Abstract
Coxsackievirus 16 (CA16) is one of the major pathogens associated with hand, foot, and mouth disease (HFMD) in infants and young children. In recent years, CA16 and human enterovirus 71 (EV71) have often circulated alternatively or together in the Western Pacific region, which has become an important public health problem in this region. HFMD caused by CA16 infection is generally thought to be mild and self-limiting. However, recently several severe and fatal cases involving CA16 have been reported. Studies have shown that co-infection with CA16 and EV71 can cause serious complications in the central nervous system (CNS) and increase the chance of genetic recombination, which may be responsible for the large HFMD outbreak in Mainland China in 2008. For these reasons, recent studies have focused on the virological characteristics of CA16 and the development of CA16-related diagnostic reagents and vaccines.
Introduction
CA16 was first isolated in South Africa in 1951.Citation1 It is a member of Human enterovirus A (HEV-A) species of the Enterovirus genus of Picornaviridae. CA16 is a small (diameter ~30 nm), non-enveloped, icosahedral particle that contains a single-stranded, positive-sense, polyadenylated viral RNA genome of approximately 7.4 kb. The genome contains one reading frame encoding a large polyprotein precursor, which is subsequently processed into structural protein P1 and nonstructural proteins P2 and P3. P1 can be processed by a virus-encoded proteinase, which results in viral capsid subunit proteins VP0, VP1, and VP3.Citation2,Citation3 VP0 can be cleaved further to yield VP2 and VP4. VP1, VP2, and VP3 lie on the outer part of the capsid while VP4 is situated on the inner part. The neutralization epitopes mainly reside on VP1.Citation4 The coding region is flanked by 5′- and 3′-non-coding regions. The 5′- non-coding region is comprised of ~740 nucleotides and contains sequences that control genome replication and translation, such as the internal ribosome entry site (IRES). The 3′- non-coding region contains a polyA tail that is essential for virus infectivity.
Both CA16 and EV71 are the major pathogens responsible for HFMD. While CA16 infection is generally thought to cause mild symptoms, such as blisters/ulcers on the hands and feet and in the mouth as well as pharyngitis in infants and children under five years old, a small number of patients also develop aseptic meningitis, encephalitis and even fatal myocarditis and pneumonia.Citation5-Citation7 In recent years, HFMD has been epidemical in the world, especially in the Western Pacific region. The first outbreak of HFMD caused by CA16 was described in Toronto in 1957.Citation8 CA16 infection was responsible for HFMD outbreaks in Sydney, Australia in 1991,Citation9 in England and Wales in 1994,Citation10 in Taiwan in 2002–2003,Citation11 in Singapore in 2002, 2005 and 2007,Citation12 in Vietnam in 2005,Citation13 and in Odisha, India in 2009.Citation14 In Mainland China, CA16 was the predominant pathogen causing HFMD in 2007 in BeijingCitation15 and in 2009 in Guangzhou.Citation3 Severe and fatal cases of HFMD have been mainly caused by EV71 infection; thus, studies have focused on EV71. Phase III clinical trials of EV71 inactivated vaccines have been completed, confirming their safety and protective effects.Citation16-Citation18 Although CA16 infections usually cause mild symptoms, CA16 infection caused severe and fatal HFMD cases reported in the United States,Citation19 France,Citation7 Japan,Citation5 Mainland China,Citation20 and Taiwan.Citation6 Among the 92 HFMD cases presenting with neurological symptoms in Shenyang, China, 19 were caused by CA16 infection, with 2 patients presenting with brainstem encephalitis and one with acute flaccid paralysis.Citation21 Currently-circulating CA16 genotype B might have arisen from recombination of CA16 genotype A (prototype, G10) with EV71 and CA4.Citation22 Studies have shown that humans can be co-infected with CA16 and EV71,Citation23,Citation24 and co-infection might increase the possibility of genetic recombination between CA16 and EV71.Citation25,Citation26 This phenomenon might account for the HFMD outbreak in Mainland China in 2008.Citation26 Vaccines are the most efficient measure to control HFMD epidemics. Recent studies indicated that anti-CA16 sera from animals immunized with virus-like particles (VLP) and inactivated whole virus can neutralize CA16 strains both in vivo and in vitro, and can also protect neonatal or mice against CA16 challenge.Citation27,Citation28 These results indicate the feasibility of developing a CA16 monovalent vaccine and an EV71-CA16 bivalent vaccine.
Epidemiology
HFMD outbreaks caused by CA16
In 1994 the largest HFMD outbreak in England and Wales was caused by CA16 (953 out of 614 303 cases).Citation10 Similarly, the predominant etiological agent of HFMD from 1999 to 2006 in Taiwan was also CA16 (2579 cases), followed by EV71 (1760 cases).Citation29 From 2001 to 2007, surveillance data in Singapore showed that the predominant circulating virus causing HFMD was CA16 for three epidemic years (2002, 2005, and 2007) and was EV71 for only 1 y (2006).Citation12 Tu PV et al. reported that there were 411 HFMD cases in Vietnam in 2005, among which 214 were identified as CA16-induced (52%) while 173 were EV71 (42%).Citation13 It has also been reported that an outbreak of HFMD in Odisha, India in 2009 was caused by CA16 (78 cases).Citation14 In recent years, HFMD has consistently reached epidemic levels in Mainland China.Citation30 The number of reported HFMD cases in Mainland China was 1 619 706 in 2011 and 2 168 737 in 2012, leading to 509 and 567 deaths, respectively. From January to June 2013, there were about 900 000 reported cases of HFMD, including 156 deaths.Citation31 The characteristics of these epidemics are that CA16 and EV71 were the main pathogens, circulating alternatively or together in different years.Citation12,Citation32-Citation35 The leading pathogen causing HFMD in Beijing in 2007 was CA16 (75%), though this changed to EV71 in 2008 and 2009 (56% and 36%).Citation15 In Guangzhou in 2008, the predominant pathogen inducing HFMD was EV71 (48.24%); in 2009 this became CA16 (59.26%), and was EV71 again in 2010 (42.28%)Citation3 ().
