Complexity of the Inoculum Determines the Rate of Reversion of SIV Gag CD8 T Cell Mutant Virus and Outcome of Infection

Escape mutant (EM) virus that evades CD8+ T cell recognition is frequently observed following infection with HIV-1 or SIV. This EM virus is often less replicatively “fit” compared to wild-type (WT) virus, as demonstrated by reversion to WT upon transmission of HIV to a naïve host and the association of EM virus with lower viral load in vivo in HIV-1 infection. The rate and timing of reversion is, however, highly variable. We quantified reversion to WT of a series of SIV and SHIV viruses containing minor amounts of WT virus in pigtail macaques using a sensitive PCR assay. Infection with mixes of EM and WT virus containing ≥10% WT virus results in immediate and rapid outgrowth of WT virus at SIV Gag CD8 T cell epitopes within 7 days of infection of pigtail macaques with SHIV or SIV. In contrast, infection with biologically passaged SHIVmn229 viruses with much smaller proportions of WT sequence, or a molecular clone of pure EM SIVmac239, demonstrated a delayed or slow pattern of reversion. WT virus was not detectable until ≥8 days after inoculation and took ≥8 weeks to become the dominant quasispecies. A delayed pattern of reversion was associated with significantly lower viral loads. The diversity of the infecting inoculum determines the timing of reversion to WT virus, which in turn predicts the outcome of infection. The delay in reversion of fitness-reducing CD8 T cell escape mutations in some scenarios suggests opportunities to reduce the pathogenicity of HIV during very early infection

The development and validation of a scoring tool to predict the operative duration of elective laparoscopic cholecystectomy

Background: The ability to accurately predict operative duration has the potential to optimise theatre efficiency and utilisation, thus reducing costs and increasing staff and patient satisfaction. With laparoscopic cholecystectomy being one of the most commonly performed procedures worldwide, a tool to predict operative duration could be extremely beneficial to healthcare organisations. Methods: Data collected from the CholeS study on patients undergoing cholecystectomy in UK and Irish hospitals between 04/2014 and 05/2014 were used to study operative duration. A multivariable binary logistic regression model was produced in order to identify significant independent predictors of long (> 90 min) operations. The resulting model was converted to a risk score, which was subsequently validated on second cohort of patients using ROC curves. Results: After exclusions, data were available for 7227 patients in the derivation (CholeS) cohort. The median operative duration was 60 min (interquartile range 45–85), with 17.7% of operations lasting longer than 90 min. Ten factors were found to be significant independent predictors of operative durations > 90 min, including ASA, age, previous surgical admissions, BMI, gallbladder wall thickness and CBD diameter. A risk score was then produced from these factors, and applied to a cohort of 2405 patients from a tertiary centre for external validation. This returned an area under the ROC curve of 0.708 (SE = 0.013, p  90 min increasing more than eightfold from 5.1 to 41.8% in the extremes of the score. Conclusion: The scoring tool produced in this study was found to be significantly predictive of long operative durations on validation in an external cohort. As such, the tool may have the potential to enable organisations to better organise theatre lists and deliver greater efficiencies in care

