Human metapneumovirus (HMPV) is a worldwide respiratory pathogen that belongs to the paramyxovirus family of enveloped viruses and affects primarily the pediatric, geriatric, and immunocompromised populations. Despite its prevalence and importance to human health, no therapies are available against this pathogen. For paramyxoviruses, it is believed that infection starts by attachment of the virus to the surface of the cell through the viral attachment protein followed by fusion between the viral and cellular membranes, a process mediated by the fusion (F) protein at the plasma membrane and at neutral pH. Previous work showed that HMPV infection can occur in the absence of the attachment protein and membrane fusion triggered by the F protein can be promoted by low pH. The work presented here are significant advances in our understanding of the entry process of HMPV. We confirmed that the F protein has receptorbinding functions and identified the cellular binding partner to be heparan sulfate proteoglycans (HSPGs). Additionally, we provide evidence that electrostatic interactions at two different regions play important roles for the proper folding, stability, and low pH triggering of the HMPV F protein. We confirmed the hypothesis that protonation of H435 is important for HMPV F triggering and provide additional evidence that the entry of HMPV may be occurring through endocytosis. Therefore, we hypothesize that HMPV entry occurs through endocytosis after viral binding to HSPGs through the F protein and membrane fusion occurs in an acidified compartment.

The use of PCR as a tool for the diagnostic virology and viral research laboratories has greatly increased in recent years, however the use of conventional PCR and amplicon detection systems can be a complex and relatively slow process that increases the risk of amplicon carry-over contamination. Many conventional PCR systems are unsuited for, or unable to perform as accurate diagnostic and quantitative tools because viruses are present in such a diverse variety of patient tissues and in a broad range of concentrations. Traditional viral culture, while still the gold standard for the detection of many viruses, is lengthy, expensive and often subjective. In addition, successful isolation of infectious virus is variable and dependent upon appropriate cell lines, lengthy incubations and careful transport and storage of clinical specimens. Many of the disadvantages arising from the use of traditional assays for the detection of viruses have been overcome by the development of real-time PCR. The technology has continued to develop due to the introduction of several commercial thermal cycling platforms and the appearance of numerous specific and non-specific fluorogenic chemistries. For the purpose of this thesis, human virology was sectioned into three diagnostic divisions containing the synthetic viruses, the well characterised viruses and the new or emerging viruses. This thesis proposes the hypothesis that real-time PCR could greatly improve upon traditional techniques for the detection, quantitation and characterisation of the members of these three divisions in both research and diagnostic environments. Conventional competitive quantitative PCR assays and a non-oligoprobe real-time PCR assay were constructed to detect novel synthetic gene therapy vectors developed from retroviruses. When compared to oligoprobe-based real-time PCR, it was clear that conventional molecular assays, whilst improving upon traditional methods of viral culture and immunofluorescence, were slower, more complex, less versatile and were hindered by a limited dynamic range. Synthetic control templates were developed and an improved method of assaying these template preparations was devised. The controls were used to precisely optimise each assay, create quality assurance reagents and to construct external standard curves permitting the absolute quantitation of viral templates. Real-time PCR achieved several significant goals during the studies performed for this thesis. The new assays detected human enterovirus (HEV) and the emerging pathogen, human metapneumovirus (hMPV) which were both responsible for seasonal outbreaks of serious disease that would otherwise have gone undiagnosed. These data led to the first description of hMPV outside of the Netherlands, as well as the first description of two validated rapid diagnostic RT-PCR assays which permitted the definitive classification of hMPV as a global pathogen of children and adults. Building upon its detection, an extensive molecular epidemiological study permitted the description of subtle differences between Australian and the more recently described international hMPV strains resulting in the classification of two distinct types of hMPV (A and B) and within these, four subtypes (A1, A2, B1 and B2). Real-time PCR rapidly detected, quantitated and genotyped herpes simplex viruses in a single reaction and determined the successful delivery of human and non-human genes by novel retroviral vectors in less time than any other phenotype detection assay. Additionally, these studies produced quantitative data which permitted the rapid calculation of transduction efficiency. Real-time PCR was able to quickly assess the efficiency of the PCR either in response to the titration of individual reaction components or as a result of amplification modifiers present within specimen extracts. The use of nucleotide sequencing studies ideally complemented earlier diagnostic studies of HEV and permitted the discrimination of pathogenic enterovirus 71. This thesis demonstrated that real-time PCR is more able to accommodate the demanding aspects of viral research and diagnostics than any other single method, and is now in a position to replace many of the traditional techniques still used by laboratories unfamiliar with the benefits of real-time PCR. The assays, techniques, reagents and publications resulting from these studies have benefited several areas of viral research and diagnostics and have improved the understanding of the role of real-time PCR in virology and of the technique in general, among the greater scientific community whilst successfully addressing the proposed hypothesis.

