Umbravirus-like RNA viruses are capable of independent systemic plant infection in the absence of encoded movement proteins.

The signature feature of all plant viruses is the encoding of movement proteins (MPs) that supports the movement of the viral genome into adjacent cells and through the vascular system. The recent discovery of umbravirus-like viruses (ULVs), some of which only encode replication-associated proteins, suggested that they, as with umbraviruses that lack encoded capsid proteins (CPs) and silencing suppressors, would require association with a helper virus to complete an infection cycle. We examined the infection properties of 2 ULVs: citrus yellow vein associated virus 1 (CY1), which only encodes replication proteins, and closely related CY2 from hemp, which encodes an additional protein (ORF5CY2) that was assumed to be an MP. We report that both CY1 and CY2 can independently infect the model plant Nicotiana benthamiana in a phloem-limited fashion when delivered by agroinfiltration. Unlike encoded MPs, ORF5CY2 was dispensable for infection of CY2, but was associated with faster symptom development. Examination of ORF5CY2 revealed features more similar to luteoviruses/poleroviruses/sobemovirus CPs than to 30K class MPs, which all share a similar single jelly-roll domain. In addition, only CY2-infected plants contained virus-like particles (VLPs) associated with CY2 RNA and ORF5CY2. CY1 RNA and a defective (D)-RNA that arises during infection interacted with host protein phloem protein 2 (PP2) in vitro and in vivo, and formed a high molecular weight complex with sap proteins in vitro that was partially resistant to RNase treatment. When CY1 was used as a virus-induced gene silencing (VIGS) vector to target PP2 transcripts, CY1 accumulation was reduced in systemic leaves, supporting the usage of PP2 for systemic movement. ULVs are therefore the first plant viruses encoding replication and CPs but no MPs, and whose systemic movement relies on a host MP. This explains the lack of discernable helper viruses in many ULV-infected plants and evokes comparisons with the initial viruses transferred into plants that must have similarly required host proteins for movement.

Mitigation of TDP-43 toxic phenotype by an RGNEF fragment in amyotrophic lateral sclerosis models.

Aggregation of the RNA-binding protein TAR DNA binding protein (TDP-43) is a hallmark of TDP-proteinopathies including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). As TDP-43 aggregation and dysregulation are causative of neuronal death, there is a special interest in targeting this protein as a therapeutic approach. Previously, we found that TDP-43 extensively co-aggregated with the dual function protein GEF (guanine exchange factor) and RNA-binding protein rho guanine nucleotide exchange factor (RGNEF) in ALS patients. Here, we show that an N-terminal fragment of RGNEF (NF242) interacts directly with the RNA recognition motifs of TDP-43 competing with RNA and that the IPT/TIG domain of NF242 is essential for this interaction. Genetic expression of NF242 in a fruit fly ALS model overexpressing TDP-43 suppressed the neuropathological phenotype increasing lifespan, abolishing motor defects and preventing neurodegeneration. Intracerebroventricular injections of AAV9/NF242 in a severe TDP-43 murine model (rNLS8) improved lifespan and motor phenotype, and decreased neuroinflammation markers. Our results demonstrate an innovative way to target TDP-43 proteinopathies using a protein fragment with a strong affinity for TDP-43 aggregates and a mechanism that includes competition with RNA sequestration, suggesting a promising therapeutic strategy for TDP-43 proteinopathies such as ALS and FTD.

RNAcanvas: interactive drawing and exploration of nucleic acid structures.

Two-dimensional drawing of nucleic acid structures, particularly RNA structures, is fundamental to the communication of nucleic acids research. However, manually drawing structures is laborious and infeasible for structures thousands of nucleotides long. RNAcanvas automatically arranges residues into strictly shaped stems and loops while providing robust interactive editing features, including click-and-drag layout adjustment. Drawn elements are highly customizable in a point-and-click manner, including colours, fonts, size and shading, flexible numbering, and outlining of bases. Tertiary interactions can be drawn as draggable, curved lines. Leontis-Westhof notation for depicting non-canonical base-pairs is fully supported, as well as text labels for structural features (e.g. hairpins). RNAcanvas also has many unique features and performance optimizations for large structures that cannot be correctly predicted and require manual refinement based on the researcher's own analyses and expertise. To this end, RNAcanvas has point-and-click structure editing with real-time highlighting of complementary sequences and motif search functionality, novel features that greatly aid in the identification of putative long-range tertiary interactions, de novo analysis of local structures, and phylogenetic comparisons. For ease in producing publication quality figures, drawings can be exported in both SVG and PowerPoint formats. URL: https://rnacanvas.app.

