Biology subjects

Jones, J. E.

Publications and source records attributed to Jones, J. E..

15 recordsLinked to original sources

bioRxiv2026

Enterovirus D68 VP1 and VP3 determine neurotropism in human spinal cord organoids

Enterovirus D68 (EV-D68) is a non-polio enterovirus that can cause a polio-like paralysis condition, acute flaccid myelitis (AFM). EV-D68 associated AFM cases waned in the US after 2018 and the reasons for this are unknown. It has recently been demonstrated that EV-D68 containing point mutations in viral structural proteins VP1 and VP3 resulted in decreased paralysis in different neonatal mouse models. However, phenotypes of these mutations in a human multicellular central nervous system (CNS) model are unknown. We hypothesized that mutations in VP1 and VP3 will similarly direct neurotropism in human spinal cord organoids (hSCO). To investigate this, we recreated viruses with mutations in VP3 (I88V) or VP1 (L1I/N2D/T98A/E283K or L1P/V148A/K282R) and infected hSCOs. We found that VP3 I88V and VP1 L1I/N2D/T98A/E283K resulted in decreased titer and viral protein staining, consistent with attenuated neurovirulence in previously published murine models. When these mutations were combined, their effects on neurotropism were not additive. Sequence analysis of recently circulating EV-D68 strains reveals that VP3 I88 and VP1 E283 have remained the dominant amino acid residues since 2014, whereas VP1 sites 1, 2, and 98 have higher population diversity, indicating that these residues may be contributing to newly reduced neurovirulence after 2018.

microbiology↗

bioRxiv2024

Recombinant B3 clade enterovirus D68 strains are efficiently rescued in 293T cells and infect human spinal cord organoids.

Enterovirus D68 (EV-D68) is associated with respiratory disease in children. Between 2014 and 2018, biennial EV-D68 outbreaks coincided with peaks of a polio-like neurologic condition called acute flaccid myelitis (AFM). We hypothesized that specific mutations within the currently circulating B3 clade of EV-D68 impacted neurovirulence. However, recovery of these strains from infectious clones by published methods proved unsuccessful. Therefore, we tested different cell lines, reagents, and conditions to enhance efficiency of recombinant (r)EV-D68 rescue. In this study, we present a tractable rescue system to define virulence determinants in B3 clade strains. Using this approach, we successfully rescued historic and contemporary rEV-D68 strains to high titer after limited cell culture passage. All strains in our study replicated efficiently in the parental RD cell line and in human respiratory epithelial cell lines, with BEAS-2B cells exhibiting greater permissivity than A549 cells. While B2 and B3 clade strains could infect the SH-SY5Y neuroblastoma cell line, the neurovirulent B2 clade strain rUSA/IL/2014-18952 was more dependent on neuron differentiation than B3 clade strains and replication was not sustained under multi-cycle growth conditions. Conversely, replication of rUSA/IL/2014-18952 was more efficient in human spinal cord organoids, which model the cellular heterogeneity of the spinal cord, while replication of B3 clade strains was more modest. Immunofluorescence staining confirmed infection, with viral antigen colocalized with neurons. These findings suggest shifting dynamics of EV-D68 neurovirulence and provide a critical platform for further assessment of viral determinants of neurovirulence in B3 clade strains. IMPORTANCEEnterovirus D68 (EV-D68) can cause acute flaccid myelitis (AFM), a debilitating neurological condition of the spinal cord in children. Identifying viral determinants of EV- D68 neuropathogenesis is critical to understanding recent shifts in AFM prevalence; however, these investigations are limited to a small subset of infectious clones distantly related to currently circulating B3 clade strains. In this study, we leverage improved rescue strategies to characterize recombinant (r)EV-D68 strains from the dominant B3 clade. While all rEV-D68 replicate efficiently in the parental cell line and in human respiratory epithelial cells, B3 clade strains achieved greater titers in cultured neurons than a neurovirulent B2 clade strain, with less dependence on neuronal differentiation state. All B3 clade strains established infection in human spinal cord organoids, but replication varied between strains. Therefore, our study presents a tractable rescue system to begin to dissect viral determinants of shifting neurotropism within contemporary EV-D68 clades.

