Davison, Andrew J.

Doszpoly, Andor

Hughes, J

Subramaniam, K

Vucak, M

Waltzek, Thomas B.

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 1Genome sequence of white sturgeon herpesvirus 1 isolated from farmed white sturgeon 1 (Acipenser transmontanus) 2  3 Thomas B. Waltzeka,b,#, Kuttichantran Subramaniama, Andor Doszpolyc, Joseph Hughesd, Matej 4 Vučakd, Andrew J. Davisond 5  6 aDepartment of Infectious Diseases and Immunology, College of Veterinary Medicine, University of 7 Florida, Gainesville, Florida, USA 8 bPresent address: Washington Animal Disease Diagnostic Laboratory and Department of 9 Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State 10 University, Pullman, Washington, USA 11 cVeterinary Medical Research Institute, Budapest, Hungary 12 dMRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom 13  14 Running title: Genome sequence of white sturgeon herpesvirus 1 15  16 #Address correspondence to Thomas Waltzek, tomwaltzek@gmail.com   17  2ABSTRACT 18  The genome sequence of white sturgeon herpesvirus 1, which was isolated from farmed white 19 sturgeon (Acipenser transmontanus), was determined. Comparative analyses suggest the 20 classification of this virus as a new species in a new genus in the family Alloherpesviridae. 21  22 Words = 39 23  24 Keywords: Alloherpesviridae, Acipenser transmontanus, sturgeon, genome. 25  26 ANNOUNCEMENT 27  White sturgeon herpesvirus 1 (WSHV1) was isolated on the white sturgeon skin (WSSK-1) cell 28 line from juvenile white sturgeon experiencing elevated mortality on a commercial farm in 29 California, USA in 1991 (1). Additional herpesviruses were isolated subsequently from farmed 30 white sturgeon experiencing elevated morbidity and mortality (2). Based on differences from 31 WSHV1 in serological properties and growth characteristics in cell culture, these isolates were 32 designated white sturgeon herpesvirus 2 (WSHV2). Phylogenetic analyses based on 12 core genes 33 present in all members of the family Alloherpesviridae revealed that WSHV1 is distantly related to 34 WSHV2 (3) and that its closest relative is lake sturgeon herpesvirus (LSHV) (4).  35  The WSHV1 isolate (strain UC Davis) was passaged six times in WSSK-1 cells (1), and DNA was 36 purified from the culture medium using a Blood & Cell Culture DNA mini kit (Qiagen). A sequencing 37 library prepared using a Nextera XT kit (Illumina) was sequenced on a MiSeq instrument (Illumina) 38 using a v3 600-cycle reagent kit, yielding 11,301,304 300 nt paired-end reads. The data were 39 analyzed using default options except where stated otherwise. The reads were trimmed using Trim 40 Galore v.0.6.6 (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) with the -q 25, --41 length 50 and --paired options. The trimmed reads were deduplicated using FastUniq v.1.1 (5) and 42 subsampled using Seqtk v.1.3 (https://github.com/lh3/seqtk) with the sample, -s34 and 0.03 (to 43 discard 97% of reads) options. The subsampled reads were assembled into contigs using SPAdes 44 v3.14.1 (6) with the --careful, --cov-cutoff auto and -k 33,77,127 options. Contigs related to the 45 genomes of members of the family Alloherpesviridae were identified by using BLASTX (7) against 46 the NCBI non-redundant protein database. These contigs were joined to others by extracting reads 47  3that extended their ends until overlaps were found. The genome terminI were assigned as 48 outlined below. The sizes of seven tandem reiterations were not resolved. The integrity of the 49 sequence was verified by aligning the trimmed reads using Bowtie 2 v2.3.1 (8) and Samtools v1.3 50 (9) and inspecting the alignment using Tablet v1.21.02.08 (10). The alignment incorporated 98% of 51 reads at an average coverage depth of 14,704 reads/nt. 52  The WSHV1 genome (195,072 bp; 40% G+C) consists of a long unique region (UL; 145,962 bp) 53 flanked by an inverted repeat (TRL/IRL; 26 bp), linked to a short unique region (US; 42,630 bp) also 54 flanked by an inverted repeat (TRS/IRS; 3,214 bp), yielding the overall structure 55 TRL−UL−IRL−IRS−US−TRS (Fig. 1). The left terminus was assigned from a large set of reads sharing an 56 end, and the right terminus from the adjacent sequence at the IRL-IRS junction. A total of 129 open 57 reading frames (ORFs) encoding functional proteins were predicted using approaches described 58 previously (4) (Fig. 1). 59  Although WSHV1 and LSHV (4) are each other’s closest relative, the core genes are only 76.8% 60 identical. This suggests the creation of a new genus in the family Alloherpesviridae incorporating 61 two new species. A second partially sequenced lake sturgeon herpesvirus (11) is more closely 62 related to LSHV than is WSHV1; the core genes are 84.3% identical. 