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Breastmilk protects newborns from infections through specific and nonspecific compounds. This study investigated the neutralizing activity against SARS-CoV-2 of breastmilk from SARS-CoV-2 negative, unvaccinated mothers, and compared it to that from infected nursing mothers. We enrolled women after COVID-19 swab testing results upon maternity admission, and divided them into two groups: group A, COVID-19-positive mothers, and group B, negative mothers. Breastmilk was randomly sampled at 2, 7, and 20 days postpartum. We collected 19 samples for Group A and 41 for Group B. A microneutralization assay was used to determine the 50% neutralization (NT50) titre. The presence of neutralizing antibodies was also determined. Group A had 100% neutralizing samples at T0, declining in T1 and T2. Group B samples exhibited neutralizing activity mostly at T1 (90%). Negative mothers' samples showed no correlation between NT50 titres and antibodies' presence, suggesting that non-specific breast milk components may exert antiviral action against SARS-CoV-2.

The ongoing emergency provoked by the SARS-CoV-2 pandemic demands the development of technologies to mitigate the spread of infection, and UV irradiation is a technique that can efficiently address this issue. However, proper use of UV equipment for disinfection requires an understanding of how the effects on SARS-CoV-2 are dependent on certain parameters. In this work, we determined the UV-C inactivation constant k for SARS-CoV-2 using an LED source at λ = 280 nm. Specifically, a Log3 reduction was measured after irradiation for 24 min with a delivered UV-C dose of 23 J m

Lysozyme (E.C. 3.2.1.17), an about 14 kDa protein and pI 11, widely spread in nature, is present in humans mainly in milk, saliva, and intestinal mucus as a part of innate defense mechanisms. It is endowed with antimicrobial activity due to its action as an N-acetylmuramidase, cleaving the 1-4β glycosidic linkage in the peptidoglycan layer of Gram-positive bacteria. This antimicrobial activity is exerted only against a limited number of Gram-negative bacteria. Different action mechanisms are proposed to explain its activity against Gram-negative bacteria, viruses, and fungi. The antiviral activity prompted the study of a possible application of lysozyme in the treatment of SARS-CoV-2 infections. Among the different sources of lysozyme, the chicken egg albumen was chosen, being the richest source of this protein (c-type lysozyme, 129 amino acids). Interestingly, the activity of lysozyme hydrochloride against SARS-CoV-2 was related to the heating (to about 100 °C) of this molecule. A chemical–physical characterization was required to investigate the possible modifications of native lysozyme hydrochloride by heat treatment. The FTIR analysis of the two preparations of lysozyme hydrochloride showed appreciable differences in the secondary structure of the two protein chains. HPLC and NMR analyses, as well as the enzymatic activity determination, did not show significant modifications.

Background Evidence showed the role of biomarkers in predicting severity and mortality of coronavirus disease 2019 (COVID-19). We evaluated associations between several biomarkers measured throughout the follow-up and COVID-19 mortality using the joint modelling (JM) approach, the candidate tool for this kind of data. Methods Between February and May 2020, a total of 403 COVID-19 patients were admitted. Baseline characteristics included sex and age, whereas biomarkers included lymphocytes, neutrophils, D-dimer, ferritin, C-reactive protein, glucose and lactate dehydrogenase (LDH). Hazard ratios (HR) and 95% confidence interval (CI) were estimated through JM using a Bayesian approach. We fitted univariable and multivariable JMs including a single biomarker and the set of all biomarkers, respectively. Results In univariable JMs, all biomarkers were significantly associated with COVID-19 mortality. In the multivariable JM, HRs were 1.78 (95% CI: 1.13–2.87) per doubling of neutrophils levels, 1.49 (95% CI: 1.19–1.95) per doubling of C-reactive protein levels, 2.66 (95% CI: 1.45–4.95) per an increase of 100 mg/dL of glucose, and 1.31 (95% CI: 1.12–1.55) per an increase of 100 U/L of LDH. No evidence of association was observed for ferritin and lymphocytes in the multivariable JM. Men had a higher risk of COVID-19 mortality than women (HR = 1.75; 95% CI: 1.07–2.80) and age showed the strongest effect with risk starting to rapidly increase from 60 years. Conclusions These findings using JM confirm the usefulness of biomarkers in assessing COVID-19 severity and mortality. Monitoring trend patterns of such biomarkers can provide additional help in tailoring the more appropriate care pathway.

Alongside vaccines, antiviral drugs are becoming an integral part of our response to the SARS-CoV-2 pandemic. Nirmatrelvir – an orally available inhibitor of the 3-chymotrypsin-like cysteine protease – has been shown to reduce the risk of progression to severe COVID-19. However, the impact of nirmatrelvir treatment on the development of SARS-CoV-2-specific adaptive immune responses is unknown. Here, by using a mouse model of SARS-CoV-2 infection, we show that nirmatrelvir administration early after infection blunts the development of SARS-CoV-2-specific antibody and T cell responses. Accordingly, upon secondary challenge, nirmatrelvir-treated mice recruited significantly fewer memory T and B cells to the infected lungs and to mediastinal lymph nodes, respectively. Together, the data highlight a potential negative impact of nirmatrelvir treatment with important implications for clinical management and might help explain the virological and/or symptomatic relapse after treatment completion reported in some individuals.

SARS-CoV-2 variants of concern (VOCs) represent an alarming threat as they show altered biological behavior and may escape vaccination effectiveness. Broad-spectrum antivirals could play an important role to control infections. The activity of

Current knowledge on

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was isolated from the oro/pharyngeal swabs of two Italian COVID-19 patients, physicians in a COVID-19 division hospital, with different courses of the disease. The complete genome sequences show that the two isolates belong to the B1.1 lineage, but contain a nucleotide mutation in the ORF6, leading to a stop codon and to the deletion of 6 amino acids in the C terminus. This deletion was unique, compared to the currently available sequences deposited in the GISAID and GenBank database. It did not affect the in vitro viral replication, neither the neutralizing activities of the patients' antibodies. Based on homology analysis with other Coronaviruses, the two isolated lacked the ORF6 aminoacidic portion responsible for the inhibition of the antiviral Interferon (IFN)-based host response. IFN seems to have a dual role of in SARS-CoV-2 infected patients: not only antiviral activity, but also a detrimental role in case of excessive production. A deletion in the SARS-CoV-2 ORF6 protein might have a specific, still unknown role in the viral pathogenesis.

Since the declaration of the COVID-19 pandemic, the number of kidney transplants (KT) performed worldwide has plummeted. Besides the generalised healthcare crisis, this unprecedented drop has multiple explanations such as the risk of viral transmission through the allograft, the perceived increase in SARS-CoV-2-related morbidity and mortality in immunocompromised hosts, and the virtual “safety” of dialysis while awaiting effective antiviral prophylaxis or treatment. Our institution, operating at the epicentre of the COVID-19 pandemic in Italy, has continued the KT programme without pre-set limitations. In this single-centre retrospective observational study with one-year follow-up, we assessed the outcomes of patients who had undergone KT (KTR) or remained on the transplant waiting list (TWL), before (Pre-COV) or during (COV) the pandemic. The main demographic and clinical characteristics of the patients on the TWL or receiving a KT were very similar in the two periods. The pandemic did not affect post-transplant recipient and allograft loss rates. On the contrary, there was a trend toward higher mortality among COV-TWL patients compared to Pre-COV-TWL subjects. Such a discrepancy was primarily due to SARS-CoV-2 infections. Chronic exposure to immunosuppression, incidence of delayed allograft function, and rejection rates were comparable. However, after one year, COV-KTR showed significantly higher median serum creatinine than Pre-COV-KTR. Our data confirm that KT practice could be safely maintained during the COVID-19 pandemic, with excellent patient- and allograft-related outcomes. Strict infection control strategies, aggressive follow-up monitoring, and preservation of dedicated personnel and resources are key factors for the optimisation of the results in case of future pandemics.

