ABSTRACT
We report the full-length genome sequence (compared to reference sequences) of a variant strain of Anguillid herpesvirus 1 (AngHV-1) isolated from imported Anguilla rostrata (American eel) from Canada. This should help to further identify such viruses in the North America.
ANNOUNCEMENT
Anguillid herpesvirus 1 (AngHV-1) is an important eel virus belonging to the genus Cyprinivirus of the family Alloherpesviridae which poses a serious threat to both farmed and wild eel species worldwide (1–3). AngHV-1 is an enveloped double-stranded DNA virus which causes hemorrhagic skin lesions and destruction of gill filaments (4). AngHV-1 is frequently detected in Anguilla anguilla from the Netherlands (5), Denmark (6), the United Kingdom (7), and Poland (8); in Anguilla japonica from Japan (4) and Taiwan (9); in Anguilla bicolor from South Korea (10); and recently, in Anguilla marmorata from Vietnam (11).
Taking cue from this, we conducted a virus surveillance program for the 2017–2019 period with American eel (Anguilla rostrata) imported (n = 5 eels/consignment) from Canada in oxygen-supplied bags directly to the laboratory without being exposed to South Korean water. For this, gill tissues were collected and aliquoted for DNA extraction, followed by PCR screening using a primer targeting the AngHV-1 DNA polymerase gene (5), and for virus isolation in the eel kidney cell line (EK-1). PCR-positive gill homogenates were inoculated using the limiting dilution method (5 times) in EK-1 cells. After development of a complete cytopathic effect involving rounding and enlargement of the cells, the supernatant containing AngHV-1 was collected, confirmed by PCR assay, and propagated in tissue culture flasks. The harvested AngHV-1 with a virus titer of 106.8 50% tissue culture infective dose (TCID50) was subjected to genomic DNA (gDNA) extraction using the high-pure PCR template preparation kit (Roche).
Double-stranded library preparation was performed with 1 mg sheared gDNA using the MGIEasy DNA library prep kit (MGI), followed by quantification using the QuantiFluor ONE double-stranded DNA (dsDNA) system (Promega). The library was circularized, digested, and cleaned by incubating it at 37°C for 60 min. Subsequently, a DNA nanoball (DNB) was prepared by incubating the library at 30°C for 25 min using DNB enzyme, followed by quantification using the QuantiFluor single-stranded DNA (ssDNA) system (Promega). Finally, sequencing of the prepared DNB was conducted using the MGISEQ system (MGI), yielding 29,772,167 reads (150-bp paired-end format) with a fold coverage of 31,543.4×. The raw data were trimmed using Cutadapt ver. 1.9, and a contig sequence was produced using the CLC Genomics Workbench ver. 20.0.4 de novo assembler (Qiagen) with default settings. The genome completeness was confirmed by mapping the filtered data into the contig sequence using the Map to Reference tool of Geneious ver. 2021.1.1 software, with default parameters. The detailed annotation of the completed sequence was manually corrected by referencing it against the sequences found under GenBank accession numbers FJ940765 and KX027736 using SnapGene software ver. 5.3.2 (GSL Biotech LLC).
The sequence analysis report displaying the AngHV-1 virus isolated from A. rostrata indicated a complete genome length of 249,121 bp, including a pair of 11-kb terminal direct repeats and 133 protein-coding open reading frames (ORFs). The gDNA sequence is 99.7% and 99.4% identical to the previously reported sequences of AngHV-1 isolated from A. japonica from Taiwan (GenBank accession number KX027736) and from A. anguilla from the Netherlands (FJ940765), respectively. Further, phylogenetic analysis of the gene encoding DNA polymerase showed that the isolated virus was identical to sequences of AngHV-1 from other eel species (Fig. 1). However, a BLASTN search revealed a difference matrix of 0.01% to 3.98% for some ORFs encoding different viral proteins compared with the reported sequences (Table 1). Thus, it can be inferred that AngHV-1 has low genetic diversity among the different strains with respect to the host eel species or the geography of the isolation source.
FIG 1.
