A Point Mutation in a Herpesvirus Polymerase Determines Neuropathogenicity

Abstract

Infection with equid herpesvirus type 1 (EHV-1) leads to respiratory disease, abortion, and neurologic disorders in horses. Molecular epidemiology studies have demonstrated that a single nucleotide polymorphism resulting in an amino acid variation of the EHV-1 DNA polymerase (N752/D752) is significantly associated with the neuropathogenic potential of naturally occurring strains. To test the hypothesis that this single amino acid exchange by itself influences neuropathogenicity, we generated recombinant viruses with differing polymerase sequences. Here we show that the N752 mutant virus caused no neurologic signs in the natural host, while the D752 virus was able to cause inflammation of the central nervous system and ataxia. Neurologic disease induced by the D752 virus was concomitant with significantly increased levels of viremia ( p = 0.01), but the magnitude of virus shedding from the nasal mucosa was similar between the N752 and D752 viruses. Both viruses replicated with similar kinetics in fibroblasts and epithelial cells, but exhibited differences in leukocyte tropism. Last, we observed a significant increase ( p < 0.001) in sensitivity of the N752 mutant to aphidicolin, a drug targeting the viral polymerase. Our results demonstrate that a single amino acid variation in a herpesvirus enzyme can influence neuropathogenic potential without having a major effect on virus shedding from infected animals, which is important for horizontal spread in a population. This observation is very interesting from an evolutionary standpoint and is consistent with data indicating that the N752 DNA pol genotype is predominant in the EHV-1 population, suggesting that decreased viral pathogenicity in the natural host might not be at the expense of less efficient inter-individual transmission.

Author Summary

Equid herpesvirus type 1 (EHV-1), a close relative of varicella-zoster virus and herpes simplex virus of humans, is spread by aerosol and is the causative agent of the most common neurologic disease of horses. Outbreaks of the neurologic form of EHV-1 can be devastating to individual animals and entire herds, and approximately one-third of the affected horses generally are at risk of death or suffer so extensively that euthanasia becomes necessary. Our report provides evidence for a direct causal link between the genotype of EHV-1 strains and their neurovirulence, and thereby gives a long-awaited explanation for the conundrum of the different clinical outcomes following EHV-1 infection. We proved that alteration of one amino acid in the key viral enzyme, DNA polymerase, which is conserved in all herpesviruses, renders the virus unable to cause neurologic disease. The improved clinical outcome is likely due to the reduction in virus levels in the bloodstream, ultimately resulting in less virus reaching the central nervous system. In summary, our study shows that herpesvirus virulence and tissue tropism in the natural host are linked with the function of a key virus-encoded enzyme involved in DNA replication.