Genome analysis of viral hemorrhagic septicemia virus isolated from Paralichthys olivaceus in ChinaDownload PDF Download PDF ArticleOpen accessPublished: 26 September 2025Ye Xu1,Taishan Tang2,Na Wang3,Chao Ding1,Yi Zhou1,Fengzhi Wang1 &…Hongan Duan1 Scientific Reports volume 15, Article number: 32976 (2025) Cite this articleSubjectsGeneticsMolecular biologyAbstractThis study determined the complete genomic sequence of the viral hemorrhagic septicemia virus (VHSV) strain GY-2307 isolated form farmed paralichthys olivaceus in northeast China. The whole genome length of GY-2307 comprised 11,140 nucleotides (nt), and the overall organization was typical of that observed for all other VHSV strains. The phylogenetic analysis results indicated that the GY-2307 isolate belonged to genotype Ⅳa and significantly related to other Korean and Japanese isolates. The N, P, M, NV and L genes of GY-2307 were compared with the VHSV sequences in GenBank. The results revealed that 315 nt were substituted, 71 of which exhibited amino acid change in the GY-2307 genome. In the G gene, specific nucleotides and amino acid variations of the Chinese isolates of VHSV were observed when compared with 28 isolates from other counties. Of the 23 nucleotide sites that changed, 9 resulted in amino acid substitution. The data further indicated that the genotype Ⅳa VHSV was introduced to and evolved in paralichthys olivaceus farm environments in China.IntroductionViral hemorrhagic septicemia virus (VHSV) is recognized as a major salmonid pathogen that is highly virulent in Western Europe, as well as in farmed paralichthys olivaceus in Asia1. Viral hemorrhagic septicemia (VHS) outbreaks often occur at water temperatures below 15 ℃. Depending on the fish species and size, virus strain, and environmental conditions, VHSV infection can result in a cumulative mortality of 90% in a broad range of susceptible species2. Infection with VHSV has been reported from countries in Europe, North America and North Asia3,4,5. Some countries in these regions have declared freedom from infection with VHSV. The disease has never been reported from the Southern Hemisphere. VHSV is a member of the rhabdoviridae family under the novirhabdovirus genus. As for all rhabdoviridae family members, VHSV is a linear non-segmented and single-stranded negative-sense RNA virus. The VHSV genome has approximately 11,000 nucleotides, and comprises six open reading frames (ORFs) that encode the genes for nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), non-virion protein (NV), and RNA-dependent RNA polymerase (large protein, L)6. The classification of VHSV isolates into four principal genotypes (I-IV) and nine subtypes (Ia-Ie, IVa-IVd) has been established through analysis of G-gene nucleotide sequences, revealing largely unique geographic distributions for each group. VHSV shows varied geographic distributions among its genomic types. Genotype I is widespread. Sublineage Ia contains European freshwater isolates from multiple countries, while Ib is found in marine areas like the Baltic Sea and North Sea. Ic consists of older Danish freshwater isolates, Id has a Norwegian and two Finnish brackish-water isolates, and Ie has a Georgian isolate. Genotype II is limited to the Eastern Gotland Basin in the Baltic Sea, likely due to the area’s unique environment. Genotype III is present in the North Sea, UK and Irish coastal waters, and northern France. The co-occurrence of genotypes I and III in some areas may be linked to fish migration. The North American genotype IV is distinct from European ones. These distributions imply that geographical and ecological factors play a role in VHSV’s spread and evolution. They also suggest complex relationships, with the virus adapting and diversifying in different regions over time, which has significant implications for understanding its epidemiology and controlling its impact on fish populations7.The paralichthys olivaceus rhabdovirus (PORV) was isolated from diseased flounder (Gui et al., 2007) in China, and analysis of the complete genome sequence revealed that the isolate shared