.i., and depletion of T cells or NK cells resulted in increased HMPV titers in the lungs, suggesting some immune control of viral persistence. This study shows that BALB/c mice are amenable for HMPV studies and indicates that HMPV persists as infectious virus in the lungs of normal mice for several weeks postinfection.

Fatal Human Metapneumovirus Infection in Stem Cell Transplant Recipients.

The importance of human metapneumovirus (hMPV), a recently discovered human respiratory virus, in hematopoietic cell transplant (HCT) recipients is not known. We retrospectively surveyed 211 bronchoalveolar lavage samples (BAL) from symptomatic adults with new or changing pulmonary infiltrates after HCT during five-year period using a quantitative real time RT-PCR assay. Medical records, radiographic studies, biopsy specimens, and autopsy material were reviewed. hMPV was detected in 8/211 (3.8%) BAL specimens from 5/162 (3%) patients. The median titer of hMPV was 9.0 x 106 copies/ml of BAL fluid (range 5.5 x 104 to 3.5 x 108). Persistent hMPV infection was noted in all 3 patients who had multiple samples tested. hMPV was also detected in lung tissue in 1 of 2 samples tested. Infected patients were between the ages of 24–54 years, and became symptomatic within the first 40 days post transplant. Initial symptoms included fever, cough, nasal congestion, and sore throat. Clinical findings near the time of diagnosis included respiratory failure, pulmonary hemorrhage, and culture-negative septic shock. Four patients died as a result of acute respiratory distress and pulmonary disease with clinical and autopsy diagnoses of “idiopathic pneumonia.” We conclude that hMPV infection is associated with respiratory failure in immunocompromised adults and may result in fulminant respiratory decompensation in the post transplant period. Prospective studies of the role of this virus in idiopathic pneumonia syndromes of immunocompromised patients are warranted.

Impact of human metapneumovirus in childhood: Comparison with respiratory syncytial virus and influenza viruses

This study evaluated the overall impact of human metapneumovirus (hMPV) infection in 1,505 children and their households, and compared it with infections due to respiratory syncytial virus (RSV) and influenza viruses. Nasopharyngeal swabs were used at enrollment to collect specimens for the detection of hMPV, RSV, and influenza virus RNA by reverse-transcriptase polymerase chain reaction (RT-PCR). hMPV was detected in 42 children (2.8%), RSV in 143 (9.5%; P < 0.0001 vs. hMPV), and influenza viruses in 230 (15.3%; P < 0.0001 vs. hMPV). Of the 42 hMPV-positive samples, one was also positive for RSV and six for influenza viruses, for a co-infection rate of 16.7%. Clinically, hMPV was identified only in patients with acute respiratory infection, whereas RSV and influenza viruses were also detected in patients with different clinical manifestations. Symptoms with statistically significant different proportions at presentation were fever (more frequent in the hMPV- and influenza-positive children) and wheezing with bronchiolitis or asthma exacerbation (more frequent among hMPV- and RSV-positive cases). The households of the hMPV- and the influenza-positive children had significantly more illnesses, needed significantly more medical visits, received more antipyretics, and missed significantly more work or school days than those of the RSV-positive children. Results show that hMPV is an emerging cause of acute respiratory infection in childhood, and may have a significant clinical and socioeconomic impact on children and their families.

A host-range restricted parainfluenza virus type 3 (PIV3) expressing the human metapneumovirus (hMPV) fusion protein elicits protective immunity in African green monkeys

