br Introduction Japanese encephalitis JE is an acute infecti
Introduction Japanese encephalitis (JE) is an acute infectious disease of the nervous system caused by Japanese encephalitis virus (JEV), which is primarily transmitted through mosquitoes. There are around 68,000 cases of JE annually worldwide, resulting in a mortality rate of approximately 10–30%. Among survivors, around 30–50% exhibit severe neurological sequelae . JE vaccination is the most effective and economical method of preventing JE. Since its approval in 1989, a live attenuated JE vaccine has been widely used in many countries, including China, South Korea, Nepal, India, and Thailand, playing an important role in controlling JE outbreaks. The live attenuated JEV vaccine strain, SA14-14-2, was derived from the virulent strain SA14, which was isolated from Culex pipiens mosquito larvae in Xi'an, China in 1954. The virulent strain was passaged for more than 100 generations in animals and primary hamster kidney (PHK) 2-NBDG and subjected to plaque purification multiple times, followed by several passages in the non-neural tissues of laboratory animals to obtain the attenuated vaccine strain with attenuated neurovirulence, high immunogenicity, and stable genetic characteristics [2,3]. In China, South Korea, Nepal, India, Thailand, Vietnam, Myanmar, and other Southeast Asian countries, large-scale human clinical studies have been conducted and demonstrated that the vaccine is very safe. Since its approval in 1989, more than one billion doses of the vaccine have been used, and no vaccine-related encephalitis cases have been reported [2,, , , , , , , , , ]. However, whether the large-scale use of the live attenuated JE vaccine will lead to the dissemination of the vaccine virus in the environment and whether reversion of the neuroattenuation of the virus will occur during the transmission process remain major concerns for some researchers. Therefore, this study was conducted to investigate whether immunization with the live attenuated JE vaccine could lead to the spread of the vaccine virus into the environment through mosquito biting and virus amplified in pigs resulting in adverse effects.
Material and methods
Discussion Mosquitoes are the transmission vectors of JE virus, with Cx. tritaeniorhynchus being the primary vector species. The virus spreads following proliferation in mosquitoes and direct infection of humans through mosquito bites, or by transmission and proliferation in pig, which is the main reservoir host of JEV. Virus proliferation in reservoir hosts produces viremia, and when mosquitoes bite infected pigs, the proliferated virus can be transmitted to humans through subsequent bites. In a previous study, Zhang et al. orally infected Cx. tritaeniorhynchus with the JE virus SA14-14-2 to evaluate the safety of the JE vaccine . When the virus meals containing 6.06 Log10 pfu/ml SA14-14-2 virus, no virus could be detected in Culextritaeniorhynchus and Culex pipiens quinquefascitatus of mosquitoes at any times. Even when it contained higher level of vaccine virus (6.18Log10 pfu), low titers (1.11 and 1.24 Log10 pfu respectively) of the virus could be detected in one mosquito pool in each of the###http://www.apexbt.com//media/diy/images/struct/B1406.png#### two species (34 pools Culextritaeniorhynchus and 5 pools Culex pipiens quinquefascitatus respectively). In contract, when the wild strain of JE virus was used for ingestion, high frequency of the mosquito pools (14/19) with high virus titers (3.8–4.79Log10 pfu) were detected. The results indicate that the attenuated virus is highly restricted in its ability to infect and replicate in both JE mosquito vectors. It demonstrates the safeguards against secondary spread of the vaccine virus by the JE mosquito vectors .In another study, infection of Cx. tritaeniorhynchus by intrathoracically (i.t.) resulted in replication of the SA14-14-2 strain in mosquitoes . Nevertheless, inoculation of a virus suspension from infected mosquitoes into suckling mouse brains or direct infection of mice through bites from infected mosquitoes resulted in the survival of all mice. In the latter study, among the viruses harvested from inoculated mosquitoes, only one nucleotide at position 1340 in the E-region exhibited an A→G substitution that resulted in a change in the encoded amino acid, E447 D (Asp)→G (Gly); however, this position is not a key site for mutations involved in neurovirulence attenuation . These studies showed that after the replication of JEV in mosquitoes, the virulence of SA14-14-2 was still attenuated, indicating that the JE live attenuated vaccine does not exhibit reversion of neuroattenuation after proliferation in mosquitoes. However, the previous study only provided the E gene sequence of the SA14-14-2 passaged virus in mosquitoes, and there was no comment on whether the genes in the other genome proteins of SA14-14-2 virus changed or not. Meanwhile there may be cases of amplification of the JE virus in pigs bitten by mosquitoes carrying the JE virus, followed by transmission to mosquitoes through additional mosquito bites and subsequent infection of humans. Therefore, we performed this study to simulate this JEV transmission process to evaluate the environmental safety of JE live attenuated vaccine SA14-14-2. After passaging the SA14-14-2 vaccine in mosquitoes, pigs were inoculated with the passaged virus. The virus was subsequently isolated from pigs with viremia and again inoculated into mosquitoes to investigate whether the virus can be transmitted or neuroattenuation phenotype restored via replication in mosquitoes and pigs. In order to assure the infection ability, we amplified the virus harvest from mosquitoes, pigs viremia in BHK21 cell to obtain viruses with higher titer.