Exercises: Viruses, viroids, and prions

Give three reasons why viruses are not considered living organisms according to classical criteria (cellular organization, metabolism, and reproduction) and one reason why the debate remains open.

Describe the basic structure of a virus (genome, capsid, and, in some, envelope) and explain the difference between a naked virus and an enveloped virus, with an example of each.

What are viroids and what are prions? State which organisms the former infect and give a human or animal disease caused by prions.

Explain what a bacteriophage is, describe its structure (a head with the genome, a tail and attachment fibres) and indicate what its "prey" is and why each phage infects only certain bacteria.

Distinguish between viruses with a DNA genome and those with an RNA genome, and give an example of a disease caused by each type (herpes or smallpox versus flu, measles or COVID-19), indicating what this implies for their variability.

What is a vaccine and how does it "train" the immune system without causing the disease? Name two types of vaccines (attenuated, inactivated, subunit, messenger RNA) and explain how they differ.

Explain what the host specificity of a virus is (why does the tobacco mosaic virus not infect us?) and which barriers a virus must jump to infect a different species (receptors, cellular machinery).

Name three transmission routes of viruses (respiratory, faecal-oral, sexual or blood-borne, via vectors such as mosquitoes) with an example of a disease for each one, and indicate one specific prevention measure for each route.

Describe the lytic cycle and the lysogenic cycle of a bacteriophage, indicating how they differ and what consequence it has for the bacterium when the phage enters latency.

Look at the structure of the virus in the image. a) Explain the function of structures 1, 2, and 4 during cell infection. b) Reason why structure 3 makes this type of virus particularly sensitive to soap and alcohol-based solutions. c) Is it possible to know from the image alone whether its genome is DNA or RNA? Justify your answer.

The 'One Health' approach views human, animal, and environmental health as a single interconnected system. Explain this using viral zoonoses: give two examples (rabies, avian influenza, or COVID-19) and reason how deforestation, intensive livestock farming, and the wildlife trade facilitate a virus jumping from an animal species to humans.

Order and describe the phases of the replication cycle of a virus (adsorption, penetration, replication and synthesis, assembly, release), and indicate at which steps antiviral drugs can act (blocking entry, inhibiting the polymerase or the protease).

Flu changes in two ways. Distinguish antigenic drift (annual point mutations) from antigenic shift (recombination between viruses, the origin of pandemics), and discuss why avian and swine flu are monitored as possible sources of new pandemic viruses.

Messenger RNA vaccines were rolled out massively with COVID-19. Explain how they work (a temporary instruction for our cells to manufacture the antigen), what design-speed advantage they offer, and why it is false that they can modify our DNA (where does the mRNA act and what is its half-life?).

The human papillomavirus (HPV) can cause cervical cancer and other tumours. Explain how a virus can favour cancer (its proteins altering the control of the cell cycle), why it takes years to appear, and why vaccinating teenagers — boys and girls — is a vaccine against cancer.

HIV infects CD4 T lymphocytes. Explain why the destruction of these cells disorganises the whole immune system, what AIDS is (the final phase without treatment) and why antiretrovirals turn the infection into a controlled chronic disease but do not cure it (reservoirs of latent virus).

During an outbreak of a novel respiratory virus, a basic reproduction number $R_0 = 2,5$ is estimated.

1. a) Public health authorities consider two alternative scenarios: implementing non-pharmaceutical containment measures (masks and ventilation, which reduce the transmission probability per contact by $40\,\%$) or relying exclusively on a vaccine with an efficacy of $80\,\%$ administered to $60\,\%$ of the population. Quantitatively predict which of the two strategies will independently manage to halt the expansion of the outbreak (making the effective reproduction number $R_t$ less than $1$), justifying your calculations.
2. b) A public official states: "If we manage to vaccinate $50\,\%$ of the population with a $100\,\%$ effective vaccine, the virus will become extinct immediately because half of the population will no longer transmit it". Refute this statement quantitatively and biologically by calculating the resulting value of $R_t$ and describing what will happen in the short term to the epidemic curve.

In a laboratory, two emerging viruses that infect human lymphocytes are analysed: virus Alpha (with a double-stranded DNA genome of $150\text{ kb}$) and virus Beta (with a single-stranded RNA genome of $15\text{ kb}$). Viral particles are isolated from patients after six months of infection:

• In Alpha, the sequence of the attachment glycoprotein gene shows an average divergence of $0,001\,\%$ relative to the inoculated strain.

