FUN2: 10:00-11:00Scribe: Caitlin Cox/Ryan O Neill

FUN2: 10:00-11:00Scribe: Caitlin Cox/Ryan O Neill

FUN2: 10:00-11:00Scribe: Caitlin Cox/Ryan O’Neill

Monday, November 17, 2008Proof: Joan-Marie Manolakis

Dr. LefkowitzNegative-Stranded RNA VirusesPage1 of 5

ssRNA(-) – negative single-stranded RNA, RSV – Respiratory Syncytial Virus, RdRp – RNA-dependent RNA polymerase, VSV – Vesiculovirus, HRSV – Human Respiratory Syncytial Virus, MMR – Measles, Mumps and Rubella

**The lecturer was Jackie Parker, who was filling in for Dr. Elliot Lefkowitz**

His contact information is on the website. The updated version is abbreviated from what was posted on the website because she only wanted to emphasize what is most important, which often resulted in reading directly off the slides.

This is indicated underlined when it occurred.

I.Objectives [S3]: This lecture will focus on negative strand RNA viruses.

  1. Must understand the fundamental common and distinguishing properties of RNA viruses.
  2. Understand the basic replication strategies of RNA viruses.
  3. Be able to identify human pathogens that belong to negative-strand RNA virus families, and some of their biological and pathogenic properties.

II.Reading [S4, S5]: The information in this lecture is supplemented by the listed chapters from the Medical Microbiology book, as well as the other listed references to make this presentation.

III.Viral Classification [S6, S7, S8]: The wheel illustrates the different types of viruses that are grouped based on their genetic information. We will focus on the single-stranded negative RNA viruses (ssRNA(-)).

  1. The host cells affected by ssRNA(-) viruses include vertebrates, invertebrates, and plants.

IV.Features used to classify RNA viruses [S9]:

  1. Virus particle size
  2. Genome structure
  3. External structure (whether it is enveloped or naked)
  4. Symmetry type (whether it is icosahedral symmetry in the capsid or helical symmetry)

V.Electron micrographs of different viral structures [S10]:

  1. We can compare enveloped, naked capsid, helical (like filovirus), and icosahedral capsid structures.

VI.The RNA virus genome structure [S11, S12] can be single or double stranded.

  1. The strand polarity can be positive, negative or ambisense.
  2. An ambisense strand has segments with both positive and negative sense polarity.
  3. Positive sense strand denotes the coding strand (mRNA), so it can be translated directly.
  4. The number of segments can vary
  5. Naked capsid viruses can have single segment or be multi-segmented.
  6. Enveloped viruses also can be single or multi-segmented.
  7. Example of the enveloped: Orthomyxovirus family is influenza.

VII.Negative-strand RNA & Ambi-sense virus properties [S13, S14]:

  1. Enveloped virion
  2. Helical nucleocapsid
  3. The ones with negative-sense, single segment RNA genomes include: Bornaviruses, Filoviruses, Rhabdoviruses, and Paramyxoviruses.
  4. The negative and ambi-sense, multi-segmented RNA genome viruses include: Arenaviruses, Bunyaviruses, and Orthomyxoviruses.
  5. For the most part they replicate in the cytoplasm with the exception of the Bornaviruses
  6. The genomes by themselves (as a consequence of being negative stranded) are non-infectious
  7. An initial round of transcription is therefore required to replicate the genome.
  8. The virion must therefore bring along the proteins required for transcription.

VIII. Virus phylogeny & Mononegavirales Order [S15-S16]

  1. Read directly off slides
  2. “Mono” – single strand, “neg” –negative-sense of the virus

IX.Mononegavirales Phylogeny [S17]

  1. Shows the relationship with the different ssRNA viruses with the Paramyxoviridae viruses, which are actually the largest subfamily. Also shows other virus relationships

X.Multi-segment, Negative and Ambisense ssRNA viruses [S18]

  1. Read directly off slide
  2. The two subfamilies of Bunyaviridae are strictly negative-sense and ambisense
  3. Tospoviruses affect plants and Phleboviruses one of causative agents of hemorraghic fever.
  4. The negative strand virus that has the most segments is the Orthomyxoviruses types A & B (8 negative sense RNA segments) and type C (7 segments).

