- Email: [email protected]
New variant of the human metapneumovirus (HMPV) associated with an acute and severe exacerbation of asthma bronchiale
Journal of Clinical Virology 31 (2004) 283–288
New variant of the human metapneumovirus (HMPV) associated with an acute and severe exacerbation of asthma bronchiale Oliver Schildgena,∗,1 , Tilman Geikowskia,1 , Thomas Glatzela , Arne Simonb , Anja Wilkesmannb , Michael Roggendorfc , Sergei Viazovc , Bertfried Matza a
Institute for Medical Microbiology and Immunology, University Hospital Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany b Department of Pediatrics, University Hospital Bonn, Adenauer Allee, 53105 Bonn, Germany c Institute of Virology, University Hospital Essen, Hufelandstr. 55, D-45122 Essen, Germany Received in revised form 1 June 2004; accepted 9 June 2004
Abstract Recently, an increasing number of studies demonstrated that the human metapneumovirus (HMPV) causes mild to severe respiratory infections in children and immunosuppressed adults, and may be a frequent but somewhat undervalued pathogen. Here, we report the detection of a new variant of HMPV that is not closely related to the HMPV strains described until now. The strain was detected in a 6.5-year-old girl with an acute and severe exacerbation of asthma bronchiale triggered by an infection with a newly detected HMPV variant. The presented data provide new information on genetic heterogeneity of HMPV and necessitate an optimization of diagnostic procedures for the detection of HMPV infection. © 2004 Elsevier B.V. All rights reserved. Keywords: HMPV; Asthma bronchiale; New variant Bonn-01
1. Introduction The human metapneumovirus (HMPV) was newly discovered in 2001 (van den Hoogen et al., 2001). It is distantly related to respiratory syncytial virus (RSV), and together with avian pneumoviruses (APV) serotypes A, B, C, and D, forms the Metapneumovirus genus of the Pneumovirinae subfamily of the Paramyxoviridae. HMPV is a worldwide distributed pathogen (Cuevas et al., 2003; Ebihara et al., 2004; Galiano et al., 2004; Madhi et al., 2003; Maggi et al., 2003; Nissen et al., 2002; Peret et al., 2002; van den Hoogen et al., 2001; Viazov et al., 2003; Wolf et al., 2003). In young children and occasionally in adults, this virus causes a mild ∗ 1
Corresponding author. Tel.: +49 228 287 4467; fax: +49 228 287 4433. E-mail address: [email protected] (O. Schildgen). These authors contributed equally to this work.
1386-6532/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2004.06.007
to severe acute infection of the nasopharyngeal tract with manifestations like severe cough, bronchiolitis, and pneumonia (reviewed by van den Hoogen et al., 2004). So far, four distinct genetic groups of HMPV have been reported. However, the existence of other variants of the virus cannot be excluded and, therefore, remains a matter of further investigation. While studying the prevalence of HMPV in young children with infections of the upper respiratory tract (from one region in Germany), we were able to identify a considerable number of clinical samples positive for HMPV RNA (manuscript in preparation). By nucleotide sequencing and phylogenetic analysis all but one of the obtained virus isolates could be assigned to one of the four known major genetic lineages of HMPV (Bastien et al., 2003; Biacchesi et al., 2003; Peret et al., 2002; Viazov et al., 2003). In the course of this study, we have detected a potentially new HMPV variant in a
284
O. Schildgen et al. / Journal of Clinical Virology 31 (2004) 283–288
young patient with an acute asthma bronchiale that was presumably initially triggered by the respiratory infection with HMPV. Here, we report on this patient and the newly identified HMPV isolate.
