Multiplex array analysis of circulating cytokines and chemokines in COVID-19 patients during the first wave of the SARS-CoV-2 pandemic in Milan, Italy.

BACKGROUND: The systemic inflammatory syndrome called "cytokine storm" has been described in COVID-19 pathogenesis, contributing to disease severity. The analysis of cytokine and chemokine levels in the blood of 21 SARS-CoV-2 positive patients throughout the phases of the pandemic has been studied to understand immune response dysregulation and identify potential disease biomarkers for new treatments. The present work reports the cytokine and chemokine levels in sera from a small cohort of individuals primarily infected with SARS-CoV-2 during the first wave of the COVID-19 pandemic in Milan (Italy). RESULTS: Among the 27 cytokines and chemokines investigated, a significant higher expression of Interleukin-9 (IL-9), IP-10 (CXCL10), MCP-1 (CCL2) and RANTES (CCL-5) in infected patients compared to uninfected subjects was observed. When the change in cytokine/chemokine levels was monitored over time, from the hospitalization day to discharge, only IL-6 and IP-10 showed a significant decrease. Consistent with these findings, a significant negative correlation was observed between IP-10 and anti-Spike IgG antibodies in infected individuals. In contrast, IL-17 was positively correlated with the production of IgG against SARS-CoV-2. CONCLUSIONS: The cytokine storm and the modulation of cytokine levels by SARS-CoV-2 infection are hallmarks of COVID-19. The current global immunity profile largely stems from widespread vaccination campaigns and previous infection exposures. Consequently, the immunological features and dynamic cytokine profiles of non-vaccinated and primarily-infected subjects reported here provide novel insights into the inflammatory immune landscape in the context of SARS-CoV-2 infection, and offer valuable knowledge for addressing future viral infections and the development of novel treatments.

Impact of in vitro SARS-CoV-2 infection on breast cancer cells.

The pandemic of coronavirus disease 19 (COVID-19), caused by severe respiratory syndrome coronavirus 2 (SARS-CoV-2), had severe repercussions for breast cancer patients. Increasing evidence indicates that SARS-CoV-2 infection may directly impact breast cancer biology, but the effects of SARS-CoV-2 on breast tumor cells are still unknown. Here, we analyzed the molecular events occurring in the MCF7, MDA-MB-231 and HCC1937 breast cancer cell lines, representative of the luminal A, basal B/claudin-low and basal A subtypes, respectively, upon SARS-CoV-2 infection. Viral replication was monitored over time, and gene expression profiling was conducted. We found that MCF7 cells were the most permissive to viral replication. Treatment of MCF7 cells with Tamoxifen reduced the SARS-CoV-2 replication rate, suggesting an involvement of the estrogen receptor in sustaining virus replication in malignant cells. Interestingly, a metagene signature based on genes upregulated by SARS-CoV-2 infection in all three cell lines distinguished a subgroup of premenopausal luminal A breast cancer patients with a poor prognosis. As SARS-CoV-2 still spreads among the population, it is essential to understand the impact of SARS-CoV-2 infection on breast cancer, particularly in premenopausal patients diagnosed with the luminal A subtype, and to assess the long-term impact of COVID-19 on breast cancer outcomes.

Longitudinal study of human polyomaviruses viruria in kidney transplant recipients.

INTRODUCTION: Immunosuppression after kidney transplantation (KTx) exposes recipients to Human Polyomaviruses (HPyVs) infections, whose natural history is still misunderstood. METHODS: Allograft biopsies, and urine from 58 donor-recipient pairs were collected before KTx (T0) and 1 (T1), 15 (T2), 30 (T3), 60 (T4), 90 (T5), 180 (T6), 270 (T7), 360 (T8), and 540 (T9) days after transplant. Specimens were tested for JC (JCPyV) and BK (BKPyV), by quantitative Real-Time PCR. The course of post-KTx HPyVs viruria, and the association between JCPyV viruria in recipients and donors, were evaluated. RESULTS: HPyVs were detected in 3/58 (5.2%) allograft biopsies. HPyVs viruria was present in 29/58 (50%) donors and 41/58 (70.7%) recipients. JCPyV DNA was detected in 26/58 (44.8%) donors and 25/58 recipients (43.1%), 19 of whom received kidney from JCPyV positive donor, whereas BKPyV genome was detected in 3 (5.2%) donors and 22 (37.9%) recipients. The median time of JCPyV, and BKPyV first episode of replication was 1, and 171 days post KTx, respectively. At T0, JCPyV viruria of donors was associated with increased risk of JCPyV replication post-KTx; recipients with JCPyV positive donors showed lower risk of BKPyV replication post-KTx. CONCLUSIONS: The results suggested that JCPyV may be transmitted by allograft, and that its replication post KTx might prevent BKPyV reactivation. Future investigation regarding correlation between chronic exposure to immunosuppressive agents and HPyVs urinary replication are warranted.

