Activation of protein kinase receptor (PKR) plays a pro-viral role in mammarenavirus-infected cells.

Many viruses, including mammarenaviruses, have evolved mechanisms to counteract different components of the host cell innate immunity, which is required to facilitate robust virus multiplication. The double-stranded RNA (dsRNA) sensor protein kinase receptor (PKR) pathway plays a critical role in the cell anti-viral response. Whether PKR can restrict the multiplication of the Old World mammarenavirus lymphocytic choriomeningitis virus (LCMV) and the mechanisms by which LCMV may counteract the anti-viral functions of PKR have not yet been investigated. Here we present evidence that LCMV infection results in very limited levels of PKR activation, but LCMV multiplication is enhanced in the absence of PKR. In contrast, infection with a recombinant LCMV with a mutation affecting the 3'-5' exonuclease (ExoN) activity of the viral nucleoprotein resulted in robust PKR activation in the absence of detectable levels of dsRNA, which was associated with severely restricted virus multiplication that was alleviated in the absence of PKR. However, pharmacological inhibition of PKR activation resulted in reduced levels of LCMV multiplication. These findings uncovered a complex role of the PKR pathway in LCMV-infected cells involving both pro- and anti-viral activities.IMPORTANCEAs with many other viruses, the prototypic Old World mammarenavirus LCMV can interfere with the host cell innate immune response to infection, which includes the dsRNA sensor PKR pathway. A detailed understanding of LCMV-PKR interactions can provide novel insights about mammarenavirus-host cell interactions and facilitate the development of effective anti-viral strategies against human pathogenic mammarenaviruses. In the present work, we present evidence that LCMV multiplication is enhanced in PKR-deficient cells, but pharmacological inhibition of PKR activation unexpectedly resulted in severely restricted propagation of LCMV. Likewise, we document a robust PKR activation in LCMV-infected cells in the absence of detectable levels of dsRNA. Our findings have revealed a complex role of the PKR pathway during LCMV infection and uncovered the activation of PKR as a druggable target for the development of anti-viral drugs against human pathogenic mammarenaviruses.

Functional impairment of "helpless" CD8 + memory T cells is transient and driven by prolonged but finite cognate antigen presentation.

Generation of functional CD8 + T cell memory typically requires engagement of CD4 + T cells. However, in certain scenarios, such as acutely-resolving viral infections, effector (T E ) and subsequent memory (T M ) CD8 + T cell formation appear impervious to a lack of CD4 + T cell help during priming. Nonetheless, such "helpless" CD8 + T M respond poorly to pathogen rechallenge. At present, the origin and long-term evolution of helpless CD8 + T cell memory remain incompletely understood. Here, we demonstrate that helpless CD8 + T E differentiation is largely normal but a multiplicity of helpless CD8 T M defects, consistent with impaired memory maturation, emerge as a consequence of prolonged yet finite exposure to cognate antigen. Importantly, these defects resolve over time leading to full restoration of CD8 + T M potential and recall capacity. Our findings provide a unified explanation for helpless CD8 + T cell memory and emphasize an unexpected CD8 + T M plasticity with implications for vaccination strategies and beyond.

Plasmid-Based Lassa Virus Reverse Genetics.

Several mammarenaviruses cause hemorrhagic fever (HF) disease in humans and pose a significant public health problem in their endemic regions. The Old World (OW) mammarenavirus Lassa virus (LASV) is estimated to infect several hundred thousand people yearly in West Africa, resulting in high numbers of Lassa fever (LF) cases, a disease associated with high morbidity and mortality. No licensed vaccines are available to combat LASV infection, and anti-LASV drug therapy is limited to the off-label use of ribavirin whose efficacy remains controversial. The development of reverse genetics approaches has provided investigators with a powerful approach for the investigation of the molecular, cell biology and pathogenesis of mammarenaviruses. The use of cell-based minigenome systems has allowed examining the cis- and trans-acting factors involved in viral genome replication and gene transcription, assembly, and budding, which has facilitated the identification of several anti-mammarenavirus candidate drugs. Likewise, it is possible now to rescue infectious recombinant mammarenaviruses from cloned cDNAs containing predetermined mutations in their genomes to investigate virus-host interactions and mechanisms of viral pathogenesis. Reverse genetics have also allowed the generation of mammarenaviruses expressing foreign genes to facilitate virus detection, to identify antiviral drugs, and to generate live-attenuated vaccine (LAV) candidates. Likewise, reverse genetics techniques have allowed the generation of single-cycle infectious, reporter-expressing mammarenaviruses to study some aspects of the biology of HF-causing human mammarenavirus without the need of high security biocontainment laboratories. In this chapter, we describe the experimental procedures to generate recombinant (r)LASV using state-of-the-art plasmid-based reverse genetics.

