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Genomic surveillance based on sequencing the entire genetic code of SARS-CoV-2 involves monitoring and studying genetic changes and variations in disease-causing organisms such as viruses and bacteria. By tracing the virus, it is possible to prevent epidemic spread in the community, ensuring a ‘precision public health’ strategy. A peptide-based design was applied to provide an efficacious strategy that is able to counteract any emerging viral variant of concern dynamically and promptly to affect the outcomes of a pandemic at an early stage while waiting for the production of the anti-variant-specific vaccine, which require longer times. The inhibition of the interaction between the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and one of the cellular receptors (DPP4) that its receptors routinely bind to infect human cells is an intriguing therapeutic approach to prevent the virus from entering human cells. Among the other modalities developed for this purpose, peptides surely offer unique advantages, including ease of synthesis, serum stability, low immunogenicity and toxicity, and small production and distribution chain costs. Here, we obtained a potent new inhibitor based on the rearrangement of a previously identified peptide that has been rationally designed on a cell dipeptidyl peptidase 4 (DPP4) sequence, a ubiquitous membrane protein known to bind the RBD-SPIKE domain of the virus. This novel peptide (named DPP4-derived), conceived as an endogenous “drug”, is capable of targeting the latest tested variants with a high affinity, reducing the VSV* DG-Fluc pseudovirus Omicron’s infection capacity by up to 14%, as revealed by in vitro testing in human Calu-3 cells. Surface plasmon resonance (SPR) confirmed the binding affinity of the new DPP4-derived peptide with Omicron variant RBD.

The human immunodeficiency virus (HIV-1) matrix protein p17 (p17) is released from infected cells as a protein capable of deregulating the biological activity of different cells. P17 variants (vp17s), more frequently detected in the plasma of HIV-1+ patients with rather than without lymphoma and characterized by amino acids insertions in their C-terminal region, were found to trigger B cell growth and clonogenicity. Vp17s endowed with B-cell-growth-promoting activity are drastically destabilized, whereas, in a properly folded state, reference p17 (refp17) does not exert any biological activity on B cell growth and clonogenicity. However, misfolding of refp17 is necessary to expose a masked functional epitope, interacting with the protease-activated receptor 1 (PAR-1), endowed with B cell clonogenicity. Indeed, it is worth noting that changes in the secondary structure can strongly impact the function of a protein. Here, we performed computational studies to show that the gain of function of vp17s is linked to dramatic conformational changes due to structural modification in the secondary-structure elements and in the rearrangement of the hydrogen bond (H-bond) network. In particular, all clonogenic vp17s showed the disengagement of two critical residues, namely Trp16 and Tyr29, from their hydrophobic core. Biological data showed that the mutation of Trp16 and Tyr29 to Ala in the refp17 backbone, alone or in combination, resulted in a protein endowed with B cell clonogenic activity. These data show the pivotal role of the hydrophobic component in maintaining refp17 stability and identify a novel potential therapeutic target to counteract vp17-driven lymphomagenesis in HIV-1+ patients.

Leclercia adecarboxylata is a gram-negative bacillus, a ubiquitous microorganism, which may be found in both aquatic environments and soil, it was first described by H. Leclerc in 1962 and was reclassified thanks to more sensitive testing methods. L. adecarboxylata has been recently recognized as an emerging pathogen that causes several clinical syndromes not only in immunocompromised patients.

Abstract The new coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic, which so far has caused over 6 million deaths in 2 years, despite new vaccines and antiviral medications. Drug repurposing, an approach for the potential application of existing pharmaceutical products to new therapeutic indications, could be an effective strategy to obtain quick answers to medical emergencies. Following a virtual screening campaign on the most relevant viral proteins, we identified the drug raloxifene, a known Selective Estrogen Receptor Modulator (SERM), as a new potential agent to treat mild-to-moderate COVID-19 patients. In this paper we report a comprehensive pharmacological characterization of raloxifene in relevant in vitro models of COVID-19, specifically in Vero E6 and Calu-3 cell lines infected with SARS-CoV-2. A large panel of the most common SARS-CoV-2 variants isolated in Europe, United Kingdom, Brazil, South Africa and India was tested to demonstrate the drug’s ability in contrasting the viral cytopathic effect (CPE). Literature data support a beneficial effect by raloxifene against the viral infection due to its ability to interact with viral proteins and activate protective estrogen receptor-mediated mechanisms in the host cells. Mechanistic studies here reported confirm the significant affinity of raloxifene for the Spike protein, as predicted by in silico studies, and show that the drug treatment does not directly affect Spike/ACE2 interaction or viral internalization in infected cell lines. Interestingly, raloxifene can counteract Spike-mediated ADAM17 activation in human pulmonary cells, thus providing new insights on its mechanism of action. A clinical study in mild to moderate COVID-19 patients (NCT05172050) has been recently completed. Our contribution to evaluate raloxifene results on SARS-CoV-2 variants, and the interpretation of the mechanisms of action will be key elements to better understand the trial results, and to design new clinical studies aiming to evaluate the potential development of raloxifene in this indication.

