Evolution of Viruses

Question 1

Origin of Viruses -> Theories

Answer 1

Regressive Evolution:
Viruses are derived from intracellular parasites; loss of almost all genes not required for basic replication

Cellular Origin:
Viruses developed from parts of the cell and developed the capacity for autonomous replication

Coevolution with Host:
Viruses developed from self replicating molecules in parallel to the evolution of their hosts

Question 2

New scheme to categorize organisms
-> Redefinition of “living organism”

Answer 2

Clusters of orthologous groups (COGs) of gene categories
- Translation
- Transcription
- RNA processing/mod. …

LUCA
Last Universal Common Anchestor

All living organisms contain probably 34 ribosomal protein genes which are still shared by archaeal, bacterial and eucaryotic organisms

Viruses (including Giant viruses) do not encode for ribosomal proteins

Question 3

On the Origin of Cells and Viruses: Primordial Virus World Scenario

Answer 3

“It is proposed that the pre-cellular stage of biological evolution unraveled within networks of inorganic compartments that harbored a diverse mix of virus-like genetic elements. This stage of evolution might comprise the Last Universal Cellular Ancestor (LUCA) that more appropriately could be denoted Last Universal Cellular Ancestral State (LUCAS). This scenario for the origin of cellular life recapitulates the early ideas of J. B. S. Haldane sketched in his classic 1928 essay. However, unlike in Haldane’s day, there is now considerable support for this scenario from three major lines of comparative-genomic evidence:
1. lack of homology between the core components of the DNA replication systems of the two primary lines of descent of cellular life forms, archaea and bacteria,
2. distinct membrane chemistries and lack of homology between the enzymes of lipid biosynthesis in archaea and bacteria,
3. spread of several viral hallmark genes, which encode proteins with key functions in viral replication and morphogenesis, among numerous and extremely diverse groups of viruses, in contrast to their absence in cellular life forms,
4. the extant archaeal and bacterial chromosomes appear to be shaped by accretion of diverse, smaller replicons, suggesting a continuity between the hypothetical, primordial virus stage of life’s evolution and the dynamic prokaryotic world that existed ever since.

Under the viral model of precellular evolution, the key components of cells including the replication apparatus, membranes, and molecular complexes involved in membrane transport and translocation originated as components of virus-like entities. The two surviving types of cellular life forms, archaea and bacteria, might have emerged from the LUCAS independently, along with, probably, numerous forms now extinct.“

Question 4

Origins and Evolution of the Global RNA Virile

Answer 4

Prokaryotic viruses:
- almost exclusively DNA genome
- only one family of RNA viruses (Leviviridae) and one family of dsRNA viruses (Cystoviridae)

Eukaryotic viruses:
- much more RNA viruses than DNA viruses

Evolution model for euk. RNA viruses:
Common gene/enzyme for all RNA viruses: RdRp
the highest similarity between the (+) RNA virus RdRps and the RTs of cellular group II introns

(+) RNA viruses developed from cellular RT-coding introns;
- only very distantly related to prok. Leviviridae

ds RNA viruses developed from (+) RNA viruses
(-) RNA viruses developed from dsRNA viruses

Question 5

Petabase-scale sequence alignment catalyses viral discovery

Answer 5

• database screen through 10.2 petabases of archived sequence data (petabase = 1015 bases)
• hunting for matches to the central core of the gene for RNA-dependent RNA polymerase
• uncovered partial genomes of novel 132,000 RNA viruses; increase by a factor of 9.8!
• 250 giant viruses that infect bacteria (not amoeba)

The amount of cloud-based, publicly available DNA sequences is expanding exponentially; if he did the same analysis next year, Babaian says he would expect to find hundreds of thousands more RNA viruses. “By the end of decade, I want to identify over 100 million.”

