Skip Navigation

Human Metapneumovirus Infection Induces Long-Term Pulmonary Inflammation Associated with Airway Obstruction and Hyperresponsiveness in Mice

  1. Marie-Ève Hamelin1,
  2. Gregory A. Prince3,
  3. Ana M. Gomez4,
  4. Richard Kinkead2 and
  5. Guy Boivin1
  1. 1Research Center in Infectious Diseases and
  2. 2Department of Pediatrics, Centre Hospitalier Universitaire de Québec and Laval University, Québec City, Québec, Canada;
  3. 3Virion Systems, Inc., Rockville, Maryland;
  4. 4Department of Pathology, University of Texas Southwestern Medical Center at Dallas, Dallas
  1. Reprints or correspondence: Dr. Guy Boivin, CHUQ-CHUL, Room RC-709, 2705 Blvd. Laurier, Sainte-Foy, Québec, Canada G1V 4G2 (guy.boivin{at}crchul.ulaval.ca)
 
Next Section

Abstract

BackgroundHuman metapneumovirus (hMPV) is a newly described paramyxovirus that is associated with bronchiolitis, pneumonia, and asthma exacerbation. The objective of the present work was to study the duration of pulmonary inflammation and the functional consequences of infection with hMPV by use of a BALB/c mouse model

MethodsBALB/c mice were inoculated with 1×108 TCID50 of hMPV type A (C-85473), and viral persistence in lungs was assessed by reverse-transcription polymerase chain reaction for 154 days after infection. Pulmonary inflammation was characterized in histopathological experiments by use of a validated scoring system, and periodic acid–Schiff (PAS) staining of lung sections was used to document increased mucus production, also until day 154. Finally, respiratory functions were analyzed by taking plethysmographic measurements until day 70

ResultsPersistence of viral RNA and significant pulmonary inflammation were noted until day 154, whereas the findings for PAS staining suggested that mucus production was increased only until day 12. Maximal breathing difficulties occurred on day 5, and airway obstruction and hyperresponsiveness were still significant until at least day 70

ConclusionAcute hMPV infection in BALB/c mice is associated with long-term pulmonary inflammation that leads to significant obstructive disease of the airways. This animal model will be of a great benefit in the evaluation of novel therapeutic and prophylactic modalities

Human metapneumovirus (hMPV) is a newly described member of the genus Metapneumovirus which is part of the Pneumovirinae subfamily of the Paramyxoviridae family [1]. hMPV was first identified in respiratory specimens from young children with various respiratory syndromes, including common colds, lower respiratory tract infections, and asthma exacerbation [1–5]. Several studies have revealed that hMPV is a worldwide pathogen that infects virtually all children by the age of 5–10 years and, thus, results in a significant percentage of hospitalizations in young children [1, 6–9]. Its genome consists of a single negative strand of RNA (∼13 kb) containing 8 genes that presumably code for 9 different proteins [10, 11]. hMPV isolates can be categorized into 2 major groups (A and B) and at least 4 subgroups [12–16]. Nucleotide and amino acid sequence identities between representative members of the 2 hMPV groups are 80% and 90%, respectively [10], although further investigation is required to determine whether these genotypes represent different antigenic groups [16, 17]

Experimental animal models of hMPV infection have been reported, including both primates and rodents. Viral replication has been found in the respiratory tracts of experimentally infected chimpanzees and monkeys and was associated with mild upper respiratory tract signs in some of these animals [1, 18, 19]. Some investigators have also shown that hMPV can replicate in the lungs of hamsters and cotton rats without producing recognizable clinical signs, although the latter did exhibit transient histopathological pulmonary changes [17, 19–22]. The BALB/c mouse has been described as a convenient animal model, with efficient viral replication and significant histopathological changes in the lungs associated with systemic and respiratory signs when large intranasal (inl) inocula are used [20, 23, 24]. We previously analyzed the acute stage of hMPV infection and reported that there was no longer any infectious virus in the lungs by day 21 after infection, although histopathological changes were still significant at that time, compared with those in sham-infected mice [20]. Recently, Alvarez and Tripp reported that hMPV RNA could still be detected ⩾180 days after infection in the lungs of hMPV-infected mice and that such persistence could be the result of an aberrant immune response [25]

In the present study, we sought to evaluate the duration of pulmonary inflammation associated with a single hMPV challenge and to characterize the consequences of this viral infection with respect to respiratory functions. Our results show that small amounts of viral RNA are still present in the lungs of hMPV-infected mice for at least 154 days after infection and are associated with significant peribronchiolitis and perivasculitis. Furthermore, such inflammation is responsible for chronic obstruction and hyperresponsiveness of the airways, which persist for >2 months

Previous SectionNext Section

Materials and Methods

Cell line and virusLLC-MK2 cells were maintained in MEM (Gibco/BRL) supplemented with 10% fetal bovine serum. hMPV strain C-85473 (a group A virus, as are CAN97-83 and NL/00-1) is a clinical strain that has been passed 6 times through LLC-MK2 cells in MEM supplemented with 0.2% glucose, 0.1% bovine serum albumin, 0.0002% trypsin, and 1% gentamicin (hMPV infection medium) [20]. High viral titers were obtained by infecting LLC-MK2 cells in 20 flasks (growth area, 75 cm2; Corning) until complete cytopathic effects were observed. Then, infected monolayers and supernatants were recovered by use of a cell scraper, sonicated, and concentrated by use of Centricon columns (Fisher). The preparation was centrifuged, to remove all cellular debris, and supernatants containing infectious virus were conserved at −80°C until inoculation of mice. The same protocol was used with 20 flasks of uninfected cells, for inoculation of sham-infected mice

