- Email: [email protected]
Expeditious neutralization assay for human metapneumovirus based on a recombinant virus expressing Renilla luciferase
Journal of Clinical Virology 56 (2013) 31–36
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Expeditious neutralization assay for human metapneumovirus based on a recombinant virus expressing Renilla luciferase Min Zhou a , Yoshinori Kitagawa a , Mayu Yamaguchi a , Chika Uchiyama a , Masae Itoh b , Bin Gotoh a,∗ a b
Division of Microbiology and Infectious Diseases, Department of Pathology, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga 520-2192, Japan Genetics of Life, Faculty of Bio-Science, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829 Japan
a r t i c l e
i n f o
Article history: Received 24 August 2012 Received in revised form 25 September 2012 Accepted 28 September 2012 Keywords: Human metapneumovirus Neutralization assay Renilla luciferase
a b s t r a c t Background: Human metapneumovirus (HMPV) is a common cause of respiratory diseases in persons of all ages. Because of its slow replication and weak cytopathic effect in cultured cells, conventional neutralization assays for HMPV require around one week for completion. Objectives: The purpose of this study is to establish a rapid neutralization assay based on a recombinant virus expressing Renilla luciferase (Rluc). Study design: A recombinant HMPV expressing both Rluc and green fluorescent protein (GFP) was created by reverse genetics method. Two-fold serial dilutions of human 23 sera were made in a 96-well plate and incubated with 50 pfu/well of the recombinant virus at 4 ◦ C for 1 h. The mixtures were then transferred to LLC-MK2 cells in a 96-well plate, incubated for 2 h, and replaced with trypsin-free fresh media. After incubation at 32 ◦ C for 24 h, the cells were lysed and measured for Rluc activity. The neutralization titer was defined as the reciprocal of the highest serum dilution that resulted in 50% reduction of Rluc activity. Results: The novel assay could be completed within 24 h and eliminated the requirement of trypsin supporting multistep replication in cultured cells, as well as laborious processes including the plaque assay with immunostaining. Neutralization titers correlated well with those determined by a GFP-based assay previously developed. Conclusions: The neutralization assay based on Rluc activity is the fastest and the most straightforward of all previous assays, and may be available for high throughput screening of neutralizing antibodies. © 2012 Elsevier B.V. All rights reserved.
1. Background Human metapneumovirus (HMPV) is a common cause of acute respiratory tract diseases in persons of all ages.1,2 Severe HMPV diseases can be caused in immunocompromized hosts and children with underlying conditions such as asthma, chronic lung disease due to prematurity, or congenital heart disease.3 Clinical signs are indistinguishable from those caused by human respiratory syncytial virus (HRSV) in the same subfamily Pneumovirinae.4,5 One of risk factors for severe HRSV diseases in infants is thought
Abbreviations: HMPV, human metapneumovirus; HRSV, human respiratory syncytial virus; CPE, cytopathic effect; rHMPV, recombinant HMPV; GFP, green fluorescent protein; NA-GFP, neutralization assay based on GFP expression; mAb, monoclonal antibody; Rluc, Renilla luciferase; NA-Rluc, neutralization assay based on Rluc activity; DMEM, Dulbecco’s modified Eagle’s medium; nt, nucleotide; ORF, open reading frame; rHMPV-GFP, rHMPV expressing GFP; rHMPV-Rluc/GFP, rHMPV expressing Rluc plus GFP; CAG promoter, cytomegalovirus enhancer-chicken actin hybrid promoter; moi, multiplicity of infection. ∗ Corresponding author. Tel.: +81 77 548 2176; fax: +81 77 548 2176. E-mail address: [email protected] (B. Gotoh). 1386-6532/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2012.09.014
to be low titers of maternal antibodies.6 The HRSV diseases in infants born with high levels of maternal neutralizing antibodies against HRSV were milder and occurred at an older age than infants with lower antibody levels.7 It suggests potential importance of determining titers of neutralizing antibodies to assess risk of the severity, although there is no direct evidence for correlation between the HMPV-neutralizing antibody level and HMPV disease severity. In addition to such a clinical prospective assessment, clinical diagnosis, vaccine development, and fundamental research to monitor the humoral immune response to HMPV infections require a fast and simple neutralization assay for HMPV. Neutralizing antibodies specific to HMPV have been usually quantified by classical methods such as a plaque reduction assay8 or reduction in the 50% tissue culture infectious dose.2,9,10 These conventional neutralization assays require around one week for completion, because of slow HMPV growth and weak cytopathic effect (CPE) in cultured cells. Furthermore multistep replication of most HMPV strains but a few exceptions in cultured cells depends on the presence of trypsin added in the medium, since trypsin is required for the cleavage activation of the HMPV F protein, which
32
M. Zhou et al. / Journal of Clinical Virology 56 (2013) 31–36
Fig. 1. Construction of a plasmid expressing the antigenomic RNA of Jpn03-1 (A) and the genome constructs of rHMPV-GFP and rHMPV-Rluc/GFP (B). Four overlapping cDNA fragments were assembled to construct the complete antigenomic cDNA except for four and five nucleotide substitutions, which create NheI and SnaBI sites in the M-F and M2-SH intergenic regions, respectively (A). Le, 3 non-transcribed leader region; Tr, 5 non-transcribed trailer region; T7P, T7 promoter; ␦ ribo, hepatitis delta-ribozyme sequence; T7T, T7 terminator; GS, gene start signal; GE, gene end signal.
mediates fusion between viral envelope and cell membrane.11 The trypsin requirement as well as weak CPE of HMPV makes the plaque assay difficult, because trypsin added in the medium causes cells off the bottom of the plate when cells are long incubated. Thus, fixation and immunostaining are indispensible for the plaque assay for HMPV. The neutralization assay for HMPV was modified by using recombinant HMPV (rHMPV) expressing green fluorescent protein (GFP).12,13 Neutralizing titers could be determined as the reciprocal of the highest serum dilution that resulted in 50% reduction of GFP expression12 or the number of GFP-positive plaques,13 both of which could be monitored by an automated scanner. The neutralization assay based on GFP expression (NAGFP) eliminated the immunostaining procedures and shortened the time for completion. Another assay, a microneutralization assay, which had been used for the measurement of neutralizing antibodies to RSV, was adapted to HMPV.14 It was performed by adding suspended cells to an antibody-virus mixture followed by an enzyme immunoassay with monoclonal antibody (mAb) to the HMPV N protein, and the titer was defined on the basis of 50% reduction of color development. However, any assay so far developed still needed at least 3–5 days for completion.
