2. Department of Infectious Diseases, Xi'an Children's Hospital, Xi'an 710003, China
2. 西安市儿童医院感染科，陕西 西安 710043
Enterovirus 71 (EV71) is a small RNA virus of the Enterovirus genus in the Picornaviridae family . EV71 was first isolated in 1969 from an American child with encephalitis and has been identified as one of the major pathogens responsible for outbreaks of hand, foot and mouth disease (HFMD) in children worldwide. Over the past two decades, severe outbreaks of HFMD had been reported in Asia-Pacific countries including China, Singapore, Malaysia, Japan, Korea, Vietnam and Thailand . According to the data of the Chinese National Health and Family Planning Commission of China, from 2008 to 2017, a total of 13.8 million cases of HFMD were reported in China, with an increasing trend of morbidity and severe cases, and around 45% of them were associated with EV71 and nearly all deaths (over 3700 cases) were attributed to EV71.
Although EV71 infections can produce varying clinical manifestations, from fever and herpangina in mild HFMD to central nervous system (CNS) involvement in severe cases, most of these infections can be asymptomatic. However, in some children the disease progresses rapidly with aseptic meningitis, acute flaccid paralysis, multiple organ dysfunction, and even death. The occurrence and development of HFMD have been confirmed to be associated with virus, host and environmental factors. The host factors can affect the occurrence and development of EV71-associated diseases. Recently, human gene polymorphisms have been shown to play an important role in the pathogenesis of EV71 infection . To investigate the mechanism of genetic susceptibility to EV71 infection at the molecular level is one of the main research directions.
2', 5'-Oligoadenylate synthetase 1 (OAS1), one of the major proteins induced by type Ⅰ interferon, plays a critical role in host defense against viral infections. In the presence of double-stranded RNA structures (viral genomes or single-stranded RNA transcripts that possess double-stranded characteristics), OAS1 converts ATP to 2', 5'-oligoadenylates (2-5As), which activate latent RNase L that, when activated, degrades single-stranded RNAs and inhibits viral replication. In recent years, the contributions of RNase L and OAS1 to innate immunity have become increasingly apparent.
Single nucleotide polymorphisms (SNPs) of OAS1 gene are known to modulate the expression level and enzyme activity of OAS1 to potentially influence the susceptibility to and the severity of viral diseases. So far 57 SNPs have been identified in OAS1 gene. Two functional SNPs (rs2660 and rs1131454) in OAS1 gene have been shown to be related with inhibiting viral replication in several viral diseases caused by hepatitis C virus [8, 9], hepatitis B virus, coxsackie virus 16, influenza A virus , dengue virus, West Nile virus, respiratory epithelial cell syncytial virus, ocular herpes simplex virus type 1, tick-borne encephalitis virus, and severe acute respiratory syndrome virus . The differences in antiviral immune response result in different prognoses after EV71 infection, but the mechanism is not clear. We hypothesize that the diversity of genetic background affects the host's immune response, which leads to the diversity of clinical manifestations and prognosis. In this study, we aimed to investigate the relationship between these two SNPs in OAS1 gene and EV71 infection to identify the potentially at-risk children susceptible to EV71 infection and CNS involvement following EV71 infection.PATIENTS AND METHODS Study participants
A total of 180 children with the diagnosis of HFMD associated with EV71 infection were enrolled from the Department of Infectious Diseases at the Second Affiliated Hospital of Xi'an Jiaotong University and the Department of Infectious Diseases of Xi'an Children's Hospital (Xi'an, China) between April, 2014 and December, 2015. The diagnosis of HFMD was established based on the criteria in the Hand, Foot and Mouth Disease Clinical Guide (2010 edition). Children with persistent high fever and nervous system symptoms such as brainstem encephalitis, pulmonary edema and circulation dysfunction were classified as having severe HFMD. All these 180 children were confirmed to have EV71 infection with laboratory evidence provided by the Clinical Laboratories of the above two hospitals and the Xi'an Center for Disease Control. We also recruited a healthy control group consisting of 201 children undergoing routine physical examinations during the same period. All the children in the control group were screened against a history of HFMD or herpangina. All the participants in this study were unrelated Chinese of Han Nationality and resided in the same geographical area.
