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Background: The hepatitis E virus (HEV) can cause acute viral hepatitis with or without neurological manifestations, and occasionally progresses to chronic infection in immunocompromised individuals. The management of chronic HEV infection in cancer patients may be challenging due to the complex immunological constellation. Furthermore, the diagnostic workflow and the impact on quality of life of neurological HEV manifestations in immunocompromised patients have not been sufficiently delineated previously., Case Description: A 61-year-old male with systemically treated chronic lymphocytic leukemia (CLL) experienced a slowly progressive atrophy of the spinal cord due to a chronic HEV infection. Despite continuous antiviral treatment with ribavirin, the patient's neurological condition continued to deteriorate, particularly following subsequent attempts to treat CLL. Treatment with obinutuzumab resulted in acute bowel and urinary retention and a further deterioration of motor skills, prompting the discontinuation of obinutuzumab. The patient's neurological status improved after the administration of intravenous immunoglobulins., Conclusion: This case study provides a comprehensive long-term follow-up of a cancer patient with chronic HEV infection and associated CNS involvement, which resulted in progressive neurological disability over several years. The challenges faced in diagnosing new neurological symptoms in patients undergoing immunosuppressive cancer treatment underscore the need for an interdisciplinary diagnostic approach that includes HEV testing. We propose a diagnostic pathway for future validation in immunocompromised cohorts presenting with neurological symptoms, emphasizing its potential to enhance clinical outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Ritter, Yomade, Holtz, Deinhardt-Emmer, McLean, Hartinger, Bechwar, Schwab, Huss, Mawrin, Axer, Schrenk, Reuken and Mäurer.)

During cellular senescence, persistent growth arrest and changes in protein expression programs are accompanied by a senescence-associated secretory phenotype (SASP). In this study, we detected the upregulation of the SASP-related protein dipeptidyl peptidase 4 (DDP4) in human primary lung cells rendered senescent by exposure to ionizing radiation. DPP4 is an exopeptidase that plays a crucial role in the cleavage of various proteins, resulting in the loss of N-terminal dipeptides and proinflammatory effects. Interestingly, our data revealed an association between severe coronavirus disease 2019 (COVID-19) and DDP4, namely that DPP4 levels increased in the plasma of patients with COVID-19 and were correlated with age and disease progression. Although we could not determine the direct effect of DDP4 on viral replication, mechanistic studies in cell culture revealed a negative impact on the expression of the tight junction protein zonula occludens-1 (ZO-1), which contributes to epithelial barrier function. Mass spectrometry analysis indicated that DPP4 overexpressing cells exhibited a decrease in ZO-1 and increased expression of pro-inflammatory cytokines and chemokines. By investigating the effect of DPP4 on the barrier function of human primary cells, we detected an increase in ZO-1 using DPP4 inhibitors. These results provide an important contribution to our understanding of DPP4 in the context of senescence, suggesting that DPP4 plays a major role as part of the SASP. Our results provide evidence that cellular senescence, a hallmark of aging, has an important impact on respiratory infections.

Inflammasome assembly is a potent mechanism responsible for the host protection against pathogens, including viruses. When compromised, it can allow viral replication, while when disrupted, it can perpetuate pathological responses by IL-1 signaling and pyroptotic cell death. SARS-CoV-2 infection was shown to activate inflammasome in the lungs of COVID-19 patients, however, potential mechanisms responsible for this response are not fully elucidated. In this study, we investigated the effects of ORF3a, E and M SARS-CoV-2 viroporins in the inflammasome activation in major populations of alveolar sentinel cells: macrophages, epithelial and endothelial cells. We demonstrated that each viroporin is capable of activation of the inflammasome in macrophages to trigger pyroptosis-like cell death and IL-1α release from epithelial and endothelial cells. Small molecule NLRP3 inflammasome inhibitors reduced IL-1 release but weakly affected the pyroptosis. Importantly, we discovered that while SARS-CoV-2 could not infect the pulmonary microvascular endothelial cells it induced IL-1α and IL-33 release. Together, these findings highlight the essential role of macrophages as the major inflammasome-activating cell population in the lungs and point to endothelial cell expressed IL-1α as a potential novel component driving the pulmonary immunothromobosis in COVID-19., (© 2024. The Author(s).)

