Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
1 Evidence of widespread natural recombination among field isolates of equine 2 herpesvirus 4 but not among field isolates of equine herpesvirus 1 3 4 Running title: Sequence and recombination analysis of EHV-1 and EHV-4 5 6 P.K. Vaz,1 J. Horsington,1,2 C.A. Hartley,1 G.F. Browning,1 N. P. Ficorilli,3 M.J. Studdert,3 J.R. 7 Gilkerson,3 and J.M Devlin1 8 9 1Asia-Pacific Centre for Animal Health, Faculty of Veterinary and Agricultural Sciences, The 10 University of Melbourne, Parkville, Victoria, 3010, Australia. 11 2 12 Victoria, 3220, Australia. 13 3 Centre 14 University of Melbourne, Parkville, Victoria, 3010, Australia. 15 Author for correspondence: Joanne Devlin, ph: +61 3 9035 8110, fax: +61 3 8344 7374, email: 16 [email protected] Present address: Australian Animal Health Laboratory, CSIRO, 5 Portarlington Rd, East Geelong, for Equine Infectious Diseases, Faculty of Veterinary and Agricultural Sciences, The 17 18 Main text = 2788 words. Summary = 196 words. Tables = 2. Figures = 4. 19 20 The GenBank accession numbers for the full EHV genome sequences determined in this study are 21 KT324734, KT324724, KT324733, KT324732, KT324731, KT324730, KT324729, KT324728, KT324727, 22 KT324726, KT324725, KT324748, KT324745, KT324738, KT324736, KT324743, KT324742, KT324740, 23 KT324739, KT324737, KT324747, KT324741, KT324744, KT324735 and KT324746. 1 24 SUMMARY 25 Recombination in alphaherpesviruses allows evolution to occur in viruses that have an otherwise 26 stable DNA genome with a low rate of nucleotide substitution. High-throughput sequencing of 27 complete viral genomes has recently allowed natural (field) recombination to be studied in a 28 number of different alphaherpesviruses, however such studies have not been applied to equine 29 herpesvirus 1 (EHV-1) or equine herpesvirus 4 (EHV-4). These two equine alphaherpesviruses are 30 genetically similar, but differ in their pathogenesis and epidemiology. Both cause economically 31 significant disease in horse populations worldwide. This study used high-throughput sequencing to 32 determine the full genome sequences of EHV-1 and EHV-4 isolates (11 and 14 isolates, respectively) 33 from Australian or New Zealand horses. These sequences were then analysed and examined for 34 evidence of recombination. Evidence of widespread recombination was detected in the genomes of 35 the EHV-4 isolates. Only one potential recombination event was detected in the genomes of the 36 EHV-1 isolates, even when the genomes from an additional 11 international EHV-1 isolates were 37 analysed. The results from this study reveal another fundamental difference between the biology of 38 EHV-1 and EHV-4. The results may also be used to help inform the future safe use of attenuated 39 equine herpesvirus vaccines. 40 2 41 INTRODUCTION 42 In many alphaherpesviruses, recombination is increasingly being recognised as an important 43 mechanism that plays a major role in evolution of viruses with an otherwise stable DNA genome 44 that have low rates of nucleotide substitution (Lee et al., 2013; Lee et al., 2012; Thiry et al., 2005). 45 Recent advances in high-throughput sequencing of complete viral genomes has allowed natural 46 (field) recombination to be studied in a number of different alphaherpesviruses affecting animals 47 and humans, including herpes simplex virus 1 (HSV-1), varicella-zoster virus (VZV) and infectious 48 laryngotracheitis virus (ILTV) (Kolb et al., 2013; Lee et al., 2013; Norberg et al., 2015; Norberg et al., 49 2006; Peters et al., 2006). Such studies provide insights into virus evolution and also allow the risk 50 of recombination to be assessed in the context of attenuated vaccine use. In 2012, outbreaks of 51 severe respiratory disease in poultry in Australia were attributed to natural recombination events 52 between attenuated vaccine strains of ILTV that generated virulent recombinant viruses (Lee et al., 53 2012). These outbreaks of disease highlight the importance of studying and understanding 54 herpesvirus recombination in order to protect animal health. 55 Equine herpesvirus 1 (EHV-1) and equine herpesvirus 4 (EHV-4) are closely related 56 alphaherpesviruses that cause economically significant disease in horses worldwide (Allen et al., 57 2004; Crabb & Studdert, 1995; Telford et al., 1992; Telford et al., 1998). Although EHV-1 and EHV-4 58 are genetically very similar, there are a number of important differences in their pathogenesis and 59 epidemiology. Infection with EHV-4 is most commonly associated with upper respiratory tract 60 disease, but is also occasionally associated with abortion (Allen et al., 2004; Patel & Heldens, 2005). 