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Short Communications
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Identification of novel gammaherpesviruses in ocelots (Leopardus pardalis) and bobcats
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(Lynx rufus)
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Caitlin C. Lozano,1 Linda L. Sweanor,1 Grete Wilson-Henjum,1 Roland W. Kays,2,3 Ricardo
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Moreno,3,4 Sue VandeWoude,1 and Ryan M. Troyer,1,5
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Campus Delivery, Fort Collins, Colorado 80523-1619, USA
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27601, USA
Department of Microbiology, Immunology and Pathology, Colorado State University, 1619
North Carolina Museum of Natural Sciences, 11 West Jones Street, Raleigh, North Carolina
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Ancón, Panamá, República de Panamá
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Yaguará Panamá-Sociedad Panameña de Biología, San Francisco, Calle 71, Chalet 15, Panamá
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Current address: Department of Biomedical Sciences, Oregon State University, 105 Magruder
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Hall, Corvallis, Oregon 97331, USA
Smithsonian Tropical Research Institute, Roosvelt Avenue, 401 Tupper Building, Balboa,
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Corresponding author:
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Ryan Troyer
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105 Magruder Hall, Oregon State University, Corvallis, OR 97331
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Tel: 970-690-2705
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Ryan.Troyer@oregonstate.edu
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Word count: 1885
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Abstract: Gammaherpesviruses (GHVs) have been identified in many species and are often
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associated with disease. Recently, we characterized three novel felid GHVs in domestic cats,
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bobcats, and pumas. In this study we sought to determine whether free ranging ocelots
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(Leopardus pardalis) and bobcats (Lynx rufus) are infected with additional GHVs. We screened
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DNA samples from ocelots on Barro Colorado Island, Panama and bobcats in western Colorado
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using a degenerate nested PCR that targets the GHV glycoprotein B gene. We identified a novel
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GHV glycoprotein B sequence in two ocelots and a second novel sequence in a bobcat which is
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distinct from the previously characterized bobcat GHV (Lynx rufus GHV1). Utilizing additional
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degenerate and virus-specific PCRs, we extended these sequences to include 3.4 kb of the GHV
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glycoprotein B and DNA polymerase genes. These sequences indicate the presence of the first
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GHV detected in ocelots and the second GHV detected in bobcats. These viruses were
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provisionally named Leopardus pardalis gammaherpesvirus 1 (LpaGHV1) and Lynx rufus
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gammaherpesvirus 2 (LruGHV2), respectively. Phylogenetic analysis indicates that these
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viruses are most closely related to recently identified GHVs of the Percavirus genus found in
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domestic cats (Felis catus GHV1) and bobcats (Lynx rufus GHV1), suggesting that a cluster of
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multiple felid GHVs exists within the Percavirus genus.
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Key Words: bobcat, gammaherpesvirus, herpesvirus, Leopardus pardalis, Lynx rufus, ocelot,
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virus
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The family Herpesviridae is a group of double-stranded DNA viruses with >120 kb
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genomes and the ability to establish life-long infection of host organisms. Herpesviridae is
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divided into three subfamilies: Alphaherpesvirinae, Betaherpesvirinae and
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Gammaherpesvirinae. Gammaherpesviruses (GHVs) are found in a wide range of animal
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species (Ackermann 2006). They typically favor establishment of latent infection, often in
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lymphocytes, with occasional or condition-dependent reactivation of gene expression and
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replication (Speck and Ganem 2010). GHVs can cause a range of disease conditions affecting
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human and animal health such as lymphoproliferative disorders, including lymphoma, and non-
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lymphoid cancers (Ackermann 2006). Evidence for the presence of GHVs in felid species has
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included detection of a GHV DNA sequence in an African lion (Ehlers et al. 2008) and detection
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of bovine herpesvirus 4 in domestic cats (Kruger et al. 2000). We recently were able to greatly
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expand this analysis by identifying DNA sequences of three new felid gammaherpesviruses in
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domestic cats (Felis catus gammaherpesvirus 1, FcaGHV1), bobcats (Lynx rufus
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gammaherpesvirus 1, LruGHV1), and pumas (Puma concolor gammaherpesvirus 1, PcoGHV1)
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(Troyer et al. 2014). Using real-time qPCR, the prevalence of GHV DNA in the blood of felids
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from the USA was determined. FcaGHV1 was found in 21/135 (16%) domestic cats, PcoGHV1
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was detected in 5/83 (6%) pumas, and LruGHV1 was detected in 30/64 (47%) bobcats and 11/83
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(13%) pumas screened (Troyer et al. 2014). Subsequently we detected FcaGHV1 in domestic
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cats in Singapore and Australia indicating a likely worldwide distribution (Beatty et al. 2014).
