'Myxoviruses'

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It is believed that all (-)sense RNA viruses may have evolved from a common ancestor - all replicate their genomes by a common mechanism. However, the Mononegvirales have non-segmented genomes with similar organization and control of gene expression.

Orthomyxoviruses

Morphology:

Influenza virus particles are highly pleiomorphic (variable), mostly spherical/ovoid, 80-120nm diameter, but many forms occur, including long filamentous particles (up to 2000nm long x 80-120nm diameter). Different strains of virus vary in their tendency to form filaments - this property maps to the matrix protein.

© Paul Digard, Dept Pathology, University of Cambridge.

The outer surface of the particle consists of a lipid envelope from which project prominent glycoprotein spikes of two types: haemagglutinin (HA), a 135Å trimer neuraminidase (NA), a 60Å tetramer The inner side of the envelope is lined by the matrix protein. The particles are relative labile (half-life a few hours @ R.T.), not resistant to drying, etc.

The genome segments are packaged into the core. The RNP (RNA + nucleoprotein, N) is in a helical form with the 3 polymerase polypeptides associated with each segment.

© Paul Digard, Dept Pathology, University of Cambridge.

To view an electron micrograph of negatively-stained influenza virus particles click here.

© Russell Kightley Media

Host Range:

• Influenza A viruses infect a wide variety of mammals, including man, horses, pigs, ferrets and birds. The main human pathogen, associated with epidemics and pandemics. There are 15 known haemagglutinin (H) serotypes and 9 known neuraminidase (N) serotypes. Pigs and birds are believed to be particularly important reservoirs, generating pools of genetically/antigenically diverse viruses which get transferred back to the human population via close contact between humans and animals.

• Influenza B viruses infect mammals only and cause disease, but generally not as severe as A types. Unlike influenza A viruses, influenza B viruses do not have distinguishable serotypes.

• Influenza C viruses also infect mammals only, but rarely cause disease. They are genetically and morphologically distinct from A and B types.

Species barrier:
The species which different types of influenza viruses are able to infect are determined by different forms of sialic acid present on the virus glycoproteins. In particular, this property depends predominately (but not exclusively) on the amino acid at position 226 of the haemagglutinin protein:

This provides a considerable species barrier between birds and humans which is not easily overcome. However, pigs provide a "mixing pot" - able to be infected by both types of virus & thus allowing the passage of avian viruses to humans.

Genome:

Replication:

• Entry into the cell is facilitated by binding of the HA spikes to mucoproteins containing terminal N-acetyl neuraminic acid (NANA = sialic acid) groups. This interaction can be reversed by polysaccharide cleavage by NA spikes - prevents virus being 'sequestered' by inappropriate cell types - NANA is very common on cell surfaces/in mucus and would otherwise swamp the virus particle, effectively 'neutralizing' it.
  Online Experiment: Haemagglutination

• After binding, the particle is engulfed by endocytosis via coated pits into endocytotic vesicles and finally endosomes. These are acidified by the cell; at about pH 5.0, the HA monomers are cleaved by trypsin-like enzymes in the endosome at the base of the 'stem' into the HA₁ (upper NH₂ portion) and HA₂ (trans-membrane COOH portion) polypeptides (linked by disulphide bonds). This cleavage causes a conformational change in the HA spike which activates the membrane-fusion function located in HA₂. The combination of the close proximity of the virus envelope and the membrane of the endosome and the active membrane-fusion domain of HA₂ results in fusion of the two membranes and passage of the nucleocapsid into the cytoplasm.

• pH activated ion-channels made up of M2 protein are also important in uncoating:

© Paul Digard, Dept Pathology, University of Cambridge.

• Specific nuclear targeting sequences in the NP protein result in translocation of the nucleocapsid into the nucleus. (Portela A, Digard P. The influenza virus nucleoprotein: a multifunctional RNA-binding protein pivotal to virus replication. J Gen Virol 83: 723-734, 2002)

   

• Genome segments are transcribed by the 3 polymerase polypeptides associated with each genome segment:

i) During the initial phase of infection (~2h), active host cell DNA synthesis is required and replication is prevented by U.V, mitomycin C, etc, but not thereafter. The reason is that the initial step in replication is that PB2 attaches to the m7G cap of host mRNAs. This structure is cleaved from the mRNA by PB1, remaining attached to PB2. The cap serves as a primer for RNA synthesis and 11-15 nucleotides (complementary to the conserved sequence at the 3' end of the vRNA) are added by PB1, after which PB2 dissociates from the growing strand (these structures can be isolated from infected cells). PB1 + PA then complete the synthesis of the (+)sense strand.

ii) Two classes of (+)sense RNA are made in infected cells:

• Incomplete, 3' polyadenylated transcripts which are exported to the cytoplasm and serve as mRNAs (due to the presence of a specific polyadenylation sequence ~20nt from the 5' end of the (-)sense vRNA template strand)
• cRNA = complete, non-polyadenylated (+)sense copies of the (-)sense vRNA (made by readthrough of the polyadenylation signal) which serve as template for the synthesis of progeny (-)sense vRNAs.