SRA = Sequence Read Archive

Question 6

Evolution of Viruses

Answer 6

Definition
Constant change of a virus population under selection pressure

Viruses
- infect vertebrates, invertebrates, plants, fungy, bacteria, archea
- show an enormous genetic diversity

Mutation
Inheritable, stable change of the genetic information

1. Point mutation
2. Recombination
   a. Deletion
   b. Duplication
   c. Insertion
   d. Reassortment

Question 7

Mechanisms of Virus Evolution

Answer 7

• Mutation
• Recombination
• Phenotypic moving (No stable change! No mutation!)
• Complementation (No stable change! No Mutation!)
• Reassortment
• Integration of cellular genes

Question 8

Evolution of Viruses -> Recombination

Answer 8

Rearrangement of DNA- or RNA-Molecules

Kind of Mutation
->Homologous Recombination: Recombination partners show significant sequence homologies (e.g. two poliovirus genomes)
-> Non-homologous Recombination: Recombination partners show no significant sequence homologies (e.g. viral genome and cellular mRNA)

Question 9

Molecular Basis of Evolution -> Polymerases (error rates)

Answer 9

DNA Repl.. Bac. 10-8 – 10-10
DNA Repl. Tag: 10-4
DNA Repl. Pfu 10-6
DNA Repl. Phi29: 10-7
RNA Polymerasen: 10-4 – 10-5

Question 10

Molecular Basis of Evolution -> Quasispecies

Answer 10

Term defining RNA-viruses as populations of genetic variants (sequence cloud)

“A Qß phage population is in a dynamic equilibrium with viral mutants arising at a high rate on the one hand, and being strongly selected against on the other hand. The genome of Qß can not be described as a defined unique structure, but rather as a weighted average of a large number of different individual sequences.”

Question 11

Evolution of Viruses -> Selection

Answer 11

Definition
Constant change of a virus population under selection pressure

Selection
- Environment (virostatika)
- Competition pressure
- Counteraction of host (immune response)

Maintenance of viral replication

Question 12

Evolution of viruses -> Amplification and diversity

Answer 12

Rapid amplification
- e.g. 10.000 virus particles per infected cell
- e.g. production of 1012 viruses/day/host

Capacity to generate an enormous genetic diversity

Example: Genome with 10.000 nucleotides -> 410000 exchanges, defining all possible mutations (still neglecting deletions and recombinations)

Question 13

Bottle Neck experiments

Answer 13

• Selection e.g. by neutralizing monoclonal antibodies or antivirals in cell culture supernatants
• Massive reduction of heterogeneity in population

Question 14

Evolution of Viruses -> Consequences/Importance

Answer 14

• Change of host range (HIV, Canine Parvovirus, SARS CoV, Influenzaviruses)
• Increase/decrease of virulence
  -> Rabbitpox
  -> Point mutation: Influenza- and Poliovirus
  ->Recombination: Influenza- and Pestiviruses
• “Immune escape“
  (Lentiviruses, HCV, Influenzaviruses)

Question 15

Origin of HIV-1

Answer 15

HIV-1 and -2 have different origins

HIV-2 corresponds to SIVsmm, a strain of the Simian Immunodeficiency Virus found in the Sooty mangabey (also known as the White-collared monkey), which is indigenous to Western Africa. Low prevalence in humans.

The more virulent, pandemic strain of HIV, namely HIV-1, was until recently more difficult to place. Until 1999, the closest counterpart that had been identified was SIVcpz, the SIV found in chimpanzees. However, this virus still had certain significant differences from HIV.

Question 16

HIV groups

Answer 16

SIVcpz
- HIV-1 group: M and N
- Number of human infections: 70 Mio and 22

SIVgor
- HIV-1 group: O and P
- Number of human infections: 100.000 and 2

To promote virus release from infected cells, pandemic HIV-1 group M strains evolved Vpu as a tetherin antagonist, while the Nef protein of less widespread HIV-1 group O strains acquired the ability to target the human tetherin (which Nef of SIVs can ́t target).

Question 17

Change of host range -> Parvovirinae

Answer 17

FPV feline Parvovirus (Panleucopenia)
CPV canine Parvovirus (Parvovirosis)

Feline Panleucopenia: known since more that 100 years Canine Parvovirosis: first description 1977 (USA)
-> Pandemia (world wide spread)

(Only human pathogen: Parvovirus B19 causing erythema infectiosum; fifth disease; Ringelröteln)

Heart muscle degeneration

Comparative sequence analysis: Genomic sequences of both viruses are almost identical

Question 18

Parvovirus in dogs -> Typical symptoms

Answer 18

• Vomiting
• Diarrhea
• Heart muscle degeneration
• Hemorrhagic Enteritis

Question 19

Evolution of parvoviruses of animals

Answer 19

• Change of host range mediated by few amino acid changes
• A few amino acids are sufficient to determine the host range of a parvovirus