Table 1. HFMD outbreaks caused by CA16 in the world
Co-circulation of CA16 and EV71 has increased the possibility of co-infections and viral genetic recombination, which make it difficult to control HFMD epidemics. It was reported that genetic recombination might account for the 2008 outbreak of HFMD in Mainland China.Citation26 The rate of EV71 and CA16 co-infection for Hunan Province in 2008–2010, Hangzhou in 2009, Beijing in 2010 and Foshan in 2010 was 0.62%, 14.3%, 7.4%, and 9.3%, respectively.Citation36-Citation39 It has been shown that co-infection with CA16 and EV71 can readily cause serious CNS complications, leading to a worse condition and longer disease duration.Citation40
Molecular epidemiology of CA16
At present, a well-accepted genetic-based classification of CA16 has not been established. In some studies, CA16 has been grouped into genotypes A, B, and C on the basis of VP4 nucleotide sequence. The nucleotide variety was 20.4% -27.4% between genotype A and the other genotypes, while the nucleotide variety was 7.0% -18.6% between genotypes B and C.Citation33 However, additional CA16 phylogenetic analysis suggested that CA16 should be grouped into genotypes A and B based on VP1 nucleotide sequence. The nucleotide variety between genotype A and B was 27.5–30.2%. Genotype B can be further divided into B1a, B1b, B1c, B2a, B2b, and B2c.Citation41,Citation42 The nucleotide variety between genotype B1 and B2 was 11.8%.Citation41 Genotypes B1a and B1b were the predominant circulating genotypes in Australia from 1999 to 2006,Citation43 and genotype B circulated in Malaysia from 1997–2006.Citation44 Phylogenetic analysis of CA16 isolated from 6 provinces in Mainland China from 1995 to 2008 showed that these strains were mainly subtypes B1a and B1b. However, phylogenetic analysis of CA16 isolated from Shenzhen from 1997 to 2009 showed that these strains were mainly subtypes B2a and B2b. The different results obtained from these two studies in Mainland China might be due to the methods of genotyping employed.Citation41,Citation42 Therefore, more information on the molecular characterization of CA16 should be provided and a standard genotyping method basing on the VP1 nucleotide sequence should be established to properly study the molecular epidemiology of CA16.Citation42-Citation44
Evolutionary rate analysis was performed using the sequence of VP1, and it was found that the annual evolution rate of CA16 was 0.91 × 10−2 substitutions per synonymous nucleotide/year,Citation41 slightly lower than that of EV71 at 1.35 × 10−2.Citation45 A growing body of evidence supports the existence of recombination events involving EV71 and various HEV-A, including CA16 and CA8.Citation26,Citation46 Similar to EV71, recombination is likely to have occurred in CA16 strains circulating in China. Zhao et al. conducted a phylogenetic analysis of circulating CA16 strains in China. The results of their investigation showed that the circulating CA16 strains in China are complex recombinants of multiple HEV-A strains, including CA4 and EV71.Citation22 Similar to that study, we analyzed the complete sequences of CA16 strains isolated from China from 2008 to 2011 and found that these isolates had high similarity to the EV71 P2 and P3 regions and the CA4 5′UTR (unpublished data). More attention should be paid to the change of the recombination viral characteristics, including antigenicity and pathogenesis, which are important for controlling HFMD and developing a vaccine.
Different circulating strains of CA16 vary greatly with regards to their antigenicity and virulence.Citation47 Interestingly, it has been found that antisera from patients infected with the genotype B1 strain can neutralize genotype A but not B1 strains.Citation48 Similarly, our study has also shown that while a genotype A strain can be neutralized by antisera induced by genotype B strains, genotype B stains are difficult to be neutralized by antisera induced by genotype A or other genotype B strains (unpublished data). It is possible that mutations in the antigenic epitopes have occurred during their circulation.Citation48 However, recombination of the CA16 genotype B strain has not affected the degree of animal protection induced by vaccines. Recent studies of CA16 vaccines showed that immunization with an inactivated vaccine and a virus-like particle vaccine can induce neutralizing antibodies against heterologous and homologous CA16 strains and protect mice against lethal infection.Citation27,Citation28
Seroepidemiology of CA16
CA16 has only one serotype. At present there are limited data on CA16 seroepidemiology. Zhu et al. tested 900 blood samples collected in 2005 from 6 different geographical areas in China and found that 390 samples were positive for CA16 (43.3%; GMT 1:9.5) and 288 were positive for EV71 (32.0%; GMT 1: 8), suggesting that EV71 and CA16 had widely circulated in China before the large-scale outbreaks in 2008.Citation30 A follow up study of 975 infants under 3 y old found that 143 infants presented with HFMD during the follow-up period.Citation49 The titers of neutralizing antibodies against CA16 and EV71 increased by 4-fold in 90 cases (62.9%) and 99 cases (69.2%), respectively. Fifty-four cases (37.8%) showed an increase in neutralizing antibodies against both CA16 and EV71. Only 8 cases (0.6%) did not show any increase in neutralizing antibodies against CA16 or EV71. These results further point to the high prevalence of CA16 and EV71 in China in recent years ().
Table 2. Seroepidemiology of CA16 in the world
Compared with the Western Pacific Region, where HFMD outbreaks were associated with severe symptoms and death, there were fewer reported HFMD epidemics and severe cases in Europe and America. Interestingly, however, the positive rates for neutralizing antibodies against CA16 and EV71 among adult populations in China and Germany are similar. The anti-CA16 neutralizing antibody-positive rate was found to be 83.0% for the 40–59-y-old group,Citation50 and the anti-EV71 neutralizing antibody-positive rate was about 75% in GermanyCitation51. The positive rates for CA16 and EV71 neutralizing antibodies were 89.1% and 85.3%,Citation49 respectively, for pregnant women group in Jiangsu province, and 78.4% and 83.8%,Citation48 respectively, for adults above 20 y old in Ningbo city of China.
