Defective RNAs of Citrus tristeza virus analogous to Crinivirus genomic RNAs

AbstractThe family Closteroviridae includes the genera Closterovirus and Ampelovirus with monopartite genomes and the genus Crinivirus with bipartite genomes. Plants infected with the Closterovirus, Citrus tristeza virus (CTV), often contain one or more populations of defective RNAs (dRNAs). Although most dRNAs are comparatively small (2–5 kb) consisting of the genomic RNA termini with large internal deletions, we recently characterized large dRNAs of ∼12 kb that retained the open reading frames (ORFs) 1a plus 1b. These were self-replicating RNAs and appeared to be analogous to the genomic RNA 1 of the bipartite criniviruses. The present report describes the finding of an additional group of large dRNAs (LdRNAs) that retained all or most of the 10 3′ ORFs and appeared to be analogous to genomic RNA 2 of criniviruses. Isolates associated with LdRNAs were found associated with double-recombinant dRNAs (DR-dRNAs) of various sizes (1.7 to 5.1 kb) that comprised the two termini and a noncontiguous internal sequence from ORF2. The genetic and epidemiological implications of the architectural identities of LdRNAs and DR dRNAs and their apparent analogy with the genomic RNA 2 of criniviruses are discussed

Two classes of subgenomic RNA of grapevine virus A produced by internal controller elements

AbstractGrapevine virus A (GVA), a species of the recently established genus Vitivirus, consists of an ∼7.3-kb single-stranded RNA genome of positive polarity, organized into five open reading frames (ORFs). The virus, which is closely associated with the grapevine rugose wood disease complex, has been poorly investigated genetically. We explored the production of viral RNAs in a GVA-infected Nicotiana benthamiana herbaceous host and characterized one nested set of three 5′-terminal sgRNAs of 5.1, 5.5, and 6.0 kb, and another, of three 3′-terminal sgRNAs of 2.2, 1.8, and 1.0 kb that could serve for expression of ORFs 2–3, respectively. Neither 3′- nor 5′-terminal sgRNAs, which would correspond to ORF5, was detected, suggesting that expression of this ORF occurs via a bi- or polycistronic mRNA. The 5′-terminal sgRNAs were abundant in dsRNA-enriched extracts. Cloning and sequence analysis of the 3′ end of 5.5-kb 5′-terminal sgRNA and the 5′ end of the 1.8-kb 3′-terminal sgRNA suggested that a mechanism other than specific cleavage was involved in production of these sgRNAs. Apparently, the production of the 5′- and 3′-terminal sgRNAs was controlled by sequences upstream of the 5′-terminus of each of ORFs 2–4. Detection of both plus and minus strands of the 5′- and 3′-terminal sgRNAs, though in different levels of accumulation, suggested that each of these cis-acting elements is involved in production of four RNAs: a 3′-terminal plus-strand sgRNA which could act as an mRNA, the corresponding 3′-terminal minus-strand RNA, a 5′-terminal plus-strand sgRNA, and the corresponding 5′-terminal minus-strand RNA

Characterization of the 5′- and 3′-Terminal Subgenomic RNAs Produced by a Capillovirus: Evidence for a CP Subgenomic RNA

The members of Capillovirus genus encode two overlapping open reading frames (ORFs): ORF1 encodes a large polyprotein containing the replication-associated proteins plus a coat protein (CP), and ORF2 encodes a movement protein (MP), located within ORF1 in a different reading frame. Organization of the CP sequence as part of the replicase ORF is unusual in capilloviruses. In this study, we examined the capillovirus genome expression strategy by characterizing viral RNAs produced by Citrus tatter leaf virus (CTLV), isolate ML, a Capillovirus. CTLV-ML produced a genome-length RNA of ∼6.5-kb and two 3′-terminal sgRNAs in infected tissue that contain the MP and CP coding sequences (3′-sgRNA1), and the CP coding sequence (3′-sgRNA2), respectively. Both 3′-sgRNAs initiate at a conserved octanucleotide (UUGAAAGA), and are 1826 (3′-sgRNA1) and 869 (3′-sgRNA2) nts with 119 and 15 nt leader sequences, respectively, suggesting that these two 3′- sgRNAs could serve to express the MP and CP. Additionally, accumulation of two 5′-terminal sgRNAs of 5586 (5′-sgRNA1) and 4625 (5′-sgRNA2) nts was observed, and their 3′-termini mapped to 38–44 nts upstream of the transcription start sites of 3′-sgRNAs. The presence of a separate 3′-sgRNA corresponding to the CP coding sequence and its cognate 5′-terminal sgRNA (5′-sgRNA1) suggests that CTLV-ML produces a dedicated sg mRNA for the expression of its CP

