Beluga whale and bottlenose dolphin ACE2 proteins allow cell entry mediated by spike protein from three variants of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses infect numerous non-human species. Spillover of SARS-CoV-2 into novel animal reservoirs may present a danger to host individuals of these species, particularly worrisome in populations already endangered or threatened by extinction. In addition, emergence in new reservoirs could pose spillback threats to humans, especially in the form of virus variants that further mutate when infecting other animal hosts. Previous work suggests beluga whales (Delphinapterus leucas) and bottlenose dolphins (Tursiops truncatus) may be at risk owing to their formation of social groups, contact with humans, exposure to contaminated wastewater, and structure of their angiotensin-converting enzyme 2 (ACE2) proteins, which SARS-CoV-2 uses as a cellular receptor. We examined marine-mammal susceptibility to virus infection by challenging 293T cells expressing beluga or dolphin ACE2 with pseudovirions bearing the SARS-CoV-2 spike protein. Beluga and dolphin ACE2 were sufficient to allow cell entry by an early pandemic isolate (Wuhan-Hu-1) and two evolved variants (Delta B.1.617.2 and Omicron BA.1 strains). We conclude that SARS-CoV-2 poses a potential threat to marine mammal reservoirs that should be considered in surveillance efforts.

Assembly and Annotation of the Complete Genome Sequence of T4-Like Bacteriophage 132.

Here, we describe the complete sequence of bacteriophage 132, a T4-like Escherichia coli phage. Phage 132 has a genome of 166,922-bp length, with 286 predicted genes.

Infection rate of Aedes aegypti mosquitoes with dengue virus depends on the interaction between temperature and mosquito genotype.

Dengue fever is the most prevalent arthropod-transmitted viral disease worldwide, with endemic transmission restricted to tropical and subtropical regions of different temperature profiles. Temperature is epidemiologically relevant because it affects dengue infection rates in Aedes aegypti mosquitoes, the major vector of the dengue virus (DENV). Aedes aegypti populations are also known to vary in competence for different DENV genotypes. We assessed the effects of mosquito and virus genotype on DENV infection in the context of temperature by challenging Ae. aegypti from two locations in Vietnam, which differ in temperature regimes, with two isolates of DENV-2 collected from the same two localities, followed by incubation at 25, 27 or 32°C for 10 days. Genotyping of the mosquito populations and virus isolates confirmed that each group was genetically distinct. Extrinsic incubation temperature (EIT) and DENV-2 genotype had a direct effect on the infection rate, consistent with previous studies. However, our results show that the EIT impacts the infection rate differently in each mosquito population, indicating a genotype by environment interaction. These results suggest that the magnitude of DENV epidemics may not only depend on the virus and mosquito genotypes present, but also on how they interact with local temperature. This information should be considered when estimating vector competence of local and introduced mosquito populations during disease risk evaluation.

Predicting virus evolution: the relationship between genetic robustness and evolvability of thermotolerance.

Evolutionary biologists often seek to infer historical patterns of relatedness among organisms using phylogenetic methods and to gauge the evolutionary processes that determine variation among individuals in extant populations. But relatively less effort is devoted to making evolutionary biology a truly predictive science, where future evolutionary events are precisely foreseen. Accurate predictions of evolvability would be particularly useful in the evolution of infectious diseases, such as the ability to preemptively address the challenge of pathogens newly emerging in humans and other host populations. Experimental evolution of microbes allows the possibility to rigorously test hypotheses regarding pathogen evolvability. Here, we review how genetic robustness was a useful predictor in gauging which variants of RNA virus varphi6 should evolve faster in a novel high-temperature environment. We also present new data on the relative survival of robust and brittle viruses across elevated temperatures and durations of ultraviolet exposure, to infer a possible mechanism for robustness. Our work suggests that virus adaptability in a new environment can be predicted given knowledge of virus canalization in the face of mutational input. These results hint that accurate predictions of virus evolvability are a realistic possibility, at least under circumstances of adaptive thermotolerance.

Laboratory-dependent bacterial ecology: a cautionary tale.

