I&I: week 7 Viral evolution and viral replication and antiviral

Define viral genotype

Genetic constitution, which determines the basis of the phenotype

Define viral phenotype

Biological properties resulting from the expression of the genotype but modified by environmental factors

What is a viral variant?

Virus alternatives that exist in a population with mutations that differentiate them, sometimes with a slightly different behavior

Define viral strain

A single isolate from pure cultures or distinct isolates of specific phenotypic/genotypic traits

What are viral biotypes?

Different groups within a species that have distinct biological characteristics

Define viral serotype

Organisms within a species that have the same type and number of surface antigens that the immune system can recognize

Why do viruses evolve very fast?

Large progeny numbers, short replication time, and high evolution rate

Why is the rate of evolution faster in RNA viruses compared to DNA viruses?

RNA polymerases in RNA viruses have no proofreading activity

What is phenotypic drift?

Accumulation of random genetic mutations resulting in slow and sometimes minor changes in proteins.

What types of mutations cause phenotypic drift?

Point mutations (substitutions, insertions, deletions)

Define point mutations

Mutations affecting a few nucleotides, including substitutions

What are substitutions in point mutations?

An exchange of a single nucleotide for another, also referred to as SNPs (single nucleotide polymorphisms)

What are the three types of point mutations based on their effect on the codon?

Silent (neutral), missense, and nonsense mutation

What is a silent mutation?

Codes for the same (or a sufficiently similar) amino acid not to change protein function

What is a missense mutation?

Codes for a different amino acid and can change the protein’s function

What is a nonsense mutation?

Codes for a stop codon and can truncate the protein

Define insertion

Add one or more extra nucleotides into the DNA. Can (but not always will) cause a shift in the reading frame

Define deletion

Remove one or more nucleotides from the DNA. Like insertions, these mutations can (but not always will) alter the reading frame of the gene

What are frameshift mutations?

Mutations that disrupt the reading frame sequence by indels (insertions or deletions) of a non-multiple of 3 nucleotide bases

What is the consequence of frameshift mutations?

Usually result in the translation of a completely different protein from the original and are also very likely to cause a stop codon to be read, which truncates the protein, possibly impairing the function of the resulting protein

What is phenotypic shift?

Sudden, major change in the antigenicity of a virus

What events cause phenotypic shift

Recombination, reassortment, and defective interfering particles

Define recombination in viruses

The polymerase might jump from one viral genome template to another by a copy choice mechanism

Define reassortment in viruses

Only occurs in segmented-genome viruses, constantly causing new strains

What are defective interfering particles (DI)?

Mutant viral genomes that need the help of normal viral genomes to replicate

What is the role of quasispecies in drug resistance?

In RNA viruses, every population within a host is composed of varied genome alternatives (quasispecies) whereby one clone is predominant under the existing conditions because it is better fit. This provides an evolutionary advantage, because mutations are already present and waiting to be selected, when selection pressure is applied to such a population a better suited clone and the previously prevalent clone will decrease or cease to exist

What is the approximate evolution rate of RNA viruses?

10-4

What is the approximate evolution rate of dsDNA viruses?

10-8

What is the reading frame

Defined by the initial nucleotide from which translation starts. Every sequence can thus be read in three reading frames, each of which will produce a different amino acid sequence

What defines the actual frame in which a protein sequence is translated?

A start codon, usually the first AUG codon in the mRNA sequence

What is degeneracy in the genetic code?

The redundancy of the genetic code where codons encoding one amino acid may differ in any of their three positions, allowing for silent mutations to occur

What is the importance of viral evolution?

Allows viruses to adapt to new hosts, reinfect the same host, and evade the host’s immune response

How are attenuated vaccines obtained?

Viral passage in a new host imposes tremendous pressure on the virus, and only the clones able to adapt will remain, even if this means losing virulence in the former host

What are cumulative point mutations useful for?

Epidemiological purposes to trace the source (genetic signatures)

Give examples of virus genotypes

Rabies has different genotypes

Give an example of virus biotypes

Bovine Viral Diarrhea (BVD) cytopathic vs non cytopathic

Give examples of virus serotypes

Dengue (4 serotypes) and Foot and Mouth Disease (FMD) virus (7 serotypes)

How is viral replication studied in vitro?

Using cell cultures (in suspension or monolayer) in what is known as the growth curve of viruses

What is measured over time in the growth curve of viruses?

The increase in infectious virus is measured over time by sampling the virus in the supernatant

What are the sequence of events in the growth curve of viruses?

