Two questions

1. What do we mean when we talk about SARS-CoV-2?
2. If we do know what we are talking about, what was its origin?

Taxonomy

• Viruses are real: Viral species are man made taxonomic constructs (Van Regenmortel 2003)
• Would a rose by any other name …..? (Shakespeare)
• Words are viruses (William S Burroughs)
• Could viruses resemble sentences in the Naked Lunch?

Fictitious foes

• Viruses are real.
• Mugwumps are not real.

Number of viral “species”

• Perhaps over one million viral species of vertebrates (Morse, source unclear)
• ~20 different viruses in each of the 50,000 vertebrates
• “Minimum of 320,000 mammalian viruses awaiting discovery” (Anthony et al. 2013)
• There is even the “Sin-nombre virus” (Lipkin 2008)
• But .. what is a “viral species?”

Why start with taxonomy?

• Appears esoteric. Quite literally “semantics”
• Impossible to communicate without using the words defined by taxonomists
• Once defined everyone has to use them
• Some levels of viral classification are simple monothetic, high level and totally uncontroversial
• e.g. Baltimore classification based on nucleic acid type and configuration.
• The lower the level, the greater the potential for disagreements.

Did a ballot in 2013 play a role in the pandemic?

• Two years worth of email discussions between 165 members of the International Committee on the Taxonomy of Viruses (ICTV) ended in 2013 with a vote
• 41% turnout 45:21 in favour of new definition of viral species
• Opposed by the ex-president (Regenmortel et al. 2013)
• The 14 senior virologists wanted to retain the previous definition.

What was the issue?

• Prior to the 1980s the very notion of viral “species” was rejected.
• Cannot apply Ernst Mayr’s definition(“species are groups of actually or potentially interbreeding natural populations, which are reproductively isolated from other such groups”)
• Viruses are obligate parasites with no clearly defined natural population. They do not interbreed. Reproductive isolation irrelevant, except in terms of contacts between host.
• Viral genotypes code for specific proteins (can assemble the capsid)
• Full viral phenotypes are essentially extended phenotypes (Dawkins) which are only fully manifested in the host

Definitions

• In 1982 ICTV tried a phenetic definition “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.”
• Replaced in 1991 by “A virus species is a polythetic class of viruses that constitutes a replicating lineage and occupies a particular ecological niche.”
• 2013 became “A species is a monophyletic group of viruses whose properties can be distinguished from those of other species by multiple criteria.”

The contrast

• Monophyletic vs Polythetic
• Cladistics (evolution) vs phenetics (properties): Genotype vs (extended) phenotype.
• 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.”
• “A monophyletic group of viruses (strains) whose properties can be distinguished from those of other species by multiple criteria”
• Current definition essentially defines virus species (clades) based only on their genomic sequence (but somehow still tacks on some other stuff)

Monophyletic evolution

• A group of taxa (clade) composed of a common ancestor and all of its lineal descendants.
• No crossovers (i.e recombinants) allowed
• Phylogeny traced to single root
• Not equivalent to geneaology within sexually reproducing organisms
• Tracing deep ancestry to a single point can only use sequences that are inherited from a single parent (mitochondrial DNA or the Y chromosome - maternal or paternal direct descent)

Ecological implications

• Ecologists weighed into the debate - on the side of cladism. “Defining a species is, by its nature, a statement about evolutionary history” (Peterson 2014)
• A consequence of the emphasis on evolution is to reduce the use of host specificity in the definition of the species.
• Geographic range should also be downplayed when naming
• Phenotypic traits such as temperature sensitivity are downweighted.
• Extended phenotype (pathogeneicity and effects on host) ignored.

Strains and variants

• Definitions of strains, variants and viral isolates cause yet more confusion (Alimpiev 2019)
• A single viral isolate is rather like a single rose plant (can be grown through cuttings)
• No clarity regarding the definition of strains and variants (how different do they need to be?)

Three problems

• Three issues make the definition problematic

1. The species definition muddies up thinking about an origin for SARS-CoV-2
2. Downplays genetic interchange
3. Uses arbitrary breakpoints on a continuum of genetic differences

Problem 1

• The Garden of Eden is a myth!

• The species Homo sapiens predated mitochondrial Eve (most recent common ancestor. 300k ybp vs 140k ybp)
• A viral species can also predate the viral lineage (which is rooted on a single sequence.
• SARS-CoV-2 is a sequence, not a species

Problem 2

• The tree of life is a metaphor
• Viral evolution occurs, but it does not follow a simple path
• Horizontal gene transfer can occur in viruses.
• A fully worked out viral phylogeny might look more like a complex road map than a tree

Hybrids in the wild

• Numerous examples in plants
• Many 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.
• Tigons, Ligers and edible frogs also exist in the world of viruses
• Recombinants

Hybrid viruses: Influenza

• Influenza A, B, C and D are considered as genera (Krammer et al. 2018)
• 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 (recombination) of genomic RNA segments occurs when two viruses of the same type infect the same cell.

