Plus Strand =PS, Minus Strand =MS

FUN2: 11:00-12:00 Scribe: Hunter Neill Friday, November 14, 2008 Proof: Brannon Heape Dr. Morrow Microbiology Page 1 of 6 Plus strand =PS, Minus Strand =MS Positive Sense RNA Viruses I. Introduction: going to continue with Plus Strand (PS) RNA viruses II. DNA to RNA to Protein Pathway [S1] a. These viral RNA genomes are called PS RNA because they are the same sense as a mRNA i. Their first step after entering the cell is to grab hold of the host cell translation machinery and get there genome translated ii. Then start to make viral proteins that then replicate viral RNAs 1. Next week material: (different from Negative Strand RNA viruses because they are the wrong sense and must bring in this RNA dependent RNA polymerase to make messenger RNA) III. Plus-Strand RNA Viruses [S2] a. Plus-strand RNA viruses have genomes comprised of RNA. They have no DNA in their replication cycles i. If you take the nucleus out of the cell the viruses will replicate just as well as having one b. (In most cases, the replication cycle take place in the cytoplasm of the cells) c. No 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 d. The plus-strand RNA viruses do use the host cell translation machinery to generate viral proteins. e. Many PS RNA viruses produce numerous viral proteins from a “single” gene i. Poly protein strategy with this protease that likes to cleave itself out. IV. Types of Plus-Strand RNA viruses [S3] a. No polymerase in the virion b. The RNA by itself is infectious i. This is something that virologist use that doesn’t happen in the real world and in the lab only 1. If you just isolate the RNA from the virion and place it in the cell it get translated, makes new proteins, and new virus ii. We use that to distinguish between negative sense/minus strand (MS) viruses. If you just put a minus strand RNA virus into the cell it cannot do anything because it is the wrong sense and it doesn’t have that enzyme that the virus normally brings into the cell 1. Its dead – early on that’s how to distinguish between PS and MS viruses – just by putting the genome into the cell 2. Has nothing to do with infection and is actually called transfection c. Types of RNA d. Class 1 has one mRNA whereas Class 2 has multiple mRNA e. Class 1 has long polyproteins whereas Class 2 has evolved away to make 1 mRNA for each protein f. Will talk about picornaviruses, poli virus and rhino virus, flavivirus (west nile), and coronaviruses (including SARS) V. Picornavirus Diseases [S4] a. Enteroviruses - short for enteric viruses and stable in the GI tract i. Poliomyelitis ii. Hepatitis A virus – virus you get when you eat shellfish at the beach. Resolves itself. 1. Other hepatituses (B and C) are completely different with different types of infectons iii. Coxsackieviruses iv. Enteroviruses Type 68-72 v. Echoviruses – infect kids. General don’t cause a lot of disease except in immuno-suppressed patients b. Rhinoviruses (not stable in the GI tract) – like to replicate in the nose i. Major cause of the common cold c. Numerous subtypes – we will see how viruses have evolved by using the RNA dependent RNA polymerase to find ways to prevent inhibition by host cell antibodies i. Also they reason tere are no vaccines VI. Fecal-Oral Transmission [S5] a. Many viruses are transmitted by human fecal matter, including from your hand, sewage, solid waste that gets into the water supply b. Hepatitus A – often found in shell fish that are good at concentrating these i. One concentrated easily leads to infection after being consumed VII. Enterovirus Pathogenesis Target Tissues [S6] – a. Won’t have to know all of this (will help in explaining polio) b. Echo, Coxsackie, and polio viruses are general swallowed i. Replicated in oral pharynx and sometime in your tonsils FUN2: 11:00-12:00 Scribe: Hunter Neill Friday, November 14, 2008 Proof: Brannon Heape Dr. Morrow Microbiology Page 2 of 6 ii. Then they pass through gut and shed in stool c. They are icosahedral viruses i. Made to do this so they are very stable and hard to get rid of. Can get in water and get concentrated. They are small and hard to get rid of. d. In some cases you get viremia in the blood, which allows them to go to secondary target tissues where they can cause a lot of problems. i. Will talk about later: Polio virus replicating in the brain and in the meninges 1. Hepititus A virus replicates in the liver, 2. Echo and Coxsackie replicatein the muscle and skin. 3. Once they get into the tissues they can cause a lot of problems because they are in abnormal places VIII.Polio virus Pathogenesis [S7] a. Polio is not a serious disease and has been eradicated. b. Some of the elements about polio are true for a almost all virses c. Only a small fraction of patients develop paralytic disease – Franklin D. Roosevelt 1. During that time it was a serious disease that led to paralysis ii. Asymptomatic infection – 90% iii. Abortive/minor illness – 5% iv. Non-paralytic progression to the CNS – 1-2% (specifically motor neurons) v. Paralytic poliovirus - .1% to 2% 1. 