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Hepatitis C virus (HCV) is the major causative pathogen associated with liver cirrhosis and hepatocellular carcinoma. The main virion component the core (C) protein has been implicated in several aspects of HCV pathology including oncogenesis, immune evasion, and stress responses. We and others have previously shown that the expression of C in different cell lines triggers Ca2+ signaling and alters Ca2+ homeostasis. In this study, we have identified two distinct C protein regions required for activation of the Ca2+/NFAT signaling. In the basic N-terminal domain, which has been implicated in the self-association of C, amino acids 1-68 were critical for NFAT activation. Sedimentation analysis of the four mutants in this domain revealed that association of the C protein into nucleocapsid-like particles correlated with NFAT-activated transcription. The internal, lipid droplet-targeting domain, was dispensable for NFAT-activated transcription. Finally, the C-terminal ER-targeting domain was required in extenso for the C protein function. Our results indicate that targeting of HCV C to the ER is a prerequisite, but not sufficient for inducing Ca2+/NFAT signaling. Taken together, our data are consistent with a model whereby proteolytic intermediates of C with intact transmembrane ER-anchor assemble into pore-like structures in the ER membrane.

PURPOSE: the pathophysiologic mechanisms explaining differences in clinical outcomes following COVID-19 are not completely described. This study aims to investigate antibody responses in critically ill patients with COVID-19 in relation to inflammation, organ failure and 30-day survival. METHODS: All patients with PCR-verified COVID-19 and gave consent, and who were admitted to a tertiary Intensive care unit (ICU) in Sweden during March-September 2020 were included. Demography, repeated blood samples and measures of organ function were collected. Analyses of anti-SARS-CoV-2 antibodies (IgM, IgA and IgG) in plasma were performed and correlated to patient outcome and biomarkers of inflammation and organ failure. RESULTS: A total of 115 patients (median age 62 years, 77% male) were included prospectively. All patients developed severe respiratory dysfunction, and 59% were treated with invasive ventilation. Thirty-day mortality was 22.6% for all included patients. Patients negative for any anti-SARS-CoV-2 antibody in plasma during ICU admission had higher 30-day mortality compared to patients positive for antibodies. Patients positive for IgM had more ICU-, ventilator-, renal replacement therapy- and vasoactive medication-free days. IgA antibody concentrations correlated negatively with both SAPS3 and maximal SOFA-score and IgM-levels correlated negatively with SAPS3. Patients with antibody levels below the detection limit had higher plasma levels of extracellular histones on day 1 and elevated levels of kidney and cardiac biomarkers, but showed no signs of increased inflammation, complement activation or cytokine release. After adjusting for age, positive IgM and IgG antibodies were still associated with increased 30-day survival, with odds ratio (OR) 7.1 (1.5-34.4) and 4.2 (1.1-15.7), respectively. CONCLUSION: In patients with severe COVID-19 requiring intensive care, a poor antibody response is associated with organ failure, systemic histone release and increased 30

BACKGROUND BK virus (BKV) infection after kidney transplantation leads to BKV-associated nephropathy (BKVAN) in up to 10% of recipients, and is associated with an increased risk of allograft dysfunction or loss. The objective of this study was to estimate the incidence of BKVAN and to analyze whether enhanced induction is associated with an increased risk of BKVAN, possibly justifying more intensive surveillance. MATERIAL AND METHODS This was a single-center retrospective cohort study. All patients who underwent kidney transplantation or simultaneous pancreas and kidney transplantation at the Uppsala University Hospital in Sweden between 2005 and 2014 were included, a period when BKV screening was not yet implemented. The effect of enhanced induction, defined as treatment with thymoglobulin, rituximab, and/or eculizumab, often in combination with IVIg and glycosorb, immunoadsorption and/or plasmapheresis/apheresis, was analyzed in a multivariable Cox proportional hazards model together with sex, age, cytomegalovirus mismatch (donor+/recipient-) and rejection treatment as co-predictors. Further, the effects of BKVAN on graft survival was analyzed in a univariable Cox proportional hazards model. RESULTS In total 44 of 928 (4.7%) patients developed a biopsy-verified BKVAN 4.8 (1.5-34.2) months after transplantation. Male sex was identified as a risk factor (HR 2.02, P=0.04) but not enhanced induction. Patients with BKVAN experienced a significantly higher risk of graft loss (HR 4.37, P<0.001). CONCLUSIONS Male sex, but not enhanced induction, was found to be a risk factor for BKVAN development after kidney transplantation. BKVAN is associated with an increased risk of graft loss.

