An Influenza Primer

Influenza pandemics occur in 10 to 40-year cycles and have historically affected as much as up to 40% of the population

Posted by Stephen on August 26, 2012

Influenza is derived from the Italian ‘un influenza di freddo’, meaning ‘influence of the cold’. It is an infectious disease of the respiratory system that causes regular seasonal epidemics in a number of mammalian and avian species. Human influenza pandemics occur in 10 to 40-year cycles and have historically affected as much as up to 40% of the population.

Classically it is characterised by fever, chills, coughing, sore throat, headache and generalised muscle pain. Children may present with gastrointestinal symptoms. Influenza infection usually takes 3 – 7 days for a complete recovery. Many of the clinical signs observed are a result of the bodies immune response producing large quantities of pro-inflammatory cytokines and chemokines which can lead to a ‘cytokine storm’. Fatalities usually occur due to pneumonia as a result of secondary bacterial infections. A third of people infected are asymptomatic.

Influenza A virus via Viral Zone

Structure

Influenza is a linear, negative sense, single-stranded RNA virus of the family Orthomyxoviridae (Greek orthos, proper, and myxa, mucus). It consists of 7 or 8 RNA segments; influenza A contains 8 RNA segments as seen in the figure above. Each RNA segment codes for one or two proteins.

Influenza is a spherical or pleomorphic enveloped virus that is surrounded by a lipid membrane, with helical nucleocapsids. Filamentous forms can occur and this is most common with influenza C virus. It is usually 80 to 120 nm in size. HA (haemagglutinin) and NA (neuraminidase) are two large glycoproteins which emanate from the surface of viral particles. Currently 16 HA and 9 NA glycoproteins have been identified. NA is involved in the virus budding from the cell and mobility of the virus in mucus. HA binds to cell receptors to allow entry of the virus into epithelial cells. Antibodies to HA and NA are largely responsible for virus neutrilisation.

Influenza virus has a segmented genome and genetic reassortment can occur resulting in new subtypes. The two main mechanisms of genetic change are the accumulation of point mutations, antigenic drift, and the reassortment between two subtypes, antigenic shift.

EM of influenza virus via Wikipedia

Types

In the family Orthomyxoviridae there are 5 genera: Influenza A, influenza B, influenza C, Thogotovirus and Isavirus. Influenza A virus is responsible for the majority of clinical infections. Subtypes of influenza A are typed based on H and N antigens. It causes significant infection in humans, birds, pigs and horses. Influenza B and C are primarily human pathogens. Infections of influenza B and C viruses in non-humans have been described; influenza B has been isolated from seals and influenza C from swine. Isavirus and Thogotovirus as their name suggest are not influenza viruses. In the genus Isavirus infectious salmon anemia virus (ISAV) is the only species. Thogotovirus is a tick borne arbovirus that is a zoonosis associated with fever and encephalitis.

The international standard classification system classifies influenza viruses according to their nomenclature: type, host species, place, year of isolation and H and N subtypes.

Influenza C virus via Viral Zone

Entry and Assembly

Viruses are intracellular parasites; they cannot replicate on their own without a living cell. Influenza infection usually occurs due to inhalation or ingestion of respiratory secretions that contain virus. The incubation period is generally one or two days.

Influenza viruses attach to virus specific receptors on the plasma membrane of epithelial cells. Influenza attaches by haemagglutinin (HA) protein which attaches to the sialic acid of the epithelial cells of the nose, throat and lungs of mammals or intestines of birds. Proteases cleave the HA to allow endocytosis to occur.

Influenza virus replication via Wikipedia

A drop in pH occurs as the virus approaches the nucleus causing the viral envelope to fuse with the vacuoles membrane. This drop in pH is achieved by M2 ion channels acidifying the virion core. This change induces the release of core proteins and viral RNA while this complex is transported to the nucleus. Transcription and translation of viral RNA commences resulting in the production of new viral proteins which are secreted by the golgi apparatus to the cell surface or return to the nucleus to form new viral genome particles. The viral proteins that reach the cell surface form HA and NA glycoproteins. NA cleaves sugars that bind the mature viral particles allowing release of progeny virus. The viral core then buds of the epithelial membrane taking part of it to form its on membrane with HA and NA on its surface. There is a noticeable absence of RNA proof reading enzymes which results in an error every 10 thousand nucleotides during transcription and translation. This build up of point mutations results in antigenic drift. Antigenic shift occurs as mentioned earlier due to reassortment and mixing of RNA segments. Antigenic shifts produce sudden large changes. These processes allow influenza viruses to possess high genetic diversity.

