Y chromosome shredding in Anopheles gambiae: Insight into the cellular dynamics of a novel synthetic sex ratio distorter.

Despite efforts to explore the genome of the malaria vector Anopheles gambiae, the Y chromosome of this species remains enigmatic. The large number of repetitive and heterochromatic DNA sequences makes the Y chromosome exceptionally difficult to fully assemble, hampering the progress of gene editing techniques and functional studies for this chromosome. In this study, we made use of a bioinformatic platform to identify Y-specific repetitive DNA sequences that served as a target site for a CRISPR/Cas9 system. The activity of Cas9 in the reproductive organs of males caused damage to Y-bearing sperm without affecting their fertility, leading to a strong female bias in the progeny. Cytological investigation allowed us to identify meiotic defects and investigate sperm selection in this new synthetic sex ratio distorter system. In addition, alternative promoters enable us to target the Y chromosome in specific tissues and developmental stages of male mosquitoes, enabling studies that shed light on the role of this chromosome in male gametogenesis. This work paves the way for further insight into the poorly characterised Y chromosome of Anopheles gambiae. Moreover, the sex distorter strain we have generated promises to be a valuable tool for the advancement of studies in the field of developmental biology, with the potential to support the progress of genetic strategies aimed at controlling malaria mosquitoes and other pest species.

Canonical vs. Grand Canonical Ensemble for Bosonic Gases under Harmonic Confinement.

We analyze the general relation between canonical and grand canonical ensembles in the thermodynamic limit. We begin our discussion by deriving, with an alternative approach, some standard results first obtained by Kac and coworkers in the late 1970s. Then, motivated by the Bose-Einstein condensation (BEC) of trapped gases with a fixed number of atoms, which is well described by the canonical ensemble and by the recent groundbreaking experimental realization of BEC with photons in a dye-filled optical microcavity under genuine grand canonical conditions, we apply our formalism to a system of non-interacting Bose particles confined in a two-dimensional harmonic trap. We discuss in detail the mathematical origin of the inequivalence of ensembles observed in the condensed phase, giving place to the so-called grand canonical catastrophe of density fluctuations. We also provide explicit analytical expressions for the internal energy and specific heat and compare them with available experimental data. For these quantities, we show the equivalence of ensembles in the thermodynamic limit.

Anti-CRISPR Anopheles mosquitoes inhibit gene drive spread under challenging behavioural conditions in large cages.

CRISPR-based gene drives have the potential to spread within populations and are considered as promising vector control tools. A doublesex-targeting gene drive was able to suppress laboratory Anopheles mosquito populations in small and large cages, and it is considered for field application. Challenges related to the field-use of gene drives and the evolving regulatory framework suggest that systems able to modulate or revert the action of gene drives, could be part of post-release risk-mitigation plans. In this study, we challenge an AcrIIA4-based anti-drive to inhibit gene drive spread in age-structured Anopheles gambiae population under complex feeding and behavioural conditions. A stochastic model predicts the experimentally-observed genotype dynamics in age-structured populations in medium-sized cages and highlights the necessity of large-sized cage trials. These experiments and experimental-modelling framework demonstrate the effectiveness of the anti-drive in different scenarios, providing further corroboration for its use in controlling the spread of gene drive in Anopheles.

Most probable path of active Ornstein-Uhlenbeck particles.

Using the path integral representation of the nonequilibrium dynamics, we compute the most probable path between arbitrary starting and final points that is followed by an active particle driven by persistent noise. We focus our attention on the case of active particles immersed in harmonic potentials, where the trajectory can be computed analytically. Once we consider the extended Markovian dynamics where the self-propulsive drive evolves according to an Ornstein-Uhlenbeck process, we can compute the trajectory analytically with arbitrary conditions on position and self-propulsion velocity. We test the analytical predictions against numerical simulations and we compare the analytical results with those obtained within approximated equilibriumlike dynamics.

Introgression of a synthetic sex ratio distortion transgene into different genetic backgrounds of Anopheles coluzzii.