61 Infection with EHV-1 also causes respiratory disease but infection frequently progresses beyond the 62 upper respiratory tract to result in systemic disease, including abortion and myeloencephalitis (Allen 63 et al., 2004; Edington et al., 1991; Patel & Heldens, 2005; Studdert et al., 2003). Sero- 3 64 epidemiological studies have revealed a high prevalence of antibodies to EHV-4 in horse 65 populations in different countries, including over 99% sero-positivity in mares and foals tested on a 66 large Thoroughbred stud farm in New South Wales, Australia (Gilkerson et al., 1999). Antibodies to 67 EHV-1 are consistently detected at a lower prevalence than antibodies to EHV-4, with a large sero- 68 epidemiological study in Australian horses detecting antibodies to EHV-1 in 26% of mares and 11% 69 of foals (Gilkerson et al., 1999). 70 Both EHV-1 and -4 have linear, double-stranded type ‘D’ DNA genomic structures that consist of a 71 unique long (UL) and a unique short (US) genome region, with the US region flanked by large 72 inverted repeats (internal repeat short, IRs, and terminal repeat short, TRs) (Telford et al., 1992; 73 Telford et al., 1998). The EHV-1 and EHV-4 genomes are 150 kbp and the 146 kbp in length, 74 respectively. Both encode the same 76 homologous genes, with three duplicated genes in EHV-4 75 and four duplicated genes in EHV-1 within the repeat regions. The level of amino acid sequence 76 identity between corresponding proteins encoded by the two genomes ranges from 55% to 96% 77 (Telford et al., 1998). 78 This study aimed to use high-throughput sequencing methods to determine the full genome 79 sequences of a collection of diverse EHV-1 and EHV-4 isolates from Australian and New Zealand 80 horses and to examine these sequences for evidence of recombination. We also aimed to assess the 81 genetic diversity of the EHV-1 and EHV-4 isolates and to examine the phylogenetic relationships 82 between the isolates. 83 84 RESULTS 85 86 Complete genome sequences of 11 Australian EHV-1 isolates 4 87 The full genome sequences of 11 Australian isolates of EHV-1 (Table 1) were determined by 88 mapping against the reference sequence, or by de novo assembly. Sequence alignments from the 89 former method of assembly are shown in Fig. 1. The results from de novo assembly were principally 90 consistent with those produced by mapping against the reference sequence, with variation 91 observed only in regions rich in repeats (Fig. S1). The estimated size of the EHV-1 genomes ranged 92 from 148.37 kbp (isolate 2019-02) to 148.91 kbp (isolate 970-90). Sequence analysis of the EHV-1 93 genomes revealed a low degree of heterogeneity between isolates, with the most distant isolates 94 (isolates 438-77, 3038-07, 2019-02 and 1966-02 compared to isolate 2222-03) sharing 99.9% 95 nucleotide identity, excluding large sequence gaps due to differences in short tandem repeat (STR) 96 copy numbers. Details of the predicted amino acid differences between isolates are shown in Table 97 S1. The number of single nucleotide polymorphisms (SNPs) between different isolates of EHV-1 is 98 summarised in Table S2. The average number of SNPs between EHV-1 isolates was 96. 99 100 Complete genome sequences of 14 Australian EHV-4 isolates 101 The full genome sequences of 14 Australian isolates of EHV-4 (Table 1) were determined by 102 mapping against the reference sequence, or by de novo assembly. Sequence alignments from the 103 former method of assembly are shown in Fig. 2. The results from de novo assembly were principally 104 consistent with those produced by mapping against the reference sequence, with variation 105 observed only in regions rich in repeats (Fig. S2). The estimated size of the EHV-4 full-length 106 genomes ranged from 143.16 bp (isolate 3407-77) to 144.16 kbp (isolate 1532-99). Alignment of the 107 14 EHV-4 genomes revealed a number of sequence differences between isolates. The genetically 108 most similar isolates shared 99.9% nucleotide sequence identity (isolates ER39-67 and R19-68; 109 isolates ER39-67 and 475-78; isolates 405-76 and 306-74), excluding large sequence gaps. The 110 genetically most diverse isolates shared 98.9% nucleotide identity (isolates 960-90 and 1532-99). 