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In the 2013–14 and 2015–16 influenza seasons, reduced vaccine effectiveness (VE) was observed for the H1N1 component of the FluMist quadrivalent live attenuated influenza vaccine (QLAIV) in the USA, leading to loss of Advisory Committee on Immunization Practices recommendation. Here we demonstrate in ferrets that 2015–16A/H1N1pdm09 vaccine strain A/Bolivia/559/2013 (A/BOL13) is outcompeted in trivalent (TLAIV) and QLAIV formulations, leading to reduced protection from wild-type challenge. While monovalent (MLAIV) A/BOL13 provided significant protection from wild-type virus shedding and fever at doses as low as 3.0 log10 fluorescent focus units (FFU), it failed to provide a similar level of protection in TLAIV or QLAIV formulation, even at a 6.0 log10 FFU dose. Conversely, clinically effective H1N1 strain A/New Caledonia/20/1999 provided significant protection in MLAIV, TLAIV, and QLAIV formulations. In conclusion, reduced A/BOL13 replicative fitness rendered it susceptible to inter-strain competition in QLAIV, contributing to its reduced VE in the 2015–16 season.

Graphical abstract, The high mutation rate in retroviruses is one of the leading causes of drug resistance. In human immunodeficiency virus type-1 (HIV-1), synergistic mutations in its protease and the protease substrate – the Group-specific antigen (Gag) polyprotein – work together to confer drug resistance against protease inhibitors and compensate the mutations affecting viral fitness. Some Gag mutations can restore Gag-protease binding, yet most Gag-protease correlated mutations occur outside of the Gag cleavage site. To investigate the molecular basis for this, we now report multiscale modelling approaches to investigate various sequentially cleaved Gag products in the context of clinically relevant mutations that occur outside of the cleavage sites, including simulations of the largest Gag proteolytic product in its viral membrane-bound state. We found that some mutations, such as G123E and H219Q, involve direct interaction with cleavage site residues to influence their local environment, while certain mutations in the matrix domain lead to the enrichment of lipids important for Gag targeting and assembly. Collectively, our results reveal why non-cleavage site mutations have far-reaching implications outside of Gag proteolysis, with important consequences for drugging Gag maturation intermediates and tackling protease inhibitor resistance.

Compared to the previous 2013–2014 outbreak, dengue 2016–2017 outbreak in New Caledonia was characterized by an increased number of severe forms associated with hepatic presentations. In this study, we assessed the virological factors associated with this enhanced severity. Whole-genome sequences were retrieved from dengue virus (DENV)-1 strains collected in 2013–2014 and from severe and non-severe patients in 2016–2017. Fitness, hepatic tropism and cytopathogenicity of DENV 2016–2017 strains were compared to those of 2013–2014 strains using replication kinetics in the human hepatic cell line HuH7. Whole-genome sequencing identified four amino acid substitutions specific to 2016–2017 strains and absent from 2013–2014 strains. Three of these mutations occurred in predicted T cell epitopes, among which one was also a B cell epitope. Strains retrieved from severe forms did not exhibit specific genetic features. DENV strains from 2016–2017 exhibited a trend towards reduced replicative fitness and cytopathogenicity in vitro compared to strains from 2013–2014. Overall, the 2016–2017 dengue outbreak in New Caledonia was associated with a viral genetic evolution which had limited impact on DENV hepatic tropism and cytopathogenicity. These mutations, however, may have modified DENV strains antigenicity, altering the anti-DENV immune response in some patients, in turn favoring the development of severe forms. Trial registration: ClinicalTrials.gov identifier: NCT04615364.

Dengue virus (DENV), like other viruses, closely interacts with the host cell machinery to complete its life cycle. Over the course of infection, DENV interacts with several host factors with pro-viral activities to support its infection. Meanwhile, it has to evade or counteract host factors with anti-viral activities which inhibit its infection. These molecular virus-host interactions play a crucial role in determining the success of DENV infection. Deciphering such interactions is thus paramount to understanding viral fitness in its natural hosts. While DENV-mammalian host interactions have been extensively studied, not much has been done to characterize DENV-mosquito host interactions despite its importance in controlling DENV transmission. Here, to provide a snapshot of our current understanding of DENV-mosquito interactions, we review the literature that identified host factors and cellular processes related to DENV infection in its mosquito vectors, Aedes aegypti and Aedes albopictus, with a particular focus on DENV-mosquito omics studies. This knowledge provides fundamental insights into the DENV life cycle, and could contribute to the development of novel antiviral strategies.

The emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic variants that may alter viral fitness highlights the urgency of widespread next-generation sequencing (NGS) surveillance. To profile genetic variants of the entire SARS-CoV-2 genome, we developed and clinically validated a hybridization capture SARS-CoV-2 NGS assay, integrating novel methods for panel design using double-stranded DNA (dsDNA) biotin-labeled probes, and built accompanying software. This test is the first hybrid capture-based NGS assay given Food and Drug Administration (FDA) emergency use authorization for detection of the SARS-CoV-2 virus. The positive and negative percent agreement (PPA and NPA, respectively) were defined in comparison to the results for an orthogonal real-time reverse transcription polymerase chain reaction (RT-PCR) assay (PPA and NPA, 96.7 and 100%, respectively). The limit of detection was established to be 800 copies/ml with an average fold enrichment of 46,791. Furthermore, utilizing the research-use-only analysis to profile the variants, we identified 55 novel mutations, including 11 in the functionally important spike protein. Finally, we profiled the full nasopharyngeal microbiome using metagenomics and found overrepresentation of 7 taxa and evidence of macrolide resistance in SARS-CoV-2-positive patients. This hybrid capture NGS assay, coupled with optimized software, is a powerful approach to detect and comprehensively map SARS-CoV-2 genetic variants for tracking viral evolution and guiding vaccine updates. IMPORTANCE This is the first FDA emergency-use-authorized hybridization capture-based next-generation sequencing (NGS) assay to detect the SARS-CoV-2 genome. Viral metagenomics and the novel hybrid capture NGS-based assay, along with its research-use-only analysis, can provide important genetic insights into SARS-CoV-2 and other emerging pathogens and improve surveillance and early detection, potentially preventing or mitigating new outbreaks. Better understanding of the continuously evolving SARS-CoV-2 viral genome and the impact of genetic variants may provide individual risk stratification, precision therapeutic options, improved molecular diagnostics, and population-based therapeutic solutions.

Severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) evolves and generates different variants through a continuously branching model. Four variants of concern have been the major pandemic drivers around the world. One important question is how they may evolve and generate subvariants, some of which may be even more virulent and drive the pandemic further. While investigating how γ (or P.1) variant has been evolving, I noticed the spike substitution P681H in a group of genomes encoding a new subvariant, which has been designated P.1.7. This subvariant has become the dominant P.1 sublineage in Brazil, Italy, Spain and Peru, supporting that P681H confers evolutionary advantage to P.1.7. In Brazil and Peru, P.1.7 was still responsible for ~30% and ~40% cases, respectively, in August 2021. However, it has been competed out by δ1 (a δ subvariant) in both countries, Italy and Spain, suggesting that P.1.7 is not as virulent as δ1. In addition, 160 P.1 genomes possess a related substitution, P681R, and 120 of them encode a new subvariant, designated P.1.8. This P.1 subvariant carries two additional spike substitutions, T470N and C1235F, located at the receptor-binding pocket and cytoplasmic tail of spike protein, respectively. More P.1.8 genomes have been identified than P.1 genomes that encode P681R but not T470N and C1235F, suggesting that these two substitutions improve virulence of P.1.8 subvariant. Some P.1 genomes carry other substitutions (such as N679K, V687L and C1250F) that affect the furin cleavage site or cytoplasmic tail of spike protein. Thus, to improve viral fitness and expand its evolutionary cage, γ variant acquires mutations to finetune the furin cleavage site and cytoplasmic tail of spike protein.

The understanding of the molecular mechanisms driving the fitness of the SARS-CoV-2 virus and its mutational evolution is still a critical issue. We built a simplified computational model, called SpikePro, to predict the SARS-CoV-2 fitness from the amino acid sequence and structure of the spike protein. It contains three contributions: the inter-human transmissibility of the virus predicted from the stability of the spike protein, the infectivity computed in terms of the affinity of the spike protein for the ACE2 receptor, and the ability of the virus to escape from the human immune response based on the binding affinity of the spike protein for a set of neutralizing antibodies. Our model reproduces well the available experimental, epidemiological and clinical data on the impact of variants on the biophysical characteristics of the virus. For example, it is able to identify circulating viral strains that, by increasing their fitness, recently became dominant at the population level. SpikePro is a useful, freely available instrument which predicts rapidly and with good accuracy the dangerousness of new viral strains. It can be integrated and play a fundamental role in the genomic surveillance programs of the SARS-CoV-2 virus that, despite all the efforts, remain time-consuming and expensive., SCOPUS: ar.j, info:eu-repo/semantics/published

Arming oncolytic adenoviruses with therapeutic transgenes is a well-established strategy for multimodal tumour attack. However, this strategy sometimes leads to unexpected attenuated viral replication and a loss of oncolytic effects, preventing these viruses from reaching the clinic. Previous work has shown that altering codon usage in viral genes can hamper viral fitness. Here, we have analysed how transgene codon usage impacts viral replication and oncolytic activity. We observe that, although transgenes with optimized codons show high expression levels at the first round of infection, they impair viral fitness and are therefore not expressed in a sustained manner. Conversely, transgenes encoded by suboptimal codons do not compromise viral replication and are thus stably expressed over time, allowing a greater oncolytic activity both in vitro and in vivo. Altogether, our work shows that fine-tuning codon usage leads to a concerted optimization of transgene expression and viral replication paving the way for the rational design of more efficacious oncolytic therapies.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), which has spread worldwide since it was first identified in Wuhan, China, at the end of 2019. With the global transmission of the virus, a large number of SARS-CoV-2 variants have also appeared, especially, emerging strains that have recently been discovered in the United Kingdom (variant 20I/501Y.V1, lineage B.1.1.7), South Africa (variant 20H/501Y.V2, lineage B.1.351), and Brazil (variant 20 J/501Y.V3, and lineage P.1). The common feature of these variants is that they share the N501Y mutation involving the SARS-CoV-2 spike (S) protein, which is precisely the target of most COVID-19 vaccines. Furthermore, mutations such as N501Y, E484K, and K417N in the S protein may affect viral fitness and transmissibility. However, current research on the impact of these variants on COVID-19 vaccines is still lacking. Herein, we briefly explain why most COVID-19 vaccines target the S protein, update the progress of research regarding S protein-related COVID-19 vaccines, review the latest studies concerning the effects of S protein variants on COVID-19 vaccines, and finally, propose certain strategies to deal with SARS-CoV-2 variants., Graphical Abstract

Global spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has triggered unprecedented scientific efforts, as well as containment and treatment measures. Despite these efforts, SARS-CoV-2 infections remain unmanageable in some parts of the world. Due to inherent mutability of RNA viruses, it is not surprising that the SARS-CoV-2 genome has been continuously evolving since its emergence. Recently, four functionally distinct variants, B.1.1.7, B.1.351, P.1 and CAL.20C, have been identified, and they appear to more infectious and transmissible than the original (Wuhan-Hu-1) virus. Here we provide evidence based upon a combination of bioinformatics and structural approaches that can explain the higher infectivity of the new variants. Our results show that the greater infectivity of SARS-CoV-2 than SARS-CoV can be attributed to a combination of several factors, including alternate receptors. Additionally, we show that new SARS-CoV-2 variants emerged in the background of D614G in Spike protein and P323L in RNA polymerase. The correlation analyses showed that all mutations in specific variants did not evolve simultaneously. Instead, some mutations evolved most likely to compensate for the viral fitness.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution is expected, given the nature of virus replication. Selection and establishment of variants in the human population depend on viral fitness and on molecular and immunological selection pressures. Here we discuss how mechanisms of replication and recombination may contribute to the emergence of current and future variants of SARS-CoV-2.

The recent identification and rise to dominance of the P.1 and B.1.351 SARS-CoV-2 variants have brought international concern because they may confer fitness advantages. The same three positions in the receptor-binding domain (RBD) are affected in both variants, but where the 417 substitution differs, the E484K/N501Y have co-evolved by convergent evolution. Here we characterize the functional and immune evasive consequences of the P.1 and B.1.351 RBD mutations. E484K and N501Y result in gain-of-function with two different outcomes: The N501Y confers a ten-fold affinity increase towards ACE-2, but a modest antibody evasion potential of plasma from convalescent or vaccinated individuals, whereas the E484K displays a significant antibody evasion capacity without a major impact on affinity. On the other hand, the two different 417 substitutions severely impair the RBD/ACE-2 affinity, but in the combined P.1 and B.1.351 RBD variants, this effect is partly counterbalanced by the effect of the E484K and N501Y. Our results suggest that the combination of these three mutations is a two-step forward and one step back in terms of viral fitness.

