El Departamento de Psiquiatría y Salud Mental del Hospital Central de la Defensa «Gómez Ulla» durante la pandemia por SARS-CoV-2 / The Department of Psychiatry and Mental Health of the Military Hospital «Gómez Ulla» during the SARS-CoV-2 pandemic

En el presente trabajo se exponen las medidas estructurales y logísticas, así como la práctica clínica planificada, para poder responder a la pandemia producida por el virus SARS-CoV-2 en el Departamento de Psiquiatría y Salud Mental del Hospital Central de la Defensa «Gómez Ulla». La planificación de la función asistencial se dividió en cinco grupos: los pacientes psiquiátricos ingresados en la Unidad de Hospitalización Breve; los pacientes con patología psiquiátrica ingresados en otros Servicios diferentes de Psiquiatría; los pacientes ambulatorios atendidos en Consultas Externas; los familiares de los pacientes ingresados por la COVID-19; el personal sanitario del Hospital Central de la Defensa «Gómez Ulla». En función de las necesidades de estos grupos asistenciales se realizó una planificación integral de la atención a los mismos. Durante el periodo 14 de marzo al 30 de mayo el 13% del grupo de profesionales del departamento de psiquiatría y salud mental, presentó síntomas moderados- graves de COVID-19; el 19% de los pacientes psiquiátricos ingresados en la unidad de hospitalización fueron COVID19 positivos, no falleciendo ninguno. El 74% de las interconsultas realizadas fueron sobre pacientes ingresados por COVID-19 que presentaron mayoritariamente cuadros confusionales de diversa intensidad o psicosis secundarias al uso de fármacos en el tratamiento activo del COVID-19. Se hicieron 4.185 llamadas a familiares, de las cuales el 14% (n=575) fueron a demanda de los propios familiares. Se hicieron más de 200 videollamadas y se mantuvo de forma telemática el 100% de las consultas externas

In this work the structural and logistical measures are exposed, as well as the planned clinical practice, to be able to respond to the pandemic caused by the virus SARS-CoV-2 in the Department of Psychiatry and Mental Health of the Central Defense Hospital «Gómez Ulla». The planning of the care function was divided into five groups: psychiatric patients admitted to the Brief Hospitalization Unit; patients with psychiatric pathology admitted to other different Psychiatric Services; outpatients treated in Outpatient Consultations; the relatives of the patients admitted by COVID-19; the health personnel of the Central Defense Hospital «Gómez Ulla». Based on the needs of these care groups, comprehensive care planning was carried out. During the period March 14 to May 30, 13% of the staff presented moderate-severe symptoms of COVID-19; 19% of the psychiatric patients admitted to the hospitalization unit were COVID19 positive, none of whom died. 74% of the inter-consultations carried out were on patients admitted for COVID-19 who presented mostly confusional symptoms of varying intensity or psychosis secondary to the use of drugs in the active treatment of COVID-19. 4.185 calls were made to family members, of which 14% (n = 575) were at the request of the family members themselves. More than 220 video calls were made and 100% of the external consultations were kept online

Membrane permeability changes induced in Escherichia coli by the SH protein of human respiratory syncytial virus.

The small hydrophobic (SH) protein of human respiratory syncytial virus (HRSV) has been efficiently expressed in Escherichia coli. In analogy to small hydrophobic proteins encoded by other RNA viruses, membrane permeability changes to low-molecular-weight compounds were detected in bacteria expressing HRSV SH protein. These changes implied, at least, the entry of both the protein synthesis inhibitor hygromycin B and the beta-galactoside substrate o-nitrophenyl-beta-d-galactopyranoside and the exit of preloaded [3H]uridine from bacterial cells. Site-directed mutagenesis indicated that the C-terminal end of SH is needed for induction of membrane permeability changes. In addition, amino acid substitution at residue 32 (Ile to Lys) abolished that activity. This was correlated with a drastic increase in SH electrophoretic mobility and a decrease of the predicted values of alpha-helix for all residues of the SH transmembrane domain. Other sequence changes have either partial effect or no effect on the membrane permeability changes induced by the SH protein. However, none of the mutations abrogated the association of SH protein with bacterial membranes, indicating that incorporation of SH protein to membranes is not sufficient to induce the observed changes. Membrane permeability changes then might provide a useful test for the identification of key amino acid residues in this unique HRSV gene product.