2. Objectives The purpose of this study is to establish a more rapid and straightforward neutralization assay for HMPV based on Renilla luciferase (Rluc) activity (NA-Rluc) using a newly created rHMPV.
3. Study design 3.1. Cells and viruses LLC-MK2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 2 mM l-glutamine, penicillin (100 IU/ml), streptomycin (100 g/ml), and 10% fetal bovine serum.15,16 HMPV Jpn03-1 strain and rHMPVs described below were propagated in LLC-MK2 cells in the presence of 4 g/ml of N-acetyl trypsin.17 Trypsin was added every three days to a final concentration of 4 g/ml to promote multistep replication.18
3.2. Plasmids and recovery of rHMPVs NheI and SnaBI sites were introduced into the putative M-F and M2-SH intergenic regions of the HMPV Jpn03-1 genome by four and five nucleotide substitutions, respectively, to create insertion sites for exogenous genes (Fig. 1A). This antigenomic cDNA copy [nucleotide (nt) 12-13337] was cloned in vector pT7PdT-MCS by assembling four overlapping cDNA clones generated by RT-PCR with specific primers designed from the Jpn03-1 genome sequence (Genbank accession number AB503857). pT7PdT-MCS contains a T7 promoter with three G residues, nt 1–12 of the antigenomic cDNA, multicloning sites, the hepatitis delta virus ribozyme, and a T7 terminator. The multicloning sites served to accept the cloned fragments. Transcription cassettes, which include the GFP or Rluc open reading frame (ORF) that was franked on its upstream and downstream sides by the F gene start and M gene end motifs, respectively, were cloned into the NheI site in the M–F intergenic region as shown in Fig. 1B. rHMPVs expressing GFP alone (rHMPV-GFP) or Rluc plus GFP (rHMPV-Rluc/GFP) were recovered
M. Zhou et al. / Journal of Clinical Virology 56 (2013) 31–36
33
Fig. 2. Growth kinetics of rHMPV-GFP and rHMPV-Rluc/GFP. LLC-MK2 cells in 6-well plates were infected with rHMPV-GFP or rHMPV-Rluc/GFP at a moi of 0.01, and incubated at 32 ◦ C in the presence of trypsin. Virus titers of culture media were determined by the plaque assay (A) and cells were observed under a fluorescence microscope (B).
using BSR T7/5 cells constitutively expressing T7 polymerase as described previously.18,19 A plasmid that expresses F, G, or SH with 3xFLAG epitope tag was created by insertion of the PCRamplified cDNAs encoding each protein into multi-cloning sites of pCA7,20 which contains the cytomegalovirus enhancer-chicken actin hybrid (CAG) promoter.21 The FLAG tag was appended to the N-terminal end of G and SH and to the C-terminal end of F. Sequence fidelity of all constructs was confirmed by using a PRISM 3130xl genetic analyzer (Applied Biosystems). 3.3. Plaque assay Serial 10-fold dilutions of samples containing rHMPV-GFP or rHMPV-Rluc/GFP were prepared in triplicate. Each dilution (400 or 50 l/well) was transferred to a confluent monolayer of LLCMK2 cells in a 6 or 96-well plate, respectively. After incubation at 32 ◦ C for 1 h, cells were washed twice with serum-free DMEM, and overlaid with 2 ml/well (for a 6-well plate) or 100 l/well (for a 96-well plate) of 3% methylcellulose in opti-MEM with 4 g/ml of N-acetyl trypsin. After incubation at 32 ◦ C for 7 days, the number of plaques with GFP fluorescence was counted by scanning with LAS-4000 (Fujifilm). 3.4. Measurement of Rluc activity rHMPV-Rluc/GFP-infected cells in a 6 or 96-well plate were lysed in 500 or 40 l of Renilla luciferase assay lysis buffer (Promega). Ten microliters of cell lysates were measured for Rluc activity. Rluc activity was expressed as average of relative light units derived from three independent experiments with Renilla luciferase assay system (Promega) and luminescence-PSN AB2200 (ATTO) as described previously.17 3.5. Immunoprecipitation and immunoblot analysis Human serum no. 9, which showed the highest titer, was selected by the NA-GFP from 23 human serum samples of pediatric outpatients in our university hospital. Two control samples for the serum no. 9 were prepared by mixing with
Fig. 3. Detection of Rluc activity in cells infected with rHMPV-Rluc/GFP. (A) LLCMK2 cells in 6-well plates were infected with rHMPV-Rluc/GFP at a moi of 0.01, and then incubated at 32 ◦ C in the presence or absence of trypsin. (B) LLC-MK2 cells in a 96-well plate were infected with 0, 10, 30, 50, or 100 pfu/well of rHMPVRluc/GFP and incubated in the absence of trypsin. Rluc activity was measured at 24 h post-infection.
34
M. Zhou et al. / Journal of Clinical Virology 56 (2013) 31–36
Fig. 4. Neutralization assays based on Rluc activity or GFP expression. Rluc activity (A) or GFP expression (B) was determined on one or seven day post-inoculation, respectively. Relative Rluc activity was expressed as percent ratio to the Rluc activity of the control of virus alone without sera (A and B). The cell lysate containing FLAG-tagged F, G, or SH was subjected to immunoprecipitation (IP) with anti-FLAG mAb, serum no.9, protein G-treated, or glutathione-treated serum, followed by immunoblot analysis with anti-FLAG mAb (C and D).
