Intra-host genetic heterogeneity of hepatitis C virus and biomedical implications

The HCV genome exhibits significant intra-host genetic heterogeneity as the result of accumulation of mutations during viral replication. At each point in time during the infection, the viral population is composed of a dominant master sequence and a number of sequences diverging from the master sequence to various extents (the viral quasi-species). The quasispecies is a complex, dynamic distribution of nonidentical, but related, replicons. In these populations, viral variants may undergo very large changes in their fitness (the replicative adaptability of an organism to its environment), including dramatic fitness loss and important fitness gains. The biological impact of this event may theoretically include modifications of tropism, appearance of escape mutants, changes in pathogenic potential, and resistance to antiviral agents. A growing body of molecular and clinical data currently suggests that both inter- and intra-host genetic heterogeneity of HCV have crucial biological and medical implications, influencing not only infection prevention, but also clinical progression of chronic liver disease in persistently infected subjects, HCV infection of non-liver cells, and response to the anti-viral therapy.     

Close kinship within multiple-genotype malaria parasite infections

Malaria infections containing multiple parasite genotypes are ubiquitous in nature, and play a central role in models of recombination, intra-host dynamics, virulence, sex ratio, immunity and drug resistance evolution in Plasmodium. While these multiple infections (MIs) are often assumed to result from superinfection (bites from multiple infected mosquitoes), we know remarkably little about their composition or generation. We isolated 336 parasite clones from eight patients from Malawi (high transmission) and six from Thailand (low transmission) by dilution cloning. These were genotyped using 384 single-nucleotide polymorphisms, revealing 22 independent haplotypes in Malawi (2-6 per MI) and 15 in Thailand (2-5 per MI). Surprisingly, all six patients from Thailand and six of eight from Malawi contained related haplotypes, and haplotypes were more similar within- than between-infections. These results argue against a simple superinfection model. Instead, the observed kinship patterns may be explained by inoculation of multiple related haploid sporozoites from single mosquito bites, by immune suppression of parasite subpopulations within infections, and serial transmission of related parasites between people. That relatedness is maintained in endemic areas in the face of repeated bites from infected mosquitoes has profound implications for understanding malaria transmission, immunity and intra-host dynamics of co-infecting parasite genotypes.     

Microparasite population dynamics and continuous immunity.

A mathematical model is presented for the transmission of a microparasite where the hosts occupy one of two states, uninfected or infected. In each state, the hosts are distributed over a continuous range of immunity. The immune levels vary within hosts due to the processes of waning of immunity (when uninfected), and increasing immunity (when infected), eventually resulting in recovery. Immunity level also influences the host''s ability to infect or be infected. Thus the proposed model incorporates both inter- and intra-host dynamics. It is shown from equilibrium results that this model is a general form of the susceptible-infected-resistant (SIR) and susceptible-infected-susceptible (SIS) family of models, a development that is useful for exploring multistrain pathogen transmission and use of vaccines which confer temporary protection.     

Evaluation of intra-host variants of the entire hepatitis B virus genome

Genetic analysis of hepatitis B virus (HBV) frequently involves study of intra-host variants, identification of which is commonly achieved using short regions of the HBV genome. However, the use of short sequences significantly limits evaluation of genetic relatedness among HBV strains. Although analysis of HBV complete genomes using genetic cloning has been developed, its application is highly labor intensive and practiced only infrequently. We describe here a novel approach to whole genome (WG) HBV quasispecies analysis based on end-point, limiting-dilution real-time PCR (EPLD-PCR) for amplification of single HBV genome variants, and their subsequent sequencing. EPLD-PCR was used to analyze WG quasispecies from serum samples of patients (n = 38) infected with HBV genotypes A, B, C, D, E and G. Phylogenetic analysis of the EPLD-isolated HBV-WG quasispecies showed the presence of mixed genotypes, recombinant variants and sub-populations of the virus. A critical observation was that HBV-WG consensus sequences obtained by direct sequencing of PCR fragments without EPLD are genetically close, but not always identical to the major HBV variants in the intra-host population, thus indicating that consensus sequences should be judiciously used in genetic analysis. Sequence-based studies of HBV WG quasispecies should afford a more accurate assessment of HBV evolution in various clinical and epidemiological settings.     

