The present study is the first report of the emergence of EV71 “double-recombinant” strains belonging to the newly proposed genotype D in Hong Kong. We have previously showed that intratypic and intertypic recombination events have occurred in EV71 strains, SZ/HK08-5 and SZ/HK08-6, and other subgenotype C4 strains, which represent the new genotype D . Although the functional relevance of the “double recombination” events in EV71 remains unclear, we speculate that the acquisition of replicative components, such as helicase, protease and polymerase, from EV71 genotype B strain and CVA16 strain in EV71 subgenotype C4 strain may confer evolutionary advantages on EV71 “double-recombinant” strains of “genotype D”. Further studies are warranted to investigate the role of the recombinations in the “genotype D” strains. In the present study, the incongruent phylogenetic relationships among the 5′UTR, P1, P2 and P3 regions of the subgenotype C4 strains, together with results of similarity plot and bootscan analyses, confirmed that these strains belonged to the “double-recombinant”, newly proposed genotype D. Such incongruities observed in EV71 phylogenetic trees when comparing structural and non-structural regions of the genomes were similar to those demonstrated in a recent study , which showed that a mosaic of incongruent patterns for EV71 were likely due to the independent evolutionary pathways of the two genome regions of EV71. Traditionally, many studies have reported the use of structural gene sequences [4, 24, 25], particularly VP1 gene, for EV71 genotype and subgenotype determination. However, VP1 gene sequence mainly provides information on the serotype of EV71 and it encompasses only about one-tenth of the entire genome. On the other hand, using whole genome sequence for EV71 genotyping seems to be more appropriate because it consists of not only structural region encoding sequences, but also non-structural region encoding sequences and untranslated regions at 5′ and 3' termini. In addition, recombination commonly occurred in enteroviruses [10–12, 15], such as EV71 subgenotype C4 strains, which were shown to cluster with other subgenotype C strains for 5′UTR and P1, but not for P2 and P3 regions (Figure 2). Furthermore, in other positive-sense single-stranded RNA viruses, such as human coronaviruses (HCoVs), the generation of novel genotypes of HCoV-HKU1 and HCoV-OC43 as a result of the recombination between different genotypes has been reported [26, 27]. Renaming subgenotype C4 as genotype D was also supported by another study , in which the complete genome sequences of subgenotype C4 strains were shown to have a nucleotide divergence of 17-20% that exceeded the average cut-off divergence of 14.95% for subgenotyping. Similar nucleotide divergence (17–18.9%) of the 7 subgenotype C4 strains in the present study was also demonstrated when compared to other EV71 strains of subgenotype C1, C2, C3 and C5 using complete genome sequences. A combination of VP1 and 3D gene sequences was proposed to be used for initial genotyping of EV71 isolates in the absence of complete genome sequences . In 2001, an EV71 isolate from a patient with acute flaccid paralysis in India has also been proposed to belong to genotype D [28, 29]. However, complete genome sequence data were lacking for more detailed phylogenetic and genome analysis of this Indian strain. As VP1 gene clustering closely resembles the serotype designation of enteroviruses, VP1 gene should be sequenced for EV71 genotyping in any circumstances. However, using VP1 gene alone may be insufficient for genotyping the EV71 recombinants. Thus, sequencing of at least three regions, probably one from P1, one from P2 and one from P3, is only required for genotyping the EV71 strains with recombination, and this will help future assignment of genotypes for enteroviruses.
EV71 “genotype D” strains have been persistently circulating in Hong Kong, which has experienced two EV71 epidemics in 2008 and 2010 with around 100 reported cases each year [21, 30]. Among the 22 EV71 isolates collected in this study, 20 belonged to “genotype D” and two belonged to subgenotype C2, indicating that “genotype D” was the predominant type of EV71 in our locality over the 7-year period. This is consistent with an epidemiological study showing that subgenotype C4 (“genotype D”) was the major type of EV71 circulating in Hong Kong since 1998 . Persistent circulation of subgenotype C4 strains was also noted in China during 1998–2010 . We found that the complete genome sequences of the 7 subgenotype C4 strains in Hong Kong were highly similar to those in China, with nucleotide sequence identity of >90%. As Hong Kong is well connected to China, this may facilitate the spread of subgenotype C4 strains from mainland China to Hong Kong. The reasons for the recent EV71 epidemics in Hong Kong due to the same genotype of virus, “genotype D”, remain to be determined. One possible explanation is that immune response against EV71 in individuals may decrease over time, leading to an accumulation of susceptible individuals, in turn facilitating the transmission of EV71 “genotype D” in our population. Further study is warranted to determine if lower seroprevalence is noted in a year prior to each EV71 epidemic. Another reason for a surge in the number of EV71 cases could be owing to the antigenic changes of EV71 . However, when comparing the amino acid sequences of VP1 of the 20 “genotype D” strains from Hong Kong, they were highly identical with conserved amino acid residues in the BC loop essential for interaction with neutralizing antibodies. The high degree of conservation in the immunogenic VP1 protein of “genotype D” strains raises the possibility of developing a vaccine against this predominant genotype in Hong Kong and mainland China, which may help protect young children at high risk.