The risk of HFMD is especially high for the infant population. Maternal antibodies against CA16 and EV71 were found to decline to the lowest level (CA16: 26.4%, 1:7.3; EV71: 41.3%, 1:11.3) in 7-mo-old infants from Jiangsu Province, and then gradually increase to high levels (CA16: 54.3% and 1:24.3; EV71: 56.2% and 1:35.3) at 27–30 mo of age.Citation49 In Ningbo, the positive rate of neutralizing antibodies against CA16 and EV71 was 43.2% and 13.5%, respectively, in 1-y-old infants, and increased to 67.6% and 67.6%, respectively, in the 5-y-old group.Citation48 The positive rate in the 5-y-old group was similar to the adult level. These results are in agreement with the epidemiological characteristics of HFMD, especially given that most HFMD cases are children under 5 y old. Positive rates of neutralizing antibodies against CA16 and EV71 in Frankfurt, Germany, were 27.0% and 12.0%, respectively, in the 1–4-y-old group, and 52.0% and 49.0%, respectively, in the 5–9-y-old group.Citation50 The upward trend in the neutralizing antibody-positive rate in Germany is similar to that observed in China, though the age of the infected infants in Germany is older. These results suggested that infants in Germany were also at high risk of CA16 and EV71 infection. However, there are few reported HFMD outbreaks and cases with severe complications in Germany. Further studies are required to confirm whether the low incidence is because the seroprevalence of EV71 is lower in most age groups than that of CA16 in Germany, or because the pathogenicity of circulating CA16 and/or EV71 strains is different between Germany and China. The results of seroprevalence studies of CA16 and EV71 may be used to guide immunization programs of the vaccines.
Laboratory Diagnosis
Serological diagnosis
Serological diagnosis is the routine method of detecting enteroviruses, which includes neutralizing antibody detection and enzyme-linked immunosorbent assay (ELISA).
Neutralizing antibody detection
A neutralization assay based on the inhibition of cytopathic effects is the standard method recommended by the World Health Organization (WHO) for measuring the levels of neutralizing antibodies against polioviruses (WHO, 1997) and has been widely employed to measure the titers of neutralizing antibodies against CA16 and EV71.Citation52,Citation53 It was reported that the genotype A strain isolated in 1951 and genotype B strains were used in different labs.Citation30,Citation48,Citation49 Another study reported that the titers of neutralizing antibodies against the genotype A strain were higher than those against B genotype.Citation48 Because of the genetic and antigenic differences existing between genotype A and genotype B stains, whether or not the genotype A strain could be used as the representative strain for neutralization assays evaluating vaccines requires further investigation. However, the conventional neutralization assay is labor-intensive, subjective and time-consuming (6–8 d). It is not suitable for the mass screening of protective antibodies in seroepidemiological studies. A neutralization assay using pseudovirus has been established to detect antibodies against CA16, and appears to be suitable for the rapid detection of neutralizing antibodies against CA16 in clinical trials or seroepidemiological surveys.Citation54 However, it still needs to be compared with conventional assays to confirm the accuracy.
ELISA
Virus isolation and RT-PCR require expensive and specialized equipment and trained personnel. In contrast, ELISA for CA16 is rapid and convenient for testing large numbers of specimens.Citation55 Zhu, et al. reported that the detection efficiency of EV71 and CA16 was lower in severe HFMD cases by RT-PCR; thus, simultaneous detection of IgM antibodies needs to be considered.Citation56 However, human enterovirus genes have relatively high homogeneity and there may be some common antigenic sites among different enteroviruses. For example, the nucleotide and amino acid homology between CA16 and EV71 was found to be 77% and 89%, respectively.Citation57 Therefore, cross-reaction between enteroviruses may cause false-positive results, which is a key issue with the ELISA method. Xu et al. have developed an IgM-capture ELISA for detecting CA16 infection that displays high sensitivity and specificity.Citation58 The results showed that the antibody positive rate was 56.3% within the first day after onset, 95.3% at day 5 to day 7, and 100% more than 8 d post-onset. This method can be employed for the rapid identification of acute CA16 infections in clinics.
RT-PCR
Although virus isolation remains the “golden standard” for HFMD diagnosis, some studies have indicated that the positive rate for EV71 by virus culture is about 20% or even lower than that of RT-PCR.Citation59 RT-PCR assays have been developed that employ samples from nasopharyngeal swabs, vesicular fluid, or feces; the detection rate of the various assays ranges from 48.0% to 88.4%.Citation59-Citation62 Recently, many studies have focused on improving the sensitivity and specificity of RT-PCR methods of detecting CA16. For example, multiplex real-time RT-PCR and microarray methods have been developed.Citation63,Citation64 To ensure the comparability of results from different laboratories, the external quality assessment for EV71 and CA16 detection using RT-PCR has been established.Citation65 In addition, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay has been developed for sensitive and accurate detection of CA16 and EV71 infections.Citation66
CA16 Vaccines
Clinical trials for the inactivated EV71 whole virus vaccines developed by three manufacturers in Mainland China have been completed. The results demonstrated the good safety and protective effects of the vaccines.Citation16-Citation18 Success in the development of EV71 vaccines will provide valuable experience and insights for CA16 vaccines. The specific protective effect of neutralizing antibodies against CA16 observed indicates the feasibility of developing CA16 vaccine. The titer of neutralizing antibodies against CA16 is inversely correlated with HFMD incidence, and a similar phenomenon was also observed with EV71. Populations with low neutralizing antibody levels (infants) are susceptible to CA16-associated HFMD.Citation49 Sera from infected patients or immunized animals can block CA16 virus-induced cytopathic effects in cell cultures.Citation48 The in vivo active and passive immunization studies showed that neutralizing antibodies can protect animals from a lethal viral challenge.Citation27,Citation28,Citation67
Animal model
Sickles et al. discovered in 1951 that coxsackievirus A generally has strong pathogenicity in newborn mice.Citation1 In 2012, Mao et al. used the clinically isolated BJCA08/CA16 strain to develop the first CA16 neonatal mouse model for the evaluation of the protective efficacy of vaccines. BJCA08 can cause 100% mortality in neonatal mice younger than 5 d and induce clinical symptoms in mice. This model uses survival rate as an evaluation indicator and exhibits good reproducibility. Pathological observation, immunohistochemical (IHC) staining, and quantitative RT-PCR revealed that BJCA08 has a strong tropism to skeletal and cardiac muscles. A maternal antibody protection study and anti-serum protection studies performed in this mouse model, showing that anti-CA16 neutralizing antibodies may block viral invasion. This neonatal mouse model could be used as a tool for evaluating vaccine efficacy.
