CLASS: 11:00 12:00 Scribe: Adam Baird

CLASS: 11:00 – 12:00 Scribe: Adam Baird DATE: October 29, 2010 Proof: PROFESSOR: Positive Sense RNA Viruses Page 1 of 7

I. THE DNA  RNA  PROTEIN PATHWAY [S1] a. We will continue to talk about RNA viruses. We will restrict this lecture to plus-strand RNA viruses. b. Plus-strand RNA viruses mean that the RNA in the genome is the same polarity as the mRNA. c. This slide shows the “central dogma” of molecular biology (DNA  RNA  Protein). II. PLUS-STRAND RNA VIRUSES [S2] a. These viruses do not have DNA intermediates, so we’ll only discuss RNA  Protein. They replicate in the cytoplasm of infected cells and they are of the plus-sense (meaning that they can be directed translated once they enter the cell, which impacts the replication cycle). b. They have genomes composed of RNA. c. They have no DNA in their replication cycles. (However, retroviruses are technically plus-strand RNA viruses. In the genome, they have a plus-strand RNA and they go through a DNA intermediate. There won’t be any questions about this on the exam, but a retrovirologist may ask you. What is HIV? Is it RNA or DNA? It’s actually RNA that gets converted to DNA during an infection.) d. In most cases, replication takes place in the cytoplasm of cells. e. There’s not involvement of the transcription machinery in the nucleus. So, the plus-strand RNA viruses produce their own enzymes for RNA transcription and replication, which recognize RNA as the template. They all use the enzyme called RNA-dependent RNA polymerase, which will be discussed later. f. Plus-strand RNA viruses do use the host cell’s translation machinery to generate viral proteins. This process will be discussed later. g. Many plus-strand RNA viruses produce numerous viral proteins from a single “gene”. This is the polyprotein. III. TYPES OF PLUS-STRAND RNA VIRUSES… [S3] a. This is an important slide, because it summarizes the two classes of plus-strand RNA viruses that will be discussed in the lecture. b. Plus-strand RNA viruses: 1. Do not have polymerase in virions (it’s just the nucleic acid). Note for later: this will contrast negative- strand RNA viruses, which do have polymerases in virions. 2. Has infectious RNA. If you grow the virus, isolate the nucleic acid, and then put back into cells, a virus can result. You can do that with plus-strand RNA viruses. You cannot do that with negative- strand RNA viruses. 3. Has just one mRNA. (In contrast, coronaviruses, which will be discussed later, have multiple mRNAs.) 4. Has long polyproteins. There is only one protein for each mRNA. 5. Picornaviruses and flaviviruses will be the examples that we use in today’s lecture. IV. PICORNAVIRUS DISEASES [S4] a. The name literally means “small RNA virus”. (“pico” = small) b. We’ll talk a lot about Enteroviruses. They are stable in GI tract. They are transmitted in a fecal-oral manner. We’ll especially talk about Poliovirus (Poliomyelitis) because it has historically been studied quite a bit. Viruses here that actually infect humans include Hepatitis A Virus (results in hepatitis after eating oysters from the gulf that “didn’t look so good but you ate them anyway”, for example) and several types of Echovirus. c. We’ll also talk a lot about Rhinoviruses, which is the major virus of the common cold. V. FECAL-ORAL TRANSMISSION [S5] a. Enteroviruses are transmitted in a fecal-oral manner. Human fecal matter that either gets on your hand and then is transmitted back into your mouth or through sewage-contaminated drinking water. Shellfish are bivalves that are known to concentrate microorganisms, so they are excellent at taking things that are in water, concentrating it, making shellfish potentially harmful as well. VI. ENTEROVIRUS PATHOGENESIS: TARGET TISSUES [S6] a. This is a nice summary slide. You need to remember a couple of feature about it. 1. With the exception of Rhinovirus, all of these Enteroviruses are ingested by humans, are replicated, and then are excreted by humans. 2. Sometimes, viruses can actually replicate, leading to viremia (the presence of viruses in the blood). The viruses can then go to secondary sites where they can cause disease. 3. Echoviruses and Coxsackieviruses replicate in the skin (resulting in hand-foot-and-mouth disease). This is especially prevalent in daycare centers. Echoviruses and Coxsackieviruses are especially problematic in muscles, especially in infants. 