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1 Department of Pediatrics, Hokkaido University Graduate School of Medicine, Sapporo, Japan
2 Mitsubishi Chemical Medience Corporation, Tokyo, Japan
3 Pediatric Clinic, Touei Hospital, Sapporo, Japan
Correspondence
Hideaki Kikuta
hide-ki{at}touei.or.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A table showing the sequences of the primers used in this study is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). hMPV and human respiratory syncytial virus (hRSV) are grouped in the same subfamily, Pneumovirinae, of the family Paramyxoviridae. Phylogenetic analysis of the nucleotide sequences indicated that there were two genetic groups, groups A and B, similar to the grouping of hRSV. Furthermore, each group could be subdivided into two subgroups, A1, A2, B1 and B2 (Bastien et al., 2004; Biacchesi et al., 2003; Ishiguro et al., 2004; Ludewick et al., 2005; Mackay et al., 2004; Peret et al., 2004; van den Hoogen et al., 2004).
hMPV encodes two major surface glycoproteins, the fusion (F) and attachment (G) proteins, which may be immunogenic and protective antigens. The hMPV F protein has been shown to be a major antigenic determinant that mediates effective neutralization and protection against the two genetic groups of hMPV (Ishiguro et al., 2005; Ma et al., 2005; Skiadopoulos et al., 2004, 2006). Since the amino acid sequences of hMPV F protein, as well as those of hRSV F protein, are highly conserved between the two groups, the hMPV F protein also plays a major role in the antigenic relatedness between the two groups of hMPV (Skiadopoulos et al., 2004, 2006). Our previous studies indicated that the hMPV F protein was a major antigenic determinant that mediates extensive cross-lineage neutralization and protection (Ishiguro et al., 2005; Ma et al., 2005).
On the other hand, the hMPV G gene was shown to be highly variable, particularly in the extracellular domain, as a result of nucleotide substitutions and insertions and the use of alternative termination transcription codons (Biacchesi et al., 2003; Peret et al., 2004; van den Hoogen et al., 2004). Our previous study showed that there is 31–35 % amino acid identity of the G protein between the A and B groups of hMPV (Ishiguro et al., 2004). Since the G protein is a type II membrane protein, its membrane anchor being proximal to the N terminus and its C terminus being oriented externally, the hMPV G proteins may contain an antigenic epitope of hMPV.
hMPV causes repeated infections throughout life, and two main groups of hMPV circulate worldwide (Ebihara et al., 2004a; Leung et al., 2005; van den Hoogen et al., 2001), which could be due either to incomplete immunity or to genetic heterogeneity of the virus. We have experienced a case of early reinfection with hMPV in an infant, who was infected with two different hMPV strains of heterologous virus type during a period of only one month (Ebihara et al., 2004b). If the cross-reactive immunity provided by the F protein is sufficient to overcome the effects of changes in the G protein, early reinfection with hMPV will not occur so frequently. However, it is not clear whether differences between strains within the same group affect susceptibility to infection.
In the present study, we examined the antibody responses to four hMPV G proteins in human serum to discuss the possibility that antigenic differences in hMPV G protein contribute to the repeated infections. We sequenced the G ORFs of four isolates representative of each genetic subgroup of hMPV, namely A1, A2, B1 and B2, expressed the corresponding proteins using recombinant baculoviruses and measured the antibody titres to four hMPV G proteins in serum samples from children with primary infection and reinfection with hMPV of known genetic subgroups using an immunofluorescence assay (IFA) method.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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RT-PCR, sequencing and phylogenetic tree analysis.
