Molecular Detection Of Comovirinae And Sequence Analysis Of The Complete Genome Of A Netherlands Isolate Arabis Mosaic Virus

Comovirinae belongs to the order Picornavirales and the family Secoviridae; it contains Comovirus, Fabavirus and Nepovirus, altogether fifty-three viruses. Eight viruses were determined as quarantine pests in China of the Comovirinae,25.8%of the quarantine pests in China. In this study, degenerate RT-PCR, multiplex RT-PCR, and RT-LAMP were applied for molecular detection of Comovirinae. At the same time, the complete nucleotide sequences of the genomic RNA1and RNA2of Lily isolate NL (Netherlands) of Arabis mosaic virus (ArMV) were determined.The complete nucleotide sequence of a Netherlands lily isolate of Arabis mosaic virus (ArMV-NL) was determined. The genomic RNA1of ArMV-NL comprised7142nt, excluding the poly (A) tails, and contains one long open reading frame encoding a polypeptide of2203amino acids, the5’and3’non-coding regions consisted of231nt and299nt, respectively. The genomic RNA2of ArMV-NL comprised3754nt, excluding the poly (A) tails, and contains one long open reading frame encoding a polypeptide of1098amino acids,5’and3’non-coding regions consisted of262nt and195nt, respectively. The corresponding5’and3’non-coding regions of ArMV-NL RNA1and RNA2showed83.5%and87.0%identity. The comparison of the amino acid sequence of the polyprotein encoded by RNA1between ArMV-NL and other ArMV isolates showed that the conserved amino acid motifs, characteristic for the viral protease co-factor, the NTP-binding protein, the cystein protease, and the RdRp core domains. The amino acid sequence of the polyprotein encoded by RNA2result in the production of putative2A protein, MP and CP. Amino acid sequence comparisons between the polyproteins encoded by RNA1of ArMV-NL and other isolates showed90.3%identity with the ArMV-Lv, and84.1%identity with ArMV-NW. Amino acid sequence of RNA2showed that ArMV-NL shared identities of83.8%-89.5%with five other ArMV isolates.A universal RT-PCR method based on degenerate primer to detect genus of Comovirus and Fabavirus had been established. In order to improve the stability, convenience and sensitivity of this method, two of non-complementary sequences were added to the5’termini of the forward and reverse primers. The optimized PCR protocols amplified a specific product of400bp from14isolates of seven virus species of Comovirus and two virus species of Fabavirus. The specificity assays showed that the new primers did not amplify a specific product from nepovirus and healthy plants which are susceptible to genus of Comovirus and Fabavirus. The sensitivity test showed that the detection limits of the optimized method were up to10-100times greater than before.Arabis mosaic virus, Tomato ringspot virus, Peach rosette mosaic virus, Tomato black ring virus and Tobacco ringspot virus are all quarantine pathogens in China. A universal degenerate primer and five virus species-specific primers were designed based on the conserved region in RNA dependent RNA polymerase genes of RNA1. Amplifications resulted in256bp PCR product for TBRV,293bp for PRMV,342bp for ArMV,472bp for ToRSV and695bp for TRSV. The sensitivity assays showed that the detection limits of five specific product were visible up to5-2dilutions. The specificity assays showed no specific products obtained from other viruses of Nepovirus and healthy plants. This multiplex RT-PCR system was applied for rapid detection of five quarantine pathogens of Nepovirus and it showed potential for epidemiological studies in fields.Cowpea severe mosaic virus is a quarantine pathogen in China. This study aimed to establish a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for detection of CPSMV. Under optimal reaction conditions, the RT-LAMP method could distinguish CPSMV from other comoviruses. A sensitivity test showed that this method was10times more sensitive than the standard RT-PCR assay. The method in this study is suitable for fast, specific and sensitive detection of CPSMV.