New World feline APOBEC3 potently controls inter-genus lentiviral transmission
The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; A3) gene family appears only in mammalian genomes. Some A3 proteins can be incorporated into progeny virions and inhibit lentiviral replication. In turn, the lentiviral viral infectivity factor (Vif) counteracts the A3-mediated antiviral effect by degrading A3 proteins. Recent investigations have suggested that lentiviral vif genes evolved to combat mammalian APOBEC3 proteins, and have further proposed that the Vif-A3 interaction may help determine the co-evolutionary history of cross-species lentiviral transmission in mammals.
Here we address the co-evolutionary relationship between two New World felids, the puma (Puma concolor) and the bobcat (Lynx rufus), and their lentiviruses, which are designated puma lentiviruses (PLVs). We demonstrate that PLV-A Vif counteracts the antiviral action of APOBEC3Z3 (A3Z3) of both puma and bobcat, whereas PLV-B Vif counteracts only puma A3Z3. The species specificity of PLV-B Vif is irrespective of the phylogenic relationships of feline species in the genera Puma, Lynx and Acinonyx. We reveal that the amino acid at position 178 in the puma and bobcat A3Z3 is exposed on the protein surface and determines the sensitivity to PLV-B Vif-mediated degradation. Moreover, although both the puma and bobcat A3Z3 genes are polymorphic, their sensitivity/resistance to PLV Vif-mediated degradation is conserved.
To the best of our knowledge, this is the first study suggesting that the host A3 protein potently controls inter-genus lentiviral transmission. Our findings provide the first evidence suggesting that the co-evolutionary arms race between lentiviruses and mammals has occurred in the New World.
KeywordsLentivirus FIV APOBEC3 Vif Evolutionary arms race Puma Bobcat PLV New World
apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3)
feline immunodeficiency virus
million years ago
Old World monkey
simian immunodeficiency virus
tripartite motif containing protein 5
viral infectivity factor
vesicular stomatitis virus G
The apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3; A3) proteins are cellular cytidine deaminases that are specifically encoded in mammals but not in other vertebrates [1, 2]. Mammalian A3 proteins, particularly primate A3 proteins, are considered cellular intrinsic immune factors that potently restrict lentiviral replication. To exhibit antiviral activity, some A3 proteins are incorporated into the released progeny virions and enzymatically insert guanine-to-adenine hypermutations into the viral genome, thereby halting viral replication. In turn, the lentiviral protein, viral infectivity factor (Vif), antagonizes the A3-mediated antiviral action by degrading A3 proteins via the ubiquitin/proteasome-dependent pathway (reviewed in ).
Elucidating co-evolutionary relationships between hosts and their viruses is an intriguing topic in virology and is crucial to understanding how viruses influence their hosts’ evolution and vice versa. Cell-based virological experiments are essential for a better understanding of the evolutionary conflict between mammals and their viruses, including lentiviruses. For instance, since the interaction between host A3 and lentiviral Vif is species specific, various investigations focusing on the functional relationship between A3 and Vif have recently been conducted in combination with molecular phylogenetic approaches (reviewed in ). This strategy stems from the “Red Queen hypothesis ”, which proposes that host and viral proteins have competed with one another for survival over time [6, 7, 8]. However, since most previous observations are based the Old World evolutionary events , whether the evolutionary arms race between mammals and lentiviruses has occurred in the New World is unclear.
Feline immunodeficiency virus (FIV) is a lentivirus that was first isolated in 1987 from domestic cats (Felis catus) with chronic AIDS-like disorders . Domestic cats encode multiple A3 genes, and the feline A3 protein (designated A3Z3) potently impairs FIV replication by incorporating into nascent virions [10, 11, 12, 13, 14, 15]. In response, FIV Vif antagonizes the antiviral activity of feline A3Z3 by degrading this protein [11, 12, 13, 14, 15]. To elucidate the evolutionary relationship between FIV and felids, we have recently reported that the domestic cat A3Z3 is polymorphic and that a haplotype of the domestic cat A3Z3 is resistant to Vif-mediated degradation by FIVfca, which is the FIV that infects domestic cats . Our findings suggest that the domestic cat A3Z3 is under positive selection due to evolutionary selective pressure caused by FIVfca or related ancestral viruses [12, 14].