Experimental protocolsExperiments were performed by initially infecting 136 4–6-week-old BALB/c mice (Charles Rivers Laboratories). The mice were inoculated inl with 1 × 108 TCID50 of hMPV strain C-85473 in 25 μL of hMPV infection medium, as described elsewhere [20], and the same number of mice were sham infected with the control described above. All mice were housed in groups of 5 in microisolator cages. At serial time points after infection (days 1, 3, 5, 7, 12, 21, 42, 70, 98, 126, and 154), lungs were removed from 12 mice at each time point (6 sham- and 6 hMPV-infected mice), both for detection of virus by cell culture and reverse-transcription polymerase chain reaction (RT-PCR) and for histopathological analysis. In addition, breathing patterns were characterized in unrestrained mice by use of a whole-body flow-through plethysmograph on days 3, 5, 7, 12, 21, 42, and 70 (20 mice at each time point; 10 sham- and 10 hMPV-infected mice). The present study was approved by the Animal Protection Committee of the Centre Hospitalier Universitaire de Québec

Virus titration in lungsAt the specified time points, mice were killed and their right lungs were removed and quickly frozen in liquid nitrogen. When ready for titration, lungs were weighed and then homogenized in 500 μL of hMPV infection medium; 100 μL of the homogenates were used to determine viral titers by inoculating 10-fold serial dilutions of virus into 24-well plates (Corning) containing LLC-MK2 cells. Before infection, cells were washed twice with PBS, to remove residual serum proteins that could inhibit trypsin activity. The plates were incubated at 37°C in 5% CO2 and replenished with 1 μL of fresh trypsin (0.0002%) every other day. Viral titers are reported as log10 TCID50 per lung, corresponding to ∼0.05 g. The lower limit of detection of our assay was 1×102 TCID50 per lung. TCID50 values were calculated by the Reed-Muench method

Real-time RT-PCR experimentsTwo hundred microliters of lung homogenates were used for viral RNA extraction, which was done using the QIAamp Viral RNA Mini Kit (Qiagen) in accordance with the manufacturer’s instructions, with elution in a final volume of 40 μL. Complementary DNA was synthesized by use of 10 μL of RNA eluate, random hexamer primers (Amersham Pharmacia Biotech), and an Omniscript RT Kit (Qiagen), used in accordance with the manufacturer’s instructions. PCR amplification of the hMPV nucleoprotein (N) gene was performed as described elsewhere [26]. Each 20-μL PCR included 2 μL of cDNA and 18 μL of a mixture containing 3.5 mmol/L MgCl2, 0.5 μmol/L each hMPV primer, 10 μL of LC DNA FastSTART DNA Master SYBR Green (Sigma), and 0.6 μL of dimethyl sulfoxide. Cycling conditions included a 30-s denaturation step at 95°C and then 50 PCR cycles consisting of 5 s at 95°C, 5 s at 58°C, and 25 s at 72°C. Fluorescence measurements were taken at each cycle (at the end of amplification) in the F1 channel. This real-time RT-PCR assay for the N gene had a lower limit of detection of 50 copies/reaction. Samples positive by SYBR Green melting-curve analysis were tested for another gene (L) by a second PCR, in which hMPV copy numbers were quantified with a TaqMan probe [27]. The limit of detection for this second PCR was 100 copies/reaction

Pulmonary histopathological analysisThe left lungs of sham- and hMPV-infected mice were collected at specified time points and fixed with 10% buffered formalin. Fixed lungs were subsequently embedded in paraffin, sectioned in slices of 5 μm, and stained with hematoxylin-eosin. The histopathological score was determined by 2 independent researchers at a single institution who were unaware of the infection status of the mice. Four types of pulmonary histopathological changes were scored independently for each lung section: peribronchiolitis, perivasculitis, interstitial pneumonitis, and alveolitis. Each histopathological change was scored on a scale from 0 (no change) to 4 (maximum inflammation), with a score of 4 being based on our prior observations of maximal pathological changes induced by human respiratory syncytial virus (hRSV) infection in cotton rat lungs [28]

Mucus production experimentsTo identify mucus-producing cells, lung sections were stained with periodic acid–Schiff (PAS) from day 1 to 154 after infection. By use of a semiquantitative scale (+, ++, and +++), the sections were scored by 2 pathologists for the presence of PAS-positive cells in peripheral or intermediate bronchioles and in central airways

Respiratory-function experimentsWhole-body flow-through plethysmography (EMKA Technologies) was used to monitor the respiratory dynamics of unrestrained mice in a quantitative manner before and after methacholine challenge. Recorded variables included respiratory rate; inspiratory flow, calculated as tidal volume / inspiratory time; and enhanced pause (Penh) value, which consists of the pause parameter—calculated as [(expiration time − relaxation time) / relaxation time]—multiplied by the ratio of peak expiratory flow to peak inspiratory flow. The accuracy of the Penh value as an index of airway obstruction is a matter of debate; however, this variable correlates well with pulmonary airflow resistance and obstruction and remains a useful tool for the evaluation of the functional consequences that microorganisms have on airway functions [29–35]. Penh values were also confirmed by inspiratory flow values. These parameters are increased when airway obstruction or hyperresponsiveness are observed as the mice generate a larger inspiratory effort in order to fill their lungs

