John Cunningham virus (JCPyV) is an ubiquitous human pathogen that causes disease in immunocompromised patients. The JCPyV genome is composed of an early region and a late region, which are physically separated by the non-coding control region (NCCR). The DNA sequence of the NCCR distinguishes two forms of JCPyV, the designated archetype and the prototype, which resulted from a rearrangement of the archetype sequence. To date, the cell culture systems for propagating JCPyV archetype have been very limited in their availability and robustness. Prior to this study, it was demonstrated that JCPyV archetype DNA replicates in COS-7 simian kidney cells expressing SV40 TAg and COS-7 cells expressing HIV-1 Tat. Based on these observations, the present study was conducted to reproduce an in vitro model in COS-7 cells transfected with the JCPyV archetype strain in order to study JCPyV DNA replication and analyze NCCR rearrangements during the viral life cycle. The efficiency of JCPyV replication was evaluated by quantitative PCR (Q-PCR) and by hemagglutination (HA) assay after transfection. In parallel, sequence analysis of JCPyV NCCR was performed. JCPyV efficiently replicated in kidney-derived COS-7 cells, as demonstrated by a progressive increase in viral load and virion particle production after transfection. The archetypal structure of NCCR was maintained during the viral cycle, but two characteristic point mutations were detected 28 days after transfection. This model is a useful tool for analyzing NCCR rearrangements during in vitro replication in cells that are sites of viral persistence, such as tubular epithelial cells of the kidney.

Since limited data are available about the prevalence of Merkel cell polyomavirus (MCPyV) and the genetic variability of its noncoding control region (NCCR) in the context of immunosuppression, this study aimed to investigate the distribution of MCPyV in anatomical sites other than the skin and the behavior of NCCR among an HIV-1-positive population.

Italy was one of the most affected nations by coronavirus disease 2019 outside China. The infections, initially limited to Northern Italy, spread to all other Italian regions. This study aims to provide a snapshot of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) epidemiology based on a single-center laboratory experience in Rome. The study retrospectively included 6565 subjects tested for SARS-CoV-2 at the Laboratory of Virology of Sapienza University Hospital in Rome from 6 March to 4 May. A total of 9995 clinical specimens were analyzed, including nasopharyngeal swabs, bronchoalveolar lavage fluids, gargle lavages, stools, pleural fluids, and cerebrospinal fluids. Positivity to SARS-CoV-2 was detected in 8% (527/6565) of individuals, increased with age, and was higher in male patients (P  < .001). The number of new confirmed cases reached a peak on 18 March and then decreased. The virus was detected in respiratory samples, in stool and in pleural fluids, while none of gargle lavage or cerebrospinal fluid samples gave a positive result. This analysis allowed to gather comprehensive information on SARS-CoV-2 epidemiology in our area, highlighting positivity variations over time and in different sex and age group and the need for a continuous surveillance of the infection, mostly because the pandemic evolution remains unknown.

Fragmented data are available on the human polyomaviruses (HPyVs) prevalence in the gastrointestinal tract. Rearrangements in the non-coding control region (NCCR) of JCPyV and BKPyV have been extensively studied and correlated to clinical outcome; instead, little information is available for KIPyV, WUPyV and MCPyV NCCRs. To get insights into the role of HPyVs in the gastrointestinal tract, we investigated JCPyV, BKPyV, KIPyV, WUPyV and MCPyV distribution among hematological patients in concomitance with gastrointestinal symptoms. In addition, NCCRs and VP1 sequences were examined to characterize the strains circulating among the enrolled patients. DNA was extracted from 62 stool samples and qPCR was carried out to detect and quantify JCPyV, BKPyV, KIPyV, WUPyV and MCPyV genomes. Positive samples were subsequently amplified and sequenced for NCCR and VP1 regions. A phylogenetic tree was constructed aligning the obtained VP1 sequences to a set of reference sequences. qPCR revealed low viral loads for all HPyVs searched. Mono and co-infections were detected. A significant correlation was found between gastrointestinal complications and KIPyV infection. Archetype-like NCCRs were found for JCPyV and BKPyV, and a high degree of NCCRs stability was observed for KIPyV, WUPyV and MCPyV. Analysis of the VP1 sequences revealed a 99% identity with the VP1 reference sequences. The study adds important information on HPyVs prevalence and persistence in the gastrointestinal tract. Gastrointestinal signs were correlated with the presence of KIPyV, although definitive conclusions cannot be drawn. HPyVs NCCRs showed a high degree of sequence stability, suggesting that sequence rearrangements are rare in this anatomical site.