The development of genetically modified mosquitoes (GMM) and their subsequent field release offers innovative approaches for vector control of malaria. A non-gene drive self-limiting male-bias Ag(PMB)1 strain has been developed in a 47-year-old laboratory G3 strain of Anopheles gambiae s.l. When Ag(PMB)1 males are crossed to wild-type females, expression of the endonuclease I-PpoI during spermatogenesis causes the meiotic cleavage of the X chromosome in sperm cells, leading to fertile offspring with a 95% male bias. However, World Health Organization states that the functionality of the transgene could differ when inserted in different genetic backgrounds of Anopheles coluzzii which is currently a predominant species in several West-African countries and thus a likely recipient for a potential release of self-limiting GMMs. In this study, we introgressed the transgene from the donor Ag(PMB)1 by six serial backcrosses into two recipient colonies of An. coluzzii that had been isolated in Mali and Burkina Faso. Scans of informative Single Nucleotide Polymorphism (SNP) markers and whole-genome sequencing analysis revealed a nearly complete introgression of chromosomes 3 and X, but a remarkable genomic divergence in a large region of chromosome 2 between the later backcrossed (BC6) transgenic offspring and the recipient paternal strains. These findings suggested to extend the backcrossing breeding strategy beyond BC6 generation and increasing the introgression efficiency of critical regions that have ecological and epidemiological implications through the targeted selection of specific markers. Disregarding differential introgression efficiency, we concluded that the phenotype of the sex ratio distorter is stable in the BC6 introgressed An. coluzzii strains.

Infravec2 guidelines for the design and operation of containment level 2 and 3 insectaries in Europe.

With the current expansion of vector-based research and an increasing number of facilities rearing arthropod vectors and infecting them with pathogens, common measures for containment of arthropods as well as manipulation of pathogens are becoming essential for the design and running of such research facilities to ensure safe work and reproducibility, without compromising experimental feasibility. These guidelines and comments were written by experts of the Infravec2 consortium, a Horizon 2020-funded consortium integrating the most sophisticated European infrastructures for research on arthropod vectors of human and animal diseases. They reflect current good practice across European laboratories with experience of safely handling different mosquito species and the pathogens they transmit. As such, they provide experience-based advice to assess and manage the risks to work safely with mosquitoes and the pathogens they transmit. This document can also form the basis for research with other arthropods, for example, midges, ticks or sandflies, with some modification to reflect specific requirements.

CRISPR-Mediated Cassette Exchange (CriMCE): A Method to Introduce and Isolate Precise Marker-Less Edits.

The introduction of small unmarked edits to the genome of insects is essential to study the molecular underpinnings of important biological traits, such as resistance to insecticides and genetic control strategies. Advances in CRISPR genome engineering have made this possible, but prohibitively laborious for most laboratories due to low rates of editing and the lack of a selectable marker. To facilitate the generation and isolation of precise marker-less edits we have developed a two-step method based on CRISPR-mediated cassette exchange (CriMCE) of a marked placeholder for a variant of interest. This strategy can be used to introduce a wider range of potential edits compared with previous approaches while consolidating the workflow. We present proof-of-principle that CriMCE is a powerful tool by engineering three single nucleotide polymorphism variants into the genome of Anopheles gambiae, with 5-41 × higher rates of editing than homology-directed repair or prime editing.

Impact of antigen test target failure and testing strategies on the transmission of SARS-CoV-2 variants.

Population testing remains central to COVID-19 control and surveillance, with countries increasingly using antigen tests rather than molecular tests. Here we describe a SARS-CoV-2 variant that escapes N antigen tests due to multiple disruptive amino-acid substitutions in the N protein. By fitting a multistrain compartmental model to genomic and epidemiological data, we show that widespread antigen testing in the Italian region of Veneto favored the undetected spread of the antigen-escape variant compared to the rest of Italy. We highlight novel limitations of widespread antigen testing in the absence of molecular testing for diagnostic or confirmatory purposes. Notably, we find that genomic surveillance systems which rely on antigen population testing to identify samples for sequencing will bias detection of escape antigen test variants. Together, these findings highlight the importance of retaining molecular testing for surveillance purposes, including in contexts where the use of antigen tests is widespread.

Presence of SARS-CoV-2 RNA in human corneal tissues donated in Italy during the COVID-19 pandemic.

OBJECTIVE: To analyse corneal tissues from asymptomatic donors with a postmortem nasopharyngeal swab tested positive for the presence of SARS-CoV-2 RNA, and therefore, understand the role that corneal transplantation may have in viral transmission. METHODS AND ANALYSIS: Between March 2020 and October 2021, 101 corneas (out of 8154 collected in Italy) from 51 donors (out of a total of 4155 Italian donors) positive for SARS-CoV-2 after postmortem nasopharyngeal swab tests were analysed for the presence of SARS-CoV-2 RNA through real-time RT-PCR. When available, the corneal tissue storage media were also assessed. Corneas and/or storage media with confirmed presence of SARS-CoV-2 RNA were further investigated by isolating SARS-CoV-2 virions, which were used to infect VeroE6 target cells. RESULTS: Only N=4 corneas and/or storage media out of 101 showed presence of SARS-CoV-2 RNA. No VeroE6 cell infection was detected with viral isolates, thus suggesting no presence of SARS-CoV-2 virions in corneal specimens and storage media. CONCLUSIONS: The presence of SARS-CoV-2 in cornea specimens would seem to be more likely due to prolonged detection of RNA rather than to active viral replication, with very low risk of infectivity and transmission through keratoplasty.