a close relationship with VHSV strains from Korea8. Another PORV isolate was reported in farmed Kareius bicoloratus from the Bohai Sea (Sun et al., 2009). In this study, a novel VHSV strain was isolated and identified in healthy farmed paralichthys olivaceus for surveillance purposes farmed near the Yellow Sea in 2023. This strain was designated as VHSV GY-2307. To facilitate the detailed comparison of the sequence characteristics of VHSV GY-2307 strain with other phylogenetically related VHSV strains isolated from around the world, the complete VHSV GY-2307 genomic sequences are described.With the expansion of marine fish cultivation in China’s central coastal regions, the aquaculture industry confronts a substantial challenge posed by viral infections, with the VHSV being a primary concern. To address this issue, the present study conducts a comprehensive whole-genome analysis of VHSV. The goal is to decipher its genetic characteristics, thereby facilitating the development of highly accurate diagnostic methods and robust control strategies against VHSV.Materials and methodsCell line and virus strainThe VHSV GY-2307 strain was isolated from central coastal China9. The Epithelioma Papulosum Cyprini (EPC) cell line sourced from the Danish National Veterinary Institute was used to isolate and propagate the VHSV strains. Cells were maintained in MEM (Gibco) supplemented with 10% fetal bovine serum (FBS) for routine growth. For viral replication assays, the medium was adjusted to 2% FBS, and all incubations were performed at 15 °C. Cytopathic effect (CPE), manifesting as cellular necrosis and functional disruption that highlight VHSV’s destructive impact on host cells, was observed in inoculated EPC cells within 2 to 6 days following infection with the virus. GY-2307 was passaged three times prior to sequencing.Library Preparation and high-throughput DNA sequencingRNA was extracted from virus-infected EPCs using MagMAX CORE (THERMO) following the manufacture’s instructions. Next generation sequencing library preparations were constructed using VAHTS® Universal V8 RNA-seq Library Prep Kit (Vazyme) for Illumina following the manufacturer’s protocol. 200 µg cDNA was randomly fragmented in an S220 ultra sonicator (Covaris) to average size of 300–350 bp. Briefly, the fragments were treated with End Prep Enzyme Mix (Vazyme) for end repairing, 5’ phosphorylation and 3’ adenylated, to add adaptors to both ends. Size selection of adaptor-ligated RNA was then performed by RNA cleanup beads. Each sample was then amplified by RT-PCR for 8 cycles using P5 and P7 primers, with both primers carrying sequences which can anneal with the flow cell to perform bridge PCR and P7 primer carrying a six-base index allowing for multiplexing. The PCR products were cleaned up and validated using an Agilent 2100 Bioanalyzer (Agilent Technologies) and nanoQC (https://github.com/wdecoster/nanoQC). The qualified libraries were sequenced pair end PE150 on the Illumina Novaseq System (Illumina).Assembly and annotationAfter quality control of the sequencing data, we used Velvet 1.2.10 (http://tracker.debian.org/pkg/velvet), A5-Miseq 20,160,825 (http://sourceforge.net/projects/ngopt), and SPAdes 3.15.4 (https://github.com/ablab/spades/releases) for assembly. Among these, the assembly results of SPAdes were selected as the optimal ones. Partial assembly results were gap filled with SSPACE 3.0 (http://www.baseclear.com/genomics-services/software-tools/) and GapFiller 1.10 (https://www.baseclear.com/genomics/bioinformatics/base-tools/gap-filler). Pilon 1.22 (https://github.com/broadinstitute/pilon/releases) was subsequently employed for assembly error correction. Prodigal 2.6.3 (https://github.com/hyattpd/Prodigal/wiki/introduction) was used for coding gene prediction, MISA 2.1 (http://webblast.ipk-gatersleben.de/misa/) was utilized for SSR analysis. The coding genes were annotated with National Center for Biotechnology Information (NCBI) nr database by BLAST. The full-length VHSV genomic nucleotide sequence of strain GY-2307 was deposited in the GenBank database under accession number