XI. Viral replication cycle [S19 – S21]

  1. Read directly off slide. These below points were the only extra points not listed on the slides.
  2. Important because there are a lot of similar properties (genes that encode, proteins responsible for attachment, etc).
  3. Uncoating - The ribonucleoprotein complex consists of the viral genome.
  4. Translation – encodes the viral proteins necessary for encapsidation and making the progeny viruses
  5. The first requirement for a negative strand virus is you have to synthesize a full-length positive-sense anti-genome, which serves as a template to produce the negative-sense genomic RNA, which will be packaged.Took out the statement “both the positive and negative sense genomes get encapsulated with the N or nuclear protein.”
  6. N-protein = nuclear protein
  7. The reason matrix proteins are localized to the plasma membrane is to help the budding process to occur and they associate with the ribonucleoprotein complexes (RNP). At this time you have the budding.

XII.Cartoon [S22]

  1. Shows the previously described process with RSV as the example where you have receptor-mediated attachment, followed by fusion, release of the RNP and follows the steps outlined in previous slides.
  2. The site of the ER and Golgi is where the viral glycoproteins and matrix proteins need to be aggregating.
  3. Note the budding process also.

XIII. Viral Proteins [S23]

  1. Viral proteins have very simple names that are just letters that illustrate certain properties of the virus.
  2. Read directly off slide. See slide for examples.
  3. The Hemagglutinin is found on many of these negative strand viruses. This is a receptor-binding protein to attach to moieties on the host cell.
  4. The Neuraminidase is found on a number of different RNA viruses.
  5. In the case of Influenza, it is required for efficient release of the newly synthesized viruses.
  6. Phosphoprotein is present in many of the virus families.
  7. RNA dependent RNA polymerase (L) – will be mentioned again, this is a unique feature of these viruses.

XIV.Rhabdovirus Virion [S24]

  1. Single stranded virus.
  2. Note the envelope membrane, matrix protein and ribonucleoprotein.

XV. Paramyxovirus Virion [S25]

  1. Has a different organization. It has the Hemagglutinin-Neuraminidase protein, which mediates both of the functions by initial attachment and helps the self-cleavage to release it.
  2. Also have a separate fusion protein. Some of the viruses only have the HA protein, which helps mediate the fusion.
  3. [SQ]: Which one has the HA and HN?
  4. [A]: Paramyxovirus does not have an HA, only a HN. Influenza or Orthomyxoviruses do have the HA and the HA and NA are separate proteins. Important thing is to remember is that the different glycoproteins that mediate entry and fusion can vary, but the premise and functions are the same. Sometimes they are combined in proteins or carried out by two different proteins. It is virus specific.

XVI.Genome Organization of all ssRNA viruses [S26]

  1. All have nucleocapsid proteins. All have the polymerase proteins. Some have separate F proteins and some that mediate through the G protein. Essentially, they use different strategies to do the same thing.

XVII.Virus Coding Strategies [S27]

  1. Won’t be on the exam.
  2. A lot of virus coding strategies because their genomes are so small.
  3. Later in the lecture there were a lot slides that went through the strategies of replication in the Paramyxoviridae, but this will not be covered today.
  4. [SQ]: Will we be expected to know these for the exam?
  5. [A]: No. They will not be on the exam.

XVIII.Genomic Strategies of RNA Viruses [S28-29]

  1. A main point of emphasis of the lecture.
  2. Important because of the RNA genome, they have to use different strategies than the DNA viruses.
  3. Read directly off the slide.
  4. Are required to have the RNA-dependent RNA polymerase
  5. The formation of the helical nucleocapsid is a characteristic of all negative strand viruses.
  6. P protein is a cofactor, which in the case of ssRNA viruses form a complex with the N and L and binds to RNA termini

XIX.Genome Configurations [S30]

  1. There are differences in the genome configurations of each of the categories.
  2. Examples of each are listed. Read directly off slide.