Human metapneumovirus (hMPV) infection causes respiratory tract disease similar to that observed during human respiratory syncytial virus infection (hRSV). hMPV infections have been reported across the entire age spectrum although the most severe disease occurs in young children. No vaccines, chemotherapeutics or antibodies are presently available for preventing or treating hMPV infections. In this study, a bovine/human chimeric parainfluenza virus type 3 (b/h PIV3) expressing the human parainfluenza type 3 (hPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) proteins was engineered to express hMPV fusion (F) protein from the second genome position (b/h PIV3/hMPV F2) with the goal of generating a novel hMPV vaccine. b/h PIV3/hMPV F2 was previously shown to protect hamsters from challenge with wt hMPV (Tang RS, Schickli JH, Macphail M, Fernandes F, Bicha L, Spaete J, et al. Effects of human metapneumovirus and respiratory syncytial virus antigen insertion in two 3’ proximal genome positions of bovine/human parainfluenza virus type 3 on virus replication and immunogenicity. J Virol 2003;77:10819–28) and is here further evaluated for efficacy and immunogenicity in African green monkeys (AGMs). AGMs immunized intranasally and intratracheally with b/h PIV3/hMPV F2 generated hMPV- and hPIV3-specific humoral and cellular immune responses and were protected from wt hMPV infection. In a separate study, the host-range restriction of b/h PIV3/hMPV F2 replication relative to wt hPIV3 was performed in rhesus monkeys to demonstrate attenuation. These studies showed that b/h PIV3/hMPV F2 was immunogenic, protective and attenuated in non-human primates and warrants further evaluation in humans as a vaccine candidate for prevention of hMPV-associated respiratory tract diseases.

Prospective study of human metapneumovirus infection in children less than 3 years of age.

Most lower respiratory tract infections (LRTIs) in children under the age of 3 years are due to respiratory syncytial virus (RSV). Epidemiological, host, and viral factors eventually account for the severity of LRTIs, but they do not completely explain it. Human metapneumovirus (hMPV) was recently identified in children with LRTIs. In a population-based prospective multicenter study (the PRI.DE study, conducted in Germany over 2 years), we tested 3,369 nasopharyngeal secretions from children younger than 3 years of age with LRTIs for RSV A and B, influenza viruses (IVs) A and B, and parainfluenza viruses (PIVs) 1 to 3. Of the children requiring intensive care (n = 85), 18% had hMPV infections, and 60% of these children were infected with hMPV in combination with RSV. We did not detect hMPV in a randomly selected subset of RSV-positive nasopharyngeal secretions (n = 120) from children not requiring intensive care support. hMPV was detected in <1% of virus-negative samples from patients without intensive care support (n = 620). Our data support the hypothesis that coinfections with RSV and hMPV are more severe than infections with either RSV or hMPV alone, at least in children younger than 3 years of age.

Pathology of Human Metapneumovirus Infection: Insights into the Pathogenesis of a Newly Identified Respiratory Virus

Human metapneumovirus (hMPV) is a recently discovered human virus that causes significant respiratory infections. Pathologic features of hMPV infection have not been described. A total of 1257 pediatric respiratory samples submitted for routine clinical virologic testing were additionally tested for hMPV by reverse transcriptase polymerase chain reaction (PCR). Pathology specimens, available in 6 of 53 hMPV-positive patients, were examined by light and electron microscopy and included 6 bronchoalveolar lavage (BAL) and 3 lung biopsy specimens from 6 patients (3 girls and 3 boys) ranging in age from 1 to 16 years. BAL from three patients performed within 4 days of the positive hMPV assay showed epithelial degenerative changes and eosinophilic cytoplasmic inclusions within epithelial cells, multinucleate giant cells, and histiocytes. Inclusions were not seen in three patients with BAL performed = 1 month from the time of their positive assay. Lung biopsy, performed in three patients, all = 1 month from the time of their positive assay, showed chronic airway inflammation and intraalveolar foamy and hemosiderin-laden macrophages; all three patients had an underlying pulmonary/systemic disorder. Our findings delineate the clinicopathologic features in hMPV-infected patients undergoing anatomic sampling, which may provide diagnostic guidance to a practicing pathologist. Further, they contribute toward understanding the pathogenesis of hMPV infection.

High genetic diversity of the attachment (G) protein of human metapneumovirus.

Complete genes encoding the predicted nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), M2-1protein, M2-2protein, small hydrophobic protein (SH), and attachmentprotein (G) of seven newly isolated human metapneumoviruses (hMPVs) were analyzed and compared with previously published data for hMPV genes. Phylogenetic analysis of the nucleotide sequences indicated that there were two genetic groups, tentatively named groups 1 and 2, similar to the grouping of human respiratory syncytial virus. Although the predicted amino acid sequences of N, P, M, F, and M2 were highly conserved between the two groups (amino acid identities, 96% for N, 85% for P, 97% for M, 94% for F, 95% for M2-1, and 90% for M2-2), the amino acid identities of the SH and G proteins were low (SH, 58%; G, 33%). Furthermore, each group could be subdivided into two subgroups by phylogenetic analysis, tentatively named subgroups 1A and 1B and subgroups 2A and 2B. The predicted amino acid sequences of G within members of each subgroup were highly conserved (amino acid identities, 88% for group 1A, 93% for group 1B, and 96% for group 2B). The G of hMPV is thought to be the major antigenic determinant and to play an important role in the production of neutralizing antibodies. Clarification of the antigenic diversity of G is important for epidemiological analysis and for establishment of strategies to prevent hMPV infection.

