Virus Surveys in Olive Orchards in Greece Identify Olive Virus T, a Novel Member of the Genus Tepovirus.

Field surveys were conducted in Greek olive orchards from 2017 to 2020 to collect information on the sanitary status of the trees. Using a high-throughput sequencing approach, viral sequences were identified in total RNA extracts from several trees and assembled to reconstruct the complete genomes of two isolates of a new viral species of the genus Tepovirus (Betaflexiviridae), for which the name olive virus T (OlVT) is proposed. A reverse transcription-polymerase chain reaction assay was developed which detected OlVT in samples collected in olive growing regions in Central and Northern Greece, showing a virus prevalence of 4.4% in the olive trees screened. Sequences of amplified fragments from the movement-coat protein region of OlVT isolates varied from 75.64% to 99.35%. Three olive varieties (Koroneiki, Arbequina and Frantoio) were infected with OlVT via grafting to confirm a graft-transmissible agent, but virus infections remained latent. In addition, cucumber mosaic virus, olive leaf yellowing-associated virus and cherry leaf roll virus were identified.

Investigating the Biological Relevance of In Vitro-Identified Putative Packaging Signals at the 5' Terminus of Satellite Tobacco Necrosis Virus 1 Genomic RNA.

Satellite tobacco necrosis virus 1 (STNV-1) is a model system for in vitro RNA encapsidation studies (N. Patel, E. C. Dykeman, R. H. A. Coutts, G. P. Lomonossoff, et al., Proc Natl Acad Sci U S A 112:2227-2232, 2015, https://doi.org/10.1073/pnas.1420812112; N. Patel, E. Wroblewski, G. Leonov, S. E. V. Phillips, et al., Proc Natl Acad Sci U S A 114:12255-12260, 2017, https://doi.org/10.1073/pnas.1706951114), leading to the identification of degenerate packaging signals (PSs) proposed to be involved in the recognition of its genome by the capsid protein (CP). The aim of the present work was to investigate whether these putative PSs can confer selective packaging of STNV-1 RNA in vivo and to assess the prospects of using decoy RNAs in antiviral therapy. We have developed an in planta packaging assay based on the transient expression of STNV-1 CP and have assessed the ability of the resulting virus-like particles (VLPs) to encapsidate mutant STNV-1 RNAs expected to have different encapsidation potential based on in vitro studies. The results revealed that >90% of the encapsidated RNAs are host derived, although there is some selectivity of packaging for STNV-1 RNA and certain host RNAs. Comparison of the packaging efficiencies of mutant STNV-1 RNAs showed that they are encapsidated mainly according to their abundance within the cells, rather than the presence or absence of the putative PSs previously identified from in vitro studies. In contrast, subsequent infection experiments demonstrated that host RNAs represent only <1% of virion content. Although selective encapsidation of certain host RNAs was noted, no direct correlation could be made between this preference and the presence of potential PSs in the host RNA sequences. Overall, the data illustrate that the differences in RNA packaging efficiency identified through in vitro studies are insufficient to explain the specific packaging of STNV-1 RNA.IMPORTANCE Viruses preferentially encapsidate their own genomic RNA, sometimes as a result of the presence of clearly defined packaging signals (PSs) in their genome sequence. Recently, a novel form of short degenerate PSs has been proposed (N. Patel, E. C. Dykeman, R. H. A. Coutts, G. P. Lomonossoff, et al., Proc Natl Acad Sci U S A 112:2227-2232, 2015, https://doi.org/10.1073/pnas.1420812112; N. Patel, E. Wroblewski, G. Leonov, S. E. V. Phillips, et al., Proc Natl Acad Sci U S A 114:12255-12260, 2017, https://doi.org/10.1073/pnas.1706951114) using satellite tobacco necrosis virus 1 (STNV-1) as a model system for in vitro studies. It has been suggested that competing with these putative PSs may constitute a novel therapeutic approach against pathogenic single-stranded RNA viruses. Our work demonstrates that the previously identified PSs have no discernible significance for the selective packaging of STNV-1 in vivo in the presence and absence of competition or replication: viral sequences are encapsidated mostly on the basis of their abundance within the cell, while encapsidation of host RNAs also occurs. Nevertheless, the putative PSs identified in STNV-1 RNA may still have applications in bionanotechnology, such as the in vitro selective packaging of RNA molecules.

Image-domain multimaterial decomposition for dual-energy CT based on prior information of material images.