4. We’ll a lot about Polioviruses, which tend to go through the blood and to the CNS, meninges, etc. 5. Once Hepatitis A Virus is ingested, it moves through the blood to the liver, where it replicates, ultimately causing Hepatitis. CLASS: 11:00 – 12:00 Scribe: Adam Baird DATE: October 29, 2010 Proof: PROFESSOR: Positive Sense RNA Viruses Page 2 of 7 6. The overall point: The primary infection goes straight down, through the body, and release in human feces. But when it moves to viremia and attack the secondary target organs, diseases arise. 190 7. Most of the known information about Enteroviruses comes from Poliovirus. Poliovirus has been studied since the 1940’s. It was one of the first viruses that was actually grown in the lab. It used to be a tremendous health problem too though. VII. POLIOVIRUS PATHOGENESIS [S7] a. When patients are exposed to Poliovirus, only a fraction of patients develop paralytic disease. b. You may have learned about patients infected with Poliovirus. These are patients who were in “iron lungs”, who had paralysis, and could not breath. c. Most of the time (90%), it is an asymptomatic infection. It may feel like a typical “flu bug”. It usually resolves in about 48 – 72 hours. d. Just because you are exposed to Polio doesn’t mean that you will get infected. e. 5% of the time it may result in abortive/minor illness. f. 1 – 2% of the time, the virus enters the CNS but it doesn’t cause paralysis. g. 0.1 – 2% of the time, the virus enters the CNS and causes paralysis. h. If the virus actually gets into the CNS, it infects motor neurons in the anterior horn of the spinal cord and motor cortex, impacting movement and breathing. VIII. POLIOVIRUS INFECTION: PROGRESSION TO CNS DISEASE [S8] a. This just shows elements of a Polio infection. b. Transmission: 1. Virus is ingested 2. Virus moves through the bloodstream (viremia) 3. Virus gets into the CNS, grow, and kills motor neuron c. The important part of this slide is the picture on the right: shows virus growth, then viremia, then growth and replication of the virus, and finally the virus becomes present in the stool. IX. PROTECTION BY ANTIBODIES [S9] a. The way that the body fights off Polio is through the production of antibodies. Antibodies take a little bit of time to produce though. The time difference between the viremia and the CNS infection that the antibody response will dictate (and impacts whether the Polio actually affects the CNS). This is also the basis of all Poliovirus vaccines. If the antibody can be moved so that they are readily present, then they can resist the Poliovirus from moving into the bloodstream or into the CNS. See next slide. b. Protection against Polioviruses: 1. Secretory antibodies can prevent primary infection. Immunologically, you have secretory antibodies (found in saliva and mucosal tissues), mainly IgA, IgG, or IgM; you also have serum antibodies (found in blood), mainly IgG. Secretory antibodies (in the back of your throat, for example) will neutralize the virus before it can actually infect. You would, then, just excrete a neutralized virus. However, serum antibodies cannot do this, thus allowing the virus to get into the bloodstream and replicate. You would, then, excrete the active virus. But, if the virus reaches the viremic stage (through the blood and onto the CNS), serum antibodies will then neutralize the virus, keeping you from infection of the CNS. X. POLIOVIRUS VACCINATION [S10] a. In the 1940’s and 1950’s, Polio/Poliomyelitis was a major disease. Franklin D. Roosevelt had Poliomyelitis. After WWII, there was a broad-based initiative to eliminate Polio. It was a terrible disease and something had to be done. 1. The major group of people infected was young children. (For example, kids swimming in swimming holes were uncirculated, creating a good environment for fecal-oral transmission. Swimming holes were often contaminated. Kids would come out with Poliomyelitis. At the time, transmission was unknown though. So a vaccine was the starting point for eliminating polio. In fact, the March of Dimes, which is currently focused on birth defects, specifically started to derive a Polio vaccine. If everyone sent in a dime, then millions of dollars could be dedicated to researching a cure for Polio. It actually worked quite well. It was one of the first instances where public funding enabled vaccine research.) b. There were two main characters in the development of the vaccine. 