The system was calibrated by injecting a known volume (0.1 mL) into the plethysmographic chamber by use of a glass syringe. The barometric pressure and the body weight of each mouse were recorded on the day of the experiment. The chamber temperature and humidity and the rectal temperature of the mice were also measured at the beginning and at the end of each experimental period. These data were used to express the tidal volume in BTPS (body temperature, pressure standard)–adjusted milliliters. Fresh air was delivered into the experimental chamber at a constant rate by use of a bias flow regulator (PLY1020; Buxco Electronics). Sham- and hMPV-infected mice were allowed to acclimate to the chamber for 30 min before recordings were made. Then, respiratory activity was recorded for 5 min, to establish baseline values for the variables described above, including airway-obstruction parameters. Mice were subsequently exposed to aerosolized methacholine (acetyl-β-methylcholine chloride; Sigma) (50 mg/mL) previously dissolved in 1× PBS for 2.5 min, and plethysmographic data were recorded for another 8 min. Airway hyperresponsiveness was defined as a greater change in Penh value and a greater change in inspiratory flow (determined as the difference between the maximum value after methacholine challenge and the baseline value) in the hMPV-infected mice, compared with those in the sham-infected mice [36]

Statistical analysisWith the exception of histopathological scores, all data are expressed as means±SDs. For data not normally distributed, the Mann-Whitney U test was used to compare values for the hMPV- and sham-infected mice at the same time point. P<.05 was considered to be statistically significant

Previous SectionNext Section

Results

Viral titers and hMPV persistence in lungs of BALB/c miceAll lung homogenates were positive for hMPV by cell culture from day 1 to 12 after infection, as reported elsewhere [20]. Maximal viral replication occurred on day 5, with a mean viral titer of 7×106 TCID50/lung (∼0.05 g) (figure 1A). No infectious virus was recovered on day 21 or later in the hMPV-infected mice and at any time point in the sham-infected mice. All samples from the hMPV-infected mice were also positive for hMPV by qualitative RT-PCR from day 1 to 12, whereas only 83.3%, 80.0%, 83.3%, 20.0%, 80.0%, and 33.3% were positive on days 21, 42, 70, 98, 126, and 154, respectively. Finally, quantitative RT-PCR analysis using a TaqMan probe indicated that hMPV copy numbers increased until day 5 (with a mean of 6.93×109 copies/lung) and then decreased until day 21, supporting the cell-culture data (figure 1B). Thereafter, viral copy numbers did not vary significantly in positive samples and were between 6.08×103 and 2.23×104 copies/lung from day 21 to 154. It should be noted that the latter viral loads represent only 3.80×102–1.60×103 copies/PCR

Figure 1
View larger version:
Figure 1

Human metapneumovirus (hMPV) replication and persistence in the lungs of infected mice. Six sham- and 5–6 hMPV-infected mice were killed on days 1, 3, 5, 7, 12, 21, 42, 70, 98, 126, and 154 after infection, and their right lungs were removed. Lung homogenates were serially diluted and incubated with LLC-MK2 cells for viral titration (A) or were used to determine hMPV copy numbers with a TaqMan probe (B). No virus or viral RNA were found in sham-infected mice

Chronic pulmonary inflammation in lungs of hMPV-infected micePulmonary inflammation was assessed using a scoring system that has been previously described in cotton rats infected with hRSV and that has also been used for hMPV-infected mice [20, 28]. Inflammation was observed in the hMPV-infected mice on day 3 after infection, peaked on day 5 (which was also the time of peak viral replication), and gradually decreased thereafter (figure 2). During the first 2–3 weeks, the inflammation mostly consisted of interstitial inflammation (characterized by increased thickness of the alveolar walls) and the presence of alveolitis (characterized by the presence of inflammatory cells within alveolar spaces), as reported elsewhere [20]. Over time, the inflammation became characterized mostly by a prominent peribronchiolar and perivascular infiltrate, which was still significant on day 154 (figure 3). A few infiltrating cells were found around bronchioles of the sham-infected mice over time, with similar histopathological scores at each time point

Figure 2
View larger version:
Figure 2

Histopathological scores in human metapneumovirus–infected mice. The degree of lung inflammation (mean histopathological score) was evaluated for peribronchial, perivascular, interstitial, and alveolar areas in mice. Histopathological score was defined as described elsewhere [20, 28]

Figure 3
View larger version:
Figure 3

Lung histopathological analysis of human metapneumovirus (hMPV)–infected mice. Six sham- and 5–6 hMPV-infected mice per group were killed at different time points after infection, and their left lungs were removed and fixed with 10% buffered formalin. Thin sections of paraffin-embedded lung tissues were cut and stained with hematoxylin-eosin. A representative section (original magnification, ×10) is shown for sham-infected mice on day 154 and for hMPV-infected mice at various time points from day 3 to 154. Lung histopathological analysis of sham-infected mice at different time points was similar to that shown for day 154