microbiology↗

bioRxiv2024

A sequence-specific RNA acetylation catalyst

N4-acetylcytidine (ac4C) is a ubiquitous RNA modification incorporated by cytidine acetyltransferase enzymes. Here, we report the biochemical characterization of Thermococcus kodakarensis Nat10 (TkNat10), an RNA acetyltransferase involved in archaeal thermotolerance. We demonstrate that TkNat10s catalytic activity is critical for T. kodakarensis fitness at elevated temperatures. Unlike eukaryotic homologs, TkNat10 exhibits robust stand-alone activity, modifying diverse RNA substrates in a temperature, ATP, and acetyl-CoA-dependent manner. Transcriptome-wide analysis reveals TkNat10 preferentially modifies unstructured RNAs containing a 5-CCG-3 consensus sequence. Using a high-throughput mutagenesis approach, we define sequence and structural determinants of TkNat10 substrate recognition. We find TkNat10 can be engineered to use non-native acyl-CoA donors, providing insight into its cofactor specificity. Finally, we demonstrate TkNat10s utility for site-specific acetylation of RNA oligonucleotides, enabling analysis of ac4C-dependent RNA-protein interactions. Our findings establish a framework for understanding archaeal RNA acetylation and a new tool for studying the functional consequences of ac4C in diverse RNA contexts.

biochemistry↗

bioRxiv2024

Immune cells promote paralytic disease in mice infected with enterovirus D68

Enterovirus D68 (EV-D68) is associated with acute flaccid myelitis (AFM), a poliomyelitis-like illness causing paralysis in young children. However, mechanisms of paralysis are unclear, and antiviral therapies are lacking. To better understand EV-D68 disease, we inoculated newborn mice intracranially to assess viral tropism, virulence, and immune responses. Wild-type (WT) mice inoculated intracranially with a neurovirulent strain of EV-D68 showed infection of spinal cord neurons and developed paralysis. Spinal tissue from infected mice revealed increased levels of chemokines, inflammatory monocytes, macrophages, and T cells relative to controls, suggesting that immune cell infiltration influences pathogenesis. To define the contribution of cytokine-mediated immune cell recruitment to disease, we inoculated mice lacking CCR2, a receptor for several EV-D68-upregulated cytokines, or RAG1, which is required for lymphocyte maturation. WT, Ccr2-/-, and Rag1-/- mice had comparable viral titers in spinal tissue. However, Ccr2-/- and Rag1-/- mice had significantly less paralysis relative to WT mice. Consistent with impaired T cell recruitment to sites of infection in Ccr2-/- and Rag1-/- mice, antibody-mediated depletion of CD4+ or CD8+ T cells from WT mice diminished paralysis. These results indicate that immune cell recruitment to the spinal cord promotes EV-D68-associated paralysis and illuminate new targets for therapeutic intervention. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/618341v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@10b38b5org.highwire.dtl.DTLVardef@1b80d41org.highwire.dtl.DTLVardef@6e39bforg.highwire.dtl.DTLVardef@9156ed_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Variable prevalence of protective Spiroplasma infection over time in two natural populations of Drosophila hydei

The temporal dynamics of protective symbionts have rarely been characterized outside of aphid hosts. Here, we determine the prevalence of Spiroplasma in two populations of D. hydei where Spiroplasma infection had been previously recorded (UK and Japan). We observe that infection in both populations is variable over time and confirm the persistence of Spiroplasma in the UK population for 9 years. Thus, variable prevalence over time appears to be a common feature of these symbioses.

