Molecular evidence for homologous strains of infectious spleen and kidney necrosis virus (ISKNV) genotype I infecting inland freshwater cultured Asian sea bass (Lates calcarifer) in Thailand

Abstract

Infectious spleen and kidney necrosis virus (ISKNV) is the sh pathogenic virus belonging to the genus Megalocytivirus of the family Iridoviridae. In 2018, disease occurrences (40-50% cumulative mortality) associated with ISKNV infection have been reported in grown-out Asian sea bass (Lates calcarifer) cultured in the inland freshwater system in Thailand. Clinical samples were collected from seven distinct farms located in the eastern and central regions of Thailand. The moribund sh showed various abnormal signs including lethargy, pale gill, darkened body, and skin hemorrhage, while the basophilic hypertrophied cell in gill, liver, and kidney tissue was observed microscopically. ISKNV infection was con rmed in 6 out of 7 farms using virus-speci c semi-nested PCR. MCP and ATPase genes showed 100% identity among virus isolates which also classi ed the virus into ISKNV genotype I clade. Koch's postulates were later con rmed by challenge assay and the mortality of the experimentally infected sh at 21 days post-challenge was 50-90% depending on the challenge dose. The complete genome of two ISKNV isolates, namely KU1 and KU2, was recovered directly from the infected specimens using the shotgun metagenomics approach. The genome length of ISKNV KU1 and KU2 were 111,487 and 111,610 bp, respectively. In comparison to the closely related ISKNV strains, the ISKNV KU1 and KU2 harbored nine unique genes, including caspase recruitment domaincontaining protein potentially involved in apoptosis inhibition. Collectively, this study indicated the homologous ISKNV strains affecting the inland cultured Asian sea bass which emphasized that the ISKNV genotype I should be prioritized for future vaccine research.

A Multiplexed, Tiled PCR Method for Rapid Whole-Genome Sequencing of Infectious Spleen and Kidney Necrosis Virus (ISKNV) in Tilapia

“…Even with advanced methods for identifying intra-host SNVs (iSNVs), the false discovery rate of iSNVs using Oxford Nanopore data alone was ~55% in a rapidly evolving RNA virus, and is expected to be higher in viruses that evolve more slowly, such as ISKNV. In studies where quantification of iSNVs is required, replicated Illumina sequencing libraries per sample, combined with Nanopore data, is recommended for accurate quantification of iSNVs [43][44][45][46][47]. The increased costs and loss of field-based sequencing of this approach would need to be weighed against the likelihood and importance of detecting intra-host variability.…”

Genetic characterization of infectious spleen and kidney necrosis virus in Banggai cardinalfish Pterapogon kauderni identified from eight separate cases between 2000 and 2017

“…Prior to this study, 23 fully sequenced ISKNV genomes were available in the NCBI GenBank database (Ao & Chen, 2006;Do et al, 2004;He et al, 2001;Kawato et al, 2020;Kerddee et al, 2021;Koda et al, 2018Koda et al, , 2019Koda et al, , 2021Kurita et al, 2002;Lu et al, 2005 F I G U R E 2 MAFFT alignment of 31 sequences based on the 137 bp region of the myristylated membrane protein gene (primers excluded). Each sequence name is followed by the associated GenBank accession number except for the eight Banggai cardinalfish cases from this study.…”

“…Prior to this study, 23 fully sequenced ISKNV genomes were available in the NCBI GenBank database (Ao & Chen, 2006; Do et al, 2004; He et al, 2001; Kawato et al, 2020; Kerddee et al, 2021; Koda et al, 2018, 2019, 2021; Kurita et al, 2002; Lu et al, 2005; Matsuyama et al, 2018; Puneeth et al, 2021; Shi et al, 2010; Shiu et al, 2018; Wen & Hong, 2016; Zhang et al, 2013). Eight ISKNV‐C1G genomes have been determined from infected mandarinfish Siniperca chauatsi from China (He et al, 2001), red seabream Pagrus major from Taiwan (Shiu et al, 2018), freshwater angelfish Pterophyllum scalare from Singapore (Kawato et al, 2020), barramundi from Thailand (Kerddee et al, 2021), freshwater albino rainbow sharks Epalzeorhynchos frenatum from the United States (Koda et al, 2021) and Banggai cardinalfish from the United States (BCIV‐2017; this study). The BCIV‐2012 genome from infected Banggai cardinalfish in the United States (this study) represents the earliest ISKNV‐C2G genome to be determined (Table 2).…”