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

Equine herpesvirus 1 (EHV-1) causes respiratory disease, abortion, neonatal death and neurological disease in equines and is endemic in most countries. The viral factors that influence EHV-1 disease severity are poorly understood, and this has hampered vaccine development. However, the N752D substitution in the viral DNA polymerase catalytic subunit has been shown statistically to be associated with neurological disease. This has given rise to the term "neuropathic strain," even though strains lacking the polymorphism have been recovered from cases of neurological disease. To broaden understanding of EHV-1 diversity in the field, 78 EHV-1 strains isolated over a period of 35 years were sequenced. The great majority of isolates originated from the United Kingdom and included in the collection were low passage isolates from respiratory, abortigenic and neurological outbreaks. Phylogenetic analysis of regions spanning 80% of the genome showed that up to 13 viral clades have been circulating in the United Kingdom and that most of these are continuing to circulate. Abortion isolates grouped into nine clades, and neurological isolates grouped into five. Most neurological isolates had the N752D substitution, whereas most abortion isolates did not, although three of the neurological isolates from linked outbreaks had a different polymorphism. Finally, bioinformatic analysis suggested that recombination has occurred between EHV-1 clades, between EHV-1 and equine herpesvirus 4, and between EHV-1 and equine herpesvirus 8.

Antigenic and genetic evolution of equine influenza A (H3N8) virus from 1968 to 2007.

Equine influenza virus is a major respiratory pathogen in horses, and outbreaks of disease often lead to substantial disruption to and economic losses for equestrian industries. The hemagglutinin (HA) protein is of key importance in the control of equine influenza because HA is the primary target of the protective immune response and the main component of currently licensed influenza vaccines. However, the influenza virus HA protein changes over time, a process called antigenic drift, and vaccine strains must be updated to remain effective. Antigenic drift is assessed primarily by the hemagglutination inhibition (HI) assay. We have generated HI assay data for equine influenza A (H3N8) viruses isolated between 1968 and 2007 and have used antigenic cartography to quantify antigenic differences among the isolates. The antigenic evolution of equine influenza viruses during this period was clustered: from 1968 to 1988, all isolates formed a single antigenic cluster, which then split into two cocirculating clusters in 1989, and then a third cocirculating cluster appeared in 2003. Viruses from all three clusters were isolated in 2007. In one of the three clusters, we show evidence of antigenic drift away from the vaccine strain over time. We determined that a single amino acid substitution was likely responsible for the antigenic differences among clusters.

Identification and analysis of an Autographa californica nuclear polyhedrosis virus structural protein of the occlusion-derived virus envelope: ODV-E56.

An Autographa californica nuclear polyhedrosis virus gene encoding an occlusion-derived virus (ODV) envelope protein of 56 kDa was identified and sequenced. Transcription initiates from a conserved baculovirus late motif (ATAAG) with transcripts detected from 16 through 72 hr p.i. The protein is detected in infected cell extracts from 36 hr p.i. Western blot assay of ODV, BV, viral envelope, and nucleocapsid preparations coupled with immunoelectron microscopy reveal that this protein localizes to the ODV envelope. This protein is named ODV-E56 to identify its viral origin, envelope location, and apparent molecular weight. ODV-E56 is enriched in viral induced intranuclear microvesicles as determined by immunogold labeling. A mutant was constructed with the C-terminal portion of the protein replaced with beta-galactosidase. The fusion protein, E56-beta-gal, locates to the viral nucleocapsids and not to the ODV envelope or intranuclear microvesicles. This suggests that the signals necessary for transport and/or retention into these structures lies within the C-terminal portion of ODV-E56. Additionally, both ODV-E56 and E56-beta-gal are enriched in electron dense regions that cluster around the inner nuclear membrane and within the nucleoplasm.

Inter- and intra-strain genomic variation in equine herpesvirus type 1 isolates.

Restriction enzyme digests of DNA from 22 unselected isolates of EHV-1 were analysed by hybridization with cloned DNA fragments covering the genome. In addition to a small amount of inter-strain variation, heterogeneity within strains was observed, caused by loss of specific restriction endonuclease sites in the DNA of a proportion of the virus particles of any one stock. Fifteen strains demonstrated the same intra-strain variation involving loss of the BamHI L-M site which was shown to lie within coding sequence for the large subunit of ribonucleotide reductase. This particular mutation may therefore be selected for by passage in RK13 cells.

Glycoprotein 300 is encoded by gene 28 of equine herpesvirus type 1: a new family of herpesvirus membrane proteins?

