IKKß deletion from CNS macrophages increases neuronal excitability and accelerates the onset of EAE, while from peripheral macrophages reduces disease severity.

BACKGROUND: Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease characterized by motor deficits and cognitive decline. Many immune aspects of the disease are understood through studies in the experimental autoimmune encephalomyelitis (EAE) model, including the contribution of the NF-κB transcription factor to neuroinflammation. However, the cell-specific roles of NF-κB to EAE and its cognitive comorbidities still needs further investigation. We have previously shown that the myeloid cell NF-κB plays a role in the healthy brain by exerting homeostatic regulation of neuronal excitability and synaptic plasticity and here we investigated its role in EAE. METHODS: We used constitutive MφIKKßΚΟ mice, in which depletion of IKKß, the main activating kinase of NF-κB, was global to CNS and peripheral macrophages, and ΜgΙΚΚßKO mice, in which depletion was inducible and specific to CNS macrophages by 28 days after tamoxifen administration. We subjected these mice to MOG35-55 induced EAE and cuprizone-induced demyelination. We measured pathology by immunohistochemistry, investigated molecular mechanisms by RNA sequencing analysis and studied neuronal functions by in vivo electrophysiology in awake animals. RESULTS: Global depletion of IKKß from myeloid cells in MφIKKßΚΟ mice accelerated the onset and significantly supressed chronic EAE. Knocking out IKKß only from CNS resident macrophages accelerated the onset and exacerbated chronic EAE, accompanied by earlier demyelination and immune cell infiltration but had no effect in cuprizone-induced demyelination. Peripheral T cell effector functions were not affected by myeloid cell deletion of IKKß, but CNS resident mechanisms, such as microglial activation and neuronal hyperexcitability were altered from early in EAE. Lastly, depletion of myeloid cell IKKß resulted in enhanced late long-term potentiation in EAE. CONCLUSIONS: IKKß-mediated activation of NF-κΒ in myeloid cells has opposing roles in EAE depending on the cell type and the disease stage. In CNS macrophages it is protective while in peripheral macrophages it is disease-promoting and acts mainly during chronic disease. Although clinically protective, CNS myeloid cell IKKß deletion dysregulates neuronal excitability and synaptic plasticity in EAE. These effects of IKKß on brain cognitive abilities deserve special consideration when therapeutic interventions that inhibit NF-κB are used in MS.

Influenza transmission during COVID-19 measures downscaling in Greece, August 2022: evidence for the need of continuous integrated surveillance of respiratory viruses.

After the near absence of influenza and other respiratory viruses during the first 2 years of the COVID-19 pandemic, an increased activity of mainly influenza A(H3N2) was detected at the beginning of August 2022 in Greece on three islands. Of 33 cases with respiratory symptoms testing negative for SARS-CoV-2 with rapid antigen tests, 24 were positive for influenza: 20 as A(H3N2) subtype and four as A(H1N1)pdm09 subtype. Phylogenetic analysis of selected samples from both subtypes was performed and they fell into clusters within subclades that included the 2022/23 vaccine strains. Our data suggest that influenza can be transmitted even in the presence of another highly infectious pathogen, such as SARS-CoV-2, with a similar transmission mode. We highlight the need for implementing changes in the current influenza surveillance and suggest a move from seasonal to continuous surveillance, especially in areas with a high number of tourists. Year-round surveillance would allow for a timelier start of vaccination campaigns and antiviral drugs procurement processes.

Molecular Epidemiology and Evolutionary Trajectory of Emerging Echovirus 30, Europe.

In 2018, an upsurge in echovirus 30 (E30) infections was reported in Europe. We conducted a large-scale epidemiologic and evolutionary study of 1,329 E30 strains collected in 22 countries in Europe during 2016-2018. Most E30 cases affected persons 0-4 years of age (29%) and 25-34 years of age (27%). Sequences were divided into 6 genetic clades (G1-G6). Most (53%) sequences belonged to G1, followed by G6 (23%), G2 (17%), G4 (4%), G3 (0.3%), and G5 (0.2%). Each clade encompassed unique individual recombinant forms; G1 and G4 displayed >2 unique recombinant forms. Rapid turnover of new clades and recombinant forms occurred over time. Clades G1 and G6 dominated in 2018, suggesting the E30 upsurge was caused by emergence of 2 distinct clades circulating in Europe. Investigation into the mechanisms behind the rapid turnover of E30 is crucial for clarifying the epidemiology and evolution of these enterovirus infections.

Specialist laboratory networks as preparedness and response tool - the Emerging Viral Diseases-Expert Laboratory Network and the Chikungunya outbreak, Thailand, 2019.