• In Beta, the divergence in the homologous protein is $4,2\,\%$, with a broad cloud of variants found within a single host.

1. a) Justify at the molecular and enzymatic level the cause of this huge disparity in the mutation rates observed between both viruses.
2. b) To combat both pathogens, a protein subunit vaccine based on a single variant of the attachment protein and an inhibitory drug targeting that protein are tested. Make a reasoned prediction of the medium-term effectiveness prognosis of these strategies in Alpha and in Beta.
3. c) What viral target or pharmacological strategy would you propose for virus Beta in order to reduce the likelihood of it developing resistant variants?

A research team is trying to determine the nature of the infectious agent causing a new spongiform encephalopathy. To do so, they subject samples of infectious brain tissue to different treatments before inoculating them into healthy laboratory mice:

• Treatment 1: Exhaustive digestion with DNases and RNases $\rightarrow$ $100\,\%$ of the mice fall ill.

• Treatment 2: Ultraviolet irradiation at lethal doses for nucleic acids $\rightarrow$ $100\,\%$ of the mice fall ill.

• Treatment 3: Autoclaving at $121\text{ }^\circ\text{C}$ for $20\text{ minutes}$ $\rightarrow$ $100\,\%$ of the mice fall ill.

• Treatment 4: Prolonged boiling in sodium hydroxide ($1\text{ M}$) or digestion with harsh proteases $\rightarrow$ No mice fall ill.

• Treatment 5: The intact infectious sample is inoculated into Knock-Out mice in which the $PRNP$ gene has been deleted (they do not express the normal cellular protein $\text{PrP}^\text{C}$) $\rightarrow$ No mice develop the disease or multiply the pathogen.

1. a) Interpret the results of treatments 1, 2, and 4: deduce the biochemical nature of the pathogen and justify why its discovery represented an apparent violation of the central dogma of molecular biology.
2. b) Explain at a structural level (secondary conformation and aggregation) why treatment 3 failed but treatment 4 managed to neutralize infectivity.
3. c) Deduce from treatment 5 the mechanism by which this pathogen manages to "replicate" in a healthy host and explain why mice without the $PRNP$ gene turn out to be immune.

The measles virus is one of the most transmissible pathogens known, with an $R_0 \approx 15$. In a population of $300.000$ inhabitants, a vaccine is available with an effectiveness against infection of $95\,\%$.

1. a) Derive from the epidemic control condition ($R_t < 1$) the formula for the herd immunity threshold ($H_c = 1 - 1/R_0$) and calculate the minimum actual percentage of the population that must be vaccinated to ensure herd protection, taking into account the effectiveness of the vaccine.
2. b) Following a misinformation campaign, vaccine coverage in that community drops to $88\,\%$. If a primary case enters the community, quantitatively predict whether the outbreak will die out or lead to an epidemic by calculating the value of the effective reproduction number ($R_t$).
3. c) 600 people live in that population who cannot be vaccinated due to medical contraindications (immunosuppression or severe allergies). Biologically justify the concept of "vaccination as an act of solidarity" and illustrate what difference this vulnerable group experiences if the coverage of the healthy population is $98\,\%$ compared to if it is $88\,\%$.

You are part of an epidemiological panel that must assess the biological feasibility of eradicating three viral pathogens:

• Virus A (Rabies virus): RNA virus; fatality rate close to $100\,\%$; infects multiple wild mammals (bats, foxes) and domestic mammals; a highly effective inactivated vaccine is available.

• Virus B (Poliovirus type 1): RNA virus; exclusively human reservoir; between $90\,\%$ and $95\,\%$ of infected individuals are completely asymptomatic but shed viable viral particles in feces for weeks; effective oral and inactivated vaccines are available.

• Virus C (Measles virus): RNA virus; exclusively human reservoir; virtually $100\,\%$ of infected individuals develop characteristic full-blown clinical symptoms (fever, rash) with no asymptomatic cases transmitting the disease; $R_0 \approx 12\text{-}18$; an effective attenuated vaccine is available.

1. a) Based on the concept of natural reservoir, justify why Virus A is not biologically eradicable on a global scale, unlike what was achieved with human smallpox.
2. b) Compare the epidemiological difficulties of Virus B versus Virus C: which of the two poses a greater obstacle to the early detection and isolation of transmission chains? Justify your answer.
3. c) Design a non-invasive epidemiological surveillance method that allows detecting the active circulation of Virus B in a city without the need to collect individual clinical samples from the entire population.