Adaptive modeling of viral diseases in bats with a focus on rabies

Many emerging and reemerging viruses, such as rabies, SARS, Marburg, and Ebola have bat populations as disease reservoirs. Understanding the spillover from bats to humans and other animals, and the associated health risks requires an analysis of the disease dynamics in bat populations. Traditional compartmental epizootic models, which are relatively easy to implement and analyze, usually impose unrealistic aggregation assumptions about disease-related structure and depend on parameters that frequently are not measurable in field conditions. We propose a novel combination of computational and adaptive modeling approaches that address the maintenance of emerging diseases in bat colonies through individual (intra-host) models of the response of the host to a viral challenge. The dynamics of the individual models are used to define survival, susceptibility and transmission conditions relevant to epizootics as well as to develop and parametrize models of the disease evolution into uniform and diverse populations. Applications of the proposed approach to modeling the effects of immunological heterogeneity on the dynamics of bat rabies are presented.     

The cophylogeny of populations and cultures: reconstructing the evolution of Iranian tribal craft traditions using trees and jungles

Phylogenetic approaches to culture have shed new light on the role played by population dispersals in the spread and diversification of cultural traditions. However, the fact that cultural inheritance is based on separate mechanisms from genetic inheritance means that socially transmitted traditions have the potential to diverge from population histories. Here, we suggest that associations between these two systems can be reconstructed using techniques developed to study cospeciation between hosts and parasites and related problems in biology. Relationships among the latter are patterned by four main processes: co-divergence, intra-host speciation (duplication), intra-host extinction (sorting) and horizontal transfers. We show that patterns of cultural inheritance are structured by analogous processes, and then demonstrate the applicability of the host-parasite model to culture using empirical data on Iranian tribal populations.     

Dynamics of severe acute respiratory syndrome coronavirus 2 genome variants in the feces during convalescence

In response to the current coronavirus disease 2019 (COVID-19) pandemic, it is crucial to understand the origin, transmission, and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which relies on close surveillance of genomic diversity in clinical samples. Although the mutation at the population level had been extensively investigated, how the mutations evolve at the individual level is largely unknown. Eighteen time-series fecal samples were collected from nine patients with COVID-19 during the convalescent phase. The nucleic acids of SARS-CoV-2 were enriched by the hybrid capture method. First, we demonstrated the outstanding performance of the hybrid capture method in detecting intra-host variants. We identified 229 intra-host variants at 182 sites in 18 fecal samples. Among them, nineteen variants presented frequency changes > 0.3 within 1–5 days, reflecting highly dynamic intra-host viral populations. Moreover, the evolution of the viral genome demonstrated that the virus was probably viable in the gastrointestinal tract during the convalescent period. Meanwhile, we also found that the same mutation showed a distinct pattern of frequency changes in different individuals, indicating a strong random drift. In summary, dramatic changes of the SARS-CoV-2 genome were detected in fecal samples during the convalescent period; whether the viral load in feces is sufficient to establish an infection warranted further investigation.     

Identification of forces shaping the commensal Escherichia coli genetic structure by comparing animal and human isolates

To identify forces shaping the Escherichia coli intraspecies ecological structure, we have characterized in terms of phylogenetic group (A, B1, D and B2) belonging, presence/absence of extraintestinal virulence genes (pap, sfa, hly and aer) and intra-host phylotype diversity a collection of 1898 commensal isolates originating from 387 animals (birds and mammals) sampled in the 1980s and the 2000s. These data have been compared with 760 human commensal isolates, sampled from 152 healthy subjects in the 2000s, and analysed with the same approach. The prevalence of the E. coli phylogenetic groups in birds, non-human mammals and humans is clearly different with a predominance of D/B1, A/B1 and A/B2 strains respectively. A major force shaping the ecological structure is the environment with a strong effect of domestication and the year of sampling followed by the climate. Host characteristics, as the diet and body mass, also influence the ecological structure. Human microbiota are characterized by a higher prevalence of virulence genes and a lower intra-host diversity than the non-human mammal ones. This work identifies for the first time a group of strains specific to the animals, the B1 phylogenetic group strains exhibiting the hly gene. In conclusion, a complex network of factors seems to shape the ecological structure of commensal E. coli, with anthropogenic factors playing a major role and perturbing natural niche equilibrium.     