To date, much discussion has been had on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lung infection associated with COVID-19 onset, of which the major manifestation is characterized by a “cytokine storm” [...]

During the COVID-19 pandemic, drug repurposing represented an effective strategy to obtain quick answers to medical emergencies. Based on previous data on methotrexate (MTX), we evaluated the anti-viral activity of several DHFR inhibitors in two cell lines. We observed that this class of compounds showed a significant influence on the virus-induced cytopathic effect (CPE) partly attributed to the intrinsic anti-metabolic activity of these drugs, but also to a specific anti-viral function. To elucidate the molecular mechanisms, we took advantage of our EXSCALATE platform for in-silico molecular modelling and further validated the influence of these inhibitors on nsp13 and viral entry. Interestingly, pralatrexate and trimetrexate showed superior effects in counteracting the viral infection compared to other DHFR inhibitors. Our results indicate that their higher activity is due to their polypharmacological and pleiotropic profile. These compounds can thus potentially give a clinical advantage in the management of SARS-CoV-2 infection in patients already treated with this class of drugs.

Abstract Background Since the first outbreak of SARS-CoV-2, the clinical characteristics of the Coronavirus Disease 2019 (COVID-19) have been progressively changed. Data reporting a viral intra-host and inter-host evolution favouring the appearance of mild SARS-CoV-2 strains are since being accumulating. To better understand the evolution of SARS-CoV-2 pathogenicity and its adaptation to the host, it is therefore crucial to investigate the genetic and phenotypic characteristics of SARS-CoV-2 strains circulating lately in the epidemic. Methods Nasopharyngeal swabs have been analyzed for viral load in the early (March 2020) and late (May 2020) phases of epidemic in Brescia, Italy. Isolation of SARS-CoV-2 from 2 high viral load specimens identified on March 9 (AP66) and on May 8 (GZ69) was performed on Vero E6 cells. Amount of virus released was assessed by quantitative PCR. Genotypic characterization of AP66 and GZ69 was performed by next generation sequencing followed by an in-depth in silico analysis of nucleotide mutations. Results The SARS-CoV-2 GZ69 strain, isolated in May from an asymptomatic healthcare worker, showed an unprecedented capability of replication in Vero E6 cells in the absence of any evident cytopathic effect. Vero E6 subculturing, up to passage 4, showed that SARS-CoV-2 GZ69 infection was as productive as the one sustained by the cytopathic strain AP66. Whole genome sequencing of the persistently replicating SARS-CoV-2 GZ69 has shown that this strain differs from the early AP66 variant in 9 nucleotide positions (C2939T; C3828T; G21784T; T21846C; T24631C; G28881A; G28882A; G28883C; G29810T) which lead to 6 non-synonymous substitutions spanning on ORF1ab (P892S; S1188L), S (K74N; I95T) and N (R203K, G204R) proteins. Conclusions Identification of the peculiar SARS-CoV-2 GZ69 strain in the late Italian epidemic highlights the need to better characterize viral variants circulating among asymptomatic or paucisymptomatic individuals. The current approach could unravel the ways for future studies aimed at analyzing the selection process which favours viral mutations in the human host.

Objectives: Structural aspects of HIV-1 integrase complex and role of integrase minor mutations and polymorphisms in ART effectiveness is still unknown. The objective of this study was to assess the 24 and 48 weeks (W) effectiveness of ART regimens in patients with Integrase Inhibitors (InSTI) minor mutations and polymorphisms receiving InSTI-based regimens. Methods: We enrolled all ART-naïve or InSTI-naïve HIV-infected patients, with a baseline InSTI genotypic resistances test between 2011 and 2016. We analyzed integrase resistance mutations using the Stanford University HIV Drug Resistance Database (HIVdb Program, version 6.3.0). The outcome was virological response at 24 and 48 W of follow up (FU) according to snapshot analysis. We defined virological failure as two consecutive HIV-RNA > 50 copies/ml, or one >1000 copies/ml. Patients were divided in those presenting InSTI minor mutations (Group 1), and those with only polymorphisms or wild type (Group 2). Results: We enrolled 83 patients. 81 patients reached 24 W of FU: 2/20 (10%) and 4/61 (6.5%) showed virological failure in Group 1 and 2 respectively. 66 patients reached 48 W of FU: 0/17 (0%) and 2/49 (4%) showed virological failure in Group 1 and 2 respectively. Interestingly, patients with polymorphisms G123S and R127K had higher risk of failure at 24 W (respectively, relative risk - RR − 36, IQR 2.1-613, p = 0.01; RR 36, IQR 2.1-613, p = 0.01) and patients with V72I had an higher risk of failure both at 24 W (RR 6.52, IQR 1.29-32.9, p = 0.02) and 48 W (RR 21.1, IQR 1.07-414, p = 0.04). Conclusions: Our study showed that the presence of V72I, G123S and R127K polymorphisms could play a role in reducing InSTI effectiveness.