Neutralising reactivity against SARS-CoV-2 Delta and Omicron variants by vaccination and infection history.

BACKGROUND: The continuous emergence of SARS-CoV-2 variants of concern (VOC) with immune escape properties, such as Delta (B.1.617.2) and Omicron (B.1.1.529), questions the extent of the antibody-mediated protection against the virus. Here we investigated the long-term antibody persistence in previously infected subjects and the extent of the antibody-mediated protection against B.1, B.1.617.2 and BA.1 variants in unvaccinated subjects previously infected, vaccinated naïve and vaccinated previously infected subjects. METHODS: Blood samples collected 15 months post-infection from unvaccinated (n=35) and vaccinated (n=41) previously infected subjects (Vo' cohort) were tested for the presence of antibodies against the SARS-CoV-2 spike (S) and nucleocapsid (N) antigens using the Abbott, DiaSorin, and Roche immunoassays. The serum neutralising reactivity was assessed against B.1, B.1.617.2 (Delta), and BA.1 (Omicron) SARS-CoV-2 strains through micro-neutralisation. The antibody titres were compared to those from previous timepoints, performed at 2- and 9-months post-infection on the same individuals. Two groups of naïve subjects were used as controls, one from the same cohort (unvaccinated n=29 and vaccinated n=20) and a group of vaccinated naïve healthcare workers (n=61). RESULTS: We report on the results of the third serosurvey run in the Vo' cohort. With respect to the 9-month time point, antibodies against the S antigen significantly decreased (P=0.0063) among unvaccinated subjects and increased (P<0.0001) in vaccinated individuals, whereas those against the N antigen decreased in the whole cohort. When compared with control groups (naïve Vo' inhabitants and naïve healthcare workers), vaccinated subjects that were previously infected had higher antibody levels (P<0.0001) than vaccinated naïve subjects. Two doses of vaccine elicited stronger anti-S antibody response than natural infection (P<0.0001). Finally, the neutralising reactivity of sera against B.1.617.2 and BA.1 was 4-fold and 16-fold lower than the reactivity observed against the original B.1 strain. CONCLUSIONS: These results confirm that vaccination induces strong antibody response in most individuals, and even stronger in previously infected subjects. Neutralising reactivity elicited by natural infection followed by vaccination is increasingly weakened by the recent emergence of VOCs. While immunity is not completely compromised, a change in vaccine development may be required going forward, to generate cross-protective pan-coronavirus immunity in the global population.

Longitudinal analysis of T cell receptor repertoires reveals shared patterns of antigen-specific response to SARS-CoV-2 infection.

T cells play a prominent role in orchestrating the immune response to viral diseases, but their role in the clinical presentation and subsequent immunity to SARS-CoV-2 infection remains poorly understood. As part of a population-based survey of the municipality of Vo', Italy, conducted after the initial SARS-CoV-2 outbreak, we sampled the T cell receptor (TCR) repertoires of the population 2 months after the initial PCR survey and followed up positive cases 9 and 15 months later. At 2 months, we found that 97.0% (98 of 101) of cases had elevated levels of TCRs associated with SARS-CoV-2. T cell frequency (depth) was increased in individuals with more severe disease. Both depth and diversity (breadth) of the TCR repertoire were positively associated with neutralizing antibody titers, driven mostly by CD4+ T cells directed against spike protein. At the later time points, detection of these TCRs remained high, with 90.7% (78 of 96) and 86.2% (25 of 29) of individuals having detectable signal at 9 and 15 months, respectively. Forty-three individuals were vaccinated by month 15 and showed a significant increase in TCRs directed against spike protein. Taken together, these results demonstrate the central role of T cells in mounting an immune defense against SARS-CoV-2 that persists out to 15 months.

Phylogeography and genomic epidemiology of SARS-CoV-2 in Italy and Europe with newly characterized Italian genomes between February-June 2020.

