Human Metapneumovirus Subgroups Research Articles

Distinct roles for type I and type III interferons in virulent human metapneumovirus pathogenesis.

Human metapneumovirus (HMPV) is an important cause of acute lower respiratory infection in children and adults worldwide. There are four genetic subgroups of HMPV and both neutralizing antibodies and T cells contribute to protection. However, little is known about mechanisms of pathogenesis and most published work is based on a few extensively passaged, laboratory-adapted strains of HMPV. In this study, we isolated and characterized a panel of low passage HMPV clinical isolates representing all four genetic subgroups. The clinical isolates exhibited lower levels of in vitro replication compared to a lab-adapted strain. We compared disease phenotypes using a well-established mouse model. Several virulent isolates caused severe weight loss, lung pathology, airway dysfunction, and fatal disease in mice, which was confirmed in three inbred mouse strains. Disease severity did not correlate with lung viral titer, as virulent strains exhibited restricted replication in the lower airway. Virulent HMPV isolates were associated with markedly increased proinflammatory cytokine production and neutrophil influx; however, depletion of neutrophils or genetic ablation of inflammasome components did not reverse disease. Virulent clinical isolates induced markedly increased type I and type III interferon (IFN) secretion in vitro and in vivo. STAT1/2-deficient mice lacking both type I and type III IFN signaling showed reduced disease severity and increased lung viral replication. Inhibition of type I IFN signaling using a blocking antibody or genetic ablation of the type I IFN receptor reduced pathology with minimal effect on viral replication. Conversely, blockade of type III IFN signaling with a neutralizing antibody or genetic ablation of the IFN-lambda receptor had no effect on pathogenesis but restored viral replication. Collectively, these results demonstrate distinct roles for type I and type III IFN in HMPV pathogenesis and immunity.

Development of a duplex real-time RT-PCR assay for the detection and identification of two subgroups of human metapneumovirus in a single tube

Human metapneumovirus (hMPV) is a common cause of respiratory infections in children. Many genetic diagnostic assays have been developed, but most detect hMPV regardless of the subgroup. In this study, we developed a real-time RT-PCR assay that can detect and identify the two major subgroups of hMPV (A and B) in one tube. Primers and probes were designed based on the sequences of recent clinical isolates in Japan. The assay showed comparable analytical sensitivity to a previously reported real-time RT-PCR assay and specific reactions to hMPV subgroups. The assay also showed no cross-reactivity to clinical isolates of 19 species of other respiratory viruses. In a validation assay using post-diagnosed clinical specimens, 98% (167/170) positivity was confirmed for the duplex assay, and the three specimens not detected were of low copy number. The duplex assay also successfully distinguished the two major subgroups for all 12 clinical specimens, for which the subgroup had already been determined by genomic sequencing analysis. The duplex assay described here will contribute to the rapid and accurate identification and surveillance of hMPV infections.

Human metapneumovirus epidemiological and evolutionary patterns in Coastal Kenya, 2007-11

BackgroundHuman metapneumovirus (HMPV) is an important global cause of severe acute respiratory infections in young children and the elderly. The epidemiology of HMPV in sub-Saharan Africa is poorly described and factors that allow its recurrent epidemics in communities not understood.MethodsWe undertook paediatric inpatient surveillance for HMPV in Kilifi County Hospital (KCH) of Coastal Kenya between 2007 and 2011. Nasopharyngeal samples collected from children aged 1 day–59 months admitted with severe or very severe pneumonia, were tested for HMPV using real-time polymerase chain reaction (RT-PCR). Partial nucleotide sequences of the attachment (G) and fusion (F) surface proteins of positive samples were determined and phylogenetically analyzed.ResultsHMPV was detected in 4.8 % (160/3320) of children [73.8 % (118/160) of these less than one year of age], ranging between 2.9 and 8.8 % each year over the 5 years of study. HMPV infections were seasonal in occurrence, with cases predominant in the months of November through April. These months frequently coincided with low rainfall, high temperature and low relative humidity in the location. Phylogenetic analysis of partial F and G sequences revealed three subgroups of HMPV, A2 (74 %, 91/123), B1 (3.2 %, 4/123) and B2 (22.8 %, 28/123) in circulation, with subgroup A2 predominant in majority of the epidemic seasons. Comparison of G sequences (local and global) provided a greater phylogenetic resolution over comparison of F sequences and indicated presence of probable multiple G antigenic variants within the subgroups due to differences in amino acid sequence, encoded protein length and glycosylation patterns.ConclusionThe present study reveals HMPV is an important seasonal contributor to respiratory disease hospitalization in coastal Kenya, with an evolutionary pattern closely relating to that of respiratory syncytial virus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1605-0) contains supplementary material, which is available to authorized users.

