Viral properties, disease and treatment of viral disease

Question 1

Define the nature of viruses, and summarise a generic life cycle of a virus, explaining its parasitic nature in relation to the host cell

Answer 1

Viruses are infectious, oblige, intracellular parasites (DNA or RNA genome) (IISSO)

1. Infectious; can travel from organism to organism and colonize them
2. Intracellular; they have to be inside the host to perform their cycles
3. Small 20nm (single ribosome)
4. Shape; can be enveloped encapsulated or both
5. Obligate; cannot replicate without the appropriate host. Needs host to produce its machinery (ribosomes) to then produce the proteins needed to assemble next virus

Viral life cycle;

1. Attachment to cell surface specific receptor
2. Injection; fusing the viral genome with the cell–> transfer to the cytoplasm
3. Transcription; (making of the RNA)
   
   • Early regulatory proteins
   • Late ( structural proteins) which help make the cell capsule when it is assembled
4. Replication of the genome
5. Virus is Assembled and exits the cell

example: HIV replication cycle –> binds to CD4 and CCR5 receptors and enters host –> viral RNA turns to viral DNA by reverse transcriptase –> integrates into host DNA

The virus hijacks the DNA/RNA replication/translation machinery to synthesize its own proteins

Examples of viruses: HIV, Poliovirus, EBV, Coxsackie virus, dengue, influenza

Question 2

Explain the basis for the classification of viruses, and how viruses are detected, cultivated and manipulated

Answer 2

Based on the Baltimore Classification system there are 7 types of viruses;

1. dsDNA
2. ssDNA
3. dsRNA
4. +sense ssRNA
5. -sense ssRNA
6. ssRNA usinf dsDNA as intermediate
7. ss/ds DNA using ssRNA as intermediate

Differences in the viral genome will have some consequnces:

1. •RNA viruses are called retroviruses
   
   • they use reverse transcriptase enzyme
   • Limited in size due to instability
2. DNA viruses have bigger genomes and hence more accessory genes that can help in evading hosts immune system
   
   • These segmeted genomes allow easy recombination e.g. rotavirus, flu

Ways of detecting a virus:

1. Detecting viral genome PCR (extract genome and amplify)
2. Detecting viral antigen IFA (immunofluorescent antibody), ELISA
3. Detecting virus particles EM (electron microscopy), HA (haemagglutination)
4. Detecting virus cytopathic effect in cultured cells (Virus isolation)
5. Detecting antibodies to virus (serology)

Investigating viruses in the laboratory :

• Cytopathic effect is usually a result of the virus lysing the cell (due to shut down of host protein synthesis or accumulation of viral proteins)
• Observe syncytia:
  
  • Viruses form plaques in cell monolayers –>Viruses with surface proteins that can fuse at neutral pH often fuse cells together
  • The plaque assay undergoes 10-fold dilutions –> and is observed under the microscope

Cultivation and Manipulation

Propagation:

1. Using a line of permissive, transformed cells (transformed; the virus has been mutated so that it will be attenuated (weakend) which is the basis of vaccines.
2. This accumulation of mutations help the virus with combating new hosts
3. If the virus evolves to become stronger against e.g. monkey or chicken cells than it will be weaker against human cells
4. Some viruses have no permissive cell lines and can be difficult to study in the lab.

fun fact: norovirus has not been cultured in vitro because it needs the enteric microbiome to gain access to target cells

Manipulation:

1. Small viral genomes can be synthesized de novo
2. When introduced to permissive cells they direct the evolution

This is reverse genetics since we are creating viruses with in-build mutations already

Question 3

Viral routes of infection: explain the different routes by which viruses cause infection

Answer 3

Transmission:

• Iatrogenic; HCW eg contaminated needles
• Nosocomial; Acquired in hospital
• Vertical; From parent to offspring
• Horizontal; All other forms
• Germ line; Part of the host genome (eg intergrated retrovirus)

Viral routes ( portals of entry);

1. Respiratory; influenza
2. Faeco-oral; rotavirus
3. Contact; Herpes simplex (HSV)
4. Zoonoses (from the animals); Rabies
5. Blood; HIV, Hep B/C
6. Needles
7. Insect bites; Dengue
8. Sexual; HIV, HPV
9. Maternal; Hep B
10. Germ line (to the offspring); retroviruses

Progression of viral infection;

1. Local infection (apical release)
2. Primary viraemia - viraemia = virus in the blood (not good) (basal release)
3. Amplification (systemic hematogenous spread to organs or neural spread)
4. Secondary viraemia
5. Target organ

Question 4

Define the term tropism, and explain what determines the tropism of a virus

Answer 4

Tropism; it is the preference of viruses to infect certain tissues and not others.

