It has been reported that the major EV71 strains basically have belonged to the C4 genotype in mainland China since 2004 [18, 33]. In this study, it was found that the epidemic strains of EV71 in mainland China were dominated by the C4 genotype since 2000 and mainly the C4b subgenotype from 2000 to 2004, and the proportion of the C4a subgenotype had increased significantly since 2005. From 2005 to 2018, the C4a subtype was dominant, and the C4b subtype was the second, and other subtypes appeared sporadically, this pattern was similar to previous reports [20, 21, 30, 33]. From 2004 to 2018, most of the EV71 isolates in mainland China were genotype C, and most of them were subtype C4 (especially subtype C4a), while other genotypes occurred sporadically (9 strains of genotype B in 2011, 4 strains of genotype A in 2008, 2 strains of genotype A in 2009, 1 strain of genotype A in 2011 and 1 strain of genotype A in 2012). The immune effectiveness of inactivated vaccines depends mostly on the antigenic correlation between epidemic strains and vaccine strains, which are often best for preventing the infection of the same subtype virus but poor against different subtypes . Recent studies have also shown that the EV71 vaccine (especially in children who receive 2 doses of immunization) can effectively prevent and control childhood EV71-associated HFMD but has no protective effect against coxsackievirus (CV) A6 (CVA-6) or CVA16, and there is no explanation for the effectiveness of other subtypes of EV71 (excluding C4a subtypes) . These studies show that the vaccine research and development for EV71 combined with CVA6 and CVA16 and other multivalent vaccines could better prevent EV71 infection.
Interestingly, this study found that the subtypes of EV71 epidemic strains in Taiwan were mainly B4 genotypes, which were different from those in mainland China, and EV71 epidemic strains are also constantly changing, which is consistent with early reports . A previous study indicated that the FI-EV71 vaccine (EV71vac) based on the B4 genotype from Taiwan, China, was safe and induced a high titer of neutralizing antibodies against EV71 in a human phase 1 clinical trial on adults in 2010 and was highly effective against B1, B5, and C4a strains. However, the titers of neutralizing antibodies against C4b and CVA16 were low in 20% of volunteers, and virus-neutralizing antibodies against the C2 genotype were not detected in 90% of the vaccine recipients [26-27]. These studies indicate that it is necessary to strengthen the monitoring of EV71 genotypes; new multivalent and effective vaccines that can cover local strains should be designed and applied according to the genotypes of local predominant EV71 epidemics to ensure that the vaccine plays a more accurate role in the control of HFMD epidemics.
Some studies have shown that the H22Q mutation in the VP1 protein of EV71 virus can lead to a decrease in the adsorption capacity of the C4 genotype to host cells [35-37]. The amino acid at position 22 of 78.15% of the 3712 strains isolated in China was H (histidine), which suggested that most of the viruses had strong adsorption capacity to host cells. Furthermore, H22Q was present in 10.99% of all EV71 strains in Taiwan, which was significantly more prevalent than that in mainland China and Hong Kong (5.50%), suggesting that the adsorption capacity of some strains in Taiwan to host cells was weak in comparison with that of strains in mainland China.
Studies have shown that the variation A289T in the EV71-VP1 protein is closely related to the occurrence of severe HFMD and that the neurological symptoms caused by EV71 infection are significantly increased when the amino acid at position 289 of VP1 is A (alanine); in contrast, there is low neurotoxicity when the amino acid at position 289 is T (threonine) [36, 38]. In this study, 76.45% (2838 strains) of the virus strains contained an A (alanine), suggesting that most of the EV71 viruses have high neurotoxicity. Moreover, 10.24% (380 strains) of the strains in mainland China (including Hong Kong) and 10.75% (399 strains) of those in Taiwan contained a T (threonine), suggesting low neurotoxicity. Additionally, whether these new mutations will cause the emergence of severe HFMD remains to be further studied, regarding A289V (valine)/D (aspartic acid)/I (isoleucine) mutations.
EV71 can infect human lymphocytes by binding to its receptor molecule, Pselectin glycoprotein ligand-1 (PSGL-1). When the amino acid at position 145, E (glutamic acid), in VP1 is mutated to G (glycine) or Q (glutamine), to the virus binds PSGL-1 more readily, whereas its PSGL-1-binding ability is weakened or lost if position 145 is E (glutamic acid) . In this study, the amino acid at position 145 is E in most strains, and the mutation rate of E145G/Q is 6.63%, suggesting that the emergence of this mutation may be result in a virus that is more likely to infect human lymphocytes.
It has been reported that the E98K mutation may increase the hydrophobicity of VP1, making it easy for large compounds to enter and interfere with VP1 receptor binding, suggesting that E98K mutant viruses are sensitive to larger compounds . Other studies have shown that E145G and N31D mutations are associated with increased virulence of EV71 and may increase the risk of neurological complications, while I262V mutations reduce the risk of neurological complications [41-43]. In this study, the mutation rates of E98K, E145G, N31D and I262V were 6.65%, 3.13%, 7.25% and 2.77%, respectively. These findings indicate that these mutations may play an important role in the pathogenicity of mild and severe EV71-associated HFMD.
Humans are the only natural host and source of EV71, as EV71 cannot infect rodents, which is due mainly to the incompatibility between the virus and rodent cells and the different expression of its scavenger receptor in humans and rodents [44-46]. However, some studies have found that the simultaneous substitution of K98E, E145A and L169F in VP1 of EV71 can cause infection in mice . Our study showed that among 3712 strains, the mutation frequencies of K98E, E145A and L169F were 93.24%, 0.30% and 0.03%, respectively, but no strain with three mutations was found. These findings indicate that humans are still the only host of EV71 in China, but the existence of individual mutations does not rule out the emergence of strains that can infect other mammals after a few years. Therefore, it is important to closely monitor the mutation of the key sites of the EV71-VP1 region.
EV71 is the most important pathogen of children with severe HFMD, which can lead to irreversible sequelae or death of infected children, and it is a serious threat to the health of children [4, 7]. At present, there is no specific drug for the treatment of EV71 infection. The development and marketing of an inactivated EV71 vaccine in China is crucial for the prevention of HFMD caused by EV71 infection [26, 47, 48, 49]. Phase III clinical trials of the EV71 inactivated vaccine approved in China in 2015 showed that its protective effectiveness against EV71-associated HFMD was more than 90% [47, 50, 51, 52]. However, molecular epidemiological studies of EV71 showed that EV71 gene mutations occur frequently, leading to genetic diversity [28, 29, 30, 31, 53]. These studies imply that there is still a need for strengthening surveillance of EV71 genotypes and the development of new EV71 vaccines.
This study is a retrospective study, and there are some limitations. First, the EV71-VP1 gene sequences from China analyzed in this study were downloaded from the GenBank database but were not tested by us. Due to the limited time, only the VP1 region has been analyzed and studied. In future study, we will conduct genetic research on the complete genome sequence of EV71 in China. Second, the Chinese EV71-VP1 strains registered in GenBank do not cover all provinces of China, and the data in some years are missing, which means that we may have missed some isolates of other genotypes. Third, it is not clear whether some variations in the amino acid sites found in the study are related to the severity of disease or the route of transmission.