Identification of Novel Gammaherpesviruses in Ocelots (Leopardus pardalis) and Bobcats (Lynx rufus) in Panama and Colorado, USA

Caitlin C. Lozano, Linda L. Sweanor, Grete Wilson-Henjum, Roland W. Kays, Ricardo Moreno, Sue VandeWoude, Ryan M. Troyer
2015 Journal of Wildlife Diseases  
Gammaherpesviruses (GHVs) have been identified in many species and are often 24 associated with disease. Recently, we characterized three novel felid GHVs in domestic cats, 25 bobcats, and pumas. In this study we sought to determine whether free ranging ocelots 26 (Leopardus pardalis) and bobcats (Lynx rufus) are infected with additional GHVs. We screened 27 DNA samples from ocelots on Barro Colorado Island, Panama and bobcats in western Colorado 28 using a degenerate nested PCR that targets
more » ... GHV glycoprotein B gene. We identified a novel 29 GHV glycoprotein B sequence in two ocelots and a second novel sequence in a bobcat which is 30 distinct from the previously characterized bobcat GHV (Lynx rufus GHV1). Utilizing additional 31 degenerate and virus-specific PCRs, we extended these sequences to include 3.4 kb of the GHV 32 glycoprotein B and DNA polymerase genes. These sequences indicate the presence of the first 33 GHV detected in ocelots and the second GHV detected in bobcats. These viruses were 34 provisionally named Leopardus pardalis gammaherpesvirus 1 (LpaGHV1) and Lynx rufus 35 gammaherpesvirus 2 (LruGHV2), respectively. Phylogenetic analysis indicates that these 36 viruses are most closely related to recently identified GHVs of the Percavirus genus found in 37 domestic cats (Felis catus GHV1) and bobcats (Lynx rufus GHV1), suggesting that a cluster of 38 multiple felid GHVs exists within the Percavirus genus. 39 virus 41 42 The family Herpesviridae is a group of double-stranded DNA viruses with >120 kb 43 genomes and the ability to establish life-long infection of host organisms. Herpesviridae is 44 divided into three subfamilies: Alphaherpesvirinae, Betaherpesvirinae and 45 3 Short Communications Gammaherpesvirinae. Gammaherpesviruses (GHVs) are found in a wide range of animal 46 species (Ackermann 2006). They typically favor establishment of latent infection, often in 47 lymphocytes, with occasional or condition-dependent reactivation of gene expression and 48 replication (Speck and Ganem 2010). GHVs can cause a range of disease conditions affecting 49 human and animal health such as lymphoproliferative disorders, including lymphoma, and non-50 lymphoid cancers (Ackermann 2006). Evidence for the presence of GHVs in felid species has 51 included detection of a GHV DNA sequence in an African lion (Ehlers et al. 2008) and detection 52 of bovine herpesvirus 4 in domestic cats (Kruger et al. 2000). We recently were able to greatly 53 expand this analysis by identifying DNA sequences of three new felid gammaherpesviruses in 54 domestic cats (Felis catus gammaherpesvirus 1, FcaGHV1), bobcats (Lynx rufus 55 gammaherpesvirus 1, LruGHV1), and pumas (Puma concolor gammaherpesvirus 1, PcoGHV1) 56 (Troyer et al. 2014). Using real-time qPCR, the prevalence of GHV DNA in the blood of felids 57 from the USA was determined. FcaGHV1 was found in 21/135 (16%) domestic cats, PcoGHV1 58 was detected in 5/83 (6%) pumas, and LruGHV1 was detected in 30/64 (47%) bobcats and 11/83 59 (13%) pumas screened (Troyer et al. 2014). Subsequently we detected FcaGHV1 in domestic 60 cats in Singapore and Australia indicating a likely worldwide distribution (Beatty et al. 2014). 