Determining Factors;

1. Susceptibility;
   
   • Is defined by receptor interactions
   • HIV needs CD4, CCR5 or CXR4 receptors ( this is why it binds to T-helper cells)
   • Measles (very contagious) uses CD155 or SLAM on immune cells for entry and Nectin 4 (on airway epithelia) for exit
2. Permissivity;
   
   • Is defined by the ability to use the host cell to complete replication
   • needs ribosomes, transcription factors and metabolistes
3. Accessibility;
   
   • Is defined by whether the virus can reach a tissue
   • Example; Influenza: the viruses receptor can bind to every cell, meaning that there is no susceptibility restriction, but its tropism is defined by the availability of host proteases, which are more prevalent in the airway epithelial cells, hence the respiratory route of viral infection.

Tropism of HIV determined by receptor use:

• CD4 and CCR5 or CXCR4 co receptors
• Delta 32 mutation in CCR5 confers resistance to HIV in Exposed Uninfected
• Viral attachment protein gp120

Tropism of Influenza virus:

• Morphology: surrounded by HA and NA molecules attached to cell membrane
• To enter host influenza virus needs:
  
  • HA to bind sialic acid receptors (sugar receptors) that are ubiquitous
  • Low endosomal pH triggers fusion- HA cleavage is required for exposure of fusion peptide
• To reassemble and exit the influenza virus needs to be cleaved by proteases
• Influenza tropism is extended by mutation at the HA cleavage site​

Question 5

Viral infection outcomes: list different outcomes of infection by viruses

Answer 5

1. Acute infection; influenza, rhinovirus, rotavirus, smallpox
   
   • there is infection, followed by the response of the organism and quick and complete resolution of the infection.
   • Can be a common condition such as flu, rotavirus (vomiting bug) etc. and sometimes asymptomatic.
   • Infection intensifies at first as it establishes itself, reaches the plateau phase and then the immune, adaptive response of the body will combat the virus, which will result in resolution of the infection.
   • Some of the acute infection might not go to resolution and lead to death if not treated -> smallpox, dengue hemorrhaging fever
   • There is a subtype of Acute infection during which there is accidental pathogenesis e.g. polio leading to paralysis or rubella leading to deafness, eye abnormalities and CHD (congenital heart disease)
2. Persistent infection: Chronic infections; papillomaviruses in warts, chronic carriers of Hepatitis B and C
   
   • gerally they are not cleaved from organisms completly because they have low levels of replication and can hide in tissues which regenerate.
   • Strategies for viral persistence include:
     
     • MHC downregulation
       
       • Compensation for lost MHC class I (Cytomegaloviruses)
     • CTL escape by mutation (Hep C)
     • Infecting tissues with low immune surveillance
       
       • CNS
       • Skin (warts)
3. Persistent infection: Latent reactivating infection; Herpes Simplex virus (HSV), Varicella Zoster (chickenpox or Shingles)
   
   • It is a type of Persistent infection
   • generally it is not cleared from the organism completely. They have a low level of replication and can hide in tissues which regenerate. Virons can’t be detected between reactivation periods except for LATs (latency-associated transcripts)
   • HSV hides in ganglions and re-emerges when there is reduced immune response and is able to re-establish itself

Herpes Simplex Virus Latency and Reactivation:

1. Primary site of infection : productive infection of epithelial cells
2. Secondary site of infection and site of latent infection : sensory neuron
3. Site of recurrent infection : productive infection of epithelial cells
4. Persistent infection; Slow infection; Measles, HIV, Human T-lymphotropic virus
   
   • Have a long incubation period ranging from months to years
   • Follow a slow but relentless progressive course leading to death
   • Tend to have a genetic predisposition
   • Often re-emerge from latency if the host becomes immuno-compromised
5. Oncogenesis; Hepatitis B/C causing hepatocellular carcinoma, HIV causing Kaposhi’s sarcoma, EBV causing Burkitt’s lymphoma
   
   • many encode oncogenes which interfere with the cell cycle in order to enhance their own replication
   • HIV–> causing Kaphosis sarcoma
   • HCV–> in which cancer can develop years after the infection
   • Epstein Bar Virus –> lytic infection in childhood or mononucleosis in young adults –> And then remains latent in B cells –> Passed on in saliva –> it also causes Burkitts lymphoma, Hodgkins lymphoma, Nasopharyngeal carcinoma

Question 6

Explain factors that interfere with the outcome of virus infection

Answer 6

1. Virus sequence
2. Virus load
3. Host immune response/status
4. Host co-morbidity
5. Co infections
6. Other medications
7. Host genetics
8. Host age, gender

Virus sequence:

Two strains of poliovirus might vary in their virulence. A single mutation in the genome can mean that one strain acts as a live attenuated vaccine (Sabin) whilst another invades the motor neurone and causes flaccid paralysis (poliomyelitis).

