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

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

Overlaps between SARS-CoV-1 and SARS-CoV-2

• Around 86% sequence similarity between SARS-CoV-1 and SARS-CoV-2
• However, also considerable sequence diversity within each clade
• “In terms of taxonomy, SARS-CoV-2 is (just) another virus in the species Severe acute respiratory syndrome-related coronavirus” (Gorbalenya et al. 2020)

Non-identical base pairs

• Overlaps in between and within clade sequence differences (Gorbalenya et al. 2020)

Phylogeny of coronaviruses

• Key questions regarding time scale of all divergences between all coronavirus taxa.

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 thes is one synonymous subsitution 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

Quasispecies

• Experimental approaches using in vitro and in vivo laboratory models focus on short-term intrahost evolutionary mechanisms
• May not always be relevant to natural systems.
• Comparative approach relies on the phylogenetic analysis of natural virus populations
• Consider data collected over multiple cycles of virus–host transmission. (Geoghegan and Holmes 2018)

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

Quasispecies and SARS-Cov-2?

• Coronaviruses contain the largest known RNA genomes (27 to 32 kilobases cf 7kb in polio) (Smith and Denison 2013)
• RNA viruses rely primarily on low-fidelity replication by RNA- dependent RNA polymerases
• With no proofreading and repair functions, the average mutation rate of RNA viruses is estimated to be around one mutation per genome per round of replication .
• RNA viruses risk crossing an error threshold at which there are too many deleterious mutations (limitation on size)
• Are Covs DNA “wannabes?”
• CoVs encode a proofreading exoribonuclease
• Distinct from the typical RNA Polymerase
• So possibly more conserved sequences than may be expected in other long sequence RNA viruses

Zoonosis

• Most pandemics involving viruses originate in animals
• Novel viruses are “driven to emerge by ecological, behavioural, or socioeconomic changes.” (Smith and Denison 2013)
• Sometimes claimed that humans are “coming into contact with wild animals more frequently than in the past”
• However, most likely source of novel pathogens are closely related species (particularly primates)
• Most human contact is with domestic animals

Biomass

Humans vs wildlife

• Livestock accounts for more biomass than all humans on earth; More than 50% greater than humans.
• Livestock accounts for much more than all wildlife (mammals + birds) by a factor of ten
• I.e humans + livestock ~ 15 times the total wildlife biomass (cells in which viruses can replicate)
• Even so, most viral genomic diversity is found in wildlife.
• Studies on viruses that affect neither livestock nor humans vanishingly small part of the literature

Zoonoses

• Source of most diversity is wildlife
• Most likely source of infection is livestock
• Wildlife -> livestock <-> humans
• Livestock including farmed animals + household pets

Co-infections and recombination

• Wildlife provides a large pool of unsequenced genetic material
• Co-infections between endemic viruses of domestic animals and “novel” (i.e. previously unknown sequences) likely source of zoonoses
• RNA viruses forming quasispecies particularly likely candidates.
• Coronaviruses may be DNA “wannabes” however they form subgenomic RNA making recombination more likely
• Recombination makes tracing evolution very hard
• Availability of sequences key to understanding “novel” recombinants
• Availability bias (do we have all the sequences?)

Recombination in SARS?

(Graham and Baric 2010)

Recombination

Bats, cats, rats (+ mustelids and primates)

• ~ 2000 species placed of rodents: 40% of mammals
• ~ 925 species of bats: 20%
• ~ 230 species of primates
• ~ 60 species of mustelids
• ~ 36 species of cats

Why bats?

• Most likely source of zoonoses would appear to be rodents
• However, bats are both highly diverse and highly mobile
• Proximity of insectivorous bats to humans goes largely unnoticed (nocturnal)

Bat viral diversity

(Wu et al. 2016)

Studies of bat coronavirus diversity

(Drexler, Corman, and Drosten 2014a)

Rhinolophus

Illustrative studies in bats

• Multiple examples of coronavirus diversity in bats throughout the world (see reference section for titles and links)

• Balboni et al. (2011) Balboni et al. (2012) Monchatre-Leroy et al. (2017) Hu et al. (2017) Rizzo et al. (2017) Hayman et al. (2013) Plowright et al. (2014) Mendenhall et al. (2019) Lacroix et al. (2020) Wong et al. (2019)Anthony et al. (2013) Tao and Tong (2019) Drexler, Corman, and Drosten (2014b) Woo et al. (2012) Smith et al. (2016) Woo et al. (2006) Lau et al. (2015) Corman et al. (2015) Drexler et al. (2011)

Inter genus host switches

(Latinne et al. 2020)

“Spillover”

(Cui, Li, and Shi 2019)

Cats as intermediate hosts

• Civet cats are farmed in China, but comparatively rare
• Domestic cats are ubiquitous
• Feline ACE2 reacts with SARS-Cov as well as human ACE2 (Guo et al. 2008) (Jo et al. 2020)

Cats in china

• Thousands of cats were rounded up and killed in China prior to Olympic games in Beijing

Cats and SARS-Cov-2

(Zhang et al. 2020)

(Musso et al. 2020)

Pangolins

• Pangolin theory based on a single sequence (Segreto and Deigin 2020)
• No evidence in wild populations (Lee et al. 2020)

Furin cleavage site

• Main objection to the zoonotic origin theory
• Furin cleavage sites are actually very common in betacoronavirus (Wu and Zhao 2021)
• S1/S2 occurs in three clades: MERS-CoV strains, the bat coronavirus HKU5 strains, and coronavirus Neoromicia/PML-PHE1/RSA/2011.
• MERS-CoV and bat coronavirus HKU5 are the only clades in Merbecovirus having furin cleavage site at S2′. In Embecovirus, furin recognition motif at spike S1/S2 is universal:
• Also universal in Gammacoronavirus, and occurs in two clades of Alphacorovanvirus including feline coronavirus and relatives.
• HCoV NL63 also has a furin cleavage site at spike S2′

Muth et al. (2018)

Corman et al. (2020)

Assumptions of lab origin theory

• Evolutionary rate known (comparatively slow and stepwise) so unlikely to lead to change
• Most possible sequences already known
• Furin cleavage site (which is absent in most bat sequences) could not have arisen “by chance”
• SARS-Cov-2 must have arisen in China
• Wuhan wet market origin implausible
• No intermediate host (as the virus was “designed in lab”)
• GOF experiments were known to be taking place in Wuhan labs
• Escape from labs plausible and likely
• Accidental escape as plausible as deliberate escape

Assumptions of zoonotic origin theory

• Evolutionary rates unknown
• Quasispecies evolution likely
• Recombination both possible and likely
• Most of the possible available sequences remain unknown
• Furin cleavage site is a common “lemma”" in many other coronviruses
• Intermediate host could be a common, domestic animal
• No single clearly defined “point of origin”

Implications of lab origin theory

• Current situation arose as a result of nefarious intent or human negligence
• Humans are “to blame” for the pandemic
• Existing knowledge was available that could have avoided the situation arising
• Political will to prevent the pandemic was lacking
• Fund more applied research to prevent future pandemics
• Viruses are dangerous
• Apparent “design” implies a “designer” (echos of anti-Darwinism)

Implications of the zoonotic origin theory

• Current situation was unavoidable.
• Arose through natural evolutionary processes (extended Darwinian evolution)
• Humans are not to blame for the pandemic. They are to blame for the response to it.
• Insufficient basic knowledge was available to prevent the situation.
• Fund more basic research to fill fundamental gaps in our understanding of life.
• Viruses are a fundamental part of ecosystems. Some are bad, some are good, all are part of a complex system
• We can’t live without viruses.