Replication of Negative-Sense RNA Viruses (Mutipartite)
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Replication of Replication of Negative-Sense Negative-Sense
RNA Viruses RNA Viruses (Mutipartite)(Mutipartite)
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(-)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)
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Family Orthomyxoviridae
• “normal” “mucus”
• (-)RNA• Envelope , large
peplomers, 120 nm
• Helical nucleocapsid, 15 nm; ribonucleoprotein (RNP)
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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
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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
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World Health Organization Influenza Nomenclature
(One of three strains in 2009 Vaccine)
Influenza type
Hemagglutinin subtype
Geographic source
(H3N2)A/Brisbane/10/2007
Year of isolation
Isolate number
Neuraminidase subtypeInfluenza type B does not occur as subtypes.
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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
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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
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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
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Influenza Virus: mRNAs
• Eight mRNAs transcribed
• Two smallest mRNAs (Segment 7, 8) spliced– Matrix: M1,
M2– Nonstructual:
NS1, NS2
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Influenza Virus: mRNA Translation
• Ten mRNAs (5’cap, 3’ polyA tail)
• Transport from nucleus to cytoplasm
• Translation on cell ribosome for ten viral proteins
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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
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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
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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
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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)
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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)
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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
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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
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Antigenic Drift & Shift
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• 1997 - Who’s Afraid Of The Big Bad Bird Flu (H5N1)?
• 2009 - Who’s Afraid Of The Big Bad Swine Flu (H1N1)?
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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)
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Flu Vaccine
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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
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“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
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Similar Genomes: (-) RNA Viruses
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Reading & Questions• Chapter 15: Replication Strategies
of RNA Viruses Requiring RNA-directed mRNA Transcription as the First Step in Viral Expression.
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QUESTIONS???
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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:
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• (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?
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Family Bunyaviridae• (-)RNA• Envelope, 90-120 nm• Three helical, circular,
nucleocapsids, 2.5 nm• Most are arboviruses• Infect arthropods, birds, mammals
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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
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Genus: Bunyavirus• Mosquito vector• Bunyamwera virus – Africa; fever,
rash, encephalitis• California encephalitis virus –
endemic in USA• La Crosse encephalitis virus -
endemic in USA
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Genus: Phlebovirus• “vein”• Sandfly vector• Rift valley fever virus – Africa• Often fatal hemorrhagic fever
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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)
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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
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Coding Strategy for S Gene Hantavirus: No
NS• Transcribe single mRNA for N
protein• Does not code for NSS protein
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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
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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
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Similar Genomes: (-) RNA Viruses
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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
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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
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Arenavirus: (-)RNA Genome
• Two RNA segments• Total genome = 10 kb• Both are ambisense genomes
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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
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Similar Genomes: (-) RNA Viruses
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Reading• Chapter 15: Replication
Strategies of RNA Viruses Requiring RNA-directed mRNA Transcription as the First Step in Viral Expression.
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QUESTIONS???
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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?
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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 ([email protected]) to post on Instructional1 for class study or save to computer in classroom