Vaccine Research
In recent years, researchers at Institute Pasteur of Shanghai have performed a series of CA16 vaccine-related studies, including the construction of an infectious cDNA clone of CA16,Citation68 the establishment of a western blot protocol for quantifying the yield of CA16 in Vero cells using recombinant protein VP0 as the reference standard,Citation69 and the development of inactivated and VLP CA16 vaccines.Citation27,Citation28 P1 and 3CD proteins of CA16 were co-expressed by using recombinant baculovirus-infected sf9 cells and formed spherical antigen particles (with a diameter of about 30 nm) consisting of processed CA16 VLP antigens VP0, VP1, and VP3. Antisera from mice immunized with VLP antigens can neutralize CA16 virus in cell substrates and protect mice against lethal challenge. Anti-CA16 serum can neutralize homologous (CA16/SZ05) and heterologous (CA16/GX08) strains of CA16 in vitro. Fourteen days after homologous strain challenge of neonatal mice, the survival rates for groups receiving anti-VLP sera, anti-sf9 sera and PBS were 100%, 77.8%, and 44.4%, respectively. However, the survival rates for neonatal mouse groups injected with anti-VLP sera and anti-sf9 sera were 88.9% and 0%, respectively, 11 d after challenge with a more virulent heterologous strain. These results indicate that the neutralizing antibodies played an important role in animal protection and lay the groundwork for the development of CA16 VLP vaccines. With regard to the development of inactivated vaccines, it has been shown that anti-CA16-specific antibodies as well as a T cell IFN-γ response can be induced in mice immunized with β-propiolactone-inactivated CA16 vaccines. And the amount of inactivated vaccine were quantified by western blot analysis using anti-VP1 antiserum as the detection antibody and using purified CA16 as the reference standard.Citation28 Anti-CA16 mice sera can neutralize homologous and heterologous strains of CA16 as well a mouse-adapted strain (CA16-MAV) in vitro. Neonatal mice were injected with two CA16 vaccines (CA16-SZ05, CA16-G08) to elicit anti-CA16 antibodies and then challenged with live CA16-G08 strains (1 × 107 TCID50). Twenty days after the infection, survival rates for the CA16-G08 and CA16-SZ05 groups and Vero cells were 85.7%, 93.3%, and 7.1%, respectively. Neonatal mice were inoculated with two CA16 vaccines (2 μg per dose) and challenged with the CA16-MAV strain (2.3 × 105 TCID50) 1 week post-immunization. All mice were protected in the vaccinated group, while most mice died in the control group.Citation28 However, yields for both VLP and inactivated vaccines were low (3 mg of VP0 per liter of cell culture; 0.3 mg of Virus per liter of cell culture). Therefore, it is necessary that future studies optimize the VLP vector and procedures and screen high-yield inactivated CA16 strains to meet the needs of large-scale production.
To understand the neutralizing epitopes of CA16, Shi, et al. made up a panel of 95 synthetic peptides spanning the entire VP1 protein of CA16.Citation4 Six unoverlaped linear neutralizing epitopes of CA16 were screened by ELISA for reactivity with neutralizing antisera against CA16 VLPs. All the anti-peptide antisera could neutralize both homologous and heterologous CA16 strains, indicating that these six peptides represented neutralizing epitopes. Among them one peptide (PEP71: residues 211–225) locates at a region overlapping with the epitopes (SP70: residues 208–222 and VP1–43: residues 211–220) of EV71, the other five CA16 epitopes do not overlap with known neutralizing epitopes of EV71. These findings have important implications for CA16 vaccine design, especially for subunit CA16 vaccines.
Summary
CA16 and EV71 are the main pathogens causing HFMD. The alternative and co-circulation of CA16 and EV71, as well as the emergence of new recombinant stains, have made it difficult to control the epidemic of HFMD. Therefore, there are several urgent research works need to be done, such as the studies on CA16 pathology, pathogenic mechanism, epidemiology, and the development of CA16 vaccine or EV71 and CA16 bivalent vaccine. Both animal models for CA16 vaccine evaluation and the protective effects elicited by VLP and inactivated vaccines demonstrate the prospect for the development of CA16 vaccines. At present, many institutions and companies have begun to carry out research on CA16 vaccines. In the very near future, EV71 vaccine will be available on the market and CA16 vaccine or EV71 and CA16 bivalent vaccine is expected to enter the clinical trials. These vaccines are hoped to be the important tools to effectively control the severe HFMD.
| Abbreviations: | ||
| CA16 | = | Coxsackievirus 16 |
| HFMD | = | hand, foot, and mouth disease |
| EV71 | = | human enterovirus 71 |
| CNS | = | central nervous system |
| VLP | = | virus-like particles |
| HEV-A | = | Human enterovirus A |
| ELISA | = | enzyme-linked immunosorbent assay |
| WHO | = | the World Health Organization |
| RT-LAMP | = | reverse transcription loop-mediated isothermal amplification |
| IHC | = | immunohistochemical |
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
This work was supported by the National 12th Five Major Special Projects Funding Program (No. 2012ZX10004701) and National High Technology Research and Development Program (“863” program, No. 2012AA02A402) from the Ministry of Science and Technology of the People’s Republic of China.
