Thursday 11 July 2013

Influenza viruses (IFVs)...

Influenzavirus taxonomy
GeneraInfluenzavirus A, Influenzavirus B, Influenzavirus C
SpeciesInfluenza A virus, Influenza B virus, Influenza C virus
Genome(-)ssRNA, 7 (IFCV)- 8 (IFAV/IFBV) segments (~14kb total length)
GenesPB2, PB1, PA, HA, NP, NA, M, NS
VirionEnveloped, approximately spherical, 80-120nm
Influenza viruses (IFVs) are always in the news somewhere as the 'flu season' begins, spikes or is otherwise noteworthy in one hemisphere or the other. 

The disease resulting from infection by an IFV is called influenza (the 'flu')and is usually among the most clinically severe of respiratory virus (viruses spread by sneezing, coughing or self-inoculation of the [usually] upper airways) infections. 

Signs (those things that can be measured; measurable temperature, cough, runny nose, inflamed red throat) and symptoms (those things you feel; feeling hot, cold, sore, crook) can include cough, sore throat, fever, headache, myalgia (sore muscles). 

In Australia, influenza cases increase during the cooler months of May-September

Like all respiratory viruses, repeat infection by the same "strain" is highly likely over a lifetime. Vaccines and antivirals don't stop a virus from being breathed in but they do interrupt the worst symptoms so subsequent infections with the same strain will usually manifest as less severe's the first one, or infection of a high risk person with a predisposition for severe disease, that will be the worst. And make no mistake, infection by an IFV that your body doesn't recognize is not fun. 

In the worst cases infection can kill and IFVs are notorious for precipitating a secondary bacterial infection that can lead to pneumonia. Interestingly, getting an IFV will keep you clear of other viruses for a short period of time. 

IFV infection more commonly leads to a lot of time in bed, time off work, poor function at work (and then poor function of the workplace since you've probably spread it to your workmates), doctor appointments, time off school, time off work to look after children who are off school, money spent in buying over-the-counter drugs and prescription drugs. 

It's not a common cold virus.

Influenza (the illness in humans) has many faces including seasonal (A: H3N2, H1N1; B), avian (or bird) (A: H7N7, H7N9, H5N1..), swine (or pig) and pandemic (A: H1N1, H2N2, H3N2..?).

The viral proteins are encoded by a multipartite (many bits) RNA genome.

IFVs are divided into 3 groups determined by the ribonucleoprotein (RNP) antigen.

A - This group is the cause of epidemics and pandemics and has an avian intermediate host (IH).
B - This group causes epidemics and has no IH
C - This group does not cause epidemics and causes mild disease

Virus strains are eventually named according to influenzavirus type, region where first isolated, strain number, year strain isolated and major type of important proteins e.g. Influenza B/Hong Kong/330/2001.


Haemagglutinin (HA)

