Awake prone positioning in nonintubated spontaneous breathing ICU patients with acute hypoxemic respiratory failure (PRONELIFE)-protocol for a randomized clinical trial.

BACKGROUND: It is uncertain whether awake prone positioning can prevent intubation for invasive ventilation in spontaneous breathing critically ill patients with acute hypoxemic respiratory failure. Awake prone positioning could benefit these patients for various reasons, including a reduction in direct harm to lung tissue, and prevention of tracheal intubation-related complications. DESIGN AND METHODS: The PRONELIFE study is an investigator-initiated, international, multicenter, randomized clinical trial in patients who may need invasive ventilation because of acute hypoxemic respiratory failure. Consecutive patients admitted to participating ICUs are randomly assigned to standard care with awake prone positioning, versus standard care without awake prone positioning. The primary endpoint is a composite of tracheal intubation and all-cause mortality in the first 14 days after enrolment. Secondary endpoints include time to tracheal intubation and effects of awake prone positioning on oxygenation parameters, dyspnea sensation, and complications. Other endpoints are the number of days free from ventilation and alive at 28 days, total duration of use of noninvasive respiratory support, total duration of invasive ventilation, length of stay in ICU and hospital, and mortality in ICU and hospital, and at 28, 60, and 90 days. We will also collect data regarding the tolerance of prone positioning. DISCUSSION: The PRONELIFE study is among the first randomized clinical trials investigating the effect of awake prone positioning on intubation rate in ICU patients with acute hypoxemic failure from any cause. The PRONELIFE study is sufficiently sized to determine the effect of awake prone positioning on intubation for invasive ventilation-patients are eligible in case of acute hypoxemic respiratory failure without restrictions regarding etiology. The PRONELIFE study is a pragmatic trial in which blinding is impossible-however, as around 35 ICUs worldwide will participate in this study, its findings will be highly generalizable. The findings of the PRONELIFE study have the potential to change clinical management of patients who may need invasive ventilation because of acute hypoxemic respiratory failure. TRIAL REGISTRATION: ISRCTN ISRCTN11536318 . Registered on 17 September 2021. The PRONELIFE study is registered at clinicaltrials.gov with reference number NCT04142736 (October, 2019).

Clinical risk factors for early mortality in patients with community-acquired septic shock. The importance of adequate source control.

OBJECTIVE: To evaluate the incidence and risk factors for early mortality (EM) in the ICU in patients with community-acquired septic shock (CASS). DESIGN: A retrospective cohort study of patients with CASS admitted to the ICU (2003-2016). SETTING: ICU at a University Hospital in Spain. PATIENTS: All consecutive patients admitted to the ICU with CASS. INTERVENTIONS: None. MAIN VARIABLES OF INTEREST: CASS was defined according to the Sepsis-3 definitions. EM were defined as occurring within of 72h following ICU admission. A multinomial logistic regression analysis was performed to identify the risk factors associated with early deaths. RESULTS: During the study period, 625 patients met the Sepsis-3 criteria and admitted with CASS. 14.4% of all patients died within the first 72h. Of 161 patients who died in the ICU, 90 (55.9%) died within the first 72h. The percentage of early and late mortality did not vary significantly during the study period. The need and adequacy of source control were significantly lower in patients with EM. In the multivariate analysis, ARDS, non-respiratory infections, bacteremia and severity at admission were variables independently associated with EM. The only factor that decreased EM was adequate source control in patients with infections amenable to source control. CONCLUSIONS: The incidence of EM has remained stable over time, which means that more than half of the patients who die from CASS do so within the first 72h. Infections where adequate source control can be performed have lower EM.

Clinical risk factors for early mortality in patients with community-acquired septic shock. The importance of adequate source control.

