Molecular Genotyping of Circulating Enterovirus in the Lazio Region from 2012 to 2023.

Enteroviruses (EVs) are ubiquitous viruses that circulate worldwide, causing sporadic or epidemic infections, typically during the summer and fall. They cause a broad spectrum of illnesses, ranging from an unspecified febrile clinical presentation to a severe illness. EVs are recognized to be the most frequent etiological agents of aseptic meningitis in children. However, as the infection is usually mild and self-limiting, it remains underestimated, and the epidemiology of EVs is poorly understood. To date, no vaccine or effective therapy for all types of enteroviruses is available, and EVs constitute a public health concern. Here, we investigated the molecular epidemiology of EV strains circulating in the Lazio region over a 10-year time span (2012-2023) by using a sequence-typing approach and phylogenetic analysis. The epidemiological trend of EV infection has undergone changes during the SARS-CoV-2 pandemic (2020-2021), which resulted in a modification in terms of the number of diagnosed cases and seasonality. From 2022, the circulation of EVs showed a behavior typical of the pre-pandemic period, although changes in predominantly circulating strains have been noted. Both epidemic and sporadic circulation events have been characterized in the Lazio region. Further analyses are needed to better characterize any strain with higher potential pathogenic power and to identify possible recombinant strains.

Molnupiravir increases SARS-CoV-2 genome diversity and complexity: A case-control cohort study.

Molnupiravir, an oral direct-acting antiviral effective in vitro against SARS-CoV-2, has been largely employed during the COVID-19 pandemic, since December 2021. After marketing and widespread usage, a progressive increase in SARS-CoV-2 lineages characterized by a higher transition/transversion ratio, a characteristic signature of molnupiravir action, appeared in the Global Initiative on Sharing All Influenza Data (GISAID) and International Nucleotide Sequence Database Collaboration (INSDC) databases. Here, we assessed the drug effects by SARS-CoV-2 whole-genome sequencing on 38 molnupiravir-treated persistently positive COVID-19 outpatients tested before and after treatment. Seventeen tixagevimab/cilgavimab-treated outpatients served as controls. Mutational analyses confirmed that SARS-CoV-2 exhibits an increased transition/transversion ratio seven days after initiation of molnupiravir. Moreover we observed an increased G->A ratio compared to controls, which was not related to apolipoprotein B mRNAediting enzyme, catalytic polypeptide-like (APOBEC) activity. In addition, we demonstrated for the first time an increased diversity and complexity of the viral quasispecies.

Enterovirus and Paraechovirus Meningitis in Neonates: Which Is the Difference?

Enterovirus (EV) and parechovirus (HPeV) are common viruses in the neonatal period, with similar seasonality and symptomatology. They also are the main causes of aseptic meningitis in newborns and children under 1 year of age. We compared the clinical signs, laboratory data, brain, and neurodevelopmental outcome of 10 infants with HPeV and 8 with EV meningitis. In patients with EV meningitis, serum C-reactive protein (CRP) values were significantly higher than those of patients with HPeV infection. Procalcitonin values were low in both groups. White blood cell (WBC) and lymphocyte values were significantly higher in EV patients. None of the infants had a brain lesion on cerebral ultrasound neither negative neurological outcome. Based solely on symptoms, it is not possible to distinguish HPeV from EV infection. C-reactive protein, WBC, and lymphocyte values might allow the physician to assume EV infection. The gold standard test for diagnosis remains real-time polymerase chain reaction on cerebral spinal fluid.

Concomitant Syndromic Diagnosis of Mpox and Other Vesicular Viruses in Patients with Skin and Genital Lesions.

The recent multi-country outbreak of the zoonotic monkeypox virus (MPXV) infection in humans without an epidemiological link with endemic areas has raised concerns about the route of transmission. Since the infection spread largely among men who have sex with men who, in most cases, presented primary lesions of the genital and oral mucosa, sexual transmission has been proposed. In the present study, we retrospectively evaluated specimens of vesicular lesions collected from the skin and genital tract of 35 patients (23 positive and 12 negative) presenting at our Institute for monkeypox (mpox) diagnosis by using a novel molecular syndromic vesicular virus panel (VVP) assay. All MPXV-positive samples but one was confirmed; however, the viral syndromic analysis revealed that 8.6% of them were coinfected with one or more viruses, and 17% had at least a virus different from the MPXV. The percentage of coinfections increased to more than 25% when nonviral pathogens, such as gonorrhea and syphilis, were also considered. These results show the usefulness of syndromic diagnosis in cases where MPXV is suspected (and vice versa) and at the same time highlight that the broader screening of sexually transmitted infections in the population with high-risk sexual behavior is critical to ensure a complete etiology and appropriate treatment.

