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 Vesicular stomatitis virus (VSV) Infect animals (cow, swine, horse), arthropods (mosquito, fly) Exanthem (rash) Vesicles (oral, skin), ulcerate

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

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

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

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

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

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

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

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

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

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

Similar Genomes: (-) RNA Viruses

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

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

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)

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

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.

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

Virus Cell Culture - Shell Vial

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

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 Marburg virus: Germany, 25 Lab researchers infected handling monkeys from Uganda, 7 (28%) deaths Ebola virus: Republic of Congo (Zaire), 318 (88%) deaths Reston virus: Virginia (USA), primate colony, monkeys imported from Philippines; respiratory transmission to other monkeys; most infected monkeys die, no transmission to humans

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?

Similar Genomes: (-) RNA Viruses

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

QUESTIONS???

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.

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 PowerPoint and Word file of report to Instructor to post on Instructional1 for class study or save to computer in

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: mg/dl (decrease = bacteria) –Protein: mg/dl (increase = bacteria) –Total nucleated cells: 0-3/ul (increase = bacteria, virus)