5 111 Details of the predicted amino acid differences between isolates are shown in Table S3. Large 112 regions of sequence difference (insertions/deletions, indels) were present in the repeat regions of 113 the genome, between ORFs 64-65 and ORFs 66-67 of the IR/TR, in ORF24 of the UL region, and in 114 ORF71 in the US region. Smaller indels were seen in the UL region within ORFs 24 and 61, and in the 115 US within ORFs 70, 71 and 75. The number of SNPs between different isolates of EHV-4 is 116 summarised in Table S4. The average number of SNPs between EHV-4 isolates was 179. 117 Recombination 118 Phylogenetic recombination networks for EHV-1 and EHV-4 were generated from nucleotide 119 alignments of the complete genome sequences, as well alignments of the individual UL, US and IR 120 regions, using SplitsTree4 (Huson, 1998). The multiple reticulate networks and pair-wise homoplasy 121 (Phi) test analyses (Fig. 3) indicate significant historical recombination events between the different 122 EHV-4 isolates in the UL region (P = 0.001) but not in the IR or US regions. No significant 123 recombination was detected between the 11 EHV-1 Australian/New Zealand isolates sequenced, 124 irrespective of the genomic region analysed (Fig. 4). No significant recombination events were 125 detected when the analyses were expanded to include the genome sequences from 11 additional 126 international EHV-1 isolates (Fig S3). 127 Detection of recombination breakpoints utilised six different methods: Recombination detection 128 program (RDP), GENECONV, 3Seq, Maximum Chi Square (MaxChi), SiScan and Bootscan. These 129 methods were implemented in the program RDP4 (Martin et al., 2015). Analyses were performed 130 using complete genome sequences, as well as alignments of the individual US, UL and IR regions. 131 Consistent with the results from the SplitsTree analysis, evidence of potential recombination events 132 was detected amongst EHV-4 isolates in the UL region using these different methods (Table 2). In 133 addition, evidence of potential recombination events was detected in the US and IR regions of EHV- 6 134 4, and another potential recombination event was detected in the IR region of EHV-1 (Table 2). 135 These potential recombination events had not been detected by SplitsTree and Phi test analyses. 136 137 DISCUSSION 138 The 11 isolates of EHV-1 and 14 isolates of EHV-4 analysed in this study were all collected from 139 Australian or New Zealand horses between 1967 and 2007. In Australia, EHV-4 was first isolated in 140 1967, whilst the first isolates of EHV-1 were not obtained until 1977, when it is believed EHV-1 was 141 first introduced into Australia. The sequence data generated in this study has substantially 142 increased the number of full genome sequences available for these viruses, particularly EHV-4, as 143 only one EHV-4 genome has been fully sequenced previously (Telford et al., 1998). Evidence of 144 extensive recombination was detected in the genomes of the EHV-4 isolates, but little or no 145 recombination was detected between the genomes of the EHV-1 isolates, irrespective of whether 146 only the genomes from Australian/New Zealand isolates of EHV-1 and -4 were analysed, or whether 147 genomes from an additional 11 international EHV-1 isolates were included in the analysis. 148 Analysis of the EHV-1 and EHV-4 full genome sequences showed that the genetic diversity amongst 149 EHV-4 isolates was greater than the diversity among EHV-1 isolates. This is consistent with earlier 150 studies that have assessed genetic differences between EHV-1 and EHV-4 isolates using other 151 techniques, including sequencing of selected genes, or digestion with restriction endonucleases 152 (Allen et al., 2004). This greater diversity in EHV-4 isolates, compared with EHV-1 isolates, is also 153 consistent with the hypothesis that the progenitor virus of EHV-1 was transferred from another host 154 to the ancestor of the modern horse more recently than EHV-4 (Crabb & Studdert, 1990) and has 155 therefore had less time to diversify. An alternative hypothesis, arising from the results from this 156 study, may be that the lack of genetic diversity within EHV-1 is a consequence of a low rate of 157 recombination in this virus species, given that recombination can contribute to genetic diversity. 7 158 The lower level of genetic diversity in EHV-1, compared to EHV-4, would make recombination more 159 difficult to detect in EHV-1. The total number of SNPs between any two EHV-1 isolates varied from 8 160 to 153 (excluding repeat sequences) with an average of 96 SNPs between any two isolates. It is 161 possible that EHV-1 recombination events may have occurred but were not able to be detected at 162 the required level of statistical significance because an insufficient number of SNPs specific for the 163 parental viruses were transferred to the recombinant viruses. However, evidence of frequent, 164 natural recombination has been readily detected in VZV, which also has a highly conserved genome 165 (approximately 30 – 200 SNP differences between any two strains) using similar methods to those in 166 this study (Norberg et al., 2015; Norberg et al., 2006; Zell et al., 2012). 