Natural selection in the evolution of SARS-CoV-2 in bats created a generalist virus and highly capable human pathogen

Virus host shifts are generally associated with novel adaptations to exploit the cells of the new host species optimally. Surprisingly, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has apparently required little to no significant adaptation to humans since the start of the Coronavirus Disease 2019 (COVID-19) pandemic and to October 2020. Here we assess the types of natural selection taking place in Sarbecoviruses in horseshoe bats versus the early SARS-CoV-2 evolution in humans. While there is moderate evidence of diversifying positive selection in SARS-CoV-2 in humans, it is limited to the early phase of the pandemic, and purifying selection is much weaker in SARS-CoV-2 than in related bat Sarbecoviruses. In contrast, our analysis detects evidence for significant positive episodic diversifying selection acting at the base of the bat virus lineage SARS-CoV-2 emerged from, accompanied by an adaptive depletion in CpG composition presumed to be linked to the action of antiviral mechanisms in these ancestral bat hosts. The closest bat virus to SARS-CoV-2, RmYN02 (sharing an ancestor about 1976), is a recombinant with a structure that includes differential CpG content in Spike; clear evidence of coinfection and evolution in bats without involvement of other species. While an undiscovered “facilitating” intermediate species cannot be discounted, collectively, our results support the progenitor of SARS-CoV-2 being capable of efficient human–human transmission as a consequence of its adaptive evolutionary history in bats, not humans, which created a relatively generalist virus.

A study of the natural origins of SARS-CoV-2 reveals very little adaptive evolution occurring since it emerged in humans, but strong evolutionary signals in the bat virus lineage from which SARS-CoV-2 arose. Evolution in bats involved lineage-specific depletion of CpG nucleotides (linked to host anti-viral molecules), and clear evidence of recombination across these lineages, supporting bat host species’ influence on the ancestral viruses.     

Genomic chronicle of SARS-CoV-2: a mutational analysis with over 1 million genome sequences

Use of information technologies to analyse big data on SARS-CoV-2 genome provides an insight for tracking variations and examining the evolution of the virus. Nevertheless, storing, processing, alignment and analyses of these numerous genomes are still a challenge. In this study, over 1 million SARS-CoV-2 genomes have been analysed to show distribution and relationship of variations that could enlighten development and evolution of the virus. In all genomes analysed in this study, a total of over 215M SNVs have been detected and average number of SNV per isolate was found to be 21.83. Single nucleotide variant (SNV) average is observed to reach 31.25 just in March 2021. The average variation number of isolates is increasing and compromising with total case numbers around the world. Remarkably, cytosine deamination, which is one of the most important biochemical processes in the evolutionary development of coronaviruses, accounts for 46% of all SNVs seen in SARS-CoV-2 genomes within 16 months. This study is one of the most comprehensive SARS-CoV-2 genomic analysis study in terms of number of genomes analysed in an academic publication so far, and reported results could be useful in monitoring the development of SARS-CoV-2.     

Patterns of evolution and host gene mimicry in influenza and other RNA viruses

It is well known that the dinucleotide CpG is under-represented in the genomic DNA of many vertebrates. This is commonly thought to be due to the methylation of cytosine residues in this dinucleotide and the corresponding high rate of deamination of 5-methycytosine, which lowers the frequency of this dinucleotide in DNA. Surprisingly, many single-stranded RNA viruses that replicate in these vertebrate hosts also have a very low presence of CpG dinucleotides in their genomes. Viruses are obligate intracellular parasites and the evolution of a virus is inexorably linked to the nature and fate of its host. One therefore expects that virus and host genomes should have common features. In this work, we compare evolutionary patterns in the genomes of ssRNA viruses and their hosts. In particular, we have analyzed dinucleotide patterns and found that the same patterns are pervasively over- or under-represented in many RNA viruses and their hosts suggesting that many RNA viruses evolve by mimicking some of the features of their host's genes (DNA) and likely also their corresponding mRNAs. When a virus crosses a species barrier into a different host, the pressure to replicate, survive and adapt, leaves a footprint in dinucleotide frequencies. For instance, since human genes seem to be under higher pressure to eliminate CpG dinucleotide motifs than avian genes, this pressure might be reflected in the genomes of human viruses (DNA and RNA viruses) when compared to those of the same viruses replicating in avian hosts. To test this idea we have analyzed the evolution of the influenza virus since 1918. We find that the influenza A virus, which originated from an avian reservoir and has been replicating in humans over many generations, evolves in a direction strongly selected to reduce the frequency of CpG dinucleotides in its genome. Consistent with this observation, we find that the influenza B virus, which has spent much more time in the human population, has adapted to its human host and exhibits an extremely low CpG dinucleotide content. We believe that these observations directly show that the evolution of RNA viral genomes can be shaped by pressures observed in the host genome. As a possible explanation, we suggest that the strong selection pressures acting on these RNA viruses are most likely related to the innate immune response and to nucleotide motifs in the host DNA and RNAs.     

