What is known about viruses?

• Example textbook. Field’s virology
• Well over 2000 pages per volume (2 volumes)
• Each chapter apologises for what is not included
• Uncertainties, known unknowns, unknown unknowns: but very few known knowns
• Most chapters focus on pathology and molecular biology
• Ecology of viruses remains poorly understood

Web of science

• The pandemic has brought viruses into the news
• However there is a shortage of literature on the ecology of viruses
• Search terms “virus” + “ecology”
• Search term “coronavirus”

Quick history

• Viruses not recognised until 1880s
• Adolf Meyer working on tobacco at Wageningen identified TMV
• Clearly an infectious agent, but could not fulfill Koch’s postulates as only cultivable in living cells:
• The issue with Koch’s postulate continues to muddy some debates to this day
• Beijerinck named the agent a “contagium vivum luidum”" or a contagious living liquid.
• d’Herelle developed the plaque assay in 1917
• First electron micrographs were taken of tobacco mosaic virus (TMV) in 1939.

Tobacco mosaic virus

• Remains a key experimental organism
• Plant viruses are possibly more diverse than higher animal viruses (more species of plant)
• Plant viruses have a different set of challenges (movement through an organism with cell walls)
• Plant viruses are not thought to infect animals directly (possible exceptions)

Bacteriophages

• Lytic (pathogneic) and lysogenic (non pathogenic) infections identified in 1900s
• Experiments with viruses resulted in the discovery of mRNA and the role of DNA
• First complete nucleotide sequence of an RNA virus genome was the bacteriophage MS2 (1976)
• Most abundant “organisms” on the planet
• Present at roughly \(10^7/mL\) in coastal seawater
• Around \(10^{31}\) individual tailed phage virions on the planet
• If all the tailed phages were laid end to end, they would extend for 200 million light years into intergalactic space.

Phage seen through electron microspope

Insect viruses

• Abundant, ubiquitous and highly diverse
• Mammalian and avian viruses may have evolved in insects
• Insects are also vectors for both animal and plant viruses
• Can play a role in very complex life histories
• Some extremely complex symbiotic relationships

Parasitic wasps use viruses as “venom”

Mammalian viruses

• Experiments on viruses of animals used immortalised human cell lines and monkey kidney cells
• Polio virus was an early focal virus
• Virology allowed key insights into oncology and cancer
• Cellular receptors began to be identified and studied in depth
• Attention mainly on pathogenic viruses of humans (and some of rodents and domestic animals)
• Full diversity of mammalian viruses remains unknown

Viral evolution

• Fundamental aspects of viral evolution remains unknown, contentious, or both
• Precise estimates of mutation rates unknown
• Deep origins of viruses still uncertain
• Did viruses predate cellular organisms?
• “Escaped gene theory” vs “RNA first” theory
• Did the earliest replicators resemble what we now know as viruses?
• Should viruses even be classified as life?

Evolution and phylogeny

• Viruses generally evolve at high rates, following basic and extended Darwinian principles
• Evolutionary time frame much shorter than that of any other organism.
• Species concept cannot be uncontentiously applied.
• True antiquity of currently circulating viruses still vague.
• No fossil record

Baltimore classification

Highest level of classification of viruses

• Split into 7 groups based on type of genetic material
• DNA viruses (single and double stranded)
• RNA viruses (double stranded, single stranded positive and negative sense )
• Reverse transcribing viruses

Number of viral species

• Perhaps over one million viruses of vertebrates
• ~20 different viruses in each of the 50,000 vertebrates
• “Minimum of 320,000 mammalian viruses awaiting discovery” (Anthony et al. 2013)

Species concept as applied to viruses

• Importance of species in biology derives from their importance in systematics
• Prior to the 1980s the notion of viral “species” was rejected by most of the scientific community
• Clearly could not apply Ernst Mayr’s definition(“species are groups of actually or potentially interbreeding natural populations, which are reproductively isolated from other such groups”)
• Concepts of species, strains, variants and viral isolates caused confusion (Alimpiev 2019)
• 1982 International Committee on Taxonomy of Viruses(ICTV): species = “a cluster of strains from a variety of sources, or a population of strains from a particular source, which have in common a set of stable properties that separate the cluster from other clusters of strains.”

