Human metapneumovirus (hMPV) was first reported in 2001 by a Dutch group that identified sequences of the virus after randomly primed reverse transcription-polymerase chain reaction (RT-PCR) tests of respiratory secretions from children with lower respiratory tract disease.1 Although the virus was not isolated until recently, the original report showed that as early as 1958 antibodies to hMPV were present in archived serum specimens from 100% of persons 8 years old or older. Thus the virus is not newly emerging. Genetic analysis of the virus showed that it is an RNA virus of the Paramyxoviridae family and part of the Pneumovirinae subfamily along with respiratory syncytial virus (RSV). The avian pneumoviruses (APV) (formerly turkey rhinotracheitis virus) are highly related to hMPV and have been separated by taxonomists into a separate genus, Metapneumovirus. These viruses can be distinguished from members of the Pneumovirus genus by their lack of genes encoding the nonstructural proteins NS1 and NS2 and by a slightly different gene order in the nonsegmented negative sense genomic RNA. APV, first isolated in the 1970s, has now been isolated in Europe, the United States, the Middle East and the African continent and has been classified in 4 subgroups (A through D). hMPV is most closely related by sequence homology to APV subgroup C. hMPV was not capable of infecting turkeys or chickens, suggesting that it is a human virus, not a zoonotic crossover infection from birds to humans.
CELL CULTURE
One of the reasons why the virus was detected only recently is that it replicates poorly in the conventional cell cultures used for respiratory virus diagnosis, such as HEK, HEp-2 and Madin-Darby canine kidney cells. Primary isolation is facilitated by the presence of a low concentration of trypsin, which is often not routinely used in diagnostic virology laboratory cultures. The virus was first isolated in primary monkey kidney cells from a clean primate facility and passed twice (“tertiary” monkey kidney cells). Monkey kidney cells commercially available in the United States cannot be used in this manner because most of these cell cultures contain foamy viruses that cause cytopathic effects on prolonged culture, that obscure any effect caused by hMPV. Subsequently several groups have found that the virus can be propagated in monolayer cultures of LLC-MK2 cells and some Vero cell lines. Growth is slow in these cell lines, which often require 2 weeks or more for maximal replication. Primary isolation from respiratory secretions often requires blind passage in these cell lines, and cytopathic effect is often apparent only on subsequent passages. The cytopathic effect usually appears to be more subtle than that typically associated with RSV in HEp-2 cell culture. Instead of large syncytia, hMPV often causes focal areas of rounding of cells and minor patches of cell-cell fusion in LLC-MK2 or Vero cell culture.
SEROLOGY
Serologic evidence for infection has been determined by relatively crude methods to date. Generally researchers have used microtiter plate-based enzyme-linked immunosorbent assays coated with virus-infected cell lysates or fixed and permeabilized infected cell monolayers as the viral antigen. Purified viral proteins are needed for more reproducible assays. Serum neutralizing antibody assays have been used to demonstrate the induction of functional antibodies in both humans and experimentally infected animals.