An exploratory algorithm to identify intra-host recombinant viral sequences

Since recombination leads to the generation of mosaic genomes that violate the assumption of traditional phylogenetic methods that sequence evolution can be accurately described by a single tree, results and conclusions based on phylogenetic analysis of data sets including recombinant sequences can be severely misleading. Many methods are able to adequately detect recombination between diverse sequences, for example between different HIV-1 subtypes. More problematic is the identification of recombinants among closely related sequences such as a viral population within a host. We describe a simple algorithmic procedure that enables detection of intra-host recombinants based on split-decomposition networks and a robust statistical test for recombination. By applying this algorithm to several published HIV-1 data sets we conclude that intra-host recombination was significantly underestimated in previous studies and that up to one-third of the env sequences longitudinally sampled from a given subject can be of recombinant origin. The results show that our procedure can be a valuable exploratory tool for detection of recombinant sequences before phylogenetic analysis, and also suggest that HIV-1 recombination in vivo is far more frequent and significant than previously thought.     

High-throughput proteomic analysis of formalin-fixed paraffin-embedded tissue microarrays using MALDI imaging mass spectrometry

A novel method for high-throughput proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue microarrays (TMA) is described using on-tissue tryptic digestion followed by MALDI imaging MS. A TMA section containing 112 needle core biopsies from lung-tumor patients was analyzed using MS and the data were correlated to a serial hematoxylin and eosin (H&E)-stained section having various histological regions marked, including cancer, non-cancer, and normal ones. By correlating each mass spectrum to a defined histological region, statistical classification models were generated that can sufficiently distinguish biopsies from adenocarcinoma from squamous cell carcinoma biopsies. These classification models were built using a training set of biopsies in the TMA and were then validated on the remaining biopsies. Peptide markers of interest were identified directly from the TMA section using MALDI MS/MS sequence analysis. The ability to detect and characterize tumor marker proteins for a large cohort of FFPE samples in a high-throughput approach will be of significant benefit not only to investigators studying tumor biology, but also to clinicians for diagnostic and prognostic purposes.     

Tracking Dengue Virus Intra-host Genetic Diversity during Human-to-Mosquito Transmission

Dengue virus (DENV) infection of an individual human or mosquito host produces a dynamic population of closely-related sequences. This intra-host genetic diversity is thought to offer an advantage for arboviruses to adapt as they cycle between two very different host species, but it remains poorly characterized. To track changes in viral intra-host genetic diversity during horizontal transmission, we infected Aedes aegypti mosquitoes by allowing them to feed on DENV2-infected patients. We then performed whole-genome deep-sequencing of human- and matched mosquito-derived DENV samples on the Illumina platform and used a sensitive variant-caller to detect single nucleotide variants (SNVs) within each sample. >90% of SNVs were lost upon transition from human to mosquito, as well as from mosquito abdomen to salivary glands. Levels of viral diversity were maintained, however, by the regeneration of new SNVs at each stage of transmission. We further show that SNVs maintained across transmission stages were transmitted as a unit of two at maximum, suggesting the presence of numerous variant genomes carrying only one or two SNVs each. We also present evidence for differences in selection pressures between human and mosquito hosts, particularly on the structural and NS1 genes. This analysis provides insights into how population drops during transmission shape RNA virus genetic diversity, has direct implications for virus evolution, and illustrates the value of high-coverage, whole-genome next-generation sequencing for understanding viral intra-host genetic diversity.     

Evolutionary pattern of intra-host pathogen antigenic drift: effect of cross-reactivity in immune response.