sufficient amounts of protein G or glutathione sepharose beads (GE Healthcare Bio-Science) followed by incubation at room temperature for 1 h. After centrifugation, the supernatants were used as control protein G-treated and glutathione-treated sera. HEK293T cells were transfected with a plasmid expressing F-FLAG, FLAG-SH or FLAG-G using Fugene HD transfection reagent according to the manufacturer’s instructions. At 24 h post-transfection, cells were lysed in 400 l of a lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Triton X-100) supplemented with protease inhibitor cocktail (Roche). After centrifugation, lysates were mixed with the no. 9, protein G-treated, glutathionetreated serum, or anti-FLAG (M2) mAb (Sigma) together with protein G sepharose beads, and incubated at 4 ◦ C for 2 h. After washing five times with the lysis buffer, proteins were eluted from the beads by boiling with Laemmli sample buffer, and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot analysis with anti-FLAG mAb.17,22 3.6. NA-Rluc and NA-GFP All sera were heat-inactivated at 56 ◦ C for 30 min prior to the neutralization assay to eliminate serum complement. Two-fold serial dilutions of serum samples initially diluted 1:20 in FBS-free DMEM were prepared in triplicate in a 96-well plate. Each serum
dilution was mixed with an equal volume of rHMPV-Rluc/GFP (50 pfu/well) and incubated at 4 ◦ C for 1 h. The virus-serum mixtures were then transferred to confluent monolayers of LLC-MK2 cells in a 96-well plate. After incubation at 32 ◦ C for 2 h, the cells were washed twice with serum-free DMEM, overlaid with 100 l of serum-free DMEM containing no trypsin for the NARluc or 4 g/ml of N-acetyl trypsin for the NA-GFP, and incubated at 32 ◦ C. Rluc activity was determined on day 1 post-inoculation, while GFP expression was quantified by scanning with LAS-4000 on day 7 post-inoculation. Neutralizing titers in the NA-Rluc and NA-GFP were defined as the reciprocal of the highest dilution that resulted in 50% reduction of Rluc activity or GFP expression, respectively. 4. Results 4.1. Replication kinetics of the rHMPV-Rluc/GFP rHMPV-GFP and rHMPV-Rluc/GFP, which express GFP alone and Rluc plus GFP, respectively, were created by reverse genetics method as described previously.18,19 rHMPV-Rluc/GFP was compared with rHMPV-GFP in multistep virus growth in LLCMK2 cells, since rHMPV-GFP was shown to be almost similar to wild-type HMPV in the efficiency of multistep replication in LLCMK2 cells.18 Titers were determined by the plaque assay without
M. Zhou et al. / Journal of Clinical Virology 56 (2013) 31–36
35
immunostaining. As shown in Fig. 2A, rHMPV-Rluc/GFP replicated with an efficiency that was similar to that of rHMPV-GFP. In accordance with the virus replication kinetics, GFP-positive cells spread with an almost similar speed (Fig. 2B). Thus the insertion of the additional Rluc gene into the HMPV genome had no significant effect on virus replication in cultured cells. 4.2. Time course of Rluc activity after rHMPV-Rluc/GFP infection To set the minimum time required for completing the NA-Rluc, we examined time course of Rluc activity expressed in cells after rHMPV-Rluc/GFP infection (Fig. 3A). LLC-MK2 cells (∼106 cells) were infected with rHMPV-Rluc/GFP at a multiplicity of infection (moi) of 0.01, and incubated in the presence or absence of trypsin. Exponential increase was observed in the presence of trypsin on day 1 to day 3 post-infection, whereas the increase was minimal in the absence of trypsin. The Rluc activities on day 1 post-infection under both conditions showed almost the same value (∼1 × 106 ), suggesting the multistep replication had not developed before day 1 post-infection. These results raise the possibility that the NA-Rluc is completed within one day without trypsin. The smaller is the number of input virions, the higher is the sensitivity of the neutralization assay. Thus we sought to determine the minimum number of input virions required for detection of Rluc activity on day 1 postinfection. As shown in Fig. 3B, Rluc activity was detectable even with 10 pfu/well of input virions. This indicates that around ten infected-cells are sufficient for detection of Rluc activity. 4.3. The inhibitory curve in the NA-Rluc and NA-GFP Initially we selected a serum (no. 9) exhibiting the highest HMPV-neutralizing titer in the previous assay, NA-GFP,12 out of 23 sera of pediatric outpatients picked at random in our university hospital. The serum no. 9 was treated with protein G or glutathione sepharose beads, as described in study design, to prepare control sera. The protein G-treated serum was expected to lose the IgG fraction. These control sera as well as the original serum were serially diluted, mixed with an equal volume of rHMPV-Rluc/GFP (50 pfu/well), and incubated at 4 ◦ C for 1 h. The mixtures were then transferred to LLC-MK2 cells grown in 96-well plates, incubated in the absence of trypsin, and Rluc activity was determined at 24 h post-inoculation (Fig. 4A). The no. 9 and glutathione-treated sera showed a similar inhibitory curve, whereas the protein G-treated serum exhibited no inhibitory effect. Similar results were obtained in the NA-GFP (Fig. 4B). In the NA-GFP, cells were incubated in the presence of trypsin, and the GFP expression was determined on day 7 post-inoculation. To investigate whether the serum no. 9 contained antibodies against HMPV, immunoprecipitation was carried out using cell extracts containing FLAG-tagged envelope proteins (F, G, and SH) (Fig. 4C and D). The serum no. 9 was found to recognize F, but little G or SH (Fig. 4C). Whereas the glutathione-treated serum retained the ability to recognize F, the protein G-treated serum lost it (Fig. 4D). These results suggest that the neutralizing activity in both assays is largely attributable to IgG antibodies against F rather than non-specific inhibitory factors. 4.4. Correlation between neutralizing titers determined by the NA-Rluc and NA-GFP Neutralizing titers of the 23 human sera were determined by the NA-Rluc and NA-GFP. Titers were defined as the reciprocal of the highest serum dilution that resulted in 50% reduction of Rluc activity or GFP expression (Fig. 4A and B). When the samples were scored as positive (more than 1:20) or negative (less than 1:20), there was a 100% correlation between the results from the NA-Rluc and NA-GFP (Fig. 5A). Correlation of the neutralization titers for
Fig. 5. Correlation of the neutralizing titers of 23 serum samples determined by the NA-Rluc and NA-GFP. A serum sample, the 1:20 initial dilution of which failed to show 50% reduction of Rluc activity or GFP expression, was defined as negative (A).
