Dengue is the most prevalent acute febrile disease with serious clinical consequences in the tropical and subtropical regions of Asia and America. In Peru, it represents a significant public health issue due to its hyperendemic nature, with serotype 2 (DENV-2) being the predominant serotype that leads to the most severe clinical manifestations of the disease. This study focuses on the molecular characterization and analysis of the intraserotypic diversity of DENV-2 circulating in the endemic region of Cajamarca.

A total of 3,967 blood serum samples from patients with acute febrile illness (AFI) were analyzed between 2016 and 2022 to detect DENV and DENV-2 using real-time RT-PCR. The viral envelope (E) gene was then sequenced using the Sanger method. Finally, phylogenetic reconstruction was conducted using the maximum likelihood method.

A total of 32 complete sequences of the envelope gene were obtained, and the phylogenetic and characterization analyses of the amino acid sequences revealed that, during the period from 2016 to 2022, two DENV-2 genotypes circulated: the Am/As genotype and the cosmopolitan genotype in lineages 2 and C, respectively.

Similarly, our findings showed that every studied outbreak was characterized by novel autochthonous variants of the Am/As genotype and by an imported variant of the cosmopolitan genotype; this demonstrates a temporal distribution of intraserotypic variability that indicates the displacement of the Am/As genotype around 2021 and the establishment of the cosmopolitan genotype. The need for ongoing genetic or genomic surveillance of the cosmopolitan virus arises in order to understand its distribution and diversification patterns in Peru.

The incidence of arboviral diseases like dengue, chikungunya, and yellow fever continues to rise in association with the expanding geographic ranges of their vectors, Aedes aegypti and Aedes albopictus. The distribution of these vectors is believed to be driven in part by climate change and increasing urbanization. Arboviruses navigate a wide range of temperatures as they transition from ectothermic vectors (from 15°C to 35°C) to humans (37°C) and back again, but the role that temperature plays in driving the evolution of arboviruses remains largely unknown. Here, we passaged replicate dengue serotype-2 virus populations 10 times at either 26°C (Low) or 37°C (High) in C6/36 Aedes albopictus cells to explore the differences in adaptation to these thermal environments. We then deep-sequenced the resulting passaged dengue virus populations and tested their replicative fitness in an all-cross temperature regime. We also assessed the ability of the passaged viruses to replicate in the insect vector. While viruses from both thermal regimes accumulated substitutions, only those reared in the 37°C treatments exhibited nonsynonymous changes, including several in the E, or envelope protein, and multiple non-structural genes. Passaging at the higher temperature also led to reduced replicative ability at 26°C in both cells and mosquitoes. One of the mutations in the E gene involved the loss of a glycosylation site previously shown to reduce infectivity in the vector. These findings suggest that viruses selected for growth at higher ambient temperatures may experience tradeoffs between thermostability and replication in the vector. Such associations might also have implications for the suitability of virus transmission under a changing climate.

In this study, we isolated a novel double-stranded RNA (dsRNA) mycovirus from the phytopathogenic fungus Colletotrichum cliviicola, designated as "Colletotrichum cliviicola partitivirus 1" (CcPV1). The complete genome of CcPV1 comprises two dsRNA fragments, referred to as dsRNA1 and dsRNA2, which are 1,756 bp and 1,485 bp in length, respectively. Each dsRNA segment contains an open reading frame (ORF), with the larger ORF1 in dsRNA 1 encoding the viral RNA-dependent RNA polymerase (RdRp) and the smaller ORF2 in dsRNA 2 encoding the capsid protein (CP). Sequence comparisons revealed that the RdRp of CcPV1 shares the highest amino acid sequence identity (76.35%) with the RdRp of Nigrospora sphaerica partitivirus 1 (NsPV1). Phylogenetic analysis based on both RdRp and CP sequences indicated that CcPV1 is a member of the genus Gammapartitivirus of the family Partitiviridae. This is the first report of a gammapartitivirus in C. cliviicola.

Genetic diversity of viral populations is almost unanimously attributed to the build-up of random mutations along with accidental recombination events. This passive role of viruses in the selection of viable genotypes is widely acknowledged. According to the hypothesis presented here, populations of steady-state error copies of a master viral sequence would have a dominant mutant rather than a random pool of heterogeneous viral genomes with changes scattered uniformly without any preferential distribution. It would let viruses face the selection stage of host surveillance having a preceding set of potential survivors or "guard" genomes among an ordinary cloud of random quasispecies.

Viruses are diverse biological entities found virtually in all environments on Earth. Their association with parasitic protozoans was shown in the late 1980's, followed by evidence that these viruses can influence the treatment of infections as well as influence parasite virulence. Recently, Matryoshka RNA viruses (MaRNAV) were discovered in Plasmodium vivax infected patients in Malaysia, as well as in species of the closely related avian haemosporidian genera Leucocytozoon and Haemoproteus in Oceania and North America. However, they have not been reported in other continents so far. The aim of this study was thus to screen haemosporidian infected European birds (African migrants and residents) for the presence of MaRNAV. Whole blood samples from wild birds were collected in Lithuania in May 2023. Haemosporidian parasite infections were first assessed by microscopic examination and later confirmed via PCR. RNA was isolated and tested by Reverse Transcriptase (RT) PCR for the presence of MaRNAV. Of the 12 samples that were RT-PCR-positive, only one from a common whitethroat (Curruca communis) had a sequence with 63% similarity to MARNAV-2 found in Leucocytozoon infected birds from Oceania. Total RNA from this sample was sequenced, bioinformatically analyzed, and a new virus, MaRNAV-7, was identified. At the amino acid level, it is phylogenetically closely related to MaRNAV-2, MaRNAV-3 and MaRNAV-6 RdRp sequences, all found in Leucocytozoon infected birds. This is the first report of MaRNAV in an African-European haemosporidian infected bird, and a first step in understanding MaRNAV prevalence, distribution, and specificity. However, the effects that MaRNAV can have on the parasites, modulation of the host immune response and transmission rates remain unknown.

