Evolution of viruses and the emergence of SARS-CoV-2 variants

Life implies adaptation. This is one of the fundamental principles that has permitted most living species to survive through ages in an ever-changing environment. Spontaneously occurring events have shaped also virus populations and their fitness. Thanks to their plasticity, viruses have thrived in extremely dissimilar conditions. Unsurprisingly, SARS-CoV-2, the etiological agent of COVID-19, is no exception. Thanks to an unprecedented rate of molecular tracing and sequence scrutiny, the virus was followed in all its changes and shown to evolve in such a way as to possibly determine subsequent waves of infection after the first global and massive outbreak. This review illustrates the major modifications occurred to the virus since its discovery. We describe the potential advantages that these changes conveyed as regards SARS-CoV-2 transmissibility, resistance to host innate and adaptive barriers and molecular diagnosis

TTV and other anelloviruses: The astonishingly wide spread of a viral infection

The broad family of viruses known as anelloviruses (AV) infects both humans and numerous animal species. They have a tiny, covalently closed single-stranded DNA genome and the astonishing capacity to infect a very high percentage of healthy and ill people with chronic infections that could last a lifetime. AV, and particularly the prototype Torquetenovirus, have established a successful interaction with the host's immune system and the rate at which they replicate is a gauge to measure overall immune function, even though many aspects of their life cycle and pathogenesis are still poorly understood

Performance Assessment of the LIAISON\uae SARS-CoV-2 Antigen Assay On Nasopharyngeal Swabs

The SARS-CoV-2 pandemic is ongoing worldwide, causing prolonged pressure on molecular diagnostics. Viral antigen (Ag) assays have several advantages, ranging from lower cost to shorter turnaround time to detection. Given the rare occurrence of low-load viremia, antigen assays for SARS-CoV-2 have focused on nasopharyngeal swab and saliva as biological matrices, but their effectiveness must be validated. We assayed here the performances of the novel quantitative Liaison\uae SARS-CoV-2 Ag assay on 119 nasopharyngeal swabs and obtained results were compared with Hologic Panther and Abbott m2000 RT-qPCR. The Ag assay demonstrated a good correlation with viral load, shorter turnaround time, and favorable economics. The best performance was obtained in the acute phase of disease

A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in Vero E6 cells

: We exploited a multi-scale microscopy imaging toolbox to address some major issues related to SARS-CoV-2 interactions with host cells. Our approach harnesses both conventional and super-resolution fluorescence microscopy and easily matches the spatial scale of single-virus/cell checkpoints. After its validation through the characterization of infected cells and virus morphology, we leveraged this toolbox to reveal subtle issues related to the entry phase of SARS-CoV-2 variants in Vero E6 cells. Our results show that in Vero E6 cells the B.1.1.7 strain (aka Alpha Variant of Concern) is associated with much faster kinetics of endocytic uptake compared to its ancestor B.1.177. Given the cell-entry scenario dominated by the endosomal "late pathway", the faster internalization of B.1.1.7 could be directly related to the N501Y mutation in the S protein, which is known to strengthen the binding of Spike receptor binding domain with ACE2. Remarkably, we also directly observed the central role of clathrin as a mediator of endocytosis in the late pathway of entry. In keeping with the clathrin-mediated endocytosis, we highlighted the non-raft membrane localization of ACE2. Overall, we believe that our fluorescence microscopy-based approach represents a fertile strategy to investigate the molecular features of SARS-CoV-2 interactions with cells

Studio dell'associazione fra Torque teno virus ed esosomi circolanti: un nuovo meccanismo di persistenza e diffusione del virus nell'ospite umano

