Risks of latent viruses that reside in ancient genomes under research?

Risks of latent viruses that reside in ancient genomes under research?

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Some interesting research in reactivating mammoth genetic material ( made me wonder what risks are inherent (or are not inherent) in reviving older genomes that may have integrated latent viral code?

Are there potential biohazards in this line of research, where dormant viruses that have otherwise gone extinct (along with the extinct host) and which now may have the potential to be reactivated? Not just in terms of zoonoses that infect across species, but also viruses that infect modern animals that are more closely related or have relatively common ancestry (e.g., from mammoth to elephant).

Could a disease be brought back via this kind of research? Or are the conditions for waking latent viruses from starting material of this kind just not possible to recreate in a lab setting?

Edit: To clarify what I mean by potential biohazard in relation to this question: Experimental conditions result in a latent virus going into lytic phase. The virus is a communicable pathogen; it spreads outside the laboratory environment; it causes sickness or death to living organisms to the extent that it creates a public health, ecological, or other crisis of significance. For background, please also see: Cambridge, The Free Dictionary, and Wikipedia.

First, I don't understand why you are more worried about viruses of extinct species, instead of ancient viruses of species that haven't gone extinct. Clearly the infectious potential of the latter is greater for the current species. I haven't heard a peep in published literature about the dangers of resurrecting mammoth viruses, but surely recreating (in 2005) the 1918 influenza did stir some fairly substantial safety debates.

In general, working with ancient DNA (aDNA) does require some safety precautions, but mainly because of the risk of contaminating the aDNA (with modern DNA)-.

Furthermore, I'm not aware of any research having resurrected an ancient virus from an extinct species aDNA (your specific scenario), but one ~5-million y.o. retrovirus (dubbed Phoenix) was assembled from current human DNA (in 2006); the authors were a little worried as to its infectious potential, so they engineered it so it couldn't replicate more than once.

More commonly, the genome of less ancient virus strains is being fairly routinely reassembled from e.g. from mummies, skeletons, or even packrat middens in order to study the evolution of common viral diseases. Most of this goes on without any safety controversies, as far as I can tell. Less commonly, ancient viruses of various species are found fairly intact in permafrost which does also stir some safety discussions (regarding the handling of permafrost).

So is your scenario possible in theory? Yes. Are experts worried about it? Not that I've heard…

Are there potential biohazards in this line of research, where dormant viruses that have otherwise gone extinct (along with the extinct host) and which now may have the potential to be reactivated?

There are always potential biohazards. If you dig into the unknown, you can't tell in advance what you can find. Said that I would like to point the attention to the fact that we don't know all the viruses already in existence, nor the ones that evolve every day.

You get in contact with new viruses during your everyday life anyway. How much more hazard would come from ancient viruses? I would say, not much but I can't point to any specific supporting experiment, so, it's just my opinion.

Could a disease be brought back via this kind of research?

Hardly. Lot's of parameters (an overview here) must be just right to have a virus spreading and cause disease. Viruses are usually very attuned to cells metabolism and environmental factors. Current parameters are probably very different from the original ones so, even if an ancient virus can be "revived", its ability to infect and spread would definitely be hindered.

Or are the conditions for waking latent viruses from starting material of this kind just not possible to recreate in a lab setting?

There are no standard conditions to awaken a dormant virus, or in general to produce viruses. Each virus is different and it will require specific conditions (here some examples on Lentivirus and Adenovirus). In theory, if all the required viral proteins can be expressed in a compatible cell host, then viable viral particles can be produced (Negev virus, Rhopalosiphum padi virus, Black queen-cell virus). "Reviving" an ancient virus is definitely possible with enough effort, but again, the rate of success will differ from virus to virus.

Hitchhiking virus confirms saga of ancient human migration

A study of the full genetic code of a common human virus offers a dramatic confirmation of the "out-of-Africa" pattern of human migration, which had previously been documented by anthropologists and studies of the human genome.

The virus under study, herpes simplex virus type 1 (HSV-1), usually causes nothing more severe than cold sores around the mouth, says Curtis Brandt, a professor of medical microbiology and ophthalmology at UW-Madison. Brandt is senior author of the study, now online in the journal PLOS ONE.

When Brandt and co-authors Aaron Kolb and Cécile Ané compared 31 strains of HSV-1 collected in North America, Europe, Africa and Asia, "the result was fairly stunning," says Brandt.

"The viral strains sort exactly as you would predict based on sequencing of human genomes. We found that all of the African isolates cluster together, all the virus from the Far East, Korea, Japan, China clustered together, all the viruses in Europe and America, with one exception, clustered together," he says.

"What we found follows exactly what the anthropologists have told us, and the molecular geneticists who have analyzed the human genome have told us, about where humans originated and how they spread across the planet," said Curtis Brandt.

Geneticists explore how organisms are related by studying changes in the sequence of bases, or "letters" on their genes. From knowledge of how quickly a particular genome changes, they can construct a "family tree" that shows when particular variants had their last common ancestor.

Studies of human genomes have shown that our ancestors emerged from Africa roughly 150,000 to 200,000 years ago, and then spread eastward toward Asia, and westward toward Europe.

Scientists have previously studied herpes simplex virus type 1 by looking at a single gene, or a small cluster of genes, but Brandt notes that this approach can be misleading. "Scientists have come to realize that the relationships you get back from a single gene, or a small set of genes, are not very accurate."

The PLOS ONE study used high-capacity genetic sequencing and advanced bioinformatics to analyze the massive amount of data from the 31 genomes.

"Our results clearly support the anthropological data, and other genetic data, that explain how humans came from Africa into the Middle East and started to spread from there."

The technology of simultaneously comparing the entire genomes of related viruses could also be useful in exploring why certain strains of a virus are so much more lethal than others. In a tiny percentage of cases, for example, HSV-1 can cause a deadly brain infection, Brandt notes.

"We'd like to understand why these few viruses are so dangerous, when the predominant course of herpes is so mild. We believe that a difference in the gene sequence is determining the outcome, and we are interested in sorting this out," he says.

For studies of influenza virus in particular, Brandt says, "people are trying to come up with virulence markers that will enable us to predict what a particular strain of virus will do."

The researchers broke the HSV-1 genome into 26 pieces, made family trees for each piece and then combined each of the trees into one network tree of the whole genome, Brandt says. "Cécile Ané did a great job in coming up with a new way to look at these trees, and identifying the most probable grouping." It was this grouping that paralleled existing analyses of human migration.

The new analysis could even detect some intricacies of migration. Every HSV-1 sample from the United States except one matched the European strains, but one strain that was isolated in Texas looked Asian. "How did we get an Asian-related virus in Texas?" Kolb asks. Either the sample had come from someone who had travelled from the Far East, or it came from a native American whose ancestors had crossed the "land bridge" across the Bering Strait roughly 15,000 years ago.

