An evolution-based high-fidelity method of epistasis measurement: Theory and application to influenza

Linkage effects in a multi-locus population strongly influence its evolution. The models based on the traveling wave approach enable us to predict the average speed of evolution and the statistics of phylogeny. However, predicting statistically the evolution of specific sites and pairs of sites in the multi-locus context remains a mathematical challenge. In particular, the effects of epistasis, the interaction of gene regions contributing to phenotype, is difficult to predict theoretically and detect experimentally in sequence data. A large number of false-positive interactions arises from stochastic linkage effects and indirect interactions, which mask true epistatic interactions. Here we develop a proof-of-principle method to filter out false-positive interactions. We start by demonstrating that the averaging of haplotype frequencies over multiple independent populations is necessary but not sufficient for epistatic detection, because it still leaves high numbers of false-positive interactions. To compensate for the residual stochastic noise, we develop a three-way haplotype method isolating true interactions. The fidelity of the method is confirmed analytically and on simulated genetic sequences evolved with a known epistatic network. The method is then applied to a large sequence database of neurominidase protein of influenza A H1N1 obtained from various geographic locations to infer the epistatic network responsible for the difference between the pre-pandemic virus and the pandemic strain of 2009. These results present a simple and reliable technique to measure epistatic interactions of any sign from sequence data.     

QSAR modeling and transmission electron microscopy stereology of altered mitochondrial ultrastructure of white blood cells in patients diagnosed as schizophrenic and treated with antipsychotic drugs.

Treatment of patients diagnosed as schizophrenic with antipsychotic drugs (neuroleptics) is known to cause occasional unexplained depletion of white blood cells, especially neutrophil granulocytes. It has been known for many years that neuroleptics can interfere with the mitochondrial respiratory chain in vitro. Because there has been a growing interest recently in mitochondrial targeting of drugs, and since a quantitative structure-activity relationship (QSAR) model that predicts mitochondrial accumulation of neuroleptics has been published, we investigated the effects of neuroleptics on white blood cell mitochondria. Venous blood samples were collected from both patients undergoing treatment with neuroleptics and healthy volunteers. The samples were processed for transmission electron microscopy. The resulting images of white blood cells were analyzed using stereology to compare quantitatively mitochondrial morphology in the patient and control groups. We found that in patients, but not in controls, there was swelling of mitochondria and fragmentation of the mitochondrial cristae. There also were fewer mitochondria in patients than in controls, although due to the swelling of the organelles, the volume density of mitochondria in the two groups was not significantly different. Such changes are typical of a toxic insult. Consequently, it seems plausible that, since schizophrenia is not a disease considered to affect white blood cells per se, these changes probably are due to the medication.     

Increasing sequence correlation limits the efficiency of recombination in a multisite evolution model

The accumulation of preexisting beneficial alleles in a haploid population, under selection and infrequent recombination, and in the absence of new mutation events is studied numerically by means of detailed Monte Carlo simulations. On the one hand, we confirm our previous work, in that the accumulation rate follows modified single-site kinetics, with a timescale set by an effective selection coefficient s(eff) as shown in a previous work, and we confirm the qualitative features of the dependence of s(eff) on the population size and the recombination rate reported therein. In particular, we confirm the existence of a threshold population size below which evolution stops before the emergence of best-fit individuals. On the other hand, our simulations reveal that the population dynamics is essentially shaped by the steady accumulation of pairwise sequence correlation, causing sequence congruence in excess of what one would expect from a uniformly random distribution of alleles. By sequence congruence, we understand here the opposite of genetic distance, that is, the fraction of monomorphic sites of specified allele type in a pair of genomes (individual sequences). The effective selection coefficient changes more rapidly with the recombination rate and has a higher threshold in this parameter than found in the previous work, which neglected correlation effects. We examine this phenomenon by monitoring the time dependence of sequence correlation based on a set of sequence congruence measures and verify that it is not associated with the development of linkage disequilibrium. We also discuss applications to HIV evolution in infected individuals and potential implications for drug therapy.     

Evolution of human immunodeficiency virus under selection and weak recombination

To predict emergence of drug resistance in patients undergoing antiretroviral therapy, we study accumulation of preexisting beneficial alleles in a haploid population of N genomes. The factors included in the model are selection with the coefficient s and recombination with the small rate per genome r (r < s sqrt of k, where k is the average number of less-fit loci per genome). Mutation events are neglected. To describe evolution at a large number of linked loci, we generalize the analytic method we developed recently for an asexual population. We show that the distribution of genomes over the deleterious allele number moves in time as a "solitary wave" that is quasi-deterministic in the middle (on the average) but has stochastic edges. We arrive at a single-locus expression for the average accumulation rate, in which the effects of linkage, recombination, and random drift are all accounted for by the effective selection coefficient s lnNr/lnNs(2)k/r. At large N, the effective selection coefficient approaches the single-locus value s. Below the critical size N(c) approximately 1/r, a population eventually becomes a clone, recombination cannot produce new sequences, and virus evolution stops. Taking into account finite mutation rate predicts a small, finite rate of evolution at N < N(c). We verify the accuracy of the results analytically and by Monte Carlo simulation. On the basis of our findings, we predict that partial depletion of the HIV population by combined anti-retroviral therapy can suppress emergence of drug-resistant strains.     