PURPOSE: Dual-Energy Computed Tomography (DECT) is of great interest in medical imaging, security inspection, and nondestructive testing. Most DECT reconstruction methods focus on producing two material images with different linear attenuation coefficients. However, the ability to reconstruct three or more basis materials is clinically and industrially important. Under the assumption that there are at most three materials in each pixel, there are a few methods that estimate multiple material images from DECT measurements by enforcing sum-to-one and a box constraint ([0 1]) derived from both the volume and mass conservation assumption. The recently proposed image-domain multimaterial decomposition (MMD) method introduces edge-preserving regularization for each material image. It enforces the assumption that there are at most three materials in each pixel using a time-consuming loop over all possible material triplets. However, this method neglects relations among material images. We propose a new image-domain MMD model for DECT that considers the prior information that different material images have common or complementary edges and encourages sparsity of material composition in each pixel using regularization. METHOD: The proposed PWLS-TNV-ℓ0 method uses penalized weighted least-square (PWLS) reconstruction with three regularization terms. The first term is total nuclear variation (TNV) that accounts for the image property that basis material images share common or complementary boundaries and each material image is piecewise constant. The second term is an ℓ0 norm that encourages each pixel containing a small subset of material types out of several possible materials. The third term is a characteristic function based on sum-to-one and a box constraint derived from the volume and mass conservation assumption. We apply the Alternating Direction Method of Multipliers (ADMM) to optimize the cost function of the PWLS-TNV-ℓ0 method. RESULT: We evaluated the proposed method on a simulated digital phantom, Catphan©600 phantom and patient's pelvis data. We implemented two existing image-domain MMD methods for DECT, the Direct Inversion and the PWLS-EP-LOOP method. We initialized the PWLS-TNV-ℓ0 method and the PWLS-EP-LOOP method with the results of the Direct Inversion method and compared performance of the proposed method with that of the PWLS-EP-LOOP method. The proposed method lowers the bias of decomposed material fractions by 84.47% in the digital phantom study, by 99.50% in the Catphan©600 phantom study, and by 99.64% in the pelvis patient study, respectively, compared to the PWLS-EP-LOOP method. The proposed method reduces noise standard deviation (STD) by 52.21% in the Catphan©600 phantom study, and by 16.74% in the patient's pelvis study, compared to the PWLS-EP-LOOP method. The proposed method increases volume fraction accuracy by 6.04%,20.55%, and 13.46% for the digital phantom, the Catphan©600 phantom, and the patient's pelvis study, respectively, compared to the PWLS-EP-LOOP method. Compared with the PWLS-EP-LOOP method, the root mean square percentage error [RMSE(%)] of electron densities in the Catphan©600 phantom is decreased by about 7.39%. CONCLUSIONS: We proposed an image-domain MMD method, PWLS-TNV-ℓ0 , for DECT. The PWLS-TNV-ℓ0 method takes low rank property of material image gradients, sparsity of material composition and mass and volume conservation into consideration. The proposed method suppresses noise, reduces cross contamination, and improves accuracy in the decomposed material images, compared to the PWLS-EP-LOOP method.

A degenerate pair of primers for simultaneous detection of four alpha- and betanecroviruses.

The high infection levels due to Olive latent virus 1 (OLV-1), Olive mild mosaic virus (OMMV) (alphanecrovirus) and Tobacco necrosis virus D (TNV-D) (betanecrovirus) in Portuguese olive orchards prompted us to develop a rapid PCR-based assay for the simultaneous detection of these viruses aimed at the sanitary selection and marketing of plant material in compliance with European Union regulations. A pair of degenerate oligonucleotide primers, parRdRp5' and parCoat3' was designed based on conserved regions located in the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes of these viruses and one other alphanecrovirus, Tobacco necrosis virus A. Its use in RT-PCR assays generated a product of ca. 2000 bp for the 4 viral species tested. These primers were compared with virus specific primers in multiplex RT-PCR, and identical results were obtained. Its application to dsRNA extracted from 54 olive field growing trees originated the expected ca. 2000 bp amplicon in 17 trees. The virus identity was determined by sequencing the cloned RT-PCR products. No TNV-A was found. The RT-PCR assay using the degenerate primers described in this study were shown to be reliable in detecting any of the above-mentioned alpha- and betanecroviruses, and it is as sensitive as that which uses virus specific primers in multiplex assays. Therefore, this assay is well suited for the rapid screen of virus-free plant material in selection and improvement crop programmes. Additionally, it has the potential to reveal virus diversity and the presence of new viruses, provided the RT-PCR generated amplicon is further sequenced.