Arch Virol DOI 10.1007/s00705-013-1870-9

ORIGINAL ARTICLE

Detection of respiratory syncytial virus fusion protein variants between 2009 and 2012 in China Qiuling Xia • Lili Zhou • Caijing Peng • Rui Hao • Ke Ni • Na Zang • Luo Ren • Yu Deng • Xiaohong Xie • Linli He • Daiyin Tian • Lijia Wang • Ailong Huang • Yao Zhao • Xiaodong Zhao • Zhou Fu • Wenwei Tu • Enmei Liu

Received: 22 March 2013 / Accepted: 23 September 2013 Ó Springer-Verlag Wien 2013

Abstract Respiratory syncytial virus (RSV) causes respiratory tract infection, particularly acute lower respiratory tract infection (ALRTI), in early childhood. The RSV fusion protein (F protein) is an important surface protein, and it is the target of both cytotoxic T lymphocytes (CTL) and neutralizing antibodies; thus, it may be useful as a candidate for vaccine research. This study investigated the genetic diversity of the RSV F protein. To this end, a total of 1800 nasopharyngeal aspirates from hospitalized children with ALRTI were collected for virus isolation between June 2009 and March 2012. There were 333 RSVpositive cases (277 cases of RSV A, 55 of RSV B, and 1 with both RSV A and RSV B), accounting for 18.5 % of the total cases. Next, 130 clinical strains (107 of RSV A, 23 of RSV B) were selected for F gene sequencing. Phylogenetic analysis revealed that the F gene sequence is highly conserved, with significant amino acid changes at residues 16, 25, 45, 102, 122, 124, 209, and 447. Mutations in human histocompatibility leukocyte antigen (HLA)-

The GenBank accession numbers of the nucleotide sequences of complete RSV fusion protein genes obtained in this study are JX482018-JX482038 and JX682715-JX682823.

restricted CTL epitopes were also observed. Variations in RSV A F protein at the palivizumab binding site 276 (N?S) increased between 2009 and 2012 and became predominant. Western blot analysis and microneutralization data showed a substitution at residue 276 (N?S) in RSV A that did not cause resistance to palivizumab. In conclusion, the RSV F gene is geographically and temporally conserved, but limited genetic variations were still observed. These data could be helpful for the development of vaccines against RSV infection.

Introduction Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection (ALRTI) in early childhood, accounting for at least 3.4 (2.8-4.3) million new cases annually worldwide. Furthermore, 66,000-199,000 children younger than 5 years of age die from RSV-associated ALRTI each year [18]. In China, RSV has been responsible for 19.3 % to 50.9 % of acute respiratory tract infection in children [19, 33], and these infections reach a peak in the winter and early spring, with a circulating

Q. Xia  L. Zhou  C. Peng  R. Hao  K. Ni  N. Zang  L. Ren  L. Wang Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing Medical University, Chongqing 400014, China

A. Huang Chinese Ministry of Education Key Laboratory of Molecular Biology on Infectious Diseases, Chongqing Medical University, Chongqing 400014, China

Y. Deng  X. Xie  L. He  D. Tian  Z. Fu  E. Liu (&) Department of Respiratory Medicine, Children’s Hospital of Chongqing Medical University, Chongqinq 400014, China e-mail: [email protected]