In this study, we perform cell-based virological experiments and demonstrate that the species specificity of the Vif proteins of PLV-A and PLV-B is different between each other. Through the combinational investigations of experimental virology and molecular phylogenetics, we also provide evidence suggesting that bobcat A3Z3 plays a role as the species barrier specifically against PLV-B. Moreover, we determine the amino acid residue responsible for the sensitivity to PLV-B Vif-dependent degradation. This is the first report addressing the co-evolutionary interplay between antiviral A3 of the two New World felids and feline lentiviral Vif.
Bobcat A3Z3 is resistant to PLV-B Vif-mediated degradation
To elucidate the co-evolutionary relationship between PLVs and their host felids, particularly between viral Vif and host A3Z3, first we constructed a phylogenetic tree of FIV vif gene. As shown in Fig. 1a, FIVfca and FIVple formed clusters based on their hosts. In sharp contrast, the FIVlru sequences were co-mingled with some FIVpco sequences, whereas the other FIVpco sequences formed a cluster (Fig. 1a). These results are consistent with the phylogeny of the full-length FIV sequences shown in a previous study . According to this previous report, here we designated the FIVlru and co-mingled FIVpco sequences PLV-A and the other FIVpco sequences PLV-B. The sequence identities of PLV-A Vif and PLV-B Vif were 90.1 ± 4.8 and 85.7 ± 7.1%, respectively, whereas the Vif sequence identity between PLV-A and PLV-B was 40.6 ± 1.1%. Thus, the PLV-A Vif and PLV-B Vif sequences clearly differed.
Next, we focused on the host felid evolutionary relationships. Based on comprehensive genetic information, a previous study  described the evolutionary history of the felids of interest in this study (Fig. 1b). The common ancestor of the puma (Puma concolor) and the bobcat (Lynx rufus) is estimated to have crossed the Bering Isthmus from Eurasia to the New World approximately 8.0–8.5 million years ago (Mya) and to have diverged approximately 7.2 Mya . The puma diverged from the cheetah (Acinonyx jubatus) approximately 4.9 Mya, whereas the bobcat was branched from the lynx (Lynx lynx) approximately 3.2 Mya (Fig. 1b) . Puma and bobcat resided in the New World, whereas lynx and cheetah returned to Eurasia approximately 1.2–1.6 Mya (Fig. 1b) . To assess the phylogenetic relationships of the A3Z3 genes of these felids, we collected body hairs from a bobcat and a cheetah from Japanese zoos and determined their A3Z3 sequences (see Additional file 1: Fig. S1 and Additional file 2: Table S1). As shown in Fig. 1c, the topology of the phylogenetic tree of the A3Z3 genes form these four felids corresponded to their evolutionary relationships.
Based on these observations, two scenarios are assumed. The first scenario was that both PLV-A and PLV-B co-circulated in pumas and bobcats in the New World but PLV-B became extinct only in the bobcat population (“scenario 1” in Fig. 1d). The other possibility was that PLV-A and PLV-B were specific viruses in bobcats and pumas, respectively, in the past. After co-habitation of these two feline species in the New World, PLV-A was transferred from bobcats to pumas, whereas PLV-B CST from pumas to bobcats was hampered for unknown reasons (“scenario 2” in Fig. 1d).
PLV Vif used in this study
Puma A3Z3 is uniquely sensitive to PLV-B Vif-mediated degradation
The threonine residue at position 178 of bobcat A3Z3 confers the resistance to PLV-B Vif-mediated degradation
Puma and bobcat A3Z3 are polymorphic
In this study, we identified the A3Z3 sequences of two felids, the bobcat and cheetah, and demonstrated that PLV-A Vif counteracted the A3Z3 proteins of both the puma and bobcat lineages, whereas PLV-B Vif counteracted only the puma A3Z3 (Figs. 1, 2, 3). Through loss-of-function (Fig. 5) and gain-of-function (Fig. 6) experiments, we also determined that the amino acid at position 178 of the puma and bobcat A3Z3 was responsible for the sensitivity to PLV-B Vif. In addition, structural modeling suggested that this residue was exposed to the protein surface (Fig. 4). Furthermore, although the puma and bobcat A3Z3 genes were polymorphic, the sensitivity to PLV-B Vif was conserved in each species (Fig. 7). Some previous studies have addressed the potential of OWM A3G to restrict HIV-1 infection [25, 26]. However, OWMs do not infect with HIV-1 in nature. Therefore, to the best of our knowledge, this study is the first to suggest that inter-species lentiviral transmission in nature is controlled by a host A3 protein.
