Compared with NA1 and GA2 genotypes, there are some significant amino-acid substitutions, parts of which have been found in Lanzhou and some not. Amino acid positions 215 and 230 are highly variable compared with reference strain GA2. However, this variation either in NA1 or ON1 genotypes was never shown, and the amino-acid substitutions L215P and S230P were conservatively replaced. What have been confirmed in our study is that there are 3 amino acid substitutions, that is, ON1 (232G, 253K, 290L) and NA1 (232E, 253T, 290P) viruses. Importantly, G gene in ON1 genotypes lacks 2 N-glycosylation sites due to the mutations of 2 amino acids T253K and N273Y, which has also been found in our study. Notably, compared with the ON1 prototype, because of the mutations of 2 amino acids P274S and S299N, new N-glycosylation sites occurred within the 23aa duplication and 24aa insertion of the ON1 viruses in our study, which had not been previously described.
4.5 Patient data and clinical diagnosis
In our study, we analyzed demographic and clinical data from 468 RSV-A cases and found differences between infections with ON1 versus non-ON1 strains. ON1 patients were more likely to be younger males receiving inpatient treatment (P = .044), similar to data record in an Italian report for 2011 to 2013. Infections were more severe than those caused by non-ON1 strains. ON1 infection is more commonly diagnosed with bronchiolitis and wheezing is a more common symptoms. However, for the ON1 patients, the PICU occupancy rate is not high (not statistically different from non-ON1) (P = .4), which is different from Italy and German Studies. In addition, ON1 patients spend more days in the hospital (P = .002).
HRSV is one of the most common pathogens causing severe ARTIs, accounting for 15% to 40% of pneumonia/bronchiolitis cases in children. RSV has been detected for 61 years, but a vaccine has not yet been developed due to constant changes in RSV antigens. The biggest change reported is a 20-aa insertion in the G protein in RSV-B BA strains detected in Buenos Aires in 1999, until the detection of the RSV-A ON1 genotype. Emerging RSV variants that possess a selective advantage in terms of genetic diversity can spread to neighboring areas, gradually replacing dominant genotypes over several years.
We documented circulation of 2 genotypes of NA1 (98.6% of RSV-A isolates detected from 2010 to 2013) and ON1 (97.1% of RSV-A isolates detected from 20132017) in Lanzhou. In addition, ON1 is a novel genotype, characterized by a 72-nt duplication in the C-terminal region of the G gene, first reported in Canada. All RSV-A G-gene variants described in this report are derived from the GA2 genotype and are genetically close to the recently characterized NA1 genotype, which are the most recent strains circulating worldwide.[11,16] Our findings document the presence of the novel ON1 genotype in Northwestern China in the 2012/2013 epidemic season (August 2012) and its rapid spread in 2013/2014, which is 2 years later than its prevalence in some of the foreign countries (e.g., Italy). We provide a detailed analysis on the spread and the associated demographic, clinical, and evolutionary characteristics of the novel RSV-A genotype ON1 in Lanzhou.
In addition to the epidemiological impact, significant genetic variation in circulating strains may involve different pathogenicity and virulence. A study from Kilif showed a change in the alternation of RSV subgroup dominance patterns because ON1 was introduced into this community. Previously, RSV-A predominated in up to 2 consecutive epidemics in Kilifi, whereas it predominated over RSV-B in 3 consecutive epidemics from 2012/2013 to 2014/2015. However, we did not find this phenomenon in our study. The first ON1 strain was found in Lanzhou in 2012 and in following years, RSV-A and B subtypes are still prevalent every 2 years. A study in Italy reported that the beginning and peak times of RSV were earlier than before because of the emergence of the ON1 genotype, and the phenomenon was also been found in our research.
The ON1 genotype was first detected in Ontario in the winter of 2010/2011 (November 2010). To the best of our knowledge, the earliest ON1 sequence from a non-Canadian isolate was detected in Malaysia in November 2011. In the ensuing years, the ON1 genotype has been found in South Africa, Italy, Germany, and South Korea, indicating its rapid global dissemination during the 2012/2013 epidemic season. In China, the ON1 genotype was first reported in Beijing in 2013 (detected in November 2012), and then in 2014, cities such as Chongqing, Shanghai, Shenzhen, Guangzhou, and other places also reported appearance of the novel genotype.[25,26] It can be inferred that Lanzhou, reported in our report, may be the earliest region of the emerging ON1 genotype in China (detected in August 2012 and spreading quickly in 2014/2015). Therefore, we can draw the conclusion that ON1 appeared and erupted 2 years later than abroad. These data demonstrate that ON1 was introduced into China from abroad, and in China's northwest, ON1 is spreading fast and completely replacing the previously genotypes, which may be the current situation of ON1 in China and worldwide.
The prevalence of ON1 varies from place to place. Among some countries such as China, Germany, and Ontario of Canada where ON1 was first detected, its prevalence remains <20%. However, in Italy, the United States, and South Korea, the prevalence of ON1 was reported between 20% and 70%. There is also study claiming that ON1 was the only RSVA genotype in Argentina, in 2014. These data show the different prevalence of ON1 in different places and the increasing rate over time. In our study, it can be seen ON1 has strong transmission dynamics: in Lanzhou, the prevalence of ON1 reached 100% in 2 successive years. In the past, GA2 replaced GA5 as dominant genotype for 7 years, and for 5 years with BA genotype to replace RSV-B non-BA genotype, but it only took <2 years for ON1 to completely replace RSV-A. Therefore, it is concluded that the strong transmission of BA genotype would be likely to be repeated by ON1 and even stronger, and ON1 would become the sole epidemic genotype of RSV-A in the next 10 years or more.