-1 Programmed ribosomal frameshifting in Class 2 umbravirus-like RNAs uses multiple long-distance interactions to shift between active and inactive structures and destabilize the frameshift stimulating element.

Plus-strand RNA viruses frequently employ -1 programmed ribosomal frameshifting (-1 PRF) to maximize their coding capacity. Ribosomes can frameshift at a slippery sequence if progression is impeded by a frameshift stimulating element (FSE), which is generally a stable, complex, dynamic structure with multiple conformations that contribute to the efficiency of -1 PRF. As FSE are usually analyzed separate from the viral genome, little is known about cis-acting long-distance interactions. Using full-length genomic RNA of umbravirus-like (ula)RNA citrus yellow vein associated virus (CY1) and translation in wheat germ extracts, six tertiary interactions were found associated with the CY1 FSE that span nearly three-quarters of the 2.7 kb genomic RNA. All six tertiary interactions are conserved in other Class 2 ulaRNAs and two are conserved in all ulaRNAs. Two sets of interactions comprise local and distal pseudoknots that involve overlapping FSE nucleotides and thus are structurally incompatible, suggesting that Class 2 FSEs assume multiple conformations. Importantly, two long-distance interactions connect with sequences on opposite sides of the critical FSE central stem, which would unzip the stem and destabilize the FSE. These latter interactions could allow a frameshifting ribosome to translate through a structurally disrupted upstream FSE that no longer blocks ribosome progression.

ADAR1 Biology Can Hinder Effective Antiviral RNA Interference.

Viruses constantly evolve and adapt to the antiviral defenses of their hosts. The biology of viral circumvention of these selective pressures can often be attributed to the acquisition of novel antagonistic gene products or by rapid genome change that prevents host recognition. To study viral evasion of RNA interference (RNAi)-based defenses, we established a robust antiviral system in mammalian cells using recombinant Sendai virus designed to be targeted by endogenous host microRNAs (miRNAs) with perfect complementarity. Using this system, we previously demonstrated the intrinsic ability of positive-strand RNA viruses to escape this selective pressure via homologous recombination, which was not observed in negative-strand RNA viruses. Here, we show that given extensive time, escape of miRNA-targeted Sendai virus was enabled by host adenosine deaminase acting on RNA 1 (ADAR1). Independent of the viral transcript targeted, ADAR1 editing resulted in disruption of the miRNA-silencing motif, suggesting an intolerance for extensive RNA-RNA interactions necessary for antiviral RNAi. This was further supported in Nicotiana benthamiana, where exogenous expression of ADAR1 interfered with endogenous RNAi. Together, these results suggest that ADAR1 diminishes the effectiveness of RNAi and may explain why it is absent in species that utilize this antiviral defense system. IMPORTANCE All life at the cellular level has the capacity to induce an antiviral response. Here, we examine the result of imposing the antiviral response of one branch of life onto another and find evidence for conflict. To determine the consequences of eliciting an RNAi-like defense in mammals, we applied this pressure to a recombinant Sendai virus in cell culture. We find that ADAR1, a host gene involved in regulation of the mammalian response to virus, prevented RNAi-mediated silencing and subsequently allowed for viral replication. In addition, the expression of ADAR1 in Nicotiana benthamiana, which lacks ADARs and has an endogenous RNAi system, suppresses gene silencing. These data indicate that ADAR1 is disruptive to RNAi biology and provide insight into the evolutionary relationship between ADARs and antiviral defenses in eukaryotic life.

Structural characterization of a new subclass of panicum mosaic virus-like 3' cap-independent translation enhancer.

