Unraveling the Dynamics of Omicron (BA.1, BA.2, and BA.5) Waves and Emergence of the Deltacton Variant: Genomic Epidemiology of the SARS-CoV-2 Epidemic in Cyprus (Oct 2021-Oct 2022).

Commencing in December 2019 with the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), three years of the coronavirus disease 2019 (COVID-19) pandemic have transpired. The virus has consistently demonstrated a tendency for evolutionary adaptation, resulting in mutations that impact both immune evasion and transmissibility. This ongoing process has led to successive waves of infections. This study offers a comprehensive assessment spanning genetic, phylogenetic, phylodynamic, and phylogeographic dimensions, focused on the trajectory of the SARS-CoV-2 epidemic in Cyprus. Based on a dataset comprising 4700 viral genomic sequences obtained from affected individuals between October 2021 and October 2022, our analysis is presented. Over this timeframe, a total of 167 distinct lineages and sublineages emerged, including variants such as Delta and Omicron (1, 2, and 5). Notably, during the fifth wave of infections, Omicron subvariants 1 and 2 gained prominence, followed by the ascendancy of Omicron 5 in the subsequent sixth wave. Additionally, during the fifth wave (December 2021-January 2022), a unique set of Delta sequences with genetic mutations associated with Omicron variant 1, dubbed "Deltacron", was identified. The emergence of this phenomenon initially evoked skepticism, characterized by concerns primarily centered around contamination or coinfection as plausible etiological contributors. These hypotheses were predominantly disseminated through unsubstantiated assertions within the realms of social and mass media, lacking concurrent scientific evidence to validate their claims. Nevertheless, the exhaustive molecular analyses presented in this study have demonstrated that such occurrences would likely lead to a frameshift mutation-a genetic aberration conspicuously absent in our provided sequences. This substantiates the accuracy of our initial assertion while refuting contamination or coinfection as potential etiologies. Comparable observations on a global scale dispelled doubt, eventually leading to the recognition of Delta-Omicron variants by the scientific community and their subsequent monitoring by the World Health Organization (WHO). As our investigation delved deeper into the intricate dynamics of the SARS-CoV-2 epidemic in Cyprus, a discernible pattern emerged, highlighting the major role of international connections in shaping the virus's local trajectory. Notably, the United States and the United Kingdom were the central conduits governing the entry and exit of the virus to and from Cyprus. Moreover, notable migratory routes included nations such as Greece, South Korea, France, Germany, Brazil, Spain, Australia, Denmark, Sweden, and Italy. These empirical findings underscore that the spread of SARS-CoV-2 within Cyprus was markedly influenced by the influx of new, highly transmissible variants, triggering successive waves of infection. This investigation elucidates the emergence of new waves of infection subsequent to the advent of highly contagious and transmissible viral variants, notably characterized by an abundance of mutations localized within the spike protein. Notably, this discovery decisively contradicts the hitherto hypothesis of seasonal fluctuations in the virus's epidemiological dynamics. This study emphasizes the importance of meticulously examining molecular genetics alongside virus migration patterns within a specific region. Past experiences also emphasize the substantial evolutionary potential of viruses such as SARS-CoV-2, underscoring the need for sustained vigilance. However, as the pandemic's dynamics continue to evolve, a balanced approach between caution and resilience becomes paramount. This ethos encourages an approach founded on informed prudence and self-preservation, guided by public health authorities, rather than enduring apprehension. Such an approach empowers societies to adapt and progress, fostering a poised confidence rooted in well-founded adaptation.