Breastmilk from COVID-19 negative lactating mothers shows neutralizing activity against SARS-COV-2.

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 2 days postpartum (T0), declining 7 days postpartum (T1) and 20 days postpartum (T2). Group B samples exhibited neutralizing activity mostly at 7 days postpartum (T1) (90%). Negative mothers' samples showed no correlation between NT50 titres and antibodies' presence, suggesting that non-specific breastmilk components may exert antiviral action against SARS-CoV-2.

In-vitro antiviral activity and in-silico targeted study of quinoline-3-carboxylate derivatives against SARS-Cov-2 isolate.

In recent years, the viral outbreak named COVID-19 showed that infectious diseases have a huge impact on both global health and the financial and economic sectors. The lack of efficacious antiviral drugs worsened the health problem. Based on our previous experience, we investigated in vitro and in silico a series of quinoline-3-carboxylate derivatives against a SARS-CoV-2 isolate. In the present study, the in-vitro antiviral activity of a series of quinoline-3-carboxylate compounds and the in silico target-based molecular dynamics (MD) and metabolic studies are reported. The compounds' activity against SARS-CoV-2 was evaluated using plaque assay and RT-qPCR. Moreover, from the docking scores, it appears that the most active compounds (1j and 1o) exhibit stronger binding affinity to the primary viral protease (NSP5) and the exoribonuclease domain of non structural protein 14 (NSP14). Additionally, the in-silico metabolic analysis of 1j and 1o defines CYP2C9 and CYP3A4 as the major P450 enzymes involved in their metabolism.

Digital Detection of Single Virus Particles by Multi-Spot, Label-Free Imaging Biosensor on Anti-Reflective Glass.

Rapid detection of whole virus particles in biological or environmental samples represents an unmet need for the containment of infectious diseases. Here, an optical device enabling the enumeration of single virion particles binding on antibody or aptamers immobilized on a surface with anti-reflective coating is described. In this regime, nanoparticles adhering to the sensor surface provide localized contributions to the reflected field that become detectable because of their mixing with the interfering waves in the reflection direction. Thus, these settings are exploited to realize a scan-free, label-free, micro-array-type digital assay on a disposable cartridge, in which the virion counting takes place in wide field-of-view imaging. With this approach we could quantify, by enumeration, different variants of SARS-CoV-2 virions interacting with antibodies and aptamers immobilized on different spots. For all tested variants, the aptamers showed larger affinity but lower specificity relative to the antibodies. It is found that the combination of different probes on the same surface enables increasing specificity of detection and dynamic range.

Determination of the UV Inactivation Constant Under 280 nm UV LED Irradiation for 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-2 . By multitarget model fitting, n = 2 and k = 0.32 ± 0.02 m2 J-1 were obtained. A lag time for the inactivation effect was also observed, which was attributed to the low irradiation levels used to perform the study. The combination of k and delay time allows for reliable estimation of disinfection times in small, closed environments.

Antiparasitic Drugs against SARS-CoV-2: A Comprehensive Literature Survey.

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.

In Vitro Study Evaluating the Effect of Different Immunosuppressive Agents on Human Polyomavirus BK Replication.

BACKGROUND: Human polyomavirus BK (BKPyV) is the etiologic agent of polyomavirus-associated nephropathy, a leading cause of kidney transplant dysfunction. Because of the lack of antiviral therapies, immunosuppression minimization is the recommended treatment. This strategy offers suboptimal outcomes and entails a significant risk of rejection. Our aim was to evaluate the effect of different immunosuppressive drugs (leflunomide, tacrolimus, mycophenolic acid, sirolimus, and everolimus) and their combinations in an in vitro model of BKPyV infection. METHODS: Human renal tubular epithelial cells were infected with BKPyV and treated with leflunomide, tacrolimus, mycophenolic acid, sirolimus, and everolimus, administered alone or in some combination thereof. Viral replication was assessed every 24 hours (up to 72 hours) by BKPyV-specific quantitative real-time polymerized chain reaction for the VIRAL PROTEIN 1 sequence in cell supernatants and by western blot analysis targeting the viral protein 1 and the glyceraldehyde 3-phosphate dehydrogenase on total protein lysates. Results were described as viral copies/mL and compared between treatments at any prespecified time point of the study. RESULTS: The highest inhibitory effects were observed using leflunomide or everolimus plus mycophenolic acid (mean BKPyV replication log reduction 0.28). The antiviral effect of everolimus persisted when it was used in combination with tacrolimus (mean BKPyV replication log reduction 0.27). CONCLUSIONS: Our experience confirms that everolimus has anti-BKPyV properties and prompts future research to investigate possible mechanisms of action. It also provides a rational basis for targeted clinical trials evaluating alternative immunosuppressive modification strategies.