ecology↗

bioRxiv2024

Potential pandemic risk of circulating swine H1N2 influenza viruses

Influenza A viruses in swine have considerable genetic diversity and continue to pose a pandemic threat to humans. They were the source of the most recent influenza pandemic, and since 2010, novel swine viruses have spilled over into humans more than 400 times in the United States. Although these zoonotic infections generally result in mild illness with limited onward human transmission, the potential for sustained transmission of an emerging influenza virus between individuals due to lack of population level immunity is of great concern. Compiling the literature on pandemic threat assessment, we established a pipeline to characterize and triage influenza viruses for their pandemic risk and examined the pandemic potential of two widespread swine origin viruses. Our analysis revealed that a panel of human sera collected from healthy adults in 2020 has no cross-reactive neutralizing antibodies against an -H1 clade strain but do against a {gamma}-H1 clade strain. Swine H1N2 virus from the -H1 clade (-swH1N2) replicated efficiently in human airway cultures and exhibited phenotypic signatures similar to the human H1N1 pandemic strain from 2009 (H1N1pdm09). Furthermore, -swH1N2 was capable of efficient airborne transmission to both naive ferrets and ferrets with prior seasonal influenza immunity. Ferrets with H1N1pdm09 pre-existing immunity had reduced -swH1N2 viral shedding from the upper respiratory tract and cleared the infection faster. Despite this, H1N1pdm09-immune ferrets that became infected via the air could still onward transmit -swH1N2 with an efficiency of 50%. Taken together, these results indicate that this -swH1N2 strain has a higher pandemic potential, but a moderate level of impact since there is reduced replication fitness in animals with prior immunity.

microbiology↗

bioRxiv2023

A modular plasmid toolkit applied in marine Proteobacteria reveals functional insights during bacteria-stimulated metamorphosis

A conspicuous roadblock to studying marine bacteria for fundamental research and biotechnology is a lack of modular synthetic biology tools for their genetic manipulation. Here, we applied, and generated new parts for, a modular plasmid toolkit to study marine bacteria in the context of symbioses and host-microbe interactions. To demonstrate the utility of this plasmid system, we genetically manipulated the marine bacterium Pseudoalteromonas luteoviolacea, which stimulates the metamorphosis of the model tubeworm, Hydroides elegans. Using these tools, we quantified constitutive and native promoter expression, developed reporter strains that enable the imaging of host-bacteria interactions, and used CRISPR interference (CRISPRi) to knock down a secondary metabolite and a host-associated gene. We demonstrate the broader utility of this modular system for rapidly creating and iteratively testing genetic tractability by modifying marine bacteria that are known to be associated with diverse host-microbe symbioses. These efforts enabled the successful transformation of twelve marine strains across two Proteobacteria classes, four orders and ten genera. Altogether, the present study demonstrates how synthetic biology strategies enable the investigation of marine microbes and marine host-microbe symbioses with broader implications for environmental restoration and biotechnology.

synthetic biology↗

bioRxiv2022

History matters: thermal environment before, but not during wasp attack determines the efficiency of symbiont-mediated protection

The outcome of natural enemy attack in insects is commonly impacted by the presence of defensive microbial symbionts residing within the host. Beyond their presence, the outcome of the interaction can also depend on genetic and environmental factors. The thermal environment is a key factor known to affect symbiont-mediated traits in insects, including their ability to defend against natural enemy attack. Cooler temperatures, for instance, have been previously shown to reduce Spiroplasma-mediated protection in Drosophila. Here, we dissect the effect of the thermal environment on Spiroplasma-mediated protection against Leptopilina boulardi in Drosophila melanogaster by examining the effect of temperature before, during and after wasp attack on fly survival and wasp success. We observed that the developmental temperature of the mothers of attacked larvae, and not the temperature of the attacked larvae themselves during or after wasp attack, strongly determines the protective influence of Spiroplasma. Spiroplasma-mediated fly survival was found to be weaker when parental flies were reared at 21{degrees}C before their larvae were exposed to wasps compared to larvae derived from parental flies reared at 23{degrees}C or 25{degrees}C. Contrastingly, there was no effect of thermal environment on protection when mothers were reared at 25{degrees}C, and their progeny exposed to lower temperatures during and after wasp attack. The effect of developmental temperature on Spiroplasma-mediated protection is likely mediated by reduced Spiroplasma titre combined, at cooler temperatures, with segregation of infection. These results indicate the historical thermal environment is a stronger determinant of protection than current environment, and that protective capacity is partly an epigenetic trait.

