Comparative Genome Analysis of Four Elephant Endotheliotropic Herpesviruses, EEHV3, EEHV4, EEHV5, and EEHV6, from Cases of Hemorrhagic Disease or Viremia

Abstract

The genomes of three types of novel endotheliotropic herpesviruses (elephant endotheliotropic herpesvirus 1A [EEHV1A], EEHV1B, and EEHV2) associated with lethal hemorrhagic disease in Asian elephants have been previously well characterized and assigned to a new Proboscivirus genus. Here we have generated 112 kb of DNA sequence data from segments of four more types of EEHV by direct targeted PCR from blood samples or necropsy tissue samples from six viremic elephants. Comparative phylogenetic analysis of nearly 30 protein-encoding genes of EEHV5 and EEHV6 show that they diverge uniformly by nearly 20% from their closest relatives, EEHV2 and EEHV1A, respectively, and are likely to have similar overall gene content and genome organization. In contrast, seven EEHV3 and EEHV4 genes analyzed differ from those of all other EEHVs by 37% and have a G؉C content of 63% compared to just 42% for the others. Three strains of EEHV5 analyzed clustered into two partially chimeric subgroups EEHV5A and EEHV5B that diverge by 19% within three small noncontiguous segments totaling 6.2 kb. We conclude that all six EEHV types should be designated as independent species within a proposed new fourth Deltaherpesvirinae subfamily of mammalian herpesviruses. These virus types likely initially diverged close to 100 million years ago when the ancestors of modern elephants split from all other placental mammals and then evolved into two major branches with high-or low-G؉C content about 35 million years ago. Later additional branching events subsequently generated three paired sister taxon lineages of which EEHV1 plus EEHV6, EEHV5 plus EEHV2, and EEHV4 plus EEHV3 may represent Asian and African elephant versions, respectively. Here we have used PCR DNA sequence analysis from multiple segments of DNA amplified directly from blood or necropsy tissue samples of six more selected cases of hemorrhagic disease to partially characterize four other types of EEHVs from either Asian or African elephants. We propose that all six types and two chimeric subtypes of EEHV belong to multiple lineages of both AT-rich and GC-rich branches within a new subfamily to be named the Deltaherpesvirinae, which evolved separately from all other mammalian herpesviruses about100 million years ago. IMPORTANCE

Acute death associated with Citrobacter freundii infection in an African elephant (Loxodonta africana)

“…15 Multiple examples of mixed infections with several strains of EEHV-2, EEHV-3, and EEHV-6 together have also been found in localized lung nodules or skin nodules from asymptomatic healthy adult elephants both in the wild in South Africa and in Kenya, as well as in a zoo elephant in the United States. 27 In our case, low levels of several types of EEHVs were detected in DNA extracted from the spleen, and none was detected in DNA extracted from the heart. The levels of EEHV DNA found were much lower than those usually associated with acute EEHV hemorrhagic disease, and many other genomic loci that would normally be found in such cases were evidently below the level of detection.…”

“…Therefore, these findings are unlikely to indicate lethal EEHV disease. The lymphoid lung nodules that have been observed to carry EEHVs in many otherwise healthy adult African elephants in the wild 16 as well as in at least 1 zoo African adult 27 resemble enlarged reactive hyperplastic lymphoid follicles.…”

Commentary: Trunkloads of Viruses

“…EEHV1 is currently formally recognized as the species Elephantid herpesvirus 1, the type species in the Proboscivirus genus within the Betaherpesvirinae. The new papers by Richman et al (8) and Zong et al (9) go well beyond this, showing that six distinct EEHV belong to the Proboscivirus lineage; the authors make the case that this lineage represents a novel subfamily within the Herpesviridae.…”

“…Somewhat analogous to human herpesviruses 6A and 6B (16), most of the 64 genes that span the center of the EEHV1A and EEHV1B genomes are Ͼ99% identical, with three interspersed sharply bounded regions of much greater sequence divergence (referred to as chimeric domains, or CD), as well as some sequence rearrangements in the vicinity of the genomic termini (4,8). Although less sequence is available, EEHV5A and EEHV5B appear to have a similar relationship (9). In contrast to human herpesvirus 6A (HHV-6A) and HHV-6B, the chimeric domains appear to be products of recombination with diverged versions of EEHV species that have not been detected and may no longer be extant.…”

“…1B) (9). Each pair of closely related viruses includes one whose natural host appears to be Asian elephants, and one of African elephants.…”