single strand, negative sense RNA Viruses

Elliot J. Lefkowitz

•Email

•ElliotL@uab.edu•Web Site

•http://www.genome.uab.edu•Office

•BBRB 277A

•Phone•934-1946

Contact Information: Elliot Lefkowitz, Ph.D.

Associate Professor, Microbiology

Objectives

•To understand the fundamental common and distinguishing properties of (-) ssRNA viruses

•To understand the basic replication strategies of (-) ssRNA viruses

•To be able to identify human pathogens that belong to (-) ssRNA virus families, and some of their biological and pathogenic properties

Reading

•Medical Microbiology, Murray et al. 6th Edition•General classification

•Chapter 4•RNA virus properties and replication

•Chapters 58, 59, 60, 63•Pathogenesis

•Chapters 48, 67

Slide References

•Fields Virology, 5th Edition•Viruses and Human Disease

•Strauss and Strauss•University of Leicester - Virology Online

•http://www-micro.msb.le.ac.uk/3035/index.html

•International Committee on Taxonomy of Viruses•The 9th ICTV Report

•Primary literature

Virus classification

The Virus World

The (-) RNA Virus World

RNA Virus Genome Structure

•Number of strands•Single or double stranded

•Strand polarity•Positive, negative, or ambisense (both +

and -)•Positive (Plus) sense denotes the coding

(mRNA) strand•Number of segments

•Single or multi-segmented

single strand RNA virus genome polarity

virion RNA (+) sense

virus mRNA (+) sense translation

3’5’

translationtranscriptionvirion RNA (-) sense

virus mRNA (+) sense 3’5’5’3’

(+) sense RNA virus

(-) sense RNA virus

Negative/Ambisense ssRNA Viruses

Properties of (-) sense ssRNA Viruses

• Enveloped virion• Helical nucleocapsid• Negative-sense, linear, single segment RNA genome

• Bornaviruses, Filoviruses, Rhabdoviruses, Paramyxoviruses

• Negative and Ambisense, linear, multi segment RNA genomes• Arenaviruses, Bunyaviruses, Orthomyxoviruses

• Cytoplasmic replication• Exception: Bornaviruses, Orthomyxoviruses

• Genomes are non-infectious• An initial round of transcription is required for

genome replication• Virion must contain proteins required for transcription

• Bornaviridae• Bornavirus

• Filoviridae• Marburg virus• Ebola virus

• Paramyxoviridae• Paramyxovirinae

• Henipavirus• Morbillivirus• Respirovirus• Rubulavirus

• Pneumovirinae• Pneumovirus• Metapneumovirus

• Rhabdoviridae• Vesiculovirus• Lyssavirus

Order: Mononegavirales:Single segment, (-) sense, ssRNA

Multi-Segment, (-) sense ssRNA viruses

•Orthomyxoviridae•Influenzavirus A

•8 genome segments•Influenzavirus B

•8 genome segments•Influenzavirus C

•7 genome segments•Isavirus

•8 genome segments•Thogotavirus

•6 genome segments

Multi-Segment, Negative and Ambisense ssRNA viruses

•Arenaviridae•Two ambisense RNA segments

•Bunyaviridae•Three RNA segments•Both negative-sense and ambisense

segments•Depends on genus

The Virus

Virion, Genome, Proteins

Viral Proteins•Attachment/entry

•G – Membrane glycoprotein•F – Fusion protein•H – Hemagglutinin•N – Neuraminidase

•Structural/Assembly•M – Matrix

•Underlies lipid bylayer•Replication

•N – nucleocapsid protein•P – Phosphoprotein•L – RNA dependent RNA polymerase

Rhabdovirus Virion

Virus replication Machinery•Proteins

•RNA-dependent RNA-polymerase (RdRp)•Transcription•Replication

•Nucleocapsidprotein (N)•Encapsidates RNA•Forms helical nucleocapsid

•P protein•Phosphoprotein - polymerase cofactor•Forms complexes with N and L•Binds to RNA termini

•RNA Genome

Genome Organization Mononegavirales

Filoviridae

Paramyxoviridae

Rhabdoviridae

Genome OrganizationArenaviridae

Bunyaviridae

Influenza A Genome Structure

Virus Coding Strategies•Individual ORFs

•Multiple transcripts with transcription attenuation

•Polyprotein processing•Single transcript to Large polyprotein:

Proteolytic processing•RNA Editing

•Insertion/deletion of additional residues (at a specified site) altering the reading frame

•Multiple ribosomal initiation sites•Stop codon read-through

Virus Replication

RNA-dependent RNA Polymerase(RdRp – L Protein)