2. Materials and methods 2.1. Specimen A nasopharyngeal aspirate (Bonn-01) was collected to confirm the suspected diagnosis of respiratory syncytial virus infection immediately after the patient was admitted to the emergency department of our Children’s University Hospital with an acute exacerbation of her previously diagnosed asthma bronchiale. One portion of the sample was frozen immediately at −20 ◦ C, another part was mixed with an equal volume of RNAlater buffer (Qiagen, Hilden) and frozen at −20 ◦ C, and the residual portion was used directly for isolation of viral RNA. Other specimens (Bonn 02–Bonn 12) were collected during the winter season 2002/3 and were initially tested for markers of respiratory syncytial virus and influenza virus infections due to the clinical symptoms of the patients at the time point of hospitalization. Specimen was divided into aliquots as described for the sample Bonn-01 and either stored at −20 ◦ C or used immediately for virus isolation and RTPCR.
2.2. RT-PCR for HMPV RNA RNA was extracted from nasopharyngeal aspirates with the RNeasy kit (Qiagen, Hilden), reverse transcribed and amplified in a nested PCR using the primer set described by Viazov et al. (2003) using the RT-PCR kit (Qiagen, Hilden) for the reverse transcription and the first PCR round and the Expand High Fidelity System (Roche, Mannheim) for the second round. Primer 750as, 5 -TGCTTTGCTGCCTGTAGATGATGAG was used to generate the cDNA. The cDNA product was subjected to a first round of 30-cycle PCR with primers 111s (5 -AGAGTCTCAGTACACAATMAAAAGAG) and 750as. The second round of 30-cycle PCR was run with primers 114s (5 -AGAGTCTCAGTACACAATMAAAAGRGATG) and 442as (5 -GCCATTGTTTTYCTTGCYTC). Each cycle included denaturation at 94 ◦ C for 1 min, annealing at 57 ◦ C for 1 min, and extension at 72 ◦ C for 2 min. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. 2.3. Detection of RSV and influenza virus infections Infection caused by RSV and influenza viruses were assayed by enzyme immunoassay (RS-Directigen, BD), and whenever the samples were fresh, by inoculation of Vero, LLC-MK2, MS, human embryonic fibroblasts (HEF), and MDCK cells.
Fig. 1. Chest X-ray of the 6.5-year-old girl with acute asthma and HMPV-infection showed hyperinflation and slight perihilar infiltrates (bronchitis/ peribronchitis).
O. Schildgen et al. / Journal of Clinical Virology 31 (2004) 283–288
2.4. Sequencing and phylogenetic analysis The amplified DNAs were gel purified by the QIAquick Gel Extraction kit (Qiagen), cloned in pTOPO TA cloning vector (Invitrogen), and subjected to sequencing in both directions (BigDye Terminator DNA sequencing kit, Perkin-Elmer). Newly obtained sequences were compared with HMPV sequences taken from the GenBank by means
285
of the phylogenetic analysis using programs DNADIST and NEIGHBOR from the package PHYLIP, version 3.5c (Felsenstein, J. 1993, Department of Genetics, University of Washington, Seattle, USA). The evaluation of the significance of the grouping was assessed by the bootstrap analysis (100 replicates) using the program SEQBOOT. The obtained trees were prepared using the program Treeview. Accession numbers of sequences used for phylogenetic tree analysis and
Fig. 2. Alignment of 302 nucleotide sequences of isolate Bonn-01 with representatives of all four known genetic lineages of HMPV and avian pneumovirus serotype C (APV-C).
286
O. Schildgen et al. / Journal of Clinical Virology 31 (2004) 283–288
Fig. 3. Phylogenetic analysis of HMPV isolates. Comparison of sequences of 302 bp fragments of the N gene. (A) Unrooted tree with sequences of isolates representing all four known lineages of HMPV. (B) Rooted tree that includes nine additional sequences of HMPV isolates obtained in the current study in Bonn. Sequence of the APV-C was used to root the tree.
O. Schildgen et al. / Journal of Clinical Virology 31 (2004) 283–288
sequence alignments were: APV-C: #AF176590 and Nld001: #NC 004148.
