Mixed infection of ITPase-encoding potyvirid and secovirid in Mercurialis perennis: evidences for a convergent euphorbia-specific viral counterstrike.

BACKGROUND: In cellular organisms, inosine triphosphate pyrophosphatases (ITPases) prevent the incorporation of mutagenic deaminated purines into nucleic acids. These enzymes have also been detected in the genomes of several plant RNA viruses infecting two euphorbia species. In particular, two ipomoviruses produce replicase-associated ITPases to cope with high concentration of non-canonical nucleotides found in cassava tissues. METHOD: Using high-throughput RNA sequencing on the wild euphorbia species Mercurialis perennis, two new members of the families Potyviridae and Secoviridae were identified. Both viruses encode for a putative ITPase, and were found in mixed infection with a new partitivirid. Following biological and genomic characterization of these viruses, the origin and function of the phytoviral ITPases were investigated. RESULTS: While the potyvirid was shown to be pathogenic, the secovirid and partitivirid could not be transmitted. The secovirid was found belonging to a proposed new Comovirinae genus tentatively named "Mercomovirus", which also accommodates other viruses identified through transcriptome mining, and for which an asymptomatic pollen-associated lifestyle is suspected. Homology and phylogenetic analyses inferred that the ITPases encoded by the potyvirid and secovirid were likely acquired through independent horizontal gene transfer events, forming lineages distinct from the enzymes found in cassava ipomoviruses. Possible origins from cellular organisms are discussed for these proteins. In parallel, the endogenous ITPase of M. perennis was predicted to encode for a C-terminal nuclear localization signal, which appears to be conserved among the ITPases of euphorbias but absent in other plant families. This subcellular localization is in line with the idea that nucleic acids remain protected in the nucleus, while deaminated nucleotides accumulate in the cytoplasm where they act as antiviral molecules. CONCLUSION: Three new RNA viruses infecting M. perennis are described, two of which encoding for ITPases. These enzymes have distinct origins, and are likely required by viruses to circumvent high level of cytoplasmic non-canonical nucleotides. This putative plant defense mechanism has emerged early in the evolution of euphorbias, and seems to specifically target certain groups of RNA viruses infecting perennial hosts.

New viruses of Cladosporium sp. expand considerably the taxonomic structure of Gammapartitivirus genus.

Despite the fact that Cladosporium sp. are ubiquitous fungi, their viromes have been little studied. By analysing a collection of Cladosporium fungi, two new partitiviruses named Cladosporium cladosporioides partitivirus 1 (CcPV1) and Cladosporium cladosporioides partitivirus 2 (CcPV2) co-infecting a strain of Cladosporium cladosporioides were identified. Their complete genome consists of two monocistronic dsRNA segments (RNA1 and RNA2) with a high percentage of pairwise identity on 5' and 3' end. The RNA directed RNA polymerase (RdRp) of both viruses and the capsid protein (CP) of CcPV1 display the classic characteristics required for their assignment to the Gammapartitivirus genus. In contrast, CcPV2 RNA2 encodes for a 41 KDa CP that is unusually smaller when aligned to CPs of other viruses classified in this genus. The structural role of this protein is confirmed by electrophoresis on acrylamide gel of purified viral particles. Despite the low percentage of identity between the capsid proteins of CcPV1 and CcPV2, their three-dimensional structures predicted by AlphaFold2 show strong similarities and confirm functional proximity. Fifteen similar viral sequences of unknown function were annotated using the CcPV2 CP sequence. The phylogeny of the CP was highly consistent with the phylogeny of their corresponding RdRp, supporting the organization of Gammapartitiviruses into three distinct clades despite stretching the current demarcation criteria. It is proposed that a new subgenus be created within the genus Gammapartitivirus for this new group.

A novel weevil-transmitted tymovirus found in mixed infection on hollyhock.

