Ebolavirus is evolving but not changing: No evidence for functional change in EBOV from 1976 to the 2014 outbreak

The 2014 epidemic of Ebola virus disease (EVD) has had a devastating impact in West Africa. Sequencing of ebolavirus (EBOV) from infected individuals has revealed extensive genetic variation, leading to speculation that the virus may be adapting to humans, accounting for the scale of the 2014 outbreak. We computationally analyze the variation associated with all EVD outbreaks, and find none of the amino acid replacements lead to identifiable functional changes. These changes have minimal effect on protein structure, being neither stabilizing nor destabilizing, are not found in regions of the proteins associated with known functions and tend to cluster in poorly constrained regions of proteins, specifically intrinsically disordered regions. We find no evidence that the difference between the current and previous outbreaks is due to evolutionary changes associated with transmission to humans. Instead, epidemiological factors are likely to be responsible for the unprecedented spread of EVD

Adaptive HIV-1 evolutionary trajectories are constrained by protein stability

Despite the use of combination antiretroviral drugs for the treatment of HIV-1 infection, the emergence of drug resistance remains a problem. Resistance may be conferred either by a single mutation or a concerted set of mutations. The involvement of multiple mutations can arise due to interactions between sites in the amino acid sequence as a consequence of the need to maintain protein structure. To better understand the nature of such epistatic interactions, we reconstructed the ancestral sequences of HIV-1's Pol protein, and traced the evolutionary trajectories leading to mutations associated with drug resistance. Using contemporary and ancestral sequences we modelled the effects of mutations (i.e. amino acid replacements) on protein structure to understand the functional effects of residue changes. Although the majority of resistance-associated sequences tend to destabilise the protein structure, we find there is a general tendency for protein stability to decrease across HIV-1's evolutionary history. That a similar pattern is observed in the non-drug resistance lineages indicates that non-resistant mutations, for example, associated with escape from the immune response, also impacts on protein stability. Maintenance of optimal protein structure therefore represents a major constraining factor to the evolution of HIV-1

Enhancement of CD4 Binding, Host Cell Entry, and Sensitivity to CD4bs Antibody Inhibition Conferred by a Natural but Rare Polymorphism in the HIV-1 Envelope

A rare but natural polymorphism in the HIV-1 envelope (Env) glycoprotein, lysine at position 425 was selected as a mutation conferring resistance to maraviroc (MVC) in vitro. N425K has not been identified in HIV-infected individuals failing an MVC-based treatment. This study reports that the rare K425 polymorphism in an HIV-1 subtype A Env has increased affinity for CD4, resulting in faster host cell entry kinetics and the ability to scavenge for low cell surface expression of CD4 to mediate entry. Whereas the subtype A wild-type isolate-74 Env (N425) is inhibited by soluble (s) CD4, HIV-1 with K425 A74 Env shows enhanced infection and the ability to infect CCR5+ cells when pretreated with sCD4. Upon adding K425 or N425 HIV-1 to CD4+/CCR5+ cells along with RANTES/CCL3, only K425 HIV-1 was able to infect cells when CCR5 recycled/returned to the cell surface at 12 h post-treatment. These findings suggest that upon binding to CD4, K425 Env may maintain a stable State 2 "open" conformation capable of engaging CCR5 for entry. Only K425 was significantly more sensitivity than wild-type N425 A74 to inhibition by the CD4 binding site (bs) compound, BMS-806, the CD4bs antibody, VRC01 and N6, and the single-chain CD4i antibody, SCm9. K425 A74 was also capable of activating B cells expressing the VRC01 surface immunoglobulin. In summary, despite increased replicative fitness, we propose that K425 HIV-1 may be counterselected within infected individuals if K425 HIV-1 is rapidly eliminated by CD4bs-neutralizing antibodies. IMPORTANCE Typically, a natural amino acid polymorphism is found as the wild-type sequence in the HIV-1 population if it provides a selective advantage to the virus. The natural K425 polymorphism in HIV-1 Env results in higher host cell entry efficiency and greater replicative fitness by virtue of its high binding affinity to CD4. The studies presented herein suggest that the rare K425 HIV-1, compared to the common N425 HIV-1, may be more sensitive to inhibition by CD4bs-neutralizing antibodies (i.e., antibodies that bind to the CD4 binding pocket on the HIV-1 envelope glycoprotein). If CD4bs antibodies did emerge in an infected individual, the K425 HIV-1 may be hypersensitive to inhibition, and thus this K425 virus variant may be removed from the HIV-1 swarm despite its higher replication fitness. Studies are now underway to determine whether addition of the K425 polymorphism into the Envelope-based HIV-1 vaccines could enhance protective immunity.</p

Tracking SARS-CoV-2 variants of concern in wastewater: an assessment of nine computational tools using simulated genomic data

