Fig 1.
Location of Amur tigers tested for GHVs in the Russian Far East.
Open circles indicate tigers that tested negative for GHVs. Filled circles indicate tigers infected with mustelid gammaherpesvirus 1 (MusGHV1). The inset map highlights tigers within and around the Sikhote-Alin Zapovednik (strictly protected area). This map was prepared using Natural Earth (https://www.naturalearthdata.com/), and contains Sikhote-Alin Nature Reserve layer (5475758, version 22) information from OpenStreetMap and OpenStreetMap Foundation, which is made available under the Open Database License (https://www.openstreetmap.org/copyright).
Fig 2.
PCR amplification strategy for GHV sequencing.
Pan-GHV degenerate primers were used to amplify small sections of glycoprotein B and DNA polymerase genes. Sequences from these regions were then used to design virus-specific primers for amplification and sequencing of an approximately 3.5 kb region spanning these two genes. Additional virus-specific primers targeting glycoprotein B were designed for high-sensitivity screening of samples.
Fig 3.
Nucleotide identity matrix for the carnivore host sub-group of GHV genus Percavirus.
GHV sequences from glycoprotein B through DNA polymerase (approximately 3.5 kb) were aligned using MUSCLE [33]. Nucleotide identity between each pair of sequences is indicated in the matrix.
Table 1.
PCR amplification of herpesvirus DNA from carnivore host species.
Fig 4.
Maximum-likelihood phylogenetic analysis of gammaherpesviruses based on concatenated gB and DNApol amino acid alignments.
The betaherpesvirus human cytomegalovirus (HHV5; GenBank accession no. NC006273) was used as an outgroup to root the tree but is not displayed due to space constraints. Bootstrap support based on 1000 replicates is displayed as a percentage out of 100 at each node. Phylogenetic clusters corresponding to established GHV genera are indicated by name and color. Virus abbreviations, their definitions, and NCBI accession numbers are as follows: HHV4, human herpesvirus 4 (Epstein-Barr virus), NC007605; CalGHV3, callitrichine gammaherpesvirus 3, NC004367; DeGHV1, delphinid gammaherpesvirus 1, NC035117; PcGHV1, phascolarctid gammaherpesvirus 1, MG452722; VoGHV1, vombatid gammaherpesvirus 1, MG452721; SuGHV3, suid gammaherpesvirus 3, AF478169; OvGHV2, ovine gammaherpesvirus 2, NC007646; AlGHV1, alcelaphine gammaherpesvirus 1, NC002531; SaGHV2, saimiriine gammaherpesvirus 2, NC001350; HHV8, human herpesvirus 8 (Kaposi’s sarcoma-associated herpesvirus), NC009333; BoGHV4, bovine gammaherpesvirus 4, NC002665; PcoGHV1, Puma concolor gammaherpesvirus 1, KF840717; PleoGHV1, Panthera leo gammaherpesvirus 1, DQ789370; VeGHV3, vespertilionid gammaherpesvirus 3, MF385016; UrGHV2, ursid gammaherpesvirus 2, MK089801; UrGHV3, ursid gammaherpesvirus 3, OP751953; VeGHV1, vespertilionid gammaherpesvirus 1, KU220026; MyoGHV2, Myotis ricketti gammaherpesvirus 2, JN692430; EqGHV2, equid gammaherpesvirus 2, NC001650; EqGHV5, equid gammaherpesvirus 5, NC026421; EzebGHV1, Equus zebra gammaherpesvirus 1, AY495965; CcroGHV1, Crocuta crocuta gammaherpesvirus 1, DQ789371; PhGHV3, phocid gammaherpesvirus 3, KP136799; MusGHV1, mustelid gammaherpesvirus 1, AF376034, PQ066357, PQ066358; MusGHV4, mustelid gammaherpesvirus 4, OP751954; LpaGHV1, Leopardus pardalis gammaherpesvirus 1, KP721220; FeGHV1, felid gammaherpesvirus 1, KT595939; LruGHV1, Lynx rufus gammaherpesvirus 1, KF840716; LcaGHV1, Lynx canadensis gammaherpesvirus 1, MH520115.
Table 2.
PCR primers designed in this study.