Genomic Epidemiology of the SARS-CoV-2 Epidemic in Cyprus from November 2020 to October 2021: The Passage of Waves of Alpha and Delta Variants of Concern.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in December 2019 resulted in the coronavirus disease 2019 (COVID-19) pandemic, which has had devastating repercussions for public health. Over the course of this pandemic, the virus has continuously been evolving, resulting in new, more infectious variants that have frequently led to surges of new SARS-CoV-2 infections. In the present study, we performed detailed genetic, phylogenetic, phylodynamic and phylogeographic analyses to examine the SARS-CoV-2 epidemic in Cyprus using 2352 SARS-CoV-2 sequences from infected individuals in Cyprus during November 2020 to October 2021. During this period, a total of 61 different lineages and sublineages were identified, with most falling into three groups: B.1.258 & sublineages, Alpha (B.1.1.7 & Q. sublineages), and Delta (B.1.617.2 & AY. sublineages), each encompassing a set of S gene mutations that primarily confer increased transmissibility as well as immune evasion. Specifically, these lineages were coupled with surges of new infections in Cyprus, resulting in the following: the second wave of SARS-CoV-2 infections in Cyprus, comprising B.1.258 & sublineages, during late autumn 2020/beginning of winter 2021; the third wave, comprising Alpha (B.1.1.7 & Q. sublineages), during spring 2021; and the fourth wave, comprising Delta (B.1.617.2 & AY. sublineages) during summer 2021. Additionally, it was identified that these lineages were primarily imported from and exported to the UK, Greece, and Sweden; many other migration links were also identified, including Switzerland, Denmark, Russia, and Germany. Taken together, the results of this study indicate that the SARS-CoV-2 epidemic in Cyprus was characterized by successive introduction of new lineages from a plethora of countries, resulting in the generation of waves of infection. Overall, this study highlights the importance of investigating the spatiotemporal evolution of the SARS-CoV-2 epidemic in the context of Cyprus, as well as the impact of protective measures placed to mitigate transmission of the virus, providing necessary information to safeguard public health.

A Multiallelic Molecular Beacon-Based Real-Time RT-PCR Assay for the Detection of SARS-CoV-2.

Emerging infectious viruses have led to global advances in the development of specific and sensitive detection techniques. Viruses have an inherent potential to easily mutate, presenting major hurdles for diagnostics and requiring methods capable of detecting genetically diverse viral strains. One such infectious agent is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which emerged in December 2019 and has resulted in the global coronavirus disease 2019 (COVID-19) pandemic. This study presents a real-time reverse transcription PCR (RT-PCR) detection assay for SARS-CoV-2, taking into account its intrinsic polymorphic nature that arises due to genetic drift and recombination, as well as the possibility of continuous and multiple introductions of genetically nonidentical strains into the human population. This advance was achieved by using mismatch-tolerant molecular beacons designed to specifically detect the SARS-CoV-2 S, E, M, and N genes. These were applied to create a simple and reproducible real-time RT-PCR assay, which was validated using external quality control panels (QCMD: CVOP20, WHO: SARS-CoV-2-EQAP-01) and clinical samples. This assay was designed for high target detection accuracy and specificity and can also be readily adapted for the detection of other emerging and rapidly mutating pathogens.

A Comprehensive Molecular Epidemiological Analysis of SARS-CoV-2 Infection in Cyprus from April 2020 to January 2021: Evidence of a Highly Polyphyletic and Evolving Epidemic.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in an extraordinary global public health crisis. In early 2020, Cyprus, among other European countries, was affected by the SARS-CoV-2 epidemic and adopted lockdown measures in March 2020 to limit the initial outbreak on the island. In this study, we performed a comprehensive retrospective molecular epidemiological analysis (genetic, phylogenetic, phylodynamic and phylogeographic analyses) of SARS-CoV-2 isolates in Cyprus from April 2020 to January 2021, covering the first ten months of the SARS-CoV-2 infection epidemic on the island. The primary aim of this study was to assess the transmissibility of SARS-CoV-2 lineages in Cyprus. Whole SARS-CoV-2 genomic sequences were generated from 596 clinical samples (nasopharyngeal swabs) obtained from community-based diagnostic testing centers and hospitalized patients. The phylogenetic analyses revealed a total of 34 different lineages in Cyprus, with B.1.258, B.1.1.29, B.1.177, B.1.2, B.1 and B.1.1.7 (designated a Variant of Concern 202012/01, VOC) being the most prevalent lineages on the island during the study period. Phylodynamic analysis showed a highly dynamic epidemic of SARS-CoV-2 infection, with three consecutive surges characterized by specific lineages (B.1.1.29 from April to June 2020; B.1.258 from September 2020 to January 2021; and B.1.1.7 from December 2020 to January 2021). Genetic analysis of whole SARS-CoV-2 genomic sequences of the aforementioned lineages revealed the presence of mutations within the S protein (L18F, ΔH69/V70, S898F, ΔY144, S162G, A222V, N439K, N501Y, A570D, D614G, P681H, S982A and D1118H) that confer higher transmissibility and/or antibody escape (immune evasion) upon the virus. Phylogeographic analysis indicated that the majority of imports and exports were to and from the United Kingdom (UK), although many other regions/countries were identified (southeastern Asia, southern Europe, eastern Europe, Germany, Italy, Brazil, Chile, the USA, Denmark, the Czech Republic, Slovenia, Finland, Switzerland and Pakistan). Taken together, these findings demonstrate that the SARS-CoV-2 infection epidemic in Cyprus is being maintained by a continuous influx of lineages from many countries, resulting in the establishment of an ever-evolving and polyphyletic virus on the island.

