Abstract Background The insect endosymbiotic bacterium Wolbachia is being deployed in field populations of the mosquito Aedes aegypti for biological control. This microbe prevents the replication of human disease-causing viruses inside the vector, including dengue, Zika and chikungunya. Relative Wolbachia densities may in part predict the strength of this ‘viral blocking’ effect. Additionally, Wolbachia densities may affect the strength of the reproductive manipulations it induces, including cytoplasmic incompatibility (CI), maternal inheritance rates or induced fitness effects in the insect host. High rates of CI and maternal inheritance and low rates of fitness effects are also key to the successful spreading of Wolbachia through vector populations and its successful use in biocontrol. The factors that control Wolbachia densities are not completely understood. Methods We used quantitative PCR-based methods to estimate relative density of the Wolbachia w AlbB strain in both the somatic and reproductive tissues of adult male and female mosquitoes, as well as in eggs. Using correlation analyses, we assessed whether densities in one tissue predict those in others within the same individual, but also across generations. Results We found little relationship among the relative Wolbachia densities of different tissues in the same host. The results also show that there was very little relationship between Wolbachia densities in parents and those in offspring, both in the same and different tissues. The one exception was with ovary–egg relationships, where there was a strong positive association. Relative Wolbachia densities in reproductive tissues were always greater than those in the somatic tissues. Additionally, the densities were consistent in females over their lifetime regardless of tissue, whereas they were generally higher and more variable in males, particularly in the testes. Conclusions Our results indicate that either stochastic processes or local tissue-based physiologies are more likely factors dictating Wolbachia densities in Ae. aegypti individuals, rather than shared embryonic environments or heritable genetic effects of the mosquito genome. These findings have implications for understanding how relative Wolbachia densities may evolve and/or be maintained over the long term in Ae. aegypti . Graphical Abstract
Abstract SARS-CoV-2 has initiated a global pandemic and vaccines are being rapidly developed. Using the reference strain SARS-CoV-2 USA-WA1/2020, we evaluated modes of transmission and the ability of prior infection or vaccine-induced immunity to protect against infection in ferrets. Ferrets were semi-permissive to infection with the USA-WA1/2020 isolate. When transmission was assessed via the detection of vRNA at multiple timepoints, direct contact transmission was efficient to 3/3 and 3/4 contact animals in two respective studies, while respiratory transmission was poor to only 1/4 contact animals. To assess the durability of immunity, ferrets were re-challenged 28 or 56 days post-primary infection. Following viral challenge, no infectious virus was recovered in nasal wash samples. In addition, levels of vRNA in the nasal wash were several orders of magnitude lower than during primary infection, and vRNA was rapidly cleared. To determine if intramuscular vaccination protected ferrets against infection, ferrets were vaccinated using a prime-boost strategy with the S-protein receptor-binding domain formulated with an oil-in-water adjuvant. Upon viral challenge, none of the mock or vaccinated animals were protected against infection, and there were no significant differences in vRNA or infectious virus titers in the nasal wash. Combined these studies demonstrate that in ferrets direct contact is the predominant mode of transmission of the SARS-CoV-2 USA-WA1/2020 isolate and immunity to SARS-CoV-2 is maintained for at least 56 days. Our studies also indicate protection of the upper respiratory tract against SARS-CoV-2 will require vaccine strategies that mimic natural infection or induce site-specific immunity. Importance The SARS-CoV-2 USA-WA1/2020 strain is a CDC reference strain used by multiple research laboratories. Here, we show the predominant mode of transmission of this isolate in ferrets is by direct contact. We further demonstrate ferrets are protected against re-infection for at least 56 days even when levels of neutralizing antibodies are low or undetectable. Last, we show that when ferrets were vaccinated by the intramuscular route to induce antibodies against SARS-CoV-2, ferrets remain susceptible to infection of the upper respiratory tract. Collectively, these studies suggest protection of the upper respiratory tract will require vaccine approaches that mimic natural infection.
The Welsh Government’s Well-being of Future Generations (Wales) Act 2015 is a unique piece of legislation which embeds healthy and sustainable development into Wales’ legislative framework. It commits 44 public bodies to improving social, economic, cultural and environmental well-being. The Future Generations Act introduces seven well-being goals and five ‘ways of working’, creating a shared vision for these public bodies to work towards. Alongside this top-down approach, there is an emerging intent to increase engagement of local people and to support individuals and communities in sustaining and improving the quality of their health and well-being. However, at present the implementation of well-being agenda at community level is limited. This paper examines an innovative approach to bridge this gap and engage local people in considering the health and well-being of their communities, now and into the future.
The Shape My Town community-led planning tool is designed to increase opportunities for local people to have a voice in the planning process through the creation of community-led Place Plans. Seen as a form of asset-based community development, the tool encourages communities to assess their place through a series of interlinked themes to build evidence for change. In this paper, the seven goals and five ways of working embedded in the Well-being Act are mapped against the tool before a case study of its implementation is presented. The sense of increased self-determination the process facilitates is linked to an increased sense of well-being, purpose and community cohesion (Deci and Ryan, 1985). However, questions are raised about the capacity of communities to deliver effective long-term change and the capacity of overburdened, under-resourced local authorities to help deliver on these aspirations.