•Catalytic subunit of the polymerase complex•Polymerization of nucleotides

•Transcription of mRNA•Capping•Methylation•Polyadenylation

•Genome Replication•Most conserved protein between the

mononegavirales virus families

Source of the RNA-dependent RNA Polymerase

•Host cells do not have a suitable one •Therefore the virus must provide its own•RNA viruses use 2 different strategies to

provide the RdRp:•Synthesized immediately upon entry

and unpackaging of the virion into the cell (positive-sense viruses)•Therefore protein synthesis is the first

step in the replication process•Packaged within the virion (negative-

sense viruses)•Therefore mRNA transcription is the

first step in the replication process

VSV Transcription & Replication

(-) sense ssRNA virus Human Pathogens

Major Viral Target Tissues

Arenaviruses/Bunyaviruses

Arenavirus and Bunyavirus Disease

• Arenaviruses• Mostly rodent viruses

• Human zoonoses• Junin virus

• Argentine hemorrhagic fever• Lassa Fever

• Bunyaviruses• Large group of arthropod-borne viruses

• Human pathogens – hemorrhagic fever• Hantaviruses

• Rodent-borne• Pulmonary Syndrome/Hemorrhagic fever

• Rift Valley Fever virus• Mosquito-borne virus

Filoviruses

Filovirus Disease

Rhabdoviruses

Rhabdoviruses

Rabies virus Pathogenesis

Paramyxoviruses

Paramyxoviruses

Human Respiratory Syncytial virus

•Major cause of lower respiratory tract infections•Rarely life-threatening• Individuals get repeat infections

•Highly infectious•Spread is by exchange of respiratory secretions• Infection confined to respiratory tract

•Globally: 100,000,000 infections/year•200,000 deaths/year

• In USA: All infants by age of 4 years are infected•100,000 hospitalizations/year•Estimated cost of $300,000,000/year (1985) •25-50% of hospital staff infected during

outbreaks

Measles virus•Extremely infectious

•Spreads through contact with respiratory secretions

•Victims are infectious before symptoms are evident

•Develops systemic infection•Globally: 45,000,000 infections/year

•1,000,000 deaths/year• In USA: Infections are rare•Occasional epidemic in unvaccinated

populations•MMR (Measles, mumps, and rubella) vaccine

highly effective (2 shots)

Acute Disseminated EncephalomyelitisMeasles Inclusion Body EncephalitisSubacute Sclerosing Panencephalitis

Neurologic Complications of Measles

Orthomyxoviruses

Orthomyxoviruses

• Influenza•A: Mild to severe disease involving upper and

especially lower respiratory tract•B: Similar spectrum of illness to A but generally

more mild•C: Sporadic upper respiratory illness in humans

•96% of human adults have antibodies•Thogotovirus

•Natural host: Ticks•Also infects: Humans, cattle, goats, waterfowl,

etc.• Isavirus

• Infectious salmon anemia virus

G Neumann et al. Nature 000, 1-9 (2009) doi:10.1038/nature08157

Schematic diagram of influenza A viruses

• Involved in virion uncoating

•Highly conserved

•Target for amantadine

M2 Ion Channel

Hemagglutinin

•Virion release from cell membrane

•Cleavage of sialic acid from cell membrane thus preventing binding by HA

•Target for Oseltamavir (Tamavir) and Zanamivir (Relenza)

Neuraminidase

Influenza virus

Variation and evolution

Influenza Virus Variation

•Antigenic drift•Amino acid changes

•Antigenic shift•Reassortment/exchange of genome

segments between strains•Recombination

•Detected but rare

Reassortment

G Neumann et al. Nature 000, 1-9 (2009) doi:10.1038/nature08157

Genesis of swine-origin H1N1 influenza viruses

Why Pigs?

•Susceptible to infection by influenza virus

•Express both human- and avian-like influenza virus receptors on their tracheal epithelial cells

•Swine may therefore be acting as a “mixing vessel” for the production, replication, and transmission of novel influenza virus reassortments

US Influenza Surveillance 2004-2008

US Influenza Surveillance 2008-2009

US Influenza Surveillance 2010-2011

Fighting back•Antiviral drugs

•Neuraminidase inhibitors•Oseltamavir (Tamavir) and Zanamivir

(Relenza)•Active against influenza A and B

• Ion channel blockers•Amantidine and rimantidine•Prevent release and subsequent transport

of the virus RNP•Active only against Influenza A

•Vaccines• Inactivated•Live attenuated

Antiviral Resistance

Antiviral Resistance 2010 - 2011

Antiviral Resistance 2008 - 2009

Vaccine Development•Inactivated vaccine (TIV)

•Produced from seed stocks in eggs•Live-attenuated vaccine (LAIV)

•Administered as a nasal spray•Vaccines contain three viruses

•H3N2; H1N1; B•Exact strains used change each year

•Strain choice determined by data collected by WHO on currently circulating strains

•Decision on composition made in February and September

Vaccine Strains for the2009-2010 and 2010-2011

Seasons• 2009 – 2010 Seasonal Vaccine

• A components unchanged from 2008-2009• B component changed toB/Brisbane/60/2008

• Related to B/Victoria• 2009 Supplementary Vaccine

• A/California/7/2009 (H1N1)• 2009 pandemic influenza A (H1N1) virus

• 2010 – 2011 AND 2011 – 2012 influenza A (H1N1) virus• A/California/7/2009 (H1N1)-like virus (99.8%

of 2010-2011 viruses match)• A/Perth/16/2009 (H3N2)-like (96.8% of 2010-

2011 viruses match)• B/Brisbane/60/2008 (94% of 2010-2011

viruses match)

And finally, how is influenza spread

between humans and pigs?