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Servizio di Virologia, IRCCS Policlinico San Matteo
Istituto di Chimica Biologica, Universita di Pavia, 27100 Pavia, Italy
ABSTRACT
Monoclonal antibodies to human metapneumovirus (hMPV) were developed for direct fluorescent antibody (DFA) staining of nasopharyngeal aspirates from 40 infants with respiratory infections, as well as for hMPV identification in shell vial cultures. With reference to reverse transcription-PCR, DFA staining showed sensitivity, specificity, and positive and negative predictive values of 73.9%, 94.1%, 94.4%, and 72.7%, respectively. Monoclonal antibodies are useful for direct hMPV detection.
TEXT
The recently identified human metapneumovirus (hMPV) is the only member of the genus Metapneumovirus (family Paramyxoviridae, subfamily Paramyxovirinae), which also includes avian pneumoviruses A, B, C, and D, that infects humans. hMPV is responsible for a fair proportion of respiratory infections in early infancy and childhood, and also in the elderly and in immunocompromised hosts (9, 10). Diagnosis of hMPV infections is currently achieved by reverse transcription (RT)-PCR or, to a lesser extent, by virus isolation in cell cultures, followed by virus identification by RT-PCR (2, 4, 12).
Rapid immunological diagnosis of conventional respiratory virus infections is currently performed in many centers by direct fluorescent antibody (DFA) staining of respiratory cells present in nasopharyngeal aspirate (NPA) samples using virus-specific monoclonal antibodies (MAbs) (8). Therefore, MAbs with specific reactivity to hMPV are needed, although they are not yet commercially available. In this report, we describe the development of MAbs specific for hMPV and their use for DFA staining of NPA samples and virus identification in shell vial cell cultures.
Following sequencing and phylogenetic analysis of hMPV strains circulating in northern Italy in the 2001 through 2004 winter-spring seasons (4a), prototype strains belonging to each type (A and B) and subtype (A1-A2 and B1-B2) were isolated and propagated onto LLC-MK2 cell cultures. After 5 to 10 passages, hMPV strains were released from infected cultures showing 100% cytopathic effect 5 days postinfection at titers of 107 50% tissue culture infective doses/ml. Following clarification, hMPV prototypes A and B were pelleted by ultracentrifugation, reaching titers of 109 50% tissue culture infective doses/ml. These virus preparations were inoculated intramuscularly into BALB/c mice as follows: the first virus inoculum was in complete Freund's adjuvant and the second inoculum (after 3 weeks) in incomplete Freund's adjuvant, while the third (after 5 weeks) and the fourth (after 6 weeks) inocula were in saline. Following the fusion of a mouse spleen cell suspension with Sp2/0Ag14 myeloma cells, the hybridoma supernatants were tested for reactivity with hMPV by enzyme-linked immunosorbent assay and the indirect fluorescent antibody (IFA) assay. Reactive hybridomas were cloned and subcloned twice. Cross-reactivities with conventional respiratory viruses (influenza viruses A and B, parainfluenza virus types 1 to 4, human respiratory syncytial virus, human adenoviruses, human coronaviruses 229E and OC43, and rhinoviruses) were tested by IFA assay. Some clones showed a variable degree of cross-reactivity with human respiratory syncytial virus and were excluded from diagnostic use. Viral proteins reactive with different MAbs are under investigation.
hMPV-specific MAbs showed three major IFA patterns on hMPV-infected LLC-MK2 cell cultures, as shown in Fig. 1: (i) granular (Fig. 1A and B), (ii) filamentous (Fig. 1C and D), and (iii) foamy (Fig. 1E and F). A pool of three MAbs, including clones C2C10 (immunoglobulin G1 [IgG1]), C2D11 (IgG1), and T3H11 (IgG2a), each representative of a different staining pattern and reactive by both IFA and enzyme-linked immunosorbent assays with all four hMPV subtypes, was used for diagnostic purposes. No MAb was reactive with avian metapneumoviruses A and B (kindly provided by Ilaria Capua, Istituto Zooprofilattico delle Tre Venezie, Padua, Italy).
Forty NPA samples collected during the winter-spring season of 2003-2004 from 40 infants and young children admitted to the hospital because of an episode of acute respiratory infection were retrospectively tested for hMPV with MAbs by (i) using frozen smears of NPA samples for DFA staining and (ii) inoculating frozen NPA samples, previously tested for conventional respiratory viruses, onto shell vial cell cultures.