Most of the proteins made (e.g. HA, NA) remain in the cytoplasm or become associated with the cell membrane. However, the NP protein migrates back into the nucleus, where it associates with newly-synthesized vRNA to form new nucleocapsids. These migrate back out into the cytoplasm and towards the cell membrane (mechanism unclear). The level of free nucleoprotein (NP) is thought to control whether mRNA or cRNA is produced, i.e. later in infection there is lots of NP, mRNA synthesis stops but cRNA synthesis continues. NP is thus a crucial switch in the replication cycle between expression and assembly.

~4h after infection, patches of M1 protein form on the cell membrane, which appears to thicken, incorporating HA and NA on the outside of the membrane. The nucleocapsid segments are incorporated into the particle as it buds out through the membrane. NA is thought to have a role in release of budding particles (inhibited by anti-NA Abs).

The influenza A NS1 protein represses interferon-β synthesis by preventing activation of the NF-kappa-B signaling pathway & transcription of interferon genes Wang X, et al. Influenza A virus NS1 protein prevents activation of NF-kappaB and induction of Alpha/Beta interferon J Virol. 2000 Dec;74(24):11566-73). Deletion of NS1 results in an attenuated phenotype.The filovirus VP35 protein has the same function and can substitute for influenza NS1 (no sequence similarity). Likewise, the bunyavirus NS_S protein has a similar function (no sequence similarity).

The packaging mechanism responsible for sorting eight distinct genome segments into each particle is not known. It has been suggested that the sorting of genome segments is not a purely random process, and recent structural studies support this view (Noda T, et al. Architecture of ribonucleoprotein complexes in influenza A virus particles. Nature. 2006 439: 490-492).

Virus particles are gradually released from the surface of the cell over a period of several hours. The cell does not lyse, but eventually dies (due to disturbance of normal cellular macromolecular synthesis?).

Pathogenesis:

Spread is by aerosols - very efficient (occasionally fomites). Even in epidemics, there are 3:1 - 9:1 infections:clinical case - very infectious. Primary infection involves the ciliated epithelial cells of the U.R.T. Necrosis of these cells results in the usual symptoms of the acute respiratory infection (fever, chills, muscular aching. headache, prostration, anorexia). Normally self-limited infection usually lasts 3-7 days (+convalescence). Death from primary influenza infection is very rare and appears to be determined by host factors rather than 'virulence' of virus. Damage to respiratory epithelium predisposes to secondary bacterial infections which accounts for most deaths (see below).

Chen W. et al: A novel influenza A virus mitochondrial protein that induces cell death. Nature Medicine 7: 1306-1312, 2001.
"While searching for alternative reading-frame peptides encoded by influenza A virus that are recognized by CD8+ T cells, we found an abundant immunogenic peptide encoded by the +1 reading frame of PB1. This peptide derives from a novel conserved 87-residue protein, PB1-F2, which has several unusual features compared with other influenza gene products in addition to its mode of translation. These include its absence from some animal (particularly swine) influenza virus isolates, variable expression in individual infected cells, rapid proteasome-dependent degradation and mitochondrial localization. Exposure of cells to a synthetic version of PB1-F2 induces apoptosis, and influenza viruses with targeted mutations that interfere with PB1-F2 expression induce less extensive apoptosis in human monocytic cells than those with intact PB1-F2. We propose that PB1-F2 functions to kill host immune cells responding to influenza virus infection."

Prevention/Treatment:

• The target for both drugs is the matrix protein (M2), but resistance to the drug maps to the haemagglutinin (HA) gene.
• This biphasic action results from the inability of drug-treated cells to lower the pH of the endosomal compartment (a function normally controlled by the M2 gene product), a process which is essential to induce conformational changes in the HA protein to permit membrane fusion.

Due to side effects and relatively poor efficency, amantadine is not licenced for use against influenza in the UK.
Neuraminidase inhibitors: Due to the important role played by neuraminidase in transmission of the virus throughout the host, it has recently been seen as a prospective target for anti-influenza drugs. The active site of neuraminidase is made up of 11 universally conserved amino acid residues, which have been targeted to produce new drugs. Synthetic sialic acid analogues work well against neuraminidase at lower levels than amantadine and rimantadine. Most importantly, these drugs work against all strains of influenza A and B:

• Hoffman-La Roche's Tamiflu (oseltamivir phosphate, GS4104) is an inhibitor of neuraminidase taken in pill form which has been shown to confer decreased severity and duration of symptoms in clinical trials.
• Glaxo Wellcome's Relenza (Zanamivir) must be inhaled. Both drugs are now approved for use in the UK.

