First fatality associated with elephant endotheliotropic herpesvirus 5 in an Asian elephant: pathological findings and complete viral genome sequence

Abstract

Infections of Asian elephants (Elephas maximus) with elephant endotheliotropic herpesvirus (EEHV) can cause a rapid, highly lethal, hemorrhagic disease, which primarily affects juvenile animals up to the age of four years. So far, the majority of deaths have been attributed to infections with genotype EEHV1 or, more rarely, EEHV3 and EEHV4. Here, we report the pathological characteristics of the first fatality linked to EEHV5 infection, and describe the complete viral DNA sequence. Gross post-mortem and histological findings were indistinguishable from lethal cases previously attributed to other EEHV genotypes, and the presence of characteristic herpesviral inclusions in capillary endothelial cells at several sites was consistent with the diagnosis of acute EEHV infection. Molecular analysis confirmed the presence of EEHV5 DNA and was followed by sequencing of the viral genome directly from post-mortem material. The genome is 180,800 bp in size and contains 120 predicted protein-coding genes, five of which are fragmented and presumably nonfunctional. The seven families of paralogous genes recognized in EEHV1 are also represented in EEHV5. The overall degree of divergence (37%) between the EEHV5 and EEHV1 genomes, and phylogenetic analysis of eight conserved genes, support the proposed classification of EEHV5 into a new species (Elephantid herpesvirus 5).

Figure 1. Pathology of EEHV5 infection in Vijay.

(a) Severe edema and multifocal subserosal hemorrhages in the dorsal mediastinum. (b) Hematoxylin and eosin staining of a liver section. The hepatic architecture is disrupted by hemorrhage and edema, with multifocal fibrin deposits (arrow) and diffuse mixed cellular infiltrates. The central vessel displays denuded endothelial cells, congestion, and mild granulocytostasis. Inset: Herpetic intranuclear inclusion body.

Figure 2. Map of the EEHV5/Vijay genome.

The copies of TR flanking U are shown in a thicker format. ORFs are shown by coloured arrows, with names below. Fragmented ORFs are shown in square brackets, and are depicted as intact. Introns are indicated by narrow white bars. Colour shading indicates core ORFs (present in the ancestor of the subfamilies Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae), betagamma ORFs (present in the subfamilies Betaherpesvirinae and Gammaherpesvirinae), beta ORFs (present only in the subfamily Betaherpesvirinae), and ORFs belonging to paralogous families. As well as being members of paralogous families, U54 and U4 are beta ORFs. A putative origin of DNA replication (ori) and a tandem reiteration (R1) are marked.

Figure 3. Neighbour-joining phylogenetic tree of the concatenated amino acid sequences of core genes (U38, U39, U40, U41, U57, U60, U77, and U81) from members or probable members of the subfamily Betaherpesvirinae, with recognized assignments of viruses to genera indicated.

The tree is rooted at the midpoint, a position that has been shown previously to be statistically robust for this subfamily. Confidence values are displayed as fractions. HCMV, human cytomegalovirus; CCMV, chimpanzee cytomegalovirus; GMCMV, green monkey cytomegalovirus; RhCMV, rhesus cytomegalovirus; OMCMV, owl monkey cytomegalovirus; SMCMV, squirrel monkey cytomegalovirus; GPCMV, guinea pig cytomegalovirus; MSHV, Miniopterus schreibersii herpesvirus; TuHV, tupaiid herpesvirus 1; MCMV, murine cytomegalovirus; RCMVE, rat cytomegalovirus England; RCMV, rat cytomegalovirus; HHV6A, human herpesvirus 6A; HHV6B, human herpesvirus 6B; HHV7, human herpesvirus 7; and PCMV, porcine cytomegalovirus. The scale bar shows nt differences/nt.

Figure 4. Matrix sequence comparison plot of the regions near the right termini of the EEHV5/Vijay and EEHV1A/Raman genomes that encompass the EE50 gene family.

The layout of ORFs in each sequence is illustrated, with shading indicating gene families provided in the key. Fragmented ORFs are shown in square brackets, and are depicted as intact. The position of TR is also shown. Detectable sequence similarity is indicated by diagonal lines.