OBJECTIVE: To evaluate the incidence and risk factors for early mortality (EM) in the ICU in patients with community-acquired septic shock (CASS). DESIGN: A retrospective cohort study of patients with CASS admitted to the ICU (2003-2016). SETTING: ICU at a University Hospital in Spain. PATIENTS: All consecutive patients admitted to the ICU with CASS. INTERVENTIONS: None. MAIN VARIABLES OF INTEREST: CASS was defined according to the Sepsis-3 definitions. EM were defined as occurring within of 72h following ICU admission. A multinomial logistic regression analysis was performed to identify the risk factors associated with early deaths. RESULTS: During the study period, 625 patients met the Sepsis-3 criteria and admitted with CASS. 14.4% of all patients died within the first 72h. Of 161 patients who died in the ICU, 90 (55.9%) died within the first 72h. The percentage of early and late mortality did not vary significantly during the study period. The need and adequacy of source control were significantly lower in patients with EM. In the multivariate analysis, ARDS, non-respiratory infections, bacteremia and severity at admission were variables independently associated with EM. The only factor that decreased EM was adequate source control in patients with infections amenable to source control. CONCLUSIONS: The incidence of EM has remained stable over time, which means that more than half of the patients who die from CASS do so within the first 72h. Infections where adequate source control can be performed have lower EM.

[Recommendations of the Working Groups from the Spanish Society of Intensive and Critical Care Medicine and Coronary Units (SEMICYUC) for the management of adult critically ill patients in the coronavirus disease (COVID-19)]. / Recomendaciones de «hacer¼ y «no hacer¼ en el tratamiento de los pacientes críticos ante la pandemia por coronavirus causante de COVID-19 de los Grupos de Trabajo de la Sociedad Española de Medicina Intensiva, Crítica y Unidades Coronarias (SEMICYUC).

On March 11, 2020, the Director-General of the World Health Organization (WHO) declared the disease caused by SARS-CoV-2 (COVID-19) as a pandemic. The spread and evolution of the pandemic is overwhelming the healthcare systems of dozens of countries and has led to a myriad of opinion papers, contingency plans, case series and emerging trials. Covering all this literature is complex. Briefly and synthetically, in line with the previous recommendations of the Working Groups, the Spanish Society of Intensive, Critical Medicine and Coronary Units (SEMICYUC) has prepared this series of basic recommendations for patient care in the context of the pandemic.

Do sedation and analgesia contribute to long-term cognitive dysfunction in critical care survivors?

Deep sedation during stay in the Intensive Care Unit (ICU) may have deleterious effects upon the clinical and cognitive outcomes of critically ill patients undergoing mechanical ventilation. Over the last decade a vast body of literature has been generated regarding different sedation strategies, with the aim of reducing the levels of sedation in critically ill patients. There has also been a growing interest in acute brain dysfunction, or delirium, in the ICU. However, the effect of sedation during ICU stay upon long-term cognitive deficits in ICU survivors remains unclear. Strategies for reducing sedation levels in the ICU do not seem to be associated with worse cognitive and psychological status among ICU survivors. Sedation strategy and management efforts therefore should seek to secure the best possible state in the mechanically ventilated patient and lower the prevalence of delirium, in order to prevent long-term cognitive alterations.

Renal replacement therapy in the critical patient: treatment variation over time.

OBJECTIVES: To analyze the evolution of patients subjected to renal replacement therapy (RRT), and to determine risk factors associated with mortality and the recovery of renal function. DESIGN: A prospective, observational study of critically ill patients. SETTING: Clinical-surgical Intensive Care Unit (ICU) of Sabadell Hospital (Spain). PATIENTS: Inclusion of all patients treated in our Unit due to acute renal failure (ARF) requiring RRT. PRIMARY VARIABLES OF INTEREST: We recorded epidemiological data, severity using the APACHE II score, days of the technique, ICU mortality, and renal function recovery. The study period was divided into 2 parts: part 1 (2000-2004) and part 2 (2005-2009). The 2 periods were compared using the Student t-test for continuous variables and the chi-squared test for categorical variables. Multiple regression analysis was performed to determine the risk factors for mortality and recovery of renal function. RESULTS: A total of 304 patients were treated. Sepsis was the main etiology of ARF (61%), involving principally respiratory and abdominal foci. In the second period the convective technique and community-acquired ARF were far more prevalent than in the first period. There were fewer days of therapy in the second period (19.7 versus 12.3 days; P=.015). Total ICU mortality was 52.3%, with a decrease in the last period (61.9% to 45.5%: P=.003). The risk factors associated to mortality were creatinine upon admission (odds ratio [OR] 0.77; 95% confidence interval [95%CI] 0.61-0.97) and treatment with IHD alone (OR 0.37, 95%CI 0.16-0.87). Survivors had normal renal function at ICU discharge in 56.7% of the cases in the second period, vs in 72.9% in the first period, with more patients subjected to IHD in the second period (10.4% versus 26.8%). The factors related to the recovery of renal function were creatinine upon admission (OR 1.98, 95%CI 1.12-3.48), acute renal failure (OR 0.11, 95%CI 0.04-0.34) and treatment with continuous techniques (OR 0.18, 95%CI 0.03-0.85). CONCLUSIONS: Mortality among critically ill patients subjected to RRT has improved in recent years.