1. Jonas Salk a. Developed Salk Vaccine i. Killed Poliovirus (Type I, Type II, Type III) by using formalin, which inactivated the virus, but maintains the antigenic structure. It is therefore called a “dead virus”. It was an intramuscular vaccination, which results in the production of serum antibodies (giving protection against Poliomyelitis, but not Poliovirus). CLASS: 11:00 – 12:00 Scribe: Adam Baird DATE: October 29, 2010 Proof: PROFESSOR: Positive Sense RNA Viruses Page 3 of 7 ii. First vaccine to be used that virtually eliminated Poliomyelitis. iii. It was “magical”. iv. Essentially, this vaccine created antibodies that were ready to defend you if the virus was acquired. 2. Albert Sabin a. Developed Sabin Vaccine i. Took Poliovirus (Type I, Type II, Type III) and grew them in tissue culture (cell that were derived from tissue). He used primate tissues for this study. Then he used these viruses as an oral vaccine (via sugar cube, squirt, etc.). So these are live viruses, but it’s attenuated for the disease Poliomyelitis. They do not cause the disease Poliomyelitis in humans. ii. Once taken, the virus replicates in your body, but it doesn’t cause disease. It still give you body time to make an immune response though (serum and secretory antibodies), thus protecting you from Poliovirus (usually) and Poliomyelitis (almost always). iii. There are some problems with this vaccine strategy though. The attenuated strains can revert to virulent forms (the Type III strain). About 1/3,000,000 people who have the Sabin vaccine would get infected with Poliovirus and possibly even vaccine-associate paralytic disease. iv. Another problem: Those who are vaccinated will shed Poliovirus into the environment. Initially, this was thought to be a good thing (thinking that it would lead to “herd immunity” and that everyone would become protected). A scientist in England tested this though (on his children) and found that not only were attenuated strains shed, but recombined strains that formed virulent poliovirus were being shed as well. What was actually being shed (and what people thought was leading to “herd immunity”) was actually potentially harmful. This is important because the Sabin Vaccine is still given today (mainly in children). If you are a parent with kids, and you’re changing diapers after your child has had the Sabin vaccine, you should be extra cautious (or you could potentially get infected). c. Summary of these vaccines: The Salk Vaccine is a killed vaccine given as an intramuscular shot and does not protect against infection of Poliovirus. The Sabin Vaccine is a live, attenuated virus vaccine that can potentially infect others. d. Both the Sabin and the Salk Vaccines have now been in use for over 50 years. They have a great safety record (with the exception of the 1/3,000,000 chance of reversion). e. Know the difference between the Salk Vaccine and the Sabin Vaccine. Why? We are changing the way we vaccinate people today and the strategy that Salk and Sabin used is the same strategy we use for many other viruses. XI. WHO ERADICATION OF POLIOVIRUS [S11] a. The WHO initiated a program to “eradicate” Poliovirus. However, it’s actually first eradicating Poliomyelitis and then eventually, Poliovirus will be eradicated. This is similar to what they have done with Smallpox, which has now been effectively eradicated. b. WHO is now trying to eradicate Polio from the “hot spots” area (mainly underdeveloped countries). In North America, Polio has been eradicated, except for the few times that it flares up due to the vaccines. XII. IS ERADICATION POSSIBLE [S12] a. Is total eradication of Polio possible? b. Oral Poliovirus Vaccine 1. Preferred in third world vaccination 2. Cheaper 3. No sterile needles required. There’s a reluctance to use needles in third world countries anyway. 4. Campaign of usage has drastically reduced wild poliovirus transmission. 5. Major issue is reversion of OPV strain to virulent forms. c. Laboratory Stocks 1. Poliovirus present in many laboratories around the world (for 50+ years) 2. It’s a much bigger concern in labs that store feces. Poliovirus contaminants in stock of other viruses (like Coxsackievirus, Rhinovirus) d. Bioterrorism 1. cDNA copies of Poliovirus genome exist in many labs; transfection into tissue culture cells result in virus production (it’s relatively easy to culture). 2. The Poliovirus genome has been generated on a gene synthesizer. 