More than two years have passed since the viral outbreak that led to the novel infectious respiratory disease COVID-19, caused by the SARS-CoV-2 coronavirus. Since then, the urgency for effective treatments resulted in unprecedented efforts to develop new vaccines and to accelerate the drug discovery pipeline, mainly through the repurposing of well-known compounds with broad antiviral effects. In particular, antiparasitic drugs historically used against human infections due to protozoa or helminth parasites have entered the main stage as a miracle cure in the fight against SARS-CoV-2. Despite having demonstrated promising anti-SARS-CoV-2 activities in vitro, conflicting results have made their translation into clinical practice more difficult than expected. Since many studies involving antiparasitic drugs are currently under investigation, the window of opportunity might be not closed yet. Here, we will review the (controversial) journey of these old antiparasitic drugs to combat the human infection caused by the novel coronavirus SARS-CoV-2.

To investigate the presence of genetic material of viral agents and the serum level of inflammatory cytokines in patients submitted to carotid endarterectomy having vulnerable versus stable atherosclerotic plaques.Data of patients consecutively submitted to carotid endarterectomy for a significant stenosis from July 2019 to December 2019 were prospectively collected. The genetic material of Epstein-Barr (EBV), CitoMegalo (CMV), Herpes Simplex (HSV), Varicella-Zoster (VZV) and Influenza (IV) Viruses was searched in the patient's plaques, both in the "mid" of the plaque and in an adjacent lateral portion of no-plaque area. The serum levels of TNF-α, IL-1β, IL-6, IL10 and CCL5 were determined. The obtained results were then correlated to the histologic vulnerability of the removed carotid plaque. P values0.05 were considered statistically significant.Data of 50 patients were analyzed. A vulnerable plaque was found in 31 patients (62%). The genome of CMV, HSV, VZV and IV was not found in any of the vascular samples, while the EBV genome was found in the "mid" of 2 vulnerable plaques, but not in their respective control area. Eighty-two percent of patients who did not receive anti-IV vaccination (23/28) had vulnerable carotid plaque, compared with 36% of vaccinated patients (8/22, P = 0.001). Serum levels of TNF-α and IL-6 were higher in patients with a vulnerable plaque compared to patients with a stable plaque (73.6 ± 238.2 vs. 3.9 ± 13.1 pg/ml, P= 0.01, and 45.9 ± 103.6 vs. 10.1 ± 25.3 pg/ml, P= 0.01, respectively), independent of comorbidities, viral exposure or flu vaccination.The EBV genome was found in the "core" of 2 vulnerable carotid plaques, but not in their respective adjacent control. Influenza vaccination was associated with a lower incidence of carotid plaque vulnerability. Serum levels of TNF-α and IL-6 were higher in patients with a vulnerable plaque compared to patients with a stable plaque.

The present research study reports the development of plastic antibodies based on Molecularly Imprinted Polymers (MIPs) capable of selectively binding a portion of the novel coronavirus SARS-CoV-2 spike protein. Indeed, molecular imprinting represents a very promising and attractive technology for the synthesis of MIPs characterized by specific recognition abilities for a target molecule. Given these characteristics, MIPs can be considered tailor-made synthetic antibodies obtained by a templating process. After in silico analysis, imprinted nanoparticles were synthesized by inverse microemulsion polymerization and their ability to prevent the interaction between ACE2 and the receptor-binding domain of SARS-CoV-2 was investigated. Of relevance, the developed synthetic antibodies are capable of significantly inhibiting virus replication in Vero cell culture, suggesting their potential application in the treatment, prevention and diagnosis of SARS-CoV-2 infection.

BACKGROUND: Allograft artery mycotic aneurysm (MA) represents a rare but life-threatening complication of kidney transplantation. Graftectomy is widely considered the safest option. Due to the rarity of the disease and the substantial risk of fatal consequences, experience with conservative strategies is limited. To date, only a few reports on surgical repair have been published. We describe a case of true MA successfully managed by aneurysm resection and arterial re-anastomosis. CASE SUMMARY: An 18-year-old gentleman, on post-operative day 70 after deceased donor kidney transplantation, presented with malaise, low urinary output, and worsening renal function. Screening organ preservation fluid cultures, collected at the time of surgery, were positive for Candida albicans. Doppler ultrasound and contrast-enhanced computer tomography showed a 4-cm-sized, saccular aneurysm of the iuxta-anastomotic segment of the allograft artery, suspicious for MA. The lesion was wide-necked and extended to the distal bifurcation of the main arterial branch, thus preventing endovascular stenting and embolization. After multidisciplinary discussion, the patient underwent surgical exploration, aneurysm excision, and re-anastomosis between the stump of the allograft artery and the internal iliac artery. The procedure was uneventful. Histology and microbiology evaluation of the surgical specimen confirmed the diagnosis of MA caused by Candida infection. Three years after the operation, the patient is doing very well with excellent allograft function and no signs of recurrent disease. CONCLUSION: Surgical repair represents a feasible option in carefully selected patients with allograft artery MA. Anti-fungal prophylaxis is advised when preservation fluid cultures are positive.

Human endogenous retroviruses (HERV) are remnants of exogenous retroviral infections, representing 8% of the human genome. Their regulation is based on the DNA methylation of promoters, the long terminal repeats (LTRs). Transcripts from HERV have been associated with cancers, but reports concerning HERV expression in colorectal cancer remain sporadic. Sixty-three patients with advanced stages of colorectal cancer were enrolled in this study. The expressions of HERV env gene, and HERV-H, -K, -R and -P LTRs and Alu, LINE-1 methylation levels, were investigated in the tumor, normal adjacent tissues, and, where possible, blood and plasmatic extracellular vesicles (EVs). Associations among HERV env expression, methylation status and clinical characteristics were evaluated. No differences were observed in HERV env gene expression levels among the clinical specimens, while Alu, LINE-1, HERV-H and -K LTRs were demethylated in the tumor compared to the normal adjacent tissues (p