"We found support for the land bridge hypothesis because the date of divergence from its most recent Asian ancestor was about 15,000 years ago. Brandt says. "The dates match, so we postulate that this was an Amerindian virus."

Herpes simplex virus type 1 was an ideal virus for the study because it is easy to collect, usually not lethal, and able to form lifelong latent infections. Because HSV-1 is spread by close contact, kissing or saliva, it tends to run in families. "You can think of this as a kind of external genome," Brandt says.

Furthermore, HSV-1 is much simpler than the human genome, which cuts the cost of sequencing, yet its genome is much larger than another virus that also has been used for this type of study. Genetics often comes down to a numbers game larger numbers produce stronger evidence, so a larger genome produces much more detail.

But what really jumped out of the study, Brandt says, "was clear support for the out-of-Africa hypothesis. Our results clearly support the anthropological data, and other genetic data, that explain how humans came from Africa into the Middle East and started to spread from there."

The correspondence with anthropology even extends, as before, to the details. In the virus, as in human genomes, a small human population entered the Middle East from Africa. "There is a population bottleneck between Africa and the rest of the world very few people were involved in the initial migration from Africa," Brandt says. "When you look at the phylogenetic tree from the virus, it's exactly the same as what the anthropologists have told us."


Tuberculosis, caused by organisms in the Mycobacterium tuberculosis complex (MTBC), has taken on renewed relevance and urgency in the twenty-first century due to its global distribution, its high morbidity, and the rise of antibiotic-resistant strains [1]. The difficulty in disease management and treatment, combined with the massive reservoir the pathogen maintains in human populations through latent infection [2], makes tuberculosis a pressing public health challenge. Despite this, controversy exists regarding the history of the relationship between members of the MTBC and their human hosts.

Existing literature suggests two estimates for the time to the most recent common ancestor (tMRCA) for the MTBC based on the application of Bayesian molecular dating to genome-wide Mycobacterium tuberculosis data. One estimate suggests the extant MTBC emerged through a bottleneck approximately 70,000 years ago, coincident with major migrations of humans out of Africa [3]. This estimate was reached using a large global dataset of exclusively modern M. tuberculosis genomes, with internal nodes of the MTBC calibrated by extrapolated dates for major human migrations [3]. This estimate relied on congruence between the topology of the MTBC and human mitochondrial phylogenies, but this congruence does not extend to human Y chromosome phylogeographic structure [4]. As an alternative approach, the first publication of ancient MTBC genomes utilized radiocarbon dates as direct calibration points to infer mutation rates and yielded an MRCA date for the complex of less than 6000 years [5]. This younger emergence was later supported by mutation rates estimated within the pervasive Lineage 4 (L4) of the MTBC, using four M. tuberculosis genomes from the late eighteenth and early nineteenth centuries [6].

Despite the agreement in studies that have relied on ancient DNA calibration so far, dating of the MTBC emergence remains controversial. The young age suggested by these works cannot account for purported detection of MTBC DNA in archeological material that predates the tMRCA estimate (e.g., Baker et al. [7] Hershkovitz et al. [8] Masson et al. [9] Rothschild et al. [10]), the authenticity of which has been challenged [11]. Furthermore, constancy in mutation rates of the MTBC has been questioned on account of observed rate variation in modern lineages, combined with the unquantified effects of latency [12]. The ancient genomes presented by Bos and colleagues, though isolated from human remains, were most closely related to Mycobacterium pinnipedii, a lineage of the MTBC currently associated with infections in seals and sea lions [5]. Given our unfamiliarity with the demographic history of tuberculosis in sea mammal populations [13], identical substitution rates between the pinniped lineage and human-adapted lineages of the MTBC cannot be assumed. Additionally, estimates of genetic diversity in MTBC strains from archeological specimens can be difficult given their similarities to environmental mycobacterial DNA from the depositional context, which increase the risk of false positive genetic characterization [14]. Though the ancient genomes published by Kay and colleagues belonged to human-adapted lineages of the MTBC, and the confounding environmental signals were significantly reduced by their funerary context in crypts, two of the four genomes used for molecular dating were derived from mixed-strain infections [6]. By necessity, diversity derived in each genome would have to be ignored for them to be computationally distinguished [6]. Though ancient DNA is a valuable tool for answering the question of when the MTBC emerged, the available ancient data remains sparse and subject to case-by-case challenges.

Here, we offer a higher resolution temporal estimate for the emergence of the MTBC and L4 using multiple Bayesian models of varying complexity through the analysis of a high-coverage seventeenth-century M. tuberculosis genome extracted from a calcified lung nodule. Removed from naturally mummified remains, the nodule provided an excellent preservation environment for the pathogen, and exhibited minimal infiltration by exogenous bacteria. The nodule and surrounding lung tissue also showed exceptional preservation of host DNA, thus showing promise for this tissue type in ancient DNA investigations.

Resurrecting an ancient coronavirus outbreak

Dr Soulimi and colleagues hypothesised that humans might have had ancient encounters with coronaviruses that could show up in our genome.

So they surveyed the genomes of thousands of people from around the world stored in the 1000 Genomes Project database.

Getty Images: Kyodo News/Contributor

And they found a coronavirus-related genetic signal in people from Vietnam, China and Japan, but not in people from other parts of the world.

"After we observed this signature of adaptation we used different tools to tell us how far back that adaptation might have happened," Dr Soulimi said.

"Adaptation seems to have started about 25,000 years ago."

Not only is this the earliest evidence of human exposure to coronaviruses but it also shows how long they can hang around.

The researchers found the virus appears to have stopped exerting evolutionary pressure on the genomes 5,000 years ago, which means the epidemic lasted around 20,000 years.

"We really can't tell if this was a periodic thing that occurred every winter like the flu, or slightly different viruses that jumped from animals to humans every five to 10 years like what happened in the past 20 years with SARS, MERS, and SARS-CoV-2," Dr Souilmi said.

It may have been a single virus or a series of viruses that use the same molecular machinery, an idea supported by other research that shows the viral family that SARS-CoV-2 belongs to, emerged about 23,000 years ago, he said.

But either way, what's clear from the research is humans were exposed to coronaviruses for a period of roughly 20,000 years at one point in our history.

"The adaptation of several genes around the same time and at the same rate can only be explained by the exposure to coronaviruses back in time," Dr Souilmi said.

The researchers also found evidence that the virus involved in the ancient outbreak invaded cells in a similar way to SARS-CoV-2.


Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, USA

Joaquín Martínez Martínez

Department of Physiology, Genetics, and Microbiology, University of Alicante, Alicante, Spain

Francisco Martinez-Hernandez & Manuel Martinez-Garcia

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All authors researched data for the article, contributed to the discussion of the content and reviewed and edited the manuscript before submission. M.M.-G. and J.M.M. wrote the article.

Corresponding author


It was recently shown that the major genetic risk factor associated with becoming severely ill with COVID-19 when infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is inherited from Neandertals. New, larger genetic association studies now allow additional genetic risk factors to be discovered. Using data from the Genetics of Mortality in Critical Care (GenOMICC) consortium, we show that a haplotype at a region on chromosome 12 associated with requiring intensive care when infected with the virus is inherited from Neandertals. This region encodes proteins that activate enzymes that are important during infections with RNA viruses. In contrast to the previously described Neandertal haplotype that increases the risk for severe COVID-19, this Neandertal haplotype is protective against severe disease. It also differs from the risk haplotype in that it has a more moderate effect and occurs at substantial frequencies in all regions of the world outside Africa. Among ancient human genomes in western Eurasia, the frequency of the protective Neandertal haplotype may have increased between 20,000 and 10,000 y ago and again during the past 1,000 y.

Neandertals evolved in western Eurasia about half a million years ago and subsequently lived largely separated from the ancestors of modern humans in Africa (1), although limited gene flow from Africa is likely to have occurred (2 ⇓ ⇓ –5). Neandertals as well as Denisovans, their Asian sister group, then became extinct about 40,000 y ago (6). However, they continue to have a biological impact on human physiology today through genetic contributions to modern human populations that occurred during the last tens of thousands of years of their existence (e.g., refs. 7 ⇓ ⇓ –10).

Some of these contributions may reflect adaptations to environments outside Africa where Neandertals lived over several hundred thousands of years (11). During this time, they are likely to have adapted to infectious diseases, which are known to be strong selective factors that may, at least partly, have differed between sub-Saharan Africa and Eurasia (12). Indeed, several genetic variants contributed by archaic hominins to modern humans have been shown to affect genes involved in immunity (e.g., refs. 7, 8, 13, 14). In particular, variants at several loci containing genes involved in innate immunity come from Neandertals and Denisovans (15), for example, toll-like receptor gene variants which decrease the susceptibility to Helicobacter pylori infections and the risk for allergies (16). Furthermore, proteins interacting with RNA viruses have been shown to be encoded by DNA regions introgressed from Neandertals more often than expected (17), and RNA viruses might have driven many adaptive events in humans (18).

Recently, it was shown that a haplotype in a region on chromosome 3 is associated with becoming critically ill upon infection with the novel severe acute respiratory coronavirus 2 (SARS-CoV-2) (19) and was contributed to modern humans by Neandertals (20). Each copy of this haplotype approximately doubles the risk of its carriers requiring intensive care when infected by SARS-CoV-2. It reaches carrier frequencies of up to ∼65% in South Asia and ∼16% in Europe, whereas it is almost absent in East Asia. Thus, although this haplotype is detrimental for its carriers during the current pandemic, it may have been beneficial in earlier times in South Asia (21), perhaps by conferring protection against other pathogens, whereas it may have been eliminated in East Asia by negative selection.

A new study from the Genetic of Mortality in Critical Care (GenOMICC) consortium, which includes 2,244 critically ill COVID-19 patients and controls (22), recently became available. In addition to the risk locus on chromosome 3, it identifies seven loci with genome-wide significant effects located on chromosomes 6, 12, 19, and 21. Here, we show that, at one of these loci, a haplotype associated with reduced risk of becoming severely ill upon SARS-CoV-2 infection is derived from Neandertals.


A historical view of viral evolution might suggest that the evolutionary processes of RNA and DNA viruses adhere to distinct and non-overlapping rules. RNA virus evolution, as covered elsewhere in this volume, involves error-prone polymerases, an inability to perform error-correction (except in rare cases such as the coronaviruses), the existence of viral quasispecies, and a constant interplay of mutation and fitness-based selection. In contrast, DNA virus evolution is often discussed in more sweeping historical terms, with a focus on how evolution has led to speciation through the slow accumulation of genetic drift and relatively rare fixation of recombination-based genetic shifts. However, there is actually much in common between the mechanisms of evolution for both RNA and DNA viruses. For instance, while the polymerases used by DNA viruses are less error-prone and can perform error-correction, the larger size of many DNA virus genomes still leaves room for the accumulation of genetic variation in every round of viral replication. Furthermore, evidence from multiple DNA viruses suggests that rather than being rare, recombination between DNA virus genomes is rampant. The progeny of these genetic exchanges go unnoticed when recombination occurs between identical or highly similar genomes, or if the progeny do not survive fitness-based selection. Host-linked evolution or co-divergence may also contribute to the apparent low mutation rates in DNA viruses. Understanding the factors that determine the rate at which viral genomes generate and fix mutations provides essential insights into their evolutionary mechanisms. We cover these topics in greater detail below, after introducing a number of additional considerations to the discussion of how DNA viruses evolve (see Fig. 1 for summary).

DNA virus evolution relies on molecular mechanisms (top, shaded gray) which are impacted by host biology (arrayed below). DNA viruses exist in a range of genome formats (center) and sizes, each of which has a different propensity to evolve via these mechanisms. Viral genome formats include circular and linear DNA that is either single- or double-stranded, with lengths ranging from

2 to � kbp. The molecular mechanisms that underlie DNA virus evolution include single nucleotide changes, recombination and horizontal gene transfer, fluctuations in tandem repeat length, and sequence gain or loss through insertions, deletions, and segment duplications. Host impacts on DNA virus evolution (listed clockwise) include host cell architecture (e.g., nucleated vs. non-nucleated host cells), the time frame being considered (e.g., one round of infection or many generations), the host complexity (single-cells vs. complex organisms), an acute vs. long-term persistent duration of host infection, selective pressures and bottlenecks that act on each virus population, and co-divergence with host species over millennia. Image created using and Adobe Illustrator.