Although laboratory dependence is an acknowledged problem in microbiology, it is seldom intensively studied or discussed. We demonstrate that laboratory dependence is real and quantifiable even in the popular model Escherichia coli. Here laboratory effects alter the equilibrium composition of a simple community composed of two strains of E. coli. Our data rule out changes in the bacterial strains, chemical batches, and human handling but implicate differences in growth medium, especially the water component.

Arbovirus circulation, temporal distribution, and abundance of mosquito species in two Carolina bay habitats.

Carolina bays, a type of geomorphic feature, may be important in the ecology of mosquito vectors in South Carolina. Their hydrology varies from wetland habitats with marked flooding/drying regimes to permanently flooded spring-fed lakes. Moreover, they possess characteristics that contribute to the support of a particularly abundant and diverse invertebrate fauna. Although it has been estimated that 2,700+ bays exist in South Carolina, approximately 97% have been altered; < or = 200 bays remain intact, and only 36 are protected by state-funded conservation projects. We conducted a study in two distinct Carolina bay habitats, Savage Bay Heritage Preserve (SBHP) and Woods Bay State Park (WBSP), from June 1997 to July 1998 to determine mosquito temporal distribution, species composition, and the occurrence of arbovirus activity. The largest mosquito collection was obtained at WBSP (n = 31,172) representing 25 species followed by SBHP (n = 3,940) with 24 species. Anopheles crucians complex were the most common species encountered in both bays. Two virus isolates were obtained from SBHP in 1997: Keystone (KEY) virus from Ochlerotatus atlanticus-tormentor and Cache Valley (CV) virus from Oc. canadensis canadensis. Twenty-nine (29) arbovirus-positive pools were obtained from WBSP: 28 in 1997 and one in 1998. KEY virus was isolated from three pools of Oc. atlanticus-tormentor and Tensaw (TEN) virus was isolated from two pools of An. crucians complex; 10 isolates could not be identified with the sera available. Additionally, 14 pools of An. crucians complex tested positive for Eastern equine encephalitis (EEE) virus antigen. These represent the first record of KEY and CV viruses in South Carolina. Our data indicate the presence of high mosquito density and diversity in both Carolina bay habitats, which may be influenced, in part, by seasonal changes in their hydroperiods. The study of mosquito and arbovirus ecology in Carolina Bay habitats could provide more information on the transmission dynamics of arboviruses and its impact on human and animal arboviral disease occurrence in South Carolina.

Patterns of epistasis in RNA viruses: a review of the evidence from vaccine design.

Epistasis results when the fitness effects of a mutation change depending on the presence or absence of other mutations in the genome. The predictions of many influential evolutionary hypotheses are determined by the existence and form of epistasis. One rich source of data on the interactions among deleterious mutations that has gone untapped by evolutionary biologists is the literature on the design of live, attenuated vaccine viruses. Rational vaccine design depends upon the measurement of individual and combined effects of deleterious mutations. In the current study, we have reviewed data from 29 vaccine-oriented studies using 14 different RNA viruses. Our analyses indicate that (1) no consistent tendency towards a particular form of epistasis exists across RNA viruses and (2) significant interactions among groups of mutations within individual viruses occur but are not common. RNA viruses are significant pathogens of human disease, and are tractable model systems for evolutionary studies--we discuss the relevance of our findings in both contexts.

Does experimental evolution reflect patterns in natural populations? E. coli strains from long-term studies compared with wild isolates.

Our results show that experimental evolution mimics evolution in nature. In particular, only 1,000 generations of periodic recombination with immigrant genotypes is enough for linkage disequilibrium values in experimental populations to change from a maximum linkage value to a value similar to the one observed in wild strains of E. coli. Our analysis suggests an analogy between the recombination experiment and the evolutionary history of E. coli; the E. coli genome is a patchwork of genes laterally inserted in a common backbone, and the experimental E. coli chromosome is a patchwork where some sites are highly prone to recombination and others are very clonal. In addition, we propose a population model for wild E. coli where gene flow (recombination and migration) are an important source of genetic variation, and where certain hosts act as selective sieves; i.e., the host digestive system allows only certain strains to adhere and prosper as resident strains generating a particular microbiota in each host. Therefore we suggest that the strains from a wide range of wild hosts from different regions of the world may present an ecotypic structure where adaptation to the host may play an important role in the population structure.

