Cellular receptors for mammalian viruses.

The interaction of viral surface components with cellular receptors and other entry factors determines key features of viral infection such as host range, tropism and virulence. Despite intensive research, our understanding of these interactions remains limited. Here, we report a systematic analysis of published work on mammalian virus receptors and attachment factors. We build a dataset twice the size of those available to date and specify the role of each factor in virus entry. We identify cellular proteins that are preferentially used as virus receptors, which tend to be plasma membrane proteins with a high propensity to interact with other proteins. Using machine learning, we assign cell surface proteins a score that predicts their ability to function as virus receptors. Our results also reveal common patterns of receptor usage among viruses and suggest that enveloped viruses tend to use a broader repertoire of alternative receptors than non-enveloped viruses, a feature that might confer them with higher interspecies transmissibility.

Enveloped viruses show increased propensity to cross-species transmission and zoonosis.

The transmission of viruses between different host species is a major source of emerging diseases and is of particular concern in the case of zoonotic transmission from mammals to humans. Several zoonosis risk factors have been identified, but it is currently unclear which viral traits primarily determine this process as previous work has focused on a few hundred viruses that are not representative of actual viral diversity. Here, we investigate fundamental virological traits that influence cross-species transmissibility and zoonotic propensity by interrogating a database of over 12,000 mammalian virus-host associations. Our analysis reveals that enveloped viruses tend to infect more host species and are more likely to be zoonotic than nonenveloped viruses, while other viral traits such as genome composition, structure, size, or the viral replication compartment play a less obvious role. This contrasts with the previous notion that viral envelopes did not significantly impact or even reduce zoonotic risk and should help better prioritize outbreak prevention efforts. We suggest several mechanisms by which viral envelopes could promote cross-species transmissibility, including structural flexibility of receptor-binding proteins and evasion of viral entry barriers.

Experimental Evolution Reveals a Genetic Basis for Membrane-Associated Virus Release.

Many animal viruses replicate and are released from cells in close association to membranes. However, whether this is a passive process or is controlled by the virus remains poorly understood. Importantly, the genetic basis and evolvability of membrane-associated viral shedding have not been investigated. To address this, we performed a directed evolution experiment using coxsackievirus B3, a model enterovirus, in which we repeatedly selected the free-virion or the fast-sedimenting membrane-associated viral subpopulations. The virus responded to this selection regime by reproducibly fixing a series of mutations that altered the extent of membrane-associated viral shedding, as revealed by full-genome ultra-deep sequencing. Specifically, using site-directed mutagenesis, we showed that substitution N63H in the viral capsid protein VP3 reduced the ratio of membrane-associated to free viral particles by 2 orders of magnitude. These findings open new avenues for understanding the mechanisms and implications of membrane-associated viral transmission.

The role of spatial structure in the evolution of viral innate immunity evasion: A diffusion-reaction cellular automaton model.

Most viruses have evolved strategies for preventing interferon (IFN) secretion and evading innate immunity. Recent work has shown that viral shutdown of IFN secretion can be viewed as a social trait, since the ability of a given virus to evade IFN-mediated immunity depends on the phenotype of neighbor viruses. Following this idea, we investigate the role of spatial structure in the evolution of innate immunity evasion. For this, we model IFN signaling and viral spread using a spatially explicit approximation that combines a diffusion-reaction model and cellular automaton. Our results indicate that the benefits of preventing IFN secretion for a virus are strongly determined by spatial structure through paracrine IFN signaling. Therefore, innate immunity evasion can evolve as a cooperative or even altruistic trait based on indirect fitness effects that IFN shutdown exerts on other members of the viral population. We identify key factors determining whether evasion from IFN-mediated immunity should evolve, such as population bottlenecks occurring during viral transmission, the relative speed of cellular infection and IFN secretion, and the diffusion properties of the medium.

Isolation of Four Lytic Phages Infecting Klebsiella pneumoniae K22 Clinical Isolates from Spain.