Annual variations of the plasmatic levels of glucose and amino acid and daily changes under different natural conditions of temperature and photoperiod in Gilthead Sea bream (Sparus aurata, L.).

Daily and annual changes in the plasmatic glucose and amino acid concentration have been determined in Sparus aurata L. Fish (average weight 330 g) were kept in cages under natural conditions of temperature and photoperiod and fed with a commercial diet. The months studied were chosen to establish whether there is any influence on the plasmatic glucose and amino acid concentration due to the change in temperature and photoperiod (equal photoperiod and different temperature, March and October; different photoperiod and equal temperature, May and November; and different photoperiod and temperature, June and January). To study the daily profile of glucose and amino acids concentrations, blood was extracted from six fish every 3 h during 24 h. Annual changes were determined as the average of the samples obtained during 1 day. Results show an annual rhythm with acrophase in June with a positive correlation with photoperiod for glucose and amino acids and with temperature only for amino acids. Daily profiles are rhythmical with a period of 24 h except in November with a period of 8 h for amino acids.

Electrochemical platinum coatings for improving performance of implantable microelectrode arrays.

The formation and properties of electrochemical platinum films grown on platinum contacts contained in implantable flexible microelectrodes were investigated. The resulting platinum deposits were obtained by applying cyclic voltammetry to baths containing concentrations around 70 mM of chloroplatinic acid. A pre-activation step was necessary before the platinum-electroplating step in order to achieve good adhesive properties. The benefits of this process were ascribed to higher corrosion resistance, lower impedance and improved adhesion to the sputtered platinum. These improvements can make the application of this electrochemical technique highly useful for increasing the lifetime of implantable microelectrode arrays, such as cuff structures (IEEE Trans. Biomed. Eng. 40 (1993) 640). These medical devices, obtained by semiconductor technology could be used for selective stimulation of nerve fascicles, although, poor long-term performance has been achieved with them. The dissolution rate for platinum thin-film microelectrodes under fixed corrosion test conditions was 38.8 ng/C. Lower rates were observed for electroplated microelectrodes, obtaining a dissolution rate of 7.8 ng/C under analogous experimental ageing conditions. The corrosion behaviour of the electroplated platinum during stimulation experimental conditions was estimated by electrochemical impedance spectroscopy.

Characterization of a mammalian homolog of the GCN2 eukaryotic initiation factor 2alpha kinase.

In eukaryotic cells, protein synthesis is regulated in response to various environmental stresses by phosphorylating the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha). Three different eIF2alpha kinases have been identified in mammalian cells, the heme-regulated inhibitor (HRI), the interferon-inducible RNA-dependent kinase (PKR) and the endoplasmic reticulum-resident kinase (PERK). A fourth eIF2alpha kinase, termed GCN2, was previously characterized from Saccharomyces cerevisiae, Drosophila melanogaster and Neurospora crassa. Here we describe the cloning of a mouse GCN2 cDNA (MGCN2), which represents the first mammalian GCN2 homolog. MGCN2 has a conserved motif, N-terminal to the kinase subdomain V, and a large insert of 139 amino acids located between subdomains IV and V that are characteristic of the known eIF2alpha kinases. Furthermore, MGCN2 contains a class II aminoacyl-tRNA synthetase domain and a degenerate kinase segment, downstream and upstream of the eIF2alpha kinase domain, respectively, and both are singular features of GCN2 protein kinases. MGCN2 mRNA is expressed as a single message of approximately 5.5 kb in a wide range of different tissues, with the highest levels in the liver and the brain. Specific polyclonal anti-(MGCN2) immunoprecipitated an eIF2alpha kinase activity and recognized a 190 kDa phosphoprotein in Western blots from either mouse liver or MGCN2-transfected 293 cell extracts. Interestingly, serum starvation increased eIF2alpha phosphorylation in MGCN2-transfected human 293T cells. This finding provides evidence that GCN2 is the unique eIF2alpha kinase present in all eukaryotes from yeast to mammals and underscores the role of MGCN2 kinase in translational control and its potential physiological significance.