3. Results and discussion The patient was a 6.5-year-old girl with a known history of asthma bronchiale. While being on budesonid inhalation tid treatment to control chronic symptoms, she experienced an acute exacerbation presumably from a viral respiratory infection. At the time of presentation, her rectal temperature was 39 ◦ C (102.2 ◦ F) in spite of the application of 250 mg of acetaminophen 30 min before. She was suffering from severe cough and acute tachydyspnea (50 breaths per minute) with wheezing, prolonged exspiration, subcostal retractions, and hypoxemia, confirmed with pulse oxymetry (oxygen saturation 89% while breathing ambient air). Her heart rate was 120 rhythmic beats per minute and her initial blood pressure was 73/46 mmHg indicating circulatory disturbance, probably due to a dehydration considering her sodium value of 141 mmol/L. A capillary blood gas analysis showed a normal pCO2 (38.7 mmHg) in spite of her actual profound hyperventilation. In addition, routine laboratory investigation revealed a leukocytosis (13.400/mm3 ) with 3% eosinophils, 14% band form, and 61% neutrophils. The C-reactive protein was 9.2 mg/L (<3 mg/L) and the immunoglobulin E fraction of her plasma was excessively elevated to 2760 IU/mL (<90 IU/mL). Her chest radiograph showed slight perihilar infiltrates in concordance with other cases of virally induced severe bronchitis/peribronchitis (Fig. 1) The clinical course of the illness was uneventful after rehydration, frequent additional inhalation with bronchodilators, and intravenous application of prednisolon. Oxygen had to be supplemented for 5 days. On day 7, after her admission, the patient continued her treatment as outpatient. From a clinical point of view, the course of the infection resembled an acute exacerbation of asthma caused by an infection with the human respiratory syncytial virus. Clinically, the symptoms were typical for an RSV infection. Based on the observation of the symptoms, a distinction between the infection described here and the typical RSV infection was impossible. The ELISA testing of nasopharyngial aspirate for RSV and influenza viruses A and B produced negative results. The aspirate was also used for infection of several cell lines permissive for respiratory viruses. Neither RSV nor influenza viruses were detected in infected cell cultures during the observation period of 4 weeks, and no HMPV-induced cytopathic effect was observed in any cell line. HMPV testing by RT-PCR was performed with both, fresh and frozen specimen of nasopharyngial aspirate. The HMPV RNA was detected in fresh aspirate and in material stored in combination with the RNAlater buffer (Qiagen) but not in aspirate samples kept at −20◦ . Several combinations of primers were used for RT-PCR in paral-
287
lel (2, 5, 14, 16) but positive PCR results were obtained only with a set of primers derived from the nucleocapsid (N) gene (Viazov et al., 2003). The amplified fragment was cloned in a plasmid vector and sequenced. Sequencing confirmed specificity of the PCR. Thus, the only agent that could cause the respiratory infection of the patient and exacerbation of asthma was HMPV. These results complement most recent observations that the HMPV infection, similar to infection caused by RSV, may trigger a chronic respiratory disease like asthma (Boivin et al., 2002; Greensill et al., 2003; Jartti et al., 2002; Maertzdorf et al., 2004; Rawlinson et al., 2003; van den Hoogen et al., 2003, 2004). The obtained viral isolate was subjected to further analysis. The predominant nucleotide sequence of the amplified fragment (302 nt) of new isolate (HMPV-B1) was compared with corresponding fragments of sequences of viral isolates belonging to all four known HMPV lineages available in the GenBank. Isolate HMPV Bonn-01 differed significantly from all these sequences – the observed similarity at nucleotide and amino acid levels varied within 68–78% and 57–65%, correspondingly. Fig. 2 demonstrates the alignment of nucleotide sequences of isolate Bonn-01 and representative of four major genetic groups of HMPV. Phylogenetic analysis of the nucleotide sequences demonstrated that the Bonn-01 strain was more closely related to one lineage of known HMPV isolates but its sequence differed significantly from other sequences that formed this phylogenetic group, including those isolated from other patients of the same hospital (Fig. 3A and B). Despite numerous attempts with a number of reported sets of primers derived from different genome regions of HMPV, we were unable to amplify any other fragment of viral genome of this new isolate. Due to the limited amount of material and the inability of the Bonn-01 strain to grow in cell culture, we were unable to continue our studies. Such a failure hampered our efforts to establish the exact evolutionary relatedness of the HMPV-B1 isolate to other HMPV strains belonging to four known lineages. Nevertheless, the identification of an unusual viral isolate suggests higher than recorded till now genetic heterogeneity of HMPV and necessitates further studies of this important issue. Studies of the heterogeneity of HMPV genome should also lead to an optimization of RT-PCR protocols used for diagnostic of HMPV infection. The application of these new diagnostic procedures will allow a more accurate estimate of the prevalence and incidence of HMPV infection in humans and may bring new light on clinical manifestations of these infections. These new diagnostic procedures moreover will reduce the number of suspicious RSV like infections, in which no pathogen could be found.
