Phylogenetic characteristics and molecular epidemiological analysis of novel enterovirus EV-B83 isolated from Tibet, China

Enterovirus B83 (EV-B83) is a new member of the enterovirus B group. Currently, there are only two full-length genomic sequences of EV-B83 in the GenBank database and few VP1 region sequences. The aetiology and epidemiology of EV-B83 is unclear. 24 stool specimens were collected from twelve AFP patients and 298 stool specimens were collected from 298 healthy children in support of polio eradication activities in Tibet in 1999. Two polioviruses (isolated by L20B cell) and one non-polio enterovirus (isolated by RD cell) were isolated from AFP patients and nine polioviruses (isolated by L20B cell) and 90 non-polio enteroviruses (isolated by RD cell) were isolated from health children. Through molecular typing, we confirmed that the six of non-polio enteroviruses belong to EV-B83. The sequence similarity between the VP1 region of the Tibet isolates and that of the EV-B83 prototype strain was 80%. The maximum-likelihood phylogenetic tree of the partial VP1 region in EV-B83 demonstrated that EV-B83 formed four genotypes globally during the evolution process. The six Tibet EV-B83 strains formed the D genotype alone. Recombination analysis of Tibet EV-B83 showed that CV-B4, CV-A9, EV-B80, and EV-B106 may act as recombinant donors in multiple regions. The serum neutralization test showed that the antibody-positive rate was 58.8% and GMT was 1:19.70, which was higher than the previously reported results of EV-B106 and EV-B80. Temperature sensitivity test results showed that the six Tibet EV-B83 strains were temperature-insensitive with stronger virulence and potential infectivity, which was consistent with the results of the serum neutralization test. This study enriched the genome-wide sequence, epidemiological characteristics, and provided basic data for the follow-up study of EV-B83.

Full-length genome sequence analysis of six strains. The six Tibet EV-B83 strains were 7396 nt in length with a single 6552 nt ORF encoding a polypeptide of 2184 amino acids. The 5′-UTR and 3′-UTR comprised 744 and 100 nt, respectively. The base composition of all six EV-B83 strains was 28.2-28.3% A, 23.8-23.9% T, 23.5-23.6% C, and 24.3-24.4% G. We compared the genome-wide sequences of six EV-B83 strains with that of the EV-B83 prototype strain (CA76-10392/USA/1976) and found that the nucleotide insertion and deletion sites in the six EV-B83 strains were identical and located in the 5′-UTR region, all of which had two nucleotide insertions at position 101, one at position 152, and three at position 722. There was one nucleotide deletion at position 129 and three at position 695. Pairwise comparisons of the nucleotide sequences and deduced amino acid sequences were conducted among the six Tibet EV-B83 strains, the prototype strain of all EV-B strains, and the Yunnan EV-B83 strain (246/YN/CHN/08HC) ( Table 1). We also compared the nucleotide similarity between the P1, P2, and P3 regions of the six EV-B83 strains and those of the prototype EV-B strain. In the P1 region, the six EV-B83 strains had the highest nucleotide similarity with the Yunnan EV-B83 and EV-B83 prototype strains. However, in the P2 and P3 regions, the six Tibet strains had the highest nucleotide similarity with CV-B6 (GenBank accession number AF039205) and EV-B106 (GenBank accession number KF990476), respectively. These indicate that the six Tibet strains EV-B83 may show recombination with other EV-B strains in the P2 and P3 regions. The nucleotide and amino acid similarities among the full-length genomic sequences of six Tibet EV-B83 strains were 97.9-99.9% and 99.1-100.0%, respectively, while those between the six strains and the EV-B83 prototype strain were 81.0-81.1% and 96.4-96.6%, respectively. Phylogenetic analysis of six Tibet EV-B83 strains. The phylogenetic tree based on the partial VP1 region (633 nt) ( Fig. 1) showed that the VP1 region greatly differed in genetic distance compared to other regions, and the selected EV-B83 sequences were further divided into genotypes. We named the prototype strain (CA76-10392/USA/1976) of EV-B83 isolated in the United States in 1976 as genotype A, and the rest were divided according to the time of isolation. EV-B83 isolated from Cambodia (GenBank accession number KX197452) was classified as genotype C, the six Tibet EV-B83 strains were classified as genotype D, and the remaining EV-B83 including Chinese Yunnan strains, Indian strains, and French strain composed genotype B. We verified genotype classification by analysing inter-group and intra-group distances and found that the average distance between the four groups was 21-25%, which met the criteria of 15-25% for defining genotype by nucleotide differences in enteroviruses 8,17 .