3-4 days after minor illness 2. Virus infects motor neurons in anterior horn of spinal cord and the motor cortex 3. Result of the virus entering and destroying the motor neurons that eventually leads to paralysis IX. Progression of Polio to CNS Disease [S8] a. The infection is just like all the other enteroviruses. It enters in a fecal oral manner, gets processed passage through and released into the stool occurring several days after the infection, even a primary viremia. b. Generally controlled by antibody i. Neutralizing antibodies – inhibit the virus from interacting with the receptor and the infection is then controlled. Therefore most of the time it is just minor illness. X. Enterovirus Replication and Disease Progression [SS9] a. Again summarizes the progression b. Primary infection – fecal oral c. Viremia d. Infection of secondary target tissue e. Secondary replication phase SYMPTOMS including paralysis with polio virus f. Most of the time, the primary infection simply releases the virus into the environment with fecal shedding g. Most of the time you don’t even know that you’re infected. Occurs in between 90-95% of people. XI. Protection by Antibodies [S10] a. There are only 3 serotypes of polio Type 1, 2, and 3 i. You body, the host immune system, has three targets to make antibodies to. The virus doesn’t change. ii. With those 3 antibodies you can neutralize the polio virus (important point). b. Serum antibodies percent viremic spread to target tissues c. Mucosal/ Secretory antibodies, in saliva and secretion, can prevent primary infection XII. Polio Vaccination [S11] a. Before AIDS or Ebola the nastiest virus was polio and it infected children and adults with no knowledge of ideology b. Swimming holes are breading grounds for fecal oral transmission thereby increasing the spread of the virus i. One of the main targets is children who became paralyze and required to wear iron lungs for their entire life ii. March of Dimes – one of the organizations started during the broad based funding effort to find a vaccine c. Salk virus – Type 1, 2, and 3 that have inactivate by formalin i. Killer virus (formalin inactivated) – cross links proteins on the virus while maintaining the mutenogenicity ii. If you get the viruses you will make serum antibody against each type 1. The serum antibodies protected against disease not infection 2. Protect you from the virus being transmitted from your gut in the viremic stage to the CNS 3. This the method for many of the intramuscular vaccines 4. Very efficient 5. Draw back: like a tetanus shot, and must have booster because its not long lasting 6. Doesn’t effect adults as much because hygiene is better FUN2: 11:00-12:00 Scribe: Hunter Neill Friday, November 14, 2008 Proof: Brannon Heape Dr. Morrow Microbiology Page 3 of 6 d. QUESTION: If adults go swimming in lakes and oceans why don’t they get sick since there is certainly fecal contamination? i. We’ve all been vaccinated by the net type so we don’t get it. North America the virus has been eradicated e. Sabin Vaccine – Type 1-3 and grow in tissue culture (monkey kidney cells) i. Hey grew them for an extended period of time until they became attunuated ii. Live, attenuated viruses that still replicate (do not cause the polio myelitis) iii. OPV – Oral Polio Vaccine - attenuated strains can revert to the virulent forms 1. The cultured, live virus gets into your gut replicates and then shed, but because it can’ t go from the gut to the CNS it doesn’t cause Polio Myelitis and provides long lasting immunity 2. Plusses v. minuses – any live viruses can revert back and every 1 in 3 mil doses the has the active, wild-type polio virus (type 3) a. A small number of cases the Sabin vaccine results in Polio Mylenitis b. Now you get the Salk virus first for the serum antibodies so that the few cases that do have reverted wild type virus don’t cause the disease XIII.WHO Eradication of Poliovirus [S12] a. Generally polo has been eradicated from North and South America b. Generally found in places with poor hygiene or poor economics i. Globally the WHO is trying to eradicate Polio Myelitis ii. The Vaccine is tremendously effect but only if you take it and follow up c. [S14] It is possible – the oral is preferred because it is cheaper and easy to administer i. When you give a child the oral vaccine the child is actually being infected with the attenuated strain ii. However since the virus is replicating, when shed it is wild type neurovirulent polio virus 