References
- Sickles GM, Mutterer M, Feorino P, Plager H. Recently classified types of Coxsackie virus, group A; behavior in tissue culture. Proc Soc Exp Biol Med 1955; 90:529 - 31; http://dx.doi.org/10.3181/00379727-90-22088; PMID: 13273503
- Ranganathan S, Singh S, Poh CL, Chow VT. The hand, foot and mouth disease virus capsid: sequence analysis and prediction of antigenic sites from homology modelling. Appl Bioinformatics 2002; 1:43 - 52; PMID: 15130856
- Zou XN, Zhang XZ, Wang B, Qiu YT. Etiologic and epidemiologic analysis of hand, foot, and mouth disease in Guangzhou city: a review of 4,753 cases. Braz J Infect Dis 2012; 16:457 - 65; http://dx.doi.org/10.1016/j.bjid.2012.08.001; PMID: 22964289
- Shi J, Huang X, Liu Q, Huang Z. Identification of conserved neutralizing linear epitopes within the VP1 protein of coxsackievirus A16. Vaccine 2013; 31:2130 - 6; http://dx.doi.org/10.1016/j.vaccine.2013.02.051; PMID: 23499595
- Goto K, Sanefuji M, Kusuhara K, Nishimura Y, Shimizu H, Kira R, Torisu H, Hara T. Rhombencephalitis and coxsackievirus A16. Emerg Infect Dis 2009; 15:1689 - 91; http://dx.doi.org/10.3201/eid1510.090594; PMID: 19861078
- Wang CY, Li Lu F, Wu MH, Lee CY, Huang LM. Fatal coxsackievirus A16 infection. Pediatr Infect Dis J 2004; 23:275 - 6; http://dx.doi.org/10.1097/01.inf.0000115950.63906.78; PMID: 15014311
- Legay F, Lévêque N, Gacouin A, Tattevin P, Bouet J, Thomas R, Chomelt JJ. Fatal coxsackievirus A-16 pneumonitis in adult. Emerg Infect Dis 2007; 13:1084 - 6; http://dx.doi.org/10.3201/eid1307.070295; PMID: 18214187
- Robinson CR, Doane FW, Rhodes AJ. Report of an outbreak of febrile illness with pharyngeal lesions and exanthem: Toronto, summer 1957; isolation of group A Coxsackie virus. Can Med Assoc J 1958; 79:615 - 21; PMID: 13585281
- Ferson MJ, Bell SM. Outbreak of Coxsackievirus A16 hand, foot, and mouth disease in a child day-care center. Am J Public Health 1991; 81:1675 - 6; http://dx.doi.org/10.2105/AJPH.81.12.1675; PMID: 1746672
- Bendig JW, Fleming DM. Epidemiological, virological, and clinical features of an epidemic of hand, foot, and mouth disease in England and Wales. Commun Dis Rep CDR Rev 1996; 6:R81 - 6; PMID: 8664928
- Chang LY. Enterovirus 71 in Taiwan. Pediatr Neonatol 2008; 49:103 - 12; http://dx.doi.org/10.1016/S1875-9572(08)60023-6; PMID: 19054914
- Ang LW, Koh BK, Chan KP, Chua LT, James L, Goh KT. Epidemiology and control of hand, foot and mouth disease in Singapore, 2001-2007. Ann Acad Med Singapore 2009; 38:106 - 12; PMID: 19271036
- Tu PV, Thao NT, Perera D, Huu TK, Tien NT, Thuong TC, How OM, Cardosa MJ, McMinn PC. Epidemiologic and virologic investigation of hand, foot, and mouth disease, southern Vietnam, 2005. Emerg Infect Dis 2007; 13:1733 - 41; http://dx.doi.org/10.3201/eid1311.070632; PMID: 18217559
- Kar BR, Dwibedi B, Kar SK. An outbreak of hand, foot and mouth disease in Bhubaneswar, Odisha. Indian Pediatr 2013; 50:139 - 42; http://dx.doi.org/10.1007/s13312-013-0033-0; PMID: 22728634
- Zhu J, Luo Z, Wang J, Xu Z, Chen H, Fan D, Gao N, Ping G, Zhou Z, Zhang Y, et al. Phylogenetic analysis of Enterovirus 71 circulating in Beijing, China from 2007 to 2009. PLoS One 2013; 8:e56318; http://dx.doi.org/10.1371/journal.pone.0056318; PMID: 23418551
- Clinical Trials [Internet]. A protected study of inactivated EV71 vaccine (human diploid cell, KMB-17) in Chinese infants and children. 2013. Available from: http://clinicaltrials.gov/show/NCT01569581
- Positive topline results from Phase III trial of Enterovirus 71 vaccine (Sinovac Biotech) against Hand, Foot and Mouth Disease. 2013. http://www.sinovac.com/?optionid=754&auto_id=715
- Zhu FC, Meng FY, Li JX, Li XL, Mao QY, Tao H, Zhang YT, Yao X, Chu K, Chen QH, 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; http://dx.doi.org/10.1016/S0140-6736(13)61049-1; PMID: 23726161
- Wright HT Jr., Landing BH, Lennette EH, McALLISTER RM. Fatal infection in an infant associated with Coxsackie virus group A, type 16. N Engl J Med 1963; 268:1041 - 4; http://dx.doi.org/10.1056/NEJM196305092681904; PMID: 14001956
- WS. Y. Clinical characteristic analysis of 104 cases of children severe hand, foot and mouth disease. Chongqing Med 2011; 40:6
- Xu W, Liu CF, Yan L, Li JJ, Wang LJ, Qi Y, Cheng RB, Xiong XY. Distribution of enteroviruses in hospitalized children with hand, foot and mouth disease and relationship between pathogens and nervous system complications. Virol J 2012; 9:8; http://dx.doi.org/10.1186/1743-422X-9-8; PMID: 22230340
