ABSTRACT
The Japanese encephalitis virus (JEV), a member of the Flaviviridae family and Flavivirus genus, is transmitted by mosquitoes. JEV, of which some 35,000 cases are recorded every year, is a positive RNA virus. Two types of JEV vaccines have been developed to prevent the onset of encephalitis in humans, namely formalin-inactivated and liveattenuated vaccines. JEV inactivated vaccines are usually made using the Nakayama-NIH or Beijing-1 strains of the JEV virus. In this study, the immunological response to the Nakayama-NIH and Beijing-1 strains was analyzed as part of the effort to compile basic data which could lead to the selection of a suitable vaccine strain. To this end, the virus titer of Beijing-1 was found to be two-fold higher than that of Nakayama-NIH by plaque assay. Moreover, Beijing-1-induced neutralizing antibodies showed a higher level of titers when confronted by Korean JEV isolates than Nakayama-NIH-induced neutralizing antibodies (1:320 vs. 1:160, respectively). However, as a minimum ratio of 1:10 neutralizing antibody titers are required to protect against JEV infection, both strains in effect exhibited a sufficient level of neutralizing antibody titers. What's more, Beijing-1 was found to induce a somewhat higher cytotoxic T lymphocyte (CTL) response than Nakayama-NIH. Taken together, this can be taken to mean that Beijing-1 may in fact be a more effective vaccine candidate strain when it comes to inducing a high level of protective immunity against JEV infection.
Subject(s)
Humans , Antibodies, Neutralizing , Asian People , Culicidae , Encephalitis , Encephalitis Virus, Japanese , Encephalitis, Japanese , Flaviviridae , Flavivirus , Lymphocytes , RNA Viruses , Vaccines , Vaccines, Inactivated , Viral LoadABSTRACT
The envelope (E) glycoprotein of JEV is the major antigen to elicit neutralizing antibody (NAb) against JEV infection. In order to develop a rapid and safe neutralization assay system for evaluation of the JEV vaccine strains, we constructed JEV-pseudotyped viruses with JEV env genes (Nakayama-NIH, Beijing-1). The titers of JEV-pseudotyped viruses with NK and BJ strains were 4.0x10(4) IFU/ml and 1.3x10(5) IFU/ml in Vero cell cultures, respectively. We have analyzed the neutralization activity of immunized mouse sera with JEV-NK and JEV-BJ pseudotyped viruses. The neutralizing antibody titers of NK and BJ (50% reduction of virus) were about 1:10,000 at each immunized sera. Compared with conventional plaque reduction neutralization test (PRNT), the method using JEV-pseudotyped virus has desirable advantages such as more rapid, easier, and non-biohazardous. This neutralization assay system might be useful to evaluate NAb activity against JEV vaccine strains or vaccine candidates.
Subject(s)
Animals , Humans , Mice , Antibodies, Neutralizing , Asian People , Encephalitis Virus, Japanese , Encephalitis, Japanese , Genes, env , Glycoproteins , Leukemia Virus, Murine , Neutralization Tests , Vero CellsABSTRACT
Recently the reverse genetics system contributed to the progresses in the investigation of positive-stranded RNA viruses. Here, we report the successful construction of a stable full-length infectious cDNA clone of the live attenuated JEV vaccine strain SA14-14-2. The eleven kilobase viral RNA genome was reverse transcribed, amplified as four overlapping DNA fragments and successively ligated into the low copy number plasmid pACYC184, which contains the p15A origin of replication. In vitro-transcribed RNAs had a specific infectivity of approximately 104 PFU/microgram RNA, and the resulting virus exhibited growth kinetics and plaque morphology similar to the parental virus in cell culture. The structural and functional integrity of the cDNA clone was stably maintained even after at least 150 generations in Escherichia coli strain TOP10. The cDNA clone was engineered to contain single nucleotide change to create a XhoI site and knock out a XbaI site (A to C at nt 9134) acting as a genetic marker. This genetic marker was retained in the recovered progeny virus. Our results suggest that the instability of the full-length infectious JEV cDNA clone can be overcome by employing low copy number plasmid pACYC184. This infectious JEV cDNA clone will aid future studies of pathogenesis, virulence, and replication. Furthermore, it will facilitate the development of SA14-14-2 based recombinant vaccines.
Subject(s)
Humans , Asian People , Cell Culture Techniques , Clone Cells , DNA , DNA, Complementary , Encephalitis Virus, Japanese , Encephalitis, Japanese , Escherichia coli , Family Characteristics , Genetic Markers , Genome , Kinetics , Parents , Plasmids , Replication Origin , Reverse Genetics , RNA , RNA Viruses , RNA, Viral , Vaccines, Synthetic , VirulenceABSTRACT
Recently the reverse genetics system contributed to the progresses in the investigation of positive-stranded RNA viruses. Here, we report the successful construction of a stable full-length infectious cDNA clone of the live attenuated JEV vaccine strain SA14-14-2. The eleven kilobase viral RNA genome was reverse transcribed, amplified as four overlapping DNA fragments and successively ligated into the low copy number plasmid pACYC184, which contains the p15A origin of replication. In vitro-transcribed RNAs had a specific infectivity of approximately 104 PFU/microgram RNA, and the resulting virus exhibited growth kinetics and plaque morphology similar to the parental virus in cell culture. The structural and functional integrity of the cDNA clone was stably maintained even after at least 150 generations in Escherichia coli strain TOP10. The cDNA clone was engineered to contain single nucleotide change to create a XhoI site and knock out a XbaI site (A to C at nt 9134) acting as a genetic marker. This genetic marker was retained in the recovered progeny virus. Our results suggest that the instability of the full-length infectious JEV cDNA clone can be overcome by employing low copy number plasmid pACYC184. This infectious JEV cDNA clone will aid future studies of pathogenesis, virulence, and replication. Furthermore, it will facilitate the development of SA14-14-2 based recombinant vaccines.