Transmission Bottleneck Size Estimation from De Novo Viral Genetic Variation.

Sequencing of viral infections has become increasingly common over the last decade. Deep sequencing data in particular have proven useful in characterizing the roles that genetic drift and natural selection play in shaping within-host viral populations. They have also been used to estimate transmission bottleneck sizes from identified donor-recipient pairs. These bottleneck sizes quantify the number of viral particles that establish genetic lineages in the recipient host and are important to estimate due to their impact on viral evolution. Current approaches for estimating bottleneck sizes exclusively consider the subset of viral sites that are observed as polymorphic in the donor individual. However, these approaches have the potential to substantially underestimate true transmission bottleneck sizes. Here, we present a new statistical approach for instead estimating bottleneck sizes using patterns of viral genetic variation that arise de novo within a recipient individual. Specifically, our approach makes use of the number of clonal viral variants observed in a transmission pair, defined as the number of viral sites that are monomorphic in both the donor and the recipient but carry different alleles. We first test our approach on a simulated dataset and then apply it to both influenza A virus sequence data and SARS-CoV-2 sequence data from identified transmission pairs. Our results confirm the existence of extremely tight transmission bottlenecks for these 2 respiratory viruses.

Standing Genetic Diversity and Transmission Bottleneck Size Drive Adaptation in Bacteriophage Qß.

A critical issue to understanding how populations adapt to new selective pressures is the relative contribution of the initial standing genetic diversity versus that generated de novo. RNA viruses are an excellent model to study this question, as they form highly heterogeneous populations whose genetic diversity can be modulated by factors such as the number of generations, the size of population bottlenecks, or exposure to new environment conditions. In this work, we propagated at nonoptimal temperature (43 °C) two bacteriophage Qß populations differing in their degree of heterogeneity. Deep sequencing analysis showed that, prior to the temperature change, the most heterogeneous population contained some low-frequency mutations that had previously been detected in the consensus sequences of other Qß populations adapted to 43 °C. Evolved populations with origin in this ancestor reached similar growth rates, but the adaptive pathways depended on the frequency of these standing mutations and the transmission bottleneck size. In contrast, the growth rate achieved by populations with origin in the less heterogeneous ancestor did depend on the transmission bottleneck size. The conclusion is that viral diversification in a particular environment may lead to the emergence of mutants capable of accelerating adaptation when the environment changes.

Transmission strategies in a chemosynthetic symbiosis: detection and quantification of symbionts in host tissues and their environment.

Transmission of bacteria vertically through host tissues ensures offspring acquire symbionts; however, horizontal transmission is an effective strategy for many associations and plays a role in some vertically transmitted symbioses. The bivalve Solemya velum and its gammaproteobacterial chemosynthetic symbionts exhibit evolutionary evidence of both transmission modes, but the dominant strategy on an ecological time scale is unknown. To address this, a specific primer set was developed and validated for the S. velum symbiont using a novel workflow called specific marker design (SMD). Symbionts were quantified in spawned eggs and sediment and seawater samples from S. velum habitats with qPCR. Each egg was estimated to contain 50-100 symbiont genomes. By contrast, symbiont DNA was found at low abundance/occurrence in sediment and seawater, often co-occurring with host mitochondrial DNA, obscuring its origin. To ascertain when eggs become infected, histological sections of S. velum tissues were labelled for symbiont 16S rRNA via in situ hybridization. This revealed symbionts in the ovary walls and mature oocytes, suggesting association in late oogenesis. These data support the hypothesis that S. velum symbionts are vertically transmitted every host generation, thus genetic signatures of horizontal transmission are driven by ecologically infrequent events. This knowledge furthers our understanding of vertical and horizontal mode integration and provides insights across animal-bacterial chemosynthetic symbioses.

Mother-to-Child HIV Transmission Bottleneck Selects for Consensus Virus with Lower Gag-Protease-Driven Replication Capacity.