1. Care handlers that haven’t had the vaccine run the risk of being infected iii. They stool however, does have the wild type as it is replicating and being shed d. The WHO wants to stop vaccinating once the polio virus once it has been eradicated similar to small pox e. Bioterrorism – Small pox isn’t vaccinated for anymore because it only exist in bioterrorist laboratories i. Because we have stopped vaccinating on a world scale we are now susceptible to it on a large scale ii. Polio has such a small genome that it can be generated using gene synthesizers – if we eradicate the vaccine, then the virus become a weapon XIV. Rhinovirus Structure [S16] a. Rhinoviruses – agents of the common cold i. These are picornaviruses that are icosahedra virus ii. Uses a polyprotein with long open reading frame and viral protease b. Structure [S17] – with atomic detail i. Know the structure and can replicate them in crystals with 3d structure ii. No nucleid capsid XV. Rhinovirus Life Cycle [S18] a. Lifecycle of Rhino and polio virus b. Bind to a receptor c. Goes through uncoating d. PS makes the viral proteins e. Polyproteins are cleaved f. Take the plus stand and replicate it g. IMPORTANT POINT (usually ask about) – What is the step in viral replication that is common for all PS viruses (type 1 and 2) i. Make a complete copy of the genomic plus strand called a MS ii. The MS serves as a template for the synthesis for new PS 1. PS to MS which serves as a template for many more PS but must make a complete copy of the MS iii. Get encapsulated – many of which are – and just lyse the cell XVI. Binding to Receptor [S19] a. Receptor for Rhinovirus is ICAM-1 (intracellular adhesion molecule) b. There is a lot of ICAM-1 in the nose and where the virus likes to replicate c. The ICAM binds to a regionin the virus called a canyon (whole in the virus) d. On the outer surface of the virus (ridges) the virus exposes to antibodies i. A lot of antibodies are made ii. The ridges can change therefore the antibodies don’t protect for multiple strains iii. Canyon where the receptor bind doesn’t change, but it is so small that the antibodies cannot get into the site so you don’t make any canyon antibodies FUN2: 11:00-12:00 Scribe: Hunter Neill Friday, November 14, 2008 Proof: Brannon Heape Dr. Morrow Microbiology Page 4 of 6 1. One of the reasons why there is no vaccine – the outer surface can change e. [S20] atom picture of the binding of the Rhinovirus to the ICAM receptor XVII.Rhinovirus Polyprotein Processing [S22] a. Polyprotein cleavage i. After it gets translated it make viral proteins b. Made as one long polyprotein i. The a protease that’s embedded in the polyprotein bites itself out ii. Cleaves the cahin at specific amino acids pairs that it can recognize to make functional proteins iii. Extremely effective way to make the proteins it need to replicate from a compacted genome c. After making the proteins it uses an RNA dependent RNA polymerase and take the PS RNA and makes a complete MS RNA i. Then that serves as a template for new PS RNA ii. Evolved away to make more PS RNA than MS RNA XVIII. Rhinovirus Pathogenesis [S24] a. Entry of virus into upper respiratory tract i. Hands and fomites (inanimate objects) ii. Inhalation of droplets that contain virus b. Non-enveloped so very stable in the environment c. Unable to replicate in the GI tract (not stable to acid) unlike Polio virus that is d. Receptor is ICAM-1 (intercellular adhesion molecule 1) e. Rhinovirus has evolved to replicate preferentially replicates in the nasal cavity at 33oC (not 37 body temp) f. Over 100 serotypes (unlike polio that has 3) i. The outer surface can change so much and even though each stimulates antibodies against it, it wont protect for the other serotypes ii. Reason for repeat infections throughout lifetime – impacts vaccines iii. Antigenic sites change but receptor-binding site is protected so antibodies cannot enter the canyon g. Making a vaccine for rhino virus would be impractical due to the number of serotypes h. Virus replicating in your nose does not cause all the symptoms that you get. It’s a response to the Rhino i. Virus with pro-inflammatory cytokines- Il-1, Il-6 and Il-8, ii. Secondary inflammation causes soar throat, nasal obstruction and cough iii. All are a result of your body trying to get rid of the virus not the virus causing these problems135 XIX. Targets for Rhinovirus [S26] a. Not going to make any antibodies i. One is you can prevent from entering the cell 1. Interacts with ICAM so blocking it might help 2. Cannot “tickle” the receptor to much because other problems arise ii. Inhibit the protease or viral polymerase 1. XX. New Rhinovirus Therapies [S27] a. There are 62 million Rhinovirus cases each year that cause more than 50% of the inspiratory