目的  柯萨奇病毒A组6型（Coxsackievirus A6，CVA6）是近几年来引起手足口病（Hand-foot-and-mouth disease，HFMD）的主要病原体之一，其致病因素尚未阐明，无特异性抗病毒药物及预防疫苗上市。本研究对CVA6 代表株的基因型、核苷酸与氨基酸差异、一步生长曲线、致病性、遗传稳定性、免疫原性等生物学特性进行初步探究，为CVA6疫苗的相关研究提供一定的实验依据。

方法 

1. 利用RD、Vero和KMB17细胞对2009年～2017年云南昆明地区和湖北襄阳地区327份HFMD病人的粪便及肛拭子临床标本进行病毒分离，利用MEGA7.0软件对鉴定为CVA6的分离株基于全长VP1核酸序列进行种系进化分析。

2. CVA6 RD细胞适应株分别在Vero细胞和KMB17细胞上连续传代适应并进行蚀斑纯化。

3. 以收样时间为横坐标，病毒感染性滴度为纵坐标，绘制10个CVA6毒株在KMB17细胞适应前和适应后的一步生长曲线（MOI=1）。

4. 筛选出感染性滴度较高的CVA6 RD细胞适应株及其KMB17适应株，分别以6.5 lgCCID₅₀剂量接种至1日龄BALB/c乳鼠颅内。根据乳鼠出现的临床特征初步评估毒株对乳鼠的致病性强弱。

5. 从以上筛选出致病力较强的两个CVA6毒株，分别以6.5 lgCCID₅₀、5.5 lgCCID₅₀和4.5 lgCCID₅₀剂量接种至1日龄BALB/c乳鼠颅内，根据乳鼠的平均体重、临床评分、生存率及乳鼠各组织的病理改变、免疫组化、病毒载量结果，比较毒株在KMB17细胞适应前和适应后对乳鼠的致病性差异。

6. 将CVA6毒株在KMB17细胞上传至15代，对P1、P5、P10、P15子代病毒的全基因组进行测序，分析毒株在KMB17细胞中的遗传稳定性。

7. 将KYN-A1205活病毒浓缩后与弗氏完全佐剂1：1乳化混匀后，3次免疫8周龄KM鼠，检测小鼠抗血清的中和效价。

结果

1. 经过三种细胞分离，仅从RD细胞中获得37个CVA6 毒株，均为D3基因亚型，未获得其它细胞适应株。

2. 经过Vero细胞和KMB17细胞分别传代适应，获得10个CVA6 KMB17细胞适应株，未获得Vero细胞适应株。

3. 7个感染性滴度较高的CVA6 RD细胞适应株中，RYN-A1205、RYN-N15、RYN-A13、RXY4051对乳鼠有致病性，且RXY4051毒株毒力最强；经KMB17细胞适应后，仅有KYN-A1205毒株使得乳鼠出现临床症状。

4. 经过筛选，将RXY4051、RYN-A1205 毒株在KMB17细胞适应前和适应后分别感染乳鼠；结果显示RXY4051毒株经KMB17细胞适应后丧失了对乳鼠的致病性；而RYN-A1205毒株在KMB17细胞适应前和适应后毒力无明显差别，均对乳鼠有致病性。

5. RXY4051、RYN-A1205和KYN-A1205毒株感染后，乳鼠的肾脏、肺脏、小肠、前肢肌肉和后肢肌肉都有明显的抗原分布，出现肌纤维坏死和核碎裂等明显的病理改变，肌肉组织病毒载量比其余组织高出10^3.3～10^4.3拷贝/mg。

6. 经过全基因组比对分析发现，RXY4051毒株在KMB17细胞适应后发生了多个氨基酸替换，且适应前和适应后核苷酸与氨基酸同源性分别为94.2%和97.9%；而RYN-A1205毒株在KMB17细胞适应前和适应后核苷酸与氨基酸同源性分别为99.80%和99.40%；KYN-A1205毒株的4代病毒的核苷酸与氨基酸序列同源性分别为99.97%～100%和99.90%～100%。

7. KYN-A1205毒株免疫小鼠后，小鼠的抗血清能完全中和另外9个CVA6 KMB17细胞适应株。

结论

1. 成功获得10个CVA6 KMB17细胞适应株。

2. 毒株在不同细胞适应后毒力不同。

3. 成功筛选到一株致病性强、免疫原性和遗传稳定性良好的CVA6 KMB17细胞适应株KYN-A1205，可作为实验性疫苗候选株。

Objective Coxsackievirus A6 (CVA6) is one of the main pathogens causing hand-foot -and-mouth disease (HFMD) in recent years. Its pathogenic factors have not been elucidated and no specific antiviral drugs and preventive vaccines are launched. In this study, the biological characteristics such as genotype, nucleotide and amino acid difference, one-step growth curve, pathogenicity, genetic stability, and immunogenicity of the CVA6 representative strains were preliminarily explored to provide reference for the relevant research of the CVA6 vaccine.