-> 2 AA determine the host range

Question 20

Hosts of Influenza A Viruses

Answer 20

• bird
• pig
• human
• horse
• duck

Continous evolution of Influenzaviruses in different host species

Genetic exchange between Influenza viruses: “Reassortment”

Question 21

Evolution of Influenza A Virus
-> Genetic drift
-> Genetic shift

Answer 21

GENETIC/ANTIGENIC DRIFT
- Molecular basis: Pointmutations
- Slow/minor change of properties and antigenictity

GENETIC/ANTIGENIC SHIFT
- Molecular basis: „Reassortment“
- Very rapid/massive change of properties and antigenictity

Question 22

Influenza A virus -> Epidemiology

Answer 22

Crossing of species barrier (e.g. bird -> swine or bird -> horse)

Zoonosis (swine -> human; bird -> human) (not only for influenza! “One health“ strategy!)

Pandemics, Epidemics

1918: Spanish Flu (H1N1)
1957: Asian Flu (H2N2)
1968: Hongkong Flu (H3N2)

1997: Transmission of H5N1 from poultry to humans (Hongkong); culling of all chickens, ducks and geese

Question 23

Classical avian plague

Answer 23

• Agent: Highly virulent influenza A Virus strains
• Systemic infections

Most influenza viruses are not virulent for poultry and replicate only locally in the gut
-> Molecular basis for difference in virulence?

Question 24

Influenza A virus -> Hemaglutinin (HA)

Answer 24

• Trimer
• Receptor binding molecule (Sialic acid)
• Fusionprotein
• HA cleavage is prerequisit for infectivity
• usually: extracellular cleavage (e.g. by Tryptase clara); locally restricted Mutation: intracellular cleavage (e.g. Furin); systemic spread
  -> Mutation at HA cleavage site can cause massive enhancement in virulence
• Trimeric receptor and fusion protein
• HA-cleavage is required for infectivity. The fusion peptide gets exposed by cleavage.
• Cleavage mostly extracellular: Infection is locally restricted since protease only occurs locally (previously: Clara cells in bronchiolar epithelia secrete tryptase Clara)

Exception
Influenza strain WSN/33 infectious without adding trypsin
Cause: plasmin in serum cleaves! Cause: deletion in NA (not HA!) removes glycosylation site
Result: NAbindstoplasminogen; conversion to plasmin results in cleavage of HA

Highly virulent avian influenza viruses contain poly-basic cleavage site in HA Result: intracellular cleavage by Furin and secretion of already infectious viruses
Avian Hong Kong Virus
H5N1 from 16 humans contains poly-basic cleavage site (never before observed in humans)
Ubiquitious growth without restriction? Pandemic? So far not: further adaption required
-> Role of PB1 in adaption? Yes, Nuclear import signal

Rarely: ubiquitious HA-cleavage after mutation of cleavage site ubiquitous production of infectious viral progeny systemic spread possible
Result: highly virulent virus mutants
HA-cleavage decides between local or systemic virus spread / low or high virulence

Novel results:
Membrane bound cellular proteases TMPRSS2 and HAT in airway epithelium activate influenza A virus -> Design of new inhibitors against host proteases
Laboratory: exogenic addition of trypsin to activate virus!

Question 25

Most influenza viruses are non-virulent for poultry and cause locally restricted viral infection of the intestine
-> Classical avian flu (Geflügelpest)

Answer 25

• Pathogen: highly virulent influenza A strains
• In contrast to “normal“ influenza virus which replicates in the gut the virulent form displays a systemic infection

Highly virulent avian flu virus:
Cleavage site in HA mutated (now poly-basic); thus HA can be cleaved after synthesis by the cellular protease Furin in the Golgi
- released viruses are infectious without further activation steps

Systemic infection: spleen, liver, lung, kidney, CNS -> lethal

Question 26

Evolution of Viruses -> Rabbitpox-Experiment

Answer 26

Myxomatosis virus isolated in Brasil from American tapeti (Sylvilagus brasiliensis)
Australia 1950 ies:
Release of Myxomatosis-virus to control population of European rabbit introduced by immigrants
Spread by fleas and mosquitos (passive, no replication in Arthropodes), 90 - 99% lethal for european rabbits

1. year: Mortality rate 99,8%
   Efficient killing of rabbits
2. year: Mortality rate only 25%
   Rapid selection of adaptive mutations in the virus (and the host) Attenuated virus variants / host population in between only a few years
   Later years: Mortality lower than birth rate of rabbits

Adaptation between host and virus!