W. Tu Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong SRA, Pokfulam, China

Y. Zhao  X. Zhao Department of Immunity, Children’s Hospital of Chongqing Medical University, Chongqing 400014, China

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pattern in which RSV A is predominant for two years, followed by one year in which RSV B predominates [34]. To date, prophylactic medication is the only treatment used to prevent RSV infection, as there are no effective and safe vaccines available for this purpose. RSV belongs to the family Paramyxoviridae and the subfamily Pneumovirinae. The RSV genome encodes at least 11 viral proteins. The RSV fusion protein (F protein) is an important surface protein that mediates binding of RSV to cellular receptors, which allows the virus to enter the host-cell cytoplasm. It is also responsible for the fusion of infected cells with adjacent cells [30]. The F protein is homologous in both subtypes of RSV (RSV A and RSV B), and antibodies against the F protein can neutralize virus infectivity. Host cytotoxic T lymphocytes (CTLs) target the F protein to eliminate RSV infection, and to date, many human histocompatibility leukocyte antigen (HLA)restricted CTL epitopes of the RSV F protein have been identified. For example, Shao et al. reported four peptides of the F protein that can bind to HLA-A*0201 [29], and Rock et al. identified a peptide in the F protein, F109-118 (RELPRFMNYT), that is an HLA-A*01-restricted CTL epitope [27]. In addition, Brandenburg et al. confirmed that F118–126 (RARRELPRF) and F551–559 (IAVGLLLYC) are epitopes that are present in the context of the HLAB*57 and HLA-Cw*12 alleles, respectively [6]. Moreover, it has been shown that palivizumab, a humanized RSV monoclonal antibody that binds to the RSV F protein at the amino acids sites between 256 and 276, provides passive immunity against RSV infection, and has been licensed for prevention of serious lower respiratory tract infections caused by RSV in children at high risk (particularly for those who are premature or with chronic lung disease or congenital heart disease). Thus far, it has proved to be effective for reducing the frequency of hospitalization of such children [3], and it is a useful means of reducing comorbidity and fatal outcome for high-risk patients [26]. Variations occasionally occur in the F protein. Kim et al. found that the F gene continuously evolves in a pattern that is associated with the genetic variability of the G protein under selective pressure [13]. The F proteins from different RSV strains are associated with different levels of pathogenicity in BALB/c mice [17]. Palivizumab-resistant strains have not only been successfully generated in the laboratory (e.g., with the mutations N262S, N268I, K272N/ M/T/Q, and S275F/L) [35–37] but are also found in clinical isolates (with the mutations N262S/D, K272E, K272Q and S275F/L) [21, 38]. Variants of the RSV F protein that are resistant to other monoclonal antibodies or small molecular compounds have also been discovered recently [7, 14]. Thus, investigating the genetic diversity of the RSV F protein can help in the development of effective vaccines against RSV infection.

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In this study, we isolated different strains of RSV from clinical samples and then selected some for full-length sequence analysis of the RSV F gene. We identified mutations located in T cell epitopes and analyzed the binding ability of palivizumab to these clinical strains.

Materials and methods Specimen collection Between June 2009 and March 2012, we recruited a total of 1800 children with ALRTI, (with symptoms such as cough, expectoration, tachypnea, and wheezing) from the Department of Respiratory Medicine at the Children’s Hospital of Chongqing Medical University in China. Nasopharyngeal aspirates (NPAs) were collected when the patients were admitted to our department. This study was authorized by the Ethics Committee of the Children’s Hospital of Chongqing Medical University. The guardians of patients signed informed consent forms to participate in this study. Virus isolation and nucleic acid extraction Human epidermoid carcinoma Hep-2 cells were maintained in Dulbecco modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 50 U/mL penicillin and 50 lg/mL streptomycin (all from Invitrogen, Carlsbad, CA). Cells were seeded and cultured to 70 to 80 % confluence, and clinical specimens were then inoculated onto Hep-2 cells and cultured for 2 h. Next, the inoculum was withdrawn by aspiration, and the cells were washed twice with phosphate-buffered saline (PBS) and refed with fresh DMEM supplemented with 2 % FBS. Cells were scraped when cytopathic effect (CPE) involved at least 75 % of the Hep-2 cell monolayer, after which virus-cell suspensions were collected, aliquoted, and stored at -80 °C. However, if the cells showed no CPE after being cultured for 7 days, they were scraped, and the cell suspensions were collected for a second inoculation. Two clinical strains (RSVA, CQ_May_2011/1712 and RSV B, CQ Jul_2011/1990) were selected for seven sequential passages in Hep-2 cells. The harvested virus-cell suspensions were centrifuged at 2,500 rpm for 5 min at 4 °C, and the supernatants were used for viral RNA extraction according to the manufacturer’s instructions (QIAGEN, Hilden, Germany). RNA of strain CQ Jun_2012/2818 from NPA was also isolated for the analysis. Reverse transcription-polymerase chain reaction (RT-PCR) The extracted viral RNA was reverse transcribed into cDNA using a SuperScript II Reverse Transcriptase System