Mucus hyperproductionThe hMPV-infected mice had increased numbers of PAS-positive cells in the central and peripheral airways on days 3, 5, 7, and 12 after infection, compared with those in the sham-infected mice, suggesting increased mucus production (figure 4). In addition, the respiratory epithelium of the hMPV-infected mice appeared tall and hypertrophic, compared with that of the sham-infected mice. No significant changes were observed from day 12 to 154

Figure 4
View larger version:
Figure 4

Increased mucus production in the lungs of human metapneumovirus (hMPV)–infected mice. Periodic acid–Shiff staining for mucus-producing cells (arrows) demonstrated increased mucus production in hMPV-infected mice (B) compared with that in sham-infected mice (A) on day 7 after infection

Resting ventilatory activity and airway obstruction in hMPV-infected micehMPV infection led to a significant increase in the resting respiratory rate of mice, compared with that in the sham-infected mice; the increase persisted until day 21 after infection (figure 5A). In addition, hMPV infection increased Penh values, suggesting obstruction of the airways. The increased inspiratory flow values observed in the hMPV-infected mice is consistent with the Penh data, because it indicates that the infected mice had to generate a larger inspiratory effort in order to fill their lungs. Together, these data suggest that obstruction peaked on day 5 (figure 5B and 5C), at the time of maximal viral replication and maximal histopathological score [20]. Thereafter, airway obstruction decreased but was still significant on day 70, compared with that in the sham-infected mice

Figure 5
View larger version:
Figure 5

Changes in respiratory functions of human metapneumovirus (hMPV)–infected mice. Ten sham- and 10 hMPV-infected mice were evaluated at different time points after infection. Respiratory rates (A) airway obstruction (B; reported as enhanced pause [Penh] values), and inspiratory flow (C; calculated as tidal volume / inspiratory time) were determined using whole-body unrestrained plethysmography. *P<.05, for the comparison between the sham- and the hMPV-infected mice (Mann-Whitney U test)

Airway hyperresponsiveness after hMPV infection Challenge with aerosolized methacholine increased both Penh values and inspiratory flow in the hMPV-infected mice from day 12 to 70 after infection (figure 6). Data are shown as the increase in Penh values and inspiratory flow from baseline. The nonsignificant differences seen at early time points (before day 12) might be explained by high baseline Penh and inspiratory flow values before methacholine challenge due to obstruction of the airways. Methacholine challenge also induced changes in the respiratory-function parameters of the sham-infected mice; however, these responses were markedly less pronounced than those of the hMPV-infected mice

Figure 6
View larger version:
Figure 6

Increased airway hyperresponsiveness after methacholine challenge in human metapneumovirus (hMPV)–infected mice. Ten sham- and 10 hMPV-infected mice were challenged with aerosolized methacholine (50 mg/mL) at different time points after infection. Enhanced pause (Penh) values (A) and inspiratory flow (B; calculated as tidal volume / inspiratory time) were determined using whole-body unrestrained plethysmography, to characterize airway hyperresponsiveness. Data are shown as the increase in Penh value or inspiratory flow, calculated as the difference between the maximum value after methacholine challenge and the baseline value. *P<.05, for the comparison between the sham- and the hMPV-infected mice (Mann-Whitney U test)

Previous SectionNext Section

Discussion

In this article, we provide additional information on chronic pulmonary inflammation and the dynamics of respiratory functions after hMPV infection in BALB/c mice. Although, after inl challenge of 1×108 TCID50, infectious virus could not be recovered from the lungs of mice on day 21 or later, we show here that we could still detect small amounts of viral RNA in the lungs of some mice on day 154. The chronic stage of pulmonary inflammation was mainly characterized by peribronchiolitis and perivasculitis that persisted until at least day 154. Infection was also associated with mucus hyperproduction and hyperplasia of the respiratory epithelium. Breathing difficulties were maximal on day 5 (the time of peak viral replication and inflammation) and were characterized by an increased respiratory rate and an important obstruction of the airways, which was still significant on day 70. Most importantly, the hMPV-infected mice demonstrated significant airway hyperresponsiveness after methacholine challenge, which also persisted for at least 70 days. Overall, our data suggest that hMPV infection induces long-term pulmonary inflammation that significantly alters respiratory functions in BALB/c mice

Our group and others have shown that the BALB/c mouse is a representative model of hMPV infection, because inl challenge leads to significant viral replication, pulmonary inflammation, and systemic signs [20, 23, 24]. Indeed, we previously reported important viral replication and inflammation in the lungs of mice that peaked on day 5 after infection [20], and such data were also confirmed in the present study. However, we were intrigued by the fact that the hMPV-infected mice still had significant pulmonary inflammation, compared with that in the sham-infected mice, on day 21. Determination of the impact that hMPV infection has on respiratory functions and, especially, on airway obstruction and hyperresponsiveness was a prime objective of our study, because, in some previous studies, severe respiratory tract infections with other paramyxoviruses (such as hRSV) during infancy have been associated with the subsequent development of asthma [37–40]