Diversity of DNA Virus Genome Types

A simplistic division of evolutionary mechanisms for viruses is generally split based on whether the genome being considered is RNA or DNA. While a single-stranded RNA virus and a double-stranded DNA virus might be considered typical exemplars of each group, these are by no means the only genome types – there are numerous variations on these themes. The prototypic double-stranded DNA (dsDNA) viruses exist in both linear and circular forms. These dsDNA viruses run the gamut in terms of size, from tiny (

5𠄸 kilobase pairs, kbp) papillomaviruses and polyomaviruses, to large bacteriophage, adenovirus, and herpesvirus genomes (ranging from

30� kbp), to the over-sized nucleocytoplasmic large DNA viruses (NCLDVs) such as poxviruses and phycodnaviruses (

130� kbp), and finally the giant viruses found in algae and amoeba (upwards of

1𠄲 megabase pairs, Mbp). There are also unusual genome formats among these dsDNA viruses, for instance, the covalently-closed ends of linear poxvirus genomes, or the partially gapped circular dsDNA genome of hepadnaviruses (e.g., hepatitis B virus). Also, there are abundant examples of single-stranded DNA (ssDNA) viruses, which include both linear (e.g., parvovirus and densovirus) and more numerous circular forms (e.g., circovirus, nanovirus, and geminivirus these are also known as Circular Rep-Encoding Single-Stranded or CRESS DNA viruses). In each case, these genome formats lead to particular constraints and opportunities for the evolutionary mechanisms discussed here. We describe the evolutionary mechanisms below in light of the most common dsDNA virus examples, and where possible, we note those areas where other DNA virus genome formats may differ.

Host Cell Biology and Availability of Host Enzymes Constrains Virus Evolution

It is possible – though not advisable – to discuss the mechanisms of DNA virus evolution without considering host cell biology. This simplification is enabled by the fact that all known hosts for these viruses are DNA-based life forms, with the concomitant presence of the requisite machinery of a DNA polymerase for replication, RNA polymerase for transcription, and ribosomes for translation. The most apparent distinctions among potential hosts for DNA viruses fall along the known bifurcations of the tree of life – namely bacteria, archaea, and the major groups of eukaryotes (i.e., plants, animals, fungi, and protists). In bacterial and archaeal hosts, the absence of a nucleus removes any distinction in where DNA virus replication occurs. However, in eukaryotes, many host enzymes are constrained to the nucleus, including host DNA and RNA polymerases as well as the RNA splicing machinery, whereas translation is limited to the cytoplasm. Viruses that utilize the host DNA polymerase to copy their genomes, such as members of the Polyomaviridae and Papillomaviridae, must therefore replicate in the nucleus. Likewise, while the Herpesviridae and Adenoviridae encode their own DNA polymerase, they use the host RNA polymerase and splicing functions, restricting their replication to the nucleus. In contrast, members of the Poxviridae and Mimiviridae that replicate in the cytoplasm encode their own DNA and RNA polymerases, whose fidelity can, therefore, evolve on a separate trajectory from that of the host. Finally, while ssDNA viruses use host DNA polymerases, their observed mutation rate far exceeds that detected in their host-cell genomes or in dsDNA viruses, suggesting that other sources of mutation such as oxidative damage and/or lack of DNA repair may be at play. For these reasons, knowledge of the host cell biology and the usage of host enzymes by a given virus species is a requirement for understanding the constraints on viral evolution.

Time Frames: Viral Adaptation Within a Host vs. Evolution Over Multiple Generations

Any discussion of the mechanisms of virus evolution needs to begin by defining the time scale under consideration. At the shortest end of this spectrum lies the time frame of a single round of viral infection. As noted below, the first infected cell may be anything from a single-celled organism to the first cellular entry point into a complex human host. From a clinical perspective, viral infection and disease are often considered on the time frame of a single individual’s infection – often a human or animal subject. As described below, the virus population within a given host may undergo adaptation within the relatively short time frame of the host’s infection. Mechanisms that enable diversification or speciation of a given virus usually require thousands of viral replication cycles, encompassing multiple host generations. At the grandest scale, the origins of viruses and specific lineages thereof spans the history of life on earth. The origins of viruses as we know them are covered elsewhere in this volume, so here we focus solely on the mechanisms that form the foundation of all viral adaptation and evolution. As such, we focus mostly on the time scale of an individual cell and/or host infection, which can include the contributions of virus populations that are more diverse and/or less fit than those which we see preserved over longer sweeps of evolutionary time.

DNA Virus Hosts Vary From Single Cells to Complex Multi-Cellular Organisms

An understanding of DNA virus adaptation and evolution requires a consideration of the host as a single-cell versus a complex multi-cellular organism. A basic theoretical model of viral replication would include productive viral replication in a single cell, followed by spread to nearby uninfected cells, potentially over multiple generations. This model may well apply to bacterial and archaeal cells, and to single-celled eukaryotic species such as marine alga or amoeba. However in most cases, more complex eukaryotic organisms, from plants to animals and humans, require a complicated series of steps for successful virus propagation and spread. These steps include entry via an accessible portal of the organism, dissemination within the organism to reach susceptible cells, evasion of host defensive responses (including innate and adaptive immunity), and egress to allow for potential spread to new hosts. There is ample evidence that evolution acts within a single host, although for the sake of clarity we will refer to these intra-host events as �ptation” rather than evolution. Using these terms allows us to highlight the distinction that local adaptation within a host is due to selective pressures that differ from those that impact transmission to new hosts, or that act across multiple generations of hosts. Also, the virus population within a complex organism may partition into distinct environmental niches within the host. For instance, the genomic diversity of human cytomegalovirus (HCMV) in patient samples is often analyzed from blood samples, and yet this viral population does not directly represent a common source of natural virus transmission between hosts (e.g., saliva). Studies of virus evolution need to carefully consider the source material used in examinations of viral diversity, and how this choice may influence the resulting observations of evolutionary fitness.

The Contributions of DNA Virus Persistence and Chronic Infections

We referred above to a theoretical model of DNA virus replication that involved productive replication in a single cell and spread into nearby uninfected cells, across multiple viral generations. An underlying assumption in such a model is that multiple rounds of productive infection occur sequentially. However, the lifecycle of many if not most DNA viruses exhibit other phases of existence, namely through persistence and chronic infections. For many bacteriophage and archaeal viruses, a common strategy is the well-known cycle of lysis versus lysogeny. For these viruses, the productive and often cell-destructive strategy of lytic replication is interleaved with phases of lysogeny, when the viral genome integrates into the host genome and is propagated as part of the host genome during cell division. A similar strategy exists for the large family of herpesviruses that infect most animal species and humans, with the long-term non-lytic phase being termed latency instead of lysogeny. An important distinction is that with a few notable exceptions, integration into the host genome is not a normal part of herpesvirus latency. Instead, these herpesviruses remain episomal in the host nucleus during lifelong latency. At the molecular level latency can be defined by the absence of significant viral replication and limited viral gene expression. Herpesvirus episomes can undergo sporadic reactivation to produce new viral progeny, which is followed by additional cycles of latency and reactivation. Similar to herpesviruses, certain members of the Adenoviridae can progress from a lytic infection of epithelial cells to a latent infection in T-lymphocytes of the tonsils and other adenoid tissues. The ability to establish a long-term infection is thus a vital part of the viral lifecycle of many DNA viruses, which contrasts with the acute infectious period of many RNA viruses (e.g., influenza virus or rotavirus). Persistence and chronic infections motivate the need to explore the contributions of within-host variation and adaptation to the evolutionary mechanisms of DNA viruses.

