H1N1 Influenza Virus (Swine Flu): A Comprehensive Insight into Escalating Catch-22 Scenarios

Introduction: Viruses have always been a major cause of various disastrous pandemics in mankind’s history. H1N1 became a threat when its original strain was first discovered back in the swine flu pandemic of 2009. It became highly catastrophic on a large scale because none of the therapeutic interventions and methodologies that were already present at the time were effective against the virus. Methods: A vast amount of literature and research is available regarding H1N1 influenza from different reputable sources online. The data was gathered following the contrasting and relative situations of 1918 as well as the 2009 pandemic in mind. The overall extracted material provides comprehensive insights into the ups and downs of H1N1 influenza from 1918 up to 2009. Results: H1N1 virus comprises of a huge potential to cause a pandemic of Influenza type A. The illness caused by the virus has a varying degree of severity depending on the immune function of the individual being under attack. The virus exploits droplet-based transmission mode for its spread from one host to another. The major center of escalation of the subtypes of virus mostly originates from different avian and swine species. Most notably subtypes H9N2 and H5N1 of influenza A, which aren’t easily transmissible among humans. Furthermore, the droplet-based transmission takes comparably less time to infect a population of thousands if not millions. This ultimately increases the overall death toll by several folds by initiating a constant wave of pro-inflammatory cytokine release among affected hosts. Conclusion: Since its discovery in 2009, researchers have developed antiviral drugs and vaccines to fight the virus, most of which have proven to be very successful in treating the interconnected complications. The present-day strategies are only efficacious until the current strains of influenza A do not produce resistance against these drugs. All the therapeutic techniques and methodologies that have been developed to confront the virus up until now have been described in this ample review. ULJRI | https://ir.library.louisville.edu/jri/vol5/iss1/4 1 ULJRI H1N1 Influenza Virus (Swine Flu): A Comprehensive Insight into Escalating Catch-22 Scenarios


