Viral Metagenomics Analysis Of Farmed Shrimp And Molecular Epidemiological Study Of Shrimp Hemocyte Iridescent Virus(SHIV)

Shrimp is an important economic species in the world aquaculture industry and it is a high-quality aquatic product that consumers like.Especially in some coastal developing countries,shrimp culture not only provides the basic protein source for national survival,but also creates great economic benefits.Shrimp aquaculture also plays an important role in our country.As early as the 1970 s,China started the artificial seedling cultivation of shrimp in China and formed a large-scale breeding in the 1980 s.The production of farmed shrimp was steadily improved after the introducing of Litopenaeus vannamei.Now China has become the largest shrimp farming country in the world,with aquaculture area of 230,000 hectares and more than 1 trillion breeding seedlings.However,even so,China still needs to import large amounts of shrimp each year to meet domestic consumer demand.Since 1993 when the shrimp disease broke out,the disease problem has been the main factor restricting the development of the shrimp aquaculture industry in China.Diseases emerge in an endless stream.The persistent transmission of known pathogens and frequent outbreaks of new pathogens have caused serious losses to the industry,which has caused the industry and the breeding environment to face enormous challenges.The viral pathogen has a rapid onset of disease,a strong ability to spread,a wide range of hosts,and no effective method of prevention and treatment,which has a serious impact on the industry.The new pathogens have uncertainty biological characteristics and infection patterns.At the same time,due to the limitations of traditional virological research methods,it has brought great challenges to the timely detection,and prevention and control of new pathogens.For the excavation of unknown viruses,electron microscopy can only be used for observation at present.This way of identifying new viruses through morphological observation is difficult and the accuracy is poor.Even if virus particles are observed,the classification of the virus cannot be accurately judged,and it is difficult to obtain the nucleic acid sequence of the virus,which brings difficulties to further research.Viral metagenomics is a new technique for studying the composition of viruses.It directly uses the genetic material of all viruses in the environment as the research object,and can quickly identify the composition of viruses in the environment,which has outstanding advantages in dynamic monitoring and other aspects.This technology has been applied in human medicine,veterinary medicine and some aquatic fishes.However,there are few reports on shrimp disease research.For the first time,this study do viral metagenomics processing and sequencing to the diseased farmed shrimps in China,and explores the composition of the virus in the diseased shrimp samples.The virus suspected to be pathogenic was excavated from the sequencing data,and the pathogen was isolated and identified,and the relationship between the new virus and shrimp illness was studied.It also investigates the infection and epidemic of new viruses and provides a theoretical basis for the prevention and control of these unknown pathogens in shrimp aquaculture.From 2014 to 2015,seven batchs of diseased shrimp samples were collected from the country and tested by PCR/RT-PCR methods recommended by the OIE and reported in the article.No pathogens corresponding to the onset symptoms were detected.To further explore potential pathogens in the diseased shrimp,seven batches of shrimp samples were subjected to virus purification,extraction of viral nucleic acid,reverse transcription of RNA,anchor anchoring of DNA and synthesis of the second strand,SISPA-PCR amplification,Illumina Hi Seq 2500 metagenomic sequencing,and data annotations.In the sequencing data of the treated sample,the reads which annotate the virus for about 25%-80% of the total data of each sample.Among the annotated viruses,the double-stranded DNA virus accounted for 78.7%,the single-stranded DNA virus accounted for 17%,and the RNA virus accounted for only 4%.Among them,more than 50% of the reads in the S2 sample were annotated to the family Iridoviridae.More than 70% of the reads in the S6 sample were annotated to the family Parvoviridae.The rest of the samples were similar in the composition of the virus family.The highest proportion was the family Herpesviridae.The reads of Iridoviridae in S2 samples,the reads of Herpesviridae in S1 and S3 samples,the reads of a phage in S3 samples,and the reads of Decapod penstyldensovirus 1 in S7 samples were extracted for assembling and PCR validation.After the reads of Iridoviridae were assembled,643 DNA fragments with a length of 10,223 bp to 100 bp were obtained.One of the 6 655 bp fragment regions was verified by PCR.After the reads of Herpesviridae were assembled,992 DNA fragments were obtained,ranging from 1,828 bp to 100 bp in length.The three regions of the two longest fragments were all verified by PCR.In addition,the complete sequence of a phage Vibrio phage VP93 in the S3 sample with a length of 43,640 bp,and the entire sequence of the Decapod penstyl densovirus 1 in the S7 sample with a length of 4,062 bp were obtained.This is the first time that the virus composition of farmed shrimp samples has been analyzed by virus metagenomics technology.The suspected virus sequences such as iridescent virus and herpes virus have been excavated and verified by PCR.These virus species are most likely the potential pathogens of cultured shrimp.,should attract the attention of the industry.In the