Question 27

Mayxomatosis virus evolution -> predominant form

Answer 27

Which was the predominant virus form?
Viruses that were moderately virulent.
Why?
Viruses that were too much attenuated had a too low titer in the skin. Therefore, the passive transmission by the mosquito did not work efficiently.
Highly virulent viruses kill the host too quick, so the time span arthropodes can get infected is too short for an efficient spread.

Question 28

Host selection under Myxomavirus infection pressure

Answer 28

Infection of rabbits under laboratory conditions with a defined Myxoma virus which killed about 89% of the rabbits collected at Lake Urana prior to release of the virus
Rabbits collected after the first two epidemics behaved the same
However, rabbits colleced after the fourth epidemic showed only 50% mortality
Lab experiments were sometimes missleading. Why?
Higher survival rates when ambient temperature is higher (38°C)!

Armsrace still ongoing between virus and host: viruses collected in 1990 were more virulent than the original 1950 viruses in non-resistant rabbits

Question 29

How rabbits escaped a deadly virus - at least for now

Answer 29

… in 1950, after a smallpox-like virus found in South American rabbits turned out to kill the European relative, Australian authorities released the virus into the wild, cutting the rabbit population by 99%. A few years later, the virus, called myxoma, was released in France and eventually spread to the United Kingdom.
The result was “an opportunity to trace host-pathogen arms races right in front of our eyes,” says Jia Liu…
…tracked down specimens of U.K., Australian, and French O. cuniculus collected by museums prior to the virus’s introduction. They sequenced all the genes and other DNA that might influence the body’s immune defenses and compared the results with sequences from modern rabbits living in the same places. The comparisons revealed changes in many genes, usually a shift in the frequency of particular versions, or alleles, of a gene. Strikingly, half of the changes were shared by the rabbits in all three countries—evidence of parallel evolution.
One allele shift affected the rabbits’ interferon, a protein released by immune cells that sounds the alarm about a viral attack and helps trigger an immune response. Compared with preinfection rabbits, modern rabbits make an interferon that is better at responding to the biocontrol virus, the researchers found when they added different versions of the protein to rabbit cell lines.

Calicivirus Rabbit hemorrhagic disease RHD
First description1984 in China; release in Australia by “lab accident“ in 1995

2008: rabbit numbers in Australia are increasing again

Question 30

Attenuation of viruses in cell culture

Answer 30

• Passaging of viruses on cultured cells (especially cells derived from non host species) may lead to attenuated virus variants with decreased virulence (e.g. by restricted tissue tropism).
• Inefficient replication or receptor binding
• Example: Poliovirus- live vaccine strains

Question 31

Problem with Poliovirus live vaccine strain: Reversion of Poliovirus 3 to wild type

Answer 31

Therefore only the killed Salk vaccine is used nowadays in Europe and the USA

Question 32

Modern approach to solve the Polio type 2 vaccine problem

Answer 32

• 1999 last case of poliovirus type 2 infection; since 2015 no vaccination with Sabin type 2
• in people vaccinated since, no immunity against Poliovirus type 2;
• vaccine-derived poliovirus type 2 cases are problematic in Africa, Afghanistan, Pakistan, Malaysia

Strategy
- Cre element in 5 ́UTR (not present in any wt picorna virus, therefore no genetic exchange of 5 ́UTR possible)
- Stable mutation in stem V (no reversion due to deleterious intermediate)
- Mutations lowering the error rate of 3Dpol and lowering the propensity for template switching stabilze the virus

Question 33

BVDV

Answer 33

• two different forms of infection
  1. Infection of non-pregnant animals („acute infections“)
• (fever, diarrhea, respiratory symptoms, thrombocytopenia)
• mostly clinically inapparent
  2. Diaplacentar infection:
• misscarriage, malformations
• persistent infections (PI animals)

