Deciphering the Role of Epstein-Barr Virus Latent Membrane Protein 1 in Immune Modulation: A Multifaced Signalling Perspective.

The disruption of antiviral sensors and the evasion of immune defences by various tactics are hallmarks of EBV infection. One of the EBV latent gene products, LMP1, was shown to induce the activation of signalling pathways, such as NF-κB, MAPK (JNK, ERK1/2, p38), JAK/STAT and PI3K/Akt, via three subdomains of its C-terminal domain, regulating the expression of several cytokines responsible for modulation of the immune response and therefore promoting viral persistence. The aim of this review is to summarise the current knowledge on the EBV-mediated induction of immunomodulatory molecules by the activation of signal transduction pathways with a particular focus on LMP1-mediated mechanisms. A more detailed understanding of the cytokine biology molecular landscape in EBV infections could contribute to the more complete understanding of diseases associated with this virus.

Antiviral treatment significantly reduces the levels of CXCL9, CXCL10 and CXCL11 in chronic hepatitis C.

In this study, we aimed to elucidate the changes in the immune response during antiviral treatment of patients with chronic hepatitis C, with an emphasis on the chemokine dynamics and their association with liver fibrosis. Serum concentrations of 12 chemokines. (CCL2, CCL3, CCL4, CCL11, CCL17, CCL20, CXCL1, CXCL5, CXCL8, CXCL9, CXCL10 and CXCL11) were measured in 32 patients with chronic hepatitis C before direct-acting antiviral treatment and after sustained virological response using bead-based flow cytometry. Chemokine levels were also measured in 14 sex- and age-matched healthy individuals. Concentrations of CXCL9, CXCL10, CXCL11 and CCL20 were significantly higher in chronic hepatitis C patients before direct-acting antiviral treatment compared to healthy individuals. We also observed a significant reduction in CXCL9, CXCL10 and CXCL11 levels after sustained virological response. Furthermore, we demonstrated a strong positive correlation between CXCL9, CXCL10 and CXCL11 levels before antiviral treatment. When considering liver fibrosis, we found significantly higher levels of CXCL10 and lower levels of CCL17 and CXCL5 in pre-treatment patients with severe fibrosis. None of the analysed chemokines were able to predict METAVIR fibrosis score reduction after sustained virological response. The results of this study emphasize the importance of proinflammatory pathways in liver fibrosis immunopathology during chronic hepatitis C. Finally, our results also characterized CXCL10 as the chemokine which most accurately distinguished pre-treatment CHC patients and healthy individuals.

Characterization of Human Immunodeficiency Virus-1 Transmission Clusters and Transmitted Drug-Resistant Mutations in Croatia from 2019 to 2022.

Molecular epidemiology of HIV-1 infection is challenging due to the highly diverse HIV-genome. We investigated the genetic diversity and prevalence of transmitted drug resistance (TDR) followed by phylogenetic analysis in 270 HIV-1 infected, treatment-naïve individuals from Croatia in the period 2019-2022. The results of this research confirmed a high overall prevalence of TDR of 16.7%. Resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside RTIs (NNRTIs), and protease inhibitors (PIs) was found in 9.6%, 7.4%, and 1.5% of persons, respectively. No resistance to integrase strand-transfer inhibitors (INSTIs) was found. Phylogenetic analysis revealed that 173/229 sequences (75.5%) were part of transmission clusters, and the largest identified was T215S, consisting of 45 sequences. Forward transmission was confirmed in several clusters. We compared deep sequencing (DS) with Sanger sequencing (SS) on 60 randomly selected samples and identified additional surveillance drug resistance mutations (SDRMs) in 49 of them. Our data highlight the need for baseline resistance testing in treatment-naïve persons. Although no major INSTIs were found, monitoring of SDRMs to INSTIs should be continued due to the extensive use of first- and second-generation INSTIs.

Hantavirus Pulmonary Syndrome Caused by Puumala Orthohantavirus-A Case Report and Literature Review.

