Replication of Replication of Negative-Sense Negative-Sense

RNA Viruses RNA Viruses (Mutipartite)(Mutipartite)

(-)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)

Family Orthomyxoviridae

• “normal” “mucus”

• (-)RNA• Envelope , large

peplomers, 120 nm

• Helical nucleocapsid, 15 nm; ribonucleoprotein (RNP)

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

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

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.

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

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

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

Influenza Virus: mRNAs

• Eight mRNAs transcribed

• Two smallest mRNAs (Segment 7, 8) spliced– Matrix: M1,

M2– Nonstructual:

NS1, NS2

Influenza Virus: mRNA Translation

• Ten mRNAs (5’cap, 3’ polyA tail)

• Transport from nucleus to cytoplasm

• Translation on cell ribosome for ten viral proteins

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

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

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

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)

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)

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

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

Antigenic Drift & Shift

• 1997 - Who’s Afraid Of The Big Bad Bird Flu (H5N1)?

• 2009 - Who’s Afraid Of The Big Bad Swine Flu (H1N1)?

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)

Flu Vaccine

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

“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

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 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:

• (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?

Family Bunyaviridae• (-)RNA• Envelope, 90-120 nm• Three helical, circular,

nucleocapsids, 2.5 nm• Most are arboviruses• Infect arthropods, birds, mammals

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

Genus: Bunyavirus• Mosquito vector• Bunyamwera virus – Africa; fever,

rash, encephalitis• California encephalitis virus –

endemic in USA• La Crosse encephalitis virus -

endemic in USA

Genus: Phlebovirus• “vein”• Sandfly vector• Rift valley fever virus – Africa• Often fatal hemorrhagic fever

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)

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

Coding Strategy for S Gene Hantavirus: No

NS• Transcribe single mRNA for N

protein• Does not code for NSS protein

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

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

Similar Genomes: (-) RNA Viruses

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

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

Arenavirus: (-)RNA Genome

• Two RNA segments• Total genome = 10 kb• Both are ambisense genomes

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

Similar Genomes: (-) RNA Viruses

Reading• Chapter 15: Replication

Strategies of RNA Viruses Requiring RNA-directed mRNA Transcription as the First Step in Viral Expression.

QUESTIONS???

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?

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@LABioMed.org) to post on Instructional1 for class study or save to computer in classroom