Virology: Picornaviridae (+) ssRNA

Family: Picornaviridae

What baltimore group is Picornviridae?

Group IV (+) ssRNA

What is a benefit of (+) ssRNA?

It is already an mRNA and can be immediately translated.

Pico-RNA-virus means

Small RNA virus

Picornviridae family is a non-enveloped or enveloped virus?

They are non-enveloped!

How do these picornviridae virus get out of the cell?

They do not bud (for enveloped viruses)! They lyse out of the cell. It happens this way because it is non-developed.

Are picornaviridae pathogens environmentaly stable?

Yes! Because they are non-enveloped. They can handle pH changes, alcohol and detergents due to the hard CAPSID shell.

Name the human pathogens within the picornviridae family (5 total)

• Poliovirus

• Rhinoviruses

• Hepatitis A virus

• Coxsackieviruses

• Other entero viruses

What is poliovirus?

• Dicovered in the 1940s and 50s, paralyzed or killed >500,000 people per year

• Nearly eradicated (destroyed) by vaccine

• Early model system for field of virology

What is a Rhinovirus?

• “The common cold” most often

• >50 million infections/year and ~100,000 deaths/year.

Hepatitis A virus infections/year and deaths are

Hepatitis A virus has had >100 million infections/year ~100,000 deaths/year.

Coxackieviruses (cox-ackee-viruses) is also known as…

Hand foot and mouth disease and some cardiac inflammatory diseases.

Other enteroviruses infect what?

• Infect gastointestinal tract

• Incorrectly called ‘the stomach flu’

What is a non human pathogen?

Foot and mouth disease and several others (devastating for livestock)

Related viruses of picornavirusviridae

Norovirus (“the cruise ship virus”)

• Formally a member of Calciviridae

Mention a few characteristics of Poliovirus and Poliomyelitis

• Most infections are asymptomatic

• ~0.5% of poliovirus infections result in muscle weakening (weaken muscles for breathing).

  • “Iron lung”

• Most people recover

• In come cases people die or are permanently paralyzed

• Chlorine is in swimming pools to reduce infections!

  • Poliovirus is a gastrointenstinal virus (transitted orally and fecally)

• There is also a vaccine

Poliovirus is a gastrointestinal virus, how can one get infected?

It is transmitted orally and fecally

Most poliovirus pathogenesis is caused by ‘off target’ replication of virus. What does off target mean?

• Polio virus is transmitted orally and fecally

• The virus replicates in the oropharynx

• In rare cases, the virus goes ‘off target’ and ends up in the nervous system which is NOT beneficial to the virus (~1% of the time).

  • When it ends up in other parts of the body, it can be lethal leading to eternal paralysis or death.

Who contracted poliomyelitis at age 39 and became a strong supporter of vaccine efforts?

Franklin D. Roosevelt

What are the two competing polio vaccines? Who created them?

• 1952 Salk vaccine: Jonas Salk

• 1961 Sabin vaccine: Albert Sabin

Describe the Salk vaccine

• Injected

• “Dead virus vaccine”

• Inactivated polio virus

• Virus can no longer replicate

• Induces antibodies, but not at intestinal route of infection.

Describe the Sabin Vaccine

• Orally taken (Sugar cubes)

• “Anttenuated virus vaccine”

• Exposure to weaker strain

• Natual route of infection

• Can also spread to uninfected people, exposing them to the varient (population vaccination)

Cons of the Salk vaccine (Killed/inactivated)?

• More costly and difficult ot manufacture

• Must be precise with every batch of vaccines, want to deactivate the replication cycle but not destroy it to the point the imune system cannot recognize the virus

• Needs to be injected (ouch!)

Cons of Sabin vaccine (live attenuated)

• Less stable environmentally

• Small chance of reactivation

• Must generate a great number of mutations to prevent reactivation.

Poliovirus was eradicated by which vaccine?

Sabin vaccine (sugar cubes that contained weaker strains)

Picornavirus are ____-_____ _______ viruses

Picornaviruses are non-enveloped lytic viruses!

Describe the life cycle of picornavirus

1. Receptor binding to CAPSID protein

2. Entry via endocytosis

3. Host ribosomes assist with IRES dirven translation and proteins are made

4. Polyproteins are made and do 5 things

5. One of them (most important) is that the viral replication complex make more (-) and (+) stands making more copies for packaging

6. Cell lysis occurs and viruses exit the cell

Describe attachment and genome delivery

• Poliovirus protein on CAPSID binds to receptors

• Uncoating occurs once brought in via endocytosis

• Results in CAPSID docking onto endocytic membrane and mRNA is secreted

Describe genome delivery at a ‘molecular’ level

• CAPSID protein (VP1) binds to receptors at N termini

• Pore opens up and the VP1 N-termini opens up by a confomational change

• genome ( +ssRNA is released and enters the cytoplasm

Genome structure: Doing a lot with only a little genome. Describe what the genome structure.