Codon usage bias refers to the preferential use of some synonymous codons over others and is a key evolutionary feature in a genome that provides important information for studying the organism’s evolution. Codon usage bias is also important to shed light on the determining mechanisms of viral fitness, virus-host interaction, pathogenesis, host immune escape as well as viral emergence and strategies of intervention. This chapter focuses on the evolutionary determinants of codon usage in organisms and research in Zika virus molecular evolution and codon usage. Virus codon usage is determined by virus- and host-dependent processes, and it likely evolves in response to different selective forces. Additionally, virus codon usage knowledge can be used to rationally combat them.

In 2018, baloxavir marboxil, which targets the polymerase acidic (PA) protein of influenza A and B viruses, was licensed in Japan and the United States. This anti-influenza drug is highly effective against these virus infections. During the 2018–2019 influenza season in Japan, however, influenza A viruses carrying an I38T mutation in PA that confers reduced susceptibility to baloxavir acid (the active form of baloxavir marboxil) were detected at a relatively high frequency in pediatric patients after treatment with this drug. In addition, influenza A virus PA-I38T variants were detected in patients before drug treatment. In animal models, the replicative abilities, pathogenicity, and transmissibility of influenza A virus PA-I38T variants isolated from patients have been shown to be comparable to those of wild-type isolates. In this chapter, we summarize recent findings regarding the fitness of influenza A viruses with reduced susceptibility to baloxavir acid isolated from patients.

Fusarium graminearum virus 1 (FgV1) is a positive-sense ssRNA virus that confers hypovirulence in its fungal host, Fusarium graminearum. Like most mycoviruses, FgV1 exists in fungal cells, lacks an extracellular life cycle, and is therefore transmitted during sporulation or hyphal anastomosis. To understand FgV1 evolution and/or adaptation, we conducted mutation accumulation (MA) experiments by serial passage of FgV1 alone or with FgV2, 3, or 4 in F. graminearum. We expected that the effects of positive selection would be highly limited because of repeated bottleneck events. To determine whether selection on the virus was positive, negative, or neutral, we assessed both the phenotypic traits of the host fungus and the RNA sequences of FgV1. We inferred that there was positive selection on beneficial mutations in FgV1 based on the ratio of non-synonymous to synonymous substitutions (d N /d S ), on the ratio of radical to conservation amino acid replacements (p NR /p NC ), and by changes in the predicted protein structures. In support of this inference, we found evidence of positive selection only in the open reading frame 4 (ORF4) protein of DK21/FgV1 (MA line 1); mutations at amino acids 163A and 289H in the ORF4 of MA line 1 affected the entire structure of the protein predicted to be under positive selection. We also found, however, that deleterious mutations were a major driving force in viral evolution during serial passages. Linear relationships between changes in viral fitness and the number of mutations in each MA line demonstrated that some deleterious mutations resulted in fitness decline. Several mutations in MA line 1 were not shared with any of the other four MA lines (PH-1/FgV1, PH-1/FgV1 + 2, PH-1/FgV1 + 3, and PH-1/FgV1 + 4). This suggests that evolutionary pathways of the virus could differ with respect to hosts and also with respect to co-infecting viruses. The data also suggested that the differences among MA lines might also be explained by mutational robustness and other unidentified factors. Additional research is needed to clarify the effects of virus co-infection on the adaptation or evolution of FgV1 to its environments.

Dendritic cell (DC) subsets are abundantly present in genital and intestinal mucosal tissue and are among the first innate immune cells that encounter human immunodeficiency virus type 1 (HIV-1) after sexual contact. Although DCs have specific characteristics that greatly enhance HIV-1 transmission, it is becoming evident that most DC subsets also have virus restriction mechanisms that exert selective pressure on the viruses during sexual transmission. In this review we discuss the current concepts of the immediate events following viral exposure at genital mucosal sites that lead to selection of specific HIV-1 variants called transmitted founder (TF) viruses. We highlight the importance of the TF HIV-1 phenotype and the role of different DC subsets in establishing infection. Understanding the biology of HIV-1 transmission will contribute to the design of novel treatment strategies preventing HIV-1 dissemination.

India has become the third worst-hit nation by the COVID-19 pandemic caused by the SARS-CoV-2 virus. Here, we investigated the molecular, phylogenomic, and evolutionary dynamics of SARS-CoV-2 in western India, the most affected region of the country. A total of 90 genomes were sequenced. Four nucleotide variants, namely C241T, C3037T, C14408T (Pro4715Leu), and A23403G (Asp614Gly), located at 5’UTR, Orf1a, Orf1b, and Spike protein regions of the genome, respectively, were predominant and ubiquitous (90%). Phylogenetic analysis of the genomes revealed four distinct clusters, formed owing to different variants. The major cluster (cluster 4) is distinguished by mutations C313T, C5700A, G28881A are unique patterns and observed in 45% of samples. We thus report a newly emerging pattern of linked mutations. The predominance of these linked mutations suggests that they are likely a part of the viral fitness landscape. A novel and distinct pattern of mutations in the viral strains of each of the districts was observed. The Satara district viral strains showed mutations primarily at the 3′ end of the genome, while Nashik district viral strains displayed mutations at the 5′ end of the genome. Characterization of Pune strains showed that a novel variant has overtaken the other strains. Examination of the frequency of three mutations i.e., C313T, C5700A, G28881A in symptomatic versus asymptomatic patients indicated an increased occurrence in symptomatic cases, which is more prominent in females. The age-wise specific pattern of mutation is observed. Mutations C18877T, G20326A, G24794T, G25563T, G26152T, and C26735T are found in more than 30% study samples in the age group of 10-25. Intriguingly, these mutations are not detected in the higher age range 61-80. These findings portray the prevalence of unique linked mutations in SARS-CoV-2 in western India and their prevalence in symptomatic patients.ImportanceElucidation of the SARS-CoV-2 mutational landscape within a specific geographical location, and its relationship with age and symptoms, is essential to understand its local transmission dynamics and control. Here we present the first comprehensive study on genome and mutation pattern analysis of SARS-CoV-2 from the western part of India, the worst affected region by the pandemic. Our analysis revealed three unique linked mutations, which are prevalent in most of the sequences studied. These may serve as a molecular marker to track the spread of this viral variant to different places.

