Abstract
Whole-genome sequencing of an isolate of Mandarivirus infecting the sweet orange [Citrus sinensis (L) Blanco] in the western part of India (Pune) was done. The single-stranded positive-sense RNA genome of Indian citrus ringspot virus (ICRSV) Pune has 7,560 nucleotides (nt), excluding a poly(A) tail, comprised of 27.98% (2,115 nt) A, 32.12% (2,428 nt) C, 19.68% (1,488 nt) G, and 20.22% (1,529 nt) T residues. The genome, organized into six open reading frames (ORFs), shares 97.7% sequence identity with the complete genome of the ICRSV K1 isolate (AF406744.1) infecting the kinnow (Citrus reticulate Blanco, a hybrid between King and Willow mandarins) in north India. The ICRSV Pune genome formed a complex secondary structure with a large number of unpaired cytosine-rich regions, and recombination analysis highlighted potential recombination in the ICRSV genome.
GENOME ANNOUNCEMENT
Mandarivirus is a monotypic genus with Indian citrus ringspot virus (ICRSV) as the type species. In view of the occurrence of ringspot disease in C. sinensis (L) Blanco cultivars from various parts of India (2), whole-genome sequencing of an ICRSV isolate infecting the sweet orange (C. sinensis) from Pune, India, was undertaken. Leaves showing yellow ringspot symptoms from 1-year-old grafted plants were taken for RNA isolation, amplification, and sequencing of the viral genome using overlapping primers. The complete genome of ICRSV Pune showed a sequence identity of 97.7% with previously sequenced ICRSV K1 isolate (AF406744.1) (6). The positive-sense single-stranded RNA (ssRNA) genome was comprised of 7,560 nucleotides (nt), excluding the poly(A) tail. It had 27.98% (2,115 nt) A, 32.12% (2,428 nt) C, 19.68% (1,488 nt) G, and 20.22% (1,529 nt) T residues. Potexviruses in the family Alphaflexiviridae showed the closest similarity (65 to 87%). Viral RNA genome formed a complex secondary structure with 228 stems, 135 interior loops, 124 hairpin loops, 109 multiloops, and 69 bulges. The high cytosine (32.12%) and low guanosine (19.68%) content resulted in large number of unpaired cytosine-rich regions throughout the secondary structure. Such regions may function in RNA-protein interactions (4). The genome is organized into six open reading frames (ORFs) on the positive strand with 5′ and 3′ untranslated regions (UTRs). The 5′ UTR is 78 nt starting with the sequence GAAAA, which is commonly found in potexviruses. ORF1 at the 5′ end (4,977 nt; nt 79 to 5055) has a methyl transferase (Met) at the N terminus, followed by an AlkB domain, and viral helicase 1 and RdRP_2 domains. The Met, helicase, and RdRP_2 domains in ORF1 were highly conserved across the genera in the family Alphaflexiviridae and play important roles in viral mRNA cap synthesis (1), template recruitment, and negative-strand synthesis in RNA (3), respectively. On the other hand, viral AlkBs maintain the integrity of the viral RNA genome (7). Overlapping ORFs 2, 3, and 4 encode triple-gene-block (TGB) proteins, viz TGB1 (nt 5063 to 5740 [678 nt]), TGB2 (nt 5718 to 6047 [330 nt]), and TGB3 (nt 5974 to 6156 [183 nt]). The amino acid motif GDPSHSLPFGGNYRDGSKVVHYN was found across the genera of Alfaflexiviridae in the movement protein domain of TGB2. Penultimate ORF5 (nt 6179 to 7156 [978 nt]) coded for coat protein (CP), which showed the domains of the “Flexi_CP superfamily” (E value, 8.8e−40), potexvirus and carlavirus coat protein signature KFAAFDFFHGVLSPAS, a proline-rich region, an extensin-like region, and a core binding factor-beta subunit. ORF6 (nt 6856 to 7524 [669 nt]) partially overlaps ORF5 and encodes a nucleic acid binding protein (NaBP) of 23 kDa. Viral NaBP (3.7e−53) and Carla_C4-Carlavirus putative NaBP (1.8e−05) domains were observed in this ORF. The 3′ UTR (nt 7525 to 7560) is 36 nt long followed by a poly(A) tail. Recombination analysis using RDP3 (5) revealed ICRSV Pune as a potential recombinant of Lily virus X (NC_007192) and Alternanthera mosaic virus (NC_007731) in ORF1 (breakpoint 732 to 830 nt; P value, 4.881 × 10−2 to 9.862 × 10−2) and Lily virus X and Garlic virus C (NC_003376) in TGB2 (breakpoint, 5,777 to 5,999 nt; P value, 4.121 × 10−14 to 1.103 × 10−4). Since Mandarivirus is found only on the Indian subcontinent, it is essential to generate more genome data to deliver information at evolutionary and epidemiological scales.
Nucleotide sequence accession number.
The GenBank accession number for the nucleotide sequence determined in this study is HQ324247.
ACKNOWLEDGMENTS
We are grateful to the Division of Plant Pathology, Indian Agricultural Research Institute (IARI), for providing the facility.
We thank the Indian Council of Agricultural Research (ICAR) outreach program for financial support. The first author is also thankful to IARI for providing a research fellowship.
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