Distribution of Endogenous Retroviruses in Crocodilians. Veterinary Science, RMC Gunn Building University of Sydney, NSW 2006,

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1 JVI Accepts, published online ahead of print on 15 July 2009 J. Virol. doi: /jvi Copyright 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 Distribution of Endogenous Retroviruses in Crocodilians Weerachai Jaratlerdsiri 1, Clara J. Rodríguez-Zárate 1, Sally R. Isberg 1,2, Chandramaya Siska Damayanti 1, Lee G. Miles 1, Nantarika Chansue 3, Chris Moran 1, Lorna Melville 4, and Jaime Gongora 1 *. 1 Centre for Advanced Technologies in Animal Genetics and Reproduction, Faculty of Veterinary Science, RMC Gunn Building University of Sydney, NSW 2006, Australia. 2 Porosus Pty Ltd, PO Box 86, Palmerston NT 0831, Australia. 3 Faculty of Veterinary Science, Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand. 4 OIC Berrimah Veterinary Laboratories, Department of Primary Industry, Fisheries and Mines, GPO Box 3000 Darwin 0801, Australia. *Corresponding author: j.gongora@usyd.edu.au

2 Abstract Knowledge of ERVs in crocodilians (Crocodylia) is limited and their distribution among extant species is unclear. Here we analyze the phylogenetic relationships of these retroelements in twenty species of crocodilians by studying the pro-pol gene. Results showed that crocodilian ERVs cluster into two major clades (CERV 1 and CERV 2). CERV 1 clustered as a sister group of Gammaretroviruses, while CERV 2 clustered distantly with respect to all known ERVs. Interestingly, CERV 1 was found only in crocodiles (Crocodylidae). The data generated here could assist future studies aimed at identifying orthologous and paralogous ERVs among crocodilians. Downloaded from on September 18, 2018 by guest

3 Endogenous retroviruses (ERVs) are copies or remnants of exogenous viruses derived from past infections of germ cells and subsequent integration into the host genome (7, 8). Most ERVs are defective, having accumulated random inactivating mutations, and are therefore not pathogenic. However, many intact ERVs have been associated with different host diseases (4, 21). Those from pigs (PERVs) are considered potentially hazardous during xenotransplantation due to reactivation or recombination with exogenous retroviruses (18). ERVs have been studied extensively in mammals and birds (9, 10, 16), while knowledge of ERVs in reptiles is limited to few host species (17, 24). Studies of a few full-length and partial pol gene sequences of some crocodilian species (Order Crocodylia) have shown that some ERV sequences clustered distantly from Spumavirus while others clustered closely with Gamma-like retroviruses (16, 17). However, their diversity, lineagespecificity and functionality have not been assessed across all extant crocodilian species. The extant Crocodylian lineage consists of twenty-three species classified in three families: Alligatoridae (alligators), Crocodylidae (crocodiles) and Gavialidae (gharials). Crocodylidae and Alligatoridae are unambiguously recognised, with gharials either lumped with Crocodylidae or assigned to a separate family Gavialidae (6, 12, 19). It has been estimated that the Alligatoridae and Crocodylidae lineages diverged from a common ancestor about million years ago, based on DNA and amino acid data (12, 19). Here we analyze the distribution, potential to function and phylogenetic relationships of ERVs in twenty extant crocodilian species by studying the proteasereverse transcriptase (pro-pol) gene. The pro-pol gene (0.8-1kb) was amplified using two sets of degenerate primers (23). PCR amplicons were gel purified and cloned