167 Importantly we assessed recombination in the EHV isolates using a number of different approaches. 168 Simulated and empirical studies have demonstrated the value of using multiple methods for 169 detecting recombination, rather than relying on a single approach (Posada, 2002; Posada & 170 Crandall, 2001). The pair-wise homoplasy test (Phi test) implemented by SplitsTree is a robust and 171 powerful method for detecting the presence/absence of recombination and has been shown to 172 produce a low rate of false positive results in a wide variety of circumstances (Bruen et al., 2006). 173 This test detected recombination in the UL region of EHV-4, but did not detect recombination in 174 EHV-1. Analysis of recombination breakpoints, using multiple methods within RDP4, produced some 175 similar results, but also detected evidence of other potential recombination events that were not 176 detected by the Phi test, including within the US and IR regions of EHV-4 and one within the IR 177 region of EHV-1. Although it is expected that different methods of detecting recombination will 178 yield different results (Posada, 2002; Posada & Crandall, 2001) the consistent detection of multiple 179 recombination events in EHV-4, but not in EHV-1, provides substantive evidence that recombination 180 is widespread in EHV-4, but is limited or absent in EHV-1. 8 181 The presence of widespread recombination in EHV-4, but not in EHV-1, may be due to differences in 182 the epidemiology of the two viruses (Gilkerson et al., 1999), particularly the lower prevalence of 183 EHV-1 infection and hence the lower rate of co-infection of the same animal with two or more EHV- 184 1s, which is the fundamental requirement for intraspecific herpesvirus recombination (Thiry et al., 185 2005). Intrinsic virus-specific factors could also influence the propensity for recombination. Recent 186 studies in HSV-1 have shown that the gene products of UL12 (5’-to-3’ exonuclease) and UL29 (single 187 stranded DNA binding protein) appear to function together as a two-component recombinase to 188 promote recombination in infected cells (Weller & Sawitzke, 2014). The homologues of these genes 189 in EHV-1 and EHV-4 (ORFs 50 and 31, respectively) have a high level of identity between the two 190 virus species but their function has not been directly compared. Future studies to assess and 191 compare recombination in EHV-1 and EHV-4 in cell culture are indicated. 192 Natural recombination between herpesviruses has been recognised as a safety concern for live 193 attenuated herpesvirus vaccines (Lee et al., 2012; Thiry et al., 2005). Although not currently used in 194 Australia, attenuated vaccines are available for use, or are under development, in other countries to 195 help control disease caused by infection with EHV-1 and EHV-4. The results from this study indicate 196 that for attenuated EHV-4 vaccines the risks of recombination should be considered when designing 197 and implementing vaccination programs, but that these considerations may be less of a concern for 198 EHV-1 vaccines. 199 METHODS 200 Viruses and cells used in this study 201 Eleven Australian or New Zealand isolates of EHV-1 and 14 Australian isolates of EHV-4 (Table 1) 202 were selected from our laboratory archives and propagated in cultures of equine foetal kidney cells 203 (Studdert & Gleeson, 1977) for six passages, including three plaque purifications, in order to amplify 9 204 sufficient virus for whole genome sequencing. Virions were purified by Ficoll gradient centrifugation 205 (Lee et al., 2011). Alternatively, nucleocapsids were purified as described previously (Pignatti et al., 206 1979). Viral DNA was extracted from purified nucleocapsids using standard phenol-chloroform 207 extraction methods, or from purified virions using the High Pure PCR Template Preparation Kit 208 (Roche) according to manufacturer’s instructions. 