Importance of Vaccination against SARS-CoV-2 in Patients with Interstitial Lung Disease Associated with Systemic Autoimmune Disease.

Objectives: To describe the frequency of COVID-19 and the effect of vaccination in patients with interstitial lung disease and systemic autoimmune disease (ILD-SAD) and to identify factors associated with infection and severity of COVID-19. Methods: We performed a cross-sectional multicenter study of patients with ILD-SAD followed between June and October 2021. The main variable was COVID-19 infection confirmed by a positive polymerase chain reaction (PCR) result for SARS-CoV-2. The secondary variables included severity of COVID-19, if the patient had to be admitted to hospital or died of the disease, and vaccination status. Other variables included clinical and treatment characteristics, pulmonary function and high-resolution computed tomography. Two logistic regression was performed to explore factors associated with "COVID-19" and "severe COVID-19". Results: We included 176 patients with ILD-SAD: 105 (59.7%) had rheumatoid arthritis, 49 (27.8%) systemic sclerosis, and 22 (12.54%) inflammatory myopathies. We recorded 22/179 (12.5%) SARS-CoV-2 infections, 7/22 (31.8%) of them were severe and 3/22 (13.22%) died. As to the vaccination, 163/176 (92.6%) patients received the complete doses. The factors associated with SARS-CoV-2 infection were FVC (OR (95% CI), 0.971 (0.946−0.989); p = 0.040), vaccination (OR (95% CI), 0.169 (0.030−0.570); p = 0.004), and rituximab (OR (95% CI), 3.490 (1.129−6.100); p = 0.029). The factors associated with severe COVID-19 were the protective effect of the vaccine (OR (95% CI), 0.024 (0.004−0.170); p < 0.001) and diabetes mellitus (OR (95% CI), 4.923 (1.508−19.097); p = 0.018). Conclusions: Around 13% of patients with ILD-SAD had SARS-CoV-2 infection, which was severe in approximately one-third. Most patients with severe infection were not fully vaccinated.

Actitudes y creencias de deportistas cubanos de alto rendimiento sobre el dopaje / Atitudes e crenças de atletas cubanos de alto desempenhosobre doping / Attitudes and beliefs of high-performance cuban athletes about doping

La presente investigación no experimental y descriptiva tiene como intención determinar las principales creencias y actitudes en deportistas cubanos de alto rendimiento respecto al dopaje. Se tomó una muestra de 300 deportistas pertenecientes a equipos nacionales de 37 deportes, a los que se le aplicó el instrumento Escala de actitudes y creencias sobre dopaje (Suárez y Ordoqui, 2019). Se realizó el análisis de los datos obtenidos mediante la distribución empírica de frecuencias, las medias y la desviación estándar. Se emplearon además la prueba KMO y Barlett para validar el uso del análisis factorial y la prueba Chi cuadrado de Pearson. Se pudo comprobar en más del 50% de la muestra, la presencia de creencias y actitudes de rechazo hacia el consumo de sustancias dopantes y quedaron expuestos los factores que los deportistas reconocen como los de mayor incidencia en la aparición de conductas de propensión hacia el dopaje. (AU)

The present non-experimental and descriptive research intends to determine the main beliefs and attitudes in high-performance Cuban athletes regarding doping. A sample of 300 athletes belonging to national teams of 37 sports was taken, to which the Doping Attitudes and Beliefs Scale instrument was applied (Suárez and Ordoqui, 2019). The analysis of the data obtained was carried out through the empirical distribution of frequencies, the means and the standard deviation. The KMO and Barlett test were also used to validate the use of factor analysis and Pearson's Chi-square test. It was possible to verify in more than 50% of the sample, the presence of beliefs and attitudes of rejection towards the consumption of doping substances and the factors that athletes recognize as those with the highest incidence in the appearance of propensity behaviors towards doping were exposed. (AU)