Several viruses are known to change their surface antigen types after infecting a host, thereby escaping the immune defence and ensuring persistent infection. In this paper, we theoretically study the pattern of intra-host micro-evolution of pathogen antigen variants under the antigen specific immune response. We assume that the antigen types of the pathogen can be indexed in one-dimensional space, and that a mutation can produce a new antigen variant that is one step distant from the parental type. We also assume that antibodies directed to a specific antigen can also neutralize similar antigen types with a decreased efficiency (cross-reactivity). The model reveals that the pattern of intra-host antigen evolution critically depends on the width of cross-reactivity. If the width of cross-reactivity is narrower than a certain threshold, antigen variants gradually evolve in antigen space as a travelling wave with a constant wave speed, and the total pathogen density approaches a constant. In contrast, if the width of cross-reactivity exceeds the threshold, the travelling wave loses stability and the distribution of antigen variants fluctuates both in time and in genotype space. In the latter case, the expected episodes after infection are a series of intermittent outbreaks of pathogen density, caused by distantly separated antigen types. The implication of the model to intra-host evolution of equine infectious anaemia virus and human immunodeficiency virus is discussed.     

Inter- and Intra-Host Viral Diversity in a Large Seasonal DENV2 Outbreak

Background

High genetic diversity at both inter- and intra-host level are hallmarks of RNA viruses due to the error-prone nature of their genome replication. Several groups have evaluated the extent of viral variability using different RNA virus deep sequencing methods. Although much of this effort has been dedicated to pathogens that cause chronic infections in humans, few studies investigated arthropod-borne, acute viral infections.

Methods and Principal Findings

We deep sequenced the complete genome of ten DENV2 isolates from representative classical and severe cases sampled in a large outbreak in Brazil using two different approaches. Analysis of the consensus genomes confirmed the larger extent of the 2010 epidemic in comparison to a previous epidemic caused by the same viruses in another city two years before (genetic distance = 0.002 and 0.0008 respectively). Analysis of viral populations within the host revealed a high level of conservation. After excluding homopolymer regions of 454/Roche generated sequences, we found 10 to 44 variable sites per genome population at a frequency of >1%, resulting in very low intra-host genetic diversity. While up to 60% of all variable sites at intra-host level were non-synonymous changes, only 10% of inter-host variability resulted from non-synonymous mutations, indicative of purifying selection at the population level.

Conclusions and Significance

Despite the error-prone nature of RNA-dependent RNA-polymerase, dengue viruses maintain low levels of intra-host variability.     

Within-host dynamics of the hepatitis C virus quasispecies population in HIV-1/HCV coinfected patients

HIV/HCV coinfected individuals under highly active antiretroviral therapy (HAART) represent an interesting model for the investigation of the role played by the immune system in driving the evolution of the HCV quasispecies. We prospectively studied the intra-host evolution of the HCV heterogeneity in 8 coinfected subjects, selected from a cohort of 32 patients initiating HAART: 5 immunological responders (group A) and 3 immunological non-responders (group B), and in two HCV singly infected controls not assuming drugs (group C). For all these subjects at least two serial samples obtained at the first observation (before HAART) and more than 1 year later, underwent clonal sequence analysis of partial E1/E2 sequences, encompassing the whole HVR1. Evolutionary rates, dated phylogenies and population dynamics were co-estimated by using a Bayesian Markov Chain Monte Carlo approach, and site specific selection pressures were estimated by maximum likelihood-based methods. The intra-host evolutionary rates of HCV quasispecies was 10 times higher in subjects treated with HAART than in controls without immunodeficiency (1.9 and 2.3 × 10(-3) sub/site/month in group A and B and 0.29 × 10(-3) sub/site/month in group C individuals). The within-host Bayesian Skyline plot analysis showed an exponential growth of the quasispecies populations in immunological responders, coinciding with a peak in CD4 cell counts. On the contrary, quasispecies population remained constant in group B and in group C controls. A significant positive selection pressure was detected in a half of the patients under HAART and in none of the group C controls. Several sites under significant positive selection were described, mainly included in the HVR1. Our data indicate that different forces, in addition to the selection pressure, drive an exceptionally fast evolution of HCV during HAART immune restoration. We hypothesize that an important role is played by the enlargement of the viral replicative space.     

