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Replication of Negative-Sense RNA Virus (Monopartite)

Replication of Negative-Sense RNA Virus (Monopartite). (-)RNA Virus Replication. Family Rhabdoviridae. “rod” (-)RNA viruses Coiled nucleocapsid into cylindrical structure Envelope, large spikes, 70x180 nm, “bullet-shape”. Genus: Vesiculovirus. “vesicles” – fluid filled lesions

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Replication of Negative-Sense RNA Virus (Monopartite)

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  1. Replication of Negative-Sense RNA Virus (Monopartite)

  2. (-)RNA Virus Replication

  3. Family Rhabdoviridae • “rod” • (-)RNA viruses • Coiled nucleocapsid into cylindrical structure • Envelope, large spikes, 70x180 nm, “bullet-shape”

  4. Genus: Vesiculovirus • “vesicles” – fluid filled lesions • Vesicular stomatitis virus (VSV) • Infect animals (cow, swine, horse), arthropods (mosquito, fly) • Exanthem (rash) • Vesicles (oral, skin), ulcerate

  5. Genus: Lyssavirus • “frenzy” • Rabies virus • Infect most mammals (fox, bat, dog) • Transmission by animal bite • Fatal encephalitis in humans (dead-end host)

  6. VSV: (-)RNA Genome • 11 kb, linear, non-segmented • Five ORF: • N (nucleocapsid) • P (NS; phosphoprotein) • M (matrix) • G (glycoprotein) • L (large; NS, RNA polymerase) • Cannot serve as mRNA

  7. VSV: mRNA Transcription • Partial virus uncoating, release viral RNA complex into cytoplasm • Entry site for RNA pol at 3’ end of viral RNA genome • Transcription complex: • Viral (-)RNA genome • N protein • L protein (RNA pol, replicative enzymes) • Phosphoprotein

  8. VSV: Five mRNA Transcripts • Transcribes a mRNA for each ORF of genome • 5’ methyl cap added • RNA pol “stutters” at intergenic region (poly-U sequence) for polyadenylation • Transcription complex most often falls off, starts transcription again at 3’ end of viral genome

  9. VSV: Regulation of mRNA Levels • Sometimes Replicase continues on to transcribe next ORF • Results in different amounts of mRNA (N>P>M>G>L)

  10. VSV: mRNA Translation • Translation on cell ribosome immediately follows mRNA transcription • Amounts of protein follows levels of mRNA (N>P>M>G>L)

  11. VSV: Functions of M Protein • Multifunctional • Structual matrix protein for virion • Down-regulates viral mRNA transcription (negative feed-back) • Interferes with cell protein transport in & out of nucleus, results in nonfunctional cell nucleus

  12. VSV: Transcription of Antigenome (RI-1) • (-)RNA genome serves as template • Requires adequate amounts of N protein • N protein complex with (+)RNA being transcribed • Prevents “stutter” at intergenic region • Read through “stop” and continues on to make full-length (+)RNA antigenome

  13. VSV: Replication of Genome RNA (RI-2) • (+)RNA antigenome serves as template • Transcription complex (N, P, L proteins) • (-)RNA genome synthesis • Ratio (+)RNA to (-)RNA = 1 to 6

  14. VSV: Assembly & Release • (-)RNA genome associates with N, P, L proteins • G glycoprotein inserted into plasma membrane • M protein associates with inserted G, and guides nucleocapsid to bud through virus specific plasma membrane • Release of enveloped virus

  15. Similar Genomes: (-) RNA Viruses

  16. Family Paramyxoviridae • “apart from” “mucus” • (-)RNA viruses • Pleomorphic envelope, 150x200 nm • Helical nucleocapsid, 18x1000 nm

  17. Genus: Paramyxovirus • Human parainfluenza virus – RTI, bronchitis, pneumonia • Mumps virus – parotitis (salivary gland), meningoencephalitis • Sendai virus – RTI in mice • Newcastle disease virus - RTI in chickens