Development of a new comprehensive HIV-1 genotypic drug resistance assay for all commercially available reverse transcriptase, protease and integrase inhibitors in patients infected with group M HIV-1 strains.

Comprehensive PCR assays for the genotypic drug resistance analysis of all HIV-1 antiretroviral agents (reverse transcriptase, protease and integrase inhibitors) are increasingly in demand due to introduction of integrase inhibitors in the first line regimens and the increasing presence of non-B HIV-1 clades around the world. This study focused on the development and evaluation of a new PCR-based assay for the amplification and sequencing of the entire HIV-1 pol region of major circulating group M HIV-1 strains in Europe for genotypic drug resistance analysis. The comprehensive touchdown PCR assay developed in this study utilized HIV-1 RNA extracted from the plasma of blood samples of consenting HIV-1 infected patients in Cyprus, collected from 2017 to 2019. The HIV-1 pol region was amplified by touchdown PCR for both the primary RT-PCR and the secondary PCR steps. Successful PCR amplicons were determined by population DNA sequencing, using the Sanger method and the genotypic drug resistance analysis was performed with the Stanford University HIV Drug Resistance Database Program. The newly developed assay successfully amplified the entire HIV-1 pol region (2844 nucleotides long) of 141 out of 144 samples of group M HIV-1 subtypes and recombinant strains of the Cyprus HIV-1 Transmission Cohort Study (CHICS) isolated from 2017 to 2019 and genotypic analyses were conducted for all currently available HIV-1 reverse transcriptase, protease and integrase inhibitors. The drug resistance, epidemiological and demographic data of these study subjects will be expanded upon in the CHICS (L.G. Kostrikis et al., manuscript in preparation for publication). The newly developed HIV-1 genotypic drug resistance assay would benefit clinical settings, and research focusing on the world-wide spread of HIV-1 drug-resistant strains, especially in geographic regions characterized by polyphyletic HIV-1 infections.

The development of inovirus-associated vector vaccines using phage-display technologies.

Introduction: Inovirus-associated vectors (IAVs) are derived from bacterial filamentous viruses (phages). As vaccine carriers, they have elicited both cellular and humoral responses against a variety of pathogens causing infectious diseases and other non-infectious diseases. By displaying specific antigen epitopes or proteins on their coat proteins, IAVs have merited much study, as their unique abilities are exploited for widespread vaccine development. Areas covered: The architectural traits of filamentous viruses and their derivatives, IAVs, facilitate the display of specific antigenic peptides which induce antibody production to prevent or curtail infection. Inoviruses provide a foundation for cost-efficient large-scale specific phage display. In this paper, the development of different applications of inovirus-based phage display vaccines across a broad range of pathogens and hosts is reviewed. The references cited in this review were selected from established databases based on the authors' knowledge of the study subject. Expert commentary: The importance of phage-display technology has been recently highlighted by the Nobel Prize in Chemistry 2018 awarded to George P. Smith and Sir Gregory P. Winter. Furthermore, the symbiotic nature of filamentous viruses infecting intestinal F+E. coli strains offers an attractive platform for the development of novel vaccines that stimulate mucosal immunity.

HCV Phylogeography of the General Population and High-Risk Groups in Cyprus Identifies the Island as a Global Sink for and Source of Infection.

Hepatitis C virus (HCV) genotype and subtype distribution differs according to geographic origin and transmission risk category. Previous molecular epidemiology studies suggest the presence of multiple subtypes among Cypriot subjects. To investigate HCV genotype- and subtype-specific dissemination patterns, origins, and transmission in Cyprus, we analyzed HCV sequences encoding partial Core-E1 and NS5B regions. Analyzed populations comprised the general population and high-risk cohorts in Cyprus and a globally sampled dataset. Maximum-likelihood phylogeny reconstruction with bootstrap evaluation, character reconstruction using parsimony, and bootstrap trees estimated by ML were performed to identify the geographic origin of HCV subtypes and statistically significant dispersal pathways among geographic regions. Phylogeographic analyses traced the origin of subtypes in the general population and among PWID in Cyprus to unique and overlapping globally distributed regions. Phylogenetic analysis in Core-E1 revealed that most sequences from incarcerated populations in Cyprus clustered with the general population and PWID. We estimate that HCV infections in Cyprus originate from multiple global sources while most HCV transmissions among incarcerated individuals occur locally. This analysis is one of a few studies tracing HCV dispersal patterns using global datasets, and these practices and findings should inform how HCV epidemics are targeted by future prevention policies.