Detection of RSV F protein variants Table 1 Primers used for PCR amplification of the F gene Primer

Target gene

Positiona

Primer sequence

Size of PCR product (bp)

A-5648-S

RSV A F

5648-5671

5’-GGGGCAAATAACAATGGAGTT-3’

1771

7394-7418

5’-CATTGTAAGAACATGATTAGGTGCT-3’

RSV B F

5502-5524

5’-CGAAAACACACCCAACTCCACAC-3’

7381-7402

5’-GTGGTTTTTTGTCTATTTGCTG-3’

A-7418-AN B-5502-S B-7402-AN

1901

S sense primer, AN antisense primer a

‘‘Position’’ refers to the A2 strain (M74568.1) for RSV A and wild-type B1 (AF013254.1) for RSV B

Table 2 Primers used for DNA sequencing of the F gene

S sense primer, AN antisense primer a

‘‘Position’’ refers to the A2 strain (M74568.1) for RSV A and wild-type B1 (AF013254.1) for RSV B

Primer

Target gene

Positiona

Primer sequence

A-6611-AN

RSV A F

6592-6611

5’-GTGTAGTTTCCAACAAGGAG-3’

A-6227-S

6227-6247

5’-CAGCAAAGTGTTAGACCTCAA-3’

A-6852-S

6852-6869

5’-TCAAAAACAGATGTAAGC-3’

6386-6403

5’-AGGTGTTGTTACACCTGC-3’

B-6206-S

6206-6226

5’-CTATCAAATGGGGTCAGTGTT-3’

B-6803-S

6803-6820

5’-AGCCTTTGTAACACTGAC-3’

B-6403-AN

RSV B F

(Invitrogen). Briefly, 10 ll of total RNA was mixed with 1 ll dNTP and 1 ll random primer, heated at 65 °C for 5 min, and quickly chilled on ice. Next, 4 ll of 59 buffer, 1 ll of 0.1 M DTT, 1 ll of RNase-OUT, and 1 ll of SuperScript II RT were added into the mixture, followed by incubation at 25 °C for 5 min, 50 °C for 60 min, and 70 °C for 15 min. Subgroup A and B identification was performed by multiplex PCR as described previously [34]. The mRNA of the RSV F gene was then amplified by PCR with subgroup-specific primers (Table 1). The PCR conditions for RSV A were as follows: an initial denaturation step at 94 °C for 2 min and 35 cycles at 94 °C for 30 s, 52 °C for 30 s, and 68 °C for 2 min, followed by a final extension at 68 °C for 10 min. For PCR amplification of RSV B, the thermal cycling conditions were the same as those for RSV A, except that the annealing temperature was set at 50 °C. DNA sequencing of the F gene In all, 130 clinical strains of RSV were selected for F gene sequencing. These strains were selected monthly by the sortition randomization method in proportion to the RSV isolates we had collected each month and obtained from different patients (patients did not have contact with each other). The PCR products of the RSV F gene were sequenced using their sequence primers, and the sequenced contiguous segments overlapped by at least 200 base pairs. Nucleotide sequencing was performed on an Applied Biosystems 3730xl DNA Sequencer. Subgroup-specific primers were used for sequencing the whole F gene (Table 2).