In the large majority of cases, HIV infection is established by a single variant, and understanding the characteristics of successfully transmitted variants is relevant to prevention strategies. Few studies have investigated the viral determinants of mother-to-child transmission. To determine the impact of Gag-protease-driven viral replication capacity on mother-to-child transmission, the replication capacities of 148 recombinant viruses encoding plasma-derived Gag-protease from 53 nontransmitter mothers, 48 transmitter mothers, and 47 infected infants were assayed in an HIV-1-inducible green fluorescent protein reporter cell line. All study participants were infected with HIV-1 subtype C. There was no significant difference in replication capacities between the nontransmitter (n = 53) and transmitter (n = 44) mothers (P = 0.48). Infant-derived Gag-protease NL4-3 recombinant viruses (n = 41) were found to have a significantly lower Gag-protease-driven replication capacity than that of viruses derived from the mothers (P < 0.0001 by a paired t test). High percent similarities to consensus subtype C Gag, p17, p24, and protease sequences were also found in the infants (n = 28) in comparison to their mothers (P = 0.07, P = 0.002, P = 0.03, and P = 0.02, respectively, as determined by a paired t test). These data suggest that of the viral quasispecies found in mothers, the HIV mother-to-child transmission bottleneck favors the transmission of consensus-like viruses with lower viral replication capacities.IMPORTANCE Understanding the characteristics of successfully transmitted HIV variants has important implications for preventative interventions. Little is known about the viral determinants of HIV mother-to-child transmission (MTCT). We addressed the role of viral replication capacity driven by Gag, a major structural protein that is a significant determinant of overall viral replicative ability and an important target of the host immune response, in the MTCT bottleneck. This study advances our understanding of the genetic bottleneck in MTCT by revealing that viruses transmitted to infants have a lower replicative ability as well as a higher similarity to the population consensus (in this case HIV subtype C) than those of their mothers. Furthermore, the observation that "consensus-like" virus sequences correspond to lower in vitro replication abilities yet appear to be preferentially transmitted suggests that viral characteristics favoring transmission are decoupled from those that enhance replicative capacity.

Higher risk of incident hepatitis C virus coinfection among men who have sex with men, in whom the HIV genetic bottleneck at transmission was wide.

BACKGROUND: High-risk sexual behaviors have been suggested as drivers of the recent dramatic increase of sexually transmitted hepatitis C virus (HCV) among human immunodeficiency virus (HIV)-infected men who have sex with men (MSM). METHODS: We assessed the association between the genetic bottleneck of HIV at transmission and the prevalence and incidence of HCV coinfection in HIV-infected MSM from the Swiss HIV Cohort Study (SHCS). As a proxy for the width of the transmission bottleneck, we used the fraction of ambiguous nucleotides detected by genotypic resistance tests sampled during early HIV infection. We defined a broad bottleneck as a fraction of ambiguous nucleotides exceeding a previously established threshold (0.5%). RESULTS: From the SHCS, we identified 671 MSM with available results of HCV serologic tests and with an HIV genotypic resistance test performed during early HIV infection. Of those, 161 (24.0%) exhibited a broad HIV transmission bottleneck, 38 (5.7%) had at least 1 positive HCV test result, and 26 (3.9%) had an incident HCV infection. Individuals with broad HIV transmission bottlenecks exhibited a 2-fold higher odds of having ever experienced an HCV coinfection (odds ratio, 2.2 [95% confidence interval {CI}, 1.1-4.3]) and a 3-fold higher hazard of having an incident HCV infection (hazard ratio, 3.0 [95% CI, 1.4-6.6]) than individuals with narrow HIV transmission bottlenecks. CONCLUSIONS: Our results indicate that the currently occurring sexual spread of HCV is focused on MSM who are prone to exhibit broad HIV transmission bottlenecks. This is consistent with an important role of high-risk behavior and mucosal barrier impairment in the transmission of HCV among MSM.

Enforcing Temporal Consistency in Migration History Inference.

In addition to undergoing evolution, members of biological populations may also migrate between locations. Examples include the spread of tumor cells from the primary tumor to distant metastases or the spread of pathogens from one host to another. One may represent migration histories by assigning a location label to each vertex of a given phylogenetic tree such that an edge connecting vertices with distinct locations represents a migration. Some biological populations undergo comigration, a phenomenon where multiple taxa from distinct lineages simultaneously comigrate from one location to another. In this work, we show that a previous problem statement for inferring migration histories that are parsimonious in terms of migrations and comigrations may lead to temporally inconsistent solutions. To remedy this deficiency, we introduce precise definitions of temporal consistency of comigrations in a phylogenetic tree, leading to three successive problems. First, we formulate the temporally consistent comigration problem to check if a set of comigrations is temporally consistent and provide a linear time algorithm for solving this problem. Second, we formulate the parsimonious consistent comigrations (PCC) problem, which aims to find comigrations given a location labeling of a phylogenetic tree. We show that PCC is NP-hard. Third, we formulate the parsimonious consistent comigration history (PCCH) problem, which infers the migration history given a phylogenetic tree and locations of its extant vertices only. We show that PCCH is NP-hard as well. On the positive side, we propose integer linear programming models to solve the PCC and PCCH problems. We demonstrate our algorithms on simulated and real data.