The emerging SARS-CoV-2 variants are evolving to evade human immunity and differ in their pathogenicity. While evasion of the variants from adaptive immunity is widely investigated, there is a paucity of knowledge about their interactions with innate immunity. Inflammasome assembly is one of the most potent mechanisms of the early innate response to viruses, but when it is inappropriate, it can perpetuate tissue damage. In this study, we focused on the capacity of SARS-CoV-2 Alpha and Delta variants to activate the NLRP3 inflammasome. We compared the macrophage activation, particularly the inflammasome formation, using Alpha- and Delta-spike virus-like particles (VLPs). We found that VLPs of both variants activated the inflammasome even without a priming step. Delta-spike VLPs had a significantly stronger effect on triggering pyroptosis and inflammasome assembly in THP-1 macrophages than did Alfa-spike VLPs. Cells treated with Delta VLPs showed greater cleavage of caspase-1 and IL-1β release. Furthermore, Delta VLPs induced stronger cytokine secretion from macrophages and caused essential impairment of mitochondrial respiration in comparison to Alpha VLPs. Additionally, infection of primary human monocyte-derived macrophages with the SARS-CoV-2 variants confirmed the observations in VLPs. Collectively, we revealed that SARS-CoV-2 Delta had a greater impact on the inflammasome activation, cell death and mitochondrial respiration in macrophages than did the Alpha variant. Importantly, the differential response to the SARS-CoV-2 variants can influence the efficacy of therapies targeting the host's innate immunity., Competing Interests: Declaration of competing interest None of the authors has conflict of interest related to the manuscript entitled: “SARS-CoV-2 Virus-Like Particle Variants Alpha and Delta Mimic the Native Viruses in Their Differential Inflammasome Activating Potential” On behalf of the authors., (Copyright © 2024. Published by Elsevier B.V.)

Respiratory influenza A virus (IAV) infections are major health concerns worldwide, where bacterial superinfections substantially increase morbidity and mortality. The underlying mechanisms of how IAV impairs host defense remain elusive. Macrophages are pivotal for the innate immune response and crucially regulate the entire inflammatory process, occurring as inflammatory M1- or pro-resolving M2-like phenotypes. Lipid mediators (LM), produced from polyunsaturated fatty acids by macrophages, are potent immune regulators and impact all stages of inflammation. Using LM metabololipidomics, we show that human pro-resolving M2-macrophages respond to IAV infections with specific and robust production of prostaglandin (PG)E 2 along with upregulation of cyclooxygenase-2 (COX-2), which persists after co-infection with Staphylococcus aureus . In contrast, cytokine/interferon production in macrophages was essentially unaffected by IAV infection, and the functionality of M1-macrophages was not influenced. Conclusively, IAV infection of M2-macrophages selectively elevates PGE 2 formation, suggesting inhibition of the COX-2/PGE 2 axis as strategy to limit IAV exacerbation., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Background: SARS-CoV-2 variants are constantly emerging with a variety of changes in the conformation of the spike protein, resulting in alterations of virus entry mechanisms. Solely omicron variants use the endosomal clathrin-mediated entry. Here, we investigate the influence of defined altered spike formations to study their impact on premature cellular senescence., Methods: In our study, in vitro infections of SARS-CoV-2 variants delta (B.1.617.2) and omicron (B.1.1.529) were analyzed by using human primary small alveolar epithelial cells and human ex vivo lung slices. We confirmed cellular senescence in human lungs of COVID-19 patients. Hence, global gene expression patterns of infected human primary alveolar epithelial cells were identified via mRNA sequencing., Results: Solely omicron variants of SARS-CoV-2 influenced the expression of cell cycle genes, highlighted by an increased p21 expression in human primary lung cells and human ex vivo lungs. Additionally, an upregulated senescence-associated secretory phenotype (SASP) was detected. Transcriptomic data indicate an increased gene expression of p16, and p38 in omicron-infected lung cells., Conclusions: Significant changes due to different SARS-CoV-2 infections in human primary alveolar epithelial cells with an overall impact on premature aging could be identified. A substantially different cellular response with an upregulation of cell cycle, inflammation- and integrin-associated pathways in omicron infected cells indicates premature cellular senescence.

Objective: Obesity is an independent risk factor for severe influenza virus and COVID-19 infections. There might be an interplay between adipose tissue and respiratory pathogens, although the mechanism is unknown. Proinflammatory factors secreted by the adipose tissue are often discussed to serve as indirect contributor to virus infection. However, the direct potential of adipose tissue to serve as a viral niche has not yet been investigated., Methods: Two murine obesity models (DIO and ob/ob) were infected with influenza A virus (IAV) and monitored for 3 weeks. p.i. Lung and adipose tissue were harvested, and the viral load was analysed. Direct replication of IAV in vitro was investigated in human derived primary adipocytes and macrophages. The indirect impact of the secretory products of adipocytes during infection was analysed in a co-culture system with lung fibroblasts. Moreover, lung and adipose tissue was harvested from deceased patients infected with SARS-CoV-2 omicron variant. Additionally, replication of SARS-CoV-2 alpha, delta, and omicron variants was investigated in vitro in adipocytes and macrophages., Results: Both murine obesity models presented high IAV titers compared to non-obese mice. Interestingly, adipose tissue adjacent to the lungs was a focal point for influenza virus replication in mice. We further detected IAV replication and antiviral response in human adipocytes. Co-cultivation of adipocytes and lung fibroblasts led to increased IL-8 concentration during infection. Though we observed SARS-CoV-2 in the thoracic adipose tissue of COVID-19 patients, no active replication was found in adipocytes in vitro. However, SARS-CoV-2 was detected in the macrophages and this finding was associated with increased inflammation., Conclusions: Our study revealed that thoracic adipose tissue contributes to respiratory virus infection. Besides indirect induction of proinflammatory factors during infection, adipocytes and macrophages within the tissue can directly support viral replication., (© 2023. The Author(s).)