Oncolytic adenoviruses (OAds) present limited efficacy in clinics. The insertion of therapeutic transgenes into OAds genomes, known as &ldquo, arming OAds&rdquo, has been the main strategy to improve their therapeutic potential. Different approaches were published in the decade of the 2000s, but with few comparisons. Most armed OAds have complete or partial E3 deletions, leading to a shorter half-life in vivo. We generated E3+ OAds using two insertion sites, After-fiber and After-E4, and two different splice acceptors linked to the major late promoter, either the Ad5 protein IIIa acceptor (IIIaSA) or the Ad40 long fiber acceptor (40SA). The highest transgene levels were obtained with the After-fiber location and 40SA. However, the set of codons of the transgene affected viral fitness, highlighting the relevance of transgene codon usage when arming OAds using the major late promoter.

The coronavirus (CoV) family consists of viruses that infects a variety of animals including humans with various levels of respiratory and fecal-oral transmission levels depending on the behavior of the viruses' natural hosts and optimal viral fitness. A model to classify and predict the levels of respective respiratory and fecal-oral transmission potentials of the various viruses was built before the outbreak of MERS-CoV using AI and empirically-based molecular tools to predict the disorder level of proteins. Using the percentages of intrinsic disorder (PID) of the nucleocapsid (N) and membrane (M) proteins of CoV, the model easily clustered the viruses into three groups with the SARS-CoV (M PID = 8%, N PID = 50%) falling into Category B, in which viruses have intermediate levels of both respiratory and fecal-oral transmission potentials. Later, MERS-CoV (M PID = 9%, N PID = 44%) was found to be in Category C, which consists of viruses with lower respiratory transmission potential but with higher fecal-oral transmission capabilities. Based on the peculiarities of disorder distribution, the SARS-CoV-2 (M PID = 6%, N PID = 48%) has to be placed in Category B. Our data show however, that the SARS-CoV-2 is very strange with one of the hardest protective outer shell, (M PID = 6%) among coronaviruses. This means that it might be expected to be highly resilient in saliva or other body fluids and outside the body. An infected body is likelier to shed greater numbers of viral particles since the latter is more resistant to antimicrobial enzymes in body fluids. These particles are also likelier to remain active longer. These factors could account for the greater contagiousness of the SARS-CoV-2 and have implications for efforts to prevent its spread.

Murine γ-herpesvirus-68 (MHV-68), genetically and biologically related to human γ-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, can be easily propagated in vitro allowing drug resistance studies. Previously, we described specific changes in MHV-68 protein kinase (PK) or thymidine kinase (TK) associated with resistance to various purine or pyrimidine nucleoside analogues, respectively. To investigate how specific TK and PK mutations affect viral replication capacity, we performed dual infection competition assays in which wild-type and drug-resistant virus compete in absence or presence of antivirals in Vero cells. The composition of the mixed viral population was analyzed using next-generation sequencing and relative fitness of seven MHV-68 PK or TK mutants was calculated based on the frequency of viral variants at the time of infection and after 5-days growth. A MHV-68 mutant losing the PK function due to a 2-nucleotide deletion was less fit than the wild-type virus in absence of antivirals, consistent with the essential role of viral PKs during lytic replication, but overgrew the wild-type virus under pressure of purine nucleosides. TK mutant viruses, with frameshift or missense mutations, grew equal to wild-type virus in absence of antivirals, in accordance with the viral TK function only being essential in non-replicating or in TK-deficient cells, but were more fit when treated with pyrimidine nucleosides. Moreover, TK missense mutant viruses also increased fitness under pressure of antivirals other than pyrimidine nucleosides, indicating that MHV-68 TK mutations might influence viral fitness by acting on cellular and/or viral functions that are unrelated to nucleoside activation. ispartof: ANTIVIRAL RESEARCH vol:182 ispartof: location:Netherlands status: published

Viruses span an impressive size range, with genome length varying more than a thousandfold and capsid volume nearly a millionfold. Physical constraints suggest that smaller viruses may have multiple fitness advantages, because a greater number of viral offspring can be created from limited host resources, and because smaller particles diffuse to encounter new hosts more rapidly. At the same time, a larger genome size allows for numerous additional functions that may increase fitness, such as better control of replication, transcription, translation, and host metabolism, and neutralization of host defenses. However, it is unclear whether important viral traits correlate with size, and whether this causes size to vary among host types or environmental contexts. Here we focus on viruses of aquatic unicellular organisms, which exhibit the greatest known range of virus size. We develop and synthesize theory, and analyze data where available, to consider how size affects the primary components of viral fitness. We suggest that the costs of larger size (lower burst size and diffusivity) are mitigated by the role of a larger genome in increasing infection efficiency, broadening host range, and potentially increasing attachment success and decreasing decay rate. These countervailing selective pressures may explain why such a breadth of sizes exist and can even coexist when infecting the same host populations. We argue that oligotrophic environments may be particularly enriched in unusually large or “giant” viruses, because environments with diverse, resource-limited phagotrophic eukaryotes at persistently low concentrations may select for broader host range, better control of host metabolism, lower decay rate, and a physical size that mimics bacterial prey. Finally, we describe areas where further research is needed to understand the ecology and evolution of viral size diversity.

The large dsDNA virus herpes simplex virus 1 (HSV-1) is considered to be genetically stable, yet it can rapidly evolve in response to strong selective pressures such as antiviral treatment. Deep sequencing has revealed that clinical and laboratory isolates of this virus exist as populations that contain a mixture of minor alleles or variants, similar to many RNA viruses. The classic virology approach of plaque purifying virus creates a genetically homogenous population, but it is not clear how closely this represents the mixed virus populations found in nature. We sought to study the evolution of mixed versus highly purified HSV-1 populations in controlled cell culture conditions, to examine the impact of this genetic diversity on evolution. We found that a mixed population of HSV-1 acquired more genetic diversity and underwent a more dramatic phenotypic shift than a plaque-purified population, producing a viral population that was almost entirely syncytial after just ten passages. At the genomic level, adaptation and genetic diversification occurred at the level of minor alleles or variants in the viral population. Certain genetic variants in the mixed viral population appeared to be positively selected in cell culture, and this shift was also observed in clinical samples during their first passages in vitro. In contrast, the plaque-purified viral population did not appear to change substantially in phenotype or overall quantity of minor allele diversity. These data indicate that HSV-1 is capable of evolving rapidly in a given environment, and that this evolution is facilitated by diversity in the viral population.

