Functional properties of measles virus proteins derived from a subacute sclerosing panencephalitis patient who received repeated remdesivir treatments.

Subacute sclerosing panencephalitis (SSPE) is a rare but fatal late neurological complication of measles, caused by persistent measles virus (MeV) infection of the central nervous system. There are no drugs approved for the treatment of SSPE. Here, we followed the clinical progression of a 5-year-old SSPE patient after treatment with the nucleoside analog remdesivir, conducted a post-mortem evaluation of the patient's brain, and characterized the MeV detected in the brain. The quality of life of the patient transiently improved after the first two courses of remdesivir, but a third course had no further clinical effect, and the patient eventually succumbed to his condition. Post-mortem evaluation of the brain displayed histopathological changes including loss of neurons and demyelination paired with abundant presence of MeV RNA-positive cells throughout the brain. Next-generation sequencing of RNA isolated from the brain revealed a complete MeV genome with mutations that are typically detected in SSPE, characterized by a hypermutated M gene. Additional mutations were detected in the polymerase (L) gene, which were not associated with resistance to remdesivir. Functional characterization showed that mutations in the F gene led to a hyperfusogenic phenotype predominantly mediated by N465I. Additionally, recombinant wild-type-based MeV with the SSPE-F gene or the F gene with the N465I mutation was no longer lymphotropic but instead efficiently disseminated in neural cultures. Altogether, this case encourages further investigation of remdesivir as a potential treatment of SSPE and highlights the necessity to functionally understand SSPE-causing MeV.IMPORTANCEMeasles virus (MeV) causes acute, systemic disease and remains an important cause of morbidity and mortality in humans. Despite the lack of known entry receptors in the brain, MeV can persistently infect the brain causing the rare but fatal neurological disorder subacute sclerosing panencephalitis (SSPE). SSPE-causing MeVs are characterized by a hypermutated genome and a hyperfusogenic F protein that facilitates the rapid spread of MeV throughout the brain. No treatment against SSPE is available, but the nucleoside analog remdesivir was recently demonstrated to be effective against MeV in vitro. We show that treatment of an SSPE patient with remdesivir led to transient clinical improvement and did not induce viral escape mutants, encouraging the future use of remdesivir in SSPE patients. Functional characterization of the viral proteins sheds light on the shared properties of SSPE-causing MeVs and further contributes to understanding how those viruses cause disease.

The radiation- and chemo-sensitizing capacity of diclofenac can be predicted by a decreased lactate metabolism and stress response.

BACKGROUND: An enhanced aerobic glycolysis ("Warburg effect") associated with an increase in lactic acid in the tumor microenvironment contributes to tumor aggressiveness and resistance to radiation and chemotherapy. We investigated the radiation- and chemo-sensitizing effects of the nonsteroidal anti-inflammatory drug (NSAID) diclofenac in different cancer cell types. METHODS: The effects of a non-lethal concentration of diclofenac was investigated on c-MYC and Lactate Dehydrogenase (LDH) protein expression/activity and the Heat shock Protein (HSP)/stress response in human colorectal (LS174T, LoVo), lung (A549), breast (MDA-MB-231) and pancreatic (COLO357) carcinoma cells. Radiation- and chemo-sensitization of diclofenac was determined using clonogenic cell survival assays and a murine xenograft tumor model. RESULTS: A non-lethal concentration of diclofenac decreases c-MYC protein expression and LDH activity, reduces cytosolic Heat Shock Factor 1 (HSF1), Hsp70 and Hsp27 levels and membrane Hsp70 positivity in LS174T and LoVo colorectal cancer cells, but not in A549 lung carcinoma cells, MDA-MB-231 breast cancer cells and COLO357 pancreatic adenocarcinoma cells. The impaired lactate metabolism and stress response in diclofenac-sensitive colorectal cancer cells was associated with a significantly increased sensitivity to radiation and 5Fluorouracil in vitro, and in a human colorectal cancer xenograft mouse model diclofenac causes radiosensitization. CONCLUSION: These findings suggest that a decrease in the LDH activity and/or stress response upon diclofenac treatment predicts its radiation/chemo-sensitizing capacity.

Target-based discovery of a broad spectrum flukicide.