This study was approved by the Ethics Committee of the Second Affiliated Hospital of Xi'an Jiaotong University and was carried out in line with the Declaration of Helsinki. Written informed consent was obtained from the guardians of all the participants.Identification of EV71 infection
Real-time quantitative PCR (qRT-PCR) was performed to identify EV71 infection by detecting EV71 nucleic acid in the fecal samples of the children using the upstream primer 5'-GCAGCCCAAAAGAACTTCAC-3' and the downstream primer 5'-ATTTCAGCAGCTTGGA GTGC-3', following precisely the manufacturer's protocols of the EV71 nucleic acid detection kit (Da An Gene Co., Guangzhou, China). EV71 viral load was determined by fluorescence quantitative PCR and compared with the standard system. In the negative control group, sterilized water for injection was used instead of the specimens, and the positive control was used as the template.Extraction and identification of genomic DNA
For children with EV71 infection, whole blood samples were collected and genomic DNA was extracted using the FlexiGene DNA Kit (Qiagen, Shanghai, China). For the healthy controls, buccal brush samples were collected and genomic DNA was extracted using the Centra Puregene Buccal Cell Kit (Qiagen, Shanghai, China).Selection of OAS1 gene polymorphic loci
The sites in the functional areas such as exons, 5' untranslated regions (UTRs) and 3' UTRs were preferentially considered. The minor allele frequency (MAF) in the population of China is ≥0.05, and the sites associated with other diseases in previous report were also considered as the secondary sites[16, 17]. The sites with larger MAFs were preferred. Thus rs2660 and rs1131454 of OAS1 gene were selected as the target polymorphic loci in this study.SNP genotyping
SNP genotyping was performed using the SNPscan Kit (Genesky Biotechnologies Inc., Shanghai, China) . PCR was carried out for 35 cycles with annealing temperatures of 62 and 52 ℃ for rs2660 and rs1131454, respectively. For each of rs2660 and rs1131454, we designed the 3' primers for the forward template chain (3F), for A allele on the forward template chain (AF), and for G allele on the forward template chain (GF), whose sequences are listed below (Ref. seq.: NC_ 000012.11):
Hardy-Weinberg equilibrium was examined using Chi-square test. Polymorphism frequencies between the groups were analyzed also using Chi-square test. The children with EV71 infection and healthy controls were compared to estimate the associations of the alleles and genotypes with the susceptibility to EV 71 infection; wethen compared the children with CNS involvement and those with mild EV71 infection to estimate the associations of the alleles and genotypes with CNS involvement of EV71 infection. All the associations were estimated by calculating age- and gender-adjusted odds ratios (ORs) and 95% confidence intervals (CIs), using binary logistic regression analysis. The additive model and dominant model were fitted for polymorphisms of OAS1 (rs2660 and rs1131454). P < 0.05 was considered to indicate a statistically significant difference. All the statistical analyses were performed using SPSS17.0 software (SPSS Inc., Chicago, IL, USA).RESULTS Baseline characteristics of the participants
A total of 180 children with EV71 infection (male/ female: 111/69; median age: 20.5 months; age range: 6-96 months) were enrolled. Seventy-two children had mild infections without any complications (male/female: 44/28; median age: 24.5 months; age range: 8-96 months). Their manifestations included exanthema (characterized by papulovesicular rash on the palms and soles) and multiple oral ulcers, with or without fever. The other 108 children had severe infections involving the CNS (male/female: 67/41; median age: 19.5 months; age range: 6-72 months), manifested by aseptic meningitis, encephalitis, poliomyelitis-like syndrome, or encephalomyelitis . CNS involvement could be accompanied by cardiorespiratory symptoms. The gender distribution was comparable between the mild cases and cases with CNS involvement (χ2=0.02, P= 0.90); the mean age was younger in cases with CNS involvement than in the mild cases, but the difference was not statistically significant (Mann-Whitney U test, P=0.07). In the 201 healthy controls, the age (median: 24 months; range: 8-72 months) and gender distribution (male/female: 119/82) were comparable to those of the EV71-infected children (χ2=0.24, P=0.62; MannWhitney U test, P=0.837, respectively).Hardy-Weinberg equilibrium of the polymorphisms
The genotypic distribution of rs2660 and rs1131454 in OAS1 gene was consistent with Hardy-Weinberg equilibrium in children with EV71 infection and healthy controls (data not shown), supporting the validity of these participants to represent the study population.Association between OAS1 gene polymorphisms and susceptibility to EV71 infection
The distribution of genotypes and alleles of rs2660 and rs1131454 showed no significant difference between the children with EV71 infection and the healthy controls (Tab. 1), or between the children with CNS involvement of EV71 infection and the healthy controls (Tab. 2).
