Isolation and genomic characterization of a reassortant G9P[8]-E2 rotavirus strain in China

doi:10.21203/rs.3.rs-2563661/v1

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Isolation and genomic characterization of a reassortant G9P[8]-E2 rotavirus strain in China

https://doi.org/10.21203/rs.3.rs-2563661/v1

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Objective: To isolate a prevalent G9P[8] group A rotavirus (RVA) strain (N4006) in China and investigate its genomic and evolutionary characteristics, with the goal of facilitating the development of a new rotavirus vaccine.

Methods: An RVA strain of the G9P[8] genotype from a diarrhea sample was passaged in MA104 cells. The strain was evaluated by SEM, polyacrylamide gel electrophoresis, and indirect immunofluorescence assay. The complete genome of the strain was obtained by RT-PCR and sequencing. The genomic and evolutionary characteristics of the strain were evaluated by nucleic acid sequence analysis with MEGA ver. 5.0.5 and DNASTAR software. The neutralizing epitopes of VP7 and VP4 (VP5* and VP8*) were analyzed using BioEdit ver. 7.0.9.0 and PyMOL ver. 2.5.2.

Results: The N4006 strain (G9P[8] genotype) was adapted in MA104 cells with a high titer (10^5.5 PFU/mL). Whole-genome sequence analysis showed N4006 to be a reassortant rotavirus of Wa-like G9P[8] RVA and the NSP4 gene of DS-1-like G2P[4] RVA, with the genotype constellation G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1 (G9P[8]-E2). Phylogenetic analysis indicated that N4006 had a common ancestor with Japanese G9P[8]-E2 rotavirus strains. Neutralizing epitope analysis showed that VP7, VP5*, and VP8* of N4006 had low homology with vaccine strains of the same genotype and marked differences with vaccine strains of other genotypes.

Conclusion: The G9P[8] genotype rotavirus with the G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1 (G9P[8]-E2) constellation predominates in China and may originate from reassortment between Japanese G9P[8] with Japanese DS-1-like G2P[4] rotaviruses. The antigenic variation of N4006 with the vaccine strain necessitates evaluation of the effect of the rotavirus vaccine on G9P[8]-E2 genotype rotavirus.

evolution

G9P[8]-E2

isolation

neutralizing antigen epitope

reassortment

rotavirus

Rotavirus (RV) is a major cause of diarrhea in infants and children < 5 years old. About 120,000 deaths are caused by RV annually [1], representing a huge disease and economic burden. RV vaccines can prevent and control RV-induced diarrhea and are in use worldwide.

The recombinant vaccine RotaTeq (RV5, Merck, Rahway, NJ, USA) and the Lanzhou lamb rotavirus vaccine (LLR; Lanzhou Institute of Biological Products, China) are the only two vaccines available in China. The RV5 vaccine is an attenuated reconstituted strain of a pentavalent human–bovine strain, which is recombined in vitro with WinStar calf 3 (WC3; G6P[5]) and five human epidemic virus strains: G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8] [2, 3]. The protective effectiveness of RV5 against all types of severe rotavirus gastroenteritis (RVGE) was 78.9% (95% confidence interval [CI]) according to phase III clinical trial data in China, and its protective effect against all levels of RVGE was 69.9% (95% CI) (Mo et al., 2017). The protective efficacy against severe RVGE and other severities of RVGE caused by the G9 genotype was 88.3% (95% CI) and 67.4% (95% CI), respectively [4]. The lamb attenuated RV strain with the G10P[15] genotype is the basis of the LLR vaccine. A post-marketing case–control study in Guangzhou, Zhengding, and Beijing, China, revealed that LLR had a protective efficacy of 52–88% against severe RVGE and 35% against RVGE of any severity. The vaccines had a heterotypic protective effect against severe RVGE caused by G3 and G9 of 52% and 40%, respectively [5].