Molecular Epidemiology of Human Metapneumovirus in Riyadh Province, Saudi Arabia

Human metapneumovirus (HMPV) is an important cause of respiratory tract illness in children. Two HMPV subgroups, A and B, and four genotypes, A1, A2, B1 and B2, have been identified. Concurrent circulation of the different genotypes in yearly epidemics has been recorded globally, but not in Saudi Arabia. The current report was designed to study HMPV epidemiology in Saudi children and to analyze the genetic diversity and circulation patterns. Nasopharyngeal aspirates (n = 174) were collected from hospitalized children in Riyadh (2008-2009). The screening of samples using real-time RT-PCR identified 19 HMPV strains. The majority of the strains belonged to subgroup B, while all strains of subgroup A were members of genotype A2. In 2008, only subgroup B was recognized, whereas in 2009 both subgroups were identified to be cocirculating at similar rates. The full-length attachment (G) gene and a partial sequence of the fusion (F) gene of positive samples were sequenced. The G gene showed a high degree of genetic diversity and exhibited a variable number of positively selected sites in different lineages. In contrast, the F gene demonstrated an extensive genetic stability with a higher tendency toward purifying selection. This is the first report on HMPV genotype circulation in Saudi Arabia; however, the exact circulation kinetics requires further retrospective and prospective study.

Human Metapneumovirus Infection in Jordanian Children: Epidemiology and Risk Factors for Severe Disease.

Human metapneumovirus (HMPV) is a leading cause of acute respiratory tract infection in young children. Our objectives were to define HMPV epidemiology and circulating strains and determine markers of severe disease in Jordanian children. We conducted a prospective study from March 16, 2010 to March 31, 2013 using quantitative reverse transcription-polymerase chain reaction to determine the frequency of HMPV infection among children <2 years old admitted with fever and/or acute respiratory illness to a major government hospital in Amman, Jordan. HMPV was present in 273 of 3168 (8.6%) of children presenting with acute respiratory tract infection. HMPV A2, B1 and B2, but not A1, were detected during the 3-year period. HMPV-infected children were older and more likely to be diagnosed with bronchopneumonia than HMPV-negative children. HMPV-infected children with lower respiratory tract infection had higher rates of cough and shortness of breath than children with lower respiratory tract infection infected with other or no identifiable viruses. Symptoms and severity were not different between children with HMPV only compared with HMPV coinfection. Children with HMPV subgroup A infection were more likely to require supplemental oxygen. In a multivariate analysis, HMPV subgroup A and age <6 months were independently associated with supplemental oxygen requirement. HMPV is a leading cause of acute respiratory tract disease in Jordanian children <2 years old. HMPV A and young age were associated with severe disease. Ninety percent of HMPV-infected hospitalized children were full term and otherwise healthy, in contrast to high-income nations; thus, factors contributing to disease severity likely vary depending on geographic and resource differences.

A broadly neutralizing human monoclonal antibody exhibits in vivo efficacy against both human metapneumovirus and respiratory syncytial virus.

Human metapneumovirus (HMPV) is a leading cause of acute respiratory tract infection, with significant morbidity and mortality. No licensed vaccines or therapeutic agents exist. Monoclonal antibodies (mAbs) are effective at preventing other infectious diseases and could be used against HMPV in high-risk hosts. In vitro assays were performed to assess the neutralizing activity and affinity kinetics of human mAb 54G10. A new mouse model was developed to assess prophylactic and therapeutic efficacy in vivo. The epitope of 54G10 was identified by generating mAb-resistant mutants (MARMs). At low concentrations, 54G10 neutralized all 4 subgroups of HMPV in vitro and had subnanomolar affinity for the fusion protein. DBA/2 mice were permissive for all 4 HMPV subgroups, and 54G10 was effective both prophylactically and therapeutically against HMPV in vivo. Sequencing of HMPV MARMs identified the 54G10 epitope, which was similar to an antigenic site on respiratory syncytial virus (RSV). 54G10 also exhibited in vitro neutralizing activity and in vivo protective and therapeutic efficacy against RSV. Human mAb 54G10 has broad neutralizing activity against HMPV and could have prophylactic and therapeutic utility clinically. The conserved epitope could represent a structural vaccine target for HMPV and RSV.

Human metapneumovirus: insights from a ten-year molecular and epidemiological analysis in Germany.