Introduction: The ongoing coronavirus disease 19 (COVID-19) outbreak involves the pediatric population, but to date, few reports have investigated the circulation of variants among children. Material and Methods: In this retrospective study, non-hospitalized pediatric patients with SARS-CoV-2-positive nasopharyngeal swabs (NPS) were enrolled at the Institute for Maternal and Child Health-IRCCS Burlo Garofolo, Trieste (Italy), from November 2020 to January 2022. SARS-CoV-2 variants were identified by in vitro viral isolation, amplification, automatic sequencing of the receptor binding domain (RBD) of the SARS-CoV-2 spike coding gene, and subsequent next-generation sequencing. The growth curves of the isolated strains were defined in vitro by infecting Vero-E6 cells and quantifying the viral load in the supernatants up to 72 h post-infection by qRT–PCR. The neutralization activity of sera obtained from a COVID-19 vaccinated subject, recovered (2020) patient, vaccinated and recovered (2021) patient, and seronegative subject was assessed by microneutralization assay against the different variants. Results: In total, 32 SARS-CoV-2-positive children, 16 (50%) females, with a median age of 1.4 years (range: 1 day–13 years), were enrolled. The D614G amino acid substitution was detected in all isolated and amplified viral strains. Of the 32 isolates, 4 (12.5%) carried a nonsynonymous nucleotide mutation leading to the N439K (3/4), lineage B.1.258 (∆H69/∆V70), and S477N (1/4) substitution. In 7/32 (21.8%) isolates, amino acid substitutions allowed the identification of a delta variant, lineage B.1.617.2-AY.43, and in 1/32 (3.1%), the Omicron strain (B.1.1.529.BA1) was identified. The growth curves of the B.1, B.1.258 (∆H69/∆V70), B.1.617.2-AY.43, and B.1.1.529.BA1 variants did not show any significant differences. A reduction in the serum neutralizing activity against B.1.258 (∆H69/∆V70) only in a vaccinated subject (1.7-fold difference), against B.1.617.2-AY.43 in a vaccinated subject and in recovered patients (12.7 and ≥2.5-fold differences, respectively), and against B.1.1.529.BA1 variant (57.6- and 1.4-fold differences in vaccinated and in vaccinated and recovered patients) were observed compared to the B.1 variant. Conclusions: SARS-CoV-2 variants carrying the B.1.258 (∆H69/∆V70) and S477N substitutions were reported here in a pediatric population for the first time. Although the growth rates of the isolated strains (B.1.258, B.1.617.2-AY.43, B.1.1.529.BA1) did not differ from the B.1 variant, neutralizing activity of the sera from vaccinated subjects significantly decreased against these variants. Attention should be devoted to the pediatric population to prevent the spread of new SARS-CoV-2 variants in an unvaccinated and predominantly naive population.

ObjectivesThe COVID-19 pandemic represents a difficult challenge for the whole of humanity. Sports, in which contact between athletes is essential, became impossible to practice without the risk of viral spread. Athletes of the national teams are a particular subgroup of the population for whom there is an important need for protection and the implementation of targeted preventive measures. The present report describes the protocol that was developed to answer the urgent protection need for athletes during COVID-19 pandemic. The protocol aimed at demonstrating the feasibility of a rigid prevention intervention to prevent outbreaks and infections in terms of COVID-19 as well as in other potential future pandemics from pathogens with similar path of transmission.MethodsThe study was conducted in rowing para-thletes training of the Paralympic Games in Tokyo2020. It was designed to create an anti-COVID-19 “protection bubble” with the aim to isolate para-athletes and their technical support team during pre-Olympic retreats. The “bubble” development relied on a carefully conducted protocol of repeated antigen and molecular COVID-19 tests on nasal and oropharyngeal fluids among all participants carried out before, during and at the end of each retreat.ResultsDuring the 10 months of protocol implementation there were no COVID-19 outbreaks among the para-athletes and technical personnel during the retreats. In total, 552 PCR tests and 298 antigen-based tests were performed for an average number of 42 test per athlete. The number of retreat participants was larger (n = 23) in the beginning of the year due to the Paralympic selection rounds and smaller at the end of the study period (n = 12).ConclusionThe protocol has indicated that it is possible to implement an anti-COVID-19 protection protocol where athletes and technical staff can train and compete in safe conditions. The study showed that it is feasible to implement a rigid prevention protocol for athletes and technical staff based on repeated COVID-19 antigenic and molecular tests for a long period of training with excellent participation and compliance.

The careful tailoring of the most appropriate immunosuppressive strategy for recipients of a kidney transplant (KT) regularly faces a risk of complications that may harm the actual graft and affect patient survival [...]

In the novel pandemic of Coronavirus Disease 2019, high levels of pro-inflammatory cytokines lead to endothelial activation and dysfunction, promoting a pro-coagulative state, thrombotic events, and microvasculature injuries. The aim of the present work was to investigate the effect of SARS-CoV-2 on pro-inflammatory cytokines, tissue factor, and chemokine release, with Human Microvascular Endothelial Cells (HMEC-1). ACE2 receptor expression was evaluated by western blot analysis. SARS-CoV-2 infection was assessed by one-step RT-PCR until 7 days post-infection (p.i.), and by Transmission Electron Microscopy (TEM). IL-6, TNF-α, IL-8, IFN-α, and hTF mRNA expression levels were detected by RT-PCR, while cytokine release was evaluated by ELISA. HMEC-1 expressed ACE2 receptor and SARS-CoV-2 infection showed a constant viral load. TEM analysis showed virions localized in the cytoplasm. Expression of IL-6 at 24 h and IFN-α mRNA at 24 h and 48 h p.i. was higher in infected than uninfected HMEC-1 (

In an immune-competent context nivolumab showed long-term benefit in overall survival in recurrent/metastatic head and neck squamous cell carcinoma (HNSCC); however, in special cancer population such as these patients with immunodeficiency and viral infections, data on checkpoint inhibitors (ICI) activity are scant. Herein, we report a patient with a Human papilloma virus (HPV)-related oropharyngeal cancer (OPC) and CD4 lymphocytopenia. After a first-line treatment complete remission, the patient experienced Human Polyomavirus (JCV) infection in the brain. Consequently, to the recovery from progressive multifocal leukoencephalopathy (PML) the patient metastasized and was enrolled in a single-arm trial with nivolumab (EudraCT number: 2017-000562-30). A complete and durable response (more 3 years) was observed after 10 nivolumab injections Q2wks, interrupted for persistent drug related G2 diarrhea and a syndrome of inappropriate antidiuretic hormone secretion. We describe the circulating immune profile (before-, during-, and after nivolumab), consistent with the clinical history. Moreover, during nivolumab treatment, brain MRI evidenced the presence of small punctuate areas of contrast enhancement, reflecting a mild immune response in perivascular spaces. By cytofluorimetry, we observed that during JCV infection the CD4/CD8 ratio of the patient was under the normal values. After JCV infection recovery and before nivolumab treatment, CD4/CD8 ratio reached the normality threshold, even if the CD4+ T cell count remained largely under the normal values. During ICI, gene expression xCell analyses of circulating immune cells of the patient, showed a progressive normalization of the total immune profile, with significant boost in CD4+ and CD8+ T cells and a reduction in NK T, comparable to the circulating immune profile of reference tumor-free HNSCC patients. The present case supports the activity of ICI in a population of special cancer patients; whether JCV and HPV infections (alone or together) might have a possible role as immune booster(s), require further investigations.

Current knowledge on Leishmania infection after kidney transplantation (KT) is limited. In order to offer a comprehensive guide for the management of post-transplant Leishmaniasis, we performed a systematic review following the latest PRISMA Checklist and using PubMed, Scopus, and Embase as databases. No time restrictions were applied, including all English-edited articles on Leishmaniasis in KT recipients. Selected items were assessed for methodological quality using a modified Newcastle–Ottawa Scale. Given the nature and quality of the studies (case reports and retrospective uncontrolled case series), data could not be meta-analyzed. A descriptive summary was therefore provided. Eventually, we selected 70 studies, describing a total of 159 cases of Leishmaniasis. Most of the patients were adult, male, and Caucasian. Furthermore, they were frequently living or travelling to endemic regions. The onset of the disease was variable, but more often in the late transplant course. The clinical features were basically similar to those reported in the general population. However, a generalized delay in diagnosis and treatment could be detected. Bone marrow aspiration was the preferred diagnostic modality. The main treatment options included pentavalent antimonial and liposomal amphotericin B, both showing mixed results. Overall, the outcomes appeared as concerning, with several patients dying or losing their transplant.

