ABSTRACT

Enterovirus A71 (EV-A71) infections are one of the main etiological agents of hand, foot and mouth disease (HFMD) and herpangina worldwide. EV-A71 infection is a life-threatening communicable disease and there is an urgent global need for the development of vaccines for its prevention and control. The morbidity rate of EV-A71 infection differs between countries. The pathogen’s genetic lineages are undergoing rapid evolutionary changes. An association between the occurrence of EV-A71 infection and the circulation of different genetic strains of EV-A71 virus has been identified around the world. In this review, we present and discuss the molecular epidemiology and pathogenesis of the human disease caused by EV-A71 infection, as well as current prospects for the development of an EV-A71 vaccine.

Hand, foot and mouth disease; Herpangina; Enterovirus A71; Vaccine

INTRODUCTION

Enterovirus A71 (EV-A71) is an important cause of hand, foot and mouth disease (HFMD) and herpangina ¹1. Yi EJ, Shin YJ, Kim JH, Kim TG, Chang SY. Enterovirus 71 infection and vaccines. Clin Exp Vaccine Res. 2017;6:4-14. . EV-A71 infects infants and young children and most cases of infection (>70%) are symptomatic ²2. Chen KT, Chang HL, Wang ST, Cheng YT, Yang JY. Epidemiologic features of hand-foot-mouth disease and herpangina caused by enterovirus 71 in Taiwan, 1998-2005. Pediatrics. 2007;120:e244-52.^,33. Chan KP, Goh KT, Chong CY, Teo ES, Lau G, Ling AE. Epidemic hand, foot and mouth disease caused by human enterovirus 71, Singapore. Emerg Infect Dis. 2003;9:78-85. . The predominant clinical presentation of HFMD is characterized by a febrile illness accompanied by a maculopapular rash or blisters on the hands, soles of feet and buttocks ¹1. Yi EJ, Shin YJ, Kim JH, Kim TG, Chang SY. Enterovirus 71 infection and vaccines. Clin Exp Vaccine Res. 2017;6:4-14.^,22. Chen KT, Chang HL, Wang ST, Cheng YT, Yang JY. Epidemiologic features of hand-foot-mouth disease and herpangina caused by enterovirus 71 in Taiwan, 1998-2005. Pediatrics. 2007;120:e244-52. . It is usually a self-limiting infection, but it is highly contagious through fecal-oral transmission of oropharyngeal secretions ²2. Chen KT, Chang HL, Wang ST, Cheng YT, Yang JY. Epidemiologic features of hand-foot-mouth disease and herpangina caused by enterovirus 71 in Taiwan, 1998-2005. Pediatrics. 2007;120:e244-52. . The most common involved area in severe or fatal cases of EV-A71 is the brain stem ²2. Chen KT, Chang HL, Wang ST, Cheng YT, Yang JY. Epidemiologic features of hand-foot-mouth disease and herpangina caused by enterovirus 71 in Taiwan, 1998-2005. Pediatrics. 2007;120:e244-52.^,33. Chan KP, Goh KT, Chong CY, Teo ES, Lau G, Ling AE. Epidemic hand, foot and mouth disease caused by human enterovirus 71, Singapore. Emerg Infect Dis. 2003;9:78-85. .

EV-A71 is an RNA virus that belongs to the human enterovirus A (EV-A) species, genus Enterovirus , family Picornaviridae¹1. Yi EJ, Shin YJ, Kim JH, Kim TG, Chang SY. Enterovirus 71 infection and vaccines. Clin Exp Vaccine Res. 2017;6:4-14. . Viral particles have an icosahedral shape, are not enveloped and contain a single-strand, positive-sense RNA genome. The most common method for enterovirus genotyping targets the 1D gene encoding the VP1 capsid protein containing the major neutralizing epitopes ⁴4. Bessaud M, Razafindratsimandresy R, Nougairède A, Joffret ML, Deshpande JM, Dubot-Pérès A, et al. Molecular comparison and evolutionary analyses of VP1 nucleotide sequences of new African human enterovirus 71 isolates reveal a wide genetic diversity. PLoS One. 2014;9:e90624. . Human-infecting enteroviruses species include enterovirus A (EV-A), EV-B, EV-C, and EV-D. The classification of human enteroviruses is based on each strain’s homology within the RNA region coding for the VP1 capsid protein ¹1. Yi EJ, Shin YJ, Kim JH, Kim TG, Chang SY. Enterovirus 71 infection and vaccines. Clin Exp Vaccine Res. 2017;6:4-14. .

Although the EV-A71 virus has been isolated in many countries, epidemics of EV-A71 infection are predominantly found in the Asia-Pacific region ²2. Chen KT, Chang HL, Wang ST, Cheng YT, Yang JY. Epidemiologic features of hand-foot-mouth disease and herpangina caused by enterovirus 71 in Taiwan, 1998-2005. Pediatrics. 2007;120:e244-52.^,33. Chan KP, Goh KT, Chong CY, Teo ES, Lau G, Ling AE. Epidemic hand, foot and mouth disease caused by human enterovirus 71, Singapore. Emerg Infect Dis. 2003;9:78-85.^,55. Chan LG, Parashar UD, Lye MS, Ong FG, Zaki SR, Alexander JP, et al. Deaths of children during an outbreak of hand, foot, and mouth disease in Sarawak, Malaysia: clinical and pathological characteristics of the disease. Clin Infect Dis. 2000;31:678-83.

6. McMinn P, Stratov I, Nagarajan L, Davis S. Neurological manifestations of enterovirus 71 infection in children during an outbreak of hand, foot, and mouth disease in Western Australia. Clin Infect Dis. 2001;32:236-42.

7. Van Tu P, Thao NT, Perera D, Truong KH, Tien NTK, Thuong TC, et al. Epidemiologic and virologic investigation of hand, foot, and mouth disease, southern Vietnam, 2005. Emerg Infect Dis. 2007;13:1733-41.

8. Komatsu H, Shimizu Y, Takeuchi Y, Ishiko H, Takada H. Outbreak of severe neurologic involvement associated with Enterovirus 71 infection. Pediatr Neurol. 1999;20:17-23.^-99. Zhang Y, Zhu Z, Yang W, Ren J, Tan X, Wang Y, et al. An emerging recombinant human enterovirus 71 responsible for the 2008 outbreak of hand, foot and mouth Disease in Fuyang city of China. Virol J. 2010;7:94. . The morbidity associated with EV-A71 infection varies from country to country ¹1. Yi EJ, Shin YJ, Kim JH, Kim TG, Chang SY. Enterovirus 71 infection and vaccines. Clin Exp Vaccine Res. 2017;6:4-14. . Seasonal epidemic patterns of EV-A71 infection have been observed in several countries. In Asia, a higher incidence has been observed during the summer months ¹⁰10. Chang HL, Chio CP, Su HJ, Liao CM, Lin CY, Shau WY, et al. The association between enterovirus 71 infections and meteorological parameters in Taiwan. PLoS One. 2012;7:e46845.