As EV71 subgenotype C4 should be re-designated as genotype D, we propose to change subgenotypes C4b and C4a into genotypes D1a and D1b respectively. The earliest characterized subgenotype C4 strain SHZH98 is in cluster b, but for more systematic classification (usually based on chronological order), the older and recent strains should belong to cluster a and b respectively. Since the late 1990s, EV71 subgenotype C4 strains have been frequently reported in Asian countries [22, 32–37]. The subgenotype C4 was further divided into two clusters, C4b and C4a [22, 37], which are better to be renamed as genotypes D1a and D1b respectively. In the present study, EV71 “genotype D” strains in Hong Kong have been stably evolving during 2004–2010, with a shift from “genotype D1a” (2004 and 2006) to “genotype D1b” (2004–2010). From the results of the present study, no correlation was found between genotypes of EV71 and disease severity, though a recent study from China showed that C4a strains (proposed genotype D1b) caused more severe diseases in patients with HFMD than C4b strains (proposed genotype D1a) .
In addition to “genotype D”, a distinct lineage of EV71 subgenotype C2 has emerged in Hong Kong in 2008. EV71 subgenotype C2 was first detected in Australia in 1995  and three years later, this predominant type circulating in Taiwan led to a devastating outbreak . From the phylogenetic analysis of VP1 in the present study, two EV71 strains from Hong Kong, V08-2231530 and V08-2236079, detected in 2008 shared a high nucleotide sequence identity with other subgenotype C2 strains in recent years (2006–2008), forming a cluster separate from the subgenotype C2 strains discovered in the 1990s (Figure 4). This was in line with the result of a study showing that recent subgenotype C2 strains from the Netherlands and United Kingdom formed a clade distinct from a group of “old” strains detected in the Netherlands, Taiwan and the United States, with high bootstrap support of 100% . Current classification of EV71 genotypes using VP1 gene sequences showed that the average nucleotide sequence divergences between and within subgenotypes were 10.1% and 3.6% respectively . In the present study, the average nucleotide divergence between “old” and “new” subgenotype C2 strains was 4.6%, which lies between the two mean cut-off values, suggesting the emergence of a distinct lineage of subgenotype C2 strains in Hong Kong. The minority of EV71 subgenotype C2 strains detected only in 2008 in Hong Kong could be due to the importation of this subgenotype from neighboring countries, including mainland China . Further epidemiological and genomic studies are required to identify the emergence of novel types of EV71 and assess their potential in causing epidemics in the near future.
To better understand the evolution of EV71, evolutionary rates and divergence dates of EV71 were also examined using VP1 gene sequence. VP1 gene was chosen because it shows a high degree of genetic diversity and no recombination has been mapped within VP1 gene , while recombination within 5'UTR, P2 and P3 regions is frequent in EV71 [15, 17, 22]. The evolutionary rate of EV71 was 3.1 × 10-3 s/s/y comparable to that of other enterovirus, such as EV68 (4.93 × 10-3 s/s/y) , but was lower than those of echovirus 30 (8.3 × 10-3 s/s/y)  and poliovirus (1.03 × 10-2 s/s/y for P1 region), with the latter shown to be the highest rate among picornaviruses . Molecular clock analysis using VP1 gene sequences suggested that the tMRCA of all EV71 genotypes most likely emerged in the 1900s (mean: 1905), which was similar to that shown in a study from China (mean: 1911) , but was different from that demonstrated in another study (mean: 1941) , it is probably due to different sample sizes and model parameters used in the analyses. While the tMRCA of genotype B and C in the 1920s (mean: 1925), that of genotype B in the 1950s (mean: 1953), that of genotype A in the late 1960s (mean: 1967), and that of genotype C in the early 1970s (mean: 1972). A recent study has demonstrated the tMRCA for EV71 strains of subgenotypes C4a and C4b , but authors did not notice that recombination events have occurred in these strains, which can bias estimates of the tMRCA . Although the tMRCA of the EV71 “double-recombinant” strains of “genotype D” could not be determined by molecular clock analysis, the detection of “genotype D” strains from Hong Kong and mainland China from year 1998 suggested that this genotype has emerged not later than this year. Since EV71 keeps on evolving via mutation and recombination [15, 19], molecular surveillance is of vital importance in monitoring the genetic variations of circulating EV71 strains and the emergence of new types or recombinants of EV71.