Molecular identification and Characterization of Viral Particles Present In a Shrimp Pond: A Metagenomic Approach

Abstract- Viruses are considered as very significant biological entities on the global ecosystem. Viruses control so many factors like bacterial growth, biogeochemical cycles as well as the nutrient cycles. Though many investigations have focused on the significance of viruses on the aquatic ecosystem, the information regarding their diversity, relationships andgenes in an aquaculture pond is still scarce.The present study was mainly concentrated on exploring the viral diversity present in shrimp farms employingmetagenomic approach. In the present study two samples were collected; one the water sample and another sediment sample from two different shrimp cultured ponds located in Cheppanam and Aroor areas respectively. Sediment sample was processed by filtration flocculation resuspension (FFR) method.Viral particles were purified and concentrated employing several stages of filtration and chemical flocculation. The presence of viral particles was confirmed at each step of purification and concentration employingepifluorescence microscopy using SYBR Gold. Presence of numerous viral particles confirmed the concentration and purity of the viral filtrate. Finally, viral nucleic acid was extracted using QIAampMinElute Virus Spin kitand the viral nucleic acids were subjected to Next Generation Sequencing at Genotypic Technology, Bangalore. The result from Next generation sequencing lab showed the presence of 75 % Synechococcusphages and remaining 25 % with different types of phages.Interestingly, no pathogenic viruses could be amplified from the shrimp farm, though the sample was collected from a farm that recorded a recent WSSV outbreak.

The viralnucleic acid was quantified and qualified by using Qubit 2.0 fluorometer. About 262 ug /ml DNA with high abundance of RNA could be obtained from the sample. PCR amplification was performed for both the viral nucleic acids with specially designed 37 DNA virus specifc primers and 23 RNA virus specific primers as well as eightsignificant aquacultureviral pathogens primers. Four DNA viruses could be amplified including two cyanophages, one iridophage and one phycodnavirus. The amplified products were sequenced at Scigenom, Kochi using both forward and reverse primers Sequence analysis was performed employing GenTool, BioEdit, ExPASy and MEGA 5.0 bioinformatic tools and the molecular characterization and phylogenetic relationship among those viruses were established.The present study shows that cyanophages, irridoviruses and phycodnaviruses are the abundant viral particles present in particle shrimp ponds. However, no pathogenic viruses could be amplified from the shrimp farm by this method also, though the samples were collected from farms that recorded a recent WSSV outbreak.

This is the first study conducted in shrimp aquaculture ponds located in Kerala and perhaps in India.Viral metagenomic data from aquaculture ponds will definitely give a better understandingof the common viral communities present in an aquaculture system. It will definitely pave way to identify the scenarios that cause disease outbreaks as well as rapid identification of potential pathogenic viruses before they can outbreak as a devastating agent.