| Summary: | HSV-2 (Herpes simplex virus type 2) is a critical viral agent that mainly causes genital herpes and life-long latent infection in the dorsal root ganglia. Gene modification via CRISPR/Cas9 Clustered regularly interspaced short palindromic repeat sequences/CRISPR associated 9) was used here to construct HSV-2 mutant strains through the deletion of fragments of the <i>RL1</i> (Repeat Long element 1) and/or <i>LAT</i> (Latency-associated Transcript) genes. The HSV-2 mutant strains <i>LAT-</i>HSV-2 and <i>RL1-LAT-</i>HSV-2 present different biological properties. The proliferation of <i>RL1-LAT-</i>HSV-2 in nerve cells was decreased significantly, and the plaques induced by <i>RL1-LAT-</i>HSV-2 in Vero cells were smaller than those induced by <i>LAT-</i>HSV-2 mutant and wild-type strains. The observation of mice infected with these two mutants compared to mice infected with the wild-type strain indicated that the mutant <i>RL1-LAT-</i>HSV-2 has an attenuated phenotype with reduced pathogenicity during both acute and latent infections and induces a stronger specific immune response than the wild-type strain, whereas the attenuation effect was not found in mice infected with the <i>LAT-</i>HSV-2 mutant containing the <i>LAT</i> gene deletion. However, the simultaneous mutation of both the <i>RL1</i> and <i>LAT</i> genes did not completely restrict viral proliferation in nerve cells, indicating that multiple HSV genes are involved in viral replication in the neural system. This work suggests that the HSV-2 genes RL1 and/or LAT might be involved in the virulence mechanisms in mouse infections.
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