Genotype 1 Hepatitis E Virus: Synthesis and Phenotypic Studies of Mutant Clouds and Attempts to Develop a Cell Culture System | Shiv Nadar University
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Genotype 1 Hepatitis E Virus: Synthesis and Phenotypic Studies of Mutant Clouds and Attempts to Develop a Cell Culture System

Replication of single-strand positive-sense RNA viruses known to be error-prone (ep), thereby these viruses generate a repertoire of viral genomes, which are linked genetically through mutation are known as quasispecies, also termed as mutant clouds. Genetic variants of mutant cloud interact at functional level and collectively contribute to the characteristics of the virus phenotypic behavior. Complexity and composition of mutant cloud in vivo are thought to contribute to virus adaptability, persistence and drug resistance. Despite constant efforts, no robust cell-culture system has been established for HEV genotype 1 (gt1), due to which molecular mechanisms underlying its life cycle are poorly understood. The aim of this work is to establish a simple and robust molecular method for generation of full-length - RNA mutant clouds (FL-MRC) and their phenotypic characterization. FL-MRC combines the advantages of ep-PCR with reverse genetics to obtain sufficient amounts of mutant clouds. Sar55 strain (HEV gt1) was used to develop FLMRC and study phenotypic behavior of HEV. Mutation rates have increased significantly in amplified full-genomes of imbalanced dNTP ep-PCRs (balanced dNTP vs imbalanced dNTP ep-PCR, p<0.001). Phylogenetic analysis of amplified full-length genomes and gt1- 4 reference sequences allowed us to ascertain the close genetic relationship of full-genomes to Sar55 strain. Ep-PCR amplified and genetic diversified FL HEV-genomes served as templates for the synthesis of mutant clouds. Further, we characterized phenotypic behavior of mutant clouds. When expressed in hepatoma cells, mutant cloud with high complexity and diversity (Cloud-variable) has shown significant differences in terms of viral RNA accumulation (intracellular and extracellular), viral protein expression and host responses compared to cells transfected with mutant clouds of varied levels of genetic diversity (Cloud -large, -moderate, -small) or no diversity (Cloud-null). Hepatoma cells transfected with Cloud-variable showed significantly high amounts of ORF1 protein and dsRNA in immunofluorescence assay. Also, they showed >2 to 10 fold increase in host gene expression levels compared to Cloud-null or Cloud-large. In the second phase of this study, a genotype 1 molecular clone, Kol16, was constructed based on patient fecal virus sequence with the aim to develop an in vitro cell culture system. We identified six mutations in Kol16 sequence, which are reported to be associated with fulminant hepatic failure. We attempted isolation of an adaptive and replicating clone by means of bioselection of a replicating virus among mutant population. Kol16-wt as well as ep RNA derived from Kol16 was found to be replicate transiently in A549 and Huh7.5 cells. However, none of the mutant clones constructed and analyzed promoted HEV replication.

Life Sciences
Student Name: 
Shubhra Agarwal
Faculty Advisor: