1 Short Communications
1 Identification of novel gammaherpesviruses in ocelots (Leopardus pardalis) and bobcats
2 (Lynx rufus)
3 Caitlin C. Lozano,1 Linda L. Sweanor,1 Grete Wilson-Henjum,1 Roland W. Kays,2,3 Ricardo
4 Moreno,3,4 Sue VandeWoude,1 and Ryan M. Troyer,1,5
5
6 1Department of Microbiology, Immunology and Pathology, Colorado State University, 1619
7 Campus Delivery, Fort Collins, Colorado 80523-1619, USA
8 2North Carolina Museum of Natural Sciences, 11 West Jones Street, Raleigh, North Carolina
9 27601, USA
10 3Smithsonian Tropical Research Institute, Roosvelt Avenue, 401 Tupper Building, Balboa,
11 Ancón, Panamá, República de Panamá
12 4Yaguará Panamá-Sociedad Panameña de Biología, San Francisco, Calle 71, Chalet 15, Panamá
13 5Current address: Department of Biomedical Sciences, Oregon State University, 105 Magruder
14 Hall, Corvallis, Oregon 97331, USA
15
16 Corresponding author:
17 Ryan Troyer
18 105 Magruder Hall, Oregon State University, Corvallis, OR 97331
19 Tel: 970-690-2705
21
22 Word count: 1885
23 2 Short Communications
24 Abstract: Gammaherpesviruses (GHVs) have been identified in many species and are often
25 associated with disease. Recently, we characterized three novel felid GHVs in domestic cats,
26 bobcats, and pumas. In this study we sought to determine whether free ranging ocelots
27 (Leopardus pardalis) and bobcats (Lynx rufus) are infected with additional GHVs. We screened
28 DNA samples from ocelots on Barro Colorado Island, Panama and bobcats in western Colorado
29 using a degenerate nested PCR that targets the GHV glycoprotein B gene. We identified a novel
30 GHV glycoprotein B sequence in two ocelots and a second novel sequence in a bobcat which is
31 distinct from the previously characterized bobcat GHV (Lynx rufus GHV1). Utilizing additional
32 degenerate and virus-specific PCRs, we extended these sequences to include 3.4 kb of the GHV
33 glycoprotein B and DNA polymerase genes. These sequences indicate the presence of the first
34 GHV detected in ocelots and the second GHV detected in bobcats. These viruses were
35 provisionally named Leopardus pardalis gammaherpesvirus 1 (LpaGHV1) and Lynx rufus
36 gammaherpesvirus 2 (LruGHV2), respectively. Phylogenetic analysis indicates that these
37 viruses are most closely related to recently identified GHVs of the Percavirus genus found in
38 domestic cats (Felis catus GHV1) and bobcats (Lynx rufus GHV1), suggesting that a cluster of
39 multiple felid GHVs exists within the Percavirus genus.
40 Key Words: bobcat, gammaherpesvirus, herpesvirus, Leopardus pardalis, Lynx rufus, ocelot,
41 virus
42
43 The family Herpesviridae is a group of double-stranded DNA viruses with >120 kb
44 genomes and the ability to establish life-long infection of host organisms. Herpesviridae is
45 divided into three subfamilies: Alphaherpesvirinae, Betaherpesvirinae and 3 Short Communications
46 Gammaherpesvirinae. Gammaherpesviruses (GHVs) are found in a wide range of animal
47 species (Ackermann 2006). They typically favor establishment of latent infection, often in
48 lymphocytes, with occasional or condition-dependent reactivation of gene expression and
49 replication (Speck and Ganem 2010). GHVs can cause a range of disease conditions affecting
50 human and animal health such as lymphoproliferative disorders, including lymphoma, and non-
51 lymphoid cancers (Ackermann 2006). Evidence for the presence of GHVs in felid species has
52 included detection of a GHV DNA sequence in an African lion (Ehlers et al. 2008) and detection
53 of bovine herpesvirus 4 in domestic cats (Kruger et al. 2000). We recently were able to greatly
54 expand this analysis by identifying DNA sequences of three new felid gammaherpesviruses in
55 domestic cats (Felis catus gammaherpesvirus 1, FcaGHV1), bobcats (Lynx rufus
56 gammaherpesvirus 1, LruGHV1), and pumas (Puma concolor gammaherpesvirus 1, PcoGHV1)
57 (Troyer et al. 2014). Using real-time qPCR, the prevalence of GHV DNA in the blood of felids
58 from the USA was determined. FcaGHV1 was found in 21/135 (16%) domestic cats, PcoGHV1
59 was detected in 5/83 (6%) pumas, and LruGHV1 was detected in 30/64 (47%) bobcats and 11/83
60 (13%) pumas screened (Troyer et al. 2014). Subsequently we detected FcaGHV1 in domestic
61 cats in Singapore and Australia indicating a likely worldwide distribution (Beatty et al. 2014).
