Nowadays, most of the Asian sea bass culture in Thailand has been conducted using an inland freshwater culture system, in response to the continual increase of consumer demand. According to our observation (unpublished data), the former cage culture system in the estuary has been considered by Asian sea bass farmers as a risky practice due to the unmanageable biosecurity and water qualities. The major cultivation area of grown-out Asian sea bass in Thailand, to date, is in the central and eastern regions due to the availability of fingerling supplies and abundant water resources. Moreover, the Asian sea bass farming area is expected to be on the rise since this fish species has been appraised, by the governmental bodies and private sectors in Thailand, as valuable fisheries products for future exportation. However, intensive farming would induce stress responses due to the overstocking and overfeeding which resulted in the increase disease vulnerability, possess a risk for severe disease outbreaks. In this study, ISKNV was identified from diseased Asian sea bass cultured in the inland freshwater system. The most common histopathological lesions from ISKNV-infected Asian sea bass collected in this study, i.e., severe necrosis and the appearance of basophilic inclusion bodies in gills, liver, and kidney, was consistent with the pathognomonic lesions of megalocytivirus infection (hypertrophy/megalocytosis in gill and liver) reported previously [32, 45]. In this study, the histopathological manifestations in the spleen, kidney, and liver also suggested the viral tropisms in these hematopoietic organs which, to some extent, may result in immune incompetent/suppression and increased susceptibility to opportunistic infections. Co-infection between Flavobacterium columnare and SDDV, another member of the genus Megalocytivirus, in grown-out Asian sea bass has been reported in our previous investigation . Considering that ISKNV and the potential bacterial pathogens, such as Aeromonas sp., Streptococcus sp., and Flavobacterium sp., was co-identified from 3 out of 7 disease incidences in this study, it is feasible that simultaneous infection is relatively common among natural disease occurrences in Asian sea bass. Simultaneous infection, also called a concurrent infection, can be found often in farmed fish and could outweigh a single infection . In our case, we speculated that simultaneous infection by ISKNV and other bacteria could influence the large diversity of clinical appearances, which range from inapparent signs to severe symptoms, e.g., extensive hemorrhage. In this study, it remains elusive whether ISKNV or bacterial pathogen is the primary pathogen, and the clear understanding of pathogens interrelation in vivo required further investigation.
Apart from Asian sea bass, ISKNV has been reported in farmed Nile tilapia and ornamental fish in Thailand as well [1, 10, 47]. The clinical appearances of these ISKNV-infected fishes also similar to those observed in this study. The ISKNV from ornamental fish were genetically classified as genotype I , based on the MCP gene sequence, similar to the ISKNV of this study. In the previous studies, the area of disease outbreaks was also in the same region (central Thailand) as the ISKNV-positive Asian sea bass samples collected in this study. Therefore, the cross-species transmission between these freshwater farmed fishes, although not yet been documented officially, should be aware and the biosecurity-oriented management should be of concern (particularly in the area with outbreak history) since the sharing of water resources among farms is hardly evitable.
ISKNV was also identified from various marine fish species in other Southeast Asian countries including Indonesia, Vietnam, Malaysia, and Singapore [11, 23, 39, 46]. Most of the ISKNV found in these countries were genotype I, similar to our study, which indicated the widespread of this ISKNV genotype in Southeast Asia. According to the original article describing ISKNV genotyping , ISKNV can be classified into three genotypes (I, II, and III) based on the diversity in the MCP gene. These genotypes later assigned into three clusters comprised of RSIV, ISKNV, and TRBIV groups under the same virus species, as described by the International Committee on Taxonomy of Viruses (ICTV, https://talk.ictvonline.org/ictv-reports/ictv_online_report/dsdna-viruses/w/iridoviridae/615/genus-megalocytivirus). To date, the ISKNV genotype II was reported from orbiculate batfish (Platax orbicularis), Banggai cardinalfish (Pterapogon kauderni), and marble goby (Oxyeleotris marmorata) in Indonesia, Japan, USA, and China [32, 43, 49], while Asian sea bass mortality associated with ISKNV genotype II infection was found in Southern China and Vietnam [11, 50]. According to the phylogenetic analysis conducted in this study, the genetic diversity among the current ISKNV isolates was rather small as all the samples are classified as genotype I and shared 100% sequence homology in both MCP and ATPase genes. MCP and ATPase, as well as DNA polymerase, genes were generally used to determine the genetic relationship of megalocytivirus due to their evolutionary conservation nature [30, 32]. However, the relationship between the genetically similar strains cannot be inferred, at least for the current collection of virus isolates, relying on these conserved genes alone. To date, the in-depth epidemiological regarding ISKNV genotype distributed in Thailand and neighboring countries in Southeast Asia is scarce. Thus, the development of an efficacious ‘regional’ vaccine against ISKNV distributed in Southeast Asia is hardly accomplished unless sufficient epidemiological insight is established. For further investigation, other molecular markers, such as four ankyrin repeat domains , offering higher discriminatory power would be added to the phylogenetic comparisons which will determine the intra-genotype diversity of ISKNV on a finer scale.