Arbovirus surveillance in South Carolina, 1996-98.

Arboviruses isolated and identified from mosquitoes in South Carolina (USA) are described, including new state records for eastern equine encephalitis virus (EEE), St. Louis encephalitis virus (SLE), Flanders virus, Tensaw virus (TEN), and a variant of Jamestown Canyon virus (JC). Mosquitoes were collected at 52 locations in 30 of 46 South Carolina counties beginning in June 1996, and ending in October 1998, and tested for arboviruses. Of 1,329 mosquito pools tested by virus isolation (85,806 mosquitoes representing 34 mosquito species or complexes), 15 pools were positive. Virus isolations included EEE from 1 pool each of Anopheles crucians complex and Culex erraticus; a variant of JC from 1 pool of An. crucians complex; a California serogroup virus from 1 pool of Aedes atlanticus/tormentor; TEN from 5 pools of An. crucians complex and 1 pool each of Culex salinarius and Psorophora ciliata; Flanders virus from 1 pool of Culiseta melanura; and Potosi virus from 1 pool each of Aedes vexans, Coquillettidia perturbans, and Psorophora columbiae. Of 300 mosquito pools tested by antigen-capture assay for EEE and SLE (14,303 mosquitoes representing 16 mosquito species or complexes), 21 were positive for EEE and I was positive for SLE. Positive EEE mosquito pools by antigen-capture assay included An. crucians complex (14 pools), Anopheles punctipennis (1 pool), Anopheles quadrimaculatus (1 pool), Cq. perturbans (4 pools), and Cs. melanura (1 pool). One pool of Cx. salinarius was positive for SLE by antigen-capture assay. Arbovirus-positive mosquito pools were identified from 12 South Carolina counties, all located in the Atlantic Coastal Plain, and from 4 of 8 Carolina bays surveyed.

Transmission bottlenecks and the evolution of fitness in rapidly evolving RNA viruses.

We explored the evolutionary importance of two factors in the adaptation of RNA viruses to their cellular hosts, size of viral inoculum used to initiate a new infection, and mode of transmission (horizontal versus vertical). Transmission bottlenecks should occur in natural populations of viruses and their profound effects on viral adaptation have been previously documented. However, the role of transmission mode has not received the same attention. Here we used a factorial experimental design to test the combined effects of inoculum (bottleneck) size and mode of transmission in evolution of vesicular stomatitis virus (VSV) in tissue culture, and compared our results to the predictions of a recent theoretical model. Our data were in accord with basic genetic principles concerning the balance between mutation, selection and genetic drift. In particular, attenuation of vertically transmitted viruses was a consequence of the random accumulation of deleterious mutations, whereas horizontally transmitted viruses experiencing similar bottlenecks did not suffer the same fitness losses because effective bottleneck size was actually determined by the number of host individuals. In addition, high levels of viral fitness in horizontally transmitted populations were explained by competition among viral variants.

Cost of host radiation in an RNA virus.

Although host radiation allows a parasite to expand its ecological niche, traits governing the infection of multiple host types can decrease fitness in the original or alternate host environments. Reasons for this reduction in fitness include slower replication due to added genetic material or modifications, fitness trade-offs across host environments, and weaker selection resulting from simultaneous adaptation to multiple habitats. We examined the consequences of host radiation using vesicular stomatitis virus (VSV) and mammalian host cells in tissue culture. Replicate populations of VSV were allowed to evolve for 100 generations on the original host (BHK cells), on either of two novel hosts (HeLa and MDCK cells), or in environments where the availability of novel hosts fluctuated in a predictable or random way. As expected, each experimental population showed a substantial fitness gain in its own environment, but those evolved on new hosts (constant or fluctuating) suffered reduced competitiveness on the original host. However, whereas evolution on one novel host negatively correlated with performance on the unselected novel host, adaptation in fluctuating environments led to fitness improvements in both novel habitats.

Kin selection and parasite evolution: higher and lower virulence with hard and soft selection.