Important: stage of gestation

Question 34

Pathogenesis of Mucosal Disease

Answer 34

1. Infection with noncp BVDV (day 30-100 of gestation)
2. persistent infection with noncp BVDV (immun-tolerance)
   -> Mutation (1-2 years)
3. Isolation of noncp and cp BVDV (virus pair)
   -> Mucosal Disease (100 % lethal)

-> 1-2 % of cattle population around the world

Question 35

RNA-recombination in pestiviruses
-> Characterization of recombinant cp genomes

Answer 35

A. Northern Blot analysis
B. RT-PCR
C. Nucleic Acid sequencing

Identification of cellular mRNA sequences in viral RNA genomes!

Question 36

RNA-Recombination in Pestiviruses
-> Analysis of cytopathogenic pestivirus strains

Answer 36

Identification and characterisation of cellular insertions in the viral RNA genome
- ubiquitin
- NEDD8
- GATE-1
-> Common theme: Substrates of cellular proteases!

Consequences of identified genome variations
-> altered cleavage profile of polyprotein, upregulation of replication
-> cytopathogenicity, lethal disease in PI animals

RNA-Recombination – cytopathogenicity - Mucosal Disease

Question 37

Role of Ubiquitin Insertion

Answer 37

Ubiquitin-insertion serves as processing signal for cellular ubiquitin-specific proteases in viral poly protein

Further cellular insertions with analogous function:
- Ubiquititin-like proteins (e.g. SUMO)
- Proteins with ubiquitin-like fold

NS2-3: no replicase component
NS3: essential replicase component
Effect of ubi-insertion:
- deregulated, complete NS2-3 cleavage
- deregulated RNA replication
- viral cytopathogenicity and disease

Question 38

Mechanism of RNA Recombination

Answer 38

1. “Template switching” of RdRp during viral RNA-replication
2. “breakage and ligation” of viral RNA genome: independent of viral replication!

Question 39

Defective Viruses
“Defective interfering (DI) RNA”

Answer 39

RNAs with deletions may arise by recombination

Defekte interfering virus genomes accumulate during high titer replication of (-) and (+)strand RNA viruses

Shorter RNA replicates faster
-> competes /interferes with complete genomes for RNA-synthesis machinery
-> May be packed into virus particles, if missing proteins are complemented by a complete helper virus

Beneficial: High „multiplicity of infection“ (m.o.i.)

Role in pathogenesis and chronicity (SSPE/chronic Measles virus infection)?
Problem in vaccine production (Influenza virus)
Potential antiviral strategy?

Question 40

Heterogeneity of Prestiviruses

Answer 40

Discussion: Effectiveness of vaccines and laboratory diagnostics (RT-PCR, Ag-/Ab-detection)

Question 41

RNA recombination
-> Importance for pathogenesis of viral diseases
-> Example: Feline Coronavirus (FCoV)

Answer 41

• non homologous deletion

Critical mutations: Deletions in accessory genes like ORF7a or 7b

Upon mutation in infected cat-> FIP virus (mutant) kills cat

Question 42

Feline infectious peritonitis

Answer 42

• lethal disease
• peritonitis and/or pleuritis
• or granulomatosis in multiple organs

Cross section of a kidney from a cat with dry FIP. Numerous granulomatous lesions are seen on the capsule of the kidney and extending downward into the parenchyma

Question 43

Evolution of Viruses -> “immune escape”

Answer 43

Definition
Escape from the immune response of the host (to establish chronic infections)

Examples:
Lentiviruses (e.g. HIV), HCV

Question 44

Evolution of viruses
-> Change of host range
-> Change of virulence
-> Persistent infections

Answer 44

1. Change of host range: HIV, Parvoviruses, Influenza viruses, SARS-Virus
2. Change of virulence: Influenza viruses, BVDV, FeCV/FIPV
3. Persistent infections: HIV, HCV

Question 45

RNA Recombination
1. Mechanism of virus evolution

Answer 45

1. Genetic variability: Change of virulence adaption
2. Correction of lethal mutations: genome conservation, repair of viral genomes, genetic stability
3. Generation of “novel” viruses