During the first wave of infections, neurological symptoms in Coronavirus Disease 2019 (COVID-19) patients raised particular concern, suggesting that, in a subset of patients, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could invade and damage cells of the central nervous system (CNS). Indeed, up to date several in vitro and in vivo studies have shown the ability of SARS-CoV-2 to reach the CNS. Both viral and/or host related features could explain why this occurs only in certain individuals and not in all the infected population. The aim of the present study was to evaluate if onset of neurological manifestations in COVID-19 patients was related to specific viral genomic signatures. To this end, viral genome was extracted directly from nasopharyngeal swabs of selected SARS-CoV-2 positive patients presenting a spectrum of neurological symptoms related to COVID-19, ranging from anosmia/ageusia to more severe symptoms. By adopting a whole genome sequences approach, here we describe a panel of known as well as unknown mutations detected in the analyzed SARS-CoV-2 genomes. While some of the found mutations were already associated with an improved viral fitness, no common signatures were detected when comparing viral sequences belonging to specific groups of patients. In conclusion, our data support the notion that COVID-19 neurological manifestations are mainly linked to patient-specific features more than to virus genomic peculiarities.

Severe COVID-19 is characterized by angiogenic features, such as intussusceptive angiogenesis, endothelialitis, and activation of procoagulant pathways. This pathological state can be ascribed to a direct SARS-CoV-2 infection of human lung ECs. Recently, we showed the capability of SARS-CoV-2 to infect ACE2-negative primary human lung microvascular endothelial cells (HL-mECs). This occurred through the interaction of an Arg-Gly-Asp (RGD) motif, endowed on the Spike protein at position 403–405, with αvβ3 integrin expressed on HL-mECs. HL-mEC infection promoted the remodeling of cells toward a pro-inflammatory and pro-angiogenic phenotype. The RGD motif is distinctive of SARS-CoV-2 Spike proteins up to the Omicron BA.1 subvariant. Suddenly, a dominant D405N mutation was expressed on the Spike of the most recently emerged Omicron BA.2, BA.4, and BA.5 subvariants. Here we demonstrate that the D405N mutation inhibits Omicron BA.5 infection of HL-mECs and their dysfunction because of the lack of Spike/integrins interaction. The key role of ECs in SARS-CoV-2 pathogenesis has been definitively proven. Evidence of mutations retrieving the capability of SARS-CoV-2 to infect HL-mECs highlights a new scenario for patients infected with the newly emerged SARS-CoV-2 Omicron subvariants, suggesting that they may display less severe disease manifestations than those observed with previous variants.

The BQ.1 SARS-CoV-2 variant, also known as Cerberus, is one of the most recent Omicron descendant lineages. Compared to its direct progenitor BA.5, BQ.1 has some additional spike mutations in some key antigenic sites, which confer further immune escape ability over other circulating lineages. In such a context, here, we perform a genome-based survey aimed at obtaining a complete-as-possible nuance of this rapidly evolving Omicron subvariant. Genetic data suggest that BQ.1 represents an evolutionary blind background, lacking the rapid diversification that is typical of a dangerous lineage. Indeed, the evolutionary rate of BQ.1 is very similar to that of BA.5 (7.6 × 10−4 and 7 × 10−4 subs/site/year, respectively), which has been circulating for several months. The Bayesian Skyline Plot reconstruction indicates a low level of genetic variability, suggesting that the peak was reached around 3 September 2022. Concerning the affinity for ACE2, structure analyses (also performed by comparing the properties of BQ.1 and BA.5 RBD) indicate that the impact of the BQ.1 mutations may be modest. Likewise, immunoinformatic analyses showed moderate differences between the BQ.1 and BA5 potential B-cell epitopes. In conclusion, genetic and structural analyses on SARS-CoV-2 BQ.1 suggest no evidence of a particularly dangerous or high expansion capability. Genome-based monitoring must continue uninterrupted for a better understanding of its descendants and all other lineages.

In this study, we show that BNT162b2 vaccine-elicited antibodies efficiently neutralize SARS-CoV-2 authentic viruses belonging to B.1, B.1.1.7, B.1.351, B.1.525 and P.1 lineages. Interestingly, the neutralization of B.1.1.7 and B.1.525 lineages was significantly higher, whereas the neutralization of B.1.351 and P.1 lineages was robust but significantly lower as compared to B.1 lineage. Following our findings, we consider that the BNT162b2 vaccine offers protection against the current prevailing variants of SARS-CoV-2.