CpG Dinucleotide Frequencies Reveal the Role of Host Methylation Capabilities in Parvovirus Evolution

Parvoviruses are rapidly evolving viruses that infect a wide range of hosts, including vertebrates and invertebrates. Extensive methylation of the parvovirus genome has been recently demonstrated. A global pattern of methylation of CpG dinucleotides is seen in vertebrate genomes, compared to “fractional” methylation patterns in invertebrate genomes. It remains unknown if the loss of CpG dinucleotides occurs in all viruses of a given DNA virus family that infect host species spanning across vertebrates and invertebrates. We investigated the link between the extent of CpG dinucleotide depletion among autonomous parvoviruses and the evolutionary lineage of the infected host. We demonstrate major differences in the relative abundance of CpG dinucleotides among autonomous parvoviruses which share similar genome organization and common ancestry, depending on the infected host species. Parvoviruses infecting vertebrate hosts had significantly lower relative abundance of CpG dinucleotides than parvoviruses infecting invertebrate hosts. The strong correlation of CpG dinucleotide depletion with the gain in TpG/CpA dinucleotides and the loss of TpA dinucleotides among parvoviruses suggests a major role for CpG methylation in the evolution of parvoviruses. Our data present evidence that links the relative abundance of CpG dinucleotides in parvoviruses to the methylation capabilities of the infected host. In sum, our findings support a novel perspective of host-driven evolution among autonomous parvoviruses.     

Mechanism and evolution of human ACE2 binding by SARS-CoV-2 spike

Spike glycoprotein of SARS-CoV-2 mediates viral entry into host cells by facilitating virus attachment and membrane fusion. ACE2 is the main receptor of SARS-CoV-2 and its interaction with spike has shaped the virus’ emergence from an animal reservoir and subsequent evolution in the human host. Many structural studies on the spike:ACE2 interaction have provided insights into mechanisms driving viral evolution during the on-going pandemic. This review describes the molecular basis of spike binding to ACE2, outlines mechanisms that have optimised this interaction during viral evolution, and suggests directions for future research.     

Oligonucleotide motifs that disappear during the evolution of influenza virus in humans increase alpha interferon secretion by plasmacytoid dendritic cells

CpG motifs in an A/U context have been preferentially eliminated from classical H1N1 influenza virus genomes during virus evolution in humans. The hypothesis of the current work is that CpG motifs in a uracil context represent sequence patterns with the capacity to induce an immune response, and the avoidance of this immunostimulatory signal is the reason for the observed preferential decline. To analyze the immunogenicity of these domains, we used plasmacytoid dendritic cells (pDCs). pDCs express pattern recognition receptors, including Toll-like receptor 7 (TLR7), which recognizes guanosine- and uridine-rich viral single-stranded RNA (ssRNA), including influenza virus ssRNA. The signaling through TLR7 results in the induction of inflammatory cytokines and type I interferon (IFN-I), an essential process for the induction of specific adaptive immune responses and for mounting a robust antiviral response mediated by IFN-α. Secretion of IFN-α is also linked to the activation of other immune cells, potentially amplifying the effect of an initial IFN-α secretion. We therefore also examined the role of IFN-α-driven activation of NK cells as another source of selective pressure on the viral genome. We found direct evidence that CpG RNA motifs in a U-rich context control pDC activation and IFN-α-driven activation of NK cells, likely through TLR7. These data provide a potential explanation for the loss of CpG motifs from avian influenza viruses as they adapt to mammalian hosts. The selective decrease of CpG motifs surrounded by U/A may be a viral strategy to avoid immune recognition, a strategy likely shared by highly expressed human immune genes.     

COVID-19: emergence and mutational diversification of SARS-CoV-2

The origin of the SARS-CoV-2 virus is not yet defined, but a viral zoonosis from bats – with or without an alternative animal as an intermediate host – is still the most likely hypothesis. The intensive virological and epidemiological research combined with massive sequencing efforts of whole viral genomes allowed an unprecedented analysis of an unfolding pandemic at the level of viral evolution with the documentation of extinction events, prevalence increases and rise to dominance for different viral lineages that provide not only fundamental insights into mechanisms of viral evolution, but influence also public health measures to contain the virus.     