209 210 High throughput sequencing and genome assembly 211 High-throughput sequencing was performed as described previously (Lee et al., 2011) and the 212 genome sequences for each isolate were assembled using Geneious V6.1.7 (Kearse et al., 2012) by 213 mapping against the reference sequences of EHV-1 (GenBank accession NC_001491.2) or EHV-4 214 (GenBank accession NC_001844.1). An overview of the sequencing metrics (including mean 215 coverage and read depth across each genome) is provided in Tables S5 (EHV-1) and S6 (EHV-4). The 216 complete genome sequences of the EHV-1 and EHV-4 isolates were deposited in the NCBI GenBank 217 database under the accession numbers listed in Table 1. For comparison, de novo assembly was also 218 performed for two virus isolates (EHV-1 438-77 and EHV-4 405-76) using medium-low sensitivity 219 settings in Geneious V6.1.7. Contig consensus sequences were aligned to the reference sequence to 220 aid scaffold construction. 221 222 Sequence alignment 223 Alignments of the complete genome sequences (excluding the TR regions) of the EHV-1 and EHV-4 224 isolates, as well as the individual UL, US and IR regions, were prepared using the Multiple Alignment 225 with Fast Fourier Transformation (MAFFT) version 7 plugin in Geneious V6.1.7 (Katoh & Standley, 226 2013). The laboratory isolates 438-77 and 405-76 (Studdert & Blackney, 1979) were used as the 227 reference sequences for EHV-1 and EHV-4 alignments, respectively. 10 228 To expand the analysis of EHV-1 isolates, these same analyses were repeated after including 229 publically available full genome sequence data from an additional 11 EHV-1 isolates. These included 230 five isolates from Japan (00c19, 90c16, 01c1, 89c105 and 89c25; GenBank accession numbers 231 KF644576, KF644566, KF644578, KF644577 and KF644579, respectively), three isolates from the 232 USA (FL06, NY05 and VA02; GenBank accession numbers KF644567, KF644570 and KF644572, 233 respectively) and three isolates from the UK (NMKT04, V592 and Ab4; GenBank accession numbers 234 KF644568, AY464052 and NC_001491.2, respectively). 235 236 Recombination analyses 237 Tandem repeat regions were identified and removed prior to recombination analysis. For this, 238 perfect and imperfect repeats were identified in the aligned genomes using the Phobos plugin in 239 Geneious V6.1.7 with default settings and score constraints for satellites. All repeat regions 240 identified were deleted in all viruses in the alignment, including in those viruses in which the repeat 241 region may not have been directly identified due to minor sequence variations that affected the 242 repetitive nature of the sequence. In this way, the deletion of repeat regions did not change the 243 alignment of the genomes. These alignments, and the isolated UL, US or internal repeat regions, 244 were then used to detect evidence of intraspecific recombination using two recombination 245 detection programs; SplitsTree4 (Huson, 1998) and RDP4 (Martin et al., 2015). Initially only the 246 genomes of the Australian/New Zealand EHV isolates were analyzed. To expand upon the analyses 247 of EHV1 isolates these same analyses were then repeated after including publically available full 248 genome sequence data from the additional 11 EHV-1 isolates described above. 249 In SplitsTree4, splits network trees were generated with an uncorrected P characters 250 transformation model, ignoring constant sites. Other models were tested but resulted in no 11 251 significant topological differences in the generated trees. Statistical analyses of the recombination 252 networks were performed using the Phi test (Bruen et al., 2006) as implemented by SplitsTree4. In 253 RDP4 six different methods were used to assess the sequences for recombination breakpoints: RDP, 254 GENECONV, Chimaera, SiScan, MaxChi and Bootscan. Default RDP4 settings were used throughout. 255 Only breakpoints with a Bonferroni-corrected P value <0.05 were reported. Duplicate breakpoints 256 were omitted. 257 258 ACKNOWLEDGEMENTS 259 This work was supported by the Australian Research Council (ARC, DP130103991). JMD is supported 260 by an ARC Future Fellowship (FT140101287 12 261 REFERENCES 262 263 Allen, G., Kydd, J. & Slater, J. (2004). Equid herpesvirus 1 and equid herpesvirus 4 infections. In 264 Infectious diseases of livestock, pp. 829-859. Edited by J. Coetzer & R. Tustin. Newmarket: 265 Oxford University Press. 266 267 268 269 270 271 272 Bagust, T. J. & Pascoe, R. R. (1968). Isolation of equine rhinopnemonitis virus from acute respiratory disease in a horse in queensland. Aus Vet J 44, 296. Bagust, T. J. & Pascoe, R. R. (1970). Studies on equine herpesviruses. 1. Characterisation of a strain of equine rhinopneumonitis virus isolated in Queensland. Aus Vet J 46, 421-427. Bruen, T. C., Philippe, H. & Bryant, D. (2006). A simple and robust statistical test for detecting the presence of recombination. Genetics 172, 2665-2681. Crabb, B. S. & Studdert, M. J. (1990). Comparative studies of the proteins of equine herpesviruses 4 273 and 1 and asinine herpesvirus 3: antibody response of the natural hosts. J Gen Virol 71, 274 2033-2041. 