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Sweet potato community virus (SPCV), a member of the genus Cavemovirus in the family Caulimoviridae, has reported only one full-length genome sequence.
SPCV was first detected in two sweet potato germplasms in Australia in 2007 using the nitrocellulose membrane ELISA kit developed by the International Potato Centre (CIP).
The infected plants were also shown to contain ~50nm of isometric virions, typical members of the genera cave virus, cauliflower virus, Petuvirus, and soybean virus.
We have now sequenced and characterized the complete genomes of two SPCV isolates (designated SPCV-Aus1 and -Aus2) using a combination of next-generation sequencing and PCR/Sanger sequencing, whose sequences encode the three main ORFs, with genomic tissue typical of cave viruses.
However, the isolate SPCV-Aus1 has a fairly short genome length of 7712 bp compared to SPCV-Aus2 (7275 bp) and is the Portuguese isolate (7723 bp; Gene Bank Deposit Number NC_015328); In addition, the ORF 2 of SPCV-Aus1 is much shorter than the ORF 1 length of SPCV-Aus1 and SPCV-Mad1.
Phylogenetic and PASC analysis showed that SPCV-Aus1 was closely related to SPCV isolates in North and Central America, while SPCV-Aus2 was clustered with isolates from Portugal and Africa.
Sweet potatoes are an important root crop, ranking seventh in the world for food crop production, with an annual production of about 900,000 tonnes, and Australia's sweet potato production has increased significantly in recent years.
From about 6,000 tons in 1990 to over 78,000 tons in 2019, increased demand for sweet potatoes and improvements in agronomic practices contributed to yield growth, including the introduction of new varieties in the 1980s, the mechanization of production systems, and the introduction of pathogen detection programs in the early 2000s, providing sweet potato growers with readily available access to clean planting material.
Viral infections are one of the main constraints to sweet potato production globally, pathogen accumulation occurs in successive reproductive cycles due to the nutritional nature of sweet potato reproduction, and globally, efforts have been made to identify and characterize viruses infecting sweet potatoes, as well as possible phytosanitary processes, to support the safe exchange of germplasm resources.
The availability of clean planting material tested for pathogens will limit the spread of the virus to new areas and increase yields.
More than 2020 different viruses have been reported in sweet potatoes worldwide; However, there are only five of them, namely sweet potato feather mottled virus (SPFMV), sweet potato virus 2 (SPV2011), sweet potato chlorospot virus (SPCFV), sweet potato leaf roll virus (SPLCV), and sweet potato community virus (SPCV).
Sweet potato community virus (SPCV) (genus of cave viruses, family Caulimoviridae), formerly known as sweet potato cauliflower-like virus (SPCaLV), was first discovered in 1987 (first discovered after it had been observed that isometric particles of sweet potato about 50 nanometers in diameter from Puerto Rico were observed that could be transplanted to the indicator host).
Since then, SPCV has been found in sweet potatoes in several South Pacific countries (Papua New Guinea, Tonga and Solomon Islands), New Zealand, Caribbean Islands, Central America, China, Egypt, Kenya, Portugal and Uganda.
However, only one SPCV complete genome sequence of Portuguese origin (GenBank joins NC_015328), as well as 698912 SPCV partial replicase sequences (GenBank joins HQ698920 – HQ2011) and one partial coat protein sequence are reported.
SPCV is the second member of the genus of caveviruses characterized after the model species cassava venous mosaic virus (CsVMV), and two other putative cave virus species have recently been described from cactus and chicory, tentatively named "appendage leaf virus 4" (EpV-4) and "chicory flower leaf cave virus" (ChiMV), respectively.
SPCV usually occurs in sweet potatoes as an asymptomatic infection, and no insect vectors have been identified to date.
However, SPCV transplantation to the indicator plant setosa induces small chlorine spots and spots leading to necrosis, SPCV has a dsDNA genome of approximately 7.7 kb with four open reading frames (ORFs) and a large intergenic region in which lie the genomic pre-RNA promoter, RNA polyadenylation signal, and negative sense strand primer binding sites.