Human metapneumovirus in children tested at a tertiary-care hospital.

Respiratory infections are the leading cause of outpatient visits in the United States, but the etiology of many of these infections is unknown. Human metapneumovirus (hMPV) is a recently discovered virus that causes respiratory infections. Respiratory specimens obtained from patients <or=18 years old, between 1 October 2000 and 31 August 2002, were tested for hMPV. The results of testing for other viruses and epidemiological information were obtained from the hospital databases. A logistic regression model, including sex and age of the patient and year and season in which the specimen was obtained, was used to determine the factors associated with hMPV infection. hMPV was detected in 6.2% of patients tested and was significantly more common among children 3-24 months old than in older or younger children. The seasonal occurrence of hMPV was similar to that of respiratory syncytial virus and influenza virus, with most cases occurring in the winter and spring. In this tertiary care-center population, patients with hMPV infection often had underlying chronic conditions. hMPV is common among young children with apparent respiratory infections, suggesting that it is a significant cause of symptomatic respiratory infections.

Experimental Human Metapneumovirus Infection of Cynomolgus Macaques ( Macaca fascicularis) Results in Virus Replication in Ciliated Epithelial Cells and Pneumocytes with Associated Lesions throughout the Respiratory Tract

A substantial proportion of hitherto unexplained respiratory tract illnesses is associated with human metapneumovirus (hMPV) infection. This virus also was found in patients with severe acute respiratory syndrome (SARS). To determine the dynamics and associated lesions of hMPV infection, six cynomolgus macaques (Macaca fascicularis) were inoculated with hMPV and examined by pathological and virological assays. They were euthanized at 5 (n = 2) or 9 (n = 2) days post-infection (dpi), or monitored until 14 dpi (n = 2). Viral excretion peaked at 4 dpi and decreased to zero by 10 dpi. Viral replication was restricted to the respiratory tract and associated with minimal to mild, multi-focal erosive and inflammatory changes in conducting airways, and increased numbers of macrophages in alveoli. Viral expression was seen mainly at the apical surface of ciliated epithelial cells throughout the respiratory tract, and less frequently in type 1 pneumocytes and alveolar macrophages. Both cell tropism and respiratory lesions were distinct from those of SARS-associated coronavirus infection, excluding hMPV as the primary cause of SARS. This study demonstrates that hMPV is a respiratory pathogen and indicates that viral replication is short-lived, polarized to the apical surface, and occurs primarily in ciliated respiratory epithelial cells.

本邦において初めて流行が確認された小児のhuman metapneumovirus感染症の臨床的, 疫学的解析

Human metapneumovirus (hMPV) was newly discovered as a pathogen in 2001 and is thought to be associated with respiratory disease. To elucidate the prevalence and clinical significance of hMPV among children, we investigated the positive cases of hMPV-RNA by reverse transcription-polymerase chain reaction (RT-PCR) in their nasopharyngeal specimens collected from January to August 2003 in Hiroshima Prefecture, Japan. Our prospective study revealed 77 hMPV-positive cases among 377 children with acute respiratory diseases. Clinical diagnoses of 77 hMPV-positive cases were as follows; bronchitis (33.8%), pneumonia (24.7%), acute respiratory illness (19.5%), asthmatic bronchitis (11.7%) and bronchiolitis (5.2%). The most common symptoms were cough (97.4%), high fever (94.8%) and rhinorrhea (76.6%). Most of the hMPV-positive cases were identified in the spring (between March and May), indicating the presence of an epidemic of hMPV infection in Hiroshima Prefecture. Phylogenetic analysis of the amplified F gene of hMPV isolates revealed that hMPV strains were divided into two genotypes and that their simultaneous circulation occurred within the same epidemic area of Hiroshima Prefecture.