Method

1. RD, Vero and KMB17 cells were used to isolate 327 clinical specimens of feces and anal swabs from 2009-2017 in Kunming, Yunnan and Xiangyang, Hubei, in China. MEGA7.0 software was used to performe the phylogenetic analysis of the strains identified as CVA6 based on the full-length VP1 nucleotide sequence.

2. CVA6 RD cell isolates were adapted and purified on Vero and KMB17 cells, respectively.

3. The one-step growth curves (MOI=1) of ten CVA6 strains before and after KMB17 cells adaptation were plotted using the time of sample collection as the abscissa and the viral titers as the ordinate.

4. The CVA6 RD cell-adapted strains with high infectious titers and their KMB17 cell-adapted strains were screened, and the pathogenicity were preliminarily evaluated based on the clinical features of the nursing mice after 6.5 lgCCID₅₀ dose inoculation to the cranial cavity of 1-day-old BALB/c suckling mice.

5. Two highly pathogenic CVA6 strains were screened from the above, and were inoculated into the cranial cavity of 1-day-old BALB/c at 6.5 lgCCID₅₀, 5.5 lgCCID₅₀ and 4.5 lgCCID₅₀ doses, respectively. The pathogenicity differences of CVA6 strains before and after KMB17 cells adaptation were compared according to the the results of average weight, clinical score, survival rate, pathological changes, immunohistochemical and viral loads of the suckling mice.

6. CVA6 was uploaded to KMB17 cells for 15 generations, and the whole genomes of P1, P5, P10 and P15 progeny viruses were sequenced to analyze the genetic stability of this strain in KMB17 cells.

7. After KYN-A1205 was concentrated and mix it with Freund's complete adjuvant in equal volume, then immunized 8-week-old KM mice for 3 times, and measured the neutralizing titers of anti-serum of mice.

Results

1. After three cells isolation, only 37 CVA6 RD cell-adapted strains were obtained, all of which were D3 gene subtypes，and no other cell-adapted strains were obtained.

2. Only 10 CVA6 KMB17 cell-adapted strains were obtained through passage adaptation in Vero and KMB17 cells, but no Vero cell-adapted strains were obtained.

3. Among the 7 RD cell-adapted strains with high infectious titers, RYN-A1205, RYN-N15, RYN-A13, RXY4051 were pathogenic to suckling mice, and strain RXY4051 had the strongest virulence. After KMB17 cell adaptation, only strain KYN-A1205 caused symptoms and even partial death in suckled mice.

4. After screening, strains RXY4051 and RYN-A1205 were tested before and after KMB17 cells adaptation, respectively. It was found that strain RXY4051 with different doses lost their pathogenic ability to suckling mice after the adaptation of KMB17 cell. However, strain RYN-A1205 caused disease in suckling mice before and after KMB17 cells adaptation, and there was no significant difference in virulence.

5. After strains RXY4051, RYN-A1205 and KYN-A1205 strains were challenged, the kidneys, lungs, small intestine, forelimb muscles and hindlimb muscles of the suckling mice had obvious antigen distribution, and the forelimb and hind limb muscles showed obvious pathological changes such as muscle fiber necrosis and nuclear fission. The viral load of muscle tissue is 10^3.3~10^4.3 copies/mg higher than other tissues.

6. Whole-genome comparison showed that strain RXY4051 had multiple amino acid substitutions after KMB17 cell adaptation, and the homology of nucleotides and amino acids before and after adaptation were 94.2% and 97.9%. The homology of nucleotides and amino acids of KYN-A1205 before and after adaptation were 99.80% and 99.40%. The nucleotide and amino acid sequence homology of strain KYN-A1205 was 99.97%~100% and 99.90%~100%.

7. After immunization with KYN-A1205, the antiserum of the mice can completely neutralize the other 9 CVA6 KMB17 cell-adapted strains.

Conclusion

1. Successfully obtained 10 CVA6 KMB17 cell-adapted strains.

2. The strains have different virulences after adapting to different cells.

3. A CVA6 KMB17 cell-adapted strain KYN-A1205 with strong pathogenicity, good immunogenicity and genetic stability was successfully screened, which can be used as a candidate for experimental vaccine.