Unrooted maximum likelihood (ML)-based tree of the polymerase gene DNA sequences of AngHV-1 isolated from imported Anguilla rostrata from Canada and from records retrieved from GenBank. AciHV, acipenserid herpesvirus; AngHV, anguillid herpesvirus; CyHV, cyprinid herpesvirus; IcHV, ictalurid herpesvirus; RaHV, ranid herpesvirus; SalHV, salmonid herpesvirus; SbSHV, Siberian sturgeon herpesvirus. The GenBank accession number is provided for each sequence. The phylogenetic tree was constructed using MEGA7 software with the Kimura 2-parameter model, with the highest log likelihood. The bootstrap consensus tree inferred from 1,000 replicates with values above 50% is taken to represent the evolutionary history of the taxa analyzed. A discrete gamma distribution was used to model the evolutionary rate differences among sites (2 categories [+G, parameter = 10.8325]). The analysis involved 20 nucleotide sequences. The codon positions included were 1, 2, 3, and noncoding. All positions containing gaps and missing data were eliminated. There were a total of 353 positions in the final data set. The scale bar represents the number of substitutions per nucleotide site.
TABLE 1.
Results of a BLASTN search comparing the identity percentage of the AngHV-1 sequence in the present article with reference sequences
| Protein-coding ORF | % identity of AngHV-1 isolated from A. rostrataa with that isolated from: |
|
|---|---|---|
| A. japonica b | A. anguilla c | |
| DNA packaging terminase subunit 1 | 100 | 100 |
| ORF83 | 99.87 | 99.5 |
| ORF18 | 100 | 100 |
| ORF131 | 99.93 | 99.26 |
| ORF34 | 99.39 | 99.79 |
| DNA polymerase catalytic subunit | 99.79 | 100 |
| Allo56 | 100 | 100 |
| ORF40 | 100 | 99.46 |
| Allo37 | 99.9 | 99.8 |
| ORF106 | 100 | 100 |
| Membrane protein ORF67 | 100 | 99.83 |
| ORF107 | 100 | 99.9 |
| ORF86 | 99.98 | 100 |
| ORF134 | 97.34 | 97.58 |
| Major capsid protein | 99.97 | 99.97 |
| ORF1 | 99.45 | 96.02 |
| ORF1 | 99.45 | 96.02 |
| ORF45 | 100 | 99.97 |
| Membrane protein ORF125 | 99.94 | 100 |
| ORF30 | 100 | 99.97 |
| ORF110 | 100 | 99.97 |
| ORF120 | 99.97 | 100 |
| Membrane protein ORF108 | 99.77 | 98.58 |
| ORF127 | 99.93 | 99.93 |
| ORF91 | 100 | 99.96 |
| ORF48 | 100 | 99.88 |
| ORF74 | 100 | 100 |
| Ribonucleotide reductase subunit 1 | 100 | 100 |
| ORF19 | 99.1 | 99.89 |
| ORF44 | 100 | 100 |
| Helicase-primase subunit | 100 | 100 |
| Membrane protein ORF109 | 100 | 100 |
| Allo54 | 99.96 | 100 |
| ORF23 | 100 | 99.96 |
| Membrane protein ORF65 | 99.91 | 99.95 |
| ORF31 | 100 | 100 |
| Capsid maturation protease | 99.26 | 100 |
| Membrane protein ORF80 | 99.86 | 99.91 |
| ORF87 | 99.95 | 100 |
| ORF130 | 99.9 | 99.7 |
| ORF111 | 99.95 | 99.95 |
| ORF92 | 100 | 99.94 |
| ORF113 | 99.43 | 99.94 |
| ORF6A | 98.84 | 98.78 |
| ORF6A | 98.84 | 98.78 |
| ORF39 | 100 | 100 |
| ORF20 | 100 | 100 |
| Allo64 | 100 | 100 |
| ORF88 | 99.94 | 99.94 |
| Helicase-primase helicase subunit | 100 | 100 |
| ORF89 | 99.94 | 99.94 |
| ORF28 | 100 | 100 |
| ORF47 | 99.94 | 100 |
| ORF85 | 100 | 98.03 |
| ORF97 | 100 | 100 |
| ORF61 | 100 | 100 |
| ORF81 | 99.93 | 99.93 |
| ORF99 | 99.93 | 100 |
| Membrane protein ORF71 | 99.54 | 98.15 |
| Membrane protein ORF101 | 100 | 100 |
| ORF121 | 100 | 99.92 |
| Membrane protein ORF66 | 100 | 100 |
| ORF38 | 100 | 100 |
| Membrane protein ORF95 | 99.91 | 100 |
| Capsid triplex subunit 1 | 100 | 100 |