Nasopharyngeal swab samples obtained from children were examined for the presence of the RNA sequence of hMPV using RT-PCR based on the F or the nucleocapsid (N) gene. Total RNA was extracted from each sample by using the RNAzol B (Tel-Test) method according to the manufacturer's protocol. Approximately 0.1 µg of each RNA sample was incubated in a solution containing 100 ng random hexadeoxynucleotides and 200 U Moloney murine leukemia virus reverse transcriptase (First-Strand cDNA Synthesis kit, GE Healthcare) in a final volume of 15 µl at 37 °C for 1 h to synthesize cDNA. The cDNA was subjected to PCR analysis to determine expression of the hMPV F or N gene. On the basis of published data (Ebihara et al., 2004a; Maertzdorf et al., 2004), a set of primers was designed for amplification of the hMPV F or N gene (primer sequences are shown in Supplementary Table S1, available with the online version of this paper). The PCR reaction mixture consisted of 100 µM each deoxyribonucleotide, 1.0 U AmpliTaq Gold, 50 mM potassium chloride, 10 mM Tris/HCl (pH 8.3), 1.5 mM magnesium chloride, 0.01 % gelatin, 10 pM each primer and cDNA in a final volume of 25 µl. The PCR conditions to detect the hMPV F gene were 94 °C for 9 min followed by 35 cycles of 94 °C for 1 min, 60 °C for 1 min and 72 °C for 1 min, and the PCR conditions to detect the hMPV N gene were 94 °C for 5 min followed by 45 cycles of 94 °C for 30 s and 60 °C for 1 min.
Eighty base pairs for the hMPV F gene and 104 bp for the hMPV N gene of the PCR products were sequenced directly by using a BigDye Dye terminator cycle sequencing ready reaction kit (Perkin Elmer Applied Biosystems) with an ABI PRISM 310 Genetic Analyzer (Perkin Elmer Applied Biosystems). Both sense and antisense strands of the PCR products were sequenced directly. Nucleotide sequences of amplified hMPV F and N gene products were aligned by using CLUSTAL W software. Phylogenetic trees were constructed by neighbour-joining by the use of the DNADIST and Neighbour software package of the PHYLIP 3.6 (alpha 3) program using 100 bootstraps with random sequence addition. Bootstrap values were computed for consensus trees created with the CONSENSE package by applying a 50 % majority consensus rule.
Expression of G and F proteins of hMPV in a baculovirus–insect cell system.
A baculovirus expression kit was used to prepare hMPV G proteins of four representative subgroups and hMPV F protein expressed in a baculovirus–insect cell system according to the instructions of the manufacturer (BD Pharmingen). The full-length cDNAs of hMPV G proteins were prepared from four strains of hMPV, JPS03-180, JPS03-178, JPS02-76 and JPS05-21, which represent four distinct subgroups of hMPV strain, A1, A2, B1 and B2, respectively. The full-length cDNA of hMPV F protein was prepared from strain JPY88-12 (GenBank accession no. AY622381
[GenBank]
), as previously reported (Ishiguro et al., 2005). Sequences of each hMPV G protein and hMPV F protein were amplified by PCR using the primers shown in Supplementary Table S1 (available with the online version of this paper). The PCR products were cloned into vector pCR2.1 using a TA Cloning kit (Invitrogen). For preparation of a recombinant baculovirus expressing hMPV G and F proteins, the PCR product was subcloned into the pVL1393 baculovirus transfer vector (BD Pharmingen). The recombinant plasmid pVL1393 containing the coding sequences for the hMPV G and F proteins was co-transfected with BaculoGold DNA (BD Pharmingen) into Spodoptera frugiperda (Sf9) cells. Trichoplusia ni (Tn5) insect cells cultured in Ex-cell 405 medium (JRH Biosciences) were infected with recombinant baculoviruses expressing hMPV G (GA1, GA2, GB1 and GB2) and F protein at 10 m.o.i. and incubated at 26 °C for 3 to 5 days. The cells were then collected, washed three times in PBS and used for IFAs to detect antibodies to hMPV G and F protein. The recombinant baculovirus-expressed proteins were named Bac-G (Bac-GA1, -GA2, -GB1 and -GB2) protein and Bac-F protein.
Immunofluorescence assay.
