No evidence that mutations in SARS-CoV-2 variants of concern derive from homologous fragments in gut microbiota.

Understanding the origin and evolution of mutations in SARS-CoV-2 variants of concern (VOCs) is a critical area of research. B. Cao, X. Wang, W. Yin, Z. Gao, and B. Xia (mBio 15:e03187-23, 2024, https://doi.org/10.1128/mbio.03187-23) proposed that these mutations originated from bacterial sequences incorporated into the viral genome through stochastic template-switching by the viral RNA-dependent RNA polymerase (RdRp). Their analysis suggested that 62% of the viral mutation fragments (VMFs) in key SARS-CoV-2 proteins were identical to bacterial protein sequences. Given the implications of this finding, we re-examined the methods employed and argue that they resulted in false-positive findings. Specifically, the short query length of VMFs, seven amino acids, leads to spurious matches in large protein databases, as indicated by high BLAST Expect values. Furthermore, we analyzed the nucleotide sequence of VMFs, revealing no unique homology between SARS-CoV-2 and bacterial sequences. Consequently, the evidence does not support the hypothesis that bacterial sequences contribute to the evolution of SARS-CoV-2 VOCs. Instead, the emergence of these variants is more plausibly attributed to factors intrinsic to viral replication and evolution, such as the error-prone nature of RdRp, intrahost diversity, and recombination of related viral sublineages.

Changes in body composition and average daily energy expenditure of men and women during arduous extended polar travel.

Weight and skin-fold measurements were made at five-day intervals during a 47-day expedition by six men and three women from the edge of the sea ice to the South Pole. From these, together with detailed manual records of the nutrition for individual participants, the average daily energy expenditure was determined before and after a resupply at approximately mid-point of the expedition. For all participants body weight fell during the expedition with the overall loss being much smaller for the three female participants (-4.0, -4.0, -4.4kg) than for the male participants, (mean±sd) -8.6±2.0kg. Fat weight fell approximately linearly during the expedition with a total loss of (-4.1, -6.5 and -2.5kg) for the three female participants and -6.8±1.7kg for the male participants. Individual fat-free weight changed by a smaller amount overall: (0.13, 2.5 and -1.8kg) for the three female participants; -1.8±2.0kg for the male participants who, with one exception, lost fat-free tissue All participants showed a substantial variation in fat-free tissue weight during the expedition. Analysis of the daily energy expenditure showed adequate nutrition but the intake fell for the second part of the expedition although the reasons for this are unclear, but adaptation to the cold, altitude and workload are possible explanations. The validity of this time-averaged measurement for individual participants was determined from analysing moments about the mean of time-series actigraphy data from wrist worn devices. The mean and autocorrelation function of the actigraphy data across subjects were analysed to determine whether measures could be compared between participants. The first, second and third moment about the mean of the day-to-day activity was found to be time-invariant for individual subjects (χ2, p>0.05) and the normalized mean and autocorrelation measured over a day for each participant indistinguishable from the mean of the group (χ2, p>0.05) allowing both longitudinal and cross-sectional analysis.

A new lineage nomenclature to aid genomic surveillance of dengue virus.

Dengue virus (DENV) is currently causing epidemics of unprecedented scope in endemic settings and expanding to new geographical areas. It is therefore critical to track this virus using genomic surveillance. However, the complex patterns of viral genomic diversity make it challenging to use the existing genotype classification system. Here, we propose adding 2 sub-genotypic levels of virus classification, named major and minor lineages. These lineages have high thresholds for phylogenetic distance and clade size, rendering them stable between phylogenetic studies. We present assignment tools to show that the proposed lineages are useful for regional, national, and subnational discussions of relevant DENV diversity. Moreover, the proposed lineages are robust to classification using partial genome sequences. We provide a standardized neutral descriptor of DENV diversity with which we can identify and track lineages of potential epidemiological and/or clinical importance. Information about our lineage system, including methods to assign lineages to sequence data and propose new lineages, can be found at: dengue-lineages.org.

A novel approach to exploring the dark genome and its application to mapping of the vertebrate virus fossil record.