tract infections b. We don’t have a good program to prevent it because you can take to asprin and resolve the symptoms i. Often cause infection 24 hours to large events is because your immune is lowered and you become more susceptible c. Cannot get rid of with Lysol d. Looked at because it causes a lot of economical hardship e. Soluble ICAM – you can sniff this to prevent saturation of Rhinoviruses that may be present in the air f. Protease inhibitors i. Necessary RNA dependent RNA polymerase makes mistakes including the protease ii. If you designed a perfect molecule to bind to the protease then any mistake would cause the protease inhibitor ineffectively bind iii. Revertance occurs g. Pleconaril – bind in the canyon where the receptor interacts and the virus cannot get in and infect h. Zycam – simple drug that is made of Zn to inhibit the viral protease. Not very effective because amount of Zn in the nose is not enough i. However, if you think you feel better you feel better XXI. West Nile Virus [S28] a. Another PS virus in the same class b. Transmission FUN2: 11:00-12:00 Scribe: Hunter Neill Friday, November 14, 2008 Proof: Brannon Heape Dr. Morrow Microbiology Page 5 of 6 i. Mosquito vecor and a bird reservoir as host ii. The birds occasionally die but do not cause a problem c. Can incidentally infect horses and the elderly who have lowered immune systems and can get serious disease XXII.Flavivirus Gene Structure [S29] a. More complex but don’t have to know what all these things do b. Important to understand that it is a PS RNA virus that makes a single long polyprotein XXIII. West Nile Genetic Structure [S30] i. Has evolved that some are cleaved by host cell enzymes ii. Some cleaved by viral NS3 protein 1. Cleaves itself and then the rest 2. NS stand for non-structural 3. E=envelope XXIV. West Nile Virus Virus Structure[S31] a. Illustration of the virus b. Nucleocapsule c. Has en envelope d. Imbedded in the envelope are proteins the virus uses to infect cells XXV. West Nile Replication Cycle [S32] a. Similar to polio and all PS viruses b. After the virus infects it is immediately translated c. It makes the viral proteins the replicate the RNA genome first as a plus that goes to a minus d. Minus the is template for making more Plus e. The virus become encapsulated and buds into these vacuoles and released from the cell f. In contrast to Polio that replicates in the cytoplasm and lyses the cell – this is a passive one that just buds off the cell and released into the environment XXVI. Coronavirus [S33] a. Also have members that are responsible for the common cold (not SARS) b. SARS is much more pathogenic than the rest of the coronaviruses c. Envelope virus with viral proteins sticking out of the envelope i. Doesn’t have an icosahedran but a nucleocapsid 1. RNA with viral proteins wrapped around it ii. [S34] Longest RNA virus that we know 1. Long single-stranded RNA molecule that is 30,000 Kb (base pairs) a. Most have been 1/3 that 2. First step is translation 3. Makes a complete minus strand copy 4. The MS copy serves to make a complete genomic PS copy or a multitude of smaller PSs iii. Different from Polio and West Nile Virses because make subgenomic mRNAs that code various viral proteins (polymerase and protease) that get translated that help form the virus particles 1. Buds into an intracellular vesicle and released 2. Different strategy for replication in terms of whether or not it makes a polyprotein or subgenomic mRNA – this makes subgenomic mRNAs d. The virus can be different sizes because the outermost membrane doesn’t form a defined structure i. Can be oblong ii. Because the viral RNA nucleocapsule is not constrained by an icosahedran XXVII. Coronavirus Receptor ACE-2 [S37] – angiotensin converting enzyme 2 a. Expressed in the heart, lung, kidney, GI tract (mainly respiratory viruses) b. Several strategies to inhibit the viral i. Soluble ACE-2 similar to ICAM-1 ii. Antibody to the ACE receptor to prevent infection c. All of these are theoretical and don’t particularly work XXVIII. SARS virus [S38] a. Buds into vessicles which go to the surface of the cell i. Picks up glycoproteins and then is released XXIX. Coronavirus Spike Proteins [S39] a. Virulence factors i. The Spike glycoprotein (S) (located in the outer surface) 1. Determines the receptor binding FUN2: 11:00-12:00 Scribe: Hunter Neill Friday, November 14, 2008 Proof: Brannon Heape Dr. Morrow Microbiology Page 6 of 6 2. Fusion and entry into the cell 3. Viruses neutralization by antibodies is target to this because ii. Because the virus is so big it doesn’t make as much RNA and is a little less prone to changes XXX. Targets for SARS Therapy [S40] a. Uses antibodies – Major target of the RNA dependent RNA Polymerase to prevent viral replication