We illustrate the potential for specialist laboratory networks to be used as preparedness and response tool through rapid collection and sharing of data. Here, the Emerging Viral Diseases-Expert Laboratory Network (EVD-LabNet) and a laboratory assessment of chikungunya virus (CHIKV) in returning European travellers related to an ongoing outbreak in Thailand was used for this purpose. EVD-LabNet rapidly collected data on laboratory requests, diagnosed CHIKV imported cases and sequences generated, and shared among its members and with the European Centre for Disease Prevention and Control. Data across the network showed an increase in CHIKV imported cases during 1 October 2018-30 April 2019 vs the same period in 2018 (172 vs 50), particularly an increase in cases known to be related to travel to Thailand (72 vs 1). Moreover, EVD-LabNet showed that strains were imported from Thailand that cluster with strains of the ECSA-IOL E1 A226 variant emerging in Pakistan in 2016 and involved in the 2017 outbreaks in Italy. CHIKV diagnostic requests increased by 23.6% between the two periods. The impact of using EVD-LabNet or similar networks as preparedness and response tool could be improved by standardisation of the collection, quality and mining of data in routine laboratory management systems.

A Cyclic Altered Peptide Analogue Based on Myelin Basic Protein 87-99 Provides Lasting Prophylactic and Therapeutic Protection Against Acute Experimental Autoimmune Encephalomyelitis.

In this report, amide-linked cyclic peptide analogues of the 87-99 myelin basic protein (MBP) epitope, a candidate autoantigen in multiple sclerosis (MS), are tested for therapeutic efficacy in experimental autoimmune encephalomyelitis (EAE). Cyclic altered peptide analogues of MBP87-99 with substitutions at positions 91 and/or 96 were tested for protective effects when administered using prophylactic or early therapeutic protocols in MBP72-85-induced EAE in Lewis rats. The Lys91 and Pro96 of MBP87-99 are crucial T-cell receptor (TCR) anchors and participate in the formation of trimolecular complex between the TCR-antigen (peptide)-MHC (major histocompability complex) for the stimulation of encephalitogenic T cells that are necessary for EAE induction and are implicated in MS. The cyclic peptides were synthesized using Solid Phase Peptide Synthesis (SPPS) applied on the 9-fluorenylmethyloxycarboxyl/tert-butyl Fmoc/tBu methodology and combined with the 2-chlorotrityl chloride resin (CLTR-Cl). Cyclo(91-99)[Ala96]MBP87-99, cyclo(87-99)[Ala91,96]MBP87-99 and cyclo(87-99)[Arg91, Ala96]MBP87-99, but not wild-type linear MBP87-99, strongly inhibited MBP72-85-induced EAE in Lewis rats when administered using prophylactic and early therapeutic vaccination protocols. In particular, cyclo(87-99)[Arg91, Ala96]MBP87-99 was highly effective in preventing the onset and development of clinical symptoms and spinal cord pathology and providing lasting protection against EAE induction.

Laboratory Surveillance of Polio and Other Enteroviruses in High-Risk Populations and Environmental Samples.

In the context of poliomyelitis eradication, a reinforced supplementary laboratory surveillance of enteroviruses was implemented in Greece. Between 2008 and 2014, the Hellenic Polioviruses/Enteroviruses Reference Laboratory performed detailed supplementary surveillance of circulating enteroviruses among healthy individuals in high-risk population groups, among immigrants from countries in which poliovirus is endemic, and in environmental samples. In total, 722 stool samples and 179 sewage water samples were included in the study. No wild-type polioviruses were isolated during these 7 years of surveillance, although two imported vaccine polioviruses were detected. Enterovirus presence was recorded in 25.3 and 25.1% of stool and sewage water samples, respectively. Nonpolio enteroviruses isolated from stool samples belonged to species A, B, or C; coxsackievirus A24 was the most frequently identified serotype. Only enteroviruses of species B were identified in sewage water samples, including four serotypes of echoviruses and four serotypes of coxsackie B viruses. Phylogenetic analysis revealed close genetic relationships among virus isolates from sewage water samples and stool samples, which in most cases fell into the same cluster. To the best of our knowledge, this is the first study to compare enterovirus serotypes circulating in fecal specimens of healthy individuals and environmental samples, emphasizing the burden of enterovirus circulation in asymptomatic individuals at high risk. Given that Greece continues to receive a large number of short-term arrivals, students, migrants, and refugees from countries in which poliovirus is endemic, it is important to guarantee high-quality surveillance in order to maintain its polio-free status until global eradication is achieved.IMPORTANCE This article summarizes the results of supplementary poliovirus surveillance in Greece and the subsequent characterization of enteroviral circulation in human feces and the environment. The examination of stool samples from healthy refugees and other individuals in "high-risk" groups for poliovirus enables the identification of enterovirus cases and forms the basis for further investigation of the community-level risk of viral transmission. In addition, the examination of composite human fecal samples through environmental surveillance links poliovirus and nonpoliovirus isolates from unknown individuals to populations served by the sewage or wastewater system. Supplementary surveillance is necessary to comply with the prerequisites imposed by the World Health Organization for monitoring the emergence of vaccine-derived polioviruses, reemergence of wild polioviruses, or disappearance of all vaccine-related strains in order for countries such as Greece to maintain their polio-free status and contribute to global poliovirus eradication.