Abstract Multiple Wolbachia strains can block pathogen infection, replication and/or transmission in Aedes aegypti mosquitoes under both laboratory and field conditions. However, Wolbachia effects on pathogens can be highly variable across systems and the factors governing this variability are not well understood. It is increasingly clear that the mosquito host is not a passive player in which Wolbachia governs pathogen transmission phenotypes; rather, the genetics of the host can significantly modulate Wolbachia ‐mediated pathogen blocking. Specifically, previous work linked variation in Wolbachia pathogen blocking to polymorphisms in the mosquito alpha‐mannosidase‐2 (αMan2) gene. Here we use CRISPR‐Cas9 mutagenesis to functionally test this association. We developed αMan2 knockouts and examined effects on both Wolbachia and virus levels, using dengue virus (DENV; Flaviviridae ) and Mayaro virus (MAYV; Togaviridae ). Wolbachia titres were significantly elevated in αMan2 knockout (KO) mosquitoes, but there were complex interactions with virus infection and replication. In Wolbachia ‐uninfected mosquitoes, the αMan2 KO mutation was associated with decreased DENV titres, but in a Wolbachia ‐infected background, the αMan2 KO mutation significantly increased virus titres. In contrast, the αMan2 KO mutation significantly increased MAYV replication in Wolbachia ‐uninfected mosquitoes and did not affect Wolbachia ‐mediated virus blocking. These results demonstrate that αMan2 modulates arbovirus infection in A. aegypti mosquitoes in a pathogen‐ and Wolbachia ‐specific manner, and that Wolbachia ‐mediated pathogen blocking is a complex phenotype dependent on the mosquito host genotype and the pathogen. These results have a significant impact for the design and use of Wolbachia ‐based strategies to control vector‐borne pathogens.
We include several data sets 1. 'viruscopiesperfamilyforheritability' is the calculated virus counts based on a standard curve per each mosquito in the family design. 2. 'CHIKDENVcandidategeneexpression' is the raw CT values for both the genes of interest and RPS17 as well as the calcuations. 3. 'raw_counts.csv'. Raw read counts for the RNAseq 4. All the individual BAM files for RNAseq. High and Low files are named by family number.
During the COVID-19 pandemic, wastewater surveillance was leveraged as a powerful tool for monitoring community-scale health. Further, the well-known persistence of some pharmaceuticals through wastewater treatment plants spurred concerns that increased usage of pharmaceuticals during the pandemic would increase the concentrations in wastewater treatment plant effluent. We collected weekly influent and effluent samples from May 2020 through May 2021 from two wastewater treatment plants in central Pennsylvania, the Penn State Water Reclamation Facility and the University Area Joint Authority, that provide effluent for beneficial reuse, including for irrigation. Samples were analyzed for severe acute respiratory syndrome coronavirus 2 (influent only), two over-the-counter medicines (acetaminophen and naproxen), five antibiotics (ampicillin, doxycycline, ofloxacin, sulfamethoxazole, and trimethoprim), two therapeutic agents (remdesivir and dexamethasone), and hydroxychloroquine. Although there were no correlations between pharmaceutical and virus concentration, remdesivir detection occurred when the number of hospitalized patients with COVID-19 increased, and dexamethasone detection co-occurred with the presence of patients with COVID-19 on ventilators. Additionally, Penn State decision-making regarding instruction modes explained the temporal variation of influent pharmaceutical concentrations, with detection occurring primarily when students were on campus. Risk quotients calculated for pharmaceuticals with known effective and lethal concentrations at which 50% of a population is affected for fish, daphnia, and algae were generally low in the effluent; however, some acute risks from sulfamethoxazole were high when students returned to campus. Remdesivir and dexamethasone persisted through the wastewater treatment plants, thereby introducing novel pharmaceuticals directly to soils and surface water. These results highlight connections between human health and water quality and further demonstrate the broad utility of wastewater surveillance.
The intensification of the poultry industry over the last 60 years facilitated the evolution of increased virulence and vaccine breaks in Marek's disease virus (MDV-1). Full-genome sequences are essential for understanding why and how this evolution occurred, but what is known about genome-wide variation in MDV comes from laboratory culture. To rectify this, we developed methods for obtaining high-quality genome sequences directly from field samples without the need for sequence-based enrichment strategies prior to sequencing. We applied this to the first characterization of MDV-1 genomes from the field, without prior culture. These viruses were collected from vaccinated hosts that acquired naturally circulating field strains of MDV-1, in the absence of a disease outbreak. This reflects the current issue afflicting the poultry industry, where virulent field strains continue to circulate despite vaccination and can remain undetected due to the lack of overt disease symptoms. We found that viral genomes from adjacent field sites had high levels of overall DNA identity, and despite strong evidence of purifying selection, had coding variations in proteins associated with virulence and manipulation of host immunity. Our methods empower ecological field surveillance, make it possible to determine the basis of viral virulence and vaccine breaks, and can be used to obtain full genomes from clinical samples of other large DNA viruses, known and unknown. IMPORTANCE Despite both clinical and laboratory data that show increased virulence in field isolates of MDV-1 over the last half century, we do not yet understand the genetic basis of its pathogenicity. Our knowledge of genome-wide variation between strains of this virus comes exclusively from isolates that have been cultured in the laboratory. MDV-1 isolates tend to lose virulence during repeated cycles of replication in the laboratory, raising concerns about the ability of cultured isolates to accurately reflect virus in the field. The ability to directly sequence and compare field isolates of this virus is critical to understanding the genetic basis of rising virulence in the wild. Our approaches remove the prior requirement for cell culture and allow direct measurement of viral genomic variation within and between hosts, over time, and during adaptation to changing conditions.