Virus-specific memory B cells (Bmem) play a crucial role in protecting against variant viruses. The ability to recognize these variant viruses, defined as antibody breadth, is achieved in Bmem populations by two very different pathways, germline-encoded cross-reactivity and affinity-driven, somatic evolution in germinal centers (GCs) for conserved viral epitopes. The latter class of broadly-reactive Bmem cells are not cross-reactive per se, but bind epitopes crucial for viral fitness. Although these conserved epitopes are often weakly immunogenic, the GC reaction is surprisingly permissive for the continued survival/proliferation of B cells that bind with low affinity or react to cryptic epitopes, increasing their chance of memory recruitment. In this review, we discuss the adaptive strategies of B-cell memory to viral antigenic variations.

Env-pseudotyped viruses are valuable reagents for studies of HIV-1 neutralizing antibodies. It is often assumed that all pseudovirus particles are capable of only a single round of infection, making them a safe alternative to work with live HIV-1. In this study, we show that some Env-pseudotyped virus preparations give rise to low levels of replication-competent virus. These levels did not compromise results in the TZM-bl neutralization assay; however, their presence highlights a need to adhere to the same level of biosafety when working with Env-pseudotyped viruses that are required for work with replication competent HIV-1.

The ability of viruses to infect their hosts depends on rapid dissemination following transmission. The notion that viral particles function as independent propagules has been challenged by recent observations suggesting that viral aggregates show enhanced infectivity and faster spread. However, these observations remain poorly understood. Here, we show that viral replication is a cooperative process, such that entry of multiple viral genome copies into the same cell disproportionately increases short-term viral progeny production. This cooperativity arises from the positive feedback established between replication templates and virus-encoded products involved in replication and should be a general feature of viruses. We develop a simple model that captures this effect, verify that cooperativity also emerges in more complex models for specific human viruses, validate our predictions experimentally using different mammalian viruses, and discuss the implications of cooperative replication for viral fitness., This work was funded by ERC Consolidator Grant 724519 (Vis-a-Vis) and the Spanish Ministerio de Ciencia, Innovación y Universidades (grant BFU2017-84762-R). I.A.-M. was funded by a Ph.D. fellowship from the Spanish Ministerio de Ciencia, Innovación y Universidades. J.-V.B. was funded by Prometeo Grant 2016/122 from the Generalitat Valenciana. A

Despite the high virological response rates achieved with current directly acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral response. The identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultradeep sequencing (UDS) methods, for HCV characterization and patient management. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV, which are not bona fide resistance-associated substitutions (RAS), from 220 patients who failed therapy. They were present frequently in basal and posttreatment virus of patients who failed different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. They also have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value for treatment response, are discussed., The work at CBMSO was supported by grants SAF2014-52400-R from Ministerio de Economía y Competitividad (MINECO), SAF2017-87846-R and BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MICIU), PI18/00210 from Instituto de Salud Carlos III, S2013/ABI-2906 (PLATESA from Comunidad de Madrid/FEDER), and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/00121 and CPII19/00001), cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). The work in Barcelona was supported by Instituto de Salud Carlos III, cofinanced by the European Regional Development Fund (ERDF) grant number PI19/00301 and by the Centro para el Desarrollo Tecnológico Industrial (CDTI) from the MICIU, grant number IDI-20151125. Work at CAB was supported by MINECO grant BIO2016-79618R and PID2019-104903RB-I00 (funded by the EU under the FEDER program) and by the Spanish State research agency (AEI) through project number MDM-2017-0737 Unidad de Excelencia “María de Maeztu”-Centro de Astrobiología (CSIC-INTA). Work at IBMB was supported by MICIN grant BIO2017-83906-P (funded by the EU under the FEDER program). C.G.-C. is supported by predoctoral contract PRE2018-083422 from MICIU. B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo), cofinanced by Fondo Social Europeo (FSE).

In the past decade, the discovery and characterization of broadly neutralizing antibodies (bnAbs) to the highly conserved stem region of influenza hemagglutinin (HA) have provided valuable insights for development of a universal influenza vaccine. However, the genetic barrier for resistance to stem bnAbs has not been thoroughly evaluated. Here, we performed a series of deep mutational scanning experiments to probe for resistance mutations. We found that the genetic barrier to resistance to stem bnAbs is generally very low for the H3 subtype but substantially higher for the H1 subtype. Several resistance mutations in H3 cannot be neutralized by stem bnAbs at the highest concentration tested, do not reducein vitroviral fitness andin vivopathogenicity, and are often present in circulating strains as minor variants. Thus, H3 HAs have a higher propensity than H1 HAs to escape major stem bnAbs and creates a potential challenge in the development of abona fideuniversal influenza vaccine.ONE SENTENCE SUMMARYAcquisition of resistance by influenza virus to broadly neutralizing hemagglutinin stem antibodies varies tremendously depending on subtype.

Summary Objective Viral fitness plays an important role in HIV-1 evolution, transmission and pathogenesis. However, how mutations accumulated during early infection affect viral fitness has not been well studied. Methods Paired infectious molecular clones (IMCs) for transmitted/founder (T/F) and 6-month (6-mo) viruses post infection were generated from 10 infected individuals to investigate the impact of accumulated mutations on viral fitness by comparing 6-mo viruses to their cognate T/F viruses. Results All ten 6-mo viruses were less fit than their cognate T/F viruses. Moreover, the fitness losses of the 6-mo viruses correlated with the decrease in viral loads from the peak of viremia. Conclusion These results show that the mutations accumulated during half a year post infection collectively reduce viral fitness and thereby contribute to lowering viral loads.