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FcaGHV1 infection correlated with the presence of multiple co-pathogens including feline
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immunodeficiency virus and was more common in cats classified as unhealthy, suggesting
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possible disease association for this felid GHV (Beatty et al. 2014). The finding that three
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distinct felid species tested were infected with GHVs across wide geographic ranges implies that
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GHVs may be common in other felid species. In addition, the presence of both PcoGHV1 and
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LruGHV1 in pumas suggests that felids can be infected with more than one GHV. Therefore, we
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sought to determine whether ocelots are infected with a GHV and whether bobcats are infected
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with GHVs in addition to LruGHV1. We employed a degenerate PCR strategy which has been
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successful in detecting GHV gene sequences from felids and many other host organisms to
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conduct this analysis (Ehlers et al. 2008, Troyer et al. 2014).
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Blood samples (n = 5) were previously collected from free-ranging ocelots on Barro
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Colorado Island, Panama from 2001 to 2004 (Franklin et al. 2008). Sampling protocols were
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reviewed by appropriate animal care committees and appropriate permits were obtained prior to
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collection. DNA was extracted from ocelot blood as previously described (Franklin et al. 2008).
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Bobcat spleen tissues (n = 55) were collected from hunter-killed and road-killed bobcats in
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western Colorado during 2007 and 2008, frozen at -80°C, and DNA was extracted using a
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previously described protocol (Zheng et al. 2011). We tested ocelot blood DNA and bobcat
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spleen DNA samples using a degenerate nested pan-GHV PCR (Ehlers et al. 2008) to amplify a
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portion of the glycoprotein B gene (gB) with reaction conditions as previously described (Troyer
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et al. 2014). Negative controls (water template) were consistently negative. We detected bands
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of the expected size for gB amplification (~500 bp) by agarose gel electrophoresis in 2/5 ocelots
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and 35/55 bobcats. DNAs were purified using the QIAquick PCR purification kit (Qiagen) and
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sequenced in both directions by the Colorado State University Proteomics Facility. Primer
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sequences were removed and unique 453 bp sequences were then compared to other GHV gB
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sequences using BioEdit (Hall 1999) and NBCI BLAST programs. The two GHV gB sequences
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from ocelots were identical to each other but distinct from any published sequences in GenBank
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with greatest nucleotide identity to LruGHV1 (95.8%). Of the 35 GHV gB sequences from
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bobcats, 34 matched the gB sequence of the previously identified bobcat GHV, LruGHV1.
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However, the remaining GHV gB sequence from a bobcat was distinct from LruGHV1 (88.5%
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nucleotide identity) with greatest nucleotide identity to FcaGHV1 (89.6%). The presence of
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unique GHV gB sequences likely indicates the presence of novel GHVs in these host species.
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We provisionally assigned the ocelot sequence the name Leopardus pardalis gammaherpesvirus
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1 (LpaGHV1) and the bobcat sequence the name Lynx rufus gammaherpesvirus 2 (LruGHV2).
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To obtain a larger genomic sequence for phylogenetic analysis of LpaGHV1 and
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LruGHV2, we targeted the DNA polymerase gene (Dpol) with a degenerate nested PCR and then
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performed long-distance gB to Dpol PCR (~3.4 kb) using virus-specific primers (Ehlers et al.