The parent strains of current RV vaccines were developed from strains prevalent 20 or 30 years ago. For example, the parental group A rotavirus (RVA) strains of RotaTeq with human G1–G4 and P[8] genotypes and the orally attenuated live vaccine ROTARIX (RV1; GSK, Brentford, UK) with human G1 P[8] genotypes were isolated more than 30 years ago [6]. Genetic mutation, recombination, and reassortment continuously alter the nucleotide sequences and genotypes of common RVA strains, potentially affecting the protective efficacies of vaccines.

RVAs can be classified into Wa-like (I1-R1-C1-M1-1-A1-1-N1-T1-T 1-E1-H1), DS-1-like (I2-R2-C2-M2-M 2-A2-N2-N 2-T2-E2-H2), and AU-1-like (I3-R3-C3-M3-A3/A12-N3-T3-E3-H3/H6). Currently, most RVAs are Wa-like [7–9]. Among RVAs, G1P[8], G3[8], and G9[8] genotypes are typically Wa-like, whereas G2P[4] and G8P[8] are usually DS-1-like [10]. In China, the major epidemic strains from 1998 to 2000 were of G1P[8] genotype (72.7%). From August 2003 to July 2007, strains of the G3 genotype predominated (> 50%). The proportion of G9P[8] RVA strains increased rapidly from 3.4% in 2009 to 91.56% in 2018 [11–14]. Genome-wide sequence analysis was performed on 64 samples of G9P[8] genotype RV in Shenzhen and Changchun, China, in 2018. Among the 54 strains (30 strains in Shenzhen and 24 in Changchun) whose genome-wide fragments were amplified, 27 strains were Wa-like G9P[8]-E1 with a G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 genetic constellation, and 27 strains were Wa-like G9P[8]-E2 with a G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1 genetic constellation. The G9[8]-E2 strains accounted for 60% and 33% of the totals in Shenzhen and Changchun, respectively [15]. According to surveillance data of the Chinese Center for Disease Control and Prevention (China CDC), G9P[8]-E2 has spread widely in China (data not shown).

The protective effect of RV vaccines is mediated by a homotypic immune response against the same G/P genotype strain of the vaccine, whereas the heterotypic or cross-reaction response is poor [16]. The LLR strain in China does not belong to any of the prevalent genotypes, and its reported protective effect is low. Although RV5 covers previously epidemic genotypes, sequence changes of the prevalent genotype might have reduced its immunogenicity and protective effect. It is important to isolate G9P[8] RV, which is the most common strain in China, for vaccine evaluation and development. In this study, a G9P[8] RV strain (N4006) was isolated from a child with diarrhea in China. We investigated its evolutionary, genetic, and antigenic characteristics.

Sample and RV isolation

The diarrhea sample N4006 was collected from a child aged 27 months with diarrhea hospitalized in the Children’s Hospital Capital Institute of Pediatrics (Beijing, China) in 2020. The sample was confirmed as a G9P[8] RVA by G/P genotyping [17]. The RV strain in this sample (N4006) was isolated by passaging 10 times and plaque-purifying twice in MA104 cells (stored at the Cell Culture Laboratory, NHC Key Laboratory of Medical Viruses and Viral Diseases, China CDC, Beijing, China).

RV passage culture was performed as follows. MA104 cells were seeded into roller tubes and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. When the cells reached 90–100% confluence, trypsin was used for disruption and the cells were inoculated into roller tubes in an incubator with a 5% CO₂ atmosphere at 37℃. The MA104 cells formed monolayers in 2 days. Next, RV was activated with 800 µg/mL CaCl₂ and 15 µg/mL trypsin at 37℃ for 1 h. Subsequently, MA104 cells in roller tubes were washed with serum-free DMEM to remove remaining serum, added to activated RV, and placed in a CO₂ incubator for 2 h for virus adsorption. Next, the MA104 cells were added to DMEM containing 20 µg/mL trypsin to culture RV. The culture was freeze-thawed three times and harvested by centrifugation at 5000 rpm for 5 min [18]. RV passaging was conducted under good laboratory practice conditions using previously reported methods and certified reagents.

