Introduction to Animal Virology Viruses are obligate intracellular parasites which need a host cell in which to replicate. The mature

viral particle is called the virion. Virions contain: 1. Nucleic acid, either RNA or DNA, either single

stranded or double stranded, to transfer genetic information

2. Protein coat, or capsid, to stabilize nucleic acid and transfer it into the cell 3. Lipid- and protein- containing membrane or envelope (sometimes) Virions range in size of that of a 20 nm ribosome (picornavirus) to 400 nm (poxvirus), visible by light microscopy

Introduction to Animal Virology Viruses have been around long before man has

In the beginning of mankind, with man living in small tribes, viruses of man would likely have been well-tolerated persistent viruses, such as herpes simplex virus and cytomegalovirus

These are large enveloped viruses with double-stranded DNA

or else viruses crossing over from other species, with man being an incidental host…

Ebolavirus? Lassavirus? Influenza virus?

1 virion RNA 2 virion RNAs 8 virion RNAs

filovirus arenavirus orthomyxovirus

As man became more intimate with animals, their viruses may have evolved to cross-over to man

Cowpox??

Smallpox

Camelpox??

Mummy of Ramses V, who died in his early thirties probably from smallpox in 1158 B.C. Smallpox lesions are visible on his lower face and neck.

What is clear is that smallpox occurred in ancient Egypt

The very characteristic smallpox disease was also noted in the ancient writings in China (1122 BC) and India (900 BC)

A withered leg of an Egyptian prince around 1500 BC suggests that polio has been around for a while, too (although I guess he could have been just kicked by a horse)

Viruses and other pathogens have played major roles in human history Suggested reading: Zinsser- “Rats, Lice, and History;” Oldstone- “Viruses, Plagues, and History.”

The plague of Athens may have been smallpox, typhus, or some other pathogen-hard to tell. It decimated the city, destroying Athenian life Fall of Greece and the rise of Rome

Thucydides on the plague of Athens, 430 BC

“Yet is was with those who had recovered from the disease that the sick and the dying found most compassion. These knew what is was from experience, and had now no fear for themselves, for the same person was never attacked twice - never at least fatally.”

189 AD – 2000 people a day die in Rome of smallpox mass conversions to Christianity

Crusades – repeatedly suffered massive disease and death from smallpox and other infections

16th Century: a smallpox epidemic started in Africa and spread to Europe and to the New World Smallpox and measles dramatically depopulated the native American population, who had no natural resistance to these viruses

Yellow fever, indigenous to Africa, was also imported to tropical regions of the New World One of the reasons that black African slaves were imported into the New World was that they were relatively genetically resistant to yellow fever, whereas the indigenous American population was extremely sensitive. Yellow fever virus was transmitted by mosquito vectors, which would grow well in the tropics and in buckets of water under hospital bed legs

Just a few years before the Pilgrims arrived in Massachusetts there was a massive depopulation of native Americans by smallpox, making it easier for Europeans to settle here (of course you know that the Pilgrims settled here rather than in Virginia because they ran out of beer)

At one point smallpox had eliminated 25% of the population of Europe, as well. At times things were so bad that people did not name their children until they encountered and hopefully survived smallpox-why waste a name on someone unlikely to survive? Prevention of small pox was paramount, but how?

Chinese had noted that people suffered from severe or mild forms of smallpox, and that by inoculating a naïve person with the mild form would provide immunity with a low kill rate to the severe form.

In the 18th century, Europeans adopted this strategy and called it variolation, from the Latin varus, meaning pimple.

Eventually, the virus causing smallpox was name variola virus, and the mild and severe forms of the disease were called variola minor and variola major.

Armed with the knowledge of variolation, the British could now use germ warfare on their enemies.

In the Battle of Quebec, fought between Wolf and Montcalm, it is rumored that the British released smallpox onto the French army, and that may have influenced the outcome

http://www.history.org/foundation/journal/spring04/warfare.cfm

Lord Jefferey Amherst gave native Americans blankets from smallpox victims in order to start epidemics in their tribes

George Washington, of course, knew better, having fought with the British in the French and Indian war. He took his troops out into the wilderness and had them variolated before taking them into battle with the British.