Recombinant baculovirus-infected Tn5 cells or hMPV (JPS02-76 strain)-infected LLC-MK2 cells were spotted onto slides. The cell smears were air-dried, fixed in acetone for 10 min and incubated with human serum for 1 h at 37 °C. After incubation, the slides were washed twice in PBS for 5 min each time. The cell smears were then incubated with fluorescein isothiocyanate (FITC)-conjugated polyclonal rabbit anti-human IgG (Dako) diluted at 1 : 40 for 1 h at 37 °C. After incubation, the cell smears were washed twice in PBS for 5 min each time, air-dried and mounted with PBS : glycerin (1 : 1). Stained preparations were then examined under a fluorescence microscope. The reactions of antibodies to four Bac-G proteins, Bac-F protein and hMPV-infected cells were analysed using the IFA method with various serum samples. Serum that reacted at a dilution of more than 1 : 10 was considered positive for each antibody. IFAs using Bac-G (-GA1, -GA2, -GB1 and -GB2) protein and Bac-F protein were named Bac-G (-GA1, -GA2, -GB1 and -GB2)-IFA and Bac-F-IFA, respectively. The IFA using hMPV-infected LLC-MK2 cells was named hMPV-IFA.
Serum samples.
Two sets of serum samples were used for this study: (i) serum samples obtained from children infected with hMPV and (ii) control serum samples randomly obtained from human donors. All serum samples were collected after obtaining informed consent from the patients or children's parents.
(i) Serum samples obtained from children infected with hMPV: since the RT-PCR test is the most sensitive test for detection of hMPV and diagnosis of hMPV infection, we examined nasopharyngeal swab samples obtained from children with respiratory infections for hMPV using RT-PCR. Serum samples from patients diagnosed with hMPV infection by RT-PCR were tested for IgG antibodies to four Bac-G proteins, Bac-F protein and hMPV-infected cells by Bac-G-IFA, Bac-F-IFA and hMPV-IFA, respectively. RT-PCR-positive and hMPV-IFA-negative cases in acute-phase serum were considered to represent primary infection, and RT-PCR-positive and hMPV-IFA-positive cases in acute-phase serum were considered to represent reinfection. Seventy-nine serum samples obtained from 67 children infected with hMPV during the period from 2003 to 2006, including 12 paired serum samples and 45 acute-phase serum samples, were used for detection of antibodies. We defined serum samples collected within 10 days after onset of illness as acute-phase serum samples and serum samples collected from 11 days to 2 months after onset of illness as convalescent-phase serum samples. Ten serum samples collected from children infected with hMPV after 2 months from onset of illness were also tested.
(ii) Control serum samples randomly obtained from human donors: serum samples randomly obtained from 95 Japanese people aged from 3 months to 30 years who visited Kitami Red Cross Hospital during the period from March to April in 2002 were examined for the presence of antibodies to four Bac-G proteins and hMPV-infected cells.
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RESULTS |
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Serological analysis
Serum samples obtained from children infected with hMPV: antibodies to the four Bac-G proteins, Bac-F protein and hMPV-infected cells were examined in 67 children infected with a known genotype of hMPV (Tables 1, 2, 3, 4). Twelve paired serum and 45 acute-phase serum samples from the 67 children were available. Neither serum samples negative for antibodies to hMPV-infected cells nor those negative for antibodies to Bac-F protein had antibodies to any Bac-G proteins. In the remaining 10 cases, serum samples were collected after 2 months from onset of illness.
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and Table 2, respectively. Although 11 of the 12 acute-phase serum samples in the paired samples obtained from children with primary infection or reinfection were negative for the antibody to any Bac-G proteins, all of the 12 convalescent-phase serum samples in those paired samples were positive for the antibody to only one of the four Bac-G proteins of the infecting genotype of hMPV. Antibody response to hMPV G protein occurred in the convalescent phase regardless of primary infection or reinfection with hMPV. For example, the serum samples obtained from children infected with the A1 genotype strain of hMPV reacted to Bac-GA1 protein, but not to Bac-GA2, Bac-GB1 and Bac-GB2 proteins. The reactivity to Bac-G proteins was dependent on the infecting genotype of hMPV.
Detection of four genetic subgroup-specific antibodies to Bac-G protein in 45 acute-phase serum samples from children infected with hMPV: seropositivities of each antibody to four Bac-G proteins in 45 acute-phase serum samples obtained from children infected with hMPV are shown in Table 3. Eight (17.8 %) of the 45 samples were positive for the antibody to at least one Bac-G protein. One of the eight positive samples reacted to two Bac-G proteins. Five of the eight positive samples reacted to Bac-G proteins of the non-infecting genotype of hMPV. Since antibody responses by hMPV-IFA in these eight patients indicated reinfection, the detected antibody to hMPV G protein might have remained after previous infection of hMPV.
