(-) strand segmented RNA viruses : Orthomyxoviruses (flu) Orthomyxoviruses: (-) strand segmented RNA viruses Influenza A, B, C - Flu, risk of secondary pneumonia - Genetic drift/shift - Vaccines - H5N1 (bird flu) - Novel H1N1 (swine flu) - H7N9 infections in China

Negative strand viruses: general definition 1. Genomic RNA cannot function as mRNA 2. Genomic RNA serves as a template for two kinds of RNA: mRNA and complementary, genomic RNA (cRNA) 3. Since cells do not have RNA dependent RNA polymerases, negative strand viruses package the enzyme in the virion All negative strand viruses infecting humans:  Are enveloped  Enter cells by fusion of envelope with cellular membranes  Egress the infected cell by budding  Become infectious on egress  Are sensitive to lipid solvents, drying, etc.

Differentiation of Influenza A, B, and C. Genetically related: all orthomyxoviruses Influenza A: infect humans, poultry, pigs, etc., wild aquatic birds are usually the natural host re-assort most virulent associated with pandemics Influenza B: predominantly infects humans (& seals) less virulent & common than A Influenza C: humans, dogs & pigs least common Vaccine: A – H1N1 A – H3N2 B

Differentiation of Influenza A, B, and C Genetically related: all orthomyxoviruses Influenza A: infect humans, poultry, pigs, etc., wild aquatic birds are usually the natural host re-assort most virulent associated with pandemics Influenza B: predominantly infects humans (and seals) less virulent and less common than A No reassortment so far Seasonal epidemics Co-circulates with A virus Influenza C: humans, dogs and pigs least common Seasonal Tetravalent Vaccine: A – H1N1 A – H3N2 B – Yamagata-lineage B – Victoria-lineage

Orthomyxoviruses Virion structure: Segmented (-) strand RNA genome Enveloped with two protein spikes on its surface: Hemagglutinin (H) fuses envelope to cell membranes Neuraminidase (N) cleaves terminal sialic acid

Influenza virus life cycle Fusion and uncoating Packaging Low pH Nuclear replication (unusual for RNA viruses) - cap stealing, mRNA splicing

The Influenza Hemagglutinin Binds cellular receptor, sialic acid. After acidification in endosomes, has a conformational change promoting fusion of viral and cellular membranes.

The Influenza Neuraminidase -cleaves N-acetyl neuraminic acid/ NANA Two functions: 1. Removing NANA from mucoproteins (mucin) enhances viral evasion of antiviral mucosa 2. Removing NANA from viral and cellular glycoproteins facilitiates virion release

Entry of Influenza virus into cells 1. Attachment to sialic acid Low pH results in a conformational change and exposure of fusogenic peptide pH 7.4 2. Uptake into endosomes 3. Fusion with lysosomes pH 5 - 6 4. Release of RNP Cytoplasm Nucleus

10 Proteins Orthomyxoviruses: flow of events during replication mRNAs Virion-carried polymerase Virion negative strand RNA mRNAs 10 Proteins Complementary Genomic RNA Progeny negative strand RNA Virus

The replicative cycle of influenza viruses pH 5-6 endosome coated vesicle coated pit M1 mRNA Np, Ns, Pol Synthesis and glycosylation of H, N Viral RNA synthesis Golgi RNP binding Budding Insertion of membrane proteins Entry Replication Nuclear replication (unusual for RNA viruses) - cap stealing

Assembly of Influenza virions Packaging of distinct RNPs at the membrane can result in reassortment when two types of virus are present

Influenza Pathogenesis S. aureus pneumococcus Group A streptococci H. influenzae

pandemic influenza

Some definitions: Endemic: infection/disease maintained in a population without external inputs - infections rates can be high, but are relatively constant Epidemic: new cases of infection within a population - can be localized or global Pandemic: world-wide epidemic

Why do new strains of influenza appear every year? Three reasons: Humans are infected by avian or swine influenza Genetic reassortants arise High mutation rate

Influenza A distribution

Chronology of Influenza Viruses in Humans pH1N1 H3N2 (Hong Kong flu) H2N2 (Asian flu) H1N1 (Spanish flu) H1N1 (Russian flu) H2N? 1890 1918 1940 1957 1968 1977 2009 Isolation of flu virus 1976 Swine H1N1 outbreak at Fort Dix-New Jersey - Vaccination of 40 million people in the U.S

Antigenic shift and pandemics 1. Genetic Reassortment: Antigenic shift Acquisition of a novel HA segment by reassortment between human and animal viruses (Segmented genome) No immunity in humans against new HA Pandemics are caused by antigenic shift Genetic Reassortment Parent Viruses Antigenic shift and pandemics Reassortants

2. Minor antigenic drift Prototype Duration Major Derivative Genotype Epidemics Strains A/SW/33 1932--46 1932 A/Weiss H0N1 A/PR8/34 1937 1946 A/Cam/46 1946-57 1949 A/Scan/50 H1N1 A/FM1/47 1951 A/Liverpool/51 1955 A/Sing/57 1957-68 1957 A/Eng/61 H2N2 A/Alch/57 1959 A/Eng/64 1961 A/Eng/68 (1890) 1967-8 A/Hong Kong/ 68 1968-current 1968 A/Hong Kong H3N2 (1900) 1972 A/England 1973 A/Port Chalmers 1977 A/Victoria 1979 A/Bangkok Swine Flu 1976 (1976) Hsw/Nsw A/USSR/77 1977-current 1977 H1N1 A/Hong Kong 1998 (1998) H5N1

