Viral mutations occur through two main mechanisms: antigenic drift and antigenic shift. Antigenic drift involves small mutations that accumulate over time and result in gradual changes to surface proteins. This allows the virus to evade existing immunity but results in related strains. Antigenic shift involves the reassortment of genome segments and results in an abrupt change where a virus acquires entirely new surface proteins. This can allow animal viruses to jump to humans, potentially causing pandemics by evading all existing immunity. RNA viruses typically mutate more rapidly than DNA viruses due to the lack of proofreading by RNA-dependent RNA polymerases during replication, though selection pressures and genome architecture also influence mutation rates.

1. Chapter 11: Virus Genetics and Mutation
1

2. 2
https://youtu.be/pNEvXlsudu0

3. Reassortment and recombination in viruses
Reassortment is an evolutionary mechanism of segmented RNA viruses that plays
an important role in virus emergence and interspecies transmission.
3
Define Virus Genome Reassortment
- a type of genetic replication that is exclusive to segmented RNA viruses.
- co-infection of a host cell with multiple viruses may result in the shuffling
of gene segments to generate progeny viruses with novel genome
combinations.
- It does not require physical proximity of the parental genomes during
replication.
- involves packaging of whole genome segments with different ancestry
into a single progeny virion.
Reassortment is most prominently described for influenza viruses as a primary
mechanism for interspecies transmission and the emergence of pandemic virus strains.

4. 4
Influenza virus
genome
composition

5. 5
Reassortment and Recombination of Viral Genomes

6. 6
Influenza viruses contain an RNA genome separated into eight segments.
Each segment can be exchanged with a corresponding segment from
another virus of a different strain, so that the resulting progeny viruses
can express a combination of proteins donated by both parent viruses.
The parent viruses, if of different subtypes, e.g. H6N2 and H7N7, can
produce two or more new subtypes via reassortment of their HA and NA
genes.
Other, internal genes can also be reassorted, but the exchange of these
HA and NA genes coding for the viral surface proteins usually has the
most dramatic effect, as this may change the virus antigenic specificity
and hence the host antibody response to the virus.
Such reassortment may lead to the emergence of epidemic and
pandemic influenza strains when pre-existing host immunity and/or that
induced by contemporary vaccines are no longer protective against these
new reassortant influenza strains.
Reassortment of RNA
Genomes in Influenza

7. 7
Quick Q: Anybody
out there still trusts
the WHO?
Just asking….

8. 8
https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1004902
Reassortment of two
tripartite genomes
producing a novel
reassortant/variant/mutant
A) Diagrammatic representation of the
emergence of a novel reassortant strain
with genes derived from two parents.
B) Phylogenetic discordance between
segments 1 and 3 (left) and segment 2
(right) for three tripartite strains.
Branches in bolder colors represent
parental strains, whereas lighter colors
represent the acquisition of gene
segments from different parents to form
a novel reassortant strain.

9. 9
Reassortment and recombination in viruses
• Recombination occurs through a template switch mechanism, also known as copy choice
recombination. When two viruses co-infect a single cell, the replicating viral RNA-dependant-RNA-
polymerase can disassociate from the first genome and continue replication by binding to and using a
second distinct genome as the replication template, resulting in the generation of novel mosaic-like
genomes with regions from different viruses.
• recombination in RNA viruses is a mechanistic by-product of the processivity of the viral polymerase
that is used in replication, and that it varies with genome structure.
Define Recombination
- occurs when at least two viral genomes co-infect the same host cell and
exchange of homologous genetic regions.
- accelerates the rate at which advantageous genetic combinations are
produced.
- the risk of producing deleterious mutations by recombination is lowest when
recombination breakpoints fall between rather than within genes

10. 10
a. This process can occur in both non-
segmented viruses or within a
segment of a segmented virus.
b. Co-infection of a cell by genetically
distinct strains of a retrovirus can
lead to the generation of
'heterozygous' virus particles, after
which a template-switching event
can lead to a recombinant provirus.
c. Co-infection of a cell by genetically
distinct strains of a segmented
virus can generate different
combinations of reassortant
progeny.
Recombinant viruses
produced by co-
infection of a single host
cell by genetically
distinct viral strains
https://www.nature.com/articles/nrmicro2614

11. 11
Recombination of HIV Genomes
HIV is a diploid retrovirus for which, when a
host cell is simultaneously (or sequentially)
infected with two strains of HIV and hence
harbors two different proviruses, the RNA
transcript from each of the HIV proviruses can
be incorporated into a single heterozygous
virion. When this virion then infects a new cell
and template switching occurs during reverse
transcription, recombinant retroviral DNA
sequence will be generated, and all
subsequent progeny virions will be of this
recombinant genotype.