11. Ma E, Lam T, Wong C, Chuang SK. Is hand, foot and mouth disease associated with meteorological parameters? Epidemiol Infect. 2010;138:1779-88.^-1212. Dowell SF. Seasonal variation in host susceptibility and cycles of certain infectious diseases. Emerg Infect Dis. 2001;7:369-74. . Several studies have also shown variations in the peak season between different years ¹³13. Mizuta K, Abiko C, Murata T, Matsuzaki Y, Itagaki T, Sanjoh K, et al. Frequent importation of enterovirus 71 from surrounding countries into the local community of Yamagata, Japan, between 1998 and 2003. J Clin Microbiol. 2005;43:6171-5.^,1414. Schmidt NJ, Lennette EH, Ho HH. An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis. 1974;129:304-9. . Most cases of EV-A71 infection occur in children five years of age and younger, and boys have a higher risk of EV-A71 infection than girls ²2. Chen KT, Chang HL, Wang ST, Cheng YT, Yang JY. Epidemiologic features of hand-foot-mouth disease and herpangina caused by enterovirus 71 in Taiwan, 1998-2005. Pediatrics. 2007;120:e244-52.^,55. Chan LG, Parashar UD, Lye MS, Ong FG, Zaki SR, Alexander JP, et al. Deaths of children during an outbreak of hand, foot, and mouth disease in Sarawak, Malaysia: clinical and pathological characteristics of the disease. Clin Infect Dis. 2000;31:678-83. . Because the virus has the potential to cause high morbidity and mortality among children, it is critical to understand the mechanisms of and control measures for EV-A71 infections. In this review, we present and discuss the molecular epidemiology and pathogenesis of EV-A71 infections, as well the prospects for the development of an EV-A71 vaccine.

Data sources

All manuscripts used in this review were published between January 1965 and December 2017; these reports related to EV-A71 infections were extracted by searching Medline (National Library of Medicine, Bethesda, Maryland, USA) and PubMed using the phrases “ enterovirus-A71 ” and “molecular epidemiology” or the key words “pathogenesis” or “vaccine.” The results were limited to manuscripts available in English.

Molecular epidemiology

In 1969, EV-A71 was first isolated from a child with encephalitis in the USA ¹⁴14. Schmidt NJ, Lennette EH, Ho HH. An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis. 1974;129:304-9. . Subsequently, several EV-A71 epidemics were reported in the 1970s around the world, including the Americas, Europe, Australia and Asia ¹⁵15. Hosoya M, Kawasaki Y, Sato M, Honzumi K, Kato A, Hiroshima T, et al. Genetic diversity of enterovirus 71 associated with hand, foot and mouth disease epidemics in Japan from 1983 to 2003. Pediatr Infect Dis J. 2006;25:691-4.

16. Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH. Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis. 2010;10:778-90.

17. Huang SW, Hsu YW, Smith DJ, Kiang D, Tsai HP, Lin KH, et al. Reemergence of enterovirus 71 in 2008 in Taiwan: dynamics of genetic and antigenic evolution from 1998 to 2008. J Clin Microbiol. 2009;47:3653-62.

18. Li L, He Y, Yang H, Zhu J, Xu X. Dong J, et al. Genetic characteristics of human enterovirus 71 and coxsackievirus A16 circulating from 1999 to 2004 in Shenzhen, People’s Republic of China. J Clin Microbiol. 2005;43:3835-9.

19. Zhang Y, Wang J, Guo W, Wang H, Zhu S, Wang D, et al. Emergence and transmission pathways of rapidly evolving evolutionary branch C4a strains of human enterovirus 71 in the Central Plain of China. PLoS One. 2011;6:e27895.

20. Geoghegan JL, Tan le V, Kühnert D, Halpin RA, Lin X, Simenauer A, et al. Phylodynamics of enterovirus A71-associated hand, foot, and mouth disease in Viet Nam. J Virol. 2015;89:8871-9.

21. Chumakov M, Voroshilova M, Shindarov L, Lavrova I, Gracheva L, Koroleva G, et al. Enterovirus 71 isolated from cases of epidemic poliomyelitis-like disease in Bulgaria. Arch Virol. 1979;60:329-40.

22. Nagy G, Takátsy S, Kukán E, Mihály I, Dömök I. Virological diagnosis of enterovirus type 71 infections: experiences gained during an epidemic of acute CNS disease in Hungary in 1978. Arch Virol. 1982;71:217-27.

23. Brown BA, Oberste MS, Alexander JP Jr, Kennett ML, Pallansch MA. Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998. J Virol. 1999;73:9969-75.

24. Merovitz L, Demers AM, Newby D, McDonald J. Enterovirus 71 infections at a Canadian center. Pediatr Infect Dis J. 2000;19:755-7.

25. Ortner B, Huang CW, Schmid D, Mutz I, Wewalka G, Allerberger F, et al. Epidemiology of enterovirus types causing neurological disease in Austria 1999-2007: detection of clusters of echovirus 30 and enterovirus 71 and analysis of prevalent genotypes. J Med Virol. 2009;81:317-24.

26. Bible JM, Iturriza-Gomara M, Megson B, Brown D, Pantelidis P, Earl P, et al. Molecular epidemiology of human enterovirus 71 in the United Kingdom from 1998 to 2006. J Clin Microbiol. 2008;46:3192-200.

27. van der Sanden S, van Eek J, Martin DP, van der Avoort H, Vennema H, Koopmans M. Detection of recombination breakpoints in the genomes of human enterovirus 71 strains isolated in the Netherlands in epidemic and non-epidemic years, 1963-2010. Infect Genet Evol. 2011;11:886-94.

28. Witsø E, Palacios G, Rønningen KS, Cinek O, Janowitz D, Rewers M, et al. Asymptomatic circulation of HEV71 in Norway. Virus Res. 2007;123:19-29.

29. Schuffenecker I, Mirand A, Antona D, Henquell C, Chomel JJ, Archimbaud C, et al. Epidemiology of human enterovirus 71 infections in France, 2000-2009. J Clin Virol. 2011;50:50-6.

30. Chakraborty R, Iturriza-Gómara M, Musoke R, Palakudy T, D’Agostino A, Gray J. An epidemic of enterovirus 71 infection among HIV-1-infected orphans in Nairobi. AIDS. 2004;18:1968-70.^-3131. Huaman JL, Carrion G, Ampuero JS, Ocaña V, Laguna-Torres VA, Hontz RD. Enterovirus-71 genotype C isolated in Peru between 2006 and 2009. J Clin Virol. 2016;85:40-3. .