Based on the sequences of all EV-B prototype strains in the GenBank database and those of the Yunnan EV-B83 strain and the six Tibet EV-B83 strains, we constructed phylogenetic trees based on VP1, P1, P2, and P3 coding regions, respectively (Fig. 2). Both VP1 and P1 phylogenetic trees showed that the six Tibet EV-B83 strains were clustered with the EV-B83 prototype and Yunnan strains with a 100% bootstrap value, which confirmed previous molecular typing results. However, the phylogenetic trees constructed based on the P2 and P3 coding regions showed different results. In the P2 region, the six Tibet EV-B83 strains were clustered with the CV-B6 prototype strain (GenBank accession number AF039205) and the E-12 prototype strain (GenBank accession number X79047). In the P3 region, they were clustered with EV-B106 (GenBank accession number KF990476). This is consistent with previous results regarding nucleotide similarities. It is also suggested that six Tibet EV-B83 strains may have recombination in the P2 and P3 coding regions, leading to a large difference in the nucleotide sequence between them and the previously isolated EV-B83 strains in these regions.
RDP4 showed that recombination patterns in these strains were largely alike 18   analysis results of the six Tibetan strains showed some differences at the breakpoint positions, considering the high sequence similarities, we still selected all primary and secondary potential parents (CV-B1, E-30, CV-B4, EV-B80, CV-B, CV-A, EV-B106) for recombination analysis using the SimPlot program (v3.5.1) 19 . The results of SimPlot and BootScan, the maximum-likelihood phylogenetic tree, and the high similarity of recombination sites of the CV-A9, CV-B4, and Tibet EV-B83 strains led to the determination that EV-B106, EV-B80 (GenBank   www.nature.com/scientificreports www.nature.com/scientificreports/ accession number JX644073), partial CV-A9 in the GenBank, and some CV-B4 strains were recombined with the six Tibet EV-B83 strains. Because the recombination sites of the CV-A9, CV-B4, and Tibet EV-B83 strains were very similar, we used the SimPlot program to analyse the Tibet EV-B83 strains in two methods.
First, we performed recombinant analysis while excluding CV-A9 (Fig. 4). The BootScan results showed that when Tibet EV-B83 was used as the query sequence for analysis, there was a strong recombination signal in the 5′-UTR and VP4 partial regions between EV-B106 and the Tibet EV-B83 group. In the P2 regions, the CV-B4, EV-B80, and Tibet EV-B83 groups showed a smaller range of recombination in 2B and 2 C, respectively. In the P3 regions, the CV-B4 and Tibet EV-B83 groups had high recombination in the 3D regions. This result had a high support value, which is consistent with the results of RDP4.
Second, recombination analysis was performed while excluding CV-B4 (Fig. 5). The BootScan results showed that EV-B106 and EV-B83 had a strong recombination signal in the 5′-UTR and VP4 regions when Tibet EV-B83 was used as the query sequence for analysis. In the P2 regions, the EV-B80, EV-B106, and Tibet EV-B83 groups had a smaller range of recombination signals in 2C and 3C, which also verified the above recombination results. The CV-A9 and Tibet EV-B83 groups had a wide range of reorganization, which covered most regions of 2A-2C and 3D and small regions of 3B and 3C.
In both cases, the aggregation of the maximum-likelihood phylogenetic tree in the P1, P2, and P3 regions of each sequence confirmed the results of our recombination analysis. Temperature sensitivity test of six strains. After the six Tibet strains were cultured at 36 °C and 39.5 °C, the titre test showed that they were not temperature-sensitive strains (Fig. 6). Although their replication ability  www.nature.com/scientificreports www.nature.com/scientificreports/ varied slightly at different temperatures, differences in the titre at 36 °C and 39.5 °C did not exceed two logarithms, and their logarithmic growth curves at the two temperatures were also highly similar. Unlike the previously reported temperature-sensitive EV-B106 and some temperature-sensitive EV-A90 strains, six Tibet EV-B83 strains is more temperature resistant.