the 23 sera was good with a correlation coefficient of 0.83 (Fig. 5B). These results demonstrate that the NA-Rluc is comparable to the NA-GFP in the sensitivity and specificity. 5. Discussion This report describes the novel neutralization assay for HMPV based on Rluc activity. The NA-Rluc offers several significant advantages over the previous assays. First, the NA-Rluc can be completed within 24 h, and is the most rapid neutralization assay for HMPV of all so far developed, which required a minimum of 3–5 days (usually one week) for completion. Second, the NA-Rluc has been freed from the requirement of trypsin. In the NA-Rluc, Rluc activity could be detected on day 1 even in the absence of trypsin if the inoculum contains more than ten infectious virions. Requirement of no trypsin and shortening the assay period eliminates the risk of spoiling the assay due to losing the monolayer by added trypsin. An attempt had been made to eliminate the trypsin requirement by introduction of a S101P mutation in the cleavage-activation site of F, which is responsible for the trypsin-independent cleavage.11 The mutation promoted multistep replication of HMPV in the absence of trypsin, but inefficiently.13 Trypsin was still required for forming clear and larger plaques. Third, the NA-Rluc is comparable to the NA-GFP in the sensitivity and specificity (Fig. 4 and Fig. 5) and has the potential for an increase in the sensitivity by reducing the number of input virions. Fourth, many types of cells other than virus-producing cell lines such as LLC-MK2 and Vero cells may be available for the NA-Rluc. We found that HEK293T cells, which are not a virus-producing cell line, exhibited a sufficient level of the Rluc activity on day 1 post-infection (unpublished data). Finally, the NA-Rluc retains all favorable characteristics of the NA-GFP that eliminates laborious steps including overlay of agars medium with trypsin, fixation of cells, and the immunostaining process with HMPV-specific antibodies. The neutralization assay is used for many types of studies; laboratory diagnosis, vaccine development, epidemiological study, and basic research to analyze humoral response to HMPV. These studies are often intended for a large number of samples. Such high
36
M. Zhou et al. / Journal of Clinical Virology 56 (2013) 31–36
thoroughput measurement of neutralizing antibodies could be achieved by automating the process of measurement for Rluc activity in the NA-Rluc. Funding This study was supported by Grants-in-Aid (No. 22590414 and No. 11020436) for Scientific Research from the Japan Society for the Promotion of Science and by grants from Shiga University of Medical Science, Wajinkai, and Yakult Foundation, Japan. Competing interests No conflict of interest. Ethical approval This study was conducted with the approval of the ethics committee of Shiga University of Medical Science (reference number 24–25). Acknowledgements We thank Yanagi Y. (Fukuoka) for providing pCA7, Miyazaki J. (Osaka) for his permission to use the CAG promoter of pCA7, and Conzelmann K.K. (Munich) for providing BSR T7/5. Sequence analysis was performed using the ABI PRISM 3130xl Genetic Analyzer in the Central Research Laboratory Shiga University of Medical Science. References 1. Boivin G, Abed Y, Pelletier G, Ruel L, Moisan D, Cote S, et al. 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. 2. van den Hoogen BG, de Jong JC, Groen J, Kuiken T, de Groot R, Fouchier RA, et al. A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat Med 2001;7:719–24. 3. Crowe JE, Williams JV. Metapneumoviruses. In: Samal SK, editor. The biology of pramyxoviruses. Norfolk, UK: Caister Academic Press; 2011. p. 411–34. 4. 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.
5. Konig B, Konig W, Arnold R, Werchau H, Ihorst G, Forster J. Prospective study of human metapneumovirus infection in children less than 3 years of age. J Clin Microbiol 2004;42:4632–5. 6. Collins PL. Human respiratory syncytial virus. In: Samal SK, editor. The biology of paramyxoviruses. Norfolk, UK: Caister Academic Press; 2011. p. 341–410. 7. Glezen WP, Paredes A, Allison JE, Taber LH, Frank AL. Risk of respiratory syncytial virus infection for infants from low-income families in relationship to age, sex, ethnic group, and maternal antibody level. J Pediatr 1981;98:708–15. 8. MacPhail M, Schickli JH, Tang RS, Kaur J, Robinson C, Fouchier RA, et al. Identification of small-animal and primate models for evaluation of vaccine candidates for human metapneumovirus (hMPV) and implications for hMPV vaccine design. J Gen Virol 2004;85:1655–63. 9. Skiadopoulos MH, Biacchesi S, Buchholz UJ, Riggs JM, Surman SR, AmaroCarambot E, et al. The two major human metapneumovirus genetic lineages are highly related antigenically, and the fusion (F) protein is a major contributor to this antigenic relatedness. J Virol 2004;78:6927–37. 10. Wyde PR, Chetty SN, Jewell AM, Boivin G, Piedra PA. Comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro. Antiviral Res 2003;60:51–9. 11. Schickli JH, Kaur J, Ulbrandt N, Spaete RR, Tang RS. An S101P substitution in the putative cleavage motif of the human metapneumovirus fusion protein is a major determinant for trypsin-independent growth in Vero cells and does not alter tissue tropism in hamsters. J Virol 2005;79:10678–89. 12. Biacchesi S, Skiadopoulos MH, Yang L, Murphy BR, Collins PL, Buchholz UJ. Rapid human metapneumovirus microneutralization assay based on green fluorescent protein expression. J Virol Methods 2005;128:192–7. 13. de Graaf M, Herfst S, Schrauwen EJ, van den Hoogen BG, Osterhaus AD, Fouchier RA. An improved plaque reduction virus neutralization assay for human metapneumovirus. J Virol Methods 2007;143:169–74. 14. Falsey AR, Formica MA, Walsh EE. Microneutralization assay for the measurement of neutralizing antibodies to human metapneumovirus. J Clin Virol 2009;46:314–7. 15. Kitagawa Y, Tani H, Limn CK, Matsunaga TM, Moriishi K, Matsuura Y. Liganddirected gene targeting to mammalian cells by pseudotype baculoviruses. J Virol 2005;79:3639–52. 16. Gotoh B, Takeuchi K, Komatsu T, Yokoo J. The STAT2 activation process is a crucial target of Sendai virus C protein for the blockade of alpha interferon signaling. J Virol 2003;77:3360–70. 17. Kitagawa Y, Zhou M, Yamaguchi M, Komatsu T, Takeuchi K, Itoh M, et al. Human metapneumovirus M2-2 protein inhibits viral transcription and replication. Microbes Infect 2010;12:135–45. 18. Biacchesi S, Skiadopoulos MH, Tran KC, Murphy BR, Collins PL, Buchholz UJ. Recovery of human metapneumovirus from cDNA: optimization of growth in vitro and expression of additional genes. Virology 2004;321:247–59. 19. Herfst S, de Graaf M, Schickli JH, Tang RS, Kaur J, Yang CF, et al. Recovery of human metapneumovirus genetic lineages A and B from cloned cDNA. J Virol 2004;78:8264–70. 20. Takeda M, Ohno S, Seki F, Nakatsu Y, Tahara M, Yanagi Y. Long untranslated regions of the measles virus M and F genes control virus replication and cytopathogenicity. J Virol 2005;79:14346–54. 21. Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 1991;108:193–9. 22. Kitagawa Y, Yamaguchi M, Zhou M, Komatsu T, Nishio M, Sugiyama T, et al. A tryptophan-rich motif in the human parainfluenza virus type 2 V protein is critical for the blockade of toll-like receptor 7 (TLR7)- and TLR9-dependent signaling. J Virol 2011;85:4606–11.