Detection of four genetic subgroup-specific antibodies to Bac-G protein in 10 serum samples collected from children after 2 months of hMPV infection: The titre of each antibody to four Bac-G proteins in ten serum samples obtained from children infected with hMPV after 2 months from onset of illness is shown in Table 4. All of the ten serum samples from children infected with hMPV of known genotype were positive for antibodies to hMPV-infected cells and Bac-F protein, but only three of the ten samples were positive for the antibody to Bac-G protein. All of those three positive samples reacted to Bac-G protein of the infecting genotype of hMPV. In those three positive cases, the serum samples were obtained 61 days, 73 days and 5 months from onset of illness. However, seven of the ten samples, which were obtained after 3 months from onset of illness, were negative for the antibodies to any Bac-G proteins.
Control serum samples randomly obtained from human donors: seropositivities of antibodies to hMPV-infected cells and Bac-G protein are shown in Table 5. Of the 95 serum samples, 75 were positive for the antibodies to hMPV-infected cells. All 20 serum samples negative for the antibody to hMPV-infected cells were also negative for the antibody to all four Bac-G proteins. Of the 75 serum samples positive for the antibody to hMPV-infected cells, 12 (16.0 %) were positive for the antibody to at least one Bac-G protein. Two of the 12 samples positive for the antibody to Bac-G protein reacted to two Bac-G proteins. The antibody titres to Bac-G protein in control serum samples were lower than those in the convalescent-phase serum samples from children infected with hMPV. The antibody titres to Bac-G protein were lower than 1 : 80 in 9 of the 12 control serum samples positive for the antibody to Bac-G protein (data not shown), while they were higher than 1 : 40 in 9 of the 12 convalescent-phase serum samples from children infected with hMPV (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Our previous serological study showed that over half of children infected with hMPV have antibodies against hMPV in the acute phase of illness and that reinfection with hMPV frequently occurs in children (Ebihara et al., 2004a; Ishiguro et al., 2005). Furthermore, reinfection with hMPV occurs frequently throughout life, implying that the host immune response induced by natural infection provides incomplete protection for a limited period of time, probably not lasting until the subsequent epidemic season. It has been shown that concurrent annual circulation of all four subgroups was common, with a single, usually different, hMPV subgroup predominating each year, suggesting that the change results from pre-existing immunity (Agapov et al., 2006; Ludewick et al., 2005; Mackay et al., 2006).
The immune response includes production of neutralizing antibodies and induction of T-cell-specific immunity. hMPV F and G proteins appear to be important for induction of protective humoral immunity. The ability to measure titres of antibodies to individual hMPV proteins is important for the analysis of immune responses to hMPV. Since the hMPV F genes in the two groups are highly conserved and the two groups of hMPV exhibited a high level of antigenic relatedness of F protein, antibodies to the conserved antigenic sites of F proteins contribute to the cross-protection against the two groups of hMPV. The majority of antibodies to hMPV in human sera are antibodies to the hMPV F protein, which is a major antigenic determinant. In our previous study (Ishiguro et al., 2005), the titres of human sera tested by hMPV-IFA using LLC-MK2 cells infected with one group of hMPV were the same as those of human sera tested by hMPV-IFA using LLC-MK2 cells infected with another group of hMPV. Furthermore, the Bac-F-IFA system made with the recombinant hMPV F protein of one group could also detect IgG antibodies to recombinant hMPV F protein of another group. These results indicate that the antibody to hMPV F protein exhibits cross-reactivity to the two groups and that there were no serotypes in hMPV. However, the existence of two serotypes is still controversial in animal models (van den Hoogen et al., 2004, 2007). Two antigenic groups have also been identified on the basis of patterns of reactivity of monoclonal antibodies to F protein (Gerna et al., 2005).
