Whole-genome analysis of CVA4 in children with herpangina in two cities of China

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This study successfully obtained 60 VP1 region sequences and 50 complete genome sequences of CVA4. Phylogenetic analysis based on the complete genome revealed that these strains clustered within the same evolutionary branch as CVA4 strains from other regions of China. Analysis of the VP1-based phylogenetic tree demonstrated that all 60 strains belonged to the C2 genetic subgenotype. Recombination analysis indicated high sequence identity (85.1–85.7%) between the study strains and the CVA4 prototype strain within the P1 region. In contrast, significant sequence similarities were observed in the P2 and P3 regions with prototype strains of CVA4, CVA5, CVA14, and CVA16. Bootscan analysis further identified recombination signals in the 5′ untranslated region (5′ UTR), P2, and P3 regions, suggesting recombination events with non-CVA4 enterovirus A (EV-A) prototype strains. This study provides evidence of genetic recombination in circulating CVA4 strains in Shandong Province, China, highlighting their complex evolutionary dynamics within the EV-A species. Biological sciences/Computational biology and bioinformatics Biological sciences/Evolution Biological sciences/Genetics Biological sciences/Microbiology Herpangina Coxsackievirus A4 Whole genome Gene sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Herpangina (HA) and hand-food-mouth disease (HFMD) are common acute enteroviral infectious diseases in children. HFMD is manifested as papules or herpes on the mouth, hands, feet and buttocks. Some cases can progress to encephalitis, meningitis, pulmonary hemorrhage, heart failure and other serious complications, even life-threatening. Although HA rarely causes such serious consequences, its strong infectivity and fast transmission speed make it easy to cause sporadic and outbreak epidemic in schools and kindergartens, bringing economic and psychological burden that cannot be ignored to children's families and society 1 . CVA4 belongs to Enterovirus A (EV-A), which is one of the common pathogens causing HFMD and HA. Epidemiological surveillance data show an increasing trend of HFMD cases due to CVA4 2,3 , previous etiological studies have shown that CVA2, CVA4 and CVA6 are the main pathogenic serotypes of HA 4 , 5 . CVA4 is a single-stranded RNA virus classified as Enterovirus (EV) in Picornaviridae. Its genome is about 7.4 kb in length and consists of two open reading frames (ORFs), ORF sencoding four structural proteins (VP1, VP2, VP3 and VP4) and seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D), and uORFs encoding a single protein uORFP 6 . At present, the systematic analysis and genetic evolution of the whole genome of CVA4 are still scarce. This study revealed the genetic evolution mechanism and variation characteristics of CVA4 strain by analyzing the whole genome characteristics and genetic characteristics of CVA4 strain, providing laboratory data for genetic variation of CVA4 epidemic strain in some areas of China, and having reference significance for vaccine development and prevention and control of related diseases of CVA4. Results In 2024, 256 throat swab specimens from herpangina (HA) patients in Linyi and Yantai cities (Shandong Province, China) were subjected to RD cell culture and RT-PCR screening, yielding 60 strains of CVA4.The patient cohort comprised 40 males and 20 females (male-to-female ratio: 2:1), aged 0–10 years, with 91.7% (55/60) being preschool children (≤ 6 years). High-throughput sequencing generated complete genomic sequences for all 60 CVA4 strains, designated as: YT01/SD/CVA4/2024 to YT23/SD/CVA4/2024 (abbreviated YT01-YT23)、LY01/SD/CVA4/2024 to LY33/SD/CVA4/2024 (abbreviated LY01-LY33). Sequence deficiencies were identified in: Strains YT01, YT03, YT06, YT12, YT15, YT21, LY24, LY28, LY30 and LY31 exhibited incomplete ORF due to insufficient sequencing coverage. Phylogenetic Analysis Based on VP1 Region The VP1 region of CVA4 is 915 nucleotides in length and encodes 305 amino acids in total. According to Ji 7 typing standard, the genotype difference of VP1 region of the same serotype > 15% is the subtype classification standard, and CVA4 can be divided into 4 genotypes A ~ D (Fig. 1). Genotype A includes a prototype strain of CVA4 isolated from North Carolina in the United States in 1948 (AY421762); genotype B includes a strain from Kenya in East Africa in 1999 (GQ176232); genotype D includes a strain from Japan in 2008 (AB457644); genotype C consists of strains isolated from China, India, Australia, Russia, and the United States between 1996 and 2024. Genotype C can be further divided into five sub-genotypes: C1-C5. The CVA4 strains in this study were clustered into subtype C2, and their phylogenetic relationship showed that most of the strains were mainly similar to Yunnan strain (LC865850) identified in 2023 and Yunnan strain (LC707448) identified in 2021, while LY17 strain was not clustered into a branch with other strains, and was closely related to Sichuan strain (MW178773) identified in 2019. Table 1 The year, location and quantity information of the A-D related sequences of the CVA4 VP1 sequence genotype Country A B C D Earliest record Latest record number of sequences Earliest record Latest record number of sequences Earliest record Latest record number of sequences Earliest record Lastest record number of sequences China 1996 2024 142 America 1948 1948 1 Kenya 1999 1999 1 Japan 2008 2008 1 India 2007 2008 6 Russia 2013 2014 2 Genome-wide analysis The nucleotide lengths of 50 CVA4 strains in this study ranged from 7349bp to 7434bp. Compared with the CVA4 reference strain High Point (AY421762), there were partial deletions in the 5′ untranslated regions (UTR) and 3′ UTR regions. The open reading frame (ORF) between the 5′UTR and 3′ UTR of CVA4 strains was 6606bp, encoding a polyprotein containing 2203 amino acids, the entire genome structure of the virus conforms to the characteristics. Phylogenetic analysis of 222 complete genome sequences (including 50 CVA4 strains in this study and 172 CVA4 complete genome sequences included in GenBank public database) showed that the complete genome phylogenetic tree could be divided into five groups: Group1 (n = 5), Group2 (n = 164), Group3 (n = 33), Group4 (n = 19) and Group5 (n = 1). All strains in this study were located in Group2, clustered into a group with China CVA4 strains, and most closely related to Shandong strain. LY17 strains have the closest genetic relationship with the isolate from Taian City, Shandong Province in 2019 (ON730865); the rest strains have the closest genetic relationship with the isolate from Linyi City, Shandong Province in 2024. The nucleotide and amino acid sequences of the whole genome of 50 CVA4 strains were analyzed (Table 2), and the nucleotide identity among 50 CVA4 strains was 88.9%~99.9%, and the amino acid identity was 96.3%~99.9%, and the nucleotide identity with High Point of CVA4 prototype strain was 84.1%~84.5%. Among them, LY17 strain had poor nucleotide identity with other strains, 88.9%~90.3%, The nucleotide and amino acid sequence identity analysis of ORF region between 50 strains and EV-A prototype strain showed that the nucleotide and amino acid similarities with CVA4 prototype strain in P1 region were 85.1%~85.7%, 97.9%~98.5%, respectively, and the nucleotide and amino acid similarities with other EV-A prototype strains were 60.8%~67.4%, 65.3%~75.5%, respectively. In the P2 region, the CVA4 strain in this study showed a high nucleotide consistency with the CVA4, CVA5, CVA14, and CVA16 prototype strains, reaching over 80%, the amino acid similarity was above 95%. In the P3 region, the nucleotide similarity with the CVA4, CVA14, and CVA16 prototype strains was high, reaching over 80%, and the amino acid similarity was above 90%. For further genome analysis, a phylogenetic tree of P1, P2 and P3 regions was constructed using 85 complete genome sequences (Fig. 3), including 11 EV-A prototype strains, 50 CVA4 strains, 7 CVA4 subgenotypes C4 and C3 (as can be seen from Fig. 2,complete genome sequences for genotypes B and D and C1 and C3 are currently unavailable from GenBank public databases due to limited data on CVA4 genome sequences), and 17 complete genome sequences for CVA5, CVA14 and CVA16. The 50 CVA4 strains in this study were located at different positions in the phylogenetic tree of P1, P2 and P3 regions. In the phylogenetic tree of P1 region, the CVA4 strains were closely related to C4 and C5 subgenotypes, and clustered into a large cluster; in the phylogenetic tree of P2 region, except LY17, the other CVA4 strains were closely related to a C5 subtype strain (MN964077) from Yancheng, China, LY17 was closely related to Yancheng C5 subtype strain and CVA5 strain (MZ491030); in P3 region, LY17 strain was closely related to CVA5 strain (MZ461030), and the rest strains were closely related to CVA14 strain(KP036482). Table 2 Consistency (%) between 50 CVA4 strains and each segment of the ORF region of the EV-A prototype strain genome.Due to incomplete 5’UTR sequencing, the amino acid consistency of the whole genome and the 5’utr region was not analyzed.The blue background area represents the consistency of nucleotide sequences; The blue line divides the P1, P2 and P3 regions. region EV-A prototype strain CVA2 CVA3 CVA4 CVA5 CVA6 CVA8 CVA10 CVA12 CVA14 CVA16 EV-A71 Genome nucleotide 73.4 ~ 73.8 73.8 ~ 74.2 84.1 ~ 84.5 74.4 ~ 74.7 74.0 ~ 74.5 73.6 ~ 74.3 73.5 ~ 74.3 74.0 ~ 73.6 74.8 ~ 75.4 75.1 ~ 75.4 72.1 ~ 72.6 5'UTR nucleotide 80.3 ~ 81.6 80.7 ~ 81.8 87.0 ~ 88.2 81.3 ~ 82.2 84.9 ~ 86.1 78.7 ~ 80.3 80.2 ~ 82.8 79.2 ~ 80.5 83.3 ~ 84.9 83.4 ~ 84.7 79.3 ~ 80.4 VP4 nucleotide 64.7 ~ 67.6 68.1 ~ 70.0 85.0 ~ 86.1 65.7 ~ 68.1 70.0 ~ 72.0 67.1 ~ 70.0 69.1 ~ 71.5 68.6 ~ 71.5 64.7 ~ 67.1 64.7 ~ 65.7 63.8 ~ 64.7 Amino acid 79.7 ~ 81.2 75.4 ~ 76.8 97.1 ~ 98.6 68.1 ~ 69.6 73.9 ~ 75.4 75.4 ~ 76.8 72.5 ~ 73.9 75.4 ~ 78.3 69.6 ~ 69.6 73.9 ~ 75.4 66.7 ~ 68.1 VP2 nucleotide 69.2 ~ 70.7 69.0 ~ 70.6 84.5 ~ 85.9 65.6 ~ 67.2 69.1 ~ 70.2 68.1 ~ 69.3 67.6 ~ 68.6 65.6 ~ 66.9 65.0 ~ 67.3 65.8 ~ 66.9 65.0 ~ 66.4 amino acid 77.0 ~ 78.1 79.7 ~ 80.9 98.0 ~ 99.2 74.2 ~ 75.0 81.3 ~ 82.0 77.7 ~ 78.1 79.3 ~ 80.1 74.2 ~ 75.4 77.0 ~ 78.1 73.0 ~ 73.8 72.7 ~ 73.4 VP3 nucleotide 70.7 ~ 72.5 70.4 ~ 71.9 85.8 ~ 86.8 67.5 ~ 68.5 68.3 ~ 69.6 70.0 ~ 71.9 69.3 ~ 70.7 69.0 ~ 70.4 62.9 ~ 64.3 62.7 ~ 63.8 63.8 ~ 65.0 amino acid 83.3 ~ 84.2 80.8 ~ 82.1 98.3 ~ 99.2 77.1 ~ 77.5 81.3 ~ 82.1 81.7 ~ 83.3 77.9 ~ 78.8 78.3 ~ 78.8 68.2 ~ 69.4 68.6 ~ 69.8 69.8 ~ 70.2 VP1 nucleotide 59.9 ~ 61.5 60.2 ~ 61.4 84.0 ~ 85.0 58.5 ~ 59.8 60.2 ~ 61.6 60.9 ~ 62.1 62.2 ~ 63.4 58.3 ~ 59.1 52.9 ~ 54.0 58.1 ~ 58.8 55.4 ~ 56.6 amino acid 64.9 ~ 65.5 65.3 ~ 66.3 97.0 ~ 98.0 63.3 ~ 64.3 61.6 ~ 62.6 65.0 ~ 66.0 62.9 ~ 63.5 56.7 ~ 57.0 55.4 ~ 56.0 58.7 ~ 59.1 54.0 ~ 54.4 P1 nucleotide 66.6 ~ 67.1 66.7 ~ 67.1 85.1 ~ 85.7 63.8 ~ 64.5 66.3 ~ 66.8 66.3 ~ 67.1 66.8 ~ 67.4 64.4 ~ 65.1 60.8 ~ 61.8 62.3 ~ 62.9 62.0 ~ 62.7 amino acid 75.0 ~ 75.4 75.1 ~ 75.5 97.9 ~ 98.5 71.0 ~ 71.5 73.8 ~ 74.4 74.2 ~ 74.9 72.9 ~ 73.3 69.8 ~ 70.2 65.6 ~ 65.9 67.3 ~ 67.7 65.3 ~ 65.5 2A nucleotide 74.4 ~ 75.8 76.2 ~ 78.4 78.7 ~ 82.0 76.0 ~ 79.6 76.7 ~ 79.8 76.0 ~ 78.4 74.7 ~ 78.4 76.0 ~ 78.4 80.0 ~ 83.1 77.1 ~ 80.0 76..4 ~ 78.7 amino acid 92.0 ~ 96.7 92.7 ~ 95.3 94.0 ~ 96.7 93.3 ~ 96.7 94.0 ~ 98.0 92.7 ~ 95.3 92.7 ~ 95.3 92.0 ~ 96.7 95.3 ~ 98.7 93.3 ~ 96.0 92.7 ~ 96.0 2B nucleotide 74.7 ~ 76.4 76.8 ~ 79.1 80.8 ~ 82.5 80.5 ~ 82.8 75.8 ~ 79.5 75.1 ~ 78.1 73.1 ~ 75.8 74.1 ~ 77.1 79.1 ~ 80.8 80.8 ~ 83.2 74.4 ~ 77.4 amino acid 93.9 ~ 96.0 92.9 ~ 94.9 97.0 ~ 98.0 96.0 ~ 98.0 93.9 ~ 96.0 93.9 ~ 96.0 92.9 ~ 94.9 92.9 ~ 94.9 97.0 ~ 98.0 97.0 ~ 99.0 90.9 ~ 91.9 2C nucleotide 77.5 ~ 78.6 78.2 ~ 80.6 82.7 ~ 84.3 82.9 ~ 84.1 78.2 ~ 79.1 79.1 ~ 80.1 78.5 ~ 79.8 78.3 ~ 80.2 82.8 ~ 84.5 83.1 ~ 85.1 78.2 ~ 79.5 amino acid 96.4 ~ 97.6 96.4 ~ 97.3 96.0 ~ 97.6 96.7 ~ 97.9 97.0 ~ 98.2 95.7 ~ 97.0 96.4 ~ 97.6 96.7 ~ 97.9 96.7 ~ 97.9 97.0 ~ 98.2 94.5 ~ 95.7 P2 nucleotide 76.5 ~ 77.3 77.9 ~ 79.1 81.5 ~ 83.4 81.3 ~ 82.4 78.2 ~ 78.8 78.1 ~ 78.9 76.6 ~ 78.5 77.2 ~ 79.0 81.7 ~ 83.1 81.5 ~ 83.2 77.8 ~ 78.4 amino acid 95.7 ~ 96.5 95.2 ~ 96.0 96.2 ~ 96.9 96.4 ~ 97.1 96.5 ~ 97.2 95.2 ~ 96.0 95.3 ~ 96.0 95.7 ~ 96.5 97.1 ~ 97.8 96.5 ~ 97.2 93.9 ~ 94.8 3A nucleotide 77.1 ~ 79.1 76.4 ~ 79.1 83.3 ~ 86.0 80.2 ~ 82.6 76.0 ~ 77.5 75.6 ~ 79.1 77.1 ~ 79.5 76.7 ~ 78.7 80.6 ~ 84.5 79.5 ~ 82.2 74.4 ~ 77.9 amino acid 94.2 ~ 97.7 93.0 ~ 95.3 96.5 ~ 97.7 95.3 ~ 96.5 93.0 ~ 95.3 93.0 ~ 96.5 94.2 ~ 95.3 94.2 ~ 95.3 