Nomenclature of flu strains

• Named after combination of H and N e.g (H1N1)
• Strains or variants
• Not considered to be novel nor to be species

Recombination in SIV (Simian Immunodeficiency Virus) 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 found in simians (Wittmann et al. 2007)

Coronavirus subgenomic RNA

• Coronavirus RNA synthesis involves the transcription of multiple sub-genomic RNAs
• Recombination highly likely

Recombination in coronaviruses

• Mentioned in almost all papers on the subject in our Mendeley database

Next strain

• No recombination apparent
• However this is a consequence of using the monophyletic (cladistic) paradigm to produce the trees

Spotting recombination within a clade

• Look for linkage disequilibrium (LD) decay signal
• Recombination disrupts linkage between single nucleotide polymorphisms (SNPs)
• Signal occurs in more distant SNPs.
• Has now been detected

How the press present recombination

How the press present recombination

Problem 3

• Genetic sequences form a continuum, with no naturally defined breaks
• Pairwise comparisons of numbers of single nucleotide polymorphisms (SNPs) lead to arbitrary classes

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

• Around 86% sequence similarity between SARS-CoV-1 and SARS-CoV-2
• 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)

Implications

• The origin of the virus might be traced to Wuhan, at or just before, the date of publication of the sequence.
• The origin of the species that it belongs to is unknown
• If recombination between viruses occur then the accepted definition of a species becomes unworkable (e.g flu).
• Classical monophyly might best apply to specific short sequences (e.g code for the RBD)
• But very similar short sequences occur in other “species”

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 (Duffy 2018)
• Many RNA virus populations thought to exist as quasispecies (swarms of individual viral genomes where every member is unique.) : Paradigmatic for flu.

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)

Stability of SARS-Cov-2?

• Coronaviruses contain the largest known RNA genomes (27 to 32 kilobases cf 7kb in polio)
• 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)
• CoVs encode a proofreading exoribonuclease, distinct from the typical RNA Polymerase
• Are Covs DNA “wannabes?” (E. C. Smith and Denison 2013)
• Possibly more conserved sequences than may be expected in other long sequence RNA viruses (an argument frequently made by Francois Balloux)

Summary: What do we mean by SARS-Cov-2

• The “virus” is a sequence: The species is a concept. Under the 2013 definition they appear to be similar.
• A sequence has an origin, but it is not the origin of the species.
• Emphasising monophyly has led to sequence origin being confused with species origin
• Emphasising monophyly has led to phenotypic properties of the species (pathogeny, host specificity, temperature tolerance) being downplayed
• Disease dagnosis uses phenetics!
• Recombination since the Wuhan sequence incompatible with monophyletic trees.
• Recombination is an accepted mechanism for the emergence of new strains of flu and probable origin of HIV and Ebola

Zoonosis

• Most pandemics involving viruses originate in animals
• Novel viruses are “driven to emerge by ecological, behavioural, or socioeconomic changes.” (E. C. Smith and Denison 2013)
• Sometimes claimed that humans are “coming into contact with wild animals more frequently than in the past”
• Most human contact is with domestic animals

Humans vs wildlife

• Livestock accounts for more biomass (cells) than all humans on earth; More than 50% greater than humans 10x greater than all wildlife.
• I.e humans + livestock ~ 15 times the total wildlife biomass (cells in which viruses can replicate)
• Even so, most viral genomic diversity must be found in wildlife (~ 5k mammal 10K bird species).
• Studies on viruses that affect neither livestock nor humans remain vanishingly small part of the literature

Zoonoses

• Source of most diversity is wildlife (15 thousand species vs “handful”)
• But the most likely proximal source of infection is livestock
• Wildlife -> livestock <-> humans
• Livestock including farmed animals + household pets

Co-infections and recombination

• Wildlife provides a very large pool of unsequenced genetic material
• Co-infections between endemic viruses of domestic animals and “novel” (i.e. previously unknown sequences) likely source of recombinants
• Availability of sequences key to understanding “novel” recombinants
• Availability bias: Do we have all the sequences? .. Clearly NOT

Why bats?