In Vitro SARS-CoV-2 Infection of Microvascular Endothelial Cells: Effect on Pro-Inflammatory Cytokine and Chemokine Release.

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 (p < 0.05). IL-6 levels were significantly higher in supernatants from infected HMEC-1 (p < 0.001) at 24 h, 48 h, and 72 h p.i., while IL-8 levels were significantly lower at 24 h p.i. (p < 0.001). These data indicate that in vitro microvascular endothelial cells are susceptible to SARS-CoV-2 infection but slightly contribute to viral amplification. However, SARS-CoV-2 infection might trigger the increase of pro-inflammatory mediators.

Novel antiviral activity of PAD inhibitors against human beta-coronaviruses HCoV-OC43 and SARS-CoV-2.

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.

Circulation of SARS-CoV-2 Variants among Children from November 2020 to January 2022 in Trieste (Italy).

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.

Longitudinal, virological, and serological assessment of hospitalized COVID-19 patients.

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 < 0.05) increased after 13 days. Although the limited cohort size, our report provides evidence that SARS-CoV-2 is shed through multiple routes, with important implications in healthcare settings.

Respiratory syncytial virus in pediatric influenza-like illness cases in Lombardy, Northern Italy, during seven consecutive winter seasons (from 2014-2015 to 2020-2021).

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 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 cases <15 years of age in all considered winter seasons except in the 2020-2021 season. DISCUSSION: 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.

In Vitro Activity of Monofunctional Pt-II Complex Based on 8-Aminoquinoline against Human Glioblastoma.

A new cationic Pt(II) complex bearing 8-aminoquinoline as chelating ligand (called Pt-8AQ) was evaluated against two human carcinomas, one mesothelioma, and three glioblastoma cell lines. The in vitro comparison to the clinically approved CisPt showed a minor activity of Pt-8AQ against carcinoma and mesothelioma, whereas a significant activity of Pt-8AQ was observed on the proliferation of the three glioblastoma cell lines (U87-MG IC50 = 3.68 ± 0.69 µM; U373-MG IC50 = 11.53 ± 0.16 µM; U138-MG IC50 = 8.05 ± 0.23 µM) that was higher than that observed with the clinically approved CisPt (U87-MG IC50 = 7.27 + 1.80 µM; U373-MG IC50 = 22.69 ± 0.05 µM; U138-MG IC50 = 32.1 ± 4.44 µM). Cell cycle analysis proved that Pt-8AQ significantly affected the cell cycle pattern by increasing the apoptotic cells represented by the sub G0/G1 region related with a downregulation of p53 and Bcl-2. Moreover, an NMR investigation of Pt-8AQ interaction with 9-EtG, GSH, and Mets7 excluded DNA as the main target, suggesting a novel mechanism of action. Our study demonstrated the high stability of Pt-8AQ after incubation at 37 °C and a significant antineoplastic activity on glioblastomas. These features also make Pt-8AQ a good candidate for developing a new selective advanced cell chemotherapy approach in combination with MSCs.

When the COVID-19 Pandemic Surges during Influenza Season: Lessons Learnt from the Sentinel Laboratory-Based Surveillance of Influenza-Like Illness in Lombardy during the 2019-2020 Season.

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.

Isolation of SARS-CoV-2 strains carrying a nucleotide mutation, leading to a stop codon in the ORF 6 protein.

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.

Human polyomaviruses genomes in clinical specimens of colon cancer patients.

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.

Human Endogenous Retroviruses Long Terminal Repeat Methylation, Transcription, and Protein Expression in Human Colon Cancer.

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.

Viral Genomic Characterization and Replication Pattern of Human Polyomaviruses in Kidney Transplant Recipients.

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.