Transcription of mRNA 

Translation of early proteins 

Replication of viral DNA/RNA 

Transcription of mRNA 

Translation of late proteins 

They bud through the nuclear envelope 

If there were 1 million cells in a flask and you add 1 million viral particles the multiplicity would be 1 but what is the probability that each cell would be infected?

what if if the multipliciticy of infection is 10?

multiplicity of 1 - not all cells infected 

Multiplicity of 10 - 99% of cells will be infected 

  It is easier to cleave within the cell (indicative of high virulence) 

The # of basic animo acids at clevage site within Fo 

the more sites available for cleavage - the more the virus can spread 

Which are more efficient?

Cell endoproteases found throughout the host  

OR 

External proteases found in the gut and respiratory tract 

ECPs External proteases 

 The cleavage of HA

cleavage is mediated by cell endoproteases and external proteases.  

The number of basic amino acids determines teh ease of cleavage. 

Canine Parvovirus 2 (parvoviridae) - months

Canine Adenovirus 1 (adenoviridae) - months 

Caliciviruses (caliciviridae) 

Rotaviruses (reoviridae) - months 

Orf (poxviridae) - over winter

Avian Influenza (orthomyxoviridae) 

Marek's Disease virus (herpesviridae) - wks/mons

FMDV (picornaviridae) FAD

Classical Swine Fever Virus (flaviviridae) FAD

African Swine Fever Virus (asfarviridae) FAD

What is the active ingredient in household bleach?

How much is in it?

FeLV (orthoretrovirinae)

Canine Distemper Virus (paramyxovirinae) 

Rinderpest (paramyxoviriniae 

Clean and disinfect cages and utensils with 5% bleach 

These viruses often spread only by direct contact

How well does Canine parvovirus 2 survive in the environment?

How would you deactivate it?

Canine Parvovirus 2 (parvoviridae)

is infective for months outside the host.  

To clean: Decontaminate the environment & remove organic debris. 

Physical clean up, soap, 5% bleach solution

Canine Adenovirus 1 (canine infectious hepatitis virus - adenoviridae) is able to survive for months outside the host - - 

THIS CAUSES NATURAL VACCINATION. 

Caliciviridea - Marine mammals to vesicular exanthema od swine .

The jump started in CA where seals were used for fur and meat was infective meat was fed to swine - virus very hardy and was able to survive and was infective to swine consuming it. Also garbage containing virus spread it to NJ.

Porcine circovirus 1

Rotaviruses are a leading cause of enteric disease (diarrhea) in children in the developing world. 

SV40 (Simian virus 40) 

this virus can cause cancer in baby hamsters - - damaging effect on humans unknown 

Rendering 

Burial 

Incineration 

Composting 

Hepatitis A

Hepatitis E

Rotavirus 

Norovirus

Adenovirus

Coxsackie virus 

Poliovirus

What are more susceptible to thermal inactivation, 

enveloped viruses or naked viruses ?

In the study by Feagans (2008): What methods proved effective at deactivating porcine HEV from liver meat?

A) Boiled 5 mins 

B) 56^OC for 1 hr. 

C) Stir fried @ internal temp of 71^OC

D) A & B only 

E) A & C only 

F) B & C only 

G) ALL of the above (A, B, C) 

Answer: E) A and C only 

(Boiled for 5 mins and stir fried, delicious) 

What is the least effective temperature for preserving viruses?

A) -70^OC (in liquid nitrogen) 

B) ^-2 to ^-4^OC 

C) 0^OC

D) 37^OC

Answer: C) 0^OC

This temp is very bad for preservation, denatures ice crystals 

CRYOPROTECTANTS

Proteins (serum, albumen) 

DMSO (Dimethyl Sulfoxide)

Substances that can be added to a virus to increase its stability during preservation.