Viruses encounter a range of selective pressures, but inefficiencies during replication can be masked. To uncover factors that limit viral replication, we used forward genetics to enrich for a murine norovirus (MNV) mutant with faster replication. We sequentially harvested the earliest progeny in cultured cells and identified a single amino acid change in the viral NS3 protein, K40R, that was sufficient to enhance replication speed. We found that the NS3-K40R virus induced earlier cell death and viral egress compared with wild-type virus. Mechanistically, NS3-K40R protein disrupted membranes more efficiently than wild-type NS3 protein, potentially contributing to increased mitochondrial dysfunction and cell death. Mice infected with NS3-K40R virus had increased titers, suggesting that increasing egress did not reduce fitness in vivo. Overall, by using a forward genetic approach, we identified a previously unknown inefficiency in norovirus egress and provide new insights into selective pressures that influence viral replication and evolution.

Communal respiratory syncytial virus (RSV) causes mild to severe illnesses, predominantly in older adults, or people with certain chronic medical conditions, and in children. Symptoms may include rhinorrhea, cough, fever, and dyspnea. In most cases, the infection is mild and resolves on its own, but in some cases, it can lead to more serious illness such as bronchiolitis or pneumonia. The RSV genome codes for ten proteins, NS1, NS2, N, P, M, SH, G, F, M2 and L. We aimed to identify the RSV geographical transmission pattern based on parsimony and investigate hotspot regions across the complete RSV genomes. We employed Viral Evolutionary Network Analysis System on full-length available RSV genomes and with HyPhy for elucidating type of selection pressure. These results indicated that RSV strains circulating in South and North America are not mixed to the European samples, however, genomes reported from Australia are the direct decedents of European samples. Samples reported from the United Kingdom exhibited significant diversity, spanning almost every cluster. This report provides a complete mutational analysis of all the individual RSV genes, and particularly the 31 hotspot substituting regions circulating across the globe in RSV type A samples. Further, protein G and L displayed higher level of codons experienced positive selection. This analysis of RSV type A highlights mutational frequencies across the whole genome, offering valuable insights for epidemiological control and drug development.

Positive-strand RNA viruses of the order Nidovirales have the largest known RNA genomes of vertebrate and invertebrate viruses with 36.7 and 41.1 kb, respectively. The acquisition of a proofreading exoribonuclease (ExoN) by an ancestral nidovirus enabled crossing of the 20 kb barrier. Other factors constraining genome size variations in nidoviruses remain poorly defined. We assemble 76 genome sequences of invertebrate nidoviruses from >500.000 published transcriptome experiments and triple the number of known nidoviruses with >36 kb genomes, including a 64 kb RNA genome. Many of the identified viral lineages acquired putative enzymatic and other protein domains linked to genome size, host phyla, or virus families. The inserted domains may regulate viral replication and virion formation, or modulate infection otherwise. We classify ExoN-encoding nidoviruses into seven groups and four subgroups, according to canonical and noncanonical modes of viral replicase expression by ribosomes and genomic organization (reModes). The most-represented group employing the canonical reMode comprises invertebrate and vertebrate nidoviruses, including coronaviruses. Six groups with noncanonical reModes include invertebrate nidoviruses with 31-to-64 kb genomes. Among them are viruses with segmented genomes and viruses utilizing dual ribosomal frameshifting that we validate experimentally. Moreover, largest polyprotein length and genome size in nidoviruses show reMode- and host phylum-dependent relationships. We hypothesize that the polyprotein length increase in nidoviruses may be limited by the host-inherent translation fidelity, ultimately setting a nidovirus genome size limit. Thus, expansion of ExoN-encoding RNA virus genomes, the vertebrate/invertebrate host division, the control of viral replicase expression, and translation fidelity are interconnected.

Respiratory viral infections present significant global health challenges, causing substantial morbidity and mortality, particularly among highly susceptible components of the population. The emergence of pandemics and epidemics, such as those caused by influenza viruses and coronaviruses, emphasizes the urgent need for effective antiviral therapeutics. In this review, we explore the potential of broad-spectrum antiviral agents targeting respiratory RNA viruses, including influenza viruses, coronaviruses, respiratory syncytial virus, human metapneumovirus, human parainfluenza viruses, and rhinoviruses. Various broad-spectrum direct-acting and host-targeting antivirals are discussed, including monoclonal antibodies targeting conserved regions of viral surface proteins, molecules interfering with host cell receptors or viral replication machinery, viral protease inhibitors, siRNA therapies, ribonuclease, and 3D8 scFv. Advancements in host-targeting approaches to reduce resistance and RNA-based therapeutics offer significant potential for combating respiratory viral threats. Despite challenges, broad-spectrum antiviral agents represent a crucial strategy, particularly when specific viral pathogens are unidentified or rapid intervention is essential, such as during pandemics or outbreaks.