RV identification and growth curve determination

The isolated RV was identified by scanning electron microscopy (SEM) and polyacrylamide gel electrophoresis (PAGE) [19]. RV replication was confirmed by indirect immunofluorescence assay (IFA) with a rabbit polyclonal antibody (orb241327, Biorbyt, Cambridge, UK) to RVA intermediate capsid protein VP6 and fluorescein-conjugated goat anti-rabbit IgG (H + L) (ZSGB-BIO, Beijing, China) as the primary and secondary antibodies, respectively. Growth curves of N4006 in adapted MA104 cells were generated by infecting fresh MA104 cell monolayers with RV at a multiplicity of infection (MOI) of 0.01. Cell lysates after repeated freeze-thawing were collected every 12 h during 72 h of incubation and the titers were determined by the 3,3'-diaminobenzidine (DAB) colorimetric method.

Nuclear Acid Extraction And Whole-genome Sequencing

The whole-genome sequences of N4006 in the original fecal sample (P0) and the isolated strain at passage 10 (P10) were determined. According to the manufacturer’s instructions, viral RNA was extracted from the stool sample and MA104 cell culture using the Viral Nucleic Acid Extraction Kit (Xi'an Tianlong Technology Co., Ltd., China) and Nucleic Acid Automatic Extractor (NP968, Tianlong).

Using the One-Step RT-PCR Kit (Qiagen, Hilden, Germany), RT-PCR was conducted to amplify the 11 gene segments of N4006 using end-specific primers [20]. The reverse transcription was performed at 50°C for 30 min, followed by PCR amplification at 95°C for 15 min, 35 cycles of amplification, and a final extension of 8 min at 72°C in a 5331 PCR Master Cycler (Eppendorf, Germany). The reaction conditions were 30 s at 94°C, 30 s at 55°C, and 3 min at 72°C. Nucleic acid electrophoresis was performed in a 1.5% agarose gel, and the amplified product was detected using a Bio-Rad Universal Hood II Gel Doc System (Bio-Rad, Hercules, CA, USA). Strips of the amplification products were purified using an E.Z.N.A. Gel Extraction Kit (Omega Bio-tek, Norcross, GA, USA).

To obtain accurate terminal sequences, the purified amplification products were cloned in T plasmids. Purified amplified products were ligated into pClone 007 vector at room temperature for 30 min (pClone 007 Vector Kit, Beijing TsingKe Biotech Co., Ltd., China) and transformed into chemically competent Trelief 5α cells (Beijing TsingKe Biotech Co., Ltd.). The transforming reagent was coated on LB plates containing ampicillin (Amp^r) and incubated for 12–16 h in an incubator at 37℃. Selected monoclonal colonies were sent to Beijing TsingKe Biotech Co., Ltd. for direct sequencing.

Phylogenetic And Genetic Analyses

Nucleotide sequence alignments and identifications were performed with DNASTAR software. The genotypes of all segments were determined using the RotaC tool, a web-based tool for the complete genome classification of RVA [21]. Maximum-likelihood phylogenetic trees for the 11 genes were constructed using MEGA ver. 5.0.5, with best fit models and 1000 bootstrap replicates. The following models were found to best fit the indicated genes: HKY + G + I (VP1), TN93 + G + I (VP2, VP4), T92 + G + I (VP3, NSP1), T92 + G (VP6, NSP4, NSP5), T92 + G + I (VP7), T92 + I (NSP2), and TN93 + I (NSP3). Reference sequences of recently circulating strains and vaccine strains were downloaded from GenBank. Lineage designations were defined based on previous studies [22–25]. GenBank accession numbers of the 11 gene fragments of RV strain N4006 are provided in the Supplemental data (Table S1).

Deduced Amino Acid Sequence Alignments And Structure-based Predictions

Deduced amino acid sequence alignments and identity analysis were performed with BioEdit ver. 7.0.9.0. To assess the representativeness of N4006 as a vaccine strain and its differences from current vaccine strains, the presumed neutralizing epitopes on VP7 (7-1a, 7-1b, 7 − 2) and VP4 (VP8*: 8 − 1, 8 − 2, 8 − 3, 8 − 4; VP5*: 5 − 1, 5 − 2, 5 − 3, 5 − 4, 5–5) in N4006 were compared with those in Chinese or global epidemic strains and vaccines using BioEdit ver. 7.0.9.0. Structural analysis of VP7 and VP4 was conducted in PyMOL ver. 2.5.2 with the protein data bank files 4V7Q [26], 3FMG[27], and 1KQR [28].

