Segmentation of viral genomes into multiple RNAs creates the potential for replication of incomplete viral genomes (IVGs). Here we use a single-cell approach to quantify influenza A virus IVGs and examine their fitness implications. We find that each segment of influenza A/Panama/2007/99 (H3N2) virus has a 58% probability of being replicated in a cell infected with a single virion. Theoretical methods predict that IVGs carry high costs in a well-mixed system, as 3.6 virions are required for replication of a full genome. Spatial structure is predicted to mitigate these costs, however, and experimental manipulations of spatial structure indicate that local spread facilitates complementation. A virus entirely dependent on co-infection was used to assess relevance of IVGs in vivo. This virus grows robustly in guinea pigs, but is less infectious and does not transmit. Thus, co-infection allows IVGs to contribute to within-host spread, but complete genomes may be critical for transmission.

Reassortment of gene segments between coinfecting influenza A viruses (IAVs) facilitates viral diversification and has a significant epidemiological impact on seasonal and pandemic influenza. Since 1977, human IAVs of H1N1 and H3N2 subtypes have cocirculated with relatively few documented cases of reassortment. We evaluated the potential for viruses of the 2009 pandemic H1N1 (pH1N1) and seasonal H3N2 lineages to reassort under experimental conditions. Results of heterologous coinfections with pH1N1 and H3N2 viruses were compared to those obtained following coinfection with homologous, genetically tagged, pH1N1 viruses as a control. High genotype diversity was observed among progeny of both coinfections; however, diversity was more limited following heterologous coinfection. Pairwise analysis of genotype patterns revealed that homologous reassortment was random while heterologous reassortment was characterized by specific biases. pH1N1/H3N2 reassortant genotypes produced under single-cycle coinfection conditions showed a strong preference for homologous PB2-PA combinations and general preferences for the H3N2 NA, pH1N1 M, and H3N2 PB2 except when paired with the pH1N1 PA or NP. Multicycle coinfection results corroborated these findings and revealed an additional preference for the H3N2 HA. Segment compatibility was further investigated by measuring chimeric polymerase activity and growth of selected reassortants in human tracheobronchial epithelial cells. In guinea pigs inoculated with a mixture of viruses, parental H3N2 viruses dominated but reassortants also infected and transmitted to cage mates. Taken together, our results indicate that strong intrinsic barriers to reassortment between seasonal H3N2 and pH1N1 viruses are few but that the reassortants formed are attenuated relative to parental strains. The genome of IAV is relatively simple, comprising eight RNA segments, each of which typically encodes one or two proteins. Each viral protein carries out multiple functions in coordination with other viral components and the machinery of the cell. When two IAVs coinfect a cell, they can exchange genes through reassortment. The resultant progeny viruses often suffer fitness defects due to suboptimal interactions among divergent viral components. The genetic diversity generated through reassortment can facilitate the emergence of novel outbreak strains. Thus, it is important to understand the efficiency of reassortment and the factors that limit its potential. The research described here offers new tools for studying reassortment between two strains of interest and applies those tools to viruses of the 2009 pandemic H1N1 and seasonal H3N2 lineages, which currently cocirculate in humans and therefore have the potential to give rise to novel epidemic strains.

Bacterial toxin-antitoxin systems are important factors implicated in growth inhibition and plasmid maintenance. Type II toxin-antitoxin pairs are regulated at the transcriptional level by the antitoxin itself. Here, we examined how the HigA antitoxin regulates the expression of the Proteus vulgaris higBA toxin-antitoxin operon from the Rts1 plasmid. The HigBA complex adopts a unique architecture suggesting differences in its regulation as compared to classical type II toxin-antitoxin systems. We find that the C-terminus of the HigA antitoxin is required for dimerization and transcriptional repression. Further, the HigA structure reveals that the C terminus is ordered and does not transition between disorderto-order states upon toxin binding. HigA residue Arg40 recognizes a TpG dinucleotide in higO2, an evolutionary conserved mode of recognition among prokaryotic and eukaryotic transcription factors. Comparison of the HigBA and HigA-higO2 structures reveals the distance between helix-turn-helix motifs of each HigA monomer increases by ~4 Å in order to bind to higO2. Consistent with these data, HigBA binding to each operator is twofold less tight than HigA alone. Together, these data show the HigB toxin does not act as a co-repressor suggesting potential novel regulation in this toxin-antitoxin system.

BackgroundNLRP3 inflammasomes activation has been demonstrated in both type 1 and type 2 diabetes mellitus and it has been correlated with dysfunctional organs. Although it has been shown that activation of NLRP3 inflammasomes contributes to bladder dysfunction in models of cystitis and bladder outlet obstruction, it is still unknown whether inflammasomes contribute to the development of diabetic bladder dysfunction.AimWe aimed to evaluate the contribution of NLRP3 inflammasomes in the late phase of diabetic bladder dysfunction which is known by hypo responsiveness of the detrusor smooth muscle.MethodsStrips of bladder were excised from male and female wild type (WT) and dbdb−/− mice at 14 – 16 week old and were mounted in a muscle strip myograph system. Contraction of the bladder was evaluated by a single concentration of KCl (120 mM), concentration‐response curves to carbachol (1 nM – 30 μM) and frequency‐response curves to electrical field stimulation (EFS: 1–16 Hz). Carbachol and EFS were repeated in the presence of the NLRP3 inhibitor MCC950 (1 μM).ResultsMale bladder strips presented higher magnitude of contraction than female bladder strips for all contractile agents tested. KCl‐induced contraction was similar in both strains (Female WT: 42 ± 7 mN; dbdb−/−: 40 ± 4 mN; Male WT: 54 ± 6.5 mN; dbdb−/−: 51 ± 17 mN). In males, the potency of carbachol was lower in the bladder of dbdb−/− mice (pEC50: 5.53 ± 0.09) compared to WT (pEC50: 6.11 ± 0.07) and it was not restored in the presence of MCC950 (pEC50: 5.66 ± 0.08). Similarly, the contraction induced by EFS was significantly reduced for all frequencies in the bladder of male dbdb−/− mice (at 16 Hz: 51 ± 10 mN) compared to WT (at 16 Hz: 77 ± 3 mN). However, it was restored in the presence of MCC950 (at 16 Hz: 71 ± 11 mN). In female mice, there was no difference in the contraction of the bladder of dbdb−/− mice when compared to WT and addition of MCC950 did not altered the responses neither in WT nor in dbdb−/−.ConclusionIn conclusion, our data suggest that NLRP3 inflammasomes contribute to the development of hypoactive detrusor response in the late phase of diabetic bladder dysfunction in males.Support or Funding InformationNational Institute of Health (NIH; HL‐134604) São Paulo Research Foundation (FAPESP; 2016/20592‐8)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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