So diseases have shaped American history, but one more example…..back to yellow fever……

The French had occupied Haiti, but there was an uprising of the black slave population, so Napoleon sent in 25,000 French troops to quell the rebellion. 22,000 died of disease, mostly yellow fever. Meanwhile, Napoleon was trying to conquer Europe, and Tom Jefferson convinced him to give up his territorial claims in the New World and sell the Louisiana territory to the United States!

All because of yellow fever virus?

http://www.foundersofscience.net/jenner.htm

1798 –Edward Jenner noted that milk maids, celebrated in poetry for their beauty, tended to have few pock marks and resisted smallpox. He also noted that cows had a mild form of smallpox-like disease called cowpox.

Jenner used drainage from a cowpox lesion to immunize against smallpox, and it worked, i.e., the first “vaccine,” from the Latin vacca = cow. This work may have represented the birth of the disciplines of both virology and immunology. Vaccinia virus, now used as the smallpox vaccine, is similar to but not the same as cowpox virus,

Jenner also first used the term virus, the Latin word for poison.

Louis Pasteur was a famous French biochemist known for disproving the theory of spontaneous generation with his swan neck flask.

He also was very much appreciated for developing pasteurization, which kept wine from spoiling.

1885- Pasteur took rabid dog brain, passed it in rabbits (serial passage)dehydrated it and used as a rabies vaccine—he also suggested that agents smaller than bacteria may cause disease.

Rabiesvirus-rhabdovirus group

Tobacco mosaic, an important disease of a cash crop-described by Mayer in 1887-he showed it could be transferred

1892-Ivanovski showed that bacteria-containing filters did not block transmission (doubted his own work)

1899-Beijerinck-repeated filtration study and concluded that it was a “virus” or a contagium vivum fluidum This represents a newly defined infectious entity – a plant virus

Tobacco Mosaic Virus

1898-Loeffler and Frosch transferred foot and mouth disease between calves through a filter. First defined animal virus: FMDV - a picornavirus.

1900-Walter Reed showed that yellow fever was transmitted by the mosquito, Aedes aegypti

Several investigators died in this study, which made possible the building of the Panama Canal and safe settlement of the Carribean

First demonstration of vector transmission

http://jem.rupress.org/content/201/3/320.full

1908-Ellermand and Bang transmitted leukemia between hens, indicating an infectious cause of cancer

1911- Peyton Rous (left) passed agent of chicken sarcomas through a filter. Hence, the first cancer-causing virus.

Weird cartoon of Rous sarcoma virus – a retrovirus

“In the course of these researches, at various times I noticed an anomaly, shown by some cultures of the coccocacillus which intrigued me greatly, although in fact the observation was ordinary enough., so banal indeed that many bacteriologists had certainly made it before on a variety of cultures. The anomaly consisted of clear spots, quite circular, two or three millimeters in diameter, speckling the cultures grown on agar.” – d’Herelle

1915, 1917: Twort and d’Herelle found “plaques”on lawns of bacteria. Plaques could be picked, passed through a filter, and give rise to more plaques “bacteriophage,” discovery of bacterial viruses.

1930: Theiler grew yellow fever virus in eggs (in ovo), and in doing serial passages in eggs he attenuated the virus and made one of the best vaccines, still used today and also used as a platform to insert genes of other viruses

Yellow fever virus is an enveloped, single-stranded + polarity RNA virus of the Flavivirus family

http://www.corbisimages.com/Enlargement/Enlargement.aspx?id=UKD1674INP&ext=1

Boris Karloff

Theiler looked a little like Boris Karloff

For a long time there was a debate over what was the genetic element-nucleic acid? protein?

1935- Stanley purified and crystallized TMV and showed it to be all proteinprotein is the genetic element? 1942- Stanley showed a small amount of RNA in TMV, casting some doubt

1946- Stanley showed that TMV RNA could infect plants, i.e., the nucleic acid was the genetic element

http://www.mardre.com/homepage/mic/tem/samples/bio/virus/tmv3.htm

Tobacco mosaic virus (TMV) is a small helical RNA virus with a protein coat which will spontaneously self assemble in vitro

1952: Hershey and Chase labeled T4 phage with P-32 (DNA) or S-35 (Protein) and found that DNA label was associated with infectivity the genetic element

1953: Watson and Crick published that DNA consists of two complementary strands arranged in a double helix

Crick later said that his was not a Noble Prize, it was the Nobel Prize.