Depletion of CpG Dinucleotides in Papillomaviruses and Polyomaviruses: A Role for Divergent Evolutionary Pressures

Background

Papillomaviruses and polyomaviruses are small ds-DNA viruses infecting a wide-range of vertebrate hosts. Evidence supporting co-evolution of the virus with the host does not fully explain the evolutionary path of papillomaviruses and polyomaviruses. Studies analyzing CpG dinucleotide frequencies in virus genomes have provided interesting insights on virus evolution. CpG dinucleotide depletion has not been extensively studied among papillomaviruses and polyomaviruses. We sought to analyze the relative abundance of dinucleotides and the relative roles of evolutionary pressures in papillomaviruses and polyomaviruses.

Methods

We studied 127 full-length sequences from papillomaviruses and 56 full-length sequences from polyomaviruses. We analyzed the relative abundance of dinucleotides, effective codon number (ENC), differences in synonymous codon usage. We examined the association, if any, between the extent of CpG dinucleotide depletion and the evolutionary lineage of the infected host. We also investigated the contribution of mutational pressure and translational selection to the evolution of papillomaviruses and polyomaviruses.

Results

All papillomaviruses and polyomaviruses are CpG depleted. Interestingly, the evolutionary lineage of the infected host determines the extent of CpG depletion among papillomaviruses and polyomaviruses. CpG dinucleotide depletion was more pronounced among papillomaviruses and polyomaviruses infecting human and other mammals as compared to those infecting birds. Our findings demonstrate that CpG depletion among papillomaviruses is linked to mutational pressure; while CpG depletion among polyomaviruses is linked to translational selection. We also present evidence that suggests methylation of CpG dinucleotides may explain, at least in part, the depletion of CpG dinucleotides among papillomaviruses but not polyomaviruses.

Conclusions

The extent of CpG depletion among papillomaviruses and polyomaviruses is linked to the evolutionary lineage of the infected host. Our results highlight the existence of divergent evolutionary pressures leading to CpG dinucleotide depletion among small ds-DNA viruses infecting vertebrate hosts.     

Patterns of Oligonucleotide Sequences in Viral and Host Cell RNA Identify Mediators of the Host Innate Immune System

The innate immune response provides a first line of defense against pathogens by targeting generic differential features that are present in foreign organisms but not in the host. These innate responses generate selection forces acting both in pathogens and hosts that further determine their co-evolution. Here we analyze the nucleic acid sequence fingerprints of these selection forces acting in parallel on both host innate immune genes and ssRNA viral genomes. We do this by identifying dinucleotide biases in the coding regions of innate immune response genes in plasmacytoid dendritic cells, and then use this signal to identify other significant host innate immune genes. The persistence of these biases in the orthologous groups of genes in humans and chickens is also examined. We then compare the significant motifs in highly expressed genes of the innate immune system to those in ssRNA viruses and study the evolution of these motifs in the H1N1 influenza genome. We argue that the significant under-represented motif pattern of CpG in an AU context - which is found in both the ssRNA viruses and innate genes, and has decreased throughout the history of H1N1 influenza replication in humans - is immunostimulatory and has been selected against during the co-evolution of viruses and host innate immune genes. This shows how differences in host immune biology can drive the evolution of viruses that jump into species with different immune priorities than the original host.     

The interplay of SARS-CoV-2 evolution and constraints imposed by the structure and functionality of its proteins

The unprecedented pace of the sequencing of the SARS-CoV-2 virus genomes provides us with unique information about the genetic changes in a single pathogen during ongoing pandemic. By the analysis of close to 200,000 genomes we show that the patterns of the SARS-CoV-2 virus mutations along its genome are closely correlated with the structural and functional features of the encoded proteins. Requirements of foldability of proteins’ 3D structures and the conservation of their key functional regions, such as protein-protein interaction interfaces, are the dominant factors driving evolutionary selection in protein-coding genes. At the same time, avoidance of the host immunity leads to the abundance of mutations in other regions, resulting in high variability of the missense mutation rate along the genome. “Unexplained” peaks and valleys in the mutation rate provide hints on function for yet uncharacterized genomic regions and specific protein structural and functional features they code for. Some of these observations have immediate practical implications for the selection of target regions for PCR-based COVID-19 tests and for evaluating the risk of mutations in epitopes targeted by specific antibodies and vaccine design strategies.     