Canonical eukaryotic mRNA translation requires 5'cap recognition by initiation factor 4E (eIF4E). In contrast, many positive-strand RNA virus genomes lack a 5'cap and promote translation by non-canonical mechanisms. Among plant viruses, PTEs are a major class of cap-independent translation enhancers located in/near the 3'UTR that recruit eIF4E to greatly enhance viral translation. Previous work proposed a single form of PTE characterized by a Y-shaped secondary structure with two terminal stem-loops (SL1 and SL2) atop a supporting stem containing a large, G-rich asymmetric loop that forms an essential pseudoknot (PK) involving C/U residues located between SL1 and SL2. We found that PTEs with less than three consecutive cytidylates available for PK formation have an upstream stem-loop that forms a kissing loop interaction with the apical loop of SL2, important for formation/stabilization of PK. PKs found in both subclasses of PTE assume a specific conformation with a hyperreactive guanylate (G*) in SHAPE structure probing, previously found critical for binding eIF4E. While PTE PKs were proposed to be formed by Watson-Crick base-pairing, alternative chemical probing and 3D modeling indicate that the Watson-Crick faces of G* and an adjacent guanylate have high solvent accessibilities. Thus, PTE PKs are likely composed primarily of non-canonical interactions.

Identification of Novel 5' and 3' Translation Enhancers in Umbravirus-Like Coat Protein-Deficient RNA Replicons.

Translation of plant plus-strand RNA viral genomes that lack a 5' cap frequently requires the use of cap-independent translation enhancers (CITEs) located in or near the 3' untranslated region (UTR). 3'CITEs are grouped based on secondary structure and ability to interact with different translation initiation factors or ribosomal subunits, which assemble a complex at the 3' end that is nearly always transferred to the 5' end via a long-distance kissing-loop interaction between sequences in the 3'CITE and 5' hairpins. We report here the identification of a novel 3'CITE in coat protein-deficient RNA replicons that are related to umbraviruses. Umbra-like associated RNAs (ulaRNAs), such as citrus yellow vein-associated virus (CYVaV), are a new type of subviral RNA that do not encode movement proteins, coat proteins, or silencing suppressors but can independently replicate using their encoded RNA-dependent RNA polymerase. An extended hairpin structure containing multiple internal loops in the 3' UTR of CYVaV is strongly conserved in the most closely related ulaRNAs and structurally resembles an I-shaped structure (ISS) 3'CITE. However, unlike ISS, the CYVaV structure binds to eIF4G and no long-distance interaction is discernible between the CYVaV ISS-like structure and sequences at or near the 5' end. We also report that the ∼30-nucleotide (nt) 5' terminal hairpin of CYVaV and related ulaRNAs can enhance translation of reporter constructs when associated with either the CYVaV 3'CITE or the 3'CITEs of umbravirus pea enation mosaic virus (PEMV2) and even independent of a 3'CITE. These findings introduce a new type of 3'CITE and provide the first information on translation of ulaRNAs. IMPORTANCE Umbra-like associated RNAs (ulaRNAs) are a recently discovered type of subviral RNA that use their encoded RNA-dependent RNA polymerase for replication but do not encode any coat proteins, movement proteins, or silencing suppressors yet can be found in plants in the absence of any discernible helper virus. We report the first analysis of their translation using class 2 ulaRNA citrus yellow vein-associated virus (CYVaV). CYVaV uses a novel eIF4G-binding I-shaped structure as its 3' cap-independent translation enhancer (3'CITE), which does not connect with the 5' end by a long-distance RNA:RNA interaction that is typical of 3'CITEs. ulaRNA 5' terminal hairpins can also enhance translation in association with cognate 3'CITEs or those of related ulaRNAs and, to a lesser extent, with 3'CITEs of umbraviruses, or even independent of a 3'CITE. These findings introduce a new type of 3'CITE and provide the first information on translation of ulaRNAs.

Occurrence of focal myositis during Behçet's disease: The identification of a specific vasculitis-associated focal myopathy.