This includes proteins (serum, albumen) and DMSO (Dimethyl sulfoxide)

Glutaraldehyde (curvilinear) 

formaldehyde (curvilinear)

betapropiolactone 

N-Acetylethyeneimine (linear) 

Chlorohexidine 

5% Clorox - - - Inactivated by organic material 

Detergent iodophores (Betadine)

Glutaraldehyde 2% 

70% ethanol 

Physically remove organic debris 

Soap and water 

5% Bleach 

What are viruses that are classified as FAD?

What are some examples?

Foreign Animal Diseases.

Foot and Mouth Disease Virus 

Classical Swine Fever 

African Swine Fever 

Euth any possibly infected animals that have not died 

2 weeks compost within building 

then can be moved outdoors for another 2 weeks in piles or windrows (those lines of hay) 

This term refers to the integrity of storage process from initial storing to the clinical user.  

Viruses should be controlled and preserved at a low temperature - if this "cold chain" is broken, the titer of the vaccine can be lost.  

A dose response curve of a viral vaccine will be the same across a species. 

True or False?

FALSE

There dose response curve is unique per system! 

This means that there will be curve differences between species, age, etc. 

Thermal inactivation - the effectiveness determined by the species of virus

Chemical inactivation

How would you handle dead pigs in sanitation effects to eliminate Classical Swine Fever?

CSF can remain infective in pork products, and can be spread by uncooked garbage. 

The environmental clean up is challenging because it is such a hardy virus. 

Best choice is to incinerate because CSF is an enveloped virus (which is highly susceptible to heat inactivation).  Organic waste should be removed, area cleaned and disinfected, and keep uninhabited for a while.

Why is N-Acetylethylenimine an ideal agent for use in vaccine production?

It chemically inactivates viruses for vaccines

It shows a linear relationship in concentration of the virus.  This will give you the most accurate measurement of virus to use . 

What type of method burial is approved for eliminated contamined wastes?

A) Contruction 

B) Hazardous waste

C) Municipal solid waste

Answer: C) Municipal solid waste 

This is the only approved method of burial because it is constructed to retain leachate and collect gases

Answer: AIR CURTAIN method 

This method allows the carcasses to be contained within a container that can be transported by truck.  The curtain also keeps SMOKE and ASH within the container. The curtain also keeps the heat it producing EXTREMELY HIGH TEMPERATURES.

- Replication determines pathogenicity 

- Location of replication = disease 

- Viral ressortment 

- ie. field intramolecular recombination of FMDV

- Antivirals 

- mechanisms of action against cell replication

- Viral vectors 

for vaccines, gene therapy, and cancer treatment

- Attachment 

- Penetration 

- Uncoating 

- Eclipse phase (translation and transcription of early and late phase proteins) 

- Assembly 

- Egression/ release (through lysis or budding)

It is called the eclipse phase because the coating has been removed and genetic material dispersed, so the viral particles are not visible.

(like how you cant see the sun in a solar eclipse, but you know its still there...)

- Lysis 

- Budding through a membrane 

acquire an envelope from modified membrane 

nuclear membrane, ER, cytoplasmic, and more

ie. herpes virus (nuclear)

What is a "One step" growth curve?

How is it generated?

One step curve is obtained by infecting all culture cells simultaneously, to ensure one cycle of growth, with a multiplicity of 10 or more. This ensures 99% infectivity.

The curve is generated by determining the presence of viral particles.  Since there are no intact viral particles, the curve is at zero during eclipse phase, then higher during assembly. 

RNA viruses are 10X faster than DNA - more steep curve.

SPECIFIC CELL SURFACE RECEPTORS

Some only need one, other need a coreceptor 

It attaches to the cell via Sialic acid residues that hang off of cell glycoproteins. 

α 2, 3 linkages are primarily in bird strains

α 2,6 linkages are in mammals (humans) 

(these are the linkages connecting the sialic acid to the glycoprotein)

What would happen if you dropped egg albumin in vinegar (an acidic environment)?

What relavance does this have to viral replication?

It would become opaque, because the proteins would become denatured and reorganized. 

This relates to the viral replication because it is similar to what happens to the HA in receptor mediated endocytosis.  Once the receptor binds, H+ ions rush into the area, decreasing the pH and causing reconfiguration of the HA proteins, this allows the N-terminal of the HA to be exposed and can fuse with the endosomal membrane.