Although it is difficult to distinguish primary infection and reinfection with hMPV, we assumed that cases negative and positive for IgG antibody to hMPV-infected cells in acute-phase serum samples represent primary infection and reinfection, respectively. According to our criteria, we suspected that 29 cases were primary infection and 28 cases were reinfection in 57 cases for which acute-phase serum samples were available. As shown in Table 1 and Table 2, all but one of the 12 paired serum samples obtained from children during the acute phase of infection were negative for the antibody to hMPV G protein and were seroconverted in the convalescent phase. Six of the 12 paired serum samples were thought to be primary infection and the other 6 samples were thought to be reinfection, based on the results of hMPV-IFA, indicating that the antibody to hMPV G protein was seroconverted regardless of primary infection or reinfection.
Seropositivity and antibody titre to Bac-G protein in control serum samples were lower than those in convalescent-phase serum samples from patients infected with hMPV, indicating that IgG antibody to hMPV G protein transiently appears after onset of disease. Although we do not know how long IgG antibody to hMPV G protein remains after hMPV infection because of the small number of serum samples collected long after onset of illness without reinfection, antibody response to hMPV G protein of the infecting genotype of hMPV seemed to be transient after infection. The patterns of response revealed by Bac-G-IFA were closely related to the infecting genotype, genetic subgroup-specific A1, A2, B1 or B2. Furthermore, cross-reactive antibody to Bac-G protein was not present in human serum samples after hMPV infection. The specificity of three of the four Bac-G proteins was confirmed by results of tests of three paired serum samples from children with primary infection of three different subgroups of hMPV (Table 2). The antibody to hMPV G protein in human serum seemed to recognize a different epitope of G protein in each genetic subgroup. Two antigenic groups have also been identified on the basis of patterns of reactivity with group-specific antiserum to hMPV G protein in guinea pigs (Bastien et al., 2004). In the present study, antibody response to hMPV G protein in human serum was genetic subgroup-specific and had no cross-reactivity, indicating that hMPV has four serotypes. Human serum showed significant antigenic divergence between hMPV G proteins of the four genetic subgroups.
Monoclonal antibodies to the hMPV F protein have been shown to have strong neutralizing activity against the strains of both group A and group B hMPVs in vitro (Ma et al., 2005; Skiadopoulos et al., 2004, 2006). Since neutralizing activity by an antibody to hMPV F protein in serum masks the neutralizing activity by an antibody to hMPV G protein, it was impossible to examine whether an antibody to hMPV G protein in human serum has neutralizing activity. Although the hMPV F protein is the major contributor to induction of serum-neutralizing antibodies and protective immunity, the hMPV G protein induces a detectable level of serum-neutralizing antibodies, and the contribution of hMPV G protein to protection was minor in an animal model (Biacchesi et al., 2005; Skiadopoulos et al., 2006). Furthermore, a deletion mutant lacking the hMPV G gene can replicate efficiently in cell culture (Biacchesi et al., 2004), indicating that the hMPV G protein is not essential for growth in cell culture. However, the sequence variation within the hMPV G gene has been postulated to be due to immunological pressure. If the level of antibody to the hMPV F protein is high, the cross-reactive immunity provided by the hMPV F protein may be sufficient to overcome the effects of changes in the hMPV G protein. However, efficient neutralization of hMPV may require antibodies to both the F and G proteins as for hRSV (Anderson et al., 1988; Hancock et al., 2000).
We showed that subgroup-specific antibody to hMPV G protein of the infecting genotype of hMPV could be detected in serum samples from children shortly after infection with hMPV. hMPV infection in cynomolgus macaques demonstrates that hMPV infection induces transient protective immunity and that group A and group B viruses represent different serotypes (van den Hoogen et al., 2007). The subgroup-specific antibody to hMPV G protein transiently detected after infection in human serum also might be important for protection against infection with hMPV of the same subgroup as well as a neutralizing antibody to hMPV F protein. In addition, it seems that the four genetic subgroups of hMPV represent four different serotypes in human serum. The antibody to hMPV G protein detects subgroup-specific responses to hMPV infection and may be useful for the study of immune responses to hMPV. Further studies are needed to clarify the risk of reinfection with hMPV.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 21 December 2007;
accepted 21 April 2008.
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