Viral load:
The first child in the family to contract chicken pox often has a milder illness than the second child. This may be because the second child is in closer contact and become infected by a higher dose.

Co Infections:

• HHV8 causes Kaposi’s Sarcoma in HIV infected individuals.
• Hepatitis delta virus, a small defective RNA virus that only infects people with Hepatitis B virus infection.. suppresses HBV replication but causes severe liver disease with rapid progression to cirrhosis and hepatic decompensation

Genetic resistance and susceptibility

CCR5 delta 32 mutation protects against HIV-1 infection

KIRs can determine the outcome of hepatitis C virus infection

IFITM3 associated with severe outcome in 2009 pH1N1 pandemic

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Question 7

Zoonoses: define the terms zoonosis and host range, explaining how viruses emerge and re-emerge using named examples

Answer 7

Zoonoses;

• are infectious diseases that can be naturally transmitted between animals (usually vertebrates) and humans.
• Ebola Virus, salmonella, rabies, Zika fever, west nile fever.
• Commonly occurs due to having household pets, farming, predatorial behaviour and research including animals.
• Arboviruses are viruses that are passed on by insects (Yellow fever; Dengue; West Nile; Zika; chikungunya;)

Zoonosis;

• this is the process of transmission from animals and humans.
• Can be direct through vectors such as air or reverse, when humans infect animals

Host range;

• organisms that can be infected by a disease
• Some hosts are dead-end hosts, meaning that they can’t pass the disease on.

Emerge and re-emerge of viruses;

1. West Nile Fever; re-emergence in NY; birds in Bronx Zoo; they are not sure how this came to the USA à Illegal birds, infected mosquito, viraemic human
   Horses and humans are dead end hosts for West Nile Virus
2. Zika; mosquitos in Brazil
3. Dengue:

• 3 billion people live in at risk areas
• 50-100 million cases of DF each year; 300 000 cases of DHF
• DHF case fatality 5%
• 4 serotypes of DV cross reactivity but no cross protection

4. Ebola; they can’t infect efficiently so they pop up every now and then. Chimpansees, Bats, Civets
5. SARS; (Severe acute respiratory syndrome).
   In China in 2002, 8422 case. Chinese horseshoe bats. Civets and racoon in markets
   Increasing exposure of human populations and domestic animals to bats

• Coronavirus; Large (30kb) positive sense RNA genome.
• Envelope spike protein.
• Receptor is human ACE-2 protein.
• A virus almost identical isolated from masked palm civets and raccoon dogs in wet markets.
• Chinese horseshoe bats harbour SARS-like coronaviruses that can use bat and human ACE2 as receptors.
• S protein is highly plastic and can adapt to different receptors overcoming host range barriers.

6. MERS; found in camels in Middle East,

• Health tourism and business travel spreading the virus beyond Midde East
• Closely related to HKU4 and HKU5 two bat coronaviruses
• High seropositivity rate in camels
• ARDS in older infected person but can be asymptomatic in infected contacts.

​

7. H1N1, H3N2; due to influenza virus being able to rearrange and change. Some variants are restriced to human, swines and birds.
8. Chikungunya: associated with prolonged arthralgia

Question 8

Explain why it is difficult to develop drugs which selectively act against viral infections

Answer 8

Selectivity; how specific is the action of the drug within the organism

Viruses use the machinery of the cell and therefore therapy can be detrimental to the host

Therefore, specific elements of the virus must be targeted ;

1. enzyme specific to viral protein synthesis
2. exit and entrance mechanism of the virus

Question 9

Analyze the principles of viral prophylaxis and therapy

Answer 9

Vaccines

• Prophylactic
• Live or inactive
• Herd immunity or defined target group?
• Safety > efficacy (RSV vaccine in 1970s!)
• Governments and WHO

Antiviral drugs

• Therapeutic
• Random screen or rational design
• Define target group: very sick or over the counter?
• Individuals

Eradication of smallpox:

• No animal reservoir (uniquely human)
• No latent or persistent infection
• Smallpox was an easily recognised disease
• The vaccine was effective against all strains of virus
• Vaccine properties. Potency, low cost, abundance, heat stability, easy administration
• WHO determination
• $250 million

Different types of viral vaccines:

1. Attenuation: live natural virus vaccine
2. Inactivation: inactivated viral vaccine
3. Fractionization: non-recombinant purified subunit vaccine
4. Cloning:
   
   • Live virus vector vaccine
   • DNA vaccine
   • Protein (subunit vaccine, virus-like particle vaccine)

Attenuation of virus to make a live virus vaccine (image)