Binding to the receptors and entering the cells, eclipse (replication of the genome, production of structural and non-structural proteins) assembly and exit of the cells to infect other cells where virus particles are observable once again

What is the eclipse period?

The time after the virus has penetrated the cell but cannot be observed for hours until the first progeny of virions become visible again

What is the difference between susceptible and permissive cell lines?

A susceptible cell has a receptor to the virus, a permissive cell allows the virus to replicate

How can viruses that do not grow in cell culture be studied?

By constructing an infectious clone, which means inserting the viral genome into a plasmid21 ...

What are the basic steps of viral replication?

Viral attachment, viral penetration, viral uncoating, transcription, translation, replication, assembly, and release

How do viruses attach to cells?

Receptors on the viral envelope or capsid become connected to complementary receptors and coreceptors on the cell membrane expressed in susceptible cells

What are the mechanisms of viral penetration?

Entry via membrane fusion, entry via endocytosis, and entry via genetic injection

What is viral uncoating?

The viral capsid opens and frees the genome (it can be partial or complete)

What is the function of cellular proteins called chaperones during viral replication?

May take the viral genome to the nucleus if it is a virus that replicates at that site

How do DNA viruses that replicate in the nucleus produce their mRNAs?

Use the cellular DNA-dependent RNA polymerase

How do DNA viruses that replicate in the cytoplasm produce their mRNAs?

Carry a DNA-dependent RNA polymerase of their own

What happens in most single-stranded + RNA viruses after entry into the cell?

The nucleic acid can directly bind to ribosomes and start translating either partially or fully. Some are capped and have polyA tails so they look and behave like cellular mRNAs. Once they translate the mRNA they usually code for their own viral polymerase to replicate their genome

What must single-stranded negative-sense RNA viruses carry inside their nucleocapsid?

Their own polymerase enzyme, a specific RNA-dependent RNA polymerase

What is the function of DNA polymerase?

Produces DNA

What is the function of RNA polymerase?

Produces RNA

What is the function of reverse transcriptase?

Produces DNA from an RNA template

How do simple icosahedral viruses assemble?

The structural proteins associate spontaneously to form capsomers which self-assemble to form capsids into which the viral nucleic acid is packaged

How do helical structured viruses assemble?

The RNA molecule self-assembles as a cylindrical helix bound to their structural proteins

How do most naked viruses exit the cell?

By lysis

How do enveloped viruses exit the cell?

By budding or exocytosis

How do retroviruses replicate?

Use their own reverse transcriptase (RT) enzyme with RNA-dependent DNA polymerase capability) to first produce an RNA-DNA hybrid and then a double-stranded DNA that can insert into the host genome

What are LTRs in retroviruses and what is their function?

Long terminal repeats that allow the viral genome to integrate into the host genome and act as a strong promoter

How do picornaviruses replicate?

Their genome looks like a mRNA, so after the nucleocapsid uncoating, it goes directly to the ribosomes to be translated into proteins; then the viral enzyme recently produced replicates and transcribes the viral genome

How do adenoviruses replicate?

After the nucleocapsid uncoating, it goes to the nucleus. There are early and late products of transcription and translation, then the viral genome is replicated, and all components are assembled, and new virions are released

Why can antivirals be toxic to the cell/organism?

Viruses use the metabolic pathways of the host cell to replicate, thus many agents that interfere with viral replication are toxic to the cells

What makes antiviral targets specific to viruses safer?

They render improved safety but are usually virus-specific

Why are antiviral drugs best suited for persistent infections?

Because there are more opportunities to interfere with viral replication

Why aren’t all existing antiviral drugs used in vet med?

Partly because of the cost and lack of efficacy and safety clinical trials in the intended host species

What are the main strategies to stop viral infections in animals in veterinary medicine?

Preventative vaccination (whenever there is a vaccine available), biosecurity, depopulation, and other interventions

Can antivirals be used in food animals?

No

How do purine and pyrimidine analogs work as antivirals?

Act as chain terminators and stop DNA or RNA polymerase from adding further nucleotides

How does Acyclovir work?

Is an inhibitor of herpesvirus DNA polymerase, it is delivered as a prodrug that requires another viral-coded enzyme (Tyrosine Kinase) to get phosphorylated to its active state. This activation then only occurs in infected cells, thus the drug is non-toxic for uninfected cells

How does GS-441524 work as an antiviral against FIP?

Once inside the cell, GS-441524 is phosphorylated three times to form the active nucleoside triphosphate, which is then incorporated into the genome of virions by the viral RNA-dependent RNA polymerase, leading to premature termination of the RNA chain

What is a potential side effect of GS-441524 treatment in cats?

