Environmental Research and Public Health the Current Status of the Disease Caused by Enterovirus 71 Infections: Epidemiology, Pathogenesis, Molecular Epidemiology, and Vaccine Development

Enterovirus 71 (EV71) infections have a major public health impact in the Asia-Pacific region. We reviewed the epidemiology, pathogenesis, and molecular epidemiology of EV71 infection as well as EV71 vaccine development. Previous studies were found using the search terms " enterovirus 71 " and " epidemiology " or " pathogenesis " or " molecular epidemiology " or " vaccine " in Medline and PubMed. Articles that were not published in the English language, manuscripts without an abstract, and opinion articles were excluded from the review. The reported epidemiology of cases caused by EV71 infection varied from country to country; seasonal variations in incidence were observed. Most cases of EV71 infection that resulted in hospitalization for complications occurred in children less than five years old. The brainstem was the most likely major target of EV71 infection. The emergence of the EV71 epidemic in the Asia-Pacific region has been associated with the circulation of different genetic lineages (genotypes B3, B4, C1, C2, and C4) that appear to be undergoing rapid evolutionary changes. The relationship between the gene structure of the EV71 virus and the factors that ensure its survival, circulation, and evasion of immunity is still unknown. EV71 infection has emerged as an important global public health problem. Vaccine development, including the development of inactivated whole-virus live attenuated, subviral particles, and DNA vaccines, has been progressing.

A higher incidence was observed during the summer months in Asia [63][64][65], and epidemics recur with a seasonal pattern. Some studies have also reported a variation of the peak season between different years [25,66]. In Taiwan, a surveillance system was established at the Taiwan Centers for Disease Control (Taiwan CDC) in 1998 to assess the epidemiologic features of EV71 infection [17]. Patients who were hospitalized for HFMD/herpangina were reported to the Taiwan CDC [3,17]. From March 1998 through December 2013, epidemic peaks occurred every year, with the highest number of cases occurring during the summer season ( Figure 1). Most cases of HFMD occurred in children aged five years old or younger; males had a higher incidence rate of HFMD than females.
Current hypotheses explaining the seasonal pattern of EV71 infection include host immune competence fluctuations mediated by seasonal factors, such as melatonin or vitamin D levels [67]; seasonal, behavioral factors unrelated to weather, such as school attendance and indoor crowding [68]; environmental factors [63,65], including temperature and relative humidity. However, human behavioral factors alone do not appear to account for the seasonal pattern observed for certain cases of EV71 infection, including cases that occur in school-aged children or in association with household crowding [68]. The relationship between meteorological data and the incidence of EV71 infection has been investigated [69,70]. A study by Chang et al. [70] showed that the incidence of EV71 infection reflected significant summer seasonality from April to June in Taiwan. The incidence of EV71 infection began to increase at a temperature above 13 • C; at a temperature higher than approximately 26 • C, the incidence began to decline, producing an inverted V-shaped relationship. This study indicated that warmer temperatures and elevated humidity would lead to an increased rate of EV71 infection in Taiwan. Similar findings have been found in China [71,72]. It showed that the HFMD infections occurred with two seasonal peaks, in summer (June) and winter (November or December). The major spatial-temporal clusters were from the eastern coastal and southern regions. The risk of infection was relatively high at 10 • C ≤ t < 15 • C and 15 • C ≤ t < 20 • C, and peaked at 20 The distribution of pathogens' serotypes and the level of sunshine could be risk factors for the outbreak of HFMD in China. Current hypotheses explaining the seasonal pattern of EV71 infection include host immune competence fluctuations mediated by seasonal factors, such as melatonin or vitamin D levels [67]; seasonal, behavioral factors unrelated to weather, such as school attendance and indoor crowding [68]; environmental factors [63,65], including temperature and relative humidity. However, human behavioral factors alone do not appear to account for the seasonal pattern observed for certain cases of EV71 infection, including cases that occur in school-aged children or in association with household crowding [68]. The relationship between meteorological data and the incidence of EV71 infection has been investigated [69,70]. A study by Chang et al. [70] showed that the incidence of EV71 infection reflected significant summer seasonality from April to June in Taiwan. The incidence of EV71 infection began to increase at a temperature above 13 °C; at a temperature higher than approximately 26 °C, the incidence began to decline, producing an inverted V-shaped relationship. This study indicated that warmer temperatures and elevated humidity would lead to an increased rate of EV71 infection in Taiwan. Similar findings have been found in China [71,72]. It showed that the HFMD infections occurred with two seasonal peaks, in summer (June) and winter (November or December). The major spatial-temporal clusters were from the eastern coastal and southern regions. The risk of infection was relatively high at 10 °C ≤ t < 15 °C and 15 °C ≤ t < 20 °C, and peaked at 20 °C ≤ t < 25 °C. The distribution of pathogens' serotypes and the level of sunshine could be risk factors for the outbreak of HFMD in China.
