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Molecular alterations induced by viral pathogen in crustacean cells

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Relation http://eprints.cmfri.org.in/9273/
 
Title Molecular alterations induced by
viral pathogen in crustacean cells
 
Creator Joseph, Jeswin
 
Subject Fish Biotechnology
Theses
 
Description 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.
 
Date 2012
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Language en
 
Identifier http://eprints.cmfri.org.in/9273/1/54.pdf
Joseph, Jeswin (2012) Molecular alterations induced by viral pathogen in crustacean cells. Doctoral thesis, Central Marine Fisheries Research Institute.