Torquetenovirus (TTV) è il prototipo di un vasto gruppo di piccoli virus privi di involucro esterno e con un genoma a singolo filamento circolare di DNA a polarità negativa. Classificato come membro del genere Alphatorquevirus all'interno della famiglia Anelloviridae, TTV costituisce la componente virale principale del viroma umano. E’ caratterizzato da un’elevata eterogeneità genetica (se ne conoscono almeno 29 specie principali) ed è capace di indurre viremia cronica nell'80% circa della popolazione generale infettando molti distretti dell’ospite. Nonostante i molti studi condotti negli anni, nessuna patologia dell’uomo è stata chiaramente correlata all’infezione da TTV e, ad oggi, il virus è considerato “orfano” di malattia. Molti aspetti del ciclo naturale di TTV nell’ospite infettato sono ancora poco conosciuti. In particolare non sono chiari i meccanismi che il virus adopera per superare le difese dell’ospite e persitere e le strategie utilizzate per diffondere cosi ampiamente da un tessuto all’altro. La recente dimostrazione che TTV codifica per sequenze di microRNA (miRNA) nella regione non tradotta (UTR) del suo genoma potrebbe spiegare alcuni meccanismi di evasione immunologica e di regolazione dell’infezione. TTV potrebbe utilizzare questi miRNA per superare le difese dell’ospite inibendo l’apoptosi cellulare e favorendo la proliferazione della cellula infettata. Studi recenti hanno dimostrato la presenza di miRNA codificati da TTV in vescicole extracellulari (VE) circolanti nel plasma con un’ampia variabilità individuale di espressione. L’uso delle VE da parte di TTV potrebbe inoltre essere un importante mezzo, ancora non dimostrato, di escape immunologico, persistenza e diffusione del virus. Tutto questo sugerisce la necessità di un'indagine più approfondita sulla possibile associazione in vivo TTV e VE. Le VE sono un gruppo eterogeneo di vescicole membranose di derivazione cellulare e, in base al meccanismo con cui si generano, vengono distinte in varie classi tra cui gli esosomi, focus di questo studio. Gli esosomi sono importanti intermediari di comunicazione intracellulare e sono coinvolti nella trasmissione di informazioni biologiche tra cellula e cellula. Un numero sempre maggiore di evidenze sperimentali mostrano che cellule infettate da differenti virus possono secernere esosomi contenenti diverse componenti di origine virale ma anche, in qualche caso, particelle virali infettanti. Questo meccanismo di veicolazione all’interno di esosomi rappresenta una via accessoria di trasmissione per alcuni virus che, eludendo la sorveglianza immunologica, favorisce il fenomeno della persistenza del virus all’interno dell’ospite. Non è noto dalla letteratura se particelle di TTV siano inglobate all’interno degli esosomi durante il processo infettivo. L’obiettivo del nostro studio è stato quello di indagare l’associazione fra TTV e esosomi, in particolare investigando la presenza di particelle del virus, del DNA virale e di miRNA prodotti da TTV all’interno di queste strutture cellulari circolanti. Mediante microscopia elettronica e tecniche di biologia molecolare, TTV è stato studiato negli esosomi di pazienti viremici e non e il DNA del virus, cosi come i miRNA prodotti da TTV, sono stati rilevati e quantificati all’interno degli esosomi circolanti nel plasma di soggetti sani o affetti da diverse patologie. I risultati ottenuti dallo studio forniscono, per la prima volta, una chiara indicazione dell’associazione fra TTV e esosomi, offrendo nuove conoscenze sui meccanismi che il virus usa per persistere e diffondere nell’ospite infettato

SARS-CoV-2 BA.2.86 (“Pirola”): Is it Pi or Just Another Omicron Sublineage?

The SARS-CoV-2 sublineage BA [...