MOLECULAR DIAGNOSIS
Identification of the virus in clinical samples, usually nasopharyngeal aspirates or swabs, is best performed by RT-PCR. Several of the original clinical reports used an RT-PCR test incorporating PCR primers hybridizing to the polymerase (L) gene, and the nucleotide sequence of the L gene PCR product was used to identify the virus. Subsequently various real time RT-PCR tests have been developed that offer speed and specificity, including an assay specifically designed to detect viruses from the 4 known genetic lineages, which are described later in this article.2–4 Investigators generally have tested for the presence of virus in respiratory secretions, but it has also been identified in lung tissue.5
PATHOGENESIS
Little direct experimental work has been reported to elucidate the mechanisms of pathogenesis in humans. The virus is thought to replicate in both the upper and lower respiratory tract, but the particular cell types that are affected have not been defined. Several studies have quantified secreted host factors in the respiratory secretions of infected persons, such as the chemokine interleukin-8,6,7 and hMPV appears to elicit lower mucosal levels of inflammatory cytokines than does RSV. The interpretation of these studies is not clear at this time. Recent studies in infected macaques suggest that ciliated epithelial cells are most affected, with antigen concentrated at the apical surface of polarized epithelial cells. However, antigen could be detected throughout the respiratory tract.8
Naturally acquired infection does not appear to protect against reinfection. Symptomatic lower respiratory tract infection has been documented in sequential years in children.9 Virtually every child exhibits serologic evidence of infection by age 5 years1; however, adult infections are common. Nevertheless it is likely that infection does induce partial protection against severe disease, because most serious lower respiratory tract illnesses occur in the first year of life.9
CLINICAL FEATURES OF INFECTION
The virus causes a variety of clinical syndromes in children that are typical of the paramyxoviruses, including upper and lower respiratory tract illnesses. We conducted a study of the association of the virus with prospectively collected data in a cohort of >2000 subjects age 0–5 years followed during a 25-year period at Vanderbilt University Medical Center.9 This study included medical provider-designated illnesses and showed that hMPV is associated with the common cold (complicated by otitis media about one-third of the time) and with bronchiolitis, pneumonia, croup and exacerbation of reactive airways disease. The profile of signs and symptoms caused by hMPV is very similar to that caused by the related pneumovirus RSV. We found that in ∼12% of cases, outpatient lower respiratory tract illness was associated with hMPV infection, which was second only to RSV. This study is the only one to date for which the denominator for patients at risk is known; thus it gives an estimate of the burden of disease caused by hMPV and shows that burden to be quite significant. A limitation of the study was that only otherwise healthy subjects were included (premature infants and those with underlying cardiac or pulmonary diseases were excluded). Therefore population-based studies are needed. The New Vaccine Surveillance Network study of the Centers for Disease Control reported testing for hMPV in all infants admitted to the hospital in Nashville, TN and Rochester, NY, for acute respiratory illness or fever without localizing symptoms.10 That study found that 3.9% of 668 such hospitalizations in children were associated with hMPV. hMPV-positive children received supplemental oxygen and medical intensive care in frequencies similar to those of the RSV-positive children in that study. Additional studies at tertiary care hospitals suggest that ∼5–10% of patient specimens collected in the winter for virus testing contain hMPV genome.11–16 hMPV has been found in association with respiratory tract disease in every continent and country in which testing has been performed, including Europe (the Netherlands,17 the United Kingdom,18 Italy,19,20 France,21 Germany22 and Greece23), Scandinavia (Finland6,24 and Norway25,26), Asia (Hong Kong,27 Thailand,28 Japan,29–32 and China33,34) Africa (South Africa35–37), North America (United States9–13 and Canada14,16,37,38), South America (Brazil39 and Argentina7,40), the Middle East (Israel41) and Australia.42
RISK FACTORS
Age.
Age affects the risk of serious disease after infection. Most of the cases of lower respiratory tract illness in children occur in the first year of life (one-half in the first 6 months).9 Both young adults and the elderly suffer hMPV infection that leads to medically attended illnesses, but frail elderly people with hMPV infection appear to seek medical attention more frequently.43 During some years, hMPV infection may account for a significant percentage of the hospitalizations of adults with respiratory infections.43
Immune Status.
It is likely that the immune status of infected subjects is important to their ability to clear the acute infection or to resolve the infection without severe disease. hMPV infections have been reported in immunocompromised patients,17,44 and many of the respiratory tract secretions found to contain hMPV genome in studies on samples submitted to diagnostic virology laboratories by medical providers were obtained from immunocompromised patients. hMPV has been reported in association with fatal lower respiratory tract disease in a hematopoietic stem cell transplant recipient.45 Prospective studies in this population are needed to define the burden of disease. hMPV has also been reported in children with HIV infection, but it is not clear that the disease is worse in these children.37 Given that hMPV infection is universal in childhood, the association is not unexpected.
Underlying Pulmonary Disease.
Respiratory virus infections have been associated often with recurrent wheezing in young children and with exacerbations of asthma in older persons. Ongoing research in the RSV field aims to discover whether or not severe respiratory virus infection in infancy predisposes to asthma later in life. A number of genetic polymorphisms have been associated with severe RSV infection and possibly with asthma. Therefore it was natural for investigators to investigate whether hMPV is associated with asthmatic exacerbations. To date, studies that have examined the strength of the association between hMPV infection and asthmatic exacerbation have yielded conflicting results.1,6,9,27,46 There is no question that some patients with asthmatic exacerbations have hMPV infection, but whether or not the virus is associated with asthmatic exacerbation more frequently than other viruses (eg, rhinoviruses) is not yet clear. Larger studies are needed to address this question.