Enterovirus-D68 (EV68) continues to present as a global health issue causing respiratory illness and outbreaks associated with long-lasting neurological disease, with no antivirals or specific treatment options. The development of antiviral therapeutics, such as small-molecule inhibitors that target conserved proteins like the enteroviral 3C protease, remains to be achieved. While various 3C inhibitors have been investigated, their design does not consider the potential emergence of drug resistance mutations. For other antivirals where resistance has been a challenge, we have demonstrated that the likelihood of resistance can be minimized by designing inhibitors that leverage the evolutionary constraints of the target. Here, we characterize a series of 3C inhibitors against EV68-3C protease through enzyme inhibition, protein crystallography, and structural analysis. We have determined and analyzed three high-resolution inhibitor-bound crystal structures of EV68-3C protease, which revealed possible sites of resistance mutations, a key structural water molecule conserved during ligand binding, and the conformational flexibility of the catalytic histidine H40. This structural analysis combined with enzymatic assays provides insights for the rational design of inhibitors that are robust against resistance toward developing antiviral treatments for EV68 infections.

Antigen-based rapid diagnostic tests (Ag-RDTs) provide timely results, are simple to use, and are less expensive than molecular assays. Recent studies suggest that antigen-based testing aligns with virus culture-based results (a proxy of contagiousness at the peak viral phase of illness); however, the performance of Ag-RDTs for newer SARS-CoV-2 variants is unclear. In this study, we (i) assessed the performance of Ag-RDTs and diagnostic antibodies to detect a range of SARS-CoV-2 variants and (ii) determined whether Ag-RDT results correlated with culture positivity. We noted only minor differences in the limit of detection by variant for all assays, and we demonstrated consistent antibody affinity to the N protein among the different variants. We observed moderate to high sensitivity (46.8%-83.9%) for Ag-RDTs when compared to PCR positivity (100%), and all variants were assessed on each assay. Ag-RDT sensitivity and PCR Ct showed an inverse correlation with the detection of viable virus. Collectively, our results demonstrate that commercially available Ag-RDTs offer variable sensitivity compared to PCR, show similar diagnostic validity across variants, and may predict the risk of transmissibility. These findings may be used to support more tailored SARS-CoV-2 isolation strategies, particularly if other studies clarify the direct association between Ag-RDT positivity and transmission risk. The apparent trade-off between sensitivity in the detection of any PCR-positive infection and concordance with infectious virus positivity may also inform new RDT diagnostic development strategies for SARS-CoV-2 and other epidemic respiratory pathogens.

Despite the availability of vaccines, COVID-19 continues to be a major health concern, and antigen-based rapid diagnostic tests (Ag-RDTs) are commonly used as point-of-care or at-home diagnostic tests. In this study, we evaluated the performance of two commercially available Ag-RDTs and a research Ag-RDT to detect multiple SARS-CoV-2 variants using upper respiratory tract swab samples from clinical COVID-19 cases. Furthermore, we determined whether Ag-RDT results correlated with culture positivity, a potential proxy of viral transmissibility. Our results have important implications to inform future testing and response strategies during periods of high COVID-19 transmission with new variants.

In recent years, coxsackievirus (CV) A10 has been associated with increasing sporadic hand, foot, and mouth disease (HFMD) cases and outbreaks globally. In addition to mild symptoms such as pharyngitis and herpangina, CVA10-related complications or even fatality can occur. Currently, systematic phylogenetic studies of CVA10 are limited.

In this study, we first explored the epidemiological and genetic characteristics of CVA10 in Nanchang, an inland southeastern city of China, based on the HFMD surveillance network from 2015-2023.

Among 3429 enterovirus-positive cases, 110 (3.04%) were associated with CVA10, with a male-to-female ratio of 1.62. The median age of the CVA10 patients was 2.3 years (interquartile range, IQR 1.0-4.0), with 94.55% (104/110) of the patients aged less than 5 years. Phylogenetic analyses using the full-length VP1, 5'UTR, P1, P2, P3 sequences and near full-length genomes indicated that CVA10 strains (n = 57) isolated in Nanchang belonged to genogroup C; two strains identified in 2017 belonged to C1 subtypes clustered with strains from Vietnam, Madagascar, France and Spain; and the others belonged to C2 subtypes interdigitating with CVA10 isolates from mainland China, the United States and Australia. Through extensive analysis, we identified a rare F168Y mutation in epitope 4 of VP1 in a Madagascar strain of genogroup F and a Chinese strain of genogroup C. Based on Bayesian evolutionary analyses, the average nucleotide substitution rate for the VP1 gene of CV10 strains was 3.07×10-3 substitutions/site/year. The most recent common ancestor (tMRCA) of genogroup C was dated 1990.84, and the tMRCA of CVA10 strains from Nanchang was dated approximately 2003.16, similar to strains circulating in other regions of China, suggesting that the viruses were likely introduced and cryptically circulated in China before the establishment of the HFMD surveillance network. Recombination analysis indicated intertypic recombination of the Nanchang strain with the genogroup G strain in the 3D region.

Given the shifting dominance of viral genotypes and frequent recombination events, the existing surveillance system needs to be regulated to enhance genomic surveillance efforts on a more diverse spectrum of genotypes in the future.

Comparing the in vitro fitness of dengue virus (DENV) isolates is a pivotal approach to assess the contribution of DENV strains' replicative fitness to epidemiological contexts, including serotype replacements. Competition assays are the gold standard to compare the in vitro replicative fitness of viral strains. Implementing competition assays between DENV serotypes requires an experimental setup and an appropriate read-out to quantify the viral progeny of strains belonging to different serotypes.

