Metabolic Reprogramming by the Excessive AMPK Activation Exacerbates Antigen-Specific Memory CD8⁺ T Cell Differentiation after Acute Lymphocytic Choriomeningitis Virus Infection

During virus infection, T cells must be adapted to activation and lineage differentiation states via metabolic reprogramming. Whereas effector CD8⁺ T cells preferentially use glycolysis for their rapid proliferation, memory CD8⁺ T cells utilize oxidative phosphorylation for their homeostatic maintenance. Particularly, enhanced AMP-activated protein kinase (AMPK) activity promotes the memory T cell response through different pathways. However, the level of AMPK activation required for optimal memory T cell differentiation remains unclear. A new metformin derivative, IM156, formerly known as HL156A, has been reported to ameliorate various types of fibrosis and inhibit in vitro and in vivo tumors by inducing AMPK activation more potently than metformin. Here, we evaluated the in vivo effects of IM156 on antigen-specific CD8⁺ T cells during their effector and memory differentiation after acute lymphocytic choriomeningitis virus infection. Unexpectedly, our results showed that in vivo treatment of IM156 exacerbated the memory differentiation of virus-specific CD8⁺ T cells, resulting in an increase in short-lived effector cells but decrease in memory precursor effector cells. Thus, IM156 treatment impaired the function of virus-specific memory CD8⁺ T cells, indicating that excessive AMPK activation weakens memory T cell differentiation, thereby suppressing recall immune responses. This study suggests that metabolic reprogramming of antigen-specific CD8⁺ T cells by regulating the AMPK pathway should be carefully performed and managed to improve the efficacy of T cell vaccine.

Semi-Functional Quantitative Flow Cytometry Assay for Lymphocytic Choriomeningitis Virus Titration

Quantitative PCR and plaque assay are powerful virological techniques used to measure the load of defective or infectious virus in mouse and human. However, these methods display limitations such as cross contamination and long run-time. Here, we describe a novel technique termed as semi-functional quantitative flow cytometry (SFQF) for the accurate estimation of the quantity of infectious lymphocytic choriomeningitis virus (LCMV). LCMV titration method using flow cytometry was previously developed but has technical shortcomings, owing to the less optimized parameters such as cell overgrowth, plate scale, and detection threshold. Therefore, we first established optimized conditions for SFQF assay using LCMV nucleoprotein (NP)-specific antibody to evaluate the threshold of the virus detection range in the plaque assay. We subsequently demonstrated that the optimization of the method increased the sensitivity of virus detection. We revealed several new advantages of SFQF assay, which overcomes some of the previously contentious points, and established an upgraded version of the previously reported flow cytometric titration assay. This method extends the detection scale to the level of single cell, allowing extension of its application for in vivo detection of infected cells and their phenotypic analysis. Thus, SFQF assay may serve as an alternative analytical tool for ensuring the reliability of LCMV titration and can be used with other types of viruses using target-specific antibodies.

Circulating natural killer and γδ T cells decrease soon after infection of rhesus macaques with lymphocytic choriomeningitis virus

Rhesus macaques infected with the WE strain of lymphocytic choriomeningitis virus (LCMV-WE) serve as a model for human infection with Lassa fever virus. To identify the earliest events of acute infection, rhesus macaques were monitored immediately after lethal infection for changes in peripheral blood mononuclear cells (PBMCs). Changes in CD3, CD4, CD8 and CD20 subsets did not vary outside the normal fluctuations of these blood cell populations; however, natural killer (NK) and γδ T cells increased slightly on day 1 and then decreased significantly after two days. The NK subsets responsible for the decrease were primarily CD3-CD8+ or CD3-CD16+ and not the NKT (primarily CD3+CD56+) subset. Macaques infected with a non-virulent arenavirus, LCMV-Armstrong, showed a similar drop in circulating NK and γδ T cells, indicating that this is not a pathogenic event. V³9 T cells, representing the majority of circulating γδ T cells in rhesus macaques, displayed significant apoptosis when incubated with LCMV in cell culture; however, the low amount of cell death for virus-co-cultured NK cells was insufficient to account for the observed disappearance of this subset. Our observations in primates are similar to those seen in LCMV-infected mice, where decreased circulating NK cells were attributed to margination and cell death. Thus, the disappearance of these cells during acute hemorrhagic fever in rhesus macaques may be a cytokine-induced lymphopenia common to many virus infections.

Comportamiento de cepas argentinas del virus de la Coriomeningitis Linfocitaria en roedores / Behavior of Argentine lymphocytic choriomeningitis virus strains in rodents

La actividad del virus LCM fue informada en Argentina a comienzos de la década del 70 y sólo han sido aisladas cinco cepas a partir del roedor Mus domesticus y dos de humanos. El objetivo de este trabajo consistió en investigar características biológicas de las cepas argentinas de virus LCM para compararlas entre sí y respecto a las cepas históricas WE y Armstrong. En células L 929 se obtuvieron placas bajo agarosa tanto con las cepas humanas como con las cepas de ratón, pero en células Vero sólo se obtuvieron placas con las cepas humanas. No se observó ninguna característica morfométrica de las placas que distinguiera nítidamente a las cepas históricas de las cepas argentinas, ni se observaron diferencias que se relacionen con las especies de origen de las cepas. Las cepas históricas y las cepas argentinas no fueron letales para ratón recién nacido (rrn) generando una infección persistente, según se comprobó al inocular ratones recién nacidos (rrn) por vía intracerebral con cepas de virus LCM y detectarse virus en los cerebros cosechados a diferentes días post inoculación. La única excepción fue la cepa Cba An 13065 que resultó virulenta para rrn ya que con sólo 0.026 UFP se logró 1 DL50. Todas las cepas resultaron letales en ratón adulto (rad), siendo las cepas de ratón más virulentas que las cepas de humanos. Estos resultados permitieron evidenciar el diferente comportamiento en cultivos celulares de las cepas de ratón con respecto a las cepas humanas, e identificar marcadores de virulencia mediante la respuesta a la inoculación por vía intracerebral del rad y del rrn.