96.5 ~ 97.7 95.3 ~ 98.8 90.7 ~ 94.2 3B nucleotide 68.2 ~ 74.2 65.2 ~ 75.8 75.8 ~ 84.8 77.3 ~ 83.3 68.2 ~ 74.2 71.2 ~ 75.8 72.7 ~ 77.3 68.2 ~ 74.2 81.8 ~ 92.4 78.8 ~ 86.4 74.2 ~ 80.3 amino acid 86.4 ~ 95.5 86.4 ~ 95.5 86.4 ~ 95.5 86.4 ~ 95.5 86.4 ~ 95.5 81.8 ~ 95.5 86.4 ~ 95.5 86.4 ~ 95.5 86.4 ~ 95.5 90.9 ~ 95.5 86.4 ~ 95.5 3C nucleotide 76.5 ~ 79.1 76.1 ~ 77.4 81.8 ~ 84.2 80.1 ~ 82.3 76.3 ~ 78.1 74.5 ~ 77.0 76.9 ~ 78.7 78.1 ~ 80.7 82.5 ~ 85.6 80.9 ~ 82.9 74.1 ~ 75.6 amino acid 91.9 ~ 95.7 94.5 ~ 96.7 95.1 ~ 97.8 95.1 ~ 96.7 94.5 ~ 96.7 91.8 ~ 94.5 93.4 ~ 96.2 95.1 ~ 97.3 96.2 ~ 98.9 95.6 ~ 98.4 91.8 ~ 94.5 3D nucleotide 77.6 ~ 78.9 78.0 ~ 78.9 83.8 ~ 85.0 77.6 ~ 79.3 77.5 ~ 79.1 78.3 ~ 80.7 76.9 ~ 78.5 77.9 ~ 79.1 82.8 ~ 83.8 82.4 ~ 84.8 78.1 ~ 80.4 amino acid 93.1 ~ 94.6 92.4 ~ 93.9 96.1 ~ 97.0 93.3 ~ 95.2 92.9 ~ 93.7 92.6 ~ 94.6 92.9 ~ 94.4 92.9 ~ 94.2 95.5 ~ 96.3 95.5 ~ 96.8 91.6 ~ 93.7 P3 nucleotide 77.4 ~ 78.5 77.5 ~ 78.0 83.5 ~ 84.2 79.1 ~ 80.3 77.2 ~ 78.4 77.4 ~ 79.2 77.3 ~ 78.4 78.0 ~ 79.1 83.0 ~ 84.1 81.9 ~ 83.2 76.9 ~ 78.7 amino acid 93.4 ~ 95.1 93.2 ~ 94.6 96.1 ~ 96.8 93.9 ~ 95.5 93.5 ~ 94.4 92.8 ~ 94.3 93.5 ~ 94.8 93.8 ~ 94.6 95.9 ~ 96.9 95.8 ~ 96.9 92.0 ~ 93.5 Recombination analysis To investigate the recombination of CVA4 in this study,bootscan and similarity plot analysis was performed on 50 strains using software Simplot v3.5.1 (Fig. 4). YT04, YT13, YT22 and LY17 were used as query sequences, and EV-A prototype strains were used as reference sequence for similarity and bootscan analysis. It was found that all CVA4 strains had high similarity with CVA4 prototype strain in P1 region. However, in the 5′ UTR region, all strains showed high similarity to CVA5, CVA14 and CVA16, and bootscan analysis also confirmed that all strains in this study had a potential recombination of approximately 250 bp with CVA16 in the 5′ UTR region. According to similarity plot analysis, all strains showed high similarity to CV-A4, CV-A5, CV-A14 and CVA16 in P2 and P3 regions. According to bootscan analysis, all strains except LY17 had potential recombination with CVA5 in region 2C and region P3; bootscan patterns of LY16, LY29, LY33 ~ 37 were similar to YT22, and potential recombination with CVA14 existed in region 2A. Discussion Since CVA4 was first isolated in 1948, it has been widespread worldwide for many years 8 . In most cases, CVA4 infection has no obvious symptoms, but it can cause HA, HFMD, respiratory tract infection and other diseases 9 , and even cause severe central nervous system symptoms 10 . Since the 1980s, CVA4 has become the main pathogen of HA in Japan 11 and continues to dominate 12 – 14 . In recent years, CVA4 has also become the main pathogen of HA in China 15 – 17 . The main pathogens of HFMD are EV-A71, CVA6, CVA16 and CVA10. Given that EV-A71 and CVA16 often lead to severe cases 18 , the early monitoring in China mainly focused on EV-A71, CVA16 and the "other enteroviruses" categories. Subsequent studies found that CVA6 and CVA10 were responsible for a higher proportion of HFMD cases in the category of "other enteroviruses" 19 , 20 , which led to their inclusion in routine surveillance in some regions. Although CVA4 is classified as "other enteroviruses" and is not listed as a routine serotype surveillance item for HFMD, recent studies have shown that the proportion of CVA4 in this category has increased with the increase in the detection rate of "other" enteroviruses and the improvement of laboratory detection capabilities 16 , 21 . Therefore, there is a significant need to intensify research on CVA4. In this study, 60 strains of CVA4 isolated from throat swabs of HA patients in 2024 were sequenced, and the complete genome sequences of 50 strains of CVA4 were successfully obtained. Genome-wide phylogenetic analysis showed that CVA4 strains circulating worldwide had formed significant genetic diversity, which was mainly divided into five evolutionary clades, Group 1–5. These strains were isolated from a number of different countries and regions, indicating that CVA4 has the ability to spread geographically, which may be a key factor in its global pandemic. At the same time, the strains in this study were closely related to CVA4 in Shandong Province in phylogenetic analysis, indicating that CVA4 had geographical aggregation at the same time. It should be noted that the China strains in this study mainly clustered in Group 2, suggesting that Group 2is the dominant epidemic branch of CVA4 at the whole genome level in China. VP1 region contains neutralizing antigenic determinants. At present, serotyping based on VP1 region sequence has become the main method to study the molecular epidemiology of enteroviruses. In this study, not only the complete VP1 sequence but also the full length sequence of CVA4 can be obtained by reference sequence assembly through second generation sequencing technology. In this study, the CVA4 VP1 region sequences of 60 throat swab samples from HA children in Linyi and Yantai in 2024 were successfully amplified, and phylogenetic trees were constructed with VP1 region gene representative sequences downloaded from Genebank public database for molecular evolution analysis. According to the typing criteria of Ji 7 , CVA4 strains are mainly divided into four genotypes A, B, C and D, among which genotype C is further divided into five subgenotypes: C1-C5. subgenotypes C1 and C3 were mainly prevalent between 1996 and 2007,subtype C2 became prevalent after 2006, and subgenotypes C4 and C5were discovered after 2014. Phylogenetic tree analysis based on VP1 region showed that all strains in this study belonged to C2 subgenotype, and C2 subgenotype was the dominant subgenotype in China. In addition, several unknown subgenotypes of CVA4 sequences were found in this study. During the phylogenetic analysis of the whole genome, three sequences from France (OQ091482, OQ091483, OQ091482) were found to have nucleotide sequence differences in VP1 region greater than 15% compared with other sequences, which may be new subgenotypes. According to the identity analysis, nucleotide homology between CVA 4 strains in this study and other non-CVA4 EV-A prototype strains is high in the non-P1 region, and the positions of these CVA4 strains are significantly different from those of other EV serotypes in the phylogenetic tree of P1, P2 and P3 regions.This difference suggests that recombination events between CVA4 and other EV serotypes may occur in the non-P1 region. EV is a small RNA virus, and its evolution depends mainly on gene mutation and recombination 22 .Since 1962, when poliovirus and influenza virus recombination events were first reported, studies on EV recombination have increased 23 , and many of the pathogens responsible for HFMD and HA outbreaks have resulted from recombination between different genotypes 24 – 26 . The P1 region of EV is relatively conservative, and recombination rarely occurs in this region. The CVA4 sequence in this study shows high similarity with CVA4 prototype strain in P1 region, which also indicates that P1 region is relatively conservative in EV. It is still reliable to classify EV types according to VP1 region. Non-structural protein coding regions (P2, P3) are mainly responsible for encoding proteases or active molecules related to viral genome replication and viral particle packaging. Many studies have shown that P2 and P3 regions are high recombination regions 27 , 28 . In this study, all strainsshowed high similarity with CV-A4, CV-A5, CV-A14 and CV-A16 in 5′ UTR. According to bootscan analysis, all strains except LY17 had recombination with CVA5 in 2C and P3 regions. LY16, LY26, LY29, LY33 ~ 37, YT22 had recombination with CVA14 in 2A to 2B regions. Whether these recombination changes the environmental adaptability and disease severity of CVA4 and whether the widespread spread of CVA4 in Shandong Province in recent years is related to these recombination needs further study. At present, due to the use of EV-A71 vaccine, complex dynamic evolution and co-circulation of EV, the pathogenic spectrum of related diseases has changed, and the infection rate of non-EV-A71 has greatly increased. Therefore, it is not possible to focus only on a few common serotypes, and continuous and comprehensive monitoring of EV types should be carried out. Methods Source of samples and cells Herpangina patients in sentinel monitoring hospitals of Linyi City and Yantai City in China in 2024 were selected as the study objects. The inclusion criteria of the study objects were that outpatients or inpatients had sore throat, fever, herpes and ulceration in palatopharyngeal arch, soft palate, ptosis and tonsil, but no herpes and ulceration in hands, feet and trunk were defined as HA patients. The Center for Disease Control and Prevention of the sentinel hospital collected throat swab samples from patients meeting HA inclusion criteria, and the positive nucleic acid samples were sent to Shandong Province Center for Disease Control and Prevention for use in this study. A total of 60 CVA4 strains were isolated from 256 throat swab samples from HA children, and the 60 strains were subjected to whole genome amplification and sequencing and further analysis. Among them, the screening conditions of strain were identified as CVA4 by RT-PCR and CT value ≤ 18. RD cells were provided by Shandong Province Center for Disease Control and Prevention. Virus Isolation and RNA Extraction Throat swab specimens stored at -80°C were thawed at room temperature. A 200 µL aliquot of each sample was inoculated into RD cell culture tubes exhibiting robust growth. Following viral propagation, cytopathic effect (CPE) was monitored daily until ≥ 75% of cells exhibited alteration (3 + CPE). The culture was then stored at -80°C for secondary subculture.When significant CPE reoccurred in the second passage, the P2 virus stock was archived at -80°C for subsequent analysis. Viral nucleic acids were extracted using the qEx-DNA/RNA Pathogen Kit (Xi'an Tianlong Technology) strictly according to the manufacturer's protocol. Identification and Typing of EVs EVs were identified using reverse transcription polymerase chain reaction (RT-PCR). Select samples with CT value ≤ 37 and sigmoidal amplification curves were selected for identification using the Coxsackievirus A4 Nucleic Acid Detection Kit (Real-time PCR). Reagent preparation, operating condition setting and result judgment were carried out according to reagent instructions. Library construction and sequencing A total of 60 CVA4 strains with CT value ≤ 18 were screened. Viral RNA was enriched using the Coxsackievirus A4 Nucleic Acid Enrichment Kit (multi-prider capture amplification) (Shanghai BioGerm Medical Technology). Enriched products underwent RT-PCR, followed by purification and next-generation sequencing (NGS) library preparation. Libraries were constructed with the Nextera XT DNA Library Prep Kit (Illumina, USA). Sequencing was performed on the MiSeq platform (Illumina, USA) using MiSeq Reagent Kit v2 (300-cycle) with 2×150 bp paired-end reads. Sequencing data analysis VP1 region sequences and whole genome sequences of CVA4 strains worldwide from 1948 to 2024 were downloaded from GenBank public database as reference sequences. The VP1 sequences with high homology (nucleotide identity ≥ 98%) and obvious errors were deleted. 