Cervical Cancer Screening Programs in Europe: The Transition Towards HPV Vaccination and Population-Based HPV Testing.

Cervical cancer is the fourth most frequently occurring cancer in women around the world and can affect them during their reproductive years. Since the development of the Papanicolaou (Pap) test, screening has been essential in identifying cervical cancer at a treatable stage. With the identification of the human papillomavirus (HPV) as the causative agent of essentially all cervical cancer cases, HPV molecular screening tests and HPV vaccines for primary prevention against the virus have been developed. Accordingly, comparative studies were designed to assess the performance of cervical cancer screening methods in order to devise the best screening strategy possible. This review critically assesses the current cervical cancer screening methods as well as the implementation of HPV vaccination in Europe. The most recent European Guidelines and recommendations for organized population-based programs with HPV testing as the primary screening method are also presented. Lastly, the current landscape of cervical cancer screening programs is assessed for both European Union member states and some associated countries, in regard to the transition towards population-based screening programs with primary HPV testing.

HIV-1 transmission networks across Cyprus (2010-2012).

A molecular epidemiology study of HIV-1 infection was conducted in one hundred diagnosed and untreated HIV-1-infected patients in Cyprus between 2010 and 2012, representing 65.4% of all the reported HIV-1 infections in Cyprus in this three-year period, using a previously defined enrolment strategy. Eighty-two patients were newly diagnosed (genotypic drug resistance testing within six months from diagnosis), and eighteen patients were HIV-1 diagnosed for a longer period or the diagnosis date was unknown. Phylogenetic trees of the pol sequences obtained in this study with reference sequences indicated that subtypes B and A1 were the most common subtypes present and accounted for 41.0 and 19.0% respectively, followed by subtype C (7.0%), F1 (8.0%), CRF02_AG (4.0%), A2 (2.0%), other circulating recombinant forms (CRFs) (7.0%) and unknown recombinant forms (URFs) (12%). Most of the newly-diagnosed study subjects were Cypriots (63%), males (78%) with median age 39 (Interquartile Range, IQR 33-48) reporting having sex with other men (MSM) (51%). A high rate of clustered transmission of subtype B drug-sensitive strains to reverse transcriptase and protease inhibitors was observed among MSM, twenty-eight out of forty-one MSM study subjects (68.0%) infected were implicated in five transmission clusters, two of which are sub-subtype A1 and three of which are subtype B strains. The two largest MSM subtype B clusters included nine and eight Cypriot men, respectively, living in all major cities in Cyprus. There were only three newly diagnosed patients with transmitted drug resistant HIV-1 strains, one study subject from the United Kingdom infected with subtype B strain and one from Romania with sub-subtype A2 strain, both with PI drug resistance mutation M46L and one from Greece with sub-subtype A1 with non-nucleoside reverse transcriptase inhibitors (NNRTI) drug resistance mutation K103N.

HIV-1 Infection in Cyprus, the Eastern Mediterranean European Frontier: A Densely Sampled Transmission Dynamics Analysis from 1986 to 2012.

Since HIV-1 treatment is increasingly considered an effective preventionstrategy, it is important to study local HIV-1 epidemics to formulate tailored preventionpolicies. The prevalence of HIV-1 in Cyprus was historically low until 2005. To investigatethe shift in epidemiological trends, we studied the transmission dynamics of HIV-1 in Cyprususing a densely sampled Cypriot HIV-1 transmission cohort that included 85 percent ofHIV-1-infected individuals linked to clinical care between 1986 and 2012 based on detailedclinical, epidemiological, behavioral and HIV-1 genetic information. Subtyping andtransmission cluster reconstruction were performed using maximum likelihood and Bayesianmethods, and the transmission chain network was linked to the clinical, epidemiological andbehavioral data. The results reveal that for the main HIV-1 subtype A1 and B sub-epidemics,young and drug-naïve HIV-1-infected individuals in Cyprus are driving the dynamics of thelocal HIV-1 epidemic. The results of this study provide a better understanding of thedynamics of the HIV-1 infection in Cyprus, which may impact the development of preventionstrategies. Furthermore, this methodology for analyzing densely sampled transmissiondynamics is applicable to other geographic regions to implement effective HIV-1 preventionstrategies in local settings.

