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Replication of Negative-Sense RNA Viruses (Mutipartite)

Replication of Negative-Sense RNA Viruses (Mutipartite). (-)RNA Virus Mutipartite Genome. Orthomyxoviridae 8 gene segments Bunyaviridae 3 gene segments (L, M, S; some S gene are ambisense) Arenaviridae 2 gene segments (L, S; both ambisense). Family Orthomyxoviridae. “normal” “mucus”

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Replication of Negative-Sense RNA Viruses (Mutipartite)

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  1. Replication of Negative-Sense RNA Viruses (Mutipartite)

  2. (-)RNA Virus Mutipartite Genome • Orthomyxoviridae • 8 gene segments • Bunyaviridae • 3 gene segments (L, M, S; some S gene are ambisense) • Arenaviridae • 2 gene segments (L, S; both ambisense)

  3. Family Orthomyxoviridae • “normal” “mucus” • (-)RNA • Envelope , large peplomers, 120 nm • Helical nucleocapsid, 15 nm; ribonucleoprotein (RNP)

  4. Genus: Influenza Virus • “influence” malign, supernatural • Envelope glycoproteins: • HA (1-16), NA (1-9) • Human groups (identify by capsid NP): • Type A infect humans and animals; epidemics • Type B infects humans; epidemics • Type C infects humans; mild disease

  5. Classification of Human Influenza Virus • HA: H1, H2, H3 (H5, H7, H9 rare, does not spread well human-human) • NA: N1, N2 • Type A or B • Geographic source • Isolate number • Year of isolation

  6. World Health Organization Influenza Nomenclature(One of three strains in 2009 Vaccine) Hemagglutinin subtype Year of isolation Influenza type (H3N2)A/Brisbane/10/2007 Isolate number Geographic source Neuraminidase subtype Influenza type B does not occur as subtypes.

  7. Influenza Virus: (-)RNA Genome • Eight gene segments (2.3 – 0.9 kb) • Total genome = 13.6 kb • Ten mRNAs translate for ten viral proteins (two smallest mRNAs are spliced) • Replication occurs in cell nucleus & cytoplasm

  8. Influenza Virus: Entry / Uncoating • Entry by receptor-mediated endocytosis • Release of eight separate RNP into cytoplasm • RNP transported into nucleus • Viral transcription occurs in nucleus

  9. Influenza Virus: mRNA • Transcription complex: • Viral (-)RNA genome • Three viral polymerase-associated proteins (PB1, PB2, PA) • “Cap snatching” viral endonuclease cleaves cell 5’ cap mRNA (10-13 bases) • Cell 5’ cap mRNA12-13 serves as “primer” for viral mRNA transcription

  10. Influenza Virus: mRNAs • Eight mRNAs transcribed • Two smallest mRNAs (Segment 7, 8) spliced • Matrix: M1, M2 • Nonstructual: NS1, NS2

  11. Influenza Virus: mRNA Translation • Ten mRNAs (5’cap, 3’ polyA tail) • Transport from nucleus to cytoplasm • Translation on cell ribosome for ten viral proteins

  12. Influenza Virus: Antigenome (RI-1) • (-)RNA genome serves as template • Synthesis of viral proteins in cytoplasm (NP, PB1, PB2, PA) and transport into nucleus • Increase levels of NP switch transcription to uncapped (+)RNA antigenome

  13. Influenza Virus: Genome (RI-2) • (+)RNA antigenome serves as template • (-)RNA genome copied from antigenome: • Template for viral mRNA • For progeny virus • Assembly of RNP: genome (-)RNA, NP, PB1, PB2, PA in nucleus • Transported out to cytoplasm by viral M1 and NS2

  14. Influenza Virus: Assembly & Release • HA, NA, M2 proteins glycosylated in ER / Golgi and inserted into plasma membrane • Viral RNP associates with matrix (M1) protein, guided to virus modified plasma membrane • Virus exits by budding

  15. Virus Respiratory Infections • Primary site – oral & respiratory mucosa, ±eye • Migrate to lymphatic tissue • Enters blood (fever, malaise) • Secondary site - reticuloendothelial system organs (liver, spleen, bone marrow) • Re-enters blood and infects other target organs (extremities & skin, RT, GI tract, CNS, heart)

  16. Influenza Infection/Disease • Virus replication in RT • Host defense compromised: • Destroys ciliated cells • MØ, T cells impaired • Viral or 2° bacterial pneumonia (Staphylococcus, Streptococcus, Haemophilus)

  17. Influenza Epidemiology • Endemic - Winter, peaks Dec - Jan • Epidemics every ~5 years • Pandemics every ~10 years • 1918 Spanish (H1N1) >20 M deaths • 1957 Asian (H2N2) 80 M infected, USA 88,000 deaths • 1968 Hong Kong (H3N2) USA 34,000 deaths • 1977 Russian (H1N1) • USA estimates each year • 10-20% get flu • >10,000 hospitalizations for flu-related complications • ~36,000 deaths from complications of flu

  18. Influenza Virus Epidemics • Ability of virus to change • Antigenic “drift” – gradual variation in HA, NA due to high RNA mutation rate • Antigenic “shift” – major variation due to dual infection and gene reassortment • Origin of new influenza A virus strains by exchange between different animal species i.e. avian » pigs » humans

  19. Antigenic Drift & Shift

  20. 1997 - Who’s Afraid Of The Big Bad Bird Flu (H5N1)? • 2009 - Who’s Afraid Of The Big Bad Swine Flu (H1N1)?