In the current study, we optimized an existing serotyping qRT-PCR by adapting primer/probe design and multiplexing the serotype-specific qRT-PCR reactions, allowing to accurately detect and quantify all four DENV serotypes. The qRT-PCR was specific, had a limit of detection of at least 5.08×101, 5.16×101, 7.14×101 and 1.36 ×101 genome copies/μL, an efficiency of 1.993, 1.975, 1.902, 1.898 and a linearity (R2) of 0.99975, 0.99975, 0.9985, 0.99965 for DENV-1, -2, -3 and -4 respectively. Challenge of this multiplex serotype-specific qRT-PCR on mixes of viral supernatants containing known concentrations of strains from two serotypes evidenced an accurate quantification of the amount of genome copies of each serotype. We next developed an in vitro assay to compare the replicative fitness of two DENV serotypes in the human hepatic cell line HuH7: quantification of the viral progeny of each serotype in the inoculum and the supernatant using the serotype-specific multiplex qRT-PCR unveiled an enrichment of the supernatant in DENV-1 genome copies, uncovering the enhanced replicative fitness of this DENV-1 isolate.

This optimized qRT-PCR combined to a relevant cellular model allowed to accurately quantify the viral progeny of two DENV strains belonging to two different serotypes in a competition assay, allowing to determine which strain had a replicative advantage. This reliable experimental setup is adaptable to the comparative study of the replicative fitness of any DENV serotypes.

Chikungunya virus (CHIKV) is a rapidly spreading re-emergent virus transmitted from mosquitoes to humans. The emergence of epidemic variants has been associated with changes in the viral genome, such as the duplication of repeated sequences in the 3' untranslated region (UTR). Indeed, blocks of repeated sequences seemingly favor RNA recombination, providing the virus with a unique ability to continuously change the 3'UTR architecture during host switching. In this work, we provide experimental data on the molecular mechanism of RNA recombination and describe specific sequence and structural elements in the viral 3'UTR that favor template switching of the viral RNA-dependent RNA polymerase on the 3'UTR. Furthermore, we found that a 3'UTR deletion mutant that exhibits markedly delayed replication in mosquito cells and impaired transmission in vivo, recombines in reference laboratory strains of mosquitoes. Altogether, our data provide novel experimental evidence indicating that RNA recombination can act as a nucleic acid repair mechanism to add repeated sequences that are associated to high viral fitness in mosquito during chikungunya virus replication.

Due to the error-prone nature of viral RNA-dependent RNA polymerases, the replication of RNA viruses results in a diversity of viral genomes harboring point mutations, deletions, insertions, and genome rearrangements. Citrus tristeza virus (CTV), a causal agent of diseases of economically important citrus species, shows intrinsic genetic stability. While the virus appears to have some mechanism that limits the accumulation of single-nucleotide variants, the production of defective viral genomes (DVGs) during virus infection has been reported for certain variants of CTV. The intra-host diversity generated during plant infection with variant T36 (CTV-T36) remains unclear. To address this, we analyzed the RNA species accumulated in the initially infected and systemic leaves of Nicotiana benthamiana plants inoculated with an infectious cDNA clone of CTV-T36, which warranted that infection was initiated by a known, well-defined sequence variant of the virus. CTV-T36 limited the accumulation of single-nucleotide mutants during infection. With that, four types of DVGs-deletions, insertions, and copy- and snap-backs-were found in all the samples, with deletions and insertions being the most common types. Hot-spots across the genome for DVG recombination and short direct sequence repeats suggest that sequence complementarity could mediate DVG formation. In conclusion, our study illustrates the formation of diverse DVGs during CTV-T36 infection. To the best of our knowledge, this is the first study that has analyzed the genetic variability and recombination of a well-defined sequence variant of CTV in an herbaceous host.

The global impact of the SARS-CoV-2 pandemic has underscored the need for a deeper understanding of viral evolution to anticipate new viruses or variants. Genetic recombination is a fundamental mechanism in viral evolution, yet it remains poorly understood. In this study, we conducted a comprehensive research on the genetic regions associated with genetic recombination features in SARS-CoV-2. With this aim, we implemented a two-phase transfer learning approach using genomic spectrograms of complete SARS-CoV-2 sequences. In the first phase, we utilized a pre-trained VGG-16 model with genomic spectrograms of HIV-1, and in the second phase, we applied HIV-1 VGG-16 model to SARS-CoV-2 spectrograms. The identification of key recombination hot zones was achieved using the Grad-CAM interpretability tool, and the results were analyzed by mathematical and image processing techniques. Our findings unequivocally identify the SARS-CoV-2 Spike protein (S protein) as the pivotal region in the genetic recombination feature. For non-recombinant sequences, the relevant frequencies clustered around 1/6 and 1/12. In recombinant sequences, the sharp prominence of the main hot zone in the Spike protein prominently indicated a frequency of 1/6. These findings suggest that in the arithmetic series, every 6 nucleotides (two triplets) in S may encode crucial information, potentially concealing essential details about viral characteristics, in this case, recombinant feature of a SARS-CoV-2 genetic sequence. This insight further underscores the potential presence of multifaceted information within the genome, including mathematical signatures that define an organism's unique attributes.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in pig production worldwide and responsible for enormous production and economic losses. PRRSV infection in gestating gilts and sows induces important reproductive failure. Additionally, respiratory distress is observed in infected piglets and fattening pigs, resulting in growth retardation and increased mortality. Importantly, PRRSV infection interferes with immunity in the respiratory tract, making PRRSV-infected pigs more susceptible to opportunistic secondary pathogens. Despite the availability of commercial PRRSV vaccines for more than three decades, control of the disease remains a frustrating and challenging task. This paper provides a comprehensive overview of PRRSV, covering its history, economic and scientific importance, and description of the viral structure and genetic diversity. It explores the virus's pathogenesis, including cell tropism, viral entry, replication, stages of infection and epidemiology. It reviews the porcine innate and adaptative immune responses to comprehend the modulation mechanisms employed by PRRS for immune evasion.

