Coxsackievirus A16 (CV-A16) is the leading cause of hand-foot-mouth disease (HFMD), which usually presents as mild and self-limiting symptoms in young children. Rarely, CV-A16 has been reported to cause severe and fatal neurological complications but little is known about these complications. In the present study, 1-day and 7-day old mouse models of CV-A16 were developed using a clinical strain via subcutaneous inoculation. All infected mice exhibited clinical signs of infection, including reduced mobility, limb weakness and paralysis between 3 to 6 days post-infection. Pathologically, the main organs involved were the central nervous system (CNS), skeletal muscles and brown fat. In the CNS, viral antigens as demonstrated by immunohistochemistry, were localized mainly to neurons in the brain stem and spinal cord, suggesting that CV-A16 is neurotropic although inflammation is very mild. The skeletal muscles showed necrosis and myositis due to viral infection as evidenced by the dense viral antigens. Focal viral antigens were also detected in the brown fat. These preliminary pathological findings indicate that our mouse models can be further developed to be useful models for pathogenesis studies, and vaccine and anti-viral drug evaluation.
Coxsackievirus Infections

OBJECTIVETo compare the differences of epidemiological and clinical characteristics in children with hand-foot-mouth disease (HFMD) caused by Coxsackievirus A16 (CA16) and Enterovirus 71 (EV71).

METHODSThe samples of vesicle fluid and throat swabs of 108 children with HFMD were collected and detected for enterovirus by RT-PCR. The clinical data of children with EV71 and CA16 infection were retrospectively reviewed and compared.

RESULTSThe total positive rate of enterovirus was 97.2% (105/108). Of the 105 cases, 56 cases were positive for EV71 (51.9%), 39 cases were positive for CA16 (36.1%), 2 cases were positive for other enterovirus (1.9%), and 8 cases were co-infected by EV71 and CA16 (7.4%). There were no significant differences in age and sex between EV71 and CV16 infected cases. The univariate analysis showed that the incidences of herpes of mouth, erythra of knees, and nose running in children infected by CA16 were higher than in those infected by EV71. The multivariate logistic regression analysis showed that the HFMD children who had erythra of knees had higher probability of CA16 infection.

CONCLUSIONSEV71 should be considered as the pathogen in children with HFMD who have no herpes of mouth, erythra of knees, and nose running.

Child ; Child, Preschool ; Coxsackievirus Infections ; epidemiology ; Enterovirus A, Human ; Female ; Hand, Foot and Mouth Disease ; epidemiology ; Humans ; Infant ; Logistic Models ; Male

Autopsy ; Coxsackievirus Infections ; Enterovirus ; isolation & purification ; Enterovirus B, Human ; isolation & purification ; Enterovirus Infections ; Hand, Foot and Mouth Disease ; pathology ; virology ; Humans ; Infant ; Male

