Biological and genetic properties of SA14-14-2, a live-attenuated Japanese encephalitis vaccine that is currently available for humans

Abstract

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is a major cause of acute encephalitis, a disease of significance for global public health. In the absence of antiviral therapy to treat JEV infection, vaccination is the most effective method of preventing the disease. In JE-endemic areas, the most widely used vaccine to date is SA(14)-14-2, a live-attenuated virus derived from its virulent parent SA(14). In this study, we describe the biological properties of SA(14)-14-2, both in vitro and in vivo, and report the genetic characteristics of its genomic RNA. In BHK-21 (hamster kidney) cells, SA(14)-14-2 displayed a slight delay in plaque formation and growth kinetics when compared to a virulent JEV strain, CNU/LP2, with no decrease in maximum virus production. The delay in viral growth was also observed in two other cell lines, SH-SY5Y (human neuroblastoma) and C6/36 (mosquito larva), which are potentially relevant to JEV pathogenesis and transmission. In 3-week-old ICR mice, SA(14)-14-2 did not cause any symptoms or death after either intracerebral or peripheral inoculation with a maximum dose of up to 1.5×10(3) plaque-forming units (PFU) per mouse. The SA(14)-14-2 genome consisted of 10977 nucleotides, one nucleotide longer than all the previously reported genomes of SA(14)-14-2, SA(14) and two other SA(14)-derived attenuated viruses. This difference was due to an insertion of one G nucleotide at position 10701 in the 3 noncoding region. Also, we noted a significant number of nucleotide and/or amino acid substitutions throughout the genome of SA(14)-14-2, except for the prM protein-coding region, that differed from SA(14) and/or the other two attenuated viruses. Our results, together with others', provide a foundation not only for the study of JEV virulence but also for the development of new and improved vaccines for JEV.

Genetic and Phenotypic Properties of Vero Cell-Adapted Japanese Encephalitis Virus SA14-14-2 Vaccine Strain Variants and a Recombinant Clone, Which Demonstrates Attenuation and Immunogenicity in Mice

“…Recombinant JEVmut14-14-2 also produced small plaques but had a moderately lower mean peak titer of 6.4 log 10 PFU/mL (Table 3), which is consistent with the titer reported for JEV SA14-14-2 in baby hamster kidney cells. 48 Recombinant JEVmut14-14-2-C292T, with reversion mutation C-S66L, replicated to a significantly higher mean peak titer of 7.1 log 10 PFU/mL, similar to most JEV SA14-14-2 variants (Table 3). The JEV SA14-14-2 variant 3 IFN had a single unique reversion mutation, NS2A-V40A, relative to JEV SA14-14-2 variant D ( Table 1) that significantly increased mean peak virus titer ( Table 3).…”

“…JN604986) of a commercial batch of JEV SA14-14-2 vaccine (Table 1). 48 Careful examination of the sequence chromatograms revealed that there were also minor secondary peaks at several positions throughout the genome indicating there was a mixture of nucleotides and predicted amino acid substitutions (Table 1). Biological cloning of this virus stock in Vero cells resulted in the isolation of six genetically distinct, Vero cell-adapted variants (A, B, C, D, G, and H).…”

“…The polyprotein amino acid sequence of rJEVmut14-14-2 was designed to closely match the sequence of a commercial batch of vaccine virus (Table 2). 48 Other JEV SA14-14-2 full genome sequences that have been published are shown in Table 2 for comparison and differ considerably from one another. Recombinant JEVmut14-14-2 was recovered and biologically cloned in Vero cells and the sequence of this virus matched that of the cDNA clone.…”

“…18 In addition, several laboratories have sequenced the JEV SA14-14-2 vaccine strain over the last 20 years with significantly different results 27,28,48 (Table 2), suggesting that the vaccine seed virus is not clonal, and this could affect attenuation and immunogenicity between vaccine lots. It was for these reasons that we initiated the development of a second-generation, recombinant JEV SA14-14-2 live-attenuated vaccine produced in WHO qualified Vero cells.…”

“…The cDNA clone of WT rJEV India/78, which has been described previously, 47 was modified to closely match the polyprotein amino acid sequence of the JEV SA14-14-2 GenBank reference strain (accession no. JN604986) 48 and was designated pJEVmut14-14-2. Synthesized cDNA gene segments encoding point mutations were cloned into pJEV India/78 (Figure 1).…”