ICTV Virus Taxonomy Profile: Leishbuviridae 2023.

Leishbuviridae is a family of negative-sense RNA viruses with genomes of about 8.0 kb that have been found in protists. The leishbuvirid genome consists of three monocistronic RNA segments with open reading frames (ORFs) that encode a nucleoprotein (NP), a glycoprotein (GP), and a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Leishbuviridae, which is available at ictv.global/report/leishbuviridae.

ICTV Virus Taxonomy Profile: Mypoviridae 2023.

Mypoviridae is a family of negative-sense RNA viruses with genomes of about 16.0 kb that have been found in myriapods. The mypovirid genome consists of three monocistronic RNA segments that encode a nucleoprotein (NP), a glycoprotein (GP), and a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Mypoviridae, which is available at: ictv.global/report/mypoviridae.

ICTV Virus Taxonomy Profile: Tulasviridae 2023.

Tulasviridae is a family of ambisense RNA viruses with genomes of about 12.2 kb that have been found in fungi. The tulasvirid genome is nonsegmented and contains three open reading frames (ORFs) that encode a nucleoprotein (NP), a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain, and a protein of unknown function (X). This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Tulasviridae, which is available at ictv.global/report/tulasviridae.

ICTV Virus Taxonomy Profile: Wupedeviridae 2023.

Wupedeviridae is a family of negative-sense RNA viruses with genomes of about 20.5 kb that have been found in myriapods. The wupedevirid genome consists of three monocistronic RNA segments with open reading frames (ORFs) that encode a nucleoprotein (NP), a glycoprotein (GP), and a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Wupedeviridae, which is available at ictv.global/report/wupedeviridae.

ICTV Virus Taxonomy Profile: Cruliviridae 2023.

Cruliviridae is a family of negative-sense RNA viruses with genomes of 10.8-11.5 kb that have been found in crustaceans. The crulivirid genome consists of three RNA segments with ORFs that encode a nucleoprotein (NP), a glycoprotein (GP), a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain, and in some family members, a zinc-finger (Z) protein of unknown function. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Cruliviridae, which is available at ictv.global/report/cruliviridae.

Activation of Protein Kinase R (PKR) Plays a Pro-Viral Role in Mammarenavirus Infected Cells.

Many viruses, including mammarenaviruses, have evolved mechanisms to counteract different components of the host cell innate immunity, which is required to facilitate robust virus multiplication. The double strand (ds)RNA sensor protein kinase receptor (PKR) pathway plays a critical role in the cell antiviral response. Whether PKR can restrict the multiplication of the Old World mammarenavirus lymphocytic choriomeningitis virus (LCMV) and the mechanisms by which LCMV may counteract the antiviral functions of PKR have not yet been investigated. Here we present evidence that LCMV infection results in very limited levels of PKR activation, but LCMV multiplication is enhanced in the absence of PKR. In contrast, infection with a recombinant LCMV with a mutation affecting the 3'-5' exonuclease (ExoN) activity of the viral nucleoprotein (NP) resulted in robust PKR activation in the absence of detectable levels of dsRNA, which was associated with severely restricted virus multiplication that was alleviated in the absence of PKR. However, pharmacological inhibition of PKR activation resulted in reduced levels of LCMV multiplication. These findings uncovered a complex role of the PKR pathway in LCMV-infected cells involving both pro-and antiviral activities.

Identification of In Vitro Inhibitors of Monkeypox Replication.

