关键词: 西番莲, 东亚西番莲病毒, 全基因组, TC-RT-PCR检测

Abstract:

【Objective】 East Asian Passiflora virus (EAPV) is an important virus in Passiflora edulis. The objective of this study is to determine the complete genome sequence of EAPV Fujian isolate (EAPV-FJ) in mainland China, define the genome sequence characteristics of EAPV-FJ, establish the TC-RT-PCR (tube capture RT-PCR) assay for the specific detection, and to provide theoretical basis and technical support for the detection, monitoring and control of the virus.【Method】The complete genome sequence of EAPV-FJ was determined by using small RNA deep sequencing technology, combined with segmental cloning and RACE technology. The obtained EAPV-FJ sequence was analyzed for sequence characteristics, phylogenetic relationship and recombination. The TC-RT-PCR technology for rapid detection of EAPV was established through optimization of reaction conditions and reaction system. The specificity and sensitivity of the TC-RT-PCR were determined. The established TC-RT-PCR technology was used to detect samples collected from P. edulis orchards in Fujian Province, and the conventional RT-PCR was used for verification.【Result】The full length of the obtained EAPV-FJ was 10 065 nt (excluding polyA tail), containing an open reading frame of 9 663 nt in length that encodes a polyprotein of 3 220 aa. The polyprotein was cleavaged into 10 proteins, which are P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa, NIb and CP, respectively. The results of genome sequence identity analysis showed that the nucleotide sequence identity between EAPV-FJ genome and the four EAPV representative isolates registered in GenBank was 80%-99%, among which Vietnamese isolate EAPV-GL1 (GenBank accession number MT450870) of AO strain had the highest identity (99%). The nucleotide and amino acid sequences of the polyprotein were 79%-99% and 82%-98% identical to the four EAPV representative isolates registered in GenBank, respectively. Phylogenetic analysis based on the nucleotide sequence of EAPV-FJ polyprotein showed that EAPV isolates were divided into two groups (Group I was AO strain and Group Ⅱ was IB strain), which did not show obvious geographical correlation. EAPV-FJ belonged to Group I and was most closely related to the reported Vietnamese isolate EAPV-GL1. The results of recombination analysis revealed that EAPV-FJ is not a recombinant of EAPV. The established TC-RT-PCR detection technology showed good specificity and sensitivity, and can only detect EAPV-infected samples of P. edulis, while other viruses including cucumber mosaic virus (CMV), Passiflora latent virus (PLV), hibiscus latent Fort Pierce virus (HLFPV), turnip mosaic virus (TuMV), soybean mosaic virus (SMV), telosma mosaic virus (TeMV) as well as healthy samples cannot be detected. The lowest sensitivity of the TC-RT-PCR could detect the crude leaf extract of EAPV-infected samples of P. edulis diluted by 10 times, which was similar to the sensitivity of conventional RT-PCR. A total of 13 EAPV-positive samples were detected from 60 suspected virus disease samples of P. edulis collected from P. edulis orchards in Fujian Province by using the established TC-RT-PCR assay, and the results were in good agreement with conventional RT-PCR.【Conclusion】 This is the first report of complete genome sequence of EAPV in mainland China. The genome structure of this isolate (EAPV-FJ) is consistent with other reported isolates. EAPV-FJ has the closest relative to the Vietnamese isolate EAPV-GL1 in phylogenetic relationship, and no recombination sites were detected. The established TC-RT-PCR assay has the advantages of convenient operation, strong specificity, high sensitivity and low cost, and can be effectively used for the actual detection of EAPV in P. edulis orchard samples.

Key words: Passiflora edulis, East Asian Passiflora virus (EAPV), complete genome, TC-RT-PCR detection