Leaves of hollyhock (Alcea rosea) exhibiting vein chlorosis and yellow mosaic symptoms were collected at public sites in Lausanne and Nyon, two cities of western Switzerland. Diagnostic methods untangled in samples from both sites the mixed infections of a novel isometric virus, tentatively named "Alcea yellow mosaic virus" (AYMV) with the carlavirus Gaillardia latent virus. A new potyvirus was also identified in samples from Nyon. A combination of Illumina, Nanopore and Sanger sequencing was necessary to assemble the full-length genome of AYMV, revealing an exceptionally high cytidine content and other features typically associated with members of the genus Tymovirus. The host range of AYMV was found to be restricted to mallows, including ornamentals as well as economically important plants. Phylogenetic analyses further showed that AYMV belongs to a Tymovirus subclade that also gathers the other mallow-infecting members. The virus was readily transmitted by sap inoculation, and the weevil species Aspidapion radiolus was evidenced as a vector. Transmission assays using another weevil or other insect species did not succeed, and seed transmission was not observed.

The potential impact of Covid-19 on the capacity of routine laboratory tests to detect heparin-induced thrombocytopenia.

In Covid-19, anticoagulation with heparin is often administered to prevent or treat thromboembolic events. Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin treatment, caused by heparin-dependent, platelet activating anti-platelet factor 4 (PF4)/heparin antibodies. Diagnosis of HIT is based on the combination of clinical parameters, allowing to determine the pretest probability, and laboratory testing for anti-PF4/heparin antibodies and confirmatory functional assays, such as the heparin-induced platelet activation (HIPA) test.We report the case of a patient with severe Covid-19 pneumonia requiring ECMO treatment, who developed recurrent clotting of the ECMO filter and a drop in platelet count under heparin treatment. He was therefore suspected to have HIT and the anticoagulation was switched to argatroban. Despite high clinical probability and high titres of anti-PF4/heparin antibodies, the functional HIPA test was negative. Nevertheless, argatroban was continued rather than to reinstate anticoagulation with heparin. Reevaluation 7 days later then demonstrated a strongly positive functional HIPA test and confirmed the diagnosis of HIT. Under anticoagulation with argatroban the patient gradually improved and was finally weaned off the ECMO.In conclusion, this case highlights the critical importance of clinical judgement, exploiting the 4 T score, given that Covid-19 patients may present a different pattern of routine laboratory test results in HIT diagnostics. The possibility of a false negative HIPA test has to be considered, particularly in early phases of presentation. In cases of a discrepancy with high clinical probability of HIT and/or high titre anti-PF4/heparin antibodies despite a negative HIPA test, a reevaluation within 3 to 5 days after the initial test should be considered in order to avoid precipitant reestablishment of unfractionated heparin, with potentially fatal consequences.

Effect of Sample Transportation on the Proteome of Human Circulating Blood Extracellular Vesicles.

Circulating extracellular vesicles (cEV) are released by many kinds of cells and play an important role in cellular communication, signaling, inflammation modulation, coagulation, and tumor growth. cEV are of growing interest, not only as biomarkers, but also as potential treatment targets. However, very little is known about the effect of transporting biological samples from the clinical ward to the diagnostic laboratory, notably on the protein composition. Pneumatic tube systems (PTS) and human carriers (C) are both routinely used for transport, subjecting the samples to different ranges of mechanical forces. We therefore investigated qualitatively and quantitatively the effect of transport by C and PTS on the human cEV proteome and particle size distribution. We found that samples transported by PTS were subjected to intense, irregular, and multidirectional shocks, while those that were transported by C mostly underwent oscillations at a ground frequency of approximately 4 Hz. PTS resulted in the broadening of nanoparticle size distribution in platelet-free (PFP) but not in platelet-poor plasma (PPP). Cell-type specific cEV-associated protein abundances remained largely unaffected by the transport type. Since residual material of lymphocytes, monocytes, and platelets seemed to dominate cEV proteomes in PPP, it was concluded that PFP should be preferred for any further analyses. Differential expression showed that the impact of the transport method on cEV-associated protein composition was heterogeneous and likely donor-specific. Correlation analysis was nonetheless able to detect that vibration dose, shocks, and imparted energy were associated with different terms depending on the transport, namely in C with cytoskeleton-regulated cell organization activity, and in PTS with a release of extracellular vesicles, mainly from organelle origin, and specifically from mitochondrial structures. Feature selection algorithm identified proteins which, when considered together with the correlated protein-protein interaction network, could be viewed as surrogates of network clusters.