AIMS: This study aimed to report the association of focal myositis (FM) and Behçet's disease (BD) and to analyse the main characteristics of such an association. METHODS: This is a retrospective multicentre study of patients with BD and FM (BD + FM+ group) and those without FM (BD - FM+ group). Clinical, laboratory, radiological, pathological, treatment and outcome data were analysed. RESULTS: The BD + FM+ group included 10 patients; the median [interquartile range] age at BD diagnosis was 25 [16-35] years, and at FM diagnosis, it was 30 [26-42] years. The diagnosis of BD preceded FM in the majority of cases (n = 8/10). FM occurrence was associated with BD flare-ups in three cases. The creatine kinase levels remained normal or slightly increased. Histological analyses identified relatively preserved muscle tissue, associated with vasculitis (n = 5/6). All patients required treatment; most patients relapsed (n = 9/10). The BD - FM+ group included 35 patients. A comparison of the groups identified a trend towards a younger median age at diagnosis of FM among those with BD (p = 0.063) and more frequent focal muscle swelling in the BD + FM+ group (p = 0.029). The pathological analysis identified significantly less frequent muscle alterations in the BD + FM+ group (muscle fibre size heterogeneity, p = 0.021; necrosis, p = 0.007; and fibrosis, p = 0.027). BD + FM+ patients had a higher frequency of relapse (p = 0.003) and systematic treatment (p = 0.042). CONCLUSIONS: FM occurring during BD appears to be part of the systemic vasculitis process and presents as a vasculitis-associated focal myopathy with a specific clinico-histological pattern. Patients with this association require long-term follow-up and adapted management. This case series also highlights the need for research on BD diagnostic criteria in cases of FM.

RNase III nucleases from diverse kingdoms serve as antiviral effectors.

In contrast to the DNA-based viruses in prokaryotes, the emergence of eukaryotes provided the necessary compartmentalization and membranous environment for RNA viruses to flourish, creating the need for an RNA-targeting antiviral system. Present day eukaryotes employ at least two main defence strategies that emerged as a result of this viral shift, namely antiviral RNA interference and the interferon system. Here we demonstrate that Drosha and related RNase III ribonucleases from all three domains of life also elicit a unique RNA-targeting antiviral activity. Systemic evolution of ligands by exponential enrichment of this class of proteins illustrates the recognition of unbranched RNA stem loops. Biochemical analyses reveal that, in this context, Drosha functions as an antiviral clamp, conferring steric hindrance on the RNA-dependent RNA polymerases of diverse positive-stranded RNA viruses. We present evidence for cytoplasmic translocation of RNase III nucleases in response to virus in diverse eukaryotes including plants, arthropods, fish, and mammals. These data implicate RNase III recognition of viral RNA as an antiviral defence that is independent of, and possibly predates, other known eukaryotic antiviral systems.

Opium Poppy Mosaic Virus Has an Xrn-Resistant, Translated Subgenomic RNA and a BTE 3' CITE.

Opium poppy mosaic virus (OPMV) is a recently discovered umbravirus in the family Tombusviridae OPMV has a plus-sense genomic RNA (gRNA) of 4,241 nucleotides (nt) from which replication protein p35 and p35 extension product p98, the RNA-dependent RNA polymerase (RdRp), are expressed. Movement proteins p27 (long distance) and p28 (cell to cell) are expressed from a 1,440-nt subgenomic RNA (sgRNA2). A highly conserved structure was identified just upstream from the sgRNA2 transcription start site in all umbraviruses, which includes a carmovirus consensus sequence, denoting generation by an RdRp-mediated mechanism. OPMV also has a second sgRNA of 1,554 nt (sgRNA1) that starts just downstream of a canonical exoribonuclease-resistant sequence (xrRNAD). sgRNA1 codes for a 30-kDa protein in vitro that is in frame with p28 and cannot be synthesized in other umbraviruses. Eliminating sgRNA1 or truncating the p30 open reading frame (ORF) without affecting p28 substantially reduced accumulation of OPMV gRNA, suggesting a functional role for the protein. The 652-nt 3' untranslated region of OPMV contains two 3' cap-independent translation enhancers (3' CITEs), a T-shaped structure (TSS) near its 3' end, and a Barley yellow dwarf virus-like translation element (BTE) in the central region. Only the BTE is functional in luciferase reporter constructs containing gRNA or sgRNA2 5' sequences in vivo, which differs from how umbravirus 3' CITEs were used in a previous study. Similarly to most 3' CITEs, the OPMV BTE links to the 5' end via a long-distance RNA-RNA interaction. Analysis of 14 BTEs revealed additional conserved sequences and structural features beyond the previously identified 17-nt conserved sequence.IMPORTANCEOpium poppy mosaic virus (OPMV) is an umbravirus in the family Tombusviridae We determined that OPMV accumulates two similarly sized subgenomic RNAs (sgRNAs), with the smaller known to code for proteins expressed from overlapping open reading frames. The slightly larger sgRNA1 has a 5' end just upstream from a previously predicted xrRNAD site, identifying this sgRNA as an unusually long product produced by exoribonuclease trimming. Although four umbraviruses have similar predicted xrRNAD sites, only sgRNA1 of OPMV can code for a protein that is an extension product of umbravirus ORF4. Inability to generate the sgRNA or translate this protein was associated with reduced gRNA accumulation in vivo We also characterized the OPMV BTE structure, a 3' cap-independent translation enhancer (3' CITE). Comparisons of 13 BTEs with the OPMV BTE revealed additional stretches of sequence similarity beyond the 17-nt signature sequence, as well as conserved structural features not previously recognized in these 3' CITEs.

