Genetic tracing of market wildlife and viruses at the epicenter of the COVID-19 pandemic.

Zoonotic spillovers of viruses have occurred through the animal trade worldwide. The start of the COVID-19 pandemic was traced epidemiologically to the Huanan Seafood Wholesale Market. Here, we analyze environmental qPCR and sequencing data collected in the Huanan market in early 2020. We demonstrate that market-linked severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic diversity is consistent with market emergence and find increased SARS-CoV-2 positivity near and within a wildlife stall. We identify wildlife DNA in all SARS-CoV-2-positive samples from this stall, including species such as civets, bamboo rats, and raccoon dogs, previously identified as possible intermediate hosts. We also detect animal viruses that infect raccoon dogs, civets, and bamboo rats. Combining metagenomic and phylogenetic approaches, we recover genotypes of market animals and compare them with those from farms and other markets. This analysis provides the genetic basis for a shortlist of potential intermediate hosts of SARS-CoV-2 to prioritize for serological and viral sampling.

Integrated cryoEM structure of a spumaretrovirus reveals cross-kingdom evolutionary relationships and the molecular basis for assembly and virus entry.

Foamy viruses (FVs) are an ancient lineage of retroviruses, with an evolutionary history spanning over 450 million years. Vector systems based on Prototype Foamy Virus (PFV) are promising candidates for gene and oncolytic therapies. Structural studies of PFV contribute to the understanding of the mechanisms of FV replication, cell entry and infection, and retroviral evolution. Here we combine cryoEM and cryoET to determine high-resolution in situ structures of the PFV icosahedral capsid (CA) and envelope glycoprotein (Env), including its type III transmembrane anchor and membrane-proximal external region (MPER), and show how they are organized in an integrated structure of assembled PFV particles. The atomic models reveal an ancient retroviral capsid architecture and an unexpected relationship between Env and other class 1 fusion proteins of the Mononegavirales. Our results represent the de novo structure determination of an assembled retrovirus particle.

Primate hemorrhagic fever-causing arteriviruses are poised for spillover to humans.

Simian arteriviruses are endemic in some African primates and can cause fatal hemorrhagic fevers when they cross into primate hosts of new species. We find that CD163 acts as an intracellular receptor for simian hemorrhagic fever virus (SHFV; a simian arterivirus), a rare mode of virus entry that is shared with other hemorrhagic fever-causing viruses (e.g., Ebola and Lassa viruses). Further, SHFV enters and replicates in human monocytes, indicating full functionality of all of the human cellular proteins required for viral replication. Thus, simian arteriviruses in nature may not require major adaptations to the human host. Given that at least three distinct simian arteriviruses have caused fatal infections in captive macaques after host-switching, and that humans are immunologically naive to this family of viruses, development of serology tests for human surveillance should be a priority.

Genomic Epidemiology of SARS-CoV-2 in Guangdong Province, China.

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 infection and was first reported in central China in December 2019. Extensive molecular surveillance in Guangdong, China's most populous province, during early 2020 resulted in 1,388 reported RNA-positive cases from 1.6 million tests. In order to understand the molecular epidemiology and genetic diversity of SARS-CoV-2 in China, we generated 53 genomes from infected individuals in Guangdong using a combination of metagenomic sequencing and tiling amplicon approaches. Combined epidemiological and phylogenetic analyses indicate multiple independent introductions to Guangdong, although phylogenetic clustering is uncertain because of low virus genetic variation early in the pandemic. Our results illustrate how the timing, size, and duration of putative local transmission chains were constrained by national travel restrictions and by the province's large-scale intensive surveillance and intervention measures. Despite these successes, COVID-19 surveillance in Guangdong is still required, because the number of cases imported from other countries has increased.

SARS-CoV-2 Omicron XBB lineage spike structures, conformations, antigenicity, and receptor recognition.

A recombinant lineage of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant, named XBB, appeared in late 2022 and evolved descendants that successively swept local and global populations. XBB lineage members were noted for their improved immune evasion and transmissibility. Here, we determine cryoelectron microscopy (cryo-EM) structures of XBB.1.5, XBB.1.16, EG.5, and EG.5.1 spike (S) ectodomains to reveal reinforced 3-receptor binding domain (RBD)-down receptor-inaccessible closed states mediated by interprotomer RBD interactions previously observed in BA.1 and BA.2. Improved XBB.1.5 and XBB.1.16 RBD stability compensated for stability loss caused by early Omicron mutations, while the F456L substitution reduced EG.5 RBD stability. S1 subunit mutations had long-range impacts on conformation and epitope presentation in the S2 subunit. Our results reveal continued S protein evolution via simultaneous optimization of multiple parameters, including stability, receptor binding, and immune evasion, and the dramatic effects of relatively few residue substitutions in altering the S protein conformational landscape.