Les paramyxovirus possèdent deux glycoprotéines d’enveloppe (gps): la protéine F, permettant la fusion avec la cellule hôte, et la protéine d’attachement appelée G, H ou HN. Les gps H et F d’une souche vaccinale du virus de la rougeole peuvent être incorporées sur des vecteurs lentiviraux (H/F-LV) permettant une transduction efficace des lymphocytes T et B humains non stimulés, habituellement réfractaires. Nous avons montré que les vecteurs H/F-LV sont capables de transduire des cellules B cancéreuses, activées et quiescentes, contrairement aux VSV-G-LV classiques. Leur utilisation in vivo est cependant confrontée à la présence d’anticorps neutralisants induits par la vaccination, dirigés majoritairement contre H. Après la mutation des 2 épitopes immunodominants de H, les vecteurs conservent leur tropisme et échappent à la neutralisation par les anticorps monoclonaux, mais sont toujours neutralisés par le sérum humain. Les souches émergentes de rougeole de génotype D, qui paraissent résister à la vaccination, présentent une glycosylation supplémentaire de la H. Introduite dans notre mutant, elle permet aux H/F-LV de transduire efficacement les cellules T et B en présence de sérum ou de sang total. Les pneumovirinae (le Virus Respiratoire Syncytial et le Métapneumovirus Humain (HMPV)) sont la première cause d’infections respiratoires chez le nourrisson, il n’existe pas de vaccin contre ces virus. Nous avons mis au point un système de Virus-Like Particle rétrovirales incorporant les gps F et G de HMPV (HMPV-VLPs). Injectées à des souris, les HMPV-VLP induisent une forte réponse d’anticorps neutralisants. De plus, suite à une épreuve virale, les souris sont protégées de l’infection par hMPV. / Paramyxoviruses contain two envelope glycoproteins : the F protein allowing fusion with the host cell and an attachment protein, called G, H or HN. Lentiviral vectors pseudotyped with the Edmonston measles virus hemagglutinin and fusion glycoproteins (H/F-LVs) allowed for the first time efficient transduction of quiescent human T and B cells. We showed that H/F-LVs were also able to efficiently transduce quiescent and activated cancer B cells, in contrast to the classical VSV-G-LVs. However, a major obstacle in the use of H/F-LVs in vivo is that most of the human population is vaccinated against measles inducing a humoral immune response exclusively directed against H. LVs pseudotyped with H-glycoproteins mutated in the 2 major epitopes escaped inactivation by monoclonal antibodies but were still neutralized by human serum. Consequently, we took advantage of newly emerged MV-D genotypes that were less sensitive to MV vaccination due to a different glycosylation pattern. The mutation responsible was introduced into the mutated H/F-LVs allowing efficient transduction of quiescent lymphocytes in the presence of high concentration of MV antibody-positive human serum or total blood. Pneumovirinae (Respiratory Syncitial Virus and human metapnemovirus (HMPV)) are the leading cause of respiratory infections in infants and no vaccine is available against these viruses. We designed retroviral Virus-Like Particle incorporating HMPV F and G gps (HMPV-VLPs). HMPV-VLPs injected to mice induce a strong neutralizing antibody immune response in vivo. Furthermore, upon a viral challenge, HMPV-VLP vaccinated mice are protected against hMPV infection.

Human metapneumovirus (HMPV) is a respiratory pathogen in Paramyxovirus family that demonstrates extremely high morbidity in the population, with most individuals having been infected by the age of five. Despite the prevalence of this negative-sense RNA virus in the population for decades, it was only identified in 2001. As such, there is currently no specific treatment for HMPV and the potentially severe consequences of infection for elderly and immunocompromised individuals and particularly infants make development of antivirals targeting HMPV of high significance. HMPV constitutes a quarter of all respiratory hospitalizations among infants, placing it second only to RSV, in addition to becoming a greater concern in concentrated populations of seniors. For these susceptible populations, the consequences of infection have a much greater probability of leading to pneumonia, bronchiolitis and even death. These studies investigate events throughout the infectious cycle of HMPV. They describe specific amino acids that modulate the triggering of viral fusion activity in response to low pH. They also include a report on the dynamic and variable control exercised over gene transcription by viral promoters. Finally, the interplay between viral nonstructural proteins and their distinct roles in both replication and assembly are examined. Ultimately, this work seeks to elucidate the goings-on within an HMPV-infected cell at multiple points throughout the process.

