Infectious cell culture system for concurrent propagation and purification of Megalocytivirus ISKNV and nervous necrosis virus from Asian Sea bass (Lates calcarifer)

Abstract

Megalocytivirus ISKNV and nervous necrosis virus (NNV) are two major viral pathogens affecting marine fish farms in Asia Pacific region. The present study reports an unexpected discovery of dual infections of both viruses in a single Asian sea bass (Lates calcarifer) farm experiencing ~50% cumulative fish mortality. A commercial Grunt Fin (GF) cell line was able to propagate concurrently two viruses from the clinical samples. Each virus was successfully purified from the co-infected cell culture, and its morphology was visualized by transmission electron microscopy (TEM). Subsequently, two viruses were identified as Megalocytivirus ISKNV and NNV from specific PCR assays using the extracted nucleic acids and sequencing of amplified PCR products. Approximately 104 and 103 copies of Megalocytivirus ISKNV and NNV were estimated in 200 ng DNA and RNA extracted from the co-infected cells. NNV propagation in GF cells was specifically confirmed using immunofluorescence microscopy. This is the first report for capability of NNV to propagate in the commercial GF cell line. This investigation opens up a promising in vitro model for studying concurrent infections of Megalocytivirus ISKNV and NNV and suggests a possible platform for concurrent production of Megalocytivirus ISKNV and NNV for bivalent vaccine.