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Expeditious neutralization assay for human metapneumovirus based on a recombinant virus expressing Renilla luciferase Min Zhou a , Yoshinori Kitagawa a , Mayu Yamaguchi a , Chika Uchiyama a , Masae Itoh b , Bin Gotoh a,∗ a b
Division of Microbiology and Infectious Diseases, Department of Pathology, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga 520-2192, Japan Genetics of Life, Faculty of Bio-Science, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829 Japan
a r t i c l e
i n f o
Article history: Received 24 August 2012 Received in revised form 25 September 2012 Accepted 28 September 2012 Keywords: Human metapneumovirus Neutralization assay Renilla luciferase
a b s t r a c t Background: Human metapneumovirus (HMPV) is a common cause of respiratory diseases in persons of all ages. Because of its slow replication and weak cytopathic effect in cultured cells, conventional neutralization assays for HMPV require around one week for completion. Objectives: The purpose of this study is to establish a rapid neutralization assay based on a recombinant virus expressing Renilla luciferase (Rluc). Study design: A recombinant HMPV expressing both Rluc and green fluorescent protein (GFP) was created by reverse genetics method. Two-fold serial dilutions of human 23 sera were made in a 96-well plate and incubated with 50 pfu/well of the recombinant virus at 4 ◦ C for 1 h. The mixtures were then transferred to LLC-MK2 cells in a 96-well plate, incubated for 2 h, and replaced with trypsin-free fresh media. After incubation at 32 ◦ C for 24 h, the cells were lysed and measured for Rluc activity. The neutralization titer was defined as the reciprocal of the highest serum dilution that resulted in 50% reduction of Rluc activity. Results: The novel assay could be completed within 24 h and eliminated the requirement of trypsin supporting multistep replication in cultured cells, as well as laborious processes including the plaque assay with immunostaining. Neutralization titers correlated well with those determined by a GFP-based assay previously developed. Conclusions: The neutralization assay based on Rluc activity is the fastest and the most straightforward of all previous assays, and may be available for high throughput screening of neutralizing antibodies. © 2012 Elsevier B.V. All rights reserved.
1. Background Human metapneumovirus (HMPV) is a common cause of acute respiratory tract diseases in persons of all ages.1,2 Severe HMPV diseases can be caused in immunocompromized hosts and children with underlying conditions such as asthma, chronic lung disease due to prematurity, or congenital heart disease.3 Clinical signs are indistinguishable from those caused by human respiratory syncytial virus (HRSV) in the same subfamily Pneumovirinae.4,5 One of risk factors for severe HRSV diseases in infants is thought
Abbreviations: HMPV, human metapneumovirus; HRSV, human respiratory syncytial virus; CPE, cytopathic effect; rHMPV, recombinant HMPV; GFP, green fluorescent protein; NA-GFP, neutralization assay based on GFP expression; mAb, monoclonal antibody; Rluc, Renilla luciferase; NA-Rluc, neutralization assay based on Rluc activity; DMEM, Dulbecco’s modified Eagle’s medium; nt, nucleotide; ORF, open reading frame; rHMPV-GFP, rHMPV expressing GFP; rHMPV-Rluc/GFP, rHMPV expressing Rluc plus GFP; CAG promoter, cytomegalovirus enhancer-chicken actin hybrid promoter; moi, multiplicity of infection. ∗ Corresponding author. Tel.: +81 77 548 2176; fax: +81 77 548 2176. E-mail address: [email protected] (B. Gotoh). 1386-6532/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2012.09.014
to be low titers of maternal antibodies.6 The HRSV diseases in infants born with high levels of maternal neutralizing antibodies against HRSV were milder and occurred at an older age than infants with lower antibody levels.7 It suggests potential importance of determining titers of neutralizing antibodies to assess risk of the severity, although there is no direct evidence for correlation between the HMPV-neutralizing antibody level and HMPV disease severity. In addition to such a clinical prospective assessment, clinical diagnosis, vaccine development, and fundamental research to monitor the humoral immune response to HMPV infections require a fast and simple neutralization assay for HMPV. Neutralizing antibodies specific to HMPV have been usually quantified by classical methods such as a plaque reduction assay8 or reduction in the 50% tissue culture infectious dose.2,9,10 These conventional neutralization assays require around one week for completion, because of slow HMPV growth and weak cytopathic effect (CPE) in cultured cells. Furthermore multistep replication of most HMPV strains but a few exceptions in cultured cells depends on the presence of trypsin added in the medium, since trypsin is required for the cleavage activation of the HMPV F protein, which
32
M. Zhou et al. / Journal of Clinical Virology 56 (2013) 31–36
Fig. 1. Construction of a plasmid expressing the antigenomic RNA of Jpn03-1 (A) and the genome constructs of rHMPV-GFP and rHMPV-Rluc/GFP (B). Four overlapping cDNA fragments were assembled to construct the complete antigenomic cDNA except for four and five nucleotide substitutions, which create NheI and SnaBI sites in the M-F and M2-SH intergenic regions, respectively (A). Le, 3 non-transcribed leader region; Tr, 5 non-transcribed trailer region; T7P, T7 promoter; ␦ ribo, hepatitis delta-ribozyme sequence; T7T, T7 terminator; GS, gene start signal; GE, gene end signal.
mediates fusion between viral envelope and cell membrane.11 The trypsin requirement as well as weak CPE of HMPV makes the plaque assay difficult, because trypsin added in the medium causes cells off the bottom of the plate when cells are long incubated. Thus, fixation and immunostaining are indispensible for the plaque assay for HMPV. The neutralization assay for HMPV was modified by using recombinant HMPV (rHMPV) expressing green fluorescent protein (GFP).12,13 Neutralizing titers could be determined as the reciprocal of the highest serum dilution that resulted in 50% reduction of GFP expression12 or the number of GFP-positive plaques,13 both of which could be monitored by an automated scanner. The neutralization assay based on GFP expression (NAGFP) eliminated the immunostaining procedures and shortened the time for completion. Another assay, a microneutralization assay, which had been used for the measurement of neutralizing antibodies to RSV, was adapted to HMPV.14 It was performed by adding suspended cells to an antibody-virus mixture followed by an enzyme immunoassay with monoclonal antibody (mAb) to the HMPV N protein, and the titer was defined on the basis of 50% reduction of color development. However, any assay so far developed still needed at least 3–5 days for completion.
