Establishment And Application Of Nucleic Acid Detection Techniques For Two Emerging Shrimp Viruses

Covert mortality nodavirus（CMNV）,an emerging RNA virus,is the pathogen of viral covert mortality disease（VCMD）,which caused serious loss of shrimp aquaculture in China in recent years.The result of previous investigation on the molecular epidemiology of CMNV in 2013²015 indicated that CMNV could infect the major species of crustaceans with high prevalence rate and was prevalent in the main shrimp farming areas of China.Being invertebrate,shrimp lack specific immune system and farms can not use vaccines to control the viral diseases.To develop molecular detection methods with high specificity and sensitivity and using them to detect CMNV as early as possible was considered to be the best and efficient measure to control the spread of VCMD.Firstly,a one-step TaqMan-based real-time fluorescence quantitative RT-PCR（TaqMan RT-qPCR）assay for CMNV was developed.Concentrations of the key components of the reaction and the procedures of the reaction were optimized firstly and then the sensitivity,specificity,and reproducibility of the method were evaluated along with the clinical application of the assay.The optimized 25μL reaction mixtures in TaqMan RT-qPCR included 2×One Step RT-PCR Buffer III 12.5μL,TaKaRa Ex Taq HS 0.4μL,Prime Script RT enzyme Mix II 0.4μL,CMNV-TAQ-R1（10μM）0.5μL and CMNV-TAQ-F2（10μM）0.5μL,CMNV-TAQ-P1（10μM）0.5μL,1μL template,RNase-free H₂O supplement.The optimized procedures conditioning 50.8oC incubating for 15 min,94oC incubating for 5 min,40 cycles（94oC 10 s,52.7℃30 s）.The method can detect the CMNV target gene from as low as 5.7×10⁰ copies of pMD20-CMNV standard plasmid,24.1 copies of CMNV standard RNA,and 9.6 pg total RNA of tissues from shrimp infected by CMNV.The Diagnostic sensitivity（Dse）and Diagnostic specificity（DSp）of the new deveoped assay were determined to 96.2%and 98.0%,respectively.Meanwhile,the assay could quantify the CMNV target gene in the initial templates concentrate range from 5.7×10⁰ to 5.7×10⁸.Secondly,performance parameters of the previously developed Highly Sensitive and Rapid Detection Kit for CMNV were evaluated.The test results showed that the kit could detect CMNV with no cross-reaction with other common shrimp pathogen including Yellow head virus（YHV）,White spot syndrome virus（WSSV）,Hepatopancreatic parvovirus（HPV）,Infectious hypodermal and hematopoietic necrosis virus（IHHNV）and Taura syndrome virus（TSV）.The Analytical sensitivity（Ase）of the kit was determined as 82.4 pg/μL of total RNA.The results of comparative analysis between the kit assay and the newly developed TaqMan RT-qPCR assay based on testing of 374 samples indicated that the DSp and DSe of the kit assay were 95.3%and73.6%,respectively.In addition,the repeatability and stability of the kit were appreciable for field use.In summary,the kit was suitable for the CMNV detection in filed with an acceptable disadvantage of that its sensitivity was no so high as that of the TaqMan RT-qPCR assay.Thirdly,reverse transcription loop-mediated isothermal amplification（RT-LAMP）assay was established for the detection of an emerging Nodavirus（temporarily named as Movement disorder nodavirus,MDNV）isolated from the disease shrimp.In our previous study on CMNV,a novel nodavirus was identified from the diseased farming shrimp and the partial RNA-dependent RNA polymerase（RdRp）gene of the novel nodavirus shared 78%homology with the original isolate of CMNV.The diseased shrimp showed clinical symptoms including swimming ability declining and hiding in the bottom of ponds.In order to distinguish this emerging virus from the original CMNV strain,it was named as MDNV in the study.The optimal conditions for this newly developed RT-LAMP reaction were found to be 4 mM MgSO₄,1.2 mM dNTPs,0.8μL Bst 2.0 WarmStart^?DNA polymerase and 0.8μL Eva Green.The optimized reaction procedures were determinded as 65.8oC,60 min.The RT-LAMP method was highly specific for CMNV detection with no cross-reaction with CMNV,EHP,IHHNV,WSSV,TSV and YHV.Within the range of 2.07×10⁸².07×10³ copies of standard plasmid,the logarithm of the initial template showed high correlation coefficient with the reaction cycles（R²=0.981）.The newly developed RT-LAMP assay supplied the basic method for the detection and quantification of the novel nodavirs of MDNV.Finally,national-wide survey of molecular epidemiology of CMNV and MDNV was conducted basing on analysis of shrimp samples collected from 2016 and 2017 by using the methods of the reverse transcription nested PCR（RT-nPCR）,RT-LAMP,and TaqMan RT-qPCR in present study.The results showed that CMNV-positive specimens were found in the major species of cultured crustaceans including Litopenaeus vannamei,Marsupenaeus japonicus,Fenneropenaeus chinensis,Penaeus monodon,and Macrobrachium rosenbergii.CMNV-positive specimens appeared in almost all the sampling coastal provinces such as Hebei,Shandong,Zhejiang,Fujian,Guangdong and Hainan.The results based on RT-nPCR assays showed that the prevalence rates of CMNV among the collected samples were 11.8%（30/254）,7.8%（30/387）in 2016 and2017,respectively.The results based on RT-LAMP assays showed that the prevalence rates of CMNV were 6.7%（17/254）,3.9%（15/387）in 2016 and 2017,respectively.The results based on TaqMan RT-qPCR assays showed that the prevalence rates of CMNV were 17.7%（45/254）,12.4%（48/387）in 2016 and 2017,respectively.The total prevalence rates of CMNV based on the above mentioned three methods were 26.8%（68/254）,16.3%（63/387）in 2016 and 2017,respectively.The results based on RT-LAMP assays showed that the prevalence rate of the MDNV was 9.4%（24/254）in2016.The high prevalence of CMNV in the major shrimp species and in the main farming areas revealed that CMNV still threatened the shrimp aquaculture in China during 2016²017,and the CMNV RdRp gene mutation raised the risk of false-negative of CMNV molecular tests,which deserved great concern.Meanwhile,the high prevalence rates of an emerging CMNV variant,MDNV,reminded that it was necessary to pay close attention to the high risk of MDNV transmission in farmed crustaceans.