The introduction of the polymerase chain reaction (PCR) into the diagnostic setting has provided unprecedented opportunities in the field of respiratory medicine, not only because pathogens need no longer be cultivable for detection but also through improved sensitivity, specificity and turnaround time compared with traditional methods. The recent discovery of several novel respiratory viruses, such as human metapneumovirus (HMPV), human bocavirus and human coronaviruses (HCoVs) NL63 and HKU1 has nevertheless created significant challenges in respiratory diagnostics, as identification of which pathogens should be tested for is increasingly difficult. The recent discovery of two novel respiratory coronaviruses (HCoV-HKU1 and HCoV-NL63) presented the opportunity to undertake large scale clinical and epidemiologic study of these alongside two previously known respiratory coronaviruses, HCoV-229E and HCoV-OC43. Over 12,000 samples collected over three years were screened using a novel four-way multiplex real-time reverse transcription-PCR (RTPCR). Clinically, coronaviruses were similar to viruses currently included in routine diagnostics, with the exception of HCoV-229E which was identified as an opportunistic pathogen in immunocompromised hosts. Variability in detection frequencies of HCoVHKU1 and HCoV-OC43 was evident. The low detection frequencies of HCoVs, comparable to those of parainfluenza viruses 1 and 2 (which are included in the routine diagnostic screening panel) indicate a borderline case for inclusion of these pathogens in routine respiratory diagnostics. To investigate the epidemiology and clinical correlates of HMPV in Edinburgh, large scale retrospective screening of over 7000 respiratory samples collected over two years was conducted. Nucleotide sequencing of HMPV-positive samples was undertaken to determine phylogenetic relationships of circulating HMPV strains. HMPV comprises two genotypes, A and B. Comparisons of the clinical presentations of the two genotypes revealed little difference, with only the observation that sub-genotype B2 was more frequently associated with infection of immunocompromised patients. Detection frequencies and symptomatology associated with HMPV infections were comparable to respiratory viruses currently included in the routine diagnostic panel, mandating its inclusion in future diagnostic screening. A switch of the predominantly circulating genotype of HMPV was observed between respiratory seasons. This is a phenomenon more widely reported for the closely related respiratory syncytial virus (HRSV), which also comprises two circulating groups. To further investigate subtype (HRSV)/ genotype (HMPV) switching, evolutionary analyses of nucleotide sequence data generated from isolates collected from geographically disparate referral centres was undertaken. The fusion and attachment (G) genes were targeted, as these encode major surface proteins and are immunogenic. Analyses were by MCMC analyses using Bayesian Evolutionary Analyses of Sampling Trees (BEAST) software. Identification of positively selected sites was performed using Phylogenetic Analysis Maximum Likelihood (PAML). Switching of the predominantly circulating lineage does not arise for either virus due to emergence of novel strains, but through fluctuating circulation frequencies of pre-existing lineages which have been circulating for several decades, indicated by the time since the most recent common ancestor. Two HRSV-A lineages comprising genotypes undergoing turnover and replacement were identified. This finding is agreeable with serologic studies of the 1970s which reported three HRSV serogroups, two within HRSV-A and one within HRSV-B. HMPV and HRSV have similar mutation rates. Positively selected sites identified within the HRSV G gene were incongruent with those identified in a previous study, generating the hypothesis that immune evasion occurs within linear epitopes rather than at specific sites. A great deal of clinical and epidemiologic data was generated through this work, parallel studies of other respiratory viruses and through diagnostic screening results. To provide a robust indication of where resources should be diverted in terms of diagnostics, therapeutics and vaccine development, and to inform infection control measures and public health policy planning, quantification of the relative disease burden attributable to the most commonly detected respiratory viruses was calculated using the World Health Organization- endorsed Disability Adjusted Life Year (DALY) model. Relative disease burden was calculated in an age stratified manner to reflect the differences in sampling in different age groups. HRSV and influenza A were consistently one of the greatest causes of disease regardless of sampled population, although HRSV caused more disease in children under 5 than influenza A and B combined. Rhinoviruses and PIV-3 were significant pathogens in all groups except those aged 16-64 years; rhinoviruses were the leading cause of disease in the immunocompromised patient group. The potential for patient-specific diagnostic screening and guidance of interventions such as patient cohorting were clear.

