Influenza virus which is also significantly recognized as the ‘flu’ infection spreads via airborne transmission causing infection either pandemically, epidemically or seasonally, affecting a wide range of humans regardless of age. In 2009, the first human influenza A virus pandemic outbreak occurred caused by the novel H1NI swine-origin Influenza virus (SOIV) in the 20th century leading to more than 18,000 deaths among 214 affected countries in a year. A study revealed that ever since in the year 1700, dozens of influenza pandemic infections occurred with a drastic increase in the mortality rate worldwide [1]. A drastic outbreak took place in 1918 which was detected in a military man caused by the H1N1 avian virus strain, killing almost more than 50 million people and another outbreak of the ‘Spanish Flu’ in the year 1977 [2–3]. Furthermore, two different influenza pandemics outbreaks were defined during the 20th century known to be the 1957 H2N2 and 1968 H3N2 influenza strains [4]. The influenza viruses are categorized under the classification of Orthomyxoviruses which consist of four different types such as type A, B, C, and D, but type A and B commonly identified causing recurring local and regional epidemical infection worldwide [5].
In Malaysia, an initial influenza A H1N1 case was reported on 13th May 2009 with the presence of the first case on June 15, 2009, at Pahang which was confirmed through a diagnosis by the real RT-PCR method [6]. From a surveillance study performed, Malaysia portrayed an outline of a yearly transmission of the virus with no specific seasonal clarity but certain studies revealed influenza transmissibility occurs at a low peak in the month of November-January and May-July with different variations presenting in alternative years [7]. As for the recent update on 14 January 2021 from WHO, it was reported a total number of 239 cases were detected in individuals infected with influenza avian virus (H5N1) from four different countries surrounding the Western Pacific Region ever since 2003 January [8]. Another update from WHO stated a cumulative number of 1,568 were infected with avian influenza A (H7N9) on January 14 which was confirmed based on laboratory results and 33 individuals were detected positive with HPAI A( H7N9). This resulted from mutations in the Hemagglutinin (HA) gene leading to an increase in pathogenicity level in the poultry area [8]. Epidemic infections occur commonly during the winter and autumn temperature region causing a massive increase in morbidity and mortality rate throughout a year. These viruses mainly spread from one individual to another individual by a close contact distance of < 1m via respiratory droplets produced by infected individuals through sneezing and coughing actions. According to studies, these viruses may also be transmitted through different routes of entry such as by droplet nuclei (aerosols), whereby the tiny particles, < 5µm circulating in the air is capable enough to travel along the lower respiratory tract (LRT) and accumulate on upper respiratory tract (URT) [9].
The direct and indirect contact of transmission occurs when the infected individual touches the surface of any object leading to another physical touch by a healthy individual, easing the transfer of particles from the object to the URT of the mucous membrane. These viruses multiply in the surface of epithelial cells along the respiratory tree branches in both LRT and URT than favorably attaches to the receptor surface of the cell, sialyloligosaccharides, and terminated via N-acetyl sialic acid which is bound at galactose through 𝛼(2,6)-a linkage found commonly in host respiratory tract. The Influenza infection occurs on a host cell due to genomic destruction on the major component present in the virus, Hemagglutinin (HA) and Neuraminidase (NA) which plays a crucial role in the attachment of sialyloligosaccharides either on glycolipids or glycoproteins surface [9–10]. HA, is significantly important in identifying the host tropism, as it attaches to the receptor of the host cell containing two distinct terminal linkages known to be 𝛼-2,3 and 𝛼-2,6 Sialic acid (SA) moieties that allows binding of both avian and human viruses. The avian viruses preferably attach to 𝛼-2,3-SA and reversibly, the virus adapted to humans such as H1N1 2009 pandemic and H3N2 virus consists of higher binding affinity to the 𝛼-2,6-SA, which demonstrated in the URT of humans. Hence the circulating virus in the human body easily adapts and binds efficiently to these respective linkages initiating host infection [10, 2]. Besides, this terminal linkage carries a cleavage site which is split by the host cell proteases. At this stage the presence of NA activity is equally required in aborting SA-containing receptors in the viral membrane and the host for the budding process; synergistically the release of virions directly from the host cell surface occurs [10].
The life cycle of the virus begins with the attachment of viral HA on the host cell receptors and once endocytosis is completed, the viral ribonucleoprotein, HA-mediated along with cellular membranes is liberated into cytoplasm whereby transported into the nucleus allowing the virus to be replicated and inducing transcription of the virus through viral polymerase complex [2]. The entrance and exit of the virus in a host happens through the respiratory tract which is the mouth and nose that is released during talking, sneezing, and coughing mode of action. The symptoms examined on infected patients include the presence of sudden high fever persisting 7 to 10 days, feeling dizzy, body soreness, dry cough, soreness in the throat, malaise, and runny nose. Therefore, all these symptoms easily can be differentiated from the general flu infection by the presence of various clinical presentations portrayed by an infected individual.