Most influenza A subtypes are cleaved by host proteases found specifically in the epithelial cells of the reproductive and respiratory tracts. Due to susceptibility to cleavage by proteases present in a number of different tissues it is easy for influenza A a to set up generalised infection. All avian influenza viruses that have proved fatal to humans possessed a highly cleavable HA. Post-translational cleavage of viral precursor HA molecule is required to the virus to perform optimally. Mutations of low pathogenicity avian influenza (LPAI) can result in for formation of highly pathogenic avian influenza (HPAI).

Equine influenza causes death of ciliated epithelial cells by triggering apoptosis due to replication inside the cell.

Epidemiology

Viral shedding generally occurs the day before symptoms occur and continues for up to a week. The virus can survive in the environment, depending on the surface, so can be transmitted due to contaminated surfaces. Generally winter time is peak time for influenza infection. Northern and southern hemispheres have winter at different times of the year so there is two flu seasons in any one year.

The reservoir for many forms of influenza A is birds – more specifically waterfowl. They can spread the virus across their migratory patterns and across borders. All 16 HA subtypes have been described in birds and it is transmitted through a very effective feacal-oral route. Avian influenza mainly affects poultry but can also infect man, swine, horses, seals and ferrets.

Many strains of avian influenza are asymptomatic. Sudden death is a clinical feature of avian influenza, or fowl plague as it is also known, with respiratory distress, diarrhoea, oedema of the head and neck, lacrimation, cessation of egg laying, cyanosis of the comb and wattles and encephalitis. The OIE (office International des Epizooties) has listed highly pathogenic avian influenza as a list A disease. Carbohydrate side chains on epithelial cells determine receptor specificity, and thus host range. Influenza virus binds to sialic acid-galactose disaccharides on the cell. The human trachea has mainly a-2,6 linkage, while the avian intestine and horse trachea mainly a-2,3 linkage. The respiratory epithelium of pigs have both types therefore swine can act as a ‘mixing vessel’ for genetic reassortment. Humans tend to be more susceptible to swine influenza than avian.

The main subtypes of swine influenza present are H1N1 (classical) North America/Asia, H1N1 (avian-like) Europe and H3N2 (human and avian like). Most H3N2 virus isolates occur due triple reassortment and possesses human HA and NA proteins. H1N2 (human-avain reassortments) is also a triple reassortment but possesses HA of human and NA of swine origin.

Swine influenza is spread by aerosol and has an incubation period of 24-72 hours and recovery can take up to 6 days. The main source of infection is the introduction of new pigs to a herd and is most prevalent in autumn and winter. The principal clinical signs observed are nasal discharge, paroxysmal coughing, fever, conjuctivitis, pneumonia and abortion. Mortality is generally low even though there is a high morbidity.

Spanish flu is thought to be the most severe human influenza pandemic to ever have occurred and was of H1N1 lineage. It had a mortality rate of 2 – 20% and compared to <0.1% in other influenza pandemics. It first appeared in the spring of 1918 followed by much more fatal second and third waves in the fall and winter of 1918–1919. The first 2 waves occurred at a time that is usually not optimal for influenza virus. There were distinct peak in deaths of young adults (20–40 years of age) as opposed to just the very young and elderly that normally occur wtih influenza. This resulted in a mortality-age curve that was ‘w’ rather than ‘u’ shaped.

Other human pandemics include the 1890 H2N8, 1900 H3N8, 1957 H2N2 (Asian Flu), 1968 H3N2 (Hong Kong flu), 1977 H3N2 and 2009 H1N1 (swine). Both the Honk Kong flu and Asian flu pandemic are estimated to have resulted in one million fatalities each. The most conservative estimate for the number of mortalities due to the Spanish flu of 1918 is 50 million. One third of the world population was thought to be infected by Spanish flu and one half of fatalities were ages 20-40.

Annual epidemics of human influenza result in 300,000 deaths worldwide. The H1N1 pandemic of 2009 originated from Mexico and rapidly spread to spread to 43 countries in one month. The number of deaths since it appeared in 2009 is estimated as more than 18,000 fatalities by WHO but the total mortality (including deaths unconfirmed or unreported) is considered to be vastly greater. This virus was a triple reassortment influenza virus consisting of 1 human segment, 2 avian segments and 6 segments from both American and Eurasian swine. The end of this pandemic was officially declared in August 2010.

In New Jersey 1976 an outbreak of Swine Influenza A virus occurred in soldiers stationed at Fort Dix. After a large vaccination campaign the virus did not spread outside the training base. In 1997 Hong Kong had an outbreak of highly pathogenic (H5N1) avian influenza which over 7 days resulted in 20 human cases. To date there have been 507 cases and 302 deaths as it spread to three continents. It had over 60% lethality and no vaccine was available.