PQ304143.Sequence and phylogenetic analysesDNAStar Lasergene 18 (https://www.dnastar.com/) was used to translate and conduct multiple aligments of the sequences, as well as to determine the percentage identities and similarity scores. To analyze the percentage identities and similarity scores, both nuleotide and amino acid sequence were aligned using the default options in MegAlign Pro. Phylogenetic analysis of the G gene, as well as of the whole genome, were then performed by the maximum likelihood (ML) method with 1,000 bootstrap replicates and visualized in MEGA 11 (https://www.megasoftware.net/). Table 1 presents all VHSV sequences used for analysis in this study.Table 1 Summary of VHSV strains obtained from GenBank and used in this study.Full size tableResultsGenome organization of the VHSV strain GY-2307The entire GY-2307 genome consisted of 11,140 nucleotides (nt) in length, and includes the 3’leader, 6 ORFs, and 5’ trailer. The GY-2307 genome organization is summarized as 3’-leader-N-P-M-G-NV-L-5’ trailer, which is similar to that of the published VHSV genomes. Table 2 shows the positions of the different genes and open reading frame (ORF). Between the ORF are nontranslated regions that include highly conserved sequences encompassing the polyadenylation signal for one gene, and the putative transcription initiation site of the next gene. An alignment of the conserved nontranslated sequences is shown in Table 3. The intergenic regions vary in length, from 73 nt between the G and NV ORF, to 126 nt between the NV and L ORF. This is consistent with what has been found for VHS virus isolates in other genotypes10. The polyadeylation signal is also present after the ORF of the L protein (25nt downstream), but the sequence (aga uug aaa aaa a) is slightly different from that found in the intergenic regions.Table 2 Genomic features and predicted proteins of the PQ304143 strain.Full size tableTable 3 Comparison of the conserved sequence parts of the intergenic regions within the VHS virus isolate, PQ304143 genome.Full size tableHomology and phylogenetic analysisThe complete GY-2307 genome was compared with that of PORV, VHSVLB2018, KJ2008, JF00Ehi1, and the nucleotide identities of which were 98.56%, 97.75%,98.39% and 98.37% respectively. Similarly, the complete GY-2307 genome sequence was also compared with different genotype of VHSV, and exhibited 85.96%, 85.17%, 85.83%, 86.01%, 85.03% and 95.3% identity with VHSV genotype I, Ia, Ib, II, III and IVb (Table 4).The results of phylogenetic analysis of the 29 VHSV full-length G genes, including GY-2307, evidently indicated that the VHSV strain GY-2307 belonged to the genotype IVa. The GY−2307 G gene was 98.10% and 96.06% homologous with other two Chinese strains of VHSV and 96.98%−98.23% homologous with 9 Japan/South Korea VHSV strains under the genotype IVa group (Table 4). The 29 VHSV full-length N gene sequence were analyzed, and the phylogenetic tree also showed that the GY−2307 and other two Chinese isolates belonged to the genotype IVa group.Table 4 Percent (%) nucleotide and deduced amino acid identity of PQ304143 with other VHSV strains and other rhabdovirus species.Full size tableBased on the full-length G gene sequences, 29 isolates including four genotypes were analyzed to evaluate the genetic diversity. Fifty nucleotide variations were observed and thirty-two led to amino acid substitution.Upon comparison of all six genes, 315 nucleotide variations in the GY−2307 genome were noted, 71 of which led to amino acid substitution (Table 5).The phylogenetic tree analyses of the sequences of the N, M, P, NV and L genes of various VHSV strains are show in Fig. 1. The results of phylogenetic analysis of the N gene showed that GY−2307 clustered with the Chinese PORV and maintained 99.19% nucleotide identity (Table 4). The succeeding nearest neighbor of GY−2307 was FYeosu05 and KJ2008 which had 98.65% and 98.56% nucleotide identity for the N gene (Fig. 1; Table 4). Among the five genes analyzed, GY−2307 exhibited the highest nucleotide sequence identity with KJ2008 in the G gene (98.29%), with JF00Ehi1 in the