Influenza Virus, Structure, Replication and Pathogenesis

ORTHOMYXOVIRUSES ( INFLUENZA VIRUSES)

-         affinity of the viruses to mucins ( myxa: mucus)

-         4 genera: influenza A, B, C viruses and thogotoviruses

-         Mutability and high frequency of genetic reassortment.
 resultant antigenic changes in viral surface gp.

-         Influenza type A antigenically highly variable and responsible for most cases of epidemic influenza.

Structure / composition

-         virion spherical, pleomorphic (80-120 nm diameter), helical nucleocapsid (9m) SS RNA, (-) sense (13.6 kb genome size) of A and B viruses with and separate segments; infection C → 7 segments lacking neuraminidase gene.

-         Segment
1→ PB₂                   4→HA             7→M₁ and M₂

      2→PB₁                        5→NP             8→NS₁ and NS₂

      3→PA                   6→NA

Schematic representation of Influenza Virus Structure

→ 9 structural proteins; one non- structural

→ NP+ viral RNA → RNP (ribonucleoprotein)
→ PB1, PB2 and PA bound to RNP and responsible for transcription and replication.
→ M1 (matrix) protein forms a shell underneath the envelope.
→ HA and NA (surface gp) determine Ag- variation and host immunity.
→ M2 protein ( ion channel) allows PH changes in the endosome.

CLASSIFICATION / NOMENCLATURE

-         antigenic differences exhibited by two of the internal structure proteins (NP and M), are used to divide influenza viruses into types A, B and C.

-         Ag – variation in surface gp (HA, NA) used to subtype the viruses
→ only type A has designated subtypes.

-         All isolates from non- humans belong to type A.

-         Aquatic birds appear to be primary reservoir of influenza viruses.

-         15 subtypes of HA (H1-H15) and 9- subtypes of NA (N1-N9). 
→ 4 HA (H1-H3, H5) and 2 NA (N1, N2) from humans.

Structure and function of HA

-         bind virus particles to susceptible cells ( respiratory epithelial cells and RBCs).

-         Agglutinate erythrocytes under certain conditions.

-         Cleavage of HA into HA1 and HA2 necessary for virus to be infectious; cleavage is PH dependent

-         -NH2 terminal of HA2 provides fusion activity ( fusion peptide). Fusion peptide is necessary for viral envelope and endosomal membrane to fuse, releasing nucleocapsid to cytoplasm.

-         HA interacts with cell- membrane receptors containing N-acetylneuraminic acid (sialic acid).

-         5 antigenic sites on HA molecule exhibit extensive mutation.

-         HA spike is a trimer ( composed of 3 intertwined HA1 and HA2 dimers) about 10 nm in length, mol. Wt of 225,000.

-         Cleavage to HA1 and HA2 mediated by cellular protease; viruses normally confined to respiratory tract because proteases are common to those sites.

-         Low PH activates the fusion activity.

STRUCTURE AND FUNCTION OF NA.

-         tetramer of mushroom- shaped protrusions; slender- stalk topped with a box- shaped head; each spike with 4 active sites

-         sialidase ( removes sialic acid from glycoconjugates).
- allows virus to permeate mucin and reach the target epithelial cells.
- destroys the haemagglutinin receptors on host cell.

-         Red cells acted on by the virus, not susceptible to agglutination by same strain of virus due to destruction of cell- receptors.

-         Prevents self- aggregation of virions by removing sialic acid residues from viral gp (↑ no. of free virus particles and their spread )

HAEMAGGLUTINATION / ELUTION
- when mixed with suspension of fowl erythrocytes, virus produces haemagglutination; which is followed by detachment of virus from cell surface, reversing the haemagglutination, this is k/as ‘ elution’ ( caused by NA).

-         NA also present in bacteria → culture filtrates of v. cholerae rich in NA- activity; red cells pretreated with them resistant to haemagglutination by influenza viruses, thus called ‘ Receptor Destroying Enzyme’ of v. cholerae

-         HA- titre → highest dilution of virus suspension that produces agglutination of fixed quantity of cells.