Hepatitis C virus (HCV) is the major causative pathogen associated with hepatocellular carcinoma and liver cirrhosis. The main virion component, the Core (C) protein, is involved in multiple aspects of HCV pathology including oncogenesis and immune evasion. In this study, we established a next-generation bisulfite sequencing (NGS-BS) protocol to analyze the CpG methylation profile at the tumor suppressor gene SHP-1 P2 promoter as a model system. Our data show that HCV C protein expression in the immortalized T cells correlated with a specific CpG methylation profile at the SHP-1 P2. The NGS-BS on HCV-positive (HCV+) patient-derived PBMCs revealed a considerably different CpG methylation profile compared to the HCV C protein immortalized T cells. Notably, the CpG methylation profile was very similar in healthy and HCV+ PBMCs, suggesting that the SHP-1 P2 CpG methylation profile is not altered in the HCV+ individuals. Collectively, the NGS-BS is a highly sensitive method that can be used to quantitatively characterize the CpG methylation status at the level of individual CpG position and also allows the characterization of cis-acting effects on epigenetic regulation.

Purpose: the pathophysiologic mechanisms explaining differences in clinical outcomes following COVID-19 are not completely described. This study aims to investigate antibody responses in critically ill patients with COVID-19 in relation to inflammation, organ failure and 30-day survival. Methods: All patients with PCR-verified COVID-19 and gave consent, and who were admitted to a tertiary Intensive care unit (ICU) in Sweden during March¿September 2020 were included. Demography, repeated blood samples and measures of organ function were collected. Analyses of anti-SARS-CoV-2 antibodies (IgM, IgA and IgG) in plasma were performed and correlated to patient outcome and biomarkers of inflammation and organ failure. Results: A total of 115 patients (median age 62 years, 77% male) were included prospectively. All patients developed severe respiratory dysfunction, and 59% were treated with invasive ventilation. Thirty-day mortality was 22.6% for all included patients. Patients negative for any anti-SARS-CoV-2 antibody in plasma during ICU admission had higher 30-day mortality compared to patients positive for antibodies. Patients positive for IgM had more ICU-, ventilator-, renal replacement therapy- and vasoactive medication-free days. IgA antibody concentrations correlated negatively with both SAPS3 and maximal SOFA-score and IgM-levels correlated negatively with SAPS3. Patients with antibody levels below the detection limit had higher plasma levels of extracellular histones on day 1 and elevated levels of kidney and cardiac biomarkers, but showed no signs of increased inflammation, complement activation or cytokine release. After adjusting for age, positive IgM and IgG antibodies were still associated with increased 30-day survival, with odds ratio (OR) 7.1 (1.5¿34.4) and 4.2 (1.1¿15.7), respectively. Conclusion: In patients with severe COVID-19 requiring intensive care, a poor antibody response is associated with organ failure, systemic histone release and increased 30

Coronavirus disease 19 (COVID-19) presents with disease severities of varying degree. In its most severe form, infection may lead to respiratory failure and multi-organ dysfunction. Here we study the levels and evolution of the damage associated molecular patterns (DAMPS) cell free DNA (cfDNA), extracellular histone H3 (H3) and neutrophil elastase (NE), and the immune modulators GAS6 and AXL in relation to clinical parameters, ICU scoring systems and mortality in patients (n = 100) with severe COVID-19. cfDNA, H3, NE, GAS6 and AXL were increased in COVID-19 patients compared to controls. These measures associated with occurrence of clinical events and intensive care unit acquired weakness (ICUAW). cfDNA and GAS6 decreased in time in patients surviving to 30 days post ICU admission. A decrease of 27.2 ng/mL cfDNA during ICU stay associated with patient survival, whereas levels of GAS6 decreasing more than 4.0 ng/mL associated with survival. The presence of H3 in plasma was a common feature of COVID-19 patients, detected in 38% of the patients at ICU admission. NETosis markers cfDNA, H3 and NE correlated well with parameters of tissue damage and neutrophil counts. Furthermore, cfDNA correlated with lowest p/f ratio and a lowering in cfDNA was observed in patients with ventilator-free days.

