Fundamentals 11:00-12:00 Scribe: Jake Nolen
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Fundamentals 11:00-12:00 Scribe: Jake Nolen Thursday, November 5, 2009 Proof: Susan Whitt Dr. Lefkowitz Single Strand Negative Sense RNA viruses Page 1 of 11 I. Single Strand Negative Sense RNA viruses [S1]: a. We’re going to continue talking about viruses and in this case we’re going look at RNA viruses that are single stranded but these are what we call negative sense viruses. I’ll go into their properties in a minute.
II. Contact Info [S2] a. Contact him with questions: email, phone whatever.
III. Objectives [S3] a. Goals are to reiterate some of the fundamental common and especially distinguishing properties of RNA viruses so you know the differences between some of the viruses that you’ve already had and the viruses I’m going to talk about today. b. I’m going to talk about some of the basic replication strategies of the RNA viruses that we’re dealing with, and then I’m going to talk about some of the diseases that these viruses cause. c. I’m going talk about some of their biological and pathogenic properties
IV. Reading [S4] a. In the text. The chapters are here.
V. Slide References [S5] a. Here’s a listing of where some of the slides and such came from.
VI. Virus Classification [S6] a. I first want to talk about what these viruses are and why they are different from some of the other viruses that you’ve had.
VII. Virosphere 2005 [S7] a. This is the whole world of virology categorized according to the particular genome that the virus contains and according to the host that these viruses infect.
VIII. ssRNA [S8] a. We’re interested in the minus sense RNA viruses today. b. There are species that infect plants and invertebrates. c. We’re interested in the species that affect vertebrates and especially humans.
IX. Features to classify RNA viruses [S9] a. Size of the virus particle b. Genome structure. That’s how we divide the viruses up between this lecture and the last lecture. c. Properties of the virion itself in terms of whether it has an envelope derived from the host cell and the symmetry of the nucleocapsid of the virus.
X. Virus Structures [S10] a. Viruses can have an envelope that it picks up from the host cell as it buds from the host cell. It also derives some viral proteins in the lipid envelope that had been previously inserted in the envelope that it gets from the host cell. b. Also the picornaviruses can have a naked icosahedral capsid structure that’s not part of the envelope. c. All of the viruses we’re talking about today are enveloped. None of the viruses we’re talking about today have an icosahedral shell. They have a helical symmetry and that helix may be a little bit ordered or it can be unordered. It doesn’t have the crystalline-like structure that you get from the icosahedral viruses.
XI. RNA Virus Genome Structure [S11] a. Genome structure is important in distinguishing viruses from other viruses and also features of the replication cycle are dependent on that genome structure. b. The viruses we’re talking about today are all single stranded. There are also double stranded RNA viruses. c. Strand polarity is the major criteria by which we’ve divided up the two lectures. The viruses he’ll talk about are negative sense or ambisense which have both negative and positive sense pieces to their genome. d. Positive sense is an RNA that looks like the mRNA. It has the possibility of being directly translated in the protein whereas negative sense viruses have to first be replicated. You have to transcribe a mRNA from the genomic RNA which is then translated into a protein. e. The genome can be a single segment or the genomes can be divided up into multiple segments. Fundamentals 11:00-12:00 Scribe: Jake Nolen Thursday, November 5, 2009 Proof: Susan Whitt Dr. Lefkowitz Single Strand Negative Sense RNA viruses Page 2 of 11 f. I’m going to talk about the single segment viruses or some of them can be multiple segment viruses.
XII. RNA Virus Classification [S12] a. We can distinguish each of the viral families. We’re interested in the helical capsids and they’re all enveloped viruses. The coronaviruses are not negative sense, even though they’re under the enveloped heading.
XIII. Negative/Ambi-Sense RNA viruses [S13] a. skipped
XIV. Properties of Negative-sense RNA viruses [S14] a. Enveloped b. Nucleocapsid c. Negative sense RNA genome d. Some are ambisense (arenoviruses and bunyaviruses) these are multi segmented too. e. Most of the viruses we talk about replicate solely in the cytoplasm of the host cell. f. The bornaviruses and orthomyxoviruses (influenza viruses) depend on the host cell nucleus for replication. These are dependent on the utilization of the host cell nucleus for replication. g. These genomes are not infectious. Using the right techniques you can take the genome from a virion of a picornavirus, for example poliovirus, and introduce it into a cell, and it will replicate and produce new virions. This cannot happen with any of the viruses I’m talking about. h. You have to play a number of tricks in order to get that genome to replicate either through the natural viral replication cycle or in the laboratory. The genome itself is not infectious.