Phylogenetic analysis DNA sequence contigs were spliced using SeqMan (DNASTAR Lasergene version 7.1). Multiple sequence alignment of the entire F gene was done using Clustal W in MEGA (version 5.05) and Bioedit (version 7.0.9). Prototype strain A2 (GenBank accession number, M74568.1) and wild-type B1 (GenBank accession number, AF013254.1) were used as the reference strains. Amino acid analysis, pairwise distance (P-distance) calculation, and neighbor-joining tree construction were performed using MEGA 5.05. Protein extraction and western blot analysis Hep-2 cells were infected with the A2 strain and clinical strain (CQ Feb-2011/1373) at an MOI of 0.1; negative controls had only PBS. Protein was extracted from cells 48 h after infection. After quantification, protein samples were resolved on 10 % SDS-PAGE gels by electrophoresis and then transferred to nitrocellulose membranes. The membranes were then subjected to western blot analysis using a 1:5000 dilution of palivizumab (a gift from Prof. Zhao) as the first antibody and a 1:2000 dilution of goat anti-human HRP-conjugated IgG (CWBIO, Beijing, China) as the secondary antibody, and the target protein was visualized using enhanced chemiluminescence (Beyotime, Shanghai, China). Microneutralization assay A microneutralization assay was carried out as described previously [1, 4] with some modifications. Briefly, 100

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50 % tissue culture infective doses (TCID50) of RSV strain A2 and clinical strain CQ Feb-2011/1373 that had a substitution at position 276 (N?S) were incubated with equal volumes of serially diluted palivizumab at 37 °C for 1 h and then added to Hep-2 cells. Five days later, the CCK-8 cell viability reagent (Dojindo, Japan) was added to the cell culture medium, and percent cell survival was determined based on absorbance values at 450 nm. Neutralizing ability was expressed as palivizumab concentration versus percentage cell survival compared to control wells.

Results

Fig. 1 Phylogenetic analysis of the RSV fusion gene. a Phylogenetic c tree of RSV A. b Phylogenetic tree of RSV B. To analyze phylogenetic relationships of the RSV F gene, unrooted phylogenetic trees were constructed based on the full-length F gene by using the neighbor-joining method in MGEA version 5.05. Clinical strains collected in China are indicated in bold and italics, and strains isolated from different years are indicated by symbols, (i.e., d, September 2009 - August 2010; e, September 2010 - August 2011; j, September 2011 - March 2012). To build the RSV A phylogenetic tree, a total of 74 clinical strains with different sequence variations were used. To build the RSV B phylogenetic tree, all 23 RSV B isolates were included. The accuracy was analyzed using 1,000 bootstrap replicates. Only bootstrap values equal to or more than 70 % are shown. The phylogenetic tree data suggest that the F gene is geographically and temporally conserved with little variation. For RSV A, almost all strains could be classified into one cluster, with the exception of two distinct strains (CQ Dec-2010/1148 and CQ Mar2012/3155). For RSV B, there were three separate branches. Strains isolated between 2009 and 2010 or between 2010 and 2011 were distributed into the three different branches, but strains isolated between 2011 and 2012 were in the same branch

In this study, we recruited a total of 1800 patients with ALRTI between June 2009 and March 2012. The ages of the patients ranged from 1 month to 140 months with a median age of 5 months. The male-to-female ratio was 2.03:1.00. NPAs were collected for virus isolation. Our data showed that 333 cases (18.5 %) were RSV positive (277 RSV A, 55 RSV B, and 1 with both RSV A and RSV B). However, RSV A strains were only isolated between 2010 and 2012.

phylogenetic tree (Fig. 1B). Strains isolated between 2009 and 2010 or between 2010 and 2011 were distributed between the three different branches, but strains isolated between 2011 and 2012 were all classified in the same branch (Fig. 1B).