Formation of urinary stones composed of GS 441524

What are the major groups of antivirals?

Nucleic acid chain terminators (& polymerase inhibitors), protease inhibitors, integrase inhibitors, neuraminidase inhibitors, and attachment inhibitors (monoclonal antibodies)

How can antiviral resistance in chronic infections be prevented?

By administering a cocktail of drugs targeting different steps at the same time

What is the role of viral attachment in viral replication?

Receptors on the viral envelope or capsid connect to complementary receptors and coreceptors on the cell membrane in susceptible cells

What are the different mechanisms of viral penetration into a host cell

Entry via membrane fusion, endocytosis, or genetic injection

Describe entry via membrane fusion

Viral receptors attach to receptors on the cell surface, and secondary receptors initiate fusion with the host cell membrane, releasing contents into the cell

Describe entry via endocytosis

A receptor-mediated process commonly used by naked viruses

Describe entry via genetic injection

The virus injects only its genome into the cell, leaving the rest of the virus on the surface (e.g., phages)

What is the role of viral uncoating in viral replication?

The viral capsid opens and releases its genome, which can be a partial or complete release

How do DNA viruses that replicate in the cytoplasm produce mRNA?

They carry their own DNA-dependent RNA polymerase, localized in the cytoplasm, similar in function to the cellular enzyme

How do single-stranded positive-sense RNA viruses initiate translation after entering a cell?

Their nucleic acid directly binds to ribosomes and starts translating, often coding for their own viral polymerase to replicate their genom

Why must single-stranded negative-sense RNA viruses carry their own polymerase enzyme?

Their genome doesn't resemble mRNA, so they need to transcribe genes into proteins using their RNA-dependent RNA polymerase

How do structural proteins assemble in simple icosahedral viruses

They spontaneously associate to form capsomers that self-assemble into capsids, into which the viral nucleic acid is packaged

How do enveloped viruses typically exit the cell?

By budding (acquiring the envelope from the cell membrane) or exocytosis (traveling in vesicles to the cell membrane for release)

How do retroviruses convert their RNA genome into DNA?

They use reverse transcriptase to produce an RNA-DNA hybrid and then a double-stranded DNA that integrates into the host genome

What is the significance of the eclipse period in viral replication?

It is a crucial time when antiviral drugs can interfere with viral replication, occurring after the virus penetrates the cell but before new virions are observable

What is the role of reverse transcriptase in retrovirus replication?

It uses a tRNA molecule as a primer to make a single-stranded DNA copy, eventually forming a double-stranded DNA that integrates into the host genome

What is the significance of LTRs (long terminal repeats) in retroviral DNA?

LTRs allow the viral genome to integrate into the host genome and act as a strong promoter for viral gene transcription

How do picornaviruses ensure rapid replication within a host cell?

Their RNA genome resembles mRNA, allowing it to be directly translated into proteins, which include the viral enzyme needed for genome replication

What is the general mechanism by which adenoviruses replicate their DNA genome?

After uncoating and entering the nucleus, early and late products of transcription and translation are produced, followed by viral genome replication and assembly of new virions

What are the key factors that influence the decision to use antiviral drugs in veterinary medicine?

Cost, efficacy, and the availability of safety and clinical trials in the intended host species

What is the primary mechanism of action of purine and pyrimidine analogs as antiviral drugs?

They act as chain terminators, preventing further addition of nucleotides by DNA or RNA polymerase, thus halting viral replication

What are the primary targets of antiviral drugs in the viral replication cycle?

Attachment, penetration, uncoating, replication, assembly, or release

How does administering a cocktail of antiviral drugs help prevent drug resistance?

By targeting different steps in the viral replication cycle simultaneously, the likelihood of the virus developing resistance to multiple drugs is reduced

What methods are used to study viral replication in the lab?

One-step growth curves or infectious clones

What is the difference between acute and persistent viral infections in the context of antiviral treatment strategies?

Antiviral drugs are better suited for persistent infections due to the longer duration and more opportunities to interfere with viral replication, compared to acute infections with shorter courses

How do retroviruses overcome the challenge that their single-stranded RNA genome cannot function directly as mRNA?

They use reverse transcriptase to produce a DNA copy that integrates into the host genome, from which viral mRNA and genomic RNA are transcribed

What are some mechanisms by which enveloped viruses obtain their envelope during the release phase of replication?

By budding through cellular membranes (e.g., Golgi or endoplasmic reticulum), which incorporate viral proteins into the envelope