Citrus sudden death (CSD) is a new disease that has killed approximately 1 million orange trees in Brazil.Here we report the identification of a new virus associated with the disease. RNAs isolated from CSD-affected and nonaffected trees were used to construct cDNA libraries. A set of viral sequences present exclusively in libraries of CSD-affected trees was used to obtain the complete genome sequence of the new virus. Phylogenetic analysis revealed that this virus is a new member of the genus Marafivirus. Antibodies raised against the putative viral coat proteins allowed detection of viral antigens of expected sizes in affected plants. Electron microscopy of purified virus confirmed the presence of typical isometric Marafivirus particles. The screening of 773 affected and nonaffected citrus trees for the presence of the virus showed a 99.7% correlation between disease symptoms and the presence of the virus. We also detected the virus in aphids feeding on affected trees. These results suggest that this virus is likely to be the causative agent of CSD. The virus was named Citrus sudden death-associated virus.

BackgroundCoronavirus disease 2019 (COVID-19) is a serious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The primary manifestation is respiratory insu iciency that can also be related to di use pulmonary microthrombosis in people with COVID-19. This disease also causes thromboembolic events, such as pulmonary embolism, deep venous thrombosis, arterial thrombosis, catheter thrombosis, and disseminated intravascular coagulopathy. Recent studies have indicated a worse prognosis for people with COVID-19 who developed thromboembolism.Anticoagulants are medications used in the prevention and treatment of venous or arterial thromboembolic events. Several drugs are used in the prophylaxis and treatment of thromboembolic events, such as heparinoids (heparins or pentasaccharides), vitamin K antagonists and direct anticoagulants. Besides their anticoagulant properties, heparinoids have an additional anti-inflammatory potential, that may a ect the clinical evolution of people with COVID-19. Some practical guidelines address the use of anticoagulants for thromboprophylaxis in people with COVID-19, however, the benefit of anticoagulants for people with COVID-19 is still under debate. ObjectivesTo assess the e ects of prophylactic anticoagulants versus active comparator, placebo or no intervention, on mortality and the need for respiratory support in people hospitalised with COVID-19. Search methodsWe searched CENTRAL, MEDLINE, Embase, LILACS and IBECS databases, the Cochrane COVID-19 Study Register and medRxiv preprint database from their inception to 20 June 2020. We also checked reference lists of any relevant systematic reviews identified and contacted specialists in the field for additional references to trials. Selection criteriaRandomised controlled trials (RCTs), quasi-RCTs, cluster-RCTs and cohort studies that compared prophylactic anticoagulants (heparin, vitamin K antagonists, direct anticoagulants, and pentasaccharides) versus active comparator, placebo or no intervention for the management of people hospitalised with COVID-19. We excluded studies without a comparator group. Primary outcomes were all-cause Prophylactic anticoagulants for people hospitalised with COVID-19 (Review)

Mutagenesis by homologous recombination was evaluated in Xylella fastidiosa by using the bga gene, coding for ␤-galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of bga and the endogenous gene was produced by one or two crossover events leading to ␤-galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a cvaB mutant.Xylella fastidiosa is a fastidious, gram-negative, xylem-limited bacterium (20) that causes a range of economically important plant diseases, including citrus variegated chlorosis (CVC) (2, 17); Pierce's disease (PD) of grapevine; alfalfa dwarf; leaf scorch of almond, coffee, elm, sycamore, oak, plum, mulberry, maple, and oleander; and periwinkle wilt (15,16). Despite the importance of the X. fastidiosa CVC strain in phytopathology, our understanding of the physiology and genetics of this bacterium is still poor. Genetic tools to study the biology of X. fastidiosa are limited due to the difficulty in culturing and transforming this fastidious organism. Production of mutants is an important and necessary way to identify and study genes and then the mechanisms involved in different processes, such as pathogenicity. Several methods could be used to produce mutants: insertion-duplication mutagenesis (IDM), allelic exchange (AE), and transposon mutagenesis. Recently, random mutagenesis by transposition was used to produce mutants in X. fastidiosa PD strains (7). However, this method could not be used to directly inactivate specific genes, which is achieved by homologous recombination (IDM and AE). IDM has been used to disrupt genes in a variety of other organisms, such as Mycobacterium smegmatis (1), Neisseria gonorrhoeae (8), Streptococcus pneumoniae (12), and Lactobacillus sake (13). This mutagenesis involves circular integration, by a single crossover event, between the targeted chromosomal gene and a truncated copy of this gene cloned in a transient suicide or replicative plasmid, resulting in integration of the entire plasmid and duplication of the target sequence. AE results in the replacement of the endogenous gene by its copy disrupted by a selectable marker. This approach involves homologous recombination with two crossovers. We previously reported the transformation of X. fastidiosa with artificial plasmids carrying the X. fastidiosa chromosomal origin of replication (oriC) and a kanamycin resistance gene under the control of the X. fastidiosa 16S rRNA promoter (14). These X. fastidiosa oriC plasmids were found to be integrated in the chromosome at the rRNA promoter site by homologous recombination involving one crossover (14), suggesting that gene disruption by homologous recombination is possible in X. fastidiosa. Here we report the disruption of genes by homologous recombination involving one crossover (IDM) or two crossovers (AE) as tools to produce mutants in X. fastidiosa by site-directed gene disruption.Construction of plasmi...

