Influenza Chapter 39

Causative Agents Influenza A virus Family Orthomyxoviridae Negative-stranded RNA genome Genome divided into 8 gene segments Spiked envelope H spike – hemagglutinin (subtypes H1-H16) Mediates attachment N spikes – neuraminidase (subtypes N1-N9) Cleaves H protein to allow fusion of viral and cellular membranes (i.e., entry into the cell) Requires cellular enzyme trypsin to facilitate infection Influenza B & C viruses only circulate in humans Seasonal Influenza

Influenza Genes Influenza A viruses have 8 gene segments that encode 10 polypeptides Segment 1 (2,341 nt): PB2 (RNA transcriptase) Segment 2 (2,341 nt): PB1 (RNA transcriptase) Segment 3 (2,233 nt): PA (RNA transcriptase) Segment 4 (1,778 nt): HA (hemagglutinin) - 16 known subtypes Segment 5 (1,565 nt): NP (nucleoprotein) Segment 6 (1,413 nt): NA (neuraminidase) - 9 known subtypes Segment 7 (1,027 nt): M1, M2 (matrix proteins) Segment 8 (890 nt): NS1, NS2 (inhibits mRNA splicing and IFN response; nuclear export signal for viral RNPs)

The HA and NA Polypeptides HA encodes hemagglutinin spike protein Binds to any cellular protein with a α2,6-sialic acid group Mediates penetration of viral core and RNA into target cell It is the major target of neutralizing antibodies for vaccines and successful immune responses NA encodes neuraminidase protein This enzyme cleaves the HA protein upon cell binding and facilitates penetration of the viral core Together they mediate cell infection Influenza viruses, in part, are named by their HA and NA subtypes (e.g., H5N1)

Symptoms Influenza Type A Short incubation period Averaging 2 days Headache Fever Muscle pain Dry cough Acute symptoms abate within a week Cough, fatigue and generalized weakness may linger Seasonal Influenza

Influenza A Transmission Cycle Circulates with limited pathology Transmission to domestic fowl Adaptation/ reassortment with swine influenza viruses Transmission to humans

Pathogenesis Acquired through inhalation of infected respiratory secretions Virus attaches to host cells via hemagglutinin spikes Once attached viral envelope fuses with host membrane, leading to viral replication within the cell Mature viruses bud from host cell Budding allows mature virus to pick up envelope Infected cells die and slough off Host immunity quickly controls viral spread Anti-HA neutralizing IgG is protective Mortality rate is low However, hundreds of thousands or millions of people are infected each year in the U. S. On average, about 30,000 Americans, mostly elderly and very young children, die from influenza each year Seasonal Influenza

Epidemiology Outbreaks occur in United States every year Vaccines are formulated months in advance using prominent circulating strains vaccine strains A/California/7/2009 (H1N1)-like virus (same strain as flu season) A/Victoria/361/2011 (H3N2)-like virus B/Wisconsin/1/2010-like virus Pandemics occur periodically Most infamous pandemic of 1918 (Spanish flu) Spanned the globe in 9 months Pandemics have higher than normal morbidity Type / Geographic origin / Strain/ Year isolated (H & N genes) Seasonal Influenza

Epidemiology Spread caused by major antigenic changes Antigenic drift Consists of amino acid changes in spikes (point mutations) Particularly hemagglutinin Changes minimize effectiveness of immunity to previous strains Ensures enough susceptible people are available for continued virus survival Antigenic shift Represent more dramatic changesVirus strains are drastically antigenically different from previous strains, importantly hemagglutininNew virus comes from genetic re-assortmentOccurs when two different viruses infect a cell at the same timeGenetic mixing results in new virus that is often more virulent Seasonal Influenza

Prevention and Treatment Vaccine can be 80% to 90% effective New vaccine required every year Due to antigenic drift Antiviral medications are 70% to 90% effective Include amantadine, rimantadine, and Tamiflu Must be taken early Not a substitute for vaccine Seasonal Influenza

There are hundreds, if not thousands, of influenza A viruses circulating in nature Seasonal influenza occurs from mammalian viruses Pigs in SE Asia are frequently a source of these viruses New reassortants arise every year, but most are not pathogenic to humans HA attaches to α2,6-sialic acid receptor Avian influenza viruses routinely circulate among wild birds Some species can be infected without conspicuous pathology These species often carry the viruses along migratory routes, exposing other birds HA attaches to α2,3-sialic acid receptor Avian Influenza