Human metapneumovirus (HMPV) is a cause of respiratory tract illness at all ages. In this study the epidemiological and molecular diversity among patients of different ages was investigated. Between 2000–2001 and 2009–2010, HMPV was detected in 3% (138/4,549) of samples from outpatients with influenza-like illness with a new, sensitive real-time RT-PCR assay. Several hundred (797) clinical specimens from hospitalized children below the age of 4 years with acute respiratory illness were investigated and HMPV was detected in 11.9% of them. Investigation of outpatients revealed that HMPV infections occurred in individuals of all ages but were most prevalent in children (0–4 years) and the elderly (>60 years). The most present clinical features of HMPV infections were cough, bronchitis, fever/shivers and pneumonia. About two thirds of HMPV-positive samples were detected in February and March throughout the study period. Molecular characterization of HMPV revealed a complex cyclic pattern of group dominance where HMPV subgroup A and B viruses predominated in general for three consecutive seasons. German HMPV represented all genetic lineages including A1, A2, B1, B2, sub-clusters A2a and A2b. For Germany, not only time-dependent circulation of lineages and sub-clusters was observed but also co-circulation of two or three predominant lineages. Two newly emerging amino acid substitutions (positions 223 and 280) of lineage B2 were detected in seven German HMPV sequences. Our study gives new insights into the molecular epidemiology of HMPV in in- and outpatients over a time period of 10 years for the first time. It is one of only few long-term surveillance studies in Europe, and allows comparative molecular analyses of HMPV circulating worldwide.

Human Metapneumovirus Subgroup Changes and Seasonality During Epidemics

Human metapneumovirus (HMPV) is a major cause of respiratory tract illness in young children and causes annual outbreaks in winter and spring seasons. We evaluated the subgroups of HMPV that caused annual outbreaks and its seasonal occurrence during a 21-year period. Real-time PCR was used for detection of HMPV in 3576 nasopharyngeal aspirates that had been continuously collected year-round for the years 1987 to 2008 from infants hospitalized with acute respiratory tract illness. Phylogenetic analysis was used to assess HMPV subgroups. Of the 3576 samples obtained, 202 (5.6%) tested positive for HMPV. All known HMPV subgroups (A1, A2a, A2b, B1, B2) could be identified as important respiratory tract pathogens in infants. We found that one HMPV subgroup predominated each year, and it was displaced by another subgroup every 1 to 3 years. Besides the frequent change in predominant HMPV subgroups, we observed a yearly shift in the seasonal occurrence, with a strong peak of HMPV activity in late spring-summer months every second year. HMPV activity is characterized by a periodic change in the predominant subgroup and it shows a stable seasonal rhythm of alternating winter and spring activity.

Population‐Based Incidence of Human Metapneumovirus Infection among Hospitalized Children

Background. Human metapneumovirus (HMPV) is a leading cause of acute respiratory illness (ARI) in children. Population-based incidence rates and comprehensive clinical characterizations of disease have not been established. Methods. We conducted population-based prospective surveillance for 2 years in 2 US counties of HMPV infection among children <5 years old who were hospitalized with ARI or fever. Nasal and throat specimens obtained with swabs were tested for HMPV by real-time reverse-transcription polymerase chain reaction and genotyped. Results. Forty-two (3.8%) of 1104 children tested positive for HMPV. The overall annual rate of HMPVassociated hospitalizations per 1000 children <5 years old was 1.2 (95% confidence interval [CI], 0.9–1.6). This rate was highest among infants 0–5 months old (4.9 per 1000 [95% CI, 2.9–7.2]), followed by children 6–11 months old (2.9 per 1000 [95% CI, 1.4–4.7]). The annual rate of hospitalization for HMPV infection was less than that for respiratory syncytial virus infection but similar to that for influenza and parainfluenza virus 3 infection in all age groups. The mean age of children hospitalized with HMPV infection was 6 months. Bronchiolitis, pneumonia, and asthma were the most common diagnoses among children with HMPV infection. All 4 HMPV subgroups were detected during both years at both sites. HPMV infection was most prominent from March through May. Conclusion. HMPV was detected in 3.8% of children hospitalized with ARI or fever, with a population incidence similar to that of influenza virus and parainfluenza virus 3.