Isolation of RV N4006

After 10 passages with limiting dilution and plaque purification, RVA strain N4006 with G9P[8] genotype was isolated in MA104 cells. Based on SEM, the infected MA104 cells contained various forms of RV particles, some being wheel shaped and of diameter 70–80 nm, and some of diameter 50–60 nm (Fig. 1A). By PAGE and silver staining, the N4006 strain had a long RNA migration pattern 4:2:3:2 typical of human RVA (Fig. 1C). RV replication was confirmed by IFA detection of VP6 specific epitopes (Fig. 1B). RV N4006 (P10) titer peaked (10^5.5 PFU/mL) at 48 h post-infection, and the simian RV strain SA11 (10^7.5 PFU/mL) at 24 h post-infection (Fig. 1D).

Whole-genome Sequencing

Alignment of full-length sequences showed that the sequences of N4006 from the original fecal sample and cell culture at passage 10 were identical. According to RotaC, the genomic constellation of N4006 was G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1 (G9P[8]-E2). This was an atypical genotype that contained an NSP4 gene segment of the DS-1-like RVA G2P[4] based on the Wa-like G9P[8] genotype. All obtained genomic sequences were deposited in GenBank (accession numbers OP901804- OP9018014).

Phylogenetic And Genetic Analyses

Phylogenetic trees were constructed for the 11 gene fragments of N4006 (P0) (Fig. 2). VP7 genes of G9 RVs were divided into six lineages in the phylogenetic tree of VP7 (Fig. 2A). The global G9-VP7 genes were mainly distributed in lineages III and VI, whereas other lineages encompassed older strains and vaccines. Although Chinese G9 strains were mainly in the III and VI lineages, N4006 and most G9 RVs circulating in China in recent years were in G[9]-lineage VI. N4006 was closest to the G9P[8]-E2 epidemic strains of 2019 and 2018 in China and Japan, such as Z2768, E6398, JZ1810, and Tokyo18-43. By contrast, N4006 was far from the G9 vaccine strain ROTAVAC-116E (lineage II) and the G9 vaccine strain ROTASIIL-Au32 (G9-lineage I), with 88.5% and 89.0% nucleotide sequence identities, and 92.4% and 94.5% aa identities, respectively. The VP7 identity between N4006 and sequences of lineage VI were 91.7–99.3% nt and 98.5–99.7% aa. The identities of N4006 and sequences of lineage III were 85.3–99.5% nt, and 92.2–99.7% aa. The VP7 identities between N4006 and other lineages were 82.3–90.6% nt, and 92.4–96.3% aa.

The VP4 genes of P[8] RVs were divided into four lineages [29] (Fig. 2A). Most contemporary P[8] RVs, including G3P[8], G9P[8], and G1P[8] strains from all over the world were clustered into P[8]-lineage 3, sharing 94.6–99.6% nt and 97.0–99.5% aa sequence identities with that of N4006. The VP4 gene of N4006 was closest to that of 2019 Chinese G9P[8] strains (e.g., Z2768 and Z6398), showing 99.6–99.7% nt and 99.4–99.5% aa sequence identities, and was close to that of 2018 Japanese G9P[8]-E2 strains (e.g., Tokyo18-30, Tokyo18-152, and Tokyo18-43), with 98.6–99.5% nt and 99.1–99.2% aa sequence identities. The VP4 sequence similarities between N4006 and the P[8] genotype vaccines RotaTeq (strain RotaTeq-W179-4), Rotarix (strains Rotarix and Rotarix-A41CB052A ), and Rotavin-M1(strains OP2-612 and MW2-1026) were 88.2–92.5% nt and 93.3–95.5% aa. In the VP4 phylogenetic tree, N4006 was also dissimilar to the latter strains.