Objective: To analyze the epidemiological characteristics of hand foot and mouth disease (HFMD) cases caused by Coxsackie virus A16 (Cox A16) in Guangdong province from 2012 to 2016. Methods: The data of mild HFMD cases caused by Cox A16 were collected from 8 sentinel hospitals in 8 prefecture-level cities in Guangdong to estimate Cox A16 infection status and its population and time distribution characteristics. Results: (1) The highest estimated incidence of Cox A16 infection was in 2014 (113.0/100 000), followed by 2016 (86.4/100 000) and 2012 (79.1/100 000), while the estimated incidence was lower in 2015 (29.0/100 000) and 2013 (28.8/100 000). (2) Cox A16 was confirmed to be the predominant pathogen causing HFMD outbreaks (54.6%, 89/163). The number of outbreaks in the year with high incidence (28 outbreaks) was 11.2 times higher than that in the year with low incidence (2.5 outbreaks). (3) Across all age groups, the annual estimated incidence of Cox A16 infection decreased with age (trend χ(2)=853 905.63, P<0.01). The incidence was highest in age group 1 year (1 449.2/100 000), followed by that in age group 3 years (1 097.0/100 000), in age group 2 years (1 083.5/100 000), in age group 4 years (687.8/100 000) and in age group 0 year (604.9/100 000). Among the age groups <12 months, the estimated incidence increased with age (trend χ(2)=5 541.77, P<0.01), which was highest in age group 11-months (2 105.1/100 000), followed by that in age groups 10-months (1 448.6/100 000), 9-months (938.3/100 000), 8-months (703.3/100 000) and 6-months (664.6/100 000). (4) The annual incidence peak was during May (143.9/100 000)-June (131.5/100 000). Conclusion: The prevalence of Cox A16 infection differed with year in Guangdong during 2012-2016. When the incidence of Cox A16 infection was high, more outbreaks occurred. The prevalence occurred mainly in nurseries and kindergartens from May to June each year. Children aged 0-4 years were the high risk group for Cox A16 infection, children aged 6-11 months were at high risk for Cox A16 infection.
Animals ; Child ; Child, Preschool ; China/epidemiology* ; Cities ; Coxsackievirus Infections/epidemiology* ; Disease Outbreaks ; Enterovirus A, Human/isolation & purification* ; Hand, Foot and Mouth Disease/epidemiology* ; Hospitals ; Humans ; Incidence ; Infant ; Schools

OBJECTIVETo investigate the epidemic characteristics of etiological agents in children with hand, foot and mouth disease (HFMD) and analyze the differences between the severe and mild cases with HFMD seen from 2008 to 2009 in the Children's Hospital.

METHODSA total of 154 patients with HFMD were enrolled from May 2008 to September 2008 and from May 2009 to September 2009, including 28 severe HFMD patients. Data from 80 cases with suspected herpangina were collected as control. Enterovirus universal type, enterovirus type 71 (EV71) and coxsackie virus group A 16 (CA16) were detected by real-time RT-PCR respectively.

RESULTSThe positive rate of enterovirus universal type in the 154 patients with HFMD was 81.82%(126/154). EV71 positive rate in these 126 patients with enterovirus universal type infection was 57.14%(72/126). The positive rate of enterovirus universal type in the 80 cases with suspected herpangina was 68.75%(55/80). There was no EV71 infection in these 80 cases with suspected herpangina. EV71 infection was mainly popular in 2008. Both EV71 and CA16 were prevalent in 2009. The epidemic characteristics of enterovirus infection with HFMD between 2008 and 2009 had significant differences (χ(2) = 23.50, P = 0.000) (P < 0.01). The epidemic characteristics of enterovirus infection between severe and mild HFMD patients also had significant differences (χ(2) = 29.85, P < 0.01). There were 28 cases with severe HFMD, in whom the EV71 positive rate was 92.86% (26/28). EV71 positive rate in the mild HFMD was 36.51% (46/126) (χ(2) = 29.22, P < 0.01). There was no significant difference in the gender (χ(2) = 0.135, P = 0.714) and virus load (t = 0.141, P = 0.889) between the mild and severe HFMD cases. But the age of mild and severe HFMD showed a significant difference (t = 2.926, P = 0.009). Patients who were less than 2 years of age had a proportion of 88.89% (8/9) with severe HFMD. The mean age of mild HFMD patients was 3.19 years.

CONCLUSIONHFMD showed different epidemic characteristics at different times of enterovirus infection. There was no significant difference in the gender and virus load between the mild and severe cases with HFMD. Children under 3 years of age with EV71 infection were at high risk for severe HFMD.

Child ; Child, Preschool ; China ; epidemiology ; Coxsackievirus Infections ; epidemiology ; Enterovirus ; Female ; Hand, Foot and Mouth Disease ; epidemiology ; virology ; Humans ; Infant ; Male ; Viral Load

Amino Acid Sequence ; China ; epidemiology ; Coxsackievirus Infections ; epidemiology ; Enterovirus A, Human ; genetics ; isolation & purification ; Genes, Viral ; genetics ; Hand, Foot and Mouth Disease ; virology ; Humans ; Phylogeny

OBJECTIVETo analyze the epidemiological characteristics of hand foot and mouth disease (HFMD) in Ningbo.