- Zhao K, Han X, Wang G, Hu W, Zhang W, Yu XF. Circulating coxsackievirus A16 identified as recombinant type A human enterovirus, China. Emerg Infect Dis 2011; 17:1537 - 40; PMID: 21801645
- Zhang HMLC, Li CR, Liu YJ, Liu WL, Fu D, Xu LM, Xie JJ, Tan Y, Wang H, Chen XC, et al. [To investigate pathogen of hand, foot and mouth disease in Shenzhen in 2008]. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 2009; 23:334 - 6; PMID: 20387478
- Pan H, Zhu YF, Qi X, Zhang YJ, Li L, Deng F, Wu B, Wang SJ, Zhu FC, Wang H. [Analysis on the epidemiological and genetic characteristics of enterovirus type 71 and Coxsackie A16 virus infection in Jiangsu, China]. Zhonghua Liu Xing Bing Xue Za Zhi 2009; 30:339 - 43; PMID: 19731523
- Yip CC, Lau SK, Zhou B, Zhang MX, Tsoi HW, Chan KH, Chen XC, Woo PC, Yuen KY. Emergence of enterovirus 71 “double-recombinant” strains belonging to a novel genotype D originating from southern China: first evidence for combination of intratypic and intertypic recombination events in EV71. Arch Virol 2010; 155:1413 - 24; http://dx.doi.org/10.1007/s00705-010-0722-0; PMID: 20549263
- Zhang Y, Zhu Z, Yang W, Ren J, Tan X, Wang Y, Mao N, Xu S, Zhu S, Cui A, 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; http://dx.doi.org/10.1186/1743-422X-7-94; PMID: 20459851
- Liu Q, Yan K, Feng Y, Huang X, Ku Z, Cai Y, Liu F, Shi J, Huang Z. A virus-like particle vaccine for coxsackievirus A16 potently elicits neutralizing antibodies that protect mice against lethal challenge. Vaccine 2012; 30:6642 - 8; http://dx.doi.org/10.1016/j.vaccine.2012.08.071; PMID: 22959985
- Cai Y, Liu Q, Huang X, Li D, Ku Z, Zhang Y, Huang Z. Active immunization with a Coxsackievirus A16 experimental inactivated vaccine induces neutralizing antibodies and protects mice against lethal infection. Vaccine 2013; 31:2215 - 21; http://dx.doi.org/10.1016/j.vaccine.2013.03.007; PMID: 23499596
- http://www.cdc.gov.tw.
- Zhu Z, Zhu S, Guo X, Wang J, Wang D, Yan D, Tan X, Tang L, Zhu H, Yang Z, et al. Retrospective seroepidemiology indicated that human enterovirus 71 and coxsackievirus A16 circulated wildly in central and southern China before large-scale outbreaks from 2008. Virol J 2010; 7:300; http://dx.doi.org/10.1186/1743-422X-7-300; PMID: 21050463
- http://www.chinacdc.cn/tjsj/fdcrbbg/.
- Iwai M, Masaki A, Hasegawa S, Obara M, Horimoto E, Nakamura K, Tanaka Y, Endo K, Tanaka K, Ueda J, et al. Genetic changes of coxsackievirus A16 and enterovirus 71 isolated from hand, foot, and mouth disease patients in Toyama, Japan between 1981 and 2007. Jpn J Infect Dis 2009; 62:254 - 9; PMID: 19628900
- Li L, He Y, Yang H, Zhu J, Xu X, Dong J, Zhu Y, Jin Q. 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; http://dx.doi.org/10.1128/JCM.43.8.3835-3839.2005; PMID: 16081920
- Lin TY, Twu SJ, Ho MS, Chang LY, Lee CY. Enterovirus 71 outbreaks, Taiwan: occurrence and recognition. Emerg Infect Dis 2003; 9:291 - 3; http://dx.doi.org/10.3201/eid0903.020285; PMID: 12643822
- Podin Y, Gias EL, Ong F, Leong YW, Yee SF, Yusof MA, Perera D, Teo B, Wee TY, Yao SC, et al. Sentinel surveillance for human enterovirus 71 in Sarawak, Malaysia: lessons from the first 7 years. BMC Public Health 2006; 6:180; http://dx.doi.org/10.1186/1471-2458-6-180; PMID: 16827926
- Huang W, et al. Genetic analysis and etiology study of hand-foot mouth disease in surveillance hospitals of Hunan province during the period of 2008-2010. Pract Prev Med 2011; 18:779 - 84
- Jia L, Zhao CS, Zhang L, Li S, Zhang DT, Liu BW, Wang QY, Li XY. [Comparisons of epidemiological and clinical characteristics in children with hand-foot-mouth disease caused by Enterovirus 71 and Coxackievirus A16]. Zhongguo Dang Dai Er Ke Za Zhi 2011; 13:635 - 7; PMID: 21849112
- Ye RXZY, Pan KN, Tong YX. Significance of enterovirus 71 and coxsackievirus 16 pathogens detection in preventing epidemics of hand, foot and mouth disease. Chin J Nosocomiol 2011; 21:1295 - 6
- Zhang YJBJ, Huang XL. Clinical analysis of 54 cases of hand, foot and mouth disease with central nervous system damage. Chin J CritCare Med 2010; 30:1090 - 2
- Zhang W, et al. Severe hand, foot, and mouth disease caused by mixed infection of enterovirus 71 and coxsackie A16: report of 6 cases. Chin Gen Practice 2011; 14:3341 - 6
