Transcript 06 M401 (-)RNA Virus (Mono) 2012 - Cal State LA

Replication of
NegativeSense RNA
Virus
(Monopartite)
(-)RNA Virus Replication
Family Rhabdoviridae
• “rod”
• (-)RNA viruses
• Coiled nucleocapsid into cylindrical
structure
• Envelope, large spikes, 70x180 nm, “bulletshape”
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, nonsegmented
• 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 (polyU 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, nonsegmented
• Six ORF:
–
–
–
–
–
NP (nucleocapsid)
P (phosphoprotein)
M (matrix)
FO (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
• 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
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 RNAdirected 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
• Email PowerPoint and Word file of report to
Instructor ([email protected]) to post on
Instructional1 for class study or save to computer in
classroom
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)