Equine Herpesvirus Type 1 ORF76 Encoding US9 as a Neurovirulence Factor in the Mouse Infection Model.

Equine herpesvirus type 1 (EHV-1) causes rhinopneumonitis, abortion, and neurological outbreaks (equine herpesvirus myeloencephalopathy, EHM) in horses. EHV-1 also causes lethal encephalitis in small laboratory animals such as mice and hamsters experimentally. EHV-1 ORF76 is a homolog of HSV-1 US9, which is a herpesvirus kinase. Starting with an EHV-1 bacterial artificial chromosome clone of neuropathogenic strain Ab4p (pAb4p BAC), we constructed an ORF76 deletion mutant (Ab4p∆ORF76) by replacing ORF76 with the rpsLneo gene. Deletion of ORF76 had no influence on replication, cell-to-cell spread in cultured cells, or replication in primary neuronal cells. In Western blots of EHV-1-infected cell lysates, an EHV-1 US9-specific polyclonal antibody detected multiple bands ranging from 35 to 42 kDa. In a CBA/N1 mouse infection model following intranasal inoculation, the parent and Ab4p∆ORF76 revertant caused the same histopathology in the brain and olfactory bulbs. The parent, Ab4p∆ORF76, and revertant mutant replicated similarly in the olfactory mucosa, although Ab4p∆ORF76 was not transported to the olfactory bulbs and was unable to infect the CNS. These results indicated that ORF76 (US9) plays an essential role in the anterograde spread of EHV-1.

Attenuation of the neuropathogenic equine herpesvirus type 1 strain Ab4p in hamsters by a single amino acid mutation (D752N) in viral DNA polymerase ORF30.

Equine herpesvirus type 1 (EHV-1) causes abortion, respiratory infection, and neurological diseases (equine herpesvirus myeloencephalopathy) in horses. A single nucleotide polymorphism (SNP) associated with a single amino acid in the DNA polymerase gene (ORF30, in which D752 is neuropathogenic and N752 is non-neuropathogenic) of EHV-1 has been associated with neuropathogenicity in horses. We constructed an EHV-1 Ab4p ORF30 N752 mutant and a repair virus to examine the effect of a D752N mutation on the neuropathogenicity of the virus in Syrian hamsters. The N752 mutation did not affect viral growth in cultured cells but it did attenuate the neuropathogenicity of Ab4p in the hamsters. The results suggest that D752N is involved in neuropathogenicity not only in horses but also in hamsters.

The N-terminal glycine of EHV-1 UL11 is essential for the localization of UL11 and EHV-1 replication in cultured cells.

Equine herpesvirus type 1 (EHV-1) UL11 is a 74-amino-acid (aa) protein encoded by ORF51. UL11 is modified by acylation including myristoylation and palmitoylation. Myristoylation of EHV-1 UL11 is assumed to occur on the N-terminal glycine, while palmitoylation is assumed to occur on the seventh and ninth cysteines. ORF51, which encodes the first 24 aa, overlaps ORF50 encoding UL12. We previously demonstrated that UL11 was essential for EHV-1 replication in cultured cells and that UL11 was localized at the Golgi apparatus where herpesviruses obtain their final envelope. It is unclear whether the acylation is related to the localization of EHV-1 UL11 and viral replication. In this study, we investigated the role of UL11 acylation in the intracellular localization and viral growth and replication of EHV-1. We constructed seven UL11 acylation mutant plasmids and seven UL11 acylation mutant BAC DNAs; then, we analysed the localizations of the mutant UL11s and attempted virus rescue. We found that both the N-terminal glycine and the seventh or ninth cysteine, especially N-terminal glycine, were involved in the localization of UL11 and viral replication. Taken together, these results suggest that EHV-1 viral growth requires that UL11 is modified by myristoylation of an N-terminal glycine and by palmitoylation of at least one of the cysteines, and that UL11 is localized at the Golgi apparatus. This study shows that a single amino acid in EHV-1 can determine the fate of viral replication.

Prevention of fatal equine herpesvirus type 1 encephalitis in mice by immunization with a limited-replication cycle virus.

Equine herpesvirus type 1 (EHV-1) is a devastating pathogen of horses, their natural hosts, and causes fatal encephalitis in non-natural hosts. We previously demonstrated that acylation of the tegument protein UL11 is required for viral replication in cultured cells. We created a mutant virus (EHV-1 UL12 trunc UL11 G2AC7AC9A), in which glycyl and cysteinyl residues at positions 2, 7 and 9 of UL11 that are normally acylated were replaced with alanyl residues. This virus, designated the 2/7/9 mutant, has a limited-replication cycle (LRC), in which replication stops after just a few cycles. Here, we tested whether the 2/7/9 mutant could be used as a vaccine against fatal encephalitis in a mouse model. A virulence test showed that the 2/7/9 mutant was not pathogenic in mice and elicited an antibody response. We also attempted to use the 2/7/9 mutant to immunize mice against a zebra-borne EHV-1, 94-137. Two trials were conducted, each with five immunized mice, five non-immunized and five control mice. In both trials, clinical signs and fatalities were much lower in the immunized mice than in the non-immunized mice. In addition, none of the mice in either trial developed neutralizing antibodies, indicating that the immunity induced by the 2/7/9 mutant was not due to neutralizing activity. The results indicate that the 2/7/9 LRC mutant has promise as a vaccine against EHV-1 infection non-natural hosts.