12. 12
Recombination of HIV Genomes
This diagram shows the
phylogenetic trees of HIV-1
subtypes B, C, and
CRF08_BC reconstructed
from the non-recombinant
regions (shown above in
darker, red, and lighter, blue,
in the HIV-1 genome
organization) where
CRF08_BC shows different
phylogenetic origins.
CRF08_BC strains (boxed
and highlighted in grey) are
clustered into different
lineages in the two trees.
This evidence indicates that
CRF08_BC strains are
recombinants of subtypes B
and C.

13. Viral Mutation Rates
https://jvi.asm.org/content/84/19/9733
Mutation rates are important, because they:
1. determine the probability that a mutation conferring drug resistance
e.g. the mutation rate of HIV-1 demonstrated that any single
mutation conferring drug resistance should occur within a single day
and that simultaneous treatment with multiple drugs was therefore
necessary.
2. Estimate the probability of antibody escape
3. Prediction of host range expansion
4. determine whether a virus population will be susceptible to drug-
induced lethal mutagenesis e.g. the effectiveness of the combined
ribavirin-interferon treatment against hepatitis C virus (HCV).
5. assessment of vaccination strategies and it has been shown to
influence the stability of live attenuated polio vaccines.
6. At both the epidemiological and evolutionary levels, the mutation
rate is one of the factors that can determine the risk of emergent
infectious disease, i.e., pathogens crossing the species barrier.
13

14. Mutation rates DNA vs. RNA viruses
RNA viruses typically have HIGHER mutation rates than DNA viruses
On a per-site level:
• DNA viruses typically have mutation rates that range between 10−8 to 10−6
substitutions per nucleotide site per cell infection (s/n/c).
• RNA viruses have higher mutation rates that range between 10−6 and 10−4 s/n/c.
• nucleotide substitutions are the most frequent type of spontaneous mutations,
being roughly four times more frequent than indels.
• the upper limit on mutation rate is a product of factors such as natural selection,
genomic architecture and the ability to avoid loss of viability and/or genetic
information.
Selection can bias mutation rate estimates – lethal mutations are
deleterious and are purged out of the population. 14

15. 15

16. 16

17. 17
Let’s talk about Shift and Drift

18. 18
Characteristics Antigenic Shift Antigenic Drift
Definition
Antigenic shift refers to the gene recombination occurring when influenza
viruses re-assort NA and HA antigens
Mutations causing minute changes in the NA and HA antigens on the surface of
the Influenza virus is termed as antigenic drift.
Result in Forms a new sub-type (Subtype A + Subtype B –> New Subtype). Forms a new strain of virus.
Genome changes Large change in nucleotides of RNA. Small mutation of RNA.
Results from Genome re-assortment between difference subtypes. Accumulation of point mutations in the gene.
Change type The change is sudden and drastic. The change is gradual.
Virus involved One or more viruses are involved. Only one virus is involved.
Magnitude of change The change is large at once. Changes increase with each division cycle.
Relatedness of new virus
The new form or subtype produced bears no similarity to the previous
virus.
The strains produced by antigenic drift are somewhat similar to the older
strains.
Host range May jump from one species to another, for instance animal to human. May infect animals of the same species only.
Antigen changes
The virus acquires completely new antigens—for example by reassortment
between avian strains and human strains.
Antigens are only mutated.
Virus type involved Occurs only in Influenza Virus A Occurs in Influenza Virus A, B and C
Pandemic/epidemic Leads to pandemics. Leads to mainly epidemics.
Treatment option Difficult to treat (need new vaccine) Easy to treat (antibody and drugs available)
Susceptibility
Everybody is susceptible to the virus after an antigenic shift, and the novel
influenza may thus spread uncontrollably.
Some people may still be immune and some others may be partly immune to
the new strain of virus thus leading to a milder illness.
Examples
The 1968 pandemic arose when the H3 hemagglutinin gene and one other
internal gene from an avian donor reassorted with the N2 neuraminidase
and five other genes from the H2N2 human strain that had been in
circulation.
The subtle mutations accumulated through antigenic drift of these subtypes
(e.g., H1N1, H3N2, H5N1) give rise to different strains of each subtype.
The 1918 pandemic arose when an avian H1N1 strain mutated to enable its
rapid and efficient transfer from human-to-human.
Antigenic drift is also known to occur in HIV (human immunodeficiency virus),
which causes AIDS, and in certain rhinoviruses, which cause common colds in
humans. It also has been suspected to occur in some cancer-causing viruses in
humans.

