Replication of Negative-Sense RNA Viruses (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

Hemagglutinin subtype Neuraminidase subtype World Health Organization Influenza Nomenclature (One of three strains in 2009 Vaccine) Hemagglutinin subtype Influenza type Year of isolation (H3N2)A/Brisbane/10/2007 World Health Organization (WHO) Influenza Nomenclature The WHO has devised the nomenclature system for tracking influenza virus antigenic variants and new strains of influenza. Subtypes of influenza type A are determined by surface antigens hemagglutinin (HA) and neuraminidase (NA); influenza type B has only one subtype; influenza type B surface HA and NA do not demonstrate the major variability that occurs suddenly in influenza type A (the basis for new subtypes). Influenza type A (H1N1) designates HA subtype 1 and NA subtype 1, whereas influenza type A (H3N2) designates HA subtype 3 and NA subtype 2. Geographic source Isolate number Neuraminidase subtype Influenza 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