Cells isolated from respiratory secretions had been previously used for preparation of multiple smears, which were fixed with methanol-acetone and stored at –80°C. After thawing, the smears were stained with MAbs to hMPV and, in parallel, with a high-titer guinea pig hyperimmune serum. The pool of three MAbs was used for retrospective DFA staining of the 40 frozen NPA smears from respiratory secretions (Table 1). Specimens had been previously tested by RT-PCR for hMPV genes N and F and found to be either positive (n = 23; 18 type A and 5 type B strains) or negative (n = 17). On the whole, DFA staining detected as positive 17/23 NPAs found to be positive by RT-PCR (12 subtype A2, 2 subtype B1, and 3 subtype B2), while 6 subtype A2 strains were negative. In addition, DFA staining detected as negative 16/17 NPAs found to be negative by RT-PCR (one subtype A2 strain was positive by DFA staining in very few cells). In the six RT-PCR-positive DFA-negative samples, no correlation was observed between a weak PCR signal and lack of DFA signal, suggesting the presence of extracellular virus not detectable by DFA staining in the relevant NPAs.
Due to problems of nonspecific staining, in addition to these 6, guinea pig immune serum did not detect hMPV in 7 additional samples, thus detecting only 10/23 NPAs as positive. The IFA pattern shown by DFA staining in respiratory cells was granular, as exemplified by positive respiratory cells observed in two different NPAs (Fig. 2A and B). Six of the 17 RT-PCR-negative samples were positive for respiratory syncytial virus by both RT-PCR and DFA staining but were not reactive with hMPV MAbs. Using RT-PCR as a reference method, DFA staining showed a sensitivity of 73.9%, a specificity of 94.1%, a positive predictive value of 94.4%, and a negative predictive value of 72.7% (Table 1).
In addition, four original NPAs (among those examined by DFA staining) stored frozen at –80°C and previously found to be positive by RT-PCR were inoculated onto LLC-MK2 shell vial cell cultures and incubated for 48 h at 37°C. Following fixation with methanol-acetone and immunostaining with the same pool of MAbs to hMPV, the four hMPV strains could be easily identified, as shown in Fig. 2C and D. Virus strains were detected in cell cultures as either multiple single infected cells (Fig. 2C) or small plaques with small syncytial formations (Fig. 2D).
Development of MAbs to hMPV is an important advance in the field of rapid direct diagnosis of respiratory tract viral infections. Following the introduction of hybridoma technology, MAbs to known respiratory viruses were developed and made commercially available. Since then, DFA staining using MAbs has become the most rapid technique for direct diagnosis of acute respiratory infections, taking only 2 to 3 h to perform. Expertise in reading the results of IFA assays and good-quality smears of respiratory cells are the conditions required for reliable performance of the DFA assay. In parallel, molecular assays aimed at amplifying viral genomes directly in clinical samples have been developed and used as an alternative to DFA staining for rapid diagnosis of respiratory viral infections. Currently, DFA and molecular assays, such as RT-PCR, may be used either as alternatives or in combination for detection of respiratory viruses in NPAs (8).
The recent discovery of hMPV as a major respiratory pathogen of infants and young children has been made possible by means of RT-PCR. Studies thus far published have mostly been conducted using the molecular approach. The availability of hMPV-specific MAbs now opens the door to the routine use of DFA staining for hMPV detection in NPAs. In addition, the documented ability of the tested MAbs to react with all four hMPV subtypes (1, 3, 6, 11) proves the ability of these reagents to detect all known hMPV strains. Since avian metapneumoviruses belong to four different types (A through D) and type C is the closest to hMPV (5, 7), we cannot exclude the existence of other as-yet-unidentified types of hMPV strains. In this regard, we must recall that the primer pair originally proposed by the group that discovered hMPV for virus detection in clinical samples was unable to detect type B strains (8, 10).
In conclusion, the MAbs to hMPV tested in this study have shown wide reactivity with all known hMPV subtypes on both NPA smears and LLC-MK2 shell vial cell cultures inoculated with NPAs.
ACKNOWLEDGMENTS
This work was partially supported by the Ministero della Salute, Ricerca Finalizzata IRCCS Policlinico San Matteo (convenzione no. 118 and grant 89282/A).
