Notes On Influenza Virus

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S. Saengamnatdej May14, 2009

Notes on Orthomyxoviruses (focused on influenza A virus)

Family Orthomyxoviridae Genera : Three Influenza A & Influenza B viruses Influenza A virus infects many vertebrate species including other mammals & birds. Is divided into subtypes. Influenza B virus infects human only and causes similar but milder symptoms. Influenza A virus share no antigens with Influenza B virus. Influenza C virus Influenza C virus infects children, but does not cause significant disease. Seven segments HEF: An envelope glycoprotein functions as hemagglutinin, esterase, and fusion protein. Receptor for influenza C virus is 9-O-Acetyl-N-neuraminic acid (different from that for influenza A and B viruses) Tick-borne Orthomyxoviruses Thogoto virus (6 segments) Dhori virus (7 segments)

Influenza A & Influenza B viruses Structure pleomorphic (spherical/ tubular/ filamentous) 80-120 nm enveloped two glycoproteins; hemagglutinin (HA or H), neuraminidase (NA or N) matrix proteins (M1) line under the envelope membrane proteins (M2) forming a ion channel

Genome : Segmented linear, negative sense ssRNA Influenza A & B viruses : Eight helical nucleocapsid segments. (Each has a looped-end and is associated with nucleoprotein (NP) and transcriptase (RNA polymerase components: PB1, PB2, PA) with total size 13.6 kb segments are in order of size from largest to smallest ones (2340 -890 bases). All code for one protein except for segments 7 and 8 code two proteins.

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Segment 1

Protein PB2

2 3 4 5 6 7

PB1 PA HA NP NA M1 M2

8

NS1 NS2

Function polymerase component : endonuclease cleaves 5' methylguanosine cap plus 10-13 nts from hnRNA and this is used as a primer (Cap snatching). polymerase component (transcriptase) polymerase component hemagglutinin nucleocapsid Neuraminidase Matrix protein: Viral structural protein (interacts with nucleocapsid and envelope, promote assembly) Membrane protein (forms membrane/proton channel & M2 of influenza A is target for amantadine/ rimantadine ,facilitates uncoating and HA production) Nonstructural protein (inhibits translation of cellular mRNA) Nonstructural protein (important but unknown function)

HA, NA, M1, M2 and NP HA trimer activated by a cellular protease (furin), which cleaves HA into 2 subunits held by a disulfide bond. Functions: viral attachment protein (to sialic acid), promote fusion, target of neutralizing antibody, binds and aggregates human/chicken/guinea pig RBCs. antigenicity (antigenic shifts occurs only with influenza A virus by genetic reassortment) subtypes: H1-H15 HA0 is cleaved into HA1 & HA2 as mentioned above. If there are monobasic links at HA1/ HA2 junction , it is sensitive to trypsin-like proteases . Thus, the viral spread is limited. If there are multibasic links at the junction, the virus will cause systemic spread, hence, is highly pathogenic. NA tetramer enzyme activity (cleaves SA, prevents clumping, & promotes virus release) undergoes antigenic changes subtypes: N1-N9 HA & NA of influenza A virus can undergo major & minor antigenic changes, whereas influenza B virus undergoes only minor antigenic changes. M1, M2, & NP are type-specific (differentiate A , B or C)

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Viral Entry, Penetration, and Uncoating : Cellular receptors for influenza virus virus replicates principally in ciliated columnar epithelium of respiratory tract. Human receptor for influenza virus is N-acetylneuraminic acid attached to a sugar molecule (galactose) with alpha (2,6)-linkage whereas avian receptor has the alpha (2,3)-linkage. Swine has these two forms of receptor and, thus, can serve as mixing vessels. Attachment, Penetration, and Uncoating After HA binds to its receptor (N-acetylneuraminic acid), the virus undergoes endocytosis. The acidification of the endosome causes the change in configuration of HA by pushing the globular parts to its side and exposing the fusion peptide which then initiates the fusion between the viral envelope and the endosomal membrane. As the time, the M2 ion channel pumps the proton into the viral particle causing the matrix protein M1 to dissociate. The viral segmented genome is finally released into the cytoplasm. Then, the nucleocapsid-polymerase complex is transported into the nucleus where transcription and replication of RNA occur.

Viral mRNA synthesis steals the methylated cap region of host mRNA. 5'-capped, 3'-polyadenylated mRNA. To produce two different mRNAs from segments 7 & 8, cellular enzymes are used to splice the transcripts. HA & NA glycoproteins are processed by ER & Golgi apparatus. M2 inserts into cellular membranes. Nucleocapid segments form and associate with M1 protein-lined membranes containing M2 and the HA and NA glycoproteins. The virus buds from the apical plasma membrane (~8hrs). [To ensure that one copy of each gene is packed into a virion, segments are enveloped in a random manner, with 11 segments per virion.]