Phylogenetic analysis of RSV isolates

Analysis of RSV amino acid sequences

To explore the genetic diversity of the F protein, we selected 130 RSV isolates (107 RSV A and 23 RSVB) to sequence the full-length F gene. We found that there were 74 variants for RSV A, of which similar variants could be isolated from the same or different epidemic seasons. There were 20 distinct variants for RSV B, of which identical variants were collected from the same epidemic season. Next, we compared them with prototype strains and found that the nucleotide P-distance ranged from 0.046 to 0.052 with an average of 0.050 for RSV A, and from 0.017 to 0.027 with an average of 0.024 for RSV B. However, when calculated within clinical strains, the P-distance was 0.005 for RSV A, 0.011 for RSV B, and 0.179 between A and B subgroups. To define whether RSV mutation occurs during virus passage in Hep-2 cells, we sequenced the F gene of CQ Jun_2012/2818 from NPA, the seventh passage of CQ May_2011/1712 and CQ Jul_2011/1990. Our data showed that sequences of the F gene were the same as those of CQ Jun_2012/2818, CQ May_2011/1712, and CQ Jul_2011/ 1990, respectively. Thus, although we cannot rule out the possibility that mutations occurred, it is reasonable to believe that the amount of genetic variation was minimal. A phylogenetic tree of the RSV F gene showed that almost all RSV A strains could be classified into one cluster, with the exception of two isolates; namely, CQ Dec-2010/1148 and CQ Mar-2012/3155 (Fig. 1A). In contrast, three separate branches were found in the RSV B

Next, we analyzed the F proteins of these different RSV isolates and found that the F gene had little variation at the amino acid level. In addition, all amino acid mutations were attributed to base substitutions, and not deletions, insertions, or frame shift mutations. Amino acid sequence identities of different regions were calculated according to the p-distance (Table 3). For example, for RSV A, the most variable region was peptide 27 followed by the F2 subunit, and for RSV B, the most variable region was the F2 subunit. Between subgroups, peptide 27 and the F2 subunit contained the most variations. Among RSV A clinical strains, some interesting amino acid changes were revealed, including 16 (T?A), 20 (F?L), 25 (G?S), 102 (P?A), 122 (A?T), 124 (K?N), 139 (V?G), 152 (V?I), 178(L?V), 379 (I?V), and 447 (M?V) changes, which caused these RSV A clinical strains to share the same amino acids with the wild-type B1 strain and RSV B isolates. Specifically, 16 (T?A), 25 (G?S), 102 (P?A), 122 (A?T), 124 (K?N) and 447 (M?V) variations changed the biochemical characteristics of the amino acids, which may result in secondary structure changes. Meanwhile, all RSV B clinical strains had an N?T substitution at residue 234, 82.6 % (19/23) had an F?L substitution at residue 45, and 21.7 % (5/23) had a Q?K substitution at residue 209. The mutation at residue 209 (Q?K) also changed the biochemical property of the amino acid.

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Detection of RSV F protein variants

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Q. Xia et al. Table 3 Amino acid sequence identities as a percentage in different regions of the F protein Region Locationa

F2 1-108

Peptide 27 109-136

F1 137-525

TM 525-548

CT 549-575

Total 1-575

RSV Ab

92.55

92.50

98.17

95.65

99.93

96.82

c

RSV B

97.57

99.19

99.46

95.46

100

98.95

Between subgroupsd

81.76

70.17

94.92

82.42

88.46

90.46

a

The location of each region was according to a previous study [8]

b

Identity of different regions between RSV A and prototype A2

c

Identity of different regions between RSV B and prototype wild-type B1

d

Identity of different regions between RSV A and RSV B isolates. Identity was calculated according to pairwise distance at the amino acid level F2, F2 subunit; peptide 27, cleaved peptide in the process of F protein maturation; TM, transmembrane region; CT, cytoplasmic tail

