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Muhammad Naveed Anwar, Speaker at Vaccines Conferences
Chinese Academy of Agricultural Sciences, Pakistan
Title : Phenotypic and Genotypic Comparison of a Live Attenuated Genotype I Japanese Encephalitis Virus SD12 F120 Strain with Its Virulent Parental SD12 Strain in vitro in vivo


Japanese encephalitis (JE) is a vector-borne zoonotic viral disease caused by (Japanese encephalitis virus, JEV). Vaccination is the most effective way to control JE in both humans and pigs. However, JEV genotype shift that the dominant genotype III (GIII) has been replaced by genotype I (GI) raised concerns about the effectiveness of GIII-derived vaccines against the GI strain infection. Indeed, GIII-derived vaccine showed a reduced protection against GI strain challenge, suggesting a potential need of development of GI-derived vaccine. In this study, a comparative analysis of the phenotypic and genotypic properties of an attenuated GI strain (SD12-F120) and its virulent parental strain (SD12) was performed. To characterize the attenuated GI strain SD12-F120, the phenotypic and genotypic characteristics of SD12-F120 with its virulent parental SD12 strain were compared in vitro and in vivo. SD12-F120 formed smaller plaque on BHK-21 cells and showed the reduced replication in mouse primary neuron cells and mouse brains as compared with SD12. Mice inoculated with SD12-F120 up to 105 PFU via either intraperitoneal or intracerebral route showed no clinical signs of JEV infection, indicating highly attenuated phenotype in terms of both neuroinvasiveness and neurovirulence. Immunization of mice with SD12-F120 provided a complete protection against SD12 challenge. Comparison of genome variations between SD12-F120 and SD12 revealed that SD12-F120 harbored 29 nucleotide variations, of which 20 were considered silent nucleotide mutations, while 9 resulted in eight amino acid substitutions: two in E, one in NS1, two in NS3, one in NS4B and two in NS5 proteins. Comparison of the amino acid variations of SD12-F120 vs SD12 pair with those from other four isogenic pairs of the attenuated and their virulent parental strains revealed that the numbers and positions of amino acid variations were different from each other. Out of the five pairs, the variations at E138 and E176 positions of E protein were identified in four and three pairs, respectively. The remained amino acid variations were almost unique to their respective strain pairs, suggesting that the genetic changes acquired during the attenuation process were likely to be strain specific and that the mechanisms associated with JEV attenuation/virulence were complicated. The SD12-F120 was obtained by serial passage of its virulent parental SD12 strain on BHK-21 cells, which was not appropriated for vaccine production. Therefore, SD12-F120 was further passaged on Vero cells for 20 passages and a Vero cell-adapted strain SD12-F120VC was obtained. SD12-F120VC had an increased in virus titers compared to early passages, showing an adaptation to Vero cells. The plaque morphology of SD12-F120VC was same as that of SD12-F120. The animal experiments showed that SD12-F120VC had attenuation phenotype in suckling mice. Vaccination of mice with SD12-F120VC completely protected vaccinated mice against challenge with SD12 strain, but failed to provide the vaccinated mice complete protection against the challenge of GIII N28 strain. The neutralizing antibodies titer of immunized mice against SD12-F120VC and SD12 was higher than that of heterologous N28 strain. SD12-F120VC harbored 6 amino acid substitutions in prM protein, of which 5 were present in the pr domain of prM. These amino acid substitutions may be involved in the adaptation of SD12-F120 to Vero cells. Overall, the phenotypic and genotypic characteristics of SD12-F120 with its virulent parental SD12 strain were compared in vitro and in vivo, and the protection of the attenuated strains was determined in mice. The outcome would be useful for development of GI-derived vaccine.


Muhammad Naveed Anwar belongs to Pakistan. He finished his Ph.D. in Preventive Veterinary Science (Microbiology) from the Chinese Academy of Agricultural Sciences(CAAS), Beijing, China in August 2020 and doctoral thesis title is “Characterization of Live-Attenuated genotype 1 strain of Japanese encephalitis virus”. Moreover, he also worked on several relevant projects which include: A Novel recombinant VLP vaccine displaying B and T cells epitopes of JEV; Adaptation of Live-Attenuated JEV vaccine to Vero cells in association with mutations to structural proteins, published his Ph.D. research work in "Viruses" and "Virus research" journals and his current VLP vaccine paper has been published in “Vaccines". His main research interest is in virology, immunology and virus-host interaction. He is an innovative, goal-oriented person who possesses a good analytical approach. Currently he is finding a post-doc position where he would relish his past experience for the benefit of a fruitful outcome.