- Viral Infections (RNA)
The infectious disease influenza (also known as flu) is caused by the influenza virus, which is a single-stranded RNA virus belonging to the virus family Orthomyxoviridae. The communicable influenza viruses A, B and C are subdivided into various strains based on the antigenic surface molecules. Highly infectious droplets containing the virus are globally spread and affect 10-20 % of the world population. Influenza-associated pandemics return on a seasonal base and are considered to have the highest global death toll of all infectious diseases with 500 000 deaths per year. Severe or life-threatening illness is mainly reported in high-risk patients like elderly, young and immunosuppressed patients. Non-specific constitutional symptoms of the viral infection are likely to be associated with complications affecting mainly the respiratory tract like pneumonia. The rapidly evolving virus strains are a huge challenge in drug development due to mutational evading mechanisms like antigenic drift and shift. Even though many annual vaccines and anti-influenza agents are already used in clinics, influenza remains a focus in research, where more efficient targets are being investigated.
The highly contagious and easily transmittable influenza virus causes clinical symptoms in 67% of the cases ranging from mild to severe features. The incubation time of the virus is 1 to 4 days and the illness is resolved within 3 to 7 days by healthy non high-risk patients. An uncomplicated influenza infection is characterized by an abrupt onset of constitutional and pulmonary signs, which is the main difference to a usual cold. Typical non-pathognomonic symptoms are headache, myalgia, sore throat, non-productive cough, hoarseness, reddened eyes/skin, sneezing, nasal discharge and fever. For children, nausea and vomiting has also been reported.
Constitutional features like severe weakness, persistent pain in the chest, dehydration or difficulties in breathing are also indicative factors. The most common comorbidity is viral or bacterial pneumonia. Due to the fact that these major symptoms are not specific and not solely associated with influenza infections, the clinical diagnosis of influenza remains a hard challenge in hospitals. The sensitivity of diagnosing influenza based on influenza-like-illness symptoms is 55-69 %. It has been reported that especially for elderly people the clinical indications for flu perform poorly. The positive predictive value for flu of non-hospitalized patients over 60, who complained about fever and cough is 30-40 %. This fact indicates that flu can be hard to identify in the absence of laboratory-based testing. Therefore, diagnosis of flu is often taken into consideration by clinicians when respiratory and constitutional symptoms are observed. The reduced specificity of the symptoms-based diagnosis is also related with high levels of disease-complexity in the study cohorts.
As laboratory diagnostic assay a rapid molecular test is often performed by using specimens from the upper respiratory tract. This FDA-approved method takes around 15 to 30 minutes and identifies nucleic acids with high sensitivity and specificity. Another method that is highly reliable is identification with RT-PCR (Reverse transcription polymerase chain reaction) from nasal swab specimens. After reverse transcription of the viral RNA, the
A collection of DNA which is capable of developing and maintaining an entire organism. can be amplified. Two other alternatives are immunofluorescence of viral antigens or viral culture (1-3 days), which allows a detailed genetic characterisation of the influenza strain.
The outbreak of flu is based on the viral invasion and replication of influenza virions into healthy living cells. Since the cell invasion of a virus is the crucial step to initiate the replicative circle, the adhesion mechanism can be considered as the main pathogenic factor. In the case of influenza virus, the proteases from the host cell are cleaving the viral surface molecule hemagglutinin (HA), which allows consequent binding to the sialic-acid containing receptors of the host cell. After being endocytosed, the endosomal host membrane fuses with the viral membrane, which results in the release of the viral
A collection of DNA which is capable of developing and maintaining an entire organism. in the target cell for the following replication steps. Lowly virulent strains are likely to be cleaved by proteases occurring in epithelial cells of the upper respiratory tract (throat/upper part of lung).
In contrast to that, highly virulent viruses like H5N1 are cleaved by a large variety of different proteases. This leads to infection of cells in the deep lungs, which can result in viral pneumonia. These kinds of pathogenic infections can be lethal for the infected person, but the viruses are rarely transmitted, since the virus is mainly located in the lower respiratory organs. The virions replicate in the alveolar epithelium and are likely to cause severe ruptures in the alveoli and bronchioles. The strains that are easily transmitted are only targeting the upper epithelial cells. Furthermore, mutations in the
A chemical compound consisting of at least two amino acids. sequence of HA molecules can have a huge impact on the level of pathogenicity of the viral strain. The HA molecules of the seasonal flu are likely to bind to α-2,6-sialylated glycans that are expressed on the host epithelial cells. The strain H1N1 is more pathogenic, due to a D222G substitution in the HA protein Protein
Large biomolecules, which consist of one or more long chains of amino acid residues. They perform a vast array of functions within organisms including transporting molecules, providing cellular structure and catalysising processes such as DNA replication. . The specificity change for the receptors is fatal, since these HA molecules can bind both α -2,6 and α -2,3-sialylated glycans. This turns the virion into a more efficient infector.