Summary: The efficacy of ALVAC-based HIV and SIV vaccines in humans and macaques correlates with antibodies to envelope variable region 2 (V2). We show here that vaccine-induced antibodies to SIV variable region 1 (V1) inhibit anti-V2 antibody-mediated cytotoxicity and reverse their ability to block V2 peptide interaction with the α4β7 integrin. SIV vaccines engineered to delete V1 and favor an α helix, rather than a β sheet V2 conformation, induced V2-specific ADCC correlating with decreased risk of SIV acquisition. Removal of V1 from the HIV-1 clade A/E A244 envelope resulted in decreased binding to antibodies recognizing V2 in the β sheet conformation. Thus, deletion of V1 in HIV envelope immunogens may improve antibody responses to V2 virus vulnerability sites and increase the efficacy of HIV vaccine candidates.

Integrins represent a gateway of entry for many viruses and the Arg-Gly-Asp (RGD) motif is the smallest sequence necessary for proteins to bind integrins. All Severe Acute Respiratory Syndrome Virus type 2 (SARS-CoV-2) lineages own an RGD motif (aa 403–405) in their receptor binding domain (RBD). We recently showed that SARS-CoV-2 gains access into primary human lung microvascular endothelial cells (HL-mECs) lacking Angiotensin-converting enzyme 2 (ACE2) expression through this conserved RGD motif. Following its entry, SARS-CoV-2 remodels cell phenotype and promotes angiogenesis in the absence of productive viral replication. Here, we highlight the αvβ3 integrin as the main molecule responsible for SARS-CoV-2 infection of HL-mECs via a clathrin-dependent endocytosis. Indeed, pretreatment of virus with αvβ3 integrin or pretreatment of cells with a monoclonal antibody against αvβ3 integrin was found to inhibit SARS-CoV-2 entry into HL-mECs. Surprisingly, the anti-Spike antibodies evoked by vaccination were neither able to impair Spike/integrin interaction nor to prevent SARS-CoV-2 entry into HL-mECs. Our data highlight the RGD motif in the Spike protein as a functional constraint aimed to maintain the interaction of the viral envelope with integrins. At the same time, our evidences call for the need of intervention strategies aimed to neutralize the SARS-CoV-2 integrin-mediated infection of ACE2-negative cells in the vaccine era.

Abstract Background Myroides spp. are common environmental organisms and they can be isolated predominantly in water, soil, food and in sewage treatment plants. In the last two decades, an increasing number of infections such as urinary tract infections and skin and soft tissue infections, caused by these microorganisms has been reported. Selection of appropriate antibiotic therapy to treat the infections caused by Myroides spp. is difficult due to the production of a biofilm and the organism’s intrinsic resistance to many antibiotic classes. Case presentation We report the case of a 69-year-old immunocompromised patient who presented with repeated episodes of macroscopic haematuria, from Northern Italy. A midstream urine sample cultured a Gram negative rod in significant amounts (> 105 colony-forming units (cfu)/mL), which was identified as Myroides odoratimimus. The patient was successfully treated with trimethoprim/sulfamethoxazole after antibiotic susceptibility testing confirmed its activity. Conclusion This case underlines the emergence of multidrug resistant Myroides spp. which are ubiquitous in the environment and it demands that clinicians should be more mindful about the role played by atypical pathogens, which may harbour or express multidrug resistant characteristics, in immunocompromised patients or where there is a failure of empiric antimicrobial therapy.

Genotype screening was implemented in Italy and showed a significant prevalence of new SARS-CoV-2 mutants carrying Q675H mutation, near the furin cleavage site of spike protein. Currently, this mutation, which is expressed on different SARS-CoV-2 lineages circulating worldwide, has not been thoughtfully investigated. Therefore, we performed phylogenetic and biocomputational analysis to better understand SARS-CoV-2 Q675H mutants’ evolutionary relationships with other circulating lineages and Q675H function in its molecular context. Our studies reveal that Q675H spike mutation is the result of parallel evolution because it arose independently in separate evolutionary clades. In silico data show that the Q675H mutation gives rise to a hydrogen-bonds network in the spike polar region. This results in an optimized directionality of arginine residues involved in interaction of spike with the furin binding pocket, thus improving proteolytic exposure of the viral protein. Furin was predicted to have a greater affinity for Q675H than Q675 substrate conformations. As a consequence, Q675H mutation could confer a fitness advantage to SARS-CoV-2 by promoting a more efficient viral entry. Interestingly, here we have shown that Q675H spike mutation is documented in all the VOCs. This finding highlights that VOCs are still evolving to enhance viral fitness and to adapt to the human host. At the same time, it may suggest Q675H spike mutation involvement in SARS-CoV-2 evolution.