The PurR-hypoxanthine-purF operator complex structure has been solved to 2.5 Å resolution and revealed several features key to specific DNA recognition, including an introduced kink in the DNA at the central CpG step.However, the structure does not reveal the strict conservation of certain DNA bases.Specifically, PurR binds 21 known operators, which can be described as 42 half-sites due to the pseudopalindromic nature of these binding sites; of these, 41 have an adenine at the 7 position, and one has a cytosine.Surprisingly, the PurR-hypoxanthine-purF operator structure shows no direct contacts between adenine7 and the protein.Equilibrium binding experiments using fluorescence anisotropy with substituted operators revealed a striking result, whereby substitution of adenine7 by cytidine, thymine or guanine resulted in 12, 30 and 107 fold higher Kd values, respectively.The crystal structures of the Escherichia coli PurR bound to operator sites mutated at the 7 position have been solved by molecular replacement and two separate effects were observed: mutation of the highly conserved base in the cytidine and thymine structures altered neighboring base contacts to Lysine 55 due to altered electrostatics in the minor groove.Moreover, inherent base stacking preferences of the wild type purine-purine step over the pyrimidine-purine steps of the cytidine and thymine substituted purF operators also play a role in PurR binding preference of Adenine at position 7. Observed in the lowest affinity guanine substituted operator was a distortion of the DNA backbone as a result of the sequence change: indirect readout.
Abstract One approach to control dengue virus transmission is the symbiont Wolbachia , which limits viral infection in mosquitoes. Despite plans for its widespread use in Aedes aegypti , Wolbachia 's mode of action remains poorly understood. Many studies suggest that the mechanism is likely multifaceted, involving aspects of immunity, cellular stress and nutritional competition. A previous study from our group used artificial selection to identify a new mosquito candidate gene related to viral blocking; alpha‐mannosidase‐2a ( alpha‐Mann‐2a ) with a predicted role in protein glycosylation. Protein glycosylation pathways tend to be involved in complex host–viral interactions; however, the function of alpha‐mannosidases has not been described in mosquito–virus interactions. We examined alpha‐Mann‐2a expression in response to virus and Wolbachia infections and whether reduced gene expression, caused by RNA interference, affected viral loads. We show that dengue virus (DENV) infection affects the expression of alpha‐Mann‐2a in a tissue‐ and time‐dependent manner, whereas Wolbachia infection had no effect. In the midgut, DENV prevalence increased following knockdown of alpha‐Mann‐2a expression in Wolbachia ‐free mosquitoes, suggesting that alpha‐Mann‐2a interferes with infection. Expression knockdown had the same effect on the togavirus chikungunya virus, indicating that alpha‐Mann‐2a may have broad antivirus effects in the midgut. Interestingly, we were unable to knockdown the expression in Wolbachia ‐infected mosquitoes. We also provide evidence that alpha‐Mann‐2a may affect the transcriptional level of another gene predicted to be involved in viral blocking and cell adhesion; cadherin87a . These data support the hypothesis that glycosylation and adhesion pathways may broadly be involved in viral infection in Ae. aegypti .
The mosquito Aedes aegypti is the primary vector of many disease-causing viruses, including dengue (DENV), Zika, chikungunya, and yellow fever. As consequences of climate change, we expect an increase in both global mean temperatures and extreme climatic events. When temperatures fluctuate, mosquito vectors will be increasingly exposed to temperatures beyond their upper thermal limits. Here, we examine how DENV infection alters Ae. aegypti thermotolerance by using a high-throughput physiological ‘knockdown’ assay modeled on studies in Drosophila. Such laboratory measures of thermal tolerance have previously been shown to accurately predict an insect’s distribution in the field. We show that DENV infection increases thermal sensitivity, an effect that may ultimately limit the geographic range of the virus. We also show that the endosymbiotic bacterium Wolbachia pipientis, which is currently being released globally as a biological control agent, has a similar impact on thermal sensitivity in Ae. aegypti. Surprisingly, in the coinfected state, Wolbachia did not provide protection against DENV-associated effects on thermal tolerance, nor were the effects of the two infections additive. The latter suggests that the microbes may act by similar means, potentially through activation of shared immune pathways or energetic tradeoffs. Models predicting future ranges of both virus transmission and Wolbachia’s efficacy following field release may wish to consider the effects these microbes have on host survival.
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