Challenges in antigenic characterization of circulating influenza A(H3N2) viruses during the 2011-2012 influenza season: an ongoing problem?

Genetic and antigenic characterization of 37 representative influenza A(H3N2) virus strains isolated in Greece during the 2011-2012 winter season was performed to evaluate matching of the viruses with the seasonal influenza vaccine strain A/Perth/16/2009. Hemagglutinin gene sequence analysis revealed that all Greek strains clustered within the Victoria/208 genetic clade. Furthermore, substitutions in the antigenic and glycosylation sites suggested potential antigenic drift. Our hemagglutination inhibition (HI) analysis showed that the Greek viruses were Perth/16-like; however, these viruses were characterized as Victoria/208-like when tested at the United Kingdom WHO Collaborating Centre (CC) with HI assays performed in the presence of oseltamivir, a finding consistent with the genetic characterization data. Variability in the HI test performance experienced by other European laboratories indicated that antigenic analysis of the A(H3N2) virus has limitations and, until its standardization, national influenza reference laboratories should include genetic characterization results for selection of representative viruses for detailed antigenic analysis by the WHO CCs.

Divergent and convergent synthesis of polymannosylated dibranched antigenic peptide of the immunodominant epitope MBP(83-99).

Multiple antigenic peptide (MAP) systems are dendrimeric structures bearing multiple copies of identical or different peptide epitopes, and they have been demonstrated to show enhanced immunogenicity. Herein, we report the direct (divergent) and indirect (convergent) synthesis, using contemporary synthetic approaches, of a di-branched antigenic peptide (di-BAP) containing the immunodominant epitope MBP(83-99), which is implicated in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). The direct synthesis (di-BAP 1) was performed using microwave irradiation. The indirect synthesis (di-BAP 2) was carried out performing an efficient chemoselective coupling reaction through the formation of a thioether bond. Both di-BAPs were conjugated to polysaccharide mannan since mannosylation is a promising technique to achieve modulation in immune response. The conjugation was achieved through free amino groups of both di-BAPs via the formation of Schiff bases. The mannan-conjugated di-BAPs were further evaluated in vivo in a prophylactic vaccination protocol, prior to EAE induction in Lewis rats.

Characterizing Kinetics and Avidity of SARS-CoV-2 Antibody Responses in COVID-19 Greek Patients.

In-depth understanding of the immune response provoked by SARS-CoV-2 infection is necessary, as there is a great risk of reinfection and a difficulty in achieving herd immunity due to a decline in both antibody concentration and avidity. Avidity testing, however, could overcome variability in the immune response associated with sex or clinical symptoms, and thus differentiate between recent and past infections. In this context, here, we analyzed SARS-CoV-2 antibody kinetics and avidity in Greek hospitalized (26%) and non-hospitalized (74%) COVID-19 patients (N = 71) in the course of up to 15 months after their infection to improve the accuracy of the serological diagnosis in dating the onset of the infection. The results showed that IgG-S1 levels decline significantly at four months (p = 0.0239) in both groups of patients and are higher in hospitalized ones (up to 2.1-fold, p < 0.001). Additionally, hospitalized patients' titers drop greatly and are equalized to non-hospitalized ones only at a time-point of twelve to fifteen months. Antibody levels of women in total remain more stable months after infection, compared to men. Furthermore, we examined the differential maturation of IgG avidity after SARS-CoV-2 infection, showing an incomplete maturation of avidity that results in a plateau at four months after infection. We also defined 38.2% avidity (sensitivity: 58.9%, specificity: 90.91%) as an appropriate "cut-off" that could be used to determine the stage of infection before avidity reaches a plateau.

Neuronal I kappa B kinase beta protects mice from autoimmune encephalomyelitis by mediating neuroprotective and immunosuppressive effects in the central nervous system.

