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1. Influenza

2. Avian Influenza

3. Virology

4. Pathogenesis and Immunology

5. Pandemic Preparedness

6. Vaccines

7. Laboratory Findings

8. Clinical Presentation

9. Treatment and Prophylaxis

10. Drugs

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Amedeo Influenza





Virology of Human Influenza

Lutz Gürtler

Human influenza viruses are members of the orthomyxovirus family, which consists of the genera: influenza A, B, and C virus, and Thogovirus (in ticks). In humans, only influenza A and B viruses are of epidemiological interest.

The main antigenic determinants of influenza A and B viruses are the haemagglutinin (H or HA) and neuraminidase (N or NA) transmembrane glycoproteins. Based on the antigenicity of these glycoproteins, influenza A viruses are further subdivided into sixteen H (H1-H16) and nine N (N1-N9) subtypes. The full nomenclature for influenza virus isolates requires connotation of the influenza virus type (A or B), the host species (omitted if human in origin), the geographical site, serial number, year of isolation, and lastly, the H and N variants in brackets, for example: A/goose/Guangdong/1/96 (H5N1).

Influenza viruses are usually transmitted via air droplets, and subsequently contaminate the mucosa of the respiratory tract. They are able to penetrate the mucin layer of the outer surface of the respiratory tract, entering respiratory epithelial cells, as well as other cell types. Replication is very quick: after only 6 hours the first influenza viruses are shed from infected cells. Part of the viral proteins, such as the fusion peptide and NS2, act as toxins to promote the production of influenza virus. Rapid bacterial growth, most commonly Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae, may begin in the very early phase of viral replication (for more details, see the chapter on Pathogenesis).

Structure

Influenza viruses are enveloped single-stranded RNA viruses with a pleomorphic appearance, and an average diameter of 120 nm. Projections of haemagglutinin and neuraminidase cover the surface of the particle (Figure 1).

Figure 1. Structure of an influenza A virus. Image copyright by Dr. Markus Eickmann, Institute for Virology, Marburg, Germany. Used with permission. - http://www.biografix.de

The influenza A and B virus genomes consist of 8 separate segments covered by the nucleocapsid protein. Together these build the ribonucleoprotein (RNP), and each segment codes for a functionally important protein:

  1. Polymerase B2 protein (PB2)
  2. Polymerase B1 protein (PB1)
  3. Polymerase A protein (PA)
  4. Haemagglutinin (HA or H)
  5. Nucleocapsid protein (NP)
  6. Neuraminidase (NA or N)
  7. Matrix protein (M): M1 constructs the matrix; and in influenza A viruses only, M2 acts as an ion channel pump to lower or maintain the pH of the endosome
  8. Non-structural protein (NS); the function of NS2 is hypothetical

The active RNA-RNA polymerase, which is responsible for replication and transcription, is formed from PB2, PB1 and PA. It has an endonuclease activity and is linked to the RNP. The NS1 and NS2 proteins have a regulatory function to promote the synthesis of viral components in the infected cell (see below).

The envelope of the virus is a lipid bilayer membrane which originates from the virus-producing cell and which contains prominent projections formed by HA and NA, as well as the M2 protein. The lipid layer covers the matrix formed by the M1 protein.

Influenza C virus harbours only 7 genome segments, and its surface carries only one glycoprotein. As it has a low pathogenicity in humans, it will not be discussed here in detail.

Haemagglutinin

Haemagglutinin (HA or H) is a glycoprotein containing either 2 of 3 glycosylation sites, with a molecular weight of approximately 76,000. It spans the lipid membrane so that the major part, which contains at least 5 antigenic domains, is presented at the outer surface. HA serves as a receptor by binding to sialic acid (N-acetyl-neuraminic acid) and induces penetration of the interior of the virus particle by membrane fusion. Haemagglutinin is the main influenza virus antigen; the antigenic sites being A, B (carrying the receptor binding site), C, D, and E. The antigenic sites are presented at the head of the molecule, while the feet are embedded in the lipid layer. The body of the HA molecule contains the stalk region and the fusiogenic domain which is needed for membrane fusion when the virus infects a new cell. At low pH, the fusion peptide is turned to an interior position. The HA forms trimers and several trimers form a fusion pore.

