Pathogenesis and host response

Experimental animal models of hMPV infection have been reported, including both primates and rodents. The first published experimental hMPV infection model in cynomolgus macaques (Macaca fascicularis) confirmed that hMPV is a primary pathogen of the respiratory tract in primates [45]. The hMPV-infected macaques showed mild clinical signs of rhinorrhea corresponding with a suppurative rhinitis at pathological examination. In addition, mild erosive and inflammatory changes in the mucosa and submucosa of conducting airways, and an increased number of alveolar macrophages in bronchioles and pulmonary alveoli were observed. A close association between these lesions and the specific expression of hMPV antigen was shown by immunohistochemistry. Based on the antigen expression, viral replication mainly took place at the apical surface of ciliated epithelial cells throughout the respiratory tract. Pharyngeal excretion of hMPV showed a peak at day 4 post infection (p.i.) decreasing to zero by day 10, concomitant with a reduction in the number of infected epithelial cells. The mild upper respiratory tract disease as observed in these macaques corresponds to that in immunocompetent adults. Due to the fact the hMPV can replicate in the lower respiratory tract of cynomolgus macaques, more severe disease can be expected in immunocompromised patients. Some investigators have also shown that hMPV can replicate in the lungs of hamsters and cotton rats without producing recognizable clinical signs, although transient histo-pathological pulmonary changes were noted in cotton rats [46-50].

hMPV infection in other small-animal models such as ferrets and rabbits has been reported to induce a strong immune response [10], but the level of virus replication in these animals has not been reported. The study of

Table 1. Incidence of human metapneumovirus (hMPV) infections in hospitalized children with respiratory tract disease

Country

Study period

Population

Method

hMPV-positive/ total number of patients

Prevalence

Peak age

Regev et al. [15]

Israel

Nov. 02-May 03 Nov. 03-May 04

< 5 years, RTI

RT-PCR(l)

42/338

10.8%

1-2 year

Wilkesmann et al. [23]

Germany

Oct. 02-May 03 Oct. 03-May 04

Children; RTI

RT-PCR(2)

114/637*

17.9%

<24 months

Foulongne et al. [24]

France

Nov. 03-0ct. 04

< 5 years; RTI

RT-PCR(2)

50/589

8.5%

Bouscambert-Duchamp et al. [25]

France

Sept. 01-June 02

Infants; <24 months

RT-PCR(2)

6/94

6.4%

2-6 months

IJpma et al. [22]

South Africa

June-Aug. 2002

Children; RTI

RT-PCR(2)

8/137

5.8%

2-24 months

K├Ânig et al. [26]

Germany

< 6 months; apneu admitted to ICU

PCR

15/87

18%

McAdam et al. [27]

USA

Oct. 00-Sept. 02

sl8 years

RT-PCR(l)

54/868

6.2%

3-24 months

Jartti et al. [28]

Finland

Sept. 00-June 02

3 months-16 years; acute expiratory wheezing

RT-PCR(2)

12/291

4%

3-11 months

D0llner et al. [29]

Norway

Nov. 02-Apr. 03

Children; RTI

PCR(2)

50/236

21%

sl2 months

Mullins et al. [30]

USA

Aug. 00-Sept. 01

<5 years; RTI

RT-PCR(2)

26/641

4%

6-24 months

Esper et al. [31]

USA

Nov. 01-Nov. 02

<5 years

RT-PCR(3)

54/668

8.1%

<12 months

Madhi et al. [8]

South-Africa

Mar. OO-Oct. 00

Infants

RT-PCR(3)

14/196

7.1%

van der Hoogen et al. [32]

Netherlands

Oct. 00-Feb. 02

All ages; RTI

RT-PCR(l)

48/685*

6.5%

4-6 months

Viazov et al. [18]

Germany

Jan. 02-May 02

<2 years; RTI

RT-PCR(2)

11/65

17.5%

Maggi et al. [33]

Italy

Jan. 00-May 02

<2 years; RTI

RT-PCR(4)

23/90

25%

s 3 months

Peiris et al. [9]

Hong Kong

Aug. 01-Mar. 02

s 18 years; RTI

RT-PCR(2)

32/587

5.5%

Thanasugarn et al. [34]

Thailand

Mar. 01-Sept. 02

<14 years; RTI

RT-PCR(2)

5/120

4.2%

Rawlinson et al. [35]

Australia

2 summers & 2

<12 years; URTI

PCR(2)

9/150

6%

winters 00-02

<17 years; asthma

PCR(2)

3/179

2%

Freymuth et al. [36]

France

Nov. 00-Mar. 01

Children

RT-PCR(3)

19/337*

6.6%

<1 year

Nov. 01-Feb. 02

(U)RTI: (upper) respiratory tract infection.

(1) All respiratory specimens obtained; (2) nasopharyngeal aspirates; (3) on common respiratory viruses negative nasopharyngeal aspirates; (4) nasal swabs.

* Number of samples.