Monkeypox (MPX) is a zoonotic disease similar to smallpox. Its fatality rate is about 11% and it is endemic to the Central and West African countries. In this paper, we analyze a compartmental model of MPX dynamics. Our goal is to see whether MPX can be controlled and eradicated by voluntary vaccinations. We show that there are three equilibria—disease free, fully endemic and previously neglected semi-endemic (with disease existing only among humans). The existence of semi-endemic equilibrium has severe implications should the MPX virus mutate to increased viral fitness in humans. We find that MPX is controllable and can be eradicated in a semi-endemic equilibrium by vaccination. However, in a fully endemic equilibrium, MPX cannot be eradicated by vaccination alone.

Viral fitness plays an important role in HIV-1 evolution, transmission and pathogenesis. However, how mutations accumulated during early and chronic infection affect viral fitness has not been well studied. We generated infectious molecular clones (IMCs) for the transmitted/founder (T/F), 6-month (6-mo) and 2-year (2-yr) viruses from the same infected individuals to investigate the impact of accumulated mutations on viral fitness by comparing 6-mo or 2-yr viruses to their cognate T/F viruses. We found that all ten 6-mo viruses were less fit than their cognate T/F viruses. Interestingly, 2-yr viruses that represented the clonally expanded viruses were more fit than the T/F viruses in two individuals. The fitness losses of the 6-mo viruses correlated with the decrease in viral loads from peak viremia. These results show that the mutations at month 6 of infection significantly reduce viral fitness and thereby contribute to lowering viral loads.

Mutagenesis is one of the key techniques in virus research. The recent development of deep mutational scanning allows the assessment of replication fitness effects of a large number of viral mutants in a high-throughput manner. Here, we describe a protocol for studying hepatitis C virus (HCV) using deep mutational scanning, which includes the methodologies for mutant library construction, passaging, sequencing, and data analysis.

Treatment options for influenza B virus infections are limited to neuraminidase inhibitors (NAIs), which block the neuraminidase (NA) glycoprotein on the virion surface. The development of NAI resistance would therefore result in a loss of antiviral treatment options for influenza B virus infections., Treatment options for influenza B virus infections are limited to neuraminidase inhibitors (NAIs), which block the neuraminidase (NA) glycoprotein on the virion surface. The development of NAI resistance would therefore result in a loss of antiviral treatment options for influenza B virus infections. This study characterized two contemporary influenza B viruses with known resistance-conferring NA amino acid substitutions, D197N and H273Y, detected during routine surveillance. The D197N and H273Y variants were characterized in vitro by assessing NA enzyme activity and affinity, as well as replication in cell culture compared to those of NAI-sensitive wild-type viruses. In vivo studies were also performed in ferrets to assess the replication and transmissibility of each variant. Mathematical models were used to analyze within-host and between-host fitness of variants relative to wild-type viruses. The data revealed that the H273Y variant had NA enzyme function similar to that of its wild type but had slightly reduced replication and transmission efficiency in vivo. The D197N variant had impaired NA enzyme function, but there was no evidence of reduction in replication or transmission efficiency in ferrets. Our data suggest that the influenza B virus variant with the H273Y NA substitution had a more notable reduction in fitness compared to wild-type viruses than the influenza B variant with the D197N NA substitution. Although a D197N variant is yet to become widespread, it is the most commonly detected NAI-resistant influenza B virus in surveillance studies. Our results highlight the need to carefully monitor circulating viruses for the spread of influenza B viruses with the D197N NA substitution.

Dengue virus (DENV) exists as four genetically distinct serotypes, each of which is historically assumed to be antigenically uniform. Recent analyses suggest that antigenic heterogeneity may exist within each serotype, but its source, extent and impact remain unclear. Here, we construct a sequence-based model to directly map antigenic change to underlying genetic divergence. We identify 49 specific substitutions and four colinear substitution clusters that robustly predict dengue antigenic relationships. We report moderate antigenic diversity within each serotype, resulting in genotype-specific patterns of heterotypic cross-neutralization. We also quantify the impact of antigenic variation on real-world DENV population dynamics, and find that serotype-level antigenic fitness is a dominant driver of dengue clade turnover. These results provide a more nuanced understanding of the relationship between dengue genetic and antigenic evolution, and quantify the effect of antigenic fitness on dengue evolutionary dynamics.

IMPORTANCE Human parainfluenza virus 3 is an important cause of morbidity and mortality among infants, the immunocompromised, and the elderly. Using deep genomic sequencing of HPIV-3-positive clinical material and its subsequent viral isolate, we discover a number of known and novel coding mutations in the main HPIV-3 attachment protein HN during brief exposure to immortalized cells. These mutations significantly alter function of the fusion complex, increasing fusion promotion by HN as well as generally decreasing neuraminidase activity and increasing HN-receptor engagement. These results show that viruses may evolve rapidly in culture even during primary isolation of the virus and before the first passage and reveal features of fitness for humans that are obscured by rapid adaptation to laboratory conditions., Human parainfluenza viruses cause a large burden of human respiratory illness. While much research relies upon viruses grown in cultured immortalized cells, human parainfluenza virus 3 (HPIV-3) evolves in culture. Cultured viruses differ in their properties compared to clinical strains. We present a genome-wide survey of HPIV-3 adaptations to culture using metagenomic next-generation sequencing of matched pairs of clinical samples and primary culture isolates (zero passage virus). Nonsynonymous changes arose during primary viral isolation, almost entirely in the genes encoding the two surface glycoproteins—the receptor binding protein hemagglutinin-neuraminidase (HN) or the fusion protein (F). We recovered genomes from 95 HPIV-3 primary culture isolates and 23 HPIV-3 strains directly from clinical samples. HN mutations arising during primary viral isolation resulted in substitutions at HN’s dimerization/F-interaction site, a site critical for activation of viral fusion. Alterations in HN dimer interface residues known to favor infection in culture occurred within 4 days (H552 and N556). A novel cluster of residues at a different face of the HN dimer interface emerged (P241 and R242) and imply a role in HPIV-3-mediated fusion. Functional characterization of these culture-associated HN mutations in a clinical isolate background revealed acquisition of the fusogenic phenotype associated with cultured HPIV-3; the HN-F complex showed enhanced fusion and decreased receptor-cleaving activity. These results utilize a method for identifying genome-wide changes associated with brief adaptation to culture to highlight the notion that even brief exposure to immortalized cells may affect key viral properties and underscore the balance of features of the HN-F complex required for fitness by circulating viruses.

