Identification And Related Characteristics Study Of Two Novel Endornaviruses

Endornavirus is a new group of viruses,whose hosts include plants,fungi and oomycetes.When endornaviruses infect their hosts,it will not cause abnormal symptoms of the hosts,and it can persist infection in hosts.Their genomic nucleic acid types are mostly dsRNA(double-stranded RNA),and the full length of the sequence is between 9.8-17.6 kbp,encoding a large ORF.The coding frame of ORF contains conserved domains such as RdRp,CPS,Hel,UGT,etc.There is a nick near the 5 end of the full-length genome from some endornaviruses,and a string of cytosine bases is detected at the 3 end of some endornaviruses.Endornaviruses do not form virions and do not encode viral capsid proteins.In this study,two novel different endornaviruses were discovered from two different hosts(plants and fungi),and their molecular characteristics and some biological characteristics were studied.The main results are as followsRFirstly,a new endornavirus was discovered in powdery mildew.The powdery mildew samples were obtained from the wild Lycium chinense infected with powdery mildew in Beijing.The pathogen of powdery mildew was identified as Arthrocladiella mougeotii(Lév.)Vassilk by the fungal ribosomal DNA internal transcribed spacer(ITS)region and sexual generation cleistothecium microscopic morphology.The viral dsRNA of from powdery mildew samples were extracted,and the full-length sequence of the viral genome was obtained by random primers cloning,RNA ligase mediated rapid amplification of cDNA ends(RLM-RACE)and gap sequence amplification.The full-length sequence of the viral genome was 11683 bp(accession numberR MH094752.1)in size.It contains a large ORF,encoding three conserved domains.A Hel-1(helicase 1)domain close to the genomic N-terminus and an RdRp 2(RNA-dependent RNA polymerase 2)domain near the genomic C-terminus and a CRR(cysteine-rich region)region between two domains.The 5 and 3 untranslated regions of the genomic nucleic acid are 16 bp and 50 bp,respectively,and there are no cytosine bases at the 3 end.Phylogenetic tree constructed based on viral nucleic acid sequences and RdRp amino acid sequences showed that the newly discovered virus was closely related to EcEV(Erysiphe cichoracearum endornavirus)and GeEV(Grapevine endophyte endornavirus)of Alphaendornavirus genus under Endornaviridae and formed a sub-cluster.The newly discovered viral nucleotide sequences homology with EcEV and GeEV was 66.53% and 68.55%,respectively,and the viral amino acid sequences homology of RdRp was 73.62% and 72.39%,respectively.In view of thenewly discovered virus,its genomic structure,encoded protein domain characteristics,evolutionary characteristics and other aspects are consistent with the characteristics of endornaviruses.Therefore,the newly discovered dsRNA virus from Arthrocladiella mougeotii is tentatively named AmEV(Arthrocladiella mougeotii endornavirus).Secondly,a novel endornavirus was found in Lagenaria siceraria var.hispida.The viral genome sequence was obtained by random primers cloning,terminal RACE amplification and intermediate sequencing.The full length of the dsRNA is 15098 bp(accession numberR KX377970.1),which contains a large ORF.The encoding frame contains five conserved domainsR one helicase region,two CPS(capsular polysaccharide synthesis protein)regions,one UGT(UDP-glycosyltransferase)region and one RdRp region,respectivily.The 5 and 3 untranslated regions of the full-length genome of the dsRNA virus are 111 bp and 52 bp,respectively.And there are also 13 consecutive cytosine bases at the end of the 3 untranslated region of the viral full-length genome.Phylogenetic tree constructed based on the viral nucleic acid sequences and RdRp amino acid sequences indicated that the novel dsRNA virus was closely related to LsEV-CA(Lagenaria siceraria endornavirus-California)and CmEV(Cucumis melo endornavirus)of Alphaendornavirus genus under the Endornaviridae family and formed a sub-cluster.The newly discovered viral nucleotide sequences homology with LsEV-CA and CmEV was 72.96% and 69.11%,respectively,and the viral amino acid sequences homology of RdRp was 82.56% and 68.10%,respectively.In view of the high homology to the genomic nucleic acid and the encoded protein between the newly discovered virus and the endornaviruses LsEV-CA,the authors believe that the new virus is a new strain of LsEV(Lagenaria siceraria endornavirus).Thus,the newly discovered dsRNA virus from important crop Lagenaria siceraria is tentatively named LsEV-HuB(Lagenaria siceraria endornavirus-Hubei).Finally,some biological characteristics of the LsEB-HuB in the Lagenaria siceraria plants were studied.These biological characteristics studies involved the probability of Lagenaria siceraria seeds carrying endornavirus,the dynamic changes of LsEV-HuB content in the Lagenaria siceraria of different periods and the relative content of LsEV-HuB in the Lagenaria siceraria.The results showed thatR(1)endornavirus-carrying rates of Lagenaria siceraria varieties were detected by two pairs of virus-specific primers of LsEV-HuB.The results showed that the percentage of LsEV-HuB carried by seeds of 16 Lagenaria siceraria varieties was as high as 100%.For example,early-maturing Lagenaria siceraria,Lv Tai No.1,Lugua No.2.Besides,The probability of LsEV-HuB in two Lagenaria siceraria varieties was 92.85%,such asLE731 and super-early-growing varieties,indicating that LsEV-HuB in Lagenaria siceraria is transmitted vertically through seeds;(2)The qRT-PCR method was used to detect the dynamic changes of LsEV-HuB content in different growth stages.The results showed that the content of LsEV-HuB was the highest in the growth and development stage,followed by the flowering stage,and the LsEV-HuB content in the seedling stage was the lowest,just reached about 1/7,1/6 of the growth and development stage and the flowering stage,respectively.These results indicated that LsEV-HuB in host plants is a dynamic changes process;(3)6 different kinds of virus-specific primers were used to detect the virus species of the Lagenaria siceraria plants in the vegetable planting bases in the suburb of Jingzhou.The results showed that WMV(watermelon mosaic virus)and LsEV-HuB were co-infected with Lagenaria siceraria plants in this vegetable planting base.The relative content of WMV and LsEV-HuB was detected by qRT-PCR.The results showed that the content of LsEV-HuB in Lagenaria siceraria plants was only 1/9 of WMV,indicating that the content of endornaviruses was much lower than that of pathogenic virus,and LsEV-HuB in the host is similar to latent infections.