In the phylogenetic tree of NSP4 fragments of N4006 and other reference strains (Fig. 2A), NSP4 belonged to the E1 and E2 genotypes. Chinese E1 RVs were of G3P[8] and G9P[8] genotypes. There were four lineages in genotype E2. The prevalent strains including N4006 were mainly distributed in lineage E2-IV, which was closest to Chinese and Japanese G9P[8]-E2 RVA strains emerging in 2018 or 2019. They were clustered into a small subbranch named clade A together with DS-1-like G2P[4] strains from Japan and Vietnam (e.g., Tokyo17-17, SP015, and Tokyo17-15). The identities of sequences of clade A were 94.5–99.3% nt and 97.7–98.9% aa. There are few G2P[4] RVs in China. G1P[8], G3P[8], and G2P[4] genotype RVs from Japan, Bulgaria, Italy, the Philippines, Brazil, Spain, and Thailand comprised the rest of lineage E2-IV and included reassortant G3P[8]-E2 RV strains (e.g., Tokyo18-25, IS1078, Tokyo17-119, and MI1125) in Japan from 2015 to 2018.

Phylogenetic trees were constructed for the other eight gene fragments of N4006 (Fig. 2B–D). N4006 was closest to 2019Chinese G9P[8]-E2 strains (e.g., Z2768, E6398 ,L2448, E6356, HB-45, and HB-43), and close to 2018 Japanese G9P[8]-E2 strains (e.g., Tokyo 18–39, Tokyo 18–40, Tokyo 18–43, Tokyo 18–30, and Tokyo 18–38). The identities among these E2 strains were in the range of 94.6–99.6% nt and 98.0–99.7% aa.

Sequence Alignments And Structure-based Predictions

We compared the presumed neutralizing epitopes on VP7 and VP4 (VP5* and VP8*) of N4006 with those of other G9 epidemic strains and vaccines (Tables 1 and 2).

Presumed VP7 antigenic epitope

The VP7 neutralizing antigen epitope analysis (Table 1) suggested no more than one aa variation between N4006 and G9 epidemic strains in lineage VI of the VP7 phylogenetic tree, and no more than two aa variations between N4006 and G9 epidemic strains in lineage III. N4006 belonged to lineage VI, which encompassed most Chinese epidemic RV strains.

There were three aa variations between the G9-genotype vaccine ROTASIIL (Serum Institute, India) and N4006 (Table 1, Fig. 3). These sites were in 7-1a and 7-1b. Of them, A87T and D100N might affect the immunogenicity of the vaccine. Compared with ROTASIIL, all G9P[8] epidemic strains had T242N (Fig. 3). All pedigree VI-G9P[8] epidemic strains had A87T and D100N. Most lineage III-G9P[8] epidemic strains had the same 100D as ROTASIIL, and a few had D100N (Table 1). The vast majority of lineage III-G9P[8] epidemic strains had 87T, unlike ROTASIIL, and a few had 87A (like ROTASIIL) (Table 1).

There were four aa variations between the G9-genotype vaccine ROTAVAC (Bharat Biotech, India) and N4006 (Table 1, Fig. 3). These sites were in 7-1a and 7 − 2, among which A87T, D100N, and N145D exhibited features of escape mutants. Compared with ROTAVAC, all G9P[8] circulating strains had N145D (Fig. 3). All G9P[8] epidemic strains in lineage VI had A87T, D100N, and N221S. Most G9P[8] epidemic strains in lineage III had 87T and 100D (unlike ROTAVAC), and a few had 87A and 100N. More than half in lineage III had 221S, and a few had 221G, unlike ROTAVAC (Fig. 3). T96A and A125T were present in some strains in lineage VI (Table 1). Comparison with the VP7 putative epitopes of existing vaccines with other G genotypes (RV1 (G1), ROTAVIN-M1 (G1), RV5 (G1, G2, G3, G4), and LLR(G10)) indicated 12–16 aa variations in N4006 (Table 1).