Acknowledgements Sergei Viazov was supported by a Grant from the IFORES program of the Medical Faculty of the University of Essen.
288
O. Schildgen et al. / Journal of Clinical Virology 31 (2004) 283–288
References Bastien N, Ward D, Van Caeseele P, Brandt K, Lee SH, McNabb G, Klisko B, Chan E, Li Y. Human metapneumovirus infection in the Canadian population. J Clin Microbiol 2003;41:4642–6. Biacchesi S, Skiadopoulos MH, Boivin G, Hanson CT, Murphy BR, Collins PL, Buchholz UJ. Genetic diversity between human metapneumovirus subgroups. Virology 2003;315:1–9. Boivin G, Abed Y, Pelletier G, Ruel L, Moisan D, Cote S, Peret TC, Erdman DD, Anderson LJ. 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. Cuevas LE, Nasser AM, Dove W, Gurgel RQ, Greensill J, Hart CA. Human metapneumovirus and respiratory syncytial virus, Brazil. Emerg Infect Dis 2003;9:1626–8. Ebihara T, Endo R, Kikuta H, Ishiguro N, Ishiko H, Hara M, Takahashi Y, Kobayashi K. Human metapneumovirus infection in Japanese children. J Clin Microbiol 2004;42:126–32. Galiano M, Videla C, Puch SS, Martinez A, Echavarria M, Carballal G. Evidence of human metapneumovirus in children in Argentina. J Med Virol 2004;72:299–303. Greensill J, McNamara PS, Dove W, Flanagan B, Smyth RL, Hart CA. Human metapneumovirus in severe respiratory syncytial virus bronchiolitis. Emerg Infect Dis 2003;9:372–5. Jartti T, Van den Hoogen B, Garofalo RP, Osterhaus AD, Ruuskanen O. Metapneumovirus and acute wheezing in children. Lancet 2002;360:1393–4. Madhi SA, Ludewick H, Abed Y, Klugman KP, Boivin G. Human metapneumovirus-associated lower respiratory tract infections among hospitalized human immunodeficiency virus type 1 (HIV1)-infected and HIV-1-uninfected African infants. Clin Infect Dis 2003;37:1705–10. Maertzdorf J, Wang CK, Brown JB, Quinto JD, Chu M, de Graaf M, van den Hoogen BG, Spaete R, Osterhaus AD, Fouchier RA. Realtime reverse transcriptase PCR assay for detection of human metap-
neumoviruses from all known genetic lineages. J Clin Microbiol 2004;42:981–6. Maggi F, Pifferi M, Vatteroni M, Fornai C, Tempestini E, Anzilotti S, Lanini L, Andreoli E, Ragazzo V, Pistello M, Specter S, Bendinelli M. Human metapneumovirus associated with respiratory tract infections in a 3-year study of nasal swabs from infants in Italy. J Clin Microbiol 2003;41:2987–91. Nissen MD, Siebert DJ, Mackay IM, Sloots TP, Withers SJ. Evidence of human metapneumovirus in Australian children. Med J Aust 2002;176:188. Peret TC, Boivin G, Li Y, Couillard M, Humphrey C, Osterhaus AD, Erdman DD, Anderson LJ. Characterization of human metapneumoviruses isolated from patients in North America. J Infect Dis 2002;185:1660–3. Rawlinson WD, Waliuzzaman Z, Carter IW, Belessis YC, Gilbert KM, Morton JR. Asthma exacerbations in children associated with rhinovirus but not human metapneumovirus infection. J Infect Dis 2003;187:1314–8. van den Hoogen BG, de Jong JC, Groen J, Kuiken T, de Groot R, Fouchier RA, Osterhaus AD. A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat Med 2001;7:719–24. van den Hoogen BG, Osterhaus DM, Fouchier RA. Clinical impact and diagnosis of human metapneumovirus infection. Pediatr Infect Dis J 2004;23:S25–32. van den Hoogen BG, van Doornum GJ, Fockens JC, Cornelissen JJ, Beyer WE, de Groot R, Osterhaus AD, Fouchier RA. Prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients. J Infect Dis 2003;188:1571–7. Viazov S, Ratjen F, Scheidhauer R, Fiedler M, Roggendorf M. High prevalence of human metapneumovirus infection in young children and genetic heterogeneity of the viral isolates. J Clin Microbiol 2003;41:3043–5. Wolf DG, Zakay-Rones Z, Fadeela A, Greenberg D, Dagan R. High seroprevalence of human metapneumovirus among young children in Israel. J Infect Dis 2003;188:1865–7.