Diseases caused by parasitic flatworms impart a considerable healthcare burden worldwide. Many of these diseases - for example, the parasitic blood fluke infection, schistosomiasis - are treated with the drug praziquantel (PZQ). However, PZQ is ineffective against disease caused by liver flukes from the genus Fasciola. This is due to a single amino acid change within the target of PZQ, a transient receptor potential ion channel (TRPMPZQ), in Fasciola species. Here we identify benzamidoquinazolinone analogs that are active against Fasciola TRPMPZQ. Structure-activity studies define an optimized ligand (BZQ) that caused protracted paralysis and damage to the protective tegument of these liver flukes. BZQ also retained activity against Schistosoma mansoni comparable to PZQ and was active against TRPMPZQ orthologs in all profiled species of parasitic fluke. This broad spectrum activity was manifest as BZQ adopts a pose within the binding pocket of TRPMPZQ dependent on a ubiquitously conserved residue. BZQ therefore acts as a universal activator of trematode TRPMPZQ and a first-in-class, broad spectrum flukicide.

Differential response of mesenchymal stromal cells (MSCs) to type 1 ex vivo cytokine priming: implications for MSC therapy.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are polymorphic, adherent cells with the capability to stimulate tissue regeneration and modulate immunity. MSCs have been broadly investigated for potential therapeutic applications, particularly immunomodulatory properties, wound healing and tissue regeneration. The exact physiologic role of MSCs, however, remains poorly understood, and this gap in knowledge significantly impedes the rational development of therapeutic cells. Here, we considered interferon γ (IFN-γ) and tumor necrosis factor alpha (TNF-α), two cytokines likely encountered physiologically and commonly used in cell manufacturing. For comparison, we studied interleukin-10 (IL-10) (anti-inflammatory) and interleukin-4 (IL-4) (type 2 cytokine). METHODS: We directly assessed the effects of these cytokines on bone marrow MSCs by comparing RNA Seq transcriptional profiles. Western blotting and flow cytometry were also used to evaluate effects of cytokine priming. RESULTS: The type 1 cytokines (IFN-γ and TNF-α) induced striking changes in gene expression and remarkably different profiles from one another. Importantly, priming MSCs with either of these cytokines did not increase variability among multiple donors beyond what is intrinsic to non-primed MSCs from different donors. IFN-γ-primed MSCs expressed IDO1 and chemokines that recruit activated T cells. In contrast, TNF-α-primed MSCs expressed genes in alternate pathways, namely PGE2 and matrix metalloproteinases synthesis, and chemokines that recruit neutrophils. IL-10 and IL-4 priming had little to no effect. CONCLUSIONS: Our data suggest that IFN-γ-primed MSCs may be a more efficacious immunosuppressive therapy aimed at diseases that target T cells (ie, graft-versus-host disease) compared with TNF-α-primed or non-primed MSCs, which may be better suited for therapies in other disease settings. These results contribute to our understanding of MSC bioactivity and suggest rational ex vivo cytokine priming approaches for MSC manufacturing and therapeutic applications.

The neuropathogenesis of highly pathogenic avian influenza H5Nx viruses in mammalian species including humans.

Circulation of highly pathogenic avian influenza (HPAI) H5Nx viruses of the A/Goose/Guangdong/1/96 lineage in birds regularly causes infections of mammals, including humans. In many mammalian species, infections are associated with severe neurological disease, a unique feature of HPAI H5Nx viruses compared with other influenza A viruses. Here, we provide an overview of the neuropathogenesis of HPAI H5Nx virus infection in mammals, centered on three aspects: neuroinvasion, neurotropism, and neurovirulence. We focus on in vitro studies, as well as studies on naturally or experimentally infected mammals. Additionally, we discuss the contribution of viral factors to the neuropathogenesis of HPAI H5Nx virus infections and the efficacy of intervention strategies to prevent neuroinvasion or the development of neurological disease.

Chronic inflammatory effects of in vivo irradiation of the murine heart on endothelial cells mimic mechanisms involved in atherosclerosis.

