Influenza – A contagious viral infection affecting both the upper and lower respiratory tract
Influenza continues to be a major public health concern despite significant advancement in vaccination and viral research. Influenza is a communicable viral disease that is known to affect the upper respiratory tract including lower and upper respiratory passages. In 1918, the worst pandemic of influenza on record, killing 50 million people worldwide. Influenza A is responsible for both seasonal, endemic, periodic infections and unpredictable pandemics.
Influenza A virus is a member of the Orthomyxoviridae family that is made up of 8 negative-sense single-stranded RNA segments that codes for 11 viral genes. The high mutation rates associated with the genome makes influenza incredibly difficult to prevent annual epidemics, resulting in millions of hospitalisation and thousands of deaths. Acute and early diagnosis of influenza A viral infections are important for rapid initiation of antiviral therapy to minimise viral related mortality and morbidity during both seasonal epidemics and pandemics. Currently, FDA approved rapid molecular assays are the commonly used method for the diagnosis of influenza in hospitalised patients.
Many antiviral drugs including zanamivir (Relenza), oseltamivir (Tamiflu), rimantadine and amantadine are available for the treatment, but the use of drugs results in the development of drug resistance and adverse side effects on the central nervous system. Resistance to antiviral drugs is an increasing concern in the affected population, where exponential viral replication and prolonged drug exposure leads to the formation of resistant influenza strains. Even though antiviral medications have proved to control viral replication, vaccination remains the most cost-effective and practical method to control and prevent serious complications resulting from influenza A.
Influenza, known as flu, is a respiratory infection caused by influenza A virus type in human and among the most important because they are known to cause high morbidity and mortality (Vemula et al., 2016). Influenza viruses belong to the Orthomyxoviridae family, which consists of four influenza genera: influenza virus A, B, C and D. Each of the virus type of classified based on the differences found in their nucleoprotein (NP) and the matrix (M).
It has been reported that each year, influenza A type causes seasonal widespread epidemics and pandemics, accounting for 4- 5 million cases with severe illness, 400,000-500,000 deaths and more than 200,000 hospitalisations worldwide (10-15% of the population) (Meštrović, 2018). Influenza widespread has occurred since ancient times and is associated with high mortality rates in elderly, infants and people diagnosed with chronic diseases (Hirsch, 1883).
Seasonal influenza epidemics can occur frequently in both Southern and Northern hemispheres. Seasonal epidemics occur mainly during winter, compared to a tropical region, in which it may appear throughout the year, leading to more irregular outbreaks. Not much is known about epidemics of Influenza in tropical regions but assumed that it occurs throughout the year. Influenza epidemics are distinguished from each other by the severity of the infection. In 1918, the Spanish flu caused 35-40 million deaths while Asian influenza outbreak in 1957 and Hong Kong influenza in 1968 resulted in 1.5 – 2 million and 1 million deaths (Fauci, 2006).
Influenza A structure and function
In general, all the virus types are made up of two essential components: i] a nucleic acid genome, ii] a genome protecting protein capsid. Both the essential components together known as the nucleocapsid. Unlike other virus types, influenza A is made up of an additional lipid bilayer, derived from the host cell in which the virus attaches and multiplies. The spikes found in the lipid bilayer are glycoproteins that consist of proteins attached to sugars known as hemagglutinin (HA) and neuraminidase (NA) (see figure 1).
HA is the most abundant (80%) envelope protein then followed by NA (17%) of the viral membrane (Samji, 2009). HA is responsible for the virion binding to the sialic acid found on the cells in the upper respiratory tract. Cleavage of a single precursor is crucial for the activation of the membrane fusion potential which results in the initiation of infectivity (Garten and Klenk, 1999). A single arginine residue is the site of cleavage which occurs extracellularly by the protease human airway trypsin-like protease (HAT) and transmembrane protease serine S1 member 2 (TMPRSS2) enzymes (Gamblin and Skehel, 2010) (Bottcher et al., 2006). The nucleocapsid protein (NP) functions as a facilitator for integration and reverse transcription (Soszynska-Jozwiak et al., 2017). NP along with RNA polymerase makes up the 8 viral ribonuclease protein complexes, which is later arranged into an active virion particle (Soszynska-Jozwiak et al., 2017).