Background Coronavirus disease 19 (COVID-19) is known to present with disease severities of varying degree. In its most severe form, infection may lead to respiratory failure and multi-organ dysfunction. Here we study the levels of extracellular histone H3 (H3), neutrophil elastase (NE) and cfDNA in relation to other plasma parameters, including the immune modulators GAS6 and AXL, ICU scoring systems and mortality in patients with severe COVID-19. Methods We measured plasma H3, NE, cfDNA, GAS6 and AXL concentration in plasma of 83 COVID-19-positive and 11 COVID-19-negative patients at admission to the Intensive Care Unit (ICU) at the Uppsala University hospital, a tertiary hospital in Sweden and a total of 333 samples obtained from these patients during the ICU-stay. We determined their correlation with disease severity, organ failure, mortality and other blood parameters. Results H3, NE, cfDNA, GAS6 and AXL were increased in plasma of COVID-19 patients compared to controls. cfDNA and GAS6 decreased in time in in patients surviving to 30 days post ICU admission. Plasma H3 was a common feature of COVID-19 patients, detected in 40% of the patients at ICU admission. Although these measures were not predictive of the final outcome of the disease, they correlated well with parameters of tissue damage (H3 and cfDNA) and neutrophil counts (NE). A subset of samples displayed H3 processing, possibly due to proteolysis. Conclusions Elevated H3 and cfDNA levels in COVID-19 patients illustrate the severity of the cellular damage observed in critically ill COVID-19 patients. The increase in NE indicates the important role of neutrophil response and the process of NETosis in the disease. GAS6 appears as part of an early activated mechanism of response in Covid-19.

Viruses are one of the major causes of acute and chronic infectious diseases and thus a major contributor to the global burden of disease. Several studies have shown how viruses have evolved to hijack basic cellular pathways and evade innate immune response by modulating key host factors and signaling pathways. A collective view of these multiple studies could advance our understanding of virus-host interactions and provide new therapeutic perspectives for the treatment of viral diseases. Here, we performed an integrative meta-analysis to elucidate the 17 different host-virus interactomes. Network and bioinformatics analyses showed how viruses with small genomes efficiently achieve the maximal effect by targeting multifunctional and highly connected host proteins with a high occurrence of disordered regions. We also identified the core cellular process subnetworks that are targeted by all the viruses. Integration with functional RNA interference (RNAi) datasets showed that a large proportion of the targets are required for viral replication. Furthermore, we performed an interactome-informed drug re-purposing screen and identified novel activities for broad-spectrum antiviral agents against hepatitis C virus and human metapneumovirus. Altogether, these orthogonal datasets could serve as a platform for hypothesis generation and follow-up studies to broaden our understanding of the viral evasion landscape.

With the increasing pace of global warming, it is important to understand the role of meteorological factors in influenza virus (IV) epidemics. In this study, we investigated the impact of temperature, UV index, humidity, wind speed, atmospheric pressure, and precipitation on IV activity in Norway, Sweden, Finland, Estonia, Latvia and Lithuania during 2010-2018. Both correlation and machine learning analyses revealed that low temperature and UV indexes were the most predictive meteorological factors for IV epidemics in Northern Europe. Our in vitro experiments confirmed that low temperature and UV radiation preserved IV infectivity. Associations between these meteorological factors and IV activity could improve surveillance and promote development of accurate predictive models for future influenza outbreaks in the region.

According to the WHO, there is an urgent need for better control of viral diseases. Re-positioning existing safe-inhuman antiviral agents from one viral disease to another could play a pivotal role in this process. Here, we reviewed all approved, investigational and experimental antiviral agents, which are safe in man, and identified 59 compounds that target at least three viral diseases. We tested 55 of these compounds against eight different RNA and DNA viruses. We found novel activities for dalbavancin against echovirus 1, ezetimibe against human immunodeficiency virus 1 and Zika virus, as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against Rift valley fever virus. Thus, the spectrum of antiviral activities of existing antiviral agents could be expanded towards other viral diseases.