PURPOSE: Radiotherapy is a major pillar in the treatment of solid tumors including breast cancer. However, epidemiological studies have revealed an increase in cardiac diseases approximately a decade after exposure of the thorax to ionizing irradiation, which might be related to vascular inflammation. Therefore, chronic inflammatory effects were examined in primary heart and lung endothelial cells (ECs) of mice after local heart irradiation. METHODS: Long-lasting effects on primary ECs of the heart and lung were studied 20-50 weeks after local irradiation of the heart of mice (8 and 16 Gy) in vivo by multiparameter flow cytometry using antibodies directed against cell surface markers related to proliferation, stemness, lipid metabolism, and inflammation, and compared to those induced by occlusion of the left anterior descending coronary artery. RESULTS: In vivo irradiation of the complete heart caused long-lasting persistent upregulation of inflammatory (HCAM, ICAM­1, VCAM-1), proliferation (CD105), and lipid (CD36) markers on primary heart ECs and an upregulation of ICAM­1 and VCAM­1 on primary ECs of the partially irradiated lung lobe. An artificially induced heart infarction induces similar effects with respect to inflammatory markers, albeit in a shorter time period. CONCLUSION: The long-lasting upregulation of prominent inflammatory markers on primary heart and lung ECs suggests that local heart irradiation induces chronic inflammation in the microvasculature of the heart and partially irradiated lung that leads to cardiac injury which might be related to altered lipid metabolism in the heart.

CYP19A1 mediates severe SARS-CoV-2 disease outcome in males.

Male sex represents one of the major risk factors for severe COVID-19 outcome. However, underlying mechanisms that mediate sex-dependent disease outcome are as yet unknown. Here, we identify the CYP19A1 gene encoding for the testosterone-to-estradiol metabolizing enzyme CYP19A1 (also known as aromatase) as a host factor that contributes to worsened disease outcome in SARS-CoV-2-infected males. We analyzed exome sequencing data obtained from a human COVID-19 cohort (n = 2,866) using a machine-learning approach and identify a CYP19A1-activity-increasing mutation to be associated with the development of severe disease in men but not women. We further analyzed human autopsy-derived lungs (n = 86) and detect increased pulmonary CYP19A1 expression at the time point of death in men compared with women. In the golden hamster model, we show that SARS-CoV-2 infection causes increased CYP19A1 expression in the lung that is associated with dysregulated plasma sex hormone levels and reduced long-term pulmonary function in males but not females. Treatment of SARS-CoV-2-infected hamsters with a clinically approved CYP19A1 inhibitor (letrozole) improves impaired lung function and supports recovery of imbalanced sex hormones specifically in males. Our study identifies CYP19A1 as a contributor to sex-specific SARS-CoV-2 disease outcome in males. Furthermore, inhibition of CYP19A1 by the clinically approved drug letrozole may furnish a new therapeutic strategy for individualized patient management and treatment.

Cortico-cortical feedback engages active dendrites in visual cortex.

Sensory processing in the neocortex requires both feedforward and feedback information flow between cortical areas1. In feedback processing, higher-level representations provide contextual information to lower levels, and facilitate perceptual functions such as contour integration and figure-ground segmentation2,3. However, we have limited understanding of the circuit and cellular mechanisms that mediate feedback influence. Here we use long-range all-optical connectivity mapping in mice to show that feedback influence from the lateromedial higher visual area (LM) to the primary visual cortex (V1) is spatially organized. When the source and target of feedback represent the same area of visual space, feedback is relatively suppressive. By contrast, when the source is offset from the target in visual space, feedback is relatively facilitating. Two-photon calcium imaging data show that this facilitating feedback is nonlinearly integrated in the apical tuft dendrites of V1 pyramidal neurons: retinotopically offset (surround) visual stimuli drive local dendritic calcium signals indicative of regenerative events, and two-photon optogenetic activation of LM neurons projecting to identified feedback-recipient spines in V1 can drive similar branch-specific local calcium signals. Our results show how neocortical feedback connectivity and nonlinear dendritic integration can together form a substrate to support both predictive and cooperative contextual interactions.

The pro-inflammatory response to influenza A virus infection is fueled by endothelial cells.

