Fibrinolysis versus thoracoscopy: Comparison of results in empyema management in the child.

OBJECTIVE: The objective of this study is to compare the outcome of treatment with drainage and urokinase (UK) versus thoracoscopy (TS) in pleural empyema secondary to complicated pneumonia. METHODS: This was a retrospective study of patients with complicated parapneumonic effusions between 2008 and 2019 treated with UK or TS. Epidemiological and evolutionary data compared days of fever, antibiotic, pre- and postprocedure stay, time to radiological resolution, and complications. The results were expressed as medians and the comparisons were made by the Mann-Whitney U-test. RESULTS: Of 143 patients with NC, 46 were empyemas (26 men), 25 were treated with TS, and 10 were treated with UK. The remaining 11 received combined treatment, being excluded from the study. There were no significant differences between TS versus UK in age (median 4 vs. 3 years), days of fever before the procedure (4 vs. 2) and after (2 vs. 2), days of antibiotic treatment before the procedure (4 vs. 4), overall hospital stay (15 vs. 13 days), and months until radiological normalization (2 vs. 2). The complications related to the therapy were scarce in both groups and had no impact on evolution. Patients with TS had a longer preprocedural stay (4 vs. 1; P < 0.001) and required fewer days of subsequent antibiotic after procedure (8 vs. 11; P = 0.03), and a shorter overall antibiotic treatment time (11 vs. 16; P = 0.03). They also had a shorter post-TS stay (9 vs. 12 days), although this difference did not become significant (P = 0.09). CONCLUSIONS: In our experience, the results obtained with both procedures are quite similar, although patients undergoing TS had a better evolution (fewer days of antibiotic and a tendency to less hospitalization), despite having been performed a priori in more evolved patients.

Expression of the K74 Killer Toxin from Saccharomyces paradoxus Is Modulated by the Toxin-Encoding M74 Double-Stranded RNA 5' Untranslated Terminal Region.

Yeast killer toxins are widely distributed in nature, conferring a competitive advantage to the producer yeasts over nonkiller ones when nutrients are scarce. Most of these toxins are encoded on double-stranded RNAs (dsRNAs) generically called M. L-A members of the viral family Totiviridae act as helper viruses to maintain M, providing the virion proteins that separately encapsidate and replicate L-A and M genomes. M genomes are organized in three regions, a 5' region coding the preprotoxin, followed by an internal poly(A) stretch and a 3' noncoding region. In this work, we report the characterization of K74 toxin encoded on M74 dsRNA from Saccharomyces paradoxus Q74.4. In M74, there is a 5' upstream sequence of 141 nucleotides (nt), which contains regulatory signals for internal translation of the preprotoxin open reading frame (ORF) at the second AUG codon. The first AUG close to the 5' end is not functional. For K74 analysis, M74 viruses were first introduced into laboratory strains of Saccharomyces cerevisiae. We show here that the mature toxin is an α/ß heterodimer linked by disulfide bonds. Though the toxin (or preprotoxin) confers immunity to the carrier, cells with increased K74 loads have a sick phenotype that may lead to cell death. Thus, a fine-tuning of K74 production by the upstream regulatory sequence is essential for the host cell to benefit from the toxin it produces and, at the same time, to safely avoid damage by an excess of toxin. IMPORTANCE Killer yeasts produce toxins to which they are immune by mechanisms not well understood. This self-immunity, however, is compromised in certain strains, which secrete an excess of toxin, leading to sick cells or suicidal phenotypes. Thus, a fine-tuning of toxin production has to be achieved to reach a balance between the beneficial effect of toxin production and the stress imposed on the host metabolism. K74 toxin from S. paradoxus is very active against Saccharomyces uvarum, among other yeasts, but an excess of toxin production is deleterious for the host. Here, we report that the presence of a 5' 141-nt upstream sequence downregulates K74 toxin precursor translation, decreasing toxin levels 3- to 5-fold. Thus, this is a special case of translation regulation performed by sequences on the M74 genome itself, which have been presumably incorporated into the viral RNA during evolution for that purpose.