Respiratory infections pose a significant health problem among elderly individuals, particularly during the COVID-19 pandemic. The increased mortality and morbidity rates among individuals over 65 highlight the criticality of these infections. The normal aging process in the lungs increases vulnerability to respiratory infections due to the accumulation of cellular damage and senescence. Consequently, the lung environment undergoes major changes in mechanical function and other systemic factors. This review aims to examine the influence of aging on respiratory infections from a clinical perspective by analyzing clinical studies. Additionally, the review will emphasize potential prevention and diagnostic developments to enhance therapy options available for elderly patients over 65 years of age.

Aging is a major risk factor associated with increased morbidity and mortality rates observed during respiratory infections. In this study, we investigated the role of influenza virus infections in the establishment of premature cellular senescence and paracrine macrophage-activated inflammation. We observed in our murine model a premature aging by the appearance of senescent cells in the lungs after 21 d of influenza A virus infection. By using murine ex vivo lung models, the influence of TNF-α on the establishment of cellular senescence was detectable. Our findings were proven by using conditioned media of infected human monocyte-derived macrophages on primary lung fibroblasts. Here, a distinct expression of senescence-associated parameters could be confirmed. Furthermore, senescent cells in the lungs strongly influenced subsequent viral infections. Our data demonstrated a higher viral load in senescent primary lung fibroblasts, indicating an intracellular effect on viral replication. Transcriptomic data revealed an increased regulation of JAK/STAT signaling in senescent IAV-infected cells accompanied with increased TRAIL expression. Additionally, senescent cells indicating low pH values, accelerating viral replication. Our study provides new insights into pathomechanisms of virus-induced cellular senescence. Hence, IAV infection induces premature senescence and subsequent infections in senescent cells lead to an increased viral replication.

Rapid testing for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) of patients presenting to emergency departments (EDs) facilitates the decision for isolation on admission to hospital wards. Differences in the sensitivity of molecular assays have implications for diagnostic workflows. This study evaluated the performance of the cobas ® Liat ® RT-PCR, which is routinely used as the initial test for ED patients in our hospitals, compared with the eazyplex ® RT-LAMP. A total of 378 oropharyngeal and nasal swabs with positive Liat ® results were analysed. Residual sample aliquots were tested using NeuMoDx™, cobas ® RT-PCR, and the eazyplex ® assay. Patients were divided into asymptomatic (n = 157) and symptomatic (n = 221) groups according to the WHO case definition. Overall, 14% of positive Liat ® results were not confirmed by RT-PCR. These samples were mainly attributed to 26.8% of asymptomatic patients, compared to 3.8% of the symptomatic group. Therefore, positive Liat ® results were used to provisionally isolate patients in the ED until RT-PCR results were available. The eazyplex ® assay identified 62% and 90.6% of RT-PCR-confirmed cases in asymptomatic and symptomatic patients, respectively. False-negative eazyplex ® results were associated with RT-PCR Ct values > 30, and were more frequent in the asymptomatic group than in the symptomatic group (38.1% vs. 5.1%, respectively). Both the Liat ® and eazyplex ® assays are suitable for testing symptomatic patients. Their use in screening asymptomatic patients depends on the need to exclude any infection or identify those at high risk of transmission.

We introduce a magnetic bead-based sample preparation scheme for enabling the Raman spectroscopic differentiation of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2)-positive and -negative samples. The beads were functionalized with the angiotensin-converting enzyme 2 (ACE2) receptor protein, which is used as a recognition element to selectively enrich SARS-CoV-2 on the surface of the magnetic beads. The subsequent Raman measurements directly enable discriminating SARS-CoV-2-positive and -negative samples. The proposed approach is also applicable for other virus species when the specific recognition element is exchanged. A series of Raman spectra were measured on three types of samples, namely SARS-CoV-2, Influenza A H1N1 virus and a negative control. For each sample type, eight independent replicates were considered. All of the spectra are dominated by the magnetic bead substrate and no obvious differences between the sample types are apparent. In order to address the subtle differences in the spectra, we calculated different correlation coefficients, namely the Pearson coefficient and the Normalized cross correlation coefficient. By comparing the correlation with the negative control, differentiating between SARS-CoV-2 and Influenza A virus is possible. This study provides a first step towards the detection and potential classification of different viruses with the use of conventional Raman spectroscopy.

Background: The prevalence of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin (PVL) gene is higher in Africa (≈50%) compared to Europe (< 5%). The study aimed to measure anti-PVL-antibodies in Africans and Germans in a multi-center study and to test whether detected antibodies can neutralize the cytotoxic effect of PVL on polymorphonuclear leukocytes (PMNs)., Methods: Sera from asymptomatic Africans (n = 22, Nigeria, Gabon) and Caucasians (n = 22, Germany) were used to quantify antibody titers against PVL and α-hemolysin (in arbitrary units [AU]) by ELISA. PMNs from one African and German donor were exposed to 5 nM recombinant PVL to measure the neutralizing effect of serial dilutions of pooled sera from African and Caucasian participants, or donor sera at 0.625 and 2.5% (v/v)., Results: Anti-PVL-antibodies were significantly higher in Africans than in Germans (1.9 vs. 0.7 AU, p < 0.0001). The pooled sera from the study participants neutralized the cytotoxic effect of PVL on African and German PMNs in a dose dependent manner. Also, neutralization of PVL on PMNs from the African and German donors had a stronger effect with African sera (half-maximal inhibitory concentration (IC 50 ) = 0.27 and 0.47%, respectively) compared to Caucasian sera (IC 50  = 3.51 and 3.59% respectively)., Conclusion: Africans have higher levels of neutralizing anti-PVL-antibodies. It remains unclear if or at what level these antibodies protect against PVL-related diseases., (© 2022. The Author(s).)