Discussion
The emergence of molecular typing technology has greatly changed the current situation and accelerated the discovery of novel enteroviruses, which have been increasingly reported 9,22-24 . Currently, there are only two EV-B83 full-length genomic sequences and twelve in the VP1 region with lengths over 600 bp available in GenBank. Moreover, the epidemiology of EV-B83 remain unclear, so research regarding EV-B83 is valuable.
The full-length genomic sequences of six Tibet EV-B83 strains were highly similar. Comparison of these sequences with that of the EV-B83 prototype strain showed that their insertion and deletion sites were identical and all located in the 5′-UTR region. Although the six EV-B83 strains came from different areas of Tibet, the high consistency in sequences indicates that they may share a common origin, and EV-B83 may have a relatively limited spread between the two places.
A phylogenetic tree constructed based on all available VP1 regions (633 nt in length) of EV-B83 illustrated an interesting phenomenon: the six Tibet EV-B83 strains form an independent evolutionary branch and are far from the two EV-B83 strains isolated from Yunnan, China, indicating that EV-B83 has two different evolutionary branches in China 10,25 . We divided all EV-B83 into four genotypes, A, B, C, and D, based on nucleotide diversity in the VP1 region. This division of genotypes clarifies and simplifies the evolutionary relationship of EV-B83. www.nature.com/scientificreports www.nature.com/scientificreports/ The number of EV-B types is larger than that of EV-A, and the recombination between the genotypes is also very frequent [26][27][28] . Many articles have discussed the reorganization of different novel EV-B strains 20,21,29 . We know that recombination and nucleotide mutations promote the evolution of enteroviruses and new type emergence. Recombinant analysis is also crucial in epidemiological studies. We found that six Tibet EV-B83 strains and many previously discovered new enteroviruses (EV-B80, EV-B106, etc.) have reliable recombination in multiple genomic regions.
The serum neutralization test is greatly important for studying virus prevalence. The change in serum antibody is a dynamic process that can objectively evaluate the prevalence and spread of the virus [30][31][32] . According to serum neutralization test results for children in Lhasa and Shigatse, the seroprevalence and GMT of EV-B83 are higher than those of previously reported enteroviruses, such as EV-B106, EV-A90, and EV-B80 20,21,24 , indicating that EV-B83 is more widespread than some of previously discovered enteroviruses and may result in potential outbreaks and mass epidemics. The temperature sensitivity test is a good indicator of virulence and contagiousness 33 . The six Tibet EV-B83 strains showed resistance to temperature changes, indicating that they have high virulence and potential contagiousness. This is consistent with our previous suggestion that EV-B83 is more common than other EV (EV-B106, EV-A90).
In recent years, more and more novel enteroviruses have been detected; this is closely related to the high recombination frequency between enteroviruses. This high-frequency recombination promotes the evolution of enteroviruses, but also leads to the emergence of recombinant enteroviruses with unknown aetiology and epidemiology, such as EV-B83 reported in this study, which shows a high resistance to temperature and higher prevalence than previously reported novel enteroviruses (such as EV-B106 and EV-B80). We should strengthen the monitoring of these novel enteroviruses and use a variety of emerging technologies to improve the detection rate in specimens in order to identify those that may cause outbreaks as early as possible [34][35][36][37] . www.nature.com/scientificreports www.nature.com/scientificreports/ By analysing six Tibet EV-B83 strains, this paper elucidated epidemiological characteristics of EV-B83 and enriched its full-length sequence in the GenBank database. These findings will aid in further research on EV-B83. www.nature.com/scientificreports www.nature.com/scientificreports/

Materials and Methods
Sample collection. This study involved no human experimentation. National Labortory for Poliomyelitis in China CDC has established national laboratory network for detecting pathogens from AFP cases, their contactors, and healthy children in China. Stool samples of AFP patients and healthy children in Tibet served as the experimental specimens. The main purpose of the study is public health. This study was approved by the second session of the Ethics Review Committee of the National Institute for Viral Disease Control and Prevention (IVDC), Chinese Center for Disease Control and Prevention, all experimental protocols were approved by IVDC with the certificate number IVDC2017-021, and the methods were carried out in accordance with the approved guidelines. To meet experimental ethics requirements, written informed consent for the use of their clinical samples was obtained from the parents of the children whose samples were analysed. All methods were performed in accordance with Polio laboratory manual (World Health Organization, Geneva, Switzerland).