The persistent breach between evidence and practice in the prevention of surgical site infection. Qualitative study.

BACKGROUND: Despite the dissemination of guidelines for surgical site infection (SSI) prevention, a gap between the theoretical measures and their compliance persists. Accurate estimates of the implementation of preventative measures is crucial before planning dissemination strategies. METHODS: A web-based survey was distributed to members of 11 Associations of operative nurses and surgeons. Questions aimed to determine their awareness of evidence, personal beliefs and actual use of the main preventative measures. RESULTS: Of 1105 responders, 50.5% receive no feed-back of their SSI rate. Responders show a moderate rate of awareness of the recommendations about not removing hair, hair clipping, skin antisepsis with alcoholic solutions, and normothermia. Antibiotic prophylaxis is given for more than 24 h by 18.8% of respondents. Screening for S. aureus is performed by 27.6%. Hair removal by shaving is used by 16.6% of responders. The most common antiseptic solutions are alcoholic chlorhexidine (57.2%) and aqueous povidone (23.3%). 62.8% of surgeons allow the solution to air dry before applying surgical drapes. Adhesive drapes in the surgical field are used routinely in 33.4% of cases. Perioperative normothermia, glucose control and hyperoxia are used in 84.3%, 65.9% and 23.3% of cases. Antimicrobial sutures and negative pressure therapy are used by 20.2% and 43.5% of teams, respectively. Prior to closing the incision, 83.9% replace surgical instruments always or selectively. Wound irrigation before closing is used in 78.1% of cases, mostly with saline. Check-lists, standardized orders, surveillance, feed-back and educational programs were rated most highly by respondents as a means to improve compliance with preventative guidelines, but few of these strategies were in place at their institutions. CONCLUSION: Gaps in the translation of evidence into practice remain in the prevention of SSI among different surgical specialities. Several areas for improvement have been identified, as some core prevention measures are not in common use.

Contribution of executive functions to eating behaviours in obesity and eating disorders.

BACKGROUND: Patients with eating disorders (ED) or obesity show difficulties in tasks assessing decision-making, set-shifting abilities and central coherence. AIMS: The aim of this study was to explore executive functions in eating and weight-related problems, ranging from restricting types of ED to obesity. METHOD: Two hundred and eighty-eight female participants (75 with obesity; 149 with ED: 76 with restrictive eating, 73 with bingeing-purging symptoms; and 64 healthy controls) were administered the Wisconsin Card Sorting Test, the Iowa Gambling Task, and the Group Embedded Figures Test to assess set-shifting, decision-making and central coherence, respectively. RESULTS: Participants with either obesity or ED performed poorly on tests measuring executive functioning compared with healthy controls, even after controlling for age and intelligence. Both participants with obesity and participants with ED showed a preference for global information processing. CONCLUSIONS: The findings suggest that treatments for obesity and ED would benefit from addressing difficulties in cognitive functioning, in addition to the more evident clinical symptoms related to eating, body weight and shape.

Feasibility and Effectiveness of a Workplace-Adapted Mindfulness-Based Programme to Reduce Stress in Workers at a Private Sector Logistics Company: An Exploratory Mixed Methods Study.

There is a high prevalence of stress in the logistics sector owing to very demanding, fast-paced and unpredictable tasks. Mindfulness-based programmes may reduce stress but require considerable practice. Our aim was to evaluate the feasibility and effectiveness of a shortened, workplace-adapted mindfulness-based programme for the logistics sector (WA-MBP-LS) for the purpose of reducing stress. A nonblinded, nonrandomised, two-arm controlled trial was conducted. The WA-MBP-LS (n = 32) consisted of six weekly 90-min mindfulness sessions. The control group (n = 36) attended a psycho-educational seminar. The Perceived Stress Scale (PSS) and Five Facets of Mindfulness Questionnaire (FFMQ) were measured at pretest, posttest and 6-month follow-up. Differences between groups were evaluated using mixed-effects models. Qualitative methods were used to analyse implementation issues. A 64.2% reduction was observed between initial volunteers and actual participants. Attrition at six-month follow-up was 45.6%. Participants attended a median of five sessions. Decreases in PSS favoured the WA-MBP-LS group at posttest and follow-up. FFMQ played a mediating role in PSS reductions. Barriers were disinterest, lack of programming, work overload and absences from work. Facilitators were curiosity, timing, company facilities and audio recordings. The WA-MBP-LS was feasible and effective in reducing stress, but more efforts to improve the practicalities of implementation are desirable.