Cytotoxic T lymphocyte (CTL) epitope analysis The RSV F protein is a target for CTLs, and the variation within epitopes may explain the immune evasion by the virus and the emergence of new genotypes. Thus, in this study, we aligned and compared the amino acids in HLArestricted CTL epitopes (Fig. 2) and found that among these RSV A isolates, the HLA-A*0201-restricted CTL epitopes F214-222 and F273-281 were conserved; 3.7 % (4/107) of clinical strains had point substitutions at F33–41; 98.1 % (105/107) of clinical strains had an S?A substitution, and 1.9 % (2/107) of strains had an A?V change at F559–567. However, all clinical strains were conserved at the HLA-A*01-restricted CTL epitope. At HLA-B*57-restricted epitopes, 0.9 % (1/107) of the isolates had an A?T substitution, and at HLA-Cw*12restricted CTL epitopes, 1.8 % (2/107) of the isolates had an A?V substitution. Furthermore, among RSV B strains, the HLA-A*0201-restricted CTL epitopes F214-222 and F273-281 were conserved as in RSV A clinical strains. All RSV B strains had an amino acid change (V?I) at F559–567; two to three sites with amino acid substitutions were found in the other reported HLA-restricted epitopes listed in Figure 2. RSV A F protein variation in the palivizumab-binding site at 276 In a recent study, the substitution 276 (N?S) was found in RSV A, and it was speculated that it may be because of a potential effect on palivizumab resistance [1]. Nevertheless, palivizumab has not been introduced in China; and, to date, the prevalence of 276 (N?S) in RSV A remains unknown. Our current data show that the frequency of 276

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(N?S) was 95.2 % (40/42) and 96.9 % (63/65) during 2010-2011 and 2011-2012, respectively. Several RSVA-positive specimens were collected during the 2007-2008 and 2008-2009 RSV epidemic seasons for further studies of the 276 (N?S) substitution. The frequency of N276S substitutions was 25 % (1/4) during 2007-2008, and 33.3 % (1/3) during 2008-2009. The reported palivizumabresistance mutations at other sites were not found, and other F-related drug-resistant variations [7] were not found among our clinical isolates. To assess the ability of palivizumab to bind the RSV A clinical strain containing the 276 (N?S) substitution, we performed western blot and microneutralization analyses. Our western blot data allowed the visualization of specific bands corresponding to both the RSV A2 and CQ Feb2011/1373 clinical strains (Fig. 3), indicating that palivizumab could bind sufficiently to clinical strains with the 276 (N?S) substitution. Also, the western blot revealed that the F1 bands of the CQ Feb-2011/1373 clinical strains migrated more slowly on the gel than the A2 strain. Moreover, microneutralization data revealed that palivizumab could react effectively with A2 and CQ Feb-2011/ 1373 (Fig. 4); the IC50 values were 0.68 ± 0.07 lg/ml and 0.94 ± 0.26 lg/ml, respectively. A previous study showed that CQ Feb-2011/1373 was sensitive to palivizumab [38].

Discussion The F protein is the major focus of research on prophylactic antibodies and recombinant vaccines [9, 11, 31]. Despite the conserved nature of the F protein, variations do occur among different strains or between RSV subtypes. Although there are limited molecular epidemiological studies of the F gene, most of them have monitored F gene mutations that lead to palivizumab resistance [1, 21, 37]. In the current study, we collected 1,800 clinical samples from China between June 2009 and March 2012. We then sequenced the RSV F gene in 130 clinical strains and found that the sequence identity within a subgroup was more than 98 % at the nucleotide level, consistent with a report by Agenbach et al. [2]. Analysis of the phylogenetic relationships of the RSV F gene isolates from China showed high homology in strains collected from other countries during different time periods, suggesting that the F gene is geographically and temporally conserved with little variation. However, we did find greater variability in the RSV B clinical strains compared to RSV A. According to our surveillance data from between June 2009 and March 2013, 33.93 % of NPAs from children with acute respiratory tract infection were positive for RSV. The predominant strains ware RSV B during the 2009-2010 epidemic season, and this changed dramatically to RSV A during 2010-2012

Detection of RSV F protein variants

Fig. 2 Amino acid sequence alignment of RSV F genes of RSV isolates from China. Representative strains (9 each for RSV A and RSV B) were selected, and their sequences were aligned. HLArestricted CTL epitopes are highlighted in the boxes. Between RSV A and RSV B, F214-222 and F273-281 were identical; Within F33-41,