BACKGROUND: Asthmatic patients have higher microbiome diversity and an altered composition, with more Proteobacteria and less Bacteroidetes compared with healthy control subjects. Studies comparing airway inflammation and the airway microbiome are sparse, especially in subjects not receiving anti-inflammatory treatment.OBJECTIVE: We sought to describe the relationship between the airway microbiome and patterns of airway inflammation in steroid-free patients with asthma and healthy control subjects.METHODS: Bronchoalveolar lavage fluid was collected from 23 steroid-free nonsmoking patients with asthma and 10 healthy control subjects. Bacterial DNA was extracted from and subjected to Illumina MiSeq sequencing of the 16S rDNA V4 region. Eosinophils and neutrophils in the submucosa were quantified by means of immunohistochemical identification and computerized image analysis. Induced sputum was obtained, and airway hyperresponsiveness to mannitol and fraction of exhaled nitric oxide values were measured. Relationships between airway microbial diversity and composition and inflammatory profiles were analyzed.RESULTS: In asthmatic patients airway microbial composition was associated with airway eosinophilia and AHR to mannitol but not airway neutrophilia. The overall composition of the airway microbiome of asthmatic patients with the lowest levels of eosinophils but not asthmatic patients with the highest levels of eosinophils deviated significantly from that of healthy subjects. Asthmatic patients with the lowest levels of eosinophils had an altered bacterial abundance profile, with more Neisseria, Bacteroides, and Rothia species and less Sphingomonas, Halomonas, and Aeribacillus species compared with asthmatic patients with more eosinophils and healthy control subjects.CONCLUSION: The level of eosinophilic airway inflammation correlates with variations in the microbiome across asthmatic patients, whereas neutrophilic airway inflammation do

Development of Hepatitis C virus (HCV) resistance against direct-acting antivirals (DAAs), including NS5A inhibitors, is an obstacle to successful treatment of HCV when DAAs are used in sub-optimal combinations. Furthermore, it has been shown that baseline (pre-existing) resistance against DAAs is present in treatment naive-patients and this will potentially complicate future treatment strategies in different HCV genotypes (GTs). Thus the aim was to detect low levels of NS5A resistant associated variants (RAVs) in a limited sample set of treatment-naive patients of HCV GT1a and 3a, since such polymorphisms can display in vitro resistance as high as 60000 fold. Ultra-deep single molecule real time (SMRT) sequencing with the Pacific Biosciences (PacBio) RSII instrument was used to detect these RAVs. The SMRT sequencing was conducted on ten samples; three of them positive with Sanger sequencing (GT1a Q30H and Y93N, and GT3a Y93H), five GT1a samples, and two GT3a non-positive samples. The same methods were applied to the HCV GT1a H77-plasmid in a dilution series, in order to determine the error rates of replication, which in turn was used to determine the limit of detection (LOD), as defined by mean + 3SD, of minority variants down to 0.24%. We found important baseline NS5A RAVs at levels between 0.24 and 0.5%, which could potentially have clinical relevance. This new method with low level detection of baseline RAVs could be useful in predicting the most cost-efficient combination of DAA treatment, and reduce the treatment duration for an HCV infected individual.

Surgically removed palatine tonsils provide a conveniently accessible source of T and B lymphocytes to study the interplay between foreign pathogens and the host immune system. In this study we have characterised the distribution of human adenovirus (HAdV), Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) in purified tonsillar T and B cell-enriched fractions isolated from three patient age groups diagnosed with tonsillar hypertrophy and chronic/recurrent tonsillitis. HAdV DNA was detected in 93 out of 111 patients (84%), while EBV DNA was detected in 58 patients (52%). The most abundant adenovirus type was HAdV-5 (68%). None of the patients were positive for HCMV. Furthermore, 43 patients (39%) showed a co-infection of HAdV and EBV. The majority of young patients diagnosed with tonsillar hypertrophy were positive for HAdV, whereas all adult patients diagnosed with chronic/recurrent tonsillitis were positive for either HAdV or EBV. Most of the tonsils from patients diagnosed with either tonsillar hypertrophy or chronic/recurrent tonsillitis showed a higher HAdV DNA copy number in T compared to B cell-enriched fraction. Interestingly, in the majority of the tonsils from patients with chronic/recurrent tonsillitis HAdV DNA was detected in T cells only, whereas hypertrophic tonsils demonstrated HAdV DNA in both T and B cell-enriched fractions. In contrast, the majority of EBV positive tonsils revealed a preference for EBV DNA accumulation in the B cell-enriched fraction compared to T cell fraction irrespective of the patients' age., De 2 sista författarna delar sistaförfattarskapet.