The two main equine influenza viruses are of avian influenza origin; A/equine/Prague/1/56 (H7N7) or A/equine 1 and A/equine/Miami/2/63 (H3N8) or A/equine 2. A H3N8 that more closely resembled avian influenza than equine was isolated in North East China in 1989. Two main lineages of H3N8 are Europe and American lineages. The OIE has advised that H7N7 be removed from vaccines. It is currently thought to be inactive and may be extinct. The the last outbreak occurred in 1979 and it was first isolated in Eastern Europe 1956. H3N8 was first isolated in Florida in 1963 and is now present worldwide. H3N8 is an important cause of respiratory disease. Vaccination or infection with one subtype of equine influenza does not provide protection against the other. Clinical signs include nasal discharge, enlarged lymph nodes, inappetance, limb oedema, dry deep cough and fever. Exercise can exasperate clinical signs. The incubation period is thought to be 1-3 days but this can be variable. Recovery time generally takes 3 weeks but again this can be variable. Equine influenza is absent from New Zealand and Iceland. An outbreak occurred in Australia in 2007 but they are officially free of the disease. As expected outbreaks tend to be associated with any congregation of horses such as events, shows, racing and markets.

A canine influenza outbreak occurred in Florida in January 2004 infecting Greyhounds and this virus proved to be very closely related to equine influenza virus. The initial fatility rate was as high as 36%. The main presenting signs were coughing and fever for two weeks with recovery or peracute death with haemorrhage in the respiratory tract. By 2006 the virus had spread to more than 22 states resulting in respiratory disease in 80% of those infected and causing a mortality rate close to 5%.

Virus detection

Ideally samples should be taken three days from onset of clinical symptoms. Samples that can be taken include a nasal swab, throat swab, nasopharyngeal aspirate, nasal wash, transtracheal lavage, bronchioalveolar lavage, lung biopsy and serum samples. Virus isolation is highly sensitive. Virus isolation in cell culture is becoming the gold standard but requires maintenance of several cell lines. Madin-Darby canine kidney (MDCK) cells are typically the preferred cell line in which to culture influenza viruses. The more traditional method of isolation in embryonated chicken eggs has been replaced by cell culture in many instances. However isolation in embryonated eggs is required for vaccine creation. Haemagglutination inhibition testing is used to identify haemagglutinin subtypes of viral isolates. HA causes red blood cells to agglutinate by binding to them. Antibodies against a specific HA protein can be used to bind to antigenic sites of the HA protein inhibiting haemagglutination.

Copyright World Health Organization WHO Global Influenza Surveillance Network Manual for the laboratory diagnosis and virological surveillance of influenza

The microneutralization assay is a highly sensitive and specific assay that can be used in combination with ELISA to detect virus specific neutrilizing antibodies. Molecular identification can be achieved through RT-PCR providing rapid results. Immunofluorescence antibody (IFA) testing is possible as monoclonal antibodies have become commercially available. Isolated virus rather than specimens is preferred as this allows amplification of the virus. Neutralization inhibition (NI) can be used to determine susceptibility to anti-viral drugs.

Newcastle disease (viscerotropic or velogenic) and fowl cholera should be distinguished from avian influenza. Isolation and characterisation is vital. Suitable samples can include organs, faeces as well as swabs from the trachea and cloaca. Eggs are inoculated and allantoic fluid examined for haemagglutinin activity after one week incubation. Immunodiffusion or haemagglutination (HI) can also be used to confirm presence of the virus. HI and NI are important for subtyping the virus. PCR for rapid identification is commonly used. Genomic sequencing can determine the amino acid present at the cleavage site of the HA glycoprotein. Chickens are inoculated with virus and those that that cause more than 75% mortality in 8 days are considered highly pathogenic, or if they have a IVPI (intravenous pathogenicity index) > 1.2 they are also considered highly pathogenic. Antibodies can be tested for by number of different serology techniques such as HI, agar gel immunodiffusion and ELISA. For whole flock checks in poultry commercial antigen immunoassays for influenza A virus are available. As with a lot of these commercial antigen immunoassays there is questionable sensitivity.

For swine influenza detection virus isolation from samples of nasal mucus or suitable tissue is used. Egg inoculation is followed by incubation for 72 hours. PCR can be used for rapid identification or HI or ELISA can detect a rise in antibody titres. Alternatively antigen detection can by imployed by immunofluorescence or ELISA.

Suitable swabs are required for viral isolation, of equine influenza, as discussed. Commercial diagnostic kits for detection of influenza A nucleoprotein in humans can be used for equine influenza. Equine influenza can be diagnosed through serological diagnosis or PCR.