Torquetenovirus detection in exosomes enriched vesicles circulating in human plasma samples

Background: Torquetenovirus (TTV) belongs to Anelloviridae family, infects nearly all people indefinitely without causing overt disease establishing a fine and successful interaction with the host. Increasing evidence have shown some human viruses exploit extracellular vesicles thereby helping viral persistence in the host. Here, the presence of TTV in extracellular vesicles circulating in human plasma was investigated. Methods: TTV DNA was quantified in plasma-derived exosomes from 122 samples collected from 97 diseased patients and 25 healthy donors. Exosomes enriched vesicles (EEVs) were extracted from plasma and characterized by Nanoparticle tracking analysis, by western blot for presence of tetraspanin CD63, CD81 and annexin II protein and, finally, by electron microscopy (EM). Presence and quantitation of TTV DNA were assessed with an universal single step real-time TaqMan PCR assay. Results: Preliminary investigation showed that the human plasma extracted extracellular vesicles exhibited a main size of 70 nm, had concentration of 2.5 7 109/ml, and scored positive for tetraspanin CD63, CD81 and annexin II, typical characteristic of the exosomes vesicles. EEVs extracted from pooled plasma with TTV DNA viremia of 9.7 7 104 copies/ml showed to contain 6.3 7 102 TTV copies/ml, corresponding to 0.65% of total viral load. Important, TTV yield changed significantly following freezing/thawing, detergents and DNAse treatment of plasma before EEVs extraction. EEVs purified by sucrose-density gradient centrifugation and analysis of gradient fraction positive for exosomes marker CD63 harbored 102 TTV copies/ml. Moreover, EM evidenced the presence of TTV-like particles in EEVs. Successive investigation of plasma EEVs from 122 subjects (37 HIV-positive, 20 HCV infected, 20 HBV infected, 20 kidney transplant recipients, and 25 healthy) reported TTV DNA detection in 42 (34%) of the viremic samples (37 were from diseased patients and 5 from healthy people) at a mean level of 4.8 7 103 copies/ml. The examination of EEVs selected samples reported the presence of TTV genogroup 1, 3, 4 and 5, with genogroup 3 highly observed. Conclusions: Collectively, although these observations should be confirmed by further studies, circulation of TTV particles in EEVs opens new avenues and mechanistic insights on the molecular strategies adopted by anelloviruses to persist in the host

Previous Humoral Immunity to the Endemic Seasonal Alphacoronaviruses NL63 and 229E Is Associated with Worse Clinical Outcome in COVID-19 and Suggests Original Antigenic Sin

Antibody-dependent enhancement (ADE) of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) infection has been hypothesized. However, to date, there has been no in vitro or in vivo evidence supporting this. Cross-reactivity exists between SARS CoV-2 and other Coronaviridae for both cellular and humoral immunity. We show here that IgG against nucleocapsid protein of alphacoronavirus NL63 and 229E correlate with the World Health Organization’s (WHO) clinical severity score ≥ 5 (incidence rate ratios was 1.87 and 1.80, respectively, and 1.94 for the combination). These laboratory findings suggest possible ADE of SARS CoV-2 infection by previous alphacoronavirus immunity

Assessment of prevalence and load of torquetenovirus viremia in a large cohort of healthy blood donors

Torquetenovirus (TTV) is an emerging marker of functional immune competence with the potential to predict transplant-related adverse events. A large-scale epidemiological study was performed to understand how basal values vary in healthy subjects according to age and gender

Lack of neutralizing activity in nonconvalescent sera, regardless of ABO blood group and anti-A isoagglutinin titer

Summary: Background: Several ABO blood groups have been associated with the likelihood of infection, severity, and/or outcome of COVID-19 in hospitalized cohorts, raising the hypothesis that anti-A isoagglutinins in non-A-group recipients could act as neutralizing antibodies against SARS-CoV-2. Materials and methods: We run live virus neutralization tests using sera from 58 SARS-CoV-2 seronegative blood donors (27 O-group and 31 A-group) negatives for SARS-CoV-2 IgG to investigate what degree of neutralizing activity could be detected in their sera and eventual correlation with anti-A isoagglutinin titers. Results: We could not find clinically relevant neutralizing activity in any blood group, regardless of anti-isoagglutinin titer, Discussion: Our findings suggest that mechanisms other than neutralization explain the differences in outcomes from COVID19 seen in different ABO blood groups