HIV has been reported to be cytotoxic in vitro and in lymph node infection models. Using a computational approach, we found that partial inhibition of transmissions of multiple virions per cell could lead to increased numbers of live infected cells. If the number of viral DNA copies remains above one after inhibition, then eliminating the surplus viral copies reduces cell death. Using a cell line, we observed increased numbers of live infected cells when infection was partially inhibited with the antiretroviral efavirenz or neutralizing antibody. We then used efavirenz at concentrations reported in lymph nodes to inhibit lymph node infection by partially resistant HIV mutants. We observed more live infected lymph node cells, but with fewer HIV DNA copies per cell, relative to no drug. Hence, counterintuitively, limited attenuation of HIV transmission per cell may increase live infected cell numbers in environments where the force of infection is high., eLife digest The HIVvirus infects cells of the immune system. Once inside, it hijacks the cellular molecular machineries to make more copies of itself, which are then transmitted to new host cells. HIV eventually kills most cells it infects, either in the steps leading to the infection of the cell, or after the cell is already producing virus. HIV can spread between cells in two ways, known as cell-free or cell-to-cell. In the first, individual viruses are released from infected cells and move randomly through the body in the hope of finding new cells to infect. In the second, infected cells interact directly with uninfected cells. The second method is often much more successful at infecting new cells since they are exposed to multiple virus particles. HIV infections can be controlled by using combinations of antiretroviral drugs, such as efavirenz, to prevent the virus from making more of itself. With a high enough dose, the drugs can in theory completely stop HIV infections, unless the virus becomes resistant to treatment. However, some patients continue to use these drugs even after the virus they are infected with develops resistance. It is not clear what effect taking ineffective, or partially effective, drugs has on how HIV progresses. Using efavirenz, Jackson, Hunter et al. partially limited the spread of HIV between human cells grown in the laboratory. The experiments mirrored the situation where a partially resistant HIV strain spreads through the body. The results show that the success of cell-free infection is reduced as drug dose increases. Yet paradoxically, in cell-to-cell infection, the presence of drug caused more cells to become infected. This can be explained by the fact that, in cell-to-cell spread, each cell is exposed to multiple copies of the virus. The drug dose reduced the number of viral copies per cell without stopping the virus from infecting completely. The reduced number of viral copies per cell made it more likely that infected cells would survive the infection long enough to produce virus particles themselves. Viruses that can kill cells, such as HIV, must balance the need to make more of themselves against the speed that they kill their host cell to maximize the number of infected cells. If transmission between cells is too effective and too many virus particles are delivered to the new cell, the virus may not manage to infect new hosts before killing the old ones. These findings highlight this delicate balance. They also indicate a potential issue in using drugs to treat partially resistant virus strains. Without care, these treatments could increase the number of infected cells in the body, potentially worsening the effects of living with HIV.

Geminiviruses (family Geminiviridae) possess single-stranded circular DNA genomes that are replicated by cellular polymerases in plant host cell nuclei. In their hosts, geminivirus populations behave as ensembles of mutant and recombinant genomes, known as viral quasispecies. This favors the emergence of new geminiviruses with altered host range, facilitating new or more severe diseases or overcoming resistance traits. In warm and temperate areas several whitefly-transmitted geminiviruses of the genus Begomovirus cause the tomato yellow leaf curl disease (TYLCD) with significant economic consequences. TYLCD is frequently controlled in commercial tomatoes by using the dominant Ty-1 resistance gene. Over a 45 day period we have studied the diversification of three begomoviruses causing TYLCD: tomato yellow leaf curl virus (TYLCV), tomato yellow leaf curl Sardinia virus (TYLCSV) and tomato yellow leaf curl Malaga virus (TYLCMaV, a natural recombinant between TYLCV and TYLCSV). Viral quasispecies resulting from inoculation of geminivirus infectious clones were examined in plants of susceptible tomato (ty-1/ty-1), heterozygous resistant tomato (Ty-1/ty-1), common bean, and the wild reservoir Solanum nigrum. Differences in virus fitness across hosts were observed while viral consensus sequences remained invariant. However, the complexity and heterogeneity of the quasispecies were high, especially in common bean and the wild host. Interestingly, the presence or absence of the Ty-1 allele in tomato did not lead to differences in begomovirus mutant spectra. However, the fitness decrease of TYLCSV and TYLCV in tomato at 45 dpi might be related to an increase in CP (Coat protein) mutation frequency. In Solanum nigrum the recombinant TYLCMaV, which showed lower fitness than TYLCSV, at 45 dpi actively explored Rep (Replication associated protein) ORF but not the overlapping C4. Our results underline the importance of begomovirus mutant spectra during infections. This is especially relevant in the wild reservoir of the viruses, which has the potential to maintain highly diverse mutant spectra without modifying their consensus sequences.

Recently, gene therapy with rhTrim5α, an innate restriction factor which blocks HIV-1 at a post entry step, have been shown to be applicable as treatment in vitro. However, HIV-1 might adapt to replicate in the presence of rhTrim5α due to its high mutation rate. Here we observed that two different HIV-1 isolates were able to replicate in cells expressing high levels of rhTrim5α. Escape mutations were found in the conserved regions of the viral genome, Gag and p51 RT subunit. Furthermore, the escape mutations, predominantly in the capsid and p51 RT, altered viral sensitivity to modified huTrim5α R332P and R335G variants, with only a minor effect on the replication capacity in primary PBMCs. Therefore, gene therapy with rhTrim5α might be suitable for HIV-1 treatment, however the virus will eventually escape the pressure by gaining mutations in the conserved regions of the viral genome without any severe fitness cost.

Drug resistance prevents the successful treatment of HIV-positive individuals by decreasing viral sensitivity to a drug or a class of drugs. In addition to transmitted resistant viruses, treatment-naïve individuals can be confronted with the problem of drug resistance through de novo emergence of such variants. Resistant viruses have been reported for every antiretroviral drug tested so far, including the integrase strand transfer inhibitors raltegravir, elvitegravir and dolutegravir. However, de novo resistant variants against dolutegravir have been found in treatment-experienced but not in treatment-naïve individuals, a characteristic that is unique amongst antiretroviral drugs. We review here the issue of drug resistance against integrase strand transfer inhibitors as well as both pre-clinical and clinical studies that have led to the identification of the R263K mutation in integrase as a signature resistance substitution for dolutegravir. We also discuss how the topic of drug resistance against integrase strand transfer inhibitors may have relevance in regard to the nature of the HIV reservoir and possible HIV curative strategies.