BACKGROUND: Genomic regions that remain poorly understood, often referred to as the dark genome, contain a variety of functionally relevant and biologically informative features. These include endogenous viral elements (EVEs)-virus-derived sequences that can dramatically impact host biology and serve as a virus fossil record. In this study, we introduce a database-integrated genome screening (DIGS) approach to investigate the dark genome in silico, focusing on EVEs found within vertebrate genomes. RESULTS: Using DIGS on 874 vertebrate genomes, we uncover approximately 1.1 million EVE sequences, with over 99% originating from endogenous retroviruses or transposable elements that contain EVE DNA. We show that the remaining 6038 sequences represent over a thousand distinct horizontal gene transfer events across 10 virus families, including some that have not previously been reported as EVEs. We explore the genomic and phylogenetic characteristics of non-retroviral EVEs and determine their rates of acquisition during vertebrate evolution. Our study uncovers novel virus diversity, broadens knowledge of virus distribution among vertebrate hosts, and provides new insights into the ecology and evolution of vertebrate viruses. CONCLUSIONS: We comprehensively catalog and analyze EVEs within 874 vertebrate genomes, shedding light on the distribution, diversity, and long-term evolution of viruses and reveal their extensive impact on vertebrate genome evolution. Our results demonstrate the power of linking a relational database management system to a similarity search-based screening pipeline for in silico exploration of the dark genome.

A new lineage nomenclature to aid genomic surveillance of dengue virus.

Dengue virus (DENV) is currently causing epidemics of unprecedented scope in endemic settings and expanding to new geographical areas. It is therefore critical to track this virus using genomic surveillance. However, the complex patterns of viral genomic diversity make it challenging to use the existing genotype classification system. Here we propose adding two sub-genotypic levels of virus classification, named major and minor lineages. These lineages have high thresholds for phylogenetic distance and clade size, rendering them stable between phylogenetic studies. We present an assignment tool to show that the proposed lineages are useful for regional, national and sub-national discussions of relevant DENV diversity. Moreover, the proposed lineages are robust to classification using partial genome sequences. We provide a standardized neutral descriptor of DENV diversity with which we can identify and track lineages of potential epidemiological and/or clinical importance. Information about our lineage system, including methods to assign lineages to sequence data and propose new lineages, can be found at: dengue-lineages.org.

Assessment of salivary cortisol dynamics in an infantry training exercise: a pilot study.

INTRODUCTION: Measuring cortisol during military training offers insights into physiological responses to stress. We attempted precisely timed, cortisol awakening response (CAR) and pre-sleep cortisol (PSC), and diurnal slope (peak morning minus evening cortisol), during a British Army exercise. We aimed to understand cortisol dynamics and evaluate the feasibility of CAR and PSC in this environment. METHOD: Setting: high-intensity, 10-day infantry exercise. Participants: regular infantry soldiers exercising (EX, n=25) or headquarters-based (HQ, n=6). Participants undertook PSC and WAKE and WAKE+30 min samples after 1-2 days, 5-6 days and 9-10 days. Wrist-worn GENEActiv accelerometers were used to assess sleep duration in EX only. Samples taken ±15 min from prespecified time points were deemed adherent. Validated questionnaires were used to measure resilience and perceived stress. Cortisol and cortisone were measured simultaneously by liquid chromatography tandem mass spectrometry. RESULTS: From adherent participants' samples, CAR was positive and tended to decrease as the exercise progressed. From all available data, HQ demonstrated greater diurnal slope than EX (F=7.68, p=0.02), reflecting higher morning cortisol (F=4.72, p=0.038) and lower PSC (p=0.04). No differences were seen in cortisol:cortisone ratio. 26.1% of CAR samples were adherent, with moderately strong associations between adherence and stress (r=0.41, p=0.009) but no association between adherence and day of exercise (χ2=0.27, p=0.8), sleep duration (r=-0.112, p=0.43) or resilience (r=-0.79, p=0.75). Test-retest reliability ratings for CAR were Cronbach's α of 0.48, -11.7 and 0.34 for the beginning, middle and end of the exercise, respectively. CONCLUSIONS: We observed a reduction in morning cortisol and decreased diurnal slope during a high-intensity military exercise, compared with the HQ comparator cohort in whom diurnal slope was preserved. A carefully timed CAR was not feasible in this setting.