Xylella fastidiosa causes citrus variegated chlorosis (CVC), a destructive disease of citrus. Xylella fastidiosa forms a biofilm inside plants and insect vectors. Biofilms are complex structures involving X. fastidiosa cells and an extracellular matrix which blocks water and nutrient transport in diseased plants. It is hypothesized that the matrix might be composed of an extracellular polysaccharide (EPS), coded by a cluster of nine genes closely related to the xanthan gum operon of Xanthomonas campestris pv. campestris. To understand the role of X. fastidiosa gum genes on biofilm formation and EPS biosynthesis, we produced gumB and gumF mutants. Xylella fastidiosa mutants were obtained by insertional duplication mutagenesis and recovered after triply cloning the cells. Xylella fastidiosa gumB and gumF mutants exhibited normal cell characteristics; typical colony morphology and EPS biosynthesis were not altered. It was of note that X. fastidiosa mutants showed a reduced capacity to form biofilm when BCYE was used as the sustaining medium, a difference not observed with PW medium. Unlike X. campestris pv. campestris, the expression of the X. fastidiosa gumB or gumF genes was not regulated by glucose.

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Objective: to estimate potential life expectancy gains and differences between males and females, if avoidable deaths from circulatory system diseases, neoplasms and external causes had been eliminated in São Paulo, SP, Brazil, in the period 2014-2016. Methods: this was a cross-sectional study using data from the Mortality Information System (SIM), and the Brazilian Institute of Geography and Statistics (IBGE), using multiple decrement tables. Results: of the 81,087 deaths from the diseases studied here, 75.1% were classified as avoidable; elimination of avoidable deaths due to circulatory system diseases was found to generate the greatest potential life expectancy gains, followed by neoplasms among females and external causes among males. Conclusion: magnitude of lost life expectancy due to avoidable deaths differs between males and females; sex differentials in avoidable mortality therefore persist, producing a series of challenges.

Harmless bacteria inhabiting inner plant tissues are termed endophytes. Population fluctuations in the endophytic bacterium Pantoea agglomerans associated with two species of field cultured citrus plants were monitored over a two-year period. The results demonstrated that populations of P. agglomerans fluctuated in Citrus reticulata but not C. sinensis. A cryptic plasmid pPA3.0 (2.9 kb) was identified in 35 out of 44 endophytic isolates of P. agglomerans and was subsequently sequenced. The origins of replication were identified and nine out of 18 open reading frames (ORFs) revealed homology with described proteins. Notably, two ORFs were related to cellular transport systems and plasmid maintenance. Plasmid pPA3.0 was cloned and the gfp gene inserted to generate the pPAGFP vector. The vector was introduced into P. agglomerans isolates and revealed stability was dependent on the isolate genotype, ninety-percent stability values were reached after 60 hours of bacterial cultivation in most evaluated isolates. In order to definitively establish P. agglomerans as an endophyte, the non-transformed bacterium was reintroduced into in vitro cultivated seedlings and the density of inner tissue colonization in inoculated plants was estimated by bacterium re-isolation, while the tissue niches preferred by the bacterium were investigated by scanning electronic microscopy (SEM). Cells from P. agglomerans (strain ARB18) at similar densities were re-isolated from roots, stems and leaves and colonization of parenchyma and xylem tissues were observed. Data suggested that P. agglomerans is a ubiquitous citrus endophyte harboring cryptic plasmids. These characteristics suggest the potential to use the bacterium as a vehicle to introduce new genes in host plants via endophytic bacterial transformation.

O objetivo geral deste trabalho é analisar o diferencial de mortalidade entre sexos, no sul de Minas Gerais, nos anos 2002 e 2012. Os dados de mortalidade e população foram coletados no endereço eletrônico do Departamento de Informática do Sistema Único de Saúde (DATASUS). Para medir o diferencial de mortalidade entre os sexos, foram analisados o hiato na esperança de vida ao nascer, a razão de sexo entre as taxas específicas de mortalidade e a contribuição dos diferentes grupos etários no hiato de esperança de vida ao nascer. Adicionalmente, foi realizado um estudo das principais causas de morte por sexo, que afetam a população total e por grupo etário. Os resultados mostram que, enquanto a esperança de vida ao nascer aumenta, o diferencial nas esperanças de vida ao nascer tem reduzido. Além disso, apesar das elevadas razões de sexo entre as taxas específicas de mortalidade dos jovens, os idosos são os principais responsáveis por causar aumentos nos diferenciais de mortalidade. Ademais, as principais causas de mortalidade da região são: doenças cardiovasculares, neoplasias e doenças do aparelho respiratório. De forma geral, características de uma população envelhecida e em fases avançadas de transição epidemiológica são observadas na região.