New variant of the human metapneumovirus (HMPV) associated with an acute and severe exacerbation of asthma bronchiale Oliver Schildgena,∗,1 , Tilman Geikowskia,1 , Thomas Glatzela , Arne Simonb , Anja Wilkesmannb , Michael Roggendorfc , Sergei Viazovc , Bertfried Matza a
Institute for Medical Microbiology and Immunology, University Hospital Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany b Department of Pediatrics, University Hospital Bonn, Adenauer Allee, 53105 Bonn, Germany c Institute of Virology, University Hospital Essen, Hufelandstr. 55, D-45122 Essen, Germany Received in revised form 1 June 2004; accepted 9 June 2004
Abstract Recently, an increasing number of studies demonstrated that the human metapneumovirus (HMPV) causes mild to severe respiratory infections in children and immunosuppressed adults, and may be a frequent but somewhat undervalued pathogen. Here, we report the detection of a new variant of HMPV that is not closely related to the HMPV strains described until now. The strain was detected in a 6.5-year-old girl with an acute and severe exacerbation of asthma bronchiale triggered by an infection with a newly detected HMPV variant. The presented data provide new information on genetic heterogeneity of HMPV and necessitate an optimization of diagnostic procedures for the detection of HMPV infection. © 2004 Elsevier B.V. All rights reserved. Keywords: HMPV; Asthma bronchiale; New variant Bonn-01
1. Introduction The human metapneumovirus (HMPV) was newly discovered in 2001 (van den Hoogen et al., 2001). It is distantly related to respiratory syncytial virus (RSV), and together with avian pneumoviruses (APV) serotypes A, B, C, and D, forms the Metapneumovirus genus of the Pneumovirinae subfamily of the Paramyxoviridae. HMPV is a worldwide distributed pathogen (Cuevas et al., 2003; Ebihara et al., 2004; Galiano et al., 2004; Madhi et al., 2003; Maggi et al., 2003; Nissen et al., 2002; Peret et al., 2002; van den Hoogen et al., 2001; Viazov et al., 2003; Wolf et al., 2003). In young children and occasionally in adults, this virus causes a mild ∗ 1
Corresponding author. Tel.: +49 228 287 4467; fax: +49 228 287 4433. E-mail address: [email protected] (O. Schildgen). These authors contributed equally to this work.
1386-6532/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2004.06.007
to severe acute infection of the nasopharyngeal tract with manifestations like severe cough, bronchiolitis, and pneumonia (reviewed by van den Hoogen et al., 2004). So far, four distinct genetic groups of HMPV have been reported. However, the existence of other variants of the virus cannot be excluded and, therefore, remains a matter of further investigation. While studying the prevalence of HMPV in young children with infections of the upper respiratory tract (from one region in Germany), we were able to identify a considerable number of clinical samples positive for HMPV RNA (manuscript in preparation). By nucleotide sequencing and phylogenetic analysis all but one of the obtained virus isolates could be assigned to one of the four known major genetic lineages of HMPV (Bastien et al., 2003; Biacchesi et al., 2003; Peret et al., 2002; Viazov et al., 2003). In the course of this study, we have detected a potentially new HMPV variant in a
284
O. Schildgen et al. / Journal of Clinical Virology 31 (2004) 283–288
young patient with an acute asthma bronchiale that was presumably initially triggered by the respiratory infection with HMPV. Here, we report on this patient and the newly identified HMPV isolate.