The emergence of multi-drug-resistant bacteria represents a major public-health threat. Phages constitute a promising alternative to chemical antibiotics due to their high host specificity, abundance in nature, and evolvability. However, phage host specificity means that highly diverse bacterial species are particularly difficult to target for phage therapy. This is the case of Klebsiella pneumoniae, which presents a hypervariable extracellular matrix capsule exhibiting dozens of variants. Here, we report four novel phages infecting K. pneumoniae capsular type K22 which were isolated from environmental samples in Valencia, Spain. Full genome sequencing showed that these phages belong to the Podoviridae family and encode putative depolymerases that allow digestion of specific K22 K. pneumoniae capsules. Our results confirm the capsular type-specificity of K. pneumoniae phages, as indicated by their narrow infectivity in a panel of K. pneumoniae clinical isolates. Nonetheless, this work represents a step forward in the characterization of phage diversity, which may culminate in the future use of large panels of phages for typing and/or for combating multi-drug-resistant K. pneumoniae.

Isolation and Characterization of Two Klebsiella pneumoniae Phages Encoding Divergent Depolymerases.

The emergence of multidrug-resistant bacteria is a major global health concern. The search for new therapies has brought bacteriophages into the spotlight, and new phages are being described as possible therapeutic agents. Among the bacteria that are most extensively resistant to current antibiotics is Klebsiella pneumoniae, whose hypervariable extracellular capsule makes treatment particularly difficult. Here, we describe two new K. pneumoniae phages, πVLC5 and πVLC6, isolated from environmental samples. These phages belong to the genus Drulisvirus within the family Podoviridae. Both phages encode a similar tail spike protein with putative depolymerase activity, which is shared among other related phages and probably determines their ability to specifically infect K. pneumoniae capsular types K22 and K37. In addition, we found that phage πVLC6 also infects capsular type K13 and is capable of striping the capsules of K. pneumoniae KL2 and KL3, although the phage was not infectious in these two strains. Genome sequence analysis suggested that the extended tropism of phage πVLC6 is conferred by a second, divergent depolymerase. Phage πVLC5 encodes yet another putative depolymerase, but we found no activity of this phage against capsular types other than K22 and K37, after testing a panel of 77 reference strains. Overall, our results confirm that most phages productively infected one or few Klebsiella capsular types. This constitutes an important challenge for clinical applications.

Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing.

Mutation rates vary by orders of magnitude across biological systems, being higher for simpler genomes. The simplest known genomes correspond to viroids, subviral plant replicons constituted by circular non-coding RNAs of few hundred bases. Previous work has revealed an extremely high mutation rate for chrysanthemum chlorotic mottle viroid, a chloroplast-replicating viroid. However, whether this is a general feature of viroids remains unclear. Here, we have used high-fidelity ultra-deep sequencing to determine the mutation rate in a common host (eggplant) of two viroids, each representative of one family: the chloroplastic eggplant latent viroid (ELVd, Avsunviroidae) and the nuclear potato spindle tuber viroid (PSTVd, Pospiviroidae). This revealed higher mutation frequencies in ELVd than in PSTVd, as well as marked differences in the types of mutations produced. Rates of spontaneous mutation, quantified in vivo using the lethal mutation method, ranged from 1/1000 to 1/800 for ELVd and from 1/7000 to 1/3800 for PSTVd depending on sequencing run. These results suggest that extremely high mutability is a common feature of chloroplastic viroids, whereas the mutation rates of PSTVd and potentially other nuclear viroids appear significantly lower and closer to those of some RNA viruses.

Viroids: survivors from the RNA world?

Because RNA can be a carrier of genetic information and a biocatalyst, there is a consensus that it emerged before DNA and proteins, which eventually assumed these roles and relegated RNA to intermediate functions. If such a scenario--the so-called RNA world--existed, we might hope to find its relics in our present world. The properties of viroids that make them candidates for being survivors of the RNA world include those expected for primitive RNA replicons: (a) small size imposed by error-prone replication, (b) high G + C content to increase replication fidelity, (c) circular structure for assuring complete replication without genomic tags, (d) structural periodicity for modular assembly into enlarged genomes, (e) lack of protein-coding ability consistent with a ribosome-free habitat, and (f) replication mediated in some by ribozymes, the fingerprint of the RNA world. With the advent of DNA and proteins, those protoviroids lost some abilities and became the plant parasites we now know.