XV. Order: Mononegavirales [S15] a. There’s a large group of viruses: mononegavirales. b. All of these have common properties and probably a common evolutionary origin. c. The families of viruses in this group of viruses: the bornaviridae, the filoviridae, paramyxoviridae, and rabdoviridae. d. I will talk about the human pathogens that are in these families. e. We’ll talk about some of their replication properties as well.
XVI. Multi-Segment, Negative and ambisense ssRNA viruses [S16] a. Mononegavirales are mono because they have a single strand RNA in the genome of the virion. These other viruses are segmented so the arenaviruses have two segments of RNA in their virion whereas the bunyaviruses have 3 segments of RNA in their virion..
XVII. Multi-Segment, Negative sense ssRNA viruses [S17] a. Orthomyxoviruses have multiple segments. Influenza has 8 segments. b. Others have differing number of segments. c. These are all multiple segmented negative sense single stranded RNA viruses.
XVIII. Viral Replication cycle [S18] a. The properties of the RNA genome of these viruses determine how they replicate, and there are important differences in how these viruses replicate in contrast to how the positive sense RNA viruses replicate that are determined by the genomes of these viruses.
XIX. Viral Replication Cycle: part 1 [S19] a. You can think about the overall replication process from the point at which the virus attaches to the host cell to the point at which new virions are released. b. That whole process looks a lot like the other RNA viruses that we dealt with. c. We’ll look at these details in the cartoon below.
XX. Viral Replication Cycle: part 2[S20] a. skips
XXI. Replication cartoon [S21] a. Virus attaches to the host cell. Then, it enters the host cell, and the viral RNA is released into that cell. Fundamentals 11:00-12:00 Scribe: Jake Nolen Thursday, November 5, 2009 Proof: Susan Whitt Dr. Lefkowitz Single Strand Negative Sense RNA viruses Page 3 of 11 b. We now have to do two things with this viral DNA: Have to make mRNA from the viral RNA. The mRNA is then translated into viral proteins. These proteins do different things. Some of them are involved in the replication of the virus. Other proteins are involved in the assembly of the new virions themselves. They may be inserted into the host cell lipid membrane where the virus can then drive into the envelope from that membrane and pick up these viral host proteins. c. Some of these other viral proteins are involved in replication, so the other thing that happens to this viral genome after it’s transcribed is that viral replication occurs in which you make reproducible copy of this viral genome based upon the polymerases that allow for the replication process. d. Then you have new viral genomes that can be packaged along with the other viral proteins and then the new virions can be released from the host cell. e. That’s the overall process. Some of the steps are similar to what we’ve seen before but a few of them are different and the differences will be pointed out in the next slide.
XXII. Viral Proteins [S22] a. A number of different proteins make up the virions that are part of the whole biology of these viruses. b. Depending on which family of viruses we’re talking about there will be a different set of proteins that those viruses code for. c. In general, there are some common properties among all of these viruses. There are some proteins that are absolutely necessary for the replication process. You have to have some protein on the viral surface that allows that virus to attach to the host cell. Sometimes it’s called the G membrane glycoprotein. Sometimes the protein has hemagglutination activity. It agglutinates red blood cells, but it also is involved in attachment of the virus to the host cell. Fusion proteins allow fusion between the viral envelope and the membrane of the host cell. d. Finally, there are other proteins on the surface of the virion that disrupt the action of, say, the hemagglutin protein or the membrane glycoprotein. They disrupt the interaction between those attachment proteins, and the proteins on the host cell surface. That allows for release of newly formed virions. e. In addition to these attachment proteins, there are always some sort of structural proteins that give the virion its structure. Those proteins are called the matrix proteins, and they usually underlie the lipid bilayer- the envelope of the virion surface. They provide the structure into which the viral genome is held by the virion itself. f. There are a number of different proteins for each of these viruses that are involved in replication. Usually, that includes a polymerase but also includes some other proteins that are required. We’ll talk a little bit about those proteins in a bit.
XXIII. Rhabdovirus Virion [S23] a. When you put all these proteins together into the virion. For the rhabdoviruses that happens to include the rabies virus. b. Rhabnovirus has a bullet shaped structure. You have the matrix envelope here, and you have the matrix protein that underlies the envelope here. You have a glycoprotein here that serves as the attachment for the virus. c. Internal to the virion you have RNA genome itself. That RNA genome is covered in an N protein that is part of the replication process, and then you also have attached to that a virion RNA, A polymerase protein called L in this case, and an accessory protein that’s involved in the replication of the virus called the P protein. d. All of these form the virion. e. Rhabdonoviruses are one of the simplest viruses in terms of the complement of proteins that are coded for and that are present in the virion, but variations on these proteins are present in pretty much all of the viruses that we’re talking about.
