Extraction Of Double-stranded RNA Research Articles

First Report of Hibiscus Chlorotic Ringspot Virus infecting Hibiscus rosa-sinensis in Italy.

Hibiscus chlorotic ringspot virus (HCRSV) belongs to the Betacarmovirus genus of the Tombusviridae family and is a positive-sense monopartite single-stranded RNA virus. HCRSV was first described in a hibiscus variety imported into the United States from El Salvador. The HCRSV natural host range is limited to species belonging to Malvaceae family, in particular Hibiscus rosa-sinensis, H. manihot, H. diversifolius and H. syriacus. In September 2023, leaf mottle and chlorotic spots were observed on the leaves of two H. rosa-sinensis plants located in a public garden of Ercolano (Naples city, Southern Italy). Two leaves, one from each symptomatic plant, were pooled and submitted to double-stranded RNA extraction using a Viral Gene-Spin Viral DNA/RNA Extraction Kit (iNtRON, Korea), followed by cDNA library preparation with the TruSeq Stranded Total RNA (Illumina, USA). Sequencing on the Illumina NovaSeq 6000 platform (Illumina, USA) with 150-bp paired-end reads yielded 22,578,913 raw reads. 21,413,571 clean reads were obtained by quality control on the sequencing data was performed with the software FastQC (v. 0.11.5). Then low quality bases and adapters were removed with the software BBDuk in the BBTools package setting a minimum read quality of 25 and minimum read length of 35 bp. The resulted filtered reads were used to assembly viral genome by using two different algorithms (Metaspades and RNAViral) implemented in SPAdes (v. 3.15.3). Of the total contigs de novo assembled, the two algorithms implemented identified respectively 340 and 559 contigs related to viruses. BLASTn analysis of the contigs identify one coting of 3965 nt covering 97% to 100% of the whole genome sequence of nine HCRSV isolates, with percentage of identity of 87.8-95.2%. No other plant viruses or viroids have been identified during bioinformatic analysis. To confirm the result, a full-length genomic sequence of the Italian HCRSV isolate (named Ita-1), was obtained by reverse transcription polymerase chain reaction (RT-PCR) using specific primers designed on the sequence of the assembled contigs. The PCR products were sequenced using the Sanger method (Microsynth Seqlab, Germany) in both directions. The obtained full-length genomic sequence of the HCRSV isolate (Acc. No. OR981792) was 3910 nt in length. Maximum-likelihood phylogenetic trees inferred from the whole genome sequence showed that Ita-1 clustered closely with HCRSV isolates. The leaf samples were further analyzed using a HCRSV ELISA kit (Agdia, USA). Healthy H. rosa-sinensis leaves were taken as a negative control and buffer solution as a blank control. The results showed a positive reaction for the two symptomatic plants (OD = 1.345 ± 0.010 at 405 nm) relative to the negative (OD = 0.097) and blank (OD = 0.065) controls. Overall, the results of serological and molecular analyses supported that symptoms observed in H. rosa-sinensis were strictly associated to HCRSV infection. Four viruses have been reported in H. rosa-sinensis in Italy so far: eggplant mottled dwarf nucleorhabdovirus (EMDV), hibiscus latent Fort Pierce virus (HLFPV), hibiscus latent ringspot virus (HLRSV), hibiscus latent Singapore virus (HLSV). To our knowledge, this is the first report of HCRSV in H. rosa-sinensis in Italy, where probably infected plants were accidentally introduced. At present, HCRSV has been reported in three continents (Asia, Oceania and North America) and the number of countries where the virus has been detected is likely to increase rapidly as a result of increased surveillance and availability of diagnostic methods. This study will help the management of viral diseases on H. rosa-sinensis in Italy.