F118-126, and F109-118, there were at least 3 amino acid alterations. Residues spanning F559-567, F551-559 of both RSV A and RSV B had 1-2 amino acid mutations. From residue 300 to 500, there are no reported HLA-restricted CTL epitopes, so the alignment of this region was not displayed here

epidemic seasons (our unpublished data). Hence, the higher rate of mutation among RSV B strains was potentially due to the fact that RSV A strains were collected from two

continuous RSV-A-prevalent epidemic seasons, but the RSV B strains were predominantly from one RSV B and two RSV A prevalent epidemic seasons. Phylogenetic

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Fig. 3 Ability of palivizumab to bind to an RSV A clinical strain with the 276 (N?S) substitution. Total cellular protein was extracted from infected and mock-infected cell lysates and then subjected to western blot analysis of palivizumab expression. A2, RSV strain A2; CQ Feb-2011/1373, clinical isolate CQ Feb-2011/1373 (an RSV A strain with substitution N?S at residue 276); Mock, mock infection group. Both A2 and CQ Feb-2011/1373 displayed specific bands at the target size of the F1 subunit, suggesting that palivizumab sufficiently binds to clinical strains with the 276 (N?S) substitution. The results also revealed that the F1 bands of the CQ Feb-2011/1373 clinical strains migrated more slowly in the gel than that of the A2 strain

Fig. 4 Microneutralization analysis of the ability of palivizumab to neutralize RSV A containing a 276 (N?S) substitution. Palivizumab concentrations are shown in lg/ml (x-axis), and the percentage of living cells is shown on the y-axis. The IC50 was 0.68 ± 0.07 lg/ml for A2 and 0.94 ± 0.26 lg/ml for CQ Feb-2011/1373. CQ Feb-2011/ 1373 can be effectively neutralized by palivizumab according to a previous study [38]

analysis showed that clinical isolates of RSV B collected between 2009 and 2011 were distributed among three branches, but strains isolated between 2011 and 2012 were from the same branch. These data indicate that isolates

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from between 2011 and 2012 may become the predominant RSV B strain in the next RSV-B-prevalent epidemic season. Thus, such data will provide better understanding of the prevalence of RSV and help us to predict circulating strains for future epidemic in China. The most variable region was peptide 27, followed by the F2 subunit; however, peptide 27 is cleaved in the process of F protein maturation in RSV A and RSV B. These data are consistent with those from previous studies [13, 23] that have indicated that most of the differences between subgroups A and B are located at the F2 subunit in the mature F protein. Thus, a previous study suggested that the F2 subunit may function as the determinant of RSV host specificity and may facilitate recognition of virus receptors [28]. Furthermore, previous studies have indicated that different RSV strains may differ in their ability to form syncytia in Hep-2 cells [32], viral load, and disease severity [20, 22], and there may be further distinctions between genotypes [15]. Taken together, it appears that the F2 subunit may play a key role in these differences, but further investigation is needed to confirm this. RSV has two furin cleavage sites, FCS-1 (RKRR136) and FCS-2 (RAR/KR109), and cleavage at both sites were required for the RSV- F protein to induce syncytia formation [10, 39]. For example, insertion of these two cleavage sites of the RSV F protein into Sendai virus fusion protein resulted in enhanced cell-cell fusion but a decreased dependence on the hemagglutinin-neuraminidase attachment protein for activity [24]. However, substitutions at residues 16, 25, 102, 122, 124, 209, or 447 altered the biochemical characteristics of the amino acids, which may affect the secondary structure of the fusion protein. Hence, changes in residues 102, 122, and 124 of the RSV A F protein, which are adjacent to furin cleavage sites (FCS-1 and FCS-2) may affect the progress of cleavage and in turn lead to differential pathogenesis of the virus. According to McLellan et al., anti-human D25 antibody recognizes the metastable pre-fusion conformation of the RSV F protein (residues 196-210) at the apex of the pre-fusion RSV F structure, which may and serve as a determinant of subtype-specific immunity [16]. In our current study, the 21.7 % (5/23) of RSV B clinical isolates had a Q?K substitution at residue 209. It is urgent to confirm the role of the Q?K substitution at residue 209 in the RSV A/B circulating pattern. In addition, the identified HLA-restricted CTL epitopes contained changes either within subgroups or between subgroups, except residues spanning F214–222 and F273–281 CTL epitopes. Changes within subgroups result in positive selection of the RSV F protein [5]. To date, most published studies on HLA-restricted CTL epitopes have been based on RSV A [6, 27, 29]. However, among the RSV B clinical isolates, 2-3 amino acid substitutions