Table 2. Incidence of hMPV infections in non-hospitalised children with RTI

Laham et al. [37] Principi et al. [38] Williams et al. [39] Bastien et al. [40]

Country Study period Population Method

USA 1982-2001 <5 years; URTI RT-PCR(3)

Germany Oct. 00-Apr. 01 <3 years; RTI <6 months; PCR(3)

apneu

Argentina June 02-Sept. 02 <1 year; RTI RT-PCR(4)

USA 1976-2001 <5 years; RTI or AOM RT-PCR(3)

USA Nov. 99-Apr. 00 Fit elderly > 65 years;RTI RT-PCR(2)

Nov. 00-Apr. 01 Young adults;RTI serology hMPV-positive/ total number of patients

118/2384 2/620

22/373 41/1331 49/248 66/445

Prevalence Peak age

6-12 months

(U)RTI: (upper) respiratory tract infection; AOM: acute otitis media.

(1) All respiratory specimens obtained; (2) nasopharyngeal aspirates; (3) on common respiratory viruses negative nasopharyngeal aspirates; (4) nasal swabs.

Skiadopoulos et al. [49] extended these observations to show that members of both hMPV lineages replicated efficiently in hamsters and that infection induced a high level of neutralizing antibodies and resistance to challenge that was effective against both homologous and heterologous strains. In addition, two species of nonhuman primates were also identified as useful models for the development of respiratory tract disease (chimpanzees) and for viral replication (African green monkeys). Chimpanzees developed a robust immune response, although the level of virus shedding was low. They were protected from disease following re-challenge with either strain. Therefore, chimpanzees may provide a useful nonhuman primate model for hMPV disease but are less ideal for studying virus replication. In contrast, rhesus macaques are not ideal animal models for the quantitation of hMPV replication, although they developed serum neutralizing antibodies following hMPV infection. hMPV replicated most efficiently in the respiratory tract of African green monkeys and the infected animals developed high level of hMPV serum-neutralizing antibodies effective against both lineages. A high degree of genetic relatedness and cross-protection was shown mediated by immunity to the highly conserved F protein. An human parainfluenza virus 1 (hPIV1) vector bearing the hMPV F protein provided protection against hPIV1 as well as both lineages of hMPV, indicating that such vectors might be useful as vaccines to protect against disease caused by both hPIV1 and hMPV.

BALB/c mice and cotton rats are considered a good and convenient experimental model to study the pathogenesis of human RSV, another paramyxovirus. For hMPV, 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 inocula are used [50-52]. A small animal experimental model of hMPV infections in BALB/c mice was developed to study mechanisms contributing to immunity and disease pathogenesis [51]. A biphasic kinetics of hMPV replication in lung tissue was shown with peak titers on days 7 and 14 p.i. Viable virus could be recovered from the lungs up to 60 days p.i., while genomic hMPV-RNA was detected up to 180 days p.i. The lung histopathology was modest and characterized by mono-nuclear cell infiltration in the interstitium starting on day 2, peaking on day 4 and decreasing on day 14 p.i, associated with bronchial and bronchiolar inflammation. This low pulmonary inflammatory response may contribute to the persistence of the virus. Hamelin et al. [50] did not detect any infectious virus in the lungs of BALB/c mice by day 21 after hMPV infection, although histopathological changes were still significant at that time, compared with those in sham-infected mice. Both duration and severity of inflammation around the alveoli was more limited in cotton rats compared to the BALB/c mice. Clinical symptoms of respiratory distress and weight loss were observed between days 4 and 10 p.i. in mice, but not in infected cotton rats. Recently, Alvarez and Tripp reported that hMPV RNA could still be detected a 180 days p.i. in the lungs of hMPV-infected mice and that such persistence results in an aberrant immune response [53]. The duration of pulmonary inflammation associated with a single hMPV challenge and the characterization of the consequences of this viral infection with respect to respiratory functions was further evaluated by Hamelin et al. [54]. The results showed that small amounts of viral RNA are still present in 33% in the lungs of hMPV-infected mice for at least 154 days p.i. and are associated with significant peribronchiolitis and perivasculitis. During the first 2-3 weeks, the inflammation mostly consisted of interstitial inflammation and the presence of alveolitis, as reported previously [50]. Over time, the inflammation became characterized by a prominent peribronchiolar and perivascular infiltrate, which was still significant on day 154. An increased number of PAS-positive cells in the central and peripheral airways up to day 12 p.i. were seen, suggesting increased mucus production. Concurrently with the time of maximal viral replication and histopathological score, the airway obstruction was most severe, followed by a gradually decrease but was still significant on day 70 p.i. Such inflammation seems to be responsible for chronic obstruction and hyperresponsiveness of the airways, which persist for > 2 months. These results reinforce the concept that severe paramyxo-virus infections early during childhood can be associated with the development of asthma in children.

Overall, these data suggest that BALB/c mice are more susceptible to hMPV infection than cotton rats on the basis of higher virus titers and levels of lung inflammation, combined with the absence of clinical signs. The absence of clinical signs has also been reported in hamsters and ferrets. These experimental models of hMPV infection show similarities with the pathogenesis, as far as studied, of RSV infection in humans.