The entry of reovirus into L cells is dependent on vacuolar proton-ATPase activity.

Inhibitors of vacuolar proton-ATPase activity (5 microM bafilomycin A1 or 50 nM concanamycin A) prevented infection by reovirus particles but not by infectious subviral particles (ISVPs). Neither compound affected virus attachment or internalization. However, both compounds potently blocked cleavage of the viral protein mu 1C. Finally, both reovirus particles and ISVPs efficiently translocated the toxin alpha-sarcin to the cytosol during virus entry. Bafilomycin A1 blocked translocation of alpha-sarcin by reovirus particles but not by ISVPs.

Requirement for vacuolar proton-ATPase activity during entry of influenza virus into cells.

The role that endosomal acidification plays during influenza virus entry into MDCK cells has been analyzed by using the macrolide antibiotics bafilomycin A1 and concanamycin A as selective inhibitors of vacuolar proton-ATPase (v-[H+]ATPase), the enzyme responsible for the acidification of endosomes. Bafilomycin A1 and concanamycin A, present at the low concentrations of 5 x 10(-7) and 5 x 10(-9) M, respectively, prevented the entry of influenza virus into cells when added during the first minutes of infection. Attachment of virion particles to the cell surface was not the target for the action of bafilomycin A1. N,N'-Dicyclohexylcarbodiimide, a nonspecific inhibitor of proton-ATPases, also blocked virus entry, whereas elaiophylin, an inhibitor of the plasma-proton ATPase, had no effect. The inhibitory actions of bafilomycin A1 and concanamycin A were tested in culture medium at different pHs. Both antibiotics powerfully prevented influenza virus infection when the virus was added under low-pH conditions. This inhibition was reduced if the virus was bound to cells at 4 degrees C prior to the addition of warm low-pH medium. Moreover, incubation of cells at acidic pH potently blocked influenza virus infection, even in the absence of antibiotics. These results indicate that a pH gradient, rather than low pH, is necessary for efficient entry of influenza virus into cells.

Concanamycin A blocks influenza virus entry into cells under acidic conditions.

The selective inhibitor of the vacuolar proton-ATPase, concanamycin A, powerfully blocks influenza virus entry into cells, if present during the initial times of virus infection. Attachment of virus particles to cells is not prevented by concanamycin A, rather the exit of influenza virus from endosomes is the step blocked by this macrolide antibiotic. Inhibition of influenza virus entry into cells by concanamycin A or by nigericin takes place under acidic conditions. Moreover, if the pH gradient is abolished by pre-incubation of cells in acidic pH, influenza virus entry does not occur even in the absence of any inhibitors. These results indicate that acidic conditions per se are not sufficient to promote virus entry into cells; rather this step of virus infection requires a pH gradient.

Concanamycin A: a powerful inhibitor of enveloped animal-virus entry into cells.

Concanamycin A, a selective inhibitor of the vacuolar proton ATPase, blocks the infection of animal cells by vesicular stomatitis virus, Semliki Forest virus and influenza virus even when the drug is present at the low concentration of 5 nM. Nevertheless the antibiotic prevents neither the attachment, to cells, of Semliki Forest virus nor its subsequent internalization. Under certain conditions, described in this communication, virus entry is prevented even when the pH of the medium is low, thus suggesting that a pH gradient, rather than low pH per se, is required to drive the entry, into cells, of these enveloped animal viruses.

Cross-neutralizing activity against divergent human immunodeficiency virus type 1 isolates induced by the gp41 sequence ELDKWAS.