In this article, we report on a rare case of acute respiratory distress syndrome (ARDS) caused by the Puumala orthohantavirus (PUUV), which is typically associated with hemorrhagic fever with renal syndrome (HFRS). This is the first documented case of PUUV-associated ARDS in Southeast Europe. The diagnosis was confirmed by serum RT-PCR and serology and corroborated by phylogenetic analysis and chemokine profiling. The patient was a 23-year-old male from Zagreb, Croatia, who had recently traveled throughout Europe. He presented with fever, headache, abdominal pain, and sudden onset of ARDS. Treatment involved high-flow nasal cannula oxygen therapy and glucocorticoids, which resulted in a full recovery. A systematic literature review identified 10 cases of hantavirus pulmonary syndrome (HPS) caused by PUUV in various European countries and Turkey between 2002 and 2023. The median age of patients was 53 years (range 24-73), and six of the patients were male. Most patients were treated in intensive care units, but none received antiviral therapy targeting PUUV. Eight patients survived hospitalization. The presented case highlights the importance of considering HPS in the differential diagnosis of ARDS, even in areas where HFRS is the dominant form of hantavirus infection.

Distribution of Epstein-Barr Virus LMP1 Variants in Patients with Infectious Mononucleosis and Association with Selected Biochemical and Hematological Parameters.

The molecular diversity of Epstein-Barr virus (EBV) is exceptionally complex and based on the characterization of sequences coding for several viral genes. The aim of this study was to analyze the distribution of EBV types 1 and 2 and to characterize LMP1 variants in a cohort of 73 patients with infectious mononucleosis (IM), as well as to investigate a possible association between viral diversity and relevant clinical parameters. Population-based sequencing of EBNA-2 gene showed the presence of EBV type 1 in all IM patients. Analysis of LMP1 gene found a restricted repertoire of LMP1 variants with the predominance of wild-type B95-8, China1, Mediterranean and North Carolina variants with the presence of more than one LMP1 variant in 16.4% of patients. Co-infections with different LMP1 variants were associated with significantly higher levels of C-reactive protein and lower levels of maximal neutrophil counts and minimal platelet count. The results of this study have shown a narrow repertoire of LMP1 variants and an exclusive presence of EBV type 1 in a cohort of IM from Croatia, suggesting a characteristic local molecular pattern of this virus. The clinical importance of distinct immunobiological features of IM patients with LMP1 variant co-infections needs to be investigated further.

Molecular Characterisation of Epstein-Barr Virus in Classical Hodgkin Lymphoma.

Hodgkin lymphomas (HLs) are a heterogeneous group of lymphoid neoplasia associated with Epstein-Barr virus (EBV) infection. EBV, considered to be an important etiological co-factor in approximately 1% of human malignancies, can be classified into two genotypes based on EBNA-2, EBNA-3A and EBNA-3C sequences, and into genetic variants based on the sequence variation of the gene coding for the LMP1 protein. Here, we present the results on the distribution of EBV genotypes 1 and 2 as well as LMP1 gene variants in 50 patients with EBV-positive classical HL selected from a cohort of 289 histologically verified cases collected over a 9-year period in a tertiary clinical center in the Southeast of Europe. The population-based sequencing of the EBNA-3C gene showed the exclusive presence of EBV genotype 1 in all cHL samples. The analysis of EBV LMP1 variant distribution showed a predominance of the wild-type strain B95-8 and the Mediterranean subtype with 30 bp deletion. These findings could contribute to the understanding of EBV immunobiology in cHL as well as to the development of a prophylactic and therapeutic vaccine.

Distinct Expression Patterns of Genes Coding for Biological Response Modifiers Involved in Inflammatory Responses and Development of Fibrosis in Chronic Hepatitis C: Upregulation of SMAD-6 and MMP-8 and Downregulation of CAV-1, CTGF, CEBPB, PLG, TIMP-3, MMP-1, ITGA-1, ITGA-2 and LOX.