• ~ 2000 species of rodents: ~ 40% of all mammals
• ~ 925 species of bats: ~ 20%
• 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

• Extremely high (Z. Wu et al. 2016)
• Nipah, Marburg, Hendra, Ebola all traced to bats (note nomenclature using place names)

Studies of bat coronavirus diversity

-Similar sequences to SARS found throughout the world (Drexler, Corman, and Drosten 2014a)

Illustrative studies in bats

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

• Balboni et al. (2011) (Balboni2012?) 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) P. C. Y. Woo et al. (2012) C. S. Smith et al. (2016) Patrick C. Y. Woo et al. (2006) Lau et al. (2015) V. M. Corman et al. (2015) Drexler et al. (2011) De Sabato et al. (2019) Kivistö et al. (2020) Musso et al. (2020) Pauly et al. (2017)

Example from Luxembourg

(Pauly et al. 2017)

• Severe acute respiratory syndrome (SARS)-related Betacoronavirus strains were shed by Rhinolophus ferrumequinum
• “The high similarity of the spike gene sequences of these viruses with mammalian Betacoronavirus 1 strains may be of concern. Both the SARS-related and Betacoronavirus 1 strains detected in bats in Luxembourg may cross the species barrier after a host adaptation process.”

Drexler et al. (2010)

• Rhinolophids carried severe acute respiratory syndrome (SARS)-related CoV at high frequencies and concentrations (26% of animals are positive; up to 2.4 copies per gram of feces), as well as two Alphacoronavirus clades, one novel and one related to the HKU2 clade.
• All three clades present in Miniopterus bats in China (HKU7, HKU8, and 1A related) were also present in European Miniopterus bats.

Muth et al. (2018)

• “We have previously described the detection of SARS-related CoV in Rhinolophus in Bulgaria”"
• To further investigate the presence of ORF8 in SARS-related CoV in Europe, we analyzed fecal samples from rhinolophid bats in Spain, Bulgaria, Italy, as well as Slovenia
• All five Rhinolophus species commonly encountered in Europe were tested positive for coronavirus RNA
• The variant termed rSCV8 contained a complete ORF8 as encountered in bats, civet cats, as well as in early human cases of SARS

Inter genus host switches

(Latinne et al. 2020)

“Spillover”

(Cui, Li, and Shi 2019)

Spillovers

• Highly likely in numerous other viruses
• Most go undetected
• Self limiting or asymptomatic

Flu “spillover” paradigm (Krammer et al. 2018)

Recombination in SARS intermediate host?

(Graham and Baric 2010)

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 2008 Olympic games in Beijing
• Suspected of carrying SARS

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)
• Pangolin theory may be connected to political issues surrounding trade (both in palm oil and wildlife)

Possible recombination across viral genera

• Alpha coronaviruses are very diverse source of genetic material
• Key questions regarding time scale of all divergences between all coronavirus taxa.
• ICTV and corona specialist group do not provide clear definitions of genera.
• Differences between alpha and beta coronaviruses particularly ill defined.

Corona virus phylogeny

V. Corman et al. (2020)

• The PCR test uses sequences derived from European bats!

Possible implications for PCR

• Drosten based some primers on pre-existing sequences from Europe
• PCR could be picking up material from diverse sources that hasn’t even recombined!
• Sequencing appears to rule that out
• However sequence alignments could be based on confirmation bias and conformity with expectations

Rhinolophus distribution

• Greater horseshoe bat distribution includes Europe and China

Rhinolophus distribution

Summary: Where did it come from?

• Finding the origin of the Wuhan sequence may involve looking at sequences in numerous coronaviruses
• Could be the first SARS-CoV that “went into hiding” in an intermediate host
• Could be a “novel” recombinant
• The devil is in the genetic detail
• Many unknown sequences may prevent origin being established.
• Sequences that match the PCR primers were already in Europe prior to Wuhan

Lab origin theory

• Seems to imply the origin of a chimeric sequence, not of a species.
• No evidence that chimera are restricted to labs (recombination frequent and ubiquitous)
• Why is a laboratory created chimera more terrifying than a liger?
• Here be dragons?

Furin cleavage site

• Main objection to the zoonotic origin theory
• Furin cleavage sites are actually extremely common in many betacoronavirus (Y. 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

Assumptions of lab origin theory

• SARS-Cov-2 has a single point of origin (i.e as a sequence, not a species).
• No recombinations outside a lab
• Evolutionary rate known (comparatively slow and stepwise) so unlikely to lead to arise by chance
• 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

Assumptions of deliberate lab origin

• 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
• Accidental escape just as plausible as deliberate release

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
• Viruses are dangerous and frightening
• Apparent “design” implies a “designer”

Assumptions of zoonotic origin theory

• No single clearly defined “point of origin”
• The species predated “the virus”
• 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

Implications of the zoonotic origin theory

• Current situation was unavoidable and not unique.
• Frequent occurrence. Has always happened.
• Arose through natural evolutionary processes (extended Darwinian evolution and recombination)
• 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. We have to live with them.