Influenza There are three types of influenza in humans: A,B, C 1. Flu A: Found in many animal species, in addition to humans Closely related to Type B but not Type C Main type responsible for human epidemics Demonstrates the greatest antigenic variability (“antigenic drift”) Reservoir in nature is waterfowl 2. Flus B and C: Found almost exclusively in humans Flu C can also infect swine Flu C is morphologically and antigenically distinct from A, B 3. Flu A strains designated by host from which isolated, where isolated, year of isolation, and type of HA and NA. An isolate (strain) number may also be included if there are multiple isolates. Example: A/goose/Leipzig/137/79 (H7N2)

Influenza Virus

Structure of Influenza Virus Influenza A virus is an enveloped particle that when spherical is about 120 nm in diameter Many particles are not spherical but filamentous in shape There are two glycoproteins at the surface in surface “spikes” HA (hemagglutinin) is present as homotrimers NA (neuraminidase) is present as homotetramers Protein M2 forms ion channels in the lipid bilayer The matrix protein M1 lines the inner side of the lipid bilayer The genome consists of 8 RNA segments present in helical nucleocapsids

Influenza Nucleocapsids NP is the major nucleocapsid protein. It has a major structural role It is also required for the switch from mRNA synthesis to genome replication. PA, PB1, and PB2 are minor components of the nucleocapsid and form the RNA synthesis machinery. The function of PA is unknown but may be involved in the switch from mRNA synthesis to genome replication PB1 is an endonuclease that process the mRNA primer; it also is a polymerase that catalyzes nucleotide addition PB2 recognizes the cap of host cell mRNA required for priming mRNA synthesis M is the matrix protein. It is a peripheral membrane protein that underlies the viral membrane. It interacts with the nucleocapsid and with the tails of HA, NA, and M2

Attachment & Entry The HA spike is a homotrimer with a molecular weight of 110 kDa. HA is synthesized as a 549 aa precursor called HA0 which is anchored in the membrane near the C-terminus. HA0 is cleaved into HA1 (328 aa) and HA2 (221 aa) At the N-terminus of HA is a 16 aa hydrophobic signal peptide for insertion into the ER. A single Arg separates HA1 from HA2 and cleavage is by a cellular trypsin-like proteinase HA1 and HA2 remain covalently associated after cleavage by a disulfide bridge The C terminus of HA2 contains a 26 aa uncharged membrane-spanning domain followed by a 10 aa hydrophilic cytoplasmic domain The HA polypeptide is glycosylated at specific asparagine residues

HA-mediated membrane fusion The HA trimer is stabilized by a hydrophobic core formed between the three stalk regions. Attachment sites for the cellular receptors are located near the top of each large globular region, which also contains neutralization epitopes. The exact glycoprotein(s) that serve as host cell surface receptors has not been identified, but it is known to contain sialic acid. After binding of HA to the cell surface receptor(s) the virus is internalized by endocytosis. The low pH of endosomes ( pH 5.0-6.0) results in an irreversible conformational change in HA which results in the extrusion of the highly conserved hydrophobic amino terminus of HA2 from its position in the native protein. This region, termed the ‘fusion peptide’, promotes membrane fusion. The mechanism by which the ‘fusion peptide’ promotes membrane fusion is not completely understood. The subsequent fusion of viral and endosomal membranes allows the release of the viral genome into the cellular cytoplasm

Activation of the HA Spike After acid treatment HA0 precursor Cleaved spike and proteolysis

Activation of Fusion Activity of Flu HA0 by Cleavage View of One Monomeric Unit in the Spike

Change illustrated for one monomeric unit of the trimeric spike

Model for Fusion

Neuraminidase NA spike consists of a tetramer. NA is a type 2 glycoprotein with the N terminus inside and the C terminus outside. NA removes sialic acid from oligosaccharides on cell-surface proteins and glycolipids, thus destroying receptors for the virus. Also removes sialic acid from HA so that progeny influenza virions cannot aggregate. Separates virus particles from inhibitory mucopolysaccharides in the respiratory tract allowing efficient infection.

Synthesis of mRNAs and RNA Replication

Splicing to Produce Influenza A mRNAs Since influenza RNA synthesis occurs in the nucleus, the cellular splicing machinery can be used In Flu A two mRNAs are produced from both segments 7 and 8 One mRNA is unspliced, the second is spliced

Influenza C Has an Esterase Flu C lacks NA and has only 7 segments It has HEF that performs the functions of HA and NA in Flu AB The receptor for Flu C is 9-O-acetyl-N-acetyl neuraminic acid The Flu C esterase removes the 9-O-acetyl group to destroy the receptor The HEF gene is also present in some coronaviruses, which must have obtained it by recombination with Flu C at some time in the past

M2 M2 tetramers form ion channels in viral and cellular membranes Exposure to low pH is required to dissociate the nucleocapsid from the matrix protein, allowing the nucleocapsid to be transported to the nucleus M2 also prevents premature activation of the fusion activity of HA Amantadine interferes with the function of M2 and is an effective flu antiviral

Virus Assembly Nucleocapsids assemble in the nucleus during genomic RNA synthesis The encapsidation signal is at the end of the RNA and not present in mRNAs Nucleocapsids are exported to the cytoplasm in a process that requires NS2 and M1 Glycoproteins are synthesized on the ER and transported to the plasma membrane Nucleocapsids bud through the plasma membrane to form virions More than 8 segments may be packaged: Ten segments randomly selected would result in ~3% of progeny virions having at least one each of the 8 segments Random selection of segments would mean efficient reassortment during mixed infection, which is known to occur