HFMD is spread from person to person by contact with saliva, respiratory secretions, fluid in vesicles, and feces. Transmission of HFMD can be reduced by isolation of confirmed cases of patients with viral shedding, and maintaining good hygiene, including hand-washing and disinfection of surfaces in child care settings [73].
Taken together, EV71 infection has emerged as an important public health problem in the world, especially in the Asia-Pacific region. The morbidity and mortality of EV71 infection are influenced by geographic, host, and environmental factors. Understanding the epidemiology and transmission dynamics of EV71 is helpful for leading to the control and elimination EV71 infection. HFMD is spread from person to person by contact with saliva, respiratory secretions, fluid in vesicles, and feces. Transmission of HFMD can be reduced by isolation of confirmed cases of patients with viral shedding, and maintaining good hygiene, including hand-washing and disinfection of surfaces in child care settings [73].
Taken together, EV71 infection has emerged as an important public health problem in the world, especially in the Asia-Pacific region. The morbidity and mortality of EV71 infection are influenced by geographic, host, and environmental factors. Understanding the epidemiology and transmission dynamics of EV71 is helpful for leading to the control and elimination EV71 infection.

Pathogenesis
EV71 infection that causes brainstem encephalitis, especially affecting the medulla and associated with cardiopulmonary dysfunction, has become a notable feature in EV71 epidemics in Asia and is associated with high mortality. The most common manifestation of enteroviral infection is a non-specific febrile illness lasting three days, followed by recovery. However, some cases progress to severe or fatal illness. Brainstem encephalitis, especially affecting the medulla and associated with cardiopulmonary dysfunction, has been noted as a clinical feature in EV71 epidemics in Asia and is an important cause of death [5,12,15,69,74,75].
Viremia occurred more frequently in children under the age of one year [76,77]. However, viremia did not have a clear significant effect on the clinical severity of EV71 infection. Additionally, the frequency of patients with CNS involvement was similar between patients with or without viremia [77,78]. EV71 is a highly neurotropic virus, and the brain stem is the most common target of EV71 infection [10,24,74,[79][80][81]. Similar to poliovirus, two likely routes by which the EV71 virus involves the CNS have been considered: the virus is either transmitted to the CNS from the blood across the blood-brain barrier (BBB) or enters the CNS through peripheral nerves via retrograde axonal transport [82][83][84][85].
The strong neurotropism of EV71 and retrograde axonal transport in neurons might represent the major transmission route of EV71 in mice [86,87]. In another study, mice were infected via the oral and parenteral routes with a murine-adapted virus strain that originated from a fatal human case, and the results showed that the EV71 virus entered the CNS via peripheral motor nerves after a skeletal muscle infection and spread within the CNS through motor and other neural pathways [88]. In a study of an autopsy sample in Malaysia, inflammation was found to be the most marked in the spinal cord gray matter, brainstem, hypothalamus, and subthalamic and dentate nuclei [89].
Neurological virulence is one of the most severe complications responsible for death [52]. The mechanism of the neurological complications of EV71 infection is poorly understood. Cordey et al. [90] have reported that an L97R alteration in the VP1 protein enhances the neuronal tropism of EV71. Some alterations in VP1, 5' NCR, and protease 2A affect viral virulence [91]. These results suggested that sequence variations may contribute to neural infection and neurological complications.
What is the mechanism of pulmonary edema among severe cases with EV71 infection? It may be due to the destruction of the medial, ventral, and caudal medullas, which may lead to sympathetic overactivation, causing a blood shift to the lungs [92,93]. Similar to acute respiratory distress syndrome, the pulmonary edema that occurs in children with EV71 brainstem encephalitis may be caused by abnormal cytokine activation that produces a severe inflammatory response, which in turn causes increased pulmonary vascular permeability [94]. One study showed that children with severe EV71 encephalitis were significantly more likely to have a cytotoxic T lymphocyte antigen haplotype (CTLA-4) than children who did not contract severe EV71 infection [95].
