Mycoplasma DnaK expression increases cancer development in vivo upon DNA damage.

Well-controlled repair mechanisms are involved in the maintenance of genomic stability, and their failure can precipitate DNA abnormalities and elevate tumor risk. In addition, the tumor microenvironment, enriched with factors inducing oxidative stress and affecting cell cycle checkpoints, intensifies DNA damage when repair pathways falter. Recent research has unveiled associations between certain bacteria, including Mycoplasmas, and various cancers, and the causative mechanism(s) are under active investigation. We previously showed that Mycoplasma fermentans DnaK, an HSP70 family chaperone protein, hampers the activity of proteins like PARP1 and p53, crucial for genomic integrity. Moreover, our analysis of its interactome in human cancer cell lines revealed DnaK's engagement with several components of DNA-repair machinery. Finally, in vivo experiments performed in our laboratory using a DnaK knock-in mouse model generated by our group demonstrated that DnaK exposure led to increased DNA copy number variants, indicative of genomic instability. We present here evidence that expression of DnaK is linked to increased i) incidence of tumors in vivo upon exposure to urethane, a DNA damaging agent; ii) spontaneous DNA damage ex vivo; and iii) expression of proinflammatory cytokines ex vivo, variations in reactive oxygen species levels, and increased ß-galactosidase activity across tissues. Moreover, DnaK was associated with increased centromeric instability. Overall, these findings highlight the significance of Mycoplasma DnaK in the etiology of cancer and other genetic disorders providing a promising target for prevention, diagnostics, and therapeutics.

Mycoplasma DnaK increases DNA copy number variants in vivo.

The human microbiota affects critical cellular functions, although the responsible mechanism(s) is still poorly understood. In this regard, we previously showed that Mycoplasma fermentans DnaK, an HSP70 chaperone protein, hampers the activity of important cellular proteins responsible for DNA integrity. Here, we describe a novel DnaK knock-in mouse model generated in our laboratory to study the effect of M. fermentans DnaK expression in vivo. By using an array-based comparative genomic hybridization assay, we demonstrate that exposure to DnaK was associated with a higher number of DNA copy number variants (CNVs) indicative of unbalanced chromosomal alterations, together with reduced fertility and a high rate of fetal abnormalities. Consistent with their implication in genetic disorders, one of these CNVs caused a homozygous Grid2 deletion, resulting in an aberrant ataxic phenotype that recapitulates the extensive biallelic deletion in the Grid2 gene classified in humans as autosomal recessive spinocerebellar ataxia 18. Our data highlight a connection between components of the human urogenital tract microbiota, namely Mycoplasmas, and genetic abnormalities in the form of DNA CNVs, with obvious relevant medical, diagnostic, and therapeutic implications.

Bacterial DnaK reduces the activity of anti-cancer drugs cisplatin and 5FU.

BACKGROUND: Chemotherapy is a primary treatment for cancer, but its efficacy is often limited by cancer-associated bacteria (CAB) that impair tumor suppressor functions. Our previous research found that Mycoplasma fermentans DnaK, a chaperone protein, impairs p53 activities, which are essential for most anti-cancer chemotherapeutic responses. METHODS: To investigate the role of DnaK in chemotherapy, we treated cancer cell lines with M. fermentans DnaK and then with commonly used p53-dependent anti-cancer drugs (cisplatin and 5FU). We evaluated the cells' survival in the presence or absence of a DnaK-binding peptide (ARV-1502). We also validated our findings using primary tumor cells from a novel DnaK knock-in mouse model. To provide a broader context for the clinical significance of these findings, we investigated human primary cancer sequencing datasets from The Cancer Genome Atlas (TCGA). We identified F. nucleatum as a CAB carrying DnaK with an amino acid composition highly similar to M. fermentans DnaK. Therefore, we investigated the effect of F. nucleatum DnaK on the anti-cancer activity of cisplatin and 5FU. RESULTS: Our results show that both M. fermentans and F. nucleatum DnaKs reduce the effectiveness of cisplatin and 5FU. However, the use of ARV-1502 effectively restored the drugs' anti-cancer efficacy. CONCLUSIONS: Our findings offer a practical framework for designing and implementing novel personalized anti-cancer strategies by targeting specific bacterial DnaKs in patients with poor response to chemotherapy, underscoring the potential for microbiome-based personalized cancer therapies.

