Structural and functional insights into the spike protein mutations of emerging SARS-CoV-2 variants.

Since the emergence of the first case of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2), the viral genome has constantly undergone rapid mutations for better adaptation in the host system. These newer mutations have given rise to several lineages/ variants of the virus that have resulted in high transmission and virulence rates compared to the previously circulating variants. Owing to this, the overall caseload and related mortality have tremendously increased globally to > 233 million infections and > 4.7 million deaths as of Sept. 28th, 2021. SARS-CoV-2, Spike (S) protein binds to host cells by recognizing human angiotensin-converting enzyme 2 (hACE2) receptor. The viral S protein contains S1 and S2 domains that constitute the binding and fusion machinery, respectively. Structural analysis of viral S protein reveals that the virus undergoes conformational flexibility and dynamicity to interact with the hACE2 receptor. The SARS-CoV-2 variants and mutations might be associated with affecting the conformational plasticity of S protein, potentially linked to its altered affinity, infectivity, and immunogenicity. This review focuses on the current circulating variants of SARS-CoV-2 and the structure-function analysis of key S protein mutations linked with increased affinity, higher infectivity, enhanced transmission rates, and immune escape against this infection.

SARS-CoV-2 Pandemic Tracing in Italy Highlights Lineages with Mutational Burden in Growing Subsets.

Tracing the appearance and evolution of virus variants is essential in the management of the COVID-19 pandemic. Here, we focus on SARS-CoV-2 spread in Italian patients by using viral sequences deposited in public databases and a tracing procedure which is used to monitor the evolution of the pandemic and detect the spreading, within the infected population of emergent sub-clades with a potential positive selection. Analyses of a collection of monthly samples focused on Italy highlighted the appearance and evolution of all the main viral sub-trees emerging at the end of the first year of the pandemic. It also identified additional expanding subpopulations which spread during the second year (i.e., 2021). Three-dimensional (3D) modelling of the main amino acid changes in mutated viral proteins, including ORF1ab (nsp3, nsp4, 2'-o-ribose methyltransferase, nsp6, helicase, nsp12 [RdRp]), N, ORF3a, ORF8, and spike proteins, shows the potential of the analysed structural variations to result in epistatic modulation and positive/negative selection pressure. These analyzes will be of importance to the early identification of emerging clades, which can develop into new "variants of concern" (i.e., VOC). These analyses and settings will also help SARS-CoV-2 coronet genomic centers in other countries to trace emerging worldwide variants.

Measuring disease burden of dominant variants of COVID-19 in Taiwan.

Objectives: The disability-adjusted life years (DALYs) of COVID-19 have been applied as a time-based measurement to estimate years of life lost due to premature mortality or healthy life lost in different countries. Limited information was found for DALYs among different variants of concern (VOC). Methods: Disease severities based on categories of asymptomatic, mild, moderate, severe, and critical cases were explored among different VOC by analyzing the proportions in confirmed cases. DALY or years of healthy life lost due to disability (YLD)-based annual burdens of COVID-19 on different ages, genders as well as trend analysis were also evaluated for VOC in Taiwan. Results: Different trends were observed in years of life lost due to premature mortality (YLLs) or YLD for various age or gender categories. Disease severity at critical stage had the highest percentage for overall YLDs encompassed from 2020 to 2022. Also, critical-grade cases were found to be predominantly caused by Wild-type, Alpha, and Omicron variants in 2020, 2021, and 2022, respectively. Conclusion: Precautionary measures are also suggested for policy makers to take in specific seasons, age or gender groups based on YLL and YLD analyses.

Mutational dynamics of SARS-CoV-2: Impact on future COVID-19 vaccine strategies.

The rapid emergence of multiple strains of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has sparked profound concerns regarding the ongoing evolution of the virus and its potential impact on global health. Classified by the World Health Organization (WHO) as variants of concern (VOC), these strains exhibit heightened transmissibility and pathogenicity, posing significant challenges to existing vaccine strategies. Despite widespread vaccination efforts, the continual evolution of SARS-CoV-2 variants presents a formidable obstacle to achieving herd immunity. Of particular concern is the coronavirus spike (S) protein, a pivotal viral surface protein crucial for host cell entry and infectivity. Mutations within the S protein have been shown to enhance transmissibility and confer resistance to antibody-mediated neutralization, undermining the efficacy of traditional vaccine platforms. Moreover, the S protein undergoes rapid molecular evolution under selective immune pressure, leading to the emergence of diverse variants with distinct mutation profiles. This review underscores the urgent need for vigilance and adaptation in vaccine development efforts to combat the evolving landscape of SARS-CoV-2 mutations and ensure the long-term effectiveness of global immunization campaigns.