3. Plus-strand RNA viral genomes alone are infectious! CLASS: 11:00 – 12:00 Scribe: Adam Baird DATE: October 29, 2010 Proof: PROFESSOR: Positive Sense RNA Viruses Page 4 of 7 e. Important 1. Understand pathogenesis elements 2. Understand initial infection 3. Understand viremia 4. Understand how it moves to secondary organs (like the CNS) 5. Understand the vaccines, how they work, how they are administered, what’s good about them, what’s bad about them, and how they are used today XIII.PICORNAVIRUS DISEASES [S13] a. In terms of a virus that you will see on a daily basis (and probably about 10% of the class has it right now) is Rhinovirus (“common cold”). XIV. HUMAN RHINOVIRUS STRUCTURE [S14] a. Rhinovirus has been extensively studied. We know so much about the Rhinovirus that we’ve crystallized the virus and we know the 3D structure of the capsid. b. We’ve cloned the virus. c. It’s a small RNA bases. It has about 7,200 bases. d. It has an open reading frame encoding polyprotein. XV. HUMAN RHINOVIRUS STRUCTURE [S15] a. This is a picture of Rhinovirus. It has the icosahedral shape (kind of looks like a soccer ball). b. The inside of the virus has the viral nucleic acid. (It is a plus-strand RNA virus, so it only has the nucleic acid inside of the virus.) c. Very small. Tightly packed. Stable in the environment, but not in the gut. XVI. RHINOVIRUS LIFE CYCLE [S16] a. Rhinovirus Life Cycle 1. Translation of polyprotein (which is cleaved by a protease) 2. The proteins replicate the plus-stranded RNA genome to a complementary minus-strand RNA, which then serves as a template for more plus-strand RNA 3. It’s assembled 4. Released from the virus XVII. RHINOVIRUS BINDING TO RECEPTOR AND ANTIBODIES [S17] a. Left Picture: The receptor for Rhinovirus is Intracellular Adhesion Molecule 1 (ICAM1). It’s present in cells that are found in your nose (which is where the Rhinovirus likes to infect). Rhinovirus interacts with ICAM1 in a “canyon” (basically a pore within the capsid shell). It’s from this interaction that it then is engulfed in the cell. b. Right Picture: Shows Rhinovirus that is bound to antibodies. Remember that when you are infected with Rhinovirus, you basically have a war going on inside of your nose. The antibodies bound to the Rhinovirus are going to try to neutralize the virus before it leads to an infection. c. These are the antibodies are target to the yellow regions. The pore for the ICAM1 is different. XVIII. ICAM-1 RECEPTOR BINDS TO CANYON ON RHINOVIRUS SURFACE [S18] a. This picture shows the ICAM1 receptor binding to the canyon on the Rhinovirus. b. The antibodies are binding to the ridges on the canyon. Why? This virus has evolved to protect these pores,, allowing it to interact with the ICAM1 receptor so that the antibodies can’t get inside of the canyons. Because the antibodies can’t get inside the canyons, the virus, then, can still infect, even if it’s covered with antibodies on the outside. c. The virus allows itself to have antibodies covering it, but it protects its canyon, which still allows it to infect the host cells. This is important if you’re trying to design a vaccine against Rhinovirus, because this canyon is essentially too small for antibodies (meaning that you can’t block it). XIX. RHINOVIRUS LIFE CYCLE [S19] a. Once the virus gets into the cell, it goes through replication. 1. It comes in as a single mRNA and then is translated by the host cell machinery to make a viral polyprotein. 2. This polyprotein is made as a single polyprotein. It is processed by a viral protease (called 3CPRO) into the smaller proteins (that form the capsid and the smaller viral replication proteins). a. So if it can’t get translated, or if it can’t get processed, it’s not going to produce new Rhinoviruses. Remember that these polyproteins and the viral replication proteins can result in one long translation product. 3. RNA replication – The first step is to take the plus-strand and make a complementary minus-strand. Know this. It will be on the exam. The minus-strand serves as a template for the synthesis of new plus-strands. With more plus-strands, it can either be translated or interacted with the capsid proteins to form new virus particles. (Plus-Strand  Minus-Strand  More Plus Strands) 4. Plus-strands are encapsulated. CLASS: 11:00 – 12:00 Scribe: Adam Baird DATE: October 29, 2010 Proof: PROFESSOR: Positive Sense RNA Viruses Page 5 of 7 XX. RHINOVIRUS POLYPROTEIN PROCESSING [S20] XXI. RHINOVIRUS LIFE CYCLE [S21] XXII. RHINOVIRUS PATHOGENESIS [S22] a. The pathogenesis of Rhinovirus is actually very interesting. 