Prevalence and Clinical and Molecular Characterization of Human Metapneumovirus in Children With Acute Respiratory Infection in China

Human metapneumovirus (HMPV), a newly discovered paramyxovirus, has been associated with acute respiratory tract infections (ARTIs). However, the prevalence and molecular characteristics of HMPV in China are still unclear. A total of 661 nasopharyngeal aspirates (NPA) specimens were collected from 661 children with ARTIs between December 2006 and November 2008. Specimens were screened for HMPV by reverse transcription-polymerase reaction. All positive amplification products were confirmed by sequencing. HMPV was detected in 45 patients (6.80%) of the 661 children. The HMPV-infected patients were from 29 days to 9 years of age. A high incidence of HMPV infection (84.4%) was observed during the winter-spring season. Of the 45 HMPV-positive patients, 25 (55.6%) were co-infected with other respiratory viruses, and respiratory syncytial virus (RSV) was the most common additional respiratory virus. The most common clinical diagnosis was bronchopneumonia (57.8%) and cough (88.9%) was the most common clinical symptom. Phylogenetic analysis of the F gene revealed that 80% of the HMPV detected were A2, 2.2% were A1, and 17.8% were B1. Statistical analyses showed that sex, ages, seasons, and severity of the disease did not correlate with HMPV genotype (P = 0.986, 0.347, 0.660, 0.252), but viral coinfection with HMPV increased hospitalization rates (P = 0.005). HMPV was frequently detected in the pediatric patients with ARTI in China. RSV was the most common coinfection virus and coinfection increased hospitalization rates. All HMPV subgroups except B2 cocirculated and there was no association found between HMPV genotypes and severity of disease.

Recovery of human metapneumovirus from cDNA: optimization of growth in vitro and expression of additional genes

Human metapneumovirus (HMPV) is a recently recognized causative agent of respiratory tract disease in individuals of all ages and especially young infants. HMPV remains poorly characterized and has been reported to replicate inefficiently in vitro. Complete consensus sequences were recently determined for two isolates representing the two proposed HMPV genetic subgroups (Biacchesi et al., Virology 315 (1) (2003) 1). We have developed a reverse genetic system to produce one of these isolates, CAN97-83, entirely from cDNA. We also recovered a version, rHMPV–GFP, in which the enhanced green fluorescent protein (GFP) was expressed from a transcription cassette inserted as the first gene, leaving the 41-nt leader region and first 16 nt of the N gene undisturbed. The ability to monitor GFP expression in living cells greatly facilitated the initial recovery of this slow-growing virus. In addition, the ability to express a foreign gene from an engineered transcription cassette confirmed the identification of the HMPV transcription signals and identified the F gene-end signal as being highly efficient for transcription termination. The ability to recover virus containing a foreign insert in this position indicated that the viral promoter is contained within the 3′-terminal 57 nt of the genome. Recombinant HMPV replicated in vitro as efficiently as biologically derived HMPV, whereas the kinetics and final yield of rHMPV–GFP were reduced several-fold. Conditions for trypsin treatment were investigated, providing for improved virus yields. Another version of HMPV, rHMPV+G1F23, was recovered that contained a second copy of the G gene and two extra copies of F in promoter-proximal positions in the order G1–F2–F3. Thus, this recombinant genome would encode 11 mRNAs rather than eight and would be 17.3 kb long, 30% longer than that of the natural virus. Nonetheless, the rHMPV+G1F23 virus replicated in vitro with an efficiency that was only modestly reduced compared to rHMPV and was essentially the same as rHMPV–GFP. Northern blot analysis showed that the increased number and promoter-proximal location of the added copies of the F and G genes resulted in a more than 6- and 14-fold increase in the expression of F and G mRNA, respectively, and sequence analysis confirmed the intactness of the added genes in recovered virus. Thus, it should be feasible to construct an HMPV vaccine virus containing extra copies of the G and F putative protective antigen genes to increase antigen expression or to provide representation of additional antigenic lineages or subgroups of HMPV.

Genetic diversity between human metapneumovirus subgroups

Complete consensus nucleotide sequences were determined for human metapneumovirus (HMPV) isolates CAN97-83 and CAN98-75, representing the two proposed genotypes or genetic subgroups of HMPV. The overall level of genome nucleotide sequence identity and aggregate proteome amino acid sequence identity between the two HMPV subgroups were 80 and 90%, respectively, similar to the respective values of 81 and 88% between the two antigenic subgroups of human respiratory syncytial virus (HRSV). The diversity between HMPV subgroups was greatest for the SH and G proteins (59 and 37% identity, respectively), which were even more divergent than their HRSV counterparts (72 and 55% cross-subgroup identity, respectively). It is reasonable to anticipate that the two genetic subgroups of HMPV represent antigenic subgroups approximately comparable to those of HRSV.