Measuring the neglected anxiety disorder: validation of the social anxiety spectrum-short version (SHY-SV) questionnaire.

BACKGROUND: In the recent years, a growing body of literature stressed the importance of a dimensional perspective on mental disorders. In particular, since its conceptualization, one of the main concerns in the field of Social Anxiety Disorder (SAD) has been the definition of a diagnostic threshold, leading to the suggestion that SAD may be more properly classified as a spectrum of severity rather than a discrete disorder based on subjectively determined threshold. The purpose of the current research is to evaluate the psychometric qualities of the Social Anxiety Spectrum - Short Version (SHY-SV), a novel questionnaire designed to measure the complete range of social anxiety symptoms, from overt manifestations to subthreshold ones. METHODS: 42 subjects with a clinical diagnosis of social anxiety disorder (SAD) according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-5), 43 subjects with a clinical diagnosis of Obsessive-Compulsive Disorder (OCD) and 60 individuals without current or lifetime mental disorders (HC) were recruited from the Psychiatric Clinic of the University of Pisa. Subjects were assessed with the SCID-5, Liebowitz Social Anxiety Scale (LSAS) and the SHY-SV. RESULTS: SHY-SV showed strong internal consistency, and both the total and domain scores had great test-retest reliability. The Pearson's coefficients for the SHY-SV domain scores ranged from 0.391 to 0.933, and they were positively and significantly correlated with one another (p 0.001). All the SHY-SV domain scores were highly correlated with the SHY-SV total score. Results from of the correlation coefficients between SHY-SV and alternative measures of SAD were all significant and positive. Significant differences among diagnostic groups on both SAD-SV domains and total scores were found. SAD-SV total score increased significantly and progressively from HCs, to the OCD up to the SAD group which showed the highest values. CONCLUSION: The SHY-SV demonstrated significant convergent validity with other dimensional SAD measures, great internal consistency, and test-retest reliability. With an increasing score gradient from healthy controls to patients with OCD to those with SAD, the questionnaire performed differently in each of the three diagnostic categories.

Orthorexia and autism spectrum in University workers: relationship with gender, body mass index and dietary habits.

OBJECTIVE: To date, few studies have investigated the relationship between autistic traits and emerging phenotypes of restrictive disorders, such as Orthorexia nervosa (ON). The aim of the present work was to investigate the relationship between ON symptoms and autistic traits in a population of University employees, focusing on the impact of gender, weight and type of diet. METHODS: All academic and technical/administrative workers of University of Pisa were invited by mail to fulfil through an anonymous online form the Adult Autism Sub-threshold Spectrum (AdAS Spectrum) and the ORTO-R. RESULTS: A total of 285 subjects filled out the questionnaires. Participants with significant autistic traits were included into the Broad autism phenotype (BAP) group, while others into the No BAP group. Subjects in the BAP group reported significantly higher ORTO-R scores than others, while no difference was reported for gender, work position, type of diet, age and BMI. Females showed significantly higher ORTO-R scores and lower BMI than males. Older subjects showed a higher BMI. No significant differences in ORTO-R scores were reported depending on type of diet and work position. A decision tree model, with ORTO-R score as dependent variable, revealed in the first step significantly higher ORTO-R scores in the BAP group than in the No BAP group, and in the second step significantly higher ORTO-R scores among females only in the No BAP group. CONCLUSION: Our results further confirm the association between ON and autism spectrum, which seems to overcome the impact of gender in this population. LEVEL OF EVIDENCE: Level V, descriptive study.

Evolution patterns of SARS-CoV-2: Snapshot on its genome variants.