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 experimentally infected pigs, JEV introduction into an immunologically naïve 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.

Antiviral therapy is constantly challenged by the emergence of resistant pathogens. At the same time, experimental approaches to understand and predict resistance are limited by long periods required for evolutionary processes. Here, we present a herpes simplex virus 1 mutant with impaired proofreading capacity and consequently elevated mutation rates. Comparing this hypermutator to parental wild type virus, we study the evolution of antiviral drug resistance in vitro. We model resistance development and elucidate underlying genetic changes against three antiviral substances. Our analyzes reveal no principle difference in the evolutionary behavior of both viruses, adaptive processes are overall similar, however significantly accelerated for the hypermutator. We conclude that hypermutator viruses are useful for modeling adaptation to antiviral therapy. They offer the benefit of expedited adaptation without introducing apparent bias and can therefore serve as an accelerator to predict natural evolution.

Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success. Taking advantage of the uniquely large genomic database of SARS-CoV-2, we examine the impact of mutations across the spectrum of viable amino acid sequences on the biophysical phenotypes of the highly expressed and multifunctional nucleocapsid protein. We find variation in the physicochemical parameters of its extended intrinsically disordered regions (IDRs) sufficient to allow local plasticity, but also observe functional constraints that similarly occur in related coronaviruses. In biophysical experiments with several N-protein species carrying mutations associated with major variants, we find that point mutations in the IDRs can have nonlocal impact and modulate thermodynamic stability, secondary structure, protein oligomeric state, particle formation, and liquid-liquid phase separation. In the Omicron variant, distant mutations in different IDRs have compensatory effects in shifting a delicate balance of interactions controlling protein assembly properties, and include the creation of a new protein-protein interaction interface in the N-terminal IDR through the defining P13L mutation. A picture emerges where genetic diversity is accompanied by significant variation in biophysical characteristics of functional N-protein species, in particular in the IDRs.

Hand, foot, and mouth disease (HFMD) is one of the most common class C infectious diseases, posing a serious threat to public health worldwide. Enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) have been regarded as the major pathogenic agents of HFMD; however, since an outbreak caused by coxsackievirus A6 (CV-A6) in France in 2008, CV-A6 has gradually become the predominant pathogen in many regions. CV-A6 infects not only children but also adults, and causes atypical clinical symptoms such as a more generalized rash, eczema herpeticum, high fever, and onychomadesis, which are different from the symptoms associated with EV-A71 and CV-A16. Importantly, the rate of genetic recombination of CV-A6 is high, which can lead to changes in virulence and the rapid evolution of other characteristics, thus posing a serious threat to public health. To date, no specific vaccines or therapeutics have been approved for CV-A6 prevention or treatment, hence it is essential to fully understand the relationship between recombination and evolution of this virus. Here, we systematically review the genetic recombination events of CV-A6 that have occurred worldwide and explore how these events have promoted virus evolution, thus providing important information regarding future HFMD surveillance and prevention.

Human enterovirus A71 (EV-A71) is a significant etiological agent responsible for epidemics of hand, foot, and mouth disease (HFMD) in Asia-Pacific regions. There are presently no licensed antivirals against EV-A71, and the druggable target for EV-A71 remains very limited. The phenotypic hit 10,10'-bis(trifluoromethyl) marinopyrrole A derivative, herein termed MPA-CF3, is a novel potent small-molecule inhibitor against EV-A71, but its pharmacological target(s) and antiviral mechanisms are not defined. Here, quantitative chemoproteomics deciphered the antiviral target of MAP-CF3 as host factor coatomer subunit zeta-1 (COPZ1). Mechanistically, MPA-CF3 disrupts the interaction of COPZ1 with the EV-A71 nonstructural protein 2C by destabilizing COPZ1 upon binding. The destruction of this interaction blocks the coatomer-mediated transport of 2C to endoplasmic reticulum, and ultimately inhibits EV-A71 replication. Taken together, our study disclosed that MPA-CF3 can be a structurally novel host-targeting anti-EV-A71 agent, providing a structural basis for developing the COPZ1-targeting broad-spectrum antivirals against enteroviruses. The mechanistic elucidation of MPA-CF3 against EV-A71 may offer an alternative COPZ1-involved therapeutic pathway for enterovirus infection.

RNA viruses lack proofreading in their RNA polymerases and therefore exist as genetically diverse populations. By exposing these diverse viral populations to selective pressures, viruses with mutations that confer fitness advantages can be enriched. To examine factors important for viral tropism and host restriction, we passaged murine norovirus (MNV) in a human cell line, HeLa cells, to select mutant viruses with increased fitness in non-murine cells. A major determinant of host range is expression of the MNV receptor CD300lf on mouse cells, but additional host factors may limit MNV replication in human cells. We found that viruses passaged six times in HeLa cells had enhanced replication compared with the parental virus. The passaged viruses had several mutations throughout the viral genome, which were primarily located in the viral non-structural coding regions. Although viral attachment was not altered for the passaged viruses, their replication was higher than the parental virus when the entry was bypassed, suggesting that the mutant viruses overcame a post-entry block in human cells. Three mutations in the viral NS1 protein were sufficient for enhanced post-entry replication in human cells. We found that the human cell-adapted MNV variants had reduced fitness in murine BV2 cells and infected mice, with reduced viral titers. These results suggest a fitness tradeoff, where increased fitness in a non-native host cell reduces fitness in a natural host environment. Overall, this work suggests that MNV tropism is determined by the presence of not only the viral receptor but also post-entry factors.