XXIV. Paramyxovirus Virion [S24] a. For comparison purposes, we’ll look at the paramyxoviruses. Has same set of proteins: attachment proteins, matrix proteins, polymerase proteins, but they have a few other proteins thrown in for good measure. They have a fusion protein. They have a small hydrophobic protein. They have a V cysteine-rich protein. They take that basic complement and they add to it. These other proteins are what gives the virus its unique biology.
XXV. Virus Coding Strategies [S25] a. All of these proteins are coded for by the viral genomes, but these viruses play a lot of tricks in order to derive the maximum amount of info out of the genomes that is contained within these viruses. b. You start with a ssRNA virus genome. You could have and do have for a number of these viruses individual open reading frame. Each one of those reading frames is transcribed into a messenger RNA which is then translated into a separate protein. That’s the simplest scheme. c. Some of these proteins also code for polyproteins similar to what the picornaviruses-some of the positive stranded viruses do. That is you have a large protein that is then cleaved up by proteases into individual Fundamentals 11:00-12:00 Scribe: Jake Nolen Thursday, November 5, 2009 Proof: Susan Whitt Dr. Lefkowitz Single Strand Negative Sense RNA viruses Page 4 of 11 proteins. That coding strategy is not used much at all in these viruses, but it’s occasionally used for a few proteins. d. In addition to those coding strategies, some viruses edit the RNA. When you make the mRNA, they may introduce extra bases into that mRNA, or they may actually have splicing of that mRNA to code for new open reading frames that you normally wouldn’t get for a simple open reading frame that starts with a start codon and stops with a stop codon. RNA editing is a method that some of these viruses use to get extra information (extra proteins) out of the RNA genome of these viruses. e. Some of these RNA viruses have multiple ribosome initiation sites, so there may be multiple start codons to start translation of a protein. If you use one or another, you could actually be in a different reading frame. Sometimes it’s the same reading frame, but in any case, you get multiple proteins from the same region of that RNA genome due to multiple ribosomal initiation sites. f. Then, some of them could have stop codons that are read through. That is, you get one protein that stops normally at a stop codon, but then you’ll get on occasion another protein that reads through that stop codon and you get a much larger protein. g. All of these methods are used to get extra coding capacity out of these RNA viruses.
XXVI. Genome Organization Mononegavirales [S26] a. Picture of structure of genome of mononegavirales order of viruses. b. We have rhabdoviruses and paramyxoviruses. c. Rhabdoviruses are fairly simple. You have this N or nuclear protein that wraps or encapsidates the RNA genome. The P protein and the polymerase make up the RNA-dependent RNA polymerase that’s required for replication. The matrix protein that underlies the viral envelope. And the G attachment protein. d. But then as you see here, we have variations on these themes in these other families of viruses.
XXVII. Genomic Replication strategies of RNA viruses [S27] a. Now we’ll go into greater detail on the replication strategy of these different viruses.
XXVIII. Summary of different initial activities of input genome [S28] a. I want to highlight one of the key differences between the replication of the plus sense RNA viruses and the negative sense RNA viruses. b. Plus sense RNA viruses- picornaviruses or coronoviruses are part of this group here. What happens when the virus attaches to that cell and the genome is uncoated within that cell. The first thing that happens is protein synthesis. The virion RNA itself can act as an mRNA and you can make protein. c. For the viruses that we’re talking about (the negative sense viruses) or the ambisense viruses that contains regions of the genome that are both positive and negative sense. In that case, the first thing that can happen is not translation of an mRNA because these are not mRNA sense. They have to be transcribed to make a new mRNA that was not present previously in the virion. d. The first activity in these viruses is RNA synthesis. RNA synthesis is mediated by a polymerase protein, or in most cases a complex of proteins (several proteins) that mediate polymerization and replication of the RNA genomes.
XXIX. RNA-dependent RNA Polymerase [S29] a. Usually there is one large polymerase protein sometimes called an L protein that’s the catalytic subunit of a polymerase complex. b. Also may have other activities: mRNA’s and genomic RNAs may be capped, methylated, polyadenylated. These activities are mediated by the polymerase and the polymerase complex. c. They’re also the most conserved proteins of all of these different viruses.
