Comparative Genomics Analysis between frog Virus 3-like Ranavirus from the First Canadian Reptile Mortality Event and Similar Viruses from Amphibians

doi:10.21203/rs.3.rs-943897/v1

Ranaviruses are emerging pathogens that threaten the biodiversity of wild and captive cold-blooded vertebrates. Reports of ranavirus-induced mortality events are increasing and ranavirus disease is reportable to the World Organization for Animal Health. Previous studies have suggested interclass transmission of ranaviruses and Frog virus 3 (FV3)-like viruses are of particular interest. This study presents the whole-genome assembly of a 106 kb FV3-like genome obtained from the liver tissue of a reptile (wild Chelydra serpentina, common snapping turtle) that died of ranavirus disease in Canada. The FV3-like ON turtle/2018 strain shares the highest genome-wide nucleotide identity (99.71%) with the wild-type FV3 virus detected in the USA from a Northern leopard frog and an FV3-like strain identified from a wood frog in 2017 in Alberta, Canada. The novel genome contains all 26 Iridoviridae core genes, 11 FV3-like genes, and 9 unique truncations, three of which are core Iridoviridae ORFs. Additionally, the two most closely related FV3-like strains from amphibians, were compared to a non-FV3-like amphibian infecting and a fish infecting ranavirus species that showed similar codon usage patterns. G/C-ending codons were the preferred codons for all five strains. Investigation of putative recombination events identified four potential recombination events in the FV3-like ON turtle/2018 genome consistent with this FV3-like reptile infecting strain originating from an amphibian infecting FV3-like ranavirus. Altogether, this study provides insights into the genome structure and the differences in the novel FV3-like genome compared to other ranavirus genomes.

Scientific Communication

Food Science & Technology

frog virus 3-like

reptile

ranavirus

amphibians

ranavirus codon usage

FV3-like codon usage

whole-genome sequencing

de novo hybrid genome assembly

virus recombination

Ranaviruses are large, cytoplasmic viruses with icosahedral capsids and double-stranded DNA genomes that belong to the Iridoviridae family of viruses ¹. Ranaviruses have a broad geographic distribution having been found on all continents except Antarctica, and are capable of infecting a wide range of ectothermic lower vertebrates, including amphibians, fish and reptiles ². Until the 1980s, it was thought that ranavirus infections resulted in subclinical disease, however, they are now recognized as etiological agents responsible for significant morbidity and mortality events in many ecologically and economically important amphibian and fish species ^1,3,4 According to the March 2021 revision by the International Committee on Taxonomy of Viruses (ICTV), there are seven ranavirus species including the type species, Frog virus 3 (FV3), as well as, Ambystoma tigrinum virus (ATV), European North Atlantic ranavirus, Common midwife toad virus (CMTV), Santee-Cooper ranavirus (SCRV), and Singapore grouper iridovirus (SGIV), Epizootic hematopoietic necrosis virus (EHNV)-a virus that causes epizootic hematopoietic necrosis which is an aquatic disease that is reportable to the World Organization for Animal Health (OIE). According to ICTV, other ranavirus strains characterized by whole genome sequencing (WGS) (ranavirus maximus, cod iridovirus and short-finned eel virus), may represent new species within the Ranavirus genus due to low relative genome-wide nucleotide identity to currently recognized ranavirus species ⁵.

Interclass transmission of ranavirus between ectothermic vertebrates has been reported in addition to variability in susceptibility of host species to ranaviruses; as one class may act as an infection reservoir for another class and some species act as subclinical carriers, allowing ranaviruses to persist in the environment ⁶. The first ranaviruses were isolated from northern leopard frogs in the 1960s from Midwest USA while the first case of fatal ranaviral infection of a chelonian reptile was in a mediterranean land tortoise in 1982 ². Subsequently, the virus has been reported in other reptiles such as soft-shelled turtles, Burmese star tortoises, Florida box turtles, red-eared sliders and eastern box turtles ^2,7. Mass mortality events have been well-documented in ecologically important wild amphibians since the mid-1960s, however, reports of ranaviral infection in reptiles, particularly chelonians, have only increased since 2010 ^2,8. Over the last decade in the United States, FV3-like ranaviruses have been responsible for several mass mortality events of eastern box turtles ^9–11. In Canada, the presence of ranaviruses was first reported in amphibians in 2004 and has been reported to be accountable for as high as 90-100% of mass mortality events ^12,23. In 2017, the first case of FV3-like virus-induced mortality in reptiles was recorded in Canada ¹². This increase in infection could be due to a combination of heightened awareness of the pathogenic potential of these viruses, increased surveillance, improved methods of detection, and also anthropogenic influences such as the global trade of amphibians and reptiles, habitat disruption and climate change ¹³. Comparative pathological studies have shown that the majority of ranaviruses detected in chelonians to date appear to be more similar to previously characterized amphibian ranaviruses (FV3- and CMTV-like) than ranaviruses identified from reptiles such as Burmese star tortoise ranavirus isolate and Terrapene carolina carolina ranavirus ¹⁴. This study presents the complete genome of an FV3-like viral strain isolated from a turtle in Ontario, Canada²³. Furthermore, a comparative genomic investigation was done with the FV3-like ON turtle/2018 strain and similar FV3-like strains isolated from amphibians. Results from this study give insights into the genome structure and differences of the novel FV3-like virus with other closely related viruses.