Introduction: The ongoing coronavirus disease 19 (COVID-19) outbreak involves the pediatric population, but to date, few reports have investigated the circulation of variants among children. Material and Methods: In this retrospective study, non-hospitalized pediatric patients with SARS-CoV-2-positive nasopharyngeal swabs (NPS) were enrolled at the Institute for Maternal and Child Health-IRCCS Burlo Garofolo, Trieste (Italy), from November 2020 to January 2022. SARS-CoV-2 variants were identified by in vitro viral isolation, amplification, automatic sequencing of the receptor binding domain (RBD) of the SARS-CoV-2 spike coding gene, and subsequent nextgeneration sequencing. The growth curves of the isolated strains were defined in vitro by infecting Vero-E6 cells and quantifying the viral load in the supernatants up to 72 h post-infection by qRT– PCR. The neutralization activity of sera obtained from a COVID-19 vaccinated subject, recovered (2020) patient, vaccinated and recovered (2021) patient, and seronegative subject was assessed by microneutralization assay against the different variants. Results: In total, 32 SARS-CoV-2-positive children, 16 (50%) females, with a median age of 1.4 years (range: 1 day–13 years), were enrolled. The D614G amino acid substitution was detected in all isolated and amplified viral strains. Of the 32 isolates, 4 (12.5%) carried a nonsynonymous nucleotide mutation leading to the N439K (3/4), lineage B.1.258 (∆H69/∆V70), and S477N (1/4) substitution. In 7/32 (21.8%) isolates, amino acid substitutions allowed the identification of a delta variant, lineage B.1.617.2-AY.43, and in 1/32 (3.1%), the Omicron strain (B.1.1.529.BA1) was identified. The growth curves of the B.1, B.1.258 (∆H69/∆V70), B.1.617.2-AY.43, and B.1.1.529.BA1 variants did not show any significant differences. A reduction in the serum neutralizing activity against B.1.258 (∆H69/∆V70) only in a vaccinated subject (1.7-fold difference), against B.1.617.2-AY.43 in a vaccinated subject and in recovered patients (12.7 and ≥2.5-fold differences, respectively), and against B.1.1.529.BA1 variant (57.6-and 1.4-fold differences in vaccinated and in vaccinated and recovered patients) were observed compared to the B.1 variant. Conclusions: SARS-CoV-2 variants carrying the B.1.258 (∆H69/∆V70) and S477N substitutions were reported here in a pediatric population for the first time. Although the growth rates of the isolated strains (B.1.258, B.1.617.2-AY.43, B.1.1.529.BA1) did not differ from the B.1 variant, neutralizing activity of the sera from vaccinated subjects significantly decreased against these variants. Attention should be devoted to the pediatric population to prevent the spread of new SARS-CoV-2 variants in an unvaccinated and predominantly naive population.

The COVID-19 pandemic represents a difficult challenge for the whole of humanity. Sports, in which contact between athletes is essential, became impossible to practice without the risk of viral spread. Athletes of the national teams are a particular subgroup of the population for whom there is an important need for protection and the implementation of targeted preventive measures. The present report describes the protocol that was developed to answer the urgent protection need for athletes during COVID-19 pandemic. The protocol aimed at demonstrating the feasibility of a rigid prevention intervention to prevent outbreaks and infections in terms of COVID-19 as well as in other potential future pandemics from pathogens with similar path of transmission.The study was conducted in rowing para-thletes training of the Paralympic Games in Tokyo2020. It was designed to create an anti-COVID-19 "During the 10 months of protocol implementation there were no COVID-19 outbreaks among the para-athletes and technical personnel during the retreats. In total, 552 PCR tests and 298 antigen-based tests were performed for an average number of 42 test per athlete. The number of retreat participants was larger (The protocol has indicated that it is possible to implement an anti-COVID-19 protection protocol where athletes and technical staff can train and compete in safe conditions. The study showed that it is feasible to implement a rigid prevention protocol for athletes and technical staff based on repeated COVID-19 antigenic and molecular tests for a long period of training with excellent participation and compliance.

BackgroundSARS-CoV-2 variants of concern (VOC) represent an alarming threat as they show altered biological behavior and may escape vaccination effectiveness. Some exhibit increased pathogenicity and transmissibility compared to the original wild type WUHAN (Hu-1). Broad-spectrum antivirals could complement and further enhance preventive benefits achieved through SARS-CoV-2 vaccination campaignsMethodsThe anti-coronavirus activity of Echinacea purpurea (Echinaforce® extract, EF) against (i) VOCs B1.1.7 (alpha), B.1.351.1 (beta), P.1 (gamma), B1.617.2 (delta), AV.1 (Scottish) and B1.525 (eta), (ii) SARS-CoV-2 spike (S) protein-pseudotyped viral particles and reference strain OC43 as well as (iii) wild-type SARS-CoV-2 (Hu-1) were analyzed. Molecular dynamics (MD) were applied to study interaction of Echinacea’s phytochemical markers with known pharmacological viral and host cell targets.ResultsEF extract broadly inhibited propagation of all investigated SARS-CoV-2 VOCs as well as entry of SARS-CoV-2 pseudoparticles at EC50’s ranging from 3.62 to 12.03 µg/ml. Preventive addition of 20 µg/ml EF to epithelial cells significantly reduced sequential infection with SARS-CoV-2 (Hu-1) as well as with the common human strain OC43. MD analyses showed constant binding affinities to Hu-1, B1.1.7, B.1.351, P.1 and B1.617.2-typic S protein variants for alkylamides, caftaric acidand feruoyl-tartaric acid in EF extract. They further indicated that the EF extract could possibly interact with TMPRSS-2, a serine protease required for virus endocytosis.ConclusionsEF extract demonstrated stable antiviral activity across 6 tested VOCs, which is likely due to the constant affinity of the contained phytochemical marker substances to all spike variants. A possible interaction of EF with TMPRSS-2 partially would explain cell protective benefits of the extract by inhibition of endocytosis. EF may therefore offer a supportive addition to vaccination endeavors in the control of existing and future SARS-CoV-2 virus mutations.