An acute respiratory syndrome (COVID-19), caused by a novel coronavirus (SARS-CoV-2) with a high rate of morbidity and elevate mortality, has emerged as one of the most important threats to humankind in the last centuries. Rigorous determination of SARS-CoV-2 infectivity is very difficult owing to the continuous evolution of the virus, with its single nucleotide polymorphism (SNP) variants and many lineages. However, it is urgently necessary to study the virus in depth, to understand the mechanism of its pathogenicity and virulence, and to develop effective therapeutic strategies. The present contribution summarizes in a succinct way the current knowledge on the evolutionary and structural features of the virus, with the aim of clarifying its mutational pattern and its possible role in the ongoing pandemic.

Exogenous bacterial DnaK increases protein kinases activity in human cancer cell lines.

BACKGROUND: Studies of molecular mechanisms underlying tumor cell signaling highlighted a critical role for kinases in carcinogenesis and cancer progression. To this regard, protein kinases regulates a number of critical cellular pathways by adding phosphate groups to specific substrates. For this reason, their involvement in the complex interactions between the human microbiota and cancer cells to determine therapy and tumor progression outcome is becoming increasingly relevant. Mycoplasmas are components of the normal human microbiota, and several species have also been associated to human diseases, including certain cancers. It is also important to note that Mycoplasmas and their proteins are a component of the common tumor microenvironment. In addition, several epidemiological, in vivo and in vitro studies indicate a close involvement of Mycoplasmas in cellular transformation and cancer progression. METHODS: In this study, we investigate the effect of exogenous Mycoplasma DnaK on kinases activity by treating in vitro four different eukaryotic cancer cell lines, namely lung and prostate cancer, colon adenocarcinoma, and neuroblastoma. Phosphorylation of kinases and specific substrates was measured at 20 and 60 min. RESULTS: Kinome analysis of our data indicates that Mycoplasma DnaK promotes the dysregulation of the activity of specific kinases and their substrates, with a known involvement in carcinogenesis and cancer progression. CONCLUSIONS: Given the similarity in structure and amino acid composition of this protein with other bacterial DnaKs we provide a novel mechanism whereby components of the human microbiota and present in the tumor microenvironment are able to deregulate phosphorylation events occurring during carcinogenesis and cancer progression.

Combined cART including Tenofovir Disoproxil, Emtricitabine, and Dolutegravir has potent therapeutic effects in HIV-1 infected humanized mice.

HIV-1 reservoirs persist in the presence of combined antiretroviral therapy (cART). However, cART has transformed HIV-1 infection into a chronic disease marked by control of HIV-1 viral load and mortality reduction. Major challenges remain, including viral resistance upon termination of cART and persistence and identification of tissue distribution of HIV-1 reservoirs. Thus, appropriate animal models that best mimic HIV-1 pathogenesis are important, and the current study complements our previously published validation of the CD34+ hematopoietic humanized mouse model for this purpose. Here we analyze viral suppression using the recently developed combination of antiretrovirals that include Tenofovir Disoproxil (TDF), Emtricitabine (FTC), and Dolutegravir (DTG), a choice based on recent clinical outcomes showing its improved antiretroviral potency, CD4+ T cell preservation, tolerability, and prevention of viral drug resistance compared to that of previous regimens. We used quantitative Airyscan-based super resolution confocal microscopy of selected mouse tissues. Our data allowed us to identify specific solid tissue reservoirs of human T cells expressing the HIV-1 core protein p24. In particular, lymph node, brain, spleen, and liver were visualized as reservoirs for residual infected cells. Marked reduction of viral replication was evident. Considering that detection and visualization of cryptic sites of HIV-1 infection in tissues are clearly crucial steps towards HIV-1 eradication, appropriate animal models with pseudo-human immune systems are needed. In fact, current studies with humans and non-human primates have limited sample availability at multiple stages of infection and cannot easily analyze the effects of differently administered combined antiretroviral treatments on multiple tissues. That is easier to manage when working with humanized mouse models, although we realize the limitations due to low human cell recovery and thus the number of cells available for thorough and comprehensive analyses. Nonetheless, our data further confirm that the CD34+ humanized mouse model is a potentially useful pre-clinical model to study and improve current anti-HIV-1 therapies.