Two new umbravirus-like associated RNAs (ulaRNAs) discovered in maize and johnsongrass from Ecuador.

Two new umbravirus-like associated RNAs (ulaRNAs) were found, respectively, in maize and Johnsongrass samples from Ecuador. The complete sequences consist of 3,053 and 3,025 nucleotides, respectively, and contain four open reading frames (ORFs). Their genome sequences were 58% identical to each other and 28 to 60% identical to the most closely related viruses. Phylogenetic analysis using full genome sequences and amino acid sequence of the RNA-dependent-RNA polymerase (RdRp) placed both sequences in a clade sharing the most recent common ancestor with ulaRNAs from sugarcane and maize, suggesting that they belong to a monophyletic grass-infecting lineage. Their terminal regions exhibit features common to umbraviruses and ulaRNAs.

Canal of Nuck Abnormalities in Pediatric Female Patients.

A groin lump is not an uncommon condition in girls and female infants, and US plays a fundamental role in its exploration. The main pathologic conditions are related to the failure of obliteration of the canal of Nuck. Radiologists should gain a full understanding of the embryology and US anatomy of the inguinal canal before assessing this entity for the first time. An optimal age-adjusted US technique-including examinations at rest and during straining-is essential to help assess the canal of Nuck, diagnose a hernia, and analyze its content. The radiologist must be aware of the various types of hernial content depending on the patient's age, including intestinal, omental, ovarian, or tubouterine hernia, and the US features of each. Incarcerated hernias are common in girls and mostly contain an ovary. In such cases, it is crucial to screen for US signs suggestive of ovarian ischemic damage, thereby calling for urgent surgery. US can also depict a cyst or hydrocele of the canal of Nuck and its complications. Moreover, other rare pathologic conditions involving the inguinal area may be depicted at US, which helps guide appropriate treatment. US is the ideal modality for evaluating an inguinal lump in girls and female infants. Online supplemental material is available for this article. ©RSNA, 2022.

How does magnetically controlled growing rods insertion affect sagittal alignment in ambulatory early onset scoliosis patients?

