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. 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.

Influenza viruses are the cause of yearly epidemics and occasional pandemics that represent a significant challenge to public health. Current control strategies are imperfect and there is an unmet need for new antiviral therapies. Here, we report the identification of small molecule compounds able to effectively and specifically inhibit growth of influenza A and B viruses in cultured cells through targeting an assembly interface of the viral RNA-dependent RNA polymerase. Using an existing crystal structure of the primary protein–protein interface between the PB1 and PA subunits of the influenza A virus polymerase, we conducted an in silico screen to identify potential small molecule inhibitors. Selected compounds were then screened for their ability to inhibit the interaction between PB1 and PA in vitro using an ELISA-based assay and in cells, to inhibit nuclear import of a binary PB1–PA complex as well as transcription by the full viral ribonucleoprotein complex. Two compounds emerged as effective inhibitors with IC ₅₀ values in the low micromolar range and negligible cytotoxicity. Of these, one compound also acted as a potent replication inhibitor of a variety of influenza A virus strains in Madin-Darby canine kidney (MDCK) cells, including H3N2 and H1N1 seasonal and 2009 pandemic strains. Importantly, this included an oseltamivir-resistant isolate. Furthermore, potent inhibition of influenza B viruses but not other RNA or DNA viruses was seen. Overall, these compounds provide a foundation for the development of a new generation of therapeutic agents exhibiting high specificity to influenza A and B viruses.