Wide Real-Life Data Support Reduced Sensitivity of Antigen Tests for Omicron SARS-CoV-2 Infections.

With the continuous spread of new SARS-CoV-2 variants of concern (VOCs), the monitoring of diagnostic test performances is mandatory. We evaluated the changes in antigen diagnostic tests' (ADTs) accuracy along the Delta to Omicron VOCs transition, exploring the N protein mutations possibly affecting ADT sensitivity and assessing the best sampling site for the diagnosis of Omicron infections. In total, 5175 subjects were enrolled from 1 October 2021 to 15 July 2022. The inclusion criteria were SARS-CoV-2 ADT combined with a same-day RT-PCR swab test. For the sampling site analysis, 61 patients were prospectively recruited during the Omicron period for nasal and oral swab analyses by RT-PCR. Next-Generation Sequencing data were obtained to evaluate the different sublineages. Using RT-PCR as a reference, 387 subjects resulted in becoming infected and the overall sensitivity of the ADT decreased from 63% in the Delta period to 33% in the Omicron period. This decrease was highly statistically significant (p < 0.001), and no decrease in viral load was detected at the RNA level. The nasal site presented a significantly higher viral load than the oral site during the Omicron wave. The reduced detection rate of Omicron infections by ADT should be considered in the global testing strategy to preserve accurate diagnoses across the changing SARS-CoV-2 variants.

A Repeat Pattern of Founder Events for SARS-CoV-2 Variants in Alaska.

Alaska is a unique US state because of its large size, geographically disparate population density, and physical distance from the contiguous United States. Here, we describe a pattern of SARS-CoV-2 variant emergence across Alaska reflective of these differences. Using genomic data, we found that in Alaska, the Omicron sublineage BA.2.3 overtook BA.1.1 by the week of 27 February 2022, reaching 48.5% of sequenced cases. On the contrary, in the contiguous United States, BA.1.1 dominated cases for longer, eventually being displaced by BA.2 sublineages other than BA.2.3. BA.2.3 only reached a prevalence of 10.9% in the contiguous United States. Using phylogenetics, we found evidence of potential origins of the two major clades of BA.2.3 in Alaska and with logistic regression estimated how it emerged and spread throughout the state. The combined evidence is suggestive of founder events in Alaska and is reflective of how Alaska's unique dynamics influence the emergence of SARS-CoV-2 variants.

A global scale COVID-19 variants time-series analysis across 48 countries.

Background: The coronavirus disease (COVID-19) pandemic is slowing down, and countries are discussing whether preventive measures have remained effective or not. This study aimed to investigate a particular property of the trend of COVID-19 that existed and if its variants of concern were cointegrated, determining its possible transformation into an endemic. Methods: Biweekly expected new cases by variants of COVID-19 for 48 countries from 02 May 2020 to 29 August 2022 were acquired from the GISAID database. While the case series was tested for homoscedasticity with the Breusch-Pagan test, seasonal decomposition was used to obtain a trend component of the biweekly global new case series. The percentage change of trend was then tested for zero-mean symmetry with the one-sample Wilcoxon signed rank test and zero-mean stationarity with the augmented Dickey-Fuller test to confirm a random COVID trend globally. Vector error correction models with the same seasonal adjustment were regressed to obtain a variant-cointegrated series for each country. They were tested by the augmented Dickey-Fuller test for stationarity to confirm a constant long-term stochastic intervariant interaction within the country. Results: The trend series of seasonality-adjusted global COVID-19 new cases was found to be heteroscedastic (p = 0.002), while its rate of change was indeterministic (p = 0.052) and stationary (p = 0.024). Seasonal cointegration relationships between expected new case series by variants were found in 37 out of 48 countries (p < 0.05), reflecting a constant long-term stochastic trend in new case numbers contributed from different variants of concern within most countries. Conclusion: Our results indicated that the new case long-term trends were random on a global scale and stable within most countries; therefore, the virus was unlikely to be eliminated but containable. Policymakers are currently in the process of adapting to the transformation of the pandemic into an endemic.