63  64 Words = 497 65  66 Data availability 67  The WSHV1 genome sequence is available in GenBank under accession number OR001786. The 68 sequence reads are available under BioProject accession number PRJNA1004073. 69  70 ACKNOWLEDGMENTS 71  TBW was supported by the College of Veterinary Medicine, Washington State University. 72 AD was supported by the Bolyai János Research Scholarship. AJD was supported by Medical 73 Research Council grant MC_UU_12014/3. The authors thank Dr. Ronald P. Hedrick for providing 74 WSHV1 strain UC Davis. 75  76 REFERENCES 77  41. Hedrick RP, McDowell TS, Groff JM, Yun S, Wingfield WH. 1991. Isolation of an epitheliotropic 78 herpesvirus from white sturgeon Acipenser transmontanus. Dis Aquat Org 11:49–56. 79 2. Watson LR, Yun SC, Groff JM, Hedrick RP. 1995. Characteristics and pathogenicity of a novel 80 herpesvirus isolated from adult and subadult white sturgeon Acipenser transmontanus. Dis 81 Aquat Org 22:199–210. 82 3. Waltzek TB, Kelley GO, Alfaro ME, Kurobe T, Davison AJ, Hedrick RP. 2009. Phylogenetic 83 relationships in the family Alloherpesviridae. Dis Aquat Org 84:179–194. 84 4. Walker L, Subramaniam K, Viadanna PHO, Vann JA, Marcquenski S, Godard D, Kieran E, Frasca 85 S Jr, Popov VL, Kerr K, Davison AJ, Waltzek TB. 2022. Characterization of an alloherpesvirus 86 from wild lake sturgeon Acipenser fulvescens in Wisconsin (USA). Dis Aquat Org 149:83–96. 87 5. Xu H, Luo X, Qian J, Pang X, Song J, Qian G, Chen J, Chen S. 2012. FastUniq: a fast de novo 88 duplicates removal tool for paired short reads. PLoS One 7:e52249.  89 6. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, 90 Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 91 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. 92 J Comput Biol 19:455–477. 93 7. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J 94 Mol Biol 215:403–410. 95 8. Langmead B, Salzberg SL. 2012. Fast gapped-read alignment with Bowtie 2. Nat Methods 96 9:357–359.  97 9. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 98 Genome Project Data Processing Subgroup. 2009. The Sequence Alignment/Map format and 99 SAMtools. Bioinformatics 25:2078–20792. 100 10. Milne I, Stephen G, Bayer M, Cock PJA, Pritchard L, Cardle L, Shaw PD, Marshall D. 2013. Using 101 Tablet for visual exploration of second-generation sequencing data. Brief Bioinform 14:193–102 202.  103 11. Johnston AE, Shavalier MA, Scribner KT, Soto E, Griffin MJ, Waldbieser GC, Richardson BM, 104 Winters AD, Yun S, Baker EA, Larson DL, Kiupel M, Loch TP. 2022. First isolation of a 105 herpesvirus (family Alloherpesviridae) from Great Lakes lake sturgeon (Acipenser fulvescens). 106 Animals 12:3230. 107  5 108  109  110  111  112  113  114  115  116  117  118  119  120  121  122  123  124  125  126  127  128  129  130  131  132  133  134  135 FIG 1. WSHV1 genome map. Unique regions (UL and US) are shown in a thinner format than inverted repeats 136 (TRL/IRL and IRS/TRS). Predicted functional ORFs are named to correspond with orthologues in the LSHV 137 genome and are indicated by arrows colored according to the key as belonging to gene families (sets of 138 paralogous genes), core ORFs (conserved among alloherpesviruses) and other noncore ORFs. Some ORFs are 139 shown by narrow arrows to make their locations clearer. Introns connecting ORFs are shown as narrow white 140 bars. Small, black rectangles indicate tandem repeats of undetermined size. 141 ORF0 familyPK familyORF37 familyORF39A familyIgD familyORF64 familyORF77 familyORF81 familyORF98 familyORF100 familyOther noncore ORFsCore ORFs0 5 10 15 20 25 30ORF0ORF1ORF2ORF3ORF4 ORF5 ORF6ORF7ORF8ORF9ORF10ORF11ORF12ORF13ORF14 ORF15ORF16ORF17ORF18ORF18AORF18BORF21 ORF22ORF19ORF2030 35 40 45 50 55 60ORF24ORF23 ORF25 ORF26 ORF27ORF28ORF29ORF30ORF31ORF32ORF33ORF34ORF35 ORF36 ORF3760 65 70 75 80 85 90ORF39ORF39AORF39BORF39CORF43ORF42ORF44ORF45ORF47ORF46 ORF48ORF49ORF50ORF51ORF52ORF53 ORF54ORF56ORF55 ORF58ORF57ORF60ORF59ORF61ORF63ORF62ORF6490 95 100 105 110 115 120ORF66ORF67ORF68ORF69ORF70ORF73ORF72ORF71 ORF74ORF75ORF76ORF77 ORF78ORF79ORF80ORF83ORF84ORF85ORF86ORF87 ORF88120 125 130 135 140 145 150ORF89ORF90ORF91ORF92ORF93ORF94 ORF95 ORF96ORF97ORF98ORF99ORF100ORF101 ORF102 ORF103 ORF103A150 155 160 165 170 175 180ORF109ORF109AORF109BORF109CORF110ORF111ORF111AORF113 ORF114 ORF116ORF117ORF118 ORF119 ORF120ORF121ORF122ORF123ORF124ORF125180 185 190 195 kbpORF126ORF128ORF129ORF130ORF130AORF103A

Genome sequence of white sturgeon herpesvirus 1 isolated from farmed white sturgeon (Acipenser transmontanus)

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Genome sequence of white sturgeon herpesvirus 1 isolated from farmed white sturgeon (Acipenser transmontanus)

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