2. Objectives The purpose of this study is to establish a more rapid and straightforward neutralization assay for HMPV based on Renilla luciferase (Rluc) activity (NA-Rluc) using a newly created rHMPV.
3. Study design 3.1. Cells and viruses LLC-MK2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 2 mM l-glutamine, penicillin (100 IU/ml), streptomycin (100 g/ml), and 10% fetal bovine serum.15,16 HMPV Jpn03-1 strain and rHMPVs described below were propagated in LLC-MK2 cells in the presence of 4 g/ml of N-acetyl trypsin.17 Trypsin was added every three days to a final concentration of 4 g/ml to promote multistep replication.18
3.2. Plasmids and recovery of rHMPVs NheI and SnaBI sites were introduced into the putative M-F and M2-SH intergenic regions of the HMPV Jpn03-1 genome by four and five nucleotide substitutions, respectively, to create insertion sites for exogenous genes (Fig. 1A). This antigenomic cDNA copy [nucleotide (nt) 12-13337] was cloned in vector pT7PdT-MCS by assembling four overlapping cDNA clones generated by RT-PCR with specific primers designed from the Jpn03-1 genome sequence (Genbank accession number AB503857). pT7PdT-MCS contains a T7 promoter with three G residues, nt 1–12 of the antigenomic cDNA, multicloning sites, the hepatitis delta virus ribozyme, and a T7 terminator. The multicloning sites served to accept the cloned fragments. Transcription cassettes, which include the GFP or Rluc open reading frame (ORF) that was franked on its upstream and downstream sides by the F gene start and M gene end motifs, respectively, were cloned into the NheI site in the M–F intergenic region as shown in Fig. 1B. rHMPVs expressing GFP alone (rHMPV-GFP) or Rluc plus GFP (rHMPV-Rluc/GFP) were recovered
M. Zhou et al. / Journal of Clinical Virology 56 (2013) 31–36
33
Fig. 2. Growth kinetics of rHMPV-GFP and rHMPV-Rluc/GFP. LLC-MK2 cells in 6-well plates were infected with rHMPV-GFP or rHMPV-Rluc/GFP at a moi of 0.01, and incubated at 32 ◦ C in the presence of trypsin. Virus titers of culture media were determined by the plaque assay (A) and cells were observed under a fluorescence microscope (B).
using BSR T7/5 cells constitutively expressing T7 polymerase as described previously.18,19 A plasmid that expresses F, G, or SH with 3xFLAG epitope tag was created by insertion of the PCRamplified cDNAs encoding each protein into multi-cloning sites of pCA7,20 which contains the cytomegalovirus enhancer-chicken actin hybrid (CAG) promoter.21 The FLAG tag was appended to the N-terminal end of G and SH and to the C-terminal end of F. Sequence fidelity of all constructs was confirmed by using a PRISM 3130xl genetic analyzer (Applied Biosystems). 3.3. Plaque assay Serial 10-fold dilutions of samples containing rHMPV-GFP or rHMPV-Rluc/GFP were prepared in triplicate. Each dilution (400 or 50 l/well) was transferred to a confluent monolayer of LLCMK2 cells in a 6 or 96-well plate, respectively. After incubation at 32 ◦ C for 1 h, cells were washed twice with serum-free DMEM, and overlaid with 2 ml/well (for a 6-well plate) or 100 l/well (for a 96-well plate) of 3% methylcellulose in opti-MEM with 4 g/ml of N-acetyl trypsin. After incubation at 32 ◦ C for 7 days, the number of plaques with GFP fluorescence was counted by scanning with LAS-4000 (Fujifilm). 3.4. Measurement of Rluc activity rHMPV-Rluc/GFP-infected cells in a 6 or 96-well plate were lysed in 500 or 40 l of Renilla luciferase assay lysis buffer (Promega). Ten microliters of cell lysates were measured for Rluc activity. Rluc activity was expressed as average of relative light units derived from three independent experiments with Renilla luciferase assay system (Promega) and luminescence-PSN AB2200 (ATTO) as described previously.17 3.5. Immunoprecipitation and immunoblot analysis Human serum no. 9, which showed the highest titer, was selected by the NA-GFP from 23 human serum samples of pediatric outpatients in our university hospital. Two control samples for the serum no. 9 were prepared by mixing with
Fig. 3. Detection of Rluc activity in cells infected with rHMPV-Rluc/GFP. (A) LLCMK2 cells in 6-well plates were infected with rHMPV-Rluc/GFP at a moi of 0.01, and then incubated at 32 ◦ C in the presence or absence of trypsin. (B) LLC-MK2 cells in a 96-well plate were infected with 0, 10, 30, 50, or 100 pfu/well of rHMPVRluc/GFP and incubated in the absence of trypsin. Rluc activity was measured at 24 h post-infection.
34
M. Zhou et al. / Journal of Clinical Virology 56 (2013) 31–36
Fig. 4. Neutralization assays based on Rluc activity or GFP expression. Rluc activity (A) or GFP expression (B) was determined on one or seven day post-inoculation, respectively. Relative Rluc activity was expressed as percent ratio to the Rluc activity of the control of virus alone without sera (A and B). The cell lysate containing FLAG-tagged F, G, or SH was subjected to immunoprecipitation (IP) with anti-FLAG mAb, serum no.9, protein G-treated, or glutathione-treated serum, followed by immunoblot analysis with anti-FLAG mAb (C and D).