An Antigenic Atlas of HIV-1 Escape from Broadly Neutralizing Antibodies Distinguishes Functional and Structural Epitopes.

Anti-HIV broadly neutralizing antibodies (bnAbs) have revealed vaccine targets on the virus's envelope (Env) protein and are themselves promising immunotherapies. The efficacy of bnAb-based therapies and vaccines depends in part on how readily the virus can escape neutralization. Although structural studies can define contacts between bnAbs and Env, only functional studies can define mutations that confer escape. Here, we mapped how all possible single amino acid mutations in Env affect neutralization of HIV by nine bnAbs targeting five epitopes. For most bnAbs, mutations at only a small fraction of structurally defined contact sites mediated escape, and most escape occurred at sites near, but not in direct contact with, the antibody. The Env mutations selected by two pooled bnAbs were similar to those expected from the combination of the bnAbs's independent action. Overall, our mutation-level antigenic atlas provides a comprehensive dataset for understanding viral immune escape and refining therapies and vaccines.

Genome chromatinization in giant double-stranded DNA viruses.

Giant viruses have extravagantly large double-stranded (ds)DNA genomes that are packaged into exceedingly complex virions. In two recent papers, Liu et al. and Valencia-Sánchez, Abini-Agbomson et al. show that some giant viruses encode unique histone doublets, which form nucleosomes remarkably similar to those found across the eukaryotic domain of life.

A variant-dependent molecular clock with anomalous diffusion models SARS-CoV-2 evolution in humans.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans has been monitored at an unprecedented level due to the public health crisis, yet the stochastic dynamics underlying such a process is dubious. Here, considering the number of acquired mutations as the displacement of the viral particle from the origin, we performed biostatistical analyses from numerous whole genome sequences on the basis of a time-dependent probabilistic mathematical model. We showed that a model with a constant variant-dependent evolution rate and nonlinear mutational variance with time (i.e., anomalous diffusion) explained the SARS-CoV-2 evolutionary motion in humans during the first 120 wk of the pandemic in the United Kingdom. In particular, we found subdiffusion patterns for the Primal, Alpha, and Omicron variants but a weak superdiffusion pattern for the Delta variant. Our findings indicate that non-Brownian evolutionary motions occur in nature, thereby providing insight for viral phylodynamics.

Invasion and Amplification of Endogenous Retroviruses in Dasyuridae Marsupial Genomes.

Retroviruses are an ancient viral family that have globally coevolved with vertebrates and impacted their evolution. In Australia, a continent that has been geographically isolated for millions of years, little is known about retroviruses in wildlife, despite the devastating impacts of a retrovirus on endangered koala populations. We therefore sought to identify and characterize Australian retroviruses through reconstruction of endogenous retroviruses from marsupial genomes, in particular the Tasmanian devil due to its high cancer incidence. We screened 19 marsupial genomes and identified over 80,000 endogenous retrovirus fragments which we classified into eight retrovirus clades. The retroviruses were similar to either Betaretrovirus (5/8) or Gammaretrovirus (3/8) retroviruses, but formed distinct phylogenetic clades compared to extant retroviruses. One of the clades (MEBrv 3) lost an envelope but retained retrotranspositional activity, subsequently amplifying throughout all Dasyuridae genomes. Overall, we provide insights into Australian retrovirus evolution and identify a highly active endogenous retrovirus within Dasyuridae genomes.

Genetic diversity accelerates canine distemper virus adaptation to ferrets.