A portion of equine herpesvirus type 1 (EHV-1) gene 28, which is homologous to herpes simplex virus type 1 gene UL32, was expressed using a prokaryotic system to yield a fusion protein which reacted on Western blots with P19, a monoclonal antibody (MAb) that reacts with EHV-1 glycoprotein 300 (gp300), confirming that this gene encodes gp300. Hydrophobicity analysis showed that gp300 is a glycoprotein with multiple hydrophobic domains that might interact with, or span, the membrane several times. As such, it may represent the first member of a new family of herpesvirus glycoproteins to be identified as a virus structural component. Gp300 was also shown to be modified by palmitic acid residues, and a second MAb (1G12) directed against gp300 inhibited fusion between EHV-1-infected cells.

Identification of the equine herpesvirus type 1 glycoprotein 17/18 as a homologue of herpes simplex virus glycoprotein D.

The DNA sequence of the equine herpesvirus type 1 (EHV-1) gD gene homologue has been determined for the strain Ab1 and compared with previously published sequences. A portion of the gene has been located to a region of the genome which also encodes homologues of the herpes simplex virus type 1 genes for gE and gI and is known to encode an epitope of the virion protein gp17/18. Analysis of the EHV-1 strain Kentucky A (KyA) by DNA hybridization showed the presence of a gD gene homologue and established the absence of genes for gI and gE. Western blot analysis, however, showed that KyA virus particles contain gp17/18, thus indicating that this protein is encoded by the gD gene homologue. The KyA gp17/18 was found to be smaller than that detected in other strains and this is accounted for by a frameshift mutation in the KyA sequence relative to Ab1. The mutation in the KyA strain results in an altered C-terminal sequence and could explain the apparent structural differences suggested by the reactivities with monoclonal antibodies (MAbs). We have also expressed part of the Ab1 gD gene as a fusion protein with glutathione S-transferase in Escherichia coli and shown that this reacts with the MAb 5H6 originally used to map gp17/18. These experiments establish that gp17/18 is encoded by the gD gene homologue.

Glycoprotein 60 of equine herpesvirus type 1 is a homologue of herpes simplex virus glycoprotein D and plays a major role in penetration of cells.

Monoclonal antibodies (MAbs) specific for equine herpesvirus type 1 (EHV-1) glycoprotein 60 (gp60) and gp 17/18 (F3132 and 5H6 respectively) were found to react with the same protein, which was identified as a homologue of herpes simplex virus type 1 gD. MAb F3132 strongly neutralized virus infectivity and inhibited the penetration of the virus into the cell. The effects on penetration were shared with three other MAbs against this protein (P68, F3116 and F3129), but no effect on virus penetration was found with any other anti-EHV-1 MAb tested. The level of glycosylation of gp60 was analysed using glycanase enzymes and glycosylation inhibitors, and consisted of mainly N-linked carbohydrate. The M(r) of non-N-glycosylated gp60 was 50K.

Location of open reading frames coding for equine herpesvirus type-1 glycoproteins with homology to gE and gI of herpes simplex virus.

The DNA fragments representing the entire short unique region and part of the repeat sequences of the equine herpesvirus type-1 genome were cloned into plasmid vectors. The approximate positions of the junctions between the short unique region and the inverted repeats were then located by restriction endonuclease mapping. Two open reading frames coding for potential glycoproteins have been identified within the short unique region, using DNA sequence analysis. The predicted amino acid sequences of these open reading frames had extensive homology to the herpes simplex virus glycoproteins gE and gI and the related glycoproteins of pseudorabies virus and varicella-zoster virus.

Sequence analysis of the 4.7-kb BamHI-EcoRI fragment of the equine herpesvirus type-1 short unique region.

To localize gene that may encode immunogens potentially important for recombinant vaccine design, we have analysed a region of the equine herpesvirus type-1 (EHV-1) genome where a glycoprotein-encoding gene had previously been mapped. The 4707-bp BamHI-EcoRI fragment from the short unique region of the EHV-1 genome was sequenced. This sequence contains three entire open reading frames (ORFs), and portions of two more. ORF1 codes for 161 amino acids (aa), and represents the C terminus of a possible membrane-bound protein. ORF2 (424 aa) and ORF3 (550 aa) are potential glycoprotein-encoding genes; the predicted aa sequences contain possible signal sequences, N-linked glycosylation sites and transmembrane domains; they also show homology to the glycoproteins gI and gE of herpes simplex virus type-1 (HSV-1), and the related proteins of pseudorabies virus and varicella-zoster virus. The predicted aa sequence of ORF4 shares no homology with other known herpesvirus proteins, but the nucleotide sequence shows a high level of homology with the corresponding region of the EHV-4 genome. ORF5 may be related to US9 of HSV-1.