MicrobiologyBytes: Virology: Orthomyxoviruses Updated: February 3, 2012 Search

'Myxoviruses'

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Name is from the Greek 'myxa' = mucus!

Influenza pandemics have been recognized for several centuries. In the last century, H. influenzae (and other bacteria) were cited as the causal agent, but in 1933 Smith, Andrewes and Laidlaw isolated the virus in ferrets. In the 1940's (still pre-tissue culture, primitive immunology, few other viruses known), the characteristic property of haemagglutination was observed, followed by the discovery that the virus could be propagated in embryonated hens eggs (after adaptation) - this made influenza one of the best studied viruses during this period. More viruses with similar properties were added to the group, until it was split into two families in the 1970s, the Paramyxoviridae and the Orthomyxoviridae:

Characteristic: Orthomyxoviridae: Paramyxoviridae:
Size: Particle: 80-120nm (highly pleiomorphic)

Core diameter: 9nm

Particle: 125-250nm (somewhat pleiomorphic)

Core diameter: 14-20nm

Replication: Nuclear Cytoplasmic
Genome: Segmented (-)sense RNA Non-segmented (-)sense RNA

Group V: (-)sense RNA Viruses

Family

(Subfamily)

Genus

Type Species

Hosts

Orthomyxoviridae

Influenza A virus

Influenza A virus

Vertebrates

Influenza B virus

Influenza B virus

Vertebrates

Influenza C virus

Influenza C virus

Vertebrates

Isavirus

Infectious salmon anemia virus

Vertebrates

Thogotovirus

Thogoto virus

Vertebrates

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.

Influenza particle
© 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.

Influenza RNP
© Paul Digard, Dept Pathology, University of Cambridge.
To view an electron micrograph of negatively-stained influenza virus particles click here.
Graphic of influenza particle
© Russell Kightley Media

Host Range:

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:

Human viruses:
HA226leu
Avian viruses:
HA226gln

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:

Consists of s/s (-)sense RNA in 8 segments (7 in Influenza C). The structure of the influenza virus genome is known in great detail because of the tremendous amount of genetic investigation (conventional and molecular) which has been done. The 5' and 3' terminal sequences of all the genome segments are highly conserved:

Influenza genome replication

Segment: Size(nt) Polypeptide(s) Function
1 2341 PB2 Transcriptase: cap binding
2 2341 PB1 Transcriptase: elongation
3 2233 PA Transcriptase: protease activity (?)
4 1778 HA Haemagglutinin
5 1565 NP Nucleoprotein: RNA binding; part of transcriptase complex; nuclear/cytoplasmic transport of vRNA
6 1413 NA Neuraminidase: release of virus
7 1027 M1 Matrix protein: major component of virion
M2 Integral membrane protein - ion channel
8 890 NS1 Non-structural: nucleus; effects on cellular RNA transport, splicing, translation. Anti-interferon protein.
NS2 Non-structural: nucleus+cytoplasm, function unknown

Genetics of influenza viruses. Ann Rev Genet. 2002 36: 305-332.

Replication:

Influenza replication

Uncoating
© Paul Digard, Dept Pathology, University of Cambridge.

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:

d/s (+/-) replicative intermediate structures can be isolated from the nucleus of infected cells.

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 NSS 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. (2012) Three-dimensional analysis of ribonucleoprotein complexes in influenza A virus. Nature Communications 3, Article number: 639 doi:10.1038/ncomms1647).

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?).

 

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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:

Several anti-influenza drugs exist. Amantadine and rimantadine are active against influenza A viruses (but not B viruses). The action of these closely related agents is complex and incompletely understood, but they are believed to block cellular membrane ion channels.
Influenza drugs

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:

 

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).

Antigenic change

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:

Unfortunately, because of the capricious nature of influenza virus, the right decision which gives truly effective protection is only made about one year out of two. There is much interest in other types of vaccine - especially genetically engineered/subunits and live attenuated, but neither is currently in use (the molecular basis for attenuation is not clear).

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:
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.

CoverAmerica'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 HA226gln 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 HA226leu, 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?

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



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