2. Materials and methods 2.1. Specimen A nasopharyngeal aspirate (Bonn-01) was collected to confirm the suspected diagnosis of respiratory syncytial virus infection immediately after the patient was admitted to the emergency department of our Children’s University Hospital with an acute exacerbation of her previously diagnosed asthma bronchiale. One portion of the sample was frozen immediately at −20 ◦ C, another part was mixed with an equal volume of RNAlater buffer (Qiagen, Hilden) and frozen at −20 ◦ C, and the residual portion was used directly for isolation of viral RNA. Other specimens (Bonn 02–Bonn 12) were collected during the winter season 2002/3 and were initially tested for markers of respiratory syncytial virus and influenza virus infections due to the clinical symptoms of the patients at the time point of hospitalization. Specimen was divided into aliquots as described for the sample Bonn-01 and either stored at −20 ◦ C or used immediately for virus isolation and RTPCR.
2.2. RT-PCR for HMPV RNA RNA was extracted from nasopharyngeal aspirates with the RNeasy kit (Qiagen, Hilden), reverse transcribed and amplified in a nested PCR using the primer set described by Viazov et al. (2003) using the RT-PCR kit (Qiagen, Hilden) for the reverse transcription and the first PCR round and the Expand High Fidelity System (Roche, Mannheim) for the second round. Primer 750as, 5 -TGCTTTGCTGCCTGTAGATGATGAG was used to generate the cDNA. The cDNA product was subjected to a first round of 30-cycle PCR with primers 111s (5 -AGAGTCTCAGTACACAATMAAAAGAG) and 750as. The second round of 30-cycle PCR was run with primers 114s (5 -AGAGTCTCAGTACACAATMAAAAGRGATG) and 442as (5 -GCCATTGTTTTYCTTGCYTC). Each cycle included denaturation at 94 ◦ C for 1 min, annealing at 57 ◦ C for 1 min, and extension at 72 ◦ C for 2 min. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. 2.3. Detection of RSV and influenza virus infections Infection caused by RSV and influenza viruses were assayed by enzyme immunoassay (RS-Directigen, BD), and whenever the samples were fresh, by inoculation of Vero, LLC-MK2, MS, human embryonic fibroblasts (HEF), and MDCK cells.
Fig. 1. Chest X-ray of the 6.5-year-old girl with acute asthma and HMPV-infection showed hyperinflation and slight perihilar infiltrates (bronchitis/ peribronchitis).
O. Schildgen et al. / Journal of Clinical Virology 31 (2004) 283–288
2.4. Sequencing and phylogenetic analysis The amplified DNAs were gel purified by the QIAquick Gel Extraction kit (Qiagen), cloned in pTOPO TA cloning vector (Invitrogen), and subjected to sequencing in both directions (BigDye Terminator DNA sequencing kit, Perkin-Elmer). Newly obtained sequences were compared with HMPV sequences taken from the GenBank by means
285
of the phylogenetic analysis using programs DNADIST and NEIGHBOR from the package PHYLIP, version 3.5c (Felsenstein, J. 1993, Department of Genetics, University of Washington, Seattle, USA). The evaluation of the significance of the grouping was assessed by the bootstrap analysis (100 replicates) using the program SEQBOOT. The obtained trees were prepared using the program Treeview. Accession numbers of sequences used for phylogenetic tree analysis and
Fig. 2. Alignment of 302 nucleotide sequences of isolate Bonn-01 with representatives of all four known genetic lineages of HMPV and avian pneumovirus serotype C (APV-C).
