Six novel gammaherpesviruses of Afrotheria provide insight into the early divergence of the Gammaherpesvirinae

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

Section: Discussionmentioning

confidence: 79%

mentioning

confidence: 99%

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

Zong

Latimer

Long

et al. 2014

“…In blood and/or tissue samples of each mammalian species (except the gorilla and Asian elephant), a novel DPOL sequence was found (data not shown), and by comparison with known herpesvirus sequences, each sequence was determined to originate from an unknown GHV (Table 1). In the gorilla and elephant samples, sequences were found which had been published recently by others (18,32).…”

Section: Vol 82 2008 Novel Mammalian Gammaherpesviruses and Lineagementioning

confidence: 72%

Novel Mammalian Herpesviruses and Lineages within the Gammaherpesvirinae : Cospeciation and Interspecies Transfer

Ehlers

Dural

Yasmum

et al. 2008

115

130

Novel members of the subfamily Gammaherpesvirinae, hosted by eight mammalian species from six orders (Primates, Artiodactyla, Perissodactyla, Carnivora, Scandentia, and Eulipotyphla), were discovered using PCR with pan-herpesvirus DNA polymerase (DPOL) gene primers and genus-specific glycoprotein B (gB) gene primers. The gB and DPOL sequences of each virus species were connected by long-distance PCR, and contiguous sequences of approximately 3.4 kbp were compiled. Six additional gammaherpesviruses from four mammalian host orders (Artiodactyla, Perissodactyla, Primates, and Proboscidea), for which only short DPOL sequences were known, were analyzed in the same manner. Together with available corresponding sequences for 31 other gammaherpesviruses, alignments of encoded amino acid sequences were made and used for phylogenetic analyses by maximum-likelihood and Bayesian Monte Carlo Markov chain methods to derive a tree which contained two major loci of unresolved branching details. The tree was rooted by parallel analyses that included alpha-and betaherpesvirus sequences. This gammaherpesvirus tree contains 11 major lineages and presents the widest view to date of phylogenetic relationships in any subfamily of the Herpesviridae, as well as the most complex in the number of deep lineages. The tree's branching pattern can be interpreted only in part in terms of the cospeciation of virus and host lineages, and a substantial incidence of the interspecies transfer of viruses must also be invoked.PCR assays with degenerate primers have been used for over a decade for the amplification of unknown herpesvirus DNA polymerase (DPOL) gene sequences. These methods have the potential to detect virtually every mammalian, avian, or reptilian herpesvirus (7, 31). In fact, more than 100 novel herpesviruses have been discovered with the help of such universal PCR methods (4, 5, 8-11, 14, 17, 19, 27, 28, 33), and new phylogenetic herpesvirus lineages within the Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae subfamilies of the Herpesviridae have emerged (21-24). In addition, previous studies with great apes revealed evidence for two lymphocryptovirus (LCV) lineages and two rhadinovirus (RHV) lineages in the Lymphocryptovirus and Rhadinovirus genera of the Gammaherpesvirinae, leading to speculations that a second human LCV related to EpsteinBarr virus (9) and a second human RHV related to human herpesvirus 8 (HHV-8) (14) may exist.Despite the tremendous accumulation of knowledge on the existence of hitherto unknown herpesviruses, only limited sequence information (i.e., a few hundred base pairs) became available in most of the cases. This information is sufficient to assess whether a virus is already known or novel and allows for assignment to a herpesvirus subfamily. However, a more precise phylogenetic analysis is often not possible, and more extensive sequence data are therefore desirable.In the present study, we wanted to further extend insight into gammaherpesvirus (GHV) evolution by analyzing mammalian hosts from d...

“…Considering the evidence that at least five highly diverged types of elephant gammaherpesvirus, designated elephant gammaherpesvirus 1 (EGHV1) to EGHV5, can also infect and be shed asymptomat-ically from otherwise healthy captive Asian and African elephants (3,33,34), the new Proboscivirus genotype designated EEHV7 now represents the 12th known major type of elephant herpesvirus, all of which have been detected within North American elephants and all of which would be judged worthy of species status according to the standard genetic criteria described above.…”

Section: Discussionmentioning

confidence: 99%

Detection of Quiescent Infections with Multiple Elephant Endotheliotropic Herpesviruses (EEHVs), Including EEHV2, EEHV3, EEHV6, and EEHV7, within Lymphoid Lung Nodules or Lung and Spleen Tissue Samples from Five Asymptomatic Adult African Elephants

Zong¹,

Heaggans²,

Long³

et al. 2016

More than 80 cases of lethal hemorrhagic disease associated with elephant endotheliotropic herpesviruses (EEHVs) have been identified in young Asian elephants worldwide. Diagnostic PCR tests detected six types of EEHV in blood of elephants with acute disease, although EEHV1A is the predominant pathogenic type. Previously, the presence of herpesvirus virions within benign lung and skin nodules from healthy African elephants led to suggestions that African elephants may be the source of EEHV disease in Asian elephants. Here, we used direct PCR-based DNA sequencing to detect EEHV genomes in necropsy tissue from five healthy adult African elephants. Two large lung nodules collected from culled wild South African elephants contained high levels of either EEHV3 alone or both EEHV2 and EEHV3. Similarly, a euthanized U.S. elephant proved to harbor multiple EEHV types distributed nonuniformly across four small lung nodules, including high levels of EEHV6, lower levels of EEHV3 and EEHV2, and a new GC-rich branch type, EEHV7. Several of the same EEHV types were also detected in random lung and spleen samples from two other elephants. Sanger PCR DNA sequence data comprising 100 kb were obtained from a total of 15 different strains identified, with (except for a few hypervariable genes) the EEHV2, EEHV3, and EEHV6 strains all being closely related to known genotypes from cases of acute disease, whereas the seven loci (4.0 kb) obtained from EEHV7 averaged 18% divergence from their nearest relative, EEHV3. Overall, we conclude that these four EEHV species, but probably not EEHV1, occur commonly as quiescent infections in African elephants. IMPORTANCEAcute hemorrhagic disease characterized by high-level viremia due to infection by members of the Proboscivirus genus threatens the future breeding success of endangered Asian elephants worldwide. Although the genomes of six EEHV types from acute cases have been partially or fully characterized, lethal disease predominantly involves a variety of strains of EEHV1, whose natural host has been unclear. Here, we carried out genotype analyses by partial PCR sequencing of necropsy tissue from five asymptomatic African elephants and identified multiple simultaneous infections by several different EEHV types, including high concentrations in lymphoid lung nodules. Overall, the results provide strong evidence that EEHV2, EEHV3, EEHV6, and EEHV7 represent natural ubiquitous infections in African elephants, whereas Asian elephants harbor EEHV1A, EEHV1B, EEHV4, and EEHV5. Although a single case of fatal cross-species infection by EEHV3 is known, the results do not support the previous concept that highly pathogenic EEHV1A crossed from African to Asian elephants in zoos. E lephant endotheliotropic herpesvirus 1 (EEHV1) is the prototype species of the novel Proboscivirus genus that has evidently evolved separately from all other mammalian herpesviruses over the past 100 million years within the ancestors of modern elephants (1-5). Small 250-bp terminase U60(TERex3) and DNA polymerase U38...