[Analysis of solid ovarian tumours in a Spanish paediatric population]. / Análisis de los tumores sólidos ováricos pediátricos en nuestra población.

INTRODUCTION AND OBJECTIVES: Ovarian tumours are rare in childhood, and account for 1-5% of all tumours. The aim of this study is to determine the epidemiological features, histological subtypes, and therapeutic management of ovarian solid ovarian tumours of the paediatric population of the province of Cordoba, in Spain. MATERIAL AND METHODS: A retrospective, descriptive, observational and institutional study was conducted in which a review was made of the clinical histories of patients younger than 14years-old diagnosed with ovarian tumours, excluding secondary tumours in a University Hospital between 1994 and 2017. A review was carried out on the age, clinical presentation, laterality, diagnostic methodology, treatment, histopathology, and evolution of these tumours. RESULTS: A total of 37 ovarian tumours were reviewed in 31 patients, 6 of them being bilateral. The mean age was 10.3 (0-14) years, with 58% presenting as a palpable mass. There was no predominance of laterality. The tumour markers were negative. Conservative surgery was performed in 29.7% and adnexectomy in 70.3%. Only one case required post-operative adjuvant chemotherapy treatment (stageI immature teratoma with peritoneal gliomatosis). The histological study shows a predominance of germ cell tumours (65%) against those of epithelial lineage (22%). There were 3 stromal tumours that corresponded to fibroma (Gorlin syndrome), and bilateral gonadoblastoma associated with Frasier syndrome. The most frequent type of tumour was mature cystic teratoma (35.1%). There were no complications in the follow-up. CONCLUSIONS: Given that most childhood ovarian tumours are benign, conservative surgery is considered as the first choice, being even more important in bilateral tumours. If there is a family history, it is essential to carry out molecular genetic studies, to rule out associated syndromes.

The cap-snatching reaction of yeast L-A double-stranded RNA virus is reversible and the catalytic sites on both Gag and the Gag domain of Gag-Pol are active.

The yeast L-A double-stranded RNA virus synthesizes capped transcripts by a unique cap-snatching mechanism in which the m7 Gp moiety of host mRNA (donor) is transferred to the diphosphorylated 5' end of the viral transcript (acceptor). This reaction is activated by viral transcription. Here, we show that cap snatching can be reversible. Because only m7 Gp is transferred during the reaction, the resulting decapped donor, as expected, retained diphosphates at the 5' end. We also found that the 5' terminal nucleotide of the acceptor needs to be G but not A. Interestingly, the A-initiated molecule when equipped with a cap structure (m7 GpppA…) could work as cap donor. Because the majority of host mRNAs in yeast have A after the cap structures at the 5' ends, this finding implies that cap-snatching in vivo is virtually a one-way reaction, in favor of furnishing the viral transcript with a cap. The cap-snatching sites are located on the coat protein Gag and also the Gag domain of Gag-Pol. Here, we demonstrate that both sites are functional, indicating that activation of cap snatching by transcription is not transmitted through the peptide bonding between the Gag and Pol domains of Gag-Pol.

Variation and Distribution of L-A Helper Totiviruses in Saccharomyces sensu stricto Yeasts Producing Different Killer Toxins.