(the HA must be cleaved for all of this to happen - no clevage occurs in the endosome)

Membrane bound vesicles 

Fusion from without 

receptor mediated endocytosis 

Direct entry

All members of the orthomyxoviridae family utilize receptor mediated endocytosis to enter host cell. 

True or False?

All members of the picornaviridae family utilize direct penetration to enter host cells.

True or False?

FALSE

Polioviruses enter cells through direct penetration, but FMDV enter cells though receptor mediated endocytosis, and both are in the picornaviridae family. 

At the nuclear membrane.

After fusion from without - the icosahedral nucleocapsid then is released and travels along the microtubules to towards the nucleus.  Along the way it loses its capsid and can enter the nucleus through nuclear pores.

Tropism 

Clevage of Fusion protein (F₀)

(number of basic amino acids) 

Ability to grow in absence of trypsin

NONE

Herpesviruses have a large genome that can code for all their own proteins and polymerases that are needed for replication and survival. 

Which viruses are more dependent on host cells for protein production?

A) Larger viruses 

B) Smaller viruses 

SMALLER VIRUSES 

The smaller the virus's genome, the less space there is for replication proteins (such as polymerases). These viruses will target rapidly dividing cells, and use thier polymerases.  

Which viruses are in the correct order of size from smallest to largest?

A) Herpesviridae, poxviridae, circoviridae

B) Adenoviridae, herpesviridae, parvovirdae

C) Parvoviridae, adenoviridae, poxviridae

D) Asfarviridae, poxviridae, hepadnaviridae

ANSWER: C)

Parvovirdiae = small and highly dependent on host cell for replication proteins, which is why they are found in rapidly dividing cells.

Adenoviridae = fairly large, genome not large enough for all the necessary proteins, so it uses early proteins to turn on resting cells. 

Poxviridae = VERY LARGE, have an adequate genome to code for all the proteins it needs to replicate. 

Crypt cells of the gut 

Developing fetal cells 

growing cerebellar cells

THIS IS ONLY ASSOCIATED WITH ss+ REPLICATION!

This is a negative sense strand of RNA that is generated from the virus's orginal ss+ RNA genome.  (virus specific RNA dependent RNA polymerase uses original ss+ as a template for the RI.) The negative strand is 3` to 5`.  

- Virus specific RNA dependent RNA polymerase then uses the RI as a backbone, where ss+ are formed off of this in a herring bone formation. These ss+ drop off as detached by enzymes. 

These viruses have proteins in their genome that act as virus specific RNA dependent RNA polymerase. 

This then allows for the ss- to serve as a template and a ss+ is generated. Same enzyme goes back and makes more ss-, which translate into more ss+, and so on. These strands are then assembled. 

Many DNA viruses have nuclear inclusions such as: 

Herpesviruses, parvoviruses, adenoviruses, papillomaviruses.

ALSO the exception to this is the Canine distemper virus, which is an RNA virus, and forms nuclear inclusion bodies in the brain (although it forms cytoplasmic inclusions in other tissues). 

Poxviruses and African Swine Fever Virus.

These have very large genomes and do not need the nucleus of the cell to provide anything for them. 

Inclusion bodies are typically made of accumulations of viral proteins or of verions. 

These viral proteins are remnants of scaffolding (structural) proteins, they are acidophilic. 

This type of infection would indicate that the animal is carrying the virus and whole intact virions can be found in the host, although they are not showing clinical signs.

IE. LCM virus of mice (and pet hamsters) always is shed in the urine and is a zoonotic agent. 

handwash after handling

Latent infections - you do not see whole intact virions, instead there is viral genome in the nucleus of the host.

IE. Herpesviruses, retroviridae 

The inside of the capsid has a sequence of animo acids, and there are packaging sequences of nucleotides in the DNA.  There is an affinity between these two and there is binding.  