NA is a box-shaped homo-tetramer made up of four identical subunits help together on a thin 60-100 Å long stalk, which is attached to the viral membrane (Varghese and Colman, 1991). The active site and Ca2+ binding domain are found in the head of the NA. Ca2+ binding domain stabilises the enzyme structure at low pH to prevent the enzyme from losing its function (Lawrenz et al., 2010). NA catalyses the cleavage of α-ketosidic linkage between the sialic acid and an adjacent sugar residue (Varghese and Colman, 1991) and facilitates the movement of viral particle to the target host cell by cleaving the neuraminic acid residues found in the respiratory tract mucins (Bovin, Mochalova and Shtyrya, 2009).
The viral matrix protein (M1) is found beneath the lipid membrane, providing strength and rigidity to the bilayer. The viral ion channel (M2) are small auxiliary membrane proteins, embedded into the membrane. Several studies have shown that M2 functions as a pH regulator across the viral membrane (Ciampor et al., 1992). Influenza A consists of 8 single-stranded negative-sense viral RNAs (ssRNAs), found within the interior of the virion coding for 11 genes (Vemula et al., 2016). Each vRNA segment found within the virion consists of RNA attached with PB1, PB2 and PA proteins termed as viral polymerase complex (see figure 2).
Figure 1 – Schematic representation of the structure of influenza A virus.
Influenza A has a spherical shape and has an additional lipid bilayer that protects the matrix protein. The genetic materials are found within the virion. Influenza A consists of segmented 8 single-stranded negative sense RNAs (ssRNAs), each of the RNA coding for different proteins that play an essential role in infecting the host cell. The polymerase complexes made up of three polymerase PB1, PB2 and PA proteins located at the end of the nucleocapsids. The helical capsids are surrounded by the Matrix protein (M1) and the lipid bilayer derived from the host cell. Both the Haemagglutinin (HA) and Neuraminidase (NA), as well as M2 ion channels, are embedded into the lipid bilayer. (Figure adapted from
Figure 2 – Influenza virus genome.
The vRNA segment 1 codes for the PB2 subunit of the RNA polymerase that determines the virulence of influenza. Segment 2 codes for the PB1 catalytic subunit of RNA polymerase (Kobayashi, Toyoda and Ishihama, 1996). Segment 3 codes for the PA protein which functions as RNA endonuclease to cleave the phosphodiester bond in the transcription of vRNAs (Muramoto et al., 2006). vRNA segment 4 codes for the HA, which plays a crucial role in sialic acid receptor binding. The cleavage of HA proteolytic site produces N-terminus of the fusion peptide, which is a conserved uncharged HA region, playing a role in virion and host membrane fusion (Skehel and Wiley, 2000). Segment 5 codes for NP protein, which encapsidate the genome for the RNA transcription, replication and packing. Segment 6 codes for the neuraminidase (NA) protein that enables the replicated virion to be released from the target cell. Figure adapted from
. Matrix M1 and M2 proteins are derived by using different reading frames from the same vRNA segment 7. Two distinct non-structural proteins NS1 and NEP are coded by using different reading frames from the same RNA segment 8.
Figure 2 – Schematic illustration of the life cycle of influenza A virus.