  • Encoded by RNA segment #4
  • Most antigenic surface protein so is under selective pressure to change and escape the host antibody response
  • Can agglutinate red blood cells - hence the nomenclature
  • Cleavage by host-cell protease is required (resulting in HA1 and HA2) for infection to occur
  • Influenzaviruses A occur according to minor or major changes in this gene. Influenzaviruses B change very slowly.
  • 17 HAs (16 avian, 1 bat)
  • HA requires host cell sialosides (sialic acid moieties that terminate a glycolipid or glycoprotein cellular protrusion) as their receptor as well other sugar chain structures and lengths. Sialosides alone are not the sole factor in receptor specificity.[14]
Neuraminidase (NA)
  • Encoded by RNA segment #6
  • Enzyme that uses neuraminic (sialic) acid as a substrate
  • Important in releasing mature virus from cells
  • A mushroom shaped protein that is partly exposed above the viral envelope and partly embedded (hydrophobic region) in the viral membrane
  • Inhibitors, which function against both IFAV and IFBVs, block enzyme function by competitive inhibition of binding to the natural sialic acid substrate. Once bound, the inhibitor fits more tightly and this blockage prevents the virus from releasing itself (budding) from the cell.
Infection and Replication...
  • Endocytosis follows receptor-specific virus binding to the target cell
  • HA's conformation is altered by the low (acidic) pH inside the endocytotic vacuole causing the virus to uncoat
  • The genome is transported to the nucleus
  • Influenza's negative sense RNA is transcribed to positive sense to work as mRNA. This is achieved with the virus's RNA polymerase and the resulting RNA is double-stranded. From this "intermediate form" new negative sense can be synthesized for inclusion into new virions
  • Human influenza (seasonal influenza viruses that transmit efficiently between humans) virus HA protein prefers binding to 'human-type' α2,6-sialosides (found predominantly in the upper respiratory tract and eye) - best for human-to-human transmission
  • Avian influenza viruses (and closely avian-related, zoonotic spillover viruses) prefer binding to α2,3-sialosides (found throughout humans but considered more of a pathogenicity factor in the lower respiratory tract; expressed more in children than adults) and these are found throughout birds and both sialosides are found in pigs. Pandemic IFVs begin life in birds (α2,3) with early human isolates from 1918, 1957 and 1968 adapting and being able to recognize α2,6. Conversion is a pathogenicity determinant.
Antigenic Shift and drift...

Reassortment of gene segments (antigenic shift) is a common feature of influenza A viruses, but not so common for IFBVs or IFCVs. When two different IFAV subtypes infect the same cell, their RNA segments can become mixed during replication. New subtypes produced in this way may survive due to a selective advantage within the population. This mixing is happening now between H1N1 and H5N1 in some parts of the world. Constant mutations in the RNA of influenza viruses lead to polypeptide changes which can affect the antigenic profile of the subtype, creating a variant that may be able to escape existing community immunity. Antigenic drift changes the virus less dramatically than does antigenic shift. If these mutations affect HA or NA they may cause localized epidemics.

A schematic depicting how new influenza A virus subtype
and strains of existing subtypes, emerge.
Virus spread (transmission)...

Transmission of IFVs from one host to another is by (i) droplet inhalation (e.g aerosols made by sneezing and coughing - each droplet carrying infectious viruses), (ii) touching virus-contaminated surfaces (called "fomites" e.g. cupboard or door handles, phones, kids toys, chairs, used tissues etc) or (iii) by direct contact with an infected host (e.g. kissing). So infection usually makes first contact with the upper respiratory tract however fewer virus particles are reportedly required to infect the lower respiratory tract than the upper.

You can imagine that if virus-containing droplets remain intact for longer, the the risk of picking up IFV infection will stay high. This timeframe is associated with humidity and temperature.

The immune response...

The NS1 protein of influenza viruses inhibit the interferon (IFN)-response to the virus, probably through its double-stranded RNA-binding ability. This agonist action helps hide the virus from a number of sentinel molecules that exist within our cells looking for dsRNA. Once found the trigger an early production of IFN, the first wave of which drives the cell into an antiviral state. Preventing this would yield obvious benefits for viral replication....time is virions! For H5N1 this maps to amino acid 92 of the NS1 protein.[13]

The disease (influenza)...

Virus reproduces within the epithelium and destroys the cilia.

Epidemics (cases of a disease increase beyond what is normal or expected in an area or population) and pandemics (a very widespread epidemic but not necessarily resulting in more severe illness) are due to the appearance of new IFV strains against which we have insufficient immunity.

In the 20th century there were three IFV pandemics in 1918-1919 (called the Spanish flu causing millions of deaths, many linked to bacterial secondary infections), 1957-1958 (called the Asian flu, comparatively mild illness) and 1968-1969 (Hong Kong flu, also more mild than the Spanish flu).

The 21st century saw it first pandemic due to pandemic (H1N1) influenza (2009), in 2009 (see box above).