The expression rate of SARS-CoV-2 entry genes, angiotensin-converting enzyme 2 (ACE2), the main viral receptor and the proteases, furin and transmembrane serine protease 2 (TMPRSS2) in cystic fibrosis (CF) individuals is poorly known. Hence, we examined their levels in upper respiratory samples of CF patients (n = 46) and healthy controls (n = 45). Moreover, we sought to understand the interplay of type I interferon (IFN-I) with ACE2, furin and TMPRSS2 by evaluating their gene expression with respect to ISG15, a well-known marker of IFN activation, in upper respiratory samples and after ex vivo IFNβ exposure. Lower ACE2 levels and trends toward the reduction of furin and TMPRSS2 were found in CF patients compared with the healthy controls; decreased ACE2 amounts were also detected in CF individuals with pancreatic insufficiency and in those receiving inhaled antibiotics. Moreover, there was a strong positive correlation between ISG15 and ACE2 levels. However, after ex vivo IFNβ stimulation of nasopharyngeal cells, the truncated isoform (dACE2), recently demonstrated as the IFN stimulated one with respect to the full-length isoform (flACE2), slightly augmented in cells from CF patients whereas in those from healthy donors, dACE2 levels showed variable levels of upregulation. An altered expression of SARS-COV-2 entry genes and a poor responsiveness of dACE2 to IFN-I stimulation might be crucial in the diffusion of SARS-CoV-2 infection in CF.

Progressive multifocal leukoencephalopathy (PML) caused by the JC virus is the main limitation to the use of disease modifying therapies for treatment of multiple sclerosis (MS).

The persistence of high-risk (HR) human papillomavirus (HPV) genotypes is a prerequisite of cervical cancer. It is not clear whether and how bacterial vaginosis (BV) and sexually transmitted infections (STIs) cause higher rates of persistent HPV infection. This study aimed to characterize mucosal innate immunity to HPV, comparing different conditions. Specifically, expression levels of genes coding for Toll-like receptors (TLR)7 and 9, several type III Interferon-related genes (IFNL1, 2, 3, their specific receptor subunit IFNLR1, and the IFN-stimulated gene ISG15). Chemokines CCL5 and CCL20 were measured in cervical cells positive, or not, for HPV, BV, and STIs. HPV DNA was detected in 51/120 (42.5%) enrolled women, two/third were HR-HPV genotypes. More than 50% of samples were BV- and/or STI-positive. HPV-positive women had BV, but not other STIs, more frequently than the HPV-negative. TLR9 and IFNL1 mRNAs were expressed in the LR, but much less in the HR HPV infection. Enhanced levels of TLR9, TLR7, IFNL2, and IFNLR1 were observed in HPV-positive women with BV and STI. TLR9-increased expression was associated with HPV persistence in previous studies; hence, bacterial coinfections may enhance this risk. Prospective measurements of type III IFNs and IFNLR1 are warranted to evaluate whether this response may act as a double-edged sword in infected epithelia.

Human polyomavirus 6 (HPyV6) and HPyV7 are two of the novel polyomaviruses that were originally detected in non-diseased skin. Serological studies have shown that these viruses are ubiquitous in the healthy adult population with seroprevalence up to 88% for HPyV6 and 72% for HPyV7. Both viruses are associated with pruritic skin eruption in immunocompromised patients, but a role with other diseases in immunoincompetent patients or malignancies has not been established.