The EV-A71 virus is enclosed by the capsid proteins VP1, VP2, VP3, and VP4 ¹1. Yi EJ, Shin YJ, Kim JH, Kim TG, Chang SY. Enterovirus 71 infection and vaccines. Clin Exp Vaccine Res. 2017;6:4-14. . VP1 shows the major antigenicity and has been defined as the neutralization determinant ¹1. Yi EJ, Shin YJ, Kim JH, Kim TG, Chang SY. Enterovirus 71 infection and vaccines. Clin Exp Vaccine Res. 2017;6:4-14. . Based on VP1 nucleotide sequence analysis, EV-A71 can be divided into three distinct genogroups (e.g., A, B, and C) ¹1. Yi EJ, Shin YJ, Kim JH, Kim TG, Chang SY. Enterovirus 71 infection and vaccines. Clin Exp Vaccine Res. 2017;6:4-14.^,2525. Ortner B, Huang CW, Schmid D, Mutz I, Wewalka G, Allerberger F, et al. Epidemiology of enterovirus types causing neurological disease in Austria 1999-2007: detection of clusters of echovirus 30 and enterovirus 71 and analysis of prevalent genotypes. J Med Virol. 2009;81:317-24. . Genogroup A includes the prototype EV-A71 strain (BrCr-CA-70); this strain was first isolated in 1969 in the USA ¹⁴14. Schmidt NJ, Lennette EH, Ho HH. An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis. 1974;129:304-9. , although it was not identified until 2008, in China ⁹9. Zhang Y, Zhu Z, Yang W, Ren J, Tan X, Wang Y, et al. An emerging recombinant human enterovirus 71 responsible for the 2008 outbreak of hand, foot and mouth Disease in Fuyang city of China. Virol J. 2010;7:94. . Genogroup B can be divided into subgenogroups B1-B5, and genogroup C can be further divided into subgenogroups C1-C5 ¹⁶16. Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH. Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis. 2010;10:778-90. . Recently, the C4 genogroup was once more subdivided into the C4a and C4b lineages. Genogroup D was initially identified in India ⁴4. Bessaud M, Razafindratsimandresy R, Nougairède A, Joffret ML, Deshpande JM, Dubot-Pérès A, et al. Molecular comparison and evolutionary analyses of VP1 nucleotide sequences of new African human enterovirus 71 isolates reveal a wide genetic diversity. PLoS One. 2014;9:e90624. , and genogroups E and F were initially identified in Africa ⁴4. Bessaud M, Razafindratsimandresy R, Nougairède A, Joffret ML, Deshpande JM, Dubot-Pérès A, et al. Molecular comparison and evolutionary analyses of VP1 nucleotide sequences of new African human enterovirus 71 isolates reveal a wide genetic diversity. PLoS One. 2014;9:e90624. .

Table 1 shows the phylogenetic origins of the EV-A71 strains that have been recently circulating in the Asia-Pacific region ²2. Chen KT, Chang HL, Wang ST, Cheng YT, Yang JY. Epidemiologic features of hand-foot-mouth disease and herpangina caused by enterovirus 71 in Taiwan, 1998-2005. Pediatrics. 2007;120:e244-52.^,55. Chan LG, Parashar UD, Lye MS, Ong FG, Zaki SR, Alexander JP, et al. Deaths of children during an outbreak of hand, foot, and mouth disease in Sarawak, Malaysia: clinical and pathological characteristics of the disease. Clin Infect Dis. 2000;31:678-83.^,88. Komatsu H, Shimizu Y, Takeuchi Y, Ishiko H, Takada H. Outbreak of severe neurologic involvement associated with Enterovirus 71 infection. Pediatr Neurol. 1999;20:17-23.^,99. Zhang Y, Zhu Z, Yang W, Ren J, Tan X, Wang Y, et al. An emerging recombinant human enterovirus 71 responsible for the 2008 outbreak of hand, foot and mouth Disease in Fuyang city of China. Virol J. 2010;7:94.^,1515. Hosoya M, Kawasaki Y, Sato M, Honzumi K, Kato A, Hiroshima T, et al. Genetic diversity of enterovirus 71 associated with hand, foot and mouth disease epidemics in Japan from 1983 to 2003. Pediatr Infect Dis J. 2006;25:691-4.

16. Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH. Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis. 2010;10:778-90.

17. Huang SW, Hsu YW, Smith DJ, Kiang D, Tsai HP, Lin KH, et al. Reemergence of enterovirus 71 in 2008 in Taiwan: dynamics of genetic and antigenic evolution from 1998 to 2008. J Clin Microbiol. 2009;47:3653-62.

18. Li L, He Y, Yang H, Zhu J, Xu X. Dong J, et al. Genetic characteristics of human enterovirus 71 and coxsackievirus A16 circulating from 1999 to 2004 in Shenzhen, People’s Republic of China. J Clin Microbiol. 2005;43:3835-9.

19. Zhang Y, Wang J, Guo W, Wang H, Zhu S, Wang D, et al. Emergence and transmission pathways of rapidly evolving evolutionary branch C4a strains of human enterovirus 71 in the Central Plain of China. PLoS One. 2011;6:e27895.^-2020. Geoghegan JL, Tan le V, Kühnert D, Halpin RA, Lin X, Simenauer A, et al. Phylodynamics of enterovirus A71-associated hand, foot, and mouth disease in Viet Nam. J Virol. 2015;89:8871-9. . Genogroup A was not identified in the Asia-Pacific region until 2008 ⁹9. Zhang Y, Zhu Z, Yang W, Ren J, Tan X, Wang Y, et al. An emerging recombinant human enterovirus 71 responsible for the 2008 outbreak of hand, foot and mouth Disease in Fuyang city of China. Virol J. 2010;7:94. . In contrast, genogroups B and C have been responsible for several large-scale outbreaks in the Asia region since 1997 ¹⁶16. Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH. Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis. 2010;10:778-90. . Four distinct subgenogroups (B3, B4, C1 and C2) were found to have been cocirculating in the Asia-Pacific region from 1990-2016. Subgenogroup C4, particularly the C4a lineage, has also emerged in the Asia-Pacific region. The evolutionary branch C4a has crucial nucleotides and amino acid mutations relative to the branch C4b, and these changes may be the primary reason for its increased neurovirulence, causing outbreaks in China ¹⁸18. Li L, He Y, Yang H, Zhu J, Xu X. Dong J, et al. Genetic characteristics of human enterovirus 71 and coxsackievirus A16 circulating from 1999 to 2004 in Shenzhen, People’s Republic of China. J Clin Microbiol. 2005;43:3835-9.^,1919. Zhang Y, Wang J, Guo W, Wang H, Zhu S, Wang D, et al. Emergence and transmission pathways of rapidly evolving evolutionary branch C4a strains of human enterovirus 71 in the Central Plain of China. PLoS One. 2011;6:e27895. . Through genetic and antigenic analysis, EV-A71 subgenogroup C4a has been confirmed to have spread from China to Vietnam; a subsequent large-scale outbreak occurred in Ho Chi Minh City and Southern Vietnam in 2011 ²⁰20. Geoghegan JL, Tan le V, Kühnert D, Halpin RA, Lin X, Simenauer A, et al. Phylodynamics of enterovirus A71-associated hand, foot, and mouth disease in Viet Nam. J Virol. 2015;89:8871-9. .

The phylogenetic origins of EV-A71 strains circulating outside the Asia-Pacific region are presented in Table 2 ²¹21. Chumakov M, Voroshilova M, Shindarov L, Lavrova I, Gracheva L, Koroleva G, et al. Enterovirus 71 isolated from cases of epidemic poliomyelitis-like disease in Bulgaria. Arch Virol. 1979;60:329-40.

22. Nagy G, Takátsy S, Kukán E, Mihály I, Dömök I. Virological diagnosis of enterovirus type 71 infections: experiences gained during an epidemic of acute CNS disease in Hungary in 1978. Arch Virol. 1982;71:217-27.