XXX. Source of the RNA-dependent RNA Polymerase [S30] a. What has to be different between the plus sense and the minus sense viruses in terms of their replication strategy? What has to be different in terms of provision of this RNA-dependent RNA polymerase? b. Since plus sense viruses can be directly translated, from the viral genome, you can make the RNA polymerase proteins directly from that genome that’s being translated. c. Since for negative sense RNA viruses, you can’t directly translate them, you can’t make the polymerase, so you can’t have the protein that’s absolutely required for replication. That protein can’t be made initially when the virion infects the host cell. Instead, what these viruses have to do is carry that polymerase and carry any other polymerase accessory proteins along with them in the virion. d. In essence what happens is all of the required proteins for replication are bound to the RNA genome. The virus attaches to a cell, the RNA genome is released into the cell, and then the polymerase proteins can get started Fundamentals 11:00-12:00 Scribe: Jake Nolen Thursday, November 5, 2009 Proof: Susan Whitt Dr. Lefkowitz Single Strand Negative Sense RNA viruses Page 5 of 11 transcribing the virion RNA making mRNAs. That’s the key difference. For the negative sense RNA viruses, the polymerase proteins are packaged into the virions. For positive sense RNA viruses, you don’t have to have those proteins present in the virions of the host cell.
XXXI. Negative-strand RNA Virus Transcription/Genome Replication and regulation [S32] a. When we look at the details, vesicular somatitis virus, which is a rhabdovirus similar to rabies virus, is a common lab strain that’s used to study the process of all these viruses.
XXXII. Virus replication Machinery [S33] a. It can be used to delineate the machinery of replication that is absolutely required for these viruses to make new viral genomes. b. You need the large polymerase protein but you also need this N protein (Nucleocapsid protein). That’s the protein that completely covers the viral genomic RNA. The RNA itself is encapsidated. You don’t see free naked RNA. It always contains this N protein. c. In addition you have this accessory protein-the P protein for this virus that’s also required for replication. d. Other viruses have a similar protein similar to P protein or other accessory proteins that they need for replication.
XXXIII. N-protein Encapsidated RNA [S34] a. This is a picture of what the RNA genomes look like for a number of different viruses. Influenza and rabies are negative sense RNA viruses. Sendai virus is another one. Can see here that this is a viral genomic RNA, and you can see little beads that fully coat this RNA that are the N protein. b. Naked viral RNA will not work as a median for replication of these viruses. They have to be coated.
XXXIV. The structure of the VSV RNP complex [S35] a. This is the crystal structure, which has been solved for the complex of that N protein with the viral RNA. The viral RNA is the yellow strand and these are individual N protein molecules that completely coat that viral RNA. b. That’s an absolute requirement for the replication process.
XXXV. VSV RNP Template [S36] a. This is the proteins arrayed along the vesicular stomatitis virus RNA genome. You have the N protein at one end and the polymerase L protein at the other. You also have your P, N, and G protein. b. You transcribe each one of these open reading frames producing a separate mRNA. After you make the protein, you can start the process of replication in which you first make the positive sense RNA (a complete genome) that serves as a template for making additional negative sense RNA genomes that can be packaged into the virion itself. There’s a particular order to the proteins that are present along this viral genome. That order is conserved in even some of the other viruses that are different that may include additional proteins, but the basic order of this N protein, an accessory protein for polymerase, matrix attachment, and polymerase protein. That order is conserved. That conservation of order is important because it regulates the production of mRNAs which in turn regulate the production of the amount of protein.
XXXVI. VSV Transcriptional Control [S37] a. When you look at the number of mRNAs present and the amount of proteins present, you have a lot more of the N protein than you do P protein. Lesser of the matrix protein. Finally, the polymerase protein has the lowest amount of message, and the lowest amount of protein present in an infected cell. b. If you change the order using molecular biology techniques of these proteins, if you change the amounts of these messages of proteins that are present, you get less replication of the virus. c. The order that is optimum for the replication of these viruses is the exact order that you see here. d. During the process of evolution of these viruses, you get this particular order. e. The fact that you get more mRNA at this end of the genome is a consequence of the fact that the RNA polymerase binds at this end of the viral genomic RNA. It makes its first transcript- the N messenger RNA. Then there’s a signal between the N and the P regions that says to the RNA polymerase: Let’s stop. We’ll terminate transcription of that N mRNA. Now, let’s go ahead and initiate transcription of the next message which is this P message. That process of termination and reinitiation is not 100% efficient. Perhaps only 30% only 70% (He said both percentages, so I don’t know which one he means.) move on to do P. Only 70% of the polymerase proteins that made it on to P make it on to M. That’s how the amount of messages are regulated. You get many made here. The polymerase occasionally drops off so you get fewer made here, some more polymerases drop off so you get fewer made here. So that’s a process whereby you can regulate the amount of transcription and thereby get the required amounts of proteins made that are optimal for viral replication. Fundamentals 11:00-12:00 Scribe: Jake Nolen Thursday, November 5, 2009 Proof: Susan Whitt Dr. Lefkowitz Single Strand Negative Sense RNA viruses Page 6 of 11
XXXVII.Overview of (-) sense RNA replication strategy [S38] a. The whole process of viral replication is summarized here. b. You have your genomic RNA here. It’s transcribed into viral mRNAs, which then make both structural protein, proteins that are present in the virion, and also makes proteins required for replication and once you have those proteins made you can go back to this genomic RNA and now you can replicate it to make plus sense genomic RNA that is a complete copy of this. That plus sense genomic RNA can then be replicated to produce additional minus sense RNA genome that are then packaged along with these viral proteins to make new virions that are released from the host cell. c. That’s the overall process of viral replication.