2.1 Comparison of the Genome structure of FV3-like ON turtle/2018 to wild type FV3 and other FV3-like ranaviruses

The FV3-like ON turtle/2018 genome is 106,187 base pairs in length and is within the reported size range for ranaviruses ¹⁵. It has a 55.2% genome-wide GC content, and 99 predicted open reading frames greater than 150 nucleotides which is also within the reported size range for ranaviruses (Table 1). The genome of the FV3-like ON turtle/2018 ranavirus contains all of the 26 Iridoviridae core genes ¹⁶ and all 11 ORFs unique to the FV3-like genomes reported to date and present in the wt-FV3 genome¹⁵ (Figure 1, Table 2). Also, the FV3-like ON turtle/2018 has two ORFs (ORFs 16.5L and 56.5R) encoding hypothetical proteins which are not present in the wt-FV3 genome. ORF 16.5L has been identified in the genomes of some FV3-like strains while ORF 56.5R is present in the genome of soft-shelled turtle iridovirus (STIV) (Table 2). The FV3-like ON turtle/2018 genome contains nine unique truncated ORFs compared to the wt-FV3 genome due to the presence of multiple premature stop codons (Table 2). Of the nine truncated ORFs, three were similar to core Iridoviridae genes. Together, the findings indicate the FV3-like ON turtle/2018 strain, while similar to the wt-FV-3 genome, possesses notable differences.

Table 1

Ranavirus strains used in the present study.
Origin Year Collected	Virus / Strain	Animal Host	Reported genome size (bp)	Predicted ORFs	GenBank Accession
Canadian Locations
NWT 2017	Frog Virus 3-like / Z897	Pseudacris maculata (A-Boreal chorus frog) ^a	104,643	94	MK959611
NWT 2017	Frog Virus 3-like / Z916	Lithobates sylvaticus (A-Wood frog) ^a	99,213	94	MK959612
NWT 2017	Frog Virus 3-like / Z920	Lithobates sylvaticus (A-Wood frog) ^a	104,603	94	MK959613
NWT 2017	Frog Virus 3-like / Z929	Pseudacris maculata (A-Boreal chorus frog) ^a	104,556	94	MK959614
NWT 2017	Frog Virus 3-like / Z930	Pseudacris maculata (A-Boreal chorus frog) ^a	104,245	94	MK959615
NWT 2017	Frog Virus 3-like / Z933	Pseudacris maculata (A-Boreal chorus frog) ^a	105,037	95	MK959616
NWT 2017	Frog Virus 3-like / Z936	Lithobates sylvaticus (A-Wood frog) ^a	97,848	91	MK959617
NWT	Frog Virus 3-like / Z938	Lithobates sylvaticus (A-Wood frog) ^a	104,549	94	MK959618
NWT 2017	Frog Virus 3-like / Z939	Lithobates sylvaticus (A-Wood frog) ^a	104,416	94	MK959619
NWT 2017	Frog Virus 3-like / Z942	Lithobates sylvaticus (A-Wood frog) ^a	103,673	94	MK959620
Alberta 2017	Frog Virus 3-like / Z994	Lithobates sylvaticus (A-Wood frog) ^a	104,788	95	MK959621
Ontario 2018	Frog Virus 3-like / ON turtle/2018	Chelydra serpentina (R -Common snapping turtle)^b	106,187	99	OK299175
Canada (Alberta) 2013	Ambystoma tigrinum virus / 2013-ATV-04	Ambystoma mavortium (A- Western tiger salamander ) ^h	106258	108	MK580531
Canada 2013	Ambystoma tigrinum virus / 2013-ATV-12	Ambystoma mavortium (A- Western tiger salamander ) ^h	106652	108	MK580532
Canada 2013	Ambystoma tigrinum virus / 2013-LL1	Ambystoma mavortium (A- Western tiger salamander ) ^h	106578	108	MK580533
Canada 2013	Ambystoma tigrinum virus / 2013-LL2	Ambystoma mavortium (A- Western tiger salamander ) ^h	106730	108	MK580534
Canada 2013	Ambystoma tigrinum virus / 2013-LL3	Ambystoma mavortium (A- Western tiger salamander ) ^h	106629	108	MK580535
Canada 2014	Ambystoma tigrinum virus / 2013-ATV-322	Ambystoma mavortium (A- Western tiger salamander ) ^h	106915	108	MK580536
Non-Canadian Locations
Netherlands (Imported from Nicaragua ) 2015	Frog Virus 3-like / OP/2015	Oophaga pumilio (A-Strawberry poison frogs) ^c	107,035	98	MF360246
United States 1965	Frog Virus 3-like / Wild-type	Rana pipiens (A- Northern leopard frog)	105,903	98	AY548484
Brazil-Sao Paulo 2012	Frog Virus 3-like / Ran-Bra-01	Lithobates catesbeianus (A-American Bullfrog) ^e	105,080	94	MH351268
United States 1998	Frog Virus 3-like / SSME	Frog (A-host not informed) ^f	105,070	95	KJ175144
United States-Arizona 1996	Ambystoma tigrinum virus / N/A	Ambystoma tigrinum (A- Western tiger salamander ^g	106,332	99	NC005832
Australia 2010	Epizootic haematopoietic necrosis virus / N/A	Perca fluvuatilis (F-European perch) ⁱ	127,011	100	NC028461
Spain 2011	European catfish virus / Valdeolmas	Silurus glanis (F-Wels catfish) ^j	127,732	136	NC017940
Hungary 2012	European catfish virus / 14612/2012	Ameiurus nebulosus (F-Brown bullhead) ^k	127,549	136	KT989885
Hungary 2012	European catfish virus / 13051/2012	Ameiurus nebulosus (F-Brown bullhead) ^l	127,751	136	KT989884
New Zealand 1991	Short-finned eel ranavirus / ANGA14001	Anguilla australis (F-Short-finned eel) ^m	126,965	111	ANGA14001
Denmark 1999	Ranavirus maximus / SMA15001	Scophthalmus maximus (F-Turbot) ⁿ	115,510	100	NC030842
Finland 1997	Pike perch iridovirus / SLU14001	Sander lucioperca (F-Zander) ^o	108,841	109	KX574341
Netherlands 2013	Common midwife toad virus / Pelophylax kl. esculentus/2013/NL	Pelophylax kl. Esculentus (A–Edible frog) ^p	107,772	102	NC039034
Denmark 2013	Pelophylax esculentus virus / PEV_DK1	Pelophylax Esculentus (A –Edible frog) ^q	107,469	99	MF538627
Australia 1992	Bohle iridovirus / BIV-ME 93/35	Limnodynastes ornatus (A-Ornate burrowing frog) ^r	103,531	100	NC038507
USA 2011	Terrapene carolina carolina ranavirus / TCA16001	Terrapene carolina carolina (R-Common box turtle) ^s	104,894	95	MG953518
^{NWT: Northwest Territories, A: Amphibian, R: Reptile, F: Fish}.
^a Data obtained from ²⁴, ^b Present study,^c Data obtained from ¹⁸, ^d Data obtained from ¹⁵, ^e Data obtained from ¹⁷, ^f Data obtained from ¹⁹, ^g Data obtained from ⁴³, ^h Data obtained from ⁴⁴, ^I Data obtained from ⁴⁵, ^j Data obtained from ⁴⁶, ^k Data obtained from ⁴⁷, ^l Data obtained from ⁴⁷, ^m Data obtained from ⁴⁸, ⁿ Data obtained from ⁴⁹, ^o Data obtained from ⁵⁰, ^p Data obtained from ⁵¹, ^q Data obtained from ⁵², ^r Data obtained from ⁵³, ^s Data obtained from ⁵⁴.