Control and treatment

As discussed viruses are transmitted largely through contaminated surfaces and by aerosol by coughing and sneezing. Virions are labile in environment and are sensitive to heat, changes in pH, lipid solvents, detergents, irradiation and oxidizing agents. Hand washing is a simple and important control.

The only treatment available in many instances is symptomatic treatment. Usually it is just advised to take acetaminophen (paracetomol) to reduce the fever and fatigue associated with infection. Aspirin should be avoided in children and teenagers when infected as this can result in Reye’s syndrome.

In some countries antiviral treatment is available. It should only be given to those who are likely to develop serious complications with infection. NA protein can be targeted by two neuraminidase inhibitors – the oral drug Oseltamivir (tamiflu) and the inhaled drug Zanamivir (Relenza). These drugs prevent the release of progeny virus due to blocking of active on viral enzyme NA. Neuraminidase inhibitors are active against both influenza A and influenza B. Serious respiratory effects have been reported with zanamivir used by people with respirtory illness. Few serious nervous system effects have been reported.

Antiviral adamantanes are another class of antiviral drugs containing amantadine (Symmetrel) and rimantadine (Flumadine). These interact via the M2 protein on influenza A. NA inhibitors are usually less toxic and more effective so they are the preferred choice in most instances. In 2005 the CDC found that 95% of samples were resistant to rimantadine, as opposed to only 11% the previous year. Adamantanes do not have activity against influenza B or C virus. Gastrointestinal and nervous system side effects reported. Rimantadine has fewer reported side effects than amantadine. Amantadine is teratogenic and embryo toxic.

Vaccines are the main method of influenza prevention and control. Due to the high genetic variability of influenza virus each year new subtypes have to be included in the human vaccine. Currently the vaccine consists of a trivalent vaccine of inactivated strains. Live attentuated vaccine is available for use in the United States for healthy people aged 2–49 years and in russia for some years. High risk groups such as the immunocompromised and the elderly are usually treated with the vaccine.

Vaccination of poultry is prohibited in where there is a slaughter policy due to trade restrictions and freedom of disease policies. Other countries have lower restrictions and some inactivated vaccines are allowed. Live vaccines against influenza A are never used in poultry due to the risk of reversion to virulence. Inactivated vaccines are commercially available in many countries.

Movement should be restricted in an infected horse with appropriate safety protocols in place including appropriate disinfection of the vicinity. A number of inactivated vaccines are available for horses. The can reduce the amount of shedding and severity of clinical signs. Booster injections are required as protection time is short. A canarypox recombinant vaccine with the HA gene of A/equine 2 is available that is DIVA compliant (differentiating infected from vaccinated animals). Strains must be updated fairly regularly. For horses attending shows a vaccination card showing that the horse has been vaccinated against equine influenza is required. The FEI requires vaccination every six months.

In Europe and North America vaccination is common in the swine industry. Most vaccines conating H1N1 and H3N2 swine origin influenza viruses.

Global influenza programmes and worldwide surveillance networks are constantly monitoring and evaluating findings and reports from local health care operators. The Influenza Control Surveillance Network consists of 136 National Influenza Centres, 4 WHO Collaborating Centres (WHOCCs) for Reference and Research on Influenza,1 WHOCC for Surveillance, Epidemiology and Control of Influenza, and 1 WHOCC for Studies on the Ecology of Influenza in Animals; and 4 key national reference laboratories involved in WHO influenza vaccine virus selection and development. As well as axillary surveillance methods such as Flunet and weekly epidemiological reports.

Further Reading

P. J. Quinn et al., 2011. Veterinary Microbiology and Microbial Disease. 2nd ed. Chichester, UK, Wiley-Blackwell.

Taubenberger JK, Morens DM (2006). 1918 Influenza: the Mother of All Pandemics.Emerging Infectious Diseases, 12(1):15-22. (Open-Access)

UK Influenza Pandemic Preparedness Strategy 2011
http://www.dh.gov.uk/prod_consum_dh/groups/dh_digitalassets/documents/digitalasset/dh_131040.pdf
http://en.wikipedia.org/wiki/Influenza/
https://en.wikipedia.org/wiki/Historical_annual_reformulations_of_the_influenza_vaccine
http://www.who.int/topics/influenza/en/
http://www.who.int/influenza/gisrs_laboratory/flunet/en/
Manual for the laboratory diagnosis and virological surveillance of influenza
http://whqlibdoc.who.int/publications/2011/9789241548090_eng.pdf
http://www.nature.com/nrmicro/focus/influenza/index.html
http://www.cdc.gov/flu/
http://www.hpa.org.uk/Topics/InfectiousDiseases/InfectionsAZ/SeasonalInfluenza/
http://www.virology.ws/influenza-101/
http://www.ncbi.nlm.nih.gov/genomes/FLU/flubiology.html

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