XXXVIII. Important human pathogens from the group of negative-strand RNA viruses a. Now we’ll move on to the viruses themselves and some of the diseases they cause, and some of the viral pathogens that are part of these viral families.
XXXIX. Major Target Tissues
a. This is a listing of a lot of these viruses and some of the major target tissues of these viruses. He’s highlighted the major negative sense viruses. They infect a variety of tissue types in humans and animals. b. There’s not much tissue that can’t be infected.
XL. Oral and Respiratory Viral Diseases a. Negative sense RNA viruses that are highlighted here in yellow infect pretty much all regions of the human respiratory tract. Infuenza viruses are present as causing cold like symptoms, symptoms of tonsillitis and laryngitis, bronchilitis and pneumonia. A lot of the other viruses present are paramyxoviruses. He’ll talk about those in particular later. The paramyxoviruses have a large number of human pathogens.
XLI. Arboviruses and Zoonoses a. He talked about the arenaviruses and the bunyaviruses, the segemented viruses. These viruses aren’t natural pathogens but can infect humans through insect viruses, that’s why they’re called arboviruses because they can infect insects. Or through zoonosis which includes contact with rodent vectors for example. So humans can pick these up and they can cause rather severe disease in humans when we come in contact with either an insect vector or an infected rodent.
XLII. Arenaviruses/Bunyaviruses a. He’s gonna go through each one of these families of viruses in turn, and briefly talk about the diseases that they cause.
XLIII. Arena/Bunya Classification a. Arenaviruses mostly affect rodents and when humans come in contact with the rodents, they are infected. b. Bunyaviruses occasionally cause zoonosis but they also infect insects. There’s an insect vector that can transmit the virus from an infected animal to humans.
XLIV. Arenavirus Disease a. These are the diseases caused by arenavirus. Some of the viruses that cause human disease are Lassa fever. These hemorrhagic fevers: Argentinian and Bolivian hemorrhagic fevers, and then there are a number of other viruses. LCM Lymphocytic Meningitis virus is not really a human pathogen, but it’s used in the lab to study these viruses and the immune reactivity to these viruses. b. Some of these viruses can be very pathogenic and cause fatality rates of up to 25% if infected.
XLV. Bunyavirus Genus a. There are a large number of viruses. Human pathogens are called Hantaviruses.
XLVI. Hantaviruses – a. Outbreaks of hantavirus disease especially in western US when humans come in contact with rodents infected with these viruses. Rodents or rodent droppings can cause humans to become infected or by inhaling infectious droplets.
XLVII. Arenavirus Genome Organization Fundamentals 11:00-12:00 Scribe: Jake Nolen Thursday, November 5, 2009 Proof: Susan Whitt Dr. Lefkowitz Single Strand Negative Sense RNA viruses Page 7 of 11 a. Talked about how these viruses are ambisense. Arenaviruse segments that are packaged into the virion, a large and small segment. Each one of these segments code two proteins. One protein is coded for in the negative sense direction. Another protein, Z protein, is coded for in the opposite direction. The glycoprotein is coded for in the positive sense direction and the nucleoprotein that encapsulates the RNA is encoded for in the opposite direction. b. You don’t directly transcribe the genomic RNA to make this Z and G protein. You first have to transcribe to make mRNAs before any of these proteins can be produced.
XLVIII. Bunyavirus L segment Coding a. They have 3 segments that are packaged into the virion. The large segment codes for the polymerase protein is all negative sense. Only one protein is coded for by any of the different groups of viruses that make up the bunyaviruses.
XLIX. Bunyavirus M segment Coding a. There’s an M or median segment that in most cases is only coded for by a negative sense, though some of these do code for multiple proteins. Multiple transcripts. But there is one group of viruses that is ambisense that codes for proteins in both directions.
L. Bunyavirus S segment Coding a. Small segment has a number of other groups of viruses that are ambisense though a few of them are also negative sense. b. Don’t memorize the groups of viruses whether they’re negative sense or ambisense but just know the variety of coating strategies that these viruses use are shown in the variety of ambisense and negative sense and segmentation that make up these different viruses.