Yeasts within the Saccharomyces sensu stricto cluster can produce different killer toxins. Each toxin is encoded by a medium size (1.5-2.4 Kb) M dsRNA virus, maintained by a larger helper virus generally called L-A (4.6 Kb). Different types of L-A are found associated to specific Ms: L-A in K1 strains and L-A-2 in K2 strains. Here, we extend the analysis of L-A helper viruses to yeasts other than S. cerevisiae, namely S. paradoxus, S. uvarum and S. kudriavzevii. Our sequencing data from nine new L-A variants confirm the specific association of each toxin-producing M and its helper virus, suggesting co-evolution. Their nucleotide sequences vary from 10% to 30% and the variation seems to depend on the geographical location of the hosts, suggesting cross-species transmission between species in the same habitat. Finally, we transferred by genetic methods different killer viruses from S. paradoxus into S. cerevisiae or viruses from S. cerevisiae into S. uvarum or S. kudriavzevii. In the foster hosts, we observed no impairment for their stable transmission and maintenance, indicating that the requirements for virus amplification in these species are essentially the same. We also characterized new killer toxins from S. paradoxus and constructed "superkiller" strains expressing them.

[More than 3 hours and less than 3 years: Safety of anaesthetic procedures in infants less than 3 years old subected to surgery for more the 3 hours]. / Más de 3 horas y menos de 3 años: Seguridad de procedimientos anestésicos en menores de 3 años sometidos a cirugía de más de 3 horas.

An FDA alert in December 2016 on the safety of general anesthesia and sedations in patients less than 3 years of age and pregnant women has raised doubts in relation to the attitude that professionals implicated in these procedures should adopt in relation to these specific group of patients. Confronted with this situation, the following medical scientific societies: Sociedad Española de Anestesia y Reanimación (SEDAR), Sociedad Española de Cirugía Pediátrica (SECP), Sociedad Española de Cuidados Intensivos Pediátricos (SECIP) y Sociedad Española de Neonatología (SENeo), have established a working group to analyze and clarify the safety of these techniques. In the present article we conclude that at present both general anesthesia and profound sedation are considered safe procedures because there is no evidence of the opposite in studies with human beings. However, this ascertained safety should not obviate the problem which still needs to be followed with attention, especially in patients less than 3 years of age undergoing anesthetic procedures for more than 3 hours or prolonged sedation in the Neonatal or Pediatric Intensive Care Units.

[Management of gastroesophageal reflux in children. Single centre experience in conventional and laparoscopic Nissen fundoplication in the last 15 years]. / Manejo del reflujo gastroesofágico en niños. Funduplicatura de Nissen convencional y por laparoscopia en los últimos 15 años en un centro especializado.

INTRODUCTION: Nissen fundoplication (NF) is the most used and effective technique for the treatment of gastroesophageal reflux in children. The laparoscopic approach (LNF) is safe, with low morbidity and high success rate, although some cases require a conventional approach (CNF). The aim of the study is to compare the results between LNF and CNF in our centre. MATERIAL AND METHODS: A retrospective review was performed on patients <14years after NF between 2000 and 2015. A comparison was made of the complications, hospital stay, and follow-up for both approaches. RESULTS: Of the total 75 NF performed, 49 (65.3%) were LNF, 23 (30.7%) CNF, and 3 (4.0%) reconversions. Concomitant laparoscopic gastrostomy was performed in 10.7%, and open gastrostomy in 5.3% of cases. Prior to NF, 10.7% had a gastrostomy. The mean age was 4 years and 68.7% were male. Of the diagnoses, 36% had encephalopathy, 14.7% hiatal hernia, 5.4% oesophageal atresia, and 5.4% an acute life-threatening event. No differences were found in operation time. More than two-thirds (36%) had complications, which were more frequent in the CNF (OR=3.30, 95%CI: 1.1-9.6). The hospital-stay decreased by 9 days in the LNF (95%CI: 5.5-13.5). Mean follow-up was 26 months (95%CI: 20.9-31.6). Mortality during follow-up was of 5.3% (5 respiratory failure, 1 sudden cardiac death, and 2 due to complications of the encephalopathy), 4.2% required re-fundoplication, 15.8% had symptomatic improvement, and 64.0% had absence of symptoms. CONCLUSIONS: The LNF is an effective technique for the treatment of gastroesophageal reflux, with lower morbidity and shorter hospital stay than CNF. It is recommended as the first surgical option.