275 276 277 Crabb, B. S. & Studdert, M. J. (1995). Equine herpesviruses 4 (equine rhinopneumonitis virus) and 1 (equine abortion virus). Adv Virus Res 45, 153-190. Edington, N., Smyth, B. & Griffiths, L. (1991). The role of endothelial cell infection in the 278 endometrium, placenta and foetus of equid herpesvirus 1 (EHV-1) abortions. J Comp Pathol 279 104, 379-387. 280 Gilkerson, J. R., Whalley, J. M., Drummer, H. E., Studdert, M. J. & Love, D. N. (1999). 281 Epidemiological studies of equine herpesvirus 1 (EHV-1) in Thoroughbred foals: a review of 282 studies conducted in the Hunter Valley of New South Wales between 1995 and 1997. Vet 283 Microbiol 68, 15-25. 13 284 285 286 287 288 289 Gleeson, L. J., Sullivan, N. D. & Studdert, M. J. (1976). Equine herpesviruses: type 3 as an abortigenic agent. Aust Vet J 52, 349-354. Horner, G. W. (1981). Serological relationship between abortifacient and respiratory strains of equine herpesvirus type 1 in New Zealand. New Zeal Vet J 29, 7-8. Huang Ja, J. A., Ficorilli, N., Hartley, C. A., Allen, G. P. & Studdert, M. J. (2002). Polymorphism of open reading frame 71 of equine herpesvirus-4 (EHV-4) and EHV-1. J Gen Virol 83, 525-531. 290 Huson, D. H. (1998). SplitsTree: analyzing and visualizing evolutionary data. Bioinformatics 14, 68-73. 291 Katoh, K. & Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7: 292 improvements in performance and usability. Mol Biol Evol 30, 772-780. 293 Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper, A., 294 Markowitz, S. & other authors (2012). Geneious Basic: an integrated and extendable 295 desktop software platform for the organization and analysis of sequence data. 296 Bioinformatics 28, 1647-1649. 297 298 299 Kolb, A. W., Ane, C. & Brandt, C. R. (2013). Using HSV-1 genome phylogenetics to track past human migrations. PLOS One 8, e76267. Lee, S. W., Devlin, J. M., Markham, J. F., Noormohammadi, A. H., Browning, G. F., Ficorilli, N. P., 300 Hartley, C. A. & Markham, P. F. (2013). Phylogenetic and molecular epidemiological studies 301 reveal evidence of multiple past recombination events between infectious laryngotracheitis 302 viruses. PLOS One 8, e55121. 303 Lee, S. W., Markham, P. F., Coppo, M. J., Legione, A. R., Markham, J. F., Noormohammadi, A. H., 304 Browning, G. F., Ficorilli, N., Hartley, C. A. & Devlin, J. M. (2012). Attenuated vaccines can 305 recombine to form virulent field viruses. Science 337, 188. 14 306 Lee, S. W., Markham, P. F., Markham, J. F., Petermann, I., Noormohammadi, A. H., Browning, G. F., 307 Ficorilli, N. P., Hartley, C. A. & Devlin, J. M. (2011). First complete genome sequence of 308 infectious laryngotracheitis virus. BMC Genomics 12, 197. 309 Martin, D., Murell, B., Golden, M., Khoosal, A. & Muhire, B. (2015). RDP4: Detection and analysis 310 of recombination patterns in virus genomes. Virus Evolution 1, doi: 10.1093/ve/vev003. 311 Norberg, P., Depledge, D. P., Kundu, S., Atkinson, C., Brown, J., Haque, T., Hussaini, Y., MacMahon, 312 E., Molyneaux, & other authors (2015). Recombination of Globally Circulating Varicella 313 Zoster Virus. J Virol 89, 7133-7146 314 Norberg, P., Liljeqvist, J. A., Bergstrom, T., Sammons, S., Schmid, D. S. & Loparev, V. N. (2006). 315 Complete-genome phylogenetic approach to varicella-zoster virus evolution: genetic 316 divergence and evidence for recombination. J Virol 80, 9569-9576. 317 318 319 320 Patel, J. R. & Heldens, J. (2005). Equine herpesviruses 1 (EHV-1) and 4 (EHV-4)--epidemiology, disease and immunoprophylaxis: a brief review. Vet J 170, 14-23. Peet, R. L., Coackley, W., Smith, V. W. & Main, C. (1978). Equine abortion associated with herpesvirus. Aus Vet J 54, 151. 321 Peters, G. A., Tyler, S. D., Grose, C., Severini, A., Gray, M. J., Upton, C. & Tipples, G. A. (2006). A 322 full-genome phylogenetic analysis of varicella-zoster virus reveals a novel origin of 323 replication-based genotyping scheme and evidence of recombination between major 324 circulating clades. J Virol 80, 9850-9860. 325 Pignatti, P. F., Cassai, E., Meneguzzi, G., Chenciner, N. & Milanesi, G. (1979). Herpes simplex virus 326 DNA isolation from infected cells with a novel procedure. Virology 93, 260-264. 327 Posada, D. (2002). Evaluation of methods for detecting recombination from DNA sequences: 328 empirical data. Mol Biol Evol 19, 708-717. 