Applying the theory of behavioral choice to plant-based dietary intentions.

Improving understanding of the intention to choose plant-based food is an important element of climate change mitigation. A cross-sectional survey of 454 North American adults was used to predict their dietary-change intentions from the theory of planned behavior (TPB) and the more-recently proposed theory of behavioral choice (TBC). The TPB accounted for 65 percent of the variance in intentions and the TBC accounted for a significantly greater (80 percent) proportion of variance. The strongest predictors of intention were the TBC's sense of obligation, attitude-values-affect (AVA), and habit, and the TBP's social norms. Five interactions also contributed in small but significant ways toward the accounting of the participants' food-choice intentions. Policy implications are discussed.

Sex differences in energy balance, body composition, and metabolic and endocrine markers during prolonged arduous military training.

This study investigated sex differences in energy balance, body composition, and metabolic and endocrine markers during prolonged military training. Twenty-three trainees (14 women) completed 44-wk military training (three terms of 14 wk with 2-wk adventurous training). Dietary intake and total energy expenditure were measured over 10 days during each term by weighed food and doubly labeled water. Body composition was measured by dual-energy X-ray absorptiometry (DXA) at baseline and at the end of each term. Circulating metabolic and endocrine markers were measured at baseline and at the end of terms 2 and 3. Absolute energy intake and total energy expenditure were higher, and energy balance was lower, for men than women (P ≤ 0.008). Absolute energy intake and balance were lower, and total energy expenditure was higher, during term 2 than terms 1 and 3 (P < 0.001). Lean mass did not change with training (P = 0.081). Fat mass and body fat increased from term 1 to terms 2 and 3 (P ≤ 0.045). Leptin increased from baseline to terms 2 and 3 in women (P ≤ 0.002) but not in men (P ≥ 0.251). Testosterone and free androgen index increased from baseline to term 3 (P ≤ 0.018). Free thyroxine (T4) decreased and thyroid-stimulating hormone (TSH) increased from baseline to term 2 and term 3 (P ≤ 0.031). Cortisol decreased from baseline to term 3 (P = 0.030). IGF-I and total triiodothyronine (T3) did not change with training (P ≥ 0.148). Men experienced greater energy deficits than women during military training due to higher total energy expenditure.NEW & NOTEWORTHY Energy deficits are common in military training and can result in endocrine and metabolic disturbances. This study provides first investigation of sex differences in energy balance, body composition, and endocrine and metabolic markers in response to prolonged and arduous military training. Men experienced greater energy deficits than women due to higher energy expenditure, which was not compensated for by increased energy intake. These energy deficits were not associated with decreases in fat or lean mass or metabolic or endocrine function.

Eating Disorder Risk and Common Mental Disorders in British Servicewomen: A Cross-Sectional Observational Study.

PURPOSE: Servicewomen are at increased risk of common mental disorders compared with servicemen and their female civilian counterparts. The prevalence of eating disorder risk and common mental disorders, and associated risk factors in British servicewomen are poorly understood. METHODS: All women younger than 45 yr in the UK Armed Forces were invited to complete a survey about demographics, exercise behaviors, eating behaviors, and common mental disorders. RESULTS: A total of 3022 women participated; 13% of participants were at high risk of an eating disorder based on Brief Eating Disorder in Athletes Questionnaire and Female Athlete Screening Tool scores. Twenty-five percent of participants had symptoms of anxiety (seven-item Generalized Anxiety Disorder Assessment score ≥10), and 26% had symptoms of depression (nine-item Patient Health Questionnaire score ≥10). Older age was associated with a lower risk, and heavier body mass was associated with a higher risk, of eating disorders ( P ≤ 0.043). Older age and higher rank were associated with a lower risk of symptoms of anxiety and depression ( P ≤ 0.031), and a heavier body mass was associated with a higher risk of symptoms of depression ( P ≤ 0.012). Longer habitual sleep duration was associated with a lower risk of eating disorders and symptoms of anxiety and depression ( P ≤ 0.028). A higher volume of field exercise was associated with a lower risk, and a higher volume of military physical training and personal physical training was associated with a higher risk, of eating disorders ( P ≤ 0.024). Job role and deployment history were not associated with any outcome. CONCLUSIONS: Sleeping and training habits provide potential novel targets for exploring how common mental disorders can be managed in British servicewomen.