As summarized in Fig. 1a, d, PLV-A is shared by both pumas and bobcats in North America, whereas PLV-B is detected only in pumas. Through cell-based virological experiments, here we demonstrated that PLV-A Vif degraded the A3Z3 protein of feline lineages including puma and bobcat, whereas PLV-B Vif counteracted only the puma A3Z3. Additionally, the bobcat A3Z3 exhibited a significant antiviral effect even in the presence of PLV-B Vif. Because the functionality of PLV Vif is independent of the sampling location and year (Fig. 2 and Table 1), our results suggest that the functional relationship between PLV Vif and feline A3Z3 is conserved. Moreover, we found a polymorphism in the puma A3Z3, but the sensitivity of the puma A3Z3 variants to PLV-B Vif-mediated degradation was also conserved (Fig. 6). These findings suggest that “scenario 2” reasonably explains the difference in species tropism between PLV-A and PLV-B and that bobcat A3Z3 acts as a species barrier to restrict PLV-B CST of PLV-B from pumas (Fig. 1d). To the best of our knowledge, this is the first report demonstrating that the host A3 protein is the factor that restricts non-primate lentiviral CST.
Consistent with our results, PLV-A and PLV-B form clusters by sampling location [17, 18, 19]. Moreover, the PLV-As detected in pumas (FIVpco) and bobcats (FIVlru), particularly those isolated in California, were highly co-mingled in a cluster (Fig. 1a). These observations suggest that PLV-As have been transmitted relatively recently from bobcats to pumas after geological sequestration, and we can assume that this bobcat-to-puma CST event occurs frequently at least in California.
Our findings in New World felids were reminiscent of the fact that simian immunodeficiency viruses (SIVs) in Old World monkeys (OWMs) were transferred to chimpanzees, which originated in the emergence of a novel SIV in chimpanzees (SIVcpz) (reviewed in [27, 28]). SIV CST from OWMs to chimpanzees in Africa is explained phylogenetically and is assumed to be due to the prey-predator relationship between OWMs and chimpanzees. Small OWMs are the prey of chimpanzees in the wild; thus, chimpanzees are frequently exposed to various SIVs that infect their OWM prey species [29, 30]. Similarly, pumas are a top carnivore in the New World  and bobcats are their prey [32, 33]. Therefore, pumas may be frequently exposed to PLV-A in bobcats via prey-predator interactions, leading to bobcat-to-puma CST; this possibility is consistent with a previous assumption based on phylogenetic analyses .
Although PLV-A Vif degraded all of the feline A3Z3 proteins tested in this study, PLV-B Vif counteracted only the puma A3Z3, suggesting the evolutionary convergence of the puma and PLV-B. Compton and Emerman reported that the Vif protein of an SIV infecting colobus monkey (Colobus guereza), which is an OWM in Africa, counteracted only colobus A3G . Based on findings for the A3G-Vif interaction in colobus monkeys, the authors proposed convergent evolution of colobus monkeys and SIVcor and further suggested that the convergent co-evolution of these monkeys and viruses occurred at least 12 Mya . Similarly, the puma diversified from the cheetah approximately 4.9 Mya (Fig. 1c) . Puma and cheetah habitats were separated approximately 1.2–1.6 Mya after cheetahs immigrated from the New World to Eurasia (Fig. 1b) . Since the cheetah A3Z3 was not counteracted by the PLV-B Vif (Fig. 3), our findings suggested that the co-evolutionary convergence of the puma and PLV-B arose at least 1.2–1.6 Mya. Moreover, OWMs exhibit higher viral RNA loads without any disorders . Since SIVs circumvent the immune pressure from host monkeys because they are naturally infected, SIVs are less diversified in African OWMs, which is assumed to be due to the co-existence of SIVs in OWMs over a long period . Lee et al.  recently showed that the PLV-B plasma viral RNA load in pumas was significantly higher than PLV-A loads in both pumas and bobcats, whereas PLV-B was less diversified than PLV-A. This is the first study suggesting the convergent co-evolution of a mammal (puma) and a lentivirus (PLV-B) in the New World, and PLV-B can be concluded to have co-evolved with pumas over a long period, similar to apathogenic SIV infections in OWMs.