Open Modification Searching of SARS-CoV-2–Human Protein Interaction Data Reveals Novel Viral Modification Sites

The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus 2019 disease, has led to an ongoing global pandemic since 2019. Mass spectrometry can be used to understand the molecular mechanisms of viral infection by SARS-CoV-2, for example, by determining virus–host protein–protein interactions through which SARS-CoV-2 hijacks its human hosts during infection, and to study the role of post-translational modifications. We have reanalyzed public affinity purification–mass spectrometry data using open modification searching to investigate the presence of post-translational modifications in the context of the SARS-CoV-2 virus–host protein–protein interaction network. Based on an over twofold increase in identified spectra, our detected protein interactions show a high overlap with independent mass spectrometry-based SARS-CoV-2 studies and virus–host interactions for alternative viruses, as well as previously unknown protein interactions. In addition, we identified several novel modification sites on SARS-CoV-2 proteins that we investigated in relation to their interactions with host proteins. A detailed analysis of relevant modifications, including phosphorylation, ubiquitination, and S-nitrosylation, provides important hypotheses about the functional role of these modifications during viral infection by SARS-CoV-2.     

Type I and III IFN-mediated antiviral actions counteracted by SARS-CoV-2 proteins and host inherited factors

SARS-CoV-2 is a recently identified coronavirus accountable for the current pandemic disease known as COVID-19. Different patterns of disease progression infer a diverse host immune response, with interferon (IFN) being pivotal. IFN-I and III are produced and released by virus-infected cells during the interplay with SARS-CoV-2, thus establishing an antiviral state in target cells. However, the efficacy of IFN and its role in the possible outcomes of the disease are not yet defined, as it is influenced both by factors inherent to the virus and to the host. The virus exhibits multiple strategies to counteract the innate immune response, including those shared by SARS-CoV and MERS-CoV and other novel ones. Inborn errors in the host may affect IFN-related effector proteins or decrease its levels in plasma upon neutralization by preexistent autoantibodies. This battle between the IFN response triggered upon SARS-CoV-2 infection, its magnitude and timing, and the efficacy of its antiviral tools in dispute against the viral evasion strategies together with the genetic factors of the host, generate a scenario whose fate contributes to defining the severity of COVID-19.     

Making sense of spike D614G in SARS-CoV-2 transmission

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), the etiologic agent of the current coronavirus disease 2019(COVID-19) pandemic, has evolved to adapt to human host and transmission over the past 12 months. One prominent adaptive mutation is the asparagine-to-glycine substitution at amino acid position 614 in the viral spike protein(D614G), which has become dominant in the currently circulating virus strains. Since spike protein determines host ranges, tissue tropism, and pathogenesis through binding to the cellular receptor of angiotensin converting enzyme 2(ACE2), the D614G mutation is hypothesized to enhance viral fitness in human host, leading to increased transmission during the global pandemic. Here we summarize the recent progress on the role of the D614G mutation in viral replication, pathogenesis, transmission, and vaccine and therapeutic antibody development. These findings underscore the importance in closely monitoring viral evolution and defining their functions to ensure countermeasure efficacy against newly emerging variants.     

In silico mutagenesis of human ACE2 with S protein and translational efficiency explain SARS-CoV-2 infectivity in different species

The coronavirus disease COVID-19 constitutes the most severe pandemic of the last decades having caused more than 1 million deaths worldwide. The SARS-CoV-2 virus recognizes the angiotensin converting enzyme 2 (ACE2) on the surface of human cells through its spike protein. It has been reported that the coronavirus can mildly infect cats, and ferrets, and perhaps dogs while not pigs, mice, chicken and ducks. Differences in viral infectivity among different species or individuals could be due to amino acid differences at key positions of the host proteins that interact with the virus, the immune response, expression levels of host proteins and translation efficiency of the viral proteins among other factors. Here, first we have addressed the importance that sequence variants of different animal species, human individuals and virus isolates have on the interaction between the RBD domain of the SARS-CoV-2 spike S protein and human angiotensin converting enzyme 2 (ACE2). Second, we have looked at viral translation efficiency by using the tRNA adaptation index. We find that integration of both interaction energy with ACE2 and translational efficiency explains animal infectivity. Humans are the top species in which SARS-CoV-2 is both efficiently translated as well as optimally interacting with ACE2. We have found some viral mutations that increase affinity for hACE and some hACE2 variants affecting ACE2 stability and virus binding. These variants suggest that different sensitivities to coronavirus infection in humans could arise in some cases from allelic variability affecting ACE2 stability and virus binding.     

