Introduction

Human enterovirus 71 (EV71) is a small, single-stranded, positive-sense RNA virus with a genome length of approximately 7400 nucleotides. It belongs to the species of Enterovirus A (EV-A), genus Enterovirus, family Picornaviridae [1, 2]. EV71 is the main pathogen responsible for causing hand, foot, and mouth disease (HFMD), an exanthematous illness typically observed in children < 5-years-old. It has been associated with severe clinical symptoms, including encephalitis and poliomyelitis that could lead to death [3]. Based on the VP1 encoding region, EV71 strains have been divided into genogroups A–C [4]. Genogroup A comprises of only one strain, the BrCr prototype, which was initially isolated in California, USA, in 1970. Genogroups B and C are common and consist of sub-genogroups B0–B5 and C1–C5, respectively [5].

Genetic recombination is a common phenomenon essential for the evolution of most RNA viruses [6]. The first natural recombinant RNA virus was reported in the poliovirus in the 1960s. Since then, numerous RNA virus recombinants have been reported, including picornaviruses, coronaviruses, and alphaviruses [7,8,9,10]. Previous studies on enterovirus have also suggested extensive recombination between and within enterovirus [11,34], we speculated that the 2A region for the CA8 serotype might be prone to recombination events with respect to gene fragment exchange.

The third intertypic recombination, strain Guangdong-2009 (accession number JF799986) belonged to the C4a subgenotype and was isolated in Guangdong, China, in 2009. Its major parent strain, G-14/SOA/1950 (accession number AY421769), belongs to genotype CA14, isolated from South Africa in 1950, while its predicted minor parent, the Anhui1-09-China strain (accession number GQ994988), was isolated from Anhui, China, in 2009, and belongs to the C4a subgenotype. The similarity plot and bootscan analysis revealed the recombination breakpoint at 4200–4300 in the 2C region, which was consistent with the data analyzed by RDP5. Based on the similarity plot and bootscan analysis, we speculated that the major parent is Anhui1-09-China and is the minor parent is CA14 (Fig. 2).

In this study, we identified 5 intratypic recombinant strains (EV71/P1034/2013, Henan-ZMD/CHN/2012, VR1432, 120/EV71/Wenzhou/CHN/2014, and EV71/P868/2013/China) (Fig. 3). For recombinant strain EV71/P1034/2013 (accession number KP289419), the putative major parent was Chongqing2-09-China (accession number GQ994990) isolated from Chongqing, China (the strains belong to a C4a subgenotype), and the minor parent strain was MY821-3/1997 (accession number DQ341367), which belongs to the B3 subgenotype that originated in Singapore in 1997. The initial actual breakpoint position of EV71/P1034/2013 was located at 5412, and the terminal breakpoint position was located at 7094. The breakpoint regions were located at 3C of the P3 nonstructural protein region, in which minor parent MY821-3/1997 shared greater similarity with recombinant strain EV71/P1034/2013. The identical evidence was provided by the bootscan result (Fig. 3).

Fig. 3
figure 3

Identification of intratypic recombination strains of EV71 by similarity plots and bootscan analyses. The similarity plot was conducted in a sliding window size of 200 nucleotides with 20-bp steps using the Kimura 2-parameter distance method; bootscan analysis was performed using the neighbor-joining tree model and the Kimura 2-parameter distance algorithm in a sliding window size of 200 nucleotides with 20-bp steps, applying the first recombinant strain EV71/P1034/2013. Similarity plots were conducted in a sliding window size of 1000 nucleotides with 20-bp steps using the Kimura 2-parameter distance method; bootscan analyses were performed using the neighbor-joining tree model and the Kimura 2-parameter distance algorithm in a sliding window size of 1000 nucleotides with 20-bp steps, applying the other recombinant strains

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For recombinant strain, Henan-ZMD/CHN/2012 (accession KP198624), the major parent R615/YN/CHN/2010(MF662684) was isolated from Yunnan, China, and the strain belongs to the C4a subgenotype, while the minor parent, Hubei-WH/CHN/2012 (accession KP198623), was isolated from Wuhan, China, and belongs to the C4a subgenotype. The recombinant region of the strain was detected in the VP3 region. The initial actual breakpoint position of Henan-ZMD/CHN/2012 was located at 1256, and the terminal breakpoint position was located at 2439 (Fig. 3).

