Differential replication efficiencies between Japanese encephalitis virus genotype I and III in avian cultured cells and young domestic ducklings

Japanese encephalitis virus (JEV) genotype dominance has shifted to genotype I (GI) from genotype III (GIII) in China as demonstrated by molecular epidemiological surveillance. In this study, we performed a serological survey in JEV-non-vaccinated pigs to confirm JEV genotype shift at the sero-epidemiological level. The average ratio of GI/GIII infection was 1.87, suggesting co-circulation of GI and GIII infections with GI infection being more prevalent in pigs in China. To gain an insight into the reasons for this JEV genotype shift, the replication kinetics of seven recently-isolated JEV isolates including three GI strains and four GIII strains were compared in mosquito C6/36 cells, chicken fibroblast cells (DF-1) and porcine iliac artery endothelial cells (PIEC). We observed that GI strains replicated more efficiently than GIII strains in DF-1 and PIEC cells, particularly in DF-1 cells with titers reaching 22.9–225.3 fold higher than GIII strains. This shows an enhanced replication efficiency of GI viruses in avian cells. To examine this enhanced replication efficiency in vivo, young domestic ducklings were used as the animal model and inoculated with GI and GIII strains at day 2 post-hatching. We observed that GI-inoculated ducklings developed higher viremia titers and displayed a comparatively longer viremic duration than GIII-inoculated ducklings. These results conform to the hypothesis of an enhanced replication efficiency for GI viruses in birds. There are 36 amino acid differences between GI and GIII viruses, some of which may be responsible for the enhanced replication efficiency of GI viruses in birds. Based on these findings, we speculated that the enhanced replication of GI viruses in birds would have resulted in higher exposure and therefore infection in mosquitoes, which could result in an increased transmission efficiency of GI viruses in the birds-mosquitoes-birds enzootic transmission cycle, thereby contributing to JEV genotype shift.

经分子流行病学监测证实，中国境内的日本脑炎病毒（Japanese encephalitis virus, JEV）基因型优势株已从Ⅲ型（genotype III, GIII）转变为Ⅰ型（genotype I, GI）。本研究针对未接种JEV疫苗的猪只开展血清学调查，旨在从血清流行病学层面验证JEV的基因型转换现象。GI与GIII感染的平均比值为1.87，表明GI与GIII感染在中国猪群中共同流行，且GI感染更为普遍。为探究此次JEV基因型转换的潜在原因，本研究对7株近期分离的JEV分离株（含3株GI毒株与4株GIII毒株）分别在蚊虫C6/36细胞、鸡成纤维细胞（DF-1）及猪髂动脉内皮细胞（PIEC）中的复制动力学进行了对比分析。结果显示，GI毒株在DF-1细胞与PIEC细胞中的复制效率显著高于GIII毒株，尤其在DF-1细胞中，GI毒株的病毒滴度较GIII毒株高出22.9~225.3倍。该结果表明GI型JEV在禽类细胞中的复制效率得到增强。为在体内验证该复制效率增强现象，本研究以孵化后2日龄的家鸭雏鸭作为动物模型，分别接种GI与GIII毒株。观测结果显示，接种GI毒株的鸭雏病毒血症滴度更高，且病毒血症持续时间较接种GIII毒株的鸭雏更长。上述结果印证了GI型JEV在禽类体内复制效率提升的假说。GI与GIII型JEV之间存在36个氨基酸差异，其中部分差异可能与GI型JEV在禽类体内的复制效率提升有关。基于上述研究结果，本研究推测：GI型JEV在禽类体内复制效率的增强，会使蚊虫更易接触并感染病毒，进而提升GI型JEV在"禽类-蚊虫-禽类"自然循环传播链中的传播效率，最终推动JEV的基因型转换。