Mpox as AIDS-defining event with a severe and protracted course: clinical, immunological, and virological implications.

A 59-year-old treatment-naive patient with advanced HIV infection presented with a severe and protracted course of mpox (formerly known as monkeypox) that did not respond to the current mpox treatment options. The patient worsened clinically, and developed new mucocutaneous lesions and necrotic evolution of pre-existing ones, along with multiple bilateral lung nodules and the appearance of a tracheal necrotic lesion. Although severe forms of mpox have been observed in people with severe immune system deficiency, including those with advanced HIV presentation, the immunological mechanisms underlying this observation have not yet been fully explained. To our knowledge, this is the first account of a necrotising mpox in a person living with HIV, with viral shedding for more than 11 months and a comprehensive immunological description. Moreover, we documented the virus' persistence by detecting mpox virus DNA from multiple sites and quantified anti-monkeypox virus IgA, IgM, IgG, and neutralising antibodies in serum samples. The severe HIV-driven immune depression and the presence of other co-infections might skew and impair immune responses, thus contributing to the persistence of monkeypox virus infection. Further investigations of immune responses to monkeypox virus infection in people with severe immunosuppression are required to improve management and prevention.

Outbreaks of autochthonous Dengue in Lazio region, Italy, August to September 2023: preliminary investigation.

Between August and September 2023, three distinct autochthonous dengue virus transmission events occurred in Lazio, Italy, with the main event in Rome. The events involved three different dengue serotypes. No link with previous imported cases was identified. Here we describe the epidemiological and phylogenetic analysis of the first autochthonous cases and the implemented control actions. The multiple transmission events call for a strengthening of the vector control strategies and future research to better characterise the risk in countries like Italy.

Genomic and Epidemiologic Surveillance of SARS-CoV-2 in the Pandemic Period: Sequencing Network of the Lazio Region, Italy.

Since the beginning of the COVID-19 pandemic, large-scale genomic sequencing has immediately pointed out that SARS-CoV-2 has rapidly mutated during the course of the pandemic, resulting in the emergence of variants with a public health impact. In this context, strictly monitoring the circulating strains via NGS has proven to be crucial for the early identification of new emerging variants and the study of the genomic evolution and transmission of SARS-CoV-2. Following national and international guidelines, the Lazio region has created a sequencing laboratory network (WGSnet-Lazio) that works in synergy with the reference center for epidemiological surveillance (SERESMI) to monitor the circulation of SARS-CoV-2. Sequencing was carried out with the aims of characterizing outbreak transmission dynamics, performing the genomic analysis of viruses infecting specific categories of patients (i.e., immune-depressed, travelers, and people with severe symptoms) and randomly monitoring variant circulation. Here we report data emerging from sequencing activities carried out by WGSnet-Lazio (from February 2020 to October 2022) linked with epidemiological data to correlate the circulation of variants with the clinical and demographic characteristics of patients. The model of the sequencing network developed in the Lazio region proved to be a useful tool for SARS-CoV-2 surveillance and to support public health measures for epidemic containment.

Tracking the Selective Pressure Profile and Gene Flow of SARS-CoV-2 Delta Variant in Italy from April to October 2021 and Frequencies of Key Mutations from Three Representative Italian Regions.

The SARS-CoV-2 Delta variant of concern (VOC) was often associated with serious clinical course of the COVID-19 disease. Herein, we investigated the selective pressure, gene flow and evaluation on the frequencies of mutations causing amino acid substitutions in the Delta variant in three Italian regions. A total of 1500 SARS-CoV-2 Delta genomes, collected in Italy from April to October 2021 were investigated, including a subset of 596 from three Italian regions. The selective pressure and the frequency of amino acid substitutions and the prediction of their possible impact on the stability of the proteins were investigated. Delta variant dataset, in this study, identified 68 sites under positive selection: 16 in the spike (23.5%), 11 in nsp2 (16.2%) and 10 in nsp12 (14.7%) genes. Three of the positive sites in the spike were located in the receptor-binding domain (RBD). In Delta genomes from the three regions, 6 changes were identified as very common (>83.7%), 4 as common (>64.0%), 21 at low frequency (2.1%-25.0%) and 29 rare (≤2.0%). The detection of positive selection on key mutations may represent a model to identify recurrent signature mutations of the virus.