1949: John Enders (right), Weller and Robbins cultivated poliovirus in human embryonic tissue and then in monkey kidney cells . First growth of virus in culture.

Early 50’s: Jonas Salk (left) grew up poliovirus in culture, inactivated it with formalin, and made an inactivated virus vaccine.

Poliovirus is a small single stranded positive polarity RNA virus without an envelope and is in the picornavirus group

1961: Jacob and Manod developed the genetic code showing conversion of DNA to RNA.

1964: Becker and Joklik showed that viral DNA is converted into RNA.

1970: Temin & Mitzutami and Baltimore showed that retroviral RNA is converted into DNA by way of a reverse transcriptase. This enabled the cloning of genes and the birth of modern day molecular biology.

Howard Temin (above); younger and older versions of David Baltimore (right)

Other notable achievements by virologists warranted Nobel prizes: 1976: Blumberg and Gadjuscek - Nobel prize for epidemiology of hepatitis and spongiform encephalopathies 1989: Bishop and Varmus - Nobel prize for discovery of oncogenes 1993: Sharp - Nobel prize for RNA splicing 1996: Doherty and Zinkernagel- Nobel prize for MHC restriction of T cells 1997: Prusiner- Nobel prize for prions 2008: Zur Hausen and Montagnier- Nobel prize for linking HPV to cancer and discovery of HIV

HIV – a lentivirus type of retrovirus: two + strand RNAs in the virion and reverse transcriptase

http://nobelgenius.blogspot.com/

http://newsletter.eortc.be/?p=1143

Harald zur Hausen His discovery led to the first vaccine to prevent cancer

Papillomavirus – a small ds DNA tumor virus

Introduction to Animal Virology Viruses are obligate intracellular parasites which need a host cell in which to replicate. The mature

viral particle is called the virion. Virions contain: 1. Nucleic acid, either RNA or DNA, either single

stranded or double stranded, to transfer genetic information

2. Protein coat, or capsid, to stabilize nucleic acid and transfer it into the cell 3. Lipid- and protein- containing membrane or envelope (sometimes) Virions range in size of that of a 20 nm ribosome (picornavirus) to 400 nm (poxvirus), visible by light microscopy

1956: Crick and Watson (also Casper and Klug) crystallized viruses, did x-ray diffraction, and concluded that virions consist of symmetrical arrays of identical subunits

What is a crystal? – a 3 dimensional periodic lattice built of equivalently related subunits

There is a finite number (230) of theoretical crystal designs, but virions have a lower number due to configuration of carbon atoms. Crystals are highly symmetrical structures.

There are three types of symmetry: Rotational – arranged around an axis, like spokes on a wheel, or like a clock or a star Translational – repetitive structures arranged in a role Optical – as in a mirror image, such as L and D isomers of sugars. Most amino acids are L-isomers, so this is not an issue with viruses

5-fold rotational symmetry

Translational symmetry

4-fold rotational symmetry

It is possible to make closed symmetrical structures purely by translational symmetry

By forming a 3 dimensional lattice based purely on translational symmetry, one can make a cylinder by wrapping the lattice around a central point

By moving one layer up a bit, you can create a helix

The TMV virion is arranged in a helix. The protein subunits have translational symmetry. Its ss RNA also has translational symmetry at its phosphate backbone

Protein subunit interactions with each other are hydrophobic, whereas protein-RNA interactions are hydrophilic salt bonds

Many RNA viruses have similar translational symmetry-based helical symmetry in their ribonucleoproteins.

Assembly of virions can involve a spontaneous crystallization. One can mix TMV RNA and protein together and it will spontaneously aggregate into virions.

Does this violate the second law of thermodynamics? Shouldn’t things in the absence of added energy go to their greatest state of disorder? Isn’t there the least free energy when entropy (disorder) is at its highest? What is the explanation?