Frequency of Thrombocytopenia and Platelet Factor 4/Heparin Antibodies in Patients With Cerebral Venous Sinus Thrombosis Prior to the COVID-19 Pandemic.

Importance: Cases of cerebral venous sinus thrombosis in combination with thrombocytopenia have recently been reported within 4 to 28 days of vaccination with the ChAdOx1 nCov-19 (AstraZeneca/Oxford) and Ad.26.COV2.S (Janssen/Johnson & Johnson) COVID-19 vaccines. An immune-mediated response associated with platelet factor 4/heparin antibodies has been proposed as the underlying pathomechanism. Objective: To determine the frequencies of admission thrombocytopenia, heparin-induced thrombocytopenia, and presence of platelet factor 4/heparin antibodies in patients diagnosed with cerebral venous sinus thrombosis prior to the COVID-19 pandemic. Design, Setting, and Participants: This was a descriptive analysis of a retrospective sample of consecutive patients diagnosed with cerebral venous sinus thrombosis between January 1987 and March 2018 from 7 hospitals participating in the International Cerebral Venous Sinus Thrombosis Consortium from Finland, the Netherlands, Switzerland, Sweden, Mexico, Iran, and Costa Rica. Of 952 patients, 865 with available baseline platelet count were included. In a subset of 93 patients, frozen plasma samples collected during a previous study between September 2009 and February 2016 were analyzed for the presence of platelet factor 4/heparin antibodies. Exposures: Diagnosis of cerebral venous sinus thrombosis. Main Outcomes and Measures: Frequencies of admission thrombocytopenia (platelet count <150 ×103/µL), heparin-induced thrombocytopenia (as diagnosed by the treating physician), and platelet factor 4/heparin IgG antibodies (optical density >0.4, in a subset of patients with previously collected plasma samples). Results: Of 865 patients (median age, 40 years [interquartile range, 29-53 years], 70% women), 73 (8.4%; 95% CI, 6.8%-10.5%) had thrombocytopenia, which was mild (100-149 ×103/µL) in 52 (6.0%), moderate (50-99 ×103/µL) in 17 (2.0%), and severe (<50 ×103/µL) in 4 (0.5%). Heparin-induced thrombocytopenia with platelet factor 4/heparin antibodies was diagnosed in a single patient (0.1%; 95% CI, <0.1%-0.7%). Of the convenience sample of 93 patients with cerebral venous sinus thrombosis included in the laboratory analysis, 8 (9%) had thrombocytopenia, and none (95% CI, 0%-4%) had platelet factor 4/heparin antibodies. Conclusions and Relevance: In patients with cerebral venous sinus thrombosis prior to the COVID-19 pandemic, baseline thrombocytopenia was uncommon, and heparin-induced thrombocytopenia and platelet factor 4/heparin antibodies were rare. These findings may inform investigations of the possible association between the ChAdOx1 nCoV-19 and Ad26.COV2.S COVID-19 vaccines and cerebral venous sinus thrombosis with thrombocytopenia.

Large-Scale RT-qPCR Diagnostics for Seed Potato Certification.

Every year, Agroscope examines nearly 300,000 tubers for the presence of viruses, as regulated for the certification of seed potatoes intended for Swiss growers. Since 2016, this examination has been performed via RT-qPCR on dormant tubers directly after harvest. This method offers fast results and eliminates the need for the use of Rindite, which is a toxic and polluting gaseous compound previously used in Switzerland to break the dormancy of seed tubers. The implementation of this molecular analytical method for the routine diagnosis of regulated viruses makes it possible to conduct additional analyses via Illumina sequencing to assess the conformity of the primers and probes used with the sequences of the different viral isolates. This form of quality control in routine diagnosis is a source of information that can answer more fundamental scientific questions related to the epidemiology of viral strains related to certification. The datasets produced in this framework can also be used to explore the diversity of rare or unknown virus species in potato crops. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11540-021-09491-3.