One to three days after infection symptoms mediated by cytokines released from leukocytes and damaged tissues cause classic flu symptoms:

  • Chills
  • Malaise
  • Tiredness
  • Fever
  • Muscular aches (myalgia)
  • Loss of appetite
  • Runny nose
  • Cough
Tissue damage may promote bronchitis and interstitial pneumonia.

There is an associated viraemia (virus in the blood) during severe infections when virus load rises-this is a marker disease severity.[4] This is not uncommon among respiratory virus infections (also detected in some HRV infections);

Temperature usually decreases to normal by day three;

Severity is also related to previous IFV and vaccine exposure (thus related to age also). Because IFV subtypes can change from year to year and because immunity drops off over time, last year's flu shot (or IFV infection) may not protect you from a different mix of IFV subtypes that may be circulating this year. Ask your Doctor about getting the new shot.

A twofold or greater rise in specific antibody titre in paired sera is diagnostic of recent infection and virus can be isolated from respiratory secretions during the initial stages of infection.

Complications of infection include:

  • Secondary bacterial pneumonia
  • Rarely indirect CNS disturbances (eg. encephalomyelitis)
Some specific IFVs that have infected humans...
Influenza A (H1N1) 2009 [also called H1N1pdm]

In 2009, a novel IFAV human virus was detected (mostly by PCR-based molecular methods) in symptomatic patients in 209 from >45 countries. It was genetically related to swine influenza A viruses isolated in North America, Asia and Europe. 

Influenza A (H1N1) is susceptible to standard anti-IFV antiviral medication and there is now a vaccine to prevent infection. The method of viral transmission (coughs and sneezes) and the symptoms (acute onset of fever, cough, sore throat, muscle aches, headache, chills and fatigue as well as nausea, vomiting or diarrhoea) seem to follow standard "flu" and as such Pandemic (H1N1) 2009 caused mortalities particularly among susceptible populations as the number of infected cases spread. 

One year later, fewer annual H1N1pdm-related deaths were recorded in the United States compared to those from seasonal IFV however more H1N1pdm-related deaths occurred among those <65yr of age compared to seasonal IFV from which most deaths occur in people >65yrs of age.

Early live attenuated (LAIV) and inactivated vaccines (delivering H) were based on A/California/7/2009 (H1N1)pdm.8 The LAIV also afforded some antibody-mediated protection against other IFV strains. The broadest cross-reactivity was from antibodies targeting the stem region of HA.[9]

Avian influenza A virus H7N9

The avian influenzavirus strain (now added to the list of agents causing "bird flu") called H7N9 is generally considered a low pathogenic avian influenza virus (LPAIV) least in its feathered hosts. They are also considered relatively rare causes of infection. This is an IFAV that has evolved in birds. However, when it was identified in humans in March 2013, it was linked to its first two deaths[10] in this host(an 87y-old male [sick 19.02, died 04.03; 2 family member(s) also reported ill[12] with pneumonia but H7N9 negative] and a 27y-old male butcher [sick 27.02 and died 10.03], both in Shanghai, China) and a critical illness (35y-old female in Chuzhou city). Four more cases were report 02.04.

nfluenzavirus H7N9 has its own page on VDU.

Influenzavirus Lab diagnoses...
is often diagnosed clinically during flu "season". The 2009 pandemic did raise the spectre of flu-like illness being associated with non-IFV viruses as well. Even common cold viruses (which are all respiratory viruses really) cause flu-like symptoms on occasion. Testing is a crucial part of a definitive diagnosis of IFV infection.

