Replication of Negative-Sense RNA Viruses (Mutipartite)

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Replication of Negative-Sense RNA Viruses
(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)
Hemagglutinin subtype
Year of isolation
Influenza type
(H3N2)A/Brisbane/10/2007
Isolate number
Geographic source
Neuraminidase subtype
Influenza 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 (5cap, 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) gt20 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
• gt10,000 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 - Whos Afraid Of The Big Bad Bird Flu
  (H5N1)?
• 2009 - Whos 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 Reyes Syndrome (injury to liver,
  encephalopathy)

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Flu Vaccine
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Flu Vaccine Risks vs. Benefits

• gt Million flu infections/year in USA
• gt100,000 hospitalizations/year due to flu
• gt20,000 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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Dont 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 cant 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 teams 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 (mlee_at_LABioMed.org) to post on
  Instructional1 for class study or save to
  computer in classroom