A novel foveavirus identified in wild grapevine (Vitis vinifera subsp. sylvestris).

We report the genome sequence of a putative new foveavirus infecting non-cultivated Vitis vinifera, tentatively named "grapevine foveavirus A" (GFVA). This virus was identified by high-throughput sequencing analysis of a European wild Vitis collected in Switzerland. Phylogenetic analysis revealed that this virus clustered with known grapevine virus T (GVT) isolates but was clearly distinct from any of them. If considering the International Committee of Taxonomy of Viruses (ICTV)-suggested foveavirus species demarcation criterion based on sequence similarity in the replicase gene/protein, this virus should be considered a member of a new species closely related to GVT. On the other hand, comparison of capsid gene/protein sequences using the same criteria indicates that GFVA is at the border of species demarcation. Whether this virus represents a highly divergent GVT isolate or a member of a distinct but closely related species is discussed.

Grapevine red blotch virus: Absence in Swiss Vineyards and Analysis of Potential Detrimental Effect on Viticultural Performance.

Grapevine red blotch virus (GRBV) is a recently described virus that infects grapevine. Little information is available on the possible occurrence and distribution outside North America. Therefore, we surveyed commercial vineyards from the three major grape-growing regions in Switzerland to determine the presence or absence of GRBV. In total, 3,062 vines were analyzed by polymerase chain reaction. None of the vines tested positive for GRBV, suggesting the absence of GRBV from Swiss vineyards. We also investigated whether GRBV was present in 653 grapevine accessions in the Agroscope grapevine virus collection at Nyon, including dominantly Swiss (457) but also international accessions. Only six referential accessions were infected by GRBV, all originating from the United States, whereas all others from 10 European and 8 non-European origins tested negative. High-throughput sequencing analysis of Zinfandel A2V13, in the collection since 1985, confirmed close similarity of GRBV isolate Z_A2V13 to American isolates according to genomes deposited in GenBank. Because the Zinfandel A2V13 reference was also maintained grafted on the leafroll virus indicator Vitis vinifera 'Gamay', we evaluated the effect of GRBV on viticultural performance over a 3-year period. Our results showed clear detrimental effects of GRBV on grapevine physiology (vine vigor, leaf chlorophyll content, and gas exchange) and fruit quality. These findings underscore the importance of implementation of GRBV testing worldwide in certification and quarantine programs to prevent the dissemination of this virus.

Revisiting a pollen-transmitted ilarvirus previously associated with angular mosaic of grapevine.

We report the characterization of a novel tri-segmented RNA virus infecting Mercurialis annua, a common crop weed and model species in plant science. The virus, named "Mercurialis latent virus" (MeLaV) was first identified in a mixed infection with the recently described Mercurialis orthotospovirus 1 (MerV1) on symptomatic plants grown in glasshouses in Lausanne (Switzerland). Both viruses were found to be transmitted by Thrips tabaci, which presumably help the inoculation of infected pollen in the case of MeLaV. Complete genome sequencing of the latter revealed a typical ilarviral architecture and close phylogenetic relationship with members of the Ilarvirus subgroup 1. Surprisingly, a short portion of MeLaV replicase was found to be identical to the partial sequence of grapevine angular mosaic virus (GAMV) reported in Greece in the early 1990s. However, we have compiled data that challenge the involvement of GAMV in angular mosaic of grapevine, and we propose alternative causal agents for this disorder. In parallel, three highly-conserved MeLaV isolates were identified in symptomatic leaf samples in The Netherlands, including a herbarium sample collected in 1991. The virus was also traced in diverse RNA sequencing datasets from 2013 to 2020, corresponding to transcriptomic analyses of M. annua and other plant species from five European countries, as well as metaviromics analyses of bees in Belgium. Additional hosts are thus expected for MeLaV, yet we argue that infected pollen grains have likely contaminated several sequencing datasets and may have caused the initial characterization of MeLaV as GAMV.