In severe cases, the patient would be presented with respiratory pneumonia illness which may lead to death. The incubation period is estimated from 1 to 4 days from the initial day of infection to the beginning of the first symptoms. In a healthy individual, the recovery stage of influenza is between 3–7 days, and severe complications occur in older adults, infants, pregnant women, individuals with immunocompromised and individuals diagnosed with chronic illness are highly potentially infected with influenza [11–12].
In this study, the influenza A virus along with HA structure would be emphasized more, this is because most of the pathogenicity of the infection is mainly caused by the antigenic drift by influenza A strain. Both influenza A and B compromised with one single-stranded viral RNA (vRNA) segment and eight negative-sense whereas seven genome segments were found in influenza C strains. Influenza A is mainly composed of two main glycoprotein spikes, Hemagglutinin (HA) and Neuraminidase (NA) in a portion of 4:1 ratio jutting out from the lipid membrane of the host cell-derived [13].
In influenza A structural composition, a minimal amount of matrix (M2) ion channels traversing from the lipid envelope consists of three different integral proteins such as Neuraminidase, Hemagglutinin, and M2 which overlapping on M1 matrix protein enclosing the core of a virion. Inside the M1 matrix surface, the nonstructural protein 2 (NS2) and complex of ribonucleoprotein (RNP) is found, which comprises RNA viral segment covered with two distinct components known to be RNA-dependent RNA polymerase that carries two different subunits, one acidic polymerase and two basic polymerases (PB1, PB2, and PA) and a nucleoprotein (NP) [14].
Several studies were performed to understand the relationship between HA and NA which showed an remarkable interaction between the two major surface glycoproteins in viral replication, this is because the HA is required to attach with sialic acid receptors of the targeted cells and stimulate virus infection whereas the NA is required in cleaving the sialic acid away from extracellular inhibitors and cellular receptors which promotes the releasing of virus and spreading of the virus to the surrounding cells. Hence, Ying and Yang’s mechanism is required between these glycoproteins controlling the spreading of Influenza infection in a host immune system [15].
In past years, the initial drugs approved by FDA such as Amantadine and Rimantadine as an antiviral drug in treating influenza infection drastically showed dysfunction in controlling the spread of influenza virus strains due to mutation, cross-resistance of the drug due to conformational changes in the genome and genomic variation caused by the mutagenicity in the strains leading to resistant against the drugs available [16].
Other than these two drugs, commonly used antiviral drugs for example Zanamir, Peramivir, Zoxfluza, and Oseltamivir which utilized in the medical field as an antiviral drug portrayed effectiveness yet not able to completely combat the recurrence of infection due to adaptability of the viruses against those drugs in a long term exposure as it tends to mutate and reproduce new strains resistance towards the drugs available 16. Apart from mutagenicity caused by the Influenza strains, few studies also demonstrated the side effects of antiviral drugs in patients for the long term which has reduced the effectiveness of the drug. It weakens one's immune system, decreases the production of antibodies against the virus, increases the mortality rate in patients diagnosed with chronic illness as the antiviral drugs administered trigger secondary infection such as bacterial infection and affect multiple organs [17].
Therefore, various molecular studies performed by analyzing the interaction between the targeted structure of virus and drugs via molecular computational docking which eases the studies of binding affinity, molecular properties, chemical characteristics, and potential active site informing effective drugs by using natural resources such as plants and herbs that available widely instead of utilizing machinery develop antiviral drugs. And as a replacement for the current drugs, studies suggested flavonoid derivatives such as Oleanolic acid compounds are highly potential as it consists of numerous properties which include antimicrobial, antifungal, antioxidant, anti-HIV, anti-Influenza, anti-Hepatitis and anti-Herpes in combating multiple infections. These flavonoids are mostly plant-based and consist of varieties of polyphenolic compounds carrying an underlying structure benzo-𝛾-pyrone which demonstrates enormous pharmacological properties [18–19].
Thus, Oleanolic acid as the favorable phytochemical compound selected should portray significant potential binding sites against the target protein Crystal structure Hemagglutinin H1N1 virus A with higher binding affinity energy > 7.9 kcal/mol by molecular docking analysis comparing to the current drugs available in the pharmaceutical market field for further drug analysis study.
1.1 Objectives of the Research
The objective of this research is to: design an effective antiviral drug based on phytochemical derivatives compound replacing the current drug available in combating influenza A virus, demonstrate the pharmacological and molecular properties presented by Oleanolic acid as an effective antiviral drug as a treatment for influenza infections, study the receptor-ligand interactive level, Oleanolic acid against Hemagglutinin protein structure via molecular docking studies approach, select a new form of an antiviral compound from the molecular docking approach for further drug analysis studies.