Seasonality.
hMPV causes late winter/early spring epidemics in temperate areas. The season appears to coincide with that of RSV, but most studies have found that the hMPV season tends to occur in the second half of the RSV season.9 In the first half of winter RSV epidemics, RSV is overwhelmingly the most common cause of bronchiolitis. In the second half of the RSV season, hMPV and RSV may occur with equal frequency.
Coinfection.
A coinfection rate of ∼5–10% with 1 or more respiratory viruses has been demonstrated in dozens of studies of respiratory viruses in the past and is to be expected. Because the epidemic seasonality for RSV coincides with that for hMPV, the potential exists for RSV/hMPV coinfections. Indeed several studies have identified cases of lower respiratory infection in which evidence for the presence of both RSV and hMPV has been detected.9,22,23,47 One study suggested that molecular tests for hMPV genome were positive in most of 30 cases of severe RSV bronchiolitis. Studies that depend solely on the amplification of viral genome by RT-PCR, especially nested PCR, must be interpreted with caution for several reasons: (1) PCR testing suffers notoriously high rates of false positives because of cross-contamination in laboratories that perform frequent amplifications, and amplified products should be confirmed by hybridization or preferably nucleotide sequence analysis to demonstrate diversity of isolates; (2) viral genome can be detected by RT-PCR in respiratory secretions for several weeks or more after shedding of cultivatable virus has terminated. Therefore it is difficult to know whether positive RT-PCR test results signify active infection or a recent acute infection that has been terminated. RSV genome has been detected for 100 days or more in experimentally infected animals.48
Potential coinfection with hMPV and the severe acute respiratory syndrome (SARS) coronavirus (CoV) deserves special mention. When the etiology of SARS was being sought in early investigations of the syndrome in Toronto and Hong Kong, hMPV genome was detected as the sole evidence of microbial infection in some cases and as a coinfection with a coronavirus.5,49–54 Subsequent studies showed that the novel coronavirus, designated SARS-CoV, is the cause of SARS. It is unclear whether SARS-CoV and hMPV coinfections were associated with more severe disease than infection solely with SARS-CoV.
It is currently not known whether a significant association exists between hMPV infection and bacterial infections. hMPV lower respiratory tract disease is associated with a high frequency of otitis media requiring antibiotics in children,9 suggesting that Eustachian tube dysfunction and possibly middle ear inflammation caused by hMPV predispose to this local bacterial infection. Bacterial pneumonia complicating hMPV infection has not been reported frequently, but the number of patients examined to date is too few to rule out an association. Large vaccine trials with effective bacterial vaccines can reveal significant but subtle interactions between viruses and bacterial infection that are not detected by common clinical reporting methods.55 The highly related APV has been associated with more severe disease during bacterial coinfection of poultry.56
TRANSMISSION
Formal transmission studies have not been reported, but the mode of transmission is almost certainly large particle respiratory secretions and fomites, based on the relatedness of this virus to other pneumoviruses. Nosocomial transmission does occur, suggesting that contact isolation and scrupulous hand washing by health care providers is warranted in the hospital or office setting.12 Many hospitals cohort children with bronchiolitis in the winter for purposes of preventing RSV transmission. The implementation of such policies may need to be refined, especially if a commercial diagnostic kit for hMPV becomes available, so that cohorting of RSV- and hMPV-infected infants is avoided.