15 329 330 331 332 333 334 335 Posada, D. & Crandall, K. A. (2001). Evaluation of methods for detecting recombination from DNA sequences: computer simulations. PNAS 98, 13757-13762. Studdert, M. J. (1971). Equine herpesviruses. 4. Concurrent infection in horses with strangles and conjunctivitis. Aus Vet J 47, 434-436. Studdert, M. J. & Blackney, M. H. (1979). Equine herpesviruses: on the differentiation of respiratory from foetal strains of type 1. Aus Vet J 55, 488-492. Studdert, M. J., Fitzpatrick, D. R., Horner, G. W., Westbury, H. A. & Gleeson, L. J. (1984). Molecular 336 epidemiology and pathogenesis of some equine herpesvirus type 1 (equine abortion virus) 337 and type 4 (equine rhinopneumonitis virus) isolates. Aus Vet J 61, 345-348. 338 Studdert, M. J. & Gleeson, L. J. (1977). Isolation of equine rhinovirus type 1. Aus Vet J 53, 452. 339 Studdert, M. J., Hartley, C. A., Dynon, K., Sandy, J. R., Slocombe, R. F., Charles, J. A., Milne, M. E., 340 Clarke, A. F. & El-Hage, C. (2003). Outbreak of equine herpesvirus type 1 myeloencephalitis: 341 new insights from virus identification by PCR and the application of an EHV-1-specific 342 antibody detection ELISA. Vet Rec 153, 417-423. 343 Studdert, M. J., Turner, A. J. & Peterson, J. E. (1970). Equine herpesviruses. I. Isolation and 344 characterisation of equine rhinopneumonitis virus and other equine herpesviruses from 345 horses. Aus Vet J 46, 83-89. 346 347 348 349 350 Telford, E. A., Watson, M. S., McBride, K. & Davison, A. J. (1992). The DNA sequence of equine herpesvirus-1. Virology 189, 304-316. Telford, E. A., Watson, M. S., Perry, J., Cullinane, A. A. & Davison, A. J. (1998). The DNA sequence of equine herpesvirus-4. J Gen Virol 79, 1197-1203. Thiry, E., Meurens, F., Muylkens, B., McVoy, M., Gogev, S., Thiry, J., Vanderplasschen, A., Epstein, 351 A., Keil, G. & Schynts, F. (2005). Recombination in alphaherpesviruses. Rev Med Virol 15, 89- 352 103. 16 353 Varrasso, A., Dynon, K., Ficorilli, N., Hartley, C. A., Studdert, M. J. & Drummer, H. E. (2001). 354 Identification of equine herpesviruses 1 and 4 by polymerase chain reaction. Aus Vet J 79, 355 563-569. 356 357 358 Weller, S. K. & Sawitzke, J. A. (2014). Recombination promoted by DNA viruses: phage lambda to herpes simplex virus. Annu Rev Microbiol 68, 237-258. Zell, R., Taudien, S., Pfaff, F., Wutzler, P., Platzer, M. & Sauerbrei, A. (2012). Sequencing of 21 359 varicella-zoster virus genomes reveals two novel genotypes and evidence of recombination. 360 J Gen Virol 86, 1608-1622. 361 17 362 363 Table 1. Australian and New Zealand equine herpesvirus isolates used in this study. Virus ID Year Region Disease association Site of collection Reference GenBank accession EHV-1 438-77 NZA-77 717A-82 970-90 1029-93 1074-94 1966-02 2019-02 2222-03 3038-07 3045-07 1977 1977 1982 1990 1993 1994 2002 2002 2003 2007 2007 Scone, NSW New Zealand VIC SA Wakefield, NSW Toowoomba, QLD Seymour, VIC Clayton, VIC Scone, NSW VIC NSW Abortion Abortion Neurological/abortion Abortion Abortion Abortion Abortion Abortion Abortion Neurological Abortion Foetus N/A Brain or foetus N/A Foetus Foetus Foetus Foetus Foetus Nasal septum N/A (Studdert & Blackney, 1979) (Horner, 1981) (Studdert et al., 1984) This study This study (Varrasso et al., 2001) This study This study This study This study This study KT324734 KT324724 KT324733 KT324732 KT324731 KT324730 KT324729 KT324728 KT324727 KT324726 KT324725 EHV-4 ER39-67 R19-68 157-69 RT4-70 306-74 2387-75 405-76 3407-77 3409-77 475-78 960-90 1532-99 1546-99 3056-07 1967 1968 1969 1970 1974 1975 1976 1977 1977 1978 1990 1999 1999 2007 SA Toowoomba, QLD Yarra Glen, VIC Oakey, QLD Flemington, VIC Esperance, WA Flemington, VIC N/A Gosnells, WA Mt Pleasant, TAS Diggers Rest, VIC Flemington, VIC Toolern Vale, VIC TAS Respiratory Respiratory Respiratory Respiratory Respiratory Abortion Respiratory Respiratory Abortion Abortion Respiratory Respiratory Respiratory Respiratory Respiratory tract Nasal swab Nasal swab Nasal swab Nasal swab Foetus Nasal swab N/A Foetus Foetus Nasal swab Nasal swab Nasal swab Nasal swab (Studdert et al., 1970) (Bagust & Pascoe, 1968) (Studdert, 1971) (Bagust & Pascoe, 1970) (Gleeson et al., 1976) (Peet et al., 1978) (Gleeson et al., 1976) This study (Peet et al., 1978) (Studdert & Blackney, 1979) (Varrasso et al., 2001) (Huang Ja et al., 2002) (Huang Ja et al., 2002) This study KT324748 KT324745 KT324738 KT324736 KT324743 KT324742 KT324740 KT324739 KT324737 KT324747 KT324741 KT324744 KT324735 KT324746 N/A, not available 18 364 Table 2. Recombination breakpoint analysis of EHV-1 and EHV-4 isolates 365 Virus and region Breakpoints^ Breakpoint beginning Breakpoint ending 99% CI 99% CI Possible viruses