Immortalization of shrimp lymphoid cells by hybridizing with the continuous cell line Sf9 leading to the development of 'PmLyO-Sf9 '.

Shrimp progressively gets more attention among marine invertebrates from researchers all over the world due to it being a healthy food as well as having economic importance. There were a lot of attempts to develop a continuous cell line from shrimp but none successful. In this context a novel hybrid cell line named 'PmLyO-Sf9' could be developed by fusing shrimp lymphoid organ cells with Sf9 cells after to metabolic blocking of Sf9 cells using puromycin and actinomycin D and effecting the fusion by way of PEG application. The cells are maintained and multiplied in a mixture of SCCM and TNM-FH having osmolality 550 mOsm kg-1 and pH 6.8. Transmission electron microscopy of the hybrid cells revealed the presence of two nuclei during the initial stages and a single nucleus subsequently. The cell line is with shrimp and Sf9 genomic components and shrimp specific protein and is susceptible to WSSV. Shrimp elongation factor, Sf9 beta-actin, shrimp STAT and peroxinectin could be expresses through RT-PCR in the cell line. This is the first successful report of a hybrid cell line with shrimp genomic components and envisaged to be recognized a model system for multitudes of biomedical research in vitro. The cell line is in the National Cell Line Repository of ICAR - National Bureaue of Fish Genetic Resources, Lucknow, India.

'PmLyO-Sf9 - WSSV complex' could be a platform for elucidating the mechanism of viral entry, cellular apoptosis and replication impediments.

White spot syndrome virus (WSSV) is the most devastating pathogen found in shrimp aquaculture. The lack of certified continuous/established cell lines from penaeid shrimp restricts in vitro studies on the viruses to bring out effective prophylactic and therapeutic measures. In this context, a novel hybrid cell line named, PmLyO-Sf9, consisting of shrimp and Sf9 genomes has been established and employed to study WSSV susceptibility and multiplication. The hybrid cells were exposed to the shrimp virus WSSV and cytopathic effects (CPE) such as (a) enlargement of cells, (b) cessation cell division, (c) granulation of cytoplasm, (d) rounding off of cells, shortening and disappearance of tail-like structures and (e) detachment from the flask. Expression of immediate early genes such as ie 1, dnapol, rr1, tk-tmk, and pk 1could be confirmed indicating that viral DNA replication in the PmLyO-Sf9 took place followed by the expression of late genes such as VP-28, VP-26, VP-15 and VP-19. Electron micrograph of WSSV infected cells demonstrated marginated dense zones in the nucleus with clumped chromatin, and the mid zone with virus-like particles. However, neither discrete virus particles nor the culture supernatant having infectivity could be observed suggesting that virions were not getting formed in the cells. This is the first report of the susceptibility of PmLyO-Sf9 to WSSV, and the 'PmLyO-Sf9 - WSSV Complex' formed, defined as the infected status of PmLyO-Sf9 with WSSV, could be of use for unraveling at molecular level the mechanism of viral entry, replication impediments and cellular apoptosis.