Biology subjects

Vignuzzi, M.

Publications and source records attributed to Vignuzzi, M..

18 recordsLinked to original sources

bioRxiv2025

Longitudinal analysis of influenza A virus deletion-containing viral genomes reveals key determinants of co-evolutionary dynamics and interference

The substantial genetic diversity generated during influenza A virus replication facilitates both evasion of pre-existing host immunity and cross-species emergence. A major contributor to this diversity is the ubiquitous production of deletion-containing viral genomes (DelVGs) - viral RNAs with large internal deletions that arise during replication. DelVGs directly compete with wild-type (WT) genomes, and their accumulation has been implicated in modulating disease severity. However, the specific functional and genetic interactions between DelVGs and WT genomes remain poorly understood. To examine how DelVGs and WT genomes may co-evolve in mixed populations, we serially passaged influenza A virus under sustained high multiplicity-of-infection (MOI) conditions and used next-generation sequencing to build a longitudinal profile of DelVG emergence and dynamics. Early passages yielded a highly diverse repertoire of DelVGs across multiple segments, followed by a sharp contraction in overall diversity and the rise of one to two PB2-derived DelVGs that persisted at sustained high frequency. We identified a single-nucleotide substitution within PB2-derived DelVGs that significantly enhanced their replication and interference, whereas the same mutation proved lethal in the WT background. Collectively, these findings indicate that DelVGs are not mere byproducts of replication but dynamic genomic elements shaped by selection; their capacity to acquire adaptive mutations and outcompete WT genomes underscores their active role in shaping within-population viral dynamics.

microbiology↗

bioRxiv2024

Fitness adaptations of Japanese encephalitis virus in pigs following vector-free serial passaging

Japanese encephalitis virus (JEV) is a zoonotic mosquito-transmitted Flavivirus circulating in birds and pigs. In humans, JEV can cause severe viral encephalitis with high mortality. Considering that vector-free direct virus transmission was observed in pigs, JEV introduction into an immunologically naive pig population could result in a series of direct transmissions disrupting the alternating host cycling between vertebrates and mosquitoes. To assess the potential consequences of such a realistic scenario, we passaged JEV ten times in pigs. This resulted in higher in vivo viral replication, increased shedding, and stronger innate immune responses in pigs. Nevertheless, the viral tissue tropism remained similar and frequency of direct transmission was not enhanced. Next generation sequencing showed single nucleotide deviations in 10% of the genome during passaging. In total, 25 point mutations were selected to reach a frequency of at least 35% in one of the passages. From these, six mutations resulted in amino acid changes located in the precursor of membrane, the envelope, the non-structural 3 and the non-structural 5 proteins. In a competition experiment with two lines of passaging, the mutation M374L in the envelope protein and N275D in the non-structural protein 5 showed a fitness advantage in pigs. Altogether, the interruption of the alternating host cycle of JEV caused a prominent selection of viral quasispecies as well as selection of de novo mutations associated with fitness gains in pigs, albeit without enhancing direct transmission frequency. Author summaryJapanese encephalitis virus (JEV) represents a major health threat in parts of Asia and Oceania. Primary vertebrate hosts are birds and pigs, but human infection also occurs and can cause severe encephalitis with high mortality. Like other Flaviviruses transmitted by insect bites, JEV requires replication in alternating cycles between mosquitoes on one side and birds or pigs on the other side. However, we previously reported that direct transmissions between pigs in absence of mosquitos can occur. Considering the increased risks for such events after the spread of JEV to a new region with immunologically naive pigs, the present study was performed to understand if and how a series of direct transmissions would promote JEV adaptations to pigs and change virus-host interactions. Pigs infected with JEV passaged ten times showed enhanced clinical symptoms and stronger antiviral immune response, but luckily no increase in direct transmission was observed. Nevertheless, genomic analysis demonstrated a complete change in dominant virus variants, as well as selection of six viral amino acid changes. This indicates that interruptions of the alternating lifestyle of JEV causes a strong evolutionary pressure, which through fitness adaptations can change the viral characteristics.