  18. Paramyxovirus: (-)RNA Genome • 15 kb, linear, non-segmented • Six ORF: • NP (nucleocapsid) • P (phosphoprotein) • M (matrix) • FO (fusion) • HN (hemagglutinin, neuraminidase) • L (NS, RNA polymerase)

  19. Genus:Morbillivirus • “measles”, “spot” • Rubeola virus – “red”, maculopapular rash • Annual epidemics worldwide • Developing countries often fatal for infants, underlying malnourishment (>1M deaths each year) • Small number persistent infections; present later with fatal subacute sclerosing panencephalitis (SSPE) • Canine distemper virus – RTI, systemic infection of dogs

  20. Genus: Pneumovirus • “lung” • Respiratory syncytial virus (RSV) – RTI in newborne/young children • cold, bronchitis, pneumonia • Winter epidemics, often requiring hospitalization for supportive treatment • Rarely fatal in U.S.

  21. RSV: Shell Vial Cell Culture • The “gold standard” for virus detection • Grow cells on glass slide in small vial • Patient sample (NP swab/wash of virus & infected cells) centrifuged onto cells of coverslip in shell vial • Enhance detection of virus antigen by fluorescein-tagged specific monoclonal antibody • Sensitive – fluorescence under UV microscope • Specific – antibody against RSV antigen

  22. Virus Cell Culture - Shell Vial

  23. Family Filoviridae • “thread” • (-)RNA, 19 kb • Seven ORF • Enveloped • Rigid helical nucleocapsid, 80x800 nm • Some are Biosafety Level 4 pathogen (physical barrier protection) causing fatal hemorrhagic fever

  24. Genus: Filovirus • Direct contact infected body fluids, aerosol • From site of infection into blood; rash, severe hemorrhagic fever; capillary cells infected, tissue necrosis, edema, shock • Animal host reservoir in nature unknown • 1967 - Marburg virus: Germany, 25 Lab researchers infected handling monkeys from Uganda, 7 (28%) deaths • 1976 - Ebola virus: Republic of Congo (Zaire), 318 (88%) deaths • 1989 - Reston virus: Virginia (USA), primate colony, monkeys imported from Philippines; respiratory transmission to other monkeys; most infected monkeys die, no transmission to humans

  25. Bornavirus • 1885 epidemic Borna, Germany • (-) RNA, 9 kb, six ORF • Calvary horses die following abnormal behavior; run excitedly, walk into walls, unable to chew • Encepalomyelitis of horses, sheep, cattle • Link virus in humans with neuropsychiatric disorders?

  26. Similar Genomes: (-) RNA Viruses

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

  28. QUESTIONS???

  29. Class Discussion – Lecture 6 • 1. Why does the virion of (-)RNA virus all have an RNA polymerase? • 2. How does vesicular stomatitis virus regulate its mRNA transcription? Protein synthesis? • 3. State numerous factors involved in Ebola virus being a deadly pathogen for us.

  30. Group Case Study Report • Tuesday, Oct. 23 • Group 1: Enterovirus • Group 2: Norwalk Virus • Thursday, Oct. 25 • Group 3: Dengue Virus • Group 4: Rabies Virus • Group 5: Respiratory Syncytial Virus • Ten minute oral presentation on patient case history and questions using PowerPoint • Written report for Group #1-5 due in class on Tuesday, October 30 • 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

  31. Table of Normal Values WBC Total Count: 4,000 – 12,000/ul (increase = bacteria) WBC Differential Count: Neutrophils: 50%-70% (increase = bacteria) Lymphocytes: 20%-30% (increase = some virus) Monocytes: 2%-6% (increase = some virus) Eosinophils: 1%-5% (increase = parasite) Basophils: <1% Cerebrospinal Fluid (CSF): Glucose: 50-75 mg/dl (decrease = bacteria) Protein: 15-45 mg/dl (increase = bacteria) Total nucleated cells: 0-3/ul (increase = bacteria, virus)

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