Monkeypox virus (MPXV) infections in humans have historically been restricted to regions of endemicity in Africa. However, in 2022, an alarming number of MPXV cases were reported globally, with evidence of person-to-person transmission. Because of this, the World Health Organization (WHO) declared the MPXV outbreak a public health emergency of international concern. The supply of MPXV vaccines is limited, and only two antivirals, tecovirimat and brincidofovir, approved by the U.S. Food and Drug Administration (FDA) for the treatment of smallpox, are currently available for the treatment of MPXV infection. Here, we evaluated 19 compounds previously shown to inhibit different RNA viruses for their ability to inhibit orthopoxvirus infections. We first used recombinant vaccinia virus (rVACV) expressing fluorescence (mScarlet or green fluorescent protein [GFP]) and luciferase (Nluc) reporter genes to identify compounds with antiorthopoxvirus activity. Seven compounds from the ReFRAME library (antimycin A, mycophenolic acid, AVN-944, pyrazofurin, mycophenolate mofetil, azaribine, and brequinar) and six compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) showed inhibitory activity against rVACV. Notably, the anti-VACV activity of some of the compounds in the ReFRAME library (antimycin A, mycophenolic acid, AVN-944, mycophenolate mofetil, and brequinar) and all the compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) were confirmed with MPXV, demonstrating their inhibitory activity in vitro against two orthopoxviruses. IMPORTANCE Despite the eradication of smallpox, some orthopoxviruses remain important human pathogens, as exemplified by the recent 2022 monkeypox virus (MPXV) outbreak. Although smallpox vaccines are effective against MPXV, access to those vaccines is limited. In addition, current antiviral treatment against MPXV infections is limited to the use of the FDA-approved drugs tecovirimat and brincidofovir. Thus, there is an urgent need to identify novel antivirals for the treatment of MPXV infection and other potentially zoonotic orthopoxvirus infections. Here, we show that 13 compounds, derived from two different libraries, previously found to inhibit several RNA viruses, also inhibit VACV. Notably, 11 compounds also displayed inhibitory activity against MPXV.

Antivirals against monkeypox infections.

Monkeypox virus (MPXV) infection in humans are historically restricted to endemic regions in Africa. However, in 2022, an alarming number of MPXV cases have been reported globally with evidence of person-to-person transmission. Because of this, the World Health Organization (WHO) declared the MPXV outbreak a public health emergency of international concern. MPXV vaccines are limited and only two antivirals, tecovirimat and brincidofovir, approved by the United States (US) Food and Drug Administration (FDA) for the treatment of smallpox, are currently available for the treatment of MPXV infection. Here, we evaluated 19 compounds previously shown to inhibit different RNA viruses for their ability to inhibit Orthopoxvirus infections. We first used recombinant vaccinia virus (rVACV) expressing fluorescence (Scarlet or GFP) and luciferase (Nluc) reporter genes to identify compounds with anti-Orthopoxvirus activity. Seven compounds from the ReFRAME library (antimycin A, mycophenolic acid, AVN- 944, pyrazofurin, mycophenolate mofetil, azaribine, and brequinar) and six compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) showed antiviral activity against rVACV. Notably, the anti-VACV activity of some of the compounds in the ReFRAME library (antimycin A, mycophenolic acid, AVN- 944, mycophenolate mofetil, and brequinar) and all the compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) were confirmed with MPXV, demonstrating the broad-spectrum antiviral activity against Orthopoxviruses and their potential to be used for the antiviral treatment of MPXV, or other Orthopoxvirus, infections. IMPORTANCE: Despite the eradication of smallpox, some Orthopoxviruses remain important human pathogens, as exemplified by the recent 2022 monkeypox virus (MPXV) outbreak. Although smallpox vaccines are effective against MPXV, there is presently limited access to those vaccines. In addition, current antiviral treatment against MPXV infections is limited to the use of the FDA-approved drugs tecovirimat and brincidofovir. Thus, there is an urgent need to identify novel antivirals for the treatment of MPXV, and other potentially zoonotic Orthopoxvirus infections. Here, we show that thirteen compounds, derived from two different libraries, previously found to inhibit several RNA viruses, exhibit also antiviral activity against VACV. Notably, eleven compounds also displayed antiviral activity against MPXV, demonstrating their potential to be incorporated into the therapeutic armamentarium to combat Orthopoxvirus infections.

