Molecular alterations induced by viral pathogen in crustacean cells

Joseph, Jeswin (2012) Molecular alterations induced by viral pathogen in crustacean cells. Doctoral thesis, Central Marine Fisheries Research Institute.

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    Abstract

    The most common and devastating disease affecting the cultured shrimp especially Penaeus monodon is caused by the white spot syndrome virus (WSSV), which has spread throughout the world mainly through different species of crustaceans carrying the virus. Although considerable progress has been made in the molecular characterisation of WSSV, little information is available on shrimp genes which are involved in the defence and progression of WSSV within the host. Similarly, knowledge of the virus-host cell interaction could reveal molecular pathways exploited by the virus, and targets that could be pursued. Quantitative real time PCR method was used to estimate the rate of viral progression in different tissues of P. monodon at 6, 12, 24, 36 and 48 hpi (hours post infection) following experimental injection with a known copy number of virus in the abdominal muscle. At 12 hpi the viral load was highest in haemocytes followed by pleopod, muscle and gills whereas at 48 hpi the gills, the main target of WSSV, showed the highest viral load followed by pleopod, muscle and haemocytes. Viral copy number in the haemocytes was the lowest beyond 12 hpi indicating a remarkable decrease in the rate of viral replication in haemocytes compared to other tissues. The viral load in haemocytes though, increased again beyond 36 hpi it never surpassed the load in the other tissues. This might be due to the haemocytes infiltrating back from the infected tissues carrying the virus back into circulation leading to increase in the viral load in the haemolymph. To develop a better understanding of shrimp antiviral defences and possibly develop new control measures for WSSV, differential expression of immune related genes in haemocytes following WSSV infection were measured. Transcriptomic approach involving synthesis of cDNA from RNA isolated from both the control and iv experimentally infected shrimps were carried out to study the relative expression of immune gene through semi-quantitative PCR. The specific primers targeted to amplify the immune genes were designed from the published sequence data using suitable softwares. Optimised protocol was used for PCR amplification of the cDNAs of the target genes. The study revealed differential expression of immune related genes encoding Clectin, lysozyme, prophenoloxidase, penaeidin, crustin and haemocyanin. The survivability of animals and the elevated level of immune gene expression were found to be directly linked. Animals surviving beyond 3½ days showed higher expression levels of the genes encoding lysozyme, C-lectin, penaeidin, prophenoloxidase-1 and prophenoloxidase-2 than those animals which died before 2½ days. This is indicative of genetic basis of individual variations in disease resistance. It also suggests that the gene expression level quantified through semi-quantitative PCR can be used as a biomarker for selecting broodstocks for better resistance to WSSV infection, thus promising a role in the fight against WSSV along with other management measures. To understand the mechanism of viral infection, it is very important to study how the viral infection affects the host’s protein expression. After the experimental infection with the WSSV, protein profiles of various tissues like gills, hepatopancreas and muscle tissues were studied by SDS PAGE at different time points and compared with the controls. In addition, the protein profiles of different subcellular organelles were also analyzed by SDS PAGE.

    Item Type: Thesis (Doctoral)
    Uncontrolled Keywords: Molecular alterations; viral pathogen; crustacean cells
    Subjects: Fish Biotechnology
    Theses
    Divisions: CMFRI-Kochi > Marine Biotechnology
    Subject Area > CMFRI > CMFRI-Kochi > Marine Biotechnology
    CMFRI-Kochi > Marine Biotechnology
    Subject Area > CMFRI-Kochi > Marine Biotechnology
    Depositing User: Arun Surendran
    Date Deposited: 16 Jul 2013 10:34
    Last Modified: 23 Jul 2016 09:46
    URI: http://eprints.cmfri.org.in/id/eprint/9273

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