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.

Genetic diversity of equine herpesvirus 1 isolated from neurological, abortigenic and respiratory disease outbreaks

“…Previously published work has shown no obvious difference in the processivity of either DNA polymerase variant in vitro , except that the G2254 version was sensitive to the drug aphidicolin (Goodman et al., 2007). This may indicate that the two versions of the protein adopt slightly different conformations that affect pathogenicity by a means that has not yet been identified.…”

“…This may indicate that the two versions of the protein adopt slightly different conformations that affect pathogenicity by a means that has not yet been identified. It has been suggested that G2254 strains are able to infect cell types in vivo that are different from those infected by A2254 strains, allowing these viruses to replicate and disseminate more efficiently (Goodman et al., 2007). However, work carried out in vitro has failed to identify any cellular preference due to this mutation (Ma, Lu, & Osterrieder, 2010).…”

Residue 752 in DNA polymerase of equine herpesvirus type 1 is non-essential for virus growth in vitro

“…It was found that only a minority of EHV-1 strains are capable of inducing neurological disorders, although all strains can cause respiratory disease and abortion (Mumford et al, 1987;Ostlund, 1993;Wilson, 1997). Recently, epidemiological as well as reverse-genetic studies have shown that a single-nucleotide polymorphism at position 2254 (G/A2254) of open reading frame 30 (ORF30), encoding viral DNA polymerase (Pol), will lead to a variation at amino acid position 752 (D/N752), which is not only necessary but also sufficient for the virus's neuropathogenic potential (Goodman et al, 2007;Nugent et al, 2006;Van de Walle et al, 2009). The D752 genotype, in contrast to N752, can induce higher levels of viraemia in horses in vivo.…”

Glycoprotein B of equine herpesvirus type 1 has two recognition sites for subtilisin-like proteases that are cleaved by furin

“…All viruses used in the study were recovered from the infectious bacterial artificial chromosome (BAC) clone of the EHV-1 strain Ab4 (Goodman et al, 2007). Viruses were reconstituted after Peptides that were identified are represented in separate boxes for each digest below the corresponding N-terminal sequence of gB.…”

“…The infected PBMCs were stained at 24 h p.i. with primary mouse antibodies against equine CD14 (monocytes), CD3 (T-lymphocytes) or IgM (B-lymphocytes) (Goodman et al, 2007). After washing, cells were labelled with a secondary Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen).…”