62 FcaGHV1 infection correlated with the presence of multiple co-pathogens including feline
63 immunodeficiency virus and was more common in cats classified as unhealthy, suggesting
64 possible disease association for this felid GHV (Beatty et al. 2014). The finding that three
65 distinct felid species tested were infected with GHVs across wide geographic ranges implies that
66 GHVs may be common in other felid species. In addition, the presence of both PcoGHV1 and
67 LruGHV1 in pumas suggests that felids can be infected with more than one GHV. Therefore, we
68 sought to determine whether ocelots are infected with a GHV and whether bobcats are infected 4 Short Communications
69 with GHVs in addition to LruGHV1. We employed a degenerate PCR strategy which has been
70 successful in detecting GHV gene sequences from felids and many other host organisms to
71 conduct this analysis (Ehlers et al. 2008, Troyer et al. 2014).
72 Blood samples (n = 5) were previously collected from free-ranging ocelots on Barro
73 Colorado Island, Panama from 2001 to 2004 (Franklin et al. 2008). Sampling protocols were
74 reviewed by appropriate animal care committees and appropriate permits were obtained prior to
75 collection. DNA was extracted from ocelot blood as previously described (Franklin et al. 2008).
76 Bobcat spleen tissues (n = 55) were collected from hunter-killed and road-killed bobcats in
77 western Colorado during 2007 and 2008, frozen at -80°C, and DNA was extracted using a
78 previously described protocol (Zheng et al. 2011). We tested ocelot blood DNA and bobcat
79 spleen DNA samples using a degenerate nested pan-GHV PCR (Ehlers et al. 2008) to amplify a
80 portion of the glycoprotein B gene (gB) with reaction conditions as previously described (Troyer
81 et al. 2014). Negative controls (water template) were consistently negative. We detected bands
82 of the expected size for gB amplification (~500 bp) by agarose gel electrophoresis in 2/5 ocelots
83 and 35/55 bobcats. DNAs were purified using the QIAquick PCR purification kit (Qiagen) and
84 sequenced in both directions by the Colorado State University Proteomics Facility. Primer
85 sequences were removed and unique 453 bp sequences were then compared to other GHV gB
86 sequences using BioEdit (Hall 1999) and NBCI BLAST programs. The two GHV gB sequences
87 from ocelots were identical to each other but distinct from any published sequences in GenBank
88 with greatest nucleotide identity to LruGHV1 (95.8%). Of the 35 GHV gB sequences from
89 bobcats, 34 matched the gB sequence of the previously identified bobcat GHV, LruGHV1.
90 However, the remaining GHV gB sequence from a bobcat was distinct from LruGHV1 (88.5%
91 nucleotide identity) with greatest nucleotide identity to FcaGHV1 (89.6%). The presence of 5 Short Communications
92 unique GHV gB sequences likely indicates the presence of novel GHVs in these host species.
93 We provisionally assigned the ocelot sequence the name Leopardus pardalis gammaherpesvirus
94 1 (LpaGHV1) and the bobcat sequence the name Lynx rufus gammaherpesvirus 2 (LruGHV2).
95 To obtain a larger genomic sequence for phylogenetic analysis of LpaGHV1 and
96 LruGHV2, we targeted the DNA polymerase gene (Dpol) with a degenerate nested PCR and then
97 performed long-distance gB to Dpol PCR (~3.4 kb) using virus-specific primers (Ehlers et al.