Table 2

Summary of genetic differences observed in the FV3-like turtle ON/2018 genome compared to the wt-FV3 reference genome.
ORFs present in FV3-like ON turtle/2018 and other FV3-like viruses but absent in the wild type FV3 genome	Start/Stop Position	Predicted function
16.5L	20,073-19,126	Hypothetical protein FV3_ORF 16.5L
56.5R	62,350-62,754	Hypothetical protein (Soft-shelled turtle iridovirus)
ORFs truncated in FV3-like ON turtle/2018 genome compared to the wild type FV3 and other FV3-like genomes	Start/Stop Position	Predicted function
31R	38,102 - 38,519	Secretory protein
34R	46,879 – 50,352	Human parainfluenza virus 1 L protein, Transmembrane domain
41R	50,874 – 51,628	RRV orf-2-like protein, Secretory protein
45L	52,109 – 52,578	LCDV1 Orf-88-like protein, Secretory protein
47L	53,138 – 53,556	Secretory protein
49L	53,908 – 54,796	LCDV1 Orf58-like protein, SP, SAP DNA binding domain
50L	54,772 – 55,410	Secretory protein
51R	55,488 – 57,164	Surface protein
82R	89,548 – 90,128	Secretory protein

2.2 Whole-genome phylogenetic comparison of the FV3-like ON turtle/2018 genome and other ranavirus species.

Phylogenetic reconstruction was performed using WGS of 33 different ranavirus strains, 15 of which were FV3-like ranaviruses from the following locations; Canada (Northwest Territories (NWT) and Alberta) ²², Brazil-Sao Paulo ¹⁷, Nicaragua strain (imported to the Netherlands) ¹⁸ and the United States ^15,19. The phylogenetic comparison showed that FV3-like ON turtle/2018 clusters in the FV3-like clade (Figure 2). This is consistent with the nucleotide percent identity and confirms that the FV3-like ON turtle/2018, detected in a reptile (common snapping turtle) is an FV3-like ranavirus. Subsequent phylogenetic analysis with other FV3-like genomes confirms previous findings that all the NWT strains clustered in the same subclade while the Alberta strain (Z994) clustered in a different subclade. The Ran-BRA-01 strain from Brazil and OP/2015 from Netherlands (imported from Nicaragua) clustered in the same subclade while the strains detected in the USA clustered together (Figure 2). Altogether, the FV3-like ON turtle/2018, detected in a reptile for the first time in Canada, is an FV3-like ranavirus as indicated by the whole genome nucleotide sequence percent identity and clusters in the same clade with other FV3-like ranaviruses (Figure 2).