LI. Filoviruses a. Now we’ll move to viruses that are part of this large mononegaviralis order of viruses. Contains a number of fairly different families that are different in terms of the disease that they cause.
LII. Filovirus Disease a. Filoviruses cause Ebola virus and Marburg virus disease, which has an extremely high case fatality rate approaching 100% in outbreaks usually in Africa. There are a variety of species that cause these diseases.
LIII.Picture of Viruses a. Filoviruses have characteristic morphology of their virions. Frequently, long filamentous virions. Under an electron microscope, if someone has a hemorrhagenous disease, and they see structure similar to this the first thought is that you may have an outbreak of Ebola or Marburg virus.
LIV. Picture of virus envelope a. These are the genomes of the viruses and their coding strategies similar to what we saw for vesicular stomatitis. Filoviruses are one of the viruses that use RNA editing. In this glycoprotein coating region, there’s an editing site that introduces new bases into the RNA transcript, and you can get upwards of 3 different proteins from this one region of the viral genome due to RNA editing.
LV. Filovirus Pathogenesis a. The life cycle of these viruses: once the virus infects the person, it may be infect the macrophages, the macrophages can then disseminate the virus throughout the host, and once it’s disseminated, you get the characteristic hemorrhaging in Ebola and Marburg. These are the things that are talked about in the book Hot Zone.
LVI. Bornaviruses a. skipped
LVII. Bornaviruses a. Haven’t mentioned much. b. It’s another mononegaviralis. It’s the virus that requires the nucleus for its replication. c. There have been some reports that bornaviruses are associated with neurological diseases. d. Even the association of bornaviruses with neurological diseases is controversial. No one has proved a causal relationship. Fundamentals 11:00-12:00 Scribe: Jake Nolen Thursday, November 5, 2009 Proof: Susan Whitt Dr. Lefkowitz Single Strand Negative Sense RNA viruses Page 8 of 11
LVIII. Rhabdoviruses a. He mentioned rabies and vesicular stomatitis.
LIX. Rhabdoviruses a. The pathogens are rabies virus and a few other viruses in the same group can also cause human disease.
LX. Picture of viruses a. They have a characteristic bullet shaped virion for both vesicular stomatitis and rabies virus.
LXI. Picture of Raccoon a. These viruses are unique because they can infect cells of the nervous system and once an animal is bitten by a rabid animal, once the viruses infect the nervous cell, it can be transmitted to the brain where you start to get the characteristic pathology- neurological response, rabid animals, salivation, and such.
LXII. Paramyxoviruses a. Have a lot of viruses that cause human disease. There are a large number of groups with a lot of different species that are part of this whole family.
LXIII. Paramyxoviruses a. Within this large number of different species, you have viruses such as mumps, human parainfluenza virus, measles virus, human respiratory syncytial virus, Hendra virus, Nepah virus. b. All of these infect humans and cause different kinds of disease in humans from this family of viruses.
LXIV. Paramyxoviruses: Maximizing Genetic Content a. These viruses also use some of these alternative coding strategies to maximize the coding potential from a single open reading frame for the viruses so some of the viruses use RNA editing to produce different transcripts producing different proteins. b. These viruses use ribosome choice. You get different proteins made using different ATG start codons. c. A lot of different ways in which these viruses code for their proteins.
LXV. Paramyxovirinae a. Still have the same overall organization of an N protein, a protein involved in replication, matrix protein, and an attachment set of proteins. In this case we have a fusion protein for hemaggluttin and neuriminidase and then the polymerase protein. Here this says PCV, using RNA editing and in some cases ribosome choice, you’ll get multiple proteins made from this region of the genome.
LXVI. Human Respiratory Syncytial virus a. Just to mention two of the diseases that are caused by viruses that make up parts of this family. b. Human syncytial virus is a major cause of respiratory tract infection. By the age of 4 most people have been infected or exposed to this virus. The infection you get is normally inapparent or very mild, but on occasion infants can get severe infection from this respiratory syncytial virus. c. Back in the 1960’s there was a vaccine against this virus. Unfortunately, this vaccine exacerbated the disease and caused several deaths among infants who were given the vaccines. There have been attempts to create a vaccine since then, but you can imagine there is a great deal of reticence towards that vaccine given the history.
LXVII. Measles virus a. Used to be ubiquitous. Used to infect most kids until an effective vaccine was developed but now that there is the vaccine we rarely see measles in the US except in areas where kids have not been vaccinated.
LXVIII. Neurologic Complications of Measles a. Measles can be fairly serious. Most kids get the characteristic measles rash and then recover, but measles has the possibility of causing neural disease months or even years later. b. That’s why vaccination is so important to prevent those neurological complications.