An interesting aspect of the present study was the persistence of hMPV RNA in the lungs of some mice up to day 154 after infection. The discordance between negative results for viral culture and positive results for RT-PCR at later time points after infection could be explained by lower viral loads in the lungs of these mice, compared with those observed during the acute phase of infection. Of note, the lower limit of detection for our cell-culture assay is 1×102 TCID50 per lung, because lower dilutions of lung homogenates interfere with the assessment of cell monolayers. Alvarez et al. used a cell-culture method based on detection of hMPV plaques by immunostaining and reported the presence of infectious virus until day 60 and the persistence of hMPV genomic RNA in the lungs until day 180 [23]. hMPV is a respiratory pathogen that belongs to the same subfamily (Pneumovirinae) as hRSV, and evidence suggests that the latter could also persist in the lungs for a long time. Indeed, hRSV genomic RNA as well as viral proteins have been detected in the lungs of infected guinea pigs and mice for 60–100 days after infection [41, 42]. The exact mechanisms that lead to paramyxovirus persistence in the lungs of these animals have not been elucidated, although a recent study has suggested that impaired hMPV clearance in mice could be explained by weak innate immune responses and aberrant adaptive immune responses characterized by the induction of a Th2 type cytokine response [25]

During the acute stage of hMPV infection in BALB/c mice (i.e., the first 14–21 days), we found both alveolar and interstitial pulmonary inflammation, which is consistent with pneumonia, whereas other groups have reported interstitial inflammation alone after hMPV challenge [23, 24]. Thereafter, this type of inflammation decreased and became characterized mainly by peribronchiolitis and perivasculitis (figures 2 and 3). These changes in pulmonary inflammatory persisted for at least 154 days and could be explained by the absence of viral clearance, as noted above. Such persistent inflammation has also been described for the same period of time in hRSV-infected mice and consisted of peribronchiolitis and perivasculitis [36]. It is also interesting to note that such chronic inflammatory changes of the airways, along with the presence of intraalveolar foamy and hemosiderin-laden macrophages, have been reported in a patient with severe hMPV infection >28 days after the onset of symptoms [43]. Peribronchiolitis is the type of inflammation typically associated with asthma, and the histopathological changes observed in mice in the present study support clinical findings indicating that hMPV may trigger asthma exacerbation in humans [2, 4, 5]. Excess mucus production, which we have reported (figure 4), is common in many pulmonary conditions (such as asthma and bronchiolitis) and has been documented in a mouse model of hRSV infection [36]. Mucus hyperproduction has also been associated with adverse effects (such as airflow obstruction), pathological conditions [44], and elevated levels of tumor necrosis factor–α, interleukin (IL)–4, IL-5, IL-9, and IL-13 in humans and mice [45–47]

The respiratory activity of BALB/c mice was significantly altered by hMPV infection in the present study. Notably, the respiratory rate was significantly higher than that of the sham-infected mice during the first 21 days of infection, and airway obstruction was present until at least day 70 (figure 5). Interestingly, Darniot et al. recently reported that airways were only significantly obstructed on days 1 and 2 after hMPV infection, whereas pulmonary inflammation persisted until day 6 [24]. The variation in findings on airway obstruction between the 2 studies could be explained by the different viral inocula and/or viral strains (C-85473 vs. CAN97-83) used by the groups. Our results suggest that the impact of hMPV infection extends well beyond the acute stage of pneumonitis and that it will be important to monitor such long-term effects during vaccine and therapeutic trials. Again, previous studies have reported relatively similar results in hRSV-infected BALB/c mice when a large viral inoculum (>1×106 pfu) was administered [34, 36, 48, 49]

Similarly, airway hyperresponsiveness induced by methacholine, which was significant until at least day 70 in our mouse model of hMPV infection, has been previously observed in hRSV-infected mice, although for a shorter period of time [32, 34, 36, 50, 51]. Airway hyperresponsiveness has also been reported in hMPV-infected mice, but for a much shorter period of time—that is, for only 3–4 days after challenge with a lower inoculum [24]. The Penh value used in many studies, including ours, is a parameter that increases in parallel with the severity of small airway disease, and our results indicate that hMPV infection may trigger that kind of pathological manifestation. Whether the degree of airway obstruction induced by hMPV infection is similar to that induced by hRSV infection, and whether coinfection with the 2 viruses leads to a synergistic effect on respiratory functions and disease severity (as has been noted in some studies in humans [52, 53]), need to be investigated

In conclusion, we report here an important chronic pulmonary inflammation associated with significant airway obstruction and hyperresponsiveness after hMPV infection in mice. Determination of whether this experimental model is representative of hMPV infection in humans requires additional study. Nevertheless, this BALB/c mouse model of hMPV infection reinforces the concept that severe paramyxovirus infections early during life could be associated with the development of asthma in children [37–40]. Subsequent studies should aim at better understanding the mechanism of viral persistence in the lungs of mice and at evaluating strategies for the prevention of the long-term pulmonary consequences associated with this viral infection

Previous SectionNext Section

Acknowledgment

We thank Gaspard Montandon, for his great help with the plethysmographic experiments

Previous SectionNext Section

Footnotes

  • ↵Potential conflicts of interest: none reported

    Financial support: Canadian Institutes of Health Research (research grant CIHR-MOP-62789); Le Fonds de la Recherche en Santé du Québec (FRSQ)–Respiratory Health Network (research grant to G.B.). G.B. is a senior research scholar of the FRSQ and holds the Canada Research Chair in Emerging Viruses; M.-E.H. has received a Ph.D. scholarship from the FRSQ; and R.K. holds the Canada Research Chair in Respiratory Neurobiology