A presente pesquisa não experimental e descritiva pretende determinar as principais crenças e atitudes em atletas cubanos de alto rendimento em relação ao doping. Foi retirada uma amostra de 300 atletas pertencentes a seleções nacionais de 37 modalidades desportivas, às quais foi aplicado o instrumento Doping Attitudes andBeliefs Scale (Suárez e Ordoqui, 2019). A análise dos dados obtidos foi realizada por meio da distribuição empírica de frequências, médias e desvio padrão. O KMO e o teste de Barlett também foram utilizados para validar o uso da análise fatorial e o testeQui-quadrado de Pearson. Foi possível verificar em mais de 50% da amostra, a presença de crenças e atitudes de rejeição ao consumo de substâncias dopantes e os fatores que os atletas reconhecem como os de maior incidência no aparecimento de comportamentosde propensão ao doping foram expostos. (AU)

Frequency of Polyautoimmunity in Patients With Rheumatoid Arthritis and Systemic Lupus Erythematosus.

OBJECTIVE: To describe the frequency of polyautoimmunity and multiple autoimmune syndrome in patients with rheumatoid arthritis (RA) and patients with systemic lupus erythematosus (SLE). PATIENTS AND METHODS: This was a cross-sectional observational study of patients with RA, SLE, and controls without autoimmune rheumatic disease. Cases were those with RA according to the 2010 American College of Rheumatology/European League Against Rheumatism criteria and SLE according to the 2019 American College of Rheumatology/European League Against Rheumatism criteria, consecutively recruited in a rheumatology clinic. Controls were subjects with no rheumatic autoimmune disease (AIDs) recruited in the same area. Patients filled out a questionnaire on polyautoimmunity. Variables of interest were polyautoimmunity (RA or SLE with other AIDs), whereas secondary variables were rheumatic, skin, endocrine, digestive, and neurological AIDs. Multiple autoimmune syndrome is defined as the presence of 3 or more AIDs and a family history of AIDs. Statistical analyses performed were descriptive, bivariate, and multivariate (dependent variable: polyautoimmunity). RESULTS: The study population comprised 109 patients with RA, 105 patients with SLE, and 88 controls. Polyautoimmunity was recorded in 15 patients with RA (13.8%), 43 with SLE (41%), and 2 controls (2.2%). The most frequent AID in RA was Sjögren syndrome (53.3%), followed by Hashimoto thyroiditis and psoriasis; the most frequent AIDs in SLE were Sjögren syndrome (55.8%) and antiphospholipid syndrome (30.2%), followed by Hashimoto thyroiditis. Obesity was associated with polyautoimmunity in RA (odds ratio [OR], 3.362; p = 0.034). In SLE, joint damage (OR, 2.282; p = 0.038) and anti-RNP antibodies (OR, 5.095; p = 0.028) were risk factors for polyautoimmunity, and hydroxychloroquine was a protective factor (OR, 0.190; p = 0.004). CONCLUSIONS: Polyautoimmunity is frequent in RA and even more frequent in SLE. It was associated with obesity in RA and with joint damage and anti-RNP in SLE. Hydroxychloroquine was a protector.

Incidence and case fatality rate of COVID-19 in patients with inflammatory articular diseases.

OBJECTIVE: To describe the incidence and fatality of coronavirus disease 2019 (COVID-19) and identify risk factors to fatality in patients with inflammatory articular diseases (IAD). METHODS: This is a cross-sectional observational study of IAD patients and COVID-19 with controls matched for age, sex, and RT-PCR. A control group was used to compare the cumulative incidence (CI) and case fatality rate (CFR). The main outcomes of the study were CI and CFR. Other variables included comorbidities, treatments, and characteristics of the COVID-19. Multiple logistic regression analysis was performed to investigate risk factors for fatality in patients with IAD. RESULTS: Of the 1537 patients who fulfilled the inclusion criteria, 23/1537 (1.49%) had IAD 13 (0.8%) had rheumatoid arthritis (RA), 5 psoriatic arthritis (PsA) (0.3%) and 5 axial spondyloarthritis (0.3%). There were no significant differences in CI of COVID-19 and CFR in patients with IAD compared with COVID-19 patients without IAD. In RT-PCR positive patients, the CI of COVID-19 in PsA and AS was higher. Of the 23 IAD patients, 2 RA patients (8.6%) died. The patients did no show characteristics of the COVID-19 disease different from the population. In multivariate analysis, the factor associated with fatality in patients with IAD was older age (OR [95% CI], 1.1 [1.0-1.2]). CONCLUSION: COVID-19 CI, fatality rate and other features do not seem to be increased in IAD patients. Older age was associated with fatality in patients with IAD.