Relationships and Evolution of Double-Stranded RNA Totiviruses of Yeasts Inferred from Analysis of L-A-2 and L-BC Variants in Wine Yeast Strain Populations.

Saccharomyces cerevisiae killer strains secrete a protein toxin active on nonkiller strains of the same (or other) yeast species. Different killer toxins, K1, K2, K28, and Klus, have been described. Each toxin is encoded by a medium-size (1.5- to 2.3-kb) M double-stranded RNA (dsRNA) located in the cytoplasm. M dsRNAs require L-A helper virus for maintenance. L-A belongs to the Totiviridae family, and its dsRNA genome of 4.6 kb codes for the major capsid protein Gag and a minor Gag-Pol protein, which form the virions that separately encapsidate L-A or the M satellites. Different L-A variants exist in nature; on average, 24% of their nucleotides are different. Previously, we reported that L-A-lus was specifically associated with Mlus, suggesting coevolution, and proposed a role of the toxin-encoding M dsRNAs in the appearance of new L-A variants. Here we confirm this by analyzing the helper virus in K2 killer wine strains, which we named L-A-2. L-A-2 is required for M2 maintenance, and neither L-A nor L-A-lus shows helper activity for M2 in the same genetic background. This requirement is overcome when coat proteins are provided in large amounts by a vector or in ski mutants. The genome of another totivirus, L-BC, frequently accompanying L-A in the same cells shows a lower degree of variation than does L-A (about 10% of nucleotides are different). Although L-BC has no helper activity for M dsRNAs, distinct L-BC variants are associated with a particular killer strain. The so-called L-BC-lus (in Klus strains) and L-BC-2 (in K2 strains) are analyzed. IMPORTANCE: Killer strains of S. cerevisiae secrete protein toxins that kill nonkiller yeasts. The "killer phenomenon" depends on two dsRNA viruses: L-A and M. M encodes the toxin, and L-A, the helper virus, provides the capsids for both viruses. Different killer toxins exist: K1, K2, K28, and Klus, encoded on different M viruses. Our data indicate that each M dsRNA depends on a specific helper virus; these helper viruses have nucleotide sequences that may be as much as 26% different, suggesting coevolution. In wine environments, K2 and Klus strains frequently coexist. We have previously characterized the association of Mlus and L-A-lus. Here we sequence and characterize L-A-2, the helper virus of M2, establishing the helper virus requirements of M2, which had not been completely elucidated. We also report the existence of two specific L-BC totiviruses in Klus and K2 strains with about 10% of their nucleotides different, suggesting different evolutionary histories from those of L-A viruses.

Stabilization of Transfected Cells Expressing Low-Incidence Blood Group Antigens: Novel Methods Facilitating Their Use as Reagent-Cells.

BACKGROUND: The identification of erythrocyte antibodies in the serum of patients rely on panels of human red blood cells (RBCs), which coexpress many antigens and are not easily available for low-incidence blood group phenotypes. These problems have been addressed by generating cell lines expressing unique blood group antigens, which may be used as an alternative to human RBCs. However, the use of cell lines implies several drawbacks, like the requirement of cell culture facilities and the high cost of cryopreservation. The application of cell stabilization methods could facilitate their use as reagent cells in clinical laboratories. METHODS: We generated stably-transfected cells expressing low-incidence blood group antigens (Dia and Lua). High-expresser clones were used to assess the effect of TransFix® treatment and lyophilization as cell preservation methods. Cells were kept at 4°C and cell morphology, membrane permeability and antigenic properties were evaluated at several time-points after treatment. RESULTS: TransFix® addition to cell suspensions allows cell stabilization and proper antigen detection for at least 120 days, despite an increase in membrane permeability and a reduction in antigen expression levels. Lyophilized cells showed minor morphological changes and antigen expression levels were rather conserved at days 1, 15 and 120, indicating a high stability of the freeze-dried product. These stabilized cells have been proved to react specifically with human sera containing alloantibodies. CONCLUSIONS: Both stabilization methods allow long-term preservation of the transfected cells antigenic properties and may facilitate their distribution and use as reagent-cells expressing low-incidence antigens, overcoming the limited availability of such rare RBCs.