The current SARS-CoV-2 pandemic, along with the likelihood that new coronavirus strains will appear in the nearby future, highlights the urgent need to develop new effective antiviral agents. In this scenario, emerging host-targeting antivirals (HTAs), which act on host-cell factors essential for viral replication, are a promising class of antiviral compounds. Here we show that a new class of HTAs targeting peptidylarginine deiminases (PADs), a family of calcium-dependent enzymes catalyzing protein citrullination, is endowed with a potent inhibitory activity against human beta-coronaviruses (HCoVs). Specifically, we show that infection of human fetal lung fibroblasts with HCoV-OC43 leads to enhanced protein citrullination through transcriptional activation of PAD4, and that inhibition of PAD4-mediated citrullination with either of the two pan-PAD inhibitors Cl-A and BB-Cl or the PAD4-specific inhibitor GSK199 curbs HCoV-OC43 replication. Furthermore, we show that either Cl-A or BB-Cl treatment of African green monkey kidney Vero-E6 cells, a widely used cell system to study beta-CoV replication, potently suppresses HCoV-OC43 and SARS-CoV-2 replication. Overall, our results demonstrate the potential efficacy of PAD inhibitors, in suppressing HCoV infection, which may provide the rationale for the repurposing of this class of inhibitors for the treatment of COVID-19 patients.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract illness in young children and can also cause influenza-like illness (ILI). Here we investigated the epidemiological features of RSV infection in pediatric ILI cases in Lombardy (a region in Northern Italy accounting nearly 10 million inhabitants) from 2014-2015 to 2020-2021 winter seasons.Data for this study were retrieved and statistically analyzed from the database of virological influenza surveillance of the regional reference laboratory for Lombardy within the Italian influenza surveillance network (InfluNet).RSV accounted for nearly 19% of pediatric ILI with a risk of infection nearly two-fold greater than that of individuals ≥15 years. RSV positivity rate increased to 28% considering 0-5 years old children. Although in children ≤5 years the risk of infection from influenza viruses resulted nearly two-fold higher than the risk of RSV infection, the age group 4-6 months and 7-12 months showed a five-fold greater risk of infection from RSV than from influenza. Children ≤5 years of age with pre-existing underlying health conditions had a nearly five-fold greater risk of getting RSV infection than otherwise healthy 0-5 years old children. RSV was identified in ILI cases15 years of age in all considered winter seasons except in the 2020-2021 season.Sentinel surveillance of ILI allowed us to identify groups at higher risk of RSV and influenza infection and to define the start, duration, timing, and intensity of the RSV and influenza community circulation. This surveillance approach can be implemented to assess the RSV circulation and impact in a real-time manner.

Endothelial cells represent one of the first cell types encountered by Leishmania promastigotes when inoculated into the skin of the human hosts by the bite of phlebotomine sand flies. However, little is known on their role in the early recruitment of phagocytic cells and in the establishment of the infection. Initially, neutrophils, rapidly recruited to the site of promastigotes deposition, phagocytize Leishmania promastigotes, which elude the killing mechanisms of the host cells, survive, and infect other phagocytic cells. Here, we show that Leishmania promastigotes co-incubated with HMEC-1, a microvascular endothelial cell line, exhibited significant morphological changes and loss of infectivity. Moreover, promastigotes of different Leishmania species stimulated the production of CXCL8 by HMEC-1 in a dose- and TLR4-dependent manner. Interestingly, we observed that the conditioned media from Leishmania-stimulated HMEC-1 cells attracted leukocytes, mostly neutrophils, after 2 h of incubation. After 24 h, a higher percentage of monocytes was detected in conditioned media of unstimulated HMEC-1 cells, whereas neutrophils still predominated in conditioned medium from Leishmania-stimulated cells. The same supernatants did not contain CCL5, a chemokine recruiting T cells and monocytes. On the contrary, inhibition of the production of CCL5 induced by TNF-α was seen. These data indicate that the interaction of Leishmania promastigotes with endothelial cells leads to the production of chemokines and the recruitment of neutrophils, which contribute to the establishment of Leishmania infection.

INTRODUCTION. Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract illness in young children and can also cause influenza-like illness (ILI). Here we investigated the epidemiological features of RSV infection in pediatric ILI cases in Lombardy (a region in Northern Italy accounting nearly 10-million inhabitants) from 2014-2015 to 2020-2021 winter seasons. MATERIAL AND METHODS. Data for this study were retrieved and statistically analyzed from the database of virological influenza surveillance of the regional reference laboratory for Lombardy within the Italian influenza surveillance network (InfluNet). RESULTS. RSV accounting for nearly 19% of pediatric ILI with a risk of infection nearly 2-fold greater than that of individuals ≥15 years. The RSV positivity rate increased to 28% considering 0-5 years old children. Although in children ≤5 years the risk of infection from influenza viruses resulted nearly 2-fold higher than the risk of RSV infection, the age group 4-6 months and 7-12 months showed 5-fold greater risk of infection from RSV than from influenza. Children ≤5 years of age with presence of one or more comorbidities had a nearly 5-fold greater risk of getting RSV infection than otherwise healthy 0-5 years old children. DISCUSSION. The use of the ILI sentinel surveillance allowed us to identify groups at higher risk of RSV and influenza infection and to define the start, duration, timing and intensity of the RSV and influenza community circulation, determining thresholds based on historical data. This surveillance approach can be implemented to assess the nearly real-time RSV circulation and impact.

Here we described the virological and serological assessment of 23 COVID-19 patients hospitalized and followed up in Milan, Italy, during the first wave of COVID-19 pandemic. Nasopharyngeal (NPS), anal swabs, and blood samples were collected from 23 COVID-19 patients, at hospital admission, and periodically up to discharge, for a median time of 20 days (3–83 days). RNA was isolated and tested for SARS-CoV-2 by qRT-PCR; anti-SARS-CoV-2 IgM and IgG antibody titers were evaluated in serum samples by ELISA. SARS-CoV-2 genome was detected in the NPS swabs of the 23 patients, at the admission, and 8/19 (42.1%) were still positive at the discharge. Anal swabs were positive to SARS-CoV-2 RNA detection in 20/23 (86.9%) patients; 6/19 (31.6%) were still positive at discharge. The mean time of RNA negative conversion was 17 days (4–36 days) and 33 days (4–77 days), for NPS and anal swabs, respectively. SARS-CoV-2-RNA was detected in the blood of 6/23 (26.1%) patients. Thirteen/23 (56.5%) and 17/23 (73.9%) patients were seropositive for IgM and IgG, respectively, at the admission, and the median IgM and IgG levels significantly (p

The development of a tractable small animal model faithfully reproducing human COVID-19 pathogenesis would arguably meet a pressing need in biomedical research. Thus far, most investigators have used transgenic mice expressing the human ACE2 in epithelial cells (K18-hACE2 transgenic mice) that are intranasally instilled with a liquid SARS-CoV-2 suspension under deep anesthesia. Unfortunately, this experimental approach results in disproportionate high CNS infection leading to fatal encephalitis, which is rarely observed in humans and severely limits this model’s usefulness. Here, we describe the use of an inhalation tower system that allows exposure of unanesthetized mice to aerosolized virus under controlled conditions. Aerosol exposure of K18-hACE2 transgenic mice to SARS-CoV-2 resulted in robust viral replication in the respiratory tract, anosmia, and airway obstruction, but did not lead to fatal viral neuroinvasion. When compared to intranasal inoculation, aerosol infection resulted in a more pronounced lung pathology including increased immune infiltration, fibrin deposition and a transcriptional signature comparable to that observed in SARS-CoV-2- infected patients. This model may prove useful for studies of viral transmission, disease pathogenesis (including long-term consequences of SARS-CoV-2 infection) and therapeutic interventions.