Risk factors for SARS-CoV-2 infection during the early stages of the COVID-19 pandemic: a systematic literature review.

Introduction: Identifying SARS-CoV-2 infection risk factors allows targeted public health and social measures (PHSM). As new, more transmissible variants of concern (VoC) emerge, vaccination rates increase and PHSM are eased, it is important to understand any potential change to infection risk factors. The aim of this systematic literature review is to describe the risk factors for SARS-CoV-2 infection by VoC. Methods: A literature search was performed in MEDLINE, PubMed and Embase databases on 5 May 2022. Eligibility included: observational studies published in English after 1 January 2020; any age group; the outcome of SARS-CoV-2 infection; and any potential risk factors investigated in the study. Results were synthesized into a narrative summary with respect to measures of association, by VoC. ROBINS-E tool was utilized for risk of bias assessment. Results: Of 6,197 studies retrieved, 43 studies were included after screening. Common risk factors included older age, minority ethnic group, low socioeconomic status, male gender, increased household size, occupation/lower income level, inability to work from home, public transport use, and lower education level. Most studies were undertaken when the ancestral strain was predominant. Many studies had some selection bias due to testing criteria and limited laboratory capacity. Conclusion: Understanding who is at risk enables the development of strategies that target priority groups at each of the different stages of a pandemic and helps inform vaccination strategies and other interventions which may also inform public health responses to future respiratory infection outbreaks. While it was not possible to determine changes to infection risk by recent VoC in this review, the risk factors identified will add to the overall understanding of the groups who are at greatest risk of infection in the early stages of a respiratory virus outbreak. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022330706, PROSPERO [CRD42022330706].

Analysis of SARS-CoV-2 genomic surveillance data during the Delta and Omicron waves at a Saudi tertiary referral hospital.

BACKGROUND: Studying the genomic evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may help determine outbreak clusters and virus transmission advantages to aid public health efforts during the pandemic. Thus, we tracked the evolution of SARS-CoV-2 by variant epidemiology, breakthrough infection, and patient characteristics as the virus spread during the Delta and Omicron waves. We also conducted phylogenetic analyses to assess modes of transmission. METHODS: Nasopharyngeal samples were collected from a cohort of 900 patients with positive polymerase chain reaction (PCR) test results confirming COVID-19 disease. Samples underwent real-time PCR detection using TaqPath assays. Sequencing was performed with Ion GeneStudio using the Ion AmpliSeq™ SARS-CoV-2 panel. Variant calling was performed with Torrent Suite™ on the Torrent Server. For phylogenetic analyses, the MAFFT tool was used for alignment and the maximum likelihood method with the IQ-TREE tool to build the phylogenetic tree. Data were analyzed using SAS statistical software. Analysis of variance or t tests were used to assess continuous variables, and χ2 tests were used to assess categorical variables. Univariate and multivariate logistic regression analyses were preformed to estimate odds ratios (ORs). RESULTS: The predominant variants in our cohort of 900 patients were non-variants of concern (11.1 %), followed by Alpha (4.1 %), Beta (5.6 %), Delta (21.2 %), and Omicron (58 %). The Delta wave had more male than female cases (112 vs. 78), whereas the Omicron wave had more female than male cases (311 vs. 208). The oldest patients (mean age, 43.4 years) were infected with non-variants of concern; the youngest (mean age, 33.7 years), with Omicron. Younger patients were mostly unvaccinated, whereas elderly patients were mostly vaccinated, a statistically significant difference. The highest risk for breakthrough infection by age was for patients aged 30-39 years (OR = 12.4, CI 95 %: 6.6-23.2), followed by patients aged 40-49 years (OR = 11.2, CI 95 %: 6.1-23.1) and then 20-29 years (OR = 8.2, CI 95 %: 4.4-15.4). Phylogenetic analyses suggested the interaction of multiple cases related to outbreaks for breakthrough infections, healthcare workers, and intensive care unit admission. CONCLUSION: The findings of this study highlighted several major public health ramifications, including the distribution of variants over a wide range of demographic and clinical variables and by vaccination status.

Never ending story? Evolution of SARS-CoV-2 monitored through Gibbs energies of biosynthesis and antigen-receptor binding of Omicron BQ.1, BQ.1.1, XBB and XBB.1 variants.