Pathogenesis firstly, infect local upper respiratory tract. Kill mucus-secreting, ciliated, and other epithelial cells (loss primary defense system) NA facilitates infection by cleaving sialic acid off the mucus providing access to tissue. If spreading down further, it can cause severe shedding of bronchial/alveolar epithelium down to a single-cell basal layer or to the basement membrane. Systemic symptoms are caused by the interferon & lymphokine response. Promote bacterial adhesion pneumonia may result from 2nd bacterial infection

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S. Saengamnatdej May14, 2009

a transient or low-level viremia is possible, but tissues other than the lung are rarely involved. Depresses macrophage & T-cell function. Recovery often precedes detection of antibody. The antibody is strain-specific , but cell-mediated immunity is more general (can react same type). Antibodies to HA is primarily protective, although antibodies to NA is also protective. Antigenic targets for T-cell responses include peptides from HA but also from the nucleocapsid protein (NP), PB2 and M1 protein. Innate immunity 1. mucus 2. continuously beating cilia 3. soluble mannose-binding lectins, lung surfactants, and sialylglycoproteins 4. alveolar macrophages These are suboptimal in the aged, the premature (very young), the person with an underlying respiratory problem, smoker, chronically ill, the immunocompromised, and the person with additional factors including inhaled pollutants, malnutrition, or prior or coincidence infection. Immunity to influenza virus 1. antibody from previous infection (few years ago) may protect infection from a closely related strain of the same HA subtype. 2. IgA protects the upper respiratory tract, whereas IgG may protect in the lung. 3. Activated macrophages and NK cells take their parts in recovery 4. interferon-γ and IL-2 are important cytokines. 5. CD8+ -cytotoxic T cells clear virus from the lower respiratory tract. 6. Long-lived memory T cells recognize conserved determinants on NP, M1, P or NS1, hence, can be activated by a different strain of influenza virus. 7. CD4+ -T cells can clear low-level infection or less virulent strains, but they are more important as Th cells, and as Td cells secreting cytokines that attract and activate MΦ, NK, and T cells. Note that CD4+ - T cells also contribute to the lung consolidation (pneumonia) which may kill the patient.

Epidemiology strains (WHO System of Nomenclature) Type(A, B, or C)/ host origin (omitted if human)/ Place of original isolation/ Number of the isolate/ Year of original isolation/ Subtypes in parentheses (HA and NA) Examples Incorrect reading (found in Murray, PR. et al 2005 p.613) A/Bangkok/1/79(H3N2) first isolated in BKK in January!!! 1979 and contain H3 and N2 antigens Correct reading

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A/swine/Iowa/3/70(H1N1) influenza A virus was isolated from a pig in Iowa from isolate number 3 in 1970 with subtypes H1 and N1. influenza B : antigen not specified because there is no antigenic changes. Pandemic Pandemic 1918 1947 1957 1968 1977

Subtype HswN1; probable swine flu strain H1N1 H2N2; Asian flu strain H3N2; Hong Kong flu strain H1N1

H5N1 in 1997 avian influenza virus (isolated from 18 humans, caused 6 deaths) H5N1 not a reassortment, very virulent, passed directly from birds (feces) to man, not man-toman transmission. Reassort with human influenza virus might generate a pandemic. Most current outbreak is H1N1 subtype in Mexico (see reference 4 for more details) Antigenic drift, occurs every 2 to 3 years, causing local outbreaks. Antigenic shift, occurs infrequently 1957, a shift to H2N2, to H3N2 in 1968, and H1N1 reappeared in 1977. outbreaks occur every year during winter for only short period (4-6 wks) transmission (inhale) often precedes symptoms Risk groups: seronegative people, elderly, immunocompromised, with heart& respiratory problems

Lab Diagnosis Higher isolaion rate is obtained from throat was, nasal wash, or nasopharyngeal aspirate. virus isolation in primary monkey kidney cell culture/ Madin-Darby canine kidney cell line (33-34 ºC, with trypsin), nonspecific CPE in as quick as 2 days (avg. 4 days) nonspecific tests: hemadsorption (guinea pig RBC, before CPE), hemagglutination Rapid antigen assay (tell A or B) RT-PCR (generic primers tell A or B, more specific primers distinguish strains) EIA for identification of antigen in detergent-disrupted cells using enzyme-labeled anti-NP monoclonal antibody specific for influenza type A or B. immunofluorescence for direct detection

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inhibition of hemadsorption HI test on the culture supernatant using antisera against the prevalent strains H1N1, H3N2 and type B serology for seroepidemiology, memory B cells produce antibody against earlier strains can complicates the interpretation of HI results.

References 1. White DO, Fenner FJ: Chapter 31 Orthomyxoviridae. Medical Virology, 4th ed., San Diego, 1994, Academic Press. p. 489-499. 2. Sutherland, S. Chapter 49 Orthomyxoviruses. In Greenwood, D, Slack, RCB, Peutherer, JF (eds.) Medical Microbiology: A guide to microbial infections: pathogenesis, immunity, laboratory diagnosis and control. 16th ed., Churchill Livingstone, p.468-474. 3. Murray, PR, Rosenthal, KS, Pfaller, MA. Chapter 60 Orthomyxoviruses. In Murray, PR, Rosenthal, KS, Pfaller, MA (eds.) Medical Microbiology, 5th ed., Philadelphia, 2005, Elsevier Mosby. p.609-617. 4. Fraser, C. et al. Pandemic Potential of a Strain of Influenza A (H1N1): Early Findings. published online. www.sciencexpress.org / 11 May 2009 / Page 5 / 10.1126/science.1176062