Preliminary Study On Movement Disorder Nodavirus (MDNV) And Its Detection Technology

In recent years,the widespread of a variety of new epidemics including viral covert mortality disease?VCMD?has caused serious losses to the shrimp farming industry in China.The covert mortality nodavirus?CMNV?was proved to be the pathogenic agent of VCMD.In the epidemiological investigation,we found that in addition to CMNV's widely prevalence,a novel noda virus has recently emerged in farmed shrimps in China.The RNA-dependent RNA polymerase?Rd Rp?gene sequence of this novel virus shares only 78%similar to that of CMNV,and it is a new virus different from CMNV.The cultured Litopenaeus vannamei infected with this new noda virus showed symptoms of decreased swimming ability,so it was temporarily named"movement disorder nodavirus?MDNV?".This study intends to develop MDNV one-step Taq Man real-time quantitative PCR?RT-q PCR?,which will be used for an alternative method except the reverse transcription nested PCR?RT-n PCR?and reverse transcription loop-mediated isothermal amplification technology?RT-LAMP?detection methods for MDNV,with intending to providing new technical support for the rapid and quantitative detection of MDNV.At the same time,we preliminary analyzed of the pathogenicity of MDNV through artificial infection experiments,and used the developed methods to analyze the prevalence of MDNV in farmed shrimp in China.In the first section of this study,the samples of diseased Litopenaeus vannamei shrimps were collected from a Litopenaeus vannamei farm in Shouguang of Shandong province in the epidemiological investigation.Then the samples were analysed by using the methods of RT-n PCR,tissue histopathology,and in situ hybridization?ISH?.RT-n PCR results showed that the samples were determined as positive of MDNV,and no other common pathogens?incluidng CMNV?were found in the samples.The histopathology and ISH analysis showed that some hepatopancreas tubules in the hepatopancreas tissues of the diseased Litopenaeus vannamei showed obvious pathological changes,the epithelial cells of the diseased hepatopancreatic tubules showed necrosis,and the positive hybridization signal of the MDNV RNA probe could be observed in the necrotic cell region.The hepatopancrea tissues of the diseased shrimp were homogenized and density gradient centrifuged for virus purification,and then the purified virus was observed by using the transmission electron microscopy?TEM?.A large number of MDNV-like virus particles with a diameter of about30 nanometers were observed when the purified virus was submitted to the TEM assay.The results of the above studies indicate that Litopenaeus vannamei samples from the farm of Shouguang,Shandong Province was infected with MDNV.In the second section of this study,by using the purified MDNV,in vivo injection infection experiments were conducted on healthy Litopenaeus vannamei.Individuals of shrimp in the MDNV injection group showed obvious symptoms,mainly manifested as lighter hepatopancreas,opaque abdominal joints,and softer carapace;histopathological analysis of affected individuals showed that some tubules in the hepatopancreas tissue were necrotic.Necrosis is mainly manifested in the disintegration and collapse of hepatopancreatic tubule epithelial cells;the result of ISH analysis by using the MDNV probes on hepatopancreas tissue sections shows that there is obvious MDNV probe hybridization signal in the cytoplasm of necrotic epithelial cells in the hepatopancreas;The localized necrosis of hepatopancreatic tubules caused by MDNV infection is mild,and different from the global necrosis of hepatopancreas caused by CMNV infection,reflecting that the pathogenicity of MDNV may be weakened compared to CMNV;however,the pathogenicity of MDNV needs further study for that no reliable cumulative mortality data has been produced in present survey.In the third section of the present study,oligonucleotide primers and probes were designed based on MDNV Rd Rp gene for the development of Taqman RT-PCR method.By using the p MD18-MDNV plasmid and RNA standard containing the MDNV target gene as templates,a one-step method for MDNV was established through the optimization of the reaction system and reaction program Taq Man RT-PCR detection method.The optimized reaction components are:20?L of Taq Man RT-PCR reaction solution premix contains 11?L of one-step RT-PCR buffer,0.8?L of enzyme mixture,0.3?M forward primer,0.3?M reverse primer,0.4?M probe,1?L template,5.2?L RNA-free H₂0;the optimized reaction program is:54.5?15 min,95?1 min;45 thermal cycles of amplification?95?10 s,60.3?30 s?.The specific analysis results show that the newly established method can achieve specific detection of MDNV,showing a good linear relationship between the initial template concentration and the number of reaction cycles within the standard plasmid concentration range of 1.4×10¹⁰?1.4×10¹copy number/?L;The method can detect a minimum of 5.5×10¹copies of RNA standard or 1.4×10¹copies of p MD18-MDNV standard plasmid;analysis also shows that the CV of the intra-batch Ct value and the inter-batch Ct value of this detection method are less than 1.27%and 1.83%,indicating good repeatability and stability of the newly developed method.Therefore,the MDNV Taq Man RT-PCR method established in this study is specific,rapid and sensitive,and can provide technical support for the qualitative,quantitative detection and monitoring of MDNV in shrimp farming practice.In the fourth section of the present study,three methods of RT-n PCR,RT-q PCR and ISH assays were used to analyze the prevalence of CMNV and MDNV in shrimp cultured in some inland and coastal provinces in China during 2018 and 2019.The results showed that CMNV and MDNV positive samples could be found in cultured Litopenaeus vannamei and Macrobrachium rosenbergii.MDNV epidemic was found in cultured prawns in Shandong,Hunan,Guangxi and Hainan,showing a trend of decreasing prevalence from south to north of China.And samples,collected from Shandong,Guangxi,Jiangsu,Zhejiang and other provinces,were proved to be CMNV positive in the detection tests.Overall,the prevalence of CMNV in farmed shrimps in the above provinces in 2018was 20.7%.In 2019,the prevalence ofof CMNV and MDNV in farmed shrimps in the above provinces and cities were 22.6%and 21.6%,respectively.The above epidemiological analysis results revealed the CMNV epidemics in farmed prawns in many places in China,which means that the spread and harm of VCMD can not be ignored.Meanwhile,the prevalence of MDNV in farmed shrimps is also high,its harm to the farming shrimp deserves further investigation and high attention.