Immunothrombosis and Complement Activation Contribute to Disease Severity and Adverse Outcome in COVID-19.

Severe COVID-19 is characterized by systemic inflammation and multiple organ dysfunction syndrome (MODS). Arterial and venous thrombosis are involved in the pathogenesis of MODS and fatality in COVID-19. There is evidence that complement and neutrophil activation in the form of neutrophil extracellular traps are main drivers for development of microvascular complications in COVID-19. Plasma and serum samples were collected from 83 patients infected by SARS-CoV-2 during the two first waves of COVID-19, before the availability of SARS-CoV-2 vaccination. Samples were collected at enrollment, day 11, and day 28; and patients had differing severity of disease. In this comprehensive study, we measured cell-free DNA, neutrophil activation, deoxyribonuclease I activity, complement activation, and D-dimers in longitudinal samples of COVID-19 patients. We show that all the above markers, except deoxyribonuclease I activity, increased with disease severity. Moreover, we provide evidence that in severe disease there is continued neutrophil and complement activation, as well as D-dimer formation and nucleosome release, whereas in mild and moderate disease all these markers decrease over time. These findings suggest that neutrophil and complement activation are important drivers of microvascular complications and that they reflect immunothrombosis in these patients. Neutrophil activation, complement activation, cell-free DNA, and D-dimer levels have the potential to serve as reliable biomarkers for disease severity and fatality in COVID-19. They might also serve as suitable markers with which to monitor the efficacy of therapeutic interventions in COVID-19.

Nuances of Responses to Two Sources of Grapevine Leafroll Disease on Pinot Noir Grown in the Field for 17 Years.

Grapevine leafroll disease (GLD) is one of the most economically damaging virus diseases in grapevine, with grapevine leafroll-associated virus 1 (GLRaV-1) and grapevine leafroll-associated virus 3 (GLRaV-3) as the main contributors. This study complements a previously published transcriptomic analysis and compared the impact of two different forms of GLD to a symptomless control treatment: a mildly symptomatic form infected with GLRaV-1 and a severe form with exceptionally early leafroll symptoms (up to six weeks before veraison) infected with GLRaV-1 and GLRaV-3. Vine physiology and fruit composition in 17-year-old Pinot noir vines were measured and a gradient of vigor, yield, and berry quality (sugar content and berry weight) was observed between treatments. Virome composition, confirmed by individual RT-PCR, was compared with biological indexing. Three divergent viromes were recovered, containing between four to seven viruses and two viroids. They included the first detection of grapevine asteroid mosaic-associated virus in Switzerland. This virus did not cause obvious symptoms on the indicators used in biological indexing. Moreover, the presence of grapevine virus B (GVB) did not cause the expected corky bark symptoms on the indicators, thus underlining the important limitations of the biological indexing. Transmission of GLRaV-3 alone or in combination with GVB by Planococcus comstocki mealybug did not reproduce the strong symptoms observed on the donor plant infected with a severe form of GLD. This result raises questions about the contribution of each virus to the symptomatology of the plant.

The Complete Genome of the "Flavescence Dorée" Phytoplasma Reveals Characteristics of Low Genome Plasticity.

Members of the genus 'Candidatus Phytoplasma' are obligate intracellular bacteria restricted to phloem sieve elements and are able to colonize several tissues and the hemolymph in their insect vectors. The current unfeasibility of axenic culture and the low complexity of genomic sequences are obstacles in assembling complete chromosomes. Here, a method combining pathogen DNA enrichment from infected insects and dual deep-sequencing technologies was used to obtain the complete genome of a phytoplasma causing Grapevine Flavescence dorée. The de novo assembly generated a circular chromosome of 654,223 bp containing 506 protein-coding genes. Quality assessment of the draft showed a high degree of completeness. Comparative analysis with other phytoplasmas revealed the absence of potential mobile units and a reduced amount of putative phage-derived segments, suggesting a low genome plasticity. Phylogenetic analyses identified Candidatus Phytoplasma ziziphi as the closest fully sequenced relative. The "Flavescence dorée" phytoplasma strain CH genome also encoded for several putative effector proteins potentially playing a role in pathogen virulence. The availability of this genome provides the basis for the study of the pathogenicity mechanisms and evolution of the Flavescence dorée phytoplasma.

