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single strand, negative sense RNA Viruses Elliot J. Lefkowitz

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Email ElliotL@uab.edu Web Site http://www.genome.uab.edu Office BBRB 277A Phone 934-1946 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

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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 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

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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 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

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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 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

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Virus classification

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The Virus World

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The (-) RNA Virus World

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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 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

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single strand RNA virus genome polarity virion RNA (+) sense virus mRNA (+) sense translation 3355 transcription virion RNA (-) sense virus mRNA (+) sense 3355 5533 (+) sense RNA virus (-) sense RNA virus

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Negative/Ambisense ssRNA Viruses

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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 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

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Bornaviridae Bornavirus Filoviridae Marburg virus Ebola virus Paramyxoviridae Paramyxovirinae Henipavirus Morbillivirus Respirovirus Rubulavirus Pneumovirinae Pneumovirus Metapneumovirus Rhabdoviridae Vesiculovirus Lyssavirus 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

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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 Orthomyxoviridae Influenzavirus A 8 genome segments Influenzavirus B 8 genome segments Influenzavirus C 7 genome segments Isavirus 8 genome segments Thogotavirus 6 genome segments

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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 Arenaviridae Two ambisense RNA segments Bunyaviridae Three RNA segments Both negative-sense and ambisense segments Depends on genus

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The Virus Virion, Genome, Proteins

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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 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

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Rhabdovirus Virion

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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 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

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Genome Organization Mononegavirales Filoviridae Paramyxoviridae Rhabdoviridae

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Genome Organization Arenaviridae Bunyaviridae

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Influenza A Genome Structure

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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 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

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Virus Replication

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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 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

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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 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

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VSV Transcription & Replication

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(-) sense ssRNA virus Human Pathogens

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Major Viral Target Tissues

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Arenaviruses/Bunyaviruses

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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 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

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Filoviruses

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Filovirus Disease

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Rhabdoviruses

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Rabies virus Pathogenesis

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Paramyxoviruses

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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 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

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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) 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)

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Acute Disseminated Encephalomyelitis Measles Inclusion Body Encephalitis Subacute Sclerosing Panencephalitis Neurologic Complications of Measles

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Orthomyxoviruses

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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 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

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G Neumann et al. Nature 000, 1-9 (2009) doi:10.1038/nature08157 Schematic diagram of influenza A viruses

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Involved in virion uncoating Highly conserved Target for amantadine Involved in virion uncoating Highly conserved Target for amantadine M2 Ion Channel

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Hemagglutinin

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Virion release from cell membrane Cleavage of sialic acid from cell membrane thus preventing binding by HA Target for Oseltamavir (Tamavir) and Zanamivir (Relenza) 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

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Influenza virus Variation and evolution

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Influenza Virus Variation Antigenic drift Amino acid changes Antigenic shift Reassortment/exchange of genome segments between strains Recombination Detected but rare Antigenic drift Amino acid changes Antigenic shift Reassortment/exchange of genome segments between strains Recombination Detected but rare

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Reassortment

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G Neumann et al. Nature 000, 1-9 (2009) doi:10.1038/nature08157 Genesis of swine-origin H1N1 influenza viruses

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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 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

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US Influenza Surveillance 2004- 2008

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US Influenza Surveillance 2008- 2009

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US Influenza Surveillance 2010- 2011

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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 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

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Antiviral Resistance Antiviral Resistance 2010 - 2011 Antiviral Resistance 2008 - 2009

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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 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

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Vaccine Strains for the 2009-2010 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) 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)

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And finally, how is influenza spread between humans and pigs?

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