microbiology↗

bioRxiv2022

IRF8 regulates efficacy of therapeutic anti-CD20 monoclonal antibodies

Anti-CD20 monoclonal antibodies such as Rituximab, Ofatumumab and Obinutuzumab are widely used to treat lymphomas and autoimmune diseases. They act by depleting B cells, mainly through Fc-dependent effectors functions. Some patients develop resistance to treatment but the underlying mechanisms are poorly understood. Here, we performed a genome-wide CRISPR/Cas9 screen to identify genes regulating the efficacy of anti-CD20 antibodies. We used as a model the killing of RAJI B cells by Rituximab through complement-dependent-cytotoxicity (CDC). As expected, the screen identified MS4A1, encoding CD20, the target of Rituximab. Among other identified genes, the role of Interferon Regulatory Factor 8 (IRF8) was validated in two B-cell lines. IRF8 knockout also decreased the efficacy of antibody-dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP) induced by anti-CD20 antibodies. We further show that IRF8 is necessary for efficient CD20 transcription. Levels of IRF8 and CD20 RNA or proteins correlated in normal B cells and in hundreds of malignant B cells. Therefore, IRF8 regulates CD20 expression and controls the depleting-capacity of anti-CD20 antibodies. Our results bring novel insights into the pathways underlying resistance to CD20-targeting immunotherapies.

immunology↗

bioRxiv2022

The E2 glycoprotein holds key residues for Mayaro virus adaptation to the urban Aedes aegypti mosquito

Adaptation to mosquito vectors suited for transmission in urban settings is a major driver in the emergence of arboviruses. To better anticipate future emergence events, it is crucial to assess their potential to adapt to new vector hosts. In this work, we used two different experimental evolution approaches to study the adaptation process of an emerging alphavirus, Mayaro virus (MAYV), to Aedes aegypti, an urban mosquito vector of many arboviruses. We identified E2-T179N as a key mutation increasing MAYV replication in insect cells and enhancing transmission by live Aedes aegypti. In contrast, this mutation decreased viral replication and binding in human fibroblasts, a primary cellular target of MAYV in humans. We also showed that MAYV E2-T179N was attenuated in vivo in a mouse model. We then used structural and experimental analyses to show that MAYV E2-T179N bound less efficiently to human cells, though the decrease in replication or binding was not mediated through interaction with one of the host receptors, Mxra8. When this mutation was introduced in the closely related chikungunya virus, which has caused major outbreaks globally in the past two decades, we observed increased replication in both human and insect cells, suggesting E2 position 179 is an important determinant of alphavirus host-adaptation, although in a virus-specific manner. Collectively, these results indicate that adaptation at the T179 residue in MAYV E2 may result in increased vector competence - but coming at the cost of optimal replication in humans - and may represent a first step towards a future emergence event. Author SummaryMosquito-borne viruses must replicate in both mosquito and vertebrate hosts to be maintained in nature successfully. When viruses that are typically transmitted by forest dwelling mosquitoes enter urban environments due to deforestation or travel, they must adapt to urban mosquito vectors to transmit effectively. For mosquito-borne viruses, the need to also replicate in a vertebrate host like humans constrains this adaptation process. Towards understanding how the emerging alphavirus, Mayaro virus, might adapt to transmission by the urban mosquito vector, Aedes aegypti, we used natural evolution approaches to identify several viral mutations that impacted replication in both mosquito and vertebrate hosts. We show that a single mutation in the receptor binding protein increased transmission by Aedes aegypti but simultaneously reduced replication and disease in a mouse model, suggesting that this mutation alone is unlikely to be maintained in a natural transmission cycle between mosquitoes and humans. Understanding the adaptive potential of emerging viruses is critical to preventing future pandemics.

microbiology↗

bioRxiv2022

Relevance study of vector competence and insecticide resistance in Aedes aegypti laboratory lines