In 1999, 24 stool specimens were collected from twelve AFP patients (2 specimens per person) in support of polio eradication activities in Tibet. World Health Organization requires at least 150 stool samples to be tested every year in order to maintain the detection ability of poliovirus, since there are very few AFP Cases in Tibet every year, stool specimens of healthy children need to be collected to maintain the detection ability. For this reason, 298 stool specimens were collected from 298 healthy children under 5 year old (1 specimen per child) in Tibet in 1999.
In this study, 51 serum samples were random collected for sero-epidemiology study by using serum neutralization tests. Serum samples were obtained from children aged 2.5-5 in Tibet, 39 of which were from Lhasa and 12 from Shigatse, from healthy children showing no signs of disease during the collection process. Serum samples used in this study were collected by the Tibet Center for Disease Control and Prevention, and written informed consent for the use of their clinical samples was obtained from the parents of the children whose samples were analysed. All methods were performed in accordance with Polio laboratory manual (World Health Organization, Geneva, Switzerland).

Virus isolation and plaque purification.
We processed stool samples of AFP patients and healthy children according to standard procedures 38 ; then, samples were inoculated into human rhabdomyosarcoma (RD) cells and a mouse cell line with the human poliovirus receptor (L20B). Both cell lines were supplied by the WHO Global Poliovirus Specialized Laboratory in the United States and purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were continuously observed after inoculation for seven days, and the culture was harvested if cells showed a complete EV-like cytopathic effect (CPE). If no CPE appears after 7 days, perform a blind passage and continue examination for a further 7 days.
The harvested culture was diluted with 4 gradients (10 −2 , 10 −3 , 10 −4 , 10 −5 ), and the diluted culture was inoculated into 6-well culture plates containing RD cells. Incubate at 35 °C for 2 hours, add agar medium without neutral red solution, and invert at 37 °C for 2 days, then add agar medium with neutral red solution, culture in the dark (35 °C) after staining, and observe for seven days. After the appearance of white spots, appropriate plaques were selected and inoculated into RD cell culture tubes, after CPE appeared, they were passaged twice and cultures were collected. Molecular typing. Viral RNA was extracted from cultured cells using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). Then one-step RT-PCR Kit (Perfect Real Time, TaKaRa, Dalian) was used to detect RNA of enteroviruses by enterovirus-specific real-time polymerase chain reaction (Real-Time PCR). If the Real-Time PCR result is positive, the partial VP1 region sequence was obtained by reverse transcription polymerase chain reaction (RT-PCR) amplification using the PrimScript One-Step RT-PCR Kit Ver. 2 (TaKaRa, Dalian, China) with primers E486/E488 (EV-A universal primers), E490/E492 (EV-B universal primers)and E494/E496 (EV-C universal primers) 39 . The amplified products were further purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Each strand was sequenced from both directions at least once using the ABI 3130 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). The obtained sequences were identified by the Online Enterovirus Genotyping Tool 16 , and the obtained partial VP1 sequences were analysed by the Basic Local Alignment Search Tool (BLAST). Sequences were compared with those in the GenBank database to confirm the enterovirus type isolated in Tibet.
Full-length genome sequencing. The 5′ end of the viral genome sequence was amplified by the 5′-Full RACE Kit (Takara, Shiga, Japan) according to the manufacturer's instructions, and the 3′ end was amplified using an oligo-dT primer (primer 7500 A) 40 . The VP4 sequence was amplified by special primers (EVP4 and OL68-1) 41 . The other part of genomic sequences were obtained by designing specific primers using the primer walking method ( Table 2). The RT-PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and sequenced using an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
Bioinformatics analysis. The full-length genomic and amino acid sequences of the six EV-B83 were aligned with those of the EV-B prototype strains using the Muscle algorithm in MEGA7 [42][43][44] . Following alignment, sequences were imported to BioEdit to obtain the identity matrix 45,46 .