Genome-Wide Estimation of the Spontaneous Mutation Rate of Human Adenovirus 5 by High-Fidelity Deep Sequencing.

Rates of spontaneous mutation determine the ability of viruses to evolve, infect new hosts, evade immunity and undergo drug resistance. Contrarily to RNA viruses, few mutation rate estimates have been obtained for DNA viruses, because their high replication fidelity implies that new mutations typically fall below the detection limits of Sanger and standard next-generation sequencing. Here, we have used a recently developed high-fidelity deep sequencing technique (Duplex Sequencing) to score spontaneous mutations in human adenovirus 5 under conditions of minimal selection. Based on >200 single-base spontaneous mutations detected throughout the entire viral genome, we infer an average mutation rate of 1.3 × 10-7 per base per cell infection cycle. This value is similar to those of other, large double-stranded DNA viruses, but an order of magnitude lower than those of single-stranded DNA viruses, consistent with the possible action of post-replicative repair. Although the mutation rate did not vary strongly along the adenovirus genome, we found several sources of mutation rate heterogeneity. First, two regions mapping to transcription units L3 and E1B-IVa2 were significantly depleted for mutations. Second, several point insertions/deletions located within low-complexity sequence contexts appeared recurrently, suggesting mutational hotspots. Third, mutation probability increased at GpC dinucleotides. Our findings suggest that host factors may influence the distribution of spontaneous mutations in human adenoviruses and potentially other nuclear DNA viruses.

Extremely High Mutation Rate of HIV-1 In Vivo.

Rates of spontaneous mutation critically determine the genetic diversity and evolution of RNA viruses. Although these rates have been characterized in vitro and in cell culture models, they have seldom been determined in vivo for human viruses. Here, we use the intrapatient frequency of premature stop codons to quantify the HIV-1 genome-wide rate of spontaneous mutation in DNA sequences from peripheral blood mononuclear cells. This reveals an extremely high mutation rate of (4.1 ± 1.7) × 10-3 per base per cell, the highest reported for any biological entity. Sequencing of plasma-derived sequences yielded a mutation frequency 44 times lower, indicating that a large fraction of viral genomes are lethally mutated and fail to reach plasma. We show that the HIV-1 reverse transcriptase contributes only 2% of mutations, whereas 98% result from editing by host cytidine deaminases of the A3 family. Hypermutated viral sequences are less abundant in patients showing rapid disease progression compared to normal progressors, highlighting the antiviral role of A3 proteins. However, the amount of A3-mediated editing varies broadly, and we find that low-edited sequences are more abundant among rapid progressors, suggesting that suboptimal A3 activity might enhance HIV-1 genetic diversity and pathogenesis.

Role of APOBEC3H in the Viral Control of HIV Elite Controller Patients.

Background APOBEC3H (A3H) gene presents variation at 2 positions (rs139297 and rs79323350) leading to a non-functional protein. So far, there is no information on the role played by A3H in spontaneous control of HIV. The aim of this study was to evaluate the A3H polymorphisms distribution in a well-characterized group of Elite Controller (EC) subjects. Methods We analyzed the genotype distribution of two different SNPs (rs139297 and rs79323350) of A3H in 30 EC patients and compared with 11 non-controller (NC) HIV patients. Genotyping was performed by PCR, cloning and Sanger sequencing. Both polymorphisms were analyzed jointly in order to adequately attribute the active or inactive status of A3H protein. Results EC subjects included in this study were able to maintain a long-term sustained spontaneous HIV-viral control and optimal CD4-T-cell counts; however, haplotypes leading to an active protein were very poorly represented in these patients. We found that the majority of EC subjects (23/30; 77%) presented allelic combinations leading to an inactive A3H protein, a frequency slightly lower than that observed for NC studied patients (10/11; 91%). Conclusions The high prevalence of non-functional protein coding-genotypes in EC subjects seems to indicate that other innate restriction factors different from APOBEC3H could be implicated in the replication control exhibited by these subjects.

Mechanisms of viral mutation.