23. Brown BA, Oberste MS, Alexander JP Jr, Kennett ML, Pallansch MA. Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998. J Virol. 1999;73:9969-75.

24. Merovitz L, Demers AM, Newby D, McDonald J. Enterovirus 71 infections at a Canadian center. Pediatr Infect Dis J. 2000;19:755-7.

25. Ortner B, Huang CW, Schmid D, Mutz I, Wewalka G, Allerberger F, et al. Epidemiology of enterovirus types causing neurological disease in Austria 1999-2007: detection of clusters of echovirus 30 and enterovirus 71 and analysis of prevalent genotypes. J Med Virol. 2009;81:317-24.

26. Bible JM, Iturriza-Gomara M, Megson B, Brown D, Pantelidis P, Earl P, et al. Molecular epidemiology of human enterovirus 71 in the United Kingdom from 1998 to 2006. J Clin Microbiol. 2008;46:3192-200.

27. van der Sanden S, van Eek J, Martin DP, van der Avoort H, Vennema H, Koopmans M. Detection of recombination breakpoints in the genomes of human enterovirus 71 strains isolated in the Netherlands in epidemic and non-epidemic years, 1963-2010. Infect Genet Evol. 2011;11:886-94.

28. Witsø E, Palacios G, Rønningen KS, Cinek O, Janowitz D, Rewers M, et al. Asymptomatic circulation of HEV71 in Norway. Virus Res. 2007;123:19-29.

29. Schuffenecker I, Mirand A, Antona D, Henquell C, Chomel JJ, Archimbaud C, et al. Epidemiology of human enterovirus 71 infections in France, 2000-2009. J Clin Virol. 2011;50:50-6.

30. Chakraborty R, Iturriza-Gómara M, Musoke R, Palakudy T, D’Agostino A, Gray J. An epidemic of enterovirus 71 infection among HIV-1-infected orphans in Nairobi. AIDS. 2004;18:1968-70.^-3131. Huaman JL, Carrion G, Ampuero JS, Ocaña V, Laguna-Torres VA, Hontz RD. Enterovirus-71 genotype C isolated in Peru between 2006 and 2009. J Clin Virol. 2016;85:40-3. . From 1963 to 1986, strains of EV-A71 belonging to subgenogoup B0, B1 and B2 were isolated in the Netherlands ²⁷27. van der Sanden S, van Eek J, Martin DP, van der Avoort H, Vennema H, Koopmans M. Detection of recombination breakpoints in the genomes of human enterovirus 71 strains isolated in the Netherlands in epidemic and non-epidemic years, 1963-2010. Infect Genet Evol. 2011;11:886-94. . In contrast, after 1987, genogroup B was replaced by genogroup C, lineages C1 and C2 ²³23. Brown BA, Oberste MS, Alexander JP Jr, Kennett ML, Pallansch MA. Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998. J Virol. 1999;73:9969-75.

24. Merovitz L, Demers AM, Newby D, McDonald J. Enterovirus 71 infections at a Canadian center. Pediatr Infect Dis J. 2000;19:755-7.

25. Ortner B, Huang CW, Schmid D, Mutz I, Wewalka G, Allerberger F, et al. Epidemiology of enterovirus types causing neurological disease in Austria 1999-2007: detection of clusters of echovirus 30 and enterovirus 71 and analysis of prevalent genotypes. J Med Virol. 2009;81:317-24.

26. Bible JM, Iturriza-Gomara M, Megson B, Brown D, Pantelidis P, Earl P, et al. Molecular epidemiology of human enterovirus 71 in the United Kingdom from 1998 to 2006. J Clin Microbiol. 2008;46:3192-200.

27. van der Sanden S, van Eek J, Martin DP, van der Avoort H, Vennema H, Koopmans M. Detection of recombination breakpoints in the genomes of human enterovirus 71 strains isolated in the Netherlands in epidemic and non-epidemic years, 1963-2010. Infect Genet Evol. 2011;11:886-94.

28. Witsø E, Palacios G, Rønningen KS, Cinek O, Janowitz D, Rewers M, et al. Asymptomatic circulation of HEV71 in Norway. Virus Res. 2007;123:19-29.

29. Schuffenecker I, Mirand A, Antona D, Henquell C, Chomel JJ, Archimbaud C, et al. Epidemiology of human enterovirus 71 infections in France, 2000-2009. J Clin Virol. 2011;50:50-6.^-3030. Chakraborty R, Iturriza-Gómara M, Musoke R, Palakudy T, D’Agostino A, Gray J. An epidemic of enterovirus 71 infection among HIV-1-infected orphans in Nairobi. AIDS. 2004;18:1968-70. . The emergence of C2 was associated with a peak in hospitalizations in 2007. According to an epidemiological study, the EV-A71 subgenogroups B1, B2, C1 and C2 circulated simultaneously in Europe, Australia and the USA ²¹21. Chumakov M, Voroshilova M, Shindarov L, Lavrova I, Gracheva L, Koroleva G, et al. Enterovirus 71 isolated from cases of epidemic poliomyelitis-like disease in Bulgaria. Arch Virol. 1979;60:329-40.

22. Nagy G, Takátsy S, Kukán E, Mihály I, Dömök I. Virological diagnosis of enterovirus type 71 infections: experiences gained during an epidemic of acute CNS disease in Hungary in 1978. Arch Virol. 1982;71:217-27.

23. Brown BA, Oberste MS, Alexander JP Jr, Kennett ML, Pallansch MA. Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998. J Virol. 1999;73:9969-75.

24. Merovitz L, Demers AM, Newby D, McDonald J. Enterovirus 71 infections at a Canadian center. Pediatr Infect Dis J. 2000;19:755-7.

25. Ortner B, Huang CW, Schmid D, Mutz I, Wewalka G, Allerberger F, et al. Epidemiology of enterovirus types causing neurological disease in Austria 1999-2007: detection of clusters of echovirus 30 and enterovirus 71 and analysis of prevalent genotypes. J Med Virol. 2009;81:317-24.

26. Bible JM, Iturriza-Gomara M, Megson B, Brown D, Pantelidis P, Earl P, et al. Molecular epidemiology of human enterovirus 71 in the United Kingdom from 1998 to 2006. J Clin Microbiol. 2008;46:3192-200.

27. van der Sanden S, van Eek J, Martin DP, van der Avoort H, Vennema H, Koopmans M. Detection of recombination breakpoints in the genomes of human enterovirus 71 strains isolated in the Netherlands in epidemic and non-epidemic years, 1963-2010. Infect Genet Evol. 2011;11:886-94.

28. Witsø E, Palacios G, Rønningen KS, Cinek O, Janowitz D, Rewers M, et al. Asymptomatic circulation of HEV71 in Norway. Virus Res. 2007;123:19-29.^-2929. Schuffenecker I, Mirand A, Antona D, Henquell C, Chomel JJ, Archimbaud C, et al. Epidemiology of human enterovirus 71 infections in France, 2000-2009. J Clin Virol. 2011;50:50-6. . B3-B5, C4and C5, which have caused large epidemics in the Asia-Pacific region since 1997, have not been observed outside the Asia-Pacific region ( Table 2 ).