Characterization of the hemin-sensitive eukaryotic initiation factor 2alpha kinase from mouse nonerythroid cells.

The heme-regulated eukaryotic initiation factor 2alpha (eIF2alpha) kinase (heme-regulated inhibitor (HRI)) is activated by heme deficiency in reticulocytes and plays an important role in translational control in these cells. Previously, HRI was cloned from rabbit reticulocytes and rat brain, but a heme-regulated eIF2alpha kinase activity has only been purified from erythroid cells. In this study, we report the purification of a heme-sensitive eIF2alpha kinase activity from both mouse liver and NIH 3T3 cell extracts. Furthermore, we have cloned and characterized this mouse liver eIF2alpha kinase (mHRI), which exhibits 83 and 94% identities to rabbit and rat HRIs, respectively. Both the purified enzyme and recombinant mHRI exhibited an autokinase and an eIF2alpha kinase activity, and both activities were inhibited in vitro by hemin. In addition, wild-type mHRI, but not the inactive mHRI-K196R mutant, was autophosphorylated in vivo when it was expressed in 293 cells. Quantitation of mHRI mRNA expression in various mouse tissues by reverse transcription-polymerase chain reaction revealed relatively high levels in liver, kidney, and testis. These results provide strong evidence that mHRI is a ubiquitous eIF2alpha kinase of mammalian cells, suggesting that it could play important roles in the translational regulation of nonerythroid tissues.

Cloning and characterization of a cDNA encoding a protein synthesis initiation factor-2alpha (eIF-2alpha) kinase from Drosophila melanogaster. Homology To yeast GCN2 protein kinase.

Phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF-2alpha) is one of the best-characterized mechanisms for downregulating protein synthesis in mammalian cells in response to various stress conditions. In Drosophila, such a regulatory mechanism has not been elucidated. We report the molecular cloning and characterization of DGCN2, a Drosophila eIF-2alpha kinase related to yeast GCN2 protein kinase. DGCN2 contains all of the 12 catalytic subdomains characteristic of eukaryotic Ser/Thr protein kinases and the conserved sequence of eIF-2alpha kinases in subdomain V. A large insert of 94 amino acids, which is characteristic of eIF-2alpha kinases, is also present between subdomains IV and V. It is particularly notable that DGCN2 possesses an amino acid sequence related to class II aminoacyl-tRNA synthetases, a unique feature of yeast GCN2 protein kinase. DGCN2 expression is developmentally regulated. During embryogenesis, DGCN2 mRNA is dynamically expressed in several tissues. Interestingly, at later stages this expression becomes restricted to a few cells of the central nervous system. Affinity-purified antibodies, raised against a synthetic peptide based on the predicted DGCN2 sequence, specifically immunoprecipitated an eIF-2alpha kinase activity and recognized an approximately 175 kDa phosphoprotein in Western blots of Drosophila embryo extracts.

The eIF-2alpha kinases and the control of protein synthesis.

Protein synthesis is regulated in response to environmental stimuli by covalent modification, primarily phosphorylation, of components of the translational machinery. Phosphorylation of the alpha subunit of eIF-2 is one of the best-characterized mechanisms for down-regulating protein synthesis in higher eukaryotes in response to various stress conditions. Three distinct protein kinases regulate protein synthesis in eukaryotic cells by phosphorylating the alpha subunit of eIF-2 at serine-51. There are two mammalian eIF-2alpha kinases: the double-stranded RNA-dependent kinase (PKR) and heme-regulated inhibitor kinase (HRI), and the yeast GCN2. The regulatory mechanisms and the molecular sizes of these eIF-2alpha kinases are different. The expression of PKR is induced by interferon, and the kinase activity is stimulated by low concentrations of double-stranded RNA. HRI is activated under heme-deficient conditions. Yeast GCN2 is activated by amino acid starvation. The phosphorylation of eIF-2alpha results in the shutdown of protein synthesis. Nevertheless, the eIF-2alpha kinases can regulate both global as well as specific mRNA translation. Inhibition of protein synthesis correlates with eIF-2alpha phosphorylation in response to a wide variety of different stimuli, including heat shock, serum deprivation, glucose starvation, amino acid starvation, exposure to heavy metal ions, and viral infection. Finally, recent studies suggest a role for eIF-2alpha phosphorylation in the control of cell growth and differentiation.

