Mutations in the NS3 gene and 3′-NCR of Japanese encephalitis virus isolated from an unconventional ecosystem and implications for natural attenuation of the virus

The T1P1 strain of Japanese encephalitis (JE) virus was recently isolated from paddy-free Liu-Chiu Islet in which natural JE antibody has been prevalent. In mouse neuroblastoma-derived Neuro-2a cells, T1P1 appeared significantly lower in virus productivity than another local isolate, CH 1392. It implied that this new isolate possesses a characteristic viral replication pattern other than that of CH 1392. T1P1 has also shown lower neurovirulence, which was reflected by a significantly higher LD₅₀ (2.44 × 10⁶ PFU) than CH 1392 (2.87 × 10² PFU). In comparison of the full-length RNA sequences between T1P1 and CH 1392, a total of 7 nucleotides, including 1 in preM/M and 2 each in NS3, NS5, and the 3′-end noncoding region (NCR), appeared different. Of them, only the changes in NS3 (position 325, T for CH 1392, A for T1P1; and position 364, G for CH 1392 and A for T1P1) resulted in substitutions of deduced amino acids. There were two additional nucleotide changes appearing in the 3′-NCR. The amino acids 109 Phe and 122 Glu in NS3 of CH 1392 were substituted by Ile and Lys, respectively, in T1P1. The unique growth properties and low virulence of T1P1 presented in this report were likely related to abnormal enzymatic activity due to mutations of the NS3 gene (especially position 364) and possibly to the mutations in the 3′-NCR. The natural attenuation of T1P1 that has been circulating in paddy-free Liu-Chiu Islet may account for the absence of clinical JE cases in past years.