In a tropical rainforest region, $60.000\text{ hectares}$ of primary forest are cleared to establish monocultures and intensive pig farms. Two years later, an outbreak of acute respiratory syndrome and encephalitis is recorded among farm workers, caused by a novel RNA virus related to bat pathogens.

1. a) Formulate an ecological hypothesis describing the transmission chain of the zoonotic spillover, explaining the role of ecosystem disturbance, the bat as a wild reservoir, and pigs as an amplifying host.
2. b) Biological sampling reveals that in the degraded forest fragments, bat species richness has decreased by $65\,\%$, but the density of an opportunistic reservoir species for the virus has tripled. Relate this pattern to the ecological concept of the "dilution effect" and argue why the loss of biodiversity increases the risk of zoonoses.
3. c) From the multidisciplinary One Health approach, design two preventive measures applicable to the environment or livestock management to prevent future viral emergence events in the area.

Potato spindle tuber viroid (PSTVd) consists of a single circular single-stranded RNA molecule of 359 nucleotides. Sequence analysis reveals that it completely lacks AUG start codons and any open reading frame (ORF), so it does not synthesize any protein.

1. a) When PSTVd is incubated with ribosomes and translation factors in a test tube, no peptide is synthesized. However, when inoculated into potato cell nuclei, the viroid replicates profusely. Justify how a non-coding nucleic acid manages to multiply inside the plant cell and deduce which plant nuclear enzyme is hijacked for this purpose.
2. b) If the viroid produces neither toxins nor capsid proteins, how does it cause severe necrosis and malformations in the plant? Explain the pathogenic mechanism at the molecular level based on the cellular processing of exogenous RNA by Dicer-like enzymes and the RNA interference (siRNA) pathway.
3. c) Certain viroids possess domains with ribozyme activity capable of catalyzing their own cleavage and ligation during replication. Justify why viroids are considered candidates for "molecular fossils" from the early stages of the origin of cellular life.

An untreated HIV-infected patient has a viral load with a daily production of $10^9$ virions. The virus's reverse transcriptase lacks proofreading activity and makes $1$ error for every $10^4$ nucleotides (its genome size is about $10^4$ bases).

1. a) It is estimated that the probability of a viral particle randomly acquiring a mutation conferring resistance to drug A is $10^{-5}$, to drug B is $10^{-5}$, and to drug C is $10^{-6}$ (independent mutations):

• Calculate how many virions resistant to drug A are generated per day in this patient and predict what will happen to the viral load if treated exclusively with drug A as monotherapy.

• Calculate the theoretical probability of a single virion simultaneously accumulating resistance to drugs A, B, and C combined. Based on that value, justify the therapeutic success of combination antiretroviral therapy (cART or triple therapy).

1. b) If the patient follows triple therapy but misses doses irregularly (frequent oversights), predict the evolutionary consequences on the virus's quasispecies diversity and explain why this behavior triggers multi-drug resistant therapeutic failure.
2. c) Why have the existence of quasispecies and the high mutation rate prevented obtaining an effective vaccine against HIV, whereas sterilizing and long-lasting vaccines do exist for viruses with stable genomes like smallpox or mumps?

Unit challenge. Imagine you are advising the Department of Health and Agriculture. Your mission is to evaluate the threat of acellular forms in your environment and design a technical biosecurity guide.

1. Real data collection: Access publications from the Carlos III Health Institute (ISCIII) or the Ministry of Agriculture (MAPA). Record the recent incidence or prevalence of a viral infection (for example, influenza with a rate of $18,4$ cases per $100.000$ inhabitants), a plant viroid (such as tomato apical stunt viroid), and a livestock prion disease.

2. Comparative analysis: Prepare a table comparing the three agents according to their composition ($ARN$, $ADN$ or the $PrP^{Sc}$ protein isoform), presence or absence of capsid/envelope, and approximate size in nanometres ($nm$).

3. Reasoned report: Write an action proposal ($1$ page) explaining which physical or chemical treatments (autoclave, UV radiation, bleach) are effective against each, justifying why the resistance of a prion or a viroid differs drastically from that of a virus with a lipid envelope.

Assessment criteria:

• Conceptual rigour when differentiating the molecular structure and pathogenesis of viruses, viroids, and prions.

• Accurate collection and appropriate citation of epidemiological data from official sources.

• Scientific consistency in the physicochemical justification of the proposed disinfection methods.