The rapid spread of the pandemic caused by the SARS-CoV-2 virus has created an unusual situation, with rapid searches for compounds to interfere with the biological processes exploited by the virus. Doxycycline, with its pleiotropic effects, including anti-viral activity, has been proposed as a therapeutic candidate for COVID-19 and about twenty clinical trials have started since the beginning of the pandemic. To gain information on the activity of doxycycline against SARS-CoV-2 infection and clarify some of the conflicting clinical data published, we designed in vitro binding tests and infection studies with a pseudotyped virus expressing the spike protein, as well as a clinically isolated SARS-CoV-2 strain. Doxycycline inhibited the transduction of the pseudotyped virus in Vero E6 and HEK-293 T cells stably expressing human receptor angiotensin-converting enzyme 2 but did not affect the entry and replication of SARS-CoV-2. Although this conclusion is apparently disappointing, it is paradigmatic of an experimental approach aimed at developing an integrated multidisciplinary platform which can shed light on the mechanisms of action of potential anti-COVID-19 compounds. To avoid wasting precious time and resources, we believe very stringent experimental criteria are needed in the preclinical phase, including infectivity studies with clinically isolated SARS-CoV-2, before moving on to (futile) clinical trials.

(1) Background: Acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent for the coronavirus disease (COVID-19) that has led to a pandemic that began in March 2020. The role of the SARS-CoV-2 components on innate and adaptive immunity is still unknown. We investigated the possible implication of pathogen-associated molecular patterns (PAMPs)–pattern recognition receptors (PRRs) interaction. (2) Methods: We infected Calu-3/MRC-5 multicellular spheroids (MTCSs) with a SARS-CoV-2 clinical strain and evaluated the activation of RNA sensors, transcription factors, and cytokines/interferons (IFN) secretion, by quantitative real-time PCR, immunofluorescence, and ELISA. (3) Results: Our results showed that the SARS-CoV-2 infection of Calu-3/MRC-5 multicellular spheroids induced the activation of the TLR3 and TLR7 RNA sensor pathways. In particular, TLR3 might act via IRF3, producing interleukin (IL)-1α, IL-1β, IL-4, IL-6, and IFN-α and IFN-β, during the first 24 h post-infection. Then, TLR3 activates the NFκB transduction pathway, leading to pro-inflammatory cytokine secretion. Conversely, TLR7 seems to mainly act via NFκB, inducing type 1 IFN, IFN-γ, and IFN-λ3, starting from the 48 h post-infection. (4) Conclusion: We showed that both TLR3 and TLR7 are involved in the control of innate immunity during lung SARS-CoV-2 infection. The activation of TLRs induced pro-inflammatory cytokines, such as IL-1α, IL-1β, IL-4, and IL-6, as well as interferons. TLRs could be a potential target in controlling the infection in the early stages of the disease.

SARS-CoV-2-associated acute respiratory distress syndrome (ARDS) and acute lung injury are life-threatening manifestations of severe viral infection. The pathogenic mechanisms that lead to respiratory complications, such as endothelialitis, intussusceptive angiogenesis, and vascular leakage remain unclear. In this study, by using an immunofluorescence assay and in situ RNA-hybridization, we demonstrate the capability of SARS-CoV-2 to infect human primary lung microvascular endothelial cells (HL-mECs) in the absence of cytopathic effects and release of infectious particles. Preliminary data point to the role of integrins in SARS-CoV-2 entry into HL-mECs in the absence of detectable ACE2 expression. Following infection, HL-mECs were found to release a plethora of pro-inflammatory and pro-angiogenic molecules, as assessed by microarray analyses. This conditioned microenvironment stimulated HL-mECs to acquire an angiogenic phenotype. Proteome analysis confirmed a remodeling of SARS-CoV-2-infected HL-mECs to inflammatory and angiogenic responses and highlighted the expression of antiviral molecules as annexin A6 and MX1. These results support the hypothesis of a direct role of SARS-CoV-2-infected HL-mECs in sustaining vascular dysfunction during the early phases of infection. The construction of virus-host interactomes will be instrumental to identify potential therapeutic targets for COVID-19 aimed to inhibit HL-mEC-sustained inflammation and angiogenesis upon SARS-CoV-2 infection.