Previously we identified the highly conserved amino acids Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ecto-domain of gp41 as the epitope of a neutralizing monoclonal antibody (2F5) directed against human immunodeficiency virus type 1. In the present study, the sequence defining the epitope was introduced into the loop of antigenic site B of the influenza virus hemagglutinin. The resulting chimeric virus was able to elicit ELDKWA-specific immunoglobulins G and A in antisera of mice. Moreover, the distantly related human immunodeficiency virus type 1 isolates MN, RF, and IIIB were neutralized by these antisera. These data suggest that this conserved B-cell epitope is a promising candidate for inclusion in a vaccine against AIDS. The results also show that influenza virus can be used to effectively present the antigenic structure of this B-cell epitope.

Influenza virus M2 protein modifies membrane permeability in E. coli cells.

The M2 protein of influenza virus is an integral membrane protein with ion channel activity. This protein has been expressed in E. coli cells in an inducible manner. Expression of the M2 protein causes rapid lysis of BL21(DE3) pLysS E. coli cells upon induction with IPTB. M2 protein increases membrane permeability to a number of hydrophylic molecules, such as ONPG, uridine or impermeant translation inhibitors. The behaviour of M2 in bacteria resembles that of other viral proteins, such as poliovirus 3A and Semliki Forest virus 6K.

Chimeric influenza virus induces neutralizing antibodies and cytotoxic T cells against human immunodeficiency virus type 1.

Expression vectors based on DNA or plus-stranded RNA viruses are being developed as vaccine carriers directed against various pathogens. Less is known about the use of negative-stranded RNA viruses, whose genomes have been refractory to direct genetic manipulation. Using a recently described reverse genetics method, we investigated whether influenza virus is able to present antigenic structures from other infectious agents. We engineered a chimeric influenza virus which expresses a 12-amino-acid peptide derived from the V3 loop of gp120 of human immunodeficiency virus type 1 (HIV-1) MN. This peptide was inserted into the loop of antigenic site B of the influenza A/WSN/33 virus hemagglutinin (HA). The resulting chimeric virus was recognized by specific anti-V3 peptide antibodies and a human anti-gp120 monoclonal antibody in both hemagglutination inhibition and neutralization assays. Mice immunized with the chimeric influenza virus produced anti-HIV antibodies which were able to bind to synthetic V3 peptide, to precipitate gp120, and to neutralize MN virus in human T-cell culture system. In addition, the chimeric virus was also capable of inducing cytotoxic T cells which specifically recognize the HIV sequence. These results suggest that influenza virus can be used as an expression vector for inducing both B- and T-cell-mediated immunity against other infectious agents.

Cloning and inducible synthesis of poliovirus nonstructural proteins.

The poliovirus nonstructural protein-encoding genes have been cloned and expressed in Escherichia coli using the inducible system described by Studier and Moffat [J. Mol. Biol. 189 (1986) 113-130] and Studier [J. Mol. Biol. 219 (1991) 37-44]. The two genes encoding the poliovirus proteases, 2Apro and 3Cpro, were cloned together with their flanking regions in order to test the ability of the polyprotein precursors synthesized to cause proteolytic cleavage and generate mature forms. Both proteases were synthesized and showed activity upon induction in this system. Previously, it had not been possible to produce the three poliovirus nonstructural proteins, 2B, 2C and 3A, and some of their precursors, 2C3AB, 2C3A and 3AB, at high levels in E. coli cells. We report the cloning of their genes using PCR techniques and their efficient expression from pET vectors upon induction with IPTG (isopropyl-beta-D-thiogalactopyranoside). Moreover, some of these proteins, e.g., 3AB, 3A and 2B, are quite toxic for E. coli cells and lysed them upon production. Our results demonstrate the usefulness of this inducible system using the pET vectors to express these toxic poliovirus proteins.

Effects of fatty acids on lipid synthesis and viral RNA replication in poliovirus-infected cells.

