Amplification of the whole genome of PNRSV by one-step and two-step RT and mRT-PCR
The whole genome of PNRSV, i.e., RNA1, RNA2, and RNA3, was successfully amplified employing one-step RT-PCR along with mRT-PCR (Fig. 1a, Supplementary Fig. S1a) and two-step RT-PCR along with mRT-PCR (Fig. 1b, Supplementary Fig. S1b).
Sensitivity of one-step RT-PCR and mRT-PCR
In one-step RT-PCR, RNA1 was identified up to 10–2 (4 ng), RNA2 up to 10–4 (0.04 ng), and RNA3 up to 10–3 (0.4 ng) dilution (Fig. 2a-c, Supplementary Fig. S2a–c). In one-step mRT-PCR, RNA1 had a detection limit up to 10–3 (0.4 ng), whereas RNA2 and RNA3 had a detection limit of up to 10–4 (0.04 ng) (Fig. 2d, Supplementary Fig. S2d).
Sensitivity of two-step RT and mRT-PCR
A ten-fold serial dilution of RNA (100–10–5) was used to assess the detection limit of two-step RT and mRT-PCR. In both two-step RT-PCR and mRT-PCR, RNA1, RNA2, and RNA3 could be identified clearly up to 10–3 (0.4 ng) dilution in RNA dilution (Fig. 3a–d, Supplementary Fig. S3a–d). In RT-PCR and mRT-PCR, there were no significant changes in the detection limits of all three PNRSV RNA fragments.
Characterization of the whole genome of PNRSV (Indian Isolate)
After sequencing analysis, RNA1, RNA2 and RNA3 nucleotide sequences of PNRSV obtained from infected apricot leaf samples were found to be 3.332 kb, 2.591 kb and 1.952 kb, respectively (Fig. 4). The obtained sequences were submitted to DDBJ with Accession Numbers viz. LC382449 (RNA1), LC382467 (RNA2) and LC382468 (RNA3). While the characteristic features of the genomic RNA sequences are mentioned in Table 2, RNA1 and RNA3 have an equal propensity of % GC content (45%), while RNA2 has 41% GC content. The open reading frames (ORFs) analysis and sequence alignment comparisons showed that ORF1a encodes Replicase1 (1045 aa), ORF2a Replicas2 (799 aa), ORF3a movement protein (283 aa), and ORF3b encodes the coat protein (226 aa) (Fig. 4 and Table 2). The RNA1 and RNA2 do not have any intergenic region (IR), while RNA3 has a short (77 nt) IR between movement protein (177–1028 nt) and coat protein (1104–1784 nt) ORFs of the PNRSV viruses.
Sequence demarcation tool (SDT) analysis
The SDT (version 1.2) analysis displayed the percentage pair-wise identity of understudy Indian isolate of PNRSV RNA1 sequence with other RNA1 sequences from different geographical boundaries, which varies from 91 to 99% (Fig. 5a). The Indian isolate showed nucleotide identity of 99% with RNA1 of the Czech Republic and Australian isolates obtained from cherry and peach respectively. Likewise, 98% of nucleotide identities were exhibited with eight other genomic RNA1 sequences, Candian Peach, Chinese Sweet cherry, Chinese Peach, Chinese Myrobalan plum, Canada Cherry and Australian cherry, respectively. The minimum pair-wise identity of 91% was shown with two PNRSV RNA1 sequences of Cherry and Sour Cherry submitted from the USA and Chez Republic (Fig. 5a).
Similarly, the SDT analysis unveiled that PNRSV genomic RNA2 under study showed a maximum nucleotide identity of 99% with eight other RNA2 sequences from different geographical locations and host species viz., Australian cherry, Canadian Peach, Czech Republic Sour cherry, China Peach, Australian Peach and USA Cherry. While the sequences China_ Myrobalan plum, and Czech Republic Sour Cherry showed a minimum pair-wise identity of 91% with the India Apricot understudy (Fig. 5b).
Likewise, The SDT analysis showed the percentage pair-wise identity of PNRSV RNA3 understudy with other RNA3 sequences from different geographical locations, which varies from 87 to 98% (Fig. 5c). The Indian isolate shared maximum nucleotide identity of 98% with twelve other PNRSV RNA3 sequences- Canadian Peach, USA Peach, Canadian Peach, Mexican Peach, China Sweet cherry, China Cherry, China Myrobalan plum, China Sweet cherry, Indian Rose, Spanish Apple, Czech Republic Sour cherry and, China Rose respectively (Fig. 5c). While one sequence, USA Peach, showed a minimum pair-wise identity of 87% with the India Apricot sequence understudy (Fig. 5c). This SDT analysis shows that similarities are higher between groups than among groups of RNA 1, RNA2 and RNA3 genomes when the three genomes were concatenated. Figure 5d clearly shows that the three genomic RNA sequences show a significant nucleotide identity among them.
Recombination detection analysis
The RDP version 4.97 has detected twelve recombination signals in fourteen unique events in PNRSV RNA1 (Table 3). The PNRSV RNA1 sequence has GenBank Acc. No. MH727235 (China) was found most potential recombinant sequence isolated from cheery, which was detected by six methods (GENECONV, BootScan, MaxChi, Chimaera, Siscan and 3Seq) of RDP version 4.97. Compared to PNRSV RNA1 understudy, the other RNA1 sequences MH727235, MH727236, KY883318, and KY883333 may be recombinant (Table 3). The recombinant sequence MH727235 may be potentially originated due to recombination between MH727233 (major) and AF278534 (minor) sequences (Fig. 6). Similarly, MH727236 may be originated by the recombination of AF278534 (major) and MH727235 (minor) sequences. The Australian recombinant sequence KY883333 may be evolved by the recombination of KY883332 (major) and AF278534 (minor) sequences (Fig. 6). Meanwhile, RDP version 4.97 identified the LC382449 (India, Apricot) RNA1 sequence as a minor parent for the KY883318 (ApMV, Australia) RNA1 sequence, although with an exceptionally low likelihood of RDP, GENECONV and 3Seq methods (Table 3 and Fig. 6). Interestingly, it was found that AF278534 is acting as a major parent of two recombinant sequences- MH727236 and KY883318: meanwhile minor parent for MH727235 and KY883333 (Fig. 6). Overall, the PNRSV RNA1 understudy (LC382449) did not exhibit any strong sign of recombinant origin assisted as a minor parent.