Viruses are the cause of a considerable burden to human, animal and plant health, while on the other hand playing an important role in regulating entire ecosystems. The power of new sequencing technologies combined with new tools for processing "Big Data" offers unprecedented opportunities to answer fundamental questions in virology. Virologists have an urgent need for virus-specific bioinformatics tools. These developments have led to the formation of the European Virus Bioinformatics Center, a network of experts in virology and bioinformatics who are joining forces to enable extensive exchange and collaboration between these research areas. The EVBC strives to provide talented researchers with a supportive environment free of gender bias, but the gender gap in science, especially in math-intensive fields such as computer science, persists. To bring more talented women into research and keep them there, we need to highlight role models to spark their interest, and we need to ensure that female scientists are not kept at lower levels but are given the opportunity to lead the field. Here we showcase the work of the EVBC and highlight the achievements of some outstanding women experts in virology and viral bioinformatics.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the coronavirus disease-19 (COVID-19) is still challenging healthcare systems and societies worldwide. While vaccines are available, therapeutic strategies are developing and need to be adapted to each patient. Many clinical approaches focus on the repurposing of approved therapeutics against other diseases. However, the efficacy of these compounds on viral infection or even harmful secondary effects in the context of SARS-CoV-2 infection are sparsely investigated. Similarly, adverse effects of commonly used therapeutics against lifestyle diseases have not been studied in detail. Using mono cell culture systems and a more complex chip model, we investigated the effects of the acetylsalicylic acid (ASA) salt D,L-lysine-acetylsalicylate + glycine (LASAG) on SARS-CoV-2 infection in vitro. ASA is commonly known as Aspirin ® and is one of the most frequently used medications worldwide. Our data indicate an inhibitory effect of LASAG on SARS-CoV-2 replication and SARS-CoV-2-induced expression of pro-inflammatory cytokines and coagulation factors. Remarkably, our data point to an additive effect of the combination of LASAG and the antiviral acting drug remdesivir on SARS-CoV-2 replication in vitro.

Purpose: Nasal screening is performed to avoid the complications of postoperative surgical site infections (SSI), especially those due to antimicrobial-resistant species such as methicillin-resistant Staphylococcus aureus (MRSA). This study examined the relationship between bacterial isolates from the conjunctiva and the nasal cavity., Methods: All patients were diagnosed with ocular surface infections, and the organisms in the conjunctiva and the nasal cavity were isolated. We investigated the relationship of the following antimicrobial-resistant bacteria between the conjunctiva and the nose: MRSA, methicillin-resistant CNS (MRCNS), levofloxacin-resistant (LVFX-R) Corynebacterium spp. Data were analyzed using Fisher's exact test, and the odds ratio was examined., Results: This study included 188 eyes of 188 subjects (87 males and 101 females; mean age 58.5 years, range 11-97 years). MRSA (4 eyes), MRCNS (29 eyes), and LVFX-R Corynebacterium spp. (41 eyes) were identified from the conjunctiva, and MRSA (6 eyes), MRCNS (38 eyes), and LVFX-R Corynebacterium spp. (41 eyes) were identified from the nasal cavity. There was a significant relationship detected between the conjunctiva and the nose for MRSA, MRCNS, and LVFX-R Corynebacterium spp. MRSA displayed high sensitivity (0.750, 95% confidence interval [CI]; 0.301 to 0.987) and specificity (0.984, 95% CI; 0.953 to 0.996) in nasal cavity cultures, and the odds ratio was 181.00 times (95% CI; 18.41 to 2320)., Conclusion: This study showed a significant relationship between conjunctival and nasal cultures of MRSA, MRCNS, and LVFX-R Corynebacterium spp., suggesting that nasal cavity culture is a potentially useful screening method for detecting resistant bacteria, especially MRSA, in the conjunctiva., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Humoral immunity after infection or after vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been attributed a key part in mitigating the further transmission of the virus. In this study, we used a commercial anti-Spike immunoglobulin G (S-IgG) assay and developed a cell culture-based neutralization assay to understand the longitudinal course of neutralizing antibodies in both SARS-CoV2 infected or vaccinated individuals. We show that even more than one year after infection, about 78% of observed study participants remained seropositive concerning S-IgG antibodies. In addition, the serum of the individuals had stable neutralization capacity in a neutralization assay against a SARS-CoV-2 patient isolate from March 2020. We also examined volunteers after either homologous BNT162b2 prime-boost vaccination or heterologous AZD1222 prime/mRNA-based booster vaccination. Both the heterologous and the homologous vaccination regimens induced higher levels of neutralizing antibodies in healthy subjects when compared to subjects after a mild infection, showing the high effectiveness of available vaccines. In addition, we could demonstrate the reliability of S-IgG levels in predicting neutralization capacity, with 94.8% of seropositive samples showing a neutralization titer of ≥10, making it a viable yet cheap and easy-to-determine surrogate parameter for neutralization capacity.