Through mutagenesis experiments, we demonstrated that the amino acid at position 178 determined the sensitivity to PLV-B Vif-dependent degradation (Fig. 5). Since both the gain- and loss-of-function experiments indicated the importance of this residue, the species specificity of PLV-B Vif against puma and bobcat A3Z3 was determined by this single amino acid. In addition, the structural biology investigation revealed that this amino acid residue was exposed on the A3Z3 protein surface (Fig. 4). In our previous study , we revealed that there are at least seven haplotypes in the domestic cat A3Z3 and that a domestic cat A3Z3 haplotype (designated haplotype V in our previous study) was resistant to FIVfca Vif-mediated degradation. Furthermore, we demonstrated that amino acid residue 65 of the domestic cat A3Z3 determines the sensitivity to FIVfca Vif . Similar to this study, residue 65 in the domestic cat A3Z3 was also exposed on the protein surface, which suggested that this residue was associated with the interaction with FIV Vif . However, the amino acid residue at position 65 was positioned at alpha-helix 2 in the domestic cat A3Z3 , whereas the residue 178 was located at alpha-helix 6 in the puma and bobcat A3Z3 (Fig. 5). Therefore, our findings suggest that these residues render resistance to FIV Vif in a different manner.
In addition to A3, other cellular proteins such as tripartite motif containing protein 5 (TRIM5)  and tetherin [37, 38] are known as restriction factors that inhibit lentiviral replication in primates. Therefore, it might be possible that these restriction factors in pumas and/or bobcats may also play roles in preventing FIV CST. However, feline TRIM5 is not functional because of the insertion of premature stop codon , and feline tetherin is unable to restrict spreading FIV infection . Furthermore, although FIV Vif is a functional antagonist against feline A3, FIV lacks functional proteins that potently counteract TRIM5 and tetherin [39, 40]. Primates encode several restriction factors against lentiviruses; however, only A3 is convincing to be a restriction factor in felids. Therefore, feline A3 may play a pivotal role in controlling CST in felids.
In summary, we demonstrated that bobcat A3Z3 was resistant to PLV-B Vif-dependent degradation, whereas PLV-A Vif overcame the antiviral action mediated by both the puma and bobcat A3Z3. The co-evolutionary relationship between primate A3 proteins and their lentiviral Vifs has been rigorously investigated [20, 22, 34, 41, 42, 43, 44]; however, few studies, including ours, have addressed the evolutionary dynamics of non-primate A3 and non-primate lentiviruses [4, 12, 14]. Here, we provided evidence suggesting that lentiviral CST between different genera (Puma and Lynx) was controlled by the Vif-A3 interaction. To the best of our knowledge, this is the first report providing evidence of the co-evolutionary arms race between mammals and lentiviruses in the New World.
To determine the feline A3Z3 sequences, blood, body hair or cryopreserved muscle tissue of pumas, bobcats and cheetahs were kindly provided by the following facilities: Tennoji zoo, Osaka, Japan; Kobe City Oji zoo, Hyogo, Japan; Omoriyama zoo, Akita, Japan; Tama Zoological Park, Tokyo, Japan; Izu Animal Kingdom, Shizuoka, Japan; and Shizuoka Municipal Nihondaira zoo, Shizuoka, Japan. Sampling was performed in accordance with the guideline of Tokyo University of Agriculture, Japan. All experimental protocols were approved by a committee at Tokyo University of Agriculture, Japan.
Sequencing PCR of the feline A3Z3 genes
Sequencing PCR of feline A3Z3 was performed as previously described [45, 46]. Briefly, genomic DNA was extracted from the samples described above using the DNA Extractor FM kit (Wako) or the DNeasy Blood & Tissue it (Qiagen). PCR was performed using the PrimeSTAR GXL DNA polymerase (Takara) and the primers are listed in Additional file 2: Table S1. The obtained PCR products were purified by gel extraction using the QIAquick gel extraction kit (Qiagen). The nucleotide sequences were determined by a DNA sequencing service (Fasmac, Kanagawa, Japan) and the data were analyzed using Sequencher v5.1 software (Gene Codes Corporation).