The remarkable capacity of some viruses to adapt to new hosts and environments is highly dependent on their ability to generate de novo diversity in a short period of time. Rates of spontaneous mutation vary amply among viruses. RNA viruses mutate faster than DNA viruses, single-stranded viruses mutate faster than double-strand virus, and genome size appears to correlate negatively with mutation rate. Viral mutation rates are modulated at different levels, including polymerase fidelity, sequence context, template secondary structure, cellular microenvironment, replication mechanisms, proofreading, and access to post-replicative repair. Additionally, massive numbers of mutations can be introduced by some virus-encoded diversity-generating elements, as well as by host-encoded cytidine/adenine deaminases. Our current knowledge of viral mutation rates indicates that viral genetic diversity is determined by multiple virus- and host-dependent processes, and that viral mutation rates can evolve in response to specific selective pressures.

Variation in RNA virus mutation rates across host cells.

It is well established that RNA viruses exhibit higher rates of spontaneous mutation than DNA viruses and microorganisms. However, their mutation rates vary amply, from 10(-6) to 10(-4) substitutions per nucleotide per round of copying (s/n/r) and the causes of this variability remain poorly understood. In addition to differences in intrinsic fidelity or error correction capability, viral mutation rates may be dependent on host factors. Here, we assessed the effect of the cellular environment on the rate of spontaneous mutation of the vesicular stomatitis virus (VSV), which has a broad host range and cell tropism. Luria-Delbrück fluctuation tests and sequencing showed that VSV mutated similarly in baby hamster kidney, murine embryonic fibroblasts, colon cancer, and neuroblastoma cells (approx. 10(-5) s/n/r). Cell immortalization through p53 inactivation and oxygen levels (1-21%) did not have a significant impact on viral replication fidelity. This shows that previously published mutation rates can be considered reliable despite being based on a narrow and artificial set of laboratory conditions. Interestingly, we also found that VSV mutated approximately four times more slowly in various insect cells compared with mammalian cells. This may contribute to explaining the relatively slow evolution of VSV and other arthropod-borne viruses in nature.

Immune activation promotes evolutionary conservation of T-cell epitopes in HIV-1.

The immune system should constitute a strong selective pressure promoting viral genetic diversity and evolution. However, HIV shows lower sequence variability at T-cell epitopes than elsewhere in the genome, in contrast with other human RNA viruses. Here, we propose that epitope conservation is a consequence of the particular interactions established between HIV and the immune system. On one hand, epitope recognition triggers an anti-HIV response mediated by cytotoxic T-lymphocytes (CTLs), but on the other hand, activation of CD4(+) helper T lymphocytes (TH cells) promotes HIV replication. Mathematical modeling of these opposite selective forces revealed that selection at the intrapatient level can promote either T-cell epitope conservation or escape. We predict greater conservation for epitopes contributing significantly to total immune activation levels (immunodominance), and when TH cell infection is concomitant to epitope recognition (trans-infection). We suggest that HIV-driven immune activation in the lymph nodes during the chronic stage of the disease may offer a favorable scenario for epitope conservation. Our results also support the view that some pathogens draw benefits from the immune response and suggest that vaccination strategies based on conserved TH epitopes may be counterproductive.

Delayed lysis confers resistance to the nucleoside analogue 5-fluorouracil and alleviates mutation accumulation in the single-stranded DNA bacteriophage ϕX174.