Virus Yellows and Syndrome "Basses Richesses" in Western Switzerland: A Dramatic 2020 Season Calls for Urgent Control Measures.

Massive outbreaks of virus yellows (VY) and syndrome "basses richesses" (SBR) are thought to be responsible for the major loss of sugar beet yields in 2020 in western cantons of Switzerland. Typical yellowing symptoms were visible during field inspections, and control measures were reportedly ineffective or even absent. Both diseases induce yellowing but have distinct etiologies; while VY is caused by aphid-transmitted RNA viruses, SBR is caused by the cixiid-transmitted γ-proteobacterium Candidatus Arsenophonus phytopathogenicus. To clarify the situation, samples from diseased plants across the country were screened for the causal agents of VY and SBR at the end of the season. Beet yellows virus (BYV) and Beet chlorosis virus (BChV) showed high incidence nationwide, and were frequently found together in SBR-infected fields in the West. Beet mild yellowing virus (BMYV) was detected in two sites in the West, while there was no detection of Beet western yellows virus or Beet mosaic virus. The nucleotide diversity of the detected viruses was then investigated using classic and high-throughput sequencing. For both diseases, outbreaks were analyzed in light of monitoring of the respective vectors, and symptoms were reproduced in greenhouse conditions by means of insect-mediated inoculations. Novel quantification tools were designed for BYV, BChV and Ca. A. phytopathogenicus, leading to the identification of specific tissues tropism for these pathogens.

Thrombophilia Impact on Treatment Decisions, Subsequent Venous or Arterial Thrombosis and Pregnancy-Related Morbidity: A Retrospective Single-Center Cohort Study.

(1) Background: Thrombophilia testing utility has remained controversial since its clinical introduction, because data on its influence on treatment decisions are limited. (2) Methods: We conducted a single-center retrospective cohort study of 3550 unselected patients referred for thrombophilia consultation at the Bern University Hospital in Switzerland from January 2010 to October 2020. We studied the influence of thrombophilia testing results on treatment decisions and evaluated the association between thrombophilia and thromboembolic and pregnancy-related morbidity events after testing up to 03/2021. (3) Results: In 1192/3550 patients (34%), at least one case of thrombophilia was found and 366 (10%) had high-risk thrombophilia. A total of 211/3550 (6%) work-ups (111/826 (13%) with low-risk thrombophilia and 100/366 (27%) with high-risk thrombophilia) led to an appropriate decision to extend or initiate anticoagulation, and 189 (5%) negative results led to the withholding of anticoagulation therapy inappropriately. A total of 2492 patients (69%) were followed up for >30 days, with a median follow-up of 49 months (range, 1−183 months). Patients with high-risk thrombophilia had a higher risk of subsequent venous thromboembolic events and pregnancy-related morbidity compared to those without thrombophilia. (4) Conclusions: Our study demonstrated the limited usefulness of thrombophilia work-up in clinical decision-making. High-risk thrombophilia was associated with subsequent venous thromboembolism and pregnancy-related morbidity.

Accuracy of the functional, flow cytometer-based Emo-Test HIT Confirm® for the diagnosis of heparin-induced thrombocytopenia.