In summary, some EV71 strains are often related with neurological manifestations but EV71 also causes aseptic meningitis and fever without a source with good outcomes in very young infants. Some of the epidemiologic studies include cases of children with non-severe manifestations [96,97]. Moreover, there is a bias when analyzing some of the clinical data due to the fact that severe outbreaks are often more easily reported [98]. Based on the data from autopsy studies of fatal cases with EV71 infection in Taiwan [74,[99][100][101], peninsular Malaysia [24], and Hong Kong [100], there is evidence that brainstem encephalitis due to EV71 infection is sufficient to cause neurogenic pulmonary edema. During the viral life cycle, enteroviruses are influenced by viral factors and multiple host factors. The combination of the interactive effect of the virus and the host is vital for viral replication, virulence, and pathogenicity [102]. However, tissue-specific viral virulence remains unclear in both cell-based systems and animal models and requires further investigation in the future [102].

Molecular Epidemiology
The phylogenetic origins of the EV71 strains recently circulating in the Asia-Pacific region have been studied (Table 1) [3,9,10,14,15,. Using the VP1 protein for analysis, EV71 can be divided into four distinct genogroups (A, B, C, and D) [29,38]. Genogroups B and C can be further divided into genogroups B1-B5 and C1-C5, respectively [39]. Recently, genogroup D was identified in India, and genogroups E and F were identified in Africa [38,40]. Genogroup A comprises the prototype EV71 strain (BrCr-CA-70), which was isolated in 1969 in the United States [4] but had not been detected subsequently until 2008 [15]; however, the source of the virus in this outbreak is unclear [103]. In contrast, genogroup B and C viruses have been causing large-scale epidemics in Asia since 1997 and are targeted for vaccine development [41,42]. The co-circulation of four distinct genogroups (B3, B4, C1, and C2) in Malaysia from 1997-2000 has been well documented [36]. Singapore [43] and Western Australia [104] were also affected by the B3 genogroup from 1997-1999. In Taiwan [41,44,45].
In South Korea, an EV71 outbreak was reported during 2009 [28]. The predominant genotype was C4, particularly C4a, which was associated with relatively low severity and a low case-fatality rate [28]. However, in China, it has been reported that the mean evolution rate of C4a EV71 is faster than all other EV71 genotypes [48]. The evolutionary branch C4a has some crucial nucleotide or amino acid mutations relative to branch C4b, and these changes may be responsible for its increased neurovirulence and the epidemic of large-scale outbreaks of HFMD in China [48]. EV71 genotype C4a viruses also spread from China to Vietnam and caused a large-scale epidemic in Ho Chi Minh City and southern Vietnam in 2011, which was confirmed through genetic and antigenic analysis [4]. Based on phylogenetic analyses of the VP1 sequences, a comprehensive evolutionary dynamic study of EV71 from 1994-2013 was conducted in the Asia-Pacific areas [49]. They showed that C4, C1, C2, and B4 are the predominant strains, and the polymorphisms and divergence of the VP1 gene of the EV71 strains evolve very slowly, which may be one of the reasons for periodic outbreaks in this area.
EV71 has a high mutability and is in constant evolution, similar to poliovirus [29]. However, to what extent genetic exchanges explain the variations in biological or epidemic behavior is open to debate. Genogroups B and C have been associated with both complicated and uncomplicated disease [35,99], thus making it difficult to pinpoint a virus-specific marker of virulence [53,105]. EV71 lacks the use of a DNA template for correcting mismatches, resulting in an average of one mutation per new genome copy [106]. Furthermore, genomic recombination is frequently used among enteroviruses as a mechanism to produce variants [107], presumably as a response to selection pressure [108]. These observations suggest that recombination and mutation may promote the spread of EV71 in the human population [109].