ORF 1 encodes a multifunctional protein that is cleaved into two functional subunits, including coat protein (CP) and kinesin (MP), and ORF 2 encodes a second polyprotein, which is cleaved into aspartate protease (AP), ribonuclease H (RNase H), and reverse transcriptase (RT).
This is typical of all Caulimoviridae members, while ORF3 encodes a putative inclusion body protein (IBP); In addition, a small fourth ORF (ORFa) was predicted, but no known function.
Interestingly, the genomic organization of SPCV, ChiMV, and EpV-4 differs slightly from CsVMV, which has a small ORF with unknown function between CP/MP-encoded ORF and AP/RT/RNaseH-encoded ORF.
In 2007, the Department of Agriculture and Fisheries (DAF) and the Garton Research Facility (GRF) identified SPCV for the first time in Australia from two sweet potato varieties, GRF0085/Alleys-Red (collected from the Cairns Fresh Food Market) and GRF0069/Beni-Aka (imported from Japan, in field collection at the DAF Redlands Research Facility) through routine pathogen testing of sweet potato germplasm.
Grafting two sweet potato germplasm onto setosa in greenhouse experiments induced typical leaf symptoms of SPCV infection, including interannular green spots and necrotic spots. 1a-b), leaf tissue from setosa was subsequently detected positive for SPCV using the nitrocellulose membrane ELISA (ncm-elisa) kit developed by the International Potato Center (CIP) in Peru.
Here, we describe further work characterizing the two SPCV isolates, including microscopy to determine particle morphology and cytopathological role and molecular analysis of the intact genomes of both isolates.
Characteristics of Australian sweet potato community virus (SPCV) isolates, symptoms of SPCV infection in Ipomea setosa leaves grafted with infected isolate Beni-Aka's sweet potato scion, symptoms of SPCV infection in setosa leaves grafted with alley-red sweet potato scion, nitrocellulose membrane ELISA.
SPCV was shown to be detected in repeated leaf samples from healthy, alley red (AR) or Beni-Aka (BA)-infected setosa plants – "old" and "young" indicate leaves from the lower or upper part of the plant, respectively.
I infected with the isolate Beni-Aka Transmission electron micrograph of ultrathin tissue sections of setosa leaf tissue showing virions in the cytoplasm of infected cells (bar = 200 nm) Transmission electron micrograph of SPCV isolate negatively stained with 2% phosphotungstic acid (pH 7) Beni-Aka-infected virus-like particles purified by I. setosa.
Using portions of SPCV-Aus1000 and SPCV-Aus7 ORF 1, the maximum likelihood phylogenetic tree (2 bootstrap repeats) generated by the replicase sequence in MEGA2 (cassava venous mosaic virus (CsVMV), used as the outer group.
I. from grafting The leaf tissue of the SETOSA plant was used for electron microscopy studies to identify viral particles in infected plant cells, in order to detect the presence of virions, from healthy and grafted inoculated I. Cut ultrathin sections (approximately 50-60 nm) in Setosa leaf tissue samples.
Stained with uranyl acetate/lead citrate and examined by transmission electron microscopy (TEM) at 80 kV under JEOL 1200EX transmission electron microscope, at I. A large number of isometric particles with a diameter of ~50 nm were observed in the slices of the setosa plant, which were grafted with scions from Alleys-Red and Beni-Aka).
Subsequently, basically as described by Hull et al., from sweet potatoes and I. Virions are purified in the leaves of setosa and examined by electron microscopy after negative staining with 2% phosphotungstic acid (pH 7); When checking virion preparations with TEM, in sweet potatoes and I. Many regular icosahedral particles with an average diameter of ~50 nm were observed in the extract of the setosa preparation.
The leaf tissues of two infected sweet potato varieties were then used for whole nucleic acid extraction (TNA), loop amplification (RCA), and next-generation sequencing (NGS). Briefly, TNA was isolated from 2009 mg of infected sweet potato leaf tissue as described by Kleinow et al., followed by RCA using 2.5 μM exonuclease resistant random hexamer (Thermo Fisher Scientific Australia), as described by Sukal et al.