sufficient amounts of protein G or glutathione sepharose beads (GE Healthcare Bio-Science) followed by incubation at room temperature for 1 h. After centrifugation, the supernatants were used as control protein G-treated and glutathione-treated sera. HEK293T cells were transfected with a plasmid expressing F-FLAG, FLAG-SH or FLAG-G using Fugene HD transfection reagent according to the manufacturer’s instructions. At 24 h post-transfection, cells were lysed in 400 l of a lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Triton X-100) supplemented with protease inhibitor cocktail (Roche). After centrifugation, lysates were mixed with the no. 9, protein G-treated, glutathionetreated serum, or anti-FLAG (M2) mAb (Sigma) together with protein G sepharose beads, and incubated at 4 ◦ C for 2 h. After washing five times with the lysis buffer, proteins were eluted from the beads by boiling with Laemmli sample buffer, and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot analysis with anti-FLAG mAb.17,22 3.6. NA-Rluc and NA-GFP All sera were heat-inactivated at 56 ◦ C for 30 min prior to the neutralization assay to eliminate serum complement. Two-fold serial dilutions of serum samples initially diluted 1:20 in FBS-free DMEM were prepared in triplicate in a 96-well plate. Each serum
dilution was mixed with an equal volume of rHMPV-Rluc/GFP (50 pfu/well) and incubated at 4 ◦ C for 1 h. The virus-serum mixtures were then transferred to confluent monolayers of LLC-MK2 cells in a 96-well plate. After incubation at 32 ◦ C for 2 h, the cells were washed twice with serum-free DMEM, overlaid with 100 l of serum-free DMEM containing no trypsin for the NARluc or 4 g/ml of N-acetyl trypsin for the NA-GFP, and incubated at 32 ◦ C. Rluc activity was determined on day 1 post-inoculation, while GFP expression was quantified by scanning with LAS-4000 on day 7 post-inoculation. Neutralizing titers in the NA-Rluc and NA-GFP were defined as the reciprocal of the highest dilution that resulted in 50% reduction of Rluc activity or GFP expression, respectively. 4. Results 4.1. Replication kinetics of the rHMPV-Rluc/GFP rHMPV-GFP and rHMPV-Rluc/GFP, which express GFP alone and Rluc plus GFP, respectively, were created by reverse genetics method as described previously.18,19 rHMPV-Rluc/GFP was compared with rHMPV-GFP in multistep virus growth in LLCMK2 cells, since rHMPV-GFP was shown to be almost similar to wild-type HMPV in the efficiency of multistep replication in LLCMK2 cells.18 Titers were determined by the plaque assay without
M. Zhou et al. / Journal of Clinical Virology 56 (2013) 31–36
35
immunostaining. As shown in Fig. 2A, rHMPV-Rluc/GFP replicated with an efficiency that was similar to that of rHMPV-GFP. In accordance with the virus replication kinetics, GFP-positive cells spread with an almost similar speed (Fig. 2B). Thus the insertion of the additional Rluc gene into the HMPV genome had no significant effect on virus replication in cultured cells. 4.2. Time course of Rluc activity after rHMPV-Rluc/GFP infection To set the minimum time required for completing the NA-Rluc, we examined time course of Rluc activity expressed in cells after rHMPV-Rluc/GFP infection (Fig. 3A). LLC-MK2 cells (∼106 cells) were infected with rHMPV-Rluc/GFP at a multiplicity of infection (moi) of 0.01, and incubated in the presence or absence of trypsin. Exponential increase was observed in the presence of trypsin on day 1 to day 3 post-infection, whereas the increase was minimal in the absence of trypsin. The Rluc activities on day 1 post-infection under both conditions showed almost the same value (∼1 × 106 ), suggesting the multistep replication had not developed before day 1 post-infection. These results raise the possibility that the NA-Rluc is completed within one day without trypsin. The smaller is the number of input virions, the higher is the sensitivity of the neutralization assay. Thus we sought to determine the minimum number of input virions required for detection of Rluc activity on day 1 postinfection. As shown in Fig. 3B, Rluc activity was detectable even with 10 pfu/well of input virions. This indicates that around ten infected-cells are sufficient for detection of Rluc activity. 4.3. The inhibitory curve in the NA-Rluc and NA-GFP Initially we selected a serum (no. 9) exhibiting the highest HMPV-neutralizing titer in the previous assay, NA-GFP,12 out of 23 sera of pediatric outpatients picked at random in our university hospital. The serum no. 9 was treated with protein G or glutathione sepharose beads, as described in study design, to prepare control sera. The protein G-treated serum was expected to lose the IgG fraction. These control sera as well as the original serum were serially diluted, mixed with an equal volume of rHMPV-Rluc/GFP (50 pfu/well), and incubated at 4 ◦ C for 1 h. The mixtures were then transferred to LLC-MK2 cells grown in 96-well plates, incubated in the absence of trypsin, and Rluc activity was determined at 24 h post-inoculation (Fig. 4A). The no. 9 and glutathione-treated sera showed a similar inhibitory curve, whereas the protein G-treated serum exhibited no inhibitory effect. Similar results were obtained in the NA-GFP (Fig. 4B). In the NA-GFP, cells were incubated in the presence of trypsin, and the GFP expression was determined on day 7 post-inoculation. To investigate whether the serum no. 9 contained antibodies against HMPV, immunoprecipitation was carried out using cell extracts containing FLAG-tagged envelope proteins (F, G, and SH) (Fig. 4C and D). The serum no. 9 was found to recognize F, but little G or SH (Fig. 4C). Whereas the glutathione-treated serum retained the ability to recognize F, the protein G-treated serum lost it (Fig. 4D). These results suggest that the neutralizing activity in both assays is largely attributable to IgG antibodies against F rather than non-specific inhibitory factors. 4.4. Correlation between neutralizing titers determined by the NA-Rluc and NA-GFP Neutralizing titers of the 23 human sera were determined by the NA-Rluc and NA-GFP. Titers were defined as the reciprocal of the highest serum dilution that resulted in 50% reduction of Rluc activity or GFP expression (Fig. 4A and B). When the samples were scored as positive (more than 1:20) or negative (less than 1:20), there was a 100% correlation between the results from the NA-Rluc and NA-GFP (Fig. 5A). Correlation of the neutralization titers for
Fig. 5. Correlation of the neutralizing titers of 23 serum samples determined by the NA-Rluc and NA-GFP. A serum sample, the 1:20 initial dilution of which failed to show 50% reduction of Rluc activity or GFP expression, was defined as negative (A).