213 VP1 gene sequences (including 60 CVA4 strains in this study and 154 strains from other countries and provinces in China) were included. A total of 222 CVA4 complete genome sequences (including 50 strains in this study and 172 CVA4 complete genome sequences from other countries and provinces in China) were included. The nucleotide and amino acid sequences were aligned by MAFFT software (v7.505). Genious Prime 2025 software was used to analyze the sequence identity of CVA4 strains and EV-A prototype strain in this study. MEGA11 software was used to construct phylogenetic tree of VP1 region by Neighbor-Joining (NJ) method. Maximum Likelihood (ML) trees for P1, P2 and P3 regions were constructed using Iqtree software (v1.6.12), with Bootstrap values set to 1000, results were visualized and trees beautified using software Figtree (v1.4.4) and ITOL (v7) websites, and potential recombination between CVA 4 strains and other EV-A prototypes was analyzed using the software Similarity Plots (v3.5.1). Declarations Author contributions statement All authors have contributed significantly, and that all authors reviewed the manuscript. Wenqing Ma: Conceptualization, Investigation, Formal analysis, Writing Original Draft, Zhen Dong: Investigation, Xiaodong Mou and Fengtao Zhang: Investigation (early experiments), Data Curation, Resources, Xiaolin Liu and Xiaolin Yu: Data Curation, Resources,Jianxing Wang: Conceptualization, Methodology, Resources,Zengqiang Kou: Resources, Methodology. Competing interests The authors declare no competing interests. Ethical approval The samples used in the study were taken after the approval of the ethical committees from the respective laboratories. This study was approved by institutional review board of Shandong center for disease control and prevention(approval number: SDJK(K)2024-012-01). All methods were performed in accordance with the relevant guidelines and regulations. Written informed consent was taken from the patients. Funding This work was supported by Medical and Health Science and Technology Development Program Fund of Shandong Province(202012061392). Title: Study on the Epidemic Pattern and Pathogen Composition of Severe Hand-Foot-Mouth Disease Cases in Shandong Province. Author Contribution All authors have contributed significantly, and that all authors reviewed the manuscript. Wenqing Ma: Conceptualization, Investigation, Formal analysis, Writing Original Draft, Zhen Dong: Investigation, Xiaodong Mou and Fengtao Zhang: Investigation (early experiments), Data Curation, Resources, Xiaolin Liu and Xiaolin Yu: Data Curation, Resources,Jianxing Wang: Conceptualization, Methodology, Resources,Zengqiang Kou: Resources, Methodology. Data Availability The datasets, are available, supporting the conclusions of this article with the accession numbers PX227139-PX227198 are available in the GenBank [National Center for Biotechnology Information] repository.Take PX227139 as an example, the link is：https://www.ncbi.nlm.nih.gov/nuccore/PX227139. References Fu, Y. L. et al. Analysis of the epidemic characteristics and disease burden of herpangina in hospitalized children in China from 2016 to 2020. Chin. J. Exp. Clin. Virol. 37 , 7–12. 10.3760/cma.j.cn112866-20220607-00095 (2023). Li, J. S. et al. Outbreak of febrile illness caused by coxsackievirus A4 in a nursery school in Beijing, China. Virol. J. 12 , 92. 10.1186/s12985-015-0325-1 (2015). Chu, P. Y. et al. Spatiotemporal phylogenetic analysis and molecular characterization of coxsackievirus A4. Infect. Genet. Evol. 11 , 1426–1435. 10.1016/j.meegid.2011.05.010 (2011). Zhao, T. S. et al. Research progress in epidemiology and etiology of herpangina. Chin. J. Viral Dis. 10 , 385–390. 10.16505/j.2095-0136.2020.0022 (2020). Li, R. et al. Epidemiological characteristics of herpetic angina and analysis of gene characteristics of related enterovirus VP1 detected in a sentinel hospital in Shanghai, 2020–2021. Dis. Surveill . 39 , 439–445. 10.3784/jbjc.202305290252 (2024). Lulla, V. et al. An upstream protein-coding region in enteroviruses modulates virus infection in gut epithelial cells. Nat. Microbiol. 4 , 280–292. 10.1038/s41564-018-0297-1 (2019). Ji, T. J. et al. 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Prevalence of enterovirus from patients with herpangina and hand, foot and mouth disease in Nagano Prefecture, Japan, 2007. Jpn J. Infect. Dis. 61 , 247–248 (2008). Yamashita, T., Ito, M., Taniguchi, A. & Sakae, K. Prevalence of coxsackievirus A5, A6, and A10 in patients with herpangina in Aichi Prefecture, 2005. Jpn J. Infect. Dis. 58 , 390–391 (2005). Sano, T. et al. Enterovirus detection status of patients with herpangina and hand, foot and mouth disease in epidemic season 2007, Kanagawa Prefecture, Japan. Jpn J. Infect. Dis. 61 , 162–163 (2008). Xiao, M. et al. Etiology and Clinical Characteristics of Herpangina and Hand, Foot,and Mouth Disease in Chengde, China in 2021. Bing Du Xue Bao . 39 , 735–742. 10.13242/j.cnki.bingduxuebao.004306 (2023). Zhang, W. T. et al. [Pathogen spectra of hand-foot and mouth disease and herpangina in Xuzhou, Jiangsu, 2021. Dis. Surveill . 39 , 863–868 (2024). Zhang, J. et al. Investigation of an outbreak of herpangina caused by coxsackievirus A4 in a kindergartens in Sichuan Province. J. Occup. Health Damage . 39 , 21–25. 10.19973/j.cnki.1006-172x.23.103 (2024). Yang, Y. Q. et al. Clinical risk of severe cases of HFMD with CV-A6 serotype. J. Trop. Med. 24 , 1224–1229 (2024). Li, Y. et al. Emerging enteroviruses causing hand, foot and mouth disease, China, 2010–2016. Emerg. Infect. Dis. 24 , 1902–1906. 10.3201/eid2410.171953 (2018). Yang, Q. et al. Molecular epidemiology and clinical characteristics of enteroviruses associated HFMD in Chengdu, China, 2013–2022. Virol. J. 20 , 202. 10.1186/s12985-023-02169-x (2023). Weng, Y. et al. Serotyping and genetic characterization of hand, foot, and mouth disease (HFMD)-associated enteroviruses of no-EV71 and non-CVA16 circulating in Fujian, China, 2011–2015. Med. Sci. Monit. 23 , 2508–2518. 10.12659/MSM.901364 (2017). Muslin, C., Mac Kain, A., Bessaud, M., Blondel, B. & Delpeyroux, F. Recombination in enteroviruses, a multi-step modular evolutionary process. Viruses 11 , 859. 10.3390/v11090859 (2019). Nikolaidis, M. et al. Large-scale genomic analysis reveals recurrent patterns of intertypic recombination in human enteroviruses. Virology 526 , 72–80. 10.1016/j.virol.2018.10.006 (2019). Hu, Y. F. et al. Complete genome analysis of coxsackievirus A2, A4, A5, and A10 strains isolated from hand, foot, and mouth disease patients in China revealing frequent recombination of human enterovirus A. J. Clin. Microbiol. 49 , 2426–2434. 10.1128/JCM.00007-11 (2011). Li, J. et al. An outbreak of a novel recombinant Coxsackievirus A4 in a kindergarten, Shandong province, China, 2021. Emerg. Microbes Infect. 11 , 2207–2210 (2022). 10.1080/22221751.2022.2114855 Wei, W. et al. Circulating HFMD-associated coxsackievirus A16 is genetically and phenotypically distinct from the prototype CV-A16. PLoS ONE . 9 , e94746. 10.1371/journal.pone.0094746 (2014). Song, J. et al. [Genome-wide analysis reveals complex patterns of intertypic recombination in human enterovirus A]. Bing Du Xue Bao . 35 , 454–460. 10.13242/j.cnki.bingduxuebao.003537 (2019). Lv, T. et al. Genome analysis of coxsackievirus A4 identified from herpangina children in northern China. Curr. Microbiol. 82 , 83. 10.1007/s00284-025-04075-7 (2025). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 09 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 16 Dec, 2025 Reviews received at journal 16 Dec, 2025 Reviewers agreed at journal 03 Dec, 2025 Reviewers agreed at journal 24 Nov, 2025 Reviews received at journal 04 Oct, 2025 Reviewers agreed at journal 24 Sep, 2025 Reviewers agreed at journal 21 Sep, 2025 Reviewers invited by journal 15 Sep, 2025 Editor assigned by journal 10 Sep, 2025 Editor invited by journal 10 Sep, 2025 Submission checks completed at journal 09 Sep, 2025 First submitted to journal 09 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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13:02:25","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126520,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7407631/v1/48d4ffd56956420cbcb715cf.html"},{"id":92086239,"identity":"6f527084-c3a9-43a1-919a-0f5713b105e3","added_by":"auto","created_at":"2025-09-24 13:02:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5266189,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree (Neighbor-Joining method) based on the VP1 gene (915 bp) of representative strains of CVA4 collected from 1948 to 2024.The isolated strains of CVA4 in this study are indicated in bold font.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7407631/v1/820b6d0af4e1115cbbc3c74c.png"},{"id":92086243,"identity":"72bd61c1-6fde-431b-91bc-466e32789070","added_by":"auto","created_at":"2025-09-24 13:02:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":37760415,"visible":true,"origin":"","legend":"\u003cp\u003eThe phylogenetic tree of the CVA4 virus was constructed based on the whole-genome sequence.The blue fonts are the strains studied in this paper.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7407631/v1/a1cb6bad845a2824bbdab956.png"},{"id":92086230,"identity":"218997b7-3b0e-4d4a-81ad-a111f5593eb5","added_by":"auto","created_at":"2025-09-24 13:02:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3200852,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7407631/v1/ec52d81d1f56bf15d7ac36d5.png"},{"id":92086232,"identity":"a6b2781c-06c9-485b-b87b-fe4d162bccc9","added_by":"auto","created_at":"2025-09-24 13:02:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1087018,"visible":true,"origin":"","legend":"\u003cp\u003eThe similarity diagrams and bootscan analysis diagrams of 5 representative strains of CVA4 and the prototype strains of EV-A in this study.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7407631/v1/5aa296c81f93955361ff8209.png"},{"id":102785322,"identity":"25692546-8dcc-4dbf-a139-d24abb8ed312","added_by":"auto","created_at":"2026-02-16 16:05:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":51310280,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7407631/v1/daa9cc49-2ac3-47d2-8164-0da21ed9f90e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Whole-genome analysis of CVA4 in children with herpangina in two cities of China","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHerpangina (HA) and hand-food-mouth disease (HFMD) are common acute enteroviral infectious diseases in children. HFMD is manifested as papules or herpes on the mouth, hands, feet and buttocks. Some cases can progress to encephalitis, meningitis, pulmonary hemorrhage, heart failure and other serious complications, even life-threatening. Although HA rarely causes such serious consequences, its strong infectivity and fast transmission speed make it easy to cause sporadic and outbreak epidemic in schools and kindergartens, bringing economic and psychological burden that cannot be ignored to children's families and society\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. CVA4 belongs to Enterovirus A (EV-A), which is one of the common pathogens causing HFMD and HA. Epidemiological surveillance data show an increasing trend of HFMD cases due to CVA4\u003csup\u003e2,3\u003c/sup\u003e, previous etiological studies have shown that CVA2, CVA4 and CVA6 are the main pathogenic serotypes of HA\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCVA4 is a single-stranded RNA virus classified as Enterovirus (EV) in Picornaviridae. Its genome is about 7.4 kb in length and consists of two open reading frames (ORFs), ORF sencoding four structural proteins (VP1, VP2, VP3 and VP4) and seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D), and uORFs encoding a single protein uORFP\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. At present, the systematic analysis and genetic evolution of the whole genome of CVA4 are still scarce. This study revealed the genetic evolution mechanism and variation characteristics of CVA4 strain by analyzing the whole genome characteristics and genetic characteristics of CVA4 strain, providing laboratory data for genetic variation of CVA4 epidemic strain in some areas of China, and having reference significance for vaccine development and prevention and control of related diseases of CVA4.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn 2024, 256 throat swab specimens from herpangina (HA) patients in Linyi and Yantai cities (Shandong Province, China) were subjected to RD cell culture and RT-PCR screening, yielding 60 strains of CVA4.The patient cohort comprised 40 males and 20 females (male-to-female ratio: 2:1), aged 0\u0026ndash;10 years, with 91.7% (55/60) being preschool children (\u0026le;\u0026thinsp;6 years).\u003c/p\u003e\n\u003cp\u003eHigh-throughput sequencing generated complete genomic sequences for all 60 CVA4 strains, designated as: YT01/SD/CVA4/2024 to YT23/SD/CVA4/2024 (abbreviated YT01-YT23)、LY01/SD/CVA4/2024 to LY33/SD/CVA4/2024 (abbreviated LY01-LY33).\u003c/p\u003e\n\u003cp\u003eSequence deficiencies were identified in:\u003c/p\u003e\n\u003cp\u003eStrains YT01, YT03, YT06, YT12, YT15, YT21, LY24, LY28, LY30 and LY31 exhibited incomplete ORF due to insufficient sequencing coverage.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003ePhylogenetic Analysis Based on VP1 Region\u003c/h2\u003e\n\u003cp\u003eThe VP1 region of CVA4 is 915 nucleotides in length and encodes 305 amino acids in total. According to Ji \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003etyping standard, the genotype difference of VP1 region of the same serotype\u0026thinsp;\u0026gt;\u0026thinsp;15% is the subtype classification standard, and CVA4 can be divided into 4 genotypes A\u0026thinsp;~\u0026thinsp;D (Fig.