Molecular phylogenetic analysis of the FIV vif and feline A3Z3 genes
Molecular phylogenetic analyses were performed as previously described [12, 14, 45, 46, 47]. Briefly, the sequences of PLV Vif and feline A3Z3, some of which were newly identified in this study (Figs. 1a, c, 7a), were aligned using ClustalW implemented in MEGA7 . The alignments were verified manually at the amino acid level. Phylogenetic trees (Figs. 1a, c, 7a) were reconstructed using the maximum likelihood (ML) method with PhyML . To calculate the amino acid sequence diversity of PLV Vif, 26 and 34 amino acid sequences from PLV-A and PLV-B Vif (summarized in Fig. 1a) are used, respectively. A multiple alignment was generated using L-INS-i in MAFFT . The gapped regions were removed using trimAl with the nogaps option , and 227 amino acid sites were used for the analysis. Then, we performed pairwise comparisons of 60 amino acid sequences to calculate the amino acid sequence identity using MEGA7 .
The expressing plasmids for HA-tagged puma and lynx A3Z3 were kindly provided by Dr. Carsten Münk . The expressing plasmids for HA-tagged bobcat and cheetah A3Z3 were constructed by using the genomic DNA fragments and the primers listed in Additional file 2: Table S1. The point mutants of HA-tagged puma and bobcat A3Z3 were constructed by using a GeneArt site-directed mutagenesis system (Thermo Fisher Scientific). Each wild-type plasmid was used as the template, and the primers used are listed in Additional file 2: Table S1. The His-tagged PLV-A Vif (strains Lru1, Lru20 and Pco5) and PLV-B (strains Pco14, Pco16 and Pco27) were obtained from GeneArt gene synthesis service (Thermo Fisher Scientific). The obtained DNA fragments were digested with BamHI and SalI and inserted into the BamHI-SalI site of pDON-AI plasmid (Takara). The nucleotide sequences were determined by a DNA sequencing service (Fasmac, Kanagawa, Japan) and the data were analyzed by Sequencher v5.1 software (Gene Codes Corporation).
Cell culture and transfection
HEK293T cells (CRL-11268; ATCC) were cultured in Dulbecco’s modified Eagle medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum and antibiotics (Thermo Fisher Scientific). Transfection was performed by using PEI Max (GE Healthcare) in accordance with the manufacturers procedures and described previously [12, 14, 45, 46, 47, 52]. To analyze the dose-dependent anti-FIV activity of feline A3Z3, pFP93 (pFIVgagpolΔvif; a replication incompetent vif-deficient FIV packaging construct derived from clone FIV-34TF10 [GenBank accession number M25381]; kindly provided by Dr. Eric M. Poeschla) (200 ng), pTiger-luc (pFIVΨ-luc) (150 ng), and pMD.G (pVSVg; a vesicular stomatitis virus G [VSVg] expression plasmid) (50 ng) were co-transfected into HEK293T cells (1 × 105 cells) with feline A3Z3 expression plasmid (50, 100, 200, or 400 ng). To analyze the functional relationship between feline A3Z3 and PLV-Vif, feline A3Z3 expression plasmid (200 ng), pFP93 (200 ng), pTiger-luc (150 ng), and pMD.G (50 ng) were co-transfected into HEK293T cells with or without His-tagged PLV Vif expression plasmid (400 ng). After 48 h post-transfection, the transfected cells and culture supernatants were harvested as previously described [12, 13, 14, 45, 46, 47, 52].