For recombinant strain VR1432, the putative major and minor parent strains were C4b-SHZH98 (accession number AF302996) and Anhui1-09-China (accession number GQ994988), respectively. The recombinant regions were detected in the VP3 and VP1 regions. The actual initial breakpoint position of VR1432 was located at 1596, and the terminal breakpoint position was located at 2567, and the recombinant region was located in VP1–VP3 of the P1 region (Fig. 3). Based on the results of recombination analysis, it was determined that the strain VR1432 is “double recombinant” and involves both intratypic and intertypic recombination events. Interestingly, in the upstream region of the genome, the recombinant strain VR1432 was similar to the subgenotype C4b, while downstream of the genome, a high degree of nucleotide identity with serotype CA8 was established.

For recombinant strain 120/EV71/Wenzhou/CHN/2014 (accession KT345959), the major parent Hubei-WH/CHN/2012 (accession KP198623) strain was isolated from Wuhan, China in 2012 (belongs to C4a subgenotype), and the minor parent was SHAPHC5218/SH/CHN/14 (accession KU936120), isolated from Shanghai, China, in 2014 (belongs to the C4a subgenotype). The recombinant region was determined in the VP3 and VP1 regions. The actual initial breakpoint position of 120/EV71/Wenzhou/CHN/2014 was located in 1930, and the terminal breakpoint position was located at 3212 (Fig. 3).

For recombinant strain EV71/P868/2013/China (accession KP289430), the major parent, Puyang-251-2012 (accession KM211579), was isolated from Puyang, China in 2012, while the minor parent, EV71/P40/2013/China (accession KP289426), was isolated from China in 2013. Both parent strains belong to the C4a subgenotype, and the recombinant region was detected at the P2 region. The initial actual breakpoint position of EV71/P868/2013/China was located at 3762, and the terminal breakpoint position was located at 5183 (Fig. 3).

Recombinant data indicated that using complete genome analysis or sequencing of multiple regions of EV71 for circulating strains is an effective method for viral genetics research. Some publications reported that the high nucleotide sequence similarity in nonstructural regions (P2 and P3) might favor intertypic recombination across homologous regions in poliovirus [35, 36]. Intertypic recombination of CV-A16 and EV-A71 genotype C4 has been reported in Central China [16, 24]. Intertypic recombination enables EV71 to obtain the gene fragment from the other types of viruses; consequently, a highly pathogenic EV71 strain with increased virulence is generated through natural recombination [5, 16, 19, 27, 37,38,39], which facilitates the recombinant virus to adapt to the new epidemic environment. Notably, the nonstructural protein regions had a high probability of recombination as described previously [16, 22], and that the P1 region encoding structural protein is a vital region for intratypic recombination. Intratypic recombination of the C4a subgenotype was observed in the VP1 region [40]. The VP1 capsid region has been reported to possess several antigenic determinants and induce neutralizing antibodies. The VP1 encodes the capsid protein with the maximal variation, which allows the virus to escape neutralizing antibodies. Thus, the antigenicity was changed to adapt to the subsequent prevalence of HFMD.

The current study indicates that the genome of EV71 undergoes recombination, which might be a key factor in determining the high virulence of some strains. However, the exact mechanism of the recombination events observed in this study is not yet clarified. To understand the recombination process, it will be necessary to develop the experimental model of in vitro selection of recombinant viruses. In addition to recombined strains introduced to China, C4a circulating strains may also be exported to other countries [23, 41]. Local strains and C4a subtypes from other countries might simultaneously go through intra- and inter-genotype recombination events, which produce new pandemic strains. Therefore, the surveillance of the emergence and epidemic of the EV71 recombinant virus might prevent the high epidemic of HFMD.

In conclusion, in this study, we identified 3 intertypic recombinants and 5 intratypic recombinants, including one “double-recombinant” strain based on the analysis of 196 complete genomes of the EV71 in mainland China during 2009–2018. The results indicated that homologous recombination contributes to the emergence of various pathogenic variants. Recombination is not only the evolutionary mechanism of EV71 but also a common pattern of genetic plasticity that leads to viral diversity [42]. Recombination between circulating strains of EV71 and coxsackievirus A of different genotypes may play a role in the evolution of the strains by improving the overall fitness of the virus, such as increased virulence and/or transmissibility [16, 19], which requires further clinical monitoring.

Taken together, this study improves the understanding of the evolution and epidemic of EV71 and facilitates the surveillance and control of the virus. Although there is an EV71 vaccine with a C4 genotype in China, understanding the virus may facilitate the development of a broad-spectrum vaccine that can be used against different genotypes. Therefore, continuous surveillance of EV71 is needed to elucidate its epidemiology and virus evolution.