A novel tamanavirus (Flaviviridae) of the European common frog (Rana temporaria) from the UK.

Flavivirids are small, enveloped, positive-sense RNA viruses from the family Flaviviridae with genomes of ~9-13 kb. Metatranscriptomic analyses of metazoan organisms have revealed a diversity of flavivirus-like or flavivirid viral sequences in fish and marine invertebrate groups. However, no flavivirus-like virus has been identified in amphibians. To remedy this, we investigated the virome of the European common frog (Rana temporaria) in the UK, utilizing high-throughput sequencing at six catch locations. De novo assembly revealed a coding-complete virus contig of a novel flavivirid ~11.2 kb in length. The virus encodes a single ORF of 3456 aa and 5' and 3' untranslated regions (UTRs) of 227 and 666 nt, respectively. We named this virus Rana tamanavirus (RaTV), as BLASTp analysis of the polyprotein showed the closest relationships to Tamana bat virus (TABV) and Cyclopterus lumpus virus from Pteronotus parnellii and Cyclopterus lumpus, respectively. Phylogenetic analysis of the RaTV polyprotein compared to Flavivirus and Flavivirus-like members indicated that RaTV was sufficiently divergent and basal to the vertebrate Tamanavirus clade. In addition to the Mitcham strain, partial but divergent RaTV, sharing 95.64-97.39 % pairwise nucleotide identity, were also obtained from the Poole and Deal samples, indicating that RaTV is widespread in UK frog samples. Bioinformatic analyses of predicted secondary structures in the 3'UTR of RaTV showed the presence of an exoribonuclease-resistant RNA (xrRNA) structure standard in flaviviruses and TABV. To examine this biochemically, we conducted an in vitro Xrn1 digestion assay showing that RaTV probably forms a functional Xrn1-resistant xrRNA.

An emerging clade of Chikungunya West African genotype discovered in real-time during 2023 outbreak in Senegal.

Chikungunya (CHIKV) is a re-emerging endemic arbovirus in West Africa. Since July 2023, Senegal and Burkina Faso have been experiencing an ongoing outbreak, with over 300 confirmed cases detected so far in the regions of Kédougou and Tambacounda in Senegal, the largest recorded outbreak yet. CHIKV is typically maintained in a sylvatic cycle in Senegal but its evolution and factors contributing to re-emergence are so far unknown in West Africa, leaving a gap in understanding and responding to recurrent epidemics. We produced, in real-time, the first locally-generated and publicly available CHIKV whole genomes in West Africa, to characterize the genetic diversity of circulating strains, along with phylodynamic analysis to estimate time of emergence and population growth dynamics. A novel strain of the West African genotype, phylogenetically distinct from strains circulating in previous outbreaks, was identified. This suggests a likely new spillover from sylvatic cycles in rural Senegal and potential of seeding larger epidemics in urban settings in Senegal and elsewhere.

Whole Genome Sequencing for Rapid Characterization of Rabies Virus Using Nanopore Technology.

Genomic data can be used to track the transmission and geographic spread of infectious diseases. However, the sequencing capacity required for genomic surveillance remains limited in many low- and middle-income countries (LMICs), where dog-mediated rabies and/or rabies transmitted by wildlife such as vampire bats pose major public health and economic concerns. We present here a rapid and affordable sample-to-sequence-to-interpretation workflow using nanopore technology. Protocols for sample collection and the diagnosis of rabies are briefly described, followed by details of the optimized whole genome sequencing workflow, including primer design and optimization for multiplex polymerase chain reaction (PCR), a modified, low-cost sequencing library preparation, sequencing with live and offline base calling, genetic lineage designation, and phylogenetic analysis. Implementation of the workflow is demonstrated, and critical steps are highlighted for local deployment, such as pipeline validation, primer optimization, inclusion of negative controls, and the use of publicly available data and genomic tools (GLUE, MADDOG) for classification and placement within regional and global phylogenies. The turnaround time for the workflow is 2-3 days, and the cost ranges from $25 per sample for a 96 sample run to $80 per sample for a 12 sample run. We conclude that setting up rabies virus genomic surveillance in LMICs is feasible and can support progress toward the global goal of zero dog-mediated human rabies deaths by 2030, as well as enhanced monitoring of wildlife rabies spread. Moreover, the platform can be adapted for other pathogens, helping to build a versatile genomic capacity that contributes to epidemic and pandemic preparedness.