In the last years, the treatment of multiple sclerosis (MS) patients with natalizumab has been associated with the occurrence of progressive multifocal leukoencephalopathy (PML) caused by human polyomavirus JC (JCV). Here, we have shown a significant correlation between patients with JC viruria and positive JC-specific antibody response and patients without JCV-specific antibodies after 1 year of natalizumab (p = 0.0006). Furthermore, JCV-specific quantitative PCR on urine and plasma samples, collected at the enrollment (t0) and every 4 months (t1, t2, t3) in the first year and at two time points (t4 and t5) in the second year of natalizumab treatment, indicated the prevalence of JC viremia rather than JC viruria only in the second year of treatment (p = 0.04). Moreover, the analysis of JCV non-coding control region (NCCR) sequences in peripheral blood mononuclear cells of patients with JC-specific antibodies after 12 natalizumab infusions (t3) revealed the presence of rearranged sequences, whereas the prevalence of genotypes 1A, 1B, and 4 was detected in these patients by VP1 sequence analysis. In summary, JC viruria evaluation seems to be useful to identify early those patients who do not already develop a humoral immune response against JCV. It may also be interesting to study the JCV NCCR rearrangements since they could give us new insights on the onset of neuro-invasive viral variants.

As their name indicates, polyomaviruses (PyVs) can induce tumors. Mouse PyV, hamster PyV and raccoon PyV have been shown to cause tumors in their natural host. During the last 30 years, 15 PyVs have been isolated from humans. From these, Merkel cell PyV is classified as a Group 2A carcinogenic pathogen (probably carcinogenic to humans), whereas BKPyV and JCPyV are class 2B (possibly carcinogenic to humans) by the International Agency for Research on Cancer. Although the other PyVs recently detected in humans (referred to here as novel HPyV; nHPyV) share many common features with PyVs, including the viral oncoproteins large tumor antigen and small tumor antigen, as their role in cancer is questioned. This review discusses whether the nHPyVs may play a role in cancer based on predicted and experimentally proven functions of their early proteins in oncogenic processes. The functional domains that mediate the oncogenic properties of early proteins of known PyVs, that can cause cancer in their natural host or animal models, have been well characterized and we examined whether these functional domains are conserved in the early proteins of the nHPyVs and presented experimental evidence that these conserved domains are functional. Furthermore, we reviewed the literature describing the detection of nHPyV in human tumors.

The aim of the study was to investigate the changes of matrix metalloproteinase (MMP)-2 and MMP-9 plasma levels during natalizumab treatment and their correlation with JC virus (JCV) reactivation and T-lymphocyte phenotypic modifications in peripheral blood samples from 34 relapsing-remitting multiple sclerosis (RRMS) patients. MMP-9 levels were assessed by zymography in plasma samples. JCV-DNA was detected through quantitative real time PCR in plasma samples. T-lymphocyte phenotype was assessed with flow cytometry. MMP-9 plasma levels resulted increased from 12 to 24 natalizumab infusions. Stratifying plasma samples according to JCV-DNA detection, MMP-9 plasma levels were significantly increased in JCV-DNA positive than JCV-DNA negative samples. MMP-9 plasma levels resulted positively correlated with JCV viral load. CD4 immune senescence, CD8 immune activation and CD8 effector percentages were positively correlated to MMP-9 plasma levels, whereas a negative correlation between CD8 naïve percentages and MMP-9 plasma levels was found. Our data indicate an increase of MMP-9 plasma levels between 12 and 24 natalizumab infusions and a correlation with JCV-DNA detection in plasma, T-lymphocyte immune activation and senescence. These findings could contribute to understand PML pathogenesis under natalizumab treatment, suggesting a potential role of MMP-9 as a predictive marker of PML in RRMS patients.

