Elephant Endotheliotropic Herpesviruses EEHV1A, EEHV1B, and EEHV2 from Cases of Hemorrhagic Disease Are Highly Diverged from Other Mammalian Herpesviruses and May Form a New Subfamily

Abstract

A family of novel endotheliotropic herpesviruses (EEHVs) assigned to the genus Proboscivirus have been identified as the cause of fatal hemorrhagic disease in 70 young Asian elephants worldwide. Although EEHV cannot be grown in cell culture, we have determined a total of 378 kb of viral genomic DNA sequence directly from clinical tissue samples from six lethal cases and two survivors. Overall, the data obtained encompass 57 genes, including orthologues of 32 core genes common to all herpesviruses, 14 genes found in some other herpesviruses, plus 10 novel genes, including a single large putative transcriptional regulatory protein (ORF-L). On the basis of differences in gene content and organization plus phylogenetic analyses of conserved core proteins that have just 20% to 50% or less identity to orthologues in other herpesviruses, we propose that EEHV1A, EEHV1B, and EEHV2 could be considered a new Deltaherpesvirinae subfamily of mammalian herpesviruses that evolved as an intermediate branch between the Betaherpesvirinae and Gammaherpesvirinae. Unlike cytomegaloviruses, EEHV genomes encode ribonucleotide kinase B subunit (RRB), thymidine kinase (TK), and UL9-like origin binding protein (OBP) proteins and have an alphaherpesvirus-like dyad symmetry Ori-Lyt domain. They also differ from all known betaherpesviruses by having a 40-kb large-scale inversion of core gene blocks I, II, and III. EEHV1 and EEHV2 DNA differ uniformly by more than 25%, but EEHV1 clusters into two major subgroups designated EEHV1A and EEHV1B with ancient partially chimeric features. Whereas large segments are nearly identical, three nonadjacent loci totaling 15 kb diverge by between 21 and 37%. One strain of EEHV1B analyzed is interpreted to be a modern partial recombinant with EEHV1A. IMPORTANCEAsian elephants are an endangered species whose survival is under extreme pressure in wild range countries and whose captive breeding populations in zoos are not self-sustaining. In 1999, a novel class of herpesviruses called EEHVs was discovered. These viruses have caused a rapidly lethal hemorrhagic disease in 20% of all captive Asian elephant calves born in zoos in the United States and Europe since 1980. The disease is increasingly being recognized in Asian range countries as well. These viruses cannot be grown in cell culture, but by direct PCR DNA sequence analysis from segments totaling 15 to 30% of the genomes from blood or necropsy tissue from eight different cases, we have determined that they fall into multiple types and chimeric subtypes of a novel Proboscivirus genus, and we propose that they should also be classified as the first examples of a new mammalian herpesvirus subfamily named the Deltaherpesvirinae.

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

“…The six cases of viremic EEHV in captive-born North American juvenile elephants for which we have carried out detailed DNA sequence analysis here are summarized in Table 1. The sources of five other reference EEHV genomes used extensively for comparisons, namely, EEHV1A(Kala,NAP18), EEHV1A(Kumari,NAP11), EEHV1B(Kiba, NAP14), EEHV1B(Haji, NAP19), and EEHV2(Kijana, NAP12), were described in the preceding accompanying paper (10), and the source of the complete genome of EEHV1A(Kimba, NAP23) was given by Ling et al (13).…”

“…Because of these unique characteristics, the EEHVs have been officially classified as members of a new Proboscivirus genus that was originally placed within the Betaherpesvirinae subfamily (15,16). However, we have proposed that their deeply diverged phylogenetic position and overall gene organization might make it more appropriate to instead assign EEHV1A, EEHV1B, and EEHV2 to a separate subfamily of mammalian herpeviruses that would logically be designated the Deltaherpesvirinae (10,13).…”

“…Sanger PCR-based DNA sequence data for between 20 to 70 kb each from seven representative strains of either EEHV1A and EEHV1B, as well as for 59 kb from a single prototype strain of EEHV2, have been generated and described in the preceding accompanying paper (10), adding to two earlier less extensive studies (11,12). Furthermore, the complete intact 180-kb genomes of three more EEHV1 strains were determined recently by de novo next-generation sequencing of necropsy tissue samples (13,14).…”

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.…”

“…As a group, EEHV display forms and levels of sequence diversity that are collectively unique with respect to forms of variation seen for other groups of herpesviruses. 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).…”