98 2008, Troyer et al. 2014). To PCR amplify Dpol, we used the “Perca-DNApol” primers and
99 reaction conditions which were previously used to amplify Dpol for FcaGHV1 (Troyer et al.
100 2014). Long-distance nested PCR was then employed to close the gap between the gB sequence
101 and the Dpol sequence. For LpaGHV1, reaction conditions were the same as those used
102 previously for FcaGHV1 and LruGHV1 (Troyer et al. 2014) with first round primers Lpa-F1 (5’-
103 AGCATGAGAGTCCAGGTCCA-3’) and Lpa-R1 (5’-CCTGAAACTGGCATCATAGGC-3’)
104 and second round primers Lpa-F2 (5’-ACTTTCAGTCAGGGCACCAAA-3’) and Lpa-R2 (5’-
105 ATGCGCCTCCCTTCATAAGTT-3’). Conditions for long-distance PCR of LruGHV2 were
106 identical to those used previously for PcoGHV1 (Troyer et al. 2014) with first round primers
107 Lru-F1 (5’-CTCCGCCGTTCATTACTTTC-3’) and Lru-R1 (5’-
108 TACTCTGAAGTTTGCATCATA-3’) and second round primers Lru-F2 (5’-
109 CATAAAAGCACCACAGACATT-3’) and Lru-R2 (5’-CAATATCAATAGGAGTGATGC-3’).
110 Nucleotide sequences were acquired from the long distance products by primer walking. This
111 resulted in unique 3.4 kb sequences for LpaGHV1 and LruGHV2 including partial open reading
112 frames for gB and Dpol genes. These sequences were deposited in the GenBank database under
113 the following accession numbers: LpaGHV1, KP721220 and LruGHV2, KP721221. 6 Short Communications
114 We performed a maximum likelihood phylogenetic analysis on the novel felid GHV
115 sequences along with a set of diverse GHVs using concatenated gB and Dpol amino acid
116 alignments. LpaGHV1 and LruGHV2 cluster with a clade of viruses corresponding to the
117 Percavirus genus of GHVs (Figure 1). The percaviruses are split into two strongly supported
118 sub-clades. The first includes equine herpesviruses EHV2 and EHV5. The second includes
119 mustelid herpesvirus 1 (MusHV1) of badgers and four felid GHVs: LruGHV2, LpaGHV1,
120 FcaGHV1 and LruGHV1. Within this second group, the felid GHVs form their own well-
121 supported clade apart from MusHV1. Within the felid GHVs, LruGHV1 of bobcats is more
122 closely related to the domestic cat and ocelot GHVs than to the new GHV found in bobcats,
123 LruGHV2. There are many GHVs for which the gB-Dpol region has not been completely
124 sequenced and typically only a small fragment (160 to 220 bp) of the Dpol is available. Of these,
125 GHVs found in sea otter (Tseng et al. 2012), fisher (Gagnon et al. 2011), oriental small-clawed
126 otter (GenBank accession no. FJ797657), and Darwin’s fox (Cabello et al. 2013) would likely
127 also cluster with MusHV1 and the felid percaviruses based on high nucleotide homology of the
128 short Dpol region (75-84%). This suggests that the felid percaviruses are part of a subgroup
129 including GHVs found in hosts of the order Carnivora.
130 LpaGHV1 is the first GHV described in ocelots while LruGHV2 is the second GHV
131 identified in bobcats. LpaGHV1 was detected in 2 of 5 ocelots surveyed from a small population
132 of approximately 30 animals on Barro Colorado, Panama. Future research will be required to
133 determine whether ocelots in other locations are infected with LpaGHV1 or whether this virus
134 can infect additional species. We detected a higher prevalence of the LruGHV1 sequence in
135 bobcat spleen tissues (62%) versus blood (25%), suggesting that LruGHV1 has higher titer in
136 spleen compared to blood (Troyer et al. 2014). In contrast, the novel LruGHV2 sequence was 7 Short Communications
137 detected in the spleen of only one adult male bobcat. Additional study will be necessary to
138 determine whether LruGHV2 is widely present in bobcats or other North American felid species.
