In this study, we segregated CV-A8 into five genotypes, namely, A, B, C, D and E, and described the whole-genomic diversity of CV-A8. Among the HFMD samples collected since 2008 from our lab, eleven samples isolated between 2013 and 2018 were identified as CV-A8, and among these 11 samples, eight of them were identified as genotype E strains. From 2008 to 2012, no CV-A8 strains were identified in our lab, maybe because of their lower transmissibility; from 2013 onwards, 11 CV-A8 samples had been identified, probably because the transmissibility of CV-A8 started to increase around that time. Furthermore, 8 of the 11 strains belonged to genotype E. We were aware that, with so many labs doing HFMD-related EVs detection in China, different laboratory capabilities, transporting distances, commercial kits, etc. would lead to inhomogeneity of the strains received by us; nevertheless, such a result would still somehow imply that the genotype E strains from China were newly arisen and might have higher transmissibility. It is intriguing that the newly emerging genotype E strains presented highly divergent VP1
capsid sequences among the CV-A8 variants; the nine specific aa residues of genotype E, especially 100N, 103Y, 240T and 241V embedded in the assumed surface-exposed loops, might enhance genotype E adaption to the human hosts as surface exposed loops’ structures in the VP1
capsid always serve as potential viral-neutralizing epitopes for EVs. Further experiments are needed to verify this conjecture. Furthermore, recombination is a frequently observed phenomenon among EVs and, more importantly, has long been recognized to act as a driving force of EV evolution by eradicating deleterious mutations. Because the 3Dpol
error-prone RNA-dependent RNA polymerases (RdRps
) of EV always lead to misincorporations during genome replication, ongoing recombination may be the main process preventing EV genomes from deleterious mutation accumulation. In addition, recombination creates chimeric molecules from parental genomes with different phylogenetic origins and may also help EVs attain combined advantageous features from various genomes during the process of evolution [30
]. This may generate new recombinants with higher virulence and transmissibility, such as vaccine-derived polioviruses and the C4a evolutionary branch of EV-A71 [33
]. In this study, we found that all the Chinese strains from genotype E, i.e., lineage 5, were likely to emerge recently from recombining with European CV-A6 strains in the whole P3
region. It is interesting that CV-A6 recombinants were reportedly associated with more severe HFMD clinical manifestations of eczema herpeticum than typical HFMD cases [37
], which might be influenced by the non-capsid features conjectured by scholars. Some advantageous non-capsid features might be obtained by the CV-A8 strains of recombinant lineage 5 and somehow impart higher transmissibility. Another interesting phenomenon was observed within Chinese CV-A8 strains of lineage 2, genotype D; this lineage was found to be recombined with two types of Chinese CV-A6 recombinants [38
]. These two types of recombinants were associated with clinical features of more widespread skin lesions and severe HFMD [38
], and the non-capsid features of lineage 2 CV-A8 may affect the pathogenicity of these CV-A6 recombinants. Although CV-A8 strains have composed the minority of HFMD pathogens, they may serve as an important recombination interchange within the EV-A gene pool and help themselves or other EVs attain advantageous features.
Overall, based on the above results, potential reasons for the emergence of genotype E could be that: (i) during the evolution of genotype E, critical aa residues on the capsid region, especially those on the potential exposed loops, underwent mutations to change the capsid structures, making it easier to interact with the receptors and adapt to the host; (ii) recombination in the non-capsid genes might play an important role in the transmissibility of genotype E, in particular in the newly emerging recombinants of lineage 5 detected in China; (iii) due to CV-A8 being a minor component of the HFMD and HA pathogen spectra, few studies have focused on the detection and sequencing of CV-A8 over these years, leading to public resource limitations; (iv) the circulation of CV-A8 strains might have regional differences, especially in China, as most of the complete VP1 Chinese sequences from GenBank were from southern areas (Guangdong and Zhejiang provinces), whereas the sequences provided by this study were from distributed regions. Even though the first two conclusions were drawn from the results of genetic analyses, the data limitations could not be neglected, as we mentioned in reasons “iii” and “iv”. Therefore, future surveillance and further sequencing of CV-A8 is important for more accurate analyses.
Mainland China incorporated HFMD into the National Notifiable Disease Surveillance System (NNDSS) in 2008, and HFMD has had the highest yearly incidence among all national notifiable diseases since 2010, with over 1.5 million cases annually reported [40
]. Although EV-A71, CV-A16 and CV-A6 have been commonly regarded as the leading pathogens of HFMD worldwide, other EVs have been frequently reported in recent years, including CV-A8 [41
]. CV-A8 was also reported to cause HA, but HA was not enrolled in the disease surveillance reporting system of China; in addition, multiple clinical phenotypes, including AFP, lamellar ichthyosis, respiratory disease, and other features, have been reported to be associated with CV-A8 infection; therefore, the burden of CV-A8-related diseases might have been underestimated, posing a threat to public health. CV-A8 is a less important HFMD pathogen, and such capsid diversity (multiple genotypes) and frequent recombination (different intra-genotypic recombinant lineages) observed in CV-A8 indicate that the CV-A8 quasispecies is still going through variable dynamic changes. Seemingly, CV-A8 strains are still selecting a type of strain that can adapt mostly to the host environment. It is possible that CV-A8 could become an important pathogen in the HFMD spectrum in the future. Further surveillance of CV-A8 is greatly needed.