286
O. Schildgen et al. / Journal of Clinical Virology 31 (2004) 283–288
Fig. 3. Phylogenetic analysis of HMPV isolates. Comparison of sequences of 302 bp fragments of the N gene. (A) Unrooted tree with sequences of isolates representing all four known lineages of HMPV. (B) Rooted tree that includes nine additional sequences of HMPV isolates obtained in the current study in Bonn. Sequence of the APV-C was used to root the tree.
O. Schildgen et al. / Journal of Clinical Virology 31 (2004) 283–288
sequence alignments were: APV-C: #AF176590 and Nld001: #NC 004148.
3. Results and discussion The patient was a 6.5-year-old girl with a known history of asthma bronchiale. While being on budesonid inhalation tid treatment to control chronic symptoms, she experienced an acute exacerbation presumably from a viral respiratory infection. At the time of presentation, her rectal temperature was 39 ◦ C (102.2 ◦ F) in spite of the application of 250 mg of acetaminophen 30 min before. She was suffering from severe cough and acute tachydyspnea (50 breaths per minute) with wheezing, prolonged exspiration, subcostal retractions, and hypoxemia, confirmed with pulse oxymetry (oxygen saturation 89% while breathing ambient air). Her heart rate was 120 rhythmic beats per minute and her initial blood pressure was 73/46 mmHg indicating circulatory disturbance, probably due to a dehydration considering her sodium value of 141 mmol/L. A capillary blood gas analysis showed a normal pCO2 (38.7 mmHg) in spite of her actual profound hyperventilation. In addition, routine laboratory investigation revealed a leukocytosis (13.400/mm3 ) with 3% eosinophils, 14% band form, and 61% neutrophils. The C-reactive protein was 9.2 mg/L (<3 mg/L) and the immunoglobulin E fraction of her plasma was excessively elevated to 2760 IU/mL (<90 IU/mL). Her chest radiograph showed slight perihilar infiltrates in concordance with other cases of virally induced severe bronchitis/peribronchitis (Fig. 1) The clinical course of the illness was uneventful after rehydration, frequent additional inhalation with bronchodilators, and intravenous application of prednisolon. Oxygen had to be supplemented for 5 days. On day 7, after her admission, the patient continued her treatment as outpatient. From a clinical point of view, the course of the infection resembled an acute exacerbation of asthma caused by an infection with the human respiratory syncytial virus. Clinically, the symptoms were typical for an RSV infection. Based on the observation of the symptoms, a distinction between the infection described here and the typical RSV infection was impossible. The ELISA testing of nasopharyngial aspirate for RSV and influenza viruses A and B produced negative results. The aspirate was also used for infection of several cell lines permissive for respiratory viruses. Neither RSV nor influenza viruses were detected in infected cell cultures during the observation period of 4 weeks, and no HMPV-induced cytopathic effect was observed in any cell line. HMPV testing by RT-PCR was performed with both, fresh and frozen specimen of nasopharyngial aspirate. The HMPV RNA was detected in fresh aspirate and in material stored in combination with the RNAlater buffer (Qiagen) but not in aspirate samples kept at −20◦ . Several combinations of primers were used for RT-PCR in paral-
287
lel (2, 5, 14, 16) but positive PCR results were obtained only with a set of primers derived from the nucleocapsid (N) gene (Viazov et al., 2003). The amplified fragment was cloned in a plasmid vector and sequenced. Sequencing confirmed specificity of the PCR. Thus, the only agent that could cause the respiratory infection of the patient and exacerbation of asthma was HMPV. These results complement most recent observations that the HMPV infection, similar to infection caused by RSV, may trigger a chronic respiratory disease like asthma (Boivin et al., 2002; Greensill et al., 2003; Jartti et al., 2002; Maertzdorf et al., 2004; Rawlinson et al., 2003; van den Hoogen et al., 2003, 2004). The obtained viral isolate was subjected to