Comparing the epidemics that have occurred over the past four decades, the epidemiologic features of EV-A71 infections appear to be changing. EV-A71 infection has emerged as an important public health problem around the world. No association has been established between genotype and disease severity ³²32. Yip CC, Lau SK, Woo PC, Yuen KY. Human enterovirus 71 epidemics: what’s next? Emerg Health Threats J. 2013;6:19780. . From 1900 to 2016, infections were successively caused by viruses of subgenogroup B0, B1and B2, followed by a shift in this predominance to those belonging to subgenogroups C1, C2, C3, C4a and C4b. A molecular epidemiological study suggested that the evolution of the EV-A71 virus has global characteristics. Global herd immunity against C1 and C2 viruses could possibly explain why epidemics caused by subgenogroups B4 and C4 are restricted to the Asia-Pacific region ²⁷27. van der Sanden S, van Eek J, Martin DP, van der Avoort H, Vennema H, Koopmans M. Detection of recombination breakpoints in the genomes of human enterovirus 71 strains isolated in the Netherlands in epidemic and non-epidemic years, 1963-2010. Infect Genet Evol. 2011;11:886-94. . Subgenogroup B5 has been reported to be antigenically distinct from B1, B4, C2 and C4 ¹⁷17. Huang SW, Hsu YW, Smith DJ, Kiang D, Tsai HP, Lin KH, et al. Reemergence of enterovirus 71 in 2008 in Taiwan: dynamics of genetic and antigenic evolution from 1998 to 2008. J Clin Microbiol. 2009;47:3653-62. and could therefore pose a potential risk for epidemic spread outside the Asia region.

Pathogenesis

The clinical spectrum of EV-A71 infection presentations is quite wide, including skin eruptions, internal organs and neurological manifestations and even death ¹1. Yi EJ, Shin YJ, Kim JH, Kim TG, Chang SY. Enterovirus 71 infection and vaccines. Clin Exp Vaccine Res. 2017;6:4-14.^,22. Chen KT, Chang HL, Wang ST, Cheng YT, Yang JY. Epidemiologic features of hand-foot-mouth disease and herpangina caused by enterovirus 71 in Taiwan, 1998-2005. Pediatrics. 2007;120:e244-52. .

EV-A71 is a highly neurotropic virus ³³33. Chang LY, Lin TY, Hsu KH, Huang YC, Lin KL, Hsueh C, et al. Clinical features and risk factors of pulmonary oedema after enterovirus 71-related hand, foot, and mouth disease. Lancet. 1999;354:1682-6. . The most common internal organ involved in EV-A71 infections is the brain stem ³³33. Chang LY, Lin TY, Hsu KH, Huang YC, Lin KL, Hsueh C, et al. Clinical features and risk factors of pulmonary oedema after enterovirus 71-related hand, foot, and mouth disease. Lancet. 1999;354:1682-6. . According to data derived from a study developed in mice, the strong neurotropism of EV-A71 and its retrograde axonal transport in neurons could underlie its major transmission route ³⁴34. Chen CS, Yao YC, Lin SC, Lee YP, Wang YF, Wang JR, et al. Retrograde axonal transport: a major transmission route of enterovirus 71 in mice. J Virol. 2007;81:8996-9003. . In previous studies ³⁴34. Chen CS, Yao YC, Lin SC, Lee YP, Wang YF, Wang JR, et al. Retrograde axonal transport: a major transmission route of enterovirus 71 in mice. J Virol. 2007;81:8996-9003.^,3535. Cordey S, Petty TJ, Schibler M, Martinez Y, Gerlach D, van Belle S, et al. Identification of site-specific adaptations conferring increased neural cell tropism during human enterovirus 71 infection. PLoS Pathog. 2012;8:e1002826. in which mice were infected via oral and parenteral routes with a murine-adapted virus strain that originated from a fatal human case, the EV-A71 virus entered the CNS via peripheral motor nerves after skeletal muscle infection and continued to spread within the CNS through motor and other neuronal pathways. Inflammation was the most profound in the spinal cord gray matter, brainstem, hypothalamus, subthalamic and dentate nuclei in autopsy samples investigated in Malaysia ³⁵35. Cordey S, Petty TJ, Schibler M, Martinez Y, Gerlach D, van Belle S, et al. Identification of site-specific adaptations conferring increased neural cell tropism during human enterovirus 71 infection. PLoS Pathog. 2012;8:e1002826. . Previous studies ³⁶36. Huang HI, Weng KF, Shih SR. Viral and host factors that contribute to pathogenicity of enterovirus 71. Future Microbiol. 2012;7:467-79.^,3737. Zhao T, Zhang Z, Zhang Y, Feng M, Fan S, Wang L, et al. Dynamic interaction of enterovirus 71 and dendritic cells in infected neonatal rhesus macaques. Front Cell Infect Microbiol. 2017;7:171. have found that the EV-A71 virus mechanism of infection is primarily focused on the respiratory tract epithelium, from which it is subsequently able to enter a pre-existing population of dendritic cells at the infection site; these cells could potentially transmit the virus from local sites to other organs through the blood stream during the infectious process.

In a previous study ³⁸38. Liu K, Zhou Y, Cui S, Song J, Ye P, Xiang W, et al. Clinical value of dorsal medulla oblongata involvement detected with conventional MRI for prediction of outcome in children with enterovirus 71-related brainstem encephalitis. Pediatr Infect Dis J. 2018 Apr 5. [Epub ahead of print]. , a total of 46 children with EV-A71-related brainstem encephalitis (EBE) were enrolled and subsequently underwent 1.5 Tesla magnetic resonance (MR) examinations of the brain. Among these 46 children, 35 had MR images evidencing dorsal medulla oblongata involvement, 32 had evidence of pons involvement, 10 had evidence of midbrain involvement and 7 had evidence of dentate nucleus involvement. Patients with dorsal medulla oblongata involvement or multiple areas involvement were significantly more likely to have poorer outcomes than patients without these features.

Additional noteworthy findings include clinical manifestations of viremia. Viremia reportedly occurs more frequently in children one year of age or younger ¹⁷17. Huang SW, Hsu YW, Smith DJ, Kiang D, Tsai HP, Lin KH, et al. Reemergence of enterovirus 71 in 2008 in Taiwan: dynamics of genetic and antigenic evolution from 1998 to 2008. J Clin Microbiol. 2009;47:3653-62.^,3939. Cheng HY, Huang YC, Yen TY, Hsia SH, Hsieh YC, Li CC, et al. The correlation between the presence of viremia and clinical severity in patients with enterovirus 71 infection: a multi-center cohort study. BMC Infect Dis. 2014;14:417. . Most patients with viremia do not show severe clinical manifestations of EV-A71 infection. In addition, the occurrence of CNS involvement is not reportedly different between patients with and without viremia ³⁹39. Cheng HY, Huang YC, Yen TY, Hsia SH, Hsieh YC, Li CC, et al. The correlation between the presence of viremia and clinical severity in patients with enterovirus 71 infection: a multi-center cohort study. BMC Infect Dis. 2014;14:417. .

