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During the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), positive-sense genomic RNA and subgenomic RNAs (sgRNAs) are synthesized by a discontinuous process of transcription characterized by a template switch, regulated by transcription-regulating sequences (TRS). Although poorly known about makeup and dynamics of sgRNAs population and function of its constituents, next-generation sequencing approaches with the help of bioinformatics tools have made a significant contribution to expand the knowledge of sgRNAs in SARS-CoV-2. For this scope to date, Periscope, LeTRS, sgDI-tector, and CORONATATOR have been developed. However, limited number of studies are available to compare the performance of such tools. To this purpose, we compared Periscope, LeTRS, and sgDI-tector in the identification of canonical (c-) and noncanonical (nc-) sgRNA species in the data obtained with the Illumina ARTIC sequencing protocol applied to SARS-CoV-2-infected Caco-2 cells, sampled at different time points. The three software showed a high concordance rate in the identification and in the quantification of c-sgRNA, whereas more differences were observed in nc-sgRNA. Overall, LeTRS and sgDI-tector result to be adequate alternatives to Periscope to analyze Fastq data from sequencing platforms other than Nanopore., (© 2023 Lavezzari et al.)

Angiotensin-converting enzyme 2 receptor (ACE2R) is a transmembrane protein expressed in various tissues throughout the body that plays a key role in the regulation of blood pressure. Recently, ACE2R has gained significant attention due to its involvement in the pathogenesis of COVID-19, the disease caused by the Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2). While ACE2 receptors serve as entry points for the novel coronavirus, Transmembrane Serine Protease 2 (TMPRSS2), an enzyme located on the cell membrane, is required for SARS-CoV-2 S protein priming. Even though numerous studies have assessed the effects of COVID-19 on the brain, very little information is available concerning the distribution of ACE2R and TMPRSS2 in the human brain, with particular regard to their topographical expression in the brainstem. In this study, we investigated the expression of ACE2R and TMPRSS2 in the brainstem of 18 adult subjects who died due to pneumonia/respiratory insufficiency. Our findings indicate that ACE2R and TMPRSS2 are expressed in neuronal and glial cells of the brainstem, particularly at the level of the vagal nuclei of the medulla and the midbrain tegmentum, thus confirming the expression and anatomical localization of these proteins within specific human brainstem nuclei. Furthermore, our findings help to define anatomically susceptible regions to SARS-CoV-2 infection in the brainstem, advancing knowledge on the neuropathological underpinnings of neurological manifestations in COVID-19.

In SARS-CoV-2 pandemic scenario, the identification of rapid methods to detect antibodies against coronavirus has been a wide and urgent issue. Epitope mapping on peptide microarrays is a rapid way to identify sequences with a high immunoreactivity. The process begins with a proteome-wide screening, based on immune affinity; the use of a high-density microarray is followed by a validation phase, where a restricted panel of probes is tested using peptide microarrays; peptide sequences are immobilized through a click-based strategy.COVID-19-positive sera are tested and immuno-domains regions are identified on SARS-CoV-2 spike (S), nucleocapsid (N) protein, and Orf1ab polyprotein. An epitope on N protein (region 155-171) provided good diagnostic performance in discriminating COVID-19-positive vs. healthy individuals. Using this sequence, 92% sensitivity and 100% specificity are reached for IgG detection in COVID-19 samples, and no cross-reactivity with common cold coronaviruses is detected. Overall, epitope 155-171 from N protein represents a promising candidate for further development and rapid implementation in serological tests., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: SARS-CoV-2 induces a spectrum of clinical conditions ranging from asymptomatic infection to life threatening severe disease. Host microRNAs have been involved in the cytokine storm driven by SARS-CoV-2 infection and proposed as candidate biomarkers for COVID-19., Methods: To discover signatures of circulating miRNAs associated with COVID-19, disease severity and mortality, small RNA-sequencing was performed on serum samples collected from 89 COVID-19 patients (34 severe, 29 moderate, 26 mild) at hospital admission and from 45 healthy controls (HC). To search for possible sources of miRNAs, investigation of differentially expressed (DE) miRNAs in relevant human cell types in vitro ., Results: COVID-19 patients showed upregulation of miRNAs associated with lung disease, vascular damage and inflammation and downregulation of miRNAs that inhibit pro-inflammatory cytokines and chemokines, angiogenesis, and stress response. Compared with mild/moderate disease, patients with severe COVID-19 had a miRNA signature indicating a profound impairment of innate and adaptive immune responses, inflammation, lung fibrosis and heart failure. A subset of the DE miRNAs predicted mortality. In particular, a combination of high serum miR-22-3p and miR-21-5p, which target antiviral response genes, and low miR-224-5p and miR-155-5p, targeting pro-inflammatory factors, discriminated severe from mild/moderate COVID-19 (AUROC 0.88, 95% CI 0.80-0.95, p<0.0001), while high leukocyte count and low levels of miR-1-3p, miR-23b-3p, miR-141-3p, miR-155-5p and miR-4433b-5p predicted mortality with high sensitivity and specificity (AUROC 0.95, 95% CI 0.89-1.00, p<0.0001). In vitro experiments showed that some of the DE miRNAs were modulated directly by SARS-CoV-2 infection in permissive lung epithelial cells., Conclusions: We discovered circulating miRNAs associated with COVID-19 severity and mortality. The identified DE miRNAs provided clues on COVID-19 pathogenesis, highlighting signatures of impaired interferon and antiviral responses, inflammation, organ damage and cardiovascular failure as associated with severe disease and death., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Giannella, Riccetti, Sinigaglia, Piubelli, Razzaboni, Di Battista, Agostini, Dal Molin, Manganelli, Gobbi, Ceolotto and Barzon.)