RNA viruses exhibit a great tendency to mutate. Mutations occur in the parts of the genome that encode the spike glycoprotein and less often in the rest of the genome. This is why Gibbs energy of binding changes more than that of biosynthesis. Starting from 2019, the wild type that was labeled Hu-1 has during the last 3 years evolved to produce several dozen new variants, as a consequence of mutations. Mutations cause changes in empirical formulas of new virus strains, which lead to change in thermodynamic properties of biosynthesis and binding. These changes cause changes in the rate of reactions of binding of virus antigen to the host cell receptor and the rate of virus multiplication in the host cell. Changes in thermodynamic and kinetic parameters lead to changes in biological parameters of infectivity and pathogenicity. Since the beginning of the COVID-19 pandemic, SARS-CoV-2 has been evolving towards increase in infectivity and maintaining constant pathogenicity, or for some variants a slight decrease in pathogenicity. In the case of Omicron BQ.1, BQ.1.1, XBB and XBB.1 variants pathogenicity is identical as in the Omicron BA.2.75 variant. On the other hand, infectivity of the Omicron BQ.1, BQ.1.1, XBB and XBB.1 variants is greater than those of previous variants. This will most likely result in the phenomenon of asymmetric coinfection, that is circulation of several variants in the population, some being dominant.

Rapid Detection of SARS-CoV-2 RNA Using Reverse Transcription Recombinase Polymerase Amplification (RT-RPA) with Lateral Flow for N-Protein Gene and Variant-Specific Deletion-Insertion Mutation in S-Protein Gene.

Rapid molecular testing for severe acute respiratory coronavirus 2 (SARS-CoV-2) variants may contribute to the development of public health measures, particularly in resource-limited areas. Reverse transcription recombinase polymerase amplification using a lateral flow assay (RT-RPA-LF) allows rapid RNA detection without thermal cyclers. In this study, we developed two assays to detect SARS-CoV-2 nucleocapsid (N) gene and Omicron BA.1 spike (S) gene-specific deletion-insertion mutations (del211/ins214). Both tests had a detection limit of 10 copies/µL in vitro and the detection time was approximately 35 min from incubation to detection. The sensitivities of SARS-CoV-2 (N) RT-RPA-LF by viral load categories were 100% for clinical samples with high (>9015.7 copies/µL, cycle quantification (Cq): < 25) and moderate (385.5-9015.7 copies/µL, Cq: 25-29.9) viral load, 83.3% for low (16.5-385.5 copies/µL, Cq: 30-34.9), and 14.3% for very low (<16.5 copies/µL, Cq: 35-40). The sensitivities of the Omicron BA.1 (S) RT-RPA-LF were 94.9%, 78%, 23.8%, and 0%, respectively, and the specificity against non-BA.1 SARS-CoV-2-positive samples was 96%. The assays seemed more sensitive than rapid antigen detection in moderate viral load samples. Although implementation in resource-limited settings requires additional improvements, deletion-insertion mutations were successfully detected by the RT-RPA-LF technique.

A DNA Vaccine Encoding the Full-Length Spike Protein of Beta Variant (B.1.351) Elicited Broader Cross-Reactive Immune Responses against Other SARS-CoV-2 Variants.

The SARS-CoV-2 pandemic remains an ongoing threat to global health with emerging variants, especially the Omicron variant and its sub-lineages. Although large-scale vaccination worldwide has delivered outstanding achievements for COVID-19 prevention, a declining effectiveness to a different extent in emerging SARS-CoV-2 variants was observed in the vaccinated population. Vaccines eliciting broader spectrum neutralizing antibodies and cellular immune responses are urgently needed and important. To achieve this goal, rational vaccine design, including antigen modeling, screening and combination, vaccine pipelines, and delivery, are keys to developing a next-generation COVID-19 vaccine. In this study, we designed several DNA constructs based on codon-optimized spike coding regions of several SARS-CoV-2 variants and analyzed their cross-reactive antibodies, including neutralizing antibodies, and cellular immune responses against several VOCs in C57BL/6 mice. The results revealed that different SARS-CoV-2 VOCs induced different cross-reactivity; pBeta, a DNA vaccine encoding the spike protein of the Beta variant, elicited broader cross-reactive neutralizing antibodies against other variants including the Omicron variants BA.1 and BA.4/5. This result demonstrates that the spike antigen from the Beta variant potentially serves as one of the antigens for multivalent vaccine design and development against variants of SARS-CoV-2.