Viruses infect specific species and cell types, which is dictated by the expression of host factors required for viral entry as well as downstream replication steps. Murine norovirus (MNV) infects mouse cells, but not human cells. However, human cells expressing the murine CD300lf receptor support MNV replication, suggesting that receptor expression is a major determinant of MNV tropism. To determine whether other factors influence MNV tropism, we selected for variants with enhanced replication in human cells. We identified mutations that enhance MNV replication in human cells and demonstrated that these mutations enhance infection at a post-entry replication step. Therefore, MNV infection of human cells is restricted at both entry and post-entry stages. These results shed new light on factors that influence viral tropism and host range.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations are rapidly emerging. In particular, beneficial mutations in the spike (S) protein, which can either make a person more infectious or enable immunological escape, are providing a significant obstacle to the prevention and treatment of pandemics. However, how the virus acquires a high number of beneficial mutations in a short time remains a mystery. We demonstrate here that variations of concern may be mutated due in part to the influence of the human microbiome. We searched the National Center for Biotechnology Information database for homologous fragments (HFs) after finding a mutation and the six neighboring amino acids in a viral mutation fragment. Among the approximate 8,000 HFs obtained, 61 mutations in S and other outer membrane proteins were found in bacteria, accounting for 62% of all mutation sources, which is 12-fold higher than the natural variable proportion. A significant proportion of these bacterial species-roughly 70%-come from the human microbiota, are mainly found in the lung or gut, and share a composition pattern with COVID-19 patients. Importantly, SARS-CoV-2 RNA-dependent RNA polymerase replicates corresponding bacterial mRNAs harboring mutations, producing chimeric RNAs. SARS-CoV-2 may collectively pick up mutations from the human microbiota that change the original virus's binding sites or antigenic determinants. Our study clarifies the evolving mutational mechanisms of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations are rapidly emerging, in particular advantageous mutations in the spike (S) protein, which either increase transmissibility or lead to immune escape and are posing a major challenge to pandemic prevention and treatment. However, how the virus acquires a high number of advantageous mutations in a short time remains a mystery. Here, we provide evidence that the human microbiota is a reservoir of advantageous mutations and aids mutational evolution and host adaptation of SARS-CoV-2. Our findings demonstrate a conceptual breakthrough on the mutational evolution mechanisms of SARS-CoV-2 for human adaptation. SARS-CoV-2 may grab advantageous mutations from the widely existing microorganisms in the host, which is undoubtedly an "efficient" manner. Our study might open a new perspective to understand the evolution of virus mutation, which has enormous implications for comprehending the trajectory of the COVID-19 pandemic.

The pandemic and tremendous impact of severe acute respiratory syndrome coronavirus 2 alert us, despite great achievements in prevention and control of infectious diseases, we still lack universal and powerful antiviral strategies to rapidly respond to the potential threat of serious infectious disease. Various highly contagious and pathogenic viruses, as well as other unknown viruses may appear or reappear in human society at any time, causing a catastrophic epidemic. Developing broad-spectrum antiviral drugs with high security and efficiency is of great significance for timely meeting public health emergency and protecting the lives and health of the people. Hence, in this review, we summarized diverse broad-spectrum antiviral targets and corresponding agents from a medicinal chemistry prospective, compared the pharmacological advantages and disadvantages of different targets, listed representative agents, showed their structures, pharmacodynamics and pharmacokinetics characteristics, and conducted a critical discussion on their development potential, in the hope of providing up-to-date guidance for the development of broad-spectrum antivirals and perspectives for applications of antiviral therapy.

The eradication of smallpox was officially declared by the WHO in 1980, leading to discontinuation of the vaccination campaign against the virus. Consequently, immunity against smallpox and related orthopoxviruses like Monkeypox virus gradually declines, highlighting the need for efficient countermeasures not only for the prevention, but also for the treatment of already exposed individuals. We have recently developed human-like monoclonal antibodies (mAbs) from vaccinia virus-immunized non-human primates. Two mAbs, MV33 and EV42, targeting the two infectious forms of the virus, were selected for in vivo evaluation, based on their in vitro neutralization potency. A single dose of either MV33 or EV42 administered three days post-infection (dpi) to BALB/c female mice provides full protection against lethal ectromelia virus challenge. Importantly, a combination of both mAbs confers full protection even when provided five dpi. Whole-body bioimaging and viral load analysis reveal that combination of the two mAbs allows for faster and more efficient clearance of the virus from target organs compared to either MV33 or EV42 separately. The combined mAbs treatment further confers post-exposure protection against the currently circulating Monkeypox virus in Cast/EiJ female mice, highlighting their therapeutic potential against other orthopoxviruses.

Drug resistance in HIV type 1 (HIV-1) is a pervasive problem that affects the lives of millions of people worldwide. Although records of drug-resistant mutations (DRMs) have been extensively tabulated within public repositories, our understanding of the evolutionary kinetics of DRMs and how they evolve together remains limited. Epistasis, the interaction between a DRM and other residues in HIV-1 protein sequences, is key to the temporal evolution of drug resistance. We use a Potts sequence-covariation statistical-energy model of HIV-1 protein fitness under drug selection pressure, which captures epistatic interactions between all positions, combined with kinetic Monte-Carlo simulations of sequence evolutionary trajectories, to explore the acquisition of DRMs as they arise in an ensemble of drug-naive patient protein sequences. We follow the time course of 52 DRMs in the enzymes protease, RT, and integrase, the primary targets of antiretroviral therapy. The rates at which DRMs emerge are highly correlated with their observed acquisition rates reported in the literature when drug pressure is applied. This result highlights the central role of epistasis in determining the kinetics governing DRM emergence. Whereas rapidly acquired DRMs begin to accumulate as soon as drug pressure is applied, slowly acquired DRMs are contingent on accessory mutations that appear only after prolonged drug pressure. We provide a foundation for using computational methods to determine the temporal evolution of drug resistance using Potts statistical potentials, which can be used to gain mechanistic insights into drug resistance pathways in HIV-1 and other infectious agents.