SARS-CoV-2 variants with reduced infectivity and varied sensitivity to the BNT162b2 vaccine are developed during the course of infection

In-depth analysis of SARS-CoV-2 quasispecies is pivotal for a thorough understating of its evolution during infection. The recent deployment of COVID-19 vaccines, which elicit protective anti-spike neutralizing antibodies, has stressed the importance of uncovering and characterizing SARS-CoV-2 variants with mutated spike proteins. Sequencing databases have allowed to follow the spread of SARS-CoV-2 variants that are circulating in the human population, and several experimental platforms were developed to study these variants. However, less is known about the SARS-CoV-2 variants that are developed in the respiratory system of the infected individual. To gain further insight on SARS-CoV-2 mutagenesis during natural infection, we preformed single-genome sequencing of SARS-CoV-2 isolated from nose-throat swabs of infected individuals. Interestingly, intra-host SARS-CoV-2 variants with mutated S genes or N genes were detected in all individuals who were analyzed. These intra-host variants were present in low frequencies in the swab samples and were rarely documented in current sequencing databases. Further examination of representative spike variants identified by our analysis showed that these variants have impaired infectivity capacity and that the mutated variants showed varied sensitivity to neutralization by convalescent plasma and to plasma from vaccinated individuals. Notably, analysis of the plasma neutralization activity against these variants showed that the L1197I mutation at the S2 subunit of the spike can affect the plasma neutralization activity. Together, these results suggest that SARS-CoV-2 intra-host variants should be further analyzed for a more thorough characterization of potential circulating variants.     

Elucidating relationships between P.falciparum prevalence and measures of genetic diversity with a combined genetic-epidemiological model of malaria

There is an abundance of malaria genetic data being collected from the field, yet using these data to understand the drivers of regional epidemiology remains a challenge. A key issue is the lack of models that relate parasite genetic diversity to epidemiological parameters. Classical models in population genetics characterize changes in genetic diversity in relation to demographic parameters, but fail to account for the unique features of the malaria life cycle. In contrast, epidemiological models, such as the Ross-Macdonald model, capture malaria transmission dynamics but do not consider genetics. Here, we have developed an integrated model encompassing both parasite evolution and regional epidemiology. We achieve this by combining the Ross-Macdonald model with an intra-host continuous-time Moran model, thus explicitly representing the evolution of individual parasite genomes in a traditional epidemiological framework. Implemented as a stochastic simulation, we use the model to explore relationships between measures of parasite genetic diversity and parasite prevalence, a widely-used metric of transmission intensity. First, we explore how varying parasite prevalence influences genetic diversity at equilibrium. We find that multiple genetic diversity statistics are correlated with prevalence, but the strength of the relationships depends on whether variation in prevalence is driven by host- or vector-related factors. Next, we assess the responsiveness of a variety of statistics to malaria control interventions, finding that those related to mixed infections respond quickly (∼months) whereas other statistics, such as nucleotide diversity, may take decades to respond. These findings provide insights into the opportunities and challenges associated with using genetic data to monitor malaria epidemiology.     

Sociobiological Control of Plasmid Copy Number in Bacteria

All genes critical for plasmid replication regulation are located on the plasmid rather than on the host chromosome. It is possible therefore that there can be copy-up “cheater” mutants. In spite of this possibility, low copy number plasmids appear to exist stably in host populations. We examined this paradox using a multilevel selection model. Simulations showed that, a slightly higher copy number mutant could out-compete the wild type. Consequently, another mutant with still higher copy number could invade the first invader. However, the realized benefit of increasing intra-host fitness was saturating whereas that of inter-host fitness was exponential. As a result, above a threshold, intra-host selection was overcompensated by inter-host selection and the low copy number wild type plasmid could back invade a very high copy number plasmid. This led to a rock-paper-scissor (RPS) like situation that allowed the coexistence of plasmids with varied copy numbers. Furthermore, another type of cheater that had lost the genes required for conjugation but could hitchhike on a conjugal plasmid, could further reduce the advantage of copy-up mutants. These sociobiological interactions may compliment molecular mechanisms of replication regulation in stabilizing the copy numbers.     