\u0026nbsp;1). Genotype A includes a prototype strain of CVA4 isolated from North Carolina in the United States in 1948 (AY421762); genotype B includes a strain from Kenya in East Africa in 1999 (GQ176232); genotype D includes a strain from Japan in 2008 (AB457644); genotype C consists of strains isolated from China, India, Australia, Russia, and the United States between 1996 and 2024. Genotype C can be further divided into five sub-genotypes: C1-C5. The CVA4 strains in this study were clustered into subtype C2, and their phylogenetic relationship showed that most of the strains were mainly similar to Yunnan strain (LC865850) identified in 2023 and Yunnan strain (LC707448) identified in 2021, while LY17 strain was not clustered into a branch with other strains, and was closely related to Sichuan strain (MW178773) identified in 2019.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Table 1 The year, location and quantity information of the A-D related sequences of the CVA4 VP1 sequence genotype\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Taba\" style=\"width: 1003px;\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth style=\"width: 48px;\" rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eCountry\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 212px;\" colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eA\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 212px;\" colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eB\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 332.34px;\" colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eC\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 52.8588px;\" align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth style=\"width: 181.118px;\" colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eD\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth style=\"width: 64px;\" align=\"left\"\u003e\n\u003cp\u003eEarliest record\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 56px;\" align=\"left\"\u003e\n\u003cp\u003eLatest record\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 92px;\" align=\"left\"\u003e\n\u003cp\u003enumber of sequences\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 64px;\" align=\"left\"\u003e\n\u003cp\u003eEarliest record\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 56px;\" align=\"left\"\u003e\n\u003cp\u003eLatest record\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 92px;\" align=\"left\"\u003e\n\u003cp\u003enumber of sequences\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 63.1365px;\" align=\"left\"\u003e\n\u003cp\u003eEarliest record\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 56px;\" align=\"left\"\u003e\n\u003cp\u003eLatest record\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 92px;\" align=\"left\"\u003e\n\u003cp\u003enumber of sequences\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 69.5949px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eEarliest record\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 62px;\" align=\"left\"\u003e\n\u003cp\u003eLastest record\u003c/p\u003e\n\u003c/th\u003e\n\u003cth style=\"width: 92px;\" align=\"left\"\u003e\n\u003cp\u003enumber of sequences\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 48px;\" align=\"left\"\u003e\n\u003cp\u003eChina\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 64px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 56px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 64px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 56px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 63.1365px;\" align=\"left\"\u003e\n\u003cp\u003e1996\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 56px;\" align=\"left\"\u003e\n\u003cp\u003e2024\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\n\u003cp\u003e142\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 69.5949px;\" colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 62px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 48px;\" align=\"left\"\u003e\n\u003cp\u003eAmerica\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 64px;\" align=\"left\"\u003e\n\u003cp\u003e1948\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 56px;\" align=\"left\"\u003e\n\u003cp\u003e1948\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 64px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 56px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 63.1365px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 56px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 69.5949px;\" colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 62px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 48px;\" align=\"left\"\u003e\n\u003cp\u003eKenya\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 64px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 56px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 64px;\" align=\"left\"\u003e\n\u003cp\u003e1999\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 56px;\" align=\"left\"\u003e\n\u003cp\u003e1999\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 63.1365px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 56px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 69.5949px;\" colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 62px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 48px;\" align=\"left\"\u003e\n\u003cp\u003eJapan\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 64px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 56px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 64px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 56px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 63.1365px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 56px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 69.5949px;\" colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e2008\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 62px;\" align=\"left\"\u003e\n\u003cp\u003e2008\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 48px;\" align=\"left\"\u003e\n\u003cp\u003eIndia\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 64px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 56px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 64px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 56px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 63.1365px;\" align=\"left\"\u003e\n\u003cp\u003e2007\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 56px;\" align=\"left\"\u003e\n\u003cp\u003e2008\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 69.5949px;\" colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 62px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 48px;\" align=\"left\"\u003e\n\u003cp\u003eRussia\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 64px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 56px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 64px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 56px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 63.1365px;\" align=\"left\"\u003e\n\u003cp\u003e2013\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 56px;\" align=\"left\"\u003e\n\u003cp\u003e2014\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 69.5949px;\" colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 62px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 92px;\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003eGenome-wide analysis\u003c/h3\u003e\n\u003cp\u003eThe nucleotide lengths of 50 CVA4 strains in this study ranged from 7349bp to 7434bp. Compared with the CVA4 reference strain High Point (AY421762), there were partial deletions in the 5\u0026prime; untranslated regions (UTR) and 3\u0026prime; UTR regions. The open reading frame (ORF) between the 5\u0026prime;UTR and 3\u0026prime; UTR of CVA4 strains was 6606bp, encoding a polyprotein containing 2203 amino acids, the entire genome structure of the virus conforms to the characteristics.\u003c/p\u003e\n\u003cp\u003ePhylogenetic analysis of 222 complete genome sequences (including 50 CVA4 strains in this study and 172 CVA4 complete genome sequences included in GenBank public database) showed that the complete genome phylogenetic tree could be divided into five groups: Group1 (n\u0026thinsp;=\u0026thinsp;5), Group2 (n\u0026thinsp;=\u0026thinsp;164), Group3 (n\u0026thinsp;=\u0026thinsp;33), Group4 (n\u0026thinsp;=\u0026thinsp;19) and Group5 (n\u0026thinsp;=\u0026thinsp;1). All strains in this study were located in Group2, clustered into a group with China CVA4 strains, and most closely related to Shandong strain. LY17 strains have the closest genetic relationship with the isolate from Taian City, Shandong Province in 2019 (ON730865); the rest strains have the closest genetic relationship with the isolate from Linyi City, Shandong Province in 2024.\u003c/p\u003e\n\u003cp\u003eThe nucleotide and amino acid sequences of the whole genome of 50 CVA4 strains were analyzed (Table\u0026nbsp;2), and the nucleotide identity among 50 CVA4 strains was 88.9%~99.9%, and the amino acid identity was 96.3%~99.9%, and the nucleotide identity with High Point of CVA4 prototype strain was 84.1%~84.5%. Among them, LY17 strain had poor nucleotide identity with other strains, 88.9%~90.3%, The nucleotide and amino acid sequence identity analysis of ORF region between 50 strains and EV-A prototype strain showed that the nucleotide and amino acid similarities with CVA4 prototype strain in P1 region were 85.1%~85.7%, 97.9%~98.5%, respectively, and the nucleotide and amino acid similarities with other EV-A prototype strains were 60.8%~67.4%, 65.3%~75.5%, respectively. In the P2 region, the CVA4 strain in this study showed a high nucleotide consistency with the CVA4, CVA5, CVA14, and CVA16 prototype strains, reaching over 80%, the amino acid similarity was above 95%. In the P3 region, the nucleotide similarity with the CVA4, CVA14, and CVA16 prototype strains was high, reaching over 80%, and the amino acid similarity was above 90%.\u003c/p\u003e\n\u003cp\u003eFor further genome analysis, a phylogenetic tree of P1, P2 and P3 regions was constructed using 85 complete genome sequences (Fig.\u0026nbsp;3), including 11 EV-A prototype strains, 50 CVA4 strains, 7 CVA4 subgenotypes C4 and C3 (as can be seen from Fig.\u0026nbsp;2,complete genome sequences for genotypes B and D and C1 and C3 are currently unavailable from GenBank public databases due to limited data on CVA4 genome sequences), and 17 complete genome sequences for CVA5, CVA14 and CVA16. The 50 CVA4 strains in this study were located at different positions in the phylogenetic tree of P1, P2 and P3 regions. In the phylogenetic tree of P1 region, the CVA4 strains were closely related to C4 and C5 subgenotypes, and clustered into a large cluster; in the phylogenetic tree of P2 region, except LY17, the other CVA4 strains were closely related to a C5 subtype strain (MN964077) from Yancheng, China, LY17 was closely related to Yancheng C5 subtype strain and CVA5 strain (MZ491030); in P3 region, LY17 strain was closely related to CVA5 strain (MZ461030), and the rest strains were closely related to CVA14 strain(KP036482).\u003c/p\u003e\n\u003cp\u003eTable 2 Consistency (%) between 50 CVA4 strains and each segment of the ORF region of the EV-A prototype strain genome.Due to incomplete 5\u0026rsquo;UTR sequencing, the amino acid consistency of the whole genome and the 5\u0026rsquo;utr region was not analyzed.The blue background area represents the consistency of nucleotide sequences; The blue line divides the P1, P2 and P3 regions.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tabb\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eregion\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"12\" align=\"left\"\u003e\n\u003cp\u003eEV-A prototype strain\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCVA2\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCVA3\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCVA4\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCVA5\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCVA6\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCVA8\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCVA10\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCVA12\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCVA14\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCVA16\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eEV-A71\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGenome\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enucleotide\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e73.4\u0026thinsp;~\u0026thinsp;73.