Improved Double-Stranded RNA Extraction and Hyphal Tip-Touch Techniques for the Detection of Mycoviruses

Mycoviruses infect a wide range of fungal species, and many of them have double-stranded RNA (dsRNA) genomes. Using a dsRNA technique makes it easier to detect unknown mycoviruses in fungi. Previously, a few studies reported dsRNA extraction methods from fungal tissues, but the procedures required a significant amount of starting material and time, particularly in growing fungal cultures. In this study, we report an efficient and quick dsRNA extraction method that is reproducible to extract quality dsRNA of mycoviruses from a small amount of fungal culture in a short time. Extracted dsRNA was subsequently used in Illumina sequencing and reverse transcription-polymerase chain reaction assays, followed by Sanger sequencing, to confirm the identity of the mycovirus. Additionally, a rapid and efficient technique named hyphal tip-touch (HTT) was developed to identify known mycoviruses without the need for dsRNA extraction. The HTT technique uses the edge of mycelia as a template for preparing complementary DNA in the reverse transcription reaction, which was used in the polymerase chain reaction assay followed by sequencing. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Do Totiviruses of the veil of Flor yeasts provide an evolutionary advantage?

The main objective of this work was to isolate and characterize viruses that infect both Saccharomyces and other Flor veil-forming genera and suggest the possible consequences they can have on the processes of formation of the Flor veil of typical D.O. Jerez-Xèrés-Sherry wines. Results obtained show that different Flor veil yeast strains isolated in Fino wines and in Amontillados presented a great resistance to ethanol and all of them presented infection by Totiviruses. These were identified after the extraction of double-stranded RNA and amplified by multiplex PCR with specific primers for the most common viruses involved in the killer factor. Subsequently, the same strains of both the S. cerevisiae species and Wickerhamomyces anomalus were subjected to an antiviral treatment with ribavirin. After microbiological analysis with Methylene Blue (MB) plates and by rt-PCR, it was determined that most of them did not show killer factor and the viruses involved in it had been correctly eliminated under the treatment with ribavirin. Subsequently, competition and implantation experiments of the treated and untreated yeasts were carried out against their controls. The wines inoculated with yeasts infected by Totiviruses showed to be dominant in all cases.

Complete Genome Sequencing and Infectious cDNA Clone Construction of Soybean Mosaic Virus Isolated from Shanxi.

Soybean mosaic virus (SMV) is the predominant viral pathogen that affects the yield and quality of soybean. The natural host range for SMV is very narrow, and generally limited to Leguminosae. However, we found that SMV can naturally infect Pinellia ternata and Atractylodes macrocephala. In order to clarify the molecular mechanisms underlying the cross-family infection of SMV, we used double-stranded RNA extraction, rapid amplification of cDNA ends polymerase chain reaction and Gibson assembly techniques to carry out SMV full-length genome amplification from susceptible soybeans and constructed an infectious cDNA clone for SMV. The genome of the SMV Shanxi isolate (SMV-SX) consists of 9,587 nt and encodes a polyprotein consisting of 3,067 aa. SMV-SX and SMV-XFQ008 had the highest nucleotide and amino acid sequence identities of 97.03% and 98.50%, respectively. A phylogenetic tree indicated that SMV-SX and SMV-XFQ018 were clustered together, sharing the closest relationship. We then constructed a pSMV-SX infectious cDNA clone by Gibson assembly technology and used this clone to inoculate soybean and Ailanthus altissima; the symptoms of these hosts were similar to those caused by the virus isolated from natural infected plant tissue. This method of construction not only makes up for the time-consuming and laborious defect of traditional methods used to construct infectious cDNA clones, but also avoids the toxicity of the Potyvirus special sequence to Escherichia coli, thus providing a useful cloning strategy for the construction of infectious cDNA clones for other viruses and laying down a foundation for the further investigation of SMV cross-family infection mechanisms.

Investigation of Enteric Viruses in the Feces of Neotropical Migratory Birds Captured on the Coast of the State of Pará, Brazil

ABSTRACT Migratory birds can become long-distance vectors for a wide range of microorganisms and can cause human disease, being the Brazilian coast a gateway for northern migratory birds. These animals are considered natural reservoirs of different viruses that cause important diseases, being relevant research of viral pathogens in migratory birds to epidemiology surveillance. The objective of the study was to investigate the presence of avian rotavirus (AvRV), avian reovirus (ARV) and picobirnavirus (PBV) in Neotropical migratory birds captured on the coast of Brazil. A total of 23 individual fecal samples of the migratory birds species Calidris pusilla (20 birds), Numenius phaeopus (1 bird) and Charadrius semipalmatus (2 birds) were collected. Fecal suspensions were prepared from the collected samples for subsequent extraction of double-stranded RNA (dsRNA), which was subjected to polyacrylamide gel electrophoresis (PAGE) and reverse transcription polymerase chain reaction (RT-PCR). The electrophoretic profiles were not detected by PAGE, and the amplification for the studied viruses PBV, ARV and AvRV (specie D, gene VP6 and NSP4) were negative. Positivity for AvRVD, VP7 gene was of 4.35% (1/23) for the migratory bird Calidris pusilla. After sequencing and building the tree of phylogenetic relationships avian Rotavirus Group D identified in this study was phylogenetically related and grouped into one branch, together to previously reported AvRVD from Brazil in chicken flocks with 99.8% nucleotide and 100% amino acid similarities.