evolutionary biology↗

bioRxiv2022

Robustness of the ferret model for influenza risk assessment studies: a cross-laboratory exercise

Ferrets represent the preferred animal model for assessing the transmission potential of newly emerged zoonotic influenza viruses. However, heterogeneity among established experimental protocols and facilities across different laboratories may lead to variable results, complicating interpretation of transmission experimental data. Between 2018-2020, a global exercise was conducted by 11 participating laboratories to assess the range of variation in ferret transmission experiments using two common stock H1N1 influenza viruses that possess different transmission characteristics in ferrets. Inoculation route, dose, and volume were standardized, and all participating laboratories followed the same experimental conditions for respiratory droplet transmission, including a strict 1:1 donor:contact ratio. Additional host and environmental parameters likely to affect influenza transmission kinetics were monitored throughout. Overall transmission outcomes for both viruses across 11 laboratories were concordant, suggesting the robustness of the ferret model for zoonotic influenza risk assessment. To attain high confidence in identifying zoonotic influenza viruses with moderate-to-high or low transmissibility, our analyses support that as few as three but as many as five laboratories, respectively, would need to independently perform viral transmission experiments with concordant results. This exercise facilitates the development of a more homogenous protocol for ferret transmission experiments that are employed for the purposes of risk assessment.

microbiology↗

bioRxiv2022

Host origin is a determinant of parallel evolution between influenza virus gene segments.

Several emerging influenza viruses, including H7N9 and H5N6 viruses, trace their origins to reassortment with H9N2 viruses that contributed internal gene segments. However, the evolutionary constraints governing reassortment of H9N2 viruses remain unknown. In seasonal human influenza A viruses, gene segments evolve in parallel at both the gene and protein levels. Here, we demonstrate that parallel evolution in human H3N2 viruses differs from avian H9 viruses, with both genes and proteins of avian H9 viruses characterized by high phylogenetic divergence. Strikingly, protein trees corresponding to avian H9 polymerase subunits diverge despite known functional constraints on polymerase evolution. Gene divergence was consistent across avian H9 isolates from different continents, suggesting that parallel evolution between H9 gene segments is not dependent on regionally defined lineages. Instead, parallel evolution in H9 viruses was dependent upon host origin. Our study reveals the role of the host in parallel evolution of influenza gene segments and suggests that high reassortment potential in avian species may be a consequence of evolutionary flexibility between gene segments.

evolutionary biology↗

bioRxiv2021

Parallel evolution between genomic segments of seasonal human influenza viruses reveals RNA-RNA relationships

The influenza A virus (IAV) genome consists of eight negative-sense viral RNA (vRNA) segments that are selectively assembled into progeny virus particles through RNA-RNA interactions. To identify relationships between vRNA segments, we examined parallel evolution between vRNA segments of seasonal human IAV, finding that evolutionary relationships between vRNA segments differ between subtypes and antigenically-shifted strains. Intersegmental relationships were distinct between H3N2 and H1N1 viruses, but largely conserved over time in H3N2 viruses. However, parallel evolution of vRNA segments diverged between H1N1 strains isolated before and after the 2009 pandemic. Surprisingly, intersegmental relationships were not driven solely by protein sequence, which is potentially indicative of RNA-RNA driven coevolution. Colocalization of highly coevolved vRNA segments was enriched over other pairs at the nuclear periphery during a productive viral infection. This study illustrates how phylogenetics can be applied to interrogate putative RNA interactions underlying selective assembly of IAV.

microbiology↗

bioRxiv2020

Coinfection of Streptococcus pneumoniae reduces airborne transmission of influenza virus