P gene (98.35%), and with JF−09 in the M gene (98.18%). Additionally, GY−2307 showed significant nucleotide identity with PORV and FYeosou5 in the L gene (98.47%) and with PORV in the NV gene (98.92%). These results demonstrate a high degree of genetic similarity between GY−2307 and the reference strains across multiple gene segments. VHSV belongs to Novirhabdovirus in Gammarhabdovirinae. Phylogenetic analysis (Fig. 2) shows it clusters with other Novirhabdovirus members like IHNV, with close relationships (small branch lengths, e.g., ≤ 0.102 among VHSV strains). Novirhabdovirus (Gammarhabdovirinae) and Sprivivirus (Alpharhabdovirinae) form distinct branches with independent evolution. VHSV has the genus-specific NV gene (between G and L) to evade host interferon, infects teleosts causing severe hemorrhagic disease via water and potential egg transmission, differing from Sprivivirus (lacks NV, with simpler genomes of five structural genes).Fig. 1Phylogenetic tree analysis of sequences of Glycoprotein (A), Nucleoprotein (B), Phosphoprotein (C), Matrix protein (D), Non-virion (E), and Polymerase protein(F) of various VHSV strains. VHSV GY-2307 strain is marked with a black star. Phylogenetic tree analysis was conducted by using the neighbor-joining method with 1000 bootstrap replications. The scale at the bottom indicates the number of substitution events, whereas bootstrap confidence values are shown at the branch nodes.Full size imageFig. 2The phylogenetic tree of fish rhabdoviruses displays genetic relationships and classifications. Branch values represent genetic distances. Gammarhabdovirinae includes all annotated viruses, while Alpharhabdovirinae is a divergent outgroup. Sprivivirus and Novirhabdovirus form parallel branches with independent evolution, and relationships within Novirhabdovirus are closer. VHSV GY-2307 strain is marked with a black star.Full size imageAnalysis of six genes (N, P, M, G, NV and L genes)The start of the N gene is localized downstream of the conserved transcription start sequence “uugaa” (positions 108–112). The transcription stop sequence, which contains the putative polyadenylation signal (AGAAAAAAA), is located at nucleotide 1407. The results of the phylogenetic analysis of the N protein showed that GY-2307 is clustered with the Chinese VHSV strains PORV and VHSVLB2018, with 99.19% and 95.39%. The nucleotide (and amino acid) identity of N gene had 87.03–88.11% (89.70-90.79%), 87.48% (92.14%), 87.93–88.38% (90.51–91.33%) and 87.75–99.19% (90.51–99.19%) identity with the corresponding sequences of the I, II, III and IV genotype isolates, respectively(Table 4). Thirty-five nucleotide variations were detected in the N gene between the GY-2307 and twenty-eight other published VHSV strains, thirteen of which led to amino acid substitution (Table 5).The phosphoprotein nucleotide sequence of GY-2307 has the highest sequence identity with JF00Ehi1 (98.35%) and PORV (98.05%). The P gene nucleotide sequence identity with other genotype isolates is shown in Table 4. The 20 nt substitutions were identified in P gene of GY-2307 compared with other twenty-eight VHSV isolates, 3 of these nucleotide sites led to amino acid substitution, as shown in Table 5.Table 5 Ratio of synonymous to non-synonymous substitutions in coding regions of PQ304143.Full size tableThe GY-2307 M gene homology has more than 98.18% identity with other VHSV strains. The M nucleotide (and amino acid) sequence has 85.97–87.13% (94.03–95.52%), 88.94% (93.53%), 85.64–87.13% (94.53%) and 86.96–98.18% (94.53–98.51%) identity with the corresponding sequences of the four genotypes of VHSV isolates, respectively (Table 4). In the M gene, 21 nt replacements were observed between GY-2307 and other VHSV isolates, and 4 of these nucleotide substitutions led to amino acid change (Table 5).In the G gene, the GY-2307 nucleotide (and amino acid) sequences have 85.43–98.36% (91.52–94.28%), 84.78% (90.14%), 84.51–85.63% (91.52–92.11%) and 85.37–98.29% (91.31–97.83%) identity with the corresponding sequences of the I, II, III and IV genotype isolates, respectively (Table 4). The