Molecular characterization of two recombinant isolates of telosma mosaic virus infecting Passiflora edulis from Fujian Province in China

Sample collection, electron microscopy and serological detection

Two passion fruit samples showing mosaic and crinkle symptoms on the leaves (Fig. 1A) were collected in 2017 from a commercial orchard in Fujian Province, China (Xie et al., 2017). After negatively staining with 2% phosphotungstic acid (pH 6.7), crude sap from the passion fruit sample was placed onto formvar-coated copper grids, and then examined using an H-7650 transmission electron microscope (Hitachi, Tokyo, Japan) operating at 80 kV. Fresh leaf samples of passion fruit were manually homogenized in pestles for homogenization with 0.05 M sodium carbonate buffer, pH 9.6. The antigen-coated indirect ELISA protocol was performed by using universal potyvirus antiserum (Agdia, Elkhart, IN, USA) according to the manufacturer’s instructions. All samples were tested in duplicate wells in microtiter plates. Absorbance values at 405 nm were measured with an automatic ELISA reader (Infinite M200, Tecan, Männedorf, Switzerland). Sample with absorbance value at least twice that of healthy control was considered positive.

RNA extraction and cDNA synthesis

Total RNA was extracted from the leaf tissue which was positive with virus infection by ELISA using an RNA extraction kit (Qiagen, Hilden, Germany). The quantity and quality of the extracted RNA were determined by measuring absorptions at 260–280 nm with a NanoDrop 2000c (Thermo Scientific, Waltham, MA, USA). The first-strand cDNA was synthesized using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Madison, WI, USA) following the manufacturer’s protocol. For the reaction, in total of 11 μl mixture containing three μl RNA (~1 μg), one μl random primer (100 μm) and seven μl DEPC-treated water were incubated at 70 °C for 10 min. Then, the mixture was transferred immediately to an ice bath for 5 min. Finally, five μl 5× buffer (Promega, Madison, WI, USA), two μl dNTP mix (Promega, Madison, WI, USA), one μl RNAsin Plus RNase inhibitor (Promega, Madison, WI, USA) and one μl M-MLV reverse transcriptase (Promega, Madison, WI, USA) were added to the mixture of primer and RNA. The RT reaction was carried out at 42 °C for 60 min followed by at 70 °C for 10 min. The cDNA was chilled on ice and stored at −70 °C.

Molecular detection of virus using polymerase chain reaction

For the detection of the genus Potyvirus, a set of universal potyvirus primer LegPotyF 5′-GCWKCHATGATYGARGCHTGGG-3′ and LegPotyR 5′-AYYTGYTYMTCHCCATCCATC-3′ (Wylie et al., 2010) was used to amplify a fragment of approximately 680 bp. Polymerase chain reaction (PCR) reactions were performed in a 25 µl volume containing 12.5 μl of 2× Taq PCR mix (Promega, Madison, WI, USA), one µl of each primer (10 µM), two µl of cDNA and 8.5 µl DEPC-treated water. PCR conditions were as follows: initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 45 °C for 45 s and an extension step at 72 °C for 1 min. The amplification program was followed by a final extension step at 72 °C for 10 min.

Telosma mosaic virus was detected by using specific primers TeMV-CPf 5′-TCAAGTAAGGTGGATGATGTT-3′ and TeMV-CPr 5′-CTGCACAGAGCCAACCCCAA-3′ as previously described (Xie et al., 2017). The primer pair TeMV-CPf/TeMV-CPr was designed to amplify the full length of the coat protein (CP) gene (~816 bp). PCR was performed in a final volume of 25 μl in a reaction mixture consisting of 2.5 μl of 10× PCR Buffer (TaKaRa, Dalian, China), 2.5 µl of MgCl₂ (25 mM), one µl of dNTPs (2.5 mM), one µl of each primer (10 µM), 0.2 µl of Taq DNA polymerase (5 U/μl), three µl of cDNA and 13.8 µl DEPC-treated water. PCR conditions were as follows: initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 1 min and an extension step at 72 °C for 1 min. A final 10 min elongation step at 72 °C was performed at the end of the 35 cycles.