A] influenza A is made up of a lipid bilayer envelope, within which 8 single-stranded RNA segments are found. Each RNA segment is associated with PB1, PB2 and PA RNA polymerases. B] the HA glycoprotein binds to the sialic acid receptors found in the host cell and the virion is transported into the cell via an endosome. The low pH triggers a conformational change of HA, which leads to viral and endosomal membrane fusion. C] In the nucleus, viral RNA polymerases initiate mRNA synthesis. D] The synthesised mRNAs are transported to the cytoplasm for translation into proteins. NA, HA and M2 are processed in the ER and glycosylated in the Golgi apparatus and transported to the membrane. E] The NS1 protein inhibits the production of the host cell’s mRNA by preventing the 3’ end of host pre-mRNAs. The viral mRNAs do not require 3’ processing by the cell machinery, unlike the host cell’s mRNAs. This helps viral mRNAs to be transported to the cytoplasm. F] The viral polymerase carries out the un-primed replication of vRNAs in steps vRNA(-) to cRNA(+) to vRNA(-). M1-NS2 complex mediates the transportation of vRNPs to the cytoplasm. G] The replicated vRNPs move to the cell membrane to be budded from the host cell. Figure adapted from Das et al., 2010
The clinical course of influenza
Respiratory transmission of influenza depends upon the production of aerosol containing virus particles. Influenza is mainly transmitted through direct contact with the infected individuals, through contact with contaminated fomites and by inhaling virus-laden aerosols. The droplets sized 1-5 microns remain in the air for a long period and can travel to long distances. When the droplets are inhaled, influenza situates in the lower and upper respiratory tract where its life cycle begins (see figure 2).
Influenza is characterised by the common gastrointestinal symptoms including nausea, diarrhoea and vomiting and sudden onset of high fever, headache, fever, sore throat, nasal congestion and malaise (Minodier et al., 2015) (Lam, Green and Coleman, 2016). Weakness and fatigue may last for weeks after the detection of infection. The incubation period of influenza lasts for 1 to 3 days (Lessler et al., 2009). Influenza affects people of all ages, but the prevalence is greatest in children (Nitsch-Osuch et al., 2013). Influenza replication in both the upper and lower respiratory track peaks approximately 48 hours after the inoculation into the nasopharynx and the replication declines slowly after 6 days (Taubenberger and Morens, 2008). Viral antigen can still be detected in cells for several days even after influenza can no longer be recovered (Kim and Poudel, 2013).
People with cardiac, pulmonary or diabetes have a high risk of progressing to severe complications from influenza. This may include the development of pneumonia, haemorrhagic bronchitis within hours (Jeganathan et al., 2013). Few studies have suggested that occasionally fulminant fatal influenza viral pneumonia can occur (Cunha, Syed and Mickail, 2010). Pulmonary oedema, cyanosis and death may occur in 48 hours after the onset of symptoms (Taubenberger and Morens, 2008).
Current approaches for diagnosis of influenza in human
Diagnosis of influenza is important for the initiation of influenza treatment because it allows clinical management to decide whether to start antiviral treatment, perform more diagnostic testing or carry out influenza control measures. Influenza testing is highly recommended for anyone being admitted to hospital. Viral culture has been considered as the gold standard approach since its introduction in the 1940s (Kim and Poudel, 2013) but It is a time-consuming approach, takes around 10-15 days for the results to arrive, which delays the antiviral therapy or the initiation of infection control measures (George, 2012) (Cox and Subbarao, 1999).
Respiratory specimens are taken from the admitted patients are tested using various molecular assays including reverse transcriptase PCR (RT-PCR), nucleic acid amplification tests and rapid molecular assays (Ghebrehewet, MacPherson and Ho, 2016). FDA approved rapid molecular assays can detect the presence of influenza in the upper respiratory tract in 15- 20 minutes with a high sensitivity of 85-95% (Otto et al., 2017). It can also detect specific influenza A subtypes, making it an accurate method for the diagnosis of influenza.
Antigen detection tests such as immunofluorescence assays and rapid influenza diagnostic tests (RIDTs) are also used for the influenza diagnosis purpose (Pianciola et al., 2010). Specificity (60-70%) and the sensitivity of these tests vary slightly by the type of the diagnosis method and quality of the respiratory specimen (Cdc.gov, 2019). An analyser reader device is used along with RIDTs to boost sensitivity (75-80%) compared to standard RT-PCR. However, RIDTs cannot distinguish different subtypes of influenza A. RIDTs performance heavily depend on the prevalence of circulating viruses in the population.