The response of the human host cells can be characterised by two main features. First the release of proinflammatory cytokines and chemokines (IFN/TNF) by leukocytes, which is mainly responsible for causing symptoms. Severe infections in high risk patients can also lead to a so-called
A broad category of small proteins released by a range of cells (including immune cells like macrophages, B lymphocytes, T lymphocytes) that act through receptors and are important for a range of processes including cell signaling, inhibition, maturation and proliferation. storm, which is the over-production of inflammatory cytokines, which can easily result in death. It is claimed that cytokine Cytokine
A broad category of small proteins released by a range of cells (including immune cells like macrophages, B lymphocytes, T lymphocytes) that act through receptors and are important for a range of processes including cell signaling, inhibition, maturation and proliferation. storms were the main influenza-associated complication and death causing mechanism during the pandemic in 1918. The second feature is the triggering of apoptosis or cell destruction by cytotoxic T-cells due to increased viral replication. This leads to tissue damages in the epithelium, which need up to one month to be recovered.
The human immune system that is attacked by influenza viruses is triggering both the innate and the adaptive immune response to tackle the viral infection efficiently. The innate immune response, which is characterized by rapid but rather unspecific reactions is likely to recognize pathogenic associated molecular structures (PAMP) by corresponding
A biological agent such as viruses, bacteria or fungi that cause a disease. recognition receptors (PRR). During an influenza infection the RIG-I receptor recognizes single stranded RNA as non-self-molecules and as a pathogenic cellular threat. Consequently, a cellular downstream cascade is induced, where transcription factors like NFκB or IRF3 are activated to induce the transcription of cytokines (especially interferons) in the nucleus. The cytokines are responsible for recruiting immune cells like natural killer cells, monocytes, macrophages, dendritic cells to the infection site.
Dendritic cells function as important mediators between the innate and the adaptive immune system. They are presenting viral antigens on their cell surface with MHC II receptors, which are bound by the T-cell receptors of CD4+ or CD8+ cells. CD8+ T-cells can mature to cytotoxic T-cells that excrete cytokines and kill infected cells by inducing apoptosis with secreted granzymes. CD4+ cells can mature to Th1 or Th2 T-cells, which induce either cytotoxic T-cell or B-cell activation. The B-cell activation in turn is inducing the secretion of antibodies. The most important major neutralizing antibodies in the respiratory mucosa are IgA and IgM that are hindering the viral entry. There is also an event called “antigenic sin” that has been reported which describes the process of developing memory B-cells after a primary infection with flu. The secreted antibodies from these cells have a low affinity against new secondary antigens. The primary antibodies are considered to supress naïve B-cells to produce modified antibodies with adapted specificity.
The average recovery time of influenza infected patients is 3-7 days, whereas especially high-risk patients develop in many cases life-threatening complications. The most frequently occurring comorbidities affect the pulmonary tract, which results in diseases like pneumonia, croup, asthma, bronchitis, ortitis media or chronic obstructive pulmonary disease (COPD). Primary viral pneumonia is caused by viruses that wander from the upper to the lower respiratory tract to infect cells in the lung tissue. The invasion leads to apoptosis and consequently to destruction of alveoli. Secondary bacterial pneumonia takes place when bacteria are inhaled in the lower respiratory tract. The invasion of alveoli triggers the immune system by releasing neutrophils and cytokines. Fever and impaired oxygen transport in the alveoli are the result. Cardiac complications of flu include myocarditis and pericarditis. Neurological manifestations can also be associated with flu infections, namely Reye’s syndrome, confusion, convulsion, psychosis, encephalomyelitis, Guillain-Barré syndrome, coma and transverse myelitis. Moreover, influenza during pregnancies can lead to prematurity, low neonatal size and weight, perinatal mortality and maternal complications.