Molecular Mechanisms Controlling the Disease Cycle in the Vascular Pathogen Verticillium dahliae Characterized Through Forward Genetics and Transcriptomics.

The soil-borne pathogen Verticillium dahliae has a worldwide distribution and a plethora of hosts of agronomic value. Molecular analysis of virulence processes can identify targets for disease control. In this work, we compared the global gene transcription profile of random T-DNA insertion mutant strain D-10-8F, which exhibits reduced virulence and alterations in microsclerotium formation and polar growth, with that of the wild-type strain. Three genes identified as differentially expressed were selected for functional characterization. To produce deletion mutants, we developed an updated version of one-step construction of Agrobacterium-recombination-ready plasmids (OSCAR) that included the negative selection marker HSVtk (herpes simplex virus thymidine kinase gene) to prevent ectopic integration of the deletion constructs. Deletion of VdRGS1 (VDAG_00683), encoding a regulator of G protein signaling (RGS) protein and highly upregulated in the wild type versus D-10-8F, resulted in phenotypic alterations in development and virulence that were indistinguishable from those of the random T-DNA insertion mutant. In contrast, deletion of the other two genes selected, vrg1 (VDAG_07039) and vvs1 (VDAG_01858), showed that they do not play major roles in morphogenesis or virulence in V. dahliae. Taken together the results presented here on the transcriptomic analysis and phenotypic characterization of D-10-8F and ∆VdRGS1 strains provide evidence that variations in G protein signaling control the progression of the disease cycle in V. dahliae. We propose that G protein-mediated signals induce the expression of multiple virulence factors during biotrophic growth, whereas massive production of microsclerotia at late stages of infection requires repression of G protein signaling via upregulation of VdRGS1 activity.

Mycoviruses in Biological Control: From Basic Research to Field Implementation.

Mycoviruses from plant pathogens can induce hypovirulence (reduced virulence) in their host fungi and have gained considerable attention as potential biocontrol tools. An increasing number of mycoviruses that induce fungal hypovirulence, from a wide variety of taxonomic groups, are currently being reported. Successful application of these viruses in disease management is greatly dependent on their ability to spread in the natural populations of the pathogen. Mycoviruses generally lack extracellular routes of transmission. Hyphal anastomosis is the main route of horizontal mycovirus transmission to other isolates, and conidia of vertical transmission to the progeny. Transmission efficiencies are influenced by both the fungal host and the infecting virus. Interestingly, artificial transfection methods have shown that potential biocontrol mycoviruses often have the ability to infect a variety of fungi. This expands their possible use to the control of pathogens others than those where they were identified. Mycovirus research is also focused on gaining insights into their complex molecular biology and the molecular bases of fungus-virus interactions. This knowledge could be exploited to manipulate the mycovirus and/or the host and generate combinations with enhanced properties in biological control. Finally, when exploring the use of mycoviruses in field conditions, the pathogen life style and the characteristics of the disease and crops affected will deeply impact the specific challenges to overcome, and the development of biocontrol formulations and delivery methods.

Mycovirus Fusarium oxysporum f. sp. dianthi Virus 1 Decreases the Colonizing Efficiency of Its Fungal Host.