PURPOSE: The importance of sagittal alignment restoration in early onset scoliosis (EOS) management has rarely been investigated to date. The aim was to report the influence of magnetically controlled growing rods (MCGR) insertion on the sagittal alignment of EOS patients. METHODS: All consecutive ambulatory patients operated with MCGR rods between 2011 and 2018 were retrospectively included in four institutions. Standing biplanar radiographs were performed preoperatively, in the early postoperative period and at latest follow-up. Global and local sagittal parameters, spinal global shape and harmony were investigated. RESULTS: A total of 37 ambulatory EOS patients were included (mean age at surgery 8.5 (± 2) years). 70% had a balanced construct postoperatively. Both MaxTK (- 17°, p = 0.02) and MaxLL (- 15°, p = 0.001) were significantly reduced, particularly at the instrumented levels. The number of vertebrae included in the lumbar lordosis significantly increased (+ 2 levels, p = 0.02), as well as the thoraco-lumbar inflexion point (+ 2 levels, p < 0.001) and the kyphosis apex (+ 1 level, p < 0.001). Overall mechanical failure rate was 40.5%, and radiological PJK was observed in 43% of the patients, with 11 remaining asymptomatic. Patients with initial hyperkyphosis (> 50°) developed more complications (62% vs. 28%, p = 0.04). CONCLUSION: MCGR insertion flattened the spine in EOS, at both instrumented and non-instrumented levels. Overall spinal harmony was modified, with a cranial shift of the thoraco-lumbar inflexion point and the thoracic kyphosis apex, associated with a lengthening of the lumbar lordosis. The rate of complication remained high, some explanations being found in the radiological changes reported such as the preoperative location of the TK apex. LEVEL OF EVIDENCE: IV.

Does the use of intrawound povidone-iodine irrigation and local vancomycin powder impact surgical site infection rate in adolescent idiopathic scoliosis surgery?

PURPOSE: Surgical site infection (SSI) is a major complication after adolescent idiopathic scoliosis (AIS) surgery, with an incidence ranging from 0.5 to 7%. Intraoperative wound decontamination with povidone-iodine (PVP-I) irrigation and/or vancomycin powder in adult spinal surgery has gained attention in the literature with controversial results. The aim of this study was to investigate the impact of using intrawound PVP-I irrigation and local vancomycin powder (LVP) on the incidence of early SSI in AIS surgery. METHODS: All AIS patients who underwent posterior spinal fusion between October 2016 and December 2019 were retrospectively reviewed. The incidence of early SSI was reported and compared between 2 groups defined by the treating spinal surgeons' preferences: group 1-intrawound irrigation with 2L of PVP-I and application of 3 g LVP before closure and control group 2-patients that did not receive either of these measures. RESULTS: Nine early cases of SSI (2.9%) were reported among the 307 AIS posterior spinal fusion patients. Incidence of SSI in group 1 (2/178 = 1.1%) was significantly lower than in group 2 (7/129 = 5.4%; p = 0.04). There were no adverse reactions to the use of PVP-I and LVP in our study. At latest follow-up, rate of surgical revision for mechanical failure with pseudarthrosis was significantly lower in group 1 (2/178 = 1.1%) than in group 2 (9/129 = 7.0%; p = 0.01). CONCLUSION: Intraoperative use of intrawound PVP-I irrigation and vancomycin powder is associated with a significant reduction of early SSI after AIS spine surgery. LEVEL OF EVIDENCE IV: Retrospective study.

Atherosclerotic Cardiovascular Events in Patients Infected With Human Immunodeficiency Virus and Hepatitis C Virus.

BACKGROUND: An increased risk of cardiovascular disease (CVD) was reported in patients coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV), without identifying factors associated with atherosclerotic CVD (ASCVD) events. METHODS: HIV-HCV coinfected patients were enrolled in the Agence Nationale de Recherches sur le Sida et les hépatites virales (ANRS) CO13 HEPAVIH nationwide cohort. Primary outcome was total ASCVD events. Secondary outcomes were coronary and/or cerebral ASCVD events, and peripheral artery disease (PAD) ASCVD events. Incidences were estimated using the Aalen-Johansen method. Factors associated with ASCVD were identified using cause-specific Cox proportional hazards models. RESULTS: At baseline, median age of the study population (N = 1213) was 45.4 (interquartile range [IQR] 42.1-49.0) years and 70.3% were men. After a median follow-up of 5.1 (IQR 3.9-7.0) years, the incidence was 6.98 (95% confidence interval [CI], 5.19-9.38) per 1000 person-years for total ASCVD events, 4.01 (2.78-6.00) for coronary and/or cerebral events, and 3.17 (2.05-4.92) for PAD ASCVD events. Aging (hazard ratio [HR] 1.06; 95% CI, 1.01-1.12), prior CVD (HR 8.48; 95% CI, 3.14-22.91), high total cholesterol (HR 1.43; 95% CI, 1.11-1.83), high-density lipoprotein cholesterol (HR 0.22; 95% CI, 0.08-0.63), statin use (HR 3.31; 95% CI, 1.31-8.38), and high alcohol intake (HR 3.18; 95% CI, 1.35-7.52) were independently associated with total ASCVD events, whereas undetectable baseline viral load (HR 0.41, 95% CI, 0.18-0.96) was associated with coronary and/or cerebral events. CONCLUSIONS: HIV-HCV coinfected patients experienced a high incidence of ASCVD events. Some traditional cardiovascular risk factors were the main determinants of ASCVD. Controlling cholesterol abnormalities and maintaining undetectable HIV RNA are essential to control cardiovascular risk.