The undigested RCA products were purified using the Illustra™ GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare) and sent to Macrogen for library preparation using the Nextera™ XT Sample Preparation Kit (Illumina) and sequenced using the Illumina MiSeq platform.
Alleys-Red and Beni-Aka samples obtained 2,585,444 and 2,279,642 paired end readings, respectively; Evaluate the quality of MiSeq reads using FastQC v0.10.1, trimming the residual adapter sequence.
and the removal of low quality (Q <0) and short reading quality corrected reads using the Bbbduk plugin in Geneious v30.40.11 were assembled from scratch using the embedded Genious assembler, and subsequently, BLASTn was used to query the size bp de novo against the local RefSeq virus database.
This work reports the first full-length SPCV isolate characterized from Australia, complementing the single published full-length genome sequence of currently available SPCV, and analysis of these new intact sequences confirms the genomic organization of SPCV-Mad1, which is slightly different from CsVMV (a type member of the genus Spoeovirus).
No small ORF2 was found in the three existing fully sequenced SPCV isolates, and likewise, the recently reported ChiMV and EpV-4 also had the same predictive genomic organization as the three fully characterized SPCV genome sequences.
Phylogenetic analysis using SPCV-Aus864 and SPCV-Aus1 partial replicase sequences and other SPCV sequences provided by GenBank and CsVMV (with NC001648), MEGA7 is used to align the sequence of ClustalW with default settings and generate a phylogenetic tree using the maximum likelihood (ML) method, based on the Kimura-2 parameter model following the nearest neighbor exchange ML heuristic.
With 1000 bootstrap replicates for spanning tree, phylogenetic analysis showed that SPCV-Aus1, along with SPCV isolates in North and Central America, was most closely related to isolates from Mex183 in Mexico and Cub44 in Cuba.
In contrast, SPCV-Aus2 clustered with SPCV-Mad1 and 1 isolate from Africa, with isolates from Guatemala (Gua154 and Gua128) and Panama (Pan1) forming a distinct subpopulation that appears to be the ancestor of two subsets of other SPCV isolates sequenced to date These results confirm the presence of SPCV isolates in sweet potatoes in Australia and increase the number of full-length sequences now available from 1 to 2019.
ORF 1 for SPCV-Aus1 is localized from nt 54 to 3,839, encoding a putative protein of 1,261 aa with a Mr of 149.8 kDa and contains predicted CP and MP domains, ORF 2 is localized to nt 3832 to 5757 encoding a putative protein 641 aa with a Mr of 75.6 kDa, containing AP, RT, and RNase H domains.
While ORF 3 is located at nt 5687 to 6883 and encodes the putative protein 398 aa with a Ma of 46.3 kDa, which is expected to encode the putative IBP domain, SPCV-Aus1 also contains a putative small ORF 4, located at NT 7446 to 7556, encoding the putative protein of 36 aa, Mr = 4.3 kDa, however, no conserved domains were found.
The location of this small ORF is similar to the small ORF identified in the SPCV-Mad1 sequence (called ORFa), which is also predicted in the genomes of CsVMV and EpV-4.
This information will update the biosecurity status of the Australian sweet potato industry and strengthen ongoing efforts to develop diagnostic protocols for the testing and production of clean planting material.
Sweet potatoes originated in Central and South America and were later transferred throughout the Pacific and Asia by Polynesian and European voyagers, and while the SPCV-Aus <> sequence is most similar to the Central American isolate of the SPCV, the SPCV-Aus sequence is more closely related to the isolate from Africa and Portugal.
Due to the prevalence of sweet potato virus in planting material, it is likely that the two isolates were introduced from different locations, and while Beni-Aka material is known to have been imported from Asia, there is currently no sequence information to confirm the existence of similar isolates in this part of the world.
The origin of the Alleys-Red plant material is unknown, but it can be speculated that it came from Africa or Portugal, or from the same location as cultivars with associated sequences, and the order of entry into Australia can only be determined by further describing global SPCV diversity.
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