• Chemotherapeutic control of influenza. Stephenson I, Nicholson KG. J. Antimicrob. Chemother. 1999 44: 6-10
• Medinfo: Problems with Relenza

Vaccines: Isolated HA gives good serological protection (estimated at 60-80%). Vaccines are produced by reassortment of egg-adapted strains with strains with the required HA type. Large amounts of virus are then grown in embryonated eggs (cheap and efficient), purified and formalin inactivated. The vaccine is given sub-cutaneously: if of a new antigenic type, 2 doses are necessary for adequate protection (i.e. depends on age of patient).

The Snag: In order to give time for adequate vaccine stocks to be produced, a decision must be made, usually in about August, as to which HA type to use for this years vaccine (for the winter season). There is an elaborate and sophisticated epidemiological monitoring system worldwide, which helps these decisions:

• World Health Organization Influenza Surveillance

• UK Department of Health current vaccination guidelines

A naked DNA vaccine against influenza virus using the nucleoprotein (core) gene of the virus has been developed and is currently in clinical trials. The nucleoprotein is similar across many strains of influenza, unlike envelope or surface proteins which change extensively from one strain to another. Nucleoproteins are therefore not subject to the same humoral immune pressure with antigenic drift as are the surface glycoproteins targeted by conventional vaccines.

History of Influenza:

The 1918 "Spanish flu" pandemic killed between 20-40 million people. In recent years, Jeffery Taubenberger & colleagues have tried to find out why this virus was so pathogenic. Unfortunately, sequencing and reverse genetic studies have shown that neither the HA nor the NS1 proteins appear to be responsible, so the reason currently remains a mystery.

America's Forgotten Pandemic: The Influenza of 1918
by Alfred W. Crosby.

Imagine that the world is gripped in the throes of the lengthy stalemate of a senseless war that has depleted Europe of most of its young men and resources, and those that remain are destitute, dispirited, starving, and suffering from the lost of loved ones. In the midst of this war, a formerly rather innocuous disease suddenly mutates into a new killer strain which infects all corners of the globe, from Alaska to Africa, within a matter of weeks. This new disease is not only remarkably contagious, but it is so lethal and destroys so many lives in such a short time-frame that even the ghastly global war pales in comparison. The scariest aspect of this tale is that it is not fiction.
(Amazon.co.UK)

Killer 'flu is coming?

In May 1997, a three year-old boy died of complications of influenza in the intensive care unit of a Hong Kong hospital. This case was the first isolation of an influenza A subtype H5N1 in a human. Subtype H5 influenza viruses can cause lethal avian influenza, a disease that can decimate flocks of domestic poultry.
Fortunately, this outbreak eventually fizzled out, but not before there had been 18 confirmed human cases and 6 deaths. Why did the outbreak go away? The H5 haemagglutinin has an HA226_gln sequence - i.e. was an avian virus poorly adapted to humans. There is clear evidence that between its emergence in 1997 and 2004, H5N1 isolates have become significantly more pathogenic for mammals (Guan Y, et al. H5N1 influenza: a protean pandemic threat. Proc Natl Acad Sci USA. 2004 101: 8156-8161;Seo SH, et al. The NS1 gene of H5N1 influenza viruses circumvents the host anti-viral cytokine responses. Virus Res. 2004 103: 107-113). All that is required now is for the virus to acquire the ability for direct human transmission and we will be in big trouble. H5N1 influenza re-emerged in Vietnam late in 2003. Also worrying, in March 1999, H9N2 viruses were isolated from two hospitalized children in Hong Kong. Molecular analyses indicated that the HA and NA genes of the human H9N2 isolates are avian in origin and prevalent in poultry in Hong Kong. This virus has HA226_leu, i.e. is a human-adapted virus, giving it the potential for rapid (pandemic?) spread in a human population which has never seen this virus before. Is THIS the next pandemic strain? OTOH, it is now approaching 50 years since the 1957 H2N2 "Asian flu" pandemic - is THIS the virus waiting in the wings to make a comeback?

So what's the worst that could happen?

• A pandemic of human-adapted avian influenza such as the 1997 H5N1 virus.
• Such a reassortant could easily have a mortality rate of 30-40%.
• Within a few months 10-25% of the world's population could have been infected.
• 6.3 billion * 0.4 * 0.25 = over half a billion deaths.
• or worse ...

Latest:

Evolution of H5N1 Avian Influenza Viruses in Asia

Kuiken T, et al. Host species barriers to influenza virus infections. Science. 2006 21: 312: 394-397

• Receptor Binding & Membrane Fusion In Virus Entry: The Influenza Hemagglutinin. Annu. Rev. Biochem. 2000. 69: 531-569

• Medscape: Influenza January 2006

• Emerging Infectious Diseases: Influenza Special Issue January 2006

• Search the WWW for more information on this topic

• Search MEDLINE for the latest publications on this topic

© MicrobiologyBytes 2007.