Pros and cons of live vs inactivated vaccines:

Live vaccine :

• Rapid broad, long lived immunity
• Dose sparing
• Cellular immunity
  BUT
• Requires attenuation
• May revert

Inactivated vaccine:

• Safe
• Can be made from wild type virus
  BUT
• Frequent boosting required
• High doses needed

Examples of viruses for which both live and inactivated vaccines are available:

Influenza

• Inactivated virus or HA subunit
• Updated regularly
• DOES NOT give the recipient the flu!!!
• LAIV is cold adapted
• FluMist delivered intranasally
• Updated regularly
• Introduced for children in the UK 2013

Poliovirus

• Salk inactivated vaccine
• Sabin live attenuated vaccine
• 1 in 7 million vaccinations associated with poliomyelitis
• Persisting in immunosuppressed individuals

Other vaccines:

Rotavirus vaccine

• Rotarix is a live attenuated rotavirus reassortant virus.
• In the developing world it can massively reduce deaths due to rotavirus infection that leads to dehydration from vomitting and diarrhoea.

Subunit vaccines

• Hepatitis B virus –> sAg cloned and expressed in yeast
• Papillomavirus –> Virus Like Particles from recombinant coat proteins - Gardasil and Cervarix

Shingles vaccine

• Shingles is a painful rash resulting from the reactivation of a latent varicella zoster virus infection (chicken pox).
• Shingles occurs in people after stress, and is more common and more serious in the elderly as their immune system wanes.
• The live attenuated vaccine is similar but distinct from the chick pox vaccine given to children in some countries

Ebola vaccines

GSK: Chimpanzee adenovirus vectored vaccine that expresses Ebola G protein

Merck : Vesicular Stomatitis Virus vectored vaccine expressing Ebola virus G protein

Question 10

Summarise the general strategy of designing drugs which target infectious agents but not the patient’s own cells, and identify stages in the life cycle of viruses which could potentially allow therapeutic intervention

Answer 10

General strategy (rational drug design):

1. Identify elements of viral life cycle that differ from host’s
2. Identify the site of drug action (usually viral enzymes)
3. Design the drug to be highly specific for the site
4. Block spread/replication/dissemination of the virus

Stages in viral life which could be targeted for theraputic inctervations.

1. Viral entry and binding;
   
   • this is connected to tropism and the need for a virus to have specific cell receptors or binding site.
   • If this is modified the viron can’t enter the cell, which is crucial for it to be able to exploit the cellular machinery of the host (REMEMBER about the obligate parasitic nature)
2. Viral replication;
   
   • ​​ Biggest area for intervention; viral genetic material will encode some of its own enzymes, which can be targeted (e.g. nucleoside analogue such as Acyclovir).
   • Other processes such as reverse transcription (for retroviruses) can also be blocked.
3. Viral Assembly;
   
   • made out of the inner genetic material and outer protein coat, which have to be assembled.
4. Viral Exit;
   
   • some viruses need to be enzymatically cleaved to be released from the hosts cells after the assembly e.g Influenza virus.

Question 11

Antiviral therapy: list examples of classes of drugs which have been used successfully in antiviral therapy, and explain the specific strategy of using acyclovir and other nucleoside analogues as antiviral drugs

Answer 11

1. Interferons; help to induce hosts natural immune response to viral infection
2. Nucleoside analogues; interfere with replication e.g. Acyclovir
3. Fusion/entry inhibitors
4. Non-nucleoside reverse transcriptase inhibitors (NNRTIs)
5. Integrase inhibitors
6. Protease inhibitors (viral exit)

Active retroviral therapy for HIV (image)

Antiviral therapy for hepatitis C virus

• HCV is a hepatotropic flavivirus that was spread widely in the 1970s in blood products before screening was put in place.​
• HCV protease inhibitors
• nucleoside polymerase inhibitor
• non-nucleoside polymerase inhibitor
• Agents that inhibit NS5A
• part of fixed-dose combinations

Question 12

Acyclovir

Answer 12

Acyclovir acts as a chain terminator on the viral thymidine kinase (an enzyme that phosphorylates)

1. It acts as biochemical confidence tricks
2. viral thymidine kinase has a high affinity for acyclovir (unlike cellular thymidine kinase) and it converts it to acycloGTP ( there are large amounts in infected cells)
3. It is an excellent target for the viral DNA polymerase but since AcycloGTP blocks further polymerization of DNA because no 3 OH group is availible for the production of a phosphate bond viral replication is blocked (hence generation of new virus is prevented)
4. acycloGTP is a poor substrate for the host cell DNA polymerase and it is left often incoporated into the cells replicating DNA ( spared from the potent effects of the drug)

Picture; Acyclovir