2.3 Codon usage comparison of FV3-like ON turtle/2018, amphibian and fish infecting ranaviruses.

2.3.1 Nucleotide Composition Analysis of five ranavirus strains and species.

The overall nucleotide composition and codon usage of the genomes of turtle ON/2018, the most closely related FV3 and FV3-like genomes, an amphibian infecting ATV strain and a fish infecting EHNC strain were investigated and compared (Table 3, S. Table 3). Analysis of the nucleotide composition of the third position of codons (T3, C3, A3, G3) and GC of silent third codon position showed C3 (0.48) and G3 (0.42 to 0.43) were the highest for all five strains, followed by A3 (0.15 to 0.16) and T3 (0.15 to 0.16). The GC of the silent third codon position was approximately 0.73 (Table 3). GC content has been proposed to be a major factor driving codon usage bias ²⁰. Therefore, the average GC content of genes was determined to be approximately 58-59% for all five strains (Table 3). Parameters utilized for relative synonymous codon usage (RSCU) analysis (Figure 3) were the number of synonymous codons (from 25,542 to 29,476) and the total number of amino acids (from 26,791 to 30,856) (Table 3).

Table 3

Average nucleotide composition analysis of predicted genes in the FV3-like turtle ON/2018 strain compared to the FV3 wild type, FV3-like Z994, EHNC (NC_028461) and ATV 2013-040 strains.
Strain	T3	C3	A3	G3	Effective Number of Codons	GC3	GC content of genes	Number of synonymous codons	Total number of amino acids	Hydropathicity of protein	Aromaticity of protein
turtle ON /2018	0.1585	0.4814	0.1627	0.4233	41.99	0.735	0.592	25542	26791	-0.322620	0.078123
Wild type	0.1600	0.4824	0.1620	0.4234	41.75	0.734	0.590	26829	28154	-0.319155	0.078568
Z994	0.1641	0.4780	0.1634	0.4217	42.00	0.730	0.589	27057	28355	-0.322144	0.076812
EHNC NC_028461	0.1483	0.4869	0.1542	0.4354	40.34	0.75	0.597	29476	30856	-0.329712	0.07697
ATV 2013-ATV-040	0.1623	0.4768	0.1657	0.4254	41.96	0.73	0.583	25901	27104	-0.301155	0.079398
^{The effective number of codons is a framework for determining the rate of codon usage bias in coding sequences that is independent of the length of the gene and the number of amino acids which ranged from 25542 to 29476 for the five strains studied. Amino acid usage is influenced by factors such as hydrophobicity and aromaticity approximately −0.31 and 0.07 respectively and were similar for all five strains}.

2.3.2 Relative synonymous codon usage analysis of five ranavirus species and strains

Relative synonymous codon usage (RSCU) analysis was performed to gain insight into the differences in codon usage variation in the FV3-like ON turtle/2018 strain and to describe the codon usage bias of FV3-like ON turtle/2018 compared to other ranaviruses. RSCU of ON turtle/2018 was compared using the two strains with the highest nucleic acid sequence identity (wt-FV3 and FV3-like strain Z994), an amphibian infecting ATV-like strain (ATV 2013-ATV-040) and a fish infecting EHNC-like ranavirus strain (EHNC NC_028461). RSCU values >1.60 were categorized as overrepresented codons (n=15), values <0.60 were categorized as underrepresented codons (n=24); while values >1.00 were classified as more frequently used codon (n=15), and values < 1.00 (n=9) were classified as less frequently used codons. Among all the codons, AGG which codes for the amino acid, Arginine was the most overrepresented codon for all five strains (Figure 3, S. Table 2) (RSCU values: 3.79 for ON turtle/2018, 3.87 for wt-FV3, 3.84 for FV3-like strain Z994, 3.91 for EHNC NC_028461 and 4.01 for ATV 2013-ATV-04).

2.4 Recombination analysis of the frog virus 3-like ON turtle/2018 genome

Four statistically significant potential recombination events (RE22, RE11, RE5, and RE4) were detected in the FV3-like ON turtle/2018 genome (Table 4, Figure 4, S. Figure 1). For RE22, the potential minor parent (a closely related sequence from which the proposed recombinant region may have originated) was Op/2015 strain detected in strawberry poison frogs in the Netherlands (imported from Nicaragua) and the potential major parent (a closely related sequence from which the greater part of the recombinant sequence may have originated) was the wt-FV3 detected in a frog in the USA. For RE11, the potential minor parent was strain FV3-like Z897 strain detected in boreal chorus frog from NWT, Canada and the potential major parent was strain Ran-Bra-01 detected in an American bullfrog in Brazil. RE4 and 5 both had unknown potential minor parents and the wt-FV3 was the potential major parent (Table 4 and Figure 4). It is of note that core Iridoviridae, Ranavirus and FV3-like ORFs were present within RE4 (Figure 4). Also, ORFs identified to be truncated in the FV3-like ON turtle/2018 genome were present in recombination events 22, 4, and 5 (Figure 4). ORF 34R that encodes Human parainfluenza virus 1 L protein is present in recombination event 22. RE4 contained five ORFs; ORFs 45L (LCDV1 Orf-88-like protein), 47L (secretory protein), 49L (LCDV1 Orf58-like protein, SP, SAP DNA binding domain), 50L (a secretory protein) and 51R (a surface protein). ORF 82R putatively encodes immediate-early protein ICP-18 and was present in recombination event 5 ^15,21. In summary, our analysis revealed that the FV3-like ON turtle/2018 ranavirus possesses four potential recombination events that contain core Iridoviridae, Ranavirus and FV3-like ORFs.