results indicate that the regions in the GY-2307 amino acid sequences between positions 55–66, 88–93, 137–155, 175–182, 184–209, 211–234, 243–269, 271–309, 313–322, 324–329, 331–344, and 350–357 nt are highly conserved compared with the G protein of 28 VHSV strains form different regions of the world (data not shown). In contrast, the regions between amino acids 42–49, 117–127, and 364–375 show more genetically diverse than any other G protein parts.The GY-2307 NV gene exhibited 72.63–98.92% identity with the other VHSV strains. The nucleotide (and amino acid) sequences of the NV gene have 74.53–77.78% (73.91–74.78%), 77.78% (71.30%), 72.63–76.15% (73.04–73.91%), and 76.15–98.92% (73.04–100%)identity with the corresponding sequences of the I, II, III and IV genotype isolates, respectively (Table 4). A comparison of the NV gene sequences of GY-2307 with 28 published sequences showed eighteen nucleotide changes that led to six amino acid changes (Table 5).The nucleotide (and amino acid) sequence of L gene had 85.71–98.47% (95.16–99.24%) identity with the corresponding sequences of other VHSV isolates. The identity of L gene and protein was 85.71–86.48% (95.16–96.12%), 86.77% (95.61%), 86.28–86.72% (95.77–96.07%), and 86.15–98.47% (95.82–99.24%) compared with the four genotypes of VHSV sequences (Table 4). A comparison of the L gene sequence of the GY-2307 isolate with the other 28 VHSV sequences exhibited 171 nucleotide changes, which led to 27 aa changes, as shown in Table 5.DiscussionVHSV has caused significant economic losses in the global aquaculture industry. Outbreaks of VHSV not only lead to mass mortalities but also trigger bans on fish exports in affected regions. Understanding the VHSV genome is crucial for developing cost-effective prevention and treatment measures, safeguarding the economic viability of the aquaculture industry. Phylogenetic analysis of VHSV glycoprotein (G) gene reveals European freshwater rainbow trout isolates and Baltic Sea/Skagerrak/Kattegat/North Sea/English Channel marine isolates share a recent common ancestor. European freshwater lineage emerged about 70 years ago (matching 1950s Danish trout farm outbreaks), likely derived from marine reservoirs7. European and North American marine isolates diverged about 500 years ago. Freshwater isolates exhibit 2.5 times faster codon substitution rates, reflecting aquaculture adaptation pressures. Data confirm marine environment as original VHSV reservoir, with multiple independent host range expansions enabling rainbow trout infection, sustaining aquaculture threat11. VHSV infections were not found in Asian countries until 1996, when VHSV was first isolated in Japan from cultured Japanese flounder Paralichthys olivaceus12. Phylogenetic analysis indicated that most of Asian VHSV belong to the genotype IVa group, suggesting that they originated from a common ancestral virus13. Isolates of VHSV have been analyzed at the sequence level and four genotypes of the virus appear to be distributed geographically, rather than by host or year of isolation14. As of now, only three cases of VHSV infection have been reported in China, involving Paralichthys olivaceus in 2007, Platichthys stellatus in 2009, and Micropterus salmoides in 201915,16,17. In this study, a novel VHSV strain was isolated and identified in healthy farmed paralichthys olivaceus for surveillance purposes farmed near the Yellow Sea in 2023. The complete genomic sequence analysis of the Chinese isolates of VHSV is necessary to understand the VHSV evolution in China and to develop Chinese VHSV vaccines. However, only three complete genomic sequences of Chinese isolates are available in Genbank, including PORV, VHSVLB2018, and GY-2307. The three isolates were sourced from Northeast, Southeast and Central area of China respectively. Their geographical distribution and isolation times were different. Therefore, they are likely different isolates.All of the six genes phlyogenetic analysis showed that GY-2307 belonged to the Asian genotype IVa, together with the other Chinese isolates PORV and VHSVLB2018. The N, L, and NV gene