Treatment-Emergent Cilgavimab Resistance Was Uncommon in Vaccinated Omicron BA.4/5 Outpatients.

Mutations in the SARS-CoV-2 Spike glycoprotein can affect monoclonal antibody efficacy. Recent findings report the occurrence of resistant mutations in immunocompromised patients after tixagevimab/cilgavimab treatment. More recently, the Food and Drug Agency revoked the authorization for tixagevimab/cilgavimab, while this monoclonal antibody cocktail is currently recommended by the European Medical Agency. We retrospectively reviewed 22 immunocompetent patients at high risk for disease progression who received intramuscular tixagevimab/cilgavimab as early COVID-19 treatment and presented a prolonged high viral load. Complete SARS-CoV-2 genome sequences were obtained for a deep investigation of mutation frequencies in Spike protein before and during treatment. At seven days, only one patient showed evidence of treatment-emergent cilgavimab resistance. Quasispecies analysis revealed two different deletions on the Spike protein (S:del138-144 or S:del141-145) in combination with the resistance S:K444N mutation. The structural and dynamic impact of the two quasispecies was characterized by using molecular dynamics simulations, showing the conservation of the principal functional movements in the mutated systems and their capabilities to alter the structure and dynamics of the RBD, responsible for the interaction with the ACE2 human receptor. Our study underlines the importance of prompting an early virological investigation to prevent drug resistance or clinical failures in immunocompetent patients.

Detection of SARS-CoV-2 Variants via Different Diagnostics Assays Based on Single-Nucleotide Polymorphism Analysis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by fast evolution with the appearance of several variants. Next-Generation Sequencing (NGS) technology is considered the gold standard for monitoring known and new SARS-CoV-2 variants. However, the complexity of this technology renders this approach impracticable in laboratories located in areas with limited resources. We analyzed the capability of the ThermoFisher TaqPath COVID-19 RT-PCR (TaqPath) and the Seegene Novaplex SARS-CoV-2 Variant assay (Novaplex) to detect Omicron variants; the Allplex VariantII (Allplex) was also evaluated for Delta variants. Sanger sequencing (SaS) was the reference method. The results obtained with n = 355 nasopharyngeal samples were: negative with TaqPath, although positive with other qualitative molecular assays (n = 35); undetermined (n = 40) with both the assays; negative for the ∆69/70 mutation and confirmed as the Delta variant via SaS (n = 100); positive for ∆69/70 and confirmed as Omicron BA.1 via SaS (n = 80); negative for ∆69/70 and typed as Omicron BA.2 via SaS (n = 80). Novaplex typed 27.5% of samples as undetermined with TaqPath, 11.4% of samples as negative with TaqPath, and confirmed 100% of samples were Omicron subtypes. In total, 99/100 samples were confirmed as the Delta variant with Allplex with a positive per cent agreement (PPA) of 98% compared to SaS. As undermined samples with Novaplex showed RdRp median Ct values (Ct = 35.4) statistically higher than those of typed samples (median Ct value = 22.0; p < 0.0001, Mann-Whitney test), the inability to establish SARS-CoV-2 variants was probably linked to the low viral load. No amplification was obtained with SaS among all 35 negative TaqPath samples. Overall, 20% of samples which were typed as negative or undetermined with TaqPath, and among them, twelve were not typed even by SaS, but they were instead correctly identified with Novaplex. Although full-genome sequencing remains the elected method to characterize new strains, our data show the high ability of a SNP-based assay to identify VOCs, also resolving samples typed as undetermined with TaqPath.

Temporal intra-host variability of mpox virus genomes in multiple body tissues.

Whole-genome sequencing (WGS) has been widely used for the genomic characterization and the phylogenesis of mpox virus (MPXV) 2022 multi-country outbreak. To date, no evidence has been reported on intra-host evolution within samples collected over time from a single patient with long-term infection. Fifty-one samples were collected from five patients at different time points post-symptom onset. All samples were confirmed as MPXV DNA positive, amplified by a multiplexed PCR amplicon, and sequenced by WGS. Complete MPXV genomes were assembled by reference mapping and then aligned to perform phylogenetic and hierarchical clustering analysis. Large intra-host variability was observed among the MPXV genomes sequenced from samples of two immunocompromised with advanced HIV-1 infection patients with prolonged MPXV shedding. Overall, 20 nucleotide mutations were identified in the 32 genomes from HIV patients, differently distributed in samples collected from different tissues and at different time points. No sequence compartmentalization nor variation was observed in the three patients with rapid viral clearance. MPXV exhibits adaptation to changing environments within the infected host and consequently demonstrates tissue compartmentalization. Further studies are needed to elucidate the role of this adaptation in forming a pool of genetic variability and contributing to viral persistence and its clinical implications.