sample S2(No.20141215),the ratio of family Iridoviridae was high,and many assembly sequences were obtained,and there is most likely a potential pathogen that have not been reported.This batch of samples was collected from a shrimp farm in Zhejiang Province on December 15,2014.The symptom of diseased shrimp include slow growth,hepatopancreas pale atrophy,and a large number of deaths.Histopathological sectioning and HE staining of the fixed cephalothorax showed that the cells in the hematopoietic tissue,gills,hepatopancreatic sinusoids,and appendages exhibited nuclear pyknotic phenomenon,while the cytoplasm contained basophilic inclusions.These pathological symptoms did not appear in the previously reported shrimp disease.By BLASTX alignment of the assembly sequences,we obtained a MCP gene sequence and partial ATP enzyme sequence of a suspected iridescent virus.Based on phylogenetic analysis of MCP and ATPase gene sequences with homologous species of family Iridoviridae,it was found that the virus is in a new branch of the Iridiviridae and does not belong to the five virus genera that have already been divided.Further,through the rough extraction of virus particles and the ultrathin sections of the infected tissues,transmission electron microscopy revealed that a large number of large-particle icosahedral viruses exist in infected shrimp tissues with a diagonal diameter of 158.6±12.5 nm(n=30)and a side diameter of 143.6±10.8 nm(n=30)and the virus contained a core with a diameter of 85.8±6.0 nm(n=30).By artificially infecttion using natural non-invasive way and intramuscularly,it was found that all the symptoms of the original sample could be reproduced,and mortality rate of 100% occurred after 15 days of infection.Observation of the ultrathin section confirmed that this virus infects shrimp hemolymph and that a large amount of virus free in the sinusoid of the tissue.The assembly DNA sequence was further verified by PCR and one-generation sequencing.A complete genome sequence with a length of 165,809 bp was obtained.The G+C content of its genome accounted for 34.6%.According to Dot plot analysis,the longest repeat sequence was 320 bp,and there were also some direct,reverse and palindromic repeat sequences.A total of 170 ORFs were predicted,102 were positive,68 were reversed,and 11 pairs of ORFs overlapped each other.63 ORFs predicted the corresponding gene functions through alignment,including ATPase,DNA polymerase and MCP.By phylogenetic analysis of 27 genes and 16 genes,it was found that this strain of iridescent virus belongs to a new virus genus of the family Iridiviridae.Based on the above results,this study isolated and identified a novel iridescent virus that infects L.vannamei and is tentatively named Shrimp hemocyte iridescent virus(SHIV),and it is proposed to divide it into a new virus genus—Xiairidovirus.Haemolymph was collected from artificial infected shrimp and hemocyte were removed by centrifugation.Supernatant were filtered and centrifuged at high speed.After the crude extract was centrifuged by sucrose density gradient centrifugation,four visible bands appeared in the centrifuge tube,distributed in the 30%,40%,and 50% regions,respectively.Among them,band 3 is the thickest and the distribution area is also the most concentrated.The OD280 and buoyancy densities of the suspensions at each position were measured,and the absorbance and buoyant density curves were obtained.Among them,four peaks corresponded to the bands in the absorbance curve,and the buoyant densities at the four peaks were 1.07 g/cm3,1.17 g/cm3,1.23 g/cm3 and 1.32 g/cm3,respectively.After negative staining with phosphotungstic acid and transmission electron microscopy observations,it was found that SHIV particles were most dense in band 3.SHIV particles were typical icosahedron,and the inner limiting membrane and core were observed inside the nucleocapsid.The purified virions was subjected to SDS-PAGE.After staining and decoloration,20 macroscopic protein bands with sizes ranging from 180 k Da to 20 k Da were obtained.The 20 bands were respectively subjected to gel-cutting and protein profiling analysis,and the annotated results of the alignments of the various bands were obtained,including the hemocyanin of the L.vannamei and the MCP protein of SHIV.Through the above experiments,it was proved that the purified virus particles are the SHIV obtained by assembling sequencing data.In situ hybridization probe was designed according to the SHIV MCP gene sequence.In situ hybridization was performed on the original sample and the artificially infected sample.As a result,blue hybridization was found in the hematopoietic tissue,gills,hepaticpancreatic sinusoids,and appendages.The signal confirms the infection of SHIV in shrimp and also determines the site of infection.Based on ATPase gene,2 pair of primers were designed and a highly specific and sensitive nested PCR assay was established.The detection limit can reach 36 fg total DNA of infected shrimp.Compared with the real time PCR method established in our laboratory,the detection sensitivity of the established nested PCR method was 97.6%,and the detection specificity was 98.3%.This nested PCR method was applied to the detection of SHIV in farmed shrimp samples.It was found that SHIV was positive in both Fenneropenaeus chinensis and Macrobrachium rosenbergii with the exception of Litopenaeus vannamei.The total positive rate of all samples was 15.8%.The positive samples were distributed widely in Hebei Province,Shandong Province,Zhejiang Province and Guangdong Province.It can be seen that this pathogen has spread widely in domestic coastal farms and should be given enough attention.