Structures of the Hepaci-, Pegi-, and Pestiviruses envelope proteins suggest a novel membrane fusion mechanism.

Enveloped viruses encode specialised glycoproteins that mediate fusion of viral and host membranes. Discovery and understanding of the molecular mechanisms of fusion have been achieved through structural analyses of glycoproteins from many different viruses, and yet the fusion mechanisms of some viral genera remain unknown. We have employed systematic genome annotation and AlphaFold modelling to predict the structures of the E1E2 glycoproteins from 60 viral species in the Hepacivirus, Pegivirus, and Pestivirus genera. While the predicted structure of E2 varied widely, E1 exhibited a very consistent fold across genera, despite little or no similarity at the sequence level. Critically, the structure of E1 is unlike any other known viral glycoprotein. This suggests that the Hepaci-, Pegi-, and Pestiviruses may possess a common and novel membrane fusion mechanism. Comparison of E1E2 models from various species reveals recurrent features that are likely to be mechanistically important and sheds light on the evolution of membrane fusion in these viral genera. These findings provide new fundamental understanding of viral membrane fusion and are relevant to structure-guided vaccinology.

Antiviral Activity of an Endogenous Parvoviral Element.

Endogenous viral elements (EVEs) are genomic DNA sequences derived from viruses. Some EVEs have open reading frames (ORFs) that can express proteins with physiological roles in their host. Furthermore, some EVEs exhibit a protective role against exogenous viral infection in their host. Endogenous parvoviral elements (EPVs) are highly represented in mammalian genomes, and although some of them contain ORFs, their function is unknown. We have shown that the locus EPV-Dependo.43-ODegus, an EPV with an intact ORF, is transcribed in Octodon degus (degu). Here we examine the antiviral activity of the protein encoded in this EPV, named DeRep. DeRep was produced in bacteria and used to generate antibodies that recognize DeRep in western blots of degu tissue. To test if DeRep could protect against exogenous parvovirus, we challenged cells with the minute virus of mice (MVM), a model autonomous parvovirus. We observed that MVM protein expression, DNA damage induced by replication, viral DNA, and cytopathic effects are reduced when DeRep is expressed in cells. The results of this study demonstrate that DeRep is expressed in degu and can inhibit parvovirus replication. This is the first time that an EPV has been shown to have antiviral activity against an exogenous virus.

Co-option of endogenous retroviruses through genetic escape from TRIM28 repression.

Endogenous retroviruses (ERVs) have rewired host gene networks. To explore the origins of co-option, we employed an active murine ERV, IAPEz, and an embryonic stem cell (ESC) to neural progenitor cell (NPC) differentiation model. Transcriptional silencing via TRIM28 maps to a 190 bp sequence encoding the intracisternal A-type particle (IAP) signal peptide, which confers retrotransposition activity. A subset of "escapee" IAPs (∼15%) exhibits significant genetic divergence from this sequence. Canonical repressed IAPs succumb to a previously undocumented demarcation by H3K9me3 and H3K27me3 in NPCs. Escapee IAPs, in contrast, evade repression in both cell types, resulting in their transcriptional derepression, particularly in NPCs. We validate the enhancer function of a 47 bp sequence within the U3 region of the long terminal repeat (LTR) and show that escapee IAPs convey an activating effect on nearby neural genes. In sum, co-opted ERVs stem from genetic escapees that have lost vital sequences required for both TRIM28 restriction and autonomous retrotransposition.

Emergence and pandemic spread of small ruminant lentiviruses.