High homology among wild-type NA1 and ON1 genotypes in Lanzhou and isolates from GenBank reveals the global distribution of these 2 genotypes. Site-specific evolutionary analysis of the C-terminal hypervariable region of the G protein among Ontario NA1 isolates revealed strong evolutionary selection pressure, resulting in 19 positively selected sites compared with the NA1 reference genotype. For ON1 genotype, the substitutions at amino acids 274 and 290 resulted in loss of group- and strain-specific epitopes.[33,34] The variability and apparent evolution seen at the positively selected site 274 in the ON1 cluster of sequences is particularly interesting. The aa 237 mutation, presenting in 56% of the Ontario NA1 isolates, suggested the gain of a potential N-glycosylation site. These reverted mutations, particularly in the epitope regions, may decrease the antigen avidity to the current circulating strain-specific antibodies. The longer attachment protein of the 72-nt duplication in ON1 viruses seems to offer more opportunities for variable changes and thus greater diversity and increased fitness over previous group A genotypes. Notably, new N-glycosylation sites were discovered in our study, 2 in 23aa in 2015/2016 epidemic season and 1 in 24-aa duplication area in 2016/2017. Overall, a variety of genetic changes could be responsible for the spread of ON1 strains, conferring low cross-protection by preexisting antibodies against RSV-A strains previously circulating in Italy: the 24aa insertion, the loss of a further potential glycosylation site due to the T253K substitution, and even other amino acid changes.
In our study, ON1-infected cases have the following characteristics: predominantly male young and more severe clinical symptoms such as more wheezing. However, studies in other countries report different results. According to Duvvuri's study, the infected subjects are mostly women, which is different from our conclusion. In Cyprus, it is reported that the clinical symptoms of ON1 was milder compared with other RSV infections. In Vitenam, the upper respiratory infection caused by RSV-ON1 was closely related to clinically severe manifestations such as wheezing, tachypnea, and difficulty in breathing, according to Yoshihara's reports. There is report in Germany that it is more common for ON1 infectors to admit into PICU than cases caused by other RSV-A strains. But researchers suggest that it is possibly related to the epidemic peak resulted from the sudden diffusion of ON1 due to genetic differences. Moreover, it is also reported that there are no differences among clinical symptoms from different RSV-A genotypes. But it is clear that differences in results may be resulted from differences in analyses, study designs, inadequate sample sizes, differences between viruses in different locations, host/environmental differences, or even different viral characteristics all could lead to different results. In our study, the clinical symptoms of ON1 infection in children are more severe; the infection-lasting time is longer but PICU admission rate is lower and the prognosis was good. But there is another possibility that ON1 is more easily combined with other pathogens, which lead to severe symptoms, rather than ON1. However, in our study, there are no evidences of mixed infection. Therefore, a more comprehensive and standardized research is needed for further investigation of clinical features of ON1.
The possibility exists that ON1 and other similar new RSV variants (e.g., the BA genotype) gain dominance by evading host immunity. The appearance of ON1 makes RSV-A “reset to zero,” which provides a new possibility for further development of an RSV vaccine. ON1 may be similar to the BA genotype and be the predominant strain of RSV-A for decades. Therefore, it is reasonable to assume the appearance of ON1 could lead to evasion of future vaccine-induced protection, lessening the herd immunity potential of vaccination, yet to provide a new possibility for manufacture of an RSV vaccine. Because of the emergence of ON1, RSV-A “reset to zero” and human immunity set to zero, the vaccine development can also been seen as zero to some extent. In conclusion, the emergence of ON1 is a challenge and an opportunity for the development of an RSV vaccine, and has important public health significance. Continued surveillance for cases and collection of detailed standardized clinical data are warranted.
We thank our colleagues of the Laboratory of Clinical Virology for helpful comments and critical reading of the manuscript.
XL, D-HL, and R-FZ conceived and designed the experiments. XL, D-HL, DC, LG, HY, YX, G-RL, Y-SS, Y-JW, W-KW, Z-PX, H-CG, and Z-JD performed the experiments and analyzed the data.
Conceptualization: Xuan Liang, Rong-Fang Zhang.
Data curation: Dong-Hai Liu, De Chen, Li Guo, Hui Yang, Yong-Sheng Shi, Yong-Jun Wang, Wei-Kai Wang, Zhi-Ping Xie, Han-Chun Gao, Zhao-Jun Duan, Rong-Fang Zhang.
Formal analysis: Dong-Hai Liu, Rong-Fang Zhang.
Funding acquisition: Rong-Fang Zhang.
Investigation: Xuan Liang, Dong-Hai Liu, De Chen, Hui Yang.
Resources: Yong-Jun Wang, Wei-Kai Wang.
Software: Xuan Liang, De Chen, Li Guo, Yong-Sheng Shi.
Supervision: Zhi-Ping Xie, Han-Chun Gao, Zhao-Jun Duan, Rong-Fang Zhang.
Validation: Xuan Liang, Rong-Fang Zhang.
Visualization: Xuan Liang.
Writing – original draft: Xuan Liang, Rong-Fang Zhang.
Writing – review and editing: Xuan Liang, Dong-Hai Liu, De Chen, Li Guo, Hui Yang, Yong-Sheng Shi, Yong-Jun Wang, Wei-Kai Wang, Zhi-Ping Xie, Han-Chun Gao, Zhao-Jun Duan, Rong-Fang Zhang.
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Keywords:Copyright © 2019 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.
Lanzhou; ON1 genotype; respiratory syncytial virus A