Secondary bacterial infection, especially with Streptococcus pneumoniae (Spn), is a common complication in fatal and ICU cases of influenza virus infection. During the H1N1 pandemic of 2009 (H1N1pdm09), there was higher mortality in healthy young adults due to secondary bacterial pneumonia, with Spn being the most frequent bacterial species. Previous studies in mice and ferrets have suggested a synergistic relationship between Spn and influenza viruses. In this study, we used the ferret model to study whether airborne transmission of H1N1pdm09 was influenced by coinfection with two Spn serotypes: type 2 (D39) and type 19F (BHN97). We found that coinfected animals experienced more severe clinical symptoms as well as increased bacterial colonization of the upper respiratory tract. In contrast, we observed that coinfection resulted in reduced airborne transmission of influenza virus. Only 1/3 animals coinfected with D39 transmitted H1N1pdm09 virus to a naive recipient compared to 3/3 transmission efficiency in animals infected with influenza virus alone. A similar trend was seen in coinfection with BHN97, suggesting that coinfection with Spn reduces influenza virus airborne transmission. The decrease in transmission does not appear to be caused by decreased stability of H1N1pdm09 in expelled droplets in the presence of Spn. Rather, coinfection resulted in decreased viral shedding in the ferret upper respiratory tract. Thus, we conclude that coinfection enhances colonization and airborne transmission of Spn but decreases replication and transmission of H1N1pdm09. Our data points to an asymmetrical relationship between these two pathogens rather than a synergistic one. SignificanceAirborne transmission of respiratory viruses is influenced by many host and environmental parameters. The complex interplay between bacterial and viral coinfections on transmission of respiratory viruses has been understudied. We demonstrate that coinfection with Streptococcus pneumoniae reduces airborne transmission of influenza A viruses by decreasing viral titers in the upper respiratory tract. Instead of implicating a synergistic relationship between bacteria and virus, our work demonstrates an asymmetric relationship where bacteria benefit from the virus but where the fitness of influenza A viruses is negatively impacted by coinfection. The implications of exploring how microbial communities can influence the fitness of pathogenic organisms is a novel avenue for transmission control of pandemic respiratory viruses.

microbiology↗

bioRxiv2020

Symbiont-mediated fly survival is independent of defensive symbiont genotype in the Drosophila melanogaster-Spiroplasma-wasp interaction

When a parasite attacks an insect, the outcome is commonly modulated by the presence of defensive heritable symbionts residing within the insect host. Previous studies noted markedly different strengths of Spiroplasma-mediated fly survival following attack by the same strain of wasp. One difference between the two studies was the strain of Spiroplasma used. We therefore performed a common garden laboratory experiment to assess whether Spiroplasma-mediated protection depends upon the strain of Spiroplasma. We perform this analysis using the two strains of male-killing Spiroplasma used previously, and examined response to challenge by two strains of Leptopilina boulardi and two strains of Leptopilina heterotoma wasp. We found no evidence Spiroplasma strain affected fly survival following wasp attack. In contrast, analysis of the overall level of protection, including the fecundity of survivors of wasp attack, did indicate the two Spiroplasma strains tested varied in protective efficiency against three of the four wasp strains tested. These data highlight the sensitivity of symbiont-mediated protection phenotypes to laboratory conditions, and the importance of common garden comparison. Our results also indicate that Spiroplasma strains can vary in protective capacity in Drosophila, but these differences may exist in the relative performance of survivors of wasp attack, rather than in survival of attack per se.

evolutionary biology↗

bioRxiv2020

Thermal sensitivity of the Spiroplasma-Drosophila hydei protective symbiosis: The best of climes, the worst of climes.