The global spread of HIV-1 subtype B epidemic.

Human immunodeficiency virus type 1 (HIV-1) was discovered in the early 1980s when the virus had already established a pandemic. For at least three decades the epidemic in the Western World has been dominated by subtype B infections, as part of a sub-epidemic that traveled from Africa through Haiti to United States. However, the pattern of the subsequent spread still remains poorly understood. Here we analyze a large dataset of globally representative HIV-1 subtype B strains to map their spread around the world over the last 50years and describe significant spread patterns. We show that subtype B travelled from North America to Western Europe in different occasions, while Central/Eastern Europe remained isolated for the most part of the early epidemic. Looking with more detail in European countries we see that the United Kingdom, France and Switzerland exchanged viral isolates with non-European countries than with European ones. The observed pattern is likely to mirror geopolitical landmarks in the post-World War II era, namely the rise and the fall of the Iron Curtain and the European colonialism. In conclusion, HIV-1 spread through specific migration routes which are consistent with geopolitical factors that affected human activities during the last 50years, such as migration, tourism and trade. Our findings support the argument that epidemic control policies should be global and incorporate political and socioeconomic factors.

Architectural insight into inovirus-associated vectors (IAVs) and development of IAV-based vaccines inducing humoral and cellular responses: implications in HIV-1 vaccines.

Inovirus-associated vectors (IAVs) are engineered, non-lytic, filamentous bacteriophages that are assembled primarily from thousands of copies of the major coat protein gp8 and just five copies of each of the four minor coat proteins gp3, gp6, gp7 and gp9. Inovirus display studies have shown that the architecture of inoviruses makes all coat proteins of the inoviral particle accessible to the outside. This particular feature of IAVs allows foreign antigenic peptides to be displayed on the outer surface of the virion fused to its coat proteins and for more than two decades has been exploited in many applications including antibody or peptide display libraries, drug design, and vaccine development against infectious and non-infectious diseases. As vaccine carriers, IAVs have been shown to elicit both a cellular and humoral response against various pathogens through the display of antibody epitopes on their coat proteins. Despite their high immunogenicity, the goal of developing an effective vaccine against HIV-1 has not yet materialized. One possible limitation of previous efforts was the use of broadly neutralizing antibodies, which exhibited autoreactivity properties. In the past five years, however, new, more potent broadly neutralizing antibodies that do not exhibit autoreactivity properties have been isolated from HIV-1 infected individuals, suggesting that vaccination strategies aimed at producing such broadly neutralizing antibodies may confer protection against infection. The utilization of these new, broadly neutralizing antibodies in combination with the architectural traits of IAVs have driven the current developments in the design of an inovirus-based vaccine against HIV-1. This article reviews the applications of IAVs in vaccine development, with particular emphasis on the design of inoviral-based vaccines against HIV-1.

Effect of FGF-binding protein 3 on vascular permeability.

Fibroblast growth factor-binding protein 1 (FGF-BP1 is BP1) is involved in the regulation of embryonic development, tumor growth, and angiogenesis by mobilizing endogenous FGFs from their extracellular matrix storage. Here we describe a new member of the FGF-BP family, human BP3. We show that the hBP3 protein is secreted from cells, binds to FGF2 in vitro and in intact cells, and inhibits FGF2 binding to heparin. To determine the function of hBP3 in vivo, hBP3 was transiently expressed in chicken embryos and resulted in > 50% lethality within 24 h because of vascular leakage. The onset of vascular permeability was monitored by recording the extravasation kinetics of FITC-labeled 40-kDa dextran microperfused into the vitelline vein of 3-day-old embryos. Vascular permeability increased as early as 8 h after expression of hBP3. The increased vascular permeability caused by hBP3 was prevented by treatment of embryos with PD173074, a selective FGFR kinase inhibitor. Interestingly, a C-terminal 66-amino acid fragment (C66) of hBP3, which contains the predicted FGF binding domain, still inhibited binding of FGF2 to heparin similar to full-length hBP3. However, expression of the C66 fragment did not increase vascular permeability on its own, but required the administration of exogenous FGF2 protein. We conclude that the FGF binding domain and the heparin binding domain are necessary for the hBP3 interaction with endogenous FGF and the activation of FGFR signaling in vivo.