BACKGROUND To date, there are no guidelines on the treatment of solid neoplasms in the transplanted kidney. Historically, allograft nephrectomy has been considered the only reasonable option. More recently, nephron-sparing surgery (NSS) and ablative therapy (AT) have been proposed as alternative procedures in selected cases. AIM To review outcomes of AT for the treatment of renal allograft tumours. METHODS We conducted a systematic review according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2009 Checklist. PubMed was searched in March 2019 without time restrictions for all papers reporting on radiofrequency ablation (RFA), cryoablation (CA), microwave ablation (MWA), high-intensity focused ultrasound (HIFU), and irreversible electroporation (IRE) of solid tumours of the kidney allograft. Only original manuscripts describing actual cases and edited in English were considered. All relevant articles were accessed in full text. Additional searches included all pertinent references. Selected studies were also assessed for methodological quality using a tool based on a modification of the Newcastle Ottawa scale. Data on recipient characteristics, transplant characteristics, disease characteristics, treatment protocols, and treatment outcomes were extracted and analysed. Given the nature and the quality of the studies available (mostly retrospective case reports and small retrospective uncontrolled case series), a descriptive summary was provided. RESULTS Twenty-eight relevant studies were selected describing a total of 100 AT procedures in 92 patients. Recipient age at diagnosis ranged from 21 to 71 years whereas time from transplant to diagnosis ranged from 0.1 to 312 mo. Most of the neoplasms were asymptomatic and diagnosed incidentally during imaging carried out for screening purposes or for other clinical reasons. Preferred diagnostic modality was Doppler-ultrasound scan followed by computed tomography scan, and magnetic resonance imaging. Main tumour types were: papillary renal cell carcinoma (RCC) and clear cell RCC. Maximal tumour diameter ranged from 5 to 55 mm. The vast majority of neoplasms were T1a N0 M0 with only 2 lesions staged T1b N0 M0. Neoplasms were managed by RFA (n = 78), CA (n = 15), MWA (n = 3), HIFU (n = 3), and IRE (n = 1). Overall, 3 episodes of primary treatment failure were reported. A single case of recurrence was identified. Follow-up ranged from 1 to 81 mo. No cancer-related deaths were observed. Complication rate was extremely low (mostly < 10%). Graft function remained stable in the majority of recipients. Due to the limited sample size, no clear benefit of a single procedure over the other ones could be demonstrated. CONCLUSION AT for renal allograft neoplasms represents a promising alternative to radical nephrectomy and NSS in carefully selected patients. Properly designed clinical trials are needed to validate this therapeutic approach.

This paper outlines the role of Lombardy’s regional influenza reference laboratory (Northern Italy) in the surveillance of influenza-like illnesses (ILIs) in monitoring SARS-CoV-2 circulation by analyzing 631 consecutive nasopharyngeal swabs (NPSs) collected from ILI outpatients by sentinel physicians during the 2019–2020 season. The samples were tested by specific real-time RT-PCRs targeting SARS-CoV-2, influenza viruses, and RSVs. Results: Of these NPSs, 31% tested positive for influenza viruses, 10% for SARS-CoV-2, and 7% for RSV. No coinfections were detected. Influenza viruses and RSVs circulated throughout the surveillance period until the end of February (week 9-2020), when they suddenly ceased to circulate seven weeks earlier than during the previous five influenza seasons. After the first detection of SARS-CoV-2 in our ILI outpatients at the beginning of March (week 10-2020), SARS-CoV-2 remained the only virus identified throughout the surveillance period. Patients ≥ 65 years had a 3.2-fold greater risk of being infected with SARS-CoV-2, while school-age children (5–14 years) and children < 5 years proved to be the age groups most at risk of contracting influenza viruses and RSV, respectively. Our experience demonstrates that laboratory-based ILI surveillance networks are essential for identifying SARS-CoV-2 cases that would otherwise remain undetected, in order to stop their spread within our communities.

The prevalence and the factors related to cytomegalovirus (CMV) disease (CMVd) during the 1st year of renal transplantation (RTx) and the relationship between CMVd and early and long-term graft and RTx-patient (RTx-p) survival were evaluated. In 505 RTx-p, followed up for 8(5–11) years, data were recorded after 1-(T1) and 12-(T12) months of RTx. CMVd was defined either by CMV replication without clinical signs of disease (CMVr, 43%), or CMV replication with signs of disease (CMVs, 57%). During the 1st year of RTx, 45% of RTx-p had CMVd (CMVd+). CMVd+ patients were older than CMVd− patients. Female gender and Donor CMV-IgG+ (CMV IgG−D+)/recipient IgG- (CMV IgG−R-) status were more prevalent in CMVd+. At T1, CMVd+ had lower albumin, haemoglobin, and higher uric-acid and reactive C-protein than CMVd− and, at T1 and T12, received more steroids. Albumin-T1 was the unique factor in determining CMVd+, maintaining its significance also after the inclusion of IgG−D+/IgG−R− status to the model. CMVs had higher prevalence of CMV IgG-D+/IgG-R- than CMVr. CMVd, CMVr, and CMVs had no impact on graft loss (11% of RTx-p) and RTx-p death (8% of RTx-p). CMVd is highly prevalent during the 1st year of RTx. Albumin-T1 influences CMVd insurgence. CMVd did not impact on RTx and RTx-p loss.

This paper outlines the role of Lombardy’s regional influenza reference laboratory (Northern Italy) in the surveillance of influenza-like illnesses (ILIs) in monitoring SARS-CoV-2 circulation by analyzing 631 consecutive nasopharyngeal swabs (NPSs) collected from ILI outpatients by sentinel physicians during the 2019–2020 season. The samples were tested by specific real-time RT-PCRs targeting SARS-CoV-2, influenza viruses, and RSVs. Results: Of these NPSs, 31% tested positive for influenza viruses, 10% for SARS-CoV-2, and 7% for RSV. No coinfections were detected. Influenza viruses and RSVs circulated throughout the surveillance period until the end of February (week 9-2020), when they suddenly ceased to circulate seven weeks earlier than during the previous five influenza seasons. After the first detection of SARS-CoV-2 in our ILI outpatients at the beginning of March (week 10-2020), SARS-CoV-2 remained the only virus identified throughout the surveillance period. Patients ≥ 65 years had a 3.2-fold greater risk of being infected with SARS-CoV-2, while school-age children (5–14 years) and children < 5 years proved to be the age groups most at risk of contracting influenza viruses and RSV, respectively. Our experience demonstrates that laboratory-based ILI surveillance networks are essential for identifying SARS-CoV-2 cases that would otherwise remain undetected, in order to stop their spread within our communities.

Colon cancer is the third cause of cancer death in the developed countries. Some environmental factors are involved in its pathogenesis, including viral infections. The possible involvement of human polyomaviruses (HPyVs) in colon cancer pathogenesis has been previously reported, leading to inconsistent conclusions. Clinical specimens were collected from 125 colon cancer patients. Specifically, 110 tumor tissues, 55 negative surgical margins, and 39 peripheral blood samples were analyzed for the presence of six HPyVs: JC polyomavirus (JCPyV), BK polyomavirus (BKPyV), Merkel cell PyV (MCPyV), HPyV -6, -7, and -9 by means of DNA isolation and subsequent duplex Real Time quantitative polymerase chain reaction. HPyVs genome was detected in 33/204 samples (16.2%): the significant higher positivity was found in tumor tissues (26/110, 23.6%), followed by negative surgical margins (3/55, 5.5%, p < .05), and peripheral blood mononuclear cells (PBMCs) (4/39; 10.3%). HPyVs load was statistically higher only in the tumor tissues compared to negative surgical margins (p < .05). Specifically, MCPyV was detected in 19.1% (21/110) of tumor tissues, 3.6% (2/55) of negative surgical margins (p < .05), and 7.7% (3/39) of PBMCs; HPyV-6 in 2.7% (3/110) of tumor tissues, and 1.8% (1/55) of negative surgical margins; one tumor tissue (1/110, 0.9%) and one PBMCs sample (1/39, 2.6%) were positive for BKPyV; JCPyV was present in 0.9% (1/110) of tumor tissues. HPyV-7 and 9 were not detected in any sample. High prevalence and load of MCPyV genome in the tumor tissues might be indicative of a relevant rather than bystander role of the virus in the colon tumorigenesis.

