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) –F O (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 classroommlee@LABioMed.org

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)