Transgenerational effects of maternal sexual interactions in seed beetles

Mating often bears large costs to females, especially in species with high levels of sexual conflict over mating rates. Given the direct costs to females associated with multiple mating, which include reductions in lifespan and lifetime reproductive success, past research focused on identifying potential indirect benefits (through increases in offspring fitness) that females may accrue. Far less attention has, however, been devoted to understanding how costs of sexual interactions to females may extend across generations. Hence, little is known about the transgenerational implications of variation in mating rates, or the net consequences of maternal sexual activities across generations. Using the seed beetle, Callosobruchus maculatus, a model system for the study of sexual conflict, we investigate the effects of mating with multiple males versus a single male, and tease apart effects due to sexual harassment and those due to mating per se, over three generations. A multigenerational analysis indicated that females that were exposed to ongoing sexual harassment and who also were permitted to mate with multiple males showed no difference in net fitness compared to females that mated just once without ongoing harassment. Intriguingly, however, females that were continually harassed, but permitted to mate just once, suffered a severe decline in net fitness compared to females that were singly (not harassed) or multiply mated (harassed, but potentially gaining benefits via mating with multiple males). Overall, the enhanced fitness in multiply mated compared to harassed females may indicate that multiple mating confers transgenerational benefits. These benefits may counteract, but do not exceed (i.e., we found no difference between singly and multiply mated females), the large transgenerational costs of harassment. Our study highlights the importance of examining transgenerational effects from an inclusive (looking at both indirect benefits but also costs) perspective, and the need to investigate transgenerational effects across several generations if we are to fully understand the consequences of sexual interactions, sexual conflict evolution, and the interplay of sexual conflict and multi-generational costs and benefits

Reversion to WT for different viruses and percentages of WT in the inocula.

<p>(A–F) Shows WT (squares) and EM (triangles) plasma viral loads over time by qRT-PCR from individual pigtail macaques inoculated with different viruses over 11 weeks. (A) SHIV_mn229 stock with 11.2% WT virus at KP9 CD8 T cell epitope (four animals) (B) <i>In vivo</i> passage of SHIV_SF162P3 with 50% WT virus at AF9 CD8 T cell epitope (2 animals). (C) <i>In vivo</i> passage of SHIV_mn229 with 4.0% WT virus at KP9 (2 animals). (D) <i>In vivo</i> passage of SHIV_mn229 with 0.34% WT virus at KP9 (two animals). (E) Mix of SIV_mac239 molecular clones containing 10% WT virus and 90% K165R EM virus. (F) Pure SIV_mac239 molecular clone of 100% K165R EM virus (0% WT, 3 animals). (G) Mean (±SEM) of WT (upper panels) or EM (lower panels) viral loads of groups of animals given the same virus. Animals administered mixes of EM and WT virus with ≥10% WT have similar WT and EM viral loads and are grouped together (left panels) in comparison to animals administered viruses with <10% WT content (right panels). The first 10 days are shaded to indicate the differences in WT virus expansion between the two types of viruses.</p

Reduced pathogenicity of passaged SHIV_mn229.

<p>(A) Comparison of mean (±SEM) viral load of 21 animals infected with the same original SHIV_mn229 stock (89% EM, 11% WT) to four animals inoculated with passaged SHIV_mn229 isolates containing less WT virus (4% or 0.3%). (B) Comparison of individual peak viral loads. (C) Comparison of individual set point viral loads.</p

Reversion of KP9 and AF9 mutant viruses.

*<p>≤indicates WT virus detected at first time point sampled.</p>†<p>>indicates WT virus levels did not exceed EM virus levels by last time point sampled.</p>**<p>Reversion rates at 50% for these animals were determined by extrapolation.</p

Dependence of reversion dynamics on the percentage of WT in the inoculum.

<p>(A) The time needed to reach 50% WT in total viral load depends on the fraction of WT in the inoculum, and starts to increase rapidly with the decrease in initial percentage below approximately 10%. (B) Dependence of reversion rate at 50% WT on the fraction of WT in the inoculum. In (A) and (B): full circle symbols, experimental data; open circles, obtained by extrapolation; line, results of the model for <i>r_W</i>−<i>r_M</i> = 3×10⁻⁴ µL/cell/day. (C) The observed reversion rate is proportional to the average target cell number (Equation 3). The dashed line represents target cells in time. The red and the green full lines show how % WT in total viral load grows if it is initially 10% or 0.25%, respectively. If WT does not reach 50% before target cells are depleted, then it will take much longer to overtake EM. (D) Experimentally observed CD4+ T cell levels and % WT in the SHIV_mn229-infected animal H20 (with initial 11% WT at KP9) and (E) in animal 6255, infected with the passaged SHIV_mn229 with 0.34% WT at KP9 conform to the theoretical pattern in (C).</p