Casein kinase II is implicated in the regulation of heme-controlled translational inhibitor of reticulocyte lysates.

The hemin-controlled inhibitor, or HCI, is a cyclic nucleotide-independent protein kinase which specifically phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF-2) leading to potential limitations in functional eIF-2 and decreases in protein synthesis initiation. We have recently demonstrated that hemin regulates eIF-2 alpha kinase activity by promoting formation of the inactive heterodimer between HCI and the 90-kDa heat shock protein (hsp90). The formation of the inactive form of HCI by hemin is prevented by treatment with sulfhydryl reagents such as N-ethylmaleimide or when hsp90 is previously phosphorylated (Méndez, R., Moreno, A., and de Haro, C. (1992) J. Biol. Chem. 267, 11500-11507). In this report, using isoelectric focusing and Western blot analyses with antibodies against a synthetic HCI peptide, we have demonstrated that HCI was also phosphorylated when a heme-reversible HCI preparation was preincubated with ATP. Furthermore, our results indicate that casein kinase II (CK II) is the enzyme involved in the regulation of HCI. Thus, the synthetic peptide RRREEETEEE, which is a specific substrate for CK II, acts as a competitive inhibitor of HCI and hsp90 phosphorylation and, at the same time, inhibits the activation of HCI, whereas a synthetic peptide which corresponds to residues 45-59 of the alpha subunit of eIF-2, including the Ser51 phosphorylated by HCI, only inhibits competitively the phosphorylation of eIF-2 alpha. In addition, treatments which modify hsp90 disabling the formation of the inactive dimer with HCI make unnecessary the presence of CK II for activation of HCI. The data strongly suggest that hemin promotes formation of an inactive HCI.hsp90 dimer preventing phosphorylation by CK II. Interestingly, the addition of the CK II peptide substrate also prevents the activation of HCI in a heme-deficient reticulocyte lysate. We hypothesize, therefore, that under physiological conditions, CK II activity appears to be necessary for activation of HCI.

Regulation of heme-controlled eukaryotic polypeptide chain initiation factor 2 alpha-subunit kinase of reticulocyte lysates.

We have obtained highly purified preparations of the heme-controlled eukaryotic initiation factor 2 alpha-subunit (eIF-2 alpha) kinase (HCI) from rabbit reticulocyte lysates containing five different polypeptides. One of these is a 87-kDa (p87) phosphopeptide which appears to show an autokinase activity. The controlled digestion with trypsin of HCI preparations leads to the suggestion that phosphorylation of p87 is not needed for kinase activity and, furthermore, that another 89-kDa polypeptide could be the kinase catalytic subunit. In agreement with this, monoclonal antibodies directed against p87 do not interfere with eIF-2 alpha kinase activity. Moreover, the anti-p87 antibodies and those directed against the mammalian 90-kDa heat shock protein recognize the same p87 polypeptide from rabbit reticulocyte lysates. Upon incubation of the HCI preparation with hemin (5-10 microM), the eIF-2 alpha kinase is converted into an inactive form and appears to become associated with related peptides forming high molecular weight complexes which can be reversibly activated by 2-mercaptoethanol. The maintenance of the integrity of the porphyrin ring is absolutely required for kinase inactivation and although the presence of metal ion is not essential, the iron and cobalt metalloporphyrins are more effective than protoporphyrin IX. The formation of the inactive form of HCI by hemin is prevented by either N-ethylmaleimide, monoclonal antibodies directed against p87, or phosphorylation of p87. The data strongly suggest that hemin regulates eIF-2 alpha kinase activity by promoting formation of the inactive dimer HCI.p87 via disulfide bonds and direct binding of hemin. A model of HCI regulation is discussed.

Characterization of cell-free protein-synthesis systems from undeveloped and developing Artemia embryos.