INTRODUCTION: Rapid functional assays have been proposed to overcome the limitations of washed platelet assays in the work-up of patients with suspected heparin-induced thrombocytopenia (HIT). Data on the diagnostic accuracy are, however, scarce and conflicting. We aimed to study the diagnostic accuracy of a rapid, flow cytometer-based assay and to explore sources of variability. MATERIAL AND METHODS: Frozen serum samples of 103 consecutive patients, evaluated for suspected HIT at our institution in 2017, and characterized with 4Ts score, IgG-PF4/heparin ELISA (GTI), HemosIL®Acustar (IgG), as well as heparin-induced platelet activation test (HIPA), were further tested using HIT Confirm, determining P-selectin release of donor platelets after incubation with patient's serum. The diagnosis of HIT was defined as a positive HIPA result. RESULTS: HIT was confirmed in 15 out of 103 patients corresponding to a prevalence of 14.6%. HIT Confirm was positive in 11 patients (10.7%), negative in 88 patients (85.4%), and inconclusive in 4 patients (3.9%). According to the intention-to-diagnose principle, the number of true positives was 9, the number of true negatives 83, the number of false negatives was 6, the number of false positives 5. This corresponds to a sensitivity of 60.0%, and a specificity of 94.3%. Modifications of the test did not improve sensitivity. CONCLUSIONS: The rapid, flow cytometer-based assay HIT Confirm is able to verify HIT in positive patient samples but cannot rule-out HIT in clinical practice. Other rapid functional assays shall be studies in appropriately designed diagnostic accuracy studies.

Resistance to thrombomodulin correlates with liver stiffness in chronic liver disease a prospective single-center cohort study.

INTRODUCTION: Chronic liver disease (CLD) is characterized by changes in haemostasis, embracing both hypo- and hypercoagulability. Global hemostatic tests such as thrombin generation assays evaluate the hemostatic balance, to better assess bleeding and thrombotic risks. In addition, procoagulant state in patients with CLD has been demonstrated using modified thrombin generation assays with thrombomodulin, a cofactor for protein C activation. In this study, we prospectively determined thrombin generation and thrombomodulin resistance in patients with CLD staged with liver stiffness measurement (LSM), using both the fully automated analyzer ST Genesia® Thrombin Generation System (STG) and the calibrated automated thrombogram assay (CAT). MATERIALS AND METHODS: Demographic, clinical and laboratory characteristics, and blood samples were collected from 65 patients with CLD. Liver stiffness was measured by transient elastography, and thrombin generation and thrombomodulin resistance, by STG and CAT. RESULTS: Patients were separated based on LSM of <21 and ≥21 kilopascals (kPa). The propagation rate of thrombin generation was higher in patients with LSM ≥21 kPa and the thrombin generation rate increased as LSM increased. In addition, thrombomodulin resistance assessed by STG and CAT was higher in patients with LSM ≥21 kPa. However, ETP inhibition by activated protein C was comparable in patients with LSM <21 and ≥21 kPa. Finally, LSM correlated with most thrombin generation parameters. CONCLUSION: The STG automated system may have value in the assessment of patients with chronic liver disease in the routine coagulation laboratory. LSM ≥21 kPa identify a procoagulant phenotype in these patients, including thrombomodulin resistance.

COVID-19 patients often show high-titer non-platelet-activating anti-PF4/heparin IgG antibodies.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a severe adverse reaction to heparin caused by heparin-dependent, platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies. Heparin is a cornerstone of treatment in critically ill COVID-19 patients. HIT antibodies can be detected by antigen tests and functional tests. Often strong reactivity in the antigen test is used as a surrogate marker for the presence of clinically relevant, platelet-activating antibodies. We observed an unexpectedly high percentage of COVID-19 patients, clinically suspected to have HIT, with high titer anti-PF4/heparin antibodies, but a negative functional test. OBJECTIVE: We investigated whether in COVID-19 patients a serum-derived factor inhibits the heparin-induced platelet activation test (HIPA). METHODS AND RESULTS: Twelve COVID-19 patients with suspected HIT were tested. Three samples tested negative in all assays; nine samples tested positive by antigen tests, among which only three tested also positive by HIPA. When we spiked COVID-19 serum or control serum with the human HIT antibody like monoclonal antibody 5B9, reactivity of 5B9 remained the same. Also, the purified IgG fractions of COVID-19 sera testing strongly positive in the PF4/heparin antigen test but negative in the functional test did not show increased reactivity in the functional test in comparison to the original serum. Both results make a functionally inhibitory factor in the serum/plasma of COVID-19 patients highly unlikely. CONCLUSION: COVID-19 patients often present with strong reactivity in PF4/heparin antigen tests without the presence of platelet-activating antibodies. Diagnosis of HIT requires confirmation of heparin-dependent, platelets activating antibodies to avoid overdiagnosis and overtreatment with non-heparin anticoagulants.