• VPg is a protein that replaces the 5’ CAP found in all of our mRNAs.

• 5’ non-coding region

  • Internal ribosome entry site

  • Binding site for replicase complex

• P1 ecodes for capsid proteins

• P2 and P3 encode foe non-structural proteins (critical for the replication cycle)

  • Protein processing

  • Host shutoff

  • RNA replication

• 3’ end non-coding region

  • Binding site for replicase complex

• Followed by poly-A tail

An IRES must be present for (+) ssRNA translation, what does it recruit?

• A virus does not need a 5’ cap, a VPg is there instead to avoid the RNA from being chewed up by nucleases.

• IRES is a complicated RNA structure that recruits ribosomes (40s ribosomal subunit)

Describe the viruses strategies used to disrupt host protein synthesis

• They cleave or degrade eIF4F comonents to block host synthesis

• They also hijack the ribosomal 40s subunit to attach to the IRES to initiate viral translation

Viral proteins are all translated as a single polyprotein and then cleaved by …

• Proteases! A pair of molecular scissors.

• Proteases are essential to be able to process things to their individual pieces.

• We have vaccines that inhibit these proteases.

Where does replication occur?

At the membrane!

• Allows the virus to localize proteins, production, etc

• Also allows the virus to ESCAPE immune detection

Cool stuff weve learned from studying members of the picornaviridae

One of the best studied group of virsues

• First animal virus discovered (foot and mouth disease virus)

• First evidence of RNA dependent RNA polymerase

• Proteins are expressed as a single polyprotein and cleaved by viral protease.

• IRES mediated by cap-independent translation

• Vaccine research

• Evolution of RNA viruses (e.g error catastrophe, quasi species, viral recombination)

What is error catastrophe?

• When a virus replicates so much and introduces many more errors to the point it dies out

• Ribavirin is used a a mutagenic drug to drive —> error catastrophe '

  • Ribavirin is a nucleoside that causes mutations and pushes the viral replication over the error threshold to the point the virus cant survive

What is R0? (“R naught”)

It is the basic reproductive number.

• One person is infected. How many more people will they infect in a naive group?

Give me the R naught equation, tell me the signifigance on R naught values.

R0 = number of contacts in a given time X transmission probability per contact X Duration of infection

R0>1 = Endemic continues (bad)

R)<1 = Endemic dies out (good)

Give me the estimates for some epidemic viruses:

MERS, Measles, Polio, Influenza, HIV, Ebola, SARS-Cov-2 (original), SARS-CoV-2 (varient)

MERS: <1

Measles :12-18

Polio: 5-7

Influenza: 1-3

HIV: 2-5

Ebola: 1.5-2.5

SARS-Cov-2 (original): 2-3

SARS-CoV-2 (varient): >5?

What are two ways to reduce R0?

• Cure the infection faster

• Distance yourself, reduces number of contacts in given time

• Masking yourself, lower transmission probability

Efective R equation is…

Effective R = R0 X Fraction of susceptible population

Effective R should = to 1

• Tells us how much of the population needs to be vaccinated

What is one way to reduce the effective R?

Vaccinate!

Immune individuals reduce effective R

Reverse genetics in RNA viruses. What does it do?

Help us hypothesize about viruses by introducing mutations

Describe “forward genetics”

Done by randomly mutating the virus, looking for a phenotype and sequencing that virus.

Describe “reverse genetics”

We make individual mutations and test whether it produces a given phenotype.

Which Baltimore group of RNA viruses do you think will be easiest to do reverse genetics on?

IV, (+) ssRNA. This is because it is readily available to translate due to being (+) readily acts as an mRNA.

Reverse genetics in (+) ssRNA, decribe the steps

1. Reverse trascribe viral genome and insert into vector (mutations)

• Can make mutations at this step via standar molecular biology techniques

2. Transcribe the RNA using a DNA dependent RNA polymerase

• Often use a phage polymerase (T7) becuase they are small, robust, and have little regulatory requirements.

2. Get mRNA into cells and recover virus.

• Either transfect or electroporate to get mRNA inside mammalian cell.

Pros and Cons of many different viruses

What are the types COVID 19 vaccines?

Whole inactivated, Live attenuated, Synthetic peptide or recombinant subunit, Recombinant viral vectors, DNA or RNA.

Decription of each COVID 19 vaccines