RSV is a single-stranded, negative-sense RNA virus, and viral genome consists of 10 genes coding 11 proteins (NS1-NS2-N-P-M-SH-G-F-M2.1-M2.2-L) [1]. Glycoprotein (G) contains two hypervariable regions (HVR-1 and HVR-2) exhibiting the greatest genetic diversity and a central conserved domain (CCD) including a conformationally constrained CX3C (182-CWAIC-186) [2]. The coronavirus disease 2019 (COVID-19) pandemic and following strict non-pharmaceutical interventions (NPIs) have changed pandemic of RSV. With the relaxation of NPIs, there was resurgent RSV infections in Beijing during April 2023. Our study aims to identify the dominant prevalent genotypes of RSV and the molecular evolution of the G gene specifically the HVR-2.
Clinical specimens from 107 children hospitalized for acute respiratory tract infections (ARTIs) in Beijing Children’s Hospital were collected from April 1st to 30th, 2023. All specimens were tested by the multiplex assay of 16 respiratory viruses (xTAG RVP Kit, Luminex, Toronto, Ontario, Canada). Nucleic acid was extracted through viral nucleic acid isolation kits (Magnetic Beads, BioPerfectus, China, SDK60104), and partial G gene was amplified by PrimerScriptTM One-Step RT-PCR Kit Ver.2 (Takara, Japan, RR055A) as same as our previous study [3]. Totally 11 partial G gene sequences including six RSVA and five RSVB were obtained through Sequencer software version 5.0 (Gene Codes, Ann Arbor, MI, USA). These sequences were submitted to GenBank database (accession numbers: OR840699-OR840710). Additionally, sequences of referenced 71 RSVA G genes and 82 of RSVB obtained during 2020-2022 were aligned together using MEGA 7.0 software (Sudhir Kumar, Arizona State University, Tempe, AZ, USA) for phylogenetic analyses. Phylogenetic tree was generated with the neighbor-joining method and the Kimura 2-parameter model with pairwise deletion treatment for the missing and gap sequences. The reliability was estimated by using the bootstrap method with 1000 replicates, with only values exceeding 70% displayed on the tree. Amino acid homology and mutations of the G protein were analyzed by comparing with the reference sequences of ON1 genotype (JN257693) and BA9 genotype (DQ227395), respectively.
The data was analyzed using GraphPad Prism software (version 5.00, GraphPad, La Jolla, CA, USA). Categorical variables were described with frequency and percentages. Differences in RSV-positive rate of various groups were evaluated using the Chi-squared Test. A P-value < 0.05 was considered indicative of a statistically significant difference. This study was approved by the Ethics Committee of Beijing Children’s Hospital, affiliated with Capital Medical University (ethics number 2014-99 and 2017-k-15).
The detection rate of RSV-positive samples in this study was 25.23% (27/107). There was no differences between genders and the detection rates (c2=0.253, P=0.615). RSV-positive children’s onset age was almost less than 5 years (26/27, 96.30%), especially aged 3 -≤ 5 years (c2=9.483, P=0.050). The detection rates of RSV-associated pneumonia, bronchitis, and bronchial asthma were 29.03%, 18.18%, and 14.29% respectively. No statistical differences were obtained between diagnoses and detection rates (URTIs vs LRTIs: c2=0.114, P=0.735; pneumonia vs bronchitis vs bronchial asthma: c2=0.824, P=0.737). More details was shown in table 1. According to phylogenetic analysis, six RSVA and five RSVB isolates’ nucleotide identity was 94.4%-99.8% and 97.1%-100.0%, and clustered into ON1 and BA9 genotype, respectively. In RSVA G protein, one isolate (OR840709) had an amino acid substitution of S174N in CX3C motif. Whether this change could affect the recognition of T cells and the synthesis of antibodies remained unknown and needed further studies. In RSVB, P237L, P247T, K258N/S, H259Y, T266I, N296Y, T302I, and E305K were found newly but not in our previous study. More details was shown in table 2 and figure 1.
The declaration of COVID-19 as a global public health emergency prompted the adoption of NPIs and profoundly impacted RSV prevalence [4]. Daniel et al has revealed that RSV prevalence was significantly lower during the lockdown period (5.3%) than pre-COVID-19 period (25.6%), but with the relaxation of NPIs, RSV outbreaks have returned and even increased, with the prevalence of 42.02% [5]. Macro et al also noted that RSV-positive had variability, ranging from 23.8% (2001-2002) to 40.6% (2019-2020) [6]. In our study, the detection rate of RSV-positive was 25.23%, similar to the rate detected during pre-COVID-19, but far lower than 42.02% and 40.6%. The detection rate difference could be explained by study designs, sample populations, and diverse diagnostic approaches.
RSVA genotype ON1 with a 72-nucleotide-duplication in the C-terminal region of G protein, was first identified in Ontario, Canada in 2010 [7]. In China, ON1 was first detected in 2011, and it completely replaced GA2 to become the predominant genotype after 2016 [8]. BA-like genotype of RSVB had a 60-nucleotide-duplication in the HVR-2 of G protein, first appeared in Buenos Aires, 1999 [9]. Nowadays, BA9 had been still the dominant genotype of RSVB in China. In this study, six RSVA and five RSVB isolates were clustered into the ON1 and BA9 genotypes, respectively. As same as the results of our previous study, ON1 and BA9 remained the dominant genotypes in Beijing during 2015-2019 [3]. Although the subtypes of isolates remained, there were still several nucleotide and amino acid variations. Our previous study about the molecular epidemiology of RSV during 2015-2019 showed that two substitutions H258Q and H266L co-occurred, and isolates with T290I and T312I were all from 2018-2019. In the RSV outbreak in 2020, Taiwan, China, T113I, V131D, N178G, H258Q, and H266L were co-occurred [10]. In our study, four coincided amino acid substitutions T113I, V131D, H258Q, and H266L were found together in four isolates. Moreover, the T cell epitope was conserved but one strain had a change for S174N. For RSVB, amino acid variations L267S, I281T, T290I, F297S, and T312I co-existed in all five isolates. Among them, A131T, T137I, T290I, and T312I had appeared since 2017. Continuous surveillance of circulated RSV genotypes and amino acid variations allows researchers to identify the drug-resistant strains, the new strains, or mutations that might escape immune responses provided by existing vaccines early on.
In conclusion, genotyping and analyzing the evolution of RSV is quite important for the first short outbreak of RSV after the end of NPIs. Early detection of emerging viral strains enables the timely implementation of containment measure and release disease burden.