Identification And Characterization Of A Novel Emaravirus Infecting Pear Trees

Small RNA deep sequencing and bioinformatics analysis provide a means for discovering new viruses.In this study,it was found that pear plants had severe chlorotic spots and chlorotic symptoms.Small RNA sequencing and RT-PCR were used in identifying the virus infecting the pear,and a novel virus of Emaravirus was found.This virus was temporarily named Pear chlorotic leaf spot virus(PCLSaV).The obtained results are as follows:1.PCLSaV is a multi-segment negative-stranded RNA virus,and the genome subsist of five RNA segments.The size of the RNA1-RNA5 was 7100 nt,2045 nt,1296 nt,1543 nt and 1263 nt,respectively.The complementary strand of each genomic RNA strand contains an open reading frame(ORF),encoding protein p1 ~ p5,followed by RNA-dependent RNA polymerase(RdRp),glycoprotein(GP)and coat protein(CP),Movement protein(MP)and p5 with indefinite function.The RdRp of the virus has five domains(motif A-E)conserved in the virus of the Emaraviru and Bunyavirales.The amino acid sequence of the proteins encoded by PCLSaV have different similarities with the virus of Emaravirus,RdRp has the highest similarity with that of PPSMV-2,which is 31.5%,glycoprote in precursor has the highest similarity with that of WMoV,which is 20.3%,CP has the highes t similarity with that of RLBV,p5 protein has the highest similarity with the p7 of WMoV,which is 22.9%.The amino acid sequence of p5 protein had the highest similarity with p7 of WMoV,which is 20.6%.Based on the phylogenetic tree analysis of the amino acid sequence of p1-p3 protein and the corresponding amino acid sequence of different species of Bunyavirales,PCLSaV and Emaravirus were clustered in the same branch.These results indicate that PCLSaV is a new species of the Emaravirus genus.2.According to the RNA3 and RNA5 of PCLSaV,two pairs of specific primers were designed.The 286 pear trees in Jiangxi and Hubei provinces were tested by RT-PCR.The positive rate of 165 samples with chlorotic spots symptoms is 98.9%,three PCLSaV isolates were detected in 121 pear trees without obvious symptoms of the virus.These results indicate that PCLSaV is associated to the severe chlorotic spots and chlorotic symptoms of pear.3.A total of 102 CP clones were selected from 93 isolates tested for PCLVS positive.The nucleotide sequence similarity was 91.3% ~ 99.9%,amino acid similarity was 97.4%~100%.