Establishment of quantitative PCR detection methods and epidemiological investigation of ranavirus and megalocytivirus infections in hybrid snakehead (Northern Snakehead ♀ × Blotched Snakehead <i>Channa maculata</i> ♂)

OBJECTIVE: In China, the incidence of iridovirus infections in economically valuable fish species is increasing, but there is a paucity of reports documenting ranavirus infections and there are no reports concerning megalocytivirus in hybrid snakehead (Northern Snakehead Channa argus ♀ × Blotched Snakehead C. maculata ♂). METHODS: In this study, the major capsid protein DNA fragments of two iridoviruses were amplified from diseased hybrid snakehead, and two specific primers and TaqMan probes were designed based on the conserved sequences. Using the constructed recombinant plasmid as the standard for real-time fluorescent quantitative PCR, two real-time TaqMan quantitative detection methods for the iridoviruses were established, and the sensitivity, repeatability, and specificity of these two methods were evaluated. RESULTS: The standard curves for both methods exhibited strong linear correlations (R2 > 0.99), with slopes of -3.28 and -3.231, respectively, and the detection limits were 28 and 27 viral genome copies, which were 100 times more sensitive than conventional PCR. Applying these methods to 272 hybrid snakehead, we found an average positive rate of 9.93% (27/272) for ranavirus and 3.31% (9/272) for megalocytivirus. During the breeding stage (parental generation and fry), megalocytivirus was detected at a rate of 2.67% (2/75), while ranavirus was absent. However, ranavirus was present from April to November during the cultivation stage, and megalocytivirus was primarily detected in July and August. Fry up to 10 cm in length were most susceptible to both viruses, but no cross infections were detected. CONCLUSIONS: These findings offer valuable insights into the epidemiology of ranavirus and megalocytivirus in hybrid snakehead.