- Zhang Y, Wang D, Yan D, Zhu S, Liu J, Wang H, Zhao S, Yu D, Nan L, An J, et al. Molecular evidence of persistent epidemic and evolution of subgenotype B1 coxsackievirus A16-associated hand, foot, and mouth disease in China. J Clin Microbiol 2010; 48:619 - 22; http://dx.doi.org/10.1128/JCM.02338-09; PMID: 20018819
- Zong W, He Y, Yu S, Yang H, Xian H, Liao Y, Hu G. Molecular phylogeny of Coxsackievirus A16 in Shenzhen, China, from 2005 to 2009. J Clin Microbiol 2011; 49:1659 - 61; http://dx.doi.org/10.1128/JCM.00010-11; PMID: 21325543
- Zhou F, Kong F, Wang B, McPhie K, Gilbert GL, Dwyer DE. Molecular characterization of enterovirus 71 and coxsackievirus A16 using the 5′ untranslated region and VP1 region. J Med Microbiol 2011; 60:349 - 58; http://dx.doi.org/10.1099/jmm.0.025056-0; PMID: 21148280
- Perera D, Yusof MA, Podin Y, Ooi MH, Thao NT, Wong KK, Zaki A, Chua KB, Malik YA, Tu PV, et al. Molecular phylogeny of modern coxsackievirus A16. Arch Virol 2007; 152:1201 - 8; http://dx.doi.org/10.1007/s00705-006-0934-5; PMID: 17308978
- 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; PMID: 10559310
- Phuektes P, Chua BH, Sanders S, Bek EJ, Kok CC, McMinn PC. Mapping genetic determinants of the cell-culture growth phenotype of enterovirus 71. J Gen Virol 2011; 92:1380 - 90; http://dx.doi.org/10.1099/vir.0.029371-0; PMID: 21346025
- Yang E, Zhao H, Zhang Y, Liu J, Liao Y, Wang L, Cui P, Yang L, Liu L, Dong C, et al. A comparative study of the characteristics of two Coxsackie A virus type 16 strains (genotype B). Science China. Life Sci 2012; 55:336 - 42; http://dx.doi.org/10.1007/s11427-012-4313-z
- Ni H, Yi B, Yin J, Fang T, He T, Du Y, Wang J, Zhang H, Xie L, Ding Y, et al. Epidemiological and etiological characteristics of hand, foot, and mouth disease in Ningbo, China, 2008-2011. J Clin Virol 2012; 54:342 - 8; http://dx.doi.org/10.1016/j.jcv.2012.04.021; PMID: 22652041
- Zhu FC, Liang ZL, Meng FY, Zeng Y, Mao QY, Chu K, Song XF, Yao X, Li JX, Ji H, et al. Retrospective study of the incidence of HFMD and seroepidemiology of antibodies against EV71 and CoxA16 in prenatal women and their infants. PLoS One 2012; 7:e37206; http://dx.doi.org/10.1371/journal.pone.0037206; PMID: 22662137
- Rabenau HF, Richter M, Doerr HW. Hand, foot and mouth disease: seroprevalence of Coxsackie A16 and Enterovirus 71 in Germany. Med Microbiol Immunol 2010; 199:45 - 51; http://dx.doi.org/10.1007/s00430-009-0133-6; PMID: 19941005
- Diedrich S, Weinbrecht A, Schreier E. Seroprevalence and molecular epidemiology of enterovirus 71 in Germany. Arch Virol 2009; 154:1139 - 42; http://dx.doi.org/10.1007/s00705-009-0413-x; PMID: 19506798
- Chang LY, King CC, Hsu KH, Ning HC, Tsao KC, Li CC, Huang YC, Shih SR, Chiou ST, Chen PY, et al. Risk factors of enterovirus 71 infection and associated hand, foot, and mouth disease/herpangina in children during an epidemic in Taiwan. Pediatrics 2002; 109:e88; http://dx.doi.org/10.1542/peds.109.6.e88; PMID: 12042582
- Kung SH, Wang SF, Huang CW, Hsu CC, Liu HF, Yang JY. Genetic and antigenic analyses of enterovirus 71 isolates in Taiwan during 1998-2005. Clin Microbiol Infect 2007; 13:782 - 7; http://dx.doi.org/10.1111/j.1469-0691.2007.01745.x; PMID: 17488328
- Jin J, Ma H, Xu L, An D, Sun S, Huang X, Kong W, Jiang C. Development of a Coxsackievirus A16 neutralization assay based on pseudoviruses for measurement of neutralizing antibody titer in human serum. J Virol Methods 2013; 187:362 - 7; http://dx.doi.org/10.1016/j.jviromet.2012.11.014; PMID: 23178532
- Yu N, Guo M, He SJ, Pan YX, Chen XX, Ding XX, Hao W, Wang YD, Ge SX, Xia NS, et al. Evaluation of human enterovirus 71 and coxsackievirus A16 specific immunoglobulin M antibodies for diagnosis of hand-foot-and-mouth disease. Virol J 2012; 9:12; http://dx.doi.org/10.1186/1743-422X-9-12; PMID: 22234169
- Zhu D, Zhao XY, Yao Y, Dai FF, He H, Li RQ, Jin RH, Liang LC, Li N. A new factor influencing pathogen detection by molecular assay in children with both mild and severe hand, foot, and mouth disease. Diagn Microbiol Infect Dis 2013; 76:162 - 7; http://dx.doi.org/10.1016/j.diagmicrobio.2013.02.011; PMID: 23535205
- Brown BA, Pallansch MA. Complete nucleotide sequence of enterovirus 71 is distinct from poliovirus. Virus Res 1995; 39:195 - 205; http://dx.doi.org/10.1016/0168-1702(95)00087-9; PMID: 8837884