Direct fluorescent antibody test (DFA) is an antigen-based test which involves direct respiratory epithelial cell staining derived from nasopharyngeal aspirates with fluorescently labelled influenza-specific antibodies, followed by fluorescent microscope examination (Bakerman et al., 2011). The sensitivity of the DFA test decreases with patients aged above 30 but increased with patients aged below 12 (Lee, Shin and Cho, 2011). The sensitivity of DFA tests varies between 70-100%, higher than that of rapid antigen detection kits but lower than PCR tests (Kim and Poudel, 2013).
Vaccination has proven to be the most effective way to prevent influenza and control from developing further complications. Several studies have shown that vaccines are 80% effective in preventing death, 30-40% effective in preventing the spread of influenza and 50% effective in preventing hospitalisation particularly in elderly people (aged 65 or over) (Keyser et al., 2000).
The seasonal vaccine safety is well accepted worldwide. Currently, the licensed vaccines mainly focus on the antibody production against the viral HA protein that binds to the receptors of the host cells to initiate entry (Houser and Subbarao, 2015). These strain-specific antibodies designed against the HA protein to neutralise influenza, resulting in prevention.
Inactivated (IIV), live attenuated (LAIV) and recombinant HA vaccines are the three types of seasonal vaccines available (Fiore, Bridges and Cox, 2009). All three types consist of components representing influenza virus anticipated to spread in the next season. IIV contains 9μg of purified HA administered intradermally or 15μg of purified HA that is administered intramuscularly (Houser and Subbarao, 2015) (Bresee et al., 2014). People aged 60 and over, can administer a higher dose (60μg) of HA to increase vaccine immunogenicity (Houser and Subbarao, 2015). The LAIV contains a mixture of two temperature sensitive influenza B lineages along with H1N1 and H3N2 influenza A subtypes, administered as an intranasal spray (Houser and Subbarao, 2015) (Coelingh et al., 2014). FluBlok is a type of recombinant HA vaccine, containing HA proteins expressed in insect cells, recommended for adults aged 18-45 (Bresee et al., 2014).
However, the main challenge with every seasonal vaccine is that annual re-evaluation and development of new formulations required to keep up with antigenic drift associated with circulating viral strains (Houser and Subbarao, 2015). Error-prone viral RNA-dependent RNA polymerase lacks proof-reading function, leading to mutations in NA, HA, M1 and other viral proteins, resulting in antigenic drift (Boivin et al., 2010). Therefore, the selection of vaccines for the upcoming seasonal influenza must occur 6 to 8 months in advance before the outbreak. Other challenges in optimising influenza vaccine include embryonated eggs dependency for production, lengthy production timeline and the need for annual vaccination.
Figure 3 – Targets of the influenza viral vaccines.
Many stages of viral infection can be targeted by influenza vaccines. Vaccines that cause immune response activation to blocking the stages of the influenza life cycle stages (Highlighted in red). Vaccines that initiate the activation of a specific host response (Highlighted in green). Figure adapted from (Houser and Subbarao, 2015).
Influenza treatment – Antiviral Therapy
Influenza is a self-limiting in healthy individuals. Usually nothing more than bed rest and adequate fluid intake until 24 to 48 hours after resolution of fever required to prevent spread to other individuals (Ghebrehewet, MacPherson and Ho, 2016). In some cases, antiviral drugs are often indicated during the confirmed influenza outbreak because of the limited effectiveness of influenza vaccines. Since untreated influenza may lead to pneumonia which is a lower respiratory lung infection causing inflammation in both lungs, it is important that antiviral drugs are administered as soon as symptoms emerge to prevent not only pneumonia but also other adverse outcomes. Compare to late treatment, early treatment within 48 hours of symptom onset of hospitalised individuals with complicated influenza reduces the odds of mortality by 52%.