involved in recombination event Method of breakpoint detection* EHV-1 IR/TR 116122 - 120856 120865 - 123600 2019-02, 438-77, Unknown GENECONV, 3Seq, BootScan EHV-4 UL UL UL IR/TR US Undetermined (28) Undetermined (20689) Undetermined (96512) Undetermined (121749) 122044 - 126553 28 – 6954 77657 - 98051 Undetermined (112236) Undetermined (122035) 126555 - 128536 306-74, 405-76, 475-78 3056-07, 475-78, RT4-70 475-78, 1546-99, 960-90, 3056-07, 306-74 3409-77, 3407-77, Unknown 960-90, 2387-75, 3056-07, Unknown GENECONV, BootScan MaxChi, Chimaera, SiScan RDP, MaxChi, BootScan GENECONV, 3Seq, BootScan, GENECONV, 3Seq, SiScan, BootScan 366 367 ^Nucleotide number according to 438/77 (EHV-1) or 405/76 (EHV-4) reference sequences 368 *Algorithm used to detect recombination, as implemented in RDP4 369 19 370 FIGURE LEGENDS 371 372 Fig. 1. Nucleotide sequence alignment for the complete genomes of Australasian EHV-1 isolates. 373 Alignment of the complete genome sequences of EHV-1 isolates was performed using MAFFT. Our 374 prototype strain 438-77 was used as the reference sequence. Vertical black lines indicate SNPs 375 compared to the reference and dashes indicate sequence gaps. 376 377 Fig. 2. Nucleotide sequence alignment for the complete genomes of Australasian EHV-4 isolates. 378 Alignment of the complete genome sequences of EHV-4 isolates was performed using MAFFT. Our 379 prototype strain EHV-4.405-76 was used as the reference sequence. Vertical black lines indicate 380 SNPs compared to the reference and dashes indicate sequence gaps. 381 382 Fig. 3. Recombination network trees generated from Australasian EHV-4 nucleotide alignments 383 (excluding sequence repeats) using SplitsTree4. (a) Complete genome sequences, (b) unique long 384 region, (c) unique short region and (d) repeat region. The multiple reticulate networks indicate 385 recombination events between the different isolates. The bar indicates the rate of evolution in 386 sequence substitutions per site. The Phi test for detecting recombination, as implemented in 387 SplitsTree4, was highly significant for the whole genome, and for the unique long region, but not for 388 the unique short region or the repeat region. 389 390 Fig. 4. Recombination network trees generated from Australasian EHV-1 nucleotide alignments 391 (excluding sequence repeats) using SplitsTree4. (a) Complete genome sequences, (b) unique long 392 region, (c) unique short region and (d) repeat region. The bar indicates the rate of evolution in 20 393 sequence substitutions per site. The Phi test for detecting recombination, as implemented in 394 SplitsTree4, was not significant for the whole genome, or for any of the individual genome regions. 395 396 397 Fig. S1. Comparison of the genome sequence of our prototype laboratory EHV-1 isolate 438-77 as 398 determined by de novo assembly (438-77 de novo), or by mapping the sequence reads to the EHV-1 399 reference sequence in GenBank accession NC_001491.2 (438-77). Vertical black lines indicate SNPs 400 and dashes indicate sequence gaps. The alignment shows a high level of identity (green) between 401 the sequences generated by the two different methods with the exception of regions of the 402 genome containing repeat sequences (dark blue). These repeat sequences were identified and 403 removed prior to recombination analyses. 404 405 Fig. S2. Comparison of the genome sequence of our prototype laboratory EHV-4 isolate 405-76 as 406 determined by de novo assembly (405-76 de novo) or by mapping the sequence reads to the EHV-4 407 reference sequence in GenBank accession NC_001844.1 (405-76). Vertical black lines indicate SNPs 408 and dashes indicate sequence gaps. The alignment shows a high level of identity (green) between 409 the sequences generated by the two different methods with the exception of regions of the 410 genome containing repeat sequences (dark blue). These repeat sequences were identified and 411 removed prior to recombination analyses. 412 413 Fig. S3. Recombination network trees generated from 11 Australasian and 11 international EHV-1 414 nucleotide alignments (excluding sequence repeats) using SplitsTree4. (a) Complete genome 415 sequences, (b) unique long region, (c) unique short region and (d) repeat region. The multiple 416 reticulate networks indicate recombination events between the different isolates. The bar indicates 21 417 the rate of evolution in sequence substitutions per site. The Phi test for detecting recombination, as 418 implemented in SplitsTree4, was not significant for the whole genome, or for any of the individual 419 genome regions. 420 22