Background: In August 2020, in the context of COVID-19 pandemics, an autochthonous dengue outbreak was identified for the first time in Italy., Methods: Following the reporting of the index case of autochthonous dengue, epidemiological investigation, vector control and substances of human origin safety measures were immediately activated, according to the national arbovirus surveillance plan. Dengue cases were followed-up with weekly visits and laboratory tests until recovery and clearance of viral RNA from blood., Results: The primary dengue case was identified in a young woman, who developed fever after returning from Indonesia to northern Italy, on 27 July 2020. She spent the mandatory quarantine for COVID-19 at home with relatives, six of whom developed dengue within two weeks. Epidemiological investigation identified further five autochthonous dengue cases among people who lived or stayed near the residence of the primary case. The last case of the outbreak developed fever on 29 September 2020. Dengue cases had a mild febrile illness, except one with persistent asthenia and myalgia. DENV-1 RNA was detected in blood and/or urine in all autochthonous cases, up to 35 days after fever onset. All cases developed IgM and IgG antibodies which cross-reacted with West Nile virus (WNV) and other flaviviruses. Sequencing of the full viral genome from blood samples showed over 99% nucleotide identity with DENV-1 strains isolated in China in 2014-2015; phylogenetic analysis classified the virus within Genotype I. Entomological site inspection identified a high density of Aedes albopictus mosquitoes, which conceivably sustained local DENV-1 transmission. Aedes koreicus mosquitoes were also collected in the site., Conclusions: Areas in Europe with high density of Aedes mosquitoes should be considered at risk for dengue transmission. The presence of endemic flaviviruses, such as WNV, might pose problems in the laboratory diagnosis., (© The Author(s) 2021. Published by Oxford University Press on behalf of International Society of Travel Medicine.)

Background: The immune response plays a pivotal role in dictating the clinical outcome in severe acute respiratory syndrome coronavirus 2 (SARS - CoV - 2)-infected adults, but it is still poorly investigated in the pediatric population., Methods: Of 209 enrolled subjects, 155 patients were confirmed by PCR and/or serology as having coronavirus disease 2019 (COVID-19). Blood samples were obtained at a median of 2.8 (interquartile, 2.1-3.7) and 6.1 (5.3-7.2) months after baseline (symptom onset and/or first positive virus detection). The immune profiles of activation, senescence, exhaustion, and regulatory cells were analyzed by flow cytometry. Neutralizing antibodies (nAbs) were detected by a plaque reduction neutralization test. In available nasopharyngeal swabs at baseline, SARS-CoV-2 levels were quantified by digital droplet PCR (ddPCR)., Results: Overall, COVID-19 patients had higher levels of immune activation, exhaustion, and regulatory cells compared to non-COVID-19 subjects. Within the COVID-19 group, activated and senescent cells were higher in adults than in children and inversely correlated with the nAbs levels. Conversely, Tregs and Bregs regulatory cells were higher in COVID-19 children compared to adults and positively correlated with nAbs. Higher immune activation still persisted in adults after 6 months of infection, while children maintained higher levels of regulatory cells. SARS-CoV-2 levels did not differ among age classes., Conclusions: Adults displayed higher immune activation and lower production of anti-SARS-CoV-2 nAbs than children. The different immune response was not related to different viral load. The higher expression of regulatory cells in children may contribute to reduce the immune activation, thus leading to a greater specific response against the virus., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Petrara, Bonfante, Costenaro, Cantarutti, Carmona, Ruffoni, Di Chiara, Zanchetta, Barzon, Donà, Da Dalt, Bortolami, Pagliari, Plebani, Rossi, Cotugno, Palma, Giaquinto and De Rossi.)