4 using standard protocols and about three ERV inserts were sequenced from each species using the sequencing service at the Australian Genome Research Facility. Sixty-five ERV DNA sequences were generated and translated to putative amino acid sequences using universal translation codes in the Molecular Toolkit interface ( Open reading frames and similarity with other available ERV sequences were determined using the blastx tool available through NCBI ( Two aligned datasets were generated using the CLUSTALW program (22); the first consisted of 286 predicted amino acids from sixty-five novel (Supplementary Table 1) and nine published (16, 17) crocodilian ERVs, and the second comprised 258 predicted amino acids from the novel crocodilian sequences and seventy-three published endogenous and exogenous retroviral sequences after exclusion of gaps. These known retroviruses included: Avian Leukosis Virus (ALV), Alpharetrovirus; Murine Leukemia Virus (MuLV), Gammaretrovirus; Mouse Mammary Tumor Virus (MMTV) and Jaagsiekte Sheep Retrovirus (JSRV), Betaretrovirus; Bovine Leukemia Virus (BLV) and Human T-lymphotropic virus (HTLV), Deltaretrovirus; Human Immunodeficiency Virus 1 (HIV-1), Equine Infectious Anemia virus (EIAV) and Visna, Lentivirus; Human Foamy Virus (HFV), Spumavirus; Walleye Dermal Sarcoma Virus (WDSV), Epsilonretrovirus; four avian, three reptilian, two amphibian and fourteen mammalian ERVs including HERVs (13); three chicken ERVs (GGERV) (10); six avian viruses similar to the alpha/beta group (7); twenty two Gamma-like ERVs (9, 16). The bestfit model (JTT matrix model with parameter α = 1.61) for the two data sets was selected by ProtTest software (1) to implement Neighbour-Joining (NJ) in the MEGA 4 program (20). In addition, a Maximum Parsimony (MP) analysis was performed.

5 Amino acid similarity between crocodilian and other ERVs sequences was also ascertained in the MEGA 4 program using the p-distance option. ERVs were found in all crocodilian lineages examined (Figures 1). All sixtyfive sequences show deletions and twenty eight of these contain in-frame stop codons (Figure 1). These mutations indicate that all crocodilian ERVs generated in this study are defective and, therefore, non-functional, as has been observed in other vertebrate hosts (2, 3, 11, 15). Although only non-functional sequences were identified in Alligatoridae and Crocodylidae, our study does not exclude the possibility of functional ERVs in these lineages. NJ and MP phylogenetic trees were consistent and showed that crocodilian ERVs cluster into two distinct major clades (Figure 1) named CERV 1 and 2. CERV 1 consists of forty-four sequences from twelve species of Crocodylidae, revealing for the first time the existence of a host lineage-specific ERV clade for crocodiles. CERV 2 consists of thirty sequences representing eight Alligatoridae species and the only Gavialidae species. Four sequences from three Crocodylidae (Crocodylus niloticus, Crocodylus palustris and Mecistops cataphractus) also clustered within CERV 2. Interestingly, sequences from both CERV 1 and 2 were found in a single Crocodylidae species (Crocodylus niloticus). Pairwise genetic distances show that the variation within CERV 1 (d = 0.070;±0.006) is very much lower than that within CERV 2 (0.459;±0.020). Analyses also show that all CERV 2 sequences are distinct, revealing additional diversity and new minor clades within this unusual clade (Figures 1), not reported previously. In contrast, CERV 1 sequences show a high degree of similarity between species and some of them are identical within species including C. intermedius, C. siamensis and C. moreletii.

6 Phylogenetic relationships with known ERVs showed that CERV 1 clusters closely with two clades of Gammaretrovirus (birds and reptiles/ mammals/ amphibians/ human) showing polytomy and long branch lengths with respect to each other (Figure 2). Clade CERV 2 clustered distantly from all known ERVs (Figure 2). These relationships are also supported by the amino acid similarity between a known representative of Gammaretrovirus, MuLV, and crocodilian ERV clades. In agreement with previous crocodilian ERV studies (17), the amino acid similarly was lower for CERV 2 (24%) and higher for CERV 1 (47%) (Table 1). Comparison of crocodilian ERV and host species phylogenies (6, 12, 19) showed discordance. While crocodilian host phylogenies based on DNA sequence data are quite well defined, this was not the case within the crocodilian ERV clades. Both CERV 1 and 2 showed a mixture of internal topologies from symmetric (bushlike) to random, to asymmetric with differential branch lengths, making it difficult to assess any coevolutionary patterns. Given that CERV 1 appeared to be Crocodylidae-specific and its internal branch lengths were considerably shorter than those observed in the crocodile host phylogeny (5) and CERV 2, it is plausible that CERV 1 has a lower mutation rate and represents a relatively recent retroviral infection after divergence of Crocodylidae and Alligatoridae from the common ancestor. The current investigation has confirmed the existence of two groups of ERVs and revealed additional distribution and diversity among extant Crocodylia. Interestingly we found a host lineage-specific clade which could have potential for use in the identification of Crocodylidae at the family level. The data generated here will assist future studies to identify orthologous and paralagous ERVs among