RNA viruses adapt rapidly to new host environments by generating highly diverse genome sets, so-called "quasispecies." Minor genetic variants promote their rapid adaptation, allowing for the emergence of drug-resistance or immune-escape mutants. Understanding these adaptation processes is highly relevant to assessing the risk of cross-species transmission and the safety and efficacy of vaccines and antivirals. We hypothesized that genetic memory within a viral genome population facilitates rapid adaptation. To test this, we investigated the adaptation of the Morbillivirus canine distemper virus to ferrets and compared an attenuated, Vero cell-adapted virus isolate with its recombinant derivative over consecutive ferret passages. Although both viruses adapted to the new host, the reduced initial genetic diversity of the recombinant virus resulted in delayed disease onset. The non-recombinant virus gradually increased the frequencies of beneficial mutations already present at very low frequencies in the input virus. In contrast, the recombinant virus first evolved de novo mutations to compensate for the initial fitness impairments. Importantly, while both viruses evolved different sets of mutations, most mutations found in the adapted non-recombinant virus were identical to those found in a previous ferret adaptation experiment with the same isolate, indicating that mutations present at low frequency in the original virus stock serve as genetic memory. An arginine residue at position 519 in the carboxy terminus of the nucleoprotein shared by all adapted viruses was found to contribute to pathogenesis in ferrets. Our work illustrates the importance of genetic diversity for adaptation to new environments and identifies regions with functional relevance.IMPORTANCEWhen viruses encounter a new host, they can rapidly adapt to this host and cause disease. How these adaptation processes occur remains understudied. Morbilliviruses have high clinical and veterinary relevance and are attractive model systems to study these adaptation processes. The canine distemper virus is of particular interest, as it exhibits a broader host range than other morbilliviruses and frequently crosses species barriers. Here, we compared the adaptation of an attenuated virus and its recombinant derivative to that of ferrets. Pre-existing mutations present at low frequency allowed faster adaptation of the non-recombinant virus compared to the recombinant virus. We identified a common point mutation in the nucleoprotein that affected the pathogenesis of both viruses. Our study shows that genetic memory facilitates environmental adaptation and that erasing this genetic memory by genetic engineering results in delayed and different adaptation to new environments, providing an important safety aspect for the generation of live-attenuated vaccines.

Revisiting the recombinant history of HIV-1 group M with dynamic network community detection.

The prevailing abundance of full-length HIV type 1 (HIV-1) genome sequences provides an opportunity to revisit the standard model of HIV-1 group M (HIV-1/M) diversity that clusters genomes into largely nonrecombinant subtypes, which is not consistent with recent evidence of deep recombinant histories for simian immunodeficiency virus (SIV) and other HIV-1 groups. Here we develop an unsupervised nonparametric clustering approach, which does not rely on predefined nonrecombinant genomes, by adapting a community detection method developed for dynamic social network analysis. We show that this method (dynamic stochastic block model [DSBM]) attains a significantly lower mean error rate in detecting recombinant breakpoints in simulated data (quasibinomial generalized linear model (GLM), P<8×10−8), compared to other reference-free recombination detection programs (genetic algorithm for recombination detection [GARD], recombination detection program 4 [RDP4], and RDP5). When this method was applied to a representative sample of n = 525 actual HIV-1 genomes, we determined k = 29 as the optimal number of DSBM clusters and used change-point detection to estimate that at least 95% of these genomes are recombinant. Further, we identified both known and undocumented recombination hotspots in the HIV-1 genome and evidence of intersubtype recombination in HIV-1 subtype reference genomes. We propose that clusters generated by DSBM can provide an informative framework for HIV-1 classification.

Transcriptomic insights into the epigenetic modulation of turnip mosaic virus evolution in Arabidopsis thaliana.

BACKGROUND: Plant-virus interaction models propose that a virus's ability to infect a host genotype depends on the compatibility between virulence and resistance genes. Recently, we conducted an evolution experiment in which lineages of turnip mosaic virus (TuMV) were passaged in Arabidopsis thaliana genotypes carrying mutations in components of the DNA methylation and the histone demethylation epigenetic pathways. All evolved lineages increased infectivity, virulence and viral load in a host genotype-dependent manner. RESULTS: To better understand the underlying reasons for these evolved relationships, we delved into the transcriptomic responses of mutant and WT plant genotypes in mock conditions and infected with either the ancestral or evolved viruses. Such a comparison allowed us to classify every gene into nine basic expression profiles. Regarding the targets of viral adaptation, our analyses allowed the identification of common viral targets as well as host genotype-specific genes and categories of biological processes. As expected, immune response-related genes were found to be altered upon infection. However, we also noticed the pervasive over-representation of other functional groups, suggesting that viral adaptation was not solely driven by the level of expression of plant resistance genes. In addition, a significant association between the presence of transposable elements within or upstream the differentially expressed genes was observed. Finally, integration of transcriptomic data into a virus-host protein-protein interaction network highlighted the most impactful interactions. CONCLUSIONS: These findings shed extra light on the complex dynamics between plants and viruses, indicating that viral infectivity depends on various factors beyond just the plant's resistance genes.

Fast Track Adaptation of Oncolytic Coxsackie B3 Virus to Resistant Colorectal Cancer Cells - a Method to Personalize Virotherapy.