XX. Different Initial Activities of Input Genome [S31]

  1. Once the genome is released into the cytoplasm different initial activities occur.
  2. Positive strand RNA viruses – first activity is protein synthesis
  3. Negative strand RNA viruses – first activity is RNA synthesis
  4. Why is this? [S32] The RNA-dependent RNA Polymerase is required for initial transcription and synthesis of the positive strand, has multiple functions. It is not only responsible for transcription, but also for replication. This is the most conserved protein between the mononegaviralesvirus families.
  5. [S33] Host cells do not have a RNA-dependent RNA Polymerase, therefore the virus must provide its own. The virus uses two different strategies to provide its own RdRp:
  6. packaged within the virion (along with the genomic information)
  7. gets synthesized immediately
  8. [S34]: Solution #1 (ACTUALLY SOLUTION #2 FROM PREVIOUS SLIDE. IT’S OUT OF ORDER)
  9. Synthesized immediately
  10. When the virus enters the cell, the genome is already positive-sense so it can be translated
  11. You get the synthesis of a large polyprotein, which gets cleaved and releases the RdRp which is then able to replicate the RNA genome
  12. [S35] This process is shown schematically here
  13. Remember, for positive strand RNA viruses you have the positive sense RNA genome which immediately is translated into polyprotein…subsequent release of the RdRp allows for the synthesis of the negative strand template which is used to synthesize more positive strand templates (genomes), which gets packaged into the progeny
  14. [S36-37] Solution #2 The previous process is not the case for negative sense RNA viruses because they are starting out with a negative sense virus. They way they get around this (not being able to make a protein from the negative sense template) is they bring along the RdRp with it into the cell.
  15. Both the negative sense genome AND the RdRp get carried into the cell and the very first activity of the polymerase is to synthesize positive strand mRNAs.
  16. [S38] Here you have your negative genome, input, polymerase then is able to synthesize the positive strand template which is then used to synthesize the message and translate your different proteins
  17. The positive sense genome is also used as a template to make more negative genomes that gets packaged into the virus.

XXI.Skipped slides [S39] – [S45]

XXII.Important Human Pathogens from the Group of Negative-Strand RNA Viruses [S46, S47]

  1. The second major emphasis of the lecture starts here.
  2. To understand the human pathogens from this particular group of negative strand RNA viruses
  3. Different targets of the negative strand RNA viruses are shown here with regards to infections in humans…different family members like the Bunyaviruses cause encephalitis, meningitis-related viruses such as mumps, measles/rabies viruses are also pathogens that affect these organs.
  4. Measles can have ocular infections
  5. Respiratory related diseases are associated with negative strand RNA viruses as well
  6. [S48] Oral and Respiratory Diseases
  7. Everything highlighted in yellow are all from the family of negative-strand RNA viruses and cause these different diseases
  8. [S49] Arboviruses and Zoonoses
  9. Harbored in other animal species that can be transmitted to humans
  10. Abroviruses are viruses that use arthropod or insect hosts
  11. Bunya and Arena viruses are very common viruses in this family that are normally are present in reservoirs in small animals such as rats or mice and can also be transmitted through mosquitoes
  12. Arena viruses including Lymphocytic choriomeningitis virus (LCMV) is another family of viruses whose reservoir is in small animals
  13. Lassa fever virus is more of an old world infection can get transmitted through mice and rat droppings and it is important to remember that a lot of these (with the exception of the rabies virus) are highly infectious and can be transmitted through contact of contaminated feces.
  14. With rabies, the virus is transmitted through saliva upon getting bitten by an animal
  15. A lot of these can be acquired through aerosol and they are highly infectious!

XXIII.Arenaviruses/Bunyaviruses [S50]

  1. There is a lot of family members, so we will go through them quickly to give you an introduction to them talking about classification and reiterating some things that have already been said.

XXIV.Arena/Bunya Classification [S51]

  1. Read directly off slide
  2. Human zoonoses – humans get virus through other animals via a vector…the reservoir species are not affected by these viruses, they just carry them around
  3. Hantavirus, Nairovirus and Phlebovirus are all associatedwith hemorrhagic disease

XXV.Arenavirus Disease [S52]

  1. Illustrates types of diseases from a clinical standpoint and a geographical standpoint
  2. Most have already been mentioned

XXVI.Bunyavirus Genus [S53]

  1. Point of the table is to show the large number of family members in the Bunyavirus Genus
  2. Remember most of them are associated with mosquitoes or other animals

XXVII.Hantavirus [S54]

  1. Best known Bunyavirus

XXVIII.Arenavirus Genome Organization [S55 – S56]

  1. Arenavirus is one of the segmented negative strand RNA viruses
  2. Has 2 genomic segments – L RNA and S RNA
  3. Contrasting with Bunyaviruses that code for 3 segments (L,M & S – long, medium and short)

XXIX.Bunyavirus L segment Coding [S57]

  1. This diagram shows the different genuses and different L segment, organization and their lengths
  2. All of the coding strategies are negative sense on the L segment (for comparison see next point)

XXX.Bunyavirus M segment Coding [S58]

  1. In this M segment coding, one family has a different strategy than just all negative sense
  2. Ex: Tospovirus - have an ambisense coding strategy.