Barcoded SARS-CoV-2 viruses define the impact of time and route of transmission on the transmission bottleneck in a Syrian hamster model.

The transmission bottleneck, defined as the number of viruses that transmit from one host to infect another, is an important determinant of the rate of virus evolution and the level of immunity required to protect against virus transmission. Despite its importance, SARS-CoV-2's transmission bottleneck remains poorly characterized, in part due to a lack of quantitative measurement tools. To address this, we adapted a SARS-CoV-2 reverse genetics system to generate a pool of >200 isogenic SARS-CoV-2 viruses harboring specific 6-nucleotide barcodes inserted in ORF10, a non-translated ORF. We directly inoculated donor Syrian hamsters intranasally with this barcoded virus pool and exposed a paired naïve contact hamster to each donor. Following exposure, the nasal turbinates, trachea, and lungs were collected, viral titers were measured, and the number of barcodes in each tissue were enumerated to quantify the transmission bottleneck. The duration and route (airborne, direct contact, and fomite) of exposure were varied to assess their impact on the transmission bottleneck. In airborne-exposed hamsters, the transmission bottleneck increased with longer exposure durations. We found that direct contact exposure produced the largest transmission bottleneck (average 27 BCs), followed by airborne exposure (average 16 BCs) then fomite exposure (average 8 BCs). Interestingly, we detected unique BCs in both the upper and lower respiratory tract of contact animals from all routes of exposure, suggesting that SARS-CoV-2 can directly infect hamster lungs. Altogether, these findings highlight the utility of barcoded viruses as tools to rigorously study virus transmission. In the future, barcoded SARS-CoV-2 will strengthen studies of immune factors that influence virus transmission.

Re-evaluating the mosquito RNAi pathway's influence on arbovirus transmission.

With new RNA interference (RNAi) pathway-deficient mosquitoes, Merkling et al. invite a shift in our view of this pathway's role in regulating arthropod-borne virus (arbovirus) transmission. While loss of RNAi function enhanced early viral replication, little impact on transmission was observed, inviting renewed exploration of molecular barriers to arbovirus transmission.

Intra- and inter-host evolution of H9N2 influenza A virus in Japanese quail.

Influenza A viruses (IAVs) are constantly evolving. Crucial steps in the infection cycle, such as sialic acid (SA) receptor binding on the host cell surface, can either promote or hamper the emergence of new variants. We previously assessed the relative fitness in Japanese quail of H9N2 variant viruses differing at a single amino acid position, residue 216 in the hemagglutinin (HA) viral surface protein. This site is known to modulate SA recognition. Our prior study generated a valuable set of longitudinal samples from quail transmission groups where the inoculum comprised different mixed populations of HA 216 variant viruses. Here, we leveraged these samples to examine the evolutionary dynamics of viral populations within and between inoculated and naïve contact quails. We found that positive selection dominated HA gene evolution, but fixation of the fittest variant depended on the competition mixture. Analysis of the whole genome revealed further evidence of positive selection acting both within and between hosts. Positive selection drove fixation of variants in non-HA segments within inoculated and contact quails. Importantly, transmission bottlenecks were modulated by the molecular signature at HA 216, revealing viral receptor usage as a determinant of transmitted diversity. Overall, we show that selection strongly shaped the evolutionary dynamics within and between quails. These findings support the notion that selective processes act effectively on IAV populations in poultry hosts, facilitating rapid viral evolution in this ecological niche.

Narrow transmission bottlenecks and limited within-host viral diversity during a SARS-CoV-2 outbreak on a fishing boat.

The long-term evolution of viruses is ultimately due to viral mutants that arise within infected individuals and transmit to other individuals. Here, we use deep sequencing to investigate the transmission of viral genetic variation among individuals during a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak that infected the vast majority of crew members on a fishing boat. We deep-sequenced nasal swabs to characterize the within-host viral population of infected crew members, using experimental duplicates and strict computational filters to ensure accurate variant calling. We find that within-host viral diversity is low in infected crew members. The mutations that did fix in some crew members during the outbreak are not observed at detectable frequencies in any of the sampled crew members in which they are not fixed, suggesting that viral evolution involves occasional fixation of low-frequency mutations during transmission rather than persistent maintenance of within-host viral diversity. Overall, our results show that strong transmission bottlenecks dominate viral evolution even during a superspreading event with a very high attack rate.