LXIX. Orthomyxoviruses a. We’ll finish with a group of viruses that have gotten a lot of publicity lately (influenza). Fundamentals 11:00-12:00 Scribe: Jake Nolen Thursday, November 5, 2009 Proof: Susan Whitt Dr. Lefkowitz Single Strand Negative Sense RNA viruses Page 9 of 11 LXX. Orthomyxoviruses a. There are 3 types of orthomyxoviruses: A,B,C. We’re mostly concerned with A and B because they cause the most disease. A causes the most severe disease. That also includes viruses that infect other animals like ticks and salmon.
LXXI. Schematic diagram of influenza A viruses a. This is a cartoon of the influenza A virus virion. b. You have your surface glycoproteins. You have the envelope that they glycoproteins are inserted into, you have a matrix like protein that underlies the virion.
LXXII. Schematic diagram of the influenza viral life cycle a. Key: Influenza encodes it genetic material on 8 different segments of RNA. All 8 must be packaged by the virion in order for that virion to be infectious. This is the life cycle which is similar to the life cycle I showed you before, but again I want to emphasize that to be infectious, all 8 of these virion genomic RNAs must be packaged into the virion. b. There do seem to be specific mechanisms and signals that ensure that all 8 of those segments are packaged into these influenza virus virions.
LXXIII. Influenza A Genome Structure a. Each virion codes for one or more proteins, and you have the same group of proteins that you might expect that are required and that we talked about with the other viruses. Proteins that are involved in replication, transcription of the viral genome, attachment proteins HA hemaggluttin protein in this case and a neuriminidase protein for the release of the virion from the cell. b. There is a nuclear protein that encapsulates the RNA, a matrix protein that underlies the virion envelope, and a few other accessory proteins thrown in for good measure that are required for viral biology.
LXXIV. Hemagglutinin a. This is a picture of hemagglutinin. Part of hamagglutinin is inserted in the virion membrane, part sticks out from the membrane, and acts as the attachment site for the virus to the host cell. The polymerase protein here is composed of a number of different proteins.
LXXV. Polymerase Complex a. I mentioned each of these in turn because each of these proteins are also possible targets for intervention. Development of drugs and vaccines that can target these proteins and help alleviate infection.
LXXVI. M2 Ion Channel a. Another protein called the M2 protein forms an ion channel. This is important because one of the current drugs against flu targets this ion channel. When it targets this channel, the virus is no longer infectious.
LXXVII. Neuraminidase a. Used to release virus from host cell. The virion has a hemagglutinin protein which attaches to host cell receptors. When the virus is released, if you don’t disrupt that attachment between the hemagglutinin and the host cell receptor, the virus can’t be released and go on to infect another round of cells. b. Virus includes a neuriminidase which breaks that interaction between the hemagglutinin and the attachment protein. The neuriminidase is required for the virion’s replication. Therefore there are drugs Tamavir and Relenza, which have been useful against the swine H1N1 influenza. They target the neuriminidase.
LXXVIII. Variation and evolution a. When we talk about influenza, when we talk about host response and pandemics of influenza, we’re always talking about the variation and evolution of the influenza virus.
LXXIX. Influenza Virus Variation a. Can vary and evolve using a number of different strategies b. Antigenic drift is due to single or multiple amino acid changes, so small changes in the amino acids of the proteins that the viruses code for that cause changes in the antigenicity of the virus. c. This may cause someone who was previously immune to a virus to no longer be immune if there are enough small changes to produce an antigenic change in the virus. d. Antigenic shift is when you pick up a segment of one of those 8 mRNA viruses, when you exchange one of those 8 segments with a completely different type of influenza virus. Fundamentals 11:00-12:00 Scribe: Jake Nolen Thursday, November 5, 2009 Proof: Susan Whitt Dr. Lefkowitz Single Strand Negative Sense RNA viruses Page 10 of 11 e. You have the H1N1 virus and the H3N2 virus. You can take the H3, introduce it into a virus that has the H1. Now you may have an H3N1 virus. f. By mixing and matching the mRNA, you have antigenic shift, which can cause large changes in the immunogenicity and our reaction to these viruses.
LXXX. Reassortment a. Reassortment is called antigenic shift. b. Here’s 2 viruses. If both of those viruses infect the same cell, you can mix and match those segments resulting in different viruses with different antigenic properties and also, different disease causing potential. Some may be more infectious or some may be more pathogenic than the original viruses that we started with. LXXXI. Emergence of pandemic influenza virus a. Model of evolution of the influenza virus since the early part of the 20th century. b. 1918 pandemic caused severe disease and severe mortality. Derived from a bird disease H1N1. c. This virus that were derived from this virus then gave rise to what we currently see circulating in our H1N1 virus. d. Some of the history is unknown, but if you go back to 1977, there was an outbreak of H1N1 virus that has slowly evolved via antigenic drift to viruses that we see circulating today for seasonal flu. This is not the swine derived flu that has been in the news. e. In addition to dealing with an H1N1 seasonal flu, we also deal with a circulating H3N2 seasonal flu virus that also may have parents derived from the Spanish flu outbreak, but it also has other components derived from other avian viruses that have mixed and matched between all of these viruses to form the virus that we see circulating today. f. There is a fairly complex history.