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2008, Troyer et al. 2014). To PCR amplify Dpol, we used the “Perca-DNApol” primers and
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reaction conditions which were previously used to amplify Dpol for FcaGHV1 (Troyer et al.
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2014). Long-distance nested PCR was then employed to close the gap between the gB sequence
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and the Dpol sequence. For LpaGHV1, reaction conditions were the same as those used
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previously for FcaGHV1 and LruGHV1 (Troyer et al. 2014) with first round primers Lpa-F1 (5’-
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AGCATGAGAGTCCAGGTCCA-3’) and Lpa-R1 (5’-CCTGAAACTGGCATCATAGGC-3’)
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and second round primers Lpa-F2 (5’-ACTTTCAGTCAGGGCACCAAA-3’) and Lpa-R2 (5’-
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ATGCGCCTCCCTTCATAAGTT-3’). Conditions for long-distance PCR of LruGHV2 were
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identical to those used previously for PcoGHV1 (Troyer et al. 2014) with first round primers
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Lru-F1 (5’-CTCCGCCGTTCATTACTTTC-3’) and Lru-R1 (5’-
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TACTCTGAAGTTTGCATCATA-3’) and second round primers Lru-F2 (5’-
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CATAAAAGCACCACAGACATT-3’) and Lru-R2 (5’-CAATATCAATAGGAGTGATGC-3’).
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Nucleotide sequences were acquired from the long distance products by primer walking. This
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resulted in unique 3.4 kb sequences for LpaGHV1 and LruGHV2 including partial open reading
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frames for gB and Dpol genes. These sequences were deposited in the GenBank database under
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the following accession numbers: LpaGHV1, KP721220 and LruGHV2, KP721221.
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We performed a maximum likelihood phylogenetic analysis on the novel felid GHV
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sequences along with a set of diverse GHVs using concatenated gB and Dpol amino acid
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alignments. LpaGHV1 and LruGHV2 cluster with a clade of viruses corresponding to the
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Percavirus genus of GHVs (Figure 1). The percaviruses are split into two strongly supported
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sub-clades. The first includes equine herpesviruses EHV2 and EHV5. The second includes
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mustelid herpesvirus 1 (MusHV1) of badgers and four felid GHVs: LruGHV2, LpaGHV1,
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FcaGHV1 and LruGHV1. Within this second group, the felid GHVs form their own well-
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supported clade apart from MusHV1. Within the felid GHVs, LruGHV1 of bobcats is more
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closely related to the domestic cat and ocelot GHVs than to the new GHV found in bobcats,
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LruGHV2. There are many GHVs for which the gB-Dpol region has not been completely
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sequenced and typically only a small fragment (160 to 220 bp) of the Dpol is available. Of these,
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GHVs found in sea otter (Tseng et al. 2012), fisher (Gagnon et al. 2011), oriental small-clawed
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otter (GenBank accession no. FJ797657), and Darwin’s fox (Cabello et al. 2013) would likely
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also cluster with MusHV1 and the felid percaviruses based on high nucleotide homology of the
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short Dpol region (75-84%). This suggests that the felid percaviruses are part of a subgroup
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including GHVs found in hosts of the order Carnivora.
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LpaGHV1 is the first GHV described in ocelots while LruGHV2 is the second GHV
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identified in bobcats. LpaGHV1 was detected in 2 of 5 ocelots surveyed from a small population
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of approximately 30 animals on Barro Colorado, Panama. Future research will be required to
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determine whether ocelots in other locations are infected with LpaGHV1 or whether this virus
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can infect additional species. We detected a higher prevalence of the LruGHV1 sequence in
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bobcat spleen tissues (62%) versus blood (25%), suggesting that LruGHV1 has higher titer in
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spleen compared to blood (Troyer et al. 2014). In contrast, the novel LruGHV2 sequence was
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detected in the spleen of only one adult male bobcat. Additional study will be necessary to
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determine whether LruGHV2 is widely present in bobcats or other North American felid species.