The aims of this study were to characterize new SARS-CoV-2 genomes sampled all over Italy and to reconstruct the origin and the evolutionary dynamics in Italy and Europe between February and June 2020. The cluster analysis showed only small clusters including < 80 Italian isolates, while most of the Italian strains were intermixed in the whole tree. Pure Italian clusters were observed mainly after the lockdown and distancing measures were adopted. Lineage B and B.1 spread between late January and early February 2020, from China to Veneto and Lombardy, respectively. Lineage B.1.1 (20B) most probably evolved within Italy and spread from central to south Italian regions, and to European countries. The lineage B.1.1.1 (20D) developed most probably in other European countries entering Italy only in the second half of March and remained localized in Piedmont until June 2020. In conclusion, within the limitations of phylogeographical reconstruction, the estimated ancestral scenario suggests an important role of China and Italy in the widespread diffusion of the D614G variant in Europe in the early phase of the pandemic and more dispersed exchanges involving several European countries from the second half of March 2020.

Rapid SARS-CoV-2 Intra-Host and Within-Household Emergence of Novel Haplotypes.

In February 2020, the municipality of Vo', a small town near Padua (Italy) was quarantined due to the first coronavirus disease 19 (COVID-19)-related death detected in Italy. To investigate the viral prevalence and clinical features, the entire population was swab tested in two sequential surveys. Here we report the analysis of 87 viral genomes, which revealed that the unique ancestor haplotype introduced in Vo' belongs to lineage B, carrying the mutations G11083T and G26144T. The viral sequences allowed us to investigate the viral evolution while being transmitted within and across households and the effectiveness of the non-pharmaceutical interventions implemented in Vo'. We report, for the first time, evidence that novel viral haplotypes can naturally arise intra-host within an interval as short as two weeks, in approximately 30% of the infected individuals, regardless of symptom severity or immune system deficiencies. Moreover, both phylogenetic and minimum spanning network analyses converge on the hypothesis that the viral sequences evolved from a unique common ancestor haplotype that was carried by an index case. The lockdown extinguished both the viral spread and the emergence of new variants.

Prospective epidemiological, molecular, and genetic characterization of a novel coronavirus disease in the Val Venosta/Vinschgau: the CHRIS COVID-19 study protocol.

The COVID-19 pandemic has been threatening the healthcare and socioeconomic systems of entire nations. While population-based surveys to assess the distribution of SARS-CoV-2 infection have become a priority, pre-existing longitudinal studies are ideally suited to assess the determinants of COVID-19 onset and severity.The Cooperative Health Research In South Tyrol (CHRIS) study completed the baseline recruitment of 13,393 adults from the Venosta/Vinschgau rural district in 2018, collecting extensive phenotypic and biomarker data, metabolomic data, densely imputed genotype and whole-exome sequencing data.Based on CHRIS, we designed a prospective study, called CHRIS COVID-19, aimed at: 1) estimating the incidence of SARS-CoV-2 infections; 2) screening for and investigating the determinants of incident infection among CHRIS participants and their household members; 3) monitoring the immune response of infected participants prospectively.An online screening questionnaire was sent to all CHRIS participants and their household members. A random sample of 1450 participants representative of the district population was invited to assess active (nasopharyngeal swab) or past (serum antibody test) infections. We prospectively invited for complete SARS-CoV-2 testing all questionnaire completers gauged as possible cases of past infection and their household members. In positive tested individuals, antibody response is monitored quarterly for one year. Untested and negative participants receive the screening questionnaire every four weeks until gauged as possible incident cases or till the study end.Originated from a collaboration between researchers and community stakeholders, the CHRIS COVID-19 study aims at generating knowledge about the epidemiological, molecular, and genetic characterization of COVID-19 and its long-term sequelae.

A single-agent extension of the SIR model describes the impact of mobility restrictions on the COVID-19 epidemic.

Mobility restrictions are successfully used to contain the diffusion of epidemics. In this work we explore their effect on the epidemic growth by investigating an extension of the Susceptible-Infected-Removed (SIR) model in which individual mobility is taken into account. In the model individual agents move on a chessboard with a Lévy walk and, within each square, epidemic spreading follows the standard SIR model. These simple rules allow to reproduce the sub-exponential growth of the epidemic evolution observed during the Covid-19 epidemic waves in several countries and which cannot be captured by the standard SIR model. We show that we can tune the slowing-down of the epidemic spreading by changing the dynamics of the agents from Lévy to Brownian and we investigate how the interplay among different containment strategies mitigate the epidemic spreading. Finally we demonstrate that we can reproduce the epidemic evolution of the first and second COVID-19 waves in Italy using only 3 parameters, i.e , the infection rate, the removing rate, and the mobility in the country. We provide an estimate of the peak reduction due to imposed mobility restrictions, i. e., the so-called flattening the curve effect. Although based on few ingredients, the model captures the kinetic of the epidemic waves, returning mobility values that are consistent with a lock-down intervention during the first wave and milder limitations, associated to a weaker peak reduction, during the second wave.

Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation.

CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading to suppression of mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying ('homing') therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is often a reliable indicator of functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel adult lethal gene drive (ALGD) in the malaria vector Anopheles gambiae, targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development, which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations.

Disease Severity and Prognosis of SARS-CoV-2 Infection in Hospitalized Patients Is Not Associated With Viral Load in Nasopharyngeal Swab.

Background: The impact of viral burden on severity and prognosis of patients hospitalized for Coronavirus Disease 2019 (COVID-19) is still a matter of debate due to controversial results. Herein, we sought to assess viral load in the nasopharyngeal swab and its association with severity score indexes and prognostic parameters. Methods: We included 127 symptomatic patients and 21 asymptomatic subjects with a diagnosis of SARS-CoV-2 infection obtained by reverse transcription polymerase chain reaction and presence of cycle threshold. According to the level of care needed during hospitalization, the population was categorized as high-intensity (HIMC, n = 76) or low intensity medical care setting (LIMC, n = 51). Results: Viral load did not differ among asymptomatic, LIMC, and HIMC SARS-CoV-2 positive patients [4.4 (2.9-5.3) vs. 4.8 (3.6-6.1) vs. 4.6 (3.9-5.7) log10 copies/ml, respectively; p = 0.31]. Similar results were observed when asymptomatic individuals were compared to hospitalized patients [4.4 (2.9-5.3) vs. 4.68 (3.8-5.9) log10 copies/ml; p = 0.13]. When the study population was divided in High (HVL, n = 64) and Low Viral Load (LVL, n = 63) group no differences were observed in disease severity at diagnosis. Furthermore, LVL and HVL groups did not differ with regard to duration of hospital stay, number of bacterial co-infections, need for high-intensity medical care and number of deaths. The viral load was not an independent risk factor for HIMC in an adjusted multivariate regression model (OR: 1.59; 95% CI: 0.46-5.55, p = 0.46). Conclusions: Viral load at diagnosis is similar in asymptomatic and hospitalized patients and is not associated with either worse outcomes during hospitalization. SARS CoV-2 viral load might not be the right tool to assist clinicians in risk-stratifying hospitalized patients.

SARS-CoV-2 antibody dynamics and transmission from community-wide serological testing in the Italian municipality of Vo'.

In February and March 2020, two mass swab testing campaigns were conducted in Vo', Italy. In May 2020, we tested 86% of the Vo' population with three immuno-assays detecting antibodies against the spike and nucleocapsid antigens, a neutralisation assay and Polymerase Chain Reaction (PCR). Subjects testing positive to PCR in February/March or a serological assay in May were tested again in November. Here we report on the results of the analysis of the May and November surveys. We estimate a seroprevalence of 3.5% (95% Credible Interval (CrI): 2.8-4.3%) in May. In November, 98.8% (95% Confidence Interval (CI): 93.7-100.0%) of sera which tested positive in May still reacted against at least one antigen; 18.6% (95% CI: 11.0-28.5%) showed an increase of antibody or neutralisation reactivity from May. Analysis of the serostatus of the members of 1,118 households indicates a 26.0% (95% CrI: 17.2-36.9%) Susceptible-Infectious Transmission Probability. Contact tracing had limited impact on epidemic suppression.

Gene-drive suppression of mosquito populations in large cages as a bridge between lab and field.

CRISPR-based gene-drives targeting the gene doublesex in the malaria vector Anopheles gambiae effectively suppressed the reproductive capability of mosquito populations reared in small laboratory cages. To bridge the gap between laboratory and the field, this gene-drive technology must be challenged with vector ecology.Here we report the suppressive activity of the gene-drive in age-structured An. gambiae populations in large indoor cages that permit complex feeding and reproductive behaviours.The gene-drive element spreads rapidly through the populations, fully supresses the population within one year and without selecting for resistance to the gene drive. Approximate Bayesian computation allowed retrospective inference of life-history parameters from the large cages and a more accurate prediction of gene-drive behaviour under more ecologically-relevant settings.Generating data to bridge laboratory and field studies for invasive technologies is challenging. Our study represents a paradigm for the stepwise and sound development of vector control tools based on gene-drive.