Histopathological assessment of hMPV infection on lung tissue obtained by open or transbronchial biopsies from five immunocompromised patients showed acute and organizing lung injury [55]. More specifically, areas of diffuse alveolar damage with hyaline membrane formation and foci of bronchiolitis obliterans/organizing pneumonia-like reactions were seen. In each sample, enlarged type II pneumocytes with smudged hyperchromatic nuclei resembling smudge cells found in adenovirus infection were detected. In contrast, smudge cells were not detected in lung tissue samples of four patients with lower RTIs due to RSV, rhinovirus, or parainfluenza virus. This might be a characteristic histopathological pattern of hMPV lower RTI. The histopathological pattern shown in this study with humans was distinct from those found in experimental infection of nonhuman primates, in which erosive and inflammatory changes were confined to the conducting airways [45].

Little is known about the nature of cytokine responses to hMPV. Human peripheral blood mononuclear cells in culture stimulated by hMPV revealed that classical CD4 T cell activation depending on antigen presentation and CD86-mediated co-stimulation occurred, comparable to stimulation by RSV [56]. In a study using BALB/c mice, it was shown that the indolent pulmonary inflammatory response was characterized by minimal innate immune and CD4 T cell trafficking, with low-level interferon (IFN)-y expression, induction of Th2-type interleukin (IL)-10 expression later during the infection, and delayed cytotoxic lymphocyte (CTL) activity [53]. Peak expression of macrophage inflammatory protein 1a, IFN-y, IL-4 and RANTES (regulated upon activation, normal T cell expressed and secreted) was related to the severity of the pulmonary inflammation in BALB/c mice [50]. hMPV was a weaker inducer of IFN-y, IL-10 and CCL5 than RSV, but induced higher levels of IL-6 instead. When looking at cytokine releases at the respiratory epithelial surfaces, hMPV, in contrast with RSV, seemed to be a poor inducer but elicited identical symptoms of similar severity [37]. Levels of the inflammatory cytokines IL-ip, TNF-a, IL-6, IL-8, IL-10, and IL-12 in respiratory secretions of infants < 1 year with an acute RTI, were two- to sixfold lower in those infected with hMPV compared to RSV. The higher levels of IL-6, inhibiting Th1 differentiation, combined with the lower levels of IFN-y induced by hMPV, are responsible for a weaker antiviral response leading to lower memory cells upon viral recall. This mechanism underlies the life-long, typically symptomatic re-infection with hMPV. IL-8 and RANTES in nasal secretions of children < 16 year admitted to hospital with acute expiratory wheezing were different from that reported in infections with RSV [57]. Patients with RSV infection had high concentrations of RANTES and varying levels of IL-8, whereas children with hMPV infection had lower concentrations of RANTES and higher levels of IL-8. It seems that mechanisms other than those known for RSV elicit symptomatic disease after infection with hMPV. Other mechanisms may include, although they are not limited to, (1) direct viral damage to the airways; (2) Th1 vs. Th2 polarization of the pulmonary immune response, leading to different clinical symptoms; and (3) chemokine-mediated inflammation. Further research is needed to elucidate the exact mechanisms of illnesses caused by hMPV.

The fusion F surface glycoprotein has been identified as a major cross-protective antigen [48, 49]. In addition to the F protein, the subfamily Pneumovirinae of the paramyxoviruses also have a separate surface glycoprotein that is involved in attachment and is called the G protein. The F and G surface glycoproteins are the only significant neutralization antigens, and are major independent protective antigens [58]. hMPV virions appear to have three surface glycoproteins, the F, G and SH protein [59]. To analyze the contribution of these three glycoproteins in neutralizing and protective antibodies, hamsters were immunized intranasally with recombinant PIV type 1 expressing each glycoprotein individually from an added gene [60]. The F glycoprotein was shown to be the major contributor to the induction of neutralizing antibodies and protective immunity. The G and SH glyco-proteins did not induce detectable neutralizing antibodies, and the contributions to protection were minor or negligible, respectively. This is in contrast with other paramyxoviruses (including RSV) in which the G protein stimulates high levels of neutralizing and protective antibodies.

Cleavage of the precursor of the F glycoprotein is a prerequisite for infectivity and is an important determinant of virulence for most Paramyxoviridae. The contribution of the trypsin-dependent cleavage site R-Q-S-R in hMPV to its growth in vitro is well known. This requirement for trypsin in vitro raises the possibility that hMPV virulence is restricted by the inefficient cleavage of the F protein. Using recombinant hMPV in which the naturally occurring cleavage sequence was replaced by sequences not depending upon added trypsin in vitro, it was shown that replication in hamsters and African green monkeys was not changed. These results suggest that cleavage activation is not a major determinant of hMPV virulence

[61]. Similar results were reported by others using a point mutation in the F gene that conferred intracellular cleavability of hMPV in a hamster model

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