139 Phylogenetic analysis of LpaGHV1 and LruGHV2 sequences contributes support for the
140 presence of a felid GHV sub-clade within the Percavirus genus of GHVs including sequences
141 from domestic cats, bobcats and ocelots. These felid viruses also appear to be part of a
142 carnivore-associated lineage within the percaviruses. LpaGHV1 and LruGHV2 were detected in
143 animals without a known disease condition, though GHVs have been associated with
144 lymphoproliferative disease and a variety of other disease conditions (Ackermann 2006).
145 Notably among the percaviruses, EHV2 and EHV5 have been linked to equine respiratory
146 disease and poor performance (Fortier et al. 2010). Among the carnivore-associated
147 percaviruses, fisher herpesvirus and mustelid herpesvirus 2 were isolated from animals with
148 ulcerative lesions (Gagnon et al. 2011, Tseng et al. 2012) and FcaGHV1 was associated with
149 poor health condition and increased incidence of FIV infection in cats (Beatty et al. 2014). Thus,
150 it is plausible that LpaGHV1 and LruGHV2 may play a role in the health of their felid hosts,
151 although this remains to be determined. Molecular identification of these novel GHVs
152 contributes to our understanding of herpesvirus diversity as well as felid health and ecology.
153 We would like to acknowledge Sam Franklin for assistance in providing ocelot blood
154 samples. Collection of bobcat samples was supported by a grant to SV from the NSF-NIH
155 Ecology of Infectious Disease Program (EF0723676).
156
157 Literature Cited
158 Ackermann M. 2006. Pathogenesis of gammaherpesvirus infections. Vet Microbiol 113:211-222. 8 Short Communications
159 Beatty JA, Troyer RM, Carver S, Barrs VR, Espinasse F, Conradi O, Stutzman-Rodriguez K, 160 Chan CC, Tasker S, Lappin MR, Vandewoude S. 2014. Felis catus gammaherpesvirus 1; 161 a widely endemic potential pathogen of domestic cats. Virology 460-461:100-107. 162 Cabello J, Esperon F, Napolitano C, Hidalgo E, Davila JA, Millan J. 2013. Molecular 163 identification of a novel gammaherpesvirus in the endangered Darwin's fox (Lycalopex 164 fulvipes). J Gen Virol 94:2745-2749. 165 Ehlers B, Dural G, Yasmum N, Lembo T, De Thoisy B, Ryser-Degiorgis MP, Ulrich RG, 166 Mcgeoch DJ. 2008. Novel mammalian herpesviruses and lineages within the 167 Gammaherpesvirinae: cospeciation and interspecies transfer. J Virol 82:3509-3516. 168 Fortier G, Van Erck E, Pronost S, Lekeux P, Thiry E. 2010. Equine gammaherpesviruses: 169 pathogenesis, epidemiology and diagnosis. Vet J 186:148-156. 170 Franklin SP, Kays RW, Moreno R, Terwee JA, Troyer JL, Vandewoude S. 2008. Ocelots on 171 Barro Colorado Island are infected with feline immunodeficiency virus but not other 172 common feline and canine viruses. J Wildl Dis 44:760-765. 173 Gagnon CA, Tremblay J, Larochelle D, Music N, Tremblay D. 2011. Identification of a novel 174 herpesvirus associated with cutaneous ulcers in a fisher (Martes pennanti). J Vet Diagn 175 Invest 23:986-990. 176 Guindon S, Gascuel O. 2003. A simple, fast, and accurate algorithm to estimate large 177 phylogenies by maximum likelihood. Syst Biol 52:696-704. 178 Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis 179 program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95-98. 180 Kruger JM, Venta PJ, Swenson CL, Syring R, Gibbons-Burgener SN, Richter M, Maes RK. 181 2000. Prevalence of bovine herpesvirus-4 infection in cats in Central Michigan. J Vet 182 Intern Med 14:593-597. 