We have developed and characterized translationally active cell-free systems from Artemia embryos at different developmental times. The optimized lysates from 16 h-developed embryos incorporated radiolabelled amino acids into polypeptides for up to 120 min. The polypeptides synthesized ranged in Mr from 150,000 to 10,000, suggesting that the endogenous mRNA was capable of directing the synthesis of complete polypeptides. Similar results were obtained by using lysates from early developmental stages; even the cell-free system prepared from 1 h-developed embryos was partially active in protein synthesis. Furthermore, all these lysates were capable of re-initiation, as demonstrated by inhibition of initiation with the inhibitors edeine and 7-methylguanosine 5'-triphosphate. Because we found no endogenous protein-synthetic activity in the corresponding lysates from undeveloped embryos, we have used cell-free translation systems from 0 h- and 16 h-developed Artemia embryos to analyse the mechanisms limiting protein synthesis at very early developmental stages. Undeveloped-embryo lysates supplemented with nuclease-treated reticulocyte lysate were capable of translating endogenous mRNAs to give products with a wide spectrum of Mr values, but lysates of 16 h-developed embryos supplemented in this way were not further stimulated. The nuclease-treated lysate appeared to be unnecessary 5 h after resumption of development. These results suggested that a component(s) limiting translation in the undeveloped-embryo lysate was provided by the nuclease-treated reticulocyte lysate, and that this component(s) no longer limited protein synthesis after development. In view of these results, partially fractionated reticulocyte lysates were tested for restoration of protein-synthetic activity in the undeveloped embryo lysate. A high-salt ribosomal wash devoid of ribosomal subunits, which is considered a crude polypeptide-initiation-factor preparation, also restored translation activity in the undeveloped embryo lysate and made it capable of directing the synthesis of both endogenous mRNAs and exogenous (globin) mRNA.

Primary structure and inhibition of protein synthesis in eukaryotic cell-free system of a novel thionin, gamma-hordothionin, from barley endosperm.

A new sulfur-rich and basic polypeptide, designated as gamma-hordothionin, has been isolated from barley endosperm by a semi-preparative purification consisting of extraction with a volatile salt solution followed by high-performance liquid chromatography using a reversed-phase C4 column. The isolated polypeptide was found to be homogeneous by micro-two-dimensional gel electrophoresis in the presence of sodium dodecyl sulfate. The complete primary structure of gamma-hordothionin was determined by automatic degradation of the intact, S-carboxymethylated and S-pyridylethylated gamma-hordothionin and fragments obtained by proteolytic cleavage. gamma-Hordothionin consists of a single polypeptide chain of 47 amino acids with a calculated molecular mass of 5250 Da and contains four disulfide bridges. gamma-Hordothionin inhibits translation in cell-free systems derived from mammalian (rabbit reticulocyte, mouse liver) as well as non-mammalian (Artemia embryo) cells, at several levels. At low concentrations (1-10 microM) the protein seems to affect mainly the polypeptide-chain-initiation process, although it might also act at the elongation level. At higher concentrations (20-80 microM) this inhibitor induces activation of an eukaryotic polypeptide-chain initiation factor 2 alpha-subunit (eIF-2 alpha) kinase in hemin-supplemented reticulocyte lysates, as does hemin deficiency. The presence of the disulfide bridges in gamma-hordothionin appears to be essential for the eIF-2 alpha kinase activation. Based on its similarity at both the structural and functional level with the different genetic variants of thionins (alpha and beta-thionins, from wheat and barley), gamma-hordothionin is a putative member of the thionin family.

Permanent cell lines that show temperature-dependent expression of adenovirus virus-associated RNA.

Temperature-sensitive COS cells, clone E540, have been stably transformed at a restrictive temperature with plasmid pVA1, which contains the adenovirus type 5 virus-associated (VA) genes in addition to the Neor marker. Transformed cell clones, named EVA cells, contained adenovirus DNA in an integrated form while grown at restrictive temperature but accumulated up to 100 to 200 copies of the input plasmid per cell after temperature shift down. Concomitant with this gene amplification, an accumulation of VA RNA was observed, reaching average concentrations of 10(4) to 10(5) copies per cell. The VA RNA synthesized in EVA cells is functional, as judged by inhibition of in vitro eucaryotic initiation factor-2 phosphorylation and enhancement of reporter gene expression. These EVA cell lines may be of use to study the mechanism of VA RNA function in the absence of adenovirus infection.