In addition to latency and lysogeny, virus persistence or chronic infection includes a whole class of DNA virus infections where viral replication is readily detected in the host, but the infection is not cleared for a significant length of time. Many smaller DNA viruses such as papillomaviruses, polyomaviruses, and certain members of the Circoviridae use this “low-and-slow” approach. These viruses replicate in actively dividing cells, but have evolved to avoid detection by the host immune system. Interestingly, many of these viruses appear to be pathogenic only if the virus persists for an extraordinarily long time. For example, in most cases, the host immune system will eventually clear human papillomavirus infections. This process typically spans several months if not years. However, a long-term infection (> 2 years) dramatically increases the risk of virus-induced cancer. Similarly, while polyomavirus infections in humans are typically asymptomatic, long-term persistence of JC polyomavirus causes complications in immunocompromised hosts. In these hosts, the otherwise benign infection can spread into the nervous system, where the viral infection can then induce significant damage (as discussed further below). The duration of animal lifespans, as opposed to single-celled hosts, means that long-term persistent viruses of animal cells have evolved to have significantly more interactions with their host’s immune system during lifelong latency, than are observed during bacteriophage or archaeal virus lysogeny. Recent advances in high-throughput sequencing technology are now enabling researchers to interrogate whether mutations in viral genomes are specifically correlated with disease progression in these chronic infection settings.

Co-Divergence With Hosts as a Driver of DNA Diversification

A common perception is that RNA viruses mutate rapidly while DNA viruses are slow and stable. This may stem from the view that the diversity of many DNA viruses can be explained by co-divergence with host species, thus placing viral evolution on a timescale of millions of years. Long-term co-divergence and consequently low rates of nucleotide substitution have been supported in some DNA viruses however, this is likely only part of the equation. The development of new sequencing technologies and the ability to include temporal information into molecular clock models allows us to estimate the rate and timescale of virus evolution independent of the (strong) assumption of co-divergence. Indeed, many DNA viruses show evolutionary rates close to those of RNA viruses, which themselves span a range of mutation rates. It is important to note that time-structured sequence data spanning years or decades often contain short-lived polymorphisms. Researchers should thus use caution when comparing mutation rates at such distinct evolutionary scales.

Nonetheless, for many viruses, it is essential to acknowledge that both short and long timescales may provide valuable information. While there is strong evidence supporting co-divergence of the Polyomaviridae with their hosts, recent studies have demonstrated the need to account for faster evolution within this virus family. In immunocompromised patients, mutations in the JC polyomavirus capsid protein allow it to escape neutralizing antibodies and invade the central nervous system, causing an opportunistic brain disease called progressive multifocal leukoencephalopathy (PML). The ability to evade the immune system – while remaining extraordinarily stable over longer timeframes – suggests that the Polyomaviridae evolve at two distinct rates. In the case of ssDNA parvoviruses, researchers seeking to understand the determinants of host range variation have tested the outcome of culturing several closely related viruses (㺘% nucleotide identity) in cells derived from phylogenetically distinct hosts. The authors found that canine parvovirus (CPV-2) underwent extensive mutation during passage in non-native host cells, while no mutations arose in cells from the native host. These data indicate that the virus was well-adapted to its current host species, but that multiple mutations in its surface protein were needed for it to infect diverse host species efficiently. These data illustrate how long-term host dependency may constrain evolutionary rates in many DNA viruses.

Single Nucleotide Differences as a Measure of Evolutionary Change

Specific mutations such as single nucleotide polymorphisms (SNPs), insertions, and deletions (together termed in/dels) are likely to experience different selection dynamics, which impact the chances that these variations become fixed in the population. However, unlike for nucleotide substitutions (i.e., SNPs), the methods for measuring the evolutionary rate of insertions and deletions (in/dels) are not well developed. Because of this limitation, our understanding of viral evolution is primarily based on measuring the accumulation of SNPs over time, which ignores the potentially critical influence of other sources of variation, such as in/dels, tandem repeat fluctuations, and recombination (discussed further below). Recent studies have also provided evidence that viral evolutionary rate estimates decrease as their measurement timescales increase. This is evident in the field of paleovirology. For example, 𠇏ossilized” hepadnavirus DNA integrated into bird genomes suggests that these viruses are at least 19 million years old. In turn, this implies a significantly slower evolutionary rate than what was predicted based solely on extant viruses.

Early studies on the mutation rate of DNA viruses using single-gene or single-locus analyzes estimated a mutation rate on the order of 1 × 10 – 7 to 1 × 10 𠄸 substitutions/site/year. These values have been further supported by genome-wide comparisons for a handful of large DNA viruses. For instance, a recent study used a high-fidelity high-throughput sequencing (HTSeq) technique called duplex sequencing to detect spontaneous mutations in clonal lineages of human adenovirus 5, and the authors found that these occurred at a rate of 1.3 × 10 𠄷 per base, per infection cycle. This rate matches well to a genome-wide estimate of the in vitro and in vivo mutation rates for murine CMV, which was obtained by shotgun Sanger approaches just before the development of HTSeq (

1 × 10 𠄷 mutations per bp per day). These low mutation rates are often cited by those wishing to contrast DNA virus stability with RNA virus diversity. However, data from both modeling and newer HTSeq-based comparative genomics studies have indicated that large DNA viruses may have mutation rates closer to 1 × 10 𠄵 or 1 × 10 𠄶 . In our comparisons of sub-clones generated from a parental population of herpes simplex virus 1 (HSV-1), we observed 3%𠄴% variation between sub-clones, genome-wide. Other studies of HSV have shown that antiviral drug resistance mutations can be selected from a naïve virus population in just one round of viral passage in vitro. These data suggest that at least under certain circumstances, standing variation is maintained in DNA virus populations. An alternative or additional theory is that de novo mutations may occur at specific genomic regions more often than others (e.g., hot spots). The wider application of genome-wide measurements of viral variation will help to elucidate these possibilities.