The significance of minor antigenic drift. Viruses are sequential isolations of A/Hong Kong-68. Table shows serum neutralization titers obtained in ferrets. Virus Hong Eng./72 Port Victoria Kong Chalmers Hong Kong -68 2,560 2,560 320 20 Eng. 72 40 1,280 320 40 Port Chalmers-C73 20 640 640 40 Victoria-75 <20 20 <20 1,280

Treatment Neuraminidase activity is essential for virion release Oseltamivir (Tamiflu) and Zanamivir inhibits NA activity Effective only if taken within 3 days of infection

Vaccines are quadrivalent: Prevention Yolk sac Allantoic fluid Majority of the vaccines are made in embryonated eggs Vaccines are quadrivalent: Influenza A H1N1 Influenza A H3N2 Influenza B Yamagata Influenza B Victoria Strains for making vaccines are selected in the Spring by WHO Committee; vaccines are made during Summer for administration in the Fall Beware of egg allergies

Vaccines Currently licensed Effectiveness Cost* Killed vaccine Poor $10 Live vaccine High $45 attenuated (Flumist) - Recommended for high risk individuals, adults >65, healthcare workers, etc. - FDA approved for uses in all age groups - Poorly immunogenic; requires a good adjuvant - FDA approved for use in Healthy 2 to 49 years old - Does not require an adjuvant *2003 prices, currently higher

Selection of annual vaccine by genetic reassortment Attenuated parent Virulent new virus X  6:2 Selection Progeny

Revisiting the 1918 Spanish Flu 50-100 million dead in 1918-1919 Unusual: lethal in young adults

Resurrecting the 1918 virus Jurassic park??? Virus did not reassort in pigs - it jumped directly from birds

National Preparedness $7.1 billion flu pandemic plan $251 million to help foreign partners train medical personnel, develop monitoring capabilities to detect outbreaks and draw up preparedness plans. $1.2 billion to purchase enough vaccine developed by the National Institutes of Health against current bird flu strain to vaccinate 20 million people. $1 billion to stockpile additional antiviral medications to provide enough to treat first responders and other key personnel. $2.8 billion for a "crash program" to accelerate cell culture technology, which Bush said would produce enough vaccine for all Americans within six months. Relief from the "burden of litigation" for vaccine manufacturers. $583 million for pandemic preparedness, including $100 million to help states complete and exercise their pandemic plans.

2009 H1N1 (Swine Flu) Why called swine flu? Quandruple reassortment: genes from influenza viruses infecting Asian/European pigs, avian, and humans further reassorted in pig Now thought to have emerged in Asia and traveled to N. America in infected human

2009 H1N1 timeline Mid March- 60% of La Gloria, Veracruz Mexico get respiratory illness Mar. 17- confirmed novel H1N1 Mar. 28- New H1N1 in U.S. April 1- H1N1 isolated from boy is later fully sequenced April 22, 2009 - U.S. declares public health emergency June 11, 2009 - Level 6 pandemic (reflects virus spread, not disease severity), >70 countries affected Aug. 2010. Pandemic declared over: ~15% of population infected, ~ 0.02% mortality Replaced the older H1N1 virus Now the 2009 virus circulates as a seasonal virus

Transmission

Age groups infected Elderly have pre-existing immunity?

Old H1N1 viruses protect against 2009 H1N1 virus 1940 1977 1918 1957 1968 2009 H1N1 (Spanish flu) H1N1 (Russian flu) Protection No Protection 1918 -1943 Complete Protection 1977 – 2007 (Partial Protection) Manicassamy et al, PLoS Pathogens, 2010

Treatment & Prevention rest, supportive care call doctor if trouble breathing, chest pain, dizziness, persistent nausea, dizziness Medication: oseltamivir (most flu now resistant), zanamivir Vaccine Hygiene: wash hands, cover nose & mouth when sneeze, don’t touch mouth, eyes, or nose stay home until 24 hours after fever subsides.

Entry of new viruses into human population

Definition of Pandemic Interepidemic period Current Pandemic alert period Pandemic

The Onion on Bird Flu Preparations Tamiflu: stockpiled by countries & individuals for fear of pandemic flu - questionable efficacy and limited production. October 26, 2005 - November 1, 2005 | Issue 41•43

Avian Influenza viruses H5N1: Detected in humans during a domestic poultry outbreak (HK 1997) Human cases from direct contact; >350 deaths (mortality rate >60%) Transmission studies: 4-5 mutations sufficient for mammals HPAIV: Highly pathogenic avian influenza viruses (H5N1 and H7N7) Multibasic cleavage site: cleaved by ubiquitous furin-like proteases Systemic dissemination of HPAIV due to ubiquitous cleavage of HA Seasonal viruses (H1N1, H3N2) restricted to upper respiratory tract - Trypsin dependent cleavage of HA LPAIV: H9N2: few human cases Trypsin-like cleavage site: proteases restricted to the respiratory tract

Will H5N1 become pandemic? Seroprevalence suggests bird-human H5N1transmission for decades without significant human-to-human spread Human-to-human transmission is low H5N1 only infects cells of lower respiratory tract in humans due to receptor specificity; transmission much better from upper respiratory tract Can acquire mammalian adaptive mutations or reassort with circulating human strain; recent studies indicate that 4-5 mutations are sufficient for transmission in ferrets

Human infection with Influenza A (H7N9) virus in China Reassortant of three avian viruses Reservoir not known Virus has been isolated from poultry and pigeons Limited human-to-human transmission Acquiring mammalian adaptive mutations Sensitive to Tamiflu