Taken together, the dynamics of the genetic and antigenic evolution of EV71 in the past decade showed a genotype shift with an antigenic property change and genome recombination. Almost all HFMD outbreaks were correlated with genetic variations caused by EV71 switches. Recombination of EV71 in the region encoding the nonstructural proteins is frequently observed, and numerous recombination crossover breakpoints have been identified within the non-structural genes, particularly of the more recent EV71 subgenotypes [44,110,111]. Recombination has also been observed between EV71 and coxsackievirus A16 [110,112] and other enterovirus A species [44]. Interestingly, recent studies have identified several subgenotype-specific recombination events [60,113] that appear to act as founding events in subgenotype emergence and global expansion [60], suggesting that recombination has played a crucial role in EV71 evolution. Thus, continual monitoring of antigenic variation and genetic evolution is critical for epidemic control and vaccine design.

Vaccine Development
EV71 is most commonly transmitted via close person-to-person contact; however, the majority of EV71 infections are asymptomatic or result in mild disease, which limits the effectiveness of public health interventions such as hand washing. The development of an effective vaccine may be the best way to control EV71 infection [114]. Many EV71 vaccines have been investigated, including an inactivated virus vaccine [115][116][117], a virus-like particle vaccine [118], DNA vaccines [119], a subunit vaccine [120], and a live attenuated vaccine [121].
Inactivated whole-virus EV71 vaccines are the most advanced candidates among the current EV71 vaccines. Inspired by previous experiences in developing inactivated vaccines, the development of inactivated whole-virus EV71 vaccines is progressing rapidly [122]. The formaldehyde-inactivated EV71 vaccines effectively protected animals from lethal virus challenge in several animal model studies [123,124]. Additionally, a formalin-inactivated EV71 virion formulated in alum-adjuvant vaccine resulted in satisfactory cross-neutralizing antibody responses in a phase III trial [115]. Phase III clinical trials of inactivated EV71 vaccines have been completed in China, involving more than 30,000 infants and children [104,[125][126][127]. Their results have shown that the safety of the EV71 vaccine is satisfactory in infants and children and can prevent over 90% of EV71-associated HFMD and 80% of EV71-associated disease [125,127]. In December 2015, China's Food and Drug Administration approved two inactivated EV-A71 vaccines for preventing severe HFMD [128]. Similar vaccines (e.g., formalin-inactivated EV71 vaccines) are being developed in Taiwan and Singapore [126,128], both of them have entered Phase I clinical trials. Inactivated EV71 vaccines, due to their inability to replicate, are preferred over the live attenuated vaccines for safety reasons. However, the cost of production of inactivated vaccines and potential supply problems cause substantial frustration in practical implementations [129].
The development of virus-like particles (VLPs) vaccines is a different approach from the inactivated whole-virus EV71 vaccines. VLPs vaccines provide the advantage of presenting all surface epitopes of the EV71 capsid proteins in their native conformations at once [128,130]. Although VLPs vaccines have a lower efficacy than inactivated vaccines, experimental animal model studies have indicated that VLPs can generate protective neutralizing antibodies and are cross-reactive against multiple subtypes that are not in the vaccine [130]. The main problem associated with VLPs is their stability, purification, and cost of manufacturing. The other types of EV71 vaccines (DNA vaccines, subunit vaccine, and live attenuated vaccine) are in the early stages of development, with the most advanced undergoing preclinical trials in mice and non-human primates [128].
In summary, the formalin-inactivated EV71 vaccines have reported to have high efficacy for preventing EV71 infection. EV71-related HFMD could become a vaccine-preventable disease in the world. However, no formalin-inactivated EV71 vaccines protect against CAV16, which is predominantly responsible for annual HFMD outbreaks. The development of vaccines that cover multiple species of enterovirus is a prospective option. Live attenuated vaccines, subunits vaccine, synthetic peptides, and DNA vaccines have been approached by researchers, but these approaches are in the early stage of vaccine development.

Conclusions
EV71 has emerged as a significant infection in the Asia-Pacific region. It is a potentially fatal neurotropic virus that may be considered the new polio. Several factors have been indicated to regulate EV71 replication. However, specific factors that contribute to neural pathogenesis remain unclear. Further study on the mechanism of EV71 infection is still needed. Although inactivated EV71 vaccines have been rapidly developed in the last few years, these vaccines have some limitations. Other types of vaccines are being developed to prevent EV71 infection. A global EV71 infection surveillance network should be established, and continuous epidemiological surveillance is important for identifying and detecting the potential emergence of new EV71 variants.