First Report of OXA-181-Producing Enterobacterales Isolates in Latin America.

We characterized five carbapenemase-producing Enterobacterales (CPE) isolates from two health care institutions in Lima, Peru. The isolates were identified as Klebsiella pneumoniae (n = 3), Citrobacter portucalensis (n = 1), and Escherichia coli (n = 1). All were identified as blaOXA-48-like gene carriers using conventional PCR. Whole-genome sequencing found the presence of the blaOXA-181 gene as the only carbapenemase gene in all isolates. Genes associated with resistance to aminoglycosides, quinolones, amphenicols, fosfomycins, macrolides, tetracyclines, sulfonamides, and trimethoprim were also found. The plasmid incompatibility group IncX3 was identified in all genomes in a truncated Tn6361 transposon flanked by ΔIS26 insertion sequences. The qnrS1 gene was also found downstream of blaOXA-181, conferring fluoroquinolone resistance to all isolates. CPE isolates harboring blaOXA-like genes are an increasing public health problem in health care settings worldwide. The IncX3 plasmid is involved in the worldwide dissemination of blaOXA-181, and its presence in these CPE isolates suggests the wide dissemination of blaOXA-181 in Peru. IMPORTANCE Reports of carbapenemase-producing Enterobacterales (CPE) isolates are increasing worldwide. Accurate detection of the ß-lactamase OXA-181 (a variant of OXA-48) is important to initiate therapy and preventive measures in the clinic. OXA-181 has been described in CPE isolates in many countries, often associated with nosocomial outbreaks. However, the circulation of this carbapenemase has yet to be reported in Peru. Here, we report the detection of five multidrug-resistant CPE clinical isolates harboring blaOXA-181 in the IncX3-type plasmid, a potential driver of dissemination in Peru.

Cerebral Cryptococcosis Associated with CD4+ T-lymphocytopenia in Non-HIV Patients after SARS-CoV-2 Infection: Case Series in a Specialized Institute in Lima, Peru.

Cases of cryptococcosis have been reported in patients with COVID-19. The majority are in patients with severe symptoms or who received immunosuppressants. However, there is still no clear association between COVID-19 and cryptococcosis. We report eight cases of cerebral cryptococcosis associated with CD4+ T lymphocytopenia in non-HIV patients after SARS-CoV-2 infection. The median age was 57 years and 5/8 were male. In addition, 2/8 of patients had diabetes, and 8/8 had a history of mild COVID-19, with a median of 75 days before diagnosis of cerebral cryptococcosis. All patients denied having received prior immunosuppressive therapy. The most frequent symptoms were confusion (8/8), headache (7/8), vomiting (6/8), and nausea (6/8) All patients were diagnosed by isolating Cryptococcus in cerebrospinal fluid. The median CD4+ and CD8+ T lymphocytes were 247 and 173.5, respectively. Other causes of immunosuppression, such as HIV or HTLV infection, were excluded in all patients. Finally, three patients died, and one presented long-term visual and auditory sequelae. The CD4+/CD8+ T lymphocyte count normalized during follow-up in those patients who survived. We hypothesize that CD4+ T lymphocytopenia in the patients in this case series could increase the risk of cryptococcosis after SARS-CoV-2 infection.

Characteristics and factors associated with mortality in tracheostomized patients with COVID-19: a retrospective cohort study in a hospital in Tacna, Peru. / Características y factores asociados a la mortalidad en pacientes con COVID-19 traqueostomizados: estudio de cohorte retrospectivo en un hospital de Tacna, Perú.