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e73.8\u0026thinsp;~\u0026thinsp;74.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e84.1\u0026thinsp;~\u0026thinsp;84.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e74.4\u0026thinsp;~\u0026thinsp;74.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e74.0\u0026thinsp;~\u0026thinsp;74.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e73.6\u0026thinsp;~\u0026thinsp;74.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e73.5\u0026thinsp;~\u0026thinsp;74.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e74.0\u0026thinsp;~\u0026thinsp;73.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e74.8\u0026thinsp;~\u0026thinsp;75.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e75.1\u0026thinsp;~\u0026thinsp;75.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e72.1\u0026thinsp;~\u0026thinsp;72.6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5'UTR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enucleotide\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e80.3\u0026thinsp;~\u0026thinsp;81.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e80.7\u0026thinsp;~\u0026thinsp;81.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e87.0\u0026thinsp;~\u0026thinsp;88.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e81.3\u0026thinsp;~\u0026thinsp;82.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e84.9\u0026thinsp;~\u0026thinsp;86.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e78.7\u0026thinsp;~\u0026thinsp;80.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e80.2\u0026thinsp;~\u0026thinsp;82.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e79.2\u0026thinsp;~\u0026thinsp;80.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e83.3\u0026thinsp;~\u0026thinsp;84.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e83.4\u0026thinsp;~\u0026thinsp;84.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e79.3\u0026thinsp;~\u0026thinsp;80.4\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eVP4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enucleotide\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e64.7\u0026thinsp;~\u0026thinsp;67.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e68.1\u0026thinsp;~\u0026thinsp;70.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e85.0\u0026thinsp;~\u0026thinsp;86.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e65.7\u0026thinsp;~\u0026thinsp;68.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e70.0\u0026thinsp;~\u0026thinsp;72.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e67.1\u0026thinsp;~\u0026thinsp;70.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e69.1\u0026thinsp;~\u0026thinsp;71.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e68.6\u0026thinsp;~\u0026thinsp;71.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e64.7\u0026thinsp;~\u0026thinsp;67.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e64.7\u0026thinsp;~\u0026thinsp;65.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e63.8\u0026thinsp;~\u0026thinsp;64.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAmino acid\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e79.7\u0026thinsp;~\u0026thinsp;81.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e75.4\u0026thinsp;~\u0026thinsp;76.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e97.1\u0026thinsp;~\u0026thinsp;98.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e68.1\u0026thinsp;~\u0026thinsp;69.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e73.9\u0026thinsp;~\u0026thinsp;75.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e75.4\u0026thinsp;~\u0026thinsp;76.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e72.5\u0026thinsp;~\u0026thinsp;73.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e75.4\u0026thinsp;~\u0026thinsp;78.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e69.6\u0026thinsp;~\u0026thinsp;69.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e73.9\u0026thinsp;~\u0026thinsp;75.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e66.7\u0026thinsp;~\u0026thinsp;68.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eVP2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enucleotide\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e69.2\u0026thinsp;~\u0026thinsp;70.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e69.0\u0026thinsp;~\u0026thinsp;70.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e84.5\u0026thinsp;~\u0026thinsp;85.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e65.6\u0026thinsp;~\u0026thinsp;67.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e69.1\u0026thinsp;~\u0026thinsp;70.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e68.1\u0026thinsp;~\u0026thinsp;69.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e67.6\u0026thinsp;~\u0026thinsp;68.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e65.6\u0026thinsp;~\u0026thinsp;66.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e65.0\u0026thinsp;~\u0026thinsp;67.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e65.8\u0026thinsp;~\u0026thinsp;66.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e65.0\u0026thinsp;~\u0026thinsp;66.4\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eamino acid\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.0\u0026thinsp;~\u0026thinsp;78.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e79.7\u0026thinsp;~\u0026thinsp;80.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e98.0\u0026thinsp;~\u0026thinsp;99.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e74.2\u0026thinsp;~\u0026thinsp;75.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e81.3\u0026thinsp;~\u0026thinsp;82.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.7\u0026thinsp;~\u0026thinsp;78.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e79.3\u0026thinsp;~\u0026thinsp;80.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e74.2\u0026thinsp;~\u0026thinsp;75.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.0\u0026thinsp;~\u0026thinsp;78.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e73.0\u0026thinsp;~\u0026thinsp;73.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e72.7\u0026thinsp;~\u0026thinsp;73.4\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eVP3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enucleotide\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e70.7\u0026thinsp;~\u0026thinsp;72.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e70.4\u0026thinsp;~\u0026thinsp;71.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e85.8\u0026thinsp;~\u0026thinsp;86.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e67.5\u0026thinsp;~\u0026thinsp;68.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e68.3\u0026thinsp;~\u0026thinsp;69.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e70.0\u0026thinsp;~\u0026thinsp;71.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e69.3\u0026thinsp;~\u0026thinsp;70.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e69.0\u0026thinsp;~\u0026thinsp;70.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e62.9\u0026thinsp;~\u0026thinsp;64.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e62.7\u0026thinsp;~\u0026thinsp;63.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e63.8\u0026thinsp;~\u0026thinsp;65.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eamino acid\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e83.3\u0026thinsp;~\u0026thinsp;84.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e80.8\u0026thinsp;~\u0026thinsp;82.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e98.3\u0026thinsp;~\u0026thinsp;99.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.1\u0026thinsp;~\u0026thinsp;77.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e81.3\u0026thinsp;~\u0026thinsp;82.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e81.7\u0026thinsp;~\u0026thinsp;83.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.9\u0026thinsp;~\u0026thinsp;78.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e78.3\u0026thinsp;~\u0026thinsp;78.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e68.2\u0026thinsp;~\u0026thinsp;69.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e68.6\u0026thinsp;~\u0026thinsp;69.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e69.8\u0026thinsp;~\u0026thinsp;70.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eVP1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enucleotide\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e59.9\u0026thinsp;~\u0026thinsp;61.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e60.2\u0026thinsp;~\u0026thinsp;61.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e84.0\u0026thinsp;~\u0026thinsp;85.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e58.5\u0026thinsp;~\u0026thinsp;59.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e60.2\u0026thinsp;~\u0026thinsp;61.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e60.9\u0026thinsp;~\u0026thinsp;62.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e62.2\u0026thinsp;~\u0026thinsp;63.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e58.3\u0026thinsp;~\u0026thinsp;59.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e52.9\u0026thinsp;~\u0026thinsp;54.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e58.1\u0026thinsp;~\u0026thinsp;58.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e55.4\u0026thinsp;~\u0026thinsp;56.6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eamino acid\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e64.9\u0026thinsp;~\u0026thinsp;65.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e65.3\u0026thinsp;~\u0026thinsp;66.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e97.0\u0026thinsp;~\u0026thinsp;98.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e63.3\u0026thinsp;~\u0026thinsp;64.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e61.6\u0026thinsp;~\u0026thinsp;62.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e65.0\u0026thinsp;~\u0026thinsp;66.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e62.9\u0026thinsp;~\u0026thinsp;63.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e56.7\u0026thinsp;~\u0026thinsp;57.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e55.4\u0026thinsp;~\u0026thinsp;56.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e58.7\u0026thinsp;~\u0026thinsp;59.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e54.0\u0026thinsp;~\u0026thinsp;54.4\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eP1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enucleotide\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e66.6\u0026thinsp;~\u0026thinsp;67.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e66.7\u0026thinsp;~\u0026thinsp;67.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e85.1\u0026thinsp;~\u0026thinsp;85.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e63.8\u0026thinsp;~\u0026thinsp;64.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e66.3\u0026thinsp;~\u0026thinsp;66.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e66.3\u0026thinsp;~\u0026thinsp;67.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e66.8\u0026thinsp;~\u0026thinsp;67.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e64.4\u0026thinsp;~\u0026thinsp;65.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e60.8\u0026thinsp;~\u0026thinsp;61.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e62.3\u0026thinsp;~\u0026thinsp;62.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e62.0\u0026thinsp;~\u0026thinsp;62.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eamino acid\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e75.0\u0026thinsp;~\u0026thinsp;75.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e75.1\u0026thinsp;~\u0026thinsp;75.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e97.9\u0026thinsp;~\u0026thinsp;98.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e71.0\u0026thinsp;~\u0026thinsp;71.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e73.8\u0026thinsp;~\u0026thinsp;74.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e74.2\u0026thinsp;~\u0026thinsp;74.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e72.9\u0026thinsp;~\u0026thinsp;73.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e69.8\u0026thinsp;~\u0026thinsp;70.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e65.6\u0026thinsp;~\u0026thinsp;65.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e67.3\u0026thinsp;~\u0026thinsp;67.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e65.3\u0026thinsp;~\u0026thinsp;65.5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e2A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enucleotide\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e74.4\u0026thinsp;~\u0026thinsp;75.