VP7 presumed neutralizing antigen epitopes (7-1a, 7-1b, 7 − 2) of N4006 were compared with vaccine strains and worldwide RVA G9 epidemic strains. ^a Epidemic strains of lineage VI in the VP7 phylogenetic tree; ^b epidemic strains of lineage III; ^c G9 genotype vaccines; ^d vaccines of other G genotypes; bold numbers, antigen-escape-related neutralizing epitopes; bold letters, Changes in antigen epitopes of reference strains compared with N4006.

Presumed VP4 antigenic epitopes

VP4 consists of two subunits, VP8* and VP5*, which have neutralizing epitope regions. The presumed neutralizing antigen epitopes in VP8* (8 − 1, 8 − 2, 8 − 3, 8 − 4) and VP5* (5 − 1, 5 − 2, 5 − 3, 5 − 4, 5–5) of N4006 were compared with those of vaccine strains and RVA P[8] epidemic strains. ^a Prevalent strains of G9P[8] genotype in VP4 phylogenetic tree lineage 3; ^b prevalent strains of P[8] of other G genotypes in lineage 3; ^c P[8] genotype vaccines; ^d vaccines of other P genotypes; bold numbers, antigen-escape-related neutralizing epitopes; bold letters, changes in antigen epitopes of reference strains compared with N4006. For prevalent strains of P[8] belonging to other G genotypes in lineage 3, only a few strains were used as references.

We also analyzed presumed antigen epitopes on VP4 (VP8* and VP5*) (Table 2). N4006 was almost identical not only to G9 but also to other G genotype Wa-like epidemic strains of P[8] genotype in China and worldwide, such as Moscow-40 (G3P[8]), Dhaka16 (G1P[8]), and CAU17L-103 (G8P[8]). All P[8] genotype Wa-like epidemic strains had almost identical aa sequences in the eight epitope regions (8 − 1, 8 − 2, 8 − 3, 8 − 4, 5 − 1, 5 − 2, 5 − 3, 5 − 4, and 5–5), except for a few with 195D, 113N, and 135N, which were different from the majority (195G, 113D, and 135D).

Compared with P[8] epidemic strains, there were six aa variations in P[8]-genotype vaccine RV1 in VP4 antigenic epitopes at sites 150, 195, 113, 125, 131, and 135 in epitope regions 8 − 1 and 8 − 3 (Table 2, Fig. 4). Sites 150, 195, 125, and 131 were different from the epidemic strains. Among the six differences, D150E and D135N in epitopes 8 − 1 and 8 − 3, respectively, might affect vaccine immunogenicity (Table 2).

Compared with P[8] epidemic strains, there were four aa variations in P[8]-RV5 at sites 150, 195, 113, and 388 in VP4 antigenic epitopes (Table 2, Fig. 4). Sites 150 and 388 were different from epidemic strains. D150E and I388L showed characteristics of escape mutants (Table 2).

There were three aa variations in the P[8]-genotype vaccine ROTAVIN-M1 at aa sites 150, 195, and 113 in VP4 antigenic epitopes (Table 2, Fig. 4). Sites 150 and 113 were different from the epidemic strains. D150E might show characteristics of escape mutants (Table 2).

Comparison with the VP4 putative epitopes of existing vaccines of other P genotypes such as LLR (P[15]), ROTAVAC (P[11]), and RV5 (P[5]) indicated 19–22 aa variations in N4006 (Table 2).

RV was first isolated by Wyatt and named Wa [30]. Laboratory RV strains, such as SA11 and RRV, can be continuously propagated in cell lines to 10⁷–10⁸ PFU/mL. However, the success rate of RV isolation from human diarrhea stool is low, and the titer of adaptive culture is lower than that of model RV [18]. In this study, RV N4006 was isolated from a child aged 1 year, 3 months, and 24 days with diarrhea in MA104 cells at a high titer (10^5.5 PFU/mL). RV particles of diameter 70–80 nm are mature and have a three-layer capsid. RVs of diameter 50–60 nm are immature and lack an outer capsid, having only the intermediate and inner capsids (VP6 and VP2) and cannot infect host cells. The 11 RNA fragments of RVA were separated by PAGE, and the aggregation mode of 4:2:3:2 was displayed. RVs with other aggregation patterns might be from birds or non-group A RV, or have rearranged genes. N4006 had a nucleotide electrophoresis migration pattern identical to RVA and the sequences of N4006 (P0) and N4006 (P10) were identical. The RV titer was low after more than 10 generations of adaptation in MA104 cells [31]. The high titer of N4006 (10^5.5 PFU/mL) with no adaptation mutation implies its adaptation to a high titer in subsequent passages.