The urban mosquito species Aedes aegypti is the main vector of arboviruses worldwide. Mosquito control with insecticides is the most prevalent method for preventing transmission in the absence of effective vaccines and available treatments; however, the extensive use of insecticides has led to the development of resistance in mosquito populations throughout the world, and the number of epidemics caused by arboviruses has increased. Three mosquito lines with different resistance profiles to deltamethrin were isolated in French Guiana, including one with the I1016 knock-down resistant allele. Significant differences were observed in the cumulative proportion of mosquitoes with a disseminated chikungunya virus infection over time. In addition, certain genes (CYP6BB2, CYP6N12, GST2, trypsin) were variably overexpressed in the midgut at 7 days after an infectious blood meal in these three lines. Therefore, detoxification enzymes and kdr mutations may contribute to an enhanced midgut barrier and reduced dissemination rate. Our work shows that vector competence for chikungunya virus varied between Ae. aegypti laboratory lines with different deltamethrin-resistance profiles. More accurate verification of the functional association between insecticide resistance and vector competence remains to be demonstrated. ImportanceThree Ae. aegypti lines, isolated from the same collection site, underwent different insecticide selection pressures against deltamethrin under laboratory conditions. As a result, they developed different resistant profiles. In this study, when these lines were fed an artificial infectious blood meal containing chikungunya virus, all three lines including the reference strain showed a high infection rate. There was no statistical difference in infection rate found; however, the dissemination rate of the virus from midgut to head were significantly different. A higher resistance level detected by the WHO test was correlated with a lower viral dissemination rate for each strain. This study presented evidence that the insecticide selection pressure or the existence of insecticide resistance could lead to differences in viral dissemination or even transmission in mosquito populations. We hope that our study can give more insights into understanding the roles of mosquito insecticide resistance on viral transmission.

molecular biology↗

bioRxiv2021

A virus-encoded microRNA contributes to evade innate immune response during SARS-CoV-2 infection

SARS-CoV-2 infection results in impaired interferon response in severe COVID-19 patients. However, how SARS-CoV-2 interferes with host immune response is incompletely understood. Here, we sequenced small RNAs from SARS-CoV-2-infected human cells and identified a micro-RNA (miRNA) encoded in a recently evolved region of the viral genome. We show that the virus-encoded miRNA produces two miRNA isoforms in infected cells by the enzyme Dicer and they are loaded into Argonaute proteins. Moreover, the predominant miRNA isoform targets the 3UTR of interferon-stimulated genes and represses their expression in a miRNA-like fashion. Finally, the two viral miRNA isoforms were detected in nasopharyngeal swabs from COVID-19 patients. We propose that SARS-CoV-2 employs a virus-encoded miRNA to hijack the host miRNA machinery and evade the interferon-mediated immune response.

molecular biology↗

bioRxiv2021

Identification of DAXX As A Restriction Factor Of SARS-CoV-2 Through A CRISPR/Cas9 Screen

Interferon restricts SARS-CoV-2 replication in cell culture, but only a handful of Interferon Stimulated Genes with antiviral activity against SARS-CoV-2 have been identified. Here, we describe a functional CRISPR/Cas9 screen aiming at identifying SARS-CoV-2 restriction factors. We identified DAXX, a scaffold protein residing in PML nuclear bodies known to limit the replication of DNA viruses and retroviruses, as a potent inhibitor of SARS-CoV-2 and SARS-CoV replication in human cells. Basal expression of DAXX was sufficient to limit the replication of SARS-CoV-2, and DAXX over-expression further restricted infection. In contrast with most of its previously described antiviral activities, DAXX-mediated restriction of SARS-CoV-2 was independent of the SUMOylation pathway. SARS-CoV-2 infection triggered the re-localization of DAXX to cytoplasmic sites and promoted its degradation. Mechanistically, this process was mediated by the viral papain-like protease (PLpro) and the proteasome. Together, these results demonstrate that DAXX restricts SARS-CoV-2, which in turn has evolved a mechanism to counteract its action.

microbiology↗

bioRxiv2021

Exploring zebrafish larvae as a COVID-19 model: probable SARS-COV-2 replication in the swim bladder