Human metapneumovirus (HMPV) is a respiratory pathogen in the Paramyxoviridae family that infects nearly 100% of the world population. This enveloped RNA virus causes severe viral respiratory disease in infants, the elderly, and immunocompromised patients worldwide. Despite its prevalence and importance to human health, no therapies are available against this pathogen. Entry of paramyxoviruses into host cells generally requires the coordinated activity of the attachment glycoprotein, G, which interacts with a cell receptor, and the fusion glycoprotein, F, which promotes subsequent fusion of viral and cellular membranes. However, HMPV F is the primary viral protein mediating both binding and fusion for HMPV. Previous work that showed HMPV F mediates attachment to heparan sulfate proteoglycans (HSPGs), and some HMPV F fusion activity can be promoted by acidic pH. The work presented here provides significant advances in our understanding of the fusion and binding events during HMPV infection. We demonstrated that low pH promotes fusion in HMPV F proteins from diverse clades, challenging previously reported requirements and identifying a critical residue that enhances low pH promoted fusion. These results support our hypothesis that electrostatic interactions play a key role in HMPV F triggering and further elucidate the complexity of viral fusion proteins. Additionally, we characterized the key features of the binding interaction between HMPV and HSPGs using heparan sulfate mimetics, identifying an important sulfate modification, and demonstrated that these interactions occur at the apical surface of polarized airways tissues. We identified differences in particle binding related to the presence or absence of the HMPV G and SH glycoproteins. Lastly, we characterized paramyxovirus infection in cystic fibrosis bronchial epithelial cells, identifying a potential specific susceptibility to HMPV infection in these individuals. The work presented here contributes to our understanding of HMPV infection, from mechanisms of early events of entry to clinical scenarios.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O metapneumovÃrus humano (MPVh) à um vÃrus que tem se destacado como um dos agentes mais freqÃentes de infecÃÃes respiratÃrias agudas (IRA) virais na infÃncia. Este estudo teve como objetivos: observar a freqÃÃncia das infecÃÃes causadas pelo MPVh em crianÃas atendidas por IRA no Hospital Infantil Albert Sabin, hospital pediÃtrico de referÃncia do estado do CearÃ, no perÃodo de janeiro de 2006 a dezembro de 2007; descrever aspectos de sazonalidade dessas infecÃÃes relacionando-as com a ocorrÃncia de chuvas e a circulaÃÃo de outros vÃrus respiratÃrios; descrever as caracterÃsticas clÃnico-epidemiolÃgicas dos pacientes infectados pelo MPVh, comparando com os pacientes negativos e com os positivos para outros vÃrus e avaliar a tÃcnica de IFI como mÃtodo de diagnÃstico para a detecÃÃo do MPVh. Amostras de secreÃÃo de nasofaringe foram coletadas de crianÃas com sintomas de IRA e submetidas à tÃcnica de imunofluorescÃncia indireta para detecÃÃo dos seguintes vÃrus respiratÃrios: MPVh, vÃrus sincicial respiratÃrio (VSR), influenza A e B, adenovÃrus e parainfluenza 1, 2 e 3. Durante os 24 meses de estudo, foram colhidas amostras de 1276 crianÃas sendo detectado algum vÃrus respiratÃrio em 380 (29,78%) amostras. O MPVh foi o segundo vÃrus respiratÃrio mais encontrado representando um total de 8,69% de todas as amostras e de 29% dentre as amostras positivas para os vÃrus pesquisados. NÃo foi observado para o MPVh um padrÃo de sazonalidade nem correlaÃÃo com perÃodo chuvoso. A maioria dos pacientes positivos para MPVh foram atendidos na emergÃncia (89,2%). A mÃdia de idade dos pacientes positivos para o MPVh foi de 27 meses sendo significativamente superior que a das crianÃas infectadas pelo VSR (15 meses), adenovÃrus (14 meses) e vÃrus parainfluenza 3 (18 meses). Dentre os pacientes infectados pelo MPVh, 53,2% tiveram o diagnÃstico de infecÃÃes das vias aÃreas superiores e 46,7% tiveram o diagnÃstico de infecÃÃes das vias aÃreas inferiores. As infecÃÃes por MPVh apresentaram o mesmo espectro de infecÃÃes causadas pelos demais vÃrus pesquisados. O MPVh associou-se mais a casos de pneumonia que levaram à hospitalizaÃÃo das crianÃas infectadas do que outros vÃrus analisados. Mais da metade dos pacientes infectados pelo MPVh