Western blotting and virus reporter assay
Western blotting and reporter assay were performed as previously described [12, 13, 14, 45, 46, 47, 52]. For the Western blotting of virus particles, 340 μl of the culture supernatant was ultracentrifuged at 100,000×g for 1 h at 4 °C using a TL-100 instrument (Beckman), and the pellet was lysed with 1 × SDS buffer. For the Western blotting of transfected cells, the cells were lysed with RIPA buffer (50 mM Tris–HCl buffer [pH 7.6], 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) with protease inhibitor cocktail (Roche). The following antibodies for Western blotting: anti-His polyclonal antibody (OGHis; Medical and Biological Laboratories), anti-HA antibody (3F10; Roche), anti-FIV p24 Capsid antibody (PAK3-2C1; Santa Cruz Biotechnology); anti-alpha-tubulin (TUBA) antibody (DM1A; Sigma), and anti-VSVg antibody (P5DA; Roche). For FIV reporter assays, HEK293T cells were used for the target of infection. Ten microliter of the culture supernatant of transfected cells was inoculated into HEK293T cells in a 96-well plate (Nunc), and the firefly luciferase activity was measured by using the BrillianStar-LT assay system (Toyo-b-net) and the 2030 ARVO X multilabel counter instrument (PerkinElmer) according to the manufacturers’ procedures.
Protein homology modeling
Homology modeling was performed using the SWISS-MODEL server [53, 54, 55, 56]. After BLAST searches  of the bobcat and puma A3Z3 amino acid sequences against protein data bank sequence entries (http://www.rcsb.org/pdb/), the crystal structure of human APOBEC3A (PDB: 5KEG)  was selected as the best template for homology modeling per the Global Model Quality Estimations, QMEAN statistical parameters, and modeled sequence length. Each generated model was minimized and refined using Discovery Studio (Dassault Systèmes BIOVIA, Discovery Studio Modeling Environment, Release 4.1, San Diego: Dassault Systèmes, 2007). Mutant models and 3D images were generated with PyMOL (The PyMOL Molecular Graphics System, version 1.8 Schrödinger, LLC).
The data are expressed as averages with the standard deviations (SDs), and statistically significant differences were determined using Student’s t test.
The A3Z3 sequences of five pumas, two bobcats and two cheetahs were submitted to DDBJ (accession numbers LC376039-LC376042).
KS conceived the study and designed experiments; Y.Konno, S.Nagaoka, IK, KY, Y.Kagawa, RK, HA and TK performed experiments; Y.Konno, S.Nagaoka, IK, KY and HA analyzed data; MTU and S.Nakagawa constructed protein homology models; S.Nakagawa and KS performed phylogenetic analyses; KS wrote the paper; and Y.Konno, S.Nagaoka, IK, MTU, S.Nakagawa, Y.Koyanagi and KS edited the manuscript. All authors read and approved the final manuscript.
We thank Carsten Münk (Heinrich-Heine-Universität, Düsseldorf, Germany) and Eric M. Poeschla (University of Colorado, USA) for providing experimental materials and Tennoji zoo (Osaka, Japan), Kobe City Oji zoo (Hyogo, Japan), Omoriyama zoo (Akita, Japan), Tama Zoological Park (Tokyo, Japan), Izu Animal Kingdom (Shizuoka, Japan), and Shizuoka Municipal Nihondaira zoo (Shizuoka, Japan) for kindly providing feline samples.
The authors declare that no competing interests exist.
Consent for publication
Ethics approval and consent to participate
This study was supported in part by AMED J-PRIDE 17fm0208006h0001 (to KS); JST CREST (to KS); JSPS KAKENHI Grants-in-Aid for Scientific Research C 15K07166 (to KS), Scientific Research B (Generative Research Fields) 16KT0111 (to KS), and Scientific Research on Innovative Areas 16H06429 (to S. Nakagawa and KS), 16K21723 (to S.Nakagawa and KS), 17H05823 (to S. Nakagawa) and 17H05813 (to KS); Takeda Science Foundation (to KS); Salt Science Research Foundation (to KS); Smoking Research Foundation (to KS); Chube Ito Foundation (to KS); Fordays Self-Reliance Support in Japan (to KS); Mishima Kaiun Memorial Foundation (to KS); Tobemaki Foundation (to KS); ONO Medical Research Foundation (to KS); MEXT-Supported Program for the Strategic Research Foundation at Private Universities (to S.Nakagawa and MTU); Joint Usage/Research Center program of Institute for Frontier Life and Medical Sciences Kyoto University (to S.Nakagawa); JSPS Core-to-Core program, A. Advanced Research Networks (to Y.Koyanagi); and AMED Research on HIV/AIDS 16fk0410203h002 (to Y.Koyanagi).
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