19. 19
Antigenic Drift
Antigenic Shift
https://www.cdc.gov/flu/about/viruses/change.htm

20. https://jvi.asm.org/content/92/14/e01031-17/figures-only 20

21. 21
Types and Sources of
Mutations

22. dsDNA helix instability
22
Also a source of
mutation….but
not so much for
viruses

23. These include host antiviral enzymes, spontaneous chemical reactions and environmental
mutagens such as ultraviolet irradiation.
Sources of mutation in viruses
https://www.nature.com/articles/nrg2323
Deamination of unpaired nitrogenous bases
• Several cellular deaminating enzymes exist e.g. apolipoprotein B-editing catalytic polypeptide-like subunit
(APOBEC) family, which add further transition mutations to polymerase errors.
• This enzymatic deamination is thought to be an intrinsic antiviral host defence mechanism and can lead to
long stretches of transitions, termed hypermutation, the products of which are usually non-functional
because of the acquisition of multiple deleterious mutations.
• Deamination can also be chemically induced and can occur spontaneously, especially if the genome spends a
significant amount of time in a single-stranded state.
23

24. Many viruses have high rates of evolution. These high evolutionary rates have been attributed to the
large population sizes, short generation times, and high mutation rates of viruses.
• The mutation rate is the rate at which errors are made during replication of the viral genome;
captures only those mutations that are able to persist in the population (non-lethal).
• The substitution rate, which is the rate at which mutations become fixed, or present within all
individuals, in a population.
• Mutation frequency refers to the proportion of mutants identified in a virus sample or population.
• Viral mutation rates vary over five orders of magnitude, whereas viral substitution rates vary over
six orders of magnitude.
Mutation rates are used to estimate the amount of genetic diversity generated within a
population of offspring, substitution rates are used to estimate the rate of evolution for
a particular lineage or taxon
Definitions
24

25. • A reduced substitution rate is seen for some latent viruses primarily undergoing
replication as integrated dsDNA within primate genomes.
E.g. Retroviruses: simian foamy virus (SFV) and human T-cell lymphotropic virus type II
(HTLV-II).
• A reduced rate of replication associated with latency has also been proposed to
explain the low rate of approximately 10–7 subs/site/year.
• Low substitution rates are thought to be associated with low rates of inter-host
transmission and correspondingly long periods of time within a single host, so that
viruses largely spread through the clonal expansion of infected cells (in which the virus
is integrated into host DNA), rather than active replication as in non-integrated
viruses.
Why a lower substitution rate for latent/retroviruses?
25

26. Drivers of mutations in viruses
1. Fidelity of genome replication
a) The higher per-site mutation rates of RNA viruses can be
explained in part by the RNA-dependent RNA polymerases (RdRp)
that replicate their genomes. Unlike many DNA polymerases, RdRp
do not have proofreading activity and are thus unable to correct
mistakes during replication.
https://jvi.asm.org/content/92/14/e01031-17
26
b) DNA polymerases have proof-reading ability so the mutation rate in DNA viruses would be lower.
This proofreading is thought to be a key factor in explaining how DNA viruses have much larger genomes (>26
kb) compared to other RNA viruses.
Retroviruses also have high mutation rates, because reverse transcriptase, like most RdRp, lacks proofreading
activity.

27. 3. Trade off: Speed vs. accuracy of replication
• Viruses must replicate faster than the host immune system can detect them. The speed of
replication is important and at the sacrifice of accuracy of replication.
• increasing replication fidelity might be expected to slow replication speed and thus be
increase time available for immune system to recognise virus.
Drivers of mutations in viruses
27
2. DNA repair mechanisms
DNA viruses have access to DNA repair mechanisms of the
host cell. These viruses would have a lower mutation rate
than RNA viruses.
Base-excision repair on mispaired bases and mutated
dsDNA can be done for DNA viruses, but unable to fix such
mistakes in dsRNA or RNA–DNA heteroduplexes.