Mycoviruses that induce hypovirulence in phytopathogenic fungi are interesting because their potential use as biological control agents of the plant diseases caused by their fungal hosts. The recently identified chrysovirus Fusarium oxysporum f. sp. dianthi virus 1 (FodV1) has been associated to the induction of hypovirulence in Fusarium oxysporum f. sp. dianthi, the forma specialis of F. oxysporum that causes vascular wilt in carnation (Dianthus caryophyllus). In this work, we have used confocal laser scanner microscopy and two isogenic GFP-labeled strains of F. oxysporum f. sp. dianthi infected (V+) and not infected (V-) with the Fusarium oxysporum f. sp. dianthi virus 1, respectively, to analyze the effect of mycovirus FodV1 on the plant colonization pattern of its fungal host. Results demonstrate that FodV1-viral infection affects the speed and spatial distribution of fungal colonization into the plant. Initial stages of external root colonization were similar for both strains, but the virus-free strain colonized the internal plant tissues faster than the virus-infected strain. In addition, other differences related to the specific zone colonized and the density of colonization were observed between both F. oxysporum f. sp. dianthi strains. The hyphae of both V- and V+ strains progressed up through the xylem vessels but differences in the number of vessels colonized and of hyphae inside them were found. Moreover, as colonization progressed, V- and V+ hyphae propagated horizontally reaching the central medulla but, while the virus-free strain V- densely colonized the interior of the medulla cells, the virus-infected strain V+ appeared mainly in the intercellular spaces and with a lower density of colonization. Finally, the incidence of FodV1-viral infections in a collection of 221 isolates sampled between 2008 and 2012 in the geographic area where the originally infected isolate was obtained has been also analyzed. The very low (<2%) incidence of viral infections is discussed here. To the best of our knowledge, this work provides the first microscopic evidence about the effect of a hypovirulence-inducing mycovirus on the pattern of plant colonization by its fungal host.

Molecular and Biological Characterization of the First Hypovirus Identified in Fusarium oxysporum.

A novel mycovirus named Fusarium oxysporum f. sp. dianthi hypovirus 2 (FodHV2) has been identified infecting isolates Fod 408 and Fod 409 of Fusarium oxysporum f. sp. dianthi from Morocco. The genome of FodHV2 is 9,444 nucleotides long excluding the poly(A) tail, and has a single open reading frame encoding a polyprotein. The polyprotein contains three highly conserved domains of UDP glucose/sterol glucosyltransferase, RNA-dependent RNA polymerase, and viral RNA helicase. In addition, particular residues of Cys, Hys, and Gly detected in the N-terminal region suggest the presence of the catalytic site of a highly diverged papain-like protease. Genomic organization, presence of particular conserved motifs, and phylogenetic analyses based on multiple alignments clearly grouped FodHV2 with the members of the family Hypoviridae. FodHV2 was transferred by hyphal anastomosis to a recipient HygR-tagged virus-free strain. The comparison of the infected and non-infected isogenic strains showed that FodHV2 did not alter the vegetative growth, neither the conidiation nor the virulence of its fungal host. Efficiency of FodHV2 transmission through the conidia was 100% in both the original and the recipient infected-isolates. To the best of our knowledge, this is the first report of a hypovirus infecting the plant pathogen F. oxysporum, and also the first one of a hypovirus detected in a fungal strain from the African continent.

Vólvulo de ciego secundario a brida epiploica entre colon transverso y anillo inguinal interno izquierdo / Cecal volvulus secondary to epiploic bridle between transverse colon and left internal inguinal ring

El vólvulo cecal es una causa poco frecuente de oclusión intestinal. Generalmente, es causado por exceso de movilidad del ciego. Aunque existen múltiples causas desencadenantes, la presencia de una brida epiploíca sin antecedentes de cirugía abdominal, constituye una etiología excepcional. La presentación clínica es inespecífica y el diagnóstico se confirma mediante TC de abdomen. Presenta un alto índice de mortalidad si no se diagnostica de manera precoz. El abordaje mínimamente invasivo constituye el trata-miento de elección. Presentamos el caso de una paciente de 96 años de edad sin antecedentes de cirugía abdominal, que presentó un cuadro de oclusión intestinal debido a vólvulo cecal secundario a brida epiploíca entre colon transverso y anillo inguinal profundo izquierdo, que requirió una hemicolectomía derecha abierta de urgencia. Realizamos una revisión de los aspectos relacionados con la etiología, fisiopatología, manifestaciones clínicas, diagnóstico y tratamiento

Cecal volvulus is a rare cause of intestinal occlusion. Generally, it is caused by excess mobility of the blind. Although there are multiple triggers causes, the presence of an epiploic bridle without a history of abdominal surgery constitutes an exceptional etio-logy. The clinical presentation is nonspecific and the diagnosis is confirmed by abdominal CT. It has a high mortality rate if it is not diagnosed early. The minimally invasive approach constitutes the treatment of choice. We present the case of a 96-year-old patient withouth a history of abdominal surgery, who presented an acute cecal volvulus secondary to an epiploic bridle between the transverse colon and left internal inguinal ring, which required an emergency right hemicolectomy. We review the aspects related to etiology, physiopathology, clinical manifestations, diagnosis and treatment

Fusarium oxysporum f. sp. dianthi virus 1 Accumulation Is Correlated with Changes in Virulence and Other Phenotypic Traits of Its Fungal Host.