The variants question: What is the problem?

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originated in Wuhan, China in early December 2019 has rapidly widespread worldwide. Over the course of the pandemic, due to the advance of whole-genome sequencing technologies, an unprecedented number of genomes have been generated, providing both invaluable insights into the ongoing evolution and epidemiology of the virus and allowing the identification of hundreds of circulating genetic variants during the pandemic. In recent months variants of SARS-CoV-2 that have an increased number of mutations on the Spike protein have brought concern all over the world. These have been called "variants of concerns" (VOCs), and/or "variants of interests" (VOIs) as it has been suggested that their genome mutations might impact transmission, immune control, and virulence. Tracking the spread of emerging SARS-CoV-2 variants is crucial to inform public health efforts and control the ongoing pandemic. In this review, a concise characterization of the SARS-CoV-2 mutational patterns of the main VOCs and VOIs circulating and cocirculating worldwide has been presented to determine the magnitude of the SARS-CoV-2 threat to better understand the virus genetic diversity and its potential impact on vaccination strategy.

SARS-CoV-2 B.1.617 Indian variants: Are electrostatic potential changes responsible for a higher transmission rate?

Lineage B.1.617+, also known as G/452R.V3 and now denoted by WHO with the Greek letters δ and κ, is a recently described SARS-CoV-2 variant under investigation first identified in October 2020 in India. As of May 2021, three sublineages labeled as B.1.617.1 (κ), B.1.617.2 (δ), and B.1.617.3 have been already identified, and their potential impact on the current pandemic is being studied. This variant has 13 amino acid changes, three in its spike protein, which are currently of particular concern: E484Q, L452R, and P681R. Here, we report a major effect of the mutations characterizing this lineage, represented by a marked alteration of the surface electrostatic potential (EP) of the receptor-binding domain (RBD) of the spike protein. Enhanced RBD-EP is particularly noticeable in the B.1.617.2 (δ) sublineage, which shows multiple replacements of neutral or negatively charged amino acids with positively charged amino acids. We here hypothesize that this EP change can favor the interaction between the B.1.617+ RBD and the negatively charged ACE2, thus conferring a potential increase in the virus transmission.

SARS-CoV-2 Infection and the COVID-19 Pandemic Emergency: The Importance of Diagnostic Methods.

BACKGROUND: Currently, a pandemic of coronavirus disease 2019 (COVID-19) caused by the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is underway, resulting in high morbidity and mortality across the globe. SUMMARY: A prompt and effective diagnosis is crucial to identify infected individuals, to monitor the infection, to perform contact tracing, and to limit the spread of the virus. Since the announcement of this public health emergency, several diagnostic methods have been developed including molecular and serological assays, and more recently biosensors. Here, we present the use of these assays as well as their main technical features, advantages, and limits. Key Messages: The development of reliable diagnostic assays is crucial not only for a correct diagnosis and containment of COVID-19 pandemic, but also for the decision-making process that is behind the clinical decisions, eventually contributing to the improvement of patient management. Furthermore, with the advent of vaccine and therapeutic monoclonal antibodies against SARS-CoV-2, serological assays will be instrumental for the validation of these new therapeutic options.

Analysis of Three Mutations in Italian Strains of SARS-CoV-2: Implications for Pathogenesis.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus initially detected in Wuhan in December 2019, responsible for coronavirus disease 2019 (COVID-19), a respiratory syndrome currently affecting >220 countries around the world, with >80 million cases registered and >1.8 million deaths. OBJECTIVE: As several vaccines are still being developed and 2 have been approved, it is particularly important to perform evolutionary surveillance to identify mutations potentially affecting vaccine efficacy. METHODS: DynaMut server has been used to evaluate the impact of the mutation found on SARS-CoV-2 isolates available on GISAID. RESULTS: In this article, we analyze whole genomes sequenced from Italian patients, and we report the characterization of 3 mutations, one of which presents in the spike protein. CONCLUSION: The mutations analyzed in this article can be useful to evaluate the evolution of SARS-CoV-2.