In Vivo Observations of Within-Host Diversity and Adaptation of DNA Viruses

Recent advances in high-throughput sequencing have now enabled the detection of minor variants within a single viral isolate or patient. These minor alleles can manifest as a new dominant allele or genotype after population bottlenecks or selective pressures such as antiviral therapy. Evidence of sequential takeover by distinct HCMV strains has been observed in immunocompromised adult patients, demonstrating both the existence of co-infections as well as the opportunities for recombination and/or subsequent selection. Studies of vaccine-associated rashes for varicella-zoster virus (VZV), and of congenital infections by HCMV, have demonstrated the potential for niche-specific adaptation or segregation of viral variants within specific body sites of infected hosts. For human papillomavirus 16 (HPV16), a recent study of several thousand women used a combination of PCR and Illumina-based HTSeq to reveal an unexpectedly high level of viral genetic variability. Of note, there was higher HPV16 genetic variability between patients than within a single patient, suggesting that many of the identified sequence differences were specific to each patient. Interestingly, women with pre-cancerous lesions had significantly less variation than women with a productive (early stage) HPV16 infection, confirming that cellular transformation by HPV represents a genetic bottleneck. This high level of inter-patient variability demonstrates that, at least within some settings, the mutation rate for HPV must be significantly higher than the previously estimated 2 × 10 𠄸 nucleotide substitutions/site/year for the viral coding genome. Importantly, the higher-than-expected rate of inter-host evolution argues against the notion that a subset of (oncogenic) human papillomaviruses were acquired by archaic hominins during their migration out of Africa. Together these data indicate that many DNA virus populations may contain and/or generate standing variation following infection. It also appears that this variation is not often transmitted to a new host. The lack of successful transmission of these minor variants suggests that the standing variation in viral populations only becomes phenotypically apparent after population bottlenecks or selection. Importantly, these studies provide corroborating evidence that the molecular mechanisms of DNA virus evolution which have been demonstrated in vitro , also operate in vivo.

Fluctuations in Tandem Repeat Copy Number as a Mechanism of Evolution

Changes in the length or copy number of tandem repeats (TRs) provide another mechanism of virus evolution. Short TRs are usually categorized into three groups: homopolymers, which are sequential repeats of a single base (e.g., 5 or more C׳s in a row) microsatellites, which have a repeating unit of 㰐 base pairs (bp) and mini- or macrosatellites, which include repeating units of 10� bp. The mechanisms of repeat expansion or contraction vary by the repeat size. Homopolymer-based length variants are presumed to arise primarily through polymerase slippage, whereas larger TRs may arise either by template looping during polymerase progression or through recombination as discussed below. The repeating units of TRs may be perfect copies or include minor imperfections in the repeating sequence, and these repeats can occur in both coding and non-coding regions. In coding sequences, repeated elements may contribute to structural units of protein folding (e.g., turns of an alpha-helix) or provide variable lengths of unstructured regions within a multi-domain protein. Noncoding repeats have been shown to include promoter elements, chromatin or insulator binding motifs, as well as secondary structural elements such as quadruplexes and other motifs.

For many tandem repeats, the only viral data available is their conservation of position in the genome of a given species, and perhaps data on the degree to which a given TR varies in length across different virus isolates of the species. Functional roles have been demonstrated for select TRs in just a handful of DNA viruses. In the few herpesvirus species that have been shown to integrate into a host genome, there are viral telomeric repeats that function in their integration into the host. In other non-integrating herpesviruses, length variations at homopolymeric tracts in the thymidine kinase (TK) and polymerase genes are a common route of viral escape from the antiviral drug acyclovir. Ribosomal frameshifting of defective transcripts in these drug-resistant genomes allows the translation of a low level of functional TK or polymerase, enabling viral survival even in the face of an otherwise disabling mutation. Fluctuations in TR lengths have also been described for JC polyomavirus populations in patients. In this case a predominant polyomavirus genotype, or archetype, is shed in the urine of most infected individuals, while rearranged forms with deletions and TR variations are found in the brains of patients with PML disease. For poxviruses and other large DNA viruses, restriction fragment length polymorphisms (RFLPs) have often been used to track changes in the dominant virus genotypes and TRs over time. In a recent study of myxoma virus (a Poxviridae member), the predominant RFLP type was observed to change each year. Expansion of the inverted terminal repeat boundaries appears to provide myxoma virus with an opportunity for evolution. Likewise, the genome of the vaccinia poxvirus shows similar heterogeneity of the terminal repeats. Repeated plaque based purifications have shown that heterogeneity in the terminal repeats can evolve rapidly from the DNA of a single vaccinia virion. As technologies to track fluctuations in the length of TRs improve, it will no doubt become easier to examine these changes and gain a better understanding of their contribution to virus adaptation and evolution.

Large DNA Viruses Undergo Frequent Recombination

Recombination can serve as a driving force for evolutionary shifts in DNA viruses, akin to the genetic shifts that result from reassortment in segmented RNA viruses. Recombination can be classified as homologous recombination – between like sequences – or as illegitimate or non-homologous recombination. For most large DNA viruses, the potential of the viral genome to recombine has been studied by analyzing phylogenetic relationships between naturally circulating viral genomes. Among the adenoviruses, which include seven species (human adenovirus A-G) and multiple serotypes, recent studies applying HTSeq-based comparative genomics have demonstrated both intra-species and interspecies recombinants – often in association with pathogenic infections. For instance, a naturally circulating intratypic recombinant of human adenovirus subtype C was found to be the etiologic agent of severe acute respiratory infections in children in China. There are also examples of both historical and recent isolates of pathogenic adenoviruses that appear to have arisen from zoonotic transmission and recombination between simian and human adenoviruses. For the beta-herpesvirus HCMV, multiple studies have demonstrated a history of rampant recombination between the genomes of different isolates. Particular sections or islands of the HCMV genome appear to have co-segregated, while widespread recombination between strains has created a mixture of alleles elsewhere in the genome. It is thought that genes in these islands are co-dependent, thus placing a fitness cost on any recombination events that occur inside these regions. Similar levels of within-species recombination have been shown for most herpesviruses with sufficient genome sequence availability to make these comparisons. Recently, data supporting potential inter-species recombination among these viruses have been observed as well, with HSV-1-like DNA detected in several loci of the HSV-2 genome. Likewise, a virulent avian herpesvirus that created an outbreak in Australian poultry was revealed to be a spontaneous recombinant derived from two live-attenuated vaccines in use in the area. For large DNA viruses such as herpesviruses and poxviruses, laboratory co-infection studies and analysis of recombinant progeny by HTSeq have further defined the genome-wide potential for recombination and begun to define hot spots or regions with a higher propensity to recombine. Together these data demonstrate the extensive role of recombination in the evolution of both nuclear- and cytoplasmic-replicating large DNA virus genomes.