Detection of RSV F protein variants

were found in epitopes spanning F33–41, F109-118, F118–126, and F551–559. Further studies are needed to determine if these variations affect recognition of host CTL cells. Our current data indicate that future studies should include both subgroups of F-protein CTL epitopes and that analysis of amino acids at the CTL epitopes will be valuable for epitope-based RSV vaccine design [29] and for investigation of the mechanism by which RSV escapes immunosurveillance. Palivizumab is a monoclonal antibody produced by recombinant DNA technology to prevent RSV infections in high-risk infants, such as premature infants or those with congenital heart disease. However, Adams et al. reported that an amino acid change in the RSV A F protein at residue 276 (N?S) alters the ability of palivizumab to bind to the F protein, resulting in palivizumab-resistant clinical RSV strains [1]. Later, however, Zhu et al. indicated that the amino acid change in N276S of RSV F did not confer resistance to palivizumab and the observations of Adams et al. were instead attributed to a viral subpopulation with a secondary K272E mutation which becomes apparent under in vitro selection pressure in the presence of palivizumab [38]. Our current western blot and microneutralization analyses showed that the substitution at residue 276 (N?S) in the RSV A F protein does not alter palivizumab binding or cause resistance to palivizumab, which is consistent with the data reported by Zhu et al. [38] and Papenburg et al. [21]. Moreover, palivizumab has been not introduced to China, although alterations at this site frequently occur in Chinese RSV isolates. These data support the conclusion that this site indeed has genetic polymorphism. On the other hand, the percentage of isolates with alterations in the F protein at 276 (N?S) is increasing, and this mutation became prevalent during 2010-2012 in China, with a similar tendency in Canada [21]. This may be related to the antigenic drift for viruses to become a dominant strain in a given human population [12, 25]. Furthermore, western blot data showed different migration patterns of F1 bands between CQ Feb-2011/1373 clinical strains and the A2 strain, suggesting the existence of an inherent size/mobility difference between the RSV F from A2 and CQ clinical isolates. However, further investigation is needed to clarify whether the 276 (N?S) strain also contains crucial changes at other antigenic sites and to explain the difference in RSV F between A2 and the CQ clinical isolates. In conclusion, the current study further demonstrates that RSV F genes share great identity geographically and temporally worldwide, including in China. However, genetic variations were also observed, especially the topology changes in the RSV B phylogenetic tree, amino acids variations in CTL epitopes, and the alterations of 276 (N?S) in RSV A. This is the first large-scale molecular epidemiology study of the RSV F gene in China. The data

on F gene may provide information that will aid in the understanding of RSV and help in the future development of novel vaccines and drugs against RSV infection. Acknowledgements We thank the staff at Department of Respiratory Medicine for assistance in collection of clinical specimens. We thank all the patients or individuals for their enrollment and participation for this study. This work was supported in part by a China Special Grant of the Prevention and Control of Infection Diseases [2009ZX10004-204] and a grant from the Second Colleges and Universities Excellent Talents Program in Chongqing (2011.12012.12). Conflict of interest of interest.

The authors declared that there are no conflicts

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Detection of respiratory syncytial virus fusion protein variants between 2009 and 2012 in China.

Respiratory syncytial virus (RSV) causes respiratory tract infection, particularly acute lower respiratory tract infection (ALRTI), in early childhood...
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