Introduction
H1N1 is classified as the serotype of the species Influenza A virus, a descendant of the family Orthomyxoviridae. H1N1 caused the disastrous swine flu pandemic of 2009 that infected 60.8 million people along with 12,469 deaths and 274,304 hospitalizations just within a span of a little more than 1.5 years. It is the same type that caused the infamous influenza pandemic of 1918. [1] Other serotypes of Influenza A are H3N1, H2N3, H1N2, and H3N2. Influenza B and C do not have serotypes. H5N1, H7N5, and other serotypes cause Influenza in avian species. Other strains include H4N6 and H9N2 that infect swine species just like H1N1. he classification for subtypes of Influenza A is based on the presence of the glycoprotein Hemagglutinin and Neuraminidase. In the reproductive cycle of the virus, Hemagglutinin performs the clumping action on the red blood cells that helps the virus to bind and infect the cells effortlessly.
[2] Locomotion and the budding action in the infected cell are executed with the help of Neuraminidase. Neuraminidase belongs to the category of glycoside hydroxylase enzymes. In general, the H1N1 influenza A does not always lead to the viral infection upon any type of contact to the swine population but if the contact leads to the infection, it is termed as zoonotic swine flu.
[3] Experimentally, it has been observed that the virus generally shows its symptoms in swine within four days and can be easily transmitted among the individuals of different swine species, mostly through proper contact. [4][5][6] There are different variants of the virus based on the genotype. For example, the recently discovered strain in China is G4 H1N1. It is a genotype 4 variant of the original strain that mainly affects the swine and has a striking resemblance with the Eurasian avian H1N1 strain. There is little evidence about this variant causing infection in humans. Severe cases of the infection of H1N1 mostly happen with pregnant women. [7,8] Fetus related hormonal changes in the body of pregnant women cause a drop in the function of the immune system that paves the way for the virus to cause infection. This is the reason it is mostly recommended for pregnant women to get vaccinated to avoid any H1N1related complications. [9][10][11][12] Humanity has already faced this kind of H1N1 influenza-related flu pandemic in the past. 1917-18 marks the era of the great disastrous influenza pandemic in the pages of history with an approximate death toll of over 50 million people worldwide. The advanced medical and therapeutic innovations that we've today weren't present back then. So, the medical copping facilities at that time were minute relatively. This was one of the main reasons why the severity of the 1918 pandemic was enormous as compared to the 2009 influenza pandemic. The mortality rate was far too high in 1918 but dropped exponentially as time passed away. However, the original strains of 1918 H1N1 are still circulating in the population today. The status of the immune system of the affected hosts was far better in 2009 than it was in 1918. Lower immune health with low cellular immunity as well as a low number of antiviral cross-reactive antibodies contributed to the severe pro-inflammatory cytokine storm along with the spread of H1N1 in the affected hosts of the 1918 pandemic. Today, we're also facing an exponential increase in the new strain of influenza (like H5N1 and H7N9) that have evolved since 1918 but the medical advancements are also on a whole different level. [13][14][15] 2009 Swine Flu: A Brief Overview During the swine flu pandemic of 2009, cases of pneumonia were showing up consistently because of bacterial co-infection along with the virus. A study was performed during the pandemic to quantify the ratio of coinfection. Although several proportions were reported, approximately 25% of the total cases were appearing to have a co-infection. Dealing with this co-infection situation, was a challenge in 2009 due to the severely compromised immune systems of the patients.
[16] Later on, experiments were performed by taking frequent samples from herds of swine to map out the lineage of the viruses that were evolving. It was concluded that swine are a "mixing vessel" of several serotypes that ultimately helps the virus to become unresponsive to a lot of newly developed human vaccines. A total of 17 sequenced whole genomes were used in this particular spatiotemporal distribution study. [17][18] Similarly, another study demonstrated in 2011 that the swine flu virus is antigenically evolved as compared to the viral strain of the 2009 pandemic. The experiments were performed using ferrets that were immune to the original strain but were least immune to this new strain. The concept is the same in the case of humans who were immune to an old strain, but are vulnerable to this mutated strain. [19][20][21][22] Real-time Polymerase Chain Reaction (PCR) techniques were approved by WHO (World Health Organization) in 2020 to detect the presence of the virus. Many new methods, like the use of biosensors (FET biosensors, PEDOT with galactose, Surface plasmon resonance, and AuNP immunosensor), have shown high sensitivity to the presence of DNA (Deoxyribonucleic Acid). The RT-LAMP (Reverse Transcription Loop-mediated Isothermal Amplification) technique that investigates antigen-antibody related interactions to confirm the presence of the virus was also utilized.
[23] Biosensors are a recent development in this field and are very efficient as compared to other conventional detection methods. Biosensors usually exploit immune-chromatographic techniques. The most modern technology uses a bacterial quench-body that is an immuno-protein adhering to different antibodies labeled with specific fluorophores. The detec- tion is based on the interaction between antigen and the quench-body. Examples of quench-body include anti-HA Fab with ATTO520 (attachment of a Cys-tag at locus N-terminus of heavy chain in the expression vector of Fab), anti-BGP Fab, anti-Flag Fab, and anti-His Fab. [24][25][26][27] All of these detection methods were developed to cope with the mutated strains of Influenza A that arose as a result of the phenomenon of divergence. Here, divergence specifies the outward spread of the mutated strains from the center of origin. Recent studies have observed the presence of the virus in Australians with divergent origins from the main human population. The diversity in the genotype of different wholegenome sequenced samples showed that all strains are somewhat different from each other. The samples that were taken between 2012 and 2016 revealed that limited diversity is the main hurdle for risk assessment as well as for the control procedures. The diversity problem poses new challenges in the further development of new treatment methods.
[28] Although the 2009 pandemic had disastrous effects on the population as the cases rise with time (Figure 1), it led to an improvement of strategies to manage future infections. In Mexico, the health shocks of the pandemic directed to better health control measures that helped health authorities to regulate the causes of diarrhea in young adults. Furthermore, aftershocks of the pandemic motivated some people to quit smoking and improve their hy-giene habits.[29-32] One phylodynamic study also displayed that after the pandemic, there was a reduction in the diversity of the virus globally due to its transitioning towards immune-driven selection instead of hostadaptation mechanisms. [33] There are some sequence-based studies with strong evidence that 3 out of 5 pandemics of flu have a possible avian origin. These studies utilized polymerase derived gene segments that showed that diverse lineages in the swine population of North America are due to the re-introduction of the original American avian strains. There was a 70% average match of antigenic sites with the original strain of the 2009 pandemic, suggesting that the original strain had an avian origin. This circulation and mutation of strains in wild avian species can play a significant role in future pandemics.[35-37] Another study displayed that the original strain of the 2009 pandemic was still circulating in Russia. Randomized trials of healthy and influenza-infected people showed that a small number of neuraminidase antibodies were present even in the healthy individuals, which supports the hypothesis of circulation of strains in the general population. It was also observed in swine that antibody responses were extremely different in different individuals of the species.[38-41] Climate also had a very crucial role in driving seasonal influenza, as described. The analysis of different Influenza serotypes affecting several regions of Australia revealed that the seasonal drives Triangles indicate samples from which no neuraminidase sequence could be obtained. The dashed red line indicates the lower limit of detection of influenza A semi-quantitative RT-PCR (Real-Time Polymerase Chain Reaction) assay. The duration of oseltamivir monotherapy and zanamivir monotherapy is indicated by light-gray and dark-gray shading, respectively, and the duration of triple therapy.] [58] of the virus are most active in subtropical areas. Although the pattern of all inter-seasonal transmissions is far more complex to comprehend using simple techniques, it is believed that climate is also a major factor in the drive of seasonal infections.[42-44] Epidemiological techniques on a molecular level were used in Europe to quantify the different strains of the virus that were in circulation after the pandemic of 2009. All the strains within four years after the pandemic were analyzed. It was revealed that most of the strains diverged from the original strain and made the vaccination process more challenging. The reason behind this is that some of the lineages of the strains were unresponsive to vaccines and antivirals that were developed for the virus.[45-47]

Advancement of Modern Antivirals
One of the main potent actions of an antiviral drug is the inhibition of strain-specific binding receptors. The blocking of these receptor sites disrupts the infection cycle and ultimately stops the spread of the virus within its host. One of these compounds that have strain-specific activity against the virus is iminosugars such as NN-DNJ (N-Nonyldeoxynojirimycin) and NB-DNJ (N-Butyldeoxynojirimycin). Both of these have shown a high strain-specific potency when their activity was observed by experimenting on a large number of Influenza A viruses. NN-DNJ is an α-glucosidase inhibitor that acts on influenza viruses by disrupting the main glycan processing between Neuraminidase and Hemagglutinin. Hemagglutinin is an essential factor for the proper inhibition of the cycle.
[48] Common antiviral treatment options include oseltamivir and zanamivir. Both of these antiviral drugs are neuraminidase inhibitors and are highly effective against H1N1 related infections. [49][50] Recent studies have shown that most of the modern strains have developed a significant amount of resistance to both of these drugs. Other drugs like rimantadine and amantadine (adamantanes) have also been developed but these drugs come with their sideeffects like embryonic toxicity and teratogenic abnormalities in the developing fetus during