No difference in HIV-1 integrase inhibitor resistance between CSF and blood compartments.

BACKGROUND: Little is known about HIV-1 integrase inhibitor resistance in the CNS. OBJECTIVES: This study aimed to evaluate integrase inhibitor resistance in CSF, as a marker of the CNS, and compare it with the resistance in plasma. METHODS: HIV integrase was sequenced both in plasma and CSF for 59 HIV-1 patients. The clinical and biological data were collected from clinical routine care. RESULTS: Among the 59 HIV-1 patients, 32 (54.2%) were under antiretroviral (ARV) treatment. The median (IQR) HIV-1 RNA in the plasma of viraemic patients was 5.32 (3.85-5.80) and 3.59 (2.16-4.50) log10 copies/mL versus 4.79 (3.56-5.25) and 3.80 (2.68-4.33) log10 copies/mL in the CSF of ARV-naive and ARV-treated patients, respectively. The patients were mainly infected with non-B subtypes (72.2%) with the most prevalent recombinant form being CRF02_AG (42.4%). The HIV-1 integrase sequences from CSF presented resistance mutations for 9/27 (33.3%) and 8/32 (25.0%) for ARV-naive (L74I, n = 3; L74I/M, n = 1; T97A, n = 1; E157Q, n = 4) and ARV-treated (L74I, n = 6; L74M, n = 1; T97A, n = 1; N155H, n = 1) patients, respectively. Integrase inhibitor resistance mutations in CSF were similar to those in plasma, except for 1/59 patients. CONCLUSIONS: This work shows similar integrase inhibitor resistance profiles in the CNS and plasma in a population of HIV-1 viraemic patients.

Intermittent two-drug antiretroviral therapies maintain long-term viral suppression in real life in highly experienced HIV-infected patients.

OBJECTIVES: To assess in real life whether two-drug regimens (2-DRs) given 4-5 days a week in virally suppressed patients can maintain viral suppression over 48 and 96 weeks. METHODS: This observational single-centre study enrolled all patients who initiated an intermittent 2-DR between 01/01/2016 and 30/06/2019. The primary outcome was the rate of virological failure (VF), defined as confirmed plasma viral load (pVL) ≥50 copies/mL or single pVL ≥50 copies/mL followed by ART change at week 48 (W48) and W96. Secondary outcomes were the 2-DR intermittent strategy success rate (pVL <50 copies/mL with no ART change), change in CD4 count, CD4/CD8 ratio and rate of residual viraemia. RESULTS: Eighty-five patients were included; 67/85 (79%) were men, median age = 57 years (IQR = 50-63), CD4 nadir = 233 cells/mm3 (110-327), ART duration = 21 years (13-24), duration of virological suppression = 6.5 years (3.7-10.8) and CD4 count = 658 cells/mm3 (519-867). Intermittent 2-DRs consisted of integrase strand transfer inhibitor (INSTI)/NNRTI (58%), INSTI/NRTI (13%), two NRTIs (11%), PI/NRTI (7%) and other combinations (11%). The median follow-up was 90 weeks (IQR = 64-111). Overall, four VFs occurred, leading to a virological success rate of 98.8% (95% CI = 93.6-100) at W48 and 95.3% (95% CI = 88.4-98.7) at W96. Resuming the same 2-DR 7 days a week led to viral resuppression in three patients, whereas the M184V mutation emerged in one patient, leading to ART modification. There was no significant change in the CD4 count or residual viraemia rate, but a small increase in the CD4/CD8 ratio (P = 0.009) occurred over the study period. CONCLUSIONS: This observational study shows the potential for intermittent 2-DRs to maintain a high virological success rate, which should be assessed in larger prospective randomized studies.