1. Virus enters the URT (usually by hands, fomites, or inhalation). In contrast to Enteroviruses, these viruses do not replicate in your GI Tract. They are not stable to acid. They will disintegrate. 2. The receptor is ICAM1. 3. The virus has evolved to preferentially replicate at 33 degrees Celsius. Typical body temperature is 37 degrees Celsius. Your nose, however, is a bit colder than the rest of your body and is actually pretty close to 33 degree Celsius. 4. One of the problems with Rhinovirus – and the reason we don’t have a vaccine for Rhinovirus – is that it has over 100 different serotypes (contrasted to Poliovirus, which just has Type I, Type II, Type III). That means that you can get repeatedly infected with different serotypes of Rhinovirus. Why? Rhinovirus can’t be neutralized. The antigenic sites change, but the receptor sites remain protected. (See picture.) 5. This is why many people are constantly getting infected with the “common cold”. This is also why a vaccine can’t be made against it. 6. When we discuss HIV, we’ll encounter a similar problem. HIV has multiple strains with multiple variants, so it’s also difficult to make a vaccine that can battle it. 7. Viruses that have limited serotypes, then, can be handled with a vaccine. Viruses that have multiple serotypes, however, are harder to be handled – and sometimes a vaccine can’t even be developed for viruses like those. XXIII. RHINOVIRUS PATHOGENESIS [S23] a. Pathogenesis of Rhinovirus follows what we discussed yesterday. It’s really not the virus that’s causing the symptoms. It’s the reaction of the immune system to the virus that is causing the symptoms. b. You can see that here in the virus infection of the nasal epithelium, which results in the synthesis of proinflammatory cytokines (IL-1, IL-6, IL-8). This results in sore throat, sneezing, nasal obstruction, cough, etc. Essentially, your immune system is physically trying to get rid of the virus. This is actually a good form of defense. XXIV. TARGETS FOR RHINOVIRUS THERAPY [S24] a. This is multi-billion dollar industry. There are many people who are researching targets for Rhinovirus therapy in hopes to reduce the common infection of Rhinovirus. b. Researchers have actually found several targets. 1. Through preventing interaction with the receptor 2. Protein cleavage 3. Inhibiting RNA replication 4. Inhibiting virion development XXV. NEW RHINOVIRUS THERAPIES [S25] a. 62 million cases of Rhinovirus in the US (that’s probably an underestimate) b. Cause more than 50% of respiratory tract infections c. Traditional vaccine approaches as with poliovirus not possible (too many serotypes), so antiviral therapies must exploit unique properties of the virus d. Soluble ICAM1 blocks virus from binding to its receptor, which would confuse the virus and keep you from getting infected i. It’s difficult to get enough ICAM1 in your body to actually result in a good defense against Rhinovirus e. AG7088: 3C protease inhibitor (if you can inhibit it, it becomes a very effective Rhinovirus agent). It’s being formulated for nasal delivery. i. Zinc was thought to inhibit protease as well. Over the last year or so, this has turned out to be not a very good idea, because in taking zinc, it also impact your sense of smell and can possible cause a little more disease. f. Pleconaril binds a pocket in the capsid, interfering with attachment and uncoating. It has promising early clinical studies, but it wasn’t approved due to possible drug-interactions; reformulations are being developed. XXVI. WEST NILE VIRUS [S26] a. We’ll take a look at some new viruses now. b. Most of you have heard of the West Nile Virus. c. A mosquito transmits it and most of the time, its reservoir is birds. Birds can actually die because of it. d. It can be incidentally transmitted to humans or horses and can then become quite serious. XXVII. TYPES OF PLUS-STRAND RNA VIRUSES…[S27] a. Considered a Class I plus-stranded RNA virus CLASS: 11:00 – 12:00 Scribe: Adam Baird DATE: October 29, 2010 Proof: PROFESSOR: Positive Sense RNA Viruses Page 6 of 7 XXVIII. FLAVIVIRUS GENE STRUCTURE [S28] a. Has some of the features that Enterovirus and Picornavirus have, but there are some slight differences. 