SARS-CoV-2 Lineages and Sub-Lineages Circulating Worldwide: A Dynamic Overview.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originated in Wuhan, China, in early December 2019 has rapidly widespread worldwide, becoming one of the major global public health issues of the last centuries. Key Messages: Over the course of the pandemic, due to the advanced whole-genome sequencing technologies, an unprecedented amount of genomes have been generated, providing invaluable insights into the ongoing evolution and epidemiology of the virus during the pandemic. Therefore, this large amount of data played an important role in the SARS-CoV-2 mitigation and control strategies. Key Messages: The active monitoring and characterization of the SARS-CoV-2 lineages circulating worldwide is useful for a more specific diagnosis, better care, and timely treatment. In this review, a concise characterization of all the lineages and sub-lineages circulating and co-circulating across the world has been presented in order to determine the magnitude of the SARS-CoV-2 threat and to better understand the virus genetic diversity and its dispersion dynamics.

End-of-life care for children with complex congenital heart disease: Parents' and medical care givers' perceptions.

AIM: In complex congenital heart diseases (CHD), patients may remain affected by significant morbidity and mortality after surgery. We analysed the end-of-life (EoL) care in children with severe CHD who died in our institution and investigated perspectives of parents and health-care professionals (HCPs). METHODS: Medical records of all children (age < 18 years old) affected by a severe CHD who died in a tertiary cardiac care centre were reviewed. Subsequently, a cross-sectional questionnaire-based study of parents and HCPs of children involved in the study was designed. RESULTS: In total, 30 children died (median age: 45 days; range: 15 days to 3.4 years). Of them, 97% (31/32) died in an intensive care unit setting and were intubated and sedated at EoL. A total of 77% (23/30) died without parents being present at bedside. Eighteen families and 10 HCPs were interviewed. For 61% of the parents (11/18) and 70% of the clinicians (7/10), the goal of therapy at the EoL was 'to lessen your child's suffering as much as possible'. Overall, 44% of parents (8/18) and 50% of HCPs recognised that their child had no chance of survival 'a few days before the child died'. CONCLUSIONS: We believe that these data suggest an unconscious reluctance to change goals of care in EoL, shifting from intensive care to comfort and quality of life.

Mycoplasma promotes malignant transformation in vivo, and its DnaK, a bacterial chaperone protein, has broad oncogenic properties.

We isolated a strain of human mycoplasma that promotes lymphomagenesis in SCID mice, pointing to a p53-dependent mechanism similar to lymphomagenesis in uninfected p53-/- SCID mice. Additionally, mycoplasma infection in vitro reduces p53 activity. Immunoprecipitation of p53 in mycoplasma-infected cells identified several mycoplasma proteins, including DnaK, a member of the Hsp70 chaperon family. We focused on DnaK because of its ability to interact with proteins. We demonstrate that mycoplasma DnaK interacts with and reduces the activities of human proteins involved in critical cellular pathways, including DNA-PK and PARP1, which are required for efficient DNA repair, and binds to USP10 (a key p53 regulator), impairing p53-dependent anticancer functions. This also reduced the efficacy of anticancer drugs that depend on p53 to exert their effect. mycoplasma was detected early in the infected mice, but only low copy numbers of mycoplasma DnaK DNA sequences were found in some primary and secondary tumors, pointing toward a hit-and-run/hide mechanism of transformation. Uninfected bystander cells took up exogenous DnaK, suggesting a possible paracrine function in promoting malignant transformation, over and above cells infected with the mycoplasma. Phylogenetic amino acid analysis shows that other bacteria associated with human cancers have similar DnaKs, consistent with a common mechanism of cellular transformation mediated through disruption of DNA-repair mechanisms, as well as p53 dysregulation, that also results in cancer-drug resistance. This suggests that the oncogenic properties of certain bacteria are DnaK-mediated.