  • Received September 8, 2005.
  • Accepted January 19, 2006.
Previous Section
 

References

  1. ↵
    1. van den Hoogen BG,
    2. de Jong JC,
    3. Groen J,
    4. et al
    . A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat Med 2001;7:719-24.
    CrossRefMedlineWeb of Science
  2. ↵
    1. Bosis S,
    2. Esposito S,
    3. Niesters HG,
    4. Crovari P,
    5. Osterhaus AD
    . Impact of human metapneumovirus in childhood: comparison with respiratory syncytial virus and influenza viruses. J Med Virol 2005;75:101-4.
    CrossRefMedlineWeb of Science
  3. ↵
    1. Hamelin ME,
    2. Abed Y,
    3. Boivin G
    . Human metapneumovirus: a new player among respiratory viruses. Clin Infect Dis 2004;38:983-90.
    Abstract/FREE Full Text
  4. ↵
    1. Jartti T,
    2. van den Hoogen B,
    3. Garofalo RP,
    4. Osterhaus AD,
    5. Ruuskanen O
    . Metapneumovirus and acute wheezing in children. Lancet 2002;360:1393-4.
    CrossRefMedlineWeb of Science
  5. ↵
    1. Peiris JS,
    2. Tang WH,
    3. Chan KH,
    4. et al
    . Children with respiratory disease associated with metapneumovirus in Hong Kong. Emerg Infect Dis 2003;9:628-33.
    MedlineWeb of Science
  6. ↵
    1. Boivin G,
    2. De Serres G,
    3. Cote S,
    4. et al
    . Human metapneumovirus infections in hospitalized children. Emerg Infect Dis 2003;9:634-40.
    MedlineWeb of Science
  7. ↵
    1. Esper F,
    2. Boucher D,
    3. Weibel C,
    4. Martinello RA,
    5. Kahn JS
    . Human metapneumovirus infection in the United States: clinical manifestations associated with a newly emerging respiratory infection in children. Pediatrics 2003;111:1407-10.
    Abstract/FREE Full Text
  8. ↵
    1. Freymouth F,
    2. Vabret A,
    3. Legrand L,
    4. et al
    . Presence of the new human metapneumovirus in French children with bronchiolitis. Pediatr Infect Dis J 2003;22:92-4.
    CrossRefMedlineWeb of Science
  9. ↵
    1. Williams JV,
    2. Harris PA,
    3. Tollefson SJ,
    4. et al
    . Human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children. N Engl J Med 2004;350:443-50.
    CrossRefMedlineWeb of Science
  10. ↵
    1. Biacchesi S,
    2. Skiadopoulos MH,
    3. Boivin G,
    4. et al
    . Genetic diversity between human metapneumovirus subgroups. Virology 2003;315:1-9.
    CrossRefMedlineWeb of Science
  11. ↵
    1. van den Hoogen BG,
    2. Bestebroer TM,
    3. Osterhaus AD,
    4. Fouchier RA
    . Analysis of the genomic sequence of a human metapneumovirus. Virology 2002;295:119-32.
    CrossRefMedlineWeb of Science
  12. ↵
    1. Boivin G,
    2. Abed Y,
    3. Pelletier G,
    4. et al
    . Virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups. J Infect Dis 2002;186:1330-4.
    Abstract/FREE Full Text
  13. ↵
    1. Boivin G,
    2. Mackay I,
    3. Sloots TP,
    4. et al
    . Global genetic diversity of human metapneumovirus fusion gene. Emerg Infect Dis 2004;10:1154-7.
    MedlineWeb of Science
  14. ↵
    1. Ishiguro N,
    2. Ebihara T,
    3. Endo R,
    4. et al
    . High genetic diversity of the attachment (G) protein of human metapneumovirus. J Clin Microbiol 2004;42:3406-14.
    Abstract/FREE Full Text
  15. ↵
    1. Peret TC,
    2. Boivin G,
    3. Li Y,
    4. et al
    . Characterization of human metapneumoviruses isolated from patients in North America. J Infect Dis 2002;185:1660-3.
    Abstract/FREE Full Text
  16. ↵
    1. van den Hoogen BG,
    2. Herfst S,
    3. Sprong L,
    4. et al
    . Antigenic and genetic variability of human metapneumoviruses. Emerg Infect Dis 2004;10:658-66.
    MedlineWeb of Science
  17. ↵
    1. Skiadopoulos MH,
    2. Biacchesi S,
    3. Buchholz UJ,
    4. et al
    . The two major human metapneumovirus genetic lineages are highly related antigenically, and the fusion (F) protein is a major contributor to this antigenic relatedness. J Virol 2004;78:6927-37.
    Abstract/FREE Full Text
  18. ↵
    1. Kuiken T,
    2. van den Hoogen BG,
    3. van Riel DA,
    4. et al
    . 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. Am J Pathol 2004;164:1893-900.
    MedlineWeb of Science
  19. ↵
    1. MacPhail M,
    2. Schickli JH,
    3. Tang RS,
    4. et al
    . Identification of small-animal and primate models for evaluation of vaccine candidates for human metapneumovirus (hMPV) and implications for hMPV vaccine design. J Gen Virol 2004;85:1655-63.