SARS-CoV-2 cell receptor gene ACE2 -mediated immunomodulation in breast cancer subtypes

The recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has impacted the world severely. The binding of the SARS-CoV-2 virus to the angiotensin-converting enzyme 2 (ACE2) and its intake by the host cell is a necessary step for infection. ACE2 has garnered widespread therapeutic possibility as it is entry/interactive point for SARS-CoV-2, responsible for coronavirus disease 2019 (COVID-19) pandemic and providing a critical regulator for immune modulation in various disease. Patients with suffering from cancer always being on the verge of being immune compromised therefore gaining knowledge about how SARS-CoV-2 viruses affecting immune cells in human cancers will provides us new opportunities for preventing or treating virus-associated cancers. Despite COVID-19 pandemic got center stage at present time, however very little research being explores, which increase our knowledge in context with how SARS-CoV-2 infection affect cancer a cellular level. Therefore, in light of the ACE-2 as an important contributor of COVID-19 global, we analyzed correlation between ACE2 and tumor immune infiltration (TIL) level and the type markers of immune cells were investigated in breast cancer subtypes by using TIMER database. Our findings shed light on the immunomodulatory role of ACE2 in the luminal A subtype which may play crucial role in imparting therapeutic resistance in this cancer subtype.     

SARS-CoV-2 biology and variants: anticipation of viral evolution and what needs to be done

The global propagation of SARS-CoV-2 and the detection of a large number of variants, some of which have replaced the original clade to become dominant, underscores the fact that the virus is actively exploring its evolutionary space. The longer high levels of viral multiplication occur – permitted by high levels of transmission –, the more the virus can adapt to the human host and find ways to success. The third wave of the COVID-19 pandemic is starting in different parts of the world, emphasizing that transmission containment measures that are being imposed are not adequate. Part of the consideration in determining containment measures is the rationale that vaccination will soon stop transmission and allow a return to normality. However, vaccines themselves represent a selection pressure for evolution of vaccine-resistant variants, so the coupling of a policy of permitting high levels of transmission/virus multiplication during vaccine roll-out with the expectation that vaccines will deal with the pandemic, is unrealistic. In the absence of effective antivirals, it is not improbable that SARS-CoV-2 infection prophylaxis will involve an annual vaccination campaign against ‘dominant’ viral variants, similar to influenza prophylaxis. Living with COVID-19 will be an issue of SARS-CoV-2 variants and evolution. It is therefore crucial to understand how SARS-CoV-2 evolves and what constrains its evolution, in order to anticipate the variants that will emerge. Thus far, the focus has been on the receptor-binding spike protein, but the virus is complex, encoding 26 proteins which interact with a large number of host factors, so the possibilities for evolution are manifold and not predictable a priori. However, if we are to mount the best defence against COVID-19, we must mount it against the variants, and to do this, we must have knowledge about the evolutionary possibilities of the virus. In addition to the generic cellular interactions of the virus, there are extensive polymorphisms in humans (e.g. Lewis, HLA, etc.), some distributed within most or all populations, some restricted to specific ethnic populations and these variations pose additional opportunities for/constraints on viral evolution. We now have the wherewithal – viral genome sequencing, protein structure determination/modelling, protein interaction analysis – to functionally characterize viral variants, but access to comprehensive genome data is extremely uneven. Yet, to develop an understanding of the impacts of such evolution on transmission and disease, we must link it to transmission (viral epidemiology) and disease data (patient clinical data), and the population granularities of these. In this editorial, we explore key facets of viral biology and the influence of relevant aspects of human polymorphisms, human behaviour, geography and climate and, based on this, derive a series of recommendations to monitor viral evolution and predict the types of variants that are likely to arise.     

Bioinformatic analysis and identification of single-stranded RNA sequences recognized by TLR7/8 in the SARS-CoV-2, SARS-CoV,and MERS-CoV genomes

During virus infection, host toll-like receptors (TLRs) can recognize different pathogen-associated molecular patterns and trigger the innate immune response. TLR7/8 can identify the single-stranded RNA (ssRNA) of the virus. This study aimed to search ssRNA sequences recognized by TLR7/8 from the SARS-CoV-2, SARS-CoV, and MERS-CoV whole genomes by a bioinformatic technique. The immunoinformatic approach showed that the SARS-CoV-2 genome has more ssRNA fragments that could be recognized by TLR7/8 than the SARS-CoV genome. These findings suggest innate immune hyperactivation by SARS-CoV-2. This activity is possibly able to provoke a robust proinflammatory response via TLR7/8 recognition and cause acute lung injury.