Analysis of DnaK Expression from a Strain of Mycoplasma fermentans in Infected HCT116 Human Colon Carcinoma Cells.

Several species of mycoplasmas, including Mycoplasma fermentans, are associated with certain human cancers. We previously isolated and characterized in our laboratory a strain of human mycoplasma M. fermentans subtype incognitus (MF-I1) able to induce lymphoma in a Severe Combined Immuno-Deficient (SCID) mouse model, and we demonstrated that its chaperone protein, DnaK, binds and reduces functions of human poly-ADP ribose polymerase-1 (PARP1) and ubiquitin carboxyl-terminal hydrolase protein-10 (USP10), which are required for efficient DNA repair and proper p53 activities, respectively. We also showed that other bacteria associated with human cancers (including Mycoplasmapneumoniae, Helicobacterpylori, Fusobacteriumnucleatum, Chlamydiathrachomatis, and Chlamydia pneumoniae) have closely related DnaK proteins, indicating a potential common mechanism of cellular transformation. Here, we quantify dnaK mRNA copy number by RT-qPCR analysis in different cellular compartments following intracellular MF-I1 infection of HCT116 human colon carcinoma cells. DnaK protein expression in infected cells was also detected and quantified by Western blot. The amount of viable intracellular mycoplasma reached a steady state after an initial phase of growth and was mostly localized in the cytoplasm of the invaded cells, while we detected a logarithmically increased number of viable extracellular bacteria. Our data indicate that, after invasion, MF-I1 is able to establish a chronic intracellular infection. Extracellular replication was more efficient while MF-I1 cultured in cell-free axenic medium showed a markedly reduced growth rate. We also identified modifications of important regulatory regions and heterogeneous lengths of dnaK mRNA transcripts isolated from intracellular and extracellular MF-I1. Both characteristics were less evident in dnaK mRNA transcripts isolated from MF-I1 grown in cell-free axenic media. Taken together, our data indicate that MF-I1, after establishing a chronic infection in eukaryotic cells, accumulates different forms of dnaK with efficient RNA turnover.

Emerging of a SARS-CoV-2 viral strain with a deletion in nsp1.

BACKGROUND: The new Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which was first detected in Wuhan (China) in December of 2019 is responsible for the current global pandemic. Phylogenetic analysis revealed that it is similar to other betacoronaviruses, such as SARS-CoV and Middle-Eastern Respiratory Syndrome, MERS-CoV. Its genome is ∼ 30 kb in length and contains two large overlapping polyproteins, ORF1a and ORF1ab that encode for several structural and non-structural proteins. The non-structural protein 1 (nsp1) is arguably the most important pathogenic determinant, and previous studies on SARS-CoV indicate that it is both involved in viral replication and hampering the innate immune system response. Detailed experiments of site-specific mutagenesis and in vitro reconstitution studies determined that the mechanisms of action are mediated by (a) the presence of specific amino acid residues of nsp1 and (b) the interaction between the protein and the host's small ribosomal unit. In fact, substitution of certain amino acids resulted in reduction of its negative effects. METHODS: A total of 17,928 genome sequences were obtained from the GISAID database (December 2019 to July 2020) from patients infected by SARS-CoV-2 from different areas around the world. Genomes alignment was performed using MAFFT (REFF) and the nsp1 genomic regions were identified using BioEdit and verified using BLAST. Nsp1 protein of SARS-CoV-2 with and without deletion have been subsequently modelled using I-TASSER. RESULTS: We identified SARS-CoV-2 genome sequences, from several Countries, carrying a previously unknown deletion of 9 nucleotides in position 686-694, corresponding to the AA position 241-243 (KSF). This deletion was found in different geographical areas. Structural prediction modelling suggests an effect on the C-terminal tail structure. CONCLUSIONS: Modelling analysis of a newly identified deletion of 3 amino acids (KSF) of SARS-CoV-2 nsp1 suggests that this deletion could affect the structure of the C-terminal region of the protein, important for regulation of viral replication and negative effect on host's gene expression. In addition, substitution of the two amino acids (KS) from nsp1 of SARS-CoV was previously reported to revert loss of interferon-alpha expression. The deletion that we describe indicates that SARS-CoV-2 is undergoing profound genomic changes. It is important to: (i) confirm the spreading of this particular viral strain, and potentially of strains with other deletions in the nsp1 protein, both in the population of asymptomatic and pauci-symptomatic subjects, and (ii) correlate these changes in nsp1 with potential decreased viral pathogenicity.