METHODSA descriptive analysis was conducted through the surveillance data of HFMD in Ningbo, Zhejiang province, from 2008 to 2011. Genes on EV71 and Cox A16 were amplified with RT-PCT from the stool samples of HFMD patients. Sequences were analyzed by bioinformatics software.

RESULTS37 524 cases of HFMD were reported from 2008 to 2011, including 196 severe cases and 12 deaths. The reported incidence was 145.26 per 100 000 and the case fatality was 0.03%. Cases in children aged 5 or younger accounted for 95.89%, and the scattered cases accounted for 64.10%. Xiangshan and Ninghai counties had the highest incidence rates in Ningbo. The peak of incidence was from April to July. The number of male patients was obviously higher than females. 2394 cases of HFMD were laboratory confirmed and EV71 with the predominant epidemic strain. Data from phylogenetic analysis revealed that EV71 isolated from HFMD patients in Ningbo belonged to C4a evolution branch of C4 sub-genotype, with several transmission chains. Cox A16 belonged to B1 evolution branch. 53.48% of the healthy children in Ningbo showed EV71 antibody positive. The geometric mean of the antibody titer (GMT) was 11.23 (8.33 - 14.98) in healthy children. Cox A16 antibody was detected at 63.18% of the healthy children in Ningbo. GMT in healthy children was 12.61 (6.70 - 16.52).

CONCLUSIONHFMD was highly endemic in Ningbo, with children under 5 years old were at high-risk. The major etiologic agent was EV71 which belonged to C4a in the C4 sub-genotypes. Cox A16 belonged to the B1 evolution branch, which were in line with the predominant virus circulating in the mainland of China.

Child ; Child, Preschool ; China ; epidemiology ; Coxsackievirus Infections ; epidemiology ; Enterovirus ; isolation & purification ; Female ; Hand, Foot and Mouth Disease ; epidemiology ; virology ; Humans ; Incidence ; Infant ; Infant, Newborn ; Male

OBJECTIVETo understand the etiology of hand, foot and mouth disease (HFMD) in Guangzhou area in 2008.

METHODTotally 1023 clinical specimens were collected from pediatric patients suspected of HFMD in 2008. TaqMan real-time RT-PCR were used for detection of enterovirus 71 (EV71), Coxsackievirus A16 (CA16) and other enteroviruses. The specimens which were enterovirus positive by RT-PCR method with universal primer but EV71 and CA16 negative, were amplified and sequenced for 5'untranslated region.

RESULTEnterovirus was identified from 434 of 1023 samples and detection rate of enterovirus was 42.42%; of the 434 samples, 276 were positive for EV71 (63.6%), 126 for CA16 (29%), 4 samples for enterovirus 84, 3 for Echovirus 11, 2 for Echovirus 9, 3 for Coxsackievirus B3, 4 for Coxsackievirus A10, 3 for Coxsackievirus A6, 6 for Coxsackievirus A12 or A5, and for 7 samples typing was difficult.

CONCLUSIONThe major causative agents of HFMD in Guangzhou were EV71 and CA16 in 2008, and EV84, CA10, CA12, CA6, COSB3, ECHV11, ECHV9 were also the pathogens for smaller proportions of patients.

Child ; Child, Preschool ; China ; epidemiology ; Coxsackievirus Infections ; epidemiology ; DNA Primers ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; Female ; Hand, Foot and Mouth Disease ; epidemiology ; virology ; Humans ; Infant ; Male ; RNA, Viral ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the myocardial protective effect of L-carnitine in children with hand, foot and mouth disease (HFMD) caused by Coxsackie A16 virus and possible mechanisms.

METHODSA total of 60 HFMD children with abnormal myocardial enzyme after Coxsackie A16 virus infection were enrolled and randomly divided into L-carnitine group and fructose-1,6-diphosphate group (fructose group), with 30 children in each group. The two groups were given L-carnitine or fructose diphosphate in addition to antiviral and heat clearance treatment. Another 30 healthy children who underwent physical examination were enrolled as control group. The changes in myocardial zymogram, malondialdehyde (MDA), superoxide dismutase (SOD), and apoptosis factors sFas and sFasL after treatment were compared between groups.