Today's testing focuses mainly on IFAV and IFBV...IFCV is a more rare beast. Isolating virus from patient samples (including nasopharyngeal swab/wash, throat swab, bronchial wash, nasal/endotracheal aspirate or sputum) in culture is the longest method (days)and is not recommended2 as the screening tool of choice. Ii still though of as a gold standard because you have shown actively replicating virus in the host...and culture usually identifies it at levels that are likely to be causal. But it can miss lead-in or lead-out infections (where viral loads are lower) and can be hit and miss if sample quality issues (cold chain in transport, storage, freeze-thawing etc) arise. Direct and indirect immunofluorescent methods on he patient cells can be completed in hours as can reverse transcription polymerase chain reaction testing (RT-PCR).

Rapid IFAV and/or IFBV diagnostic tests (RIDTs) that target the nucleoprotein (NP) can be completed at the bedside/doctor's office in minutes but rely on good specimen collection within 72hrs after illness start and can miss a third of IFV-positive cases. They generally don't subtype the IFVs.

Antibody (Ab) testing detects the response of the host to they require an early (acute) sample and a convalescent sample (from the recovering host) a couple of weeks later. A twofold or greater rise in specific antibody titre in paired sera is diagnostic of "recent" infection (its all relative in the age of RIDT and PCR). Mostly used for public health epidemiology studies but not for screening.

Prophylactic and therapeutic options for IFVs...

Antiviral drugs exist to treat (amantadine, rimantadine, zanamavir[Relenza] and oseltamivir[Tamiflu; works best if administered within 48 hours after signs and symptoms begin]) and prevent (amantadine, rimantadine, and oseltamivir) IFV disease. Oseltamavir and zanamavir are neuraminidase inhibitors. More and more however, we are noting single amino acid mutations which confer resistance to antivirals driving new research into different viral targets and mechanisms to treat infection once it has been acquired. Those benefiting most from the use of antivirals include people 65 years or older, pregnant women, indigenous people 17 years or older, nursing home/aged care facility residents, and anyone older than 6 months of age with an underlying medical condition (cardiac or kidney disease, severe asthma, diabetes, impaired immunity or neuromuscular disease).

A table listing a few key nucleotide changes/mutations which lead to viral changes that have an important impact on the infected host. It is not all-inclusive and only some of the scientific literature is listed, but enough to get you started. I recommend a longer and more detailed list compiled and hosted by the US Centers for Disease Control and Prevention and its collaborators.
Vaccines exist to prevent severe disease by influenza viruses. A conventional vaccine strain takes time to grow, and some strains do not grow well using traditional methods.
  • Smallpox vaccines made in eggs (using chick embryos) were used in 1935[6] and egg-based influenza vaccines quickly followed helped by additives (called adjuvants) which boost our response to the injected virus[7] by acting as an irritant (recruiting a bigger amplifying response) or as a base (called the depot effect) from which vaccine antigen is slowly released/presented to the host immune system over time (e.g. oil emulsions or aluminium hydroxide).
  • When a new pandemic threat emerges, a high growth seed strain is sought quickly
  • New methods do not need to grow "live" (infectious) virus, but use reverse engineered viruses made through recombinant DNA technologies, which grow well in insect cells[5], removing the need for eggs, improving scalability and expressing just those parts of the virus which give us protection from infection (the HAs from multiple IFVs for example).
  • Vaccines mostly provide IFV components that protect you from three different influenza virus strains (trivalent vaccine; 2x FluA,1x FluB) that are expected to be circulating for the next season. Quadrivalent vaccines are on the way (four strains)
  • In people who are already "primed", a smaller dose could be used to boost immunity.
The literature...

  1. Fedson,DS, Pandemic Influenza and the Global Vaccine Supply.CID 2003;36:1552-
  3. Beck E, MMWR. 2012;64(43):873-.
  4. Tse H, PLoSONE. 2011;e22534.
  7. Francis T, Bull World Health Org. 1953.p725-.
  9. Li G-M., PNAS. 2012;109:p9047-
  11. Palese P, PNAS. 2012;109:p2211-
  13. Seo et al. Nat Med 2002 8(9):950-
  14. Ge & Wang. Crit Rev Microbiol. 2011. 37(2):157-

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