- Xu F, He D, He S, Wu B, Guan L, Niu J, Li L, Li C, Weng Z, Yan Q, et al. Development of an IgM-capture ELISA for Coxsackievirus A16 infection. J Virol Methods 2011; 171:107 - 10; http://dx.doi.org/10.1016/j.jviromet.2010.10.009; PMID: 20970457
- Singh S, Chow VT, Phoon MC, Chan KP, Poh CL. Direct detection of enterovirus 71 (EV71) in clinical specimens from a hand, foot, and mouth disease outbreak in Singapore by reverse transcription-PCR with universal enterovirus and EV71-specific primers. J Clin Microbiol 2002; 40:2823 - 7; http://dx.doi.org/10.1128/JCM.40.8.2823-2827.2002; PMID: 12149336
- Chen HMJ, Ma XM. Analysis of etiology detection result of HFMD cases in Ningxia in2008. Ningxia Med J 2009; 33:515 - 6
- Jiang B, Zhang J, You X, Dong C, Cheng X, Dai X, Meng J. Diagnosis of hand, foot, and mouth disease caused by EV71 and other enteroviruses by a one-step, single tube, duplex RT-PCR. J Med Virol 2012; 84:1803 - 8; http://dx.doi.org/10.1002/jmv.23391; PMID: 22997084
- Yang F, Zhang T, Hu Y, Wang X, Du J, Li Y, Sun S, Sun X, Li Z, Jin Q. Survey of enterovirus infections from hand, foot and mouth disease outbreak in China, 2009. Virol J 2011; 8:508; http://dx.doi.org/10.1186/1743-422X-8-508; PMID: 22054534
- Tan EL, Chow VT, Quak SH, Yeo WC, Poh CL. Development of multiplex real-time hybridization probe reverse transcriptase polymerase chain reaction for specific detection and differentiation of Enterovirus 71 and Coxsackievirus A16. Diagn Microbiol Infect Dis 2008; 61:294 - 301; http://dx.doi.org/10.1016/j.diagmicrobio.2008.02.009; PMID: 18394844
- Chen TC, Chen GW, Hsiung CA, Yang JY, Shih SR, Lai YK, Juang JL. Combining multiplex reverse transcription-PCR and a diagnostic microarray to detect and differentiate enterovirus 71 and coxsackievirus A16. J Clin Microbiol 2006; 44:2212 - 9; PMID: 16757623
- Song L, Sun S, Li B, Pan Y, Li W, Zhang K, Li J. External quality assessment for enterovirus 71 and coxsackievirus A16 detection by reverse transcription-PCR using armored RNA as a virus surrogate. J Clin Microbiol 2011; 49:3591 - 5; http://dx.doi.org/10.1128/JCM.00686-11; PMID: 21865426
- Wang X, Zhu JP, Zhang Q, Xu ZG, Zhang F, Zhao ZH, Zheng WZ, Zheng LS. Detection of enterovirus 71 using reverse transcription loop-mediated isothermal amplification (RT-LAMP). J Virol Methods 2012; 179:330 - 4; http://dx.doi.org/10.1016/j.jviromet.2011.11.019; PMID: 22155579
- Mao Q, Wang Y, Gao R, Shao J, Yao X, Lang S, Wang C, Mao P, Liang Z, Wang J. A neonatal mouse model of coxsackievirus A16 for vaccine evaluation. J Virol 2012; 86:11967 - 76; http://dx.doi.org/10.1128/JVI.00902-12; PMID: 22951825
- Liu F, Liu Q, Cai Y, Leng Q, Huang Z. Construction and characterization of an infectious clone of coxsackievirus A16. Virol J 2011; 8:534; http://dx.doi.org/10.1186/1743-422X-8-534; PMID: 22165961
- Liu Q, Ku Z, Cai Y, Sun B, Leng Q, Huang Z. Detection, characterization and quantitation of coxsackievirus A16 using polyclonal antibodies against recombinant capsid subunit proteins. J Virol Methods 2011; 173:115 - 20; http://dx.doi.org/10.1016/j.jviromet.2011.01.016; PMID: 21295614
- Puenpa J, Theamboonlers A, Korkong S, Linsuwanon P, Thongmee C, Chatproedprai S, Poovorawan Y. Molecular characterization and complete genome analysis of human enterovirus 71 and coxsackievirus A16 from children with hand, foot and mouth disease in Thailand during 2008-2011. Arch Virol 2011; 156:2007 - 13; http://dx.doi.org/10.1007/s00705-011-1098-5; PMID: 21898187
- Qiaoyun F, Xiongfei J, Lihuan L, Angao X. Epidemiology and etiological characteristics of hand, foot and mouth disease in Huizhou City between 2008 and 2011. Arch Virol 2013; 158:895 - 9; http://dx.doi.org/10.1007/s00705-012-1566-6; PMID: 23229012
- Zhou X, Fan G, Xia W, He F, Hu M, Ni X, Zhang Y, Chen H. Molecular epidemiology of human enterovirus 71 strains in the Nanchang region of China in 2011. Jpn J Infect Dis 2013; 66:149 - 50; http://dx.doi.org/10.7883/yoken.66.149; PMID: 23514913
- Urquhart GE. A survey of coxsackie A16 virus antibodies in human sera. J Hyg (Lond) 1984; 93:205 - 12; http://dx.doi.org/10.1017/S002217240006472X; PMID: 6094661
- Lin Y, Wen K, Pan Y, Wang Y, Che X, Wang B. Cross-reactivity of anti-EV71 IgM and neutralizing antibody in series sera of patients infected with Enterovirus 71 and Coxsackievirus A 16. J Immunoassay Immunochem 2011; 32:233 - 43; http://dx.doi.org/10.1080/15321819.2011.559297; PMID: 21574094