Mucosal immunization with Ad5-based vaccines protects Syrian hamsters from challenge with omicron and delta variants of SARS-CoV-2.

SARS-CoV-2 variant clades continue to circumvent antibody responses elicited by vaccination or infection. Current parenteral vaccination strategies reduce illness and hospitalization, yet do not significantly protect against infection by the more recent variants. It is thought that mucosal vaccination strategies may better protect against infection by inducing immunity at the sites of infection, blocking viral transmission more effectively, and significantly inhibiting the evolution of new variants of concern (VOCs). In this study, we evaluated the immunogenicity and efficacy of a mucosally-delivered, non-replicating, adenovirus type 5-vectored vaccine that expresses the spike (S) gene of Wuhan (rAd5-S-Wuhan), delta (rAd5-S-delta), or omicron (rAd5-S-omicron) SARS-CoV-2 VOCs. Hamsters were immunized with these vaccines intranasally prior to challenge with omicron or delta variants. Additionally, one group was vaccinated by oral gavage with rAd5-S-Wuhan prior to challenge with the delta variant. Both intranasal and oral administration of rAd5-S-Wuhan generated cross-reactive serum IgG and mucosal IgA to all variant spike and RBD proteins tested. rAd5-S-omicron and rAd5-S-delta additionally elicited cross-reactive antibodies, though rAd5-S-omicron had significantly lower binding antibody levels except against its matched antigens. Two weeks after the final vaccination, hamsters were challenged with a SARS-CoV-2 variant; omicron or delta. Whether matched to the challenge or with rAd5-S-Wuhan, all vaccines protected hamsters from weight loss and lung pathology caused by challenge and significantly reduced viral shedding compared to placebo. Vaccination with rAd5-S-Wuhan provided significant protection, although there was an improved reduction in shedding and disease pathology in groups protected by the matched VOC vaccines. Nevertheless, Wuhan-based vaccination elicited the most cross-reactive antibody responses generally. Overall, heterologous vaccination via mucosal routes may be advantageous for second-generation vaccines.

Impact of SARS-CoV-2 Omicron and Delta variants in patients requiring intensive care unit (ICU) admission for COVID-19, Northern Italy, December 2021 to January 2022.

This multicenter observational study included 171 COVID-19 adult patients hospitalized in the ICUs of nine hospitals in Lombardy (Northern Italy) from December, 1st 2021, to February, 9th 2022. During the study period, the Delta/Omicron variant ratio of cases decreased with a delay of two weeks in ICU patients compared to that in the community; a higher proportion of COVID-19 unvaccinated patients was infected by Delta than by Omicron whereas a higher rate of COVID-19 boosted patients was Omicron-infected. A higher number of comorbidities and a higher comorbidity score in ICU critically COVID-19 inpatients was positively associated with the Omicron infection as well in vaccinated individuals. Although people infected by Omicron have a lower risk of severe disease than those infected by Delta variant, the outcome, including the risk of ICU admission and the need for mechanical ventilation due to infection by Omicron versus Delta, remains uncertain. The continuous monitoring of the circulating SARS-CoV-2 variants remains a milestone to counteract this pandemic.

A tool for the cheap and rapid screening of SARS-CoV-2 variants of concern (VoCs) by Sanger sequencing.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging virus that, since March 2020, has been responsible for a global and ongoing pandemic. Its rapid spread over the past nearly 3 years has caused novel variants to arise. To monitor the circulation and emergence of SARS-CoV-2 variants, surveillance systems based on nucleotide mutations are required. In this regard, we searched in the spike, ORF8, and nucleocapsid genes to detect variable sites among SARS-CoV-2 variants. We describe polymorphic genetic regions that enable us to differentiate between the Alpha, Beta, Gamma, Delta, and Omicron variants of concern (VoCs). We found 21 relevant mutations, 13 of which are unique for Omicron lineages BA.1/BA.1.1, BA.2, BA.3, BA.4, and BA.5. This genetic profile enables the discrimination between VoCs using only four reverse transcription PCR fragments and Sanger sequencing, offering a cheaper and faster alternative to whole-genome sequencing for SARS-CoV-2 surveillance. IMPORTANCE Our work describes a new (Sanger sequencing-based) screening methodology for SARS-CoV-2, performing PCR amplifications of a few target regions to detect diagnostic mutations between virus variants. Using the methodology developed in this work, we were able to discriminate between the following VoCs: Alpha, Beta, Gamma, Delta, and Omicron (BA.1/BA.1.1, BA.2, BA.3, BA.4, and BA.5). This becomes important, especially in low-income countries where current methodologies like next-generation sequencing have prohibitive costs. Furthermore, rapid detection would allow sanitary authorities to take rapid measures to limit the spread of the virus and therefore reduce the probability of new virus dispersion. With this methodological approach, 13 previously unreported diagnostic mutations among several Omicron lineages were found.