Inverse correlation between average monthly high temperatures and COVID-19-related death rates in different geographical areas.

BACKGROUND: With the aim of providing a dynamic evaluation of the effects of basic environmental parameters on COVID-19-related death rate, we assessed the correlation between average monthly high temperatures and population density, with death/rate (monthly number of deaths/1 M people) for the months of March (start of the analysis and beginning of local epidemic in most of the Western World, except in Italy where it started in February) and April 2020 (continuation of the epidemic). Different geographical areas of the Northern Hemisphere in the United States and in Europe were selected in order to provide a wide range among the different parameters. The death rates were gathered from an available dataset. As a further control, we also included latitude, as a proxy for temperature. METHODS: Utilizing a publicly available dataset, we retrieved data for the months of March and April 2020 for 25 areas in Europe and in the US. We computed the monthly number of deaths/1 M people of confirmed COVID-19 cases and calculated the average monthly high temperatures and population density for all these areas. We determined the correlation between number of deaths/1 M people and the average monthly high temperatures, the latitude and the population density. RESULTS: We divided our analysis in two parts: analysis of the correlation among the different variables in the month of March and subsequent analysis in the month of April. The differences were then evaluated. In the month of March there was no statistical correlation between average monthly high temperatures of the considered geographical areas and number of deaths/1 M people. However, a statistically significant inverse correlation became significant in the month of April between average monthly high temperatures (p = 0.0043) and latitude (p = 0.0253) with number of deaths/1 M people. We also observed a statistically significant correlation between population density and number of deaths/1 M people both in the month of March (p = 0.0297) and in the month of April (p = 0.0116), when three areas extremely populated (NYC, Los Angeles and Washington DC) were included in the calculation. Once these three areas were removed, the correlation was not statistically significant (p = 0.1695 in the month of March, and p = 0.7076 in the month of April). CONCLUSIONS: The number of COVID-19-related deaths/1 M people was essentially the same during the month of March for all the geographical areas considered, indicating essentially that the infection was circulating quite uniformly except for Lombardy, Italy, where it started earlier. Lockdown measures were implemented between the end of March and beginning of April, except for Italy which started March 9th. We observed a strong, statistically significant inverse correlation between average monthly high temperatures with the number of deaths/1 M people. We confirmed the data by analyzing the correlation with the latitude, which can be considered a proxy for high temperature. Previous studies indicated a negative effect of high climate temperatures on Sars-COV-2 spreading. Our data indicate that social distancing measure are more successful in the presence of higher average monthly temperatures in reducing COVID-19-related death rate, and a high level of population density seems to negatively impact the effect of lockdown measures.

Impact of lockdown on Covid-19 case fatality rate and viral mutations spread in 7 countries in Europe and North America.