Animal models are essential to understand COVID-19 pathophysiology and for pre-clinical assessment of drugs and other therapeutic or prophylactic interventions. We explored the small, cheap and transparent zebrafish larva as a potential host for SARS-CoV-2. Bath exposure, as well as microinjection in the coelom, pericardium, brain ventricle, bloodstream, or yolk, did not result in detectable SARS-CoV-2 replication in wild-type larvae. However, when the virus was inoculated in the swim bladder, a modest increase in viral RNA was observed after 24 hours, suggesting a successful infection in some animals. This was confirmed by immunohistochemistry, with cells positive for SARS-CoV-2 nucleoprotein observed in the swim bladder. Several variants of concern were also tested with no evidence of increased infectivity in our model. Low infectivity of SARS-CoV-2 in zebrafish larvae was not due to the host type I interferon response, as comparable viral loads were detected in type I interferon-deficient animals. Mosaic overexpression of human ACE2 was not sufficient to increase SARS-CoV-2 infectivity in zebrafish embryos or in fish cells in vitro. In conclusion, wild-type zebrafish larvae appear mostly non-permissive to SARS-CoV-2, except in the swim bladder, an aerial organ sharing similarities with the mammalian lung.

microbiology↗

bioRxiv2021

Phospholipidosis is a shared mechanism underlying the in vitro antiviral activity of many repurposed drugs against SARS-CoV-2

Repurposing drugs as treatments for COVID-19 has drawn much attention. A common strategy has been to screen for established drugs, typically developed for other indications, that are antiviral in cells or organisms. Intriguingly, most of the drugs that have emerged from these campaigns, though diverse in structure, share a common physical property: cationic amphiphilicity. Provoked by the similarity of these repurposed drugs to those inducing phospholipidosis, a well-known drug side effect, we investigated phospholipidosis as a mechanism for antiviral activity. We tested 23 cationic amphiphilic drugs--including those from phenotypic screens and others that we ourselves had found--for induction of phospholipidosis in cell culture. We found that most of the repurposed drugs, which included hydroxychloroquine, azithromycin, amiodarone, and four others that have already progressed to clinical trials, induced phospholipidosis in the same concentration range as their antiviral activity; indeed, there was a strong monotonic correlation between antiviral efficacy and the magnitude of the phospholipidosis. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the gross physical properties of drugs, and does not reflect specific target-based activities, rather it may be considered a confound in early drug discovery. Understanding its role in infection, and detecting its effects rapidly, will allow the community to better distinguish between drugs and lead compounds that more directly impact COVID-19 from the large proportion of molecules that manifest this confounding effect, saving much time, effort and cost. One Sentence SummaryDrug-induced phospholipidosis is a single mechanism that may explain the in vitro efficacy of a wide-variety of therapeutics repurposed for COVID-19.

pharmacology and toxicology↗

bioRxiv2021

Host PDZ-containing proteins targeted by SARS-Cov-2

Small linear motif targeting protein interacting domains called PDZ have been identified at the C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins E, 3a, and N. Using a high-throughput approach of affinity-profiling against the full human PDZome, we identified sixteen human PDZ binders of SARS-CoV-2 proteins E, 3A and N showing significant interactions with dissociation constants values ranging from 3 M to 82 M. Six of them (TJP1, PTPN13, HTRA1, PARD3, MLLT4, LNX2) are also recognized by SARS-CoV while three (NHERF1, MAST2, RADIL) are specific to SARS-CoV-2 E protein. Most of these SARS-CoV-2 protein partners are involved in cellular junctions/polarity and could be also linked to evasion mechanisms of the immune responses during viral infection. Seven of the PDZ-containing proteins among binders of the SARS-CoV-2 proteins E, 3a or N affect significantly viral replication under knock-down gene expression in infected cells. This PDZ profiling identifying human proteins potentially targeted by SARS-CoV-2 can help to understand the multifactorial severity of COVID19 and to conceive effective anti-coronaviral agents for therapeutic purposes.

biochemistry↗

bioRxiv2021

BRD2 inhibition blocks SARS-CoV-2 infection in vitro by reducing transcription of the host cell receptor ACE2

SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted a targeted CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. We found that the protein BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells, including those of human nasal epithelia. Moreover, pharmacological BRD2 inhibition with the drug ABBV-744 inhibited SARS-CoV-2 replication in Syrian hamsters. We also found that BRD2 controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates the antiviral response. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a novel therapeutic target for COVID-19.

cell biology↗

bioRxiv2020

Characterisation of protease activity during SARS-CoV-2 infection identifies novel viral cleavage sites and cellular targets for drug repurposing