Background and Objectives: The aim of this study was to analyze the expression of genes on transcriptomic levels involved in inflammatory immune responses and the development of fibrosis in patients with chronic hepatitis C. Materials and Methods: Expression patterns of 84 selected genes were analyzed with real-time quantitative RT PCR arrays in the peripheral blood of treatment-naive patients with chronic hepatitis C and healthy controls. The panel included pro- and anti-fibrotic genes, genes coding for extracellular matrix (EMC) structural constituents and remodeling enzymes, cell adhesion molecules, inflammatory cytokines, chemokines and growth factors, signal transduction members of the transforming growth factor- beta (TGF-ß) superfamily, transcription factors, and genes involved in epithelial to mesenchymal transition. Results: The expression of SMAD-6 coding for a signal transduction TGF-beta superfamily member as well as MMP-8 coding for an ECM protein were significantly increased in CHC patients compared with controls. Conclusions: Chronic hepatitis C was also characterized by a significant downregulation of a set of genes including CAV-1, CTGF, TIMP-3, MMP-1, ITGA-1, LOX, ITGA-2, PLG and CEBPB encoding various biological response modifiers and transcription factors. Our results suggest that chronic hepatitis C is associated with distinct patterns of gene expression modulation in pathways associated with the regulation of immune responses and development of fibrosis.

The Effect of Treatment-Induced Viral Eradication on Cytokine and Growth Factor Expression in Chronic Hepatitis C.

In this study, we evaluated the effect of hepatitis C virus eradication using direct-acting antivirals (DAA) on the serum cytokine and growth factor profiles of chronic hepatitis C patients (CHC). Serum concentrations of 12 cytokines and 13 growth factors were measured in 56 patients with CHC before, during the DAA treatment and after sustained virological response using bead-based flow cytometry. Cytokine and growth factor levels were also measured in 15 healthy individuals. The majority of the selected cytokines and growth factors exhibited similar concentrations before, during and after successful DAA treatment, the exceptions being IL-10, EGF, HGF and VEGF. Significantly lower concentrations of IL-10, IL-13, IL-4, IL-4, IL-9, TNF- α and higher levels of Ang-2, HGF and SCF were observed in patients with CHC before and after DAA treatment compared with healthy individuals. Patients with severe fibrosis stages exhibited higher levels of Ang-2 and lower levels of EGF, PDGF-AA and VEGF. Furthermore, IL-4, IL-5 and SCF were characterized as potential biomarkers of DAA treatment using random forest. Additionally, logistic regression characterized EGF as a potential biomarker of severe CHC. Our results suggest inhibition of pro-inflammatory processes and promotion of liver regeneration in CHC patients during DAA treatment.

Molecular Epidemiology and Baseline Resistance of Hepatitis C Virus to Direct Acting Antivirals in Croatia.

Molecular epidemiology of hepatitis C virus (HCV) is exceptionally complex due to the highly diverse HCV genome. Genetic diversity, transmission dynamics, and epidemic history of the most common HCV genotypes were inferred by population sequencing of the HCV NS3, NS5A, and NS5B region followed by phylogenetic and phylodynamic analysis. The results of this research suggest high overall prevalence of baseline NS3 resistance associate substitutions (RAS) (33.0%), moderate prevalence of NS5A RAS (13.7%), and low prevalence of nucleoside inhibitor NS5B RAS (8.3%). Prevalence of RAS significantly differed according to HCV genotype, with the highest prevalence of baseline resistance to NS3 inhibitors and NS5A inhibitors observed in HCV subtype 1a (68.8%) and subtype 1b (21.3%), respectively. Phylogenetic tree reconstructions showed two distinct clades within the subtype 1a, clade I (62.4%) and clade II (37.6%). NS3 RAS were preferentially associated with clade I. Phylogenetic analysis demonstrated that 27 (9.0%) HCV sequences had a presumed epidemiological link with another sequence and classified into 13 transmission pairs or clusters which were predominantly comprised of subtype 3a viruses and commonly detected among intravenous drug users (IDU). Phylodynamic analyses highlighted an exponential increase in subtype 1a and 3a effective population size in the late 20th century, which is a period associated with an explosive increase in the number of IDU in Croatia.