<div class="gridtable">&nbsp;<table border="1" id="Tab4">
        <caption language="En">
            <div class="CaptionNumber">Table 4</div>
            <div class="CaptionContent">
                <p>Summary of significant recombination events identified between the whole genome sequence of the FV3-like ON turtle/2018 and the other 15 FV3-like ranaviruses used in this study. Breakpoint position in the alignment, Potential parental identity (strain name) and average p-values of the detection methods used are indicated.</p>
            </div>
        </caption>
        <thead>
            <tr>
                <th align="left" colspan="3">
                    <p>Breakpoint position in Alignment in the FV3-like turtle ON/2018</p>
                </th>
                <th align="left" colspan="2">
                    <p>Potential Parental Strain Identity</p>
                </th>
                <th align="left" colspan="6">
                    <p>Detection Methods (Average <em>p-values)</em></p>
                </th>
            </tr>
        </thead>
        <tbody>
            <tr>
                <td align="left">
                    <p><strong>Recombination Event number</strong></p>
                </td>
                <td align="left">
                    <p><em>Begin</em></p>
                </td>
                <td align="left">
                    <p><em>End</em></p>
                </td>
                <td align="left">
                    <p><em>Minor</em></p>
                </td>
                <td align="left">
                    <p><em>Major</em></p>
                </td>
                <td align="left">
                    <p><em>RDP</em></p>
                </td>
                <td align="left">
                    <p><em>GENECONV</em></p>
                </td>
                <td align="left">
                    <p><em>Bootscan</em></p>
                </td>
                <td align="left">
                    <p><em>MaxChi</em></p>
                </td>
                <td align="left">
                    <p><em>Chimaera</em></p>
                </td>
                <td align="left">
                    <p><em>SisScan</em></p>
                </td>
            </tr>
            <tr>
                <td align="left">
                    <p>22</p>
                </td>
                <td align="left">
                    <p>40609</p>
                </td>
                <td align="left">
                    <p>41031</p>
                </td>
                <td align="left">
                    <p>OP/2015</p>
                </td>
                <td align="left">
                    <p>wild type</p>
                </td>
                <td align="left">
                    <p>7.443E-17</p>
                </td>
                <td align="left">
                    <p>1.282E-17</p>
                </td>
                <td align="left">
                    <p>6.965E-17</p>
                </td>
                <td align="left">
                    <p>4.306E-06</p>
                </td>
                <td align="left">
                    <p>1.873E-06</p>
                </td>
                <td align="left">
                    <p>2.996E-03</p>
                </td>
            </tr>
            <tr>
                <td align="left">
                    <p>4</p>
                </td>
                <td align="left">
                    <p>50524</p>
                </td>
                <td align="left">
                    <p>58806</p>
                </td>
                <td align="left">
                    <p>Unknown</p>
                </td>
                <td align="left">
                    <p>wild type</p>
                </td>
                <td align="left">
                    <p>8.601E-61</p>
                </td>
                <td align="left">
                    <p>2.904E-80</p>
                </td>
                <td align="left">
                    <p>1.473E-63</p>
                </td>
                <td align="left">
                    <p>2.045E-32</p>
                </td>
                <td align="left">
                    <p>8.411E-30</p>
                </td>
                <td align="left">
                    <p>5.779E-19</p>
                </td>
            </tr>
            <tr>
                <td align="left">
                    <p>11</p>
                </td>
                <td align="left">
                    <p>58807</p>
                </td>
                <td align="left">
                    <p>72657</p>
                </td>
                <td align="left">
                    <p>Z897</p>
                </td>
                <td align="left">
                    <p>Ran-Bra-01</p>
                </td>
                <td align="left">
                    <p>7.100E-29</p>
                </td>
                <td align="left">
                    <p>6.079E-26</p>
                </td>
                <td align="left">
                    <p>2.434E-15</p>
                </td>
                <td align="left">
                    <p>1.502E-15</p>
                </td>
                <td align="left">
                    <p>2.541E-16</p>
                </td>
                <td align="left">
                    <p>6.217E-14</p>
                </td>
            </tr>
            <tr>
                <td align="left">
                    <p>5</p>
                </td>
                <td align="left">
                    <p>80910</p>
                </td>
                <td align="left">
                    <p>96262</p>
                </td>
                <td align="left">
                    <p>Unknown</p>
                </td>
                <td align="left">
                    <p>wild type</p>
                </td>
                <td align="left">
                    <p>8.402E-79</p>
                </td>
                <td align="left">
                    <p>1.623E-77</p>
                </td>
                <td align="left">
                    <p>7.016E-78</p>
                </td>
                <td align="left">
                    <p>4.930E-33</p>
                </td>
                <td align="left">
                    <p>3.273E-33</p>
                </td>
                <td align="left">
                    <p>4.595E-36</p>
                </td>
            </tr>
        </tbody>
    </table>
</div>

In recent years, reports of ranavirus epizootics have increased with viruses such as the European North Atlantic ranavirus, emerging as a novel species ²². It has also been reported that outbreaks of FV3-like ranavirus can result in high mortality for animals both in the wild and in captivity ². FV3-like ranavirus is the only member of the Iridoviridae reported in turtles as its presence is enhanced by anthropogenic activities such as importing or exporting infected animals as pets or for food, and agricultural activities ². The FV3-like ON turtle/2018 ranavirus sequenced in this study is the first confirmed ranavirus-induced mortality in a reptile (snapping turtle) in Canada ²³ and our analysis offers new insight into the genome of an FV3-like virus derived from a dead reptile host.