XXXI.Bunyavirus S segment Coding [S59]

  1. Ambisense coding in Tospovirus and Phlebovirus in S segment

XXXII.Filoviruses [S60, S61]

  1. They look like filaments/fibers
  2. Ebola virus & Marburg Virus
  3. Reston Virus outbreak in Virginia due to infected monkeys
  4. [S62] – EM of Filovirus
  5. [S63] - skipped

XXXIII.Filovirus Pathogenesis [S64]

  1. VERY hemorrhagic viruses
  2. Infect the macrophages, especially those that circulate in the blood stream (your immune cells) and this is the primary target of the filovirus replication…because of this they get distributed all over the body (lungs, kidneys, endothelial cells spleen, etc.) and you have total organ infection with rapid progression

XXXIV.Bornaviruses [S65, S66]

  1. Is the exception to the other single stranded RNA viruses in that they replicate in the nucleus and some of its mRNA transcripts are spliced.
  2. Read directly off slide
  3. There is still question as to their affect in humans.

XXXV.Rhabdoviruses [S67, S68]

  1. Divided into two family members: VSV and Lyssavirus (which includes rabies)
  2. These vectors are found in mammals and mosquitoes and different insect populations
  3. Rabies virus are present in bats, raccoons and rats that can be transmitted to other animals and/or humans
  4. [S69] – EM of Rhabdovirus;look like bullets
  5. [S70] Pathogenesis
  6. Read steps directly off slide
  7. Know when the signs start to occur – when it reaches the brain

XXXVI.Paramyxoviruses [S71 – S73]

  1. Single stranded genome
  2. Includes:
  3. Sendai Virus
  4. Human Parainfluenza Virus types 1 and 3
  5. Simian Virus 5
  6. New Castle Disease Virus
  7. Measles Virus
  8. Canine Distemper Virus
  9. Respiratory Syncytial Virus – big problem for infants in that it can cause severe illness/death
  10. As adults, we show no other serious symptoms
  11. [S74] - skipped
  12. [S75] Genomic Organization of different Paramyxovirus members
  13. presence of proteins conserved across the family members (especially the L protein, matrix, fusion and nuclear proteins)
  14. [S76] HRSV
  15. Read directly off slide
  16. Most deaths (0.2%) are in infants or immunocompromised
  17. Economic impact – developing a vaccine to this virus is a high priority

XXXVII. Measles Virus [S77]

  1. Read directly off slide
  2. Total body rash occurs-evidence of a systemic infection
  3. Don’t hear about Measles in the U.S. too much anymore because we have developed a vaccine (MMR) for it, but in other parts of the world it is a big problem
  4. [S78] Neurological Complications
  5. The onset of the virus can be many years later (as many as 12 years later)
  6. But remember, we have a vaccine!

XXXVIII.Why Do We Care? (see slide 79)

  1. Influenza wasn’t focused on a lot in this lecture because it is a whole lecture in itself. With influenza you have a constant change with the dominant strain circulating and therefore there is no long-standing vaccine against it that is effective.
  2. [S79] NIH List of Re-emerging Infectious Diseases
  3. Not all of these are ssRNA viruses, but just take a look over this slide at some viruses that have shown up previously in the lecture

[SQ] How do we need to approach the material?

Understand why negative strand are different from other viruses, how they replicate (RdRp)

Unique characteristics (vs. positive stranded RNA viruses and DNA viruses)

Common themes (e.g. hemorrhagic diseases)

Also, know which are associated with hemorrhagic conditions

How they are acquired (from secretions, animals)

Know different viral proteins that mediate transcription.

Respiratory associated viruses (common theme of proteins across families)

Very similar symptoms among some of these viruses…know those similarities

Understand replication and common theme of proteins.

Exam questions focus on replication, different proteins that are unique to these viruses and the diseases associated with it. Be able to pick out something that doesn’t belong.

[end 53:05]

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