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.2\u0026thinsp;~\u0026thinsp;78.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e78.7\u0026thinsp;~\u0026thinsp;82.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.0\u0026thinsp;~\u0026thinsp;79.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.7\u0026thinsp;~\u0026thinsp;79.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.0\u0026thinsp;~\u0026thinsp;78.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e74.7\u0026thinsp;~\u0026thinsp;78.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.0\u0026thinsp;~\u0026thinsp;78.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e80.0\u0026thinsp;~\u0026thinsp;83.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.1\u0026thinsp;~\u0026thinsp;80.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76..4\u0026thinsp;~\u0026thinsp;78.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eamino acid\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92.0\u0026thinsp;~\u0026thinsp;96.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92.7\u0026thinsp;~\u0026thinsp;95.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e94.0\u0026thinsp;~\u0026thinsp;96.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.3\u0026thinsp;~\u0026thinsp;96.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e94.0\u0026thinsp;~\u0026thinsp;98.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92.7\u0026thinsp;~\u0026thinsp;95.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92.7\u0026thinsp;~\u0026thinsp;95.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92.0\u0026thinsp;~\u0026thinsp;96.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.3\u0026thinsp;~\u0026thinsp;98.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.3\u0026thinsp;~\u0026thinsp;96.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92.7\u0026thinsp;~\u0026thinsp;96.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e2B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enucleotide\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e74.7\u0026thinsp;~\u0026thinsp;76.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.8\u0026thinsp;~\u0026thinsp;79.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e80.8\u0026thinsp;~\u0026thinsp;82.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e80.5\u0026thinsp;~\u0026thinsp;82.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e75.8\u0026thinsp;~\u0026thinsp;79.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e75.1\u0026thinsp;~\u0026thinsp;78.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e73.1\u0026thinsp;~\u0026thinsp;75.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e74.1\u0026thinsp;~\u0026thinsp;77.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e79.1\u0026thinsp;~\u0026thinsp;80.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e80.8\u0026thinsp;~\u0026thinsp;83.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e74.4\u0026thinsp;~\u0026thinsp;77.4\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eamino acid\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.9\u0026thinsp;~\u0026thinsp;96.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92.9\u0026thinsp;~\u0026thinsp;94.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e97.0\u0026thinsp;~\u0026thinsp;98.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e96.0\u0026thinsp;~\u0026thinsp;98.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.9\u0026thinsp;~\u0026thinsp;96.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.9\u0026thinsp;~\u0026thinsp;96.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92.9\u0026thinsp;~\u0026thinsp;94.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92.9\u0026thinsp;~\u0026thinsp;94.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e97.0\u0026thinsp;~\u0026thinsp;98.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e97.0\u0026thinsp;~\u0026thinsp;99.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e90.9\u0026thinsp;~\u0026thinsp;91.9\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e2C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enucleotide\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.5\u0026thinsp;~\u0026thinsp;78.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e78.2\u0026thinsp;~\u0026thinsp;80.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e82.7\u0026thinsp;~\u0026thinsp;84.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e82.9\u0026thinsp;~\u0026thinsp;84.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e78.2\u0026thinsp;~\u0026thinsp;79.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e79.1\u0026thinsp;~\u0026thinsp;80.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e78.5\u0026thinsp;~\u0026thinsp;79.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e78.3\u0026thinsp;~\u0026thinsp;80.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e82.8\u0026thinsp;~\u0026thinsp;84.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e83.1\u0026thinsp;~\u0026thinsp;85.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e78.2\u0026thinsp;~\u0026thinsp;79.5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eamino acid\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e96.4\u0026thinsp;~\u0026thinsp;97.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e96.4\u0026thinsp;~\u0026thinsp;97.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e96.0\u0026thinsp;~\u0026thinsp;97.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e96.7\u0026thinsp;~\u0026thinsp;97.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e97.0\u0026thinsp;~\u0026thinsp;98.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.7\u0026thinsp;~\u0026thinsp;97.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e96.4\u0026thinsp;~\u0026thinsp;97.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e96.7\u0026thinsp;~\u0026thinsp;97.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e96.7\u0026thinsp;~\u0026thinsp;97.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e97.0\u0026thinsp;~\u0026thinsp;98.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e94.5\u0026thinsp;~\u0026thinsp;95.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eP2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enucleotide\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.5\u0026thinsp;~\u0026thinsp;77.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.9\u0026thinsp;~\u0026thinsp;79.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e81.5\u0026thinsp;~\u0026thinsp;83.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e81.3\u0026thinsp;~\u0026thinsp;82.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e78.2\u0026thinsp;~\u0026thinsp;78.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e78.1\u0026thinsp;~\u0026thinsp;78.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.6\u0026thinsp;~\u0026thinsp;78.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.2\u0026thinsp;~\u0026thinsp;79.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e81.7\u0026thinsp;~\u0026thinsp;83.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e81.5\u0026thinsp;~\u0026thinsp;83.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.8\u0026thinsp;~\u0026thinsp;78.4\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eamino acid\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.7\u0026thinsp;~\u0026thinsp;96.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.2\u0026thinsp;~\u0026thinsp;96.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e96.2\u0026thinsp;~\u0026thinsp;96.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e96.4\u0026thinsp;~\u0026thinsp;97.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e96.5\u0026thinsp;~\u0026thinsp;97.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.2\u0026thinsp;~\u0026thinsp;96.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.3\u0026thinsp;~\u0026thinsp;96.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.7\u0026thinsp;~\u0026thinsp;96.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e97.1\u0026thinsp;~\u0026thinsp;97.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e96.5\u0026thinsp;~\u0026thinsp;97.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.9\u0026thinsp;~\u0026thinsp;94.8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e3A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enucleotide\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.1\u0026thinsp;~\u0026thinsp;79.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.4\u0026thinsp;~\u0026thinsp;79.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e83.3\u0026thinsp;~\u0026thinsp;86.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e80.2\u0026thinsp;~\u0026thinsp;82.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.0\u0026thinsp;~\u0026thinsp;77.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e75.6\u0026thinsp;~\u0026thinsp;79.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.1\u0026thinsp;~\u0026thinsp;79.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.7\u0026thinsp;~\u0026thinsp;78.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e80.6\u0026thinsp;~\u0026thinsp;84.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e79.5\u0026thinsp;~\u0026thinsp;82.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e74.4\u0026thinsp;~\u0026thinsp;77.9\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eamino acid\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e94.2\u0026thinsp;~\u0026thinsp;97.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.0\u0026thinsp;~\u0026thinsp;95.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e96.5\u0026thinsp;~\u0026thinsp;97.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.3\u0026thinsp;~\u0026thinsp;96.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.0\u0026thinsp;~\u0026thinsp;95.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.0\u0026thinsp;~\u0026thinsp;96.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e94.2\u0026thinsp;~\u0026thinsp;95.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e94.2\u0026thinsp;~\u0026thinsp;95.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e96.5\u0026thinsp;~\u0026thinsp;97.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.3\u0026thinsp;~\u0026thinsp;98.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e90.7\u0026thinsp;~\u0026thinsp;94.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e3B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enucleotide\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e68.2\u0026thinsp;~\u0026thinsp;74.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e65.2\u0026thinsp;~\u0026thinsp;75.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e75.8\u0026thinsp;~\u0026thinsp;84.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.3\u0026thinsp;~\u0026thinsp;83.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e68.2\u0026thinsp;~\u0026thinsp;74.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e71.2\u0026thinsp;~\u0026thinsp;75.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e72.7\u0026thinsp;~\u0026thinsp;77.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e68.2\u0026thinsp;~\u0026thinsp;74.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e81.8\u0026thinsp;~\u0026thinsp;92.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e78.8\u0026thinsp;~\u0026thinsp;86.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e74.2\u0026thinsp;~\u0026thinsp;80.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eamino acid\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e86.4\u0026thinsp;~\u0026thinsp;95.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e86.4\u0026thinsp;~\u0026thinsp;95.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e86.4\u0026thinsp;~\u0026thinsp;95.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e86.4\u0026thinsp;~\u0026thinsp;95.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e86.4\u0026thinsp;~\u0026thinsp;95.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e81.8\u0026thinsp;~\u0026thinsp;95.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e86.4\u0026thinsp;~\u0026thinsp;95.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e86.4\u0026thinsp;~\u0026thinsp;95.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e86.4\u0026thinsp;~\u0026thinsp;95.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e90.9\u0026thinsp;~\u0026thinsp;95.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e86.4\u0026thinsp;~\u0026thinsp;95.5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e3C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enucleotide\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.5\u0026thinsp;~\u0026thinsp;79.