Selective adaptation of SARS-CoV-2 Omicron under booster vaccine pressure: a multicentre observational study.

BACKGROUND: High rates of vaccination and natural infection drive immunity and redirect selective viral adaptation. Updated boosters are installed to cope with drifted viruses, yet data on adaptive evolution under increasing immune pressure in a real-world situation are lacking. METHODS: Cross-sectional study to characterise SARS-CoV-2 mutational dynamics and selective adaptation over >1 year in relation to vaccine status, viral phylogenetics, and associated clinical and demographic variables. FINDINGS: The study of >5400 SARS-CoV-2 infections between July 2021 and August 2022 in metropolitan New York portrayed the evolutionary transition from Delta to Omicron BA.1-BA.5 variants. Booster vaccinations were implemented during the Delta wave, yet booster breakthrough infections and SARS-CoV-2 re-infections were almost exclusive to Omicron. In adjusted logistic regression analyses, BA.1, BA.2, and BA.5 had a significant growth advantage over co-occurring lineages in the boosted population, unlike BA.2.12.1 or BA.4. Selection pressure by booster shots translated into diffuse adaptive evolution in Delta spike, contrasting with strong, receptor-binding motif-focused adaptive evolution in BA.2-BA.5 spike (Fisher Exact tests; non-synonymous/synonymous mutation rates per site). Convergent evolution has become common in Omicron, engaging spike positions crucial for immune escape, receptor binding, or cleavage. INTERPRETATION: Booster shots are required to cope with gaps in immunity. Their discriminative immune pressure contributes to their effectiveness but also requires monitoring of selective viral adaptation processes. Omicron BA.2 and BA.5 had a selective advantage under booster vaccination pressure, contributing to the evolution of BA.2 and BA.5 sublineages and recombinant forms that predominate in 2023. FUNDING: The study was supported by NYU institutional funds and partly by the Cancer Center Support Grant P30CA016087 at the Laura and Isaac Perlmutter Cancer Center.

Trends in the COVID-19 Pandemic in Italy during the Summers of 2020 (before Mass Vaccination), 2021 (after Primary Mass Vaccination) and 2022 (after Booster Mass Vaccination): A Real-World Nationwide Study Based on a Population of 58.85 Million People.

Like all RNA viruses, SARS-CoV-2 shows a high mutation rate, which has led to the emergence of new variants. Among them, Gamma and Delta developed at the turn of 2020-2021 in Amazonas and India, two ecoregions characterized by hot-humid weather, very similar to that of the summer season in Italy due to climate change, the first Western country to be hit hard by COVID-19 and to experience lockdown restrictions in a democratic framework of 58.85 million people. The aim of our research has been to evaluate the impact of climate on the COVID-19 pandemic in Italy during the summers of 2020 (before mass vaccination), 2021 (after primary mass vaccination) and 2022 (after booster mass vaccination), also taking into account the emergence of these two variants. METHODS: During the state of national health emergency and the Draghi government, the Civil Defense Department released the aggregate data coming from the Ministry of Health, the Higher Institute of Health, the Independent Provinces and the Italian Regions daily, in order to inform about the pandemic situation in Italy. Among these data there were the number of deaths, hospitalizations in intensive care units (ICU), non-ICU patients, contagions and performed swabs. By means of a team effort, we have collected and elaborated all these data, comparing the COVID-19 pandemic in Italy during the summers of 2020 (following the nationwide lockdown), 2021 and 2022. RESULTS: from the summer of 2020 to the summers of 2021 and 2022 all pandemic trend indicators have shown a sharp worsening in Italy. COVID-19 deaths increased by ≈298% and ≈834%, ICU hospitalizations by ≈386% and ≈310%, non-ICU hospitalizations by ≈224% and ≈600%, contagions by ≈627% and ≈6850% (i.e., ≈68.50 times), swabs by ≈354% and ≈370%, and the mean positivity rate passed from ≈1% to ≈2% and ≈20%, respectively. CONCLUSIONS: SARS-CoV-2 can be transmitted in any climate, including areas with hot and humid weather, and the emergence of variants adapted to hot-humid climates may result in summer COVID-19 outbreaks, even in neither tropical nor subtropical countries. Although COVID-19 vaccines can confer cross-protection against newly emerging variants, this cross-immunity is naturally not absolute but limited, considering that vaccine protection wanes significantly after 6 months. It follows that a subject vaccinated at the beginning of the winter will not be completely covered in the height of the summer, and we should not forget the unvaccinated. As a final remark, the long and strict nationwide lockdown made it possible to flatten SARS-CoV-2 circulation and, therefore, its negative impact on Italy during the summer of 2020.