The elliptically shaped virions (80-120nm) are enveloped by a capsid consisting of a lipid membrane. The inside of the virions contains helical ribonucleoprotein (RNP) complexes with a width of 10 nm. The compounds of the RNPs are defining the type of strain, since it includes the viral
A collection of DNA which is capable of developing and maintaining an entire organism. as well as the polymerase machinery proteins. The negative single stranded RNA builds the core of the RNP associated virion core. The genome consists of eight viral RNA segments with 13,5 kilobases in total, which encode 11 proteins including hemagglutinin (HA), neuraminidase (NA), nucleoprotein, matrix proteins (M1/2), non-structural proteins (NS1/2) and the polymerase proteins (PA, PB1/2). The genome Genome
A collection of DNA which is capable of developing and maintaining an entire organism. sequence has terminal repeats at both ends with 9 to 13 nucleotides each. The 500 spike-like surface molecules include the highly antigenic proteins NA and HA, which are expressed with a ratio of 4:1.
The first step of the viral replication process is the attachment to the host cell. The viral surface molecule hemagglutinin is cleaved by host proteases, which allows consequent binding to the sialic-acid containing receptors of the epithelial cells. After being endocytosed clathrin-dependently, the endosomal host membrane fuses with the viral membrane, which results in the release of Viral
A collection of DNA which is capable of developing and maintaining an entire organism. and polymerase proteins in the target cytosol. The fusion is facilitated by the viral membrane protein Protein
Large biomolecules, which consist of one or more long chains of amino acid residues. They perform a vast array of functions within organisms including transporting molecules, providing cellular structure and catalysising processes such as DNA replication. M2, that acidifies the virion by H+ transport. The viral RNA is transported into the nucleus, where it is transcribed by the RNA polymerase into positive stranded circular RNA (cRNA). After being translated in the cytosolic ribosomes, the surface proteins wander to the Golgi and are transported to the cell surface. The other proteins go back to the nucleus to bind the vRNA and to form the new RNPs and to be packed into the nascent virions, which bud off at the host membrane. Viral proteins degrade the host mRNA during the whole process and they also inhibit the translational host system. The viral enzyme neuraminidase cleaves the sialic acid group from the hemagglutinin receptors, which helps the new virions to escape the host cell during the budding process. After the release of the virions the host cell dies.
Due to the fact that the RNA polymerase does not have a proofreading function, an insertion error occurs per 10 000 nucleotides, which means that every virion includes at least one mutation. The important feature of RNA viruses is the replication in the nucleus, which means that they cannot produce mRNA independently. They make use of cap snatching, where the RNA polymerase
Large biomolecules, which consist of one or more long chains of amino acid residues. They perform a vast array of functions within organisms including transporting molecules, providing cellular structure and catalysising processes such as DNA replication. PB3 binds to cellular pre-mRNA at the 5’ capped end. The PA protein Protein
Large biomolecules, which consist of one or more long chains of amino acid residues. They perform a vast array of functions within organisms including transporting molecules, providing cellular structure and catalysising processes such as DNA replication. cleaves off the mRNA 5’ end. The gained cap fragment is used for starting the transcription of the viral RNA. The cap is also crucial for the recognition of the RNA by ribosomes for the following translation.
Influenza virus belongs to the virus family Orthomyxoviridae. The lipid membrane of the virions covers the ribonucleoprotein (RNP) complexes (10 nm width). The RNPs consist of three major compounds: The viral
A collection of DNA which is capable of developing and maintaining an entire organism. , viral structural proteins (M1, NP) for RNA binding and heterotrimeric replication-associated proteins (PA, PB1/2). The genome Genome
A collection of DNA which is capable of developing and maintaining an entire organism. is characterised by negative single stranded RNA. The genome Genome
A collection of DNA which is capable of developing and maintaining an entire organism. consists of eight viral RNA segments with 13,5 kilobases in total (influenza C only seven RNA strands). The influenza virus can be subdivided into three types: Influenza virus A, B and C, whereby type A is the most common type infecting humans. The RNA strands are encoding 10 to 11 viral proteins that are essential for the viral replication and multiplication. The genomic subdivision into RNA strands is building the base for genetic reassorting of the gene segments (antigenic shift) that results in a vast variability of genetic combinations. In case of a double infection of a host cell the virions can exchange gene segments, which results in new reassorted genomes that reflect another antigenicity and pathogenicity. Viral RNA polymerase doesn’t have an exonuclease function, which leads to increased point mutation rates in the RNA genes. Mutations due to that inefficient polymerase activity results in antigenic drift. Approximately between 1×10−3 and 8×10−3 substitutions per site per year are occurring during replication. Since these mutations affect the structure of the surface glycoproteins NA and HA, the antigens can seldom be attacked antibodies or cytotoxic T-cells. For that reason, vaccines that include antigens of conserved viral molecules are of huge interest. These two genetic factors of the flu virus are the major efficient evading mechanisms to escape the host immune response.
