Analysis Of The Pathogen Pathology And Screen, Expression Of Immune-related Factor For Hyriopsis Cumingii Plague

Hyriopsis cumingii is one of the most important endemic freshwater pearl species in China. Hyriopsis cumingii Plague is an infectious disease and spreads fast. It severely affects the production of freshwater pearl mussels by causing high mortality with no affective treatments. Little known about the immune defense system and lacking of knowledge of immune-reaction mechanism in shellfish, and their bivalves morphology make it almost impossible to diagnose and prevent the disease timely. The epidemics of this disease placed severe problematic consequences impacts on freshwater pearl industries, generating rapid decreases in shellfish production in China.Studies on Hyriopsis cumingii plague pathogen, including pathogenic bacteriology and virology, has reported since 1980s. In this study, mussel samples with typical symptoms of Hyriopsis cumingii plague were collected in September 2005 and used for isolation and purification of the virus from the mussel tissues based on the morphology and molecular structure of the Hyriopsis cumingii plague virus (HcPV). The pathohistochemical observations on tissue samples collected from the healthy Hyriopsis cumingii infected by HcPV were carried out and results were discussed. To establish technology and methodology of genetic breeding resistant to Hyriopsis cumingii plagues, the newly developed technique of suppression subtractive hybridization (SSH) was used to construct a cDNA library of the Hyriopsis cumingii liver and screened for immune related factors associated with Hyriopsis cumingii plagues. The major objectives and analyses are outlined as follows:1. Morphological and molecular structure of HcPVTiusses taken from Hyriopsis cumingii with typical HcPVA collected in Changde were treated in a sterile condition, analyzed by differential sucrose density gradient centrifugation, staining and distaining to observe the virus particles observed under electron microscope. It showed that the particles are shaped round or oval with a diameter of about 20-310 nm, and no envelopes were evidence while tenuous tubers scatter over the surface. Virus particles were mainly found in the sucrose density of 30% -50% in the gradient, and clearly shown, under an electron microscope, on the ultrathin tissue slice of the Hyriopsis cumingii that had obvious pathological symptoms. Reproduced for three generations continuously, these virus particles were still able to cause a high mortality rate of greater than 90%. The finding of this study provided more concise identity for HcPV than previous investigations.The molecular biology study of the virus extracted by differential centrifugation revealed that the virus composed of double-stranded RNA with 0.2-3.0 kb molecular mass in three domains. The virus RNA was transcribed into complementary DNA to give raise of a virus cDNA library constructed by random hexamer primers. 45 clones were randomly chosen, sequenced and found 5 novel positive clones, with E values≥-6, among 38 validated sequences. These 5 sequences were identified for the first time and showed no significant homologous sequences in the databases, implicating that they are potential candidates for HcPV.2. The pathohistochemical characteristics of Hyriopsis cumingii plagueHyriopsis cumingii plague pathological tissues are observed by Optical microscopy and electron microscopy to mutually compensate the limitations of their observation visions. Pathological changes in tissues at the 3^rd, 5^th, 7^th, 9^th, 11^th day of infection are examined under electron microscope, showed structural effects on the tissue, including karyon, mitochondria, endoplasmic reticulum swell and deform, fractured membrane structures, lyses and formation of vacuoles. The Optical microscopic observations of the infected tissues showed that the epithelial cells in the tissue swelled and deformed, irregularly arrayed, even shed or lysed, increased basophils and mucus cells, ruptured and lysed connective tissue and muscle fiber cells, which created cavities. The observations on tissue pathological changes revealed that the virus had the fast duplication and became effective quickly, resulted in serious damages to the tissues, in the liver, stomach and intestine. Slower duplications and affections were observed in the mantle, gill and axe foot.3. Construction of subtracted liver cDNA library and sequence analysis of positive clones in Hyriopsis cumingiiSubtracted liver cDNA library of healthy and disease-resistant Hyriopsis cumingii (survivors after infected) were constructed using the suppression subtractive hybridization technology (SSHT). 87 out of the 107 positive cDNA clones randomly selected for sequencing were effective in obtaining of cDNA sequences.Sequence homology search showed that 58 out of the 87 effective cDNA sequences were homologous with high sequence identities to known sequences of functional proteins in the GenBank (E value≤6). Sequences of 4 positive clones had high similarities with open reading frame (ORF) of molecules of unknown function referred as hypothetical protein in the database. Only 1 sequence showed no significant similarity to any other sequences, which might be a novel EST sequence and a potential 5'- or 3'- UTR sequence in the genome that had been reported previously. 82 known function EST for 23 different genes were reported in Hyriopsis cumingii.for the first time in this study.According to the classification of functional genes by Adam, the identified EST in Hyriopsis cumingii fell into eight categories relevant to cell division, cellular structure and movement, metabolism, signal transduction, cell immune-action, gene and protein expression associates (including transcriptional regulation, translation, ribosome structure and the protein post-translational processing) and other proteins with unknown functions. 46 sequences, 52.87% of the total analyzed clones, reported in this study are related to the immune factors of cell recovery, metabolism and functional protein expression.4. Sequence analysis and tissue Specificity expression of Hyriopsis cumingii homolog similar to PIT 54Deduced amino acid sequence of the Hyriopsis cumingii homolog to PIT 54 (EX828677) was aligned to the PIT 54 or HP sequences Gallus gallus, Ctenopharyngodon idellus, Mus musculus, Bos taurus, Sus scrofa and Homo sapiens using Clustal W1.83 software. The multiple alignment showed that amino acids sequence of Hyriopsis cumingii homolog to PIT 54 shared high similarity with other related proteins involved in the comparison, the highest similarity between Hyriopsis cumingii and Gallus gallus (35.04%), then the Homo sapiens (13.87%), Ctenopharyngodon idellus (13.56%), Mus musculus (13.14%), Bos taurus and Sus scrofa (11.68% respectively). The phylogenetic tree constructed using MEGA4.0 software confirms the consentaneous evolutionary development of the PIT 54 homologous including Hyriopsis cumingii.Gene expression studies using the semi-quantitative TR-PCR showed that Hyriopsis cumingii homolog to PIT 54 is transcribed in different adult tissues. A constant expression was detected in the liver (both induced and non-induced). However, the expression is stronger in the induced tissue than non-induced, indicating that the expression level of the PIT 54 homolog in Hyriopsis cumingii is under regulation of the infactions in a dosage manner.The results of this paper had laid a solid theoretical foundation for a further systemic studies on pathology, molecular biology and disease-resistant genetics breeding of Hyriopsis cumingii plague, and provided instructions and practical methodology for studies on prevention of the Hyriopsis cumingii plague.