1. It makes a single, long polyproteind 2. It has a capsid 3. It has an envelope protein (contrasted to Picornaviruses) 4. Has NS1, NS3, and NS5 proteins, which are just structural proteins 5. In contrast to Picornaviruses, which use only a viral encoated protease, West Nile Virus uses a protease found in the host. It also has a viral protease (called NS3). So, it uses both a viral and a host cell protease. XXIX. WEST NILE VIRUS GENETIC STRUCTURE AND PROTEIN EXPRESSION [S29] XXX. WEST NILE VIRUS STRUCTURE [S30] a. This is a picture of West Nile Virus. It is too big to crystallize, but 3D reconstructions have been done. b. It’s an icosohedral virus surrounded by a virion envelope (derived from the plasma cell membrane). Embedded in this envelope are viral proteins called E-Proteins. c. Similar to envelope viruses, this virus is easily inactivated. It doesn’t exist for long periods of time in the environment. It needs to be in fluids. If the envelope is broken, the virus is no longer effective. XXXI. WEST NILE VIRUS REPLICATION CYCLE [S31] a. This is replication cycle, which mimics that occur for the Picornavirus. After the virus interacts with the receptor and is release, translation occurs (because it’s a plus-stranded RNA virus), it makes viral proteins, which replicate the plus-strand genome to make complementary minus-strand, which serves as a template for the new plus-strand RNA. This plus-strand RNA then interacts with viral proteins. This virus actually forms on intracellular membranes and buds into vesicles and finally, is fused with the membrane and release into the environment (not via cell lysis though). Very important. XXXII. CORONAVIRUSES [S32] a. You have heard of SARS b. Acute respiratory virus c. Potentially a new and harmful disease d. Part of the Coronavirus family e. XXXIII. TYPES OF PLUS-STRAND RNA…[S33] a. Type II class b. Very large, plus-stranded RNA virus c. Has some of the same features though d. No polymerase in virions e. RNA itself is infectious f. In contrast to Picornaviruses and West Nile Virus that have just a single RNA, Coronaviruses have multiple RNA, one for each mRNA (so it doesn’t have a lot of polyproteins). The mRNA produce the proteins that the Coronavirus uses. XXXIV. CORONAVIRUSES [S34] a. Replication Cycle 1. After the virus infects, then the next step is replication. 2. Then translation of viral proteins (which are very important) 3. Then a complementary minus-strand is made 4. From the complementary minus-strand, subgenomic mRNAs are made that encode the various viral proteins 5. This virus is 30,000 bp (3.5 times bigger than West Nile Virus). It’s so big that it can’t make a huge polyprotein, so it’s evolved to make small mRNAs, which encode the viral proteins. 6. The complementary minus-strand also serves as a template for new genomic RNA, which then interacts with the viral capsid proteins. The virions are then formed in the Golgi and intracellular vesicles, which take it to the surface of the cell. 7. Finally, it is release from the cell. b. What’s important to notice: no polyprotiens, just subgenomic mRNAs. c. (Plus-Strand  Minus-Strand  More Plus Strands) XXXV. CORONAVIRUSES [S35] a. Picture showing Coronavirus. b. Has a lipid bilayer c. Glycoprotiens imbedded in the lipid bilayer d. It’s the glycoprotiens that interact with the host cell. XXXVI. CORONAVIRUSES [S36] CLASS: 11:00 – 12:00 Scribe: Adam Baird DATE: October 29, 2010 Proof: PROFESSOR: Positive Sense RNA Viruses Page 7 of 7 a. We now know the receptor for Coronaviruses XXXVII.CORONAVIRUS RECEPTOR = ACE-2 [S37] a. Angiotensin Converting Enzyme-2 b. Expressed in heart, kidney, lung, and GI Tract c. Main point of infection is in the lung XXXVIII. SARS VIRUS BUDDING [S38] a. Assembly process of SARS b. Actually occurs in intracellular vesicles c. The vesicles then move to the PM and fuse d. In fusing, it allows the virus to be released into the environment XXXIX. TARGETS FOR SAS THERAPY [S39] a. Vaccines are always a possibility, but we don’t know how many strains there are b. Similar to what we discussed with Rhinovirus, there’s a potential to use antibodies and there’s a potential to somehow disrupt the cell with its receptor c. Some developments can be made against the RNA-dependent RNA polymerase (which replicates the virus) d. Eventually, other targeted proteins may attempt to disrupt the budding process e. Important summary points: 1. They have subgenomic RNA 2. (Plus-Strand  Minus-Strand  More Plus Strands) [End 54:54 mins]