Diphosphates at the 5' end of the positive strand of yeast L-A double-stranded RNA virus as a molecular self-identity tag.

The 5'end of RNA conveys important information on self-identity. In mammalian cells, double-stranded RNA (dsRNA) with 5'di- or triphosphates generated during virus infection is recognized as foreign and elicits the host innate immune response. Here, we analyze the 5' ends of the dsRNA genome of the yeast L-A virus. The positive strand has largely diphosphates with a minor amount of triphosphates, while the negative strand has only diphosphates. Although the virus can produce capped transcripts by cap snatching, neither strand carried a cap structure, suggesting that only non-capped transcripts serve as genomic RNA for encapsidation. We also found that the 5' diphosphates of the positive but not the negative strand within the dsRNA genome are crucial for transcription in vitro. Furthermore, the presence of a cap structure in the dsRNA abrogated its template activity. Given that the 5' diphosphates of the transcripts are also essential for cap acquisition and that host cytosolic RNAs (mRNA, rRNA, and tRNA) are uniformly devoid of 5' pp-structures, the L-A virus takes advantage of its 5' terminal diphosphates, using them as a self-identity tag to propagate in the host cytoplasm.

Testicular and paratesticular tumors in children. / Tumores testiculares y paratesticulares en la edad pediátrica.

OBJECTIVES: Testicular (TT) and paratesticular (PT) tumors account for 1-2%of all infant solid tumors. Due to the increased frequency of benign tumors, conservative management is recommended. Our experience and the therapeutic approach adopted considering testis-sparing surgery, was reviewed. METHODS: A retrospective observational study concerning testicular and paratesticular tumors in our hospital between 1998 and 2016, was performed. Age, side, symptoms, imaging, treatment methods, histological findings and evolution were reviewed. RESULTS: Nineteen cases of TT and PT were reviewed in 17 patients. A painless scrotal mass was found in most cases as the initial presentation (79%). Tumor markers were normal in all cases. Similar distribution between germ cell and stromal testicular tumors was found Nevertheless, benign and malignant PT proportion was similar. Testis preserving surgery was performed in 58% of TT and in 57% of PT. CONCLUSIONS: Due to the high incidence of the benign histological findings, testicular sparing surgery should be considered as a first therapeutic option, especially in those cases with normal tumor markers.

Estimation of scavenging capacity of melatonin and other antioxidants: contribution and evaluation in germinated seeds.

Seven edible seeds for the levels of melatonin, phenolic compounds and their antioxidant capacity were evaluated during germination process. Radical scavenging parameters were also studied in standard antioxidants to understand their antiradical actions. Germination brought about significant increases of total phenol compounds in all edible seeds, showing red cabbage, radish and broccoli the highest contents (21.6, 20.4 and 16.4 mg GAE/g DW, respectively). The concentration of melatonin is greatly variable in edible seeds, exhibiting significant increases during germination. The highest levels were found in red cabbage (857 pg/g DW) radish (536 pg/g DW) and broccoli (439 pg/g DW). The germinated seeds which had the highest levels of polyphenols and melatonin were those that showed the most relevant antiradical activities (>97%). This information is valuable for the incorporation of red cabbage, radish and broccoli germinated seeds into the diet to promote potential health benefits.

Resting complexes of the persistent yeast 20S RNA Narnavirus consist solely of the 20S RNA viral genome and its RNA polymerase p91.