-         Haemagglutination inhibition test (HAI) → for detection and quantitation of antibody to the virus.
→ disadvantage is frequent presence of non-specific inhibitors of haemagglutination in sera eg α (Francis), ß (Chu), γ (Shimoja) inhibitors.
→ such sera treated with RDE, trypsin, K- periodate, Kaolin and CO₂ etc for inactivating the inhibitors without affecting Ab- content of sera.

-         Plasma membrane of tissue culture cells in which virus is multiplying contain HA; red cells adsorb onto the surface of such cells → haemadsorption.
→ growth of virus in cell cultures can be identified.

ANTIGEN- DRIFT / ANTIGEN – SHIFT
- the internal RNP Ag and M- protein are stable; but both surface Ag: HA and NA undergo independent Ag- variation.

-         antigen drift due to accumulation of point mutations in the gene; gradual change
→ new Ag react with antisera to the predecessor strains to varying degrees.
→ a variant must sustain two or more mutations before a new epidemiologically significant strain emerges.

-         Antigen shift is an abrupt, drastic, discontinuous variation; too extreme to be explained by mutation.
→ mechanism for shift is ‘genetic reassortment’ between human and avian viruses; segmented genomes reassort readily in doubly infected cells; frequency → 40%.
→ influenza B and C viruses don’t exhibit shift as only few related viruses exist in animals.

REPLICATION
- all of its RNA transcription and replication occurs in nucleus.

-         new progeny viruses produced within 8-10 hrs.

1. attachment / penetration/ uncoating
   - attaches to cell surface sialic acid via the receptor site ( located on the top of the large globule of HA).
   - virus particles internalized within endosomes by ‘receptor – mediated endocytosis’.
   - ↓ PH within endosome → virus mediated membrane fusion → uncoating and release of RNP to cytosol
   → conformational change in HA structure to bring HA2 ‘usion peptide’ in correct contact with the membrane; forms and fusion pore which inserts into cell lipid layer.
2. Transcription / Translation
   - virus encoded polymerase primarily responsible for transcription ; its action primed by cellular transcripts ( newly synthesized by cellular RNA pol II).
   - thus viral replication inhibited by dactinomycin and α – amanitin which block cellular transcription.
   - transcription produces 10 mRNA species as segments 7 and 8 both have two reading frames.
3. RNA replication
   - accomplished by same virus- encoded polymerase involved in transcription.
   - templates for viral RNA synthesis remain coated with nucleoproteins.
   - 1^st step is production of (+) strand (antigenome) copies of each segment, these serve as templates for synthesis of copies of genomic RNAs.
4. Maturation
   - by budding from the surface of the cell.
   - nucleocapsids assembled in nucleus and move out to cell surface.
   - HA and NA synthesized in the ER and inserted into plasma membrane.
   - HA is cleaved into HA₁ and HA₂.
   - NA removes terminal sialic acid from cellular and viral surface gp.
   - many of the particles are not infectious at they fail to encapsidate the complete complement of genome segments.

PATHOGENESIS / PATHOLOGY
- spreads by air- borne droplets or by contact with contaminated hands / surfaces.

-         few cells of respiratory epithelium infected, if deposited virus avoid removal by cough reflex and escape neutralization by pre- existing IgA or inactivation by non- specific inhibitors.
- progeny virions soon produced and spread to adjacent cells.

-         Viral NA lowers the viscosity of the mucous film, promoting the spread of virus- containing fluid to lower portions of the tract.

-         The ciliated cells of respiratory tract are main cites of infection; these cells are damaged and shed, laying bare the basal cells in trachea and bronchi.

-         Cell damage initiates on acute inflammation response with oedema and attraction of phagocytes.

-         Release of IFN from infected cells protect both adjacent and distal cells; the IFNs cause many of the systemic features of ‘ flu- like’ syndrome’, IFN response contributes to host recovery from infection.

-         Incubation period varies from 1-4 days, depending on viral dose and hosts immune status.

-         Complete reparation of cellular damage takes upto 1 month.

-         Viral damage to respiratory epithelium lowers its resistance to 2⁰ – bacterial invaders.