Influenza - Some History Oldest record of an epidemic probably caused by flu: Hippocrates, 412 BC. Epidemics have occurred relatively frequently but at irregular intervals Epidemics vary in severity but the very young and elderly are most at risk. Epidemics appear to radiate from specific locations Example: 1781 epidemic that spread across Russia from Asia. Influenza has killed untold millions throughout the centuries 1. 1918-1919 epidemic was particularly severe 2. 20-1000 million people died, more than died in World War I. 3. 80% of US WWI deaths were due to influenza 4. A significant factor in the German loss was influenza First human influenza virus was isolated in 1933. Different strains cause different epidemics, but human strains can recirculate

Antigenic Shift and Drift in Flu A HA and NA are the major surface antigens of the virus Antigenic drift describes the selection of variants by the immune system Relatively slow Resistance is only partial Antigenic shift describes the results of recombination (reassortment) There are 15 different subtypes of HA There are 9 different subtypes of HN Subtypes differ by 30% or more in amino acid sequence A reassortant with a different HA and/or HN may cause a pandemic Only a few of the subtypes have been isolated from humans

Influenza A in Birds The reservoir of influenza A in nature is birds All 15 HA and 9 NA have been found in aquatic birds In particular, migratory ducks are important in the maintenance and spread of influenza Influenza infection of birds is usually asymtomatic Influenza replicates in the respiratory tract and the intestinal tract of birds It is excreted in the feces and high concentrations have been found in waters in which migratory ducks congregate The virus appears to be in equilibrium in birds--little or no sequence drift has been found in bird viruses and disease seldom results from infection In contrast, the virus drifts rapidly in humans and vaccines must be reformulated yearly, and serious illness is produced

Epidemic Influenza Strains Year Virus Common Name 1889 H2N2 1900 H3N8 1918 H1N1 Spanish 1957 H2N2 Asian 1968 H3N2 Hong Kong 1977 H1N1 Russian When a new strain appears the previous strain usually dies out At present, H3N2 and H1N1 continue to cocirculate in humans An H1N1 strains has circulated continuously in pigs in the U.S. since 1918

Sialic Acid (N-Acetyl Neuraminic Acid) Terminal NANA is attached to galactose by a2,3 or a2,6 linkages Different HAs prefer one or the other linkage Avian intestine contains predominantly a2,3 linkages Human trachea contains predominately a2,6 linkages Pig trachea contains both linkages and serves as an efficient intermediate host in which reassortment can take place--pigs are often referred to as mixing chambers Other components also contribute to host specificity, best studied for NP

U.S. Life Expectancy

1918 Influenza Deaths

The 1918 Flu in America

Influenza affects 10-20% of U. S Influenza affects 10-20% of U.S. population each year, causing up to 70,000 deaths. Average death rate in people over 65 is 1/2200 but in 1957-8 it was 1/300.

Illness Induced by Influenza Virus Influenza virus infects superficial cells throughout the respiratory tract. There is little or no spread to other organs. High temperatures often accompany the infection, 38-41 C, that last 3-6 days. Cough and weakness can last 1-2 weeks longer. Extensive destruction of epithelial cells of the LRT can result in primary viral pneumonia. Influenza infection can result in secondary bacterial infection of the LRT resulting in bacterial pneumonia. Death following influenza infection is usually due to pneumonia, whether viral or bacterial or combined. Immunity following influenza infection is incomplete and appears to fade in time. It has been suggested that the high death in young adults in the 1918 pandemic could have resulted from a more active immune response to the virus.

Bird Influenza An epidemic of influenza in chickens occurred in Hong Kong in 1997 The virus was highly virulent, killing 70-100% of infected chickens Bird viruses are not normally transmitted to humans but the 1997 Hong Kong virus resulted in 18 humans becoming infected This virus was highly virulent in humans--6 of 18 infected people died The virus was H5N1 and did not spread in humans--no person to person transmission occurred To eradicate the virus and to prevent new reassortants from arising that might give rise to epidemic virus by direct person to person transmission, 1.6 million chickens were slaughtered

Defenses against Influenza Antivirals Amantadine and Rimantadine licensed for use and ameliorate symptoms Inhibitors of NA being developed Vaccines Inactivated vaccines are in widespread use These vaccines must be reformulated every year because of shift and drift They are 60-80% effective Attempts being made to develop attenuated virus vaccines that could be reformulated yearly by reassortment An emergency response to swine flu in 1976 demonstrates the difficulties in preparedness decisions

Bunyaviridae Sin Nombre Virus La Crosse Virus

Arenaviridae Budding Machupo Virion Lassa Virions Tacaribe Virion

Representative Arenaviruses Rodent Host Disease Where Found Old World Lymphocytic Mus musculus Meningitis Worldwide choriomeningitis Lassa Mastomys sp. HF West Africa Mobala Praomys sp. ? CAR New World Tamiami Sigmodon hispidus None? Florida Whitewater Arroyo Neotoma albigula 3 fatal ARDS Western U.S. Pichinde Oryzomys albigularis None? Colombia Guanarito Zygodontomys brevicauda Venezuelan HF Venezuela Junin Calomys musculinus Argentine HF Argentina Machupo Calomys callosus Bolivian HF Bolivia Sabia ? 3 severe cases Brazil Tacaribe ? ? Trinidad

Arenaviruses in the New World

Some Viruses Causing Hemorrhagic fever

Representative Viruses Causing Encephalitis Flaviviridae Alphaviruses St. Louis encephalitis Eastern equine enceph. Japanese encephalitis Western equine enceph. West Nile Venezuelan equine enceph. Murray Valley enceph. Herpesviridae Tick-borne enceph. Herpes simplex Bunyaviridae Paramyxoviridae La Crosse Mumps California enceph. Measles