RESULTSThere was no significant difference in treatment response between the L-carnitine group and the fructose group (P>0.05). One child in the fructose group progressed to critical HFMD, which was not observed in the L-carnitine group. Before treatment, the L-carnitine group and the fructose group had significantly higher indices of myocardial zymogram and levels of MDA, sFas, and sFasL and a significantly lower level of SOD than the control group (P<0.05), while there were no significant differences in these indices between the L-carnitine group and the fructose group (P>0.05). After treatment, the L-carnitine group and the fructose group had significant reductions in the indices of myocardial zymogram and levels of MDA, sFas, and sFasL and a significant increase in the level of SOD (P<0.05); the fructose group had a significantly higher level of creatine kinase (CK) than the control group and the L-carnitine group, and there were no significant differences in other myocardial enzyme indices, MDA, sFas, and sFasL between the L-carnitine group and the fructose group, as well as between the L-carnitine and fructose groups and the control group (P>0.05). SOD level was negatively correlated with aspartate aminotransferase, lactate dehydrogenase (LDH), CK, and creatine kinase-MB (CK-MB) (r=-0.437, -0.364, -0.397, and -0.519 respectively; P<0.05), and MDA level was positively correlated with LDH and CK-MB (r=0.382 and 0.411 respectively; P<0.05).

CONCLUSIONSL-carnitine exerts a good myocardial protective effect in children with HFMD caused by Coxsackie A16 virus, possibly by clearing oxygen radicals and inhibiting cardiomyocyte apoptosis.

Carnitine ; therapeutic use ; Child, Preschool ; Coxsackievirus Infections ; complications ; Female ; Hand, Foot and Mouth Disease ; drug therapy ; etiology ; metabolism ; Heart ; drug effects ; Humans ; Infant ; Male ; Malondialdehyde ; analysis ; Myocardium ; metabolism ; pathology ; Superoxide Dismutase ; metabolism

Coxsackievirus A16 belongs to the family Picornaviridae, and is a major agent of hand-foot-and-mouth disease that infects mostly children, and to date no vaccines or antiviral therapies are available. 2A protease of enterovirus is a nonstructural protein and possesses both self-cleavage activity and the ability to cleave the eukaryotic translation initiation factor 4G. Here we present the crystal structure of coxsackievirus A16 2A protease, which interestingly forms hexamers in crystal as well as in solution. This structure shows an open conformation, with its active site accessible, ready for substrate binding and cleavage activity. In conjunction with a previously reported "closed" state structure of human rhinovirus 2, we were able to develop a detailed hypothesis for the conformational conversion triggered by two "switcher" residues Glu88 and Tyr89 located within the bll2-cII loop. Substrate recognition assays revealed that amino acid residues P1', P2 and P4 are essential for substrate specificity, which was verified by our substrate binding model. In addition, we compared the in vitro cleavage efficiency of 2A proteases from coxsackievirus A16 and enterovirus 71 upon the same substrates by fluorescence resonance energy transfer (FRET), and observed higher protease activity of enterovirus 71 compared to that of coxsackievirus A16. In conclusion, our study shows an open conformation of coxsackievirus A16 2A protease and the underlying mechanisms for conformational conversion and substrate specificity. These new insights should facilitate the future rational design of efficient 2A protease inhibitors.
Coxsackievirus Infections ; virology ; Crystallography, X-Ray ; Cysteine Endopeptidases ; chemistry ; genetics ; Fluorescence Resonance Energy Transfer ; Hand, Foot and Mouth Disease ; enzymology ; pathology ; virology ; Humans ; Picornaviridae ; chemistry ; enzymology ; genetics ; Protein Conformation ; Structure-Activity Relationship ; Substrate Specificity ; Viral Proteins ; chemistry ; genetics