BACKGROUND: The efficacy of oncolytic viruses (OV) in cancer treatment depends on their ability to successfully infect and destroy tumor cells. However, patients' tumors vary, and in the case of individual insensitivity to an OV, therapeutic efficacy is limited. Here, we present a protocol for rapid generation of tumor cell-specific adapted oncolytic coxsackievirus B3 (CVB3) with enhanced oncolytic potential and a satisfactory safety profile. This is achieved by combining directed viral evolution (DVE) with genetic modification of the viral genome and the use of a microRNA-dependent regulatory tool. METHODS: The oncolytic CVB3 variant PD-H was adapted to the refractory colorectal carcinoma cell line Colo320 through serial passaging. XTT assays and virus plaque assays were used to determine virus cytotoxicity and virus replication in vitro. Recombinant PD-H variants were generated through virus mutagenesis. Apoptosis was detected by Western blots, Caspase 3/7 assays, and DAPI staining. The therapeutic efficacy and safety of the adapted recombinant OV PD-SK-375TS were assessed in vivo using a subcutaneous Colo320 xenograft mouse model. RESULTS: PD-H was adapted to the colorectal cancer cell line Colo320 within 10 passages. Sequencing of passage 10 virus P-10 revealed a heterogenous virus population with five nucleotide mutations resulting in amino acid substitutions. The genotypically homogeneous OV PD-SK was generated by inserting the five detected mutations of P-10 into the genome of PD-H. PD-SK showed significantly stronger replication and cytotoxicity than PD-H in Colo320 cells, but not in other colorectal carcinoma cell lines. Increase of apoptosis induction was detected as key mechanisms of Colo320 cell-specific adaptation of PD-SK. For in vivo safety PD-SK was engineered with target sites of the miR-375 (miR-375TS) to exclude virus replication in normal tissues. PD-SK-375TS, unlike the PD-H-375TS not adapted homolog suppressed the growth of subcutaneous Colo320 tumors in nude mice without causing any side effects. CONCLUSION: Taken together, here we present an optimized protocol for the rapid generation of tumor cell-specific adapted oncolytic CVB3 based on the oncolytic CVB3 strain PD-H. The protocol is promising for the generation of personalized OV for tumor therapy and has the potential to be applied to other OV.

Amino acid substitution L232F in non-structural protein 6 identified as a possible human-adaptive mutation in clade B MERS coronaviruses.

IMPORTANCE: Viral host adaptation plays an important role in inter-species transmission of coronaviruses and influenza viruses. Multiple human-adaptive mutations have been identified in influenza viruses but not so far in MERS-CoV that circulates widely in dromedary camels in the Arabian Peninsula leading to zoonotic transmission. Here, we analyzed clade B MERS-CoV sequences and identified an amino acid substitution L232F in nsp6 that repeatedly occurs in human MERS-CoV. Using a loss-of-function reverse genetics approach, we found the nsp6 L232F conferred increased viral replication competence in vitro, in cultures of the upper human respiratory tract ex vivo, and in lungs of mice infected in vivo. Our results showed that nsp6 L232F may be an adaptive mutation associated with zoonotic transmission of MERS-CoV. This study highlighted the capacity of MERS-CoV to adapt to transmission to humans and also the need for continued surveillance of MERS-CoV in camels.

Amino acid substitutions in norovirus VP1 dictate host dissemination via variations in cellular attachment.

IMPORTANCE: All viruses initiate infection by utilizing receptors to attach to target host cells. These virus-receptor interactions can therefore dictate viral replication and pathogenesis. Understanding the nature of virus-receptor interactions could also be important for the development of novel therapies. Noroviruses are non-enveloped icosahedral viruses of medical importance. They are a common cause of acute gastroenteritis with no approved vaccine or therapy and are a tractable model for studying fundamental virus biology. In this study, we utilized the murine norovirus model system to show that variation in a single amino acid of the major capsid protein alone can affect viral infectivity through improved attachment to suspension cells. Modulating plasma membrane mobility reduced infectivity, suggesting an importance of membrane mobility for receptor recruitment and/or receptor conformation. Furthermore, different substitutions at this site altered viral tissue distribution in a murine model, illustrating how in-host capsid evolution could influence viral infectivity and/or immune evasion.

The Role of Extensive Recombination in the Evolution of Geminiviruses.