We thank Daniela Sartori for preparing the manuscript and Daniele Lilleri for help in picture processing. We are also indebted to Massimo Fabbi for preparing guinea pig hyperimmune sera.
REFERENCES
Bastien, N., S. Normand, T. Taylor, D. Ward, C. Peret, G. Boivin, L. J. Anderson, and Y. Li. 2003. Sequence analysis of the N, P, M and F genes of Canadian human metapneumovirus strains. Virus Res. 93:51-62.
Boivin, G., Y. Abed, G. Pelletier, L. Ruel, D. Moisan, S. Cote, T. C. T. Peret, D. D. Erdman, and L. J. Anderson. 2002. 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. 186:1330-1334.
Boivin, G., I. Mackay, T. P. Sloots, S. Madhi, F. Freymuth, D. Wolf, Y. Shemer-Avni, H. Ludewick, G. C. Gray, and E. LeBlanc. 2004. Global genetic diversity of human metapneumovirus fusion gene. Emerg. Infect. Dis. 10:1154-1157.
Chan, P. K. S., J. S. Tam, C.-W. Lam, E. Chan, A. Wu, C.-K. Li, T. A. Buckley, K.-C. Ng, G. M. Joynt, F. W. T. Cheng, K.-F. To, N. Lee, D. S. C. Hui, J. L. K. Cheung, I. Chu, E. Liu, S. S. C. Chung, and J. J. Y. Sung. 2003. Human metapneumovirus detection in patients with severe acute respiratory syndrome. Emerg. Infect. Dis. 9:1058-1063.
Gerna, G., G. Campanini, F. Rouida, A. Sarasini, S. Paolucci, A. Marchi, F. Baldantin, and M. G. Revello. Changing circulation rate of human metapneuvirus strains and styles among hospitalized pediatric patients during three consecutive winter-spring seasons. Arch. Virol., in press.
Jacobs, J. A., M. K. Njenga, R. Alvarez, K. Mawditt, P. Britton, D. Cavanagh, and B. S. Seal. 2003. Subtype B avian metapneumovirus resembles subtype A more closely than subtype C or human metapneumovirus with respect to the phosphoprotein, and second matrix and small hydrophobic proteins. Virus Res. 92:171-178.
Mackay, I. M., S. Bialasiewicz, Z. Waliuzzaman, G. R. Chidlow, D. C. Fegredo, S. Laingam, P. Adamson, G. B. Harnet, W. Rawlinson, M. D. Nissen, and T. P. Sloots. 2004. Use of the P gene to genotype human metapneumovirus identifies 4 viral subtypes. J. Infect. Dis. 190:1913-1918.
Njenga, M. K., H. M. Lwamba, and B. S. Seal. 2003. Metapneumoviruses in birds and humans. Virus Res. 91:163-169.
Rovida, F., E. Percivalle, M. Zavattoni, M. Torsellini, A. Sarasini, G. Campanini, S. Paolucci, F. Baldanti, M. G. Revello, and G. Gerna. 2005. Monoclonal antibodies versus reverse transcription-PCR for detection of respiratory viruses in a patient population with respiratory tract infections admitted to hospital. J. Med. Virol. 75:336-347.
van den Hoogen, B. G., J. C. de Jong, J. Groen, T. Kuiken, R. deGroot, R. A. M. Fouchier, and A. D. M. E. Osterhaus. 2001. A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat. Med. 7:719-724.
van den Hoogen, B. G., G. J. J. van Doornum, J. C. Fockens, J. J. Cornelissen, W. E. P. Beyer, R. de Groot, A. D. M. E. Osterhaus, and R. A. M. Fouchier. 2003. Prevalence and clinical symptoms of human metapneumovirus infection in hospitalised patients. J. Infect. Dis. 188:1571-1577.
van den Hoogen, B. G., S. Herfst, L. Sprong, P. A. Cane, E. Forleo-Neto, R. L. de Swart, A. D. M. E. Osterhaus, and R. A. M. Fouchier. 2004. Antigenic and genetic variability of human metapneumoviruses. Emerg. Infect. Dis. 10:658-665.
Williams, J. V., P. A. Harris, S. J. Tollefson, L. L. Halburnt-Rush, J. M. Pingsterhaus, K. N. M. Edwards, P. F. Wright, and J. E. Crowe. 2004. Human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children. N. Engl. J. Med. 350:443-450.