Fusarium oxysporum f. sp. dianthi virus 1 (FodV1) was detected in isolate 116 (116V+) of Fusarium oxysporum f. sp. dianthi, reaching such a high accumulation level that it was clearly visible after agarose gel electrophoresis of total DNA extracts. FodV1 consists of four double-stranded RNA segments that correspond to a new mycovirus in the Chrysoviridae family. We obtained an isolate of F. oxysporum f. sp. dianthi 116 (116V-) with only a residual level of FodV1 RNA accumulation by single-conidia selection. Compared with 116V-, isolate 116V+ showed significant phenotypic alterations in vegetative growth and virulence. Thus, the presence of a high titer of mycovirus FodV1 was associated with a modified morphology and a reduced growth of the colonies on solid medium, and with a diminished conidiation in liquid medium. Inoculation of four susceptible carnation cultivars with either 116V- or 116V+ showed that the presence of a high titer of FodV1 was also correlated with a significantly reduced virulence of its fungal host. All of the results suggest that FodV1 could be associated with hypovirulence, identifying it as a potential biocontrol agent for Fusarium wilt of carnation. This is the first report of a mycovirus potentially associated with the induction of hypovirulence in the species F. oxysporum.

Characterization of a novel single-stranded RNA mycovirus related to invertebrate viruses from the plant pathogen Verticillium dahliae.

Fungal viruses, also known as mycoviruses, are widespread in all major groups of fungi. Mycoviruses from plant pathogens can reduce the virulence of their host fungus and have therefore potential as biological control agents. This has spurred the identification of novel mycoviruses in plant pathogens, research which is greatly contributing to our understanding of these organisms. In this work, we report the characterization of a novel monopartite mycovirus from Verticillium dahliae, the main causal agent of Verticillium wilt. This novel mycovirus, which we termed Verticillium dahliae RNA virus 1 (VdRV1), was identified in three different isolates of V. dahliae collected in olive growing areas of the Guadalquivir valley, southern Spain. We determined that the VdRV1 genome is a positive (+) single-stranded (ss) RNA, 2631 nucleotides in length, containing two open reading frames. VdRV1 showed few similarities with known mycoviruses, only with a group of unassigned (+) ssRNA mycoviruses which are related to plant viruses classified within the family Tombusviridae. However, phylogenetic analysis revealed that VdRV1 and the unassigned (+) ssRNA mycoviruses have a closer relationship with recently reported invertebrate viruses. This result indicates that as more viral sequences become available, the relationships of mycoviruses with viruses from other hosts should be reexamined. Additionally, the work supports the hypothesis of a heterogeneous origin for mycoviruses.

The Heterologous Expression of the p22 RNA Silencing Suppressor of the Crinivirus Tomato Chlorosis Virus from Tobacco Rattle Virus and Potato Virus X Enhances Disease Severity but Does Not Complement Suppressor-Defective Mutant Viruses.

To counteract host antiviral RNA silencing, plant viruses express suppressor proteins that function as pathogenicity enhancers. The genome of the Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae) encodes an RNA silencing suppressor, the protein p22, that has been described as having one of the longest lasting local suppressor activities when assayed in Nicotiana benthamiana. Since suppression of RNA silencing and the ability to enhance disease severity are closely associated, we analyzed the effect of expressing p22 in heterologous viral contexts. Thus, we studied the effect of the expression of ToCV p22 from viral vectors Tobacco rattle virus (TRV) and Potato virus X (PVX), and from attenuated suppressor mutants in N. benthamiana plants. Our results show that although an exacerbation of disease symptoms leading to plant death was observed in the heterologous expression of ToCV p22 from both viruses, only in the case of TRV did increased viral accumulation occur. The heterologous expression of ToCV p22 could not complement suppressor-defective mutant viruses.