LXXXII. Influenza 2008-2009 a. If you look at the outbreak of influenza for last year’s season, at the end of January as we were approaching the peak of the season, you see sporadic outbreaks in some states, whereas other states have large regional outbreaks like in AL. b. There is a mix in terms of how severe flu was last year.
LXXXIII. Influenza October 2009 a. Today in October, this is what the flu outbreak looks like now. It’s widespread over the whole US.
LXXXIV. US Influenza Surveillance 2008-2009 a. If you look at the viruses that cause these outbreaks, here’s the outbreak from last season of the normal seasonal flu outbreak that we would expect. This outbreak is caused by a combination of usually H1N1, the seasonal one, and the circulating H3N2 virus. A different strain of virus, the B strain also was causing an outbreak at that time. Those peaked around Jan and Feb of last year. Then, we have an unusual spike that we haven’t seen before. This is a spike of non-seasonal influenza caused by the swine origin H1N1 virus that’s very different from the H1N1 virus that we saw here. It’s enough like the H1N1 that we still call it H1N1. The virus’s amino acid that the virus codes for in the hemagglutinin and neurininidase are different enough that those who are exposed to the seasonal H1N1 don’t have immunity to the swine H1N1. b. There was an outbreak in the spring, and now we’re in another outbreak. The data is from the middle of October, so we’re still going up even though it looks like it has gone down.
LXXXV.Reconstruction of the sequence of reassortment events leading up to the emergence of S-OIV a. You can look at the origins of the swine derived H1N1. This is the virus here but if you trace it back you see that it has origins from a number of different viruses in a number of different species.
LXXXVI. Genesis of swine-origin H1N1 influenza viruses a. Just to simplify that, here’s the virus we have today the swine H1N1 no seasonal. It can originally from a group of avian viruses and swine viruses. There was a North American avian virus, an H3N2 human virus that resulted in a triple reassortment. Then you picked up another swine derived segment that caused additional reassortment and eventually gave rise to the virus we see today. b. This shows the complicated evolutionary history of these viruses.
LXXXVII. Fighting back a. We have drugs and vaccines to combat these viruses. We talked about the neuriminidase inhibitors Tamavir and Relenza that are effective against flu A and B. The ion blocking drug amantidine is also effective. Fundamentals 11:00-12:00 Scribe: Jake Nolen Thursday, November 5, 2009 Proof: Susan Whitt Dr. Lefkowitz Single Strand Negative Sense RNA viruses Page 11 of 11 LXXXVIII. Antiviral Resistance 2008-2009 Season a. Even though these drugs work, they don’t always work. The virus that was circulating last year was resistant to Tamavir, but luckily they were still sensitive to Relenza and Amantidine. The H3N2 was sensitive to Tamavir and Relenza but all were resistant to Amantidine. b. The flu B viruses are sensitive to Tamavir and Relenza. c. This is a continuing problem. The viruses of this year may have different sensitivities to these drugs. d. We must make new drugs to combat viruses that are increasingly resistant.
LXXXIX. Vaccine Development a. You’ve heard about the different vaccines that are in use against viruses against the seasonal and against the swine H1N1. There is an inactivated vaccine that’s produced in eggs, and there is a live attenuated vaccine. This is similar to the different vaccines available for polio. b. Every year at the end of the outbreak, the WHO looks at the circulating viruses and determines what viruses will be included in the vaccine for the subsequent year.
XC. Production of Attenuated Vaccines a. When they produce those vaccines, last year they had a strain of H1N1 and a strain of H3N2. There is always a strain of both of those. There is also a strain of a B virus in the vaccine. That’s a guess from the previous year. b. As it turns out the H1N1 and H3N2 in the previous season were what was circulating in the 2008-2009 season. The Influenza B Virus was a new one that wasn’t part of the vaccine that was circulating. For this year, they replaced the B component but kept the A component. c. We also have the new vaccine against the swine H1N1.
XCI. Vaccine Strains for the 2008-2009 Season
XCII. And what about the future? a. There are new viruses circulating all the time. He just kind of clicked through these last couple slides without talking about them.
XCIII. Viruses on the NIH List of Emerging and Re-emerging Infectious Diseases
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