The FV3-like ON turtle/2018 genome was compared to the published wt-FV3 genome, along with previously identified FV3-like genomes and other ranavirus genome sequences. Phylogenetic analysis placed the FV3-like ON turtle/2018 strain in the FV3-like clade, consistent with its high nucleotide sequence identity to other FV3-like viral strains. The FV3-like ON turtle/2018 strain showed the highest nucleotide identity (99.71%) to the genome of the wt-FV3 and an FV3-like strain detected in Alberta, Canada (S. Table 1). Based on the genome structure and organization, the FV3-like ON turtle/2018 genome possesses genetic features that are present in the wt-FV3 genome including all of the core Iridoviridae and FV3-like genes. Nine ORFs (31R, 34R, 41R, 45L, 47L, 49L, 50L, 51R and 82R) were identified to be truncated in the FV3-like ON turtle/2018 genome compared to the wt-FV3 and other reported FV3-like genomes. Three of the nine truncated ORFs were Iridoviridae core ORFs (31R (a secretory protein), 34R (Human parainfluenza virus 1 L protein, transmembrane domain), and 82R (secretory protein)) suggesting these genes may be critical for viral replication and transmission. Many viruses utilize secretory pathways to facilitate viral replication ²⁴, while transmembrane domains may play a role in the co-localization of viruses to the endoplasmic reticulum ^24,25. Previous work identified truncations in ORFs of newly characterized FV3-like genomes in two (34R and 41R) of the nine ORFs in question in the Ran-Bra-01 strain, although the significance of these identified truncations remains unknown ²⁵. Thus future studies that focus on the functional analysis of ranavirus and FV3-like core genes are essential to elucidate the specific roles and phenotypic consequences of these truncated ORFs. Our findings demonstrate and identify genetic variations present between closely related FV3-like strains in coding regions ²⁴. Altogether, the genomic differences identified in the FV3-like ON turtle/2018 genome compared to the wt-FV3 genome may contribute to, or be a result of the adaptation of the FV3-like ON turtle/2018 strain to infecting a novel host-turtle.

The present study is the first to investigate the nucleotide comparison of all ORFs and relative synonymous codon usage analysis of FV3-like strains. Results of the codon usage analysis revealed that the FV3-like turtle ON/2018 shares a similar relative synonymous codon usage to the wt-FV3, the Z994 FV3-like strain, an ATV strain and an EHNC strain. Also, the nucleotide composition analysis showed that the FV3-like genomes have a higher G/C-ending codon compared to A/T–ending codons. From these analyses, it can be concluded that the G/C-ending codons may be the preferred codons over the A/T-ending codons in the five ranavirus genomes analyzed from different hosts representing different taxonomic classes. A study by Tian et al. investigated codon usage bias of the major capsid protein-coding genes of five groups of ranavirus and reported that amphibian-like ranaviruses prefer C- and G- ending codons (Tian et al., 2020). In addition, the nucleotide composition of the FV3-like turtle ON/2018 genome is similar to other FV3-like strains. These findings support the hypothesis by McKenzie et al., ²³ that the FV3-like ON turtle/2018 ranavirus likely emerged in snapping turtles from amphibians sharing the same environment ^10,26, since results of this study show that whole-genome sequence comparison and codon usage in this reptile derived strain was similar to that of the amphibian derived strains. Interclass transmission of ranaviruses was demonstrated in a controlled laboratory setting ⁶ in a study by Brenes et al, using FV3-like ranavirus infected gray tree frog larvae, mosquito fish and read-eared slider. It was shown that the FV3-like exposed gray tree frog larvae were capable of transmitting ranavirus to unexposed larvae and turtles. Their findings support the hypothesis that interclass transmission may occur in wild populations for FV3-like ranaviruses ⁶.

Previous studies have shown that some ranavirus species evolve from different localities through recombination ^17,27. Recombination events in viruses are also an important mechanism involved in viral-host adaptation to new environments ²⁸. These events commonly occur in ranaviruses and our analysis of the FV3-like ON turtle/2018 genome further supports these findings. Our analysis revealed four potential recombination events in the newly isolated FV3-like ON turtle/2018 strain when compared to FV3-like strains and these events were located in breakpoints that contain multiple core ORFs. Potential major parental FV3-like strains were identified as the wt-FV3 virus from the United States for three of the four recombination events. The potential parental lineage of the fourth recombination event was identified as either the Ran-Bra-01 - Brazil strain, the Op/2015 strain detected in the Netherlands (imported from Nicaragua), or the FV3-like Z897 strain from NWT, Canada. This suggests that the FV3-like ON turtle/2018 strain may be a mosaic derived from recombination events involving ranaviruses introduced by anthropogenic factors such as the global commercial exotic pet trade. Investigations of recombination events, such as these, are important since recombinant strains may alter the phenotype and lead to changes in pathogenicity and/or the hosts range. For example, the recombinant CMTV-like RCV-Z2 bullfrog isolate, an FV3 x CMTV recombinant ranavirus that was more pathogenic than wt-FV3 ²⁷. Furthermore, investigating recombination events has the potential to study the anthropogenic factors driving the evolution of viruses.