Intra-host mathematical model of chronic wasting disease dynamics in deer (Odocoileus)

Bioassays of native cervid hosts have established the presence of infectious chronic wasting disease (CWD) prions in saliva, blood, urine, and feces of clinically diseased and pre-clinical infected deer. The intra-host trafficking of prions from the time of initial infection to shedding has been less well defined. We created a discrete-time compartmentalized model to simulate the misfolding catalysis, trafficking, and shedding of infectious prions throughout the organ systems of CWD-infected cervids. Using parameter values derived from experimental infections of North American deer (Odocoileus spp.), the exponential-based model predicts prion deposition over time with: 1) nervous tissues containing the highest deposition of prions at 20 months post-infection and 2) excreted fluids containing low levels of prions throughout infection with the highest numbers of prions predicted to be shed in saliva and feces (as high as 10 lethal doses (1.34 × 10²⁹ prions) in 11–15 months). These findings are comparable to prion deposition described in literature as assayed by conventional and ultrasensitive amplification assays. The comparison of our model to published data suggests that highly sensitive assays (sPMCA, RT-QuIC, and bioassay) are appropriate for early prion detection in bodily fluids and secretions. The model provides a view of intra-host prion catalysis leading to pre-clinical shedding and provides a framework for continued development of antemortem diagnostic methods.     

Genetic Relatedness among Hepatitis A Virus Strains Associated with Food-Borne Outbreaks

The genetic characterization of hepatitis A virus (HAV) strains is commonly accomplished by sequencing subgenomic regions, such as the VP1/P2B junction. HAV genome is not extensively variable, thus presenting opportunity for sharing sequences of subgenomic regions among genetically unrelated isolates. The degree of misrepresentation of phylogenetic relationships by subgenomic regions is especially important for tracking transmissions. Here, we analyzed whole-genome (WG) sequences of 101 HAV strains identified from 4 major multi-state, food-borne outbreaks of hepatitis A in the Unites States and from 14 non-outbreak-related HAV strains that shared identical VP1/P2B sequences with the outbreak strains. Although HAV strains with an identical VP1/P2B sequence were specific to each outbreak, WG were different, with genetic diversity reaching 0.31% (mean 0.09%). Evaluation of different subgenomic regions did not identify any other section of the HAV genome that could accurately represent phylogenetic relationships observed using WG sequences. The identification of 2–3 dominant HAV strains in 3 out of 4 outbreaks indicates contamination of the implicated food items with a heterogeneous HAV population. However, analysis of intra-host HAV variants from eight patients involved in one outbreak showed that only a single sequence variant established infection in each patient. Four non-outbreak strains were found closely related to strains from 2 outbreaks, whereas ten were genetically different from the outbreak strains. Thus, accurate tracking of HAV strains can be accomplished using HAV WG sequences, while short subgenomic regions are useful for identification of transmissions only among cases with known epidemiological association.     

Systematic Position of Allium macrostemon Based on nrDNA ITS and cpDNA trnL-F Sequence Data

Allium macrostemon is an important medicinal and edible plant. Its systematic position and taxonmical classification remain controversial to date. To explore this issue, we reconstructed phylogenetic relationships among Amacrostemon and other related taxa using nrDNA ITS and cpDNA trnL F markers. The phylogenetic trees derived from Bayesian inference and maximum parsimony analysis showed that Amacrostemon is monophyletic, and has a close relationship with some species of polyphyletic sections Caerulea and Pallasia instead of sections Codonoprasum and Allium. The including of Amacrostemon within section Allium was not supported by both molecular data and morphological characters of spathe, filaments and ovary. Allium macrostemon should be included in a new section, however further studies using additional samples (especially those from Central Asia) is necessary. In addition, we also provided a discussion on the phylogenetic relationships among four original plants (Amacrostemon, Achinense, Acaeruleum and Aneriniflorum) of Allii Macrostemonis Bulbus and systematic position of partial species of section Pallasia.