The positive strand 20S RNA narnavirus persistently infects Saccharomyces cerevisiae. The 20S RNA genome has a single gene that encodes the RNA-dependent RNA polymerase (p91). 20S RNA forms ribonucleoprotein resting complexes (RNPs) with p91 and resides in the cytoplasm. Here we found no host proteins stoichiometrically associated with the RNP by pull-down experiments. Furthermore, 20S RNA, when expressed from a vector in Escherichia coli, formed RNPs with p91 in the absence of yeast proteins. This interaction required the 3' cis signal for complex formation. Moreover, when 23S RNA, the genome of another narnavirus, was expressed in E. coli, it also formed RNPs with its RNA polymerase p104. Finally, when both RNAs were expressed in the same E. coli cell, they formed RNPs only with their cognate RNA polymerases. These results altogether indicate that narnaviruses RNPs consist of only the viral genomes and their cognate RNA polymerases. Because the copy number of the RNPs can be induced almost equivalent to those of rRNAs in some yeast strains, the absence of host proteins may alleviate the burden on the host by not sequestering proteins into the RNPs. It may also contribute to the persistent infection of narnaviruses by decreasing their visibility.

Cap snatching in yeast L-BC double-stranded RNA totivirus.

Yeast L-A double-stranded RNA virus furnishes its transcript with a 5' cap structure by a novel cap-snatching mechanism in which m(7)Gp from a host mRNA cap structure is transferred to the 5'-diphosphate terminus of the viral transcript. His-154 of the coat protein Gag forms an m(7)Gp adduct, and the H154R mutation abolishes both m(7)Gp adduct formation and cap snatching. Here we show that L-BC, another totivirus closely related to L-A, also synthesizes 5'-diphosphorylated transcripts and transfers m(7)Gp from mRNA to the 5' termini of the transcripts. L-BC Gag also covalently binds to the cap structure and the mutation H156R, which corresponds to H154R of L-A Gag, abolishes cap adduct formation. Cap snatching of the L-BC virus is very similar to that of L-A; N7 methylation of the mRNA cap is essential for cap donor activity, and only 5'-diphosphorylated RNA is used as cap acceptor. L-BC cap snatching is also activated by viral transcription. Furthermore, both viruses require Mg(2+) and Mn(2+) for cap snatching. These cations are not only required for transcription activation but also directly involved in the cap transfer process. These findings support our previous proposal that the cap-snatching mechanism of the L-A virus is shared by fungal totiviruses closely related to L-A. Interestingly, L-A and L-BC viruses accept either viral transcript as cap acceptor in vitro. Because L-A and L-BC viruses cohabit in many yeast strains, it raises the possibility that their cohabitation in the same host may be beneficial for their mutual cap acquisition.

L-A-lus, a new variant of the L-A totivirus found in wine yeasts with Klus killer toxin-encoding Mlus double-stranded RNA: possible role of killer toxin-encoding satellite RNAs in the evolution of their helper viruses.

Yeast killer viruses are widely distributed in nature. Several toxins encoded in double-stranded RNA (dsRNA) satellites of the L-A totivirus have been described, including K1, K2, K28, and Klus. The 4.6-kb L-A genome encodes the Gag major structural protein that forms a 39-nm icosahedral virion and Gag-Pol, a minor fusion protein. Gag-Pol has transcriptase and replicase activities responsible for maintenance of L-A (or its satellite RNAs). Recently we reported a new killer toxin, Klus. The L-A virus in Klus strains showed poor hybridization to known L-A probes, suggesting substantial differences in their sequences. Here we report the characterization of this new L-A variant named L-A-lus. At the nucleotide level, L-A and L-A-lus showed only 73% identity, a value that increases to 86% in the amino acid composition of Gag or Gag-Pol. Two regions in their genomes, however, the frameshifting region between Gag and Pol and the encapsidation signal, are 100% identical, implying the importance of these two cis signals in the virus life cycle. L-A-lus shows higher resistance than L-A to growth at high temperature or to in vivo expression of endo- or exonucleases. L-A-lus also has wider helper activity, being able to maintain not only Mlus but also M1 or a satellite RNA of L-A called X. In a screening of 31 wine strains, we found that none of them had L-A; they carried either L-A-lus or a different L-A variant in K2 strains. Our data show that distinct M killer viruses are specifically associated with L-As with different nucleotide compositions, suggesting coevolution.