Presence of HIV-1 G-to-A mutations linked to APOBEC editing is more prevalent in non-B HIV-1 subtypes and is associated with lower HIV-1 reservoir.

OBJECTIVES: APOBEC3 editing activity contributes to sequences variation and viral diversification. We aimed to characterize virological and clinical factors associated with G-to-A mutations and stop codons in the HIV-1 reservoir, markers of APOBEC3 footprints, in order to better understand HIV-1 diversity among virologically suppressed HIV-1-infected patients. METHODS: Immuno-virological and clinical factors were compared between 92 patients harbouring G-to-A mutations and stop codons (APOBEC+) in the reverse transcriptase gene and 92 patients without G-to-A mutations (APOBEC-) and stop codons in their DNA genotypes. RESULTS: Patients were predominantly men (74.5%) and were mostly infected by B-subtype (69.0%), with 44.1% and 55.9% in APOBEC+ and APOBEC- groups, respectively. At time of HIV DNA genotypes, the total cell-associated HIV-1 DNA load was 2.34 log10 copies/106 cells (IQR 1.85-2.67) and 33.2% of them had a detectable ultrasensitive plasma viral load. Hypermutated sequences were identified in 28.2% of the APOBEC+ group. The median total cell-associated HIV-1 DNA level was significantly lower in APOBEC+ than APOBEC- group: 2.13 log10 copies/106 cells (IQR 1.60-2.60) versus 2.52 log10 copies/106 cells (IQR 2.19-2.71) (P < 0.001), respectively. Presence of G-to-A mutations and stop codon was independently associated with HIV-1 subtype non-B (P = 0.017). CONCLUSIONS: These results show an independent association between the presence of G-to-A mutations and stop codons with HIV-1 subtype non-B and low proviral DNA that could be explained by the APOBEC3 footprints and restriction of DNA synthesis and integration. However, further investigations are needed to study the contribution of Vif amino acid variability among HIV-1 subtypes.

Genome characterization of fig umbra-like virus.

The complete genome of a new umbra-like virus from edible fig (Ficus carica) was identified by high-throughput sequencing. Based on its similarity to umbra-like virus genome sequences available in GenBank, the proposed name of this new virus is "fig umbra-like virus" (FULV). The genome of full-length FULV-1 consists of 3049 nucleotides organized into three open reading frames (ORFs). Pairwise comparisons showed that the complete nucleotide sequence of the virus had the highest identity (71.3%) to citrus yellow vein-associated virus (CYVaV). In addition, phylogenetic trees based on whole-genome nucleotide sequences and amino acid sequences of the RNA-dependent RNA polymerase showed that FULV forms a monophyletic lineage with CYVaV and other umbra-like viruses. Based on the demarcation criteria of the genus Umbravirus, and lack of two umbravirus ORFs, we propose that FULV is a putative new member of the umbra-like virus clade within the family Tombusviridae.

Drosophila melanogaster Stress Odorant (dSO) Displays the Characteristics of an Interspecific Alarm Cue.

Organisms depend on visual, auditory, and olfactory cues to signal the presence of danger that could impact survival and reproduction. Drosophila melanogaster emits an olfactory alarm signal, termed the Drosophila stress odorant (dSO), in response to mechanical agitation or electric shock. While it has been shown that conspecifics avoid areas previously occupied by stressed individuals, the contextual underpinnings of the emission of, and response to dSO, have received little attention. Using a binary choice assay, we determined that neither age and sex of emitters, nor the time of the day, affected the emission or avoidance of dSO. However, both sex and mating status affected the response to dSO. We also demonstrated that while D. melanogaster, D. simulans, and D. suzukii, have different dSO profiles, its avoidance was not species-specific. Thus, dSO should not be considered a pheromone but a general alarm signal for Drosophila. However, the response levels to both intra- and inter-specific cues differed between Drosophila species and possible reasons for these differences are discussed.