Limited genomic reconstruction of SARS-CoV-2 transmission history within local epidemiological clusters.

A detailed understanding of how and when severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission occurs is crucial for designing effective prevention measures. Other than contact tracing, genome sequencing provides information to help infer who infected whom. However, the effectiveness of the genomic approach in this context depends on both (high enough) mutation and (low enough) transmission rates. Today, the level of resolution that we can obtain when describing SARS-CoV-2 outbreaks using just genomic information alone remains unclear. In order to answer this question, we sequenced forty-nine SARS-CoV-2 patient samples from ten local clusters in NW Spain for which partial epidemiological information was available and inferred transmission history using genomic variants. Importantly, we obtained high-quality genomic data, sequencing each sample twice and using unique barcodes to exclude cross-sample contamination. Phylogenetic and cluster analyses showed that consensus genomes were generally sufficient to discriminate among independent transmission clusters. However, levels of intrahost variation were low, which prevented in most cases the unambiguous identification of direct transmission events. After filtering out recurrent variants across clusters, the genomic data were generally compatible with the epidemiological information but did not support specific transmission events over possible alternatives. We estimated the effective transmission bottleneck size to be one to two viral particles for sample pairs whose donor-recipient relationship was likely. Our analyses suggest that intrahost genomic variation in SARS-CoV-2 might be generally limited and that homoplasy and recurrent errors complicate identifying shared intrahost variants. Reliable reconstruction of direct SARS-CoV-2 transmission based solely on genomic data seems hindered by a slow mutation rate, potential convergent events, and technical artifacts. Detailed contact tracing seems essential in most cases to study SARS-CoV-2 transmission at high resolution.

Direct transmission of within-host Mycobacterium tuberculosis diversity to secondary cases can lead to variable between-host heterogeneity without de novo mutation: A genomic investigation.

BACKGROUND: Whole genome sequencing (WGS) has enabled the development of new approaches to track Mycobacterium tuberculosis (Mtb) transmission between tuberculosis (TB) cases but its utility may be challenged by the discovery that Mtb diversifies within hosts. Nevertheless, there is limited data on the presence and degree of within-host evolution. METHODS: We profiled a well-documented Mtb transmission cluster with three pulmonary TB cases to investigate within-host evolution and describe its impact on recent transmission estimates. We used deep sequencing to track minority allele frequencies (<50·0% abundance) during transmission and standard treatment. FINDINGS: Pre-treatment (n = 3) and serial samples collected over 2 months of antibiotic treatment (n = 16) from all three cases were analysed. Consistent with the epidemiological data, zero fixed SNP separated all genomes. However, we identified six subclones between the three cases with an allele frequency ranging from 35·0% to 100·0% across sampling intervals. Five subclones were identified within the index case pre-treatment and shared with one secondary case, while only the dominant clone was observed in the other secondary case. By tracking the frequency of these heterogeneous alleles over the two-month therapy, we observed distinct signatures of drift and negative selection, but limited evidence for de novo mutations, even under drug pressure. INTERPRETATION: We document within-host Mtb diversity in an index case, which led to transmission of minority alleles to a secondary case. Incorporating data on heterogeneous alleles may refine our understanding of Mtb transmission dynamics. However, more evidence is needed on the role of transmission bottleneck on observed heterogeneity between cases.

High-resolution Genomic Surveillance of 2014 Ebolavirus Using Shared Subclonal Variants.

BACKGROUND: Viral outbreaks, such as the 2014 ebolavirus, can spread rapidly and have complex evolutionary dynamics, including coinfection and bulk transmission of multiple viral populations. Genomic surveillance can be hindered when the spread of the outbreak exceeds the evolutionary rate, in which case consensus approaches will have limited resolution. Deep sequencing of infected patients can identify genomic variants present in intrahost populations at subclonal frequencies (i.e. <50%). Shared subclonal variants (SSVs) can provide additional phylogenetic resolution and inform about disease transmission patterns. METHODS: We use metrics from population genetics to analyze data from the 2014 ebolavirus outbreak in Sierra Leone and identify phylogenetic signal arising from SSVs. We use methods derived from information theory to measure a lower bound on transmission bottleneck size. RESULTS AND CONCLUSIONS: We identify several SSV that shed light on phylogenetic relationships not captured by consensus-based analyses. We find that transmission bottleneck size is larger than one founder population, yet significantly smaller than the intrahost effective population. Our results demonstrate the important role of shared subclonal variants in genomic surveillance.