The Identification Of Two Novel Negative Viruses Infecting Fruits By Metatranscriptomics

Fruits like apples,citrus are nutritious and good for health,they are with important market economic value throughout the world.Virus diseases can affect the normal growth of fruit trees and even cause serious damage to related industries.There are many types of fruit tree virus diseases,some pathogens of which have not been confirmed so far.Therefore,the detection and identification of fruit virus diseases is of great importance for production and prevention.However,the viruses distribution in trees is seasonal and uneven,and some are latent,traditional methods are limited on virus identification.Metatranscriptomics technology on plant virus identification can break the limit without any homologous reference sequences,accelerating the detection and identification of unknown virus pathogens.Total RNA sample contains a large amount of rRNA.There are two methods to create a library in the process of transcriptome sequencing in ordor to avoid subsequent sequencing resource wasting on repeated rRNA sequences.Firstly,we used the poly(A)-enriching method to sequence a periwinkle sample that showed leaf curling,vein yellowing,and dwarfing and obtained virus fragments,which were identified as Lettuce chrolosis virus(LCV),which had not been reported in China.Sequence alignment analysis showed the isolate LCV-PW and the California isolates genome share nucleotide sequence similarity of 85%(RNA1)and 88%(RNA2)and amino acids' of 11 ORFs are 62%-100%,indicating it might be a new strain.The LCV genome does not contain poly(A)tail but come fragments could be blocked by others with poly(A)tail during the enriching.The Poly(A)-enriching method is not perfectly suitable for detecting of unknown pathogens,because it could overlook new viruses that do not contain poly(A)tail or only provide some fragment sequences.Thus,we used the rRNA removing method to build the library.A new latent virus from the leaves of an asymptomatic apple was analyzed and named Apple flat limb virus(AFLV).AFLV is a three-stranded negative RNA virus,RNA1-RNA3 encoding RdRp,MP,and CP,respectively.AFLV was detected in five apple production areas,and the infection rates were 9.4%-83.7%.Remapping raw data sequences to the whole genome revealed that the rRNA removing method can not only break the limit of poly(A)-enriching methods,but also obtain near full length of sequences.We also used the rRNA removing method to analyze another new virus from Fina-Clementine with leaves flecking,tentatively named Citrus leaf flecking-associated virus(CLFaV).CLFaV consists of two single-stranded RNAs and contains three open reading frames.RNA1 codes for an RdRp with tipycal signatures of nsRNA viruses;RNA2 is an ambisense RNA with an intergenic region containing transcription termination signals(TTS)reported previously in phlebovirus.Grafting experiments revealed thatCLFaV could cause gum in albedo on Dweet and Eureka lemon,suggesting that it might be the pathogen of citrus concave gum disease.Althrough CLFaV has homology with WCLaV,it's not clear that whether it can infect watermelon.In this study,we detected and identified AFLV and CLFaV using Metatranscriptomics.They share some similarities,and are highly homologous to the genus Phlebovirus.Therefore,they were classified in different new genera under the Phenuiviridae.