Conventional models predict that low genetic relatedness among parasites that coinfect the same host leads to the evolution of high parasite virulence. Such models assume adaptive responses to hard selection only. We show that if soft selection is allowed to operate, low relatedness leads instead to the evolution of low virulence. With both hard and soft selection, low relatedness increases the conflict among coinfecting parasites. Although parasites can only respond to hard selection by evolving higher virulence and overexploiting their host, they can respond to soft selection by evolving other adaptations, such as interference, that prevent overexploitation. Because interference can entail a cost, the host may actually be underexploited, and virulence will decrease as a result of soft selection. Our analysis also shows that responses to soft selection can have a much stronger effect than responses to hard selection. After hard selection has raised virulence to a level that is an evolutionarily stable strategy, the population, as expected, cannot be invaded by more virulent phenotypes that respond only to hard selection. The population remains susceptible to invasion by a less virulent phenotype that responds to soft selection, however. Thus, hard and soft selection are not just alternatives. Rather, soft selection is expected to prevail and often thwart the evolution of virulence in parasites. We review evidence from several parasite systems and find support for soft selection. Most of the examples involve interference mechanisms that indirectly prevent the evolution of higher virulence. We recognize that hard selection for virulence is more difficult to document, but we take our results to suggest that a kin selection model with soft selection may have general applicability.

The two faces of mutation: extinction and adaptation in RNA viruses.

From a population standpoint, two main features characterize the replication of RNA viruses and viruses that use RNA as a replicative intermediate: high genetic variability, and enormous fluctuations in population size. Their genetic variability mainly reflects a lack of the proof-reading and post-replicative error correction mechanisms that operate during cellular DNA replication, but recombination and segment exchange can also play an important role. Viral population size can change tremendously as a consequence of transmission between hosts or between different tissues within an infected host. A new infection can be initiated with very few particles that subsequently expand many trillion-fold. Repeated bottleneck events can lead to drastic fitness losses or even to viral extinction, whereas continuously large population sizes result in fitness gains and adaptation. Here we review experimental evidence for the effects of mutation, selection, and genetic drift on the adaptation and extinction of RNA viruses.

Prisoner's dilemma in an RNA virus.

The evolution of competitive interactions among viruses was studied in the RNA phage phi6 at high and low multiplicities of infection (that is, at high and low ratios of infecting phage to host cells). At high multiplicities, many phage infect and reproduce in the same host cell, whereas at low multiplicities the viruses reproduce mainly as clones. An unexpected result of this study was that phage grown at high rates of co-infection increased in fitness initially, but then evolved lowered fitness. Here we show that the fitness of the high-multiplicity phage relative to their ancestors generates a pay-off matrix conforming to the prisoner's dilemma strategy of game theory. In this strategy, defection (selfishness) evolves, despite the greater fitness pay-off that would result if all players were to cooperate. Viral cooperation and defection can be defined as, respectively, the manufacturing and sequestering of diffusible (shared) intracellular products. Because the low-multiplicity phage did not evolve lowered fitness, we attribute the evolution of selfishness to the lack of clonal structure and the mixing of unrelated genotypes at high multiplicity.

Hybrid frequencies confirm limit to coinfection in the RNA bacteriophage phi6.

Coinfection of the same host cell by multiple viruses may lead to increased competition for limited cellular resources, thus reducing the fitness of an individual virus. Selection should favor viruses that can limit or prevent coinfection, and it is not surprising that many viruses have evolved mechanisms to do so. Here we explore whether coinfection is limited in the RNA bacteriophage phi6 that infects Pseudomonas phaseolicola. We estimated the limit to coinfection in phi6 by comparing the frequency of hybrids produced by two marked phage strains to that predicted by a mathematical model based on differing limits to coinfection. Our results provide an alternative method for estimating the limit to coinfection and confirm a previous estimate between two to three phages per host cell. In addition, our data reveal that the rate of coinfection at low phage densities may exceed that expected through random Poisson sampling. We discuss whether phage phi6 has evolved an optimal limit that balances the costly and beneficial fitness effects associated with multiple infections.

Sex and the evolution of intrahost competition in RNA virus phi6.

