Generation Of Mutant Viruses Research Articles

Accurate Measurement of the Effects of All Amino-Acid Mutations on Influenza Hemagglutinin.

Influenza genes evolve mostly via point mutations, and so knowing the effect of every amino-acid mutation provides information about evolutionary paths available to the virus. We and others have combined high-throughput mutagenesis with deep sequencing to estimate the effects of large numbers of mutations to influenza genes. However, these measurements have suffered from substantial experimental noise due to a variety of technical problems, the most prominent of which is bottlenecking during the generation of mutant viruses from plasmids. Here we describe advances that ameliorate these problems, enabling us to measure with greatly improved accuracy and reproducibility the effects of all amino-acid mutations to an H1 influenza hemagglutinin on viral replication in cell culture. The largest improvements come from using a helper virus to reduce bottlenecks when generating viruses from plasmids. Our measurements confirm at much higher resolution the results of previous studies suggesting that antigenic sites on the globular head of hemagglutinin are highly tolerant of mutations. We also show that other regions of hemagglutinin—including the stalk epitopes targeted by broadly neutralizing antibodies—have a much lower inherent capacity to tolerate point mutations. The ability to accurately measure the effects of all influenza mutations should enhance efforts to understand and predict viral evolution.

Role of a single amino acid substitution of VP3 H142D for increased acid resistance of foot-and-mouth disease virus serotype A.

Foot-and-mouth disease virus (FMDV) particles lose infectivity due to their dissociation into pentamers at pH value below 6.5. After the uptake of FMDV by receptor-mediated endocytosis, the acid-dependent dissociation process is required for the release of FMDV genome inside endosomes. Nevertheless, dissociation of FMDV particles in mildly acidic conditions renders the inactivated FMD vaccine less effective. To improve the acid stability of inactivated FMD vaccine during the manufacturing process, a serotype A IND 40/2000 (in-use vaccine strain) mutant with increased resistance to acid inactivation was generated through reverse genetics approach. Based upon the earlier reports, the crucial amino acid residue, H142 of VP3 capsid protein was substituted separately to various amino acid residues Arg (R), Phe (F), Ala (A), and Asp (D) on the full-genome length cDNA clone. While the H142→R or H142→F or H142→A substitutions resulted in non-infectious FMDV, H142→D mutation on VP3 protein (H3142D) resulted in the generation of mutant virus with enhanced resistance to acid-induced inactivation. In addition, H3142D substitution did not alter the replication ability and antigenicity of mutant as compared to the parental virus. However, the virus competition experiments revealed that the H3142D substitution conferred a loss of fitness for the mutant virus. Results from this study demonstrate that the H3142D substitution is the molecular determinant of acid-resistant phenotype in FMDV serotype A.

Vaccination Alters the Balance between Protective Immunity, Exhaustion, Escape, and Death in Chronic Infections

While T cell-based vaccines have the potential to provide protection against chronic virus infections, they also have the potential to generate immunopathology following subsequent virus infection. We develop a mathematical model to investigate the conditions under which T cells lead to protection versus adverse pathology. The model illustrates how the balance between virus clearance and immune exhaustion may be disrupted when vaccination generates intermediate numbers of specific CD8 T cells. Surprisingly, our model suggests that this adverse effect of vaccination is largely unaffected by the generation of mutant viruses that evade T cell recognition and cannot be avoided by simply increasing the quality (affinity) or diversity of the T cell response. These findings should be taken into account when developing vaccines against persistent infections.

Cloning of herpesviral genomes as bacterial artificial chromosomes.

Herpesviruses, which are important pathogens for both animals and humans, have large and complex genomes with a coding capacity for up to 225 open reading frames (ORFs). Due to the large genome size and the slow replication kinetics in vitro of some herpesviruses, mutagenesis of viral genes in the context of the viral genome by conventional recombination methods in cell culture has been difficult. Given that mutagenesis of viral genes is the basic strategy to investigate function, many of the herpesvirus ORFs could not be defined functionally. Recently, a completely new approach for the construction of herpesvirus mutants has been developed, based on cloning of the virus genome as a bacterial artificial chromosome (BAC) in E. coli. This technique allows the maintenance of viral genomes as a plasmid in E. coli and the reconstitution of viral progeny by transfection of the BAC plasmid into eukaryotic cells. Any genetic modification of the viral genome in E. coli using prokaryotic recombination proteins is possible, thereby allowing the generation of mutant viruses and facilitating the analysis of herpesvirus genomes cloned as infectious BACs. In this review, we describe the principle of cloning a viral genome as a BAC using murine gammaherpesvirus 68 (MHV-68), a mouse model for gammaherpesvirus infections, as an example.