What might cause severe or fatal clinical presentations of EV-A71 infection? Based on the results of one autopsy, EV-A71 infection can lead to severe or fatal disease due to pulmonary edema. The mechanism of pulmonary edema among patients with severe EV-A71 infection is currently unclear. Evidences ⁴⁰40. Hsia SH, Wu CT, Chang JJ, Lin TY, Chung HT, Lin KL, et al. Predictors of unfavorable outcomes in enterovirus 71-related cardiopulmonary failure in children. Pediatr Infect Dis J. 2005;24:331-4. suggest that the brainstem involvement characteristic of EV-A71 infection may be an important etiological mechanism of neurogenic pulmonary edema. Virus-host interactions significantly influence viral replication, virulence and pathogenicity during the viral life cycle ⁴¹41. Zhang B, Wu X, Huang K, Li L, Zheng L, Wan C, et al. The variations of VP1 protein might be associated with nervous system symptoms caused by enterovirus 71 infection. BMC Infect Dis. 2014;14:243. . However, tissue-specific virulence is still not well understood in both, cell-based assays and animal models, therefore further studies are needed.

Vaccine development

Close person-to-person contact is considered the most common route of EV-A71 transmission. In areas where EV-A71 circulation is present, especially because the clinical manifestations of the majority of EV-A71 infections are mild or asymptomatic, public health interventions, such as the promotion of hand washing, are necessary for the effective prevention of EV-A71 infection. Because effective treatment may not always be available, the best way to control and eradicate EV-A71 infection is to develop an effective vaccine. Several EV-A71 vaccines have been produced ¹1. Yi EJ, Shin YJ, Kim JH, Kim TG, Chang SY. Enterovirus 71 infection and vaccines. Clin Exp Vaccine Res. 2017;6:4-14. , including a subunit vaccine, a virus-like particle vaccine, DNA vaccines, a live-attenuated vaccine and an inactivated virus vaccine. The potential vaccines targeting EV-A71 are discussed in greater detail below.

Subunit vaccine

Currently, there are no effective antiviral drugs or vaccines for prevention of EV-A71 infection. A previous study has suggested the potential of the VP1 protein as a candidate antigen for an EV-A71 vaccine ⁴²42. Wu CN, Lin YC, Fann C, Liao NS, Shih SR, Ho MS. Protection against lethal enterovirus 71 infection in newborn mice by passive immunization with subunit VP1 vaccines and inactivated virus. Vaccine. 2001;20:895-904.^,4343. Wang M, Jiang S, Wang Y. Recombinant VP1 protein expressed in Pichia pastoris induces protective immune responses against EV71 in mice. Biochem Biophys Res Commun 2013;430:387-93. . A recombinant VP1 protein of the EV-A71 virus has been produced in Escherichia coli , yeast and the baculovirus system ⁴²42. Wu CN, Lin YC, Fann C, Liao NS, Shih SR, Ho MS. Protection against lethal enterovirus 71 infection in newborn mice by passive immunization with subunit VP1 vaccines and inactivated virus. Vaccine. 2001;20:895-904.

43. Wang M, Jiang S, Wang Y. Recombinant VP1 protein expressed in Pichia pastoris induces protective immune responses against EV71 in mice. Biochem Biophys Res Commun 2013;430:387-93.^-4444. Premanand B, Kiener TK, Meng T, Tan YR, Jia Q, Chow VT, et al. Induction of protective immune responses against EV71 in mice by baculovirus encoding a novel expression cassette for capsid protein VP1. Antiviral Res 2012;95:311-5. . Vaccination with a VP1 protein vaccine can induce neutralizing antibodies to protect against EV-A71 infection ⁴²42. Wu CN, Lin YC, Fann C, Liao NS, Shih SR, Ho MS. Protection against lethal enterovirus 71 infection in newborn mice by passive immunization with subunit VP1 vaccines and inactivated virus. Vaccine. 2001;20:895-904.

43. Wang M, Jiang S, Wang Y. Recombinant VP1 protein expressed in Pichia pastoris induces protective immune responses against EV71 in mice. Biochem Biophys Res Commun 2013;430:387-93.^-4444. Premanand B, Kiener TK, Meng T, Tan YR, Jia Q, Chow VT, et al. Induction of protective immune responses against EV71 in mice by baculovirus encoding a novel expression cassette for capsid protein VP1. Antiviral Res 2012;95:311-5. . However, another study showed that vaccination with a recombinant VP1 induced a lower titer of EV-A71-specific IgG antibodies than inoculation with the inactivated virus ⁴²42. Wu CN, Lin YC, Fann C, Liao NS, Shih SR, Ho MS. Protection against lethal enterovirus 71 infection in newborn mice by passive immunization with subunit VP1 vaccines and inactivated virus. Vaccine. 2001;20:895-904.^,4343. Wang M, Jiang S, Wang Y. Recombinant VP1 protein expressed in Pichia pastoris induces protective immune responses against EV71 in mice. Biochem Biophys Res Commun 2013;430:387-93. . Although recombinant VP1 vaccine can elicit similar levels of neutralizing antibodies, it provided effective protection only at a low challenge dose of EV-A71 ⁴²42. Wu CN, Lin YC, Fann C, Liao NS, Shih SR, Ho MS. Protection against lethal enterovirus 71 infection in newborn mice by passive immunization with subunit VP1 vaccines and inactivated virus. Vaccine. 2001;20:895-904. .

In another study ⁴⁵45. Foo DG, Alonso S, Phoon MC, Ramachandran NP, Chow VT, Poh CL. Identification of neutralizing linear epitopes from the VP1 capsid protein of enterovirus 71 using synthetic peptides. Virus Res. 2007;125:61-8. , antiserum was produced in mice against overlapping synthetic peptides elicited by the VP1 capsid protein of EV-A71. Peptides SP55 (amino acids 163-177 of VP1) and SP70 (amino acids 208-222 of VP1) could elicit neutralizing antibodies against EV-A71 in vitro . SP70 was identified as particularly potent in eliciting a neutralizing antibody titer compared to that obtained with whole virion-immune serum. The amino acid residues of epitope SP70 are more conserved than the VP1 sequences of various subgenogroups of EV-A71. However, obtaining superior synthetic antigens requires the use of more effective adjuvants. Although the yeast-expressed VP1 protein provides good immunogenicity, VP1 subunit vaccines require further refinement to contribute significantly to an effective vaccine strategy ⁴³43. Wang M, Jiang S, Wang Y. Recombinant VP1 protein expressed in Pichia pastoris induces protective immune responses against EV71 in mice. Biochem Biophys Res Commun 2013;430:387-93. .

Virus-like particle vaccine

Virus-like particles (VLPs) for EV-A71 are similar in morphology to the natural viral capsid structure and have been developed as potential vaccines ⁴⁶46. Sun S, Gao F, Mao Q, Shao J, Jiang L, Liu D, et al. Immunogenicity and protective efficacy of an EV71 virus-like particle vaccine against lethal challenge in newborn mice. Hum Vaccin Immunother. 2015;11:2406-13. . Vaccination with an EV-A71 VLP vaccine showed significant potency against lethal challenge in newborn mice. However, an ideal enterovirus vaccine should be effective against both EV-A71 and Coxsackie virus-A16 (CV-A16) infections. Consequently, a bivalent enterovirus vaccine based on chimeric EV-A71 virus-like particles (ChiEV-A71 VLPs) has been proposed ⁴⁷47. Zhao H, Li HY, Han JF, Deng YQ, Zhu SY, Li XF, et al. Novel recombinant chimeric virus-like particle is immunogenic and protective against both enterovirus 71 and coxsackievirus A16 in mice. Sci Rep. 2015;5:7878. . In chimeric EV-A71 VLPs, the neutralizing epitope SP70 within the capsid protein VP1 of EV-A71 was replaced by CV-A16. Chimeric VLPs are able to elicit protective neutralizing antibodies against EV-A71 and CV-A16 in mice ⁴⁷47. Zhao H, Li HY, Han JF, Deng YQ, Zhu SY, Li XF, et al. Novel recombinant chimeric virus-like particle is immunogenic and protective against both enterovirus 71 and coxsackievirus A16 in mice. Sci Rep. 2015;5:7878. . Although studies have demonstrated that VLP vaccines can induce protective neutralizing antibodies and show cross-protective efficacy against different EV-A71 subtypes not present in other experimental vaccines, these vaccines have a lower efficacy than inactivated vaccines. They must also be further studied.

DNA vaccines

DNA vaccines and recombinant vector DNA vaccines have been studied in EV-A71 vaccine development. Several research groups have designed and constructed a DNA vaccine against EV-A71 infection using its viral capsid protein (VP1) gene in order to verify the functionality of this vaccine in vitro and/or in vivo⁴⁸48. Tung WS, Bakar SA, Sekawi Z, Rosli R. DNA vaccine constructs against enterovirus 71 elicit immune response in mice. Genet Vaccines Ther. 2007;5:6.

49. Chiu CH, Chu C, He CC, Lin TY. Protection of neonatal mice from lethal enterovirus 71 infection by maternal immunization with attenuated Salmonella enterica serovar Typhimurium expressing VP1 of enterovirus 71. Microbes Infect. 2006;8:1671-8.^-5050. Tsou YL, Lin YW, Shao HY, Yu SL, Wu SR, Lin HY, et al. Recombinant adeno-vaccine expressing enterovirus 71-like particles against hand, foot, and mouth disease. PLoS Negl Trop Dis. 2015;9:e0003692. . One study showed that vaccination with an EV-A71 DNA vaccine could elicit VP1-specific IgG titers and neutralizing antibodies against EV-A71, although it resulted only in low levels of antigenicity ⁴⁸48. Tung WS, Bakar SA, Sekawi Z, Rosli R. DNA vaccine constructs against enterovirus 71 elicit immune response in mice. Genet Vaccines Ther. 2007;5:6. . Another study by Chiu et al . ⁴⁹49. Chiu CH, Chu C, He CC, Lin TY. Protection of neonatal mice from lethal enterovirus 71 infection by maternal immunization with attenuated Salmonella enterica serovar Typhimurium expressing VP1 of enterovirus 71. Microbes Infect. 2006;8:1671-8. investigated the potential use of attenuated Salmonella enterica serovar Typhimurium strains to express and deliver VP1 of EV-A71 as a vaccine to prevent EV-A71 infections in mice. They showed that offspring born to female mice immunized with Salmonella -based VP1 vaccine had higher survival rates (50-60%) than offspring of unvaccinated control mice (0%) ⁴⁹49. Chiu CH, Chu C, He CC, Lin TY. Protection of neonatal mice from lethal enterovirus 71 infection by maternal immunization with attenuated Salmonella enterica serovar Typhimurium expressing VP1 of enterovirus 71. Microbes Infect. 2006;8:1671-8. . Another study indicated that recombinant adenoviruses, expressing EV-A71 P1 and 3CD genes, could elicit production of neutralizing antibodies and protect against EV-A71 infection, both useful characteristics for the prevention of EV-A71 infections ⁵⁰50. Tsou YL, Lin YW, Shao HY, Yu SL, Wu SR, Lin HY, et al. Recombinant adeno-vaccine expressing enterovirus 71-like particles against hand, foot, and mouth disease. PLoS Negl Trop Dis. 2015;9:e0003692. . Although promising preliminar results have been shown, further investigation on the immunogenicity of this potential EV-A71 DNA vaccine is needed.

Live-attenuated virus vaccines

Cynomolgus monkeys inoculated with an attenuated EV-A71 vaccine showed mild neurological symptoms but survived lethal challenge by virulent EV-A71 (BrCr-TR) without exacerbation of symptoms ⁵¹51. Arita M, Nagata N, Iwata N, Ami Y, Suzaki Y, Mizuta K, et al. An attenuated strain of enterovirus 71 belonging to genotype a showed a broad spectrum of antigenicity with attenuated neurovirulence in cynomolgus monkeys. J Virol. 2007;81:9386-95. . Although this study indicated that the monkeys’ immunization with attenuated EV-A71 vaccine had the potential to produce significant titers of neutralized antibodies against different genogroups of EV-A71, including A, B1, B4, C2 and C4 ⁵¹51. Arita M, Nagata N, Iwata N, Ami Y, Suzaki Y, Mizuta K, et al. An attenuated strain of enterovirus 71 belonging to genotype a showed a broad spectrum of antigenicity with attenuated neurovirulence in cynomolgus monkeys. J Virol. 2007;81:9386-95. , several safety issues concerning live-attenuated virus vaccines need to be overcome. For example, immunization with the attenuated strain might cause mild neurological symptoms when inoculated via the intravenous route. However, a high-fidelity variant of EV-A71 exhibited an attenuated phenotype and showed an intriguing potential as a live-attenuated EV-A71 vaccine ⁵²52. Meng T, Kwang J. Attenuation of human enterovirus 71 high-replication-fidelity variants in AG129 mice. J Virol. 2014;88:5803-15. .

Inactivated virus vaccines

Compared to the various vaccine candidates, inactivated EV-A71 vaccines are more capable of fulfilling the demand for prevention of EV-A71 infections. Inspired by previous developments of inactivated vaccines, the development of inactivated EV-A71 vaccines has progressed rapidly in recent decades.