OAS1 rs2660 polymorphism was significantly different between children with CNS involvement and those with mild EV71 infection. The genotype AG frequency was significantly higher (OR=2.27, 95% CI: 1.07-4.85) and the genotype GG frequency was significantly lower (OR= 0.27, 95% CI: 0.08-0.97) in children with CNS involvement. The distribution of the genotypes and alleles of rs1131454 showed no significant difference between the children with CNS involvement and those with mild EV71 infection (Tab. 3).
HFMD is an acute infectious disease mainly caused by EV71 and coxsackievirus 16. Differences in antiviral immune responses result in different prognoses after EV71 infection, but the mechanism is not clear. OAS1 is an antiviral protein induced by interferon and also an important kinase in cell signaling pathways that respond to cellular and environmental stress. Considering the effects of OAS1 gene polymorphism on the antiviral activity of OAS1 protease, several studies examined the association between OAS1 gene and HFMD. In this study we selected two functional OAS1 polymorphic markers (rs2660 and rs1131454) that are associated with multiple viral infections, including hepatitis C virus and West Nile virus[8, 9, 11, 16]. OAS1 rs1131454 SNP is located in the third exon of the OAS1 gene, and affects the synthesis of OAS1 protein. It has been shown that patients with EV71 infection with different genotypes of OAS1 rs1131454 have significantly different blood leukocyte count, C-reactive protein level, and blood glucose concentration[20, 21], which reflect the severity of EV71 infection. In this study we found no significant difference in the genotype or allele distribution of rs1131454 between the children with EV71 infection and the healthy controls, or between children with CNS involvement and those with mild EV71 infection. As wedid not analyze the levels of OAS1 in the peripheral blood or cerebrospinal fluid of the patients, and all the samples were collected from Xi'an with a limited geographical coverage, it remains unclear whether OAS1 rs1131454 affects the expression of OAS1 in children with EV71 infection.
We did not observe significant differences in OAS1 rs2660 genotype or allele frequencies between EV71-infected children and the healthy controls. But in the children with EV71 infection, we found that OAS1 rs2660 AG genotype frequency was significantly higher and GG genotype frequency was lower in children with CNS involvement than in those with mild EV71 infection. These results suggest that OAS1 gene polymorphism is not significantly correlated to the susceptibility to EV71 infection. In general, the rs2660 AA genotype promotes disease susceptibility, whereas GG genotype confers protection[22-24], possibly because AA genotype results in the lowest, AG the intermediate and GG the highest OAS1 enzyme activity. Our results are consistent with these observations and suggest that the rs2660 AG genotype may not increase the risk of EV71 infection, but can be associated with an increased possibility of CNS involvement after infection. But given the retrospective nature of this study and the geographically localized sampling, we need to further validate our results in a large epidemiological screening study.
In summary, our results confirm that OAS1 rs1131454 and rs2660 polymorphisms are not associated with the risk of EV71 infection, but children carrying the OAS1 rs2660 AG genotype are more likely to have CNS involvement after EV71 infection. Further studies are needed to investigate the mechanism by which EV71 infections result in different clinical phenotypes, especially in the context of genetic predisposing factors of CNS infection.
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