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.1\u0026thinsp;~\u0026thinsp;77.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e81.8\u0026thinsp;~\u0026thinsp;84.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e80.1\u0026thinsp;~\u0026thinsp;82.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.3\u0026thinsp;~\u0026thinsp;78.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e74.5\u0026thinsp;~\u0026thinsp;77.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.9\u0026thinsp;~\u0026thinsp;78.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e78.1\u0026thinsp;~\u0026thinsp;80.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e82.5\u0026thinsp;~\u0026thinsp;85.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e80.9\u0026thinsp;~\u0026thinsp;82.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e74.1\u0026thinsp;~\u0026thinsp;75.6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eamino acid\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e91.9\u0026thinsp;~\u0026thinsp;95.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e94.5\u0026thinsp;~\u0026thinsp;96.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.1\u0026thinsp;~\u0026thinsp;97.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.1\u0026thinsp;~\u0026thinsp;96.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e94.5\u0026thinsp;~\u0026thinsp;96.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e91.8\u0026thinsp;~\u0026thinsp;94.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.4\u0026thinsp;~\u0026thinsp;96.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.1\u0026thinsp;~\u0026thinsp;97.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e96.2\u0026thinsp;~\u0026thinsp;98.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.6\u0026thinsp;~\u0026thinsp;98.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e91.8\u0026thinsp;~\u0026thinsp;94.5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e3D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enucleotide\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.6\u0026thinsp;~\u0026thinsp;78.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e78.0\u0026thinsp;~\u0026thinsp;78.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e83.8\u0026thinsp;~\u0026thinsp;85.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.6\u0026thinsp;~\u0026thinsp;79.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.5\u0026thinsp;~\u0026thinsp;79.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e78.3\u0026thinsp;~\u0026thinsp;80.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.9\u0026thinsp;~\u0026thinsp;78.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.9\u0026thinsp;~\u0026thinsp;79.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e82.8\u0026thinsp;~\u0026thinsp;83.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e82.4\u0026thinsp;~\u0026thinsp;84.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e78.1\u0026thinsp;~\u0026thinsp;80.4\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eamino acid\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.1\u0026thinsp;~\u0026thinsp;94.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92.4\u0026thinsp;~\u0026thinsp;93.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e96.1\u0026thinsp;~\u0026thinsp;97.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.3\u0026thinsp;~\u0026thinsp;95.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92.9\u0026thinsp;~\u0026thinsp;93.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92.6\u0026thinsp;~\u0026thinsp;94.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92.9\u0026thinsp;~\u0026thinsp;94.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92.9\u0026thinsp;~\u0026thinsp;94.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.5\u0026thinsp;~\u0026thinsp;96.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.5\u0026thinsp;~\u0026thinsp;96.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e91.6\u0026thinsp;~\u0026thinsp;93.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eP3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enucleotide\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.4\u0026thinsp;~\u0026thinsp;78.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.5\u0026thinsp;~\u0026thinsp;78.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e83.5\u0026thinsp;~\u0026thinsp;84.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e79.1\u0026thinsp;~\u0026thinsp;80.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.2\u0026thinsp;~\u0026thinsp;78.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.4\u0026thinsp;~\u0026thinsp;79.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e77.3\u0026thinsp;~\u0026thinsp;78.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e78.0\u0026thinsp;~\u0026thinsp;79.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e83.0\u0026thinsp;~\u0026thinsp;84.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e81.9\u0026thinsp;~\u0026thinsp;83.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.9\u0026thinsp;~\u0026thinsp;78.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eamino acid\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.4\u0026thinsp;~\u0026thinsp;95.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.2\u0026thinsp;~\u0026thinsp;94.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e96.1\u0026thinsp;~\u0026thinsp;96.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.9\u0026thinsp;~\u0026thinsp;95.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.5\u0026thinsp;~\u0026thinsp;94.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92.8\u0026thinsp;~\u0026thinsp;94.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.5\u0026thinsp;~\u0026thinsp;94.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e93.8\u0026thinsp;~\u0026thinsp;94.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.9\u0026thinsp;~\u0026thinsp;96.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e95.8\u0026thinsp;~\u0026thinsp;96.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92.0\u0026thinsp;~\u0026thinsp;93.5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003ch3\u003eRecombination analysis\u003c/h3\u003e\n\u003cp\u003eTo investigate the recombination of CVA4 in this study,bootscan and similarity plot analysis was performed on 50 strains using software Simplot v3.5.1 (Fig.\u0026nbsp;4). YT04, YT13, YT22 and LY17 were used as query sequences, and EV-A prototype strains were used as reference sequence for similarity and bootscan analysis. It was found that all CVA4 strains had high similarity with CVA4 prototype strain in P1 region. However, in the 5\u0026prime; UTR region, all strains showed high similarity to CVA5, CVA14 and CVA16, and bootscan analysis also confirmed that all strains in this study had a potential recombination of approximately 250 bp with CVA16 in the 5\u0026prime; UTR region. According to similarity plot analysis, all strains showed high similarity to CV-A4, CV-A5, CV-A14 and CVA16 in P2 and P3 regions. According to bootscan analysis, all strains except LY17 had potential recombination with CVA5 in region 2C and region P3; bootscan patterns of LY16, LY29, LY33\u0026thinsp;~\u0026thinsp;37 were similar to YT22, and potential recombination with CVA14 existed in region 2A.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSince CVA4 was first isolated in 1948, it has been widespread worldwide for many years\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In most cases, CVA4 infection has no obvious symptoms, but it can cause HA, HFMD, respiratory tract infection and other diseases\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, and even cause severe central nervous system symptoms\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Since the 1980s, CVA4 has become the main pathogen of HA in Japan\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and continues to dominate\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In recent years, CVA4 has also become the main pathogen of HA in China\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The main pathogens of HFMD are EV-A71, CVA6, CVA16 and CVA10. Given that EV-A71 and CVA16 often lead to severe cases \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, the early monitoring in China mainly focused on EV-A71, CVA16 and the \"other enteroviruses\" categories. Subsequent studies found that CVA6 and CVA10 were responsible for a higher proportion of HFMD cases in the category of \"other enteroviruses\"\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, which led to their inclusion in routine surveillance in some regions. Although CVA4 is classified as \"other enteroviruses\" and is not listed as a routine serotype surveillance item for HFMD, recent studies have shown that the proportion of CVA4 in this category has increased with the increase in the detection rate of \"other\" enteroviruses and the improvement of laboratory detection capabilities\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Therefore, there is a significant need to intensify research on CVA4.\u003c/p\u003e\u003cp\u003eIn this study, 60 strains of CVA4 isolated from throat swabs of HA patients in 2024 were sequenced, and the complete genome sequences of 50 strains of CVA4 were successfully obtained. Genome-wide phylogenetic analysis showed that CVA4 strains circulating worldwide had formed significant genetic diversity, which was mainly divided into five evolutionary clades, Group 1\u0026ndash;5. These strains were isolated from a number of different countries and regions, indicating that CVA4 has the ability to spread geographically, which may be a key factor in its global pandemic. At the same time, the strains in this study were closely related to CVA4 in Shandong Province in phylogenetic analysis, indicating that CVA4 had geographical aggregation at the same time. It should be noted that the China strains in this study mainly clustered in Group 2, suggesting that Group 2is the dominant epidemic branch of CVA4 at the whole genome level in China.\u003c/p\u003e\u003cp\u003eVP1 region contains neutralizing antigenic determinants. At present, serotyping based on VP1 region sequence has become the main method to study the molecular epidemiology of enteroviruses. In this study, not only the complete VP1 sequence but also the full length sequence of CVA4 can be obtained by reference sequence assembly through second generation sequencing technology. In this study, the CVA4 VP1 region sequences of 60 throat swab samples from HA children in Linyi and Yantai in 2024 were successfully amplified, and phylogenetic trees were constructed with VP1 region gene representative sequences downloaded from Genebank public database for molecular evolution analysis. According to the typing criteria of Ji\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, CVA4 strains are mainly divided into four genotypes A, B, C and D, among which genotype C is further divided into five subgenotypes: C1-C5. subgenotypes C1 and C3 were mainly prevalent between 1996 and 2007,subtype C2 became prevalent after 2006, and subgenotypes C4 and C5were discovered after 2014. Phylogenetic tree analysis based on VP1 region showed that all strains in this study belonged to C2 subgenotype, and C2 subgenotype was the dominant subgenotype in China. In addition, several unknown subgenotypes of CVA4 sequences were found in this study. During the phylogenetic analysis of the whole genome, three sequences from France (OQ091482, OQ091483, OQ091482) were found to have nucleotide sequence differences in VP1 region greater than 15% compared with other sequences, which may be new subgenotypes.