The anti-CD49d monoclonal antibody natalizumab is currently an effective therapy against the relapsing-remitting form of multiple sclerosis (RRMS). Natalizumab therapeutic efficacy is limited by the reactivation of the John Cunningham polyomavirus (JCV) and development of progressive multifocal leukoencephalopathy (PML). To correlate natalizumab-induced phenotypic modifications of peripheral blood T-lymphocytes with JCV reactivation, JCV-specific antibodies (serum), JCV-DNA (blood and urine), CD49d expression and relative abundance of peripheral blood T-lymphocyte subsets were longitudinally assessed in 26 natalizumab-treated RRMS patients. Statistical analyses were performed using GraphPad Prism and R. Natalizumab treatment reduced CD49d expression on memory and effector subsets of peripheral blood T-lymphocytes. Moreover, accumulation of peripheral blood CD8+ memory and effector cells was observed after 12 and 24 months of treatment. CD4+ and CD8+ T-lymphocyte immune-activation was increased after 24 months of treatment. Higher percentages of CD8+ effectors were observed in subjects with detectable JCV-DNA. Natalizumab reduces CD49d expression on CD8+ T-lymphocyte memory and effector subsets, limiting their migration to the central nervous system and determining their accumulation in peripheral blood. Impairment of central nervous system immune surveillance and reactivation of latent JCV, can explain the increased risk of PML development in natalizumab-treated RRMS subjects.

Chronic inflammatory rheumatic diseases (CIRDs) are immune-mediated pathologies involving joints. To date, TNFα-blocking agents administration is the most promising therapy, although these treatments are associated with an increased Polyomavirus JC (JCPyV) reactivation, the etiological agent of the Progressive Multifocal Leukoencephalopathy (PML). The aim of this study was the recruitment and the analysis of a CIRDs cohort in order to investigate a possible correlation between JCPyV presence and the influence of anti-TNF-α agents on viral loads. Blood and urine samples were collected from 34 CIRDs subjects prior the first anti-TNF-α infusion (T0) and after 3 (T3), 6 (T6), 12 (T12), and 18 (T18) months. Results showed persistent JC viruria significantly higher than JC viremia throughout the 18 month follow-up study (p = 0.002). In JCPyV positive samples, the non-coding control region (NCCR) was analyzed. Results evidenced archetypal structures (type II-S) in all isolates with the exception of a sequence isolated from a plasma sample, that corresponds to the type II-R found in PML subjects. Finally, the viral protein 1 (VP1) genotyping was performed and results showed the prevalence of the European genotypes 1A, 1B, and 4. Since only few studies have been carried out to understand whether there is a PML risk in CIRDs population infected by JCPyV, this study contributes to enrich literature insight on JCPyV biology in this cluster. Further investigations are necessary in order to recognize the real impact of biologics on JCPyV life cycle and to identify possible and specific viral variants related to increased virulence in CIRDs patients.

Escherichia coli sequence type 167 (ST167), producing the metallo beta-lactamase NDM-5, has been isolated as a colonizer of patients recovered at the University Hospital Policlinico Umberto I of Rome. Phylogenesis and comparative analysis of the genomes of these strains were performed against 343 ST167 genomes available from the EnteroBase database. These analyses revealed that resistance plasmids, integrative conjugative elements (ICEs), carrying the yersiniabactin virulence trait and capsular synthesis gene clusters had variable compositions and distributions within different strains of the ST167 clone. A novel capsular synthesis gene cluster, highly similar to the K48 cluster previously described only for Klebsiella pneumoniae, was identified in phylogenetically related strains of the ST167 clone.IMPORTANCE Global dissemination of some E. coli high-risk clones has been described in the last decades. The most widespread was the ST131 clone, associated with extended-spectrum-beta-lactamase (ESBL) production. Genomics of ST131 demonstrated that one clade within the ST emerged in the early 2000s, followed by a rapid, global expansion. The E. coli ST167 clone is emerging throughout the world, being frequently reported for its association with carbapenem resistance. Our study shows that virulence features are differently represented within the ST167 population. One clade shows the K48 capsular synthesis gene cluster of K. pneumoniae, not previously described for E. coli, and is populated by NDM-5-producing strains. The combination of resistance and virulence may sustain the global expansion of this specific ST167 clade.