Abstract Background The introduction of cART has changed the morbidity and mortality patterns affecting HIV-infected (HIV+) individuals. The risk of breast cancer in HIV+ patients has now approached the general population risk. However, breast cancer has a more aggressive clinical course and poorer outcome in HIV+ patients than in general population, without correlation with the CD4 or virus particles count. These findings suggest a likely influence of HIV-1 proteins on breast cancer aggressiveness and progression. The HIV-1 matrix protein (p17) is expressed in different tissues and organs of successfully cART-treated patients and promotes migration of different cells. Variants of p17 (vp17s), characterized by mutations and amino acid insertions, differently from the prototype p17 (refp17), also promote B-cell proliferation and transformation. Methods Wound-healing assay, matrigel-based invasion assay, and anchorage-independent proliferation assay were employed to compare the biological activity exerted by refp17 and three different vp17s on the triple-negative human breast cancer cell line MDA-MB 231. Intracellular signaling was investigated by western blot analysis. Results Motility and invasiveness increased in cells treated with both refp17 and vp17s compared to untreated cells. The effects of the viral proteins were mediated by binding to the chemokine receptor CXCR2 and activation of the ERK1/2 signaling pathway. However, vp17s promoted MDA-MB 231 cell growth and proliferation in contrast to refp17-treated or not treated cells. Conclusions In the context of the emerging role of the microenvironment in promoting and supporting cancer cell growth and metastatic spreading, here we provide the first evidence that exogenous p17 may play a crucial role in sustaining breast cancer cell migration and invasiveness, whereas some p17 variants may also be involved in cancer cell growth and proliferation.

Abstract Recent data highlight the presence, in HIV-1-seropositive patients with lymphoma, of p17 variants (vp17s) endowed with B-cell clonogenicity, suggesting a role of vp17s in lymphomagenesis. We investigated the mechanisms responsible for the functional disparity on B cells between a wild-type p17 (refp17) and a vp17 named S75X. Here, we show that a single Arginine (R) to Glycine (G) mutation at position 76 in the refp17 backbone (p17R76G), as in the S75X variant, is per se sufficient to confer a B-cell clonogenic potential to the viral protein and modulate, through activation of the PTEN/PI3K/Akt signaling pathway, different molecules involved in apoptosis inhibition (CASP-9, CASP-7, DFF-45, NPM, YWHAZ, Src, PAX2, MAPK8), cell cycle promotion and cancer progression (CDK1, CDK2, CDK8, CHEK1, CHEK2, GSK-3 beta, NPM, PAK1, PP2C-alpha). Moreover, the only R to G mutation at position 76 was found to strongly impact on protein folding and oligomerization by altering the hydrogen bond network. This generates a conformational shift in the p17 R76G mutant which enables a functional epitope(s), masked in refp17, to elicit B-cell growth-promoting signals after its interaction with a still unknown receptor(s). Our findings offer new opportunities to understand the molecular mechanisms accounting for the B-cell growth-promoting activity of vp17s.

Capnocytophaga canimorsus infection was recently recognized as a zoonosis. We report the first case of fulminant septic shock in Italy caused by this pathogen. The patient, with a history of splenectomy, died at the main hospital in Brescia with a presumptive diagnosis of sepsis. PCR and sequencing on post mortem samples confirmed C. canimorsus as a causative organism. Our purpose is to alert medical professionals to the virulence of C. canimorsus in asplenic and immunocompromised patients. Keywords: Capnocytophaga canimorsus, septic shock, PCR, sequencing

Background: COVID-19 pandemic is requesting unprecedented efforts by health-care workers (HCWs) in all countries, and especially in Italy during the first semester of 2020. Methods: This is a retrospective, observational study conducted at the Spedali Civili General Hospital, in Brescia, Northern Italy during the SARS CoV-2 pandemic in the first semester of 2020. Serum samples from HCWs were tested for SARS-CoV-2 spike protein-specific antibodies. An online survey was used to collect demographic, clinical, and epidemiological data. Results: Of the 1893 HCWs included, 433 (22.9%) were found seropositive for SARS-CoV-2 IgG. The cumulative prevalence of SARS-CoV-2 infection (antibodies production or past positive RT-PCR on nasal/throat swab) was 25.1% (475/1893). Fifty-six out of 433 (13%) seropositive participants declared to have been asymptomatic during the study period. The development of COVID-19 signs or symptoms is the main determinant of seropositivity (OR: 11.3, p < 0.0001) along with their duration and severity. 40/290 (14.5%) HCWs with documented positive RT-PCR during the study period did not show any detectable antibody response. IgG levels positively correlate with age, COVID-19-compatible signs and symptoms experienced and their duration. Conclusions: In this study, carried out in one of the most affected areas in Europe, we demonstrate that most HCWs with COVID-19 related symptoms develop a spike protein-specific antibodies with potential neutralizing effect.