\u003c/p\u003e\u003cp\u003eAccording to the identity analysis, nucleotide homology between CVA 4 strains in this study and other non-CVA4 EV-A prototype strains is high in the non-P1 region, and the positions of these CVA4 strains are significantly different from those of other EV serotypes in the phylogenetic tree of P1, P2 and P3 regions.This difference suggests that recombination events between CVA4 and other EV serotypes may occur in the non-P1 region. EV is a small RNA virus, and its evolution depends mainly on gene mutation and recombination\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.Since 1962, when poliovirus and influenza virus recombination events were first reported, studies on EV recombination have increased \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, and many of the pathogens responsible for HFMD and HA outbreaks have resulted from recombination between different genotypes\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The P1 region of EV is relatively conservative, and recombination rarely occurs in this region. The CVA4 sequence in this study shows high similarity with CVA4 prototype strain in P1 region, which also indicates that P1 region is relatively conservative in EV. It is still reliable to classify EV types according to VP1 region. Non-structural protein coding regions (P2, P3) are mainly responsible for encoding proteases or active molecules related to viral genome replication and viral particle packaging. Many studies have shown that P2 and P3 regions are high recombination regions\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In this study, all strainsshowed high similarity with CV-A4, CV-A5, CV-A14 and CV-A16 in 5\u0026prime; UTR. According to bootscan analysis, all strains except LY17 had recombination with CVA5 in 2C and P3 regions. LY16, LY26, LY29, LY33\u0026thinsp;~\u0026thinsp;37, YT22 had recombination with CVA14 in 2A to 2B regions.\u003c/p\u003e\u003cp\u003eWhether these recombination changes the environmental adaptability and disease severity of CVA4 and whether the widespread spread of CVA4 in Shandong Province in recent years is related to these recombination needs further study. At present, due to the use of EV-A71 vaccine, complex dynamic evolution and co-circulation of EV, the pathogenic spectrum of related diseases has changed, and the infection rate of non-EV-A71 has greatly increased. Therefore, it is not possible to focus only on a few common serotypes, and continuous and comprehensive monitoring of EV types should be carried out.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eSource of samples and cells\u003c/h2\u003e\u003cp\u003eHerpangina patients in sentinel monitoring hospitals of Linyi City and Yantai City in China in 2024 were selected as the study objects. The inclusion criteria of the study objects were that outpatients or inpatients had sore throat, fever, herpes and ulceration in palatopharyngeal arch, soft palate, ptosis and tonsil, but no herpes and ulceration in hands, feet and trunk were defined as HA patients. The Center for Disease Control and Prevention of the sentinel hospital collected throat swab samples from patients meeting HA inclusion criteria, and the positive nucleic acid samples were sent to Shandong Province Center for Disease Control and Prevention for use in this study. A total of 60 CVA4 strains were isolated from 256 throat swab samples from HA children, and the 60 strains were subjected to whole genome amplification and sequencing and further analysis. Among them, the screening conditions of strain were identified as CVA4 by RT-PCR and CT value\u0026thinsp;\u0026le;\u0026thinsp;18. RD cells were provided by Shandong Province Center for Disease Control and Prevention.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eVirus Isolation and RNA Extraction\u003c/h3\u003e\n\u003cp\u003eThroat swab specimens stored at -80\u0026deg;C were thawed at room temperature. A 200 \u0026micro;L aliquot of each sample was inoculated into RD cell culture tubes exhibiting robust growth. Following viral propagation, cytopathic effect (CPE) was monitored daily until \u0026ge;\u0026thinsp;75% of cells exhibited alteration (3\u0026thinsp;+\u0026thinsp;CPE). The culture was then stored at -80\u0026deg;C for secondary subculture.When significant CPE reoccurred in the second passage, the P2 virus stock was archived at -80\u0026deg;C for subsequent analysis. Viral nucleic acids were extracted using the qEx-DNA/RNA Pathogen Kit (Xi'an Tianlong Technology) strictly according to the manufacturer's protocol.\u003c/p\u003e\n\u003ch3\u003eIdentification and Typing of EVs\u003c/h3\u003e\n\u003cp\u003eEVs were identified using reverse transcription polymerase chain reaction (RT-PCR). Select samples with CT value\u0026thinsp;\u0026le;\u0026thinsp;37 and sigmoidal amplification curves were selected for identification using the Coxsackievirus A4 Nucleic Acid Detection Kit (Real-time PCR). Reagent preparation, operating condition setting and result judgment were carried out according to reagent instructions.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eLibrary construction and sequencing\u003c/h2\u003e\u003cp\u003eA total of 60 CVA4 strains with CT value\u0026thinsp;\u0026le;\u0026thinsp;18 were screened. Viral RNA was enriched using the Coxsackievirus A4 Nucleic Acid Enrichment Kit (multi-prider capture amplification) (Shanghai BioGerm Medical Technology). Enriched products underwent RT-PCR, followed by purification and next-generation sequencing (NGS) library preparation. Libraries were constructed with the Nextera XT DNA Library Prep Kit (Illumina, USA). Sequencing was performed on the MiSeq platform (Illumina, USA) using MiSeq Reagent Kit v2 (300-cycle) with 2\u0026times;150 bp paired-end reads.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eSequencing data analysis\u003c/h2\u003e\u003cp\u003eVP1 region sequences and whole genome sequences of CVA4 strains worldwide from 1948 to 2024 were downloaded from GenBank public database as reference sequences. The VP1 sequences with high homology (nucleotide identity\u0026thinsp;\u0026ge;\u0026thinsp;98%) and obvious errors were deleted. 213 VP1 gene sequences (including 60 CVA4 strains in this study and 154 strains from other countries and provinces in China) were included. A total of 222 CVA4 complete genome sequences (including 50 strains in this study and 172 CVA4 complete genome sequences from other countries and provinces in China) were included. The nucleotide and amino acid sequences were aligned by MAFFT software (v7.505). Genious Prime 2025 software was used to analyze the sequence identity of CVA4 strains and EV-A prototype strain in this study. MEGA11 software was used to construct phylogenetic tree of VP1 region by Neighbor-Joining (NJ) method. Maximum Likelihood (ML) trees for P1, P2 and P3 regions were constructed using Iqtree software (v1.6.12), with Bootstrap values set to 1000, results were visualized and trees beautified using software Figtree (v1.4.4) and ITOL (v7) websites, and potential recombination between CVA 4 strains and other EV-A prototypes was analyzed using the software Similarity Plots (v3.5.1).\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eAuthor contributions statement\u003c/h2\u003e\u003cp\u003eAll authors have contributed significantly, and that all authors reviewed the manuscript. Wenqing Ma: Conceptualization, Investigation, Formal analysis, Writing Original Draft, Zhen Dong: Investigation, Xiaodong Mou and Fengtao Zhang: Investigation (early experiments), Data Curation, Resources, Xiaolin Liu and Xiaolin Yu: Data Curation, Resources,Jianxing Wang: Conceptualization, Methodology, Resources,Zengqiang Kou: Resources, Methodology.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003cp\u003eThe samples used in the study were taken after the approval of the ethical committees from the respective laboratories. This study was approved by institutional review board of Shandong center for disease control and prevention(approval number: SDJK(K)2024-012-01). All methods were performed in accordance with the relevant guidelines and regulations. Written informed consent was taken from the patients.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by Medical and Health Science and Technology Development Program Fund of Shandong Province(202012061392). Title: Study on the Epidemic Pattern and Pathogen Composition of Severe Hand-Foot-Mouth Disease Cases in Shandong Province.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors have contributed significantly, and that all authors reviewed the manuscript. Wenqing Ma: Conceptualization, Investigation, Formal analysis, Writing Original Draft, Zhen Dong: Investigation, Xiaodong Mou and Fengtao Zhang: Investigation (early experiments), Data Curation, Resources, Xiaolin Liu and Xiaolin Yu: Data Curation, Resources,Jianxing Wang: Conceptualization, Methodology, Resources,Zengqiang Kou: Resources, Methodology.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets, are available, supporting the conclusions of this article with the accession numbers PX227139-PX227198 are available in the GenBank [National Center for Biotechnology Information] repository.Take PX227139 as an example, the link is：https://www.ncbi.nlm.nih.gov/nuccore/PX227139.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFu, Y. L. et al. Analysis of the epidemic characteristics and disease burden of herpangina in hospitalized children in China from 2016 to 2020. \u003cem\u003eChin. J. Exp. Clin. 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Microbiol.\u003c/em\u003e \u003cb\u003e82\u003c/b\u003e, 83. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00284-025-04075-7\u003c/span\u003e\u003cspan address=\"10.1007/s00284-025-04075-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2025).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Herpangina, Coxsackievirus A4, Whole genome, Gene sequencing","lastPublishedDoi":"10.21203/rs.3.rs-7407631/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7407631/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSporadic epidemics of coxsackievirus A4 (CVA4) have been reported worldwide, but there are relatively few studies on CVA4. This study successfully obtained 60 VP1 region sequences and 50 complete genome sequences of CVA4. Phylogenetic analysis based on the complete genome revealed that these strains clustered within the same evolutionary branch as CVA4 strains from other regions of China. Analysis of the VP1-based phylogenetic tree demonstrated that all 60 strains belonged to the C2 genetic subgenotype. Recombination analysis indicated high sequence identity (85.1\u0026ndash;85.7%) between the study strains and the CVA4 prototype strain within the P1 region. In contrast, significant sequence similarities were observed in the P2 and P3 regions with prototype strains of CVA4, CVA5, CVA14, and CVA16. Bootscan analysis further identified recombination signals in the 5\u0026prime; untranslated region (5\u0026prime; UTR), P2, and P3 regions, suggesting recombination events with non-CVA4 enterovirus A (EV-A) prototype strains. This study provides evidence of genetic recombination in circulating CVA4 strains in Shandong Province, China, highlighting their complex evolutionary dynamics within the EV-A species.\u003c/p\u003e","manuscriptTitle":"Whole-genome analysis of CVA4 in children with herpangina in two cities of China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-24 13:02:20","doi":"10.21203/rs.3.rs-7407631/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-16T11:15:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-16T07:47:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296251778933884792847678721554769929853","date":"2025-12-03T14:55:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"141092170375393385467394918647034762222","date":"2025-11-25T00:23:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-04T20:42:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"329554864905535136194917242072337997661","date":"2025-09-24T07:06:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"304798020238103595027257811515350497796","date":"2025-09-21T09:50:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-16T03:41:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-10T10:42:58+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-10T08:57:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-09T07:04:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-09T06:57:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b40ee6fe-3d30-4e40-ab8f-acf2b88e10ff","owner":[],"postedDate":"September 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":55069380,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":55069381,"name":"Biological sciences/Evolution"},{"id":55069382,"name":"Biological sciences/Genetics"},{"id":55069383,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-02-16T16:01:46+00:00","versionOfRecord":{"articleIdentity":"rs-7407631","link":"https://doi.org/10.1038/s41598-026-39455-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-02-09 15:57:28","publishedOnDateReadable":"February 9th, 2026"},"versionCreatedAt":"2025-09-24 13:02:20","video":"","vorDoi":"10.1038/s41598-026-39455-x","vorDoiUrl":"https://doi.org/10.1038/s41598-026-39455-x","workflowStages":[]},"version":"v1","identity":"rs-7407631","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7407631","identity":"rs-7407631","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}