Genomic surveillance of SARS-CoV-2 positive passengers on flights from China to Italy, December 2022.

With numbers of COVID-19 cases having substantially increased at the end of 2022 in China, some countries have started or expanded testing and genomic surveillance of travellers. We report screening results in Italy in late December 2022 of 556 flight passengers in provenance from two Chinese provinces. Among these passengers, 126 (22.7%) tested SARS-CoV-2 positive. Whole genome sequencing of 61 passengers' positive samples revealed Omicron variants, notably sub-lineages BA.5.2.48, BF.7.14 and BQ.1.1, in line with data released from China.

SARS-CoV-2 Variants Identification: Overview of Molecular Existing Methods.

Since the beginning of COVID-19 pandemic the Real Time sharing of genome sequences of circulating virus supported the diagnostics and surveillance of SARS-CoV-2 and its transmission dynamics. SARS-CoV-2 straightaway showed its tendency to mutate and adapt to the host, culminating in the emergence of variants; so it immediately became of crucial importance to be able to detect them quickly but also to be able to monitor in depth the changes on the whole genome to early identify the new possibly emerging variants. In this scenario, this manuscript aims to provide an overview of the existing methods for the identification of SARS-CoV-2 variants (from rapid method based on identification of one or more specific mutations to Whole Genome sequencing approach-WGS), taking into account limitations, advantages and applications of them in the field of diagnosis and surveillance of SARS-CoV-2.

Molecular Characterization of Whole-Genome SARS-CoV-2 from the First Suspected Cases of the XE Variant in the Lazio Region, Italy.

We report two cases of SARS-CoV-2 recombinant variant XE detected in nasopharyngeal swabs (NPS) of hospitalized patients with no evident epidemiological link in Lazio, Central Italy. Whole-Genome Sequencing (WGS) performed on an Ion Torrent GSS5 platform according to Italian flash surveys showed genomes corresponding to the PANGOLIN unclassified lineage and the Nextclade XE clade. Further analyses were then carried out to investigate more deeply the genetic characteristics of these XE-like sequences. When phylogenetic trees, by using IQ-TREE, were built splitting the genome into two regions according to the putative XE recombination site, the upstream and downstream regions were seen to be clustered near BA.1 and BA.2 sequences, respectively. However, our XE-like sequences clustered separately, with a significant bootstrap, from the classified European and Italian XE strains, although the recombination site between BA.1 and BA.2 was identified at the nucleotide site 11556 by RDP4 software, consistent with the putative XE breakpoint. These findings show the risk of the introduction of novel recombinant variants of SARS-CoV-2 and the existence of XE-like strains, phylogenetically separated, that could make their exact taxonomy difficult. It follows the need for continued SARS-CoV-2 surveillance by WGS.

Detection of recombinant breakpoint in the genome of human enterovirus E11 strain associated with a fatal nosocomial outbreak.

BACKGROUND: The aim of this study was to characterize the genome of a recombinant Enterovirus associated with severe and fatal nosocomial infection; it was typed as Echovirus 11 (E-11) according to the VP1 gene. Enterovirus infection is generally asymptomatic and self-limited, but occasionally it may progress to a more severe clinical manifestation, as in the case described here. Recombination plays a crucial role in the evolution of Enteroviruses (EVs) and has been recognized as the main driving force behind the emergence of epidemic strains associated with severe infection. Therefore, it is of utmost importance to monitor the circulation of recombinant strains for surveillance purposes. METHODS: Enterovirus-RNA was detected in the serum and liver biopsy of patients involved in the nosocomial cluster by commercial One-Step qRT-PCR method and the Enterovirus strains were isolated in vitro. The EVs typing was determined by analyzing the partial-length of the 5'UTR and VP1 sequences with the web-based open-access Enterovirus Genotyping Tool Version 0.1. The amplicons targeting 5'UTR, VP1 and overlapping fragments of the entire genome were sequenced with the Sanger method. Phylogenetic analysis was performed comparing the VP1 and the full-genome sequences of our strains against an appropriate reference set of Enterovirus prototypes of the Picornaviridae genera and species retrieved from the Enterovirus Genotyping Tool. Recombination analysis was performed using RDP4 software. RESULTS: The Neighbor-Joining tree of the VP1 gene revealed that the 4 patients were infected with an identical molecular variant of Echovirus 11 (E-11). While the phylogenetic and the RDP4 analysis of the full-genome sequences provided evidence that it was a chimeric strain between an E-11 and a Coxsackievirus B (CV-B). CONCLUSIONS: The chimeric structure of the E-11 genome might have contributed to the severe infection and epidemic feature of the strain, but further biological characterizations are needed. The evidence reported in this study, highlights the limit of typing techniques based on the VP1 gene, as they fail to identify the emergence of recombinant strains with potentially more pathogenic or epidemic properties, thus providing only partial information on the epidemiology and pathogenesis of Enteroviruses.