Cloning and sequencing of TeMV complete genome

Polymerase chain reaction products were analyzed by 1.5% agarose gel electrophoresis, stained with GelRed and photographed under UV-light. The target fragments of PCR products were purified by using Agarose Gel DNA Purification Kit (TaKaRa, Dalian, China). The purified products were ligated to pMD18-T vector (TaKaRa, Dalian, China) and then transformed into the Escherichia coli DH5α competent cell. The positive clones containing the insert fragment were identified by PCR. Three of the positive clones were sequenced by Shanghai Sangon Biological Engineering Technology and Service Co., Ltd.

To amplify and clone the full-length genome sequences, five overlapping fragments covering the coding regions of the TeMV genome were amplified using RT-PCR with 5 pairs of specific primers designed from the highly conservative region of the TeMV genome (Table S1). RACE PCRs for the 5′ and 3′-ends of the virus genome were conducted using the SMARTer RACE 5′/3′ Kit (TaKaRa, Dalian, China) and 3′-Full RACE Core Set with PrimeScript™ RTase (TaKaRa, Dalian, China). Long PCR fragment was determined by using primer-walking method (Sangon Biotech, Shanghai, China). At least three colonies derived from each transformant were sequenced and the consensus sequences were used for genome assembling using DNAMAN version 9.0 program (Lynnon, QC, Canada). Complete genome sequences of the Fuzhou and isolate (accession number: MK340754) and Wuyishan isolate (accession number: MK340755) were deposited in GenBank.

Phylogenetic and recombination analyses

The complete genome was assembled from overlapping RT-PCR clones after removal of the vector and primer sequence. To identify the closest relatives of the Fuzhou and Wuyishan isolates, we performed a BLASTn search against the nt/nr databases and a sequence identity matrix using BioEdit. The putative cleavage site patterns in the polyprotein were identified using online website (http://www.dpvweb.net/potycleavage/index.html).

To reveal the evolutionary relationship of TeMV, the reference sequences of other potyviruses were retrieved from the NCBI GenBank database. We aligned sequences (excluding the UTRs) by codon and removed poorly aligned regions using TranslatorX (Abascal, Zardoya & Telford, 2010). Maximum-likelihood-based phylogenetic analysis was performed using IQ-TREE 1.6.6 (Nguyen et al., 2014) under the GTR + F + R5 nucleotide substitution model, which was selected by ModelFinder (Kalyaanamoorthy et al., 2017). Topological support was estimated by 5,000 Ultrafast bootstrap replicates as well as the SH-aLRT test with 1,000 replicates (Guindon et al., 2010).

Recombination analysis were conducted using seven different methods (RDP, GENECONV, BOOTSCAN, MAXCHI, CHIMAERA, SISCAN and 3SEQ) implemented in the RDP4 package (Martin et al., 2015). For each putative recombinant breakpoint, a Bonferroni-corrected p-value cutoff of 0.01 was calculated. To reduce the presence of false positives, recombination events supported by at least four methods with an associated p-value of < 10⁻⁶ were considered to be significant.

Detection of TeMV

The results of electron microscopy showed the presence of potyvirus-like flexuous rod particles of about 750–770 nm in length (Fig. 1B). Presence of potyvirus infection was confirmed by using indirect ELISA. Parts of the nuclear inclusion protein b (NIb) and CP gene of TeMV (680 bp) and the entire CP gene of TeMV (816 bp) were obtained by RT-PCR (Fig. 1C).

A BLASTn search against GenBank indicated that the RT-PCR sequences obtained here share more than 98% nucleotide identities with TeMV (accession number: KJ789129). These results suggested that TeMV was present in passion fruit plants showing mosaic and crinkle leaves from Fuzhou and Wuyishan.