GENETICS OF THE VIRUS
The initial report of the virus demonstrated it to be a single-stranded negative sense RNA virus that is most highly related genetically to avian pneumovirus; taxonomists have placed hMPV and APV in the family Paramyxoviridae, subfamily Pneumovirinae, within their own genus designated Metapneumovirus. Although APV and hMPV are related to the human and animal RSV viruses, they differ in that the gene order in the nonsegmented genome is slightly altered and APV/hMPV are lacking the 2 nonstructural proteins NS1 and NS2 located at the 3′ end of RSV genomes. These proteins counteract host interferons; therefore the lack of these genes in the metapneumoviruses may have important implications for the relative pathogenicity of these viruses compared with RSV strains. Full length sequences of at least 4 hMPV genomes have been reported.57–59 The hMPV genome is predicted to encode 9 proteins in the order 3′-N-P-M-F-M2-SH-G-L-5′ (the M2 gene is predicted to encode 2 proteins, M2-1 and M2-2, using overlapping open reading frames, as in RSV).57 The genome also contains noncoding 3′ leader, 5′ trailer and intergenic regions, consistent with the organization of RSV.58 The viral promoter is contained within the 3′-terminal 57 nucleotides of the genome.60 Sequence identity between APV and hMPV open reading frames is 56% to 88%.57 Therefore, although the function of each of the gene products has not been formally tested, it is likely that the function of the proteins can be predicted by comparison with other paramyxoviruses. The F (fusion), G (glycosylated) and SH (short hydrophobic) proteins likely are integral membrane proteins on the surface of infected cells and virion particles. The F protein appears to be a classic viral fusion protein, with a predicted nonfurin F1/F2 cleavage site near a hydrophobic fusion peptide and 2 heptad repeats in the extracellular domain that facilitate membrane fusion. The predicted G protein of hMPV exhibits the basic features of a glycosylated type II mucin-like protein but interestingly lacks the cluster of conserved cysteines sometimes termed the “cysteine noose” that is found in the RSV and APV G proteins. The N (nucleoprotein), P (phosphoprotein) and L (large, polymerase) proteins are replication proteins in the nucleocapsid, the M2-1 and M2-2 proteins are regulatory proteins and the M (matrix) protein may coordinate viral assembly of viral nucleocapsids with envelope proteins. The ability of the combination of N, P, L and M2-1 proteins encoded from cDNA copies to drive genome replication in transfected cells (using “reverse genetics,” discussed subsequently) supports the predicted function of these proteins.
GENETIC AND ANTIGENIC DIVERSITY
The classification and naming of serotypes/serogroups, genotypes, subgroups, strains, variants and isolates of hMPV are just being developed. It is important to determine the extent of diversity in circulating field strains to design optimal diagnostic, therapeutic and preventive strategies. Viral diversity can be described in several ways, principally by phylogenetic analysis of genetic sequence information or by differences in antigenicity in animals. Every study to date reporting partial sequences of the viral genome has suggested that there are at least 2 major genetic subgroups (designated A and B), and the phylogenetic groupings are concordant when F, G, N, M or L sequences are analyzed.9,59,61–63 Full length sequences of genomes from viruses representing the 2 major subgroups performed by 2 groups using isolates from Canada (strains CAN97-83 and CAN98-75) or from the Netherlands (strains NL/1/00 and NL/1/99) show that the diversity between hMPV subgroup A and B sequences is greatest for the SH and G proteins.57–59 In these genes, the hMPV subgroup viruses exhibit slightly more divergence than RSV subgroup A and B strains do, but the hMPV F protein, which is predicted to be the principal target of protective antibodies, is not particularly divergent in hMPV strains. Therefore the evidence to date suggests that the genetic diversity of circulating hMPV strains of A and B subgroups is comparable with that of RSV subgroups A and B. hMPV phylogeny studies also suggest that the virus sequences within subgroups A and B can be further divided into 2 clades per subgroup; the significance of this diversity is unclear at this time.
There is no question that genetic diversity in hMPV strains affects antigenic diversity, but to what extent has not been resolved. Studies with experimental infection of animals have suggested that under some conditions infection with subgroup A or B prototype strains induces antibodies in serum that are sufficiently different by technical definition to indicate that the viruses fall into 2 different serotypes.1,58,64 Other studies contradict these findings and are more consistent with the interpretation that viruses of the 2 major genotypes fall into 2 subgroups within a single serotype.59,65 Cross-protection and reciprocal cross-neutralization studies in experimentally infected animals show that cross-protection is induced at a high level, consistent with a single serotype.65,66 The extent of cross-protection in rodents cannot be extrapolated directly to the human situation because the animals are only semipermissive hosts for hMPV replication. The most relevant test of the importance of genetic diversity is whether or not viruses of one genotype induce greater protection against the homologous virus than against the heterologous virus. This issue is difficult to assess in humans because reinfections with paramyxoviruses reoccur throughout life, even with homologous viruses. Although difficult to assess, the extent of cross-protection is important to estimate because vaccine developers will choose to develop either a monovalent or a bivalent vaccine formulation based on this factor.