Recombination at Different Frequencies for Small DNA Virus Genomes

Large DNA viruses appear to recombine more readily than the small dsDNA viruses of the Papillomaviridae and Polyomaviridae. Even under controlled experimental conditions, no conclusive evidence for recombination within these two virus families has been described. One theory for this lack of observable recombination is that smaller viruses have fully optimized the usage of their genomic real-estate, such that recombination events would be highly likely to interrupt co-dependent genes or regulatory sequences – and thus carry too high a fitness cost to survive. However, phylogenetic analyses have identified evidence for several recombination events within the Papillomaviridae. As in HCMV, it appears that ancient recombination has segregated functional regions of the viral genome, separating the genes coding for non-structural proteins from the structural genes. Recombination does not appear to play a significant role in the short-term adaptation of the papillomaviruses, implying that recombined daughter viruses are not as fit as the parental genomes. Supporting this hypothesis, even when evidence of HPV16 recombination was detected within a single patient, these recombinant genomes were incapable of sustained replication within the host. Similarly, while phylogenetic analysis can detect evidence for ancient recombination near the root of the Polyomaviridae phylogenetic tree, recombination does not appear to be a significant component of ongoing polyomavirus evolution. However rare recombination events can and do contribute to virus evolution. For instance, conservation efforts to prevent the extinction of the western barred bandicoot have been hampered by an outbreak of the bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1), a recombinant between an ancestral papillomavirus and polyomavirus. This virus is a hybrid that appears to have recombined the structural genes of the Papillomaviridae with the non-structural genes of the Polyomaviridae. These examples illustrate how rare and unusual recombination events can enable the dramatic expansion of viral evolutionary sequence space.

Despite being roughly the same size as the Polyomaviridae, single-stranded DNA viruses recombine relatively efficiently. Single-stranded parvoviruses have shown an ability to jump to new hosts rapidly, and recombination along with a relatively high mutation rate has been hypothesized to underlie this ability. Parvoviruses have also been demonstrated to readily recombine in cell culture. Although the mechanism of parvovirus recombination is not known, a role for viral secondary structure has been proposed. Indeed, the parvovirus origin of replication forms a hairpin structure that is a recombination hot spot, potentially due to stalling of DNA polymerase at this secondary structure. Template swapping before re-initialization of replication could then result in the formation of a chimeric genome. Alternatively, parvovirus replication may create intermediate concatemers. Resolving these concatemers may activate DNA repair enzymes, leading to the creation of mosaic viruses through the homologous recombination repair system. While recombination appears to play an essential role in the evolution of ssDNA viruses, these viruses appear to have adapted to minimize combinations of incompatible regulatory elements. For example, the gene encoding the replication protein (Rep) and the cis-acting elements that interact with the replication protein are usually within 100 nucleotides of one another. This ensures that the replication machinery is highly likely to remain together and compatible following any recombination events. A detailed comparison of recombination patterns within ssDNA viruses also found that breakpoints tend to fall outside of known genes. These observations imply that viruses expressing recombinant proteins are not usually tolerated.

Duplication and Deletions of Genes and Genome Segments

The outcome of recombination within identical or highly similar genomes is rarely noticed, except for occasions where this event leads to gene duplication or loss. Evidence of gene duplication and subsequent divergence is prevalent in adenovirus genomes. Ancient incidents of gene capture presumably produced those adenoviral gene products with similarity to host genes or those of other viruses, which are found across many adenoviral genera. Other more evolutionarily-recent duplications are found in smaller subsets of adenoviral species. The phenomenon of gene loss has been well-documented in herpesviruses, where across the diverse alpha-, beta-, and gamma-subfamilies of the Herpesviridae, many examples of gene loss have been found during viral propagation in vitro. The phenomena of genetic drift and gene loss were first detected in laboratory-passaged strains of the beta-herpesvirus HCMV, where the gene regions lost in vitro were later found to have functions associated with cell tropism and immune evasion in vivo. The extremely large mimivirus dsDNA genome has also been shown to undergo gene loss from both its termini during repeated passage in an amoebal host. In mimivirus, this gene loss was associated with a phenotypic change in virions, which was visible as a loss of fibrils on the virion surface.

In contrast to gene loss, the duplication of genetic segments – a gene accordion – has been best demonstrated by a series of elegant studies in poxviruses grown in vitro. These studies showed that expansion of gene copy number could provide functional fitness recovery after deletion of a core viral gene, by driving higher expression of a less-efficient gene version. This expansion also enabled the adaptation and eventual evolution of improved function, via mutations that occurred in the redundant copies of this gene. Whether or not this type of gene accordion occurs for DNA viruses that replicate in the nucleus remains to be determined. The segregated nature of nuclear replication and transcription, followed by translation in the cytoplasm, means that nuclear-replicating viruses will complement defects in co-replicating genomes in trans , since proteins made in the cytoplasm can be utilized by all progeny genomes.

Among the small DNA viruses, a subset of human papillomaviruses is associated with recurrent respiratory papillomatosis (RRP). Interestingly, these RRP-associated viruses are not typically considered as oncogenic viruses. However while RRP is considered a benign neoplasm of the larynx, involvement of the lungs is almost invariably fatal. Whole genome sequencing efforts have implicated a duplication of the viral promoter and a subset of viral genes in the RRP progression towards lung invasion. While the expansion of these loci in the papillomaviral genome is likely not important during a normal viral lifecycle, these data illustrate how duplications can provide a powerful adaptation mechanism for otherwise slow-evolving viruses.

Host-Virus Exchange via Horizontal Gene Transfer and Transposable Elements

Horizontal gene transfer (HGT) provides another avenue for evolutionary adaptation of both viruses and their hosts. HGT has been well-documented between bacterial and archaeal host species, often vectored by large DNA bacteriophages or archaeal viruses. Recent data have demonstrated that HGT may also take place between eukaryotic hosts and their viruses. For example, transposable elements (TEs) found in the moth genome have also been detected in the genomes of baculoviruses that infect these moths. Since this baculovirus infects several species of sympatric, co-occurring moths, it may well be the historical vector that moved TEs among these different host species. Other host-derived sequences were also detected in about 5% of progeny baculovirus genomes, although the co-opted host DNA was not carried beyond a few cycles of viral replication. Most of the integrated host sequences were TEs, but others appeared to result from recombination at sites of microhomology between the host and viral genomes. Most large DNA viruses are not known to integrate into the host genome as part of their overall replication strategy. Select herpesviruses of the alpha- and gamma- subfamilies do integrate into the host genome, although for these viruses it appears to be a reversible process that can lead to later excision and non-integrative replication. Marek’s disease virus, an alpha-herpesvirus of poultry, and human herpesvirus (HHV) 6A and 6B, two gamma-herpesviruses of humans, integrate into host telomeres as a central part of their lifecycle. The germline or chromosomal integration of human herpesviruses (ciHHV), usually HHV6A, is detected in about 1% of the human population, although the clinical consequences of ciHHV are as yet unknown. These examples recommend the use of genome-wide HTSeq of viral populations as a means to detect horizontal gene transfer in action.