Because the incidence of Merkel cell carcinoma (MCC) has increased significantly during the last 10 years and it is recognized that Merkel cell polyomavirus (MCPyV) and ultraviolet (UV) radiation represent two different etiological inputs sharing clinical, histopathological, and prognostic similar features, although with different prognosis, this study investigated the detection of MCPyV in skin and lymph nodes with histological diagnosis of MCC. Formalin-fixed paraffin-embedded tissue (FFPE) were retrieved from archived specimens and MCPyV non-coding control region (NCCR) and viral capsid protein 1 (VP1) sequences were amplified and sequenced. Results provide an interesting observation concerning the discrepancy between the MCPyV DNA status in primary and metastatic sites: in fact, in all cases in which primary and metastatic lesions were investigated, MCPyV DNA was detected only in the primary lesions. Our data further support the "hit-and-run" theory, also proposed by other authors, and may lead to speculation that in some MCCs the virus is only necessary for the process of tumor initiation and that further mutations may render the tumor independent from the virus. Few point mutations were detected in the NCCR and only silent mutations were observed in the VP1 sequence compared to the MCPyV MCC350 isolate. To unequivocally establish a role of MCPyV in malignancies, additional well-controlled investigations are required, and larger cohorts should be examined.

Herpes simplex virus type-1 (HSV-1) and John Cunningham polyomavirus (JCPyV) are widely distributed DNA viruses causing mainly asymptomatic infection, but also mild to very severe diseases, especially when these viruses reach the brain. Some drugs have been developed to inhibit HSV-1 replication in host cells, but their prolonged use may induce resistance phenomena. In contrast, to date, there is no cure for JCPyV. The search for alternative drugs that can reduce viral infections without undermining the host cell is moving toward antimicrobial peptides (AMPs) of natural occurrence. These include amphibian AMPs belonging to the temporin family. Herein, we focus on temporin G (TG), showing that it strongly affects HSV-1 replication by acting either during the earliest stages of its life cycle or directly on the virion. Computational studies have revealed the ability of TG to interact with HSV-1 glycoprotein B. We also found that TG reduced JCPyV infection, probably affecting both the earliest phases of its life cycle and the viral particle, likely through an interaction with the viral capsid protein VP1. Overall, our results are promising for the development of short naturally occurring peptides as antiviral agents used to counteract diseases related to HSV-1 and JCPyV.

Merkel cell polyomavirus (MCPyV) is the major cause of Merkel cell carcinoma (MCC), an aggressive skin cancer. MCPyV large T-antigen (LTag) and small T-antigen (sTag) are the main oncoproteins involved in MCPyV-induced MCC. A hallmark of MCPyV-positive MCC cells is the expression of a C-terminal truncated LTag. Protein kinase A (PKA) plays a fundamental role in a variety of biological processes, including transcription by phosphorylating and thereby regulating the activity of transcription factors. As MCPyV LTag has been shown to be phosphorylated and acts as a transcription factor for the viral early and late promoter, we investigated whether LTag can be phosphorylayted by PKA, and whether this affects the transcript activity of LTag. Using a phosphorylation prediction algorithm, serine 191, 203, and 265 were identified as putative phosphorylation sites for PKA. Mass spectrometry of in vitro PKA-phosphorylated peptides confirmed phosphorylation of S203 and S265, but not S191. Full-length LTag inhibited early and late promoter activity of MCPyV, whereas the truncated MKL2 LTag variant stimulated both promoters. Single non-phosphorylable, as well as phosphomimicking mutations did not alter the inhibitory effect of full-length LTag. However, the non-phosphorylable mutations abrogated transactivation of the MCPyV promoters by MKL2 LTag, whereas phosphomimicking substitutions restored the ability of MKL2 LTag to activate the promoters. Triple LTag and MKL2 LTag mutants had the same effect as the single mutants. Activation of the PKA signaling pathway did not enhance MCPyV promoter activity, nor did it affect LTag expression levels in MCPyV-positive Merkel cell carcinoma (MCC) cells. Our results show that phosphorylation of truncated LTag stimulates viral promoter activity, which may contribute to higher levels of the viral oncoproteins LTag and sTag. Interfering with PKA-induced LTag phosphorylation/activity may be a therapeutic strategy to treat MCPyV-positive MCC patients.