Compared to the PNRSV RNA2 understudy, RDP version 4.97 detects fifteen recombination signals in twelve unique events in other PNRSV RNA2 sequences from different geographical locations (Table 3). The PNRSV RNA2 sequence had GenBank Acc. No. MH727231 (China, cherry) and MH727230 (China, cherry) were found potential recombinant sequence isolated from cheery, which was detected by five and four methods of RDP version 4.97 viz., RDP, GENECONV, BootScan, MaxChi, and Chimaera (Table 3). The RNA2 sequence with Acc. No. MH727231 (China, cherry) is most probably originated due to the recombination between MH727228 (China, Peach) and MH727232 (China, Mpl), as a major and minor parent, respectively (Fig. 6). Similarly, the PNRSV RNA2 sequence having Acc. No. MH727230 (China, Cherry) was found to be another potential recombinant generated by the recombination of MH727231 (China, Cherry) and MH727232 (China, Myrobalan plum) as a major and minor parent, respectively (Fig. 6). Meanwhile the lowest p-values of 3Seq, BootScan, Chimaera, MaxChi, and GENCONV of 8.409 × 10–52, 5.153 × 10–26, 3.026 × 10–23, 1.121 × 10–18 and 3.020 × 10–17 respectively reflects highest probability of MH727231 (China, cherry) is derived due to recombination between MH727228 (China, Peach) and MH727232 (China, Myrobalan plum) sequences (Table 3). There was no evidence of a recombination event in the PNRSV RNA2 understudy.
RDP version 4.97 reveals seventeen recombination signals in seventeen unique events in various PNRSV RNA3 sequences from different geographical regions, compared to the PNRSV RNA3 (LC382468, India, Apricot) understudy (Table 3). The PNRSV RNA3 sequence has GenBank Acc. No. FJ610344 (China) and KY883335 (Australia) were found to be the most potential recombinant sequence isolated from peach and rose, respectively. They were detected by six and five methods of RDP version 4.97 viz., GENECONV, BootScan, MaxChi, Chimaera, SiScan and 3Seq, respectively (Table 3). The RNA3 sequence with Acc. No. KY883335 (Australia) was originated due to the recombination between FJ546090 (Mexican, peach) and MH427285 (Australian, Apple) as a major and minor parent, respectively (Fig. 6). Similarly, the PNRSV RNA3 sequence with Acc. No. FJ610344 (China, Rose) was found to be another potential recombinant originating by the recombination of KT444703 (China, Sweet cherry) and KU977379 (China, Myrobalan plum) as a major and minor parent, respectively (Fig. 6). But the lowest p-values of 3Seq, SiScan, MaxChi, BootScan, and GENCONV, of 4.583 × 10–41, 4.722 × 10–34, 8.452 × 10–15, 1.556 × 10–14 , and 2.351 × 10–13 respectively reflects that KY883335 (Australia) is strongly recombination RNA3 sequence (Table 3). In this analysis, we found that the PNRSV RNA3 sequence under study acts as a major parent for the origination of a putative recombinant FJ546092 (Mexican, Peach) RNA3 sequence, but the chances of occurrence are low because it is detected by only one RDP method viz., GENCONV having p-value 1.093 × 10–02.
The phylogenetic NJ analysis revealed PNRSV RNA1 has two major clades, with clade 1 having the RNA1 Indian isolates from apricot, which is the closest relative of the PNRSV sequences isolated from the sweet cheery of China (Fig. 7a). This study also revealed that clade 1 has phylogenetically most similar RNA1 sequences from diverse origins, including China, Australia, the Czech Republic, Canada, and India (Fig. 7a). The clade2 has three less similar sequences belonging to China, the USA and the Czech Republic. In the phylogenetic analysis, the prune dwarf virus RNA1 is found on a distinct branch as an out-group.
Similarly, the phylogenetic NJ analysis for PNRSV RNA2 Indian isolates from apricot revealed the most closely related phylogenetic relationship with PNRSV sequences isolated from Czech Republic sour cherry (Fig. 7b). The PNRSV RNA2 sequences were similarly found to be divided into two clades. Sequences from China, the United States, Australia, the Czech Republic, Canada, and India make up the first clade of highly similar sequences (Fig. 7b). Furthermore, the PDV RNA2, shown on a distinct branch as an out-group, is close to the second clade of less comparable sequences (from China and the Czech Republic).
Meanwhile, the NJ phylogenetic analysis for the PNRSV RNA3 of Indian isolates from apricot under study clearly showed that it is phylogenetically closest to the already reported PNRSV genomic RNA3 sequence isolated from the Indian rose (Fig. 7c). The complete genomic RNA3 sequences are categorized into three clades, and the two bigger clades contain thirteen sequences each. The third and smallest clade contains three sequences close to the PDV RNA3 shown on a separate branch as an out-group. The Indian isolate comes under the first clade, with sequences from diverse origins like India, China, Mexico, Canada, the Czech Republic, Spain, and the USA.
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