Some aspects of CNS-directed autoimmunity in multiple sclerosis are modeled in mice by immunization with myelin Ags where tissue damage is driven by myelin-reactive Th1 and Th17 effector lymphocytes. Whether the CNS plays an active role in controlling such autoimmune diseases is unknown. We used mice in which IkappaB kinase beta was deleted from Ca(2+)/calmodulin-dependent kinase IIalpha-expressing neurons (nIKKbetaKO) to investigate the contribution of neuronal NF-kappaB to the development of myelin oligodendrocyte glycoprotein 35-55-induced experimental autoimmune encephalomyelitis. We show that nIKKbetaKO mice developed a severe, nonresolving disease with increased axon loss compared with controls and this was associated with significantly reduced CNS production of neuroprotective factors (vascular endothelial growth factor, CSF1-R, and FLIP) and increased production of proinflammatory cytokines (IL-6, TNF, IL-12, IL-17, and CD30L) and chemokines. The isolation of CNS-infiltrating monocytes revealed greater numbers of CD4(+) T cells, reduced numbers of NK1.1(+) cells, and a selective accumulation of Th1 cells in nIKKbetaKO CNS from early in the disease. Our results show that neurons play an important role in determining the quality and outcome of CNS immune responses, specifically that neuronal IkappaB kinase beta is required for neuroprotection, suppression of inflammation, limitation of Th1 lymphocyte accumulation, and enhancement of NK cell recruitment in experimental autoimmune encephalomyelitis-affected CNS and stress the importance of neuroprotective strategies for the treatment of multiple sclerosis.

Molecular characterization of enteroviruses among hospitalized patients in Greece, 2013-2015.

BACKGROUND: There are only sporadic data for the circulation of Enteroviruses (EVs) in Greece with previous studies reporting mainly the presence of Echoviruses (E) and Coxsackie viruses (CV) B. OBJECTIVES: We carried out a surveillance study for the molecular characterization of EVs detected in hospitalized patients throughout Greece as well as a phylogenetic analysis of the most frequently encountered serotypes. STUDY DESIGN: Stools, cerebrospinal fluids, throat swabs and blood samples were collected from hospitalized patients with suspicion of EV infection. All samples were tested for EVs by rRT-PCR targeting the 5' untranslated region of EV genome. For positive samples, PCR amplification and sequencing targeting a part of VP1 region was performed. RESULTS: We examined 831 samples and 209 were positive for EVs with Enterovirus B species being the most frequently amplified. E30, CVB5 and E9 were the most frequent serotypes of Enterovirus B species, whereas CVA6 and EV-A71 the most frequent serotypes of Enterovirus A species. Evs were significantly detected more frequently in stool samples compared to other types of specimens. Phylogenetic analysis revealed that most EV-A71 strains clustered in the subgenogroups C2 whereas all the CVA6 strains belonged to sub-genotype D3. Additionally, two different lineages of E30 and three different clusters of E9 viruses circulated simultaneously in Greece. Our data indicated that most EV strains from Greece were similar to strains circulating throughout Europe during the same period. CONCLUSIONS: We provide a comprehensive picture of EVs circulating in Greece which can be helpful to interpret trends in EV diseases by associating them with circulating serotypes.

Laboratory investigation and phylogenetic analysis of an imported Middle East respiratory syndrome coronavirus case in Greece.

Rapid and reliable laboratory diagnosis of persons suspected of Middle East respiratory syndrome coronavirus (MERS-CoV) infection is important for timely implementation of infection control practices and disease management. In addition, monitoring molecular changes in the virus can help elucidate chains of transmission and identify mutations that might influence virus transmission efficiency. This was illustrated by a recent laboratory investigation we conducted on an imported MERS-CoV case in Greece. Two oropharyngeal swab specimens were collected on the 1st and 2nd day of patient hospitalization and tested using two real-time RT-PCR (rRT-PCR) assays targeting the UpE and Orf-1a regions of the MERS-CoV genome and RT-PCR and partial sequencing of RNA-dependent RNA polymerase and nucleocapsid genes. Serum specimens were also collected and serological test were performed. Results from the first swab sample were inconclusive while the second swab was strongly positive for MERS-CoV RNA by rRT-PCR and confirmed positive by RT-PCR and partial gene sequencing. Positive serologic test results further confirmed MERS-CoV infection. Full-length nucleocapsid and spike gene coding sequences were later obtained from the positive swab sample. Phylogenetic analysis revealed that the virus was closely related to recent human-derived MERS-CoV strains obtained in Jeddah and Makkah, Saudi Arabia, in April 2014 and dromedary camels in Saudi Arabia and Qatar. These findings were consistent with the patient's history. We also identified a unique amino acid substitution in the spike receptor binding domain that may have implications for receptor binding efficiency. Our initial inconclusive rRT-PCR results highlight the importance of collecting multiple specimens from suspect MERS-CoV cases and particularly specimens from the lower respiratory tract.

Echovirus 30 outbreak associated with a high meningitis attack rate in Thrace, Greece.

Between late May and July 2012, 105 children (62 boys) originating from 2 cities of Thrace were examined because of fever, headache and abdominal pain. Thirty-three of them were hospitalized. They had normal hemograms, and mild to moderate cerebrospinal fluid pleocytosis. Echovirus 30 was isolated from fecal and cerebrospinal fluid samples. Among confirmed cases of echoviral illness, the meningitis attack rate was 51.9%.