28. 4. Error threshold or error catastrophe in RNA
viruses – a mathematical theory to be proven?
• The catastrophic effect of high error rates was originally
predicted in a mathematical model by Eigen and Schuster.
• RNA viruses are said to replicate at the edge of “error
catastrophe”. ‘error threshold’, beyond which high mutation
rates would result in a large reduction in fitness and eventual
extinction.
• Error catastrophe is a term coined to describe the supposed
inability of a genetic element to be maintained in a population
as the fidelity of its replication machinery decreases beyond a
certain threshold value.
• It is the theoretical basis for treatment of viral infection with
drugs that would push the error rate for copying of the viral
genome beyond this threshold.
• This threshold means that RNA viruses have genome sizes
relative to this error threshold.
Drivers of mutations in viruses
28

29. Drivers of mutations in viruses
29
5. Genome size
• Small viruses mutate faster than large viruses irrespective of the
nucleic acid of their genome.
• Chances of incorporating errors in a large genome during replication is higher
than for smaller genomes whether DNA or RNA virus.
• Small eukaryotic ssDNA viruses evolve fastest as measured by the number of
substitutions that become fixed per year.
• there is a negative correlation between mutation rate and genome size observed
for RNA viruses.
• The largest RNA viruses, coronaviruses, show low mutation rates due to their 3′
exonuclease proofreading activity in their replicases.
• Because even small increases in RNA-virus mutation rates have serious fitness
effects, it is likely that many RNA viruses have mutation rates close to the highest
rates they can tolerate.

30. https://www.nature.com/articles/nrg2323
Average rates of spontaneous mutation in viruses, adjusted to the rate per
genome replication
30

31. 6. Genome architecture and secondary structures on
RNA strand
• Secondary structures in the genome can cause the polymerase
to pause, increasing the chance of template slippage, which
would lead to deletions or misreads in the copied RNA strand.
• RNA folding into secondary and even tertiary structures affect
processivity of replicating enzymes.
• Replicases tend to “fall off” the template when these structures
are present which may lead to the instability of RNA
macromolecules could be one reason why RNA viruses do not
exceed 33 kb in size.
Drivers of mutations in viruses
31

32. 7. Genome polarity
• genome polarity or structure, can influence the viral mutation rate.
• dsRNA is less exposed to chemical damage than ssRNA, and ssRNA(−) viruses because
these viruses pack their genetic material densely with nucleoproteins, which might
confer protection against mutation.
Drivers of mutations in viruses
8. Genomic context
• adjacent nucleotides affect mutation rate, and the precise location
of a nucleotide in the genome can influence its mutation rate.
• Long stretches of mononucleotide repeats can lead to replicase
slippage off the template.
32
strands

33. Factors that increase viral
substitution rates are
shown in red and those
that decrease it are shown
in blue.
The baseline substitution
rate is
determined by the neutral
mutation rate, µ. Because
increasing mutation rate
can increase the
substitution rate, the
factors increasing
mutation rate are also
shown in red and those
decreasing mutation rate
in blue.
Factors affecting mutation and substitution rates in viruses
33

34. The population genetics of virus mutation rates
• Studies of viral mutational fitness effects that most mutations are lethal or
deleterious, a minority are neutral, and only a few are beneficial. Thus lethal and/or
deleterious mutations dramatically underestimate the mutation rate.
• Mutation rates determine the amount of genetic variation generated in a
population, which is the material upon which natural selection can act. For this
reason, a higher mutation rate correlates with a higher evolutionary rate, but only to
a point.
• While the high mutation rates of retroviruses and RNA viruses may explain their
higher evolutionary rates relative to those of DNA viruses, several DNA viruses
exhibit evolutionary rates comparable to those of RNA viruses.
• This highlights the importance of additional factors in determining the evolutionary
rate, such as within-host dynamics or cell tropism.
34

35. Three hypotheses for why mutation rates have not evolved to be zero: The drift-barrier hypothesis: genetic drift prevents
mutation rates from reaching zero. The physicochemical limit hypothesis: the cost of perfect polymerase function pushes
the mutation rate away from zero. The selection hypothesis: selection for adaptability and/or replicative speed drives
mutation rates higher. Figures are approximate trends and are not meant to indicate exact relationships (e.g., linear).
The main evolutionary determinants of virus mutation
35

36. Natural Selection for specific virus mutations
36
• natural selection should generally
favor decreased genomic mutation
rates.
• selection to reduce mutation rates
could be counterbalanced by
selection based on the need for new
mutations to facilitate ongoing
adaptation.
• selection to reduce mutation rates
might be counterbalanced by the
fitness cost of increasing genomic
replication fidelity or by
physicochemical limits to the accuracy
of replication and repair processes.