First Report of physostegia chlorotic mottle virus on Tomato (Solanum lycopersicum) in Germany

In September 2015, a tomato sample collected in the German state of Hesse was sent to the Julius Kuhn-Institut for analysis. While the fruits showed marbling and discoloration, the leaf samples from this plant did not show any obvious symptoms. Transmission electron microscopy (TEM) revealed the presence of bullet-shaped virus particles indicating the presence of a rhabdovirus. However, immunosorbent electron microscopy using antiserum JKI-1073 for Eggplant mottled dwarf virus (EMDV) could not confirm EMDV infection. The virus was mechanically transmitted to Nicotiana benthamiana, N. clevelandii, and Chenopodium quinoa inducing yellowing and leaf deformation, while mechanical transmission to N. occidentalis (P1 and 37b) failed. Extraction of double stranded-RNA (dsRNA) followed by random-PCR (Froussard 1992), cloning of PCR products, and sequencing failed to reveal any virus sequences. Total RNA was extracted from infected N. benthamiana, followed by ribo-depletion, library preparation and submission for next-generation sequencing (NGS) using an Illumina MiSeq platform as described by Knierim et al. (2017). De novo assembly of the trimmed reads was done with Geneious v 10.1.3 (Biomatters LTD, NZ). Using MEGA BLAST, 13 contigs showed between 95.6 and 98.5% similarity with physostegia chlorotic mottle virus (PhCMoV) isolate PV-1182 (accession no. KX636164). The complete PhCMoV genome (13,321 nt length) was assembled by mapping reads to this reference genome and used to design PhCMoV-specific RT-PCR primers for detection (HZ-343 5′-CGGTGAGTGGGGCAACTAAT-3′/HZ-344 5′-AGCGATGGGGTCTAGTGTCT-3′). RT-PCR confirmed the presence of PhCMoV in the test plants resulting in amplicons of approximately 875 bp. In August 2016, similar symptoms on tomato fruits were observed by a different grower in Hesse. The presence of PhCMoV was confirmed by TEM and RT-PCR. Additionally, the PCR products were sequenced and showed 97% identity to KX636164. Surprisingly, reanalysis of a tomato sample from 2003 that was infected by a hitherto unknown rhabdovirus using NGS also confirmed infection with PhCMoV. This sample also originated from Hesse although the original grower is unknown. The complete genome of the 2003 PhCMoV sample was assembled following the same methods described above. Pairwise comparison between the genomes of 2015 and 2003 isolates resulted in 99.7% nucleotide identity and 96.9% when compared with KX636164. These findings indicate the presence of PhCMoV in tomato in Germany for a long time albeit isolated occurrences in different production areas. PhCMoV was recently identified from Physostegia virginiana plants showing leaf deformation and severe chlorotic and mottle symptoms in Austria (Menzel et al. 2016). However, it is not known if there is a link between PhCMoV isolates infecting P. virginiana and tomato as the routes of transmission and dissemination are currently unknown. The sequences from this report were deposited in GenBank (accession nos. KY706238 and KY859866 [full-length sequences], KY882263 and KY882264 [partial sequences]). To our knowledge, this is the first host record of PhCMoV in tomato and a new country record for Germany.

A non-phenol–chloroform extraction of double-stranded RNA from plant and fungal tissues

Double-stranded RNA (dsRNA) molecules of viruses are found in nature at a very high frequency. Their detection in plants and fungi has been carried out with difficulty due to the complicated dsRNA extraction techniques used commonly which includes phenol–chloroform extractions. In this study, an extraction method for isolation of dsRNA is described that is free of phenol and chloroform. A lysis buffer, containing β-mercaptoethanol and polyvinylpolypyrrolidone (PVPP-40), was added to homogenised tissues and the subsequent supernatant was filtered through a cellulose CF-11 mini-column. DsRNA molecules were separated based on the differing affinity of nucleic acids for the cellulose CF-11 resin in 20% ethanol buffer. This easy, rapid and cheap technique has been successfully tested on fungi and plants containing different dsRNA virus molecules, indicating the possibility of a wide use of the method.