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Phylogenetic analysis of LpaGHV1 and LruGHV2 sequences contributes support for the
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presence of a felid GHV sub-clade within the Percavirus genus of GHVs including sequences
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from domestic cats, bobcats and ocelots. These felid viruses also appear to be part of a
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carnivore-associated lineage within the percaviruses. LpaGHV1 and LruGHV2 were detected in
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animals without a known disease condition, though GHVs have been associated with
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lymphoproliferative disease and a variety of other disease conditions (Ackermann 2006).
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Notably among the percaviruses, EHV2 and EHV5 have been linked to equine respiratory
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disease and poor performance (Fortier et al. 2010). Among the carnivore-associated
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percaviruses, fisher herpesvirus and mustelid herpesvirus 2 were isolated from animals with
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ulcerative lesions (Gagnon et al. 2011, Tseng et al. 2012) and FcaGHV1 was associated with
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poor health condition and increased incidence of FIV infection in cats (Beatty et al. 2014). Thus,
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it is plausible that LpaGHV1 and LruGHV2 may play a role in the health of their felid hosts,
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although this remains to be determined. Molecular identification of these novel GHVs
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contributes to our understanding of herpesvirus diversity as well as felid health and ecology.
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We would like to acknowledge Sam Franklin for assistance in providing ocelot blood
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samples. Collection of bobcat samples was supported by a grant to SV from the NSF-NIH
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Ecology of Infectious Disease Program (EF0723676).
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Figure Legends
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Figure 1. Phylogenetic analysis of gammaherpesviruses. Amino acid sequences derived from
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partial gB and Dpol gene sequences were aligned using T-Coffee (Notredame et al. 2000).
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Positions with gaps and areas of weak support for the alignment were removed. The resulting
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gB and Dpol alignments were concatenated to form a single amino acid alignment with 999
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positions for phylogenetic analysis. Maximum likelihood phylogenetic analyses were performed
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using the PhyML program (Guindon and Gascuel 2003) with the substitution model of Le and
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Gascuel (LG) and gamma distribution with five discreet categories. The betaherpesvirus human
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cytomegalovirus was used to root the tree (HHV5, NC006273) but is not displayed due to space
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constraints. Bootstrap analyses were performed with 100 iterations and support for each node is
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displayed (values <50 are not shown). Phylogenetic clusters representing GHV genera are
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shown in color and were inferred on the basis of most recent common ancestor of established
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members of each GHV genera (indicated with asterisks). Novel GHVs identified in this study
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are indicated with boxes and other felid GHVs are underlined. Virus abbreviations and GenBank
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accession numbers are as follows: HHV4, Human herpesvirus 4 (Epstein-Barr virus),
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NC007605; CalHV3, Callitrichine herpesvirus 3, NC004367; SuHV3, Suid herpesvirus 3,
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AF478169; AlHV1, Alcelaphine herpesvirus 1, NC002531; OvHV2, Ovine herpesvirus 2,
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NC007646; SaHV2, Saimiriine herpesvirus 2, NC001350; HHV8, Human herpesvirus 8
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(Kaposi’s sarcoma-associated herpesvirus), NC009333; BoHV4, Bovine herpesvirus 4,
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NC002665; MuHV4, Murid herpesvirus 4, NC001826; PcoGHV1, Puma concolor
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gammaherpesvirus 1, KF840717; PleoGHV1, Panthera leo gammaherpesvirus 1, DQ789370;
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EHV2, Equid herpesvirus 2, NC001650; EHV5, Equid herpesvirus 5, AF050671; CcroGHV1,
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Crocuta crocuta gammaherpesvirus 1, DQ789371; EzebGHV1, Equus zebra gammaherpesvirus
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1, AY495965; MusHV1, Mustelid herpesvirus 1, AF376034; LruGHV2, Lynx rufus
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gammaherpesvirus 2, KP721221; Leopardus pardalis gammaherpesvirus 1, KP721220;
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FcaGHV1, Felis catus gammaherpesvirus 1, KF840715; LruGHV1, Lynx rufus
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gammaherpesvirus 1, KF840716.