Distinct Cytokine Profiles in Severe COVID-19 and Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is identified as a risk factor for developing severe COVID-19. While NAFLD is associated with chronic low-grade inflammation, mechanisms leading to immune system hyperactivation remain unclear. The aim of this prospective observational study is to analyze cytokine profiles in patients with severe COVID-19 and NAFLD. A total of 94 patients with severe COVID-19 were included. Upon admission, clinical and laboratory data were collected, a liver ultrasound was performed to determine the presence of steatosis, and subsequently, 51 were diagnosed with NAFLD according to the current guidelines. There were no differences in age, sex, comorbidities, and baseline disease severity between the groups. Serum cytokine concentrations were analyzed using a multiplex bead-based assay by flow cytometry. Upon admission, the NAFLD group had higher C-reactive protein, procalcitonin, alanine aminotransferase, lactate dehydrogenase, and fibrinogen. Interleukins-6, -8, and -10 and CXCL10 were significantly higher, while IFN-γ was lower in NAFLD patients. Patients with NAFLD who progressed to critical illness had higher concentrations of IL-6, -8, -10, and IFN-ß, and IL-8 and IL-10 appear to be effective prognostic biomarkers associated with time to recovery. In conclusion, NAFLD is associated with distinct cytokine profiles in COVID-19, possibly associated with disease severity and adverse outcomes.

A Glimpse on the Evolution of RNA Viruses: Implications and Lessons from SARS-CoV-2.

RNA viruses are characterised by extremely high genetic variability due to fast replication, large population size, low fidelity, and (usually) a lack of proofreading mechanisms of RNA polymerases leading to high mutation rates. Furthermore, viral recombination and reassortment may act as a significant evolutionary force among viruses contributing to greater genetic diversity than obtainable by mutation alone. The above-mentioned properties allow for the rapid evolution of RNA viruses, which may result in difficulties in viral eradication, changes in virulence and pathogenicity, and lead to events such as cross-species transmissions, which are matters of great interest in the light of current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemics. In this review, we aim to explore the molecular mechanisms of the variability of viral RNA genomes, emphasising the evolutionary trajectory of SARS-CoV-2 and its variants. Furthermore, the causes and consequences of coronavirus variation are explored, along with theories on the origin of human coronaviruses and features of emergent RNA viruses in general. Finally, we summarise the current knowledge on the circulating variants of concern and highlight the many unknowns regarding SARS-CoV-2 pathogenesis.

Frequency of baseline NS5A resistance-associated substitutions in patients infected with genotype 1 of hepatitis C virus in Croatia.

The backbone of current treatment for chronic Hepatitis C virus (HCV) infection are direct-acting antivirals targeting viral nonstructural proteins (NS3, NS4A, NS5A, NS5B). To date, there are six NS5A inhibitors approved for treatment of chronic HCV infection. The presence of drug-associated resistance substitutions is mainly due to fast error-prone replication, showing differential frequency between genotypes and subtypes. The aim of this study was to determine the frequency of baseline resistance to NS5A protein inhibitors in patients with genotype 1 HCV in Croatia. Resistance-associated substitutions (RAS) were detected by Sanger sequencing of HCV NS5A region amplified from 84 patients followed by phylogenetic analysis and analysis with Geno2Pheno algorithm. The frequency of NS5A RAS was 14.3% and highly dependent on viral subtype. The overall frequency of NS5A RAS was higher in patients infected with HCV subtype 1b (24.2%) than in those infected with HCV subtype 1a (7.8%). Overall, three resistance-conferring mutations were detected (Q30R, M28T and Y93H) along with two mutations (M28V and L31I) that cause reduced susceptibility to NS5A inhibitors. Analysis of the sequences showed two distinct subtype 1a clades with RAS detected in 4.3% (1/23) clade I and 10.7% (3/28) clade II sequences. Only a few distinct NS5A RAS were detected suggesting a high degree of homogeneity of the viral population. High frequency of clinically relevant NS5A RAS in Croatia suggest that the analysis of frequency and patterns of resistance mutations in local populations and evaluation of their possible clinical impact could be beneficial.