A new method for extraction of double-stranded RNA from plants

The occurrence of high molecular weight double-stranded RNA (dsRNA) in plants is associated with the presence of RNA viruses. DsRNA is stable, can be extracted easily from the majority of plant species and provides an excellent tool for characterization of novel viruses that are recalcitrant to purification. Several protocols have been developed for dsRNA purification, the majority of which are based on extraction with phenol and chloroform. We have developed a protocol for dsRNA extraction based on a lithium salts buffer that does not require organic solvents other than alcohols. The method yields comparable amount of dsRNA to protocols described previously and yields consistently dsRNA from Vaccinium hosts that have been recalcitrant to dsRNA purification using traditional protocols. The quality of the dsRNA purified is such that it can be used for downstream enzymatic reactions including reverse transcription-polymerase chain reaction and cloning.

CHARACTERIZATION OF APHID-TRANSMITTED VIRUS ASSOCIATED WITH BLACK RASPBERRY DECLINE IN OREGON

A serious disease of black raspberry (Rubus occidentalis) was observed in Oregon in the last decade. Plants showing mosaic symptoms declined rapidly and in many cases died over a period of several years. Double-stranded RNA extraction from symptomatic black raspberry revealed the presence of two high molecular weight bands, which were cloned and partially sequenced. Sequence analysis disclosed the presence of Black raspberry virus (BRV). A newly planted field of black raspberries in Oregon was studied to assess the rate of spread of BRV. The timing of BRV infection as it relates to aphid populations and flights was also determined. Testing of nearby vegetation identified several symptomless Rubus hosts of BRV. It was determined that BRV spreads rapidly with a low aphid threshold and was associated with symptoms of decline in black raspberries in Oregon.

Identification, Characterization, and Detection of Black raspberry necrosis virus

ABSTRACT A serious disease was observed in black raspberry (Rubus occidentalis) in Oregon in the last decade. Plants showing mosaic symptoms declined rapidly and, in many cases, died after several years. Double-stranded RNA extraction from symptomatic black raspberry revealed the presence of two high molecular weight bands which were cloned and sequenced. Sequence analysis disclosed the presence of a novel virus that was tentatively named Black raspberry decline-associated virus (BRDaV). The complete sequences of the two genomic RNAs, excluding the 3' poly-adenosine tails, were 7,581 and 6,364 nucleotides, respectively. The genome organization was identical to that of Strawberry mottle virus, a member of the genus Sadwavirus. The C terminus of the RNA 1 poly-protein is unique within the genus Sadwavirus, with homology to AlkB-like domains, suggesting a role in repair of alkylation damage. A reverse-transcriptase polymerase chain reaction test was designed for the detection of BRDaV from Rubus tissue, and tests revealed that BRDaV was associated consistently with the observed decline symptoms. While this publication was under review, it came to our attention that scientists at the Scottish Crop Research Institute had molecular data on Black raspberry necrosis virus (BRNV), a virus that shared many biological properties with BRDaV. After exchange of data, we concluded that BRDaV is a strain of BRNV, a previously described yet unsequenced virus. The North American strain was vectored nonpersistently by the large raspberry aphid and the green peach aphid. Phylogenetic analysis indicates that BRNV belongs to the genus Sadwavirus.

An improved reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of two cherry flexiviruses in Prunus spp.