utilizaram o aerossol / salbutamol no seu tratamento (68,5%). A tÃcnica de IFI mostrou-se bastante eficaz como mÃtodo de diagnÃstico para a detecÃÃo do MPVh nesse estudo / The human metapneumovÃrus (hMPV) is a newly discovered virus that has been considered as one of the most common agents of acute respiratory infections (ARI) virus in childhood. The objectives of this study were: 1) to observe the frequency of infections caused by hMPV among children attending Hospital Infantil Albert Sabin, a major pediatric hospital in CearÃ, from January 2006 to December 2007; 2) to describe aspects of seasonality of these infections relating them to the occurrence of rain and the circulation of other respiratory viruses, 3) to describe the clinical and epidemiological characteristics of patients infected by hMPV, compared with positive and negative patients for other viruses; 4) to evaluate the IFI assay as a method of diagnosis for the detection of hMPV. Nasopharyngeal aspirates were collected from children with symptoms of ARI and submitted to indirect immunofluorescence assays for the detection of the following respiratory viruses: hMPV, respiratory syncytial virus (RSV), influenza A and B, adenovirus and parainfluenza 1, 2 and 3. During the 24 months of study, samples were collected from 1276 and respiratory viruses were demonstrated in 380 (29.78%) samples. The hMPV was the second most frequently detected respiratory viruses representing a total of 8.69% of all samples and 29% among the samples positive for the virus analyzed. It was not observed for hMPV a pattern of seasonality or correlation with the rainy season. Most patients positive for hMPV were attended in the emergence (89.2%). The mean age of patients infected by hMPV was 27 months, wich is significantly older than that for VSR (15 months), adenovirus (14 months) and parainfluenza virus 3 (18 months). Among patients infected by hMPV, 53.2% had a diagnosis of infections of the upper airways and 46.7% had a diagnosis of infections of the lower airways. The hMPV infections showed the same spectrum of infections caused by other viruses analyzed. The hMPV associated to more cases of pneumonia that led to the hospitalization of children infected than other viruses analyzed. More than half of these patients used the aerosol / salbutamol as conduct therapy (68.5%). The IFI assay proved to be quite effective as a method of diagnosis for the detection of hMPV in this study

IV. ABSTRACT The human metapneumovirus is a novel paramyxovirus associated with acute respiratory infections in children, adults, elderly and immunocompromised individuals. It has a worldwide distribution and the prevalence range between 1.5% to 25% in individuals with respiratory infections. Based on phylogenetic analysis 2 distinct genetic groups (A and B) that are sub-divided into four subgroups (A1, A2, B1 and B2) have been shown to circulate. Until recently, there was no information on the molecular epidemiology and the clinical characteristics of the hMPV in Africa, including South Africa, a region with a high prevalence of paediatric human immunodeficiency virus type-1 (HIV) infection. The molecular epidemiology and clinical characteristics of the hMPV in South Africa was investigated over a three period (2000-2002) in children hospitalized with lower respiratory tract infection. The children were part of a cohort participating in a phase 3 clinical trial investigating the efficacy of a 9-valent-pneumocococcal protein-polysaccharide conjugate vaccine (PCV). The objectives of the study were: i. to investigate the molecular epidemiology of hMPV in South Africa; ii. characterize the burden of hMPV disease and determine the clinical features of hMPV-LRTI in children infected and not infected by HIV; iii. probe the role of Streptococcus pneumoniae in the pathogenesis of hMPV-LRTI. The overall prevalence of hMPV in children hospitalized with lower respiratory tract infections (LRTI) was 7.4%. The mean age of children with hMPV associated LRTI (hMPV-LRTI) in South Africa was 13.3 months (range 1.4-49.2 months), with HIV infected children being older than children not infected with HIV (mean [range] 17.6 [4.5-44.3] vs. 12.3 [1.4-49.2] months; P=0.007). The incidence of hMPV-LRTI was 5.0 (95%C.I.3.3-7.5) fold greater in HIV infected children (incidence rate: 2 504 [95%C.I. 1 683-3 577] per 100 000) than in HIV