Establishment Of Molecular Epidemiological Investigation And Detection Technology Of Tilapia Lake Virus Disease In Western Guangdong And Development Of Subunit Vaccine

The disease is a problem in tilapia farming,the pathogen is mainly Streptococcus agalactiae,but the recent outbreak of tilapia lake virus disease at home and abroad has also caused large-scale tilapia deaths,resulting in huge Economic losses have severely restricted the development of tilapia aquaculture.In this study,molecular epidemiology of tilapia lake virus disease was carried out in western Guangdong,the main tilapia culture area in China,and Tilapia Lake Virus detection technology was established.The viral genome structure was analyzed.Also.We have developed the Tilapia Lake Virus disease subunit vaccine and conducted a preliminary evaluation of its immune effect.The main findings are as follows:1.Molecular epidemiological survey of Tilapia Lake Virus disease in westernIn October 2018 and October 2019,Tilapia Lake Virus infections were investigated in five areas of tilapia farming in western Guangdong-Leizhou,Huazhou,Wuchuan,Gaozhou,and Lianjiang,and tilapia was collected.150 fish samples.The results showed that the positive rate of 48 samples in Leizhou was 62.5%;the test results of 21 samples in Huazhou were all negative;the positive rate of 21 samples in Wuchuan was 19%;the positive rate of 15 samples in Gaozhou was 13.3%;The positive rate of Lianjiang's 45samples was 24.4%.The results indicate that the Tilapia Lake Virus is endemic in the tilapia culture area in western Guangdong.2.Tilapia Lake Virus gene sequencing and sequence analysisPCR amplified Tilapia Lake Virus segments 1?10 and sequenced.The results showed that the fragments were 1641 bp,1471 bp,1371 bp,1250 bp,1099 bp,1.044 bp,777 bp,657 bp,548 bp and 465 bp.Sequence comparison analysis of fragment 1 showed a protein molecular mass of 57KD,no transmembrane structure,and a tertiary structure.3.Establishment of rapid fluorescence absolute detection technology for Tilapia Lake VirusThe study designed a pair of specific primers based on the conserved region of Tilapia Lake Virus segment 8 and established a SYBR Green?real-time fluorescent quantitative RT-PCR method for detecting Luohu virus,and evaluated the specificity,sensitivity and repeatability of the method.The results show that the standard curve has a good linear relationship between 2.5×10¹⁰?2.5×10⁴ copy numbers?correlation coefficient R²=0.999?,and the detection limit is 2.5×10⁰ copy numbers.Coefficients of variation within and between experiments are 0.23%to 0.78%and 0.54%to 2.92%,respectively,with strong repeatability;no amplification reaction to other viruses and bacteria of aquatic animals,with good specificity4.Expression of viral immune proteins and preliminary study of subunit vaccineDNA recombinant technology was used to construct the prokaryotic expressionplasmid p ET28a,and the recombinant plasmid was transferred into E.coli BL21 to express and purify the recombinant protein.The immunogenicity of the recombinant protein was preliminary evaluated:The prokaryotic expression plasmid was constructed by connecting the p ET28a gene of tilapia lake virus segment 10.The expressed fusion protein was 17KD.Recombinant protein immunization of healthy tilapia on the 7th day after immunization,the specific antibody Ig M significantly increased?P<0.05?,14d,21,28 days of detection was significantly increased?P<0.01?.The protective rate of recombinant protein in the experimental group was 33%.