Modified definitions

• Van Regenmortel: “a polythetic class of viruses (strains) that constitute a replicating lineage and occupy a particular ecological niche”
• Polythetic is defined as “characteristics which occur commonly in members of a group or class, none of which is essential for membership of that group or class.”
• 2012 ICTV viral species definition based on shared ancestry. “A monophyletic group of viruses (strains) whose properties can be distinguished from those of other species by multiple criteria”
• Can be used to define any virus taxon. (I.e. not just a species)
• Current definition essentially defines virus species (clades) based only on their genomic sequence
• However genetic sequences are a continuum, with no naturally defined breaks

Time since divergence

Knowing time since divergence key to understanding viral evolution and viral “novelty.”

• Three approaches

1. Find a good match between the phylogenetic tree of viruses and that of their hosts. Assume co-divergence (not usually possible as few viruses are narrowly host specific .. can work for DNA viruses .. not generally RNA)
2. Find endogenous genome copies within the host: Use the substitution rate of the host (similar to 1)
3. Measure current evolution (i.e mutations fixed during time frame of human observation)

RNA viruses

• Particularly difficult to produce phylogenies
• Rarely strictly host specific
• Very fast rates of mutation
• Many RNA virus populations thought to exist as quasispecies
• Swarms of individual viral genomes where every member is unique.

Molecular clock

• Rate of evolution has been estimated for polio (short RNA ~7500 base pairs)
• Estimated that there is one synonymous substitution per 100 base pairs per year (Jorba et al. 2008)
• Non-synonymous substitutions constrained by purifying selection
• However, these measurements were on a population with low prevalence
• Hard to generalise, as rate of evolution depends on number of infections

Evolution involves the host

• Experimental approaches using in vitro and in vivo laboratory models focus on short-term intrahost evolutionary mechanisms
• These may not always be relevant to natural systems.
• Should consider data collected over multiple cycles of virus–host transmission. (Geoghegan and Holmes 2018)
• Evolution can take place at different rates and follow different lines in different host populations

Geoghegan and Holmes (2018)

Quasispecies evolution

• Mutation–selection balance in which a distribution of variant viral genomes is ordered around the fittest, or “master,” sequence.
• Mutation rates in RNA viruses are so high that the frequency of any variant is not only a function of its own replication rate (fitness), but also of the mutation of other variants in the population derived from the original sequences
• “Mutational coupling” leads to a distribution of evolutionarily interlinked viral genomes
• Entire mutant distribution behaves as a single unit
• Natural selection acts on the mutant distribution as a whole, rather than on individual variants.
• The “quasispecies”" as a whole evolves to maximize its average fitness.
• Has been applied to SARS-Cov-1 (Xu, Zhang, and Wang 2004) and SARS-CoV-2 (Jary et al. 2020)

Quasispecies evolution

Recombination

• Many, many examples of hybridisation in plants
• Some examples in well known animals (e.g edible frogs)
• Can be very difficult to establish as hybrids unless the sequences of all species involved are known.
• The equivalent of Tigons, Ligers and edible frogs also exist in the world of viruses:
• Known as Recombinants
• The result of simultaneous infection with different viral sequences

“Hybrid viruses”: Influenza

• Influenza A, B, C and D are genera
• Enveloped negative sense single strand RNA viruses with a segmented genome
• Cell entry through glycoproteins: haemagglutinin (HA) and neuraminidase (NA)
• 16 antigenically different HA and 9 antigenically different NA serotypes
• Interchange, of genomic RNA segments occurs when two viruses of the same type infect the same cell.

Recombination is SIV (simian IV) and Ebola

• SIVcpz represents a complex mosaic, generated by recombination of two lineages of SIVs that infect monkeys (Sharp and Hahn 2011)
• Multiple ebola recombinants in simians (Wittmann et al. 2007)

Coronavirus subgenomic RNA

• Coronavirus viral RNA synthesis involves the transcription of multiple sub-genomic RNAs
• Potential for recombination unclear, but highly likely

Putative phylogeny of SARS-CoV-2 clades

• Divergence based on assumptions regarding conserved characteristics of the sequence.
• Dates rely on assumed molecular clock
• Most sequences have gone extinct
• Extant (still circulating) sequences appear to have reduced virulence

Putative phylogeny of SARS-CoV-2 clades