Citrus cultivation plays a pivotal role, making a significant contribution to global fruit production and dietary consumption. Accurate identification of viral pathogens is imperative for the effective management of plant viral disease in citrus crops. High-throughput sequencing serves as an alternative approach, enabling comprehensive pathogen identification on a large scale without requiring pre-existing information. In this study, we employed HTS to investigate viral pathogens infecting citrus in three different regions of South Korea: Jejudo (Jeju), Wando-gun (Wando), and Dangjin-si (Dangjin). The results unveiled diverse viruses and viroids that exhibited regional variations. Notably, alongside the identification of well-known citrus viruses such as satsuma dwarf virus, citrus tatter leaf virus, and citrus leaf blotch virus (CLBV), this study also uncovered several viruses and viroids previously unreported in Korean citrus. Phylogenetic analysis revealed that majority of identified viruses exhibited the closest affilations with isolates from China or Japan. However, CLBV and citrus viroid-I-LSS displayed diverse phylogenetic positions, reflecting their regional origins. This study advances our understanding of citrus virome diversity and regional dynamics through HTS, emphasizing its potential in unraveling intricate viral pathogens in agriculture. Consequently, it significantly contributes to disease management strategies, ensuring the resilience of the citrus industry.

RNA viruses and viroids exist and evolve as quasispecies due to error-prone replication. Quasispecies consist of a few dominant master sequences alongside numerous variants that contribute to genetic diversity. Upon environmental changes, certain variants within quasispecies have the potential to become the dominant sequences, leading to the emergence of novel infectious strains. However, the emergence of new infectious variants remains unpredictable. Using mutant pools prepared by saturation mutagenesis of selected stem and loop regions, our study of potato spindle tuber viroid (PSTVd) demonstrates that mutants forming local three-dimensional (3D) structures similar to the wild type (WT) are more likely to accumulate in PSTVd quasispecies. The selection mechanisms underlying this biased accumulation are likely associated with cell-to-cell movement and long-distance trafficking. Moreover, certain trafficking-defective PSTVd mutants can be spread by functional sister genomes in the quasispecies. Our study reveals that the RNA 3D structure of stems and loops constrains the evolution of viroid quasispecies. Mutants with a structure similar to WT have a higher likelihood of being maintained within the quasispecies and can potentially give rise to novel infectious variants. These findings emphasize the potential of targeting RNA 3D structure as a more robust approach to defend against viroid infections.

RNA viruses and viroids replicate with high mutation rates, forming quasispecies, population of variants centered around dominant sequences. The mechanisms governing quasispecies remain unclear. Plasmodesmata regulate viroid movement and were hypothesized to impact viroid quasispecies. Here, we sequenced the progeny of potato spindle tuber viroid intermediate (PSTVd-I) strain from mature guard cells lacking plasmodesmal connections and from in vitro-cultivated mesophyll cell protoplasts from systemic leaves of early-infected tomato (Solanum lycopersicum) plants. Remarkably, more variants accumulated in guard cells compared to whole leaves. Similarly, after extended cell culture, we observed more variants in cultivated mesophyll protoplasts. Coinfection and single-cell sequencing experiments demonstrated that the same plant cell can be infected multiple times by the same or different PSTVd sequences. To study the impact of initial population composition on PSTVd-I quasispecies, we conducted coinfections with PSTVd-I and variants. Two inoculum ratios (10:1 or 1:10) established quasispecies with or without PSTVd-I as the master sequence. In the absence of the master sequence, the percentage of novel variants initially increased. Moreover, a 1:1 PSTVd-I/variant RNA ratio resulted in PSTVd-I dominating (>50%), while the variants reached 20%. After PSTVd-I-only infection, the variants reached around 10%, while after variant-only infection, the variants were significantly more than 10%. These results emphasize the role of cell-to-cell communication and initial population composition in shaping PSTVd quasispecies.

Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success. Taking advantage of the uniquely large genomic database of SARS-CoV-2, we examine the impact of mutations across the spectrum of viable amino acid sequences on the biophysical phenotypes of the highly expressed and multifunctional nucleocapsid protein. We find variation in the physicochemical parameters of its extended intrinsically disordered regions (IDRs) sufficient to allow local plasticity, but also exhibiting functional constraints that similarly occur in related coronaviruses. In biophysical experiments with several N-protein species carrying mutations associated with major variants, we find that point mutations in the IDRs can have nonlocal impact and modulate thermodynamic stability, secondary structure, protein oligomeric state, particle formation, and liquid-liquid phase separation. In the Omicron variant, distant mutations in different IDRs have compensatory effects in shifting a delicate balance of interactions controlling protein assembly properties, and include the creation of a new protein-protein interaction interface in the N-terminal IDR through the defining P13L mutation. A picture emerges where genetic diversity is accompanied by significant variation in biophysical characteristics of functional N-protein species, in particular in the IDRs.