7 crocodilian species, assess the variation, distribution and taxonomy of these retroelements within crocodilian species and populations. Nucleotide sequence accession numbers. GenBank accession numbers for sequences derived in this study are FJ through FJ ACKNOWLEDGEMENTS We thank Dr Travis Glenn, Dr Kent Vliet, Dr Robert Godshalk, Dr Mitch Eaton and Matthew Shirley who kindly provided us with many of the crocodilian DNA samples included in this investigation. We are also grateful to Porosus Pty Ltd for providing the Australian saltwater and Johnston's crocodile samples, and Dr Panya Youngprapakorn of Golden Crocodile Agriculture Co. Ltd., Thailand and Dr Thanida Haetrakul of Chulalongkorn University, Thailand, for providing us with samples from Siamese crocodile species. REFERENCES 1. Abascal F, R. Zardoya, and D. Posada ProtTest: Selection of best-fit models of protein evolution. Bioinformatics. 21: Belshaw, R., J. Watson, A. Katzourakis, A. Howe, J. Woolven-Allen, A. Burt, and M. Tristem Rate of recombinational deletion among human endogenous retroviruses. J. Virol. 81: Borysenko L., V. Stepanets, and A.V. Rynditch Molecular characterization of full-length MLV-related endogenous retrovirus ChiRV1 from the chicken, Gallus gallus. Virology. 376: Dolei, A. and H. Perron The multiple sclerosis-associated retrovirus and its HERV-W endogenous family: a biological interface between virology, genetics, and immunology in human physiology and disease. J. Neurovirol. 15:4-13.

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10 Tamura, K., J. Dudley, M. Nei, and S. Kumar MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Phylogenet. Evol. 24: Tarlinton, R.E., J. Meers, and P.R. Young Biology and evolution of the endogenous koala retrovirus. Cell. Mol. Life Sci. 65: Thompson, J.D., D.G. Higgins, and T.J. Gibson CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence-weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: Tristem, M Amplification of divergent retroelements by PCR. BioTechniques. 20: Tristem, M., T. Myles, and F. Hill A highly divergent retroviral sequence in the tuatara (Sphenodon). Virology. 210: Downloaded from on September 18, 2018 by guest

11 Downloaded from FIG. 1. NJ tree of 286 putative amino acids of the pol gene from crocodilian ERV using chicken ERV sequences as an outgroup (left) and the host tree based on nuclear, mitochondrial and morphological data (right), modified with author s permission (5). The column next to the viral tree shows the number of stop codons and deletions observed for the pro-pol reading frame in each crocodilian ERV. The value on the tree node indicates bootstrap branch support (1,000 replicates). Symbols represent: - Crocodylidae; - Alligatoridae; - Gavialidae; RV- crocodilian ERVs from other studies (16, 17); and, GGERV- chicken ERVs. Branch lengths are proportional to sequence differences among ERVs. on September 18, 2018 by guest

12 Downloaded from FIG. 2. Neighbour-Joining (NJ) analyses of 258 putative amino acids of the pol gene from crocodilian ERVs and representatives from seven known ERV genera. Boxed symbols represent the ERV hosts used in this study. Dashed circles show the two major crocodilian ERV clades: CERV 1 and 2. Clustering information within crocodilian ERVs is provided in Figure 1. on September 18, 2018 by guest

13 TABLE 1. Percentage of similarity between crocodilian ERV clades 1 (CERV 1) and 2 (CERV 2) with exogenous members of the Retroviridae a Similarity (%) Retroviradae representatives b Crocodilian ERVs ALV MMTV MuLV BLV HIV 1 HFV WDSV CERV CERV a Calculated from alignments of 265 amino acid alignments of the pro-pol gene. Values are based on average similarity observed in CERV 1 and CERV 2 (Pairwise deletion option in the MEGA 4 program). b Abbreviated names of Retroviridae representatives correspond to those described in the text. Downloaded from on September 18, 2018 by guest