OBJECTIVE: We aimed to describe the main demographic, clinical, laboratory and therapeutic characteristics and to identify whether they are associated with mortality in tracheostomized patients. MATERIAL AND METHODS.: Retrospective cohort study in adult patients diagnosed with COVID-19, admitted to ICU (Intensive Care Unit) and requiring tracheostomy. Demographic, clinical, laboratory and treatment data were obtained from the medical records of patients admitted to Hospital III Daniel Alcides Carrión in Tacna. The Cox proportional hazards model was used for survival analysis and hazard ratios (HR) with their 95% confidence intervals (95%CI) were calculated. RESULTS.: We evaluated 73 patients, 72.6% were men, the most common comorbidities were obesity (68.5%), type 2 diabetes mellitus (35.6%), and arterial hypertension (34.2%). Thirty-seven percent of the participants died during their stay at the ICU. The median time from intubation to tracheostomy and the duration of tracheostomy was 17 (RIC: 15-21) and 21 (RIC: 3-39) days, respectively. Multivariate analysis showed that the factors associated with mortality were procalcitonin > 0.50 ng/dL at the time of tracheostomy (HRa: 2.40 95%CI: 1.03-5.59) and a PaO2/FiO2 ratio less than or equal to 150 mmHg (HRa: 4.44 95%CI: 1.56-12.60). CONCLUSIONS.: The factors associated with mortality at the time of tracheostomy were procalcitonin > 0.50 ng/dL and a PaO2/FiO2 ratio less than or equal to 150 mmHg.

OBJETIVO.: Describir las principales características demográficas, clínicas, laboratoriales y terapéuticas e identificar si están asociados con la mortalidad en pacientes traqueostomizados. MATERIAL Y MÉTODOS.: Estudio de cohorte retrospectiva en pacientes adultos con diagnóstico de COVID-19, ingresados a UCI (Unidad de Cuidados Intensivos) y que requirieron traqueostomía. Se extrajeron datos demográficos, clínicos, laboratoriales y de tratamiento de las historias clínicas de pacientes que ingresaron al Hospital III Daniel Alcides Carrión de Tacna. Para el análisis de supervivencia se empleó el modelo de riesgos proporcionales de Cox y se calcularon los cocientes de riesgo instantáneos (HR) con sus intervalos de confianza al 95% (IC95%). RESULTADOS.: Se evaluaron 73 pacientes, el 72,6% eran hombres, las comorbilidades más comunes fueron obesidad (68,5%), diabetes mellitus tipo 2 (35,6%) e hipertensión arterial (34,2%). El 37% de los participantes fallecieron durante la estancia en UCI. La mediana de tiempo desde la intubación hasta la traqueostomía y la duración de esta fue 17 (RIC: 15−21) y 21 (RIC: 3−39) días, respectivamente. El análisis multivariado mostró que los factores asociados a mortalidad, fueron presentar un valor de procalcitonina > 0,50 ng/dL en el momento de la traqueostomía (HRa: 2,40 IC95%: 1,03−5,59) y el nivel de PaO2/FiO2 menor o igual a 150 mmHg, (HRa: 4,44 IC95%: 1,56−12,60). CONCLUSIONES.: Los factores asociados a mortalidad al momento de realizar la traqueostomía fueron presentar un valor de procalcitonina > 0,50 ng/dL y un cociente PaO2/FiO2 menor o igual a 150 mmHg.

Diagnostic performance of the time to positivity of blood cultures to distinguish true bacteremia from contaminants based on an automated system. / Utilidad diagnóstica de los tiempos de positividad de hemocultivos para distinguir verdaderas bacteriemias de contaminantes en base a un sistema automatizado.