It has been demonstrated in mice that immunization with a formalin-inactivated EV-A71 vaccine can elicit high titers of EV-A71 virus-specific neutralizing antibodies, conferring protection against an EV-A71 lethal challenge ⁵³53. Ong KC, Devi S, Cardosa MJ, Wong KT. Formaldehyde-inactivated whole-virus vaccine protects a murine model of enterovirus 71 encephalomyelitis against disease. J Virol. 2010;84:661-5. . After successful preclinical experiments, phase I and phase II clinical trials were performed to determine the efficacy of inactivated EV-A71 candidate vaccines. Immunization with inactivated EV-A71 vaccine elicited high titers of neutralizing antibody and induced specific T-cell reactions in the study population, with no significant inflammatory reaction reported ⁵⁴54. Liu L, Zhang Y, Wang J, Zhao H, Jiang L, Che Y, et al. Study of the integrated immune response induced by an inactivated EV71 vaccine. PLoS One. 2013;8:e54451. . In addition, another study indicated that inactivated EV-A71 vaccine candidates can elicit cross-neutralizing antibody responses against EV-A71 subgenogroups B1, B4, B5 and C4A ⁵⁵55. Chou AH, Liu CC, Chang JY, Jiang R, Hsieh YC, Tsao A, et al. Formalin-inactivated EV71 vaccine candidate induced cross-neutralizing antibody against subgenotypes B1, B4, B5 and C4A in adult volunteers. PLoS One. 2013;8:e79783. . Due to their stability, the research and development of inactivated EV-A71 vaccines has progressed more rapidly compared to other types of vaccines. In recent years, at least five inactivated EV-A71 vaccine candidates have been developed and advanced to clinical trials. All EV-A71 vaccines developed in China are inactivated whole-virus alum-adjuvant vaccines^1. All of them use the C4 subgenogroup virus as the vaccine strain ¹1. Yi EJ, Shin YJ, Kim JH, Kim TG, Chang SY. Enterovirus 71 infection and vaccines. Clin Exp Vaccine Res. 2017;6:4-14. . Randomized, double-blind, placebo-controlled trials of EV-A71 vaccines have been completed ⁵⁶56. Zhu FC, Meng FY, Li JX, Li XL, Mao QY, Tao H, et al. Efficacy, safety, and immunology of an inactivated alum-adjuvant enterovirus 71 vaccine in children in China: a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial. Lancet. 2013;381:2024-32. . More than 30,000 infants and children have been involved in Phase III clinical trials of inactivated EV-A71 C4 vaccines in China ⁵⁶56. Zhu FC, Meng FY, Li JX, Li XL, Mao QY, Tao H, et al. Efficacy, safety, and immunology of an inactivated alum-adjuvant enterovirus 71 vaccine in children in China: a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial. Lancet. 2013;381:2024-32. . These studies have shown that the EV-A71 vaccines can prevent more than 90% of EV-A71-associated HFMD or herpangina and 80% of other EV-A71-associated disease symptoms. It has been reported that the seroconversion rate is 100% after two vaccinations, and all C4-based vaccines prevented EV-A71-associated hospitalizations. Furthermore, preexisting antibodies due to undetected subclinical infections in young children did not interfere with vaccine efficacy against different EV-A71 genogroups ⁵⁷57. Mao Q, Cheng T, Zhu F, Li J, Wang Y, Li Y, et al. The cross-neutralizing activity of enterovirus 71 subgenotype c4 vaccines in healthy Chinese infants and children. PLoS One. 2013;8:e79599. . However, the inactivated EV-A71 vaccines did not protect against CV-A16 ¹1. Yi EJ, Shin YJ, Kim JH, Kim TG, Chang SY. Enterovirus 71 infection and vaccines. Clin Exp Vaccine Res. 2017;6:4-14.^,5757. Mao Q, Cheng T, Zhu F, Li J, Wang Y, Li Y, et al. The cross-neutralizing activity of enterovirus 71 subgenotype c4 vaccines in healthy Chinese infants and children. PLoS One. 2013;8:e79599. .

Indurations, erythema, and pain at the injection site have been the most common side effects of EV-A71 vaccination; high fever has also occasionally been observed ⁵⁸58. Zhu F, Xu W, Xia J, Liang Z, Liu Y, Zhang X, et al. Efficacy, safety, and immunogenicity of an enterovirus 71 vaccine in China. N Engl J Med. 2014;370:818-28. . The rate of rare serious adverse events (SAEs) in vaccinated groups was not significantly different from that observed in control groups, and SAEs were not causally related to vaccination ⁵⁸58. Zhu F, Xu W, Xia J, Liang Z, Liu Y, Zhang X, et al. Efficacy, safety, and immunogenicity of an enterovirus 71 vaccine in China. N Engl J Med. 2014;370:818-28. .

An estimation of the cost-effectiveness of a national EV-A71 vaccination has been performed in China ⁵⁹59. Wu JT, Jit M, Zheng Y, Leung K, Xing W, Yang J, et al. Routine pediatric enterovirus 71 vaccination in China: a cost-effectiveness analysis. PLoS Med. 2016;13:e1001975. . It was shown that the vaccination would be cost-saving or cost-effective due to prevention of EV-A71-related morbidity, mortality and use of health services among children aged five or younger, compared to anticipated costs with no vaccination. This finding bodes well for the introduction of a safe and effective EV-A71 vaccine in the near future. In December 2015, the Food and Drug Administration (FDA) of China approved an EV-A71-targeting vaccine ⁵⁹59. Wu JT, Jit M, Zheng Y, Leung K, Xing W, Yang J, et al. Routine pediatric enterovirus 71 vaccination in China: a cost-effectiveness analysis. PLoS Med. 2016;13:e1001975. . In China, inactivated EV-A71 vaccines are now commercially available products.

An inactivated EV-A71 vaccine still faces one major challenge ⁶⁰60. Lee MS, Tseng FC, Wang JR, Chi CY, Chong P, Su IJ. Challenges to licensure of enterovirus 71 vaccines. PLoS Negl Trop Dis. 2012;6:e1737. . Although both, C4-based and B4-based antibodies cross-neutralize the current circulating EV-A71 isolates ²2. Chen KT, Chang HL, Wang ST, Cheng YT, Yang JY. Epidemiologic features of hand-foot-mouth disease and herpangina caused by enterovirus 71 in Taiwan, 1998-2005. Pediatrics. 2007;120:e244-52. , the B4 vaccine poorly neutralizes an atypical C2 strain. In addition, no formalin-inactivated EV-A71 vaccines developed to date protect against CV-A16, which is a primary cause of annual HFMD outbreaks. Additionally, the humoral immunity associated with protection initially observed, appears to wane after the first 6 months of vaccination. However, inactivated EV-A71 vaccines have significant safety advantages over live-attenuated ones because of their inability to replicate.

CONCLUSIONS

EV-A71 is currently an important, threatening infectious agent in the world. It can cause severe neurological disorders and death in infected young children. Due to the lack of effective control measures, the development of effective vaccines is urgently needed for prevention and control of EV-A71 infections. Currently, several candidates for an EV-A71 vaccine exist, including a formalin-inactivated whole virus vaccine, DNA vaccines, VLP and recombinant protein vaccines. Monovalent EV-A71 vaccines are expected to be marketed soon in mainland China, if the process of production goes well. It has been estimated that EV-A71 vaccines will be effective and cost-saving in most developing countries ⁵⁹59. Wu JT, Jit M, Zheng Y, Leung K, Xing W, Yang J, et al. Routine pediatric enterovirus 71 vaccination in China: a cost-effectiveness analysis. PLoS Med. 2016;13:e1001975. . Several promising inactivated EV-A71 vaccines have been developed in the last few years; however, they have certain limitations. Currently, inactivated EV-A71 vaccines can protect against EV-A71 but not against CV-A16 infections. All of these EV-A71 vaccines remain in the initial stages of development. Stability, purity and cost of production are the primary future challenges for these vaccines.

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