Most avian influenza viruses are highly inefficient at infecting humans However, some cultures have domestic birds and pigs in close periodomestic proximity This practice increases the chance of Reassortment with mammalian influenza viruses 1957, 1967 pandemic strains were reassortant mammalian viruses with avian segments (antigenic shift) Emergence of mutant avian strains that can infect humans 1918 pandemic strain was an avian virus that adapted to efficient human to human transmission (antigenic drift) Avian Influenza

Recovery of the Spanish Flu Virus Using Reverse Genetics Spanish Flu pandemic: Estimated 20 to 50 million dead High death rate among young, health people Played a role in ending World War 1 No viable virus was saved

Rescue of the 1918 pandemic strain Virology did not exist in 1918 The virus could not be isolated, thus went extinct when the pandemic ended In 2005 a group resurrected the 1918 strain from bodies buried in Alaskan permafrost Viral genome sequencing indicated it was an avian influenza A virus Spanish Influenza

Molecular Recovery of Gene Segments Collect infected tissues from victims of the virus buried in permafrost or formalin-fixed tissues Use reverse transcription-PCR to amplify gene segments Clone into plasmids Transfect MDCK (canine kidney cell line) Recover infectious viruses

Characteristics of the Spanish Flu Virus It is an avian influenza virus (i.e., directly jumped from birds to humans) In infects cells in culture without trypsin (an indicator of pathogenicity in mammals) It was an H1N1 virus Kills chicken eggs in 4.5 days Pathology was largely confined to the lungs of experimentally- infected mice (similar to humans) Mice died in as little as 3 days post infection Substantial damage occurred in the lungs (similar to humans) Very high titers of virus were obtained from the lungs

Lungs from Mice Infected with Rescued 1918 H1N1 Pandemic Virus 1918 Strain 1918 Strain(HN)/Texas 1991 Strain hybrid 1918 Strain with Texas 1991 H segment Texas 1991 strain (control) Tumpey et al., 310: Avian Influenza

Is Another Avian Influenza Pandemic Upon Us? The current avian influenza (H5N1) virus that has been circulating since 1997 has reached Europe and Africa All human cases thus far are confined to SE Asian countries Sporadic person to person transmission has occurred Will this virus become efficient at person to person transmission?

FeatureSI 1 H5N1 AI H1N1 3 Transmission efficiencyHighVery low/noneHigh Replication site Upper and lower respiratory tract Lower RT only Likely upper & lower RT Viral CPE 4 LimitedSubstantial ImmunopathologyLimitedSubstantial Kills embyronated chicken eggs?NoYes Requires trypsin for infection of cell cultures? YesNo VaccineYesNoN/A Fatality rate0.03% (U.S.)57% (global)About 1-2% (U.S) Demographic Young children, elderly Young adults 1 Seasonal influenza 2 Currently circulating H5N1 avian influenza virus 3 Rescued 1918 pandemic H1N1 avian influenza virus 4 Cytopathic effect (damage directly caused by the virus) Red - more pathogenic feature Yellow - less pathogenic feature Avian Influenza

Will A Vaccine Be Easy to Produce? For seasonal influenza, viruses are grown in chicken eggs for 7 days to produce high-enough titers for vaccines Avian influenza viruses kills eggs in 4.5 days An insufficient titer is generated for vaccines The world’s annual vaccine capacity is 300 million doses If the vaccine can be produced by cell culture, then adequate supplies should be able to be produced A reverse genetics vaccine may be the only way to prevent an avian influenza pandemic A DNA vaccine has recently been described that protects mice from lethal challenge to the 1918 strain DNA vaccines are plasmids that express viral antigens under the control of a mammalian promoter Although many have been shown to be effective, none have been approved for use with the general public

Will A Vaccine Be Easy to Produce? For seasonal influenza, viruses are grown in chicken eggs for 7 days to produce high-enough titers for vaccines Avian influenza viruses kills eggs in 4.5 days An insufficient titer is generated for vaccines The world’s annual vaccine capacity is 300 million doses If the vaccine can be produced by cell culture, then adequate supplies should be able to be produced A reverse genetics vaccine may be the only way to prevent an avian influenza pandemic A DNA vaccine has recently been described that protects mice from lethal challenge to the 1918 strain DNA vaccines are plasmids that express viral antigens under the control of a mammalian promoter Although many have been shown to be effective, none have been approved for use with the general public