SARS-CoV-2 fine-tunes the interferon (IFN)-induced antiviral responses, which play a key role in preventing coronavirus disease 2019 (COVID-19) progression. Indeed, critically ill patients show an impaired type I IFN response accompanied by elevated inflammatory cytokine and chemokine levels, responsible for cell and tissue damage and associated multi-organ failure. Here, the early interaction between SARS-CoV-2 and immune cells was investigated by interrogating an in vitro human peripheral blood mononuclear cell (PBMC)-based experimental model. We found that, even in absence of a productive viral replication, the virus mediates a vigorous TLR7/8-dependent production of both type I and III IFNs and inflammatory cytokines and chemokines, known to contribute to the cytokine storm observed in COVID-19. Interestingly, we observed how virus-induced type I IFN secreted by PBMC enhances anti-viral response in infected lung epithelial cells, thus, inhibiting viral replication. This type I IFN was released by plasmacytoid dendritic cells (pDC) via an ACE-2-indipendent but Neuropilin-1-dependent mechanism. Viral sensing regulates pDC phenotype by inducing cell surface expression of PD-L1 marker, a feature of type I IFN producing cells. Coherently to what observed in vitro, asymptomatic SARS-CoV-2 infected subjects displayed a similar pDC phenotype associated to a very high serum type I IFN level and induction of anti-viral IFN-stimulated genes in PBMC. Conversely, hospitalized patients with severe COVID-19 display very low frequency of circulating pDC with an inflammatory phenotype and high levels of chemokines and pro-inflammatory cytokines in serum. This study further shed light on the early events resulting from the interaction between SARS-CoV-2 and immune cells occurring in vitro and confirmed ex vivo. These observations can improve our understanding on the contribution of pDC/type I IFN axis in the regulation of the anti-viral state in asymptomatic and severe COVID-19 patients., Competing Interests: The authors have declared that no competing interests exist.

Background: Recent evidence suggests that neutralizing antibodies (nAbs) to severe acute respiratory syndrome coronavirus 2 may persist over time; however, knowledge regarding pediatric subjects is limited., Methods: A single-center, prospective observational study was conducted on 57 family clusters of coronavirus disease 2019, including children of neonatal and pediatric age attending the University Hospital of Padua (Italy). For each patient, blood samples were collected for both the quantification of nAbs through a plaque reduction neutralizing test and the detection of antinucleocapsid-spike protein immunoglobulin G and/or immunoglobulin M., Results: We analyzed 283 blood samples collected from 152 confirmed coronavirus disease 2019 cases (82 parents and 70 children or older siblings of median age of 8 years, interquartile range: 4-13), presenting asymptomatic or with mildly symptomatic disease. Despite the decrease of immunoglobulin G over time, nAbs were found to persist up to 7 to 8 months in children, whereas adults recorded a modest declining trend. Interestingly, children aged <6 years, and, in particular, those aged <3 years, developed higher long-lasting levels of nAbs compared with older siblings and/or adults., Conclusions: Mild and asymptomatic severe acute respiratory syndrome coronavirus 2 infections in family clusters elicited higher nAbs among children., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2021 by the American Academy of Pediatrics.)