Graphical abstract, Objectives Despite an extensive risk of exposure to COVID-19, the residents of Giglio Island, Italy, did not develop any symptom of SARS-CoV-2. The present study aims to characterize the nature of exposure and to describe the local population dynamics underlying its apparent resistance to COVID-19. Methods Descriptive study of an islander partially-segregated population cohort based on a seroprevalence screening conducted from Aprile 29 to May 3, 2020 and including SARS-CoV-2 seroprevalence and viral prevalence in samples of saliva assessed through RT-qPCR. Serologic testing was performed using a COVID-19 IgG/IgM rapid test while molecular analyses were carried out by Allplex 2019-nCoV Assay (Seegene). Results A total of 634 residents out of 748 (84.8%) present at the time, and 89 non-residents underwent serological testing. 364 males and 359 females with a median age of 58.5 years. The serological screening identified one positive, asymptomatic subject. The Nucleic Acid Amplification Tests (NAAT) did not yield any positive result. Conclusion Despite extensive exposure to SARS-CoV-2, possibly only one new asymptomatic infection occurred in this population, as documented by IgM positivity not confirmed by RT-qPCR. This may be due to unknown protective factors or chance. On the basis of this baseline study, using its population as a reference model, further investigations will be conducted to test the advanced hypotheses, focusing on the evaluation of a possible cross-reactivity to SARS-CoV-2 from exposure to endemic viruses.

In survival analysis, time-varying covariates are endogenous when their measurements are directly related to the event status and incomplete information occur at random points during the follow-up. Consequently, the time-dependent Cox model leads to biased estimates. Joint models (JM) allow to correctly estimate these associations combining a survival and longitudinal sub-models by means of a shared parameter (i.e., random effects of the longitudinal sub-model are inserted in the survival one). This study aims at showing the use of JM to evaluate the association between a set of inflammatory biomarkers and Covid-19 mortality. During Covid-19 pandemic, physicians at Istituto Clinico di Città Studi in Milan collected biomarkers (endogenous time-varying covariates) to understand what might be used as prognostic factors for mortality. Furthermore, in the first epidemic outbreak, physicians did not have standard clinical protocols for management of Covid-19 disease and measurements of biomarkers were highly incomplete especially at the baseline. Between February and March 2020, a total of 403 COVID-19 patients were admitted. Baseline characteristics included sex and age, whereas biomarkers measurements, during hospital stay, included log-ferritin, log-lymphocytes, log-neutrophil granulocytes, log-C-reactive protein, glucose and LDH. A Bayesian approach using Markov chain Monte Carlo algorithm were used for fitting JM. Independent and non-informative priors for the fixed effects (age and sex) and for shared parameters were used. Hazard ratios (HR) from a (biased) time-dependent Cox and joint models for log-ferritin levels were 2.10 (1.67-2.64) and 1.73 (1.38-2.20), respectively. In multivariable JM, doubling of biomarker levels resulted in a significantly increase of mortality risk for log-neutrophil granulocytes, HR=1.78 (1.16-2.69); for log-C-reactive protein, HR=1.44 (1.13-1.83); and for LDH, HR=1.28 (1.09-1.49). Increasing of 100 mg/dl of glucose resulted in a HR=2.44 (1.28-4.26). Age, however, showed the strongest effect with mortality risk starting to rise from 60 years.

Objective Progressive multifocal leukoencephalopathy (PML) is still burdened by high mortality in a subset of patients, such as those affected by hematological malignancies. The aim of this study was to analyze the safety and carry out preliminary evaluation of the efficacy of polyomavirus JC (JCPyV)-specific T cell therapy in a cohort of hematological patients with PML. Methods Between 2014 and 2019, 9 patients with a diagnosis of "definite PML" according to the 2013 consensus who were showing progressive clinical deterioration received JCPyV-specific T cells. Cell lines were expanded from autologous or allogenic peripheral blood mononuclear cells by stimulation with JCPyV antigen-derived peptides. Results None of the patients experienced treatment-related adverse events. In the evaluable patients, an increase in the frequency of circulating JCPyV-specific lymphocytes was observed, with a decrease or clearance of JCPyV viral load in cerebrospinal fluid. In responsive patients, transient appearance of punctate areas of contrast enhancement within, or close to, PML lesions was observed, which was interpreted as a sign of immune control and which regressed spontaneously without the need for steroid treatment. Six of 9 patients achieved PML control, with 5 alive and in good clinical condition at their last follow-up. Interpretation Among other novel treatments, T cell therapy is emerging as a viable treatment option in patients with PML, particularly for those not amenable to restoration of specific immunity. Neurologists should be encouraged to refer PML patients to specialized centers to allow access to this treatment strategy. ANN NEUROL 2021;89:769-779.

ObjectivesIn Italy, the pandemic of COVID-19 resulted in congestion of hospitals and laboratories and probably determined an underestimation of the number of infected subjects, as the molecular diagnosis of SARS-CoV-2 infection was mainly performed on hospitalised patients. Therefore, limited data are available about the number of asymptomatic/paucisymptomatic subjects in the general population across time. To understand SARS-CoV-2 infection in the general population, we have developed a cross-sectional study (the ‘UNIversity against CORoNavirus study’) to investigate infection trends in asymptomatic/paucisymptomatic subjects in Milan (Italy), between March and June 2020.ParticipantsThe study population included 2023 subjects asymptomatic at the enrolment.Primary outcome measuresA nasal mid-turbinate swab for the detection of SARS-CoV-2 RNA and blood specimen for testing serum antibodies (immunoglobulin M (IgM) and IgG) were collected.ResultsSubjects showing positivity for the SARS-CoV-2 RNA and/or for anti-SARS-CoV-2 Ig is 237 (11.7%). Only 1.2% (n=25) of the total population had a positive nasal swab for SARS-CoV-2 and the large majority (21/25) of them were observed in March. A total of 226 subjects (11%) had IgM (n=19; 0.9%), IgG (n=155; 7.7%) or both (n=52; 2.6%) against SARS-CoV-2. Subjects with a present or past SARS-CoV-2 infection did not differ from other subjects as regards the number of cohabiting family members, travels, fever and upper and lower respiratory infection episodes.ConclusionsResults from the present study support the hypothesis that the actual spread of the virus in Lombardy was underestimated in the official records. However, as it is not known how long Ig persist, numbers should be taken cautiously.