The Emergence of Omicron: Challenging Times Are Here Again!

COVID-19 has been reported to have caused more than 286 million cases and 5.4 million deaths till date. COVID variants have appeared at regular intervals-alpha, beta, gamma, delta and now omicron. 'Omicron' is driving the current surge of cases in most countries including India and is poised to replace 'delta' the world over. This variant with more than 50 mutations is phylogenetically very different from other variants. The omicron variant spreads rapidly with an average doubling time of two days. The disease so far has been mild as compared with delta. Though previous infection and vaccination offer little or no protection against infection with omicron, they do seem to partially protect against hospitalization and severe disease. Booster vaccinations have not made any notable impact on the spread of omicron and have further worsened global vaccine equity. The indirect consequences of omicron from lockdowns, restrictions, travel bans, economic losses, health care worker infections and overwhelming of health care facilities are likely to be enormous. The direct effects of omicron on children are expected to be mild like with the previous variants. However, the indirect effects on child mental, physical, and social health may be considerable owing to school closures, missed vaccinations, neglect of other diseases, etc. It is, therefore, imperative that governments take rational decisions to navigate the world through this latest crisis.

Continued Complexity of Mutations in Omicron Sublineages.

The latest SARS-CoV-2 variant of concern (VOC), Omicron (B.1.1.529), has diversified into more than 300 sublineages. With an expanding number of newly emerging sublineages, the mutation profile is also becoming complicated. There exist mutually exclusive and revertant mutations in different sublineages. Omicron sublineages share some common mutations with previous VOCs (Alpha, Beta, Gamma, and Delta), indicating an evolutionary relationship between these VOCs. A diverse mutation profile at the spike-antibody interface, flexibility of the regions harboring mutations, mutation types, and coexisting mutations suggest that SARS-CoV-2's evolution is far from over.

Survival and detection of SARS-CoV-2 variants on dry swabs post storage.

COVID-19 has resulted in nearly 598 million infections and over 6.46 million deaths since the start of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in 2019. The rapid onset of the pandemic, combined with the emergence of viral variants, crippled many health systems particularly from the perspective of coping with massive diagnostic loads. Shortages of diagnostic kits and capacity forced laboratories to store clinical samples resulting in huge backlogs, the effects of this on diagnostic pickup have not been fully understood. Herein, we investigated the impact of storing SARS-CoV-2 inoculated dry swabs on the detection and viability of four viral strains over a period of 7 days. Viral load, as detected by qRT-PCR, displayed no significant degradation during this time for all viral loads tested. In contrast, there was a ca. 2 log reduction in viral viability as measured by the tissue culture infectious dose (TCID) assay, with 1-3 log viable virus detected on dry swabs after 7 days. When swabs were coated with 102 viral copies of the Omicron variant, no viable virus was detected after 24 hours following storage at 4°C or room temperature. However there was no loss of PCR signal over 7 days. All four strains showed comparable growth kinetics and survival when cultured in Vero E6 cells. Our data provide information on the viability of SARS-CoV-2 on stored swabs in a clinical setting with important implications for diagnostic pickup and laboratory processing protocols. Survival after 7 days of SARS-CoV-2 strains on swabs with high viral loads may impact public health and biosafety practices in diagnostic laboratories.