Influenza (flu) Virus: Classification, Structure, and Diagnosis

Influenza, commonly called “the flu,” is a contagious respiratory illness, a very important global public health problem. It causes seasonal flu epidemics yearly and is a critical disease responsible for school and job absenteeism, hospitalization, and deaths. Flu pandemics have killed millions of people worldwide; another flu pandemic is looming in our future.

Influenza (flu) is caused by influenza viruses, a member of the orthomyxovirus family. Influenza viruses infect the respiratory tract (i.e., nose, throat, lungs) and can cause mild to severe life-threatening illnesses. There are three types of influenza (flu) viruses: A, B, and C. Antigenic differences exhibited by two internal structure proteins (NP and M) are used to divide influenza viruses into types A, B, and C.

Influenza viruses A and B cause seasonal epidemics, whereas type C causes mild respiratory illness. Currently circulating influenza B viruses are from two lineages: B/Yamagata and B/Victoria. There are no subtypes of the influenza B virus.

Influenza A virus has been further divided into subtypes and strains. The mutability and high frequency of genetic reassortment in this virus result in antigenic changes in viral surface glycoprotein, making influenza type A antigenically highly variable and responsible for most cases of epidemic influenza.

Subtypes of Influenza A viruses

Influenza A viruses are divided into subtypes based on two proteins on the virus’s surface: hemagglutinin (H) and neuraminidase (N). There are 18 different hemagglutinin subtypes (H1 through H18) and 11 neuraminidase (N1 through N11) subtypes.

Strains of Influenza A viruses

Influenza A viruses can be further broken down into different strains. Some of the  most successful and virulent strains of flu are

1. H5N1 avian (bird) flu: caused outbreaks in domestic poultry in parts of Asia and the Middle East. Human infection with H5N1 is rare; nearly 650 cases of human cases of H5N1 have been reported from 15 countries since 2003.
2. H1N1 (swine flu): caused a worldwide pandemic in 2009.
3. Influenza A H3N2 variant viruses

Nomenclature (naming) of Influenza Virus 

Internationally accepted naming convention is followed for the naming of influenza viruses. The approach uses the following components:

• The antigenic type (e.g., A, B, C)
• The host of origin (e.g., swine, chicken, duck, etc. For human-origin viruses, no-host of origin designation is given.)
• Geographical origin (e.g., Hong Kong, Taiwan, etc.)

Strain number (e.g., 15, 7, etc.) Year of isolation (e.g., 57, 2009, etc.) For influenza, A viruses, the hemagglutinin, and neuraminidase antigen descriptions are in parentheses (e.g., (H1N1), (H5N1)

For example:

• A/duck/Alberta/35/76 (H1N1) for a virus of duck origin
• A/Perth/16/2009 (H3N2) for a virus of human origin

Structure of Influenza virus 

• Influenza virions are usually spherical (diameter 80-110 nm).
• An enveloped virus, the outer layer is a lipid membrane that is taken from the host cell.
• Inserted into the lipid membrane are ‘spikes,’ about 10 nm long, glycoproteins are known as HA (hemagglutinin) and NA (neuraminidase).         
  # NA is a target of the antiviral drugs Tamiflu
• HA and NA  are the important antigens that determine
  • antigenic variation of influenza viruses and host immunity.
  • subtype and strains of influenza virus (A/H1N1, for example).
• Protection against re-infection is mainly due to developing antibodies to HA (but antibodies to NA are also protective).

• M2 proteins are also embedded in the lipid membrane. M2 proteins are the targets of antiviral drugs adamantanes – amantadine and rimantadine.
• Beneath the lipid membrane is a viral protein called M1, or matrix protein. Matrix protein is a major component of the virion (about 40% of viral protein), forms a shell, and gives strength and rigidity to the lipid envelope.
• Within the interior of the virion are the genetic material of the virus; the viral single-stranded, negative-sense, viral RNAs
  • Eight of them for influenza A and B viruses; the influenza C virus has only seven segments; lacking a neuraminidase gene);
  • Most of the segments code for a single protein.
• Each RNA segment consists of RNA joined with several proteins PB1, PB2, PA, and NP (RNA Polymerase), responsible for RNA transcription and replication.
• The interior of the virion also contains another protein called NEP.

Laboratory Diagnosis

For the laboratory diagnosis of influenza virus infection, specimens should be taken as early as possible during the disease, preferably within the first 72 hours after the onset of symptoms. Routine specimens for influenza virus diagnosis include

• Nasal washings
• Nasopharyngeal aspirate
• Nasopharyngeal swab
• Nasal and throat swabs
  For successful isolation, a specimen should be placed on a viral transport medium to stabilize the virus

Cell Culture

Madin-Darby kidney cell Line (MDCK) supplemented with trypsin supports growth and multiple cycles of influenza A, B, and C. Standard virus isolation is done in cell culture tubes seeded with MDCK cells. The cultures are incubated at 34°C in a stationary rack or roller machine. Some influenza viruses cause a distinct cytopathic effect in MDCK cells several days after inoculation. Negative cultures should be checked by a hemadsorption or hemagglutination test at 2-3  days intervals.  

Detection of Infected Cells in Clinical Specimens by Immuno-fluorescence 

Demonstration of infected epithelial cells in nasopharyngeal aspirate or nasopharyngeal swab is a sensitive method (50-90% more sensitive than virus isolation) for the diagnosis of influenza. The result can be obtained within a few hours after the specimen collection. Exfoliated cells in NPA and NPS are prepared, applied to microscopic slides, and fixed. Polyclonal sera and/or monoclonal antibodies are used as a reagent for the staining process.

Detection of Influenza Virus Antigens by Immunoassays
Various tests such as radioimmunoassay, enzyme immunoassay (EIA), and fluoroimmunoassay have been developed to detect influenza virus antigens directly in clinical specimens or after amplification in cell culture. Antigens in clinical samples or cell culture material are commonly captured by specific antibodies that have previously adsorbed to the solid phase. After incubation, unbound material is washed, the bound antigen is reacted with a secondary antibody and then incubated further with a labeled anti-species antibody.  

Reverse Transcriptase PCR

The reverse transcriptase PCR has been applied to detect influenza virus in clinical specimens and cell culture or egg-grown material. The viral RNA is first transcribed into DNA, then amplified in a second step using suitable primers and DNA Polymerase.   Amplified DNA is electrophoresed in an agarose gel, and DNA is detected by staining with ethidium bromide. Molecular weight markers are included to identify the amplified DNA of the appropriate size. DNA can also be detected by molecular hybridization with specific labeled probes either in Southern blots or by spot hybridization. Appropriate selection of primers permits type-specific identification of influenza virus A, B, and C or subtype-specific identification.

Serology

Although serologic methods seldom yield a result early enough to influence the patient’s treatment, they often establish the diagnosis of influenza virus infection when other methods do not detect the virus. When the sensitivities of newly developed diagnostic tests are being evaluated, serology should be considered instead of or in combination with virus isolation as the “Gold Standard.”  Hemagglutination inhibition test (HAI), the neutralization test (NT), and the complement fixation test (CFT) are traditional methods in serodiagnosis and seroepidemiologic studies of influenza, but during recent years, EIA has found wide application. The complement fixation test measures antibodies against the NP and thus allows the type-specific detection of antibodies to influenza A or B viruses. HAI and NT are more sensitive and measure antibodies against subtype and strain-specific antigens.

Frequently Asked Questions