The development of a tractable small animal model faithfully reproducing human coronavirus disease 2019 pathogenesis would arguably meet a pressing need in biomedical research. Thus far, most investigators have used transgenic mice expressing the human ACE2 in epithelial cells (K18-hACE2 transgenic mice) that are intranasally instilled with a liquid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suspension under deep anesthesia. Unfortunately, this experimental approach results in disproportionate high central nervous system infection leading to fatal encephalitis, which is rarely observed in humans and severely limits this model’s usefulness. Here, we describe the use of an inhalation tower system that allows exposure of unanesthetized mice to aerosolized virus under controlled conditions. Aerosol exposure of K18-hACE2 transgenic mice to SARS-CoV-2 resulted in robust viral replication in the respiratory tract, anosmia, and airway obstruction but did not lead to fatal viral neuroinvasion. When compared with intranasal inoculation, aerosol infection resulted in a more pronounced lung pathology including increased immune infiltration, fibrin deposition, and a transcriptional signature comparable to that observed in SARS-CoV-2–infected patients. This model may prove useful for studies of viral transmission, disease pathogenesis (including long-term consequences of SARS-CoV-2 infection), and therapeutic interventions.

Human Polyomavirus (HPyV) infections are common, ranging from 60% to 100%. In kidney transplant (KTx) recipients, HPyVs have been associated with allograft nephropathy, progressive multifocal leukoencephalopathy, and skin cancer. Whether such complications are caused by viral reactivation or primary infection transmitted by the donor remains debated. This study aimed to investigate the replication pattern and genomic characterization of BK Polyomavirus (BKPyV), JC Polyomavirus (JCPyV), and Merkel Cell Polyomavirus (MCPyV) infections in KTx. Urine samples from 57 KTx donor/recipient pairs were collected immediately before organ retrieval/transplant and periodically up to post-operative day 540. Specimens were tested for the presence of BKPyV, JCPyV, and MCPyV genome by virus-specific Real-Time PCR and molecularly characterized. HPyVs genome was detected in 49.1% of donors and 77.2% of recipients. Sequences analysis revealed the archetypal strain for JCPyV, TU and Dunlop strains for BKPyV, and IIa-2 strain for MCPyV. VP1 genotyping showed a high frequency for JCPyV genotype 1 and BKPyV genotype I. Our experience demonstrates that after KTx, HPyVs genome remains stable over time with no emergence of quasi-species. HPyVs strains isolated in donor/recipient pairs are mostly identical, suggesting that viruses detected in the recipient may be transmitted by the allograft.

SARS-CoV-2 symptoms are non-specific and can range from asymptomatic presentation to severe pneumonia. Asymptomatic subjects carrying SARS-CoV-2 often remain undiagnosed and it is still debated whether they develop immunoglobulins (Ig) and how long they persist. The aim of this study was to investigate the development and persistence of antibodies against SARS-CoV-2 in asymptomatic subjects infected by the virus. This follow-up study was performed on the 31 asymptomatic subjects who presented a positive nasal swab or serology against SARS-CoV-2 (Ig against Spike-RBD) in the first part of the UNICORN study (March 2020) aimed at attesting previous or current contacts with the virus in the personnel of the University of Milan. Eight weeks after the first Ig measure, these subjects were invited to donate a second blood sample for testing serum antibodies (IgM, IgG and total antibodies) and to fill-in a structured questionnaire. About 80% of asymptomatic subjects did not present circulating immunoglobulins against SARS-CoV-2 after 8 weeks from a positive nasal swab against the virus. Moreover, in more than 40% of these subjects, no Ig against SARS-CoV-2 were detected at any time. Finally, about two third of subjects with immunoglobulins at baseline did not present IgG against SARS-CoV-2 after 8 weeks. The majority of subjects who developed an asymptomatic SARS-CoV-2 infection do not present antibodies against the RBD-spike protein after 8 weeks of follow-up. These data should be taken into account for the interpretation of the serological evidences on SARS-CoV-2 that are emerging nowadays.

Colon cancer is the fourth most common malignancy in both incidence and mortality in developed countries. Infectious agents are among the risk factors for colon cancer. Variations in human endogenous retrovirus (HERV) transcript and protein levels are associated with several types of cancers, but few studies address HERV expression in colon cancer. Fifty-eight patients with advanced-stage colon cancer were enrolled in this study. HERV-H, -K (HML-2), -P LTRs, Alu, and LINE-1 methylation levels and transcription of HERV-H, -K (HML-2), and -P env and HERV-K pol genes in normal adjacent and tumor tissues were investigated by pyrosequencing and RT-qPCR, respectively. Expression of the HERV-K (HML-2) Pol and Env proteins in selected tissues was examined by Western blotting. Associations between HERV transcript expression and methylation levels and between clinical characteristics and HERV expression were evaluated. Compared to adjacent normal tissues, LINE-1 was hypomethylated in tumor tissues (p < 0.05), whereas Alu, HERV-K (HML-2), and -H LTRs showed a decreasing trend in tumor tissue compared to normal tissue, though without a significant difference. The transcription levels of HERV env and pol genes were similar. However, the HERV-K (HML-2) Pol protein was more highly expressed (p < 0.01) in surrounding normal tissues, but the HERV-K (HML-2) Env protein was only expressed in tumor tissues. Although HERV LTR methylation and gene expression did not show significant differences between tumor and normal tissues, HERV protein expression differed greatly. Pol protein expression in normal cells may induce reverse transcription and subsequent integration into the host genome, likely favoring cell transformation; in contrast, the Env protein in tumor tissue may contribute to cancer progression through cell-to-cell fusion.

Aim: Merkel cell polyomavirus (MCPyV) is the etiological agent of Merkel cell carcinoma. Its genome has been detected in anatomic districts from healthy and ill subjects. Data regarding the MCPyV DNAemia in neurological patients are lacking. Materials & methods: Blood was obtained from 129 neurological patients and 181 controls (HIV positive or negative). Real-time polymerase chain reaction (Q-PCR) was conducted to quantify MCPyV loads in blood specimens. Results: MCPyV DNA was detected in 17.1% of cases and 11.0% of controls in

Background Inhaled particles have been shown to produce systemic changes in DNA methylation. Global hypomethylation has been associated to viral sequence reactivation, possibly linked to the activation of pro-inflammatory pathways occurring after exposure. This observation provides a rationale to investigate viral sequence (both exogenous and endogenous) methylation in association to metal-rich particulate matter exposure. To verify this hypothesis, we chose the Wp promoter of the Epstein-Barr Virus (EBV-Wp) and the promoter of the human-endogenous-retrovirus w (HERV-w), respectively as a paradigm of an exogenous and an endogenous retroviral sequence, to be investigated by bisulfite PCR Pyrosequencing. We enrolled 63 male workers in an electric furnace steel plant, exposed to high level of metal-rich particulate matter. Results Comparing samples obtained in the first day of a work week (time 0-baseline, after 2 days off work) and the samples obtained after 3 days of work (time 1-post exposure), the mean methylation of EBV-Wp was significantly higher at baseline compared to post-exposure (meanbaseline = 56.7%5mC; meanpost-exposure = 47.9%5mC; p-value = 0.009), whereas the mean methylation of HERV-w did not significantly differ. Individual exposure to inhalable particles and metals was estimated based on measures in all working areas and time spent by the study subjects in each area. In a regression model adjusted for age, body mass index and smoking, PM and metal components had a positive association with EBV-Wp methylation (i.e. PM10: β = 5.99, p-value Conclusions The difference observed comparing baseline and post-exposure samples may be suggestive of a rapid change in EBV methylation induced by air particles, while correlation between EBV methylation and PM/metal exposure may represent a more stable adaptive mechanism. Future studies investigating a larger panel of viral sequences could better elucidate possible mechanisms and their role in pro-inflammatory pathways leading to systemic health effects.