further analysis. The predominant nucleotide sequence of the amplified fragment (302 nt) of new isolate (HMPV-B1) was compared with corresponding fragments of sequences of viral isolates belonging to all four known HMPV lineages available in the GenBank. Isolate HMPV Bonn-01 differed significantly from all these sequences – the observed similarity at nucleotide and amino acid levels varied within 68–78% and 57–65%, correspondingly. Fig. 2 demonstrates the alignment of nucleotide sequences of isolate Bonn-01 and representative of four major genetic groups of HMPV. Phylogenetic analysis of the nucleotide sequences demonstrated that the Bonn-01 strain was more closely related to one lineage of known HMPV isolates but its sequence differed significantly from other sequences that formed this phylogenetic group, including those isolated from other patients of the same hospital (Fig. 3A and B). Despite numerous attempts with a number of reported sets of primers derived from different genome regions of HMPV, we were unable to amplify any other fragment of viral genome of this new isolate. Due to the limited amount of material and the inability of the Bonn-01 strain to grow in cell culture, we were unable to continue our studies. Such a failure hampered our efforts to establish the exact evolutionary relatedness of the HMPV-B1 isolate to other HMPV strains belonging to four known lineages. Nevertheless, the identification of an unusual viral isolate suggests higher than recorded till now genetic heterogeneity of HMPV and necessitates further studies of this important issue. Studies of the heterogeneity of HMPV genome should also lead to an optimization of RT-PCR protocols used for diagnostic of HMPV infection. The application of these new diagnostic procedures will allow a more accurate estimate of the prevalence and incidence of HMPV infection in humans and may bring new light on clinical manifestations of these infections. These new diagnostic procedures moreover will reduce the number of suspicious RSV like infections, in which no pathogen could be found.
Acknowledgements Sergei Viazov was supported by a Grant from the IFORES program of the Medical Faculty of the University of Essen.
288
O. Schildgen et al. / Journal of Clinical Virology 31 (2004) 283–288
References Bastien N, Ward D, Van Caeseele P, Brandt K, Lee SH, McNabb G, Klisko B, Chan E, Li Y. Human metapneumovirus infection in the Canadian population. J Clin Microbiol 2003;41:4642–6. Biacchesi S, Skiadopoulos MH, Boivin G, Hanson CT, Murphy BR, Collins PL, Buchholz UJ. Genetic diversity between human metapneumovirus subgroups. Virology 2003;315:1–9. Boivin G, Abed Y, Pelletier G, Ruel L, Moisan D, Cote S, Peret TC, Erdman DD, Anderson LJ. 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. Cuevas LE, Nasser AM, Dove W, Gurgel RQ, Greensill J, Hart CA. Human metapneumovirus and respiratory syncytial virus, Brazil. Emerg Infect Dis 2003;9:1626–8. Ebihara T, Endo R, Kikuta H, Ishiguro N, Ishiko H, Hara M, Takahashi Y, Kobayashi K. Human metapneumovirus infection in Japanese children. J Clin Microbiol 2004;42:126–32. Galiano M, Videla C, Puch SS, Martinez A, Echavarria M, Carballal G. Evidence of human metapneumovirus in children in Argentina. J Med Virol 2004;72:299–303. Greensill J, McNamara PS, Dove W, Flanagan B, Smyth RL, Hart CA. Human metapneumovirus in severe respiratory syncytial virus bronchiolitis. Emerg Infect Dis 2003;9:372–5. Jartti T, Van den Hoogen B, Garofalo RP, Osterhaus AD, Ruuskanen O. Metapneumovirus and acute wheezing in children. Lancet 2002;360:1393–4. Madhi SA, Ludewick H, Abed Y, Klugman KP, Boivin G. Human metapneumovirus-associated lower respiratory tract infections among hospitalized human immunodeficiency virus type 1 (HIV1)-infected and HIV-1-uninfected African infants. Clin Infect Dis 2003;37:1705–10. Maertzdorf J, Wang CK, Brown JB, Quinto JD, Chu M, de Graaf M, van den Hoogen BG, Spaete R, Osterhaus AD, Fouchier RA. Realtime reverse transcriptase PCR assay for detection of human metap-
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