The activity of LCM virus was first reported in Argentina at the beginning of the seventies and only five strains have been isolated from rodents Mus domesticus and two from humans. The objective of this paper was to find differential biological characteristics of Argentine strains of LCM virus comparing them in relation to the historical strains WE and Armstrong. Regarding the results obtained in tissue culture, when L 929 cells were used, plaque forming units (PFU) were obtained with human and mouse strains, whilst on Vero cells only human strains developed PFU. Differentials characteristics of historical and Argentine strain's plates were not found, neither differences related to the strain's origin. Neither historical nor Argentine strains were lethal to new-born mice giving a persistent infection, that was demonstrated when we inoculated new-born mouse by intracranial route with different strains of LCM virus and virus was isolated from brains harvested at different days post inoculation. The only exception was Cba An 13065 strain that exhibited virulence in new-born mice, only with 0.026 PFU was obtained 1 DL50. All the strains resulted lethal to adult mice. The mouse strains were more virulent than human strains, being Cba An 13065 the most virulent. These results demonstrate a different behavior in tissue culture between human and mouse strains and allow the identification of virulence markers by intracranial inoculation into new-born or adult mice.

Aislamiento del virus de la coriomeningitis linfocitaria de seres humanos / Isolation of lymphocytic choriomeningitis virus from human individuals

The activity of lymphocytic choriomeningitis virus (LCMv) in Argentina has been previously reported on the basis of serological evidence in rodents and humans and the isolation of only one strain of LCMv from a Mus domesticus captured in the province of Cordoba. The aim of this paper was to register patients with serological diagnosis of LCM, to isolate and to identify human strains of LCMv in Argentina. During the last 19 years, 15 cases were diagnosed as LCM by immunoflourescent indirect assay (IFI) and enzyme-linked immunosorbent assay (ELISA) but when neutralizing assay (NT) was incorporated, eight cases were classified as confirmed, three as probable and four as negative. The geographic distribution of the cases included three provinces: Cordoba, Buenos Aires and Santa Fe. Viral isolation was attempted in five patients classified as confirmed and only two resulted positive (P5226 and P8573). They were identified as LCMv by IFI and NT. The coexistence of LCMv with other arenaviruses, such as Junin and Oliveros viruses, in the same area, raises the probability of interactions between them, which could modify the virulence and/or pathogenicity for humans associated to genomic changes. Future studies of antigenic, genomic and virulence variability of different Argentine strains of LCMv, as well as the systematic search for human infection, will contribute to define the importance of this viral agent in our country and to implement control measures.

Patterns of transient expression of the arenavirus nucleocapsid protein gene in transfected cells

Los genes clonados de las proteínas de nucleocápside, N, de los arenavirus Junín y LCM (choriomeningitis linfocitaria) se insertaron en el vector de expresión pKG4 regulado por el promotor tardío del virus SV40. Cuando estas construcciones se utilizaron para transfectar las líneas celulares BHK-21 (fibroblastos de hamster lactante) y CV-1 (fibroblastos de riñón de mono verde africano) se observó la expresión transiente de un polipéptido de tamaño e inmunoreactividad indistinguible de la proteína N sintetizada durante una infección viral. El análisis por inmunofluorescencia reveló un patrón de distribución intracelular semejante al observado en células infectadas. Este patrón presentó variaciones desde una tinción citoplásmica difusa hasta gránulos citoplásmicos dispersos o concentrados en la zona perinuclear. La asociación de la proteína N con gránulos basófilos es semejante a la descripta en el efecto citópático causado por los arenavirus en las células infectadas, y podría relacionarse con las características fisicoquímicas de la proteina N, que contiene numerosas secuencias de aminoácidos básicos capaces de interactuar con ácidos ribonucleicos celulares

Infection of guinea pigs with two strains of lymphocytic choriomeningitis virus

Con el objeto de estudiar las lesiones producidas, por el virus de la coriomeningitis linfocítica (LCMV), se inocularon cobayos adultos por vía intraperitoneal con 1000 UFP de las cepas WE y Armstrong de LCMV. Los animales se scrificaron a los 3, 7,10 y 14 días postinoculación (PI) para estudios de virus en bazo, médula ósea y sangre. En los cobayos infectados con WE se observó destrucción de la pulpa roja esplénica con altos títulos de virus y diferentes grados de neumonitis. Los estudios hematolóficos mostraron linfopenia a partir del día 7 Pl y necrosis focal de la médula ósea. En los animales infectados con la cepa Armstrong sólo se observó una neutropenia moderada sin lesiones evidentes, siendo la replicación del virus menor de 2 log, en relación a WE. La presencia de numerosos polimorfonucleares (PMN) en la pulpa roja esplénica y en el infiltrado pulmonar en forma previa a las lesiones observadas, sugiere que el daño es mediado en alguna forma por esas células. Los cobayos infectados con la cepa WE podrían representar un mdoelo adecuado para estudiar el rol de los PMN en als enfermedades virales, así como los mecanismos patogenéticos no inmunes con respecto a LCMV