The G9P[8] genotype is the fifth most common RVA epidemic genotype combination worldwide after G1P[8], G2P[4], G3P[8], and G4P[8] [32]. G9P[8] RV as a cause of acute gastroenteritis in children was first reported in the United States in 1983 [33]. Although G9P[8] was later sporadically detected in Japan, Thailand, and other countries [34, 35], it was not until one or two decades later that G9P[8] RV emerged in large numbers and spread worldwide [36–38]. In China, the first human RVA G9P[8] was isolated from a child with gastroenteritis in 1997 and named T203 [39]. Thereafter, the number of G9P[8] RVA strains increased rapidly [11–13], and was detected in 74.05% of hospitalized children with acute gastroenteritis in 2016 (data not shown). However, G9P[8] RVA strains before 2016 typically had the Wa-like gene constellation: I1-R1-C1-M1-A1-N1-T1-E1-H1. Whole-genome sequence analysis showed that N4006 belonged to a recently emerged G9P[8]-E2 (G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1). The newly emerged G9P[8]-E2 was first reported in Japan in 2019 and became the prevalent strain locally [24] and spread widely in Tokyo. According to the China CDC RV monitoring network, G9P[8]-E2 has been the main RV genotype combination in China since 2018 (data not shown). Therefore, N4006, which matches the most widespread genotype, is representative of RVs in China.

In phylogenetic trees, the 11 gene fragments of N4006 had the highest identities and closest genetic relationships with G9P[8]-E2 strains from China in 2019. Except for VP6, all Chinese G9P[8]-E2 strains, including N4006, had the highest identities and closest relationships with Japanese G9P[8]-E2 strains in 2018. Therefore, Chinese G9P[8]-E2 RV strains, including N4006, had a common ancestor with Japanese G9P[8]-E2 RV strains. In addition, the NSP4 genes of N4006 and G9P[8]-E2 strains from China and Japan had the highest identities and closest relationships with DS-1-like G2P[4] strains from Japan and Vietnam. Therefore, the NSP4 gene fragments of these G9P[8]-E2 RVs originated from DS-1-like G2P[4] strains. Interestingly, the reassortment of G3P[8] with the NSP4 gene of DS-1-like RV occurred in Japan almost simultaneously with that of G9P[8]-E2. This indicated the strong adaptability of Japanese DS-1-like RV in terms of genetic reassortment with RVs of prevalent genotypes. Because there is no report of G9P[8]-E2 RVs in Vietnam and few G2P[4] strains in China at present, Chinese G9P[8]-E2 RV might originate from an introduction after reassortment between Japanese G9P[8] with Japanese DS-1-like G2P[4] RVs. In short, Chinese G9P[8]-E2 RV strains, including N4006, have a common ancestor with Japanese G9P[8]-E2 RV strains, which might originate from reassortment between Japanese G9P[8] with Japanese DS-1-like G2P[4] RVs.

Presumed neutralizing epitope analysis of VP7, VP8*, and VP5* showed that N4006 was almost identical to G9P[8] epidemic strains in VP7, including the majority of recent Chinese RV strains, and was identical to Wa-like-P[8] strains in VP8* and VP5*. Also, it had low identity with vaccines of the same genotype (e.g., ROTASIIL (G9), ROTAVAC (G9), RV1 (P[8]), RV5 (P[8]), and ROTAVIN-M1) and marked differences with vaccines of other genotypes. N4006 may have a good protective effect against currently prevalent G9P[8] RVs and can be used to develop a new RV vaccine. In conclusion, N4006 is representative of the dominant epidemic strains in China, and the differences in neutralization epitopes between N4006 and current vaccines suggest the need for vaccine evaluation and the development of a new vaccine.