What are other examples of spontaneous crystallization?

The other type of closed symmetrical structure is based on cubic symmetry, where the structure is a closed box.

Boxes can be 4, 6, 8, 12, or 20-sided, but due to the chemistry of the carbon atom, the 20-sided icosahedron is the most stable structure and commonly found with virions.

Icosahedrons are a combination of translational symmetry and 5.3.2-fold rotational symmetry

Icosahedrons have 30 edges, 12 pentameric vertices, and 12 sides.

Icosahedrons and other virions are composed of morphological subunits called capsomeres. These are repetitive subunits often arranged with translational symmetry in the icosahedrons.

Virions may have 3,4,5, or 6 capsomeres arranged on an edge. This virion has 5, including the vertex.

Why you should study virology:

Viruses are important pathogens.

Viruses, by providing high concentrations of select proteins and nucleic acids, have been instrumental in developing major concepts in biochemistry and molecular and cellular biology.

Viruses, by virtue of being replicating antigens, have helped to unravel the mystery of the immune system.

Viruses can be modified to become vectors for expressing genes in cells and in producing new age vaccines.

Linking viruses to pathogenesis and epidemiology:

Arenaviruses are enveloped viruses containing two ss RNA molecules and ribosomes in the virion. They include major human pathogens of hemorrhagic fevers, including Lassa, Junin, Machupo, as well as lymphocytic choriomeningitis virus, a major model for immunology.

All arenaviruses form persistent infections in mouse-like rodents, where man is an incidental host. LCMV and Lassa are closely related Old World arenaviruses, whereas Junin, Machupo, Guanarito, and Sabia are more distantly related New World viruses.

LCMV and Lassa virus use the same receptor: alpha-dystroglycan, which binds laminin in the extracellular matrix and is linked to a variety of intracellular proteins.

Lassa virus is indigenous to West Africa (Sierra Leone, Nigeria) and kills tens of thousands of people a year

Recent molecular epidemiology studies have found that in the Lassa-endemic area there is a high preponderance in polymorphisms in the genes encoding proteins associated with the alpha-dystroglycan receptor

This suggests a molecular identification of infection-driven in-progress genetic selection of a population

This type of thing might be going on all the time, but it is difficult to pinpoint and evaluate. Case in point: the high sensitivity of native American populations to measles virus.

Arenaviruses exist as persistent infections in rodents in nature and infect humans through shedding of rodent feces. Why doesn’t the immune response in rodents kick in and reject these viruses?

Frank MacFarland Burnet used the LCMV model to coin the theory of immunological tolerance, whereby exposure to antigen congenitally during immune system development will make one tolerant to that antigen.

This was only part of the story, but it won him the Nobel Prize in 1960.

LCMV is actually a strong inducer of immune responses in adult mice, where tolerance is less of an issue, and with this model T cells were shown to mediate either protective immunity or immune pathology, an important feature in viral pathogenesis.

Doherty and Zinkernagel used the LCMV model to demonstrate that T cells recognize virus-modified MHC antigens, and received the Nobel prize in 1996 for that.

The LCMV model has been used to unravel many of the mysteries of the immune system, including the importance of specific proteins in T and B cell responses, the demonstration of the activation and role of natural killer cells in viral infections, and the involvement of heterologous immunity in viral pathogenesis.

Peter Doherty http://www.wellsphere.com/general-medicine-article/9-x-increase-in-seizures-after-swine-flu-vaccine-australian-gov-t-investigation/1126536

Rolf Zinkernagel http://www.swissinfo.ch/eng/specials/switzerland_for_the_record/world_records/Switzerlands_Nobel_boom_bust.html?cid=686650

Further, inoculation of adult mice with very high doses of LCMV leads to an apoptotic deletion and clonal exhaustion of T cell responses and persistence rather than clearance of virus.

High dose LCMV

T cell exhaustion and viral persistence

The LCMV infection of the mouse is currently the major small animal model for persistent HCV and HIV infections, and immune system-based therapies altering LCMV persistence are being evaluated for treating these human infections.

This course will describe and evaluate immune system-pathogen interactions with other viruses.