Sequence Analysis of the Fusion Protein Gene of Human Respiratory Syncytial Virus Circulating in China from 2003 to 2014

www.nature.com/scientificreports OPEN Received: 9 May 2018 Accepted: 9 November 2018 Published: xx xx xxxx Sequence Analysis of the Fusion Protein Gene of Human Respiratory Syncytial Virus Circulating in China from 2003 to 2014 Jinhua Song1, Huiling Wang1, Teresa I. Ng2, Aili Cui1, Shuangli Zhu1, Yanzhi Huang3, Liwei Sun3, Zifeng Yang4, Deshan Yu5, Pengbo Yu6, Hong Zhang7, Yan Zhang1 & Wenbo Xu1 The human respiratory syncytial virus (HRSV) fusion (F) protein is important for HRSV infection, but few studies have examined the genetic diversity of the F gene from Chinese samples. In this study, a total of 330 HRSV F sequences collected from different regions of China between 2003 and 2014 were analyzed to understand their genetic characteristics. In addition, these sequences were compared with 1150 HRSV F sequences in Genbank from 18 other countries. In phylogenetic analysis, Chinese HRSV F sequences sorted into a number of clusters containing sequences from China as well as other countries. F sequences from different genotypes (as determined based on the G gene sequences) within a HRSV subgroup could be found in the same clusters in phylogenetic trees generated based on F gene sequences. Amino acid analysis showed that HRSV F sequences from China and other countries were highly conserved. Of interest, F protein sequences from all Chinese samples were completely conserved at the palivizumab binding site, thus predicting the susceptibility of these strains to this neutralizing antibody. In conclusion, HRSV F sequences from China between 2003 and 2014, similar to those from other countries, were highly conserved. Human respiratory syncytial virus (HRSV) is one of the leading pathogens causing lower-respiratory tract infections in infants and young children worldwide1,2. HRSV is a single-stranded, negative-sense RNA virus in the Pneumoviridae family. The attachment glycoprotein (G protein) and the fusion glycoprotein (F protein) are the two major glycoproteins on the HRSV surface. The G protein mediates the viral attachment to the host cells whereas the F protein mediates viral penetration and fusion of the infected cells3–5. HRSV could be divided into two subgroups, subgroup A (HRSVA) and subgroup B (HRSVB), based on the antigenic characteristics and the reactivity with monoclonal antibodies6. The HRSV G gene sequence is highly variable. Based on the sequences of the second hypervariable region of the G gene, HRSV strains from each subgroup are further classified into different genotypes. To date, 15 genotypes of HRSVA have been identified (GA1~7, NA1~4, ON1~2, SAA1, CBA)7,8 whereas 30 genotypes of HRSVB have been identified (GB1~4, BA1~14, BAc, SAB1~4, URU1~2, CB1(GB5), CBB, BA-CCA, BA-CCB and THB)9–14. According to the phylogenetic analysis of the G gene, the same predominant clades of HRSV circulated globally, and when different HRSV strains emerged, the distribution of the old clades could be changed15. The F protein is synthesized as a precursor F0 protein [574 amino acids (aa) in length]. When the F0 protein passes through the Golgi, it can be activated by the cleavage with a furin-like intracellular host protease at 2 sites 1 WHO WPRO Regional Reference Measles/Rubella Laboratory and Key Laboratory of Medical Virology, National Health Commission of the People’s Republic of China, National Institute for Viral Disease Control and Prevention, China Center for Disease Control and Prevention, Beijing, People’s Republic of China. 2AbbVie, Inc, North Chicago, IL, USA. 3Jilin Children’s Medical Center, Children’s Hospital of Changchun, Changchun, People’s Republic of China. 4 State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, People’s Republic of China. 5Gansu Provincial Centers for Disease Control and Prevention, Lanzhou, People’s Republic of China. 6Shaanxi Provincial Centers for Disease Control and Prevention, Xian, People’s Republic of China. 7Hunan Provincial Centers for Disease Control and Prevention, Changsha, People’s Republic of China. Correspondence and requests for materials should be addressed to Y.Z. (email: ) or W.X. (email: ) SCIeNtIFIC REPOrTS | (2018) 8:17618 | DOI:10.1038/s41598-018-35894-3 1 www.nature.com/scientificreports/ Region Dongbei Huabei Huadong Xibei Zhongnan Xinan Province/ City Jilin Beijing Hebei Shanghai Gansu Shaanxi Hunan Guangdong Chongqing 2003 — — 3(0) — — — — — — 3(0) 2004 — 6(6) — — — — — — — 6(6) Total 2008 — 5(5) — — — — — 5(0) — 10(5) 2009 1(1) 26(26) — — — — — 2(2) 2(0) 31(29) 2010 — 9(9) 1(1) 4(4) 5(5) 5(5) — 11(11) 18(0) 53(35) 2011 — 1(1) — — 8(8) 3(3) 1(1) 28(27) 81(0) 122(40) 2012 7(7) — — — — — 5(5) 2(1) 31(0) 45(13) 2013 8(8) — 4(4) — — 5(5) 8(8) 1(0) 5(0) 31(25) 2014 12(12) 10(10) 2(2) 1(0) — — 4(4) — — 29(29) Total 28(28) 57(57) 10(10) 5(4) 13(13) 13(13) 18(18) 49(41) 137(0) 330(181) Table 1. Distribution of Chinese HRSV samples by geographical region and year. Numbers shown are the sum of sequences collected in this study and those downloaded from GenBank; sequences collected in this study are shown within parentheses. after amino acid residues 109 and 136 to generate three polypeptides: F1 (aa 137–574), F2 (aa 1–109) subunits and an intervening 27 amino acid peptide, pep27, (aa 110–136)16,17. The mature F protein is a homotrimer of the F1 and F2 subunits, and the F1 subunit is essential for the protein to cause membrane fusion. The F0 precursor contains 5 or 6 predicted N-linked glycosylation sites depending on the HRSV strain. After activation, 2 predicted N-linked glycosylation sites in F2, 1 predicted N-linked glycosylation site in F1 and 2–3 predicted N-linked glycosylation sites in in the pep27 are left18,19. The F protein has been identified as having at least two dominant conformations: the prefusion and postfusion forms20. The functional F protein trimer in the virion membrane is in a metastable, prefusion form. This prefusion F protein had a ‘lollipop’ shape by electron microscopy21,22. In the prefusion form of the F1 protein, the fusion peptide at the N terminus of F1 is followed by 4 short α-helices connected by 3 non-helical peptides5. The structure of the postfusion F protein revealed a cone-shaped molecule, with a globular head and an extended stalk21. Three F2/F1 subunits that make up the trimeric molecule are tightly intertwined, with 3-fold symmetry that runs the length of the molecule. The globular head contains both the F2 and F1 subunits, as well as the cysteine-rich region. The stalk region is almost entirely helical, composed of the 6-helix bundle that is characteristic of the postfusion state of many type I viral fusion proteins5,21,23. The F protein is a target of virus-specific cytotoxic T lymphocytes (CTLs). Three related human (...truncated)