Mutation, recombination and pseudo-recombination are the major forces driving the evolution of viruses by the generation of variants upon which natural selection, genetic drift and gene flow can act to shape the genetic structure of viral populations. Recombination between related virus genomes co-infecting the same cell usually occurs via template swapping during the replication process and produces a chimeric genome. The family Geminiviridae shows the highest evolutionary success among plant virus families, and the common presence of recombination signatures in their genomes reveals a key role in their evolution. This review describes the general characteristics of members of the family Geminiviridae and associated DNA satellites, as well as the extensive occurrence of recombination at all taxonomic levels, from strain to family. The review also presents an overview of the recombination patterns observed in nature that provide some clues regarding the mechanisms involved in the generation and emergence of recombinant genomes. Moreover, the results of experimental evolution studies that support some of the conclusions obtained in descriptive or in silico works are summarized. Finally, the review uses a number of case studies to illustrate those recombination events with evolutionary and pathological implications as well as recombination events in which DNA satellites are involved.

Virus Evolution on Fitness Landscapes.

The landscape paradigm is revisited in the light of evolution in simple systems. A brief overview of different classes of fitness landscapes is followed by a more detailed discussion of the RNA model, which is currently the only evolutionary model that allows for a comprehensive molecular analysis of a fitness landscape. Neutral networks of genotypes are indispensable for the success of evolution. Important insights into the evolutionary mechanism are gained by considering the topology of sequence and shape spaces. The dynamic concept of molecular quasispecies is viewed in the light of the landscape paradigm. The distribution of fitness values in state space is mirrored by the population structures of mutant distributions. Two classes of thresholds for replication error or mutations are important: (i) the-conventional-genotypic error threshold, which separates ordered replication from random drift on neutral networks, and (ii) a phenotypic error threshold above which the molecular phenotype is lost. Empirical landscapes are reviewed and finally, the implications of the landscape concept for virus evolution are discussed.

Genetic diversity and cross-species transmissibility of bat-associated picornaviruses from Spain.

BACKGROUND: Emerging zoonotic diseases arise from cross-species transmission events between wild or domesticated animals and humans, with bats being one of the major reservoirs of zoonotic viruses. Viral metagenomics has led to the discovery of many viruses, but efforts have mainly been focused on some areas of the world and on certain viral families. METHODS: We set out to describe full-length genomes of new picorna-like viruses by collecting feces from hundreds of bats captured in different regions of Spain. Viral sequences were obtained by high-throughput Illumina sequencing and analyzed phylogenetically to classify them in the context of known viruses. Linear discriminant analysis (LDA) was performed to infer likely hosts based on genome composition. RESULTS: We found five complete or nearly complete genomes belonging to the family Picornaviridae, including a new species of the subfamily Ensavirinae. LDA suggested that these were true vertebrate viruses, rather than viruses from the bat diet. Some of these viruses were related to picornaviruses previously found in other bat species from distant geographical regions. We also found a calhevirus genome that most likely belongs to a proposed new family within the order Picornavirales, and for which genome composition analysis suggested a plant host. CONCLUSIONS: Our findings describe new picorna-like viral species and variants circulating in the Iberian Peninsula, illustrate the wide geographical distribution and interspecies transmissibility of picornaviruses, and suggest new hosts for calheviruses.

Evolutionary Invasion Analysis of Modern Epidemics Highlights the Context-Dependence of Virulence Evolution.

Models are often employed to integrate knowledge about epidemics across scales and simulate disease dynamics. While these approaches have played a central role in studying the mechanics underlying epidemics, we lack ways to reliably predict how the relationship between virulence (the harm to hosts caused by an infection) and transmission will evolve in certain virus-host contexts. In this study, we invoke evolutionary invasion analysis-a method used to identify the evolution of uninvadable strategies in dynamical systems-to examine how the virulence-transmission dichotomy can evolve in models of virus infections defined by different natural histories. We reveal peculiar patterns of virulence evolution between epidemics with different disease natural histories (SARS-CoV-2 and hepatitis C virus). We discuss the findings with regards to the public health implications of predicting virus evolution, and in broader theoretical canon involving virulence evolution in host-parasite systems.

Plant virus evolution under strong drought conditions results in a transition from parasitism to mutualism.

Environmental conditions are an important factor driving pathogens' evolution. Here, we explore the effects of drought stress in plant virus evolution. We evolved turnip mosaic potyvirus in well-watered and drought conditions in Arabidopsis thaliana accessions that differ in their response to virus infection. Virus adaptation occurred in all accessions independently of watering status. Drought-evolved viruses conferred a significantly higher drought tolerance to infected plants. By contrast, nonsignificant increases in tolerance were observed in plants infected with viruses evolved under standard watering. The magnitude of this effect was dependent on the plant accessions. Differences in tolerance were correlated to alterations in the expression of host genes, some involved in regulation of the circadian clock, as well as in deep changes in the balance of phytohormones regulating defense and growth signaling pathways. Our results show that viruses can promote host survival in situations of abiotic stress, with the magnitude of such benefit being a selectable trait.