The RNA segments 1,2 and 3 are including the genetic sequence for the RNA polymerase proteins (PB1/2). RNA strand number 4 is encoding hemagglutinin (500 molecules per virion) and strand 6 for neuraminidase (100 molecules per virion). Strands 5,7 and 8 are the template for the proteins NP, M1/2 and NS1/ NEP. The latter two proteins facilitate the export of the RNP components from the nucleus in the
Refers to all of the material within a cell which is enclosed by the cell membrane, except for the cell nucleus. The main components of the cytoplasm are cytosol and organelles (substructures within the cell). , where budding can be initiated at the host membrane.
The three classes A, B and C of influenza virus are subdivided into serotypes based on their genetic variants of the surface molecules hemagglutinin and neuraminidase. So far 18 HA antigens and 11 different NA antigens are known for influenza A virus. These proteins are the focus of the classification system, since they are the pathogenicity defining molecules for an infection and replication in the host system.
Due to the global spread of influenza and seasonal outbreaks the pathological mechanisms of influenza virus are of huge interest both in medical and in pharmaceutical research. There many FDA-approved antiviral medications as well as many other drugs which are currently in the preclinical or clinical testing phase. The most commonly used drugs in clinics directly target the viral replication process and can be subdivided in the two main groups: neuraminidase and M2 inhibitors. Aside from these medications, seasonal vaccines and MEK-inhibition based drugs are also available.
Neuraminidase (NA) is a viral enzyme that cleaves the sialic acid group at hemagglutinin receptors of respiratory epithelial cells, which helps nascent virions to escape the host cell during the budding process. This digestion of the HA receptors also prevents the virus particles from clumping after being released and allows free dispersion and reinfection of new cells. The most important representative of NA inhibitors is Tamiflu (oseltamivir). It is an antiviral agent that is effectively inhibiting influenza virus A and B. The duration of symptoms and complications can be reduced, if the medication is initiated within 48 hours of flu symptom onset. Tamiflu mimics a substrate that blocks the active site of the enzyme. The prodrug oseltamivir-phosphate is quickly metabolised to the active form oseltamivir-carboxylate by hepatic ester hydrolysis. The bioavailability of Tamiflu in the blood is 80 %. The drug is highly selective for the neuraminidase sequence of seasonal Influenza A and B molecules. Due to the chemical structure of Tamiflu, it’s quite prone to become inefficient in the next season by causing mutations in the NA genes. It has been demonstrated that another NA inhibitor (zanamivir) is not affected by viral resistance. But the disadvantage of this drug is that it is only applicable by inhalation.
Antiviral drugs such as amantadine are interfering in the viral replication process by acting as an ion channel blocker of M2. The viral
Large biomolecules, which consist of one or more long chains of amino acid residues. They perform a vast array of functions within organisms including transporting molecules, providing cellular structure and catalysising processes such as DNA replication. M2 is an integral membrane protein Protein
Large biomolecules, which consist of one or more long chains of amino acid residues. They perform a vast array of functions within organisms including transporting molecules, providing cellular structure and catalysising processes such as DNA replication. type 3 that is responsible for the transport of H+ ions. After the virions are endocytosed in an endosome, the interior of the viral particle is acidified by the H+ channel. The channel is solely activated if the outer pH value is acidic (<7), which is sensed by the N-terminus of the helical structured membrane protein Protein
Large biomolecules, which consist of one or more long chains of amino acid residues. They perform a vast array of functions within organisms including transporting molecules, providing cellular structure and catalysising processes such as DNA replication. . Amantadine is only selective for the A/M2 protein Protein
Large biomolecules, which consist of one or more long chains of amino acid residues. They perform a vast array of functions within organisms including transporting molecules, providing cellular structure and catalysising processes such as DNA replication. in influenza A.