Ranavirus infections have been identified in outbreaks of captive and free-ranging eastern box turtles and so genomic comparison was performed with the only available whole-genome sequence of a common box turtle infecting ranavirus, Terrapene carolina carolina ranavirus (TCCR). The TCCR, detected in common box turtles in the United States, has been sequenced and reported to be most closely related to an FV3-like strains ^10,29. However, FV3-like turtle ON/2018 is a distinct strain sharing only 99.17% genome-wide nucleotide identity with TCCR and TCCR was not identified as a potential parental strain in any of the significant recombination events. The mechanisms and significance of changes resulting from recombination events remain unknown; however, genetic recombination of viruses has been reported to influence the formation of novel chimeric genomes that drive the establishment of viral diversity, variation and could lead to the creation of novel viruses ³⁰. Indeed, a highly invasive chimeric ranavirus identified in the United States, as a result of multiple recombination events, was shown to decimate tadpole populations rapidly as compared to non-chimeric ranaviruses ³⁰. Therefore, additional recombination analysis of FV3-like ranavirus genomes isolated from new reptile or non-amphibian hosts may shed light on the emergence and spread of novel viral strains in different cold-blooded vertebrates.

The results presented here reinforce the need for characterization and comparison of viral strains obtained from similar or different hosts using WGS, and that strain identification, based on a single or a few genes, is insufficient. Diagnostic PCR tests that target the major capsid protein can confirm the presence or absence of the targeted gene, but offer little information for characterizing novel viral strains, such as the gene structure and genomic arrangement of the virus being studied. This study provides the first hybrid de novo whole-genome assembly of an FV3-like genome generated using both long and short-read sequencing technologies; most of the previously characterized FV3-like genomes were assembled using short reads generated using Illumina sequencers, or direct sequencing of PCR products. By using a combination of Illumina short-read and Nanopore long-read sequencing, we aimed to improve the genome completeness and accuracy of the FV3-like ON turtle/2018 assembly ³¹. Also, using both sequencing platforms has allowed us to improve the assembly of the FV3-like ON turtle/2018 genome and to study the presence of potential recombination events and codon usage bias and function of the FV3-like strain. The long read data also helped resolve repetitive sequences present in the Iridoviridae genome that have been reported to function as regulatory sequences ³⁰. It is expected that the high-quality FV3-like ON turtle/2018 genome obtained in this study will serve as a reference sequence resource for future turtle/reptile-derived FV3-like ranaviruses.

The present study reports the whole genome sequence of FV3-like ranavirus detected for the first time in a snapping turtle (reptile) in Canada. This study provides insight into a viral pathogen that causes high mortality in cold-blooded vertebrates. Our results provide evidence for the transmission of FV3-like ranaviruses to ectotherm vertebrates and shed light on the differences and similarities of the reptile FV3-like turtle ON/2018 viral genome and the amphibian FV3-like genomes. Additionally, this study is the first to investigate codon usage in Ranavirus species, which suggests that FV3-like turtle ON/2018 has a similar nucleotide composition to other amphibians and fish infecting ranavirus strains. However, future work should compare the genomes of FV3-like viruses obtained from other reptiles to understand the genetic implications, pathogenicity and host range of FV3-like transmission between different classes of vertebrates.

5.1. Case history

In Canada, the first recorded case of ranavirus-induced mortality in reptiles was confirmed in 2017 in Southern Ontario after a common snapping turtle (Chelydra serpentina) tested positive for the presence of ranavirus by a conventional PCR targeting the major capsid protein gene at the Animal Health Centre, B.C. Ministry of Agriculture, Food and Fisheries²³. A second ranavirus case from Ontario was identified in 2018 after a wood turtle (Glyptemys insculpta) also tested positive by PCR ³². DNA samples from infected liver tissue from two snapping turtles part of the original cluster and virus amplified in cell culture were provided to the Genomics Unit at the Canadian Food Inspection Agency’s National Centre for Foreign Animal Disease, Winnipeg, Manitoba, for characterization by whole genome sequencing. The objective of this study was to compare the whole genome sequence and codon usage of the first recorded FV3-like ranavirus in reptiles to other FV3-like viruses derived from amphibians in Canada and globally and a ranavirus from fish. Putative recombination events present in the reptile FV3 were also characterized.

5.2. Sample processing

Extracted DNA from three samples (two liver and one tissue culture-derived) positive for ranavirus by PCR was provided by the Animal Health Centre, B.C. Ministry of Agriculture, Food and Fisheries. Briefly, total DNA from turtle liver tissue homogenates (10% w/v) prepared in Minimum Essential Medium and EPC (human endothelial progenitor cells) cell culture supernatant were extracted using QiaAMP® DNA Mini kit (Qiagen Inc., Toronto, ON, Canada) according to the manufacturer’s instructions. The presence of ranavirus was confirmed by PCR and electron microscopy as described previously²³ .

5.3. High throughput-Sequencing

Both short-read Illumina and long-read Oxford Nanopore sequencing were performed. Short read high throughput sequencing (HTS) was performed on an Illumina MiSeq instrument after capture probe-based enrichment (Virocap) with the tissue culture-derived sample ³³. Processing and HTS were performed as previously described^34,35. Briefly, the KAPA HyperPlus library preparation kit (Roche Diagnostics) and a custom pan-vertebrate virus targeted enrichment probe panel according to the Nimblegen SeqCap EZ HyperCap Workflow User’s Guide (Roche) and the manufacturer’s recommendations were used. Enriched libraries were quantified, pooled and sequenced on a V2 flowcell using a 500 cycle kit (Illumina).