OBJECTIVE.: To determine the diagnostic performance of blood culture positivity times for distinguishing true bacteremia from contaminants in the automated "BACT/ALERT®" system. MATERIALS AND METHODS.: A cross-sectional, diagnostic test-type study was conducted from a database of blood culture samples processed between January 2016 and August 2021. All blood culture samples from patients with suspected bacteremia were included; blood culture samples were entered into the "BACT/ALERT®" system to differentiate true bacteremia from contaminants. RESULTS.: We obtained 33,951 blood cultures samples, of which 3875 were positive. Of the total number of positive blood cultures, 75.2% (n=2913) were true bacteremia and 24.8% (n=962) were contaminants. The median time to positivity in blood cultures with true bacteremia was significantly shorter (16.3 hours; IQR: 11.2 - 24.9) than the median time to positivity of blood cultures with contaminants (22.5 hours; IQR: 18.4 - 31.8; p<0.001). The positivity time showed the capacity to differentiate true bacteremia from contaminants, with an AUC-ROC of 0.73 (95%CI: 0.71 - 0.75), with 85% and 63% sensitivity and specificity respectively for the diagnosis of contaminants when the positivity time exceeds 16.5 hours. The use of antibiotics prior to sampling delayed the time to positivity, while having fever before sampling shortened the time to positivity. CONCLUSIONS.: Our results show good diagnostic performance of blood culture positivity times to differentiate true bacteremia from contaminants using the "BACT/ALERT®" system when the positivity time was longer than 16.5 hours.

OBJETIVO.: Determinar la utilidad diagnóstica de los tiempos de positividad de hemocultivos para distinguir verdaderas bacteriemias de contaminantes en el sistema automatizado «BACT/ALERT®¼. MATERIALES Y MÉTODOS.: Se realizó un estudio transversal de tipo pruebas diagnósticas, a partir de una base de datos de muestras de hemocultivos procesadas entre enero del 2016 y agosto del 2021. Se incluyeron todas las muestras de hemocultivos de pacientes con sospecha de bacteriemia, las muestras de hemocultivos fueron ingresadas al sistema «BACT/ALERT®¼ para diferenciar verdaderas bacteriemias de contaminantes. RESULTADOS.: Se analizó un total de 33 951 frascos de hemocultivos y se obtuvieron 3875 frascos positivos. El 75,2% (n=2913) del total de hemocultivos positivos fueron verdaderas bacteriemias y 24,8% (n=962) fueron contaminantes. La mediana de tiempo de positividad en los hemocultivos con verdaderas bacteriemias fue significativamente menor (16,3 horas; RIC: 11,2 - 24,9) que la mediana de tiempo de positividad de hemocultivos con contaminantes (22,5 horas; RIC: 18,4 - 31,8; p<0,001). El tiempo de positividad demostró capacidad discriminante para diferenciar verdaderas bacteriemias de contaminantes, con un AUC-ROC de 0,73 (IC95%: 0,71 - 0,75), con 85% y 63% de sensibilidad y especificidad respectivamente para el diagnóstico de contaminantes cuando el tiempo de positividad supera las 16,5 horas. La administración de antibióticos previos a la toma retrasó el tiempo de positividad, en cambio, haber presentado fiebre antes de la toma de muestra acortó el tiempo de positividad. CONCLUSIONES.: Nuestros resultados muestran un buen desempeño de los tiempos de positividad de hemocultivos para diferenciar verdaderas bacteriemias de contaminantes utilizando el sistema «BACT/ALERT®¼ cuando el tiempo de positividad fue superior a 16,5 horas.

Determination of PaO2/FiO2 after 24 h of invasive mechanical ventilation and ΔPaO2/FiO2 at 24 h as predictors of survival in patients diagnosed with ARDS due to COVID-19.

Introduction: Acute respiratory distress syndrome (ARDS) due to Coronavirus Disease 2019 (COVID-19) causes high mortality. The objective of this study is to determine whether the arterial pressure of oxygen/inspiratory fraction of oxygen (PaO2/FiO2) 24 h after invasive mechanical ventilation (IMV) and the difference between PaO2/FiO2 at 24 h after IMV and PaO2/FiO2 before admission to IMV (ΔPaO2/FiO2 24 h) are predictors of survival in patients with ARDS due to COVID-19. Methods: A retrospective cohort study was conducted that included patients with ARDS due to COVID-19 in IMV admitted to the intensive care unit (ICU) of a hospital in southern Peru from April 2020 to April 2021. The ROC curves and the Youden index were used to establish the cut-off point for PaO2/FiO2 at 24 h of IMV and ΔPaO2/FiO2 at 24 h associated with mortality. The association with mortality was determined by Cox regression, calculating the crude (cHR) and adjusted (aHR) risk ratios, with their respective 95% confidence intervals (95% CI). Results: Two hundred patients were analyzed. The average age was 54.29 years, 79% were men, and 25.5% (n = 51) died. The cut-off point calculated for PaO2/FiO2 24 h after IMV and ΔPaO2/FiO2 24 h was 222.5 and 109.5, respectively. Those participants with a value below the cut-off point of ΔPaO2/FiO2 24 h and PaO2/FiO2 24 h after IMV had higher mortality, aHR = 3.32 (CI 95% [1.82-6.07]) and aHR = 2.87 (CI 95% [1.48-5.57]) respectively. Conclusion: PaO2/FiO2 24 h after IMV and ΔPaO2/FiO2 24 h in patients diagnosed with ARDS due to COVID-19 on IMV were associated with higher hospital mortality. These findings are helpful to identify those patients with a higher risk of dying on admission to the ICU.