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, and responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication, and inhibitors targeting proteases have already shown success at inhibiting SARS-CoV-2 in cell culture models. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigenic proteins S and N, which are the main targets for vaccine and antibody testing efforts. We discovered significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases, validating a subset with in vitro assays. We showed that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, showed a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19.

microbiology↗

bioRxiv2020

An ultra-high affinity synthetic nanobody blocks SARS-CoV-2 infection by locking Spike into an inactive conformation

Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.

biochemistry↗

bioRxiv2019

High-throughput fluorescence-based screen identifies the neuronal microRNA miR-124 as a positive regulator of alphavirus infection

Micro (mi)RNAs are small regulatory RNAs, which act as guide for the RISC complex to modulate the expression of target genes. In addition to their role in maintaining essential physiological functions in the cell, miRNAs can also regulate viral infections. They can do so directly by targeting RNAs of viral origin or indirectly by targeting RNAs from the host and this can result in a positive or negative outcome for the virus. Here, we performed a miRNA genome-wide screen in order to identify cellular miRNAs involved in the regulation of arbovirus infection in human cells. We identified sixteen miRNAs showing a positive effect on the virus, among which a number of neuron-specific ones such as miR-124. We confirmed that overexpression of miR-124 increases Sindbis virus (SINV) viral production and that this effect is mediated by its seed sequence. We further demonstrated that the SINV genome possesses a binding site for miR-124-3p. Both inhibition of miR-124-3p or silent mutations to disrupt this binding site in the viral RNA abolished the positive regulation. We also proved that miR-124 inhibition reduces SINV infection in human differentiated neuronal cells. Finally, we showed that the proviral effect of miR-124 is conserved for other medically relevant alphaviruses. Indeed, inhibition of miR-124 expression reduces chikungunya virus (CHIKV) viral production in human cells. Altogether, our work expands the panel of positive regulation of the viral cycle by direct binding of host miRNAs to the viral RNA and provides new insights into the role of cellular miRNAs as regulators of alphavirus infection.\n\nSIGNIFICANCE STATEMENTArthropod-borne (arbo) viruses are part of a class of pathogens that are transmitted to their final hosts by insects. Because of climate change, the habitat of some of these insects, such as mosquitoes, is shifting, thereby facilitating the emergence of viral epidemics. Among the pathologies associated with arboviruses infection, neurological diseases like meningitis or encephalitis represent a significant health burden. Using a genome-wide miRNA screen, we identified the neuronal miR-124 as a positive regulator of the Sindbis and chikungunya alphaviruses. We also showed that this effect was in part direct, thereby opening novel avenues to treat alphaviruses infection.

molecular biology↗

bioRxiv2019

Comparative analysis of neuroinvasion by Japanese encephalitis virulent and vaccine strains in an in cellulo model of human blood-brain barrier

Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in South East Asia. It has been suggested that JEV gets access to the central nervous system (CNS) as a consequence of a preceding inflammatory process which leads to the blood-brain barrier (BBB) disruption and viral neuroinvasion. However, what happens at early times of JEV contact with the BBB is poorly understood. In the present work, we evaluated the ability of both a virulent and a vaccine strain of JEV (JEV RP9 and SA14-14-2, respectively) to cross an in cellulo human BBB model consisting of hCMEC/D3 human endothelial cells cultivated on permeable inserts above SK-N-SH human neuroblastoma cells. Using this system, we demonstrated that both JEV RP9 and SA14-14-2 are able to cross the BBB without disrupting it at early times post-addition. Furthermore, this BBB model was able to discriminate between the virulent RP9 and the vaccine SA14-14-2 strains, as demonstrated by the presence of almost 10 times more RP9 infectious particles that crossed the BBB than SA14-14 particles at a high MOI. Besides contributing to the understanding of early events in JEV neuroinvasion, this in cellulo BBB model represents a suitable and useful system to study the viral determinants of JEV neuroinvasiveness and the molecular mechanisms by which this flavivirus crosses the BBB at early times of neuroinvasion.

microbiology↗

bioRxiv2019

Modeling cell infection via virus-producing cells rather than free infectious virus significantly improves fits of an in vitro viral kinetic data