183 Notredame C, Higgins DG, Heringa J. 2000. T-Coffee: A novel method for fast and accurate 184 multiple sequence alignment. J Mol Biol 302:205-217. 185 Speck SH, Ganem D. 2010. Viral latency and its regulation: lessons from the gamma- 186 herpesviruses. Cell Host Microbe 8:100-115. 187 Troyer RM, Beatty JA, Stutzman-Rodriguez KR, Carver S, Lozano CC, Lee JS, Lappin MR, 188 Riley SP, Serieys LE, Logan KA, Sweanor LL, Boyce WM, Vickers TW, Mcbride R, 189 Crooks KR, Lewis JS, Cunningham MW, Rovnak J, Quackenbush SL, Vandewoude S. 190 2014. Novel gammaherpesviruses in north american domestic cats, bobcats, and pumas: 191 identification, prevalence, and risk factors. J Virol 88:3914-3924. 192 Tseng M, Fleetwood M, Reed A, Gill VA, Harris RK, Moeller RB, Lipscomb TP, Mazet JA, 193 Goldstein T. 2012. Mustelid herpesvirus-2, a novel herpes infection in northern sea otters 194 (Enhydra lutris kenyoni). J Wildl Dis 48:181-185. 195 Zheng X, Carver S, Troyer RM, Terwee JA, Vandewoude S. 2011. Prior virus exposure alters 196 the long-term landscape of viral replication during feline lentiviral infection. Viruses 197 3:1891-1908. 198 199
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201 9 Short Communications
202 Figure Legends
203 Figure 1. Phylogenetic analysis of gammaherpesviruses. Amino acid sequences derived from
204 partial gB and Dpol gene sequences were aligned using T-Coffee (Notredame et al. 2000).
205 Positions with gaps and areas of weak support for the alignment were removed. The resulting
206 gB and Dpol alignments were concatenated to form a single amino acid alignment with 999
207 positions for phylogenetic analysis. Maximum likelihood phylogenetic analyses were performed
208 using the PhyML program (Guindon and Gascuel 2003) with the substitution model of Le and
209 Gascuel (LG) and gamma distribution with five discreet categories. The betaherpesvirus human
210 cytomegalovirus was used to root the tree (HHV5, NC006273) but is not displayed due to space
211 constraints. Bootstrap analyses were performed with 100 iterations and support for each node is
212 displayed (values <50 are not shown). Phylogenetic clusters representing GHV genera are
213 shown in color and were inferred on the basis of most recent common ancestor of established
214 members of each GHV genera (indicated with asterisks). Novel GHVs identified in this study
215 are indicated with boxes and other felid GHVs are underlined. Virus abbreviations and GenBank
216 accession numbers are as follows: HHV4, Human herpesvirus 4 (Epstein-Barr virus),
217 NC007605; CalHV3, Callitrichine herpesvirus 3, NC004367; SuHV3, Suid herpesvirus 3,
218 AF478169; AlHV1, Alcelaphine herpesvirus 1, NC002531; OvHV2, Ovine herpesvirus 2,
219 NC007646; SaHV2, Saimiriine herpesvirus 2, NC001350; HHV8, Human herpesvirus 8
220 (Kaposi’s sarcoma-associated herpesvirus), NC009333; BoHV4, Bovine herpesvirus 4,
221 NC002665; MuHV4, Murid herpesvirus 4, NC001826; PcoGHV1, Puma concolor
222 gammaherpesvirus 1, KF840717; PleoGHV1, Panthera leo gammaherpesvirus 1, DQ789370;
223 EHV2, Equid herpesvirus 2, NC001650; EHV5, Equid herpesvirus 5, AF050671; CcroGHV1,
224 Crocuta crocuta gammaherpesvirus 1, DQ789371; EzebGHV1, Equus zebra gammaherpesvirus 10 Short Communications
225 1, AY495965; MusHV1, Mustelid herpesvirus 1, AF376034; LruGHV2, Lynx rufus
226 gammaherpesvirus 2, KP721221; Leopardus pardalis gammaherpesvirus 1, KP721220;
227 FcaGHV1, Felis catus gammaherpesvirus 1, KF840715; LruGHV1, Lynx rufus
228 gammaherpesvirus 1, KF840716.