Influenza virus (IV) infections are considered to cause severe diseases of the respiratory tract. Beyond mild symptoms, the infection can lead to respiratory distress syndrome and multiple organ failure. Occurrence of resistant seasonal and pandemic strains against the currently licensed antiviral medications points to the urgent need for new and amply available anti-influenza drugs. Interestingly, the virus-supportive function of the cellular phosphatidylinositol 3-kinase (PI3K) suggests that this signaling module may be a potential target for antiviral intervention. In the sense of repurposing existing drugs for new indications, we used Pictilisib, a known PI3K inhibitor to investigate its effect on IV infection, in mono-cell-culture studies as well as in a human chip model. Our results indicate that Pictilisib is a potent inhibitor of IV propagation already at early stages of infection. In a murine model of IV pneumonia, the in vitro key findings were verified, showing reduced viral titers as well as inflammatory response in the lung after delivery of Pictilisib. Our data identified Pictilisib as a promising drug candidate for anti-IV therapies that warrant further studying. These results further led to the conclusion that the repurposing of previously approved substances represents a cost-effective and efficient way for development of novel antiviral strategies.

Infections with SARS-CoV-2 can be asymptomatic, but they can also be accompanied by a variety of symptoms that result in mild to severe coronavirus disease-19 (COVID-19) and are sometimes associated with systemic symptoms. Although the viral infection originates in the respiratory system, it is unclear how the virus can overcome the alveolar barrier, which is observed in severe COVID-19 disease courses. To elucidate the viral effects on the barrier integrity and immune reactions, we used mono-cell culture systems and a complex human chip model composed of epithelial, endothelial, and mononuclear cells. Our data show that SARS-CoV-2 efficiently infected epithelial cells with high viral loads and inflammatory response, including interferon expression. By contrast, the adjacent endothelial layer was neither infected nor did it show productive virus replication or interferon release. With prolonged infection, both cell types were damaged, and the barrier function was deteriorated, allowing the viral particles to overbear. In our study, we demonstrate that although SARS-CoV-2 is dependent on the epithelium for efficient replication, the neighboring endothelial cells are affected, e.g., by the epithelial cytokines or components induced during infection, which further results in the damage of the epithelial/endothelial barrier function and viral dissemination. IMPORTANCE SARS-CoV-2 challenges healthcare systems and societies worldwide in unprecedented ways. Although numerous new studies have been conducted, research to better understand the molecular pathogen-host interactions are urgently needed. For this, experimental models have to be developed and adapted. In the present study we used mono cell-culture systems and we established a complex chip model, where epithelial and endothelial cells are cultured in close proximity. We demonstrate that epithelial cells can be infected with SARS-CoV-2, while the endothelium did not show any infection signs. Since SARS-CoV-2 is able to establish viremia, the link to thromboembolic events in severe COVID-19 courses is evident. However, whether the endothelial layer is damaged by the viral pathogens or whether other endothelial-independent homeostatic factors are induced by the virus is essential for understanding the disease development. Therefore, our study is important as it demonstrates that the endothelial layer could not be infected by SARS-CoV-2 in our in vitro experiments, but we were able to show the destruction of the epithelial-endothelial barrier in our chip model. From our experiments we can assume that virus-induced host factors disturbed the epithelial-endothelial barrier function and thereby promote viral spread., (Copyright © 2021 Deinhardt-Emmer et al.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global health problem. Although the respiratory system is the main impaired organ, conjunctivitis is one of its common findings. However, it is not yet understood if SARS-CoV-2 can infect the eye and if the ocular surface can be a potential route of SARS-CoV-2 transmissions. Our review focuses on the viral entry mechanisms to give a better understanding of the interaction between SARS-CoV-2 and the eye. We highlighted findings that give evidence for multiple potential receptors of SARS-CoV-2 on the ocular surface. Additionally, we focused on data concerning the detection of viral RNA and its spike protein in the various ocular tissues from patients. However, the expression level seemed to be relatively low compared to the respiratory tissues as a result of a unique environment surrounding the ocular surface and the innate immune response of SARS-CoV-2. Nevertheless, our review suggests the ocular surface as a potential route for SARS-CoV-2 transmission, and as a result of this study we strongly recommend the protection of the eyes for ophthalmologists and patients at risk.