UNLABELLED: Rates of spontaneous mutation determine viral fitness and adaptability. In RNA viruses, treatment with mutagenic nucleoside analogues selects for polymerase variants with increased fidelity, showing that viral mutation rates can be adjusted in response to imposed selective pressures. However, this type of resistance is not possible in viruses that do not encode their own polymerases, such as single-stranded DNA viruses. We previously showed that serial passaging of bacteriophage ϕX174 in the presence of the nucleoside analogue 5-fluorouracil (5-FU) favored substitutions in the lysis protein E (P. Domingo-Calap, M. Pereira-Gomez, and R. Sanjuán, J. Virol. 86:: 9640-9646, 2012, doi:10.1128/JVI.00613-12). Here, we found that approximately half (6/12) of the amino acid replacements in the N-terminal region of this protein led to delayed lysis, and two of these changes (V2A and D8A) also conferred partial resistance to 5-FU. By delaying lysis, the V2A and D8A substitutions allowed the virus to increase the burst size per cell in the presence of 5-FU. Furthermore, these substitutions tended to alleviate drug-induced mutagenesis by reducing the number of rounds of copying required for population growth, revealing a new mechanism of resistance. This form of mutation rate regulation may also be utilized by other viruses whose replication mode is similar to that of bacteriophage ϕX174. IMPORTANCE: Many viruses display high rates of spontaneous mutations due to defects in proofreading or postreplicative repair, allowing them to rapidly adapt to changing environments. Viral mutation rates may have been optimized to achieve high adaptability without incurring an excessive genetic load. Supporting this, RNA viruses subjected to chemical mutagenesis treatments have been shown to evolve higher-fidelity polymerases. However, many viruses cannot modulate replication fidelity because they do not encode their own polymerase. Here, we show a new mechanism for regulating viral mutation rates. We found that, under mutagenic conditions, the single-stranded bacteriophage ϕX174 evolved delayed lysis, and that this allowed the virus to increase the amount of progeny produced per cell. As a result, the viral population was amplified in fewer infection cycles, reducing the chances for mutation appearance.

Changes in protein domains outside the catalytic site of the bacteriophage Qß replicase reduce the mutagenic effect of 5-azacytidine.

UNLABELLED: The high genetic heterogeneity and great adaptability of RNA viruses are ultimately caused by the low replication fidelity of their polymerases. However, single amino acid substitutions that modify replication fidelity can evolve in response to mutagenic treatments with nucleoside analogues. Here, we investigated how two independent mutants of the bacteriophage Qß replicase (Thr210Ala and Tyr410His) reduce sensitivity to the nucleoside analogue 5-azacytidine (AZC). Despite being located outside the catalytic site, both mutants reduced the mutation frequency in the presence of the drug. However, they did not modify the type of AZC-induced substitutions, which was mediated mainly by ambiguous base pairing of the analogue with purines. Furthermore, the Thr210Ala and Tyr410His substitutions had little or no effect on replication fidelity in untreated viruses. Also, both substitutions were costly in the absence of AZC or when the action of the drug was suppressed by adding an excess of natural pyrimidines (uridine or cytosine). Overall, the phenotypic properties of these two mutants were highly convergent, despite the mutations being located in different domains of the Qß replicase. This suggests that treatment with a given nucleoside analogue tends to select for a unique functional response in the viral replicase. IMPORTANCE: In the last years, artificial increase of the replication error rate has been proposed as an antiviral therapy. In this study, we investigated the mechanisms by which two substitutions in the Qß replicase confer partial resistance to the mutagenic nucleoside analogue AZC. As opposed to previous work with animal viruses, where different mutations selected sequentially conferred nucleoside analogue resistance through different mechanisms, our results suggest that there are few or no alternative AZC resistance phenotypes in Qß. Also, despite resistance mutations being highly costly in the absence of the drug, there was no sequential fixation of secondary mutations. Bacteriophage Qß is the virus with the highest reported mutation rate, which should make it particularly sensitive to nucleoside analogue treatments, probably favoring resistance mutations even if they incur high costs. The results are also relevant for understanding the possible pathways by which fidelity of the replication machinery can be modified.

Urinary KIM-1, NGAL and L-FABP for the diagnosis of AKI in patients with acute coronary syndrome or heart failure undergoing coronary angiography.

Acute kidney injury (AKI) is a common complication after coronary angiography. Early biomarkers of this disease are needed since increase in serum creatinine levels is a late marker. To assess the usefulness of urinary kidney injury molecule-1 (uKIM-1), neutrophil gelatinase-associated lipocalin (uNGAL) and liver-type fatty acid-binding protein (uL-FABP) for early detection of AKI in these patients, comparing their performance with another group of cardiac surgery patients. Biomarkers were measured in 193 patients, 12 h after intervention. In the ROC analysis, AUC for KIM-1, NGAL and L-FABP was 0.713, 0.958 and 0.642, respectively, in the coronary angiography group, and 0.716, 0.916 and 0.743 in the cardiac surgery group. Urinary KIM-1 12 h after intervention is predictive of AKI in adult patients undergoing coronary angiography, but NGAL shows higher sensitivity and specificity. L-FABP provides inferior discrimination for AKI than KIM-1 or NGAL in contrast to its performance after cardiac surgery. This is the first study showing the predictive capacity of KIM-1 for AKI after coronary angiography. Further studies are still needed to answer relevant questions about the clinical utility of biomarkers for AKI in different clinical settings.