  21. Influenza Treatment • Antivirals: • Rimantadine for Flu A • Tamiflu and Relenza for Flu A & B) • Inactivated killed whole virus or subunit vaccine (HA, NA) for: • Elderly, nursing home residents • Patients with chronic diseases • Health care workers • Anyone desiring protection • Live cold adapted (25ºC) virus vaccine: • Given as nasal spray • Ages 5-50 years • Use of aspirin to treat fever due to virus infection of children contraindicated; associated with Reye’s Syndrome (injury to liver, encephalopathy)

  22. Flu Vaccine

  23. Flu Vaccine: Risks vs. Benefits • > Million flu infections/year in USA • >100,000 hospitalizations/year due to flu • >20,000 – 40,000 deaths/year due to flu or its complications • Vaccine Side Effects (What to Expect Flu Shot) • Kill inactivated, cannot get flu • Soreness, redness, swelling • Fever (low grade) • Aches • Rare serious problem – allergic reaction toegg protein

  24. “Don’t Blame Flu Shots for All Ills, Officials Say” • N. Y. Times, Sept. 28, 2009 • Dr. Harvey V. Fineberg, President, Institute of Medicine • Every year: • 1.1 million heart attacks • 795,000 strokes • 876, 000 miscarriages • 200,000 have first seizure

  25. Similar Genomes: (-) RNA Viruses

  26. Reading & Questions • Chapter 15: Replication Strategies of RNA Viruses Requiring RNA-directed mRNA Transcription as the First Step in Viral Expression.

  27. QUESTIONS???

  28. Class Discussion – Lecture 7a • 1. Why can’t influenza virus replicate in a cell where the nucleus has been removed? • 2. You lab is researching the Spring fever virus (SpFV) and the debilitating variant SpFV-4 that causes senioritis. Others have identified SpFV as an Influenza virus but your team’s research results show it may be a new genus tenatively called Procrastinovirus. The following table list properties of SpFV strains studied in your lab:

  29. (a) Which features of SpFV are similar to Influenza virus? • (b) Which features are different from Influenza virus? • (c) Which viral proteins do you predict will be different between SpFV and SpFV-4? • (d) What might account for the ability of SpFV-4 strain to produce senioritis?

  30. Family Bunyaviridae • (-)RNA • Envelope, 90-120 nm • Three helical, circular, nucleocapsids, 2.5 nm • Most are arboviruses • Infect arthropods, birds, mammals

  31. Bunyaviridae: (-)RNA Genome • Three segments of (-)RNA: • L = polymerase (RNA pol) • M = G1, G2 (envelope gp), NSM • S = RNP (nucleocapsid), ± NSS • Total: 13- 21 kb

  32. Genus: Bunyavirus • Mosquito vector • Bunyamwera virus – Africa; fever, rash, encephalitis • California encephalitis virus – endemic in USA • La Crosse encephalitis virus - endemic in USA

  33. Genus: Phlebovirus • “vein” • Sandfly vector • Rift valley fever virus – Africa • Often fatal hemorrhagic fever

  34. Genus: Hantavirus • Transmission by contact with rodent excreta • Hantaan virus – Korea; hemorrhagic fever + renal syndrome • Sin Nombre virus – S.W. USA; hantavirus adult respiratory distress syndrome (HARDS)

  35. Various Coding Strategy for Bunyaviridae S Gene • Virus replication occurs in cytoplasm • Transcribe mRNA for N, ±NSS protein • mRNA has 5’ cap, 3’ no polyA tail

  36. Coding Strategy for S Gene Hantavirus: No NS • Transcribe single mRNA for N protein • Does not code for NSS protein

  37. Coding Strategy for S Gene Bunyavirus: Overlapping ORF • Two partially overlapping ORFs • NSS ORF within N ORF • Transcription of a single mRNA • Translation for both N and NSS proteins using alternate reading frame of mRNA

  38. Coding Strategy for S Gene Phlebovirus: Ambisense Genome • S genome RNA, two ORF: • (+)NSs gene • (-)N gene • Transcribes for two subgenomic mRNAs: • N mRNA from genome • NSS from antigenome

  39. Similar Genomes: (-) RNA Viruses

  40. Family Arenaviridae • “sandy” – ribsomes in virions • (-)RNA • Envelope, 90-100 nm • Two helical, circular nucleocapsids, 9-15 nm • Natural hosts are rodents • Virus transmission by excreta

  41. Genus: Arenavirus • Lymphocytic choriomeningitis virus (LCM) – mild “flu” in mice, humans • Lassa fever virus – Africa; highly fatal hemorrhagic fever, Biosafety Level 4 pathogen • Junin virus – Argentine hemorrhagic fever • Machupo virus – Bolivian hemorrhagic fever

  42. Arenavirus: (-)RNA Genome • Two RNA segments • Total genome = 10 kb • Both are ambisense genomes

  43. LCM: Persistent Infections • Infection of host early in life • Persistent chronic infection • Viremia • Virus shedding in saliva and urine • Little or no neutralizing antibody • Model to study virus/host factors for chronic infections

  44. Similar Genomes: (-) RNA Viruses

  45. Reading • Chapter 15: Replication Strategies of RNA Viruses Requiring RNA-directed mRNA Transcription as the First Step in Viral Expression.

  46. QUESTIONS???

  47. Class Discussion – Lecture 7b • 1. How are two different ways Bunyavirus makes more than one protein from a “monocistronic” mRNA? • 2. Why are the (-)RNA viruses thought to have appeared fairly recently?

  48. Group Case Study • Tuesday, Oct. 30: • Group 6 – Influenza Virus • Group 7 – Bunyavirus • Group 8 - Prions • Ten minute oral presentation on patient case history and questions using PowerPoint • Written report due in class (also for Group #1-5) • Email PowerPoint and Word file of report to Instructor (mlee@LABioMed.org) to post on Instructional1 for class study or save to computer in classroom

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