For long-read sequencing, a total of 5 ng of DNA prepared from EPC cultured virus as used as input to generate barcoded sequencing libraries using the Rapid PCR Barcoding Kit (ONT; SQK-RPB004) according to the manufacturer’s instructions. A volume of 1 µL of each barcoded library was quantified using the Qubit 3 Fluorometer with the dsDNA High Sensitivity Kit and pooled to a final volume of 10 µL before completing library preparation and flow cell loading. Sequencing was conducted using an R9.4.1 flow cell on a MinION Mk1B for 48 hours with default settings. Raw reads were basecalled using Guppy basecaller v4.5.4 (Oxford Nanopore) and guppy_barcoder v4.5.4 was used to barcode and demultiplex the basecalled reads. The obtained fastq files were used for downstream hybrid genome assembly.

5.4. Genome assembly and Data analysis

Due to the higher quality of the results, only the tissue culture-derived Illumina reads and a random subsampling of 10% of the Nanopore reads (120,044 reads out of ~1.2 million total reads) were used for the whole genome assembly. Unicycler v0.4.4 on conservative mode was used for de novo hybrid assembly of reads obtained from the Illumina sequencing and Oxford Nanopore sequencing to produce a contig of approximately 106 Kbp. The resulting ONT + Illumina assembly was rotated and adjusted to have a similar starting position as the wild-type frog virus 3 ranavirus (AY548484) using rorate_contig, an in-house script (https://gist.github.com/peterk87/de921ea877dbad280ea7b395312f71e8). The assembled contig was subjected to a blastn query (default parameters) of the NCBI nr/nt database (downloaded May 19, 2021). The complete genome is available on NCBI under the accession OK299175. The viral genome annotation system (VGAS), designed to identify viral genes, was used for annotation of the FV3-like ON turtle/2018 genome (default settings were used, except minimum gene length parameter was changed to 120 bp)³⁶. Geneious Prime V2021.1.1 was used to confirm the predicted location of coding open reading frames (ORFs) and for functional gene, annotation using the wt-FV3 genome (Genbank accession No. AY548484) as a reference. ORF positions were confirmed based on findings from Eaton et al ¹⁶.

5.5. Codon Usage

Codon usage and relative synonymous codon usage (RSCU) values for both individual and concatenated genes were calculated using CodonW ³⁷ with default settings and the correspondence analysis (COA) set for codon usage. The codon usage indices applied were an effective number of codons (Nc), GC content of gene (G+C), GC of silent 3rd codon position (GC3), silent base composition (T3/C3/A3/G3), number of synonymous codons (L_sym), the total number of amino acids (L_aa), hydrophobicity of protein (Hydro), and aromaticity of protein (Aromo).

5.6. Phylogenetic Analysis

For phylogenetic characterization, identity analysis, and recombination analyzes of the FV3-like ON turtle/2018 genome, the complete genome sequences of 33 ranavirus species were retrieved from GenBank (FV3-like WGS: downloaded May 21, 2021, ranavirus WGS: downloaded June 1, 2021) (Table1). Maximum likelihood phylogenetic reconstruction was performed using IQ-TREE version 1.6.12 ³⁸ from MAFFT v7.450 multiple sequence alignments (MSA) using default settings ³⁹ of the reptile FV3-like genome and the 33 ranavirus strains using whole-genome nucleotide sequences. The phylogenetic tree was produced using the substitution model selected by ModelFinder ⁴⁰, with 1,000 ultrafast bootstraps ⁴¹ and visualized using shiptv, a standalone interactive phylogenetic tree viewer (https://github.com/peterk87/shiptv).

5.7. Investigation of Recombination Events

Recombination analysis was carried out using the Recombination Detection Program (RDP4) v4.101 software ⁴² and inputting the WGS of the FV3-like ON turtle/2018 and 15 FV3-like ranavirus strains from Canada (Alberta and NWT), Brazil, Netherlands and the USA. Six recombination detection methods (RDP, GENECONV, BOOTSCAN, MaxChi, Chimaera, and SiSCAN) were implemented with RDP4 for the identification of recombinant sequences and breakpoints. The default settings were used for all methods, and the highest acceptable p-value cut-off was set to 0.01. Putative recombination events were identified if observed for the same event using all six algorithms.

Author Contributions

O.L. designed the experiment and co-wrote the manuscript with all co-authors. M.T., K.S., and T.J. performed the sample processing, virus amplification, nucleic acid extraction, and PCR testing. A.B., M.N., and P.K. performed the analysis and prepared the figures and tables. C.B. and M.F. performed the library preparation and high-throughput sequencing. All authors reviewed the manuscript.

Data Availability Statement

The DNA sequence of the Frog Virus 3-like Ranavirus presented and analyzed in this study has been deposited in the GenBank under the accession number : OK299175 and reads generated from both the Illumina and Nanopore sequencing were deposited in the Sequence Read Archive (SRA), National Center for Biotechnology Information under the accession code: SAMN21442565.

Acknowledgements

The authors with to acknowledge the input provided by Dr. Chad Laing and Mr. Josip Rudar regarding the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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No competing interests reported.

Comparative Genomics Analysis between frog Virus 3-like Ranavirus from the First Canadian Reptile Mortality Event and Similar Viruses from Amphibians

Status:

Version 1

Abstract

Figures

1. Introduction

2. Results