Avian reovirus p17 (ARV p17) is a non-structural protein known to activate autophagy, interfere with gene transcription and induce a significant tumor cell growth inhibition in vitro and in vivo. In this study, we show that ARV p17 is capable of exerting potent antiangiogenic properties. The viral protein significantly inhibited the physiological angiogenesis of human endothelial cells (ECs) by affecting migration, capillary-like structure and new vessel formation. ARV p17 was not only able to suppress the EC physiological angiogenesis but also rendered ECs insensitive to two different potent proangiogenic inducers, such as VEGF-A and FGF-2 in the three-dimensional (3D) Matrigel and spheroid assay. ARV p17 was found to exert its antiangiogenic activity by upregulating transcription and release of the well-known tumor suppressor molecule dipeptidyl peptidase 4 (DPP4). The ability of ARV p17 to impact on angiogenesis is completely new and highlights the “two compartments” activity of the viral protein that is expected to hamper the tumor parenchymal/stromal crosstalk. The complex antitumor activities of ARV p17 open the way to a new promising field of research aimed to develop new therapeutic approaches for treating tumor and cancer metastasis.

Human herpesvirus 6 (HHV-6) is a β-herpesvirus that is highly prevalent in the human population. HHV-6 comprises two recognized species (HHV-6A and HHV-6B). Despite different cell tropism and disease association, HHV-6A/B show high genome homology and harbor the conserved U94 gene, which is limited to HHV-6 and absent in all the other human herpesviruses. U94 has key functions in the virus life cycle and associated diseases, having demonstrated or putative roles in virus replication, integration, and reactivation. During natural infection, U94 elicits an immune response, and the prevalence and extent of the anti-U94 response are associated with specific diseases. Notably, U94 can entirely reproduce some virus effects at the cell level, including inhibition of cell migration, induction of cytokines and HLA-G expression, and angiogenesis inhibition, supporting a direct U94 role in the development of HHV-6-associated diseases. Moreover, specific U94 properties, such as the ability to modulate angiogenesis pathways, have been exploited to counteract cancer development. Here, we review the information available on this key HHV-6 gene, highlighting its potential uses.

The aim of this study is the characterization and genomic tracing by phylogenetic analyses of 59 new SARS-CoV-2 Italian isolates obtained from patients attending clinical centres in North and Central Italy until the end of April 2020. All but one of the newly-characterized genomes belonged to the lineage B.1, the most frequently identified in European countries, including Italy. Only a single sequence was found to belong to lineage B. A mean of 6 nucleotide substitutions per viral genome was observed, without significant differences between synonymous and non-synonymous mutations, indicating genetic drift as a major source for virus evolution. tMRCA estimation confirmed the probable origin of the epidemic between the end of January and the beginning of February with a rapid increase in the number of infections between the end of February and mid-March. Since early February, an effective reproduction number (Re) greater than 1 was estimated, which then increased reaching the peak of 2.3 in early March, confirming the circulation of the virus before the first COVID-19 cases were documented. Continuous use of state-of-the-art methods for molecular surveillance is warranted to trace virus circulation and evolution and inform effective prevention and containment of future SARS-CoV-2 outbreaks.

Human Metapneumovirus (HMPV) is a major cause of lower respiratory tract infections. HMPV infection has been hypothesized to alter dendritic cell (DC) immune response; however, many questions regarding HMPV pathogenesis within the infected lung remain unanswered. Here, we show that HMPV productively infects human lung microvascular endothelial cells (L-HMVECs). The release of infectious virus occurs for up to more than 30 days of culture without producing overt cytopathic effects and medium derived from persistently HMPV-infected L-HMVECs (secretome) induced monocyte-derived DCs to prime naïve CD4 T-cells toward a Th2 phenotype. Moreover, we demonstrated that infected secretomes trigger DCs to up-regulate OX40L expression and OX40L neutralization abolished the pro-Th2 effect that is induced by HMPV-secretome. We clarified secretome from HMPV by size exclusion and ultracentrifugation with the aim to characterize the role of viral particles in the observed pro-Th2 effect. In both cases, the percentage of IL-4-producing cells and expression of OX40L returned at basal levels. Finally, we showed that HMPV, per se, could reproduce the ability of secretome to prime pro-Th2 DCs. These results suggest that HMPV, persistently released by L-HMVECs, might take part in the development of a skewed, pro-Th2 lung microenvironment.

Although the advent of combined antiretroviral therapy has substantially improved the survival of HIV-1-infected individuals, non-AIDS-related diseases are becoming increasingly prevalent in HIV-1-infected patients. Persistent abnormalities in coagulation appear to contribute to excess risk for a broad spectrum of non-AIDS defining complications. Alterations in coagulation biology in the context of HIV infection seem to be largely a consequence of a chronically inflammatory microenvironment leading to endothelial cell (EC) dysfunction. A possible direct role of HIV-1 proteins in sustaining EC dysfunction has been postulated but not yet investigated. The HIV-1 matrix protein p17 (p17) is secreted from HIV-1-infected cells and is known to sustain inflammatory processes by activating ECs. The aim of this study was to investigate the possibility that p17-driven stimulation of human ECs is associated with increased production of critical coagulation factors. Here we show the involvement of autophagy in the p17-induced accumulation and secretion of von Willebrand factor (vWF) by ECs. In vivo experiments confirmed the capability of p17 to exert a potent pro-coagulant activity soon after its intravenous administration.