Question 46

RNA recombination
-> Relevance: Genome conservation

Answer 46

Recombination is one strategy to eliminate errors occuring during RNA-synthesis (negative or lethal point mutations or deletions)
-> Repair of viral RNA genomes
especially important for viruses with large RNA genomes (e.g. coronaviruses)

Question 47

RNA Recombination
-> Relevance: generation of “novel” viruses

Answer 47

Example 1 (alphaviruses):
Eastern equine encephalitis virus (EEV) + Sindbis virus  Western equine encephalitis virus (WEEV)

Beispiel 2: FCoV-1 + CCV —> FCoV-2

Further examples
WEEV
FCoV-2
Poliovirus (inter- and intratypic)
MKS-Virus
Enterovirus
Dengue Virus
…

Question 48

RNA recombination
-> Importance: Essential Step in Virusreplication

Answer 48

Example 1:
Discontinuous transcription of subgenomic RNAs (Nidovirales)
Intramolecular RNA recombination during minus- strand-synthesis of Nidovirus subgenomic RNAs
Non contiguous genomic sequences are fused

Example 2:
Cap snatching of Orthomyxoviruses (e.g. Influenza virus)
Viral polymerase elongates cell derived mRNA fragment thereby fusing cell-derived and viral sequences

Question 49

Phage-Mediated Intergeneric Transfer of Toxin Genes

Answer 49

Because bacteriophages generally parasitize only closely related bacteria, it is assumed that phage-mediated genetic exchange occurs primarily within species. Here we report that staphylococcal pathogenenicity islands, containing superantigen genes, and other mobile elements transferred to Listeria monocytogenes at the same high frequencies as they transfer within Staphylococcus aureus.
Several staphylococcal phages transduced L. monocytogenes but could not form plaques. In an experiment modeling phage therapy for bovine mastitis, we observed pathogenicity island transfer between S. aureus and L. monocytogenes in raw milk. Thus, phages may participate in a far more expansive network of genetic information exchange among bacteria of different species than originally thought, with important implications for the evolution of human pathogens.

Question 50

Variation in the human gut virome

Answer 50

• 1 g of human feces contains more than 109 virions; many phages
• viral populations differ greatly between humans - correlates in part to differences in microbiome
• 2.5 years sampling from one person; sequencing of DNA
• 478 DNA virus sequences; 87% of the DNAs showed no overlap with previously identified viruses
• viral populations themselves undergo in those 2.5 years a rapid evolution, giving rise to new phage species

The healthy human gut virome consists mainly of long-term resident phages that can rapidly evolve, contributing to variation between individuals.

Question 51

Example for genomic exchange between virus and host

Answer 51

Example for genomic exchange between virus and host

Polydnaviridae (e.g. bracoviruses) (from polydisperse DNA virus) -> 10-30 circular dsDNAs up to 300 kbp
- virus-like particles observed in the ovaries of numerous species of parasitic wasps (parasitoids); in tens of thousands of wasp species
- present in all females of all affected species, suggesting vertical transmission
- part of the viral proteins for packaging not encoded by the virus but by the host (Bézier et al., Science 2009; 323:926ff)
- wasp host encodes for the genes missing for viral packaging: 22 putative nudivirus gene homologs in the wasp genome, including12 core genes that are also present in the related baculoviruses
- those genes are expressed in the eggs of the wasp which are injected into caterpillars, which is paralyzed, and new wasps develop
- considerable antigenic and genetic similarity between the venom genes of the wasp and polydnaviral genes
-> virus genes support the parasitoid survival in the caterpillar

So far viruses have been typically seen as either parasites or commensals; we must now recognize a potential for obligatory mutualism (kind of symbiosis).

Question 52

Example for genomic “exchange” between virus and host

Answer 52

• endogenic retroviruses (ERVs) in the genome of the eukaryotic host
  Endogenous retroviruses regulate periimplantation placental growth and differentiation
• envelope proteins of endogenous Jaagsiekte sheep retroviruses (enJSRVs) regulates trophectoderm growth and differentiation in the periimplantation conceptus (embryo/fetus and associated extraembryonic membranes)

Proof for functional role:
- antisense oligos against ERV mRNA injected into onceptus trophectoderm retarded trophectoderm outgrowth, inhibits cell differentiation and leads to loss of pregnancy

ERVs play fundamental roles in placental morphogenesis and mammalian reproduction