Another promising approach for antiviral medication are drugs that are interfering in the host cell itself and not in the virus, which has the great advantage of not being dependent on viral resistance mechanisms. Influenza virus activates the Raf/MEK/ERK signalling pathway in the host cells. In the initial step the HA
A part of an antigen that is recognised by the immune system, specifically by antibodies, B cells, or T cells. is binding and activating GTP-Ras, which in turn induces the phosphorylation of Raf. The activation of this pathway contributes to the nuclear escape of viral RNPs. This can be impaired by drugs like CI-1040 that inhibits MEK activity and therefore a crucial step in the cascade. It has been successfully shown in many preclinical studies that the compound can efficiently block host-cell mediated phosphorylation events. Efficacy against Tamiflu resistant strains has also been demonstrated by a substantial reduction of viral titers both in cell culture and in animal experiments.
Seasonal prevention with vaccination is especially recommended for high risk patients which include elderly people > 60, pregnant women, children < 5, immunosuppressed individuals and those suffering from chronic disease. Since the viral mutational mechanisms of influenza virus (antigenic drift and shift) are highly evasive, an annual reformulation for viral variants is required. Vaccines can be designed inactivated (IIV) or live attenuated (LAIV) influenza viruses. LAIV are applicated intranasally, whereas inactivated vaccines are applied by intramuscular injection. Even though the viruses of LAIV vaccines have low virulence, they are more likely to cause complications in immunosuppressed individuals and are therefore not recommended for all patient groups. IIV vaccines induce virus-specific serum antibodies that neutralize specific viral antigens, whereas LAIV depends strongly on the secondary T-cell response. IIV vaccines are trivalent or quadrivalent (H1N1, H3N2, Influenza B), which means that antigens of three different strains are mixed up to reach a protection level of 60 % by matching the respective circulating strain. Hemagglutinin is the main immunogen in inactivated vaccines, whereas neuraminidase antibodies are not efficient enough, since neuraminidase antigens are not strongly immunogenic. The most common technique for vaccine formulation is egg- based: The desired strains are injected in a chicken egg, where the genes mix up and replicate. The new reassorted strain is isolated afterwards. Other methods for vaccine design are realized by plasmid-based reverse genetics or with cloning strategies in viral vectors. This results in a recombinant expression product that leads to the desired HA
Large biomolecules, which consist of one or more long chains of amino acid residues. They perform a vast array of functions within organisms including transporting molecules, providing cellular structure and catalysising processes such as DNA replication. required for antigenicity-based antibody production. In primed individuals, IIV stimulates a cross-reactive T-cell response, whereas this event has not been measured in un-primed humans. Some implications show that the secretion of cross-reactive antibodies generated by different vaccination events could depend on the antigenic distance between the strains included in the sequential vaccines.
Influenza outbreaks are occurring globally on a seasonal basis caused by the contagious and rapidly evolving respiratory influenza virus. Pandemic outbreaks have been registered especially in winter months, whereas tropical regions are affected throughout the year. The rapid genetic evolution and the multi-strain character of the virus leads to more than 500,000 deaths per year and up to 5 million severe cases. It has the highest global death toll of all infectious diseases (year 2017/18). Antibodies that are produced by the adaptive immune system can reduce risks for complications and milder the symptoms, but they do not prevent reinfection. The spread of the virus depends on two main variables - transmission and susceptibility of the population. 10-20 % of the worldwide population is affected by influenza, whereas all age classes are representative in this group. 67% of the infected patients develop mild to severe symptoms during the illness. High-risk groups are more likely to be infected and experience severe complications and comorbidities while infected. Individuals at higher risk are pregnant women, children under 60 months, elderly people over 60 years, immunosuppressed indivuduals, health care workers and those with chronic health issues. Most of the influenza-induced or flu-associated deaths are registered in the elderly population. Influenza-associated hospitalizations are higher rated for children under 2 years than for children older than 2 years.
The shedding process is characterised by the spread of more than 500,000 virus particles per cough or sneeze of an infected person. Children and immunosuppressed individuals are infectious for a longer period compared to other groups. There are three ways of transmission that form th underlying cause of the spread of contagious virus particles:
- Direct transmission by sneezing directly in eyes, mouth or nose of another person.
- Airborne transmission which is defined by infection by inhaling contagious aerosols (0.5-5 μl). One drop is enough to cause an infection in this case.
- Indirect transmission, including hand-to-eye, hand-to-nose or hand-to-mouth transmission. Virus particles can survive up to two days on normal surfaces.
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