uninfected children (incidence rate: 505 [95%C.I. 409-618] per 100 000, P<0.0001). Human metapneumovirus was identified less frequently than RSV but more commonly than other studied respiratory viruses. The double-blind PCV-9 vs. placebo controlled trial was used to probe the role of pneumococcal co-infections contributing to the pathogenesis of severe hMPV-LRTI. The incidence of hospitalization for hMPV-LRTI was reduced by 46% (95%, CI, 25-63; P=0.0002) in PCV-9 vaccinees compared to placebo recipients. This inferred that coinfection with Streptococcus pneumoniae was integral to the pathogenesis of hMPV-LRTI requiring hospitalization. Both groups of the hMPV circulated during the three year period including concurrent circulation of multiple subtypes of the virus. There was a transition from group B to group A subtype virus as the dominant circulating virus over sequential years. Sequence analysis of the two attachment glycoproteins (F and G), showed the F gene protein to be highly conserved, in contrast the attachment protein gene (G protein) was highly variable particularly in the extracellular domain between lineages. Repeat hMPV-LRTI by either homologous or heterologous strains within 3 months of each other suggested that natural infection did not confer complete immunity to hMPV. The present study demonstrated that hMPV is a leading pathogen associated with LRTI among children in Africa and indicated that occult pneumococcal co-infections’ were integral in the pathogenesis of hMPV-LRTI requiring hospitalization. Additionally, this is the first study to have characterized the molecular epidemiology of hMPV in Africa and provides insight as to issues that may exist regarding the design of an hMPV vaccine.

No description available.

A reação em cadeia por polimerase (PCR) e suas variantes tem sido, desde o isolamento do metapneumovírus humano (hMPV), a técnica mais utilizada para a detecção do vírus em secreções respiratórias de diferentes grupos de pacientes. Entretanto, a interpretação de estudos abordando aspectos epidemiológicos e patogenéticos da infecção pelo hMPV tem sido dificultada pelo uso de uma grande variedade de técnicas de PCR \"in house\" na ausência de uma técnica \"padrão ouro\" claramente definida. A avaliação da sensibilidade, especificidade e reprodutibilidade de qualquer técnica molecular \"in house\" é um passo crucial para podermos comparar estudos realizados por diferentes grupos de pesquisa e diferentes grupos de pacientes. Este estudo teve como objetivos a padronização de duas técnicas de PCR - convencional e em tempo real - para a detecção do hMPV em secreções respiratórias e a avaliação da concordância existente entre as técnicas. Entre 228 amostras de lavado de nasofaringe coletadas de receptores de transplante de células tronco hematopoiéticas com sintomas de infecção respiratória aguda, 10 (4,4%) foram positivas para a presença do hMPV pela técnica de PCR convencional enquanto que 11 (4,8%) foram positivas pela técnica de PCR em tempo real. A concordância entre as técnicas, medida pelo índice Kappa para um intervalo de confiança de 95%, foi de 0,95, ou seja, quase perfeita. / The polymerase chain reaction (PCR) has been, since the isolation of the virus in 2001, the most used technique for detection of human metapneumovirus (hMPV) in respiratory specimens of several groups of patients. However, the interpretation of studies regarding the epidemiology and pathogenesis of hMPV infection has been hindered by the use of a great variety of PCRs techniques for hMPV detection, in the absence of a clearly defined \"gold standard\". The assessment of the sensitivity, specificity and reproducibility of any in-house molecular technique is a crucial step to allow the comparison of studies conducted in different settings and different groups of patients. The aim of the present study was to standardize two in-house PCR assays a conventional PCR and a real-time PCR for detecting hMPV in nasopharyngeal aspirates and to evaluate the agreement between the two assays. Of 228 samples of nasopharyngeal aspirates obtained from hematopoietic stem cell transplant recipients with acute respiratory symptoms, 10 (4.4%) were positive for hMPV by conventional PCR whereas 11 (4.8%) were positive by real-Time PCR. The agreement of both assays, measured by Kappa Index, was almost perfect (0.95, 95% confidence interval).