Animal viruses profoundly modify the metabolism of lipids, the synthesis of new membranes, and membrane traffic. These alterations are related to the replication of viral genomes. Addition of oleic acid from the beginning of poliovirus infection inhibits the appearance of virus polypeptides at concentrations that do not affect translation in mock-infected HeLa cells. This inhibition is due to the blockade of viral RNA synthesis. Membranes made in poliovirus-infected cells in the presence of oleic acid differ in their buoyant density from control membranes. These results suggest that the incorporation of oleic acid into membranes leads to increased membrane fluidity and decreased buoyant density, making these membranes nonfunctional for poliovirus RNA replication.

Synthesis of Semliki Forest virus RNA requires continuous lipid synthesis.

The involvement of lipid biosynthesis in the replication of Semliki Forest virus (SFV) in HeLa cells has been analyzed by the use of cerulenin, an inhibitor of lipid synthesis. The presence of this agent from the beginning of infection blocks the appearance of viral proteins. However, when the antibiotic is added at later stages of infection it has no effect on protein synthesis, the cleavage of viral proteins and their acylation by palmitic acid. Cerulenin is a powerful inhibitor of viral RNA synthesis, as analyzed by [3H]uridine incorporation, incorporation of [32P]phosphate into viral replication complexes, or Northern blot analysis of viral RNAs hybridized with minus- or plus-stranded riboprobes. Finally, analysis of phospholipids made in SFV-infected cells indicates that viral infection clearly stimulates the synthesis of phosphatidyl choline and modifies the membrane formed as analyzed by sucrose gradient centrifugation. Cerulenin blocks the synthesis of phospholipids and inhibits the formation of new membranes. These results show that, when the synthesis of lipids is blocked by cerulenin, SFV RNA replication is hampered, suggesting that the synthesis of viral RNAs needs continuous lipid synthesis and membrane formation.

Phospholipid biosynthesis and poliovirus genome replication, two coupled phenomena.

Poliovirus infection leads to an increase of phospholipid synthesis and the proliferation of new membranes, giving rise to a great number of cytoplasmic vesicles in the infected cells. Viral RNA replication is physically associated with these newly-synthesized membranes. Cerulenin, an inhibitor of lipid biosynthesis, effectively blocks the growth of poliovirus in HeLa cells. The presence of cerulenin after virus entry prevents the synthesis of poliovirus proteins. However, if this antibiotic is added at later stages of the virus replication cycle, it has no effect on viral translation itself, nor on the proteolytic processing and myristoylation of poliovirus proteins. The synthesis of viral, but not cellular RNA is selectively inhibited by cerulenin. Analysis of the viral RNA made in poliovirus-infected cells by specific minus-or plus-stranded RNA probes suggests a selective blockade by cerulenin of plus-strand RNA synthesis. Finally, the synthesis of phospholipids and the proliferation of membranes does not take place if cerulenin is added to the culture medium. These findings indicate that continuous phospholipid synthesis is required for efficient poliovirus genome replication and provide new insights towards the understanding of the molecular events that occur during poliovirus growth.

Modification of phospholipase C and phospholipase A2 activities during poliovirus infection.

The infection of HeLa cells by poliovirus leads to profound alterations in the activities of both phospholipase C and the A23187-stimulated phospholipase A2. As early as the third hour after poliovirus infection, the activity of phospholipase C is enhanced, as measured by the increase in inositol triphosphate (IP3) in the cells. By the fifth hour post-infection there is a 5-fold increase in IP3 in the infected cells. Therefore, the synthesis of the bulk of poliovirus proteins and poliovirus genomes takes place in cells containing a high and sustained increase in IP3. This augmentation in IP3 is dependent on the multiplicity of infection used. Poliovirus gene expression is required to induce the increase in phospholipase C activity, since the presence of cycloheximide or guanidine blocked it. In contrast to the activation of phospholipase C induced by poliovirus, there is a drastic blockade of the A23187-induced phospholipase A2 activity, measured as the release of [3H]arachidonic acid to the medium. This action on phospholipase A2 is dependent on poliovirus gene expression because it was prevented by cycloheximide or 3-methylquercetin. To our knowledge this is the first report analyzing these two activities in animal virus-infected cells. The findings described may help to explain the profound modifications of both membrane permeability and lipid metabolism undergone by poliovirus-infected cells.