61 FcaGHV1 infection correlated with the presence of multiple co-pathogens including feline 62 immunodeficiency virus and was more common in cats classified as unhealthy, suggesting 63 possible disease association for this felid GHV (Beatty et al. 2014). The finding that three 64 distinct felid species tested were infected with GHVs across wide geographic ranges implies that 65 GHVs may be common in other felid species. In addition, the presence of both PcoGHV1 and 66 LruGHV1 in pumas suggests that felids can be infected with more than one GHV. Therefore, we 67 sought to determine whether ocelots are infected with a GHV and whether bobcats are infected 68 4 Short Communications with GHVs in addition to LruGHV1. We employed a degenerate PCR strategy which has been 69 successful in detecting GHV gene sequences from felids and many other host organisms to 70 conduct this analysis (Ehlers et al. 2008, Troyer et al. 2014. 71 Blood samples (n = 5) were previously collected from free-ranging ocelots on Barro 72 Colorado Island, Panama from 2001 to 2004 (Franklin et al. 2008. Sampling protocols were 73 reviewed by appropriate animal care committees and appropriate permits were obtained prior to 74 collection. DNA was extracted from ocelot blood as previously described (Franklin et al. 2008). 75 Bobcat spleen tissues (n = 55) were collected from hunter-killed and road-killed bobcats in 76 western Colorado during 2007 and 2008, frozen at -80°C, and DNA was extracted using a 77 previously described protocol (Zheng et al. 2011). We tested ocelot blood DNA and bobcat 78 spleen DNA samples using a degenerate nested pan-GHV PCR (Ehlers et al. 2008) to amplify a 79 portion of the glycoprotein B gene (gB) with reaction conditions as previously described (Troyer 80 et al. 2014). Negative controls (water template) were consistently negative. We detected bands 81 of the expected size for gB amplification (~500 bp) by agarose gel electrophoresis in 2/5 ocelots 82 and 35/55 bobcats. DNAs were purified using the QIAquick PCR purification kit (Qiagen) and 83 sequenced in both directions by the Colorado State University Proteomics Facility. Primer 84 sequences were removed and unique 453 bp sequences were then compared to other GHV gB 85 sequences using BioEdit (Hall 1999) and NBCI BLAST programs. The two GHV gB sequences 86 from ocelots were identical to each other but distinct from any published sequences in GenBank 87 with greatest nucleotide identity to LruGHV1 (95.8%). Of the 35 GHV gB sequences from 88 bobcats, 34 matched the gB sequence of the previously identified bobcat GHV, LruGHV1. 89 However, the remaining GHV gB sequence from a bobcat was distinct from LruGHV1 (88.5% 90 nucleotide identity) with greatest nucleotide identity to FcaGHV1 (89.6%). The presence of 91 5 Short Communications unique GHV gB sequences likely indicates the presence of novel GHVs in these host species. 92 We provisionally assigned the ocelot sequence the name Leopardus pardalis gammaherpesvirus 93 1 (LpaGHV1) and the bobcat sequence the name Lynx rufus gammaherpesvirus 2 (LruGHV2). 94 To obtain a larger genomic sequence for phylogenetic analysis of LpaGHV1 and 95 LruGHV2, we targeted the DNA polymerase gene (Dpol) with a degenerate nested PCR and then 96 performed long-distance gB to Dpol PCR (~3.4 kb) using virus-specific primers (Ehlers et al. 97 2008, Troyer et al. 2014). To PCR amplify Dpol, we used the "Perca-DNApol" primers and 98 reaction conditions which were previously used to amplify Dpol for FcaGHV1 (Troyer et al. 99 2014). Long-distance nested PCR was then employed to close the gap between the gB sequence 100 and the Dpol sequence. For LpaGHV1, reaction conditions were the same as those used 101 previously for FcaGHV1 and LruGHV1 (Troyer et al. 2014) with first round primers Lpa-F1 (5'-102
doi:10.7589/2015-01-027 pmid:26280877 fatcat:ubrtxfjafzcpznoyu4tezaerxu