(this is the point where Dr. Platt makes an obscure reference to the Ed Sullivan show, that only James is old enough to know...)

It is assembled AFTER the late eclipse phase and in occurs IN THE NUCLEUS. 

This icosahedral capsid than buds through the host cell's nuclear membrane and acquires its envelope. 

BEFORE ATTACHMENT - - 

At this point if the HA is not cleaved, then replication is aborted!!

Cleavage of the HA occurs within the endosome.

True or False?

FALSE 

If you get this wrong - - HE WILL TAKE OFF 30 PTS 

For serious.

clevage needs to occur before attachment to the host cell or there will not be fusion within the endosome. 

In the Golgi apparatus as the proteins are being processed and packaged.  Here is there are enough basic amino acids the HA can be cleaved by cell endoproteases. 

This is in highly virulent strains.

- BLOCK M2 ion channel that allow H+ ions to flow into an endosome allowing for HA reconfirmation

- Interefere with the clevage of HA 

- Interfere with "cap snatching" so viral RNA cannot be translated

- *Interfere with neuraminidase (glycoprotein) to prevent release of progeny virus (this is important because it is what most available antivirals for influeza are).

The cell secretory pathway 

Transcription of cell DNA

Translation of cell proteins from cell mRNA.

The redirection of these functions are mediated by nonstructural proteins. 

Enterovirus  - - ie. poliovirus

Rhinovirus  - - ie. rhinovirus

Hepatovirus - - ie. hepatitis A 

Cardiovirus  - - ie. encephalomyocarditis virus 

Aphthovirus - - ie. FMDV

Polioviruses produce a single polypeptide out of its ss+ RNA.  This polypeptide is translated into just structural proteins. 

True or False?

False!! 

The polypeptide codes for both functional and nonfunctional proteins.  Of the nonfuncitonal proteins included are those involved in taking over host cell functions.  

NON STRUCTURAL 

Protein 2A (interfere with translation of cellular mRNA) 

Protein 3C (interferes with cell DNA transcription) 

2BC, 3A (Inhibition of the secretory pathway) 

Internal Ribosomal Entry Site

This can be found on Poliovirus mRNA.  

A protein will cleave off the cap of the mRNA, and because polioviruses are NOT CAP DEPENDENT, translation will occur at these sites.  This allows the poliovirus to steal the amino acid resources from the host cell that IS cap dependent. 

Initiation factors, 

Elongation factors, 

Termination factors, 

mRNA

Ribosomes

tRNA

the cap (on mRNA)

These are located at the 5' end of eukaryotic mRNA.

Host cells are cap dependent and require the cap to begin translation.

Polioviruses are NOT cap-dependent, instead they rely on IRES to initiate translation of mRNA.  So, in order to redirect resources from the cell, the virus codes for a non structural protein that cleaves the "cap" on cell mRNA.  

With the cell unable to initiate translation, amino acids and other resources can be used by the virus. 

The TATA box is an indicator of an open reading frame, and when the TATA binding protein is present, transcription initiation factors will bind and begin transcription. 

The TATA binding protein can be inhibited by non structural proteins such as those produced by polioviruses and this prevent transcription of host DNA.  

This allows for an accumulation of vesicles that the virus can now use to replicate its RNA in.  

The more vesicles a RNA virus has, the more readily it is able to replicate!

An increased number of vesicles would allow for an increased viral efficiency in replication. 

These are sites of viral RNA replication, so the more present, the more able to virus is to replicate. 

- redirecting of cellular resources to the virus for replication

- failure to generate cellular proteins 

- decreased integrity of cellular membranes 

- LYSIS 

REPLICATION AND RELEASE OF PROGENCY VIRUS!!

- Mutation 

- Genetic interaction between viruses 

This refers to RNA viruses because of their ability to mutate quite readily and frequentyl (1milX more than DNA viruses)   

RNA viruses undergo mutations very readily.  This occurs much more frequently in RNA than in DNA viruses. 

This is because RNA viruses lack the proof reading mechanism for RNA dependent RNA polymerase.