Molecular genomic characterization of TeMV isolate

The complete genome sequence (excluding the 3′ poly (A) tail) of Fuzhou is 10,050 nucleotides (nts) in length and of Wuyishan is 10,057 nts in length. Their 5′-untranslated regions (5′-UTRs) are 169 and 177 nts, respectively, while the 3′-UTRs were both 251 nts (Table 1). Both contained an open reading frame of 9,630 nts, encoding a polyprotein of 3,209 amino acids. An additional short open reading frame translated by ribosomal frameshift, called PIPO, was also identified within the P3 cistron (Chung et al., 2008); this includes the highly conserved G₂A₆ motif at nucleotide positions 2,873–3,913 in the genome of the Fuzhou isolate, and at nucleotide positions 2,880–3,920 in the genome of the Wuyishan isolate.

The polyproteins of Fuzhou and Wuyishan were predicted to be proteolytically processed into ten mature peptides. Their cleavage sites are in consensus to those of other TeMV isolates, whose dipeptides are Y/S, G/G, Q/G, Q/S, Q/S, Q/G, E/S, Q/S and Q/S (Fig. 2). Details regarding genome organization, and protein sizes are presented in Table 1.

Pairwise comparisons showed that Fuzhou shares 78%, 98% and 96% nucleotide sequences and 84%, 99% and 98% amino acid sequence identities with Hanoi, GX and PasFru, respectively at the complete genome level (Table 1A). Wuyishan shares 78%, 99% and 88% nucleotide sequences and 83%, 99% and 92% amino acid sequence identities with Hanoi, GX and PasFru, respectively at the complete genome level (Table 1A). At the individual cistron level, Fuzhou and Wuyishan share 43–100% nucleotide sequence identity and 40–100% amino acid sequence identity with Hanoi, GX and PasFru, respectively (Table 1B). However, the CP of Fuzhou and Wuyishan both share more than 85.9% nucleotide and 88.9% amino acid sequence identities with Hanoi, GX and PasFru, respectively. According to the accepted species demarcation criterion for the genus Potyvirus, Fuzhou, Wuyishan, Hanoi, GX and PasFru should be considered as one species of Potyvirus. Interestingly, the N-terminal region of P1 sequences of Fuzhou and Wuyishan are divergent to both length and amino acid sequences, especially with Hanoi (Fig. 2B).

Phylogenetic classification of TeMV

Our phylogenetic analysis indicated that Wuyishan and Fuzhou were clustered into a monophyletic clade with high confidence (UFBoot/BPs = 100), together with GX, Hanoi and PasFru (Fig. 3), suggesting that these isolates share a common ancestral origin. Notably, PasFru was not placed in a new taxon, although it was proposed to be a new member of the Potyvirus species (Yang et al., 2018). Hanoi, isolated from T. cordata in Vietnam, was clustered in an outer branch of the other TeMV isolates collected from P. edulis in China.

Recombination signals in TeMV

Recombination analyses identified a recombination region of approximately 4,000-nucleotides in the Fuzhou genome, with two recombination breakpoints within the CI and NIb regions (Fig. 3B) and PasFru and GX isolates were identified as its parents with a high level of significance by the RDP suite (GENECONV, p ≤ 7.70E × 10⁻¹³⁴, Bootscan, p ≤ 1.34 × 10⁻¹³⁸; Maxchi, p ≤ 2.19 × 10⁻⁴⁵; Chimaera, p ≤ 5.65 × 10⁻⁴⁷; Siscan, p ≤ 7.25 × 10⁻⁵⁴ and 3Seq, p ≤ 4.44 × 10⁻¹⁵) (Fig. 3C). A short recombination region within the P1 region was identified in the Wuyishan genome (Fig. 3B) and PasFru and Hanoi isolates were recognized as its parents with high degree of support by RDP (p ≤ 5.53 × 10⁻⁶), Bootscan (p ≤ 5.21 × 10⁻²³), Maxchi (p ≤ 4.12 × 10⁻¹⁸), Chimaera (p ≤ 2.13 × 10⁻¹⁰), Siscan (p ≤ 5.92 × 10⁻⁹) and 3Seq (p ≤ 1.11 × 10⁻¹⁴) (Fig. 3C).