EV-B83 sequences longer than 600 nt were obtained from GenBank database, and sequences containing the VP1 region were screened out. After comparison, some VP1 coding regions (633 nt in length and located in the EV-B83 prototype strain at nt 2528-3160) were selected to construct phylogenetic trees. For evolutionary analysis, we also selected the sequences of EV-B prototype strains in the GenBank, Yunnan EV-B83, and six Tibet EV-B83 strains to construct the maximum-likelihood phylogenetic trees of VP1, P1, P2, and P3 coding regions. The sequences were processed by ModelGenerator0.85 to confirm GTR + G model as the best model, and the maximum-likelihood tree was constructed in MEGA7 with 1000 bootstrap replicates 47 . (2020) 10:6630 | https://doi.org/10.1038/s41598-020-63691-4 www.nature.com/scientificreports www.nature.com/scientificreports/ Meanwhile, BLAST was used to analyse the P2 and P3 sequence regions of the six strains, and full-length sequences with over 85% similarity in GenBank were obtained as potential parents. We used the Recombination Detection Program 4 (RDP4, v4.46) for preliminary recombination analysis and selected RDP, GENECONV, Chimaera, MaxChi, Bootscan, SiScan, and 3Seq for recombination detection 18,48 . For recombinant analysis, we set P-values less than 0.01 to obtain more accurate results. We only considered sequences supported by at least three RDP4 methods as potential parental sequences. Since BLAST showed that the P3 region of some CV-A9 strains had high similarity with the P3 region of six Tibet EV-B83 strains, we downloaded the full-length genomic sequences of the most similar CV-A9 strains in the GenBank database as potential parents. The CV-B6 and EV-B106 strains (GenBank accession numbers AF039205 and KF990476) in the maximum-likelihood phylogenetic tree of the P2 and P3 regions were closest to six Tibet EV-B83 strains and were also used as potential parents. We continuously screened a variety of potential parents using the SimPlot program (200-nt window moving in 20-nt steps) and the maximum-likelihood phylogenetic tree to obtain more reliable potential parental sequences.
Neutralization titration test. Six Tibet EV-B83 strains were purified by plaque assay, and the titres of them were measured three times, and the average value was taken. The strain with the highest titre, 99258, was selected for the neutralization test. We selected 51 serum samples from children in Tibet for inactivation at 56 °C for 30 minutes, diluted the inactivated serum into five gradients (1:4, 1:16, 1:64, 1:256, 1:1024), and added 50 μL of each serum dilution to a 96-well plate in duplicate. Then, 50 μL of the virus dilution (100 CCID 50 ) was added to each well, and the 96-well plate was placed in a 5% CO 2 incubator at 36 °C. After neutralizing for 2 h, 100 μL RD cell solution was added to each well. Serum control (1:4 serum dilution without virus) and cell control were set up in the experiment. A back titration of challenging virus is included in each test to allow calculation of the titre of virus actually present in that test. After all cells were added, the 96-well plates were placed in a 5% CO 2 incubator and cultured at 36 °C. After continuous observation for 7 days, the highest serum dilution showing protection from CPE in 50% of cultures was determined. If a neutralizing antibody titre was observed at a serum dilution > 1:8, the serum sample was considered positive and the geometric mean titre (GMT) was calculated. We also used SPSS Statistics software (v19.0) (SPSS Inc., Chicago, IL, USA) for statistical analysis and selected the chi-square test to compare differences in seroprevalence between Lhasa and Shigatse.
Temperature sensitivity test. In 24-well plates, monolayer RD cells were used to determine the temperature sensitivity of six purified EV-B83 strains, and temperature-sensitive and temperature-resistance strains (HTPS-QDH11F/XJ/CHN/2011 and HTYT-ARL-AFP02F/XJ/CHN/2011, respectively) were selected as control strains 20,26 . Then, 50 μL of undiluted strains were inoculated into 24-well plates and cultured in incubators at optimal and supraoptimal temperatures for viral culture (36 and 39.6 °C, respectively). After 1 h, the unadsorbed virus was removed, and 100 μL maintenance medium was added to each well. The 24-well plates were incubated at 36 or 39.5 °C, and the virus was harvested at 4, 8, 16, 24, and 48 h post-infection. The 50% cell culture infectious dose (CCID 50 ) was calculated by the end-point dilution method on monolayer RD cells in 96-well plates at 36 °C. If the titre of the same virus isolate at different temperatures at the same time point were reduced by more than two logarithms, the strain was considered to be temperature-sensitive 49 . Nucleotide sequence accession number. The full-length genomic sequences of the six EV-B83 strains (99245,99258,99267,99268,99269,99275) described in this study were uploaded to the GenBank (accession numbers MN164683-MN164688).

Data availability
The nucleotide sequences of the entire genome for the six strains determined in this study have been deposited in GenBank nucleotide sequence database under accession numbers MN164683-MN164688.