Prominent mutations in the antigenic sites reduce or inhibit the binding of neutralising antibodies, thereby allowing a new subtype to spread within a non-immune population. This phenomenon is called antigenic drift. The mutations that cause the antigenic drift are the molecular explanation for the seasonal influenza epidemics during winter time in temperate climatic zones. The immune response to the HA antigenic sites is followed by the production of neutralising antibody, which is the basis for resolving infection in an individual, and is sometimes part of the cross immunity found in elderly individuals when a new pandemic virus strain occurs.

Antigenic shift - also termed genome reassortment or just reassortment - arises when the HA is exchanged in a virus, for example H1 replaced by H5 resulting in the formation of a mosaic virus. This may happen when a cell is infected by 2 different influenza viruses and their genome segments are exchanged during replication.

This phenomenon of genome reassortment is frequently seen in water birds, especially ducks. Although the birds are seldomly symptomatic after infection, the virus is shed in their faeces for several months.

Neuraminidase

Like HA, neuraminidase (NA or N) is a glycoprotein, which is also found as projections on the surface of the virus. It forms a tetrameric structure with an average molecular weight of 220,000. The NA molecule presents its main part at the outer surface of the cell, spans the lipid layer, and has a small cytoplasmic tail.

NA acts as an enzyme, cleaving sialic acid from the HA molecule, from other NA molecules and from glycoproteins and glycolipids at the cell surface. It also serves as an important antigenic site, and in addition, seems to be necessary for the penetration of the virus through the mucin layer of the respiratory epithelium.

Antigenic drift can also occur in the NA. The NA carries several important amino acid residues which, if they mutate, can lead to resistance against neuraminidase inhibitors. Mutations that have been observed include:

  • R292K

  • H274Y, R152K, E119V

The letters represent amino acids (R, arginine; K, lysine; H, histidine; Y, tyrosine; E, glutamic acid; V, valine): the former letter is the original amino acid, and the latter the amino acid after mutation occurred.

When the amino acid arginine (R) is replaced by lysine (K) at position 292 of the neuraminidase glycoprotein, complete resistance may result. The mutation of R to K is linked to a single nucleotide exchange of AGA to AAA in the N gene. Position 292 is so significant because mutation may induce resistance not only against the substance oseltamivir, but also against zanamavir and two other new prodrugs.

M2 protein

When the virus particle is taken up in the endosome, the activity of the M2 ion channel is increased so that ions flood into the particle, inducing a low pH. As a result of this, the HA-M1 linkage is disturbed, the particle opens, the fusion peptide within the HA is translocated, and the HA fuses with the inner layer of the endosome membrane. The ribonucleoproteins are liberated into the cytoplasm of the cell and transported to the nucleus, where the complex is disrupted, and viral RNA synthesis is initiated.

The activity of the M2 protein is inhibited by amantadine, rimantadine and related substances.

Possible function of NS1

Human messenger RNA carries a poly-A tail at the 5' end. NS1, with a molecular weight of 26,000, and forms a dimer that inhibits the export of poly-A containing mRNA molecules from the nucleus, thus giving preference to viral RNA which is transported to the ribosome and translated. NS1 might also inhibit splicing of pre-mRNA. In addition, NS1 is probably able to suppress the interferon response in the virus-infected cell leading to unimpaired virus production.

Possible function of NS2

NS2 is a small molecule with a molecular weight of 11,000. In the particle it might be bound to M1 protein. Its function is believed to facilitate the transport of newly synthesised RNPs from the nucleus to the cytoplasm to accelerate virus production.

 

Replication cycle

Adsorption of the virus

The influenza virus binds to the cell surface by fixing the outer top of the HA to the sialic acid of a cells glycoproteins and glycolipids. The sialic acid linkage to the penultimate galactose, either alpha 2,3 (in birds) or alpha 2,6 (in humans), determines host specificity. Since sialic acid-presenting carbohydrates are present on several cells of the organism, the binding capacity of the HA explains why multiple cell types in an organism may be infected.