Wastewater-based surveillance (WBS) is an important epidemiological and public health tool for tracking pathogens across the scale of a building, neighbourhood, city, or region. WBS gained widespread adoption globally during the SARS-CoV-2 pandemic for estimating community infection levels by qPCR. Sequencing pathogen genes or genomes from wastewater adds information about pathogen genetic diversity, which can be used to identify viral lineages (including variants of concern) that are circulating in a local population. Capturing the genetic diversity by WBS sequencing is not trivial, as wastewater samples often contain a diverse mixture of viral lineages with real mutations and sequencing errors, which must be deconvoluted computationally from short sequencing reads. In this study we assess nine different computational tools that have recently been developed to address this challenge. We simulated 100 wastewater sequence samples consisting of SARS-CoV-2 BA.1, BA.2, and Delta lineages, in various mixtures, as well as a Delta-Omicron recombinant and a synthetic 'novel' lineage. Most tools performed well in identifying the true lineages present and estimating their relative abundances and were generally robust to variation in sequencing depth and read length. While many tools identified lineages present down to 1 % frequency, results were more reliable above a 5 % threshold. The presence of an unknown synthetic lineage, which represents an unclassified SARS-CoV-2 lineage, increases the error in relative abundance estimates of other lineages, but the magnitude of this effect was small for most tools. The tools also varied in how they labelled novel synthetic lineages and recombinants. While our simulated dataset represents just one of many possible use cases for these methods, we hope it helps users understand potential sources of error or bias in wastewater sequencing analysis and to appreciate the commonalities and differences across methods.ISSN:2057-585

Effective and targeted latency reversal in CD4+ T cells from individuals on long term combined antiretroviral therapy initiated during chronic HIV-1 infection

To date, an affordable, effective treatment for an HIV-1 cure remains only a concept with most "latency reversal" agents (LRAs) lacking specificity for the latent HIV-1 reservoir and failing in early clinical trials. We assessed HIV-1 latency reversal using a multivalent HIV-1-derived virus-like particle (HLP) to treat samples from 32 people living with HIV-1 (PLWH) in Uganda, US and Canada who initiated combined antiretroviral therapy (cART) during chronic infection. Even after 5-20 years on stable cART, HLP could target CD4+ T cells harbouring latent HIV-1 reservoir resulting in 100-fold more HIV-1 release into culture supernatant than by common recall antigens, and 1000-fold more than by chemotherapeutic LRAs. HLP induced release of a divergent and replication-competent HIV-1 population from PLWH on cART. These findings suggest HLP provides a targeted approach to reactivate the majority of latent HIV-1 proviruses among individuals infected with HIV-1.</p

Adaptation of the Wound Healing Questionnaire universal-reporter outcome measure for use in global surgery trials (TALON-1 study): mixed-methods study and Rasch analysis

BackgroundThe Bluebelle Wound Healing Questionnaire (WHQ) is a universal-reporter outcome measure developed in the UK for remote detection of surgical-site infection after abdominal surgery. This study aimed to explore cross-cultural equivalence, acceptability, and content validity of the WHQ for use across low- and middle-income countries, and to make recommendations for its adaptation.MethodsThis was a mixed-methods study within a trial (SWAT) embedded in an international randomized trial, conducted according to best practice guidelines, and co-produced with community and patient partners (TALON-1). Structured interviews and focus groups were used to gather data regarding cross-cultural, cross-contextual equivalence of the individual items and scale, and conduct a translatability assessment. Translation was completed into five languages in accordance with Mapi recommendations. Next, data from a prospective cohort (SWAT) were interpreted using Rasch analysis to explore scaling and measurement properties of the WHQ. Finally, qualitative and quantitative data were triangulated using a modified, exploratory, instrumental design model.ResultsIn the qualitative phase, 10 structured interviews and six focus groups took place with a total of 47 investigators across six countries. Themes related to comprehension, response mapping, retrieval, and judgement were identified with rich cross-cultural insights. In the quantitative phase, an exploratory Rasch model was fitted to data from 537 patients (369 excluding extremes). Owing to the number of extreme (floor) values, the overall level of power was low. The single WHQ scale satisfied tests of unidimensionality, indicating validity of the ordinal total WHQ score. There was significant overall model misfit of five items (5, 9, 14, 15, 16) and local dependency in 11 item pairs. The person separation index was estimated as 0.48 suggesting weak discrimination between classes, whereas Cronbach's α was high at 0.86. Triangulation of qualitative data with the Rasch analysis supported recommendations for cross-cultural adaptation of the WHQ items 1 (redness), 3 (clear fluid), 7 (deep wound opening), 10 (pain), 11 (fever), 15 (antibiotics), 16 (debridement), 18 (drainage), and 19 (reoperation). Changes to three item response categories (1, not at all; 2, a little; 3, a lot) were adopted for symptom items 1 to 10, and two categories (0, no; 1, yes) for item 11 (fever).ConclusionThis study made recommendations for cross-cultural adaptation of the WHQ for use in global surgical research and practice, using co-produced mixed-methods data from three continents. Translations are now available for implementation into remote wound assessment pathways