| Allo60 | 100 | 100 |
| Capsid triplex subunit 2 | 99.82 | 100 |
| Membrane protein ORF94 | 99.72 | 99.91 |
| ORF54 | 98.88 | 99.72 |
| Membrane protein ORF93 | 100 | 99.9 |
| Ribonucleotide reductase subunit 2 | 100 | 100 |
| Uracil-DNA glycosylase | 100 | 100 |
| ORF112 | 99.76 | 100 |
| Thymidylate synthase | 99.89 | 100 |
| ORF3 | 100 | 100 |
| ORF3 | 100 | 99.89 |
| ORF35 | 100 | 100 |
| ORF16 | 100 | 100 |
| ORF76 | 100 | 99.88 |
| Deoxyguanosine kinase 1 | 100 | 99.88 |
| ORF14 | 99.88 | 100 |
| Membrane protein ORF124 | 100 | 100 |
| ORF68 | 100 | 99.64 |
| ORF13 | 99.88 | 100 |
| ORF53 | 99.75 | 98.89 |
| Membrane protein ORF11 | 99.75 | 100 |
| Membrane protein ORF12 | 100 | 100 |
| Deoxyguanosine kinase 2 | 100 | 100 |
| ORF24 | 100 | 100 |
| ORF4 | 100 | 96.95 |
| ORF4 | 100 | 96.95 |
| Membrane protein ORF50 | 100 | 100 |
| Membrane protein ORF64 | 99.86 | 100 |
| ORF58 | 100 | 99.86 |
| Membrane protein ORF51 | 100 | 100 |
| ORF62 | 100 | 100 |
| ORF92B | 100 | 100 |
| ORF63 | 99.86 | 100 |
| ORF17 | 100 | 100 |
| Membrane protein ORF49 | 100 | 100 |
| Dihydrofolate reductase | 99.85 | 100 |
| ORF105 | 100 | 100 |
| Thymidylate kinase | 100 | 100 |
| ORF103 | 100 | 100 |
| Membrane protein ORF8 | 100 | 100 |
| ORF126 | 100 | 96.7 |
| ORF46 | 100 | 100 |
| Nucleoside diphosphate kinase | 100 | 100 |
| Membrane protein ORF102 | 100 | 99.67 |
| ORF118 | 99.83 | 100 |
| ORF117 | 100 | 100 |
| Guanosine triphosphatase | 99.65 | 100 |
| ORF59 | 100 | 100 |
| ORF41 | 100 | 100 |
| ORF122 | 98.75 | 98.57 |
| ORF114 | 100 | 100 |
| ORF32 | 100 | 100 |
| ORF60 | 99.81 | 100 |
| ORF84 | 100 | 100 |
| Deoxyuridine triphosphatase | 100 | 100 |
| Deoxyuridine triphosphatase | 100 | 99.8 |
| ORF56 | 100 | 100 |
| Interleukin-10 | 100 | 99.8 |
| ORF26 | 100 | 100 |
| ORF43 | 100 | 100 |
| ORF27 | 100 | 100 |
| ORF33 | 100 | 100 |
| Membrane protein ORF92A | 100 | 100 |
| Membrane protein ORF78 | 100 | 100 |
| ORF69 | 100 | 100 |
| ORF73 | 100 | 99.77 |
| ORF70 | 100 | 100 |
| ORF115 | 100 | 100 |
Data availability.
The whole-genome sequence of AngHV-1 isolated from A. rostrata is available at NCBI under GenBank accession number OM936983. The raw data reads for the AngHV-1 genome sequence are available at NCBI under SRA accession number PRJNA883046. Images of diseased eels, AngHV-1 isolated in EK-1 cells, and transmission electron micrographs of AngHV-1 are available from Figshare (https://doi.org/10.6084/m9.figshare.20501703).
ACKNOWLEDGMENT
This study was financially supported by Chonnam National University (grant number 2020-3688). We acknowledge Olga Haenen, Wageningen Bioveterinary Research, Netherlands, for providing the EK-1 cell line required for conducting the present study.
Contributor Information
Sung-Ju Jung, Email: sungju@chonnam.ac.kr.
John J. Dennehy, Queens College CUNY
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Associated Data
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Data Availability Statement
The whole-genome sequence of AngHV-1 isolated from A. rostrata is available at NCBI under GenBank accession number OM936983. The raw data reads for the AngHV-1 genome sequence are available at NCBI under SRA accession number PRJNA883046. Images of diseased eels, AngHV-1 isolated in EK-1 cells, and transmission electron micrographs of AngHV-1 are available from Figshare (https://doi.org/10.6084/m9.figshare.20501703).