phylogenentic analysis demonstrated that the GY-2307 were most closely related with Chinese isolates PORV. The G, M, and P genes were most closely related with KJ2008 (South Korea), JF-09 (Japan), and JF00Ehil (Japan), respectively. Sequence comparisons of genotype IV isolates from North America had tended to show a very low level of genetic diversity, indicating an older, well-established host-pathogen relationship18. The high genetic identity of GY-2307 with other genotype IVa isolates confirmed this relationship.In recent years, the detection of VHSV in previously unaffected geographical regions has raised concerns about its potential for global dissemination. Multiple factors, including climate change, the burgeoning international trade of live fish, and the expansion of aquaculture operations, have contributed to the enhanced spread of VHSV19. VHSV encompasses various strains, each demonstrating distinct levels of virulence and host ranges20. Understanding the molecular basis underlying the emergence and spread of novel VHSV strains is crucial. Whole-genome analysis serves as a powerful tool to identify the genetic determinants associated with the emergence and dispersal of new VHSV strains.Data availabilitySequence data supporting this study have been deposited in GenBank under the accession number PQ304143.ReferencesOgut, H. & Altuntas, C. Survey of viral haemorrhagic septicaemia virus in wild fishes in the southeastern black sea. Dis. Aquat. Org. 109 (2), 99–106 (2014).Article CAS Google Scholar Olesen, N. J., Garver, K. & Kim, H. J. CHAPTER 2.3.10: infection with viral haemorrhagic septicaemia virus. In WOAH Manual of Diagnostic Tests for Aquatic Animals (2025).Al-Hussinee, L. & Lumsden, J. Detection of VHSV IVb within the gonads of great lakes fish using in situ hybridization. Dis. Aquat. Org. 95 (1), 81–86 (2011).Article CAS Google Scholar Emmenegger, E. J., Moon, C. H., Hershberger, P. K. & Kurath, G. Virulence of viral hemorrhagic septicemia virus (VHSV) genotypes ia, iva, ivb, and IVc in five fish species. Dis. 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Organ. 66 (2), 145–151 (2005).Article PubMed Google Scholar Download referencesAcknowledgementsWe thank Mingxu Shao and Yiwu Ma for collecting samples from the fish farm where the GY-2307 isolate was found.FundingThis study was supported by the Research Project of the General Administration of Customs (No. 2024HK025) and the Nanjing Customs Research Project (No. 2025KJ04).Author informationAuthors and AffiliationsComprehensive Technology Center, Lianyungang Customs, Lianyungang, 222000, Jiangsu, ChinaYe Xu, Chao Ding, Yi Zhou, Fengzhi Wang & Hongan DuanAnimal, Plant and Food Inspection Center, Nanjing Customs District, Nanjing, 210019, Jiangsu, ChinaTaishan TangChinese Academy of Quality and Inspection &Testing, Beijing, 100176, ChinaNa WangAuthorsYe XuView author publicationsSearch author on:PubMed Google ScholarTaishan TangView author publicationsSearch author on:PubMed Google ScholarNa WangView author publicationsSearch author on:PubMed Google ScholarChao DingView author publicationsSearch author on:PubMed Google ScholarYi ZhouView author publicationsSearch author on:PubMed Google ScholarFengzhi WangView author publicationsSearch author on:PubMed Google ScholarHongan DuanView author publicationsSearch author on:PubMed Google ScholarContributionsXY and DHA contributed to conceptualization, funding acquisition, writing original draft, review and editing, methodology, supervision, resources and validation. TTS and WN performed investigation and formal analysis. DC, ZY and WFZ analyzed the data. All authors contributed to the article and approved the submitted version.Corresponding authorCorrespondence to Hongan Duan.Ethics declarationsCompeting interestsThe authors declare no competing interests.Ethics statementAll experiments involving live vertebrates were approved by the Comprehensive Technology Center, Lianyungang Customs (Approval No. 2023-001). The study was conducted in strict accordance with the ARRIVE 2.0 guidelines. 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