Rapid adaptive amplification of preexisting variation in an RNA virus

The amount and nature of preexisting variation in a population of RNA viruses is an important determinant of the virus's ability to adapt rapidly to a changed environment. However, direct quantification of this preexisting variation may be cumbersome, because potentially beneficial alleles are typically rare, and isolation of a large number of subclones is required. Here, we propose a simpler method. We infer the initial population structure of vesicular stomatitis virus (VSV) by fitting a mathematical model of asexual evolution to an extensive set of measurements of VSV fitness dynamics under various conditions, including new and previously published data. The inferred variation of fitness in the initial population agrees very well with the results of direct experiments with subclone fitness quantification. From the same procedure, we also estimate the mean fitness effect of beneficial mutations (selection coefficient s), the percentage of sites in the genome that are under moderate positive or negative selection, and the percentage of sites where beneficial mutations may potentially occur. For VSV strain MARM U evolving in BHK-21 cells, the three parameters have values of 0.39, 9%, and 0.06%, respectively. The method can be generalized and applied easily to other rapidly evolving microbes, including both asexual microorganisms and those with recombination.     

QSAR modeling and transmission electron microscopy stereology of altered mitochondrial ultrastructure of white blood cells in patients diagnosed as schizophrenic and treated with antipsychotic drugs.

Treatment of patients diagnosed as schizophrenic with antipsychotic drugs (neuroleptics) is known to cause occasional unexplained depletion of white blood cells, especially neutrophil granulocytes. It has been known for many years that neuroleptics can interfere with the mitochondrial respiratory chain in vitro. Because there has been a growing interest recently in mitochondrial targeting of drugs, and since a quantitative structure-activity relationship (QSAR) model that predicts mitochondrial accumulation of neuroleptics has been published, we investigated the effects of neuroleptics on white blood cell mitochondria. Venous blood samples were collected from both patients undergoing treatment with neuroleptics and healthy volunteers. The samples were processed for transmission electron microscopy. The resulting images of white blood cells were analyzed using stereology to compare quantitatively mitochondrial morphology in the patient and control groups. We found that in patients, but not in controls, there was swelling of mitochondria and fragmentation of the mitochondrial cristae. There also were fewer mitochondria in patients than in controls, although due to the swelling of the organelles, the volume density of mitochondria in the two groups was not significantly different. Such changes are typical of a toxic insult. Consequently, it seems plausible that, since schizophrenia is not a disease considered to affect white blood cells per se, these changes probably are due to the medication.     

Transition between Stochastic Evolution and Deterministic Evolution in the Presence of Selection: General Theory and Application to Virology

We present here a self-contained analytic review of the role of stochastic factors acting on a virus population. We develop a simple one-locus, two-allele model of a haploid population of constant size including the factors of random drift, purifying selection, and random mutation. We consider different virological experiments: accumulation and reversion of deleterious mutations, competition between mutant and wild-type viruses, gene fixation, mutation frequencies at the steady state, divergence of two populations split from one population, and genetic turnover within a single population. In the first part of the review, we present all principal results in qualitative terms and illustrate them with examples obtained by computer simulation. In the second part, we derive the results formally from a diffusion equation of the Wright-Fisher type and boundary conditions, all derived from the first principles for the virus population model. We show that the leading factors and observable behavior of evolution differ significantly in three broad intervals of population size, N. The “neutral limit” is reached when N is smaller than the inverse selection coefficient. When N is larger than the inverse mutation rate per base, selection dominates and evolution is “almost” deterministic. If the selection coefficient is much larger than the mutation rate, there exists a broad interval of population sizes, in which weakly diverse populations are almost neutral while highly diverse populations are controlled by selection pressure. We discuss in detail the application of our results to human immunodeficiency virus population in vivo, sampling effects, and limitations of the model.     

RAPID SCREENING FOR SALMONELLA IN FISHMEAL BY ENRICHMENT-IMMUNOASSAY

We previously described an enrichment-immunoassay utilizing a T6 monoclonal antibody capture enzyme-linked immunosorbent assay. Here we evaluated it for the rapid screening for Salmonella in fishmeal obtained from the national Animal and Plant Quarantine service in the People's Republic of China. In this method, the number of Salmonella present is first expanded by appropriate enrichment cultures, and the pathogens are then directly detected by the T6 immunoassay. In a total of 94 enrichment cultures of fishmeal, we obtained an overall concordance of 98% between the results obtained in parallel by this method and by conventional test method. The positive prediction by this method was 92% and the negative prediction was 100%. The turn around time for the new test was 27 h which is a significant improvement from the turn around time exceeding 96 h required for the conventional test method. This test proved to be compatible with the routine work flow in the practical setting of a quarantine laboratory.