Although antibody levels progressively decrease following SARS-CoV-2 infection, the immune memory persists for months. Thus, individuals who naturally contracted SARS-CoV-2 are expected to develop a more rapid and sustained response to COVID-19 vaccines than naïve individuals. In this study, we analyzed the dynamics of the antibody response to the BNT162b2 mRNA COVID-19 vaccine in six healthcare workers who contracted SARS-CoV-2 in March 2020, in comparison to nine control subjects without a previous infection. The vaccine was well tolerated by both groups, with no significant difference in the frequency of vaccine-associated side effects, with the exception of local pain, which was more common in previously infected subjects. Overall, the titers of neutralizing antibodies were markedly higher in response to the vaccine than after natural infection. In all subjects with pre-existing immunity, a rapid increase in anti-spike receptor-binding domain (RBD) IgG antibodies and neutralizing antibody titers was observed one week after the first dose, which seemed to act as a booster. Notably, in previously infected individuals, neutralizing antibody titers 7 days after the first vaccine dose were not significantly different from those observed in naïve subjects 7 days after the second vaccine dose. These results suggest that, in previously infected people, a single dose of the vaccine might be sufficient to induce an effective response.

In this Review, we briefly describe the basic virology and pathogenesis of SARS-CoV-2, highlighting how stem cell technology and organoids can contribute to the understanding of SARS-CoV-2 cell tropisms and the mechanism of disease in the human host, supporting and clarifying findings from clinical studies in infected individuals. We summarize here the results of studies, which used these technologies to investigate SARS-CoV-2 pathogenesis in different organs. Studies with in vitro models of lung epithelia showed that alveolar epithelial type II cells, but not differentiated lung alveolar epithelial type I cells, are key targets of SARS-CoV-2, which triggers cell apoptosis and inflammation, while impairing surfactant production. Experiments with human small intestinal organoids and colonic organoids showed that the gastrointestinal tract is another relevant target for SARS-CoV-2. The virus can infect and replicate in enterocytes and cholangiocytes, inducing cell damage and inflammation. Direct viral damage was also demonstrated in in vitro models of human cardiomyocytes and choroid plexus epithelial cells. At variance, endothelial cells and neurons are poorly susceptible to viral infection, thus supporting the hypothesis that neurological symptoms and vascular damage result from the indirect effects of systemic inflammatory and immunological hyper-responses to SARS-CoV-2 infection.

SARS-CoV-2 is a novel strain of Coronavirus with important implications for the CNS. Data deriving from autopsy studies supports the neuroinvasive potential of SARS-CoV-2, even though infection appears to be limited to sparse cells within the brainstem and was not associated with the severity of neuropathological changes. We examined the neuropathological alterations of twenty-four (24) patients who died following a diagnosis of Sars-CoV-2 infection in Padova, during the COVID-19 pandemic, and 10 Age-matched controls with comparable medical conditions. The brain, cranial nerves, meninges, choroid plexus were sampled and histopathological evaluation was conducted. Immunohistochemistry for GFAP, CD3, CD8, CD20, CD61, CD68 and HLA-DR antibodies was performed. SARS-CoV-2 proteins (Spike Subunit 1 and Nucleocapsid Protein) and RNA (N2 Gene and E Gene) were investigated through immunohistochemistry, RT-PCR, in-situ hybridization and electron microscopy. Digitally assisted quantification of reactive microglia was performed on sections of the medulla, pons and mesencephalon. Neuropathological alterations include marked astrogliosis at the level of the brainstem, microgliosis and microglial nodules in different sites of the medulla oblongata, substantia nigra and basal ganglia. Haemorragic injury was found in four patients and small vessel thromboses in seven patients. Seven subjects displayed immunoreactive structures within CNS parenchima. Five subjects presented immunoreactive neurons within the anatomically defined boundaries of the solitary tract nucleus, nucleus ambiguus and substantia nigra. Viral RNA was detected through RT-PCR in all the aforementioned IHC Viral protein positive samples. Quantification of reactive microglia revealed an anatomically segregated pattern of inflammation targeting mainly the medulla oblongata and the substantia nigra, and was significantly higher when compared to matched controls. The study contributes to define the neuroinvasive potential of SARS-CoV-2 within the CNS. Unlike previous findings, we have documented several cases in which viral proteins and RNA were clearly detectable within anatomically defined regions of the CNS, testifying an important neuroinvasive potential by the virus. SARS-CoV-2 direct invasion does not appear to directly correlate with the severity of neuropathological changes. This may be ascribed to the relative short interval between infection and death, and requires further confirmation from other studies. [ABSTRACT FROM AUTHOR]