The outcome of natural enemy attack in insects has commonly been found to be influenced by the presence of protective symbionts in the host. The degree to which protection functions in natural populations, however, will depend on the robustness of the phenotype to variation in the abiotic environment. We studied the impact of a key environmental parameter - temperature - on the efficacy of the protective effect of the symbiont Spiroplasma on its host Drosophila hydei, against attack by the parasitoid wasp Leptopilina heterotoma. In addition, we investigated the thermal sensitivity of the symbionts vertical transmission, which may be a key determinant of the ability of the symbiont to persist. We found that vertical transmission was more robust than previously considered, with Spiroplasma being maintained at 25 {degrees}C, 18 {degrees}C and with 18/15 {degrees}C diurnal cycles, with rates of segregational loss only increasing at 15 {degrees}C. Protection against wasp attack was ablated before symbiont transmission was lost, with the symbiont failing to rescue the fly host at 18 {degrees}C. We conclude that the presence of a protective symbiosis in natural populations cannot be simply inferred from presence of a symbiont whose protective capacity has been tested under narrow controlled conditions. More broadly, we argue that the thermal environment is likely to represent an important determinant of the evolutionary ecology of defensive symbioses in natural environments, potentially driving seasonal, latitudinal and altitudinal variation in symbiont frequency, and modulating the strength of selection for symbiotic protective systems compared to defensive systems encoded in the nuclear genomes.

ecology↗

bioRxiv2019

Symbiont-mediated protection varies with wasp genotype in the Drosophila melanogaster-Spiroplasma interaction

The ability of an insect to survive attack by natural enemies can be modulated by the presence of defensive symbionts. Study of aphid-symbiont-enemy interactions has indicated that protection may depend on the interplay of symbiont, host and attacking parasite genotypes. However, the importance of these interactions are poorly understood outside of this model system. Here, we study interactions within a Drosophila model system, in which Spiroplasma protect their host against parasitoid wasps and nematode attack. We examine whether the strength of protection conferred by Spiroplasma to its host, Drosophila melanogaster varies with strain of attacking Leptopilina heterotoma wasp. We perform this analysis in the presence and absence of ethanol, an environmental factor that also impacts the outcome of parasitism. We observed that Spiroplasma killed all strains of wasp. However, the protection produced by Spiroplasma following wasp attack depended on attacking wasp strain. A composite measure of protection, including both the chance of the fly surviving attack and the relative fecundity/fertility of the survivors, varied from a <4% positive effect of the symbiont following attack of the fly host by the Lh14 strain of wasp to 21% for the Lh-Fr strain in the absence of ethanol. Variation in protection provided was not associated with differences in the oviposition behaviour of the different wasp strains. We observed that environmental ethanol altered the pattern of protection against wasp strains, with Spiroplasma being most protective against the Lh-Mad wasp strain in the presence of ethanol. These data indicate that the dynamics of the Spiroplasma-Drosophila-wasp tripartite interaction depend upon the genetic diversity within the attacking wasp population, and that prediction of symbiont dynamics in natural systems will thus require analysis across natural enemy genotypes and levels of environmental ethanol.\n\nImpact SummaryNatural enemies - predators, parasites and pathogens - are a common source of mortality in animals, and this has driven the evolution of an array of mechanisms for preventing and surviving attack. Recently it has been observed that microbial symbionts form a component of insect defence against attack by pathogens and parasites. Whether an individual fly dies or lives following wasp attack, for instance, is partly determined by the presence or absence of Spiroplasma bacteria in the fly blood. The evolutionary biology of these protective symbioses will in part depend on the specificity of defence - does Spiroplasma defend against all wasp strains equally, or does defence vary between wasp strains? We investigated this in the model insect, Drosophila melanogaster. We observed that the defensive symbiont killed all strains of wasps tested. However, the capacity of the symbiont to rescue the fly varied - Spiroplasma rescued the flies for some attacking wasp strains, but not for others. These data mean that the degree to which symbionts protect their host will depend on the wasp strains circulating in nature. Our results are important in terms of understanding the forces that promote symbiont mediated protection and understanding the origins of diversity of circulating wasp strains. Further, these data indicate enemy diversity and their interaction with protective symbionts should be included in evaluation of the efficiency of biocontrol programmes involving natural enemies.

evolutionary biology↗