Identification and Molecular Characterization of a Novel Hordeivirus Associated With Yellow Mosaic Disease of Privet (Ligustrum vulgare) in Europe.

Wild plants serve as a large reservoir of known and yet-unknown viruses and as a source of viral pathogens of cultivated plants. Yellow mosaic disease of forest shrub Ligustrum vulgare (privet) was recurrently observed in Europe for more than 100 years. Using a universal virus identification approach based on deep sequencing and de novo assembly of viral small interfering (si)RNAs we identified a causative agent of this disease in Switzerland and reconstructed its complete 3-segmented RNA genome. Notably, a short 3'-terminal common region (CR) attached to each segment via a ∼53-71 nucleotide poly(A) tract, as determined by RT-PCR sequencing, was initially identified as an orphan siRNA contig with conserved tRNA-like secondary structure. Phylogenomic analysis classified this virus as a novel member in the genus Hordeivirus of family Virgaviridae, which we named ligustrum mosaic virus (LigMV). Similar to other hordeiviruses, LigMV formed rod-shape virions (visualized by electron microscopy), was transmitted through seeds and could also be mechanically transmitted to herbaceous hosts Chenopodium quinoa and Nicotiana benthamiana. Blot hybridization analysis identified genomic and subgenomic RNAs, sharing the 3'-CR and likely serving as monocistronic mRNAs for seven evolutionarily-conserved viral proteins including two subunits of viral RNA-dependent RNA polymerase, coat protein, triple gene block proteins mediating viral movement and cysteine-rich suppressor of RNA silencing. Analysis of size, polarity, and hotspot profiles of viral siRNAs suggested that they are produced by the plant antiviral Dicer-like (DCL) proteins DCL2 and DCL4 processing double-stranded intermediates of genomic RNA replication. Whole genome sequencing of French and Austrian isolates of LigMV revealed its genetic stability over a wide geographic range (>99% nucleotide identity to Swiss isolates and each other), suggesting its persistence and spread in Europe via seed dispersal.

Diagnostic and Prognostic Implications of Caspase-1 and PD-L1 Co-Expression Patterns in Myelodysplastic Syndromes.

BACKGROUND: The inflammasome plays an essential role in lower risk MDS and immune subversion, with the up-regulation of immune checkpoint molecules in the progression to higher-risk disease. In this study, we explored the utility of immune-related biomarkers for the diagnosis and prognosis of MDS. METHODS: We performed an exploratory, case-control study with 20 randomly selected MDS patients and nine controls with non-inflammatory (n = 3) and inflammatory conditions (n = 6). Patients were stratified in groups of lower (n = 10) and higher risk (n = 10) using IPSS-R. For the exploration of inflammasome and immune checkpoint activities, the expression of caspase-1 (Casp1), programmed cell death protein 1 (PD-1) and its ligand (PD-L1) were assessed in bone marrow samples using immunohistochemistry. RESULTS: In multivariate analysis, we observed significant differences for Casp1 but not PD1/PD-L1 expression in our four conditions (p = 0.003). We found a discordant co-expression of Casp1/PD-L1 in MDS (rho = -0.41, p = 0.07) compared with a concordant co-expression in controls (rho = 0.64, p = 0.06). Neutrophil counts correlated directly with Casp1 (rho = 0.57, p = 0.009) but inversely with PD-L1 expression (rho = -0.58, p = 0.007). CONCLUSION: We identified characteristic discordant co-expression patterns in lower- (Casp1high/PD-L1low) and higher-risk MDS (Casp1low/PD-L1high), contrasting with concordant patterns in the non-inflammatory (Casp1low/PD-L1low) and inflammatory conditions (Casp1high/PD-L1high). Further validation is warranted in larger, prospective studies.