Small ruminant lentiviruses (SRLVs) cause chronic, persistent infections in populations of domestic sheep (Ovis aries) and goats (Capra hircus) worldwide. The vast majority of SRLV infections involve two genotypes (A and B) that spread in association with the emergence of global livestock trade. However, SRLVs have likely been present in Eurasian ruminant populations since at least the early Neolithic period. Here, we use phylogenetic and phylogeographic approaches to reconstruct the origin of pandemic SRLV strains and infer their historical pattern of global spread. We constructed an open computational resource ('Lentivirus-GLUE') via which an up-to-date database of published SRLV sequences, multiple sequence alignments (MSAs), and sequence-associated metadata can be maintained. We used data collated in Lentivirus-GLUE to perform a comprehensive phylogenetic investigation of global SRLV diversity. Phylogenies reconstructed from genome-length alignments reveal that the deep divisions in the SRLV phylogeny are consistent with an ancient split into Eastern (A-like) and Western (B-like) lineages as agricultural systems disseminated out of domestication centres during the Neolithic period. These findings are also consistent with historical and phylogeographic evidence linking the early 20th century emergence of SRLV-A to the international export of Central Asian Karakul sheep. Investigating the global diversity of SRLVs can help reveal how anthropogenic factors have impacted the ecology and evolution of livestock diseases. The open resources generated in our study can expedite these studies and can also serve more broadly to facilitate the use of genomic data in SRLV diagnostics and research.

Measurement of Energy Intake Using the Principle of Energy Balance Overcomes a Critical Limitation in the Assessment of Energy Availability.

Prolonged low energy availability, which is the underpinning aetiology of the Relative Energy Deficiency in Sport and the Female and Male Athlete Triad frameworks, can have unfavourable impacts on both health and performance in athletes. Energy availability is calculated as energy intake minus exercise energy expenditure, expressed relative to fat free mass. The current measurement of energy intake is recognized as a major limitation for assessing energy availability due to its reliance on self-report methods, in addition to its short-term nature. This article introduces the application of the energy balance method for the measurement of energy intake, within the context of energy availability. The energy balance method requires quantification of the change in body energy stores over time, with concurrent measurement of total energy expenditure. This provides an objective calculation of energy intake, which can then be used for the assessment of energy availability. This approach, the Energy Availability - Energy Balance (EAEB) method, increases the reliance on objective measurements, provides an indication of energy availability status over longer periods and removes athlete burden to self-report energy intake. Implementation of the EAEB method could be used to objectively identify and detect low energy availability, with implications for the diagnosis and management of Relative Energy Deficiency in Sport and the Female and Male Athlete Triad.

Sex, gender or occupational psychology: what matters most to preventing heat-related illnesses and improving outcomes for women in ground close combat?

Since the advent of women in ground close combat (WGCC) roles, the impact on women of the attendant risk of heat stress and heat illness has been considered. Much emphasis has been placed on sex differences in thermal physiology. This article considers the application of evidence of sex-associated thermoregulatory variation to the occupational and environmental setting of WGCC, and weighs the relative importance of physiological differences arising from biological sex, and behaviour associated with gender normatives. Quantifying the risk of heat illness to WGCC should draw on data from their real-world occupational context.

Comparative analysis of genome-encoded viral sequences reveals the evolutionary history of flavivirids (family Flaviviridae).

Flavivirids (family Flaviviridae) are a group of positive-strand ribonucleic acid (RNA) viruses that pose serious risks to human and animal health on a global scale. Here, we use flavivirid-derived deoxyribonucleic acid (DNA) sequences, identified in animal genomes, to reconstruct the long-term evolutionary history of family Flaviviridae. We demonstrate that flavivirids are >100 million years old and show that this timing can be combined with dates inferred from co-phyletic analysis to produce a cohesive overview of their evolution, distribution, and diversity wherein the main flavivirid subgroups originate in early animals and broadly co-diverge with major animal phyla. In addition, we reveal evidence that the 'classical flaviviruses' of vertebrates, most of which are transmitted via blood-feeding arthropod vectors, originally evolved in haematophagous arachnids and later acquired the capacity to be transmitted by insects. Our findings imply that the biological properties of flavivirids have been acquired gradually over the course of animal evolution. Thus, broad-scale comparative analysis will likely reveal fundamental insights into their biology. We therefore published our results via an open, extensible, database (Flavivirid-GLUE), which we constructed to facilitate the wider utilisation of genomic data and evolution-related domain knowledge in flavivirid research.