Mutations 

- misreadings 

Fracture of nucleic acids

- causes the nucleic acid to break off and reattach in the wrong location 

- No effect 

- Lethal (failure of replication) 

- Change in virulence (tropism, efficiency or replication) 

- Change in efficiency of replication at different temperatures (ie. avian vs mammalian influenzas) 

Increased numbers of basic amino acids at the clevage site increases the virulence of a virus. 

True or False?

TRUE

If you have not grasped this concept yet...

I would throw in the towel now...

he has only said it approximately a bajillion times in class

Serine will DECREASE virulence of a virus.

This is because it serves as a base for carbohydrate chains, which if close enough will interfere with access by the proteases to the cleavage site.  

Without the HA being cleaved, the virulence of a virus decreases.

Arginine

Lysine 

Glutamine 

Histadine

Intramolecular recombination 

Genetic reassortment 

- many numbers of basic amino acids in cleavage 

- has H5 and H7 subtypes 

- Replication in cell culture in the absence of Trypsin

- ability to kill 75-100% of innoculated chicks 

(these may not contain the H5 and H7 subtypes, but if it can kill that many chicks, its totally a highly pathogenic avian influenza) 

Ex.

Intramolecular recombination occurs where polymerase jumps from the HA gene to the Matrix (M) gene.  When it jumps back to the HA gene it has acquired a sequence of basic amino acids in the cleavage site.  This will increase the virulence by increasing the ease of cleavage of HA, which will initiate binding of the fusion protein allowing for virus replication.

There is cross reactivity between the major serotypes of FMDV, allowing vaccines to be effective and still nonspecific.

True or False?

FALSE 

FMDV is NOT cross reactive serotypes because of the antigenic variation and this INTERFERES with sucessful vaccination

Redistribution of 1 or more gene segments from 2 or more replicating parental viruses to progeny virions. 

This occurs within ALL segmented viral genomes 

Can result in the emergence of new subtypes, can change host range and virulence of the virus.

Orthomyxoviridae

Reoviridae

Bunyaviridae

Arenaviridae

- Alpha 2,3 and alpha 2,6 linkages of galactose

- Internal temperature difference in avian and mammalian species 

- PB2 component of polymerase complex

1) that PB2 determines host specificity 

2) that PB1 and PA determine virulence (PB2 did not) 

Intermolecular genetic reassortment between viruses can change host range and virulence (THIS IS THE BOTTOM LINE OF THESE EXPERIMENTS). 

The Dominant subspecies - 

this dominance can CHANGE over time however.

Host or Environmental pressure

this change is facilitated by mutations and intermolecular recombinations. 

Diversity of viral subspecies promotes fitness.

True or False?

TRUE 

remove diversity and the fitness of the population declines

ACUTE INFECTION

This is a self limited infection, where there is an acute phase where clinical illness corelates to the period of virus shedding. 

IE. Rotavirus

A) represents the time of virus shedding 

B) represents the episode of illness

C) time when virus can be demonstrated in tissues

Acute infection with chronic reoccurring shedding

These viruses can be demonstrated in the tissues for the majority of the animal's life, and shedding of the virus can reoccur in bouts. 

IE. FMVD

Chronic infection

These are often congenital infections, with partial immune response that still allows viral replication.  

Virus can ALWAYS be demonstrated in the tissue. 

IE. Lymphocytic choriomeningitis virus (Ab-Ag complexes form and cause glomerulonephritis) 

Latent Infection 

Here there is an initial acute infection that becomes latent, and reoccurs and periodic shedding. 

IE. Herpesvirus

African Swine Fever (Asfarviridae)

Have icosahedral capsid, DS DNA viruses 

The best response is a balanced response in regards to CMI and Humoral immunity.

True or False

True! 

Although some viruses can be eliminated with just humoral (like Canine Distemper - modified live vaccine used)

Immunocompetance of the host

Immunocompromised individuals will have severe disease because they are not able to mount proper immune response. 

In general, viruses cause more severe disease in natural hosts, and accidental host infections may go unnoticed.