Entry of the virus

After attachment, the virus is taken up by the cell via a clathrin-coated receptor-mediated endocytosis process. When internalised, the clathrin molecules are liberated and the vesicle harbouring the whole virus fuses with endosomes. The contents of the vesicle are usually digested through a stepwise lowering of the pH within the phagosome.

Uncoating of the virus

When a certain level is reached, the lowering of the pH is stopped by the action of the M2 protein which induces the partial liberation of the fusion peptide of the HA. This allows the fusion of the HA with the membrane of the vesicle and liberation of the ribonucleoproteins (RNPs) into the cytoplasm, as described above. The ion influx from the endosome to the virus particle leads to disconnection of the different viral proteins; M1-protein aggregation is disrupted and RNPs no longer adhere to the M1-protein complex. Uncoating is completed within 20-30 min of virus attachment.

Synthesis of viral RNA and viral proteins

The RNPs are transported to the nucleus, where the polymerase complex binds to viral RNA, cleaves viral RNA by its endonuclease activity, and simultaneously leads to elongation. The production of viral RNA is limited by the NP in favour of mRNA. Both are transported to the cytoplasm, where viral proteins are generated at the ribosome. Part of the viral mRNA is spliced by cellular enzymes so that finally viral proteins, such as M1 and NS2, can be synthesised without any further cleavage. Some of the newly synthesised viral proteins are transported to the nucleus where they bind to viral RNA to form RNPs. Other newly synthesised viral proteins are processed in the endoplasmic reticulum and the Golgi apparatus where glycosylation occurs. These modified proteins are transported to the cell membrane where they stick in the lipid bilayer. When they reach a high enough concentration at the plasma membrane, RNPs and M1 proteins aggregate and condense to produce the viral particle. Finally, the particle is extruded from the membrane and will be liberated by the neuraminidase activity.

The time from entry to production of new virus is on average 6 h.

Shedding of the virus and infectivity

Immunohistological pictures show that foci of virus-producing cells are clustered in the mucous layer of the respiratory tract, in the gut and even in endothelial layers, myocardium and brain. Within nasal secretions, millions of virus particles per ml are shed, so that a 0.1 l aerosol particle contains more than 100 virus particles. A single HID (human infectious dose) of influenza virus might be between 100 and 1,000 particles. At least during the early course of influenza infection, the virus can be found also in the blood and in other body fluids.

Infectivity of influenza virus particles is preserved depending on temperature, pH and salinity of the water, and UV irradiation. At 4C, the half-life of infectivity is about 2-3 weeks in water. Due to the conformation of the lipid bilayer, survival under normal environmental conditions should be shorter.

Infectivity of the influenza virus particle is easily inactivated by all alcoholic disinfectants, chlorine and aldehydes. As far as is known, temperatures above 70C will destroy infectivity in a few seconds.

 


References

Nicholson KG, Webster RG, Hay AJ. Textbook of Influenza. Blackwell Science, Oxford, 1998.

Lamb RA, Krug RM. Orthomyxoviridae: The viruses and their Replication. In: Fields Virology fourth edition, Knipe DM, Howley PM eds, Lippincott, Philadelphia 2001, pp 1487-1531

Wright PF, Webster RG. Orthomyxoviruses. In: Fields Virology fourth edition, Knipe DM, Howley PM eds, Lippincott, Philadelphia 2001, pp 1533-1579

Special reference

Wetherall NT, Trivedi T, Zeller J, Hodges-Savola C, McKimm-Breschkin JL, Zambon M, Hayden FG. Evaluation of neuraminidase enzyme assays using different substrates to measure susceptibility of influenza virus clinical isolates to neuraminidase inhibitors: report of the neuraminidase inhibitor susceptibility network. J Clin Microbiol 2003; 41: 742-750. Full text at http://jcm.asm.org/cgi/content/full/41/2/742?view=long&pmid=12574276





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