Sex allows beneficial mutations that occur in separate lineages to be fixed in the same genome. For this reason, the Fisher-Muller model predicts that adaptation to the environment is more rapid in a large sexual population than in an equally large asexual population. Sexual reproduction occurs in populations of the RNA virus phi6 when multiple bacteriophages coinfect the same host cell. Here, we tested the model's predictions by determining whether sex favors more rapid adaptation of phi6 to a bacterial host, Pseudomonas phaseolicola. Replicate populations of phi6 were allowed to evolve in either the presence or absence of sex for 250 generations. All experimental populations showed a significant increase in fitness relative to the ancestor, but sex did not increase the rate of adaptation. Rather, we found that the sexual and asexual treatments also differ because intense intrahost competition between viruses occurs during coinfection. Results showed that the derived sexual viruses were selectively favored only when coinfection is common, indicating that within-host competition detracts from the ability of viruses to exploit the host. Thus, sex was not advantageous because the cost created by intrahost competition was too strong. Our findings indicate that high levels of coinfection exceed an optimum where sex may be beneficial to populations of phi6, and suggest that genetic conflicts can evolve in RNA viruses.

Response imagery: aftereffects and reminiscence.

In two experiments, subjects imagined themselves performing a tracking task under a massed practice schedule. After interpolated rest or no rest, subjects actually performed the criterion task. Some subjects' imagery was augmented with sounds that matched the temporal characteristics of the criterion task. These subjects produced greater aftereffects than subjects who imaged without augmentation or subjects provided with imagery augmentation matching a variation of the criterion. Reminiscence (performance gain attributed to interpolated rest) was demonstrated with imagery, except when the accompanying augmentation was faster than the criterion.

Ill health retirement--guidelines for occupational physicians. Association of Local Authority Medical Advisors (working party).

These guidelines have been produced by a working party of the Association of Local Authority Medical Advisors to help doctors arrive at equitable decisions when assessing applications for ill health retirement. The general guidelines are intended to apply to all pension schemes and the specific ones to those such as The Local Government Pension Scheme where there is a requirement for the applicant to have permanent ill health.

Variability in precompetition anxiety and performance in college track and field athletes.

The influence of anxiety on sport performance was examined using the inverted-U hypothesis and Zone of Optimal Function (ZOF) theory. Sixty-seven collegiate track and field athletes completed versions of the State-Trait Anxiety Inventory (STAI) to determine baseline state anxiety, recalled best anxiety, and precompetition anxiety. Precompetition state anxiety was determined 1 h before competition in four meets. Three variants of the inverted-U hypothesis were examined: 1) task-specific anxiety, 2) individualized median precompetition anxiety, and 3) mean precompetition anxiety. For ZOF, optimal anxiety was established from anxiety scores based on recalled best performance. Subjects were grouped as either inside or outside of optimal anxiety levels according to inverted-U or ZOF criteria. Mean group performance was determined by transforming individual results using intra- and interindividual standards. ANOVA revealed that subjects possessing optimal anxiety according to inverted-U criteria performed no better (P > 0.05) than cases outside optimal. Subjects with precompetition anxiety values within their individual ZOF performed significantly (P < 0.05) better than cases with anxiety outside ZOF for all contrasts. The largest (P < 0.05) performance decrements resulted when anxiety was within a 1 SD range above or below ZOF. These results indicate that ZOF theory was more efficacious than the variants of the inverted-U hypothesis examined.

State anxiety and blood pressure following 30 min of leg ergometry or weight training.

The effect of different modes of acute exercise on state anxiety and blood pressure was examined. Collegiate varsity athletes (11 females and 15 males) completed 30-min sessions of leg cycle ergometry or weight training in a randomized order on separate days. Both exercise modes were performed within 70-80% of the maximum capacity of each subject. State anxiety, systolic (SBP) and diastolic (DBP) blood pressure were measured at baseline prior to exercise, immediately post-exercise, 20 min and 60 min following exercise. Repeated-measures ANOVA detected significant (P < 0.05) trial by condition effects for state anxiety and SBP. State anxiety increased (P < 0.001) immediately following weight training but returned to baseline for the remaining assessments. State anxiety decreased (P < 0.05) below baseline at 60 min following ergometry. SBP did not decrease following weight training but was reduced (P < 0.05) by 6.5 mm Hg below baseline at 60 min following ergometry. DBP did not change following either exercise mode. In summary, state anxiety and blood pressure responses to acute physical activity appear to be dependent on the exercise mode, with reductions in state anxiety and systolic blood pressure being associated with ergometry but not weight training.