BACKGROUND: Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) caused the first coronavirus disease 2019 (COVID-19) outbreak in China and has become a public health emergency of international concern. SARS-CoV-2 outbreak has been declared a pandemic by WHO on March 11th, 2020 and the same month several Countries put in place different lockdown restrictions and testing strategies in order to contain the spread of the virus. METHODS: The calculation of the Case Fatality Rate of SARS-CoV-2 in the Countries selected was made by using the data available at https://github.com/owid/covi-19-data/tree/master/public/data . Case fatality rate was calculated as the ratio between the death cases due to COVID-19, over the total number of SARS-CoV-2 reported cases 14 days before. Standard Case Fatality Rate values were normalized by the Country-specific ρ factor, i.e. the number of PCR tests/1 million inhabitants over the number of reported cases/1 million inhabitants. Case-fatality rates between Countries were compared using proportion test. Post-hoc analysis in the case of more than two groups was performed using pairwise comparison of proportions and p value was adjusted using Holm method. We also analyzed 487 genomic sequences from the GISAID database derived from patients infected by SARS-CoV-2 from January 2020 to April 2020 in Italy, Spain, Germany, France, Sweden, UK and USA. SARS-CoV-2 reference genome was obtained from the GenBank database (NC_045512.2). Genomes alignment was performed using Muscle and Jalview software. We, then, calculated the Case Fatality Rate of SARS-CoV-2 in the Countries selected. RESULTS: In this study we analyse how different lockdown strategies and PCR testing capability adopted by Italy, France, Germany, Spain, Sweden, UK and USA have influenced the Case Fatality Rate and the viral mutations spread. We calculated case fatality rates by dividing the death number of a specific day by the number of patients with confirmed COVID-19 infection observed 14 days before and normalized by a ρ factor which takes into account the diagnostic PCR testing capability of each Country and the number of positive cases detected. We notice the stabilization of a clear pattern of mutations at sites nt241, nt3037, nt14408 and nt23403. A novel nonsynonymous SARS-CoV-2 mutation in the spike protein (nt24368) has been found in genomes sequenced in Sweden, which enacted a soft lockdown strategy. CONCLUSIONS: Strict lockdown strategies together with a wide diagnostic PCR testing of the population were correlated with a relevant decline of the case fatality rate in different Countries. The emergence of specific patterns of mutations concomitant with the decline in case fatality rate needs further confirmation and their biological significance remains unclear.

Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant.

BACKGROUND: SARS-CoV-2 is a RNA coronavirus responsible for the pandemic of the Severe Acute Respiratory Syndrome (COVID-19). RNA viruses are characterized by a high mutation rate, up to a million times higher than that of their hosts. Virus mutagenic capability depends upon several factors, including the fidelity of viral enzymes that replicate nucleic acids, as SARS-CoV-2 RNA dependent RNA polymerase (RdRp). Mutation rate drives viral evolution and genome variability, thereby enabling viruses to escape host immunity and to develop drug resistance. METHODS: We analyzed 220 genomic sequences from the GISAID database derived from patients infected by SARS-CoV-2 worldwide from December 2019 to mid-March 2020. SARS-CoV-2 reference genome was obtained from the GenBank database. Genomes alignment was performed using Clustal Omega. Mann-Whitney and Fisher-Exact tests were used to assess statistical significance. RESULTS: We characterized 8 novel recurrent mutations of SARS-CoV-2, located at positions 1397, 2891, 14408, 17746, 17857, 18060, 23403 and 28881. Mutations in 2891, 3036, 14408, 23403 and 28881 positions are predominantly observed in Europe, whereas those located at positions 17746, 17857 and 18060 are exclusively present in North America. We noticed for the first time a silent mutation in RdRp gene in England (UK) on February 9th, 2020 while a different mutation in RdRp changing its amino acid composition emerged on February 20th, 2020 in Italy (Lombardy). Viruses with RdRp mutation have a median of 3 point mutations [range: 2-5], otherwise they have a median of 1 mutation [range: 0-3] (p value < 0.001). CONCLUSIONS: These findings suggest that the virus is evolving and European, North American and Asian strains might coexist, each of them characterized by a different mutation pattern. The contribution of the mutated RdRp to this phenomenon needs to be investigated. To date, several drugs targeting RdRp enzymes are being employed for SARS-CoV-2 infection treatment. Some of them have a predicted binding moiety in a SARS-CoV-2 RdRp hydrophobic cleft, which is adjacent to the 14408 mutation we identified. Consequently, it is important to study and characterize SARS-CoV-2 RdRp mutation in order to assess possible drug-resistance viral phenotypes. It is also important to recognize whether the presence of some mutations might correlate with different SARS-CoV-2 mortality rates.