寨卡病毒E蛋白和NS1蛋白关键氨基酸突变可促进病毒在小鼠睾丸中的复制

Key mutations in the envelope and NS1 proteins of Zika virus contribute to enhanced viral replication in mouse testis

寨卡病毒(Zika virus,ZIKV)是一种蚊媒黄病毒,也可通过性传播,同时还能感染睾丸组织并导致损伤.探究寨卡病毒在睾丸内持续复制过程中病毒基因的变化规律,发现潜在的睾丸适应性突变位点,可为寨卡病毒变异的风险评估提供重要参考.为此,本研究以寨卡病毒株FSS13025为研究对象,通过将其在A129小鼠睾丸中连续传代10轮后获得适应性传代毒株,将原始株(WT)、第5代(P5)和第10代(P10)病毒经腹腔接种A129小鼠后发现,睾丸中P5和P10的RNA载量较WT分别提高4.6和3.8倍;进一步通过深度测序分析P5和P10的基因组变异情况,结果显示二者的包膜蛋白(envelope,E)和非结构蛋白1(nonstructural protein 1,NS1)分别出现H401Y和K146E突变.在此基础上,以寨卡病毒FSS13025株为基因骨架,利用反向遗传学技术构建拯救携带上述突变的重组病毒H401Y+K146E,并评价其在细胞和小鼠体内的复制能力,结果显示病毒感染睾丸细胞15P-1和TM3后第48 h,细胞培养上清中的重组病毒RNA含量分别是WT的5.2和2.1倍;经腹腔途径感染A129小鼠后的第3和6天,睾丸中重组病毒RNA载量较WT分别提高97.5和58.4倍.上述结果表明,H401Y+K146E联合突变显著增强了寨卡病毒在睾丸细胞中的复制能力,同时明显提升了其对睾丸的嗜性和侵袭力,上述位点可作为寨卡病毒变异风险预警的监测靶点.

Zika virus(ZIKV)belongs to the Flavivirus genus within the Flaviviridae family,which includes other important mosquito-borne human pathogens,including dengue(DENV),West Nile(WNV),Japanese encephalitis(JEV),and yellow fever viruses(YFV).ZIKV was initially isolated from a sentinel rhesus monkey in the Zika forest,Uganda in 1947.It received little attention until it caused the first notable human epidemic in the Pacific Ocean in 2007 followed by another major outbreak in French Polynesia in 2013.Recently,due to its association with Guillain-Barrésyndrome(GBS)in adults and congenital Zika syndrome(CZS)in infants born to ZIKV-infected mothers,the biggest ZIKV outbreak that began in Brazil in 2015 was declared a Public Health Emergency of International Concern by the World Health Organization on 1 February 2016.During this epidemic,ZIKV infections were reported in over 80 countries worldwide with more than 1.3 million infections in the Americas alone.Currently,there are no antiviral drugs or vaccines available for ZIKV infection.ZIKV has also been shown to be transmitted among humans through male-to-female sexual contact.There is strong evidence that the male reproductive tract acts as a significant reservoir for ZIKV.Both infectious ZIKV and ZIKV RNA have been identified in the semen of ZIKV-infected men.The presence of viral RNA in human semen can persist for up to 370 days following the onset of symptoms.The relationship between the intensity and duration of viremia and the invasion of the genital tract by ZIKV,viral shedding in semen,and the subsequent risk of sexual transmission from males to females or males to males remains unclear.Exploring the changes of ZIKV genes during continuous replication in testes and identifying potential testicular adaptive mutations can provide an important reference for assessing the risk of ZIKV mutations.Here,the FSS13025 strain of ZIKV(named WT)was serially passaged in testes of A129 mice for 10 times.Analysis using qRT-PCR revealed that levels of viral RNA in testes of mice infected with passage 5(P5)or P10 were significantly 4.6 and 3.8 times higher than those of WT,respectively.Deep sequencing analysis of the genomes of P5 and P10 identified mutations H401Y in the envelope protein and K146E in the NS1 protein.To further test the biological functions of the mutations,a recombinant virus carrying both H401Y and K146E mutations(named H401Y+K146E)was rescued using the FSS13025 strain as the genetic backbone by reverse genetics.H401Y+K146E showed an increased replication in multiple cell lines including testicular cell lines 15P-1 and TM3 compared to WT.Importantly,the replication of H401Y+K146E was significantly enhanced specifically in testes of A129 mice in comparison with WT.This study identifies two testisadapted mutations of ZIKV,which confer enhanced viral replication in mouse testis.These two functional mutations have important implications for assessing the risk of ZIKV evolution in the future.