Summary: Arbovirus transmission is modulated by host, vector, virus, and environmental factors. Even though viral fitness plays a salient role in host and vector adaptation, the transmission success of individual strains in a heterogeneous population may be stochastic. Our large-scale molecular epidemiological analyses of a dengue virus type 1 population revealed that only a subset of strains (16.7%; n = 6) were able to sustain transmission, despite the population being widely dispersed, dynamic, and heterogeneous. The overall dominance was variable even among the “established” lineages, albeit sharing comparable evolutionary characteristics and replication profiles. These findings indicated that virological parameters alone were unlikely to have a profound effect on the survival of viral lineages, suggesting an important role for non-viral factors in the transmission success of lineages. Our observations, therefore, emphasize the strategic importance of a holistic understanding of vector, human host, and viral factors in the control of vector-borne diseases. : Disease; Virology; Evolutionary Biology; Phylogenetics Subject Areas: Disease, Virology, Evolutionary Biology, Phylogenetics

Determining the fitness of viral genotypes has become a standard practice in virology as it is essential to evaluate their evolutionary potential. Darwinian fitness, defined as the advantage of a given genotype with respect to a reference one, is a complex property that captures, in a single figure, differences in performance at every stage of viral infection. To what extent does viral fitness result from specific molecular interactions with host factors and regulatory networks during infection? Can we identify host genes in functional classes whose expression depends on viral fitness? Here, we compared the transcriptomes of tobacco plants infected with seven genotypes of tobacco etch potyvirus that differ in fitness. We found that the larger the fitness differences among genotypes, the more dissimilar the transcriptomic profiles are. Consistently, two different mutations, one in the viral RNA polymerase and another in the viral suppressor of RNA silencing, resulted in significantly similar gene expression profiles. Moreover, we identified host genes whose expression showed a significant correlation, positive or negative, with the virus' fitness. Differentially expressed genes which were positively correlated with viral fitness activate hormone- and RNA silencing-mediated pathways of plant defense. In contrast, those that were negatively correlated with fitness affect metabolism, reducing growth, and development. Overall, these results reveal the high information content of viral fitness and suggest its potential use to predict differences in genomic profiles of infected hosts., This research was supported by grants from Spain’s Agencia Estatal de Investigacion—FEDER (BFU2012-30805 and BFU2015-65037-P to S.F.E. and BFU2015-66894-P to G.R.) and Generalitat Valenciana (PROMETEOII/2014/021).

Integrase strand transfer inhibitors (INSTIs) are the latest class of potent anti-HIV drugs. Currently, three INSTIs have been approved by the US Food and Drug Administration: raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG). Resistance mutations to RAL and EVG emerge rapidly, and significant cross-resistance between these compounds has been documented. In addition, limited cross-resistance has been observed among DTG, a newer INSTI, and RAL and EVG even though clinical resistance to DTG, or mutations associated with DTG resistance in treatment-naïve patients, has not yet been observed. This review summarises progress in studies on understanding resistance to DTG, mechanisms of possible resistance to DTG, and reasons for the absence of DTG-associated resistance mutations when the drug has been used in first-line therapy.

Since phage-bacteria interactions are among the most abundant in nature, they have become in the object of intensive study. In particular, how phages evolved temperateness — the propensity of the phage to enter lysogeny — and how temparateness has been optimized, remains poorly understood. In this work, we study the advantages of the lysogeny over the lytic path in phages under fluctuating environments. Also we explore how multiplicity of infection (MOI) drives the decision made by the phage. We found that temperate phages might use the lysogenic path to protect themselves from long periods of bad conditions. Additionally, phages with MOI strategies might have better chances of survive long periods of detrimental conditions.

Live attenuated viral vaccines provide the most robust and longest lasting immune response. Yet designing them a priori to have reduced growth capacity and also to be robust to evolutionary reversion can be challenging. On the one hand, genome editing methods now enable us to create almost any conceivable viral genome composition. Yet understanding and predicting how engineered genomes will behave and evolve is a challenge. Here we adopt a systems approach in studying a simple attenuation design in bacteriophage T7: promoter knockout. Either or both promoters for the two most highly expressed genes were abolished. Overall fitnesses, major phenotypes and gene expression levels were measured for all initial genomes and for genomes evolved toward for fitness recovery. Initial genomes behaved broadly as expected, but the genomes showed an unexpected ability to evolve back to high fitness. Genome sequences, RNA Seq and proteomics reveal the molecular foundations of the attenuations and recoveries. Overall, the work suggests that a systems approach is ultimately yielding to understanding, if not predicting the consequences of genome editing and evolutionary recoveries of simple genomes.

Direct-acting antivirals (DAAs) have significantly improved the treatment of infection with the hepatitis C virus. A promising class of novel antiviral agents targets the HCV NS5A protein. The high potency and broad genotypic coverage are favorable properties. NS5A inhibitors are currently assessed in advanced clinical trials in combination with viral polymerase inhibitors and/or viral protease inhibitors. However, the clinical use of NS5A inhibitors is also associated with new challenges. HCV variants with decreased susceptibility to these drugs can emerge and compromise therapy. In this review, we discuss resistance patterns in NS5A with focus prevalence and implications for inhibitor binding.

Dolutegravir (DTG) is an HIV integrase inhibitor that was recently approved for therapy by the Food and Drug Administration in the United States. When used as part of first-line therapy, DTG is the only HIV drug that has not selected for resistance mutations in the clinic. We believe that this is due to the long binding time of DTG to the integrase enzyme as well as greatly diminished replication capacity on the part of viruses that might become resistant to DTG. We further speculate that DTG might be able to be used in strategies aimed at HIV eradication.

Vpr is a lentiviral accessory protein that is expressed late during the infection cycle and is packaged in significant quantities into virus particles through a specific interaction with the P6 domain of the viral Gag precursor. Characterization of the physiologically relevant function(s) of Vpr has been hampered by the fact that in many cell lines, deletion of Vpr does not significantly affect viral fitness. However, Vpr is critical for virus replication in primary macrophages and for viral pathogenesis in vivo. It is generally accepted that Vpr does not have a specific enzymatic activity but functions as a molecular adapter to modulate viral or cellular processes for the benefit of the virus. Indeed, many Vpr interacting factors have been described by now, and the goal of this review is to summarize our current knowledge of cellular proteins targeted by Vpr.