A novel group of mammalian DNA viruses called elephant endotheliotropic herpesviruses (EEHVs) belonging to the Proboscivirus genus has been associated with nearly 100 cases of highly lethal acute hemorrhagic disease in young Asian elephants worldwide. The complete 180-kb genomes of prototype strains from three AT-rich branch viruses, EEHV1A, EEHV1B, and EEHV5, have been published. However, less than 6 kb of DNA sequence each from EEHV3, EEHV4, and EEHV7 showed them to be a hugely diverged second major branch with GC-rich characteristics. Here, we determined the complete 206-kb genome of EEHV4(Baylor) directly from trunk wash DNA by next-generation sequencing and de novo assembly procedures. Among a total of 119 genes with an overall colinear organization similar to those of the AT-rich EEHVs, major features of EEHV4 include a family of 26 paralogous 7xTM and vGPCR-like genes plus 25 novel or missing genes. The genome also contains an unusual distribution of tracts of 5 to 11 successive A or T nucleotides in intergenic domains between the mostly much higher GC content protein coding regions. Furthermore, an extremely high GC-rich bias in the third wobble position of codons clearly delineates the coding regions for many but not all proteins. There are also two novel captured cellular genes, including a C-type lectin (vECTL) and an O-linked acetylglucosamine transferase (vOGT), as well as an unusually large and complex Ori-Lyt dyad symmetry domain. Finally, 30 kb from a second strain proved to include three small chimeric domains, indicating the existence of distinct EEHV4A and EEHV4B subtypes. IMPORTANCE Multiple species of herpesviruses from three different lineages of the Proboscivirus genus (EEHV1/6, EEHV2/5, and EEHV3/4/7) infect both Asian and African elephants, but lethal hemorrhagic disease is largely confined to Asian elephant calves and is predominantly associated with EEHV1. Milder disease caused by EEHV5 or EEHV4 is being increasingly recognized as well, but little is known about the latter, which is estimated to have diverged at least 35 million years ago from the others within a distinctive GC-rich branch of the Proboscivirus genus. Here, we have determined the complete genomic DNA sequence of a strain of EEHV4 obtained from a trunk wash sample collected from a surviving Asian elephant calf undergoing asymptomatic shedding during convalescence after an acute hemorrhagic disease episode. This represents the first example from among the three known GC-rich branch Proboscivirus species to be assembled and fully annotated. Several distinctive features of EEHV4 compared to AT-rich branch genomes are described.
Keywords: Elephas maximus calf; G-plus-C nucleotide content bias; acute hemorrhagic disease; elephant endotheliotropic herpesviruses; evolutionary divergence; trunk wash shedding.