Isolation of ISKNV from the infected Asian sea bass specimens using GF cell line was successful in this study. Application of GF cell for the propagation of ISKNV simultaneously with nervous necrosis virus has been reported in our previous study . However, the result of this study was slightly inconsistent with another publication in terms of CPE on-set . In the former study, CPE cannot be observed clearly until 14 dpi, whereas 5 dpi on-set was found in this study and clearly discerned at 7 dpi. The variation in CPE development possibly due to the difference in the initial inoculation dose since this study intentionally selected a specimen with potentially high viral titer (indicated by one-tubed snPCR). Recently, the alternative cell line GS-1 originated from orange-spotted grouper (Epinephelus coioides) fibroblast was reported to be ISKNV susceptible and allowed the virus to reach 105.2 TCID50/ml titer within 7 days, though direct comparison of ISKNV replication kinetic to GF cell has not been described yet .
The pathogenicity assay conducted in this study was able to fulfill Koch’s postulates and proved that ISKNV was pathogenic to freshwater reared Asian sea bass. The result suggested that the pathogenicity of ISKNV and on-set of mortality depend on the infection dose (Fig. 6). The cumulative mortalities were similar to the previous investigations (90, 77, and 85.89%), as well as the on-set of mortalities at 5–9 dpc [48, 50]. It should be mentioned that the size of the experimental animal used in this study (juvenile, 22 g weight) was different from the previous report (fingerling, 3.5 g weight) , which suggested that ISKNV was able to cause severe mortality in Asian sea bass at various life stages. Pertaining to clinical appearances, it is worth mentioning that the experimental animals exhibited only the typical darkened body and pale gill, in agreement with those described in the recent study . On the other hand, the obvious external lesions such as scale loss, muscle necrosis, and hemorrhage were observed only from the naturally infected fish. The difference in the clinical manifestations could be due to bacterial pathogens co-infected with ISKNV in natural cases (Table 2). This emphasized that diagnosis of field outbreaks should be performed cautiously and multiple approaches, including pathogen isolation, PCR, and histopathology, should be applied when possible, to get a comprehensive understanding of the disease scenario.
The metagenomic shotgun sequence was able to unveil the draft genome sequence of SDDV directly from the infected specimen, without the need of virus culture, in our recent investigation . In this study, the same analytical approach has been implemented and the complete genomes of ISKNV KU1 and KU2 were reconstructed with the acceptable coverage depth at 47–56×. The genome-scale phylogenetic network (Fig. 3C) showed the reticulation pattern within ISKNV and RSIV groups which implied the possible genetic recombination between the members of these groups. In fact, one of the ISKNV group members, RSIV-Ku, harbored 7% of genome contents similar to the GSIV-K1 (belonging to RSIV group) which indicated that this strain was ISKNV/RSIV recombinant . Herein, possible recombination between ISKNV KU1/KU2 and other megalocytiviruses was screened using the RDP4 program  but no evidence of recombination was found in their genomes (data not shown). Orthology analysis showed a surprisingly high number of unassigned orthogroups (unique genes) among the genomes of ISKNN KU1/KU2, RSIV-Ku, and reference strain. It is predictable that the strain RSIV-Ku, a natural recombinant virus, may possess numerous unique genes since its genome carried a 7.8-kb-long region similar to RSIV genotype II rather than ISKNV . In the case of the ISKNV reference strain, 18 genes were unassigned to any orthologous groups, though the core genome similarity comparing to the ISKNV KU1/KU2 was as high as 99.98%. This could be explained by the difference in genome annotation methods used for CDS prediction in the ISKNV reference strain and KU1/KU2. Protein-coding sequences of the ISKNV reference strain were identified by querying sequences through a protein domain database , whereas the unsupervised machine learning algorithm-based program (Prokka) was employed in the case of ISKNV KU1/KU2. Among the non-orthologous genes presented in ISKNV KU1/KU2, the caspase recruitment domain-containing (CARD) protein was identified. This protein was highly similar to those of Angelfish iridovirus AFIV-16, also belongs to ISKNV genotype I , isolated from angelfish Pterophyllum scalare in Southeast Asia. CARDs are well-known interaction motifs involved in inflammation and apoptosis regulation . CARD protein has been demonstrated in vitro for apoptosis inhibitory effects in Grouper iridovirus, a member of the genus Ranavirus of the family Iridoviridae . Nevertheless, the role of CARD protein on the molecular pathogenesis of ISKNV, whether it is involved in apoptosis inhibition, remains to be further elucidated.
Summarily, the homologous strains of ISKNV genotype I have been identified as a causative agent of mass mortalities in freshwater cultured Asian sea bass in the eastern and central Thailand by a combination of histopathology, molecular analyses, and pathogenicity assay. The complete genome of two ISKNV isolates, KU1 and KU2, was obtained using a metagenomics approach. The genome information, as well as the virus archive collected in this study, could be useful for evolutionary analysis and selection of potential genotype I vaccine candidates for the sustainable prevention of ISKNV outbreaks in the future.