In summary, we adapted the G9P[8] RV strain N4006 in MA104 cells, achieving a high titer (10^5.5 PFU/mL). The genotype constellation was G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1 (G9P[8]-E2) and N4006 might originate from reassortment between Japanese G9P[8] with Japanese DS-1-like G2P[4] RVs. VP7, VP5*, and VP8* of N4006 had low identities with vaccine strains of the same genotype and marked differences from vaccine strains of other genotypes. The presumed antigenic variation of N4006 with the current vaccine strain warrants evaluation of RV vaccine efficacy against the prevalent G9P[8]-E2 genotype of RV.

SEM: Scanning electron microscope; PAGE: Polyacrylamide gel electrophoresis; IFA: Indirect immunofluorescence assay; RVA: Group A rotavirus; WC3: WinStar calf 3; RVGE: Rotavirus gastroenteritis; MOI: Multiplicity of infection; amino acid: aa

Acknowledgements

The authors thank the contributions by the staff at the National Institute for Viral Diseases Control and Prevention, Chinese Centre for Disease Control and Prevention.

Authors’ contributions

Conceived and designed the experiments: Zhao-jun Duan and Saleha Shahar. Performed the experiments: Rui Peng, Meng-xuan Wang, Yu-hang Wei, and Guang-ping Xiong. Analysed the data: Rui Peng, Jin-dong Wang, Dan Liu, Li-li Pang, Xiao-man Sun. Contributed reagents/materials/analysis tools: Hui-ying Li, Xiang-yu Kong. Wrote the paper: Rui Peng and Dan-di Li. All authors reviewed the manuscript.

Funding

National key research and development program (Finance Code 2022YFC2304302).

Availability of data and materials

All sequences used in this study are available in GenBank. All other relevant information is provided in this current manuscript. If required, the data presented in this work can be shared by e-mail.

Ethics approval and consent to participate

This study was reviewed and approved by the National Institute for Viral Diseases Control and Prevention (Beijing, China). The guardians of the recruited children in this study were informed of the aims of this investigation and provided oral consent.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no conflict of interest.

Author details

¹Department of Biosciences, Faculty of Sciences, Universiti Teknologi Malaysia, Johor Bahru, 81310, Malaysia.

[email protected] (R.P.); [email protected] (S.S.)

²NHC Key Laboratory of Medical Viruses and Viral Diseases, National Health Commission, Institute of Viral Disease Prevention and Control, Chinese Centre for Disease Control and Prevention, Beijing 102206, China.

[email protected] (R.P.); [email protected] (Z.-J.D.); [email protected] (D.-D.L.); [email protected] (M.-X.W.); [email protected] (D.L.); [email protected] (L.-L.P.); [email protected] (X.-M.S.); [email protected] (H.-Y.L.); [email protected](X.-Y.K.)

³College of Life Science, Hengshui University, Hengshui 053000, China. [email protected] (R.P.)

⁴Department of Pathogenic Biology, Weifang Medical University, Weifang 261053, China. [email protected] (J.-D.W.)

⁵College of Public Health, Gansu University Of Chinese Medicine, Lanzhou 730000, China. [email protected] (Y.-H.W.); [email protected] (G.-P.X.)

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Tables 1 and 2 are available in the Supplementary Files section.

No competing interests reported.

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Isolation and genomic characterization of a reassortant G9P[8]-E2 rotavirus strain in China

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Abstract

Figures

Background

Methods

Sample and RV isolation

RV identification and growth curve determination

Nuclear Acid Extraction And Whole-genome Sequencing

Phylogenetic And Genetic Analyses

Deduced Amino Acid Sequence Alignments And Structure-based Predictions

Results

Isolation of RV N4006

Whole-genome Sequencing

Phylogenetic And Genetic Analyses

Sequence Alignments And Structure-based Predictions

Presumed VP7 antigenic epitope

Presumed VP4 antigenic epitopes

Discussion

Conclusion

Abbreviations

Declarations

References

Tables

Additional Declarations

Supplementary Files

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