    Abstract/FREE Full Text
  20. ↵
    1. Hamelin ME,
    2. Yim K,
    3. Kuhn KH,
    4. et al
    . Pathogenesis of human metapneumovirus lung infection in BALB/c mice and cotton rats. J Virol 2005;79:8894-903.
    Abstract/FREE Full Text
  21. ↵
    1. Tang RS,
    2. Schickli JH,
    3. MacPhail M,
    4. 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.
    Abstract/FREE Full Text
  22. ↵
    1. Wyde PR,
    2. Chetty SN,
    3. Jewell AM,
    4. Schoonover SL,
    5. Piedra PA
    . Development of a cotton rat-human metapneumovirus (hMPV) model for identifying and evaluating potential hMPV antivirals and vaccines. Antiviral Res 2005;66:57-66.
    CrossRefMedlineWeb of Science
  23. ↵
    1. Alvarez R,
    2. Harrod KS,
    3. Shieh WJ,
    4. Zaki S,
    5. Tripp RA
    . Human metapneumovirus persists in BALB/c mice despite the presence of neutralizing antibodies. J Virol 2004;78:14003-11.
    Abstract/FREE Full Text
  24. ↵
    1. Darniot M,
    2. Petrella T,
    3. Aho S,
    4. Pothier P,
    5. Manoha C
    . Immune response and alteration of pulmonary function after primary human metapneumovirus (hMPV) infection of BALB/c mice. Vaccine 2005;23:4473-80.
    CrossRefMedlineWeb of Science
  25. ↵
    1. Alvarez R,
    2. Tripp RA
    . The immune response to human metapneumovirus is associated with aberrant immunity and impaired virus clearance in BALB/c mice. J Virol 2005;79:5971-8.
    Abstract/FREE Full Text
  26. ↵
    1. Maertzdorf J,
    2. Wang CK,
    3. Brown JB,
    4. et al
    . Real-time reverse transcriptase PCR assay for detection of human metapneumoviruses from all known genetic lineages. J Clin Microbiol 2004;42:981-6.
    Abstract/FREE Full Text
  27. ↵
    1. Deffrasnes C,
    2. Cote S,
    3. Boivin G
    . Analysis of replication kinetics of the human metapneumovirus in different cell lines by real-time PCR. J Clin Microbiol 2005;43:488-90.
    Abstract/FREE Full Text
  28. ↵
    1. Prince GA,
    2. Prieels JP,
    3. Slaoui M,
    4. Porter DD
    . Pulmonary lesions in primary respiratory syncytial virus infection, reinfection, and vaccine-enhanced disease in the cotton rat (Sigmodon hispidus). Lab Invest 1999;79:1385-92.
    MedlineWeb of Science
  29. ↵
    1. Gonzalo JA,
    2. Lloyd CM,
    3. Wen D,
    4. et al
    . The coordinated action of CC chemokines in the lung orchestrates allergic inflammation and airway hyperresponsiveness. J Exp Med 1998;188:157-67.
    Abstract/FREE Full Text
  30. ↵
    1. Hamelmann E,
    2. Schwarze J,
    3. Takeda K,
    4. et al
    . Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography. Am J Respir Crit Care Med 1997;156:766-75.
    Abstract/FREE Full Text
  31. ↵
    1. Hardy RD,
    2. Rios AM,
    3. Chavez-Bueno S,
    4. et al
    . Antimicrobial and immunologic activities of clarithromycin in a murine model of Mycoplasma pneumoniae-induced pneumonia. Antimicrob Agents Chemother 2003;47:1614-20.
    Abstract/FREE Full Text
  32. ↵
    1. Lukacs NW,
    2. Tekkanat KK,
    3. Berlin A,
    4. et al
    . Respiratory syncytial virus predisposes mice to augmented allergic airway responses via IL-13-mediated mechanisms. J Immunol 2001;167:1060-5.
    Abstract/FREE Full Text
  33. ↵
    1. Schwarze J,
    2. Cieslewicz G,
    3. Joetham A,
    4. et al
    . Critical roles for interleukin-4 and interleukin-5 during respiratory syncytial virus infection in the development of airway hyperresponsiveness after airway sensitization. Am J Respir Crit Care Med 2000;162:380-6.
    Abstract/FREE Full Text
  34. ↵
    1. van Schaik SM,
    2. Enhorning G,
    3. Vargas I,
    4. Welliver RC
    . Respiratory syncytial virus affects pulmonary function in BALB/c mice. J Infect Dis 1998;177:269-76.
    MedlineWeb of Science
  35. ↵
    1. Schwarze J,
    2. Hamelmann E,
    3. Gelfand EW,
    4. Adler A,
    5. Cieslewicz G
    . Barometric whole body plethysmography in mice. J Appl Physiol 2005;98:1955-7. author reply 1956–7.
    Abstract/FREE Full Text
  36. ↵
    1. Jafri HS,
    2. Chavez-Bueno S,
    3. Mejias A,
    4. et al
    . Respiratory syncytial virus induces pneumonia, cytokine response, airway obstruction, and chronic inflammatory infiltrates associated with long-term airway hyperresponsiveness in mice. J Infect Dis 2004;189:1856-65.