NA inhibitors (NAIs) have been used successfully for the influenza treatment for more than a decade. NAIs prevent the release of progeny virions by blocking the active site of the neuraminidase enzyme, inhibiting the enzyme from cleaving sialic acid residues on the host cell (McKimm-Breschkin, 2012). NAIs such as oseltamivir and zanamivir, are the only antiviral drugs currently licensed globally for the treatment of influenza (von Itzstein et al., 1993). The benefit of these drugs was detected within 36 to 48 hours of symptom onset (Shahrour, 2001). Oseltamivir is administered orally, twice a day (75mg) (Oo et al., 2001) whereas zanamivir is inhaled, twice a day (10mg) for adults (Cass, Efthymiopoulos and Bye, 1999). Oseltamivir is taken orally and its level in plasma is determined to be between 400-1200 nM and its level in the upper respiratory tract is significantly lower at 150nM compared to 10,000mM for zanamivir level in the upper respiratory tract (Peng, Milleri and Stein, 2000). However, in infants (>1), safety and efficacy of both zanamivir and oseltamivir in infants have not been established, suggesting that more research is required (Allen, Aoki and Stiver, 2006).
Several new and future neuraminidase inhibitors including Laninamivir, Favipiravir and Peramivir have been developed for the influenza treatment. Laninamivir is a highly promising and long-acting NAI that effectively preventing oseltamivir-resistant viruses (Sugaya and Ohashi, 2010). Leninamivir is available for oral inhalation and single inhalation (40mg) is as effective as repeated doses of zanamivir (Sugaya and Ohashi, 2010). Favipiravir is also shown to be promising against oseltamivir-sensitive and resistant H5N1 influenza. It’s an active form, ribofuranosyl triphosphate derivative (Barik, 2012), functions as a viral RNA- dependent RNA polymerase inhibitor, preventing RNA replication (Furuta et al., 2002).
M2 inhibitors such as amantadine and rimantadine have been around for almost 50 years but rapid development of drug resistance and adverse side effects such as affecting the central nervous system (CNS) in treated patients (Keyser et al., 2000). The HIN1 influenza A subtype was already resistant and H3N2 subtype has been resistant since the 2000s (McKimm-Breschkin, 2012). H5N1 influenza strains in Southeast Asia are also resistant to M2 inhibitor, meaning their use in influenza treatment is very limited (McKimm-Breschkin, 2012).
Figure 4 – Anti-influenza drugs and their biological targets.
Neuraminidase inhibitors cleave the sialic acid receptor on the host cell membrane (Oseltamivir (Tamiflu), Zanamivir (Relenza), Laninamivir and Peramivir). M2 ion channel blockers – Amantadine and Rimantadine. HA inhibitors – Peptides Retrocyclins. Figure adapted from (Barik, 2012).
Challenges in treating influenza
There are many difficulties in the treatment of influenza that contribute to constant influenza threat. Influenza RNA polymerase lacks proofreading, resulting in relatively high mutation rates. Even a small change in the coding sequence of HA and NA (genetic drift) may lead to virion escape from the adaptive immunity of the host cell and makes the antiviral drugs ineffective. This may result in increased infectivity leading to greater vertical and horizontal spread. New influenza strains can emerge by reassortment in co-infection. For example, co-infection by human and swine influenza can result in the development of reassortment viruses that consist of RNA from two species, causing major epidemic (Webby and Webster, 2001).
Influenza can be deadly to the elderly and people with AIDS or diabetes. In fact, people aged 60 or older account for 85-90% of influenza-related death (Metersky et al., 2012). Therefore, this population group is in the need for more intensive treatment as soon as symptoms emerge. Unfortunately, the elderly population are less tolerant of more aggressive treatment such as the use of drugs, making influenza in the elderly, a difficult disease to treat.
Influenza is a contagious viral infection that remains a significant global health burden. Timely diagnosis and initiation of treatments such as antiviral drugs reduce the stigma of the disease and also results in a controlled outbreak. Many diagnostic methods are available for the detection of influenza virus, RT-PCR and viral culture approaches being the gold standard with high sensitivity and specificity. The frequent alterations in the antigenic structures of influenza viruses make it difficult to produce an effective vaccine, meaning annual re-evaluation and new formulations required to keep up with the influenza antigenic drift. It is important that influenza treatment is carried out as soon as the symptoms emerge to prevent further complications. However, rapid mutability, genomic reassortment and vulnerable population groups make influenza a difficult disease to deal with. Therefore, it is important that a clear understanding of these factors is crucial in strategizing new improved treatments.
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