Morbidity and mortality from influenza are associated with high levels of systemic inflammation. Endothelial cells play a key role in systemic inflammatory responses during severe influenza A virus (IAV) infections, despite being rarely infected in humans. How endothelial cells contribute to systemic inflammatory responses is unclear. Here, we developed a transwell system in which airway organoid-derived differentiated human lung epithelial cells were co-cultured with primary human lung microvascular endothelial cells (LMECs). We compared the susceptibility of LMECs to pandemic H1N1 virus and recent seasonal H1N1 and H3N2 viruses and assessed the associated pro-inflammatory responses. Despite the detection of IAV nucleoprotein in LMEC mono-cultures, there was no evidence for productive infection. In epithelial-endothelial co-cultures, abundant IAV infection of epithelial cells resulted in the breakdown of the epithelial barrier, but infection of LMECs was rarely detected. We observed a significantly higher secretion of pro-inflammatory cytokines in LMECs when co-cultured with IAV-infected epithelial cells than LMEC mono-cultures exposed to IAV. Taken together, our data show that LMECs are abortively infected by IAV but can fuel the inflammatory response.

Viral immunity: Basic mechanisms and therapeutic applications-a Keystone Symposia report.

Viruses infect millions of people each year. Both endemic viruses circulating throughout the population as well as novel epidemic and pandemic viruses pose ongoing threats to global public health. Developing more effective tools to address viruses requires not only in-depth knowledge of the virus itself but also of our immune system's response to infection. On June 29 to July 2, 2022, researchers met for the Keystone symposium "Viral Immunity: Basic Mechanisms and Therapeutic Applications." This report presents concise summaries from several of the symposium presenters.

Do SARS-CoV-2 Variants Differ in Their Neuropathogenicity?

Neurological complications associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections are a huge societal problem. Although the neuropathogenicity of SARS-CoV-2 is not yet fully understood, there is evidence that SARS-CoV-2 can invade and infect cells of the central nervous system. Kong et al. (https://doi.org/10.1128/mbio.02308-22) shows that the mechanism of virus entry into astrocytes in brain organoids and primary astrocytes differs from entry into respiratory epithelial cells. However, how SARS-CoV-2 enters susceptible CNS cells and whether there are differences among SARS-CoV-2 variants is still unclear. In vivo and in vitro models are useful to study these important questions and may reveal important differences among SARS-CoV-2 variants in their neuroinvasive, neurotropic, and neurovirulent potential. In this commentary we address how this study contributes to the understanding of the neuropathology of SARS-CoV-2 and its variants.

Social Play Behavior Is Critical for the Development of Prefrontal Inhibitory Synapses and Cognitive Flexibility in Rats.

Sensory driven activity during early life is critical for setting up the proper connectivity of the sensory cortices. We ask here whether social play behavior, a particular form of social interaction that is highly abundant during postweaning development, is equally important for setting up connections in the developing prefrontal cortex (PFC). Young male rats were deprived from social play with peers during the period in life when social play behavior normally peaks [postnatal day 21-42] (SPD rats), followed by resocialization until adulthood. We recorded synaptic currents in layer 5 cells in slices from medial PFC of adult SPD and control rats and observed that inhibitory synaptic currents were reduced in SPD slices, while excitatory synaptic currents were unaffected. This was associated with a decrease in perisomatic inhibitory synapses from parvalbumin-positive GABAergic cells. In parallel experiments, adult SPD rats achieved more reversals in a probabilistic reversal learning (PRL) task, which depends on the integrity of the PFC, by using a more simplified cognitive strategy than controls. Interestingly, we observed that one daily hour of play during SPD partially rescued the behavioral performance in the PRL, but did not prevent the decrease in PFC inhibitory synaptic inputs. Our data demonstrate the importance of unrestricted social play for the development of inhibitory synapses in the PFC and cognitive skills in adulthood and show that specific synaptic alterations in the PFC can result in a complex behavioral outcome.SIGNIFICANCE STATEMENT This study addressed the question whether social play behavior in juvenile rats contributes to functional development of the prefrontal cortex (PFC). We found that rats that had been deprived from juvenile social play (social play deprivation - SPD) showed a reduction in inhibitory synapses in the PFC and a simplified strategy to solve a complex behavioral task in adulthood. Providing one daily hour of play during SPD partially rescued the cognitive skills in these rats, but did not prevent the reduction in PFC inhibitory synapses. Our results demonstrate a key role for unrestricted juvenile social play in PFC development and emphasize the complex relation between PFC circuit connectivity and cognitive function.

In vitro and in vivo differences in neurovirulence between D614G, Delta And Omicron BA.1 SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with various neurological complications. Although the mechanism is not fully understood, several studies have shown that neuroinflammation occurs in the acute and post-acute phase. As these studies have predominantly been performed with isolates from 2020, it is unknown if there are differences among SARS-CoV-2 variants in their ability to cause neuroinflammation. Here, we compared the neuroinvasiveness, neurotropism and neurovirulence of the SARS-CoV-2 ancestral strain D614G, the Delta (B.1.617.2) and Omicron BA.1 (B.1.1.529) variants using in vitro and in vivo models. The Omicron BA.1 variant showed reduced neurotropism and neurovirulence compared to Delta and D614G in human induced pluripotent stem cell (hiPSC)-derived cortical neurons co-cultured with astrocytes. Similar differences were obtained in Syrian hamsters inoculated with D614G, Delta and the Omicron BA.1 variant 5 days post infection. Replication in the olfactory mucosa was observed in all hamsters, but most prominently in D614G inoculated hamsters. Furthermore, neuroinvasion into the CNS via the olfactory nerve was observed in D614G, but not Delta or Omicron BA.1 inoculated hamsters. Furthermore, neuroinvasion was associated with neuroinflammation in the olfactory bulb of hamsters inoculated with D614G. Altogether, our findings suggest differences in the neuroinvasive, neurotropic and neurovirulent potential between SARS-CoV-2 variants using in vitro hiPSC-derived neural cultures and in vivo in hamsters during the acute phase of the infection.

PHB Producing Cyanobacteria Found in the Neighborhood-Their Isolation, Purification and Performance Testing.

Cyanobacteria are a large group of prokaryotic microalgae that are able to grow photo-autotrophically by utilizing sunlight and by assimilating carbon dioxide to build new biomass. One of the most interesting among many cyanobacteria cell components is the storage biopolymer polyhydroxybutyrate (PHB), a member of the group of polyhydroxyalkanoates (PHA). Cyanobacteria occur in almost all habitats, ranging from freshwater to saltwater, freely drifting or adhered to solid surfaces or growing in the porewater of soil, they appear in meltwater of glaciers as well as in hot springs and can handle even high salinities and nutrient imbalances. The broad range of habitat conditions makes them interesting for biotechnological production in facilities located in such climate zones with the expectation of using the best adapted organisms in low-tech bioreactors instead of using "universal" strains, which require high technical effort to adapt the production conditions to the organism's need. These were the prerequisites for why and how we searched for locally adapted cyanobacteria in different habitats. Our manuscript provides insight to the sites we sampled, how we isolated and enriched, identified (morphology, 16S rDNA), tested (growth, PHB accumulation) and purified (physical and biochemical purification methods) promising PHB-producing cyanobacteria that can be used as robust production strains. Finally, we provide a guideline about how we managed to find potential production strains and prepared others for basic metabolism studies.

The neuroinvasiveness, neurotropism, and neurovirulence of SARS-CoV-2.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection is associated with a diverse spectrum of neurological complications during the acute and postacute stages. The pathogenesis of these complications is complex and dependent on many factors. For accurate and consistent interpretation of experimental data in this fast-growing field of research, it is essential to use terminology consistently. In this article, we outline the distinctions between neuroinvasiveness, neurotropism, and neurovirulence. Additionally, we discuss current knowledge of these distinct features underlying the pathogenesis of SARS-CoV-2-associated neurological complications. Lastly, we briefly discuss the advantages and limitations of different experimental models, and how these approaches can further be leveraged to advance the field.

Fluoxetine targets an allosteric site in the enterovirus 2C AAA+ ATPase and stabilizes a ring-shaped hexameric complex.

Enteroviruses are globally prevalent human pathogens responsible for many diseases. The nonstructural protein 2C is a AAA+ helicase and plays a key role in enterovirus replication. Drug repurposing screens identified 2C-targeting compounds such as fluoxetine and dibucaine, but how they inhibit 2C is unknown. Here, we present a crystal structure of the soluble and monomeric fragment of coxsackievirus B3 2C protein in complex with (S)-fluoxetine (SFX), revealing an allosteric binding site. To study the functional consequences of SFX binding, we engineered an adenosine triphosphatase (ATPase)­competent, hexameric 2C protein. Using this system, we show that SFX, dibucaine, HBB [2-(α-hydroxybenzyl)-benzimidazole], and guanidine hydrochloride inhibit 2C ATPase activity. Moreover, cryo­electron microscopy analysis demonstrated that SFX and dibucaine lock 2C in a defined hexameric state, rationalizing their mode of inhibition. Collectively, these results provide important insights into 2C inhibition and a robust engineering strategy for structural, functional, and drug-screening analysis of 2C proteins.

Bithiazole Inhibitors of Phosphatidylinositol 4-Kinase (PI4KIIIß) as Broad-Spectrum Antivirals Blocking the Replication of SARS-CoV-2, Zika Virus, and Human Rhinoviruses.

Over half a century since the description of the first antiviral drug, "old" re-emerging viruses and "new" emerging viruses still represent a serious threat to global health. Their high mutation rate and rapid selection of resistance toward common antiviral drugs, together with the increasing number of co-infections, make the war against viruses quite challenging. Herein we report a host-targeted approach, based on the inhibition of the lipid kinase PI4KIIIß, as a promising strategy for inhibiting the replication of multiple viruses hijacking this protein. We show that bithiazole inhibitors of PI4KIIIß block the replication of human rhinoviruses (hRV), Zika virus (ZIKV) and SARS-CoV-2 at low micromolar and sub-micromolar concentrations. However, while the anti-hRV/ZIKV activity can be directly linked to PI4KIIIß inhibition, the role of PI4KIIIß in SARS-CoV-2 entry/replication is debated.

Replication Kinetics, Cell Tropism, and Associated Immune Responses in SARS-CoV-2- and H5N1 Virus-Infected Human Induced Pluripotent Stem Cell-Derived Neural Models.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with a wide variety of neurological complications. Even though SARS-CoV-2 is rarely detected in the central nervous system (CNS) or cerebrospinal fluid, evidence is accumulating that SARS-CoV-2 might enter the CNS via the olfactory nerve. However, what happens after SARS-CoV-2 enters the CNS is poorly understood. Therefore, we investigated the replication kinetics, cell tropism, and associated immune responses of SARS-CoV-2 infection in different types of neural cultures derived from human induced pluripotent stem cells (hiPSCs). SARS-CoV-2 was compared to the neurotropic and highly pathogenic H5N1 influenza A virus. SARS-CoV-2 infected a minority of individual mature neurons, without subsequent virus replication and spread, despite angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and neuropilin-1 (NPR1) expression in all cultures. However, this sparse infection did result in the production of type III interferons and interleukin-8 (IL-8). In contrast, H5N1 virus replicated and spread very efficiently in all cell types in all cultures. Taken together, our findings support the hypothesis that neurological complications might result from local immune responses triggered by virus invasion, rather than abundant SARS-CoV-2 replication in the CNS. IMPORTANCE Infections with the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are often associated with neurological complications. Evidence suggests that SARS-CoV-2 enters the brain via the olfactory nerve; however, SARS-CoV-2 is only rarely detected in the central nervous system of COVID-19 patients. Here, we show that SARS-CoV-2 is able to infect neurons of human iPSC neural cultures but that this infection is abortive and does not result in virus spread to other cells. However, infection of neural cultures did result in the production of type III interferon and IL-8. This study suggests that SARS-CoV-2 might enter the CNS and infect individual neurons, triggering local immune responses that could contribute to the pathogenesis of SARS-CoV-2-associated CNS disease.

In Vivo Models to Study the Pathogenesis of Extra-Respiratory Complications of Influenza A Virus Infection.

Animal models are an inimitable method to study the systemic pathogenesis of virus-induced disease. Extra-respiratory complications of influenza A virus infections are not extensively studied even though they are often associated with severe disease and mortality. Here we review and recommend mammalian animal models that can be used to study extra-respiratory complications of the central nervous system and cardiovascular system as well as involvement of the eye, placenta, fetus, lacteal gland, liver, pancreas, intestinal tract, and lymphoid tissues during influenza A virus infections.