Identification Of The Virome Of Ectropis Grisescens Reveal Two Novel Viruses

Ectropis grisescens Warren is an important pest in tea plantations,mainly feeding on the leaves of tea plants.When the pest infestation is serious,the entire tea plant will be eaten up.In view of the particularity of tea products,green and environmentally friendly control methods were used in the prevention and control of Ectropis grisescens Warren,such as biological control and physical control.The use of Eo NPV to control Ectropis grisescens Warren has a certain effect,which is a concrete manifestation of the actual production of insect viruses.Identification and research on insect viruses can provide new biocontrol resources and scientific basis for the management of agricultural and forestry pests.In this study,the macrovirome sequencing technology and bioinformatics analysis methods were used to analyze the viral composition of Ectropis grisescens Warren,and two new viruses were identified and discovered.The main results are as follows:1.We collected mix the samples of Ectropis grisescens Warren from Hong’an County of Huanggang City and Zigui County of Yichang City,Hubei Province for macrovirus group sequencing and bioinformatics analysis.We obtained a total of 33 virus contig by BLASTn in NCBI,and these 33 contig were divided into 9 DNA or RNA viruses.Among them,12 contig and 2 contig have certain similarities with a +ss RNA virus and a-ss RNA virus,but the similarity rate is very low(39.36%-68.95%).They may be two new viruses.2.Using BLASTx analysis on NCBI,the + ss RNA virus had the highest similarity with Tni Ted V of family Metaviridae,and the +ss RNA virus was tentatively named Ectropis grisescens TED virus(Eg Ted V).The labeled probe was used to perform Northern blot with the total RNA extracted by the "column method",and the result showed a hybridization signal,indicating that Eg Ted V was a virus.Using the method of terminal cloning,the full-length nucleotide sequence of Eg Ted V is 7264 nt,which is predicted to encode four open reading frames(ORF).ORF A is 1206 nt in size,encoding EBNA-3B protein;ORF B is 1071 nt in size,encoding reverse transcriptase;ORF C is2166 nt in size,encoding Ty3 / Gypsy family ribonuclease H(RNase H),zinc Domain integrase,IS 481 family transposase,reverse transcriptase(RT);ORF D size is 1152 nt,encoding baculovirus F protein.A phylogenetic tree was constructed by combining Eg Ted V with the reported viruses in the family Metaviridae viruses,and the results showed that Eg Ted V belonged to genus Errantivirus.We designed the detection primers,and tested 52 samples of Ectropis grisescens Warren collected in Hong’an County of Huanggang City,Zigui County of Yichang City and Hangzhou City by RT-PCR.The results showed that Eg Ted V was detected in 52 samples,and the detection rate was100%.We carried out RT-PCR detection on the offspring of Ectropis grisescens Warren with the Eg Ted V,and the results showed that Eg Ted V can be transmitted vertically to the next generation,however,Eg Ted V was not detected in the leaves of Ectropis grisescens Warren after feeding.In addition,we detected Empoasca onukii Matsuda and Euproctis pseudoconspersa Strand by RT-PCR method,however,no Eg Ted V was detected.We performed ultracentrifugation on the crude virion extracts of Eg Ted V with different concentrations of sucrose layers,and analyzed samples of sucrose layers at different concentrations.The samples in the sucrose layers with different concentrations were extracted with chloroform,and the results of PAGE electrophoresis analysis and RT-PCR detection showed that the virus could be detected in each sucrose layer.3.The-ss RNA virus by sequencing analysis has two contig sequences.Using BLASTx analysis on NCBI,the-ss RNA virus had the highest similarity with Photinus pyralis orthomyxo like virus 2 of family Orthomyxoviridae,and the-ss RNA virus was tentatively named Ectropis grisescens orthomyxo virus(Eg OV).The labeled probe was used to perform Northern blot with the total RNA extracted by the "column method",and the result showed Eg OV was a virus.The family Orthomyxoviridae viruses usually have 6-8 RNA strands,but we obtained two RNA strands.Both strands have unique ORF that encode NP protein or PB 2 protein,respectively.In order to determine the taxonomic status of Eg OV and its relationship with other viruses of family Orthomyxoviridae,we carried out a phylogenetic tree analysis of Eg OV.The results showed that Eg OV clustered with members of the genus Quaranjavirus,but it is genetically distant from other members of the genus Quaranjavirus.To detect the virus-carrying status of samples from different regions,we tested 36 samples from three different regions by RT-PCR,and the detection rate was 100%.Eg OV can replicate in the body of Ectropis grisescens Warren and can also be transmitted vertically to the next generation,and Eg OV was not detected in the leaves of Ectropis grisescens Warren after feeding.Eg OV was not detected in the body of Empoasca onukii Matsuda and Euproctis pseudoconspersa Strand.