From molecular genetics to phylodynamics: evolutionary relevance of mutation rates across viruses.

Although evolution is a multifactorial process, theory posits that the speed of molecular evolution should be directly determined by the rate at which spontaneous mutations appear. To what extent these two biochemical and population-scale processes are related in nature, however, is largely unknown. Viruses are an ideal system for addressing this question because their evolution is fast enough to be observed in real time, and experimentally-determined mutation rates are abundant. This article provides statistically supported evidence that the mutation rate determines molecular evolution across all types of viruses. Properties of the viral genome such as its size and chemical composition are identified as major determinants of these rates. Furthermore, a quantitative analysis reveals that, as expected, evolution rates increase linearly with mutation rates for slowly mutating viruses. However, this relationship plateaus for fast mutating viruses. A model is proposed in which deleterious mutations impose an evolutionary speed limit and set an extinction threshold in nature. The model is consistent with data from replication kinetics, selection strength and chemical mutagenesis studies.

Temperature impacts SARS-CoV-2 spike fusogenicity and evolution.

SARS-CoV-2 infects both the upper and lower respiratory tracts, which are characterized by different temperatures (33°C and 37°C, respectively). In addition, fever is a common COVID-19 symptom. SARS-CoV-2 has been shown to replicate more efficiently at low temperatures, but the effect of temperature on different viral proteins remains poorly understood. Here, we investigate how temperature affects the SARS-CoV-2 spike function and evolution. We first observed that increasing temperature from 33°C to 37°C or 39°C increased spike-mediated cell-cell fusion. We then experimentally evolved a recombinant vesicular stomatitis virus expressing the SARS-CoV-2 spike at these different temperatures. We found that spike-mediated cell-cell fusion was maintained during evolution at 39°C but was lost in a high proportion of viruses that evolved at 33°C or 37°C. Consistently, sequencing of the spikes evolved at 33°C or 37°C revealed the accumulation of mutations around the furin cleavage site, a region that determines cell-cell fusion, whereas this did not occur in spikes evolved at 39°C. Finally, using site-directed mutagenesis, we found that disruption of the furin cleavage site had a temperature-dependent effect on spike-induced cell-cell fusion and viral fitness. Our results suggest that variations in body temperature may affect the activity and diversification of the SARS-CoV-2 spike. IMPORTANCE: When it infects humans, SARS-CoV-2 is exposed to different temperatures (e.g., replication site and fever). Temperature has been shown to strongly impact SARS-CoV-2 replication, but how it affects the activity and evolution of the spike protein remains poorly understood. Here, we first show that high temperatures increase the SARS-CoV-2 spike fusogenicity. Then, we demonstrate that the evolution of the spike activity and variants depends on temperature. Finally, we show that the functional effect of specific spike mutations is temperature-dependent. Overall, our results suggest that temperature may be a factor influencing the activity and adaptation of the SARS-CoV-2 spike in vivo, which will help understanding viral tropism, pathogenesis, and evolution.

Optimized Recovery of Viral DNA and RNA from Blood Plasma for Viral Metagenomics.

Metagenomics is vastly improving our ability to discover new viruses, as well as their possible associations with disease. However, metagenomics has also changed our understanding of viruses in general. This is because we can find viruses in healthy hosts in the absence of disease, which changes the perspective of viruses as mere pathogens and offers a new perspective in which viruses function as important components of ecosystems. In concrete, human blood metagenomics has revealed the presence of different types of viruses in apparently healthy subjects. These viruses are human anelloviruses and, to a lower extent, human pegiviruses. Viral metagenomics' major challenge is the correct isolation of the viral nucleic acids from a specific sample. For the protocol to be successful, all steps must be carefully chosen, in particular those that optimize the recovery of viral nucleic acids. Here, we present a procedure that allows the recovery of both DNA and RNA viruses from plasma samples.