A one-step reverse transcription-polymerase chain reaction (RT-PCR) procedure was developed to detect Cherry green ring mottle virus (CGRMV) and Cherry necrotic rusty mottle virus (CNRMV) in woody indicators and naturally infected Prunus spp. Viral RNA suitable for RT-PCR was obtained by a simple trapping method that did not require either extraction of double-stranded RNA (dsRNA) or total RNA, availability of virus antibodies, or purification of viral particles. Consensus primers, degenerate primers and virus-specific primers, whose designs were based on alignments of available cherry flexivirus sequences, were tested to amplify viral genomic fragments of six CGRMV isolates and one CNRMV isolate. RT-PCR allowed CGRMV detection in total RNA and viral RNA preparations equivalent to 400 μg and 4 μg of infected leaf tissue, respectively. CGRMV was detected in tender shoots, leaves, bark and root tips, and the strongest bands were obtained using young leaves. Detection was less consistent in summer when the temperature was elevated and plant tissues were old. A direct comparison of the RT-PCR and grafting assays indicated that the RT-PCR assay is sensitive, rapid and reliable. The method will improve the routine diagnosis of cherry flexiviruses in Prunus spp.

Extraction of Double-Stranded RNA from Plant Tissues Without the Use of Organic Solvents

Double-stranded RNAs (dsRNAs) are commonly isolated from plants and insects with toxic organic solvents such as phenol and chloroform that denature and remove proteins from aqueous solutions containing nucleic acids. This procedure was modified by replacing the toxic solvents with sodium dodecyl sulfate and potassium acetate (SDS/KOAc). Samples were homogenized in liquid nitrogen mixed with buffer and SDS at 65 C and then with KOAc at 0 C. A precipitate, consisting of SDS, KOAc, and proteins, was removed by centrifugation. The supernatant containing nucleic acids was collected and subjected to standard CF-11 cellulose chromatography to separate dsRNAs from other nucleic acids. The SDS/KOAc method produced comparable yields and purity of dsRNA products compared to the phenol/chloroform method and is considered a convenient, effective alternative for isolating dsRNA from plant tissues.

Rapid methods for comparing the double-stranded RNA genome profiles of bluetongue virus

Various double-stranded RNA extraction procedures, gel electrophoresis systems, and methods to detect the RNA bands in the gel were investigated to find the most rapid methods to obtain the genome profiles of bluetongue virus in small volumes (1–25 ml) of infected cell culture fluids. Rapid double-stranded RNA extraction procedures coupled with staining the acrylamide gel slabs with ethidium bromide or silver nitrate resulted in well-defined genome profiles from bluetongue virus infected cell cultures in 6–48 h. Radioactive labelling of viral RNA with 32P was time consuming, cumbersome and expensive. These techniques detect less than 0.5 μg of double-stranded RNA which can be obtained from one 1-ml well of a 24-well cluster plate of bluetongue virus infected cell monolayers. The methods were therefore suitable for rapid comparisons of the electropherotypes of multiple virus isolates.

A simple procedure for the extraction of double-stranded RNA from virus-infected plants

A simple procedure for the isolation of double-stranded (ds) RNA from virus-infected plants is described. The method is based on grinding plant tissue in 4% p-aminosalicylic acid and recovery of ds RNA by phenol extraction and precipitation with 30% ethanol. The presence of both negative and positive virus RNA strands in RNA fractionated in agarose gels was verified by Northern blot hybridization with polynucleotide kinase labelled genomic RNA or complementary DNA (cDNA) probes. The procedure enabled detection of three major ds RNA species (MWs 4.2, 1.05 and 0.48 × 10 6) and at least 4 minor bands with estimated MWs of 3.5, 2.5, 2.2 and 2.0 × 10 6 in Nicotiana tabacum plants systemically infected with tobacco mosaic virus (TMV). Cucumber mosaic virus (CMV)-infected Pachystachys coccinea plants contained 2 minor bands of MWs 0.49 and 0.35 × 10 6 in addition to the previously described 4 major ds RNAs and ds CARNA 5 (MW 0.22 × 10 6). The patterns of ds RNA are useful for diagnosing natural infections of CMV and TMV in N. glauca plants and of citrus tristeza virus in Citrus spp.

The release of viral genome RNA from virions of the cytoplasmic-polyhedrosis virus of Malacosoma disstria by acetone treatment

Acetone treatment of the CPV of Malacosoma disstria released the viral genome RNA from nonoccluded virions, virions liberated from polyhedra by alkali treatment, and virions still occluded in polyhedra; in the third case the RNA was retained within the polyhedra until they had been dissolved by alkali treatment. The components were separated by sucrose gradient centrifugation; typical 15 and 12 S viral RNA samples were obtained by this method. Acetone treatment may prove useful for the extraction of double-stranded RNA from virus-infected tissues.