The p22 RNA Silencing Suppressor of the Crinivirus Tomato chlorosis virus is Dispensable for Local Viral Replication but Important for Counteracting an Antiviral RDR6-Mediated Response during Systemic Infection.

Among the components of the RNA silencing pathway in plants, RNA-dependent RNA polymerases (RDRs) play fundamental roles in antiviral defence. Here, we demonstrate that the Nicotiana benthamiana RDR6 is involved in defence against the bipartite crinivirus (genus Crinivirus, family Closteroviridae) Tomato chlorosis virus (ToCV). Additionally, by producing a p22-deficient ToCV infectious mutant clone (ToCVΔp22), we studied the role of this viral suppressor of RNA silencing in viral infection in both wild-type and RDR6-silenced N. benthamiana (NbRDR6i) plants. We demonstrate that p22 is dispensable for the replication of ToCV, where RDR6 appears not to have any effect. Furthermore, the finding that ToCV∆p22 systemic accumulation was impaired in wild-type N. benthamiana but not in NbRDR6i plants suggests a role for p22 in counteracting an RDR6-mediated antiviral response of the plant during systemic infection.

The p22 RNA silencing suppressor of the crinivirus Tomato chlorosis virus preferentially binds long dsRNAs preventing them from cleavage.

Viruses encode silencing suppressor proteins to counteract RNA silencing. Because dsRNA plays a key role in silencing, a general silencing suppressor strategy is dsRNA binding. The p22 suppressor of the plant virus Tomato chlorosis virus (ToCV; genus Crinivirus, family Closteroviridae) has been described as having one of the longest lasting local suppressor activities. However, the mechanism of action of p22 has not been characterized. Here, we show that ToCV p22 binds long dsRNAs in vitro, thus interfering with their processing into small RNAs (sRNAs) by an RNase III-type Dicer homolog enzyme. Additionally, we have studied whether a putative zinc finger motif found in p22 has a role in dsRNA binding and suppressor function. The efficient ability of p22 to suppress RNA silencing, triggered by hairpin transcripts transiently expressed in planta, supports the relationship between its ability to bind dsRNA in vitro and its ability to inhibit RNA silencing in vivo.

Genetic diversity and silencing suppression activity of the p22 protein of Tomato chlorosis virus isolates from tomato and sweet pepper.

As for other bipartite criniviruses (genus Crinivirus, family Closteroviridae), the genome of Tomato chlorosis virus encodes an RNA silencing suppressor, the protein p22, in the 3'-proximal region of RNA1. This protein has been reported as having one of the longest lasting local suppressor activities when transiently expressed in Nicotiana benthamiana. Here, we examined the genetic diversity of the p22 gene in ToCV isolates from tomato and sweet pepper. The p22 gene sequences clearly grouped into two separated clades. However, functional analysis of both types of p22 proteins indicated no evident differences in suppressor activity. Our findings provide experimental evidence that the presence of a "strong" silencing suppressor is a conserved feature of ToCV isolates.

Complete genome sequence of a novel dsRNA mycovirus isolated from the phytopathogenic fungus Fusarium oxysporum f. sp. dianthi.

A novel double-stranded RNA (dsRNA) mycovirus, designated Fusarium oxysporum f. sp. dianthi mycovirus 1 (FodV1), was isolated from a strain of the phytopathogenic fungus F. oxysporum f. sp. dianthi. The FodV1 genome had four dsRNA segments, designated, from the largest to the smallest one, dsRNA 1, 2 3, and 4. Each one of these segments contained a single open reading frame (ORF). dsRNA 1 (3555 bp) and dsRNA 3 (2794 bp) encoded a putative RNA-dependent RNA polymerase (RdRp) and a putative coat protein (CP), respectively. dsRNA 2 (2809 bp) and dsRNA 4 (2646 bp) contained ORFs encoding hypothetical proteins (named P2 and P4, respectively) with unknown functions. Analysis of its genomic structure, homology searches of the deduced amino acid sequences, and phylogenetic analysis all indicated that FodV1 is a new member of the family Chrysoviridae. This is the first report of the complete genomic characterization of a mycovirus identified in the plant pathogen Fusarium oxysporum.