the 23 sera was good with a correlation coefficient of 0.83 (Fig. 5B). These results demonstrate that the NA-Rluc is comparable to the NA-GFP in the sensitivity and specificity. 5. Discussion This report describes the novel neutralization assay for HMPV based on Rluc activity. The NA-Rluc offers several significant advantages over the previous assays. First, the NA-Rluc can be completed within 24 h, and is the most rapid neutralization assay for HMPV of all so far developed, which required a minimum of 3–5 days (usually one week) for completion. Second, the NA-Rluc has been freed from the requirement of trypsin. In the NA-Rluc, Rluc activity could be detected on day 1 even in the absence of trypsin if the inoculum contains more than ten infectious virions. Requirement of no trypsin and shortening the assay period eliminates the risk of spoiling the assay due to losing the monolayer by added trypsin. An attempt had been made to eliminate the trypsin requirement by introduction of a S101P mutation in the cleavage-activation site of F, which is responsible for the trypsin-independent cleavage.11 The mutation promoted multistep replication of HMPV in the absence of trypsin, but inefficiently.13 Trypsin was still required for forming clear and larger plaques. Third, the NA-Rluc is comparable to the NA-GFP in the sensitivity and specificity (Fig. 4 and Fig. 5) and has the potential for an increase in the sensitivity by reducing the number of input virions. Fourth, many types of cells other than virus-producing cell lines such as LLC-MK2 and Vero cells may be available for the NA-Rluc. We found that HEK293T cells, which are not a virus-producing cell line, exhibited a sufficient level of the Rluc activity on day 1 post-infection (unpublished data). Finally, the NA-Rluc retains all favorable characteristics of the NA-GFP that eliminates laborious steps including overlay of agars medium with trypsin, fixation of cells, and the immunostaining process with HMPV-specific antibodies. The neutralization assay is used for many types of studies; laboratory diagnosis, vaccine development, epidemiological study, and basic research to analyze humoral response to HMPV. These studies are often intended for a large number of samples. Such high
36
M. Zhou et al. / Journal of Clinical Virology 56 (2013) 31–36
thoroughput measurement of neutralizing antibodies could be achieved by automating the process of measurement for Rluc activity in the NA-Rluc. Funding This study was supported by Grants-in-Aid (No. 22590414 and No. 11020436) for Scientific Research from the Japan Society for the Promotion of Science and by grants from Shiga University of Medical Science, Wajinkai, and Yakult Foundation, Japan. Competing interests No conflict of interest. Ethical approval This study was conducted with the approval of the ethics committee of Shiga University of Medical Science (reference number 24–25). Acknowledgements We thank Yanagi Y. (Fukuoka) for providing pCA7, Miyazaki J. (Osaka) for his permission to use the CAG promoter of pCA7, and Conzelmann K.K. (Munich) for providing BSR T7/5. Sequence analysis was performed using the ABI PRISM 3130xl Genetic Analyzer in the Central Research Laboratory Shiga University of Medical Science. References 1. Boivin G, Abed Y, Pelletier G, Ruel L, Moisan D, Cote S, et al. 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. 2. van den Hoogen BG, de Jong JC, Groen J, Kuiken T, de Groot R, Fouchier RA, et al. A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat Med 2001;7:719–24. 3. Crowe JE, Williams JV. Metapneumoviruses. In: Samal SK, editor. The biology of pramyxoviruses. Norfolk, UK: Caister Academic Press; 2011. p. 411–34. 4. 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.
5. Konig B, Konig W, Arnold R, Werchau H, Ihorst G, Forster J. Prospective study of human metapneumovirus infection in children less than 3 years of age. J Clin Microbiol 2004;42:4632–5. 6. Collins PL. Human respiratory syncytial virus. In: Samal SK, editor. The biology of paramyxoviruses. Norfolk, UK: Caister Academic Press; 2011. p. 341–410. 7. Glezen WP, Paredes A, Allison JE, Taber LH, Frank AL. Risk of respiratory syncytial virus infection for infants from low-income families in relationship to age, sex, ethnic group, and maternal antibody level. J Pediatr 1981;98:708–15. 8. MacPhail M, Schickli JH, Tang RS, Kaur J, Robinson C, Fouchier RA, et al. Identification of small-animal and primate models for evaluation of vaccine candidates for human metapneumovirus (hMPV) and implications for hMPV vaccine design. J Gen Virol 2004;85:1655–63. 9. Skiadopoulos MH, Biacchesi S, Buchholz UJ, Riggs JM, Surman SR, AmaroCarambot E, et al. The two major human metapneumovirus genetic lineages are highly related antigenically, and the fusion (F) protein is a major contributor to this antigenic relatedness. J Virol 2004;78:6927–37. 10. Wyde PR, Chetty SN, Jewell AM, Boivin G, Piedra PA. Comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro. Antiviral Res 2003;60:51–9. 11. Schickli JH, Kaur J, Ulbrandt N, Spaete RR, Tang RS. An S101P substitution in the putative cleavage motif of the human metapneumovirus fusion protein is a major determinant for trypsin-independent growth in Vero cells and does not alter tissue tropism in hamsters. J Virol 2005;79:10678–89. 12. Biacchesi S, Skiadopoulos MH, Yang L, Murphy BR, Collins PL, Buchholz UJ. Rapid human metapneumovirus microneutralization assay based on green fluorescent protein expression. J Virol Methods 2005;128:192–7. 13. de Graaf M, Herfst S, Schrauwen EJ, van den Hoogen BG, Osterhaus AD, Fouchier RA. An improved plaque reduction virus neutralization assay for human metapneumovirus. J Virol Methods 2007;143:169–74. 14. Falsey AR, Formica MA, Walsh EE. Microneutralization assay for the measurement of neutralizing antibodies to human metapneumovirus. J Clin Virol 2009;46:314–7. 15. Kitagawa Y, Tani H, Limn CK, Matsunaga TM, Moriishi K, Matsuura Y. Liganddirected gene targeting to mammalian cells by pseudotype baculoviruses. J Virol 2005;79:3639–52. 16. Gotoh B, Takeuchi K, Komatsu T, Yokoo J. The STAT2 activation process is a crucial target of Sendai virus C protein for the blockade of alpha interferon signaling. J Virol 2003;77:3360–70. 17. Kitagawa Y, Zhou M, Yamaguchi M, Komatsu T, Takeuchi K, Itoh M, et al. Human metapneumovirus M2-2 protein inhibits viral transcription and replication. Microbes Infect 2010;12:135–45. 18. Biacchesi S, Skiadopoulos MH, Tran KC, Murphy BR, Collins PL, Buchholz UJ. Recovery of human metapneumovirus from cDNA: optimization of growth in vitro and expression of additional genes. Virology 2004;321:247–59. 19. Herfst S, de Graaf M, Schickli JH, Tang RS, Kaur J, Yang CF, et al. Recovery of human metapneumovirus genetic lineages A and B from cloned cDNA. J Virol 2004;78:8264–70. 20. Takeda M, Ohno S, Seki F, Nakatsu Y, Tahara M, Yanagi Y. Long untranslated regions of the measles virus M and F genes control virus replication and cytopathogenicity. J Virol 2005;79:14346–54. 21. Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 1991;108:193–9. 22. Kitagawa Y, Yamaguchi M, Zhou M, Komatsu T, Nishio M, Sugiyama T, et al. A tryptophan-rich motif in the human parainfluenza virus type 2 V protein is critical for the blockade of toll-like receptor 7 (TLR7)- and TLR9-dependent signaling. J Virol 2011;85:4606–11.