The Easy-to-Use SARS-CoV-2 Assembler for Genome Sequencing: Development Study.

Background: Early sequencing and quick analysis of the SARS-CoV-2 genome have contributed to the understanding of the dynamics of COVID-19 epidemics and in designing countermeasures at a global level. Objective: Amplicon-based next-generation sequencing (NGS) methods are widely used to sequence the SARS-CoV-2 genome and to identify novel variants that are emerging in rapid succession as well as harboring multiple deletions and amino acid-changing mutations. Methods: To facilitate the analysis of NGS sequencing data obtained from amplicon-based sequencing methods, here, we propose an easy-to-use SARS-CoV-2 genome assembler: the Easy-to-use SARS-CoV-2 Assembler (ESCA) pipeline. Results: Our results have shown that ESCA could perform high-quality genome assembly from Ion Torrent and Illumina raw data and help the user in easily correct low-coverage regions. Moreover, ESCA includes the possibility of comparing assembled genomes of multisample runs through an easy table format. Conclusions: In conclusion, ESCA automatically furnished a variant table output file, fundamental to rapidly recognizing variants of interest. Our pipeline could be a useful method for obtaining a complete, rapid, and accurate analysis even with minimal knowledge in bioinformatics.

Multi-omics approach to COVID-19: a domain-based literature review.

BACKGROUND: Omics data, driven by rapid advances in laboratory techniques, have been generated very quickly during the COVID-19 pandemic. Our aim is to use omics data to highlight the involvement of specific pathways, as well as that of cell types and organs, in the pathophysiology of COVID-19, and to highlight their links with clinical phenotypes of SARS-CoV-2 infection. METHODS: The analysis was based on the domain model, where for domain it is intended a conceptual repository, useful to summarize multiple biological pathways involved at different levels. The relevant domains considered in the analysis were: virus, pathways and phenotypes. An interdisciplinary expert working group was defined for each domain, to carry out an independent literature scoping review. RESULTS: The analysis revealed that dysregulated pathways of innate immune responses, (i.e., complement activation, inflammatory responses, neutrophil activation and degranulation, platelet degranulation) can affect COVID-19 progression and outcomes. These results are consistent with several clinical studies. CONCLUSIONS: Multi-omics approach may help to further investigate unknown aspects of the disease. However, the disease mechanisms are too complex to be explained by a single molecular signature and it is necessary to consider an integrated approach to identify hallmarks of severity.

Late Relapse and Reinfection in HCV Patients Treated with Direct-Acting Antiviral (DAA) Drugs.

The risk of hepatitis C virus (HCV) recurrence after direct-acting antiviral (DAA) treatment is <0.5%. However, the distinction between HCV RNA late relapse and reinfection still represents a challenge in virological diagnostics. The aim of this study was to employ next-generation sequencing (NGS) to investigate HCV RNA recurrence in patients achieving a sustained virologic response (SVR) at least six months post-treatment. NGS was performed on plasma samples from six HCV-positive patients (Pt1-6) treated with DAA. NGS of HCV NS5B was analyzed before treatment (T0), after HCV RNA rebound (T1), and, for Pt3, after a second rebound (T2). Reinfection was confirmed for Pt5, and for the first rebound observed in Pt3. Conversely, viral relapse was observed when comparing T0 and T1 for Pt6 and T1 and T2 for Pt3. Z-scores were calculated and used to predict whether HCV-positive patient samples at different time points belonged to the same quasispecies population. A low Z-score of <2.58 confirmed that viral quasispecies detected at T0 and T1 were closely related for both Pt1 and Pt2, while the Z-score for Pt4 was suggestive of possible reinfection. NGS data analyses indicate that the Z-score may be a useful parameter for distinguishing late relapse from reinfection.