True or False?

FALSE 

As a rule, most infections are more severe in incidental hosts and mild-inappearent in natural hosts.  

IE. Herpes Simplex Virus 1 causes mild infection in humans (cold sores) and causes fatal disease in monkeys. The opposite applies to the Simian B virus of macaques, where they may be latent but shedding and cause fatal disease in humans.

Bolivian Hemorrhagic fever (an arenavirus) causes severe disease in rodents, but can also cause clinically mild disease in humans. 

True or False?

False

It causes SEVERE disease in humans (22% mortality rate) and can be inapparent in mice.  

Genetic: 

Absence of receptors (ALV)

Immune response gene (MuLV)

Host physiology:

Body temp (avian vs mammalian influenza) 

Conducive intracellular environment

Nutrition

Age 

Stress

Exposure route 

Exposure dose 

This is variolation. 

If there is an abnormal exposure route there would be immunity development instead of disease.

For example: Small pox with is normally respiratory is used on scarified skin it can be used as immunization, and doesnt typically produce severe disease. 

An increased exposure dose increases the susceptibility to infection.

True or False?

Which is more to cause an inappearent disease?

A) High exposure dose 

B) Low exposure dose 

LD₅₀ 

Viremia (time to peak titer) 

Ability to spread in host tissues 

Time to host death 

Degree of disease in host 

fever, weight loss, tissue damage

The higher the LD₅₀, the higher the virulence of a virus.

True or False?

FALSE 

A LOW LD₅₀ would have a higher virulence, because this measure would indicate it takes LESS virus to kill 50% of a population

Virus tissue tropism 

Viral proteins:

assist in replication 

facilitate spreading

are toxic 

Interfere with host immune response

How can viral replicative proteins determine virulence?

What is an example of this?

Proteins that assist in replication can make a more efficently replicating virus, which will increase the viral load, which will produce more disease = increase virulence

IE. Influenza virus 

codes for efficient polymerase complex

How does the Rabies virus get to the salivary gland?

(What is the Pathogenesis)

Rabies enters through a bite wound or skin abrasion, this then replicates in the local muscle cells before getting into local nerves via the ACh receptor.  The virus travels through these nerves to the CNS where it first effects the Limbic system (this can be the furious rabies) and causes functional lesions.  Then leaves the brain through the peripheral nerves to various organs INCLUDING salivary gland. IN salivary gland it replicates in the epithelial cells and buds out the apical surface of the cell into the ducts - - NOW virus can be found in the saliva.

Which Equine RNA virus needs to test negative before crossing state borders in the US?

What diagnostic test is used?

Equine infectious anemia

Coggins test

Specific cell receptors 

Virus requires specific cell functions to replicate 

GI and Resp endoproteases 

requiring polymerases of rapidly dividing cells (ie. parvovirus)

The viral capability to multiple in specific tissue or cells

Regulated by:

**Virus attachment protein + virus receptor binding

Route of infection

Interaction of the Virus Attachment Protein (VAP - on surface of cell) and viral proteins.

also minorly associated with route of infection

Receptor mediated endocytosis (influenza and FMDV) 

Fusion from without (Paramyxoviridae) 

Direct Entry: Polioviruses 

GET ON THAT HA - - it serves both the function of attachment receptor and it initiates uncoating, but for the later to occur it must be cleaved. In case you haven't heard...

By altering the amino acids in the clevage site of the HA there would be less opprotunity for the virus to uncoat and replicate. 

1st line: physical and chemical defences

skin, hair, clearance mechanisms 

2nd line: Intrinsic (Local) defense

PMPs/PAMPs, type I IFNs, apoptosis

3rd line: Innate and adaptive immune defense

(Organ wide)

These are the physical and chemical defenses. 

- skin 

- muco-ciliary transport system (escalade) 

- Acid and protease barrier of stomach 

Ammonia

cold temperatures 

Pattern Recognition Receptors 

found on cells - allows the cell to detect pathogen associated molecular patterns (PAMPs)