For the small DNA polyoma- and papillomaviruses, integration of all or a fragment of the viral genome into the host cell DNA is an evolutionary dead end, with an outcome that is nonetheless well-known for having the potential to induce dramatic outcomes of dysregulated cell division and tumor formation. In a recent study of HTSeq data from HPV-positive head and neck cancers, evidence was found to suggest that the HPV genome can replicate as an independent viral–human hybrid mini-chromosome, at least in some instances. These data implied that following an integration event, the viral genome may be excised from the human chromosome, creating a viral–human hybrid circular episome. Under particular circumstances, these hybrid genomes could theoretically get packaged into infectious virions. However, considering the tight regulation of papillomavirus replication, it appears unlikely that these hybrid genomes would be able to establish an infection in the next host.

Risks of latent viruses that reside in ancient genomes under research? - Biology

The distinctions between virions and viruses and modern and ancient cells are crucial to understand virus origins and evolution.

Viruses can be better defined by their generic features of genome propagation and dissemination rather than physical or biological properties of their virions or hosts.

Virus genomes are characterized by the abundance of virus-specific genes that lack detectable cellular homologs. Despite their abundance, virus-specific genes are rarely discussed in the models of virus origin and evolution.

The alignment-based methods are ill-suited for the origins of life research, especially when the objective is to place fast-evolving organisms or viruses in the tree of life.

Protein structures may provide a better alternative to resolve the very deep branches in the tree of life.

The ongoing COVID-19 pandemic has piqued public interest in the properties, evolution, and emergence of viruses. Here, we discuss how these basic questions have surprisingly remained disputed despite being increasingly within the reach of scientific analysis. We review recent data-driven efforts that shed light into the origin and evolution of viruses and explain factors that resist the widespread acceptance of new views and insights. We propose a new definition of viruses that is not restricted to the presence or absence of any genetic or physical feature, detail a scenario for how viruses likely originated from ancient cells, and explain technical and conceptual biases that limit our understanding of virus evolution. We note that the philosophical aspects of virus evolution also impact the way we might prepare for future outbreaks.

Viruses Collectively Decide Bacterial Cell's Fate

A new study suggests that bacteria-infecting viruses &ndash called phages &ndash can make collective decisions about whether to kill host cells immediately after infection or enter a latent state to remain within the host cell.

The research, published in the September 15 issue of the Biophysical Journal, shows that when multiple viruses infect a cell, this increases the number of viral genomes and therefore the overall level of viral gene expression. Changes in viral gene expression can have a dramatic nonlinear effect on gene networks that control whether viruses burst out of the host cell or enter a latent state.

"What has confounded the virology community for quite some time is the observation that the cell fate of a bacteria infected by a single virus can be dramatically different than that infected by two viruses," said Joshua Weitz, an assistant professor in the School of Biology at the Georgia Institute of Technology. "Our study suggests that viruses can collectively decide whether or not to kill a host, and that individual viruses 'talk' to each other as a result of interactions between viral genomes and viral proteins they direct the infected host to produce."

To study viral infections, Weitz teamed with postdoctoral fellow Yuriy Mileyko, graduate student Richard Joh and Eberhard Voit, who is a professor in the Wallace H. Coulter Department of Biomedical Engineering, the David D. Flanagan Chair Georgia Research Alliance Eminent Scholar in Biological Systems and director of the new Integrative BioSystems Institute at Georgia Tech.

Nearly all previous theoretical studies have claimed that switching between "lysis" and "latency" pathways depends on some change in environmental conditions or random chance. However, this new study suggests that the response to co-infection can be an evolvable feature of viral life history.

For this study, the researchers analyzed the decision circuit that determines whether a virus initially chooses the pathway that kills the host cell &ndash called the lytic pathway &ndash or the pathway where it remains dormant inside the host cell &ndash called the lysogenic pathway.

When the lytic pathway is selected, the virus utilizes bacterial resources to replicate and then destroys the host cell, releasing new viruses that can infect other cells. In contrast, in the lysogenic pathway, the viral genome inserts itself into the bacterial genome and replicates along with it, while repressing viral genes that lead to lysis. The virus remains dormant until host conditions change, which can result in a switch to the lytic pathway.

The decision of the genetic circuit that controls whether a virus initially chooses lysis or lysogeny is not random. Instead, cell fate is controlled by the number of infecting viruses in a coordinated fashion, according to the new study, which was funded by the Defense Advanced Research Projects Agency, the National Science Foundation and the Burroughs Wellcome Fund.

"In the case of perhaps the most extensively studied bacteriophage, lambda phage, experimental evidence indicates that a single infecting phage leads to host cell death and viral release, whereas if two or more phages infect a host the outcome is typically latency," explained Weitz, who is a core member of the new Integrative BioSystems Institute at Georgia Tech. "We wanted to know why two viruses would behave differently than a single virus, given that the infecting viruses possess the same genetic decision circuit."

To find out, the researchers modeled the complex gene regulatory dynamics of the lysis-lysogeny switch for lambda phage. They tracked the dynamics of three key genes &ndash cro, cI and cII &ndash and their protein production. The decision circuit involved both negative and positive feedback loops, which responded differently to changes in the total number of viral genomes inside a cell. The positive feedback loop was linked to the lysogenic pathway and the negative feedback loop was linked to the lytic pathway.

With a single virus, cro dominated and the lytic pathway prevailed. If the number of co-infecting viruses exceeded a certain threshold, the positive feedback loop associated with cI dominated, turning the switch to the lysogenic pathway. The differences in bacterial cell fate were stark and hinged upon whether or not one or two viruses were inside a given cell.

The researchers found that the cII gene acted as the gate for the system. Increasing the number of viruses drove the dynamic level of cII proteins past a critical point facilitating production of cI proteins leading to the lysogenic pathway.

"The decision circuit is a race between two pathways and in the case of a single virus, the outcome is biased toward lysis," explained Weitz. "In our model, when multiple viruses infect a given cell, the overall production of regulatory proteins increases. This transient increase is reinforced by a positive feedback loop in the latency pathway, permitting even higher production of lysogenic proteins, and ultimately the latent outcome."

The central idea in the model proposed by Weitz and collaborators is that increases in the overall amount of viral proteins produced from multiple viral genomes can have a dramatic effect on the nonlinear gene networks that control cell fate.

"Many questions still remain, including to what extent subsequent viruses can change the outcome of previously infected, but not yet committed, viruses, and to what extent microenvironments inside the host impact cell fate," added Weitz. "Nonetheless, this study proposes a mechanistic explanation to a long-standing paradox by showing that when multiple viruses infect a host cell, those viruses can make a collective decision rather than behaving as they would individually."

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Materials provided by Georgia Institute of Technology. Note: Content may be edited for style and length.


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