Mutations in the 5’ NTR and the Non-Structural Protein 3A of the Coxsackievirus B3 Selectively Attenuate Myocarditogenicity

The 5’ non-translated region (NTR) is an important molecular determinant that controls replication and virulence of coxsackievirus B (CVB)3. Previous studies have reported many nucleotide (nt) sequence differences in the Nancy strain of the virus, including changes in the 5’ NTR with varying degrees of disease severity. In our studies of CVB3-induced myocarditis, we sought to generate an infectious clone of the virus for routine in vivo experimentation. By determining the viral nt sequence, we identified three new nt substitutions in the clone that differed from the parental virus strain: C97U in the 5’ NTR; a silent mutation, A4327G, in non-structural protein 2C; and C5088U (resulting in P1449L amino acid change) in non-structural protein 3A of the virus leading us to evaluate the role of these changes in the virulence properties of the virus. We noted that the disease-inducing ability of the infectious clone-derived virus in three mouse strains was restricted to pancreatitis alone, and the incidence and severity of myocarditis were significantly reduced. We then reversed the mutations by creating three new clones, representing 1) U97C; 2) G4327A and U5088C; and 3) their combination together in the third clone. The viral titers obtained from all the clones were comparable, but the virions derived from the third clone induced myocarditis comparable to that induced by wild type virus; however, the pancreatitis-inducing ability remained unaltered, suggesting that the mutations described above selectively influence myocarditogenicity. Because the accumulation of mutations during passages is a continuous process in RNA viruses, it is possible that CVB3 viruses containing such altered nts may evolve naturally, thus favoring their survival in the environment.

5'非翻译区（non-translated region，NTR）是控制柯萨奇病毒B组3型（coxsackievirus B3，CVB3）复制与致病力的重要分子决定簇。此前已有研究报道了该病毒Nancy毒株的诸多核苷酸（nucleotide，nt）序列差异，其中包括5'NTR区域的改变，且此类改变与不同程度的疾病严重程度相关。在我们针对CVB3诱导心肌炎的研究中，我们旨在构建该病毒的感染性克隆（infectious clone）以用于常规体内实验（in vivo experimentation）。通过测定病毒的核苷酸序列，我们在克隆中发现了三处与亲本病毒毒株不同的新核苷酸替换：5'NTR区域的C97U；非结构蛋白2C（non-structural protein 2C）编码区的沉默突变（silent mutation）A4327G；以及病毒非结构蛋白3A（non-structural protein 3A）区域的C5088U（该突变导致氨基酸改变P1449L），这促使我们评估这些突变对病毒致病特性的影响。我们观察到，感染性克隆衍生的病毒在三种小鼠品系中的致病能力仅局限于胰腺炎，心肌炎的发病率与严重程度均显著降低。随后我们通过构建三个新克隆逆转了上述突变：1）仅携带U97C的克隆；2）仅携带G4327A与U5088C的克隆；3）同时携带上述全部突变的第三组克隆。所有克隆获得的病毒滴度均无显著差异，但第三组克隆衍生的病毒粒子诱导的心肌炎程度与野生型病毒相当，但其诱导胰腺炎的能力并未发生改变，这表明上述突变可选择性影响心肌炎致病力（myocarditogenicity）。由于RNA病毒在传代过程中突变的积累是一个持续过程，携带此类核苷酸改变的CVB3病毒有可能自然演化，进而更利于其在环境中存活。