Expansion of CD8+ T cell population in Lassa virus survivors with low T cell precursor frequency reveals durable immune response in most survivors.

INTRODUCTION: Lassa virus is a priority pathogen for vaccine research and development, however the duration of cellular immunity and protection in Lassa fever (LF) survivors remains unclear. METHODS: We investigated Lassa virus specific CD8+ T cell responses in 93 LF survivors. Peripheral blood mononuclear cells from these individuals were infected with recombinant vesicular stomatitis virus encoding Lassa virus antigens and virus specific T cell responses were measured after 18-hour incubation. Participants who had undetectable CD8+ T cell response underwent further analysis using a 10-day T cell proliferation assays to evaluate for low T cell precursor frequency. RESULTS: Forty-five of the 93 LF survivors did not have a Lassa virus specific CD8+ T cell response. Of those with responses and a known date of onset of LF (N = 11), 9 had LF within the last ten years. Most participants without a measurable CD8+ T cell response were more than 10 years removed from a clinical history of LF (N = 14/16). Fourteen of 21 patients (67%) with undetectable CD8+ T cell response had a measurable Lassa virus specific CD8+ T cell response with the 10-day assay. DISCUSSION: Despite reports of strong CD8+ T cell responses during acute Lassa virus infection, circulating Lassa virus-specific CD8+ T cells declined to undetectable levels in most Lassa fever survivors after ten years when evaluated with an 18-hour T cell stimulation. However, when Lassa virus-specific T cells were expanded prior to restimulation, a Lassa virus-specific CD8+ T cell response could be detected in many if the samples that were negative in the 18-hour stimulation assay, suggesting that prolonged cellular immunity does exist in Lassa fever survivors at low frequencies.

2022 taxonomic update of phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

In March 2022, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by two new families (bunyaviral Discoviridae and Tulasviridae), 41 new genera, and 98 new species. Three hundred forty-nine species were renamed and/or moved. The accidentally misspelled names of seven species were corrected. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

Proximity interactome analysis of Lassa polymerase reveals eRF3a/GSPT1 as a druggable target for host-directed antivirals.

Completion of the Lassa virus (LASV) life cycle critically depends on the activities of the virally encoded, RNA-dependent RNA polymerase in replication and transcription of the viral RNA genome in the cytoplasm of infected cells. The contribution of cellular proteins to these processes remains unclear. Here, we applied proximity proteomics to define the interactome of LASV polymerase in cells under conditions that recreate LASV RNA synthesis. We engineered a LASV polymerase-biotin ligase (TurboID) fusion protein that retained polymerase activity and successfully biotinylated the proximal proteome, which allowed the identification of 42 high-confidence LASV polymerase interactors. We subsequently performed a small interfering RNA (siRNA) screen to identify those interactors that have functional roles in authentic LASV infection. As proof of principle, we characterized eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), which we found to be a proviral factor that physically associates with LASV polymerase. Targeted degradation of GSPT1 by a small-molecule drug candidate, CC-90009, resulted in strong inhibition of LASV infection in cultured cells. Our work demonstrates the feasibility of using proximity proteomics to illuminate and characterize yet-to-be-defined host-pathogen interactome, which can reveal new biology and uncover novel targets for the development of antivirals against highly pathogenic RNA viruses.