    Abstract/FREE Full Text
  37. ↵
    1. Hogg JC
    . Childhood viral infection and the pathogenesis of asthma and chronic obstructive lung disease. Am J Respir Crit Care Med 1999;160 Suppl:S26-8.
    Abstract/FREE Full Text
  38. ↵
    1. Lemanske RF Jr.
    . The childhood origins of asthma (COAST) study. Pediatr Allergy Immunol 2002;13 Suppl 15:38-43.
    CrossRefMedlineWeb of Science
  39. ↵
    1. Martinez FD
    . Role of respiratory infection in onset of asthma and chronic obstructive pulmonary disease. Clin Exp Allergy 1999;29 Suppl 2:53-8.
  40. ↵
    1. Openshaw PJ,
    2. Dean GS,
    3. Culley FJ
    . Links between respiratory syncytial virus bronchiolitis and childhood asthma: clinical and research approaches. Pediatr Infect Dis J 2003;22:S58-64. discussion S64–5.
    CrossRefMedlineWeb of Science
  41. ↵
    1. Hegele RG,
    2. Hayashi S,
    3. Bramley AM,
    4. Hogg JC
    . Persistence of respiratory syncytial virus genome and protein after acute bronchiolitis in guinea pigs. Chest 1994;105:1848-54.
    Abstract/FREE Full Text
  42. ↵
    1. Schwarze J,
    2. O’Donnell DR,
    3. Rohwedder A,
    4. Openshaw PJ
    . Latency and persistence of respiratory syncytial virus despite T cell immunity. Am J Respir Crit Care Med 2004;169:801-5.
    Abstract/FREE Full Text
  43. ↵
    1. Vargas SO,
    2. Kozakewich HP,
    3. Perez-Atayde AR,
    4. McAdam AJ
    . Pathology of human metapneumovirus infection: insights into the pathogenesis of a newly identified respiratory virus. Pediatr Dev Pathol 2004;7:478-86.
    CrossRefMedlineWeb of Science
  44. ↵
    1. Rogers DF
    . Pulmonary mucus: pediatric perspective. Pediatr Pulmonol 2003;36:178-88.
    CrossRefMedlineWeb of Science
  45. ↵
    1. Levine SJ,
    2. Larivee P,
    3. Logun C,
    4. Angus CW,
    5. Ognibene FP
    . Tumor necrosis factor-alpha induces mucin hypersecretion and MUC-2 gene expression by human airway epithelial cells. Am J Respir Cell Mol Biol 1995;12:196-204.
    Abstract/FREE Full Text
  46. ↵
    1. Tekkanat KK,
    2. Maassab HF,
    3. Cho DS,
    4. et al
    . IL-13-induced airway hyperreactivity during respiratory syncytial virus infection is STAT6 dependent. J Immunol 2001;166:3542-8.
    Abstract/FREE Full Text
  47. ↵
    1. Wills-Karp M
    . Trophic slime, allergic slime. Am J Respir Cell Mol Biol 2000;22:637-9.
    FREE Full Text
  48. ↵
    1. Johnson JE,
    2. Mitchell DB,
    3. Graham BS
    . Respiratory syncytial virus infection prolongs methacholine-induced airway hyperresponsiveness in ovalbumin-sensitized mice. J Med Virol 1999;57:186-92.
    CrossRefMedlineWeb of Science
  49. ↵
    1. van Schaik SM,
    2. Obot N,
    3. Enhorning G,
    4. et al
    . Role of interferon gamma in the pathogenesis of primary respiratory syncytial virus infection in BALB/c mice. J Med Virol 2000;62:257-66.
    CrossRefMedlineWeb of Science
  50. ↵
    1. Schwarze J,
    2. Cieslewicz G,
    3. Hamelmann E,
    4. et al
    . IL-5 and eosinophils are essential for the development of airway hyperresponsiveness following acute respiratory syncytial virus infection. J Immunol 1999;162:2997-3004.
    Abstract/FREE Full Text
  51. ↵
    1. Schwarze J,
    2. Hamelmann E,
    3. Bradley KL,
    4. Takeda K,
    5. Gelfand EW
    . Respiratory syncytial virus infection results in airway hyperresponsiveness and enhanced airway sensitization to allergen. J Clin Invest 1997;100:226-33.
    MedlineWeb of Science
  52. ↵
    1. Greensill J,
    2. McNamara PS,
    3. Dove W,
    4. Flanagan B,
    5. Smyth RL
    . Human metapneumovirus in severe respiratory syncytial virus bronchiolitis. Emerg Infect Dis 2003;9:372-5.
    MedlineWeb of Science
  53. ↵
    1. Semple MG,
    2. Cowell A,
    3. Dove W,
    4. et al
    . Dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis. J Infect Dis 2005;191:382-6.
    Abstract/FREE Full Text
| Table of Contents

This Article

  1. J Infect Dis. (2006) 193 (12): 1634-1642. doi: 10.1086/504262
  1. AbstractFree
  2. » Full Text (HTML)Free
  3. Full Text (PDF)Free

Classifications

Share

  1. Email this article

Navigate This Article

Current Issue

  1. March 1, 2012 205 (5)
  1. Journal of Infectious Diseases
  1. Alert me to new issues

Most