Single-strand RNA (ssRNA) and single-strand DNA phages elicit host lysis using a single gene, in each case designated as sgl. Of the 11 identified Sgls, three have been shown to be specific inhibitors of different steps in the pathway that supplies lipid II to the peptidoglycan (PG) biosynthesis machinery. These Sgls have been called "protein antibiotics" because the lytic event is a septal catastrophe indistinguishable from that caused by cell wall antibiotics. Here, we designate these as type I Sgls. In this formalism, the other eight Sgls are assigned to type II, the best-studied of which is protein L of the paradigm F-specific ssRNA phage MS2. Comparisons have suggested that type II Sgls have four sequence elements distinguished by hydrophobic and polar character. Environmental metatranscriptomics has revealed thousands of new ssRNA phage genomes, each of which presumably has an Sgl. Here, we describe methods to distinguish type I and type II Sgls. Using phase contrast microscopy, we show that both classes of Sgls cause the formation of blebs prior to lysis, but the location of the blebs differs significantly. In addition, we show that L and other type II Sgls do not inhibit the net synthesis of PG, as measured by radio-labeling of PG. Finally, we provide direct evidence that the Sgl from Pseudomonas phage PP7 is a type I Sgl, in support of a recent report based on a genetic selection. This shows that the putative four-element sequence structure suggested for L is not a reliable discriminator for the operational characterization of Sgls.

The ssRNA phage world has recently undergone a metagenomic expansion upward of a thousandfold. Each genome likely carries at least one single-gene lysis (sgl) cistron encoding a protein that single-handedly induces host autolysis. Here, we initiate an approach to segregate the Sgls into operational types based on physiological analysis, as a first step toward the alluring goal of finding many new ways to induce bacterial death and the attendant expectations for new antibiotic development.

Despite extensive scientific efforts directed toward the evolutionary trajectory of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans at the beginning of the COVID-19 epidemic, it remains unclear how the virus jumped into and evolved in humans so far. Herein, we recruited almost all adult coronavirus disease 2019 (COVID-19) cases appeared locally or imported from abroad during the first 8 months of the outbreak in Shanghai. From these patients, SARS-CoV-2 genomes occupying the important phylogenetic positions in the virus phylogeny were recovered. Phylogenetic and mutational landscape analyses of viral genomes recovered here and those collected in and outside of China revealed that all known SARS-CoV-2 variants exhibited the evolutionary continuity despite the co-circulation of multiple lineages during the early period of the epidemic. Various mutations have driven the rapid SARS-CoV-2 diversification, and some of them favor its better adaptation and circulation in humans, which may have determined the waxing and waning of various lineages.

Viral pandemics and epidemics pose a significant global threat. While macaque models of viral disease are routinely used, it remains unclear how conserved antiviral responses are between macaques and humans. Therefore, we conducted a cross-species analysis of transcriptomic data from over 6,088 blood samples from macaques and humans infected with one of 31 viruses. Our findings demonstrate that irrespective of primate or viral species, there are conserved antiviral responses that are consistent across infection phase (acute, chronic, or latent) and viral genome type (DNA or RNA viruses). Leveraging longitudinal data from experimental challenges, we identify virus-specific response kinetics such as host responses to Coronaviridae and Orthomyxoviridae infections peaking 1-3 days earlier than responses to Filoviridae and Arenaviridae viral infections. Our results underscore macaque studies as a powerful tool for understanding viral pathogenesis and immune responses that translate to humans, with implications for viral therapeutic development and pandemic preparedness.

The SARS-CoV-2 virus, which is a major threat to human health, has undergone many mutations during the replication process due to errors in the replication steps and modifications in the structure of viral proteins. The XBB variant was identified for the first time in Singapore in the fall of 2022. It was then detected in other countries, including the United States, Canada, and the United Kingdom. We study the impact of sequence changes on spike protein structure on the subvariants of XBB, with particular attention to the velocity of variant diffusion and virus activity with respect to its diffusion. We examine the structural and functional distinctions of the variants in three different conformations: (i) spike glycoprotein in complex with ACE2 (1-up state), (ii) spike glycoprotein (closed-1 state), and (iii) S protein (open-1 state). We also estimate the affinity binding between the spike protein and ACE2. The market binding affinity observed in specific variants raises questions about the efficacy of current vaccines in preparing the immune system for virus variant recognition. This work may be useful in devising strategies to manage the ongoing COVID-19 pandemic. To stay ahead of the virus evolution, further research and surveillance should be carried out to adjust public health measures accordingly.

RNA viruses lack proofreading in their RNA polymerases and therefore exist as genetically diverse populations. By exposing these diverse viral populations to selective pressures, viruses with mutations that confer fitness advantages can be enriched. To examine factors important for viral tropism and host restriction, we passaged murine norovirus (MNV) in a human cell line, HeLa cells, to select for mutant viruses with increased fitness in non-murine cells. A major determinant of host range is expression of the MNV receptor CD300lf on mouse cells, but additional host factors may limit MNV replication in human cells. We found that viruses passaged six times in HeLa cells had enhanced replication compared with the parental virus. The passaged viruses had several mutations throughout the viral genome, which were primarily located in the viral non-structural coding regions. While viral attachment was not altered for the passaged viruses, their replication was higher than the parental virus when entry was bypassed, suggesting the mutant viruses overcame a post-entry block in human cells. Three mutations in the viral NS1 protein were sufficient for enhanced post-entry replication in human cells. We found that the human cell-adapted MNV variants had reduced fitness in mouse BV2 cells. Although the mutant viruses had increased fitness in HeLa cells, they did not have increased fitness in mice. Overall, this work suggests that MNV tropism is not only determined by the presence of the viral receptor but also post-entry factors.