Since the non-coding control region (NCCR) and microRNA (miRNA) could represent two different and independent modalities of regulating JC polyomavirus (JCPyV) replication at the transcriptional and post-transcriptional levels, the interplay between JC viral load based on NCCR architecture and miRNA levels, following JCPyV infection with archetypal and rearranged (rr)-NCCR JCPyV variants, was explored in COS-7 and SVGp12 cells infected by different JCPyV strains. Specifically, the involvement of JCPyV miRNA in regulating viral replication was investigated for the archetypal CY strain-which is the transmissible form-and for the rearranged MAD-1 strain, which is the first isolated variant from patients with progressive multifocal leukoencephalopathy. The JCPyV DNA viral load was low in cells infected with CY compared with that in MAD-1-infected cells. Productive viral replication was observed in both cell lines. The expression of JCPyV miRNAs was observed from 3 days after viral infection in both cell types, and miR-J1-5p expression was inversely correlated with the JCPyV replication trend. The JCPyV miRNAs in the exosomes present in the supernatants produced by the infected cells could be carried into uninfected cells. Additional investigations of the expression of JCPyV miRNAs and their presence in exosomes are necessary to shed light on their regulatory role during viral reactivation.

Although natalizumab (anti-α4 integrin) represents an effective therapy for relapsing remitting multiple sclerosis (RRMS), it is associated with an increased risk of developing progressive multifocal leukoencephalopathy (PML), caused by the polyomavirus JC (JCV). The aim of this study was to explore natalizumab-induced phenotypic changes in peripheral blood T-lymphocytes and their relationship with JCV reactivation. Forty-four patients affected by RRMS were enrolled. Blood and urine samples were classified according to natalizumab infusion number: 0 (N0), 1-12 (N12), 13-24 (N24), 25-36 (N36), and over 36 (N > 36) infusions. JCV-DNA was detected in plasma and urine. T-lymphocyte phenotype was evaluated with flow cytometry. JCV serostatus was assessed. Ten healthy donors (HD), whose ages and sexes matched with the RRMS patients of the N0 group, were enrolled. CD8 effector (CD8 E) percentages were increased in natalizumab treated patients with detectable JCV-DNA in plasma or urine compared to JCV-DNA negative patients (JCV-) (p < 0.01 and p < 0.001, resp.). Patients with CD8 E percentages above 10.4% tended to show detectable JCV-DNA in plasma and/or urine (ROC curve p = 0.001). The CD8 E was increased when JCV-DNA was detectable in plasma or urine, independently from JCV serology, for N12 and N24 groups (p < 0.01). As long as PML can affect RRMS patients under natalizumab treatment with a negative JCV serology, the assessment of CD8 E could help in the evaluation of JCV reactivation.

Merkel cell polyomavirus (MCPyV) viral protein 1 (VP1) is the capsid protein that mediates virus attachment to host cell receptors and is the major immune target. Given the limited data on MCPyV VP1 mutations, the VP1 genetic variability was examined in 100 plasma and 100 urine samples from 100 HIV+ individuals. Sequencing of VP1 DNA in 17 urine and 17 plasma specimens, simultaneously MCPyV DNA positive, revealed that 27 samples displayed sequences identical to VP1 of MCC350 strain. VP1 from two urine specimens had either Thr47Ser or Ile115Phe substitution, whereas VP1 of one plasma contained Asp69Val and Ser251Phe substitutions plus deletion (∆) of Tyr79. VP1 DNA in the remaining samples had mutations encoding truncated protein. Three-dimensional prediction models revealed that Asp69Val, Ser251Phe, and Ile115Phe caused neutral effects while Thr47Ser and Tyr79∆ produced a deleterious effect reducing VP1 stability. A549 cells infected with urine or plasma samples containing full-length VP1 variants with substitutions, sustained viral DNA replication and VP1 expression. Moreover, medium harvested from these cells was able to infect new A549 cells. In cells infected by samples with truncated VP1, MCPyV replication was hampered. In conclusion, MCPyV strains with unique mutations in the VP1 gene are circulating in HIV+ patients. These strains display altered replication efficiency compared to the MCC350 prototype strain in A549 cells.