HIV-1 diversity is increasing in European countries due to immigration flows, as well as travels and human mobility, leading to the circulation of both new viral subtypes and new recombinant forms, with important implications for public health. We analyzed 710 HIV-1 sequences comprising protease and reverse-transcriptase (PR/RT) coding regions, sampled from 2011 to 2017, from naive patients in Spedali Civili Hospital, Brescia, Italy. Subtyping was performed by using a combination of different tools; the phylogenetic analysis with a structured coalescence model and Makarov Chain Monte Carlo was used on the datasets, to determine clusters and evolution. We detected 304 (43%) patients infected with HIV-1 non-B variants, of which only 293 sequences were available, with four pure subtypes and five recombinant forms; subtype F1 (17%) and CRF02_AG (51.1%) were most common. Twenty-five transmission clusters were identified, three of which included >10 patients, belonging to subtype CRF02_AG and subtype F. Most cases of alleged transmission were between heterosexual couples. Probably due to strong migratory flows, we have identified different subtypes with particular patterns of recombination or, as in the case of the subtype G (18/293, 6.1%), to a complete lack of relationship between the sequenced strains, revealing that they are all singletons. Continued HIV molecular surveillance is most important to analyze the dynamics of the boost of transmission clusters in order to implement public health interventions aimed at controlling the HIV epidemic.

In recent years immunomodulators have gained a strong interest and represent nowadays an active expanding area of research for the control of microbial diseases and for their therapeutic potential in preventing, treating and reducing the morbidity and mortality of different diseases. Pidotimod (3-L-pyroglutamyl-L-thiaziolidine-4carboxylic acid, PDT) is a synthetic dipeptide, which possesses immunomodulatory properties and exerts a well-defined pharmacological activity against infections, but its real mechanism of action is still undefined. Here, we show that PDT is capable of activating tyrosine phosphorylation-based cell signaling in human primary monocytes and triggering rapid adhesion and chemotaxis. PDT-induced monocyte migration requires the activation of the PI3K/Akt signaling pathway and chemokine receptor CXCR3. Indeed, a mAb to CXCR3 and a specific receptor inhibitor suppressed significantly PDT-dependent chemotaxis, and CXCR3-silenced primary monocytes lost responsiveness to PDT chemoattraction. Moreover, our results highlighted that the PDT-induced migratory activity is sustained by the CXCR3A isoform, since CXCR3-transfected L1.2 cells acquired responsiveness to PDT stimulation. Finally, we show that PDT, as CXCR3 ligands, is also able to direct the migration of IL-2 activated T cells, which express the highest levels of CXCR3 among CXCR3-expressing cells. In conclusion, our study defines a chemokine-like activity for PDT through CXCR3A and points on the possible role that this synthetic dipeptide may play in leukocyte trafficking and function. Since recent studies have highlighted diverse therapeutic roles for molecules which activates CXCR3, our findings call for an exploration of using this dipeptide in different pathological processes.

Cyclin-dependent kinase-5 (Cdk5) is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p(Tyr15)Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine) resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N) kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C) as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP) vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition.

The HIV-1 matrix protein p17 is a structural protein that can act in the extracellular environment to deregulate several functions of immune cells, through the interaction of its NH(2)-terminal region with a cellular surface receptor (p17R). The intracellular events triggered by p17/p17R interaction have been not completely characterized yet. In this study we analyze the signal transduction pathways induced by p17/p17R interaction and show that in Raji cells, a human B cell line stably expressing p17R on its surface, p17 induces a transient activation of the transcriptional factor AP-1. Moreover, it was found to upregulate pERK1/2 and downregulate pAkt, which are the major intracellular signalling components involved in AP-1 activation. These effects are mediated by the COOH-terminal region of p17, which displays the capability of keeping PTEN, a phosphatase that regulates the PI3K/Akt pathway, in an active state through the serine/threonine (Ser/Thr) kinase ROCK. Indeed, the COOH-terminal truncated form of p17 (p17Δ36) induced activation of the PI3K/Akt pathway by maintaining PTEN in an inactive phosphorylated form. Interestingly, we show that among different p17s, a variant derived from a Ugandan HIV-1 strain, named S75X, triggers an activation of PI3K/Akt signalling pathway, and leads to an increased B cell proliferation and malignant transformation. In summary, this study shows the role of the COOH-terminal region in modulating the p17 signalling pathways so highlighting the complexity of p17 binding to and signalling through its receptor(s). Moreover, it provides the first evidence on the presence of a p17 natural variant mimicking the p17Δ36-induced signalling in B cells and displaying the capacity of promoting B cell growth and tumorigenesis.