Defective RNA Molecules Associated with Citrus Tristeza Virus

AbstractPreparations of single-stranded (ss) RNA extracted from particles of the Israeli VT strain of citrus tristeza virus (CTV-VT), and ss- and double-stranded (ds) RNA preparations extracted from infected Alemow (Citrus macrophylla) plants, contained a population of molecules with features that suggest that they are defective RNAs. The prototype of 2424 nt was cloned and sequenced and was found to be composed of two genomic regions corresponding to the 5′ (1151 nt) and the 3′ (1259 nt) termini of the genomic CTV-RNA, with two perfect direct repeats of eight nucleotides of unknown origin at the junction site. Northern hybridization analysis demonstrated that this 2.4-kb defective RNA is an abundant species among the other CTV-specific ss- and ds-RNAs in infected plants. The 2.4-kb RNA was found encapsidated by the CTV coat protein indicating that the CTV origin of assembly is located close to the 5′ or 3′ terminus. This is the first defective RNA to be reported for a member of the closterovirus group

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

Transcription Strategy in a \u3ci\u3eClosterovirus\u3c/i\u3e: a Novel 5\u27-Proximal Controller Element of \u3ci\u3eCitrus Tristeza Virus\u3c/i\u3e Produces 5\u27- and 3\u27-Terminal Subgenomic RNAs and Differs from 3\u27 Open Reading Frame Controller Elements

Citrus tristeza virus (CTV) produces more than thirty 3\u27- or 5\u27-terminal subgenomic RNAs (sgRNAs) that accumulate to various extents during replication in protoplasts and plants. Among the most unusual species are two abundant populations of small 5\u27-terminal sgRNAs of approximately 800 nucleotides (nt) termed low-molecular-weight tristeza (LMT1 and LMT2) RNAs. Remarkably, CTV replicons with all 10 3\u27 genes deleted produce only the larger LMT1 RNAs. These 5\u27-terminal positive-sense sgRNAs do not have corresponding negative strands and were hypothesized to be produced by premature termination during plus-strand genomic RNA synthesis. We characterized a cis-acting element that controls the production of the LMT1 RNAs. Since manipulation of this cis-acting element in its native position (the L-ProI region of replicase) was not possible because the mutations negatively affect replication, a region (5\u27TR) surrounding the putative termination sites (nt ~550 to 1000) was duplicated in the 3\u27 end of a CTV replicon to allow characterization. The duplicated sequence continued to produce a 5\u27-terminal plus-strand sgRNA, here much larger (~11 kb), apparently by termination. Surprisingly, a new 3\u27-terminal sgRNA was observed from the duplicated 5\u27TR. A large 3\u27-terminal sgRNA resulting from the putative promoter activity of the native 5\u27TR was not observed, possibly because of the down-regulation of a promoter ~19 kb from the 3\u27 terminus. However, we were able to observe a sgRNA produced from the native 5\u27TR of a small defective RNA, which placed the native 5\u27TR closer to the 3\u27 terminus, demonstrating sgRNA promoter activity of the native 5\u27TR. Deletion mutagenesis mapped the promoter and the terminator activities of the 5\u27TR (in the 3\u27 position in the CTV replicon) to a 57-nt region, which was folded by the MFOLD computer program into two stem-loops. Mutations in the putative stem-loop structures equally reduced or prevented production of both the 3\u27- and 5\u27-terminal sgRNAs. These mutations, when introduced in frame in the native 5\u27TR, similarly abolished the synthesis of the LMT1 RNAs and presumably the large 3\u27 -terminal sgRNA while having no impact on replication, demonstrating that neither 5\u27- nor 3\u27-terminal sgRNA is necessary for replication of the replicon or full-length CTV in protoplasts. Differences between the 5\u27TR, which produced two plus-strand sgRNAs, and the cis-acting elements controlling the 3\u27 open reading frames, which produced additional minus-strand sgRNAs corresponding to the 3\u27 -terminal mRNAs, suggest that the different sgRNA controller elements had different origins in the modular evolution of closteroviruses

Exotic and Emergent Citrus Viruses Relevant to the Mediterranean Region

Citrus production in the Mediterranean area is of considerable importance, in both cultural and economic terms, and the viability of the industry greatly depends on proper phytosanitary management. In this review, we focus on exotic and emerging dangerous citrus viruses that have still not been reported in the countries of the Mediterranean area, that are not yet regulated or that are restricted to certain small areas. We also discuss the contribution that old and new technologies may offer for valuable surveys aimed at promoting the adoption and sharing of better control measures and the production of pathogen-tested citrus trees and rootstocks