Chikungunya and Zika viruses are arthropod-borne viruses that pose significant threat to public health. Experimental data show that during in vitro infection both viruses exhibit qualitatively distinct replication cycle kinetics. Chikungunya viral load rapidly accumulates within the first several hours post infection whereas Zika virus begins to increase at much later times. We sought to characterize these qualitatively distinct in vitro kinetics of chikungunya and Zika viruses by fitting a family of mathematical models to time course viral load datasets. We demonstrate that the standard viral kinetic model, which considers that new infections result only from free virus penetrating susceptible cells, does not fit experimental data as well as a model in which the number of virus-infected cells is the primary determinant of infection rate. We provide biologically meaningful quantifications of the main viral kinetic parameters and show that our results support cell-to-cell or localized transmission as a significant contributor to viral infection with chikungunya and Zika viruses.\n\nImportanceMathematical modeling has become a useful tool to tease out information about virus-host interactions and thus complements experimental work in characterizing and quantifying processes within viral replication cycle. Importantly, mathematical models can fill in incomplete data sets and identify key parameters of infection, provided the appropriate model is used. The in vitro time course dynamics of mosquito transmitted viruses, such as chikungunya and Zika, have not been studied by mathematical modeling and thus limits our knowledge about quantitative description of the individual determinants of viral replication cycle. This study employs dynamical modeling framework to show that the rate at which cells become virus-infected is proportional to the number or virus-infected cells rather than free extracellular virus in the milieu, a widely accepted assumption in models of viral infections. Using the refined mathematical model in combination with viral load data, we provide quantification of the main drivers of chikungunya and Zika in vitro kinetics. Together, our results bring quantitative understanding of the basic components of chikungunya and Zika virus dynamics.

microbiology↗

bioRxiv2018

DISSEQT - DIStribution based modeling of SEQuence space Time dynamics

Rapidly evolving microbes are a challenge to model because of the volatile, complex and dynamic nature of their populations. We developed the DISSEQT pipeline (DIStribution-based SEQuence space Time dynamics) for analyzing, visualizing and predicting the evolution of heterogeneous biological populations in multidimensional genetic space, suited for population-based modeling of deep sequencing and high-throughput data. DISSEQT is openly available on GitHub (https://github.com/rasmushenningsson/DISSEQT.jl) and Synapse (https://www.synapse.org/#!Synapse:syn11425758), covering the entire workflow from read alignment to visualization of results. DISSEQT is centered around robust dimension and model reduction algorithms for analysis of genotypic data with additional capabilities for including phenotypic features to explore dynamic genotype-phenotype maps. We illustrate its utility and capacity with examples from evolving RNA virus populations, which present on of the highest degrees of population heterogeneity found in nature. Using DISSEQT, we empirically reconstruct the evolutionary trajectories of evolving populations in sequence space and genotype-phenotype fitness landscapes. We show that while sequence space is vastly multidimensional, the relevant genetic space of evolving microbial populations is of intrinsically low dimension. In addition, evolutionary trajectories of these populations can be faithfully monitored to identify the key minority genotypes contributing most to evolution. Finally, we show that empirical fitness landscapes, when reconstructed to include minority variants, can predict phenotype from genotype with high accuracy.

evolutionary biology↗

bioRxiv2018

Long term context dependent genetic adaptation of the viral genetic cloud

RNA viruses generate a cloud of genetic variants within each host. This cloud contains high frequency genotypes, and a very large number of rare variants. While the dynamics of frequent variants are affected by the fitness of each variant, the rare variants cloud is affected by more complex genetic factors, including context dependent mutations. It serves as a spearhead for the viral populations movement within the adaptive landscape. We here use an experimental evolution system to show that the genetic cloud surrounding the Coxsackie virus master sequence slowly, but steadily, evolves over hundreds of generations. The evolution of the rare variants cloud often precedes the appearance of high frequency variants. The rare variants clouds evolution is driven by a combination of a context-dependent mutation pattern and selection for and against specific nucleotide compositions.This combination affects the mutated dinucleotide distribution, and eventually leads to a non-uniform dinucleotide distribution in the main viral sequence. We then tested these conclusions on other RNA viruses with similar conclusions.

genetics↗