Study of Phylogenetic Relationships Among Fusarium oxysporum f. sp. dianthi Isolates: Confirmation of Intrarace Diversity and Development of a Practical Tool for Simple Population Analyses.

Fusarium wilt, caused by Fusarium oxysporum f. sp. dianthi, is the most important disease of carnation worldwide. Knowing the diversity of the F. oxysporum f. sp. dianthi population present in a carnation growing area is a key component of preventing dramatic losses in production. Sequence analyses of partial ß-tubulin, translation elongation factor 1α genes, and the full-length ribosomal DNA intergenic spacer (IGS) were conducted to resolve phylogenetic relationships in a wide collection of Spanish F. oxysporum f. sp. dianthi isolates, along with some representatives from Italy. We found that, among the three different gene regions, the IGS sequence was the best choice to resolve phylogenetic relationships among F. oxysporum f. sp. dianthi isolates. The phylogenetic tree generated with the complete IGS region was the only one showing a clear clustering of isolates according to the molecular group (virulence grouping) and the vegetative compatibility group. In order to develop a more practical tool based on a shorter DNA sequence to quickly analyze diversity in F. oxysporum f. sp. dianthi populations, we examined IGS nucleotide alignments and identified a region of approximately 300 bp that accumulates enough "informative" changes to resolve intraspecific relationships and determine pathogenic variants in F. oxysporum f. sp. dianthi. Moreover, the "condensed" alignment of this short IGS region showing only the informative positions revealed the existence of virulence group-discriminating positions. In addition to clarifying the phylogenetic relationships among F. oxysporum f. sp. dianthi isolates of the recently described race groups by using multigene genealogies, we have developed simple tools for the phylogenetic analyses of F. oxysporum f. sp. dianthi populations and the determination of the molecular group of uncharacterized F. oxysporum f. sp. dianthi isolates.

A sensitive method for the quantification of virion-sense and complementary-sense DNA strands of circular single-stranded DNA viruses.

Circular single-stranded DNA (ssDNA) viruses are the smallest viruses known to infect eukaryotes. High recombination and mutation rates have conferred these viruses with an evolutionary potential that has facilitated their emergence. Their damaging effects on livestock (circoviruses) and crops (geminiviruses and nanoviruses), and the ubiquity of anelloviruses in human populations and other mammalian species, have resulted in increased interest in better understanding their epidemiology and infection mechanisms. Circular ssDNA viral replication involves the synthesis of dsDNA intermediates containing complementary-sense (CS) and virion-sense (VS) strands. Precise quantification of VS and CS accumulation during viral infections can provide insights into the molecular mechanisms underlying viral replication and the host invasion process. Although qPCR protocols for quantifying viral molecules exist, none of them discriminate VS and CS strands. Here, using a two-step qPCR protocol we have quantified VS and CS molecule accumulation during the infection process of Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) (genus Begomovirus, family Geminiviridae). Our results show that the VS/CS strand ratio and overall dsDNA amounts vary throughout the infection process. Moreover, we show that these values depend on the virus-host combination, and that most CS strands are present as double-stranded molecules.

The complete nucleotide sequence of a novel partitivirus isolated from the plant pathogenic fungus Verticillium albo-atrum.

We have characterized the bisegmented genome of a novel double-stranded RNA (dsRNA) virus isolated from the plant pathogenic fungus Verticillium albo-atrum. We determined that its larger segment (dsRNA1) was 1747 base pairs in length and potentially encoded an RNA-dependent RNA polymerase of 539 amino acids, whereas the smaller segment (dsRNA2) was 1517 base pairs long and was predicted to encode a capsid protein of 435 amino acids. Homology searches and phylogenetic analysis confirmed that, as expected from its dsRNA banding profile, the identified virus was a new member of the family Partitiviridae, and we have therefore designated it V. a lbo- a trum partitivirus 1 (VaaPV1). This is the first report of a mycovirus identified in V. albo-atrum.