It is important that most of the genetic and therefore antigenic diversity resides in the G protein. hMPV F gene sequences are highly related between subgroups A and B, with amino acid identity 93–96%.62 In contrast, the G glycoprotein genes exhibit nucleotide and amino acid sequence identities ranging from 52 to 58% and from 31 to 35%, respectively, between the 2 subgroups.63
EXPERIMENTAL INFECTION OF ANIMALS
The close genetic relatedness of hMPV and APV suggested that hMPV might be a zoonotic infection crossing from poultry to humans, but early experiments showed that hMPV does not replicate in turkeys or chickens.1 Humans are the only known natural host of hMPV. Investigators have sought to identify animals that could be experimentally infected for research purposes. A wide variety of small animals have been tested, including hamsters, mice, cotton rats, guinea pigs and ferrets.1,65,66 Nonhuman primate species, including rhesus macaques, African green monkeys and chimpanzees, have also been tested.65 Hamsters, ferrets and cotton rats appear to replicate virus to reasonable levels, and the virus appeared to replicate relatively efficiently in most of the primates tested.
MECHANISMS OF IMMUNITY
Very little is known about the mechanisms of immunity to hMPV infection, but it is likely that immunity will function in a manner similar to that against other paramyxoviruses. Infection induces serum neutralizing antibodies in most experimentally infected animals that have been tested, and protection against reinfection has been induced by primary infection of hamsters.65 Although mice appear to exhibit relatively low permissivity for viral replication, they offer a tractable model for studying immune mechanisms, and experiments are ongoing in this area.
EXPERIMENTAL VACCINES
Although the virus was only recently identified, live attenuated virus vaccine development is well under way. When the virus was discovered in 2001, several groups were working with molecular systems that allow the generation (“rescue”) of recombinant paramyxoviruses (especially RSV and parainfluenza viruses) from plasmid DNA copies of virus genes and the virus genome. This technique, referred to as reverse genetics, was rapidly used to study the replication of hMPV and to generate recombinant hMPV strains. Foreign genes such as the reporter gene for green fluorescence protein were inserted into the hMPV genome and expressed, which effectively defined the transcription start and gene end signals.60 Reverse genetics has been used to rescue both strains from Canada (strain CAN97-83) and the Netherlands (strains NL/1/00 and NL/1/99) entirely from complementary DNA (cDNA).58,60 Because the viruses are made from DNA copies, chimeric viruses can be made with the use of the antigenic protein of one virus inserted into the genome of another virus. For example, 1 group has made and tested a recombinant human parainfluenza virus type 1 virus expressing the F protein of the hMPV CAN83 strain. This chimeric virus induced a serum neutralizing antibody response that protected hamsters against challenge with hMPV strains representing either subgroup A or subgroup B. Cold-adapted APV live attenuated virus vaccine strains have been tested in 1-day-old poultry and were found to be effective, and a plasmid DNA vaccine candidate encoding APV F protein induced protection.67
ANTIVIRALS
Antiviral drug therapy for respiratory viruses is difficult to implement effectively in actual practice, because viral shedding is usually already decreasing at the point in the course of infection when patients present to their medical providers. Nevertheless it is reasonable to identify agents with significant antiviral activity when new major human pathogens are discovered. Ribavirin and intravenous immunoglobulin, which have activity against RSV, were tested against hMPV in vitro and found to have equivalent antiviral activity against hMPV and RSV.68 Heparin and the sulfated sialyl lipid also have been shown to have activity against hMPV.69
DISCUSSION
Question: You reported an approximately 37% incidence of otitis media concomitant with this virus. Did you have any outpatient studies to balance the samples or anything else to determine whether it's isolated from the middle ear fluid cavity?
James Crowe Jr., MD: We have not done that, although I'm aware of a “professional” otitis media group that will be doing that. There's nothing published on it as yet, but I think that data will be forthcoming. There are better scenarios, with people looking at hMPV who have large archives of tympanocentesis samples, and they already know the bacteria and viruses involved. But we do see a large incidence of otitis media in both the upper and lower respiratory infection studies associated with hMPV.
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