Clinical observations indicate that COVID-19 is a systemic disease. An investigation of the viral distribution within the human body and its correlation with tissue damage can aid in understanding the pathophysiology of SARS-CoV-2 infection. We present a detailed mapping of the viral RNA in 61 tissues and organs of 11 deceased patients with COVID-19. The autopsies were performed within the early postmortem interval (between 1.5 and 15 hr, mean: 5.6 hr) to minimize the bias due to viral RNA and tissue degradation. Very high viral loads (>10 4 copies/ml) were detected in most patients' lungs, and the presence of intact viral particles in the lung tissue could be verified by transmission electron microscopy. Interestingly, viral RNA was detected throughout various extrapulmonary tissues and organs without visible tissue damage. The dissemination of SARS-CoV-2-RNA throughout the body supports the hypothesis that there is a maladaptive host response with viremia and multiorgan dysfunction., Competing Interests: SD, DW, JS, SK, SE, KH, VV, CH, JR, AH, CE, MB, MP, NG, SN, BL, GM No competing interests declared, (© 2021, Deinhardt-Emmer et al.)

Obesity is a globally increasing health problem, entailing diverse comorbidities such as infectious diseases. An obese weight status has marked effects on lung function that can be attributed to mechanical dysfunctions. Moreover, the alterations of adipocyte-derived signal mediators strongly influence the regulation of inflammation, resulting in chronic low-grade inflammation. Our review summarizes the known effects regarding pulmonary bacterial and viral infections. For this, we discuss model systems that allow mechanistic investigation of the interplay between obesity and lung infections. Overall, obesity gives rise to a higher susceptibility to infectious pathogens, but the pathogenetic process is not clearly defined. Whereas, viral infections often show a more severe course in obese patients, the same patients seem to have a survival benefit during bacterial infections. In particular, we summarize the main mechanical impairments in the pulmonary tract caused by obesity. Moreover, we outline the main secretory changes within the expanded adipose tissue mass, resulting in chronic low-grade inflammation. Finally, we connect these altered host factors to the influence of obesity on the development of lung infection by summarizing observations from clinical and experimental data.

Background: It is essential to avoid admission of patients with undetected corona virus disease 2019 (COVID-19) to hospitals' general wards. Even repeated negative reverse transcription polymerase chain reaction (RT-PCR) results do not rule-out COVID-19 with certainty. The study aimed to evaluate a rule-out strategy for COVID-19 using chest computed tomography (CT) in adults being admitted to the emergency department and suspected of COVID-19., Methods: In this prospective, single centre, diagnostic accuracy cohort study, consecutive adults (≥ 18 years) presenting with symptoms consistent with COVID-19 or previous contact to infected individuals, admitted to the emergency department and supposed to be referred to general ward were included in March and April 2020. All participants underwent low-dose chest CT. RT-PCR- and specific antibody tests were used as reference standard. Main outcome measures were sensitivity and specificity of chest CT. Predictive values were calculated based on the theorem of Bayes using Fagan's nomogram., Results: Of 165 participants (56.4% male, 71 ± 16 years) included in the study, the diagnosis of COVID-19 was confirmed with RT-PCR and AB tests in 13 participants (prevalence 7.9%). Sensitivity and specificity of chest CT were 84.6% (95% confidence interval [CI], 54.6-98.1) and 94.7% (95% CI, 89.9-97.7), respectively. Positive and negative likelihood ratio of chest CT were 16.1 (95% CI, 7.9-32.8) and 0.16 (95% CI, 0.05-0.58) and positive and negative predictive value were 57.9% (95% CI, 40.3-73.7) and 98.6% (95% CI, 95.3-99.6), respectively., Conclusion: At a low prevalence of COVID-19, chest CT could be used as a complement to repeated RT-PCR testing for early COVID-19 exclusion in adults with suspected infection before referral to hospital's general wards. Trial registration ClinicalTrials.gov: NCT04357938 April 22, 2020.

Tropical pyomyositis (TP) is a life-threatening bacterial infection of the skeletal muscle that occurs particularly among children, young adults and those with immunocompromised conditions. The appropriate diagnosis and treatment are often delayed due to its non-specific signs, leading to fatal consequences. Staphylococcus aureus, especially methicillin-susceptible S. aureus, is responsible for most TP cases. However, other bacteria (i.e. streptococci, Pseudomonas aeruginosa, Escherichia coli, Klebsiella spp., Candida spp., Mycobacterium spp.) have been reported. This narrative review provides an update on the epidemiology and clinical course of TP. A special focus is laid on the role of toxins (i.e. Panton-Valentine Leucocidin and α-toxin) in the pathogenesis of TP and their implication for the clinical management of infection., (© 2020 The Authors. Tropical Medicine & International Health Published by John Wiley & Sons Ltd.)

Severe influenza virus (IV) infections still represent a major challenge to public health. To combat IV infections, vaccines and antiviral compounds are available. However, vaccine efficacies vary with very limited to no protection against newly emerging zoonotic IV introductions. In addition, the development of resistant virus variants against currently available antivirals can be rapidly detected, in consequence demanding the design of novel antiviral strategies. Virus supportive cellular signaling cascades, such as the NF-κB pathway, have been identified to be promising antiviral targets against IV in in vitro and in vivo studies and clinical trials. While administration of NF-κB pathway inhibiting agents, such as LASAG results in decreased IV replication, it remained unclear whether blocking of NF-κB might sensitize cells to secondary bacterial infections, which often come along with viral infections. Thus, we examined IV and Staphylococcus aureus growth during LASAG treatment. Interestingly, our data reveal that the presence of LASAG during superinfection still leads to reduced IV titers. Furthermore, the inhibition of the NF-κB pathway resulted in decreased intracellular Staphylococcus aureus loads within epithelial cells, indicating a dependency on the pathway for bacterial uptake. Unfortunately, so far it is not entirely clear if this phenomenon might be a drawback in bacterial clearance during infection., Competing Interests: The authors have declared that no competing interests exist.

Pneumonia is the leading cause of hospitalization worldwide. Besides viruses, bacterial co-infections dramatically exacerbate infection. In general, surfactant protein-A (SP-A) represents a first line of immune defense. In this study, we analyzed whether influenza A virus (IAV) and/or Staphylococcus aureus ( S. aureus ) infections affect SP-A expression. To closely reflect the situation in the lung, we used a human alveolus-on-a-chip model and a murine pneumonia model. Our results show that S. aureus can reduce extracellular levels of SP-A, most likely attributed to bacterial proteases. Mono-epithelial cell culture experiments reveal that the expression of SP-A is not directly affected by IAV or S. aureus . Yet, the mRNA expression of SP-A is strongly down-regulated by TNF-α, which is highly produced by professional phagocytes in response to bacterial infection. By using the human alveolus-on-a-chip model, we show that the down-regulation of SP-A is strongly dependent on macrophages. In a murine model of pneumonia, we can confirm that S. aureus decreases SP-A levels in vivo. These findings indicate that (I) complex interactions of epithelial and immune cells induce down-regulation of SP-A expression and (II) bacterial mono- and super-infections reduce SP-A expression in the lung, which might contribute to a severe outcome of bacterial pneumonia.

Pneumonia is one of the most common infectious diseases worldwide. The influenza virus can cause severe epidemics, which results in significant morbidity and mortality. Beyond the virulence of the virus itself, epidemiological data suggest that bacterial co-infections are the major cause of increased mortality. In this context, Staphylococcus aureus represents a frequent causative bacterial pathogen. Currently available models have several limitations in the analysis of the pathogenesis of infections, e.g. some bacterial toxins strongly act in a species-specific manner. Human 2D mono-cell culture models often fail to maintain the differentiation of alveolus-specific functions. A detailed investigation of the underlying pathogenesis mechanisms requires a physiological interaction of alveolus-specific cell types. The aim of the present work was to establish a human in vitro alveolus model system composed of vascular and epithelial cell structures with cocultured macrophages resembling the human alveolus architecture and functions. We demonstrate that high barrier integrity maintained for up to 14 d in our model containing functional tissue-resident macrophages. We show that flow conditions and the presence of macrophages increased the barrier function. The infection of epithelial cells induced a high inflammatory response that spread to the endothelium. Although the integrity of the epithelium was not compromised by a single infection or co-infection, we demonstrated significant endothelial cell damage associated with loss of barrier function. We established a novel immune-responsive model that reflects the complex crosstalk between pathogens and host. The in vitro model allows for the monitoring of spatiotemporal spreading of the pathogens and the characterization of morphological and functional alterations attributed to infection. The alveolus-on-a-chip represents a promising platform for mechanistic studies of host-pathogen interactions and the identification of molecular and cellular targets of novel treatment strategies in pneumonia.

Staphylococcus aureus is a facultative pathogenic bacterium that colonizes the nasopharyngeal area of healthy individuals, but can also induce severe infection, such as pneumonia. Pneumonia caused by mono- or superinfected S. aureus leads to high mortality rates. To establish an infection, S. aureus disposes of a wide variety of virulence factors, which can vary between clinical isolates. Our study aimed to characterize pneumonia isolates for their virulent capacity. For this, we analyzed isolates from colonization, pneumonia due to S. aureus , and pneumonia due to S. aureus /influenza virus co-infection. A total of 70 strains were analyzed for their virulence genes and the host-pathogen interaction was analyzed through functional assays in cell culture systems. Strains from pneumonia due to S. aureus mono-infection showed enhanced invasion and cytotoxicity against professional phagocytes than colonizing and co-infecting strains. This corresponded to the high presence of cytotoxic components in pneumonia strains. By contrast, strains obtained from co-infection did not exhibit these virulence characteristics and resembled strains from colonization, although they caused the highest mortality rate in patients. Taken together, our results underline the requirement of invasion and toxins to cause pneumonia due to S. aureus mono-infection, whereas in co-infection even low-virulent strains can severely aggravate pneumonia.

Staphylococcus aureus colonizes epithelial surfaces, but it can also cause severe infections. The aim of this work was to investigate whether bacterial virulence correlates with defined types of tissue infections. For this, we collected 10⁻12 clinical S. aureus strains each from nasal colonization, and from patients with endoprosthesis infection, hematogenous osteomyelitis, and sepsis. All strains were characterized by genotypic analysis, and by the expression of virulence factors. The host⁻pathogen interaction was studied through several functional assays in osteoblast cultures. Additionally, selected strains were tested in a murine sepsis/osteomyelitis model. We did not find characteristic bacterial features for the defined infection types; rather, a wide range in all strain collections regarding cytotoxicity and invasiveness was observed. Interestingly, all strains were able to persist and to form small colony variants (SCVs). However, the low-cytotoxicity strains survived in higher numbers, and were less efficiently cleared by the host than the highly cytotoxic strains. In summary, our results indicate that not only destructive, but also low-cytotoxicity strains are able to induce infections. The low-cytotoxicity strains can successfully survive, and are less efficiently cleared from the host than the highly cytotoxic strains, which represent a source for chronic infections. The understanding of this interplay/evolution between the host and the pathogen during infection, with specific attention towards low-cytotoxicity isolates, will help to optimize treatment strategies for invasive and therapy-refractory infection courses.

Staphylococcus aureus is the most frequent pathogen causing diabetic foot infections. Here, we investigated the degree of bacterial virulence required to establish invasive tissue infections in diabetic organisms. Staphylococcal isolates from diabetic and non-diabetic foot ulcers were tested for their virulence in in vitro functional assays of host cell invasion and cytotoxicity. Isolates from diabetes mellitus type I/II patients exhibited less virulence than isolates from non-diabetic patients, but were nevertheless able to establish severe infections. In some cases, non-invasive isolates were detected deep within diabetic wounds, even though the strains were non-pathogenic in cell culture models. Testing of defined isolates in murine footpad injection models revealed that both low- and high-virulent bacterial strains persisted in higher numbers in diabetic compared to non-diabetic hosts, suggesting that hyperglycemia favors bacterial survival. Additionally, the bacterial load was higher in NOD mice, which have a compromised immune system, compared to C57Bl/6 mice. Our results reveal that high as well as low-virulent staphylococcal strains are able to cause soft tissue infections and to persist in diabetic humans and mice, suggesting a reason for the frequent and endangering infections in patients with diabetes., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Therapy or prophylaxis of herpes simplex virus type 2 (HSV-2) infections with the nucleoside analog aciclovir (ACV) can lead to the emergence of drug-resistant HSV-2 strains, particularly in immunocompromised patients. In this context, multiple amino acid (aa) changes can accumulate in the ACV-converting viral thymidine kinase (TK) which hampers sequence-based diagnostics significantly. In this study, the so far unknown or still doubted relevance of several individual aa changes for drug resistance in HSV-2 was clarified. For this purpose, ten recombinant fluorescent HSV-2 strains differing in the respective aa within their TK were constructed using the bacterial artificial chromosome (BAC) pHSV2(MS)Lox. Similar TK expression levels and similar replication behavior patterns were demonstrated for the mutants as compared to the unmodified BAC-derived HSV-2 strain. Subsequently, the resulting strains were tested for their susceptibility to ACV as well as penciclovir (PCV) in parallel to a modified cytopathic effect (CPE) inhibition assay and by determining the relative fluorescence intensity (quantified using units, RFU) as a measure for the viral replication capacity. While aa changes Y53N and R221H conferred ACV resistance with cross-resistance to PCV, the aa changes G25A, G39E, T131M, Y133F, G150D, A157T, R248W, and L342W maintained a susceptible phenotype against both antivirals. The CPE inhibition assay and the measurement of relative fluorescence intensity yielded comparable results for the phenotypic testing of recombinant viruses. The latter test showed some technical advantages. In conclusion, the significance of single aa changes in HSV-2 TK on ACV/PCV resistance was clarified by the construction and phenotypic testing of recombinant viral strains. This was facilitated by the fluorescence based method., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We describe the first isolation of Mycobacterium hassiacum , a rapid-growing, partial acid-resistant mycobacterium, in a respiratory specimen from a patient with exacerbated chronic obstructive pulmonary disease. To provide therapeutic recommendation for future cases, antibiotic susceptibility testing of 3 clinical isolates was performed by broth microdilution. All strains tested showed susceptibility to clarithromycin, imipenem, ciprofloxacin, and doxycycline. The role of M hassiacum as a respiratory pathogen remains unclear and needs to be evaluated by future reports., Competing Interests: Declaration Of Conflicting Interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Here, we describe a novel reliable method to assess the significance of individual mutations within the thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1) to nucleoside analogue resistance. Eleven defined single nucleotide polymorphisms that occur in the TK gene of clinical HSV-1 isolates and a fluorescence reporter were introduced into the HSV-1 strain 17(+) that had been cloned into a bacterial artificial chromosome. The susceptibility of these different strains to aciclovir, penciclovir, brivudin, and foscarnet was determined with a modified cytopathic effect reduction assay. The strains were also tested for their aciclovir susceptibility by measuring the relative fluorescence intensity as an indicator for HSV-1 replication and by quantifying the virus yield. Our data indicate that the amino acid substitutions R41H, R106H, A118V, L139V, K219T, S276R, L298R, S345P, and V348I represent natural polymorphisms of the TK protein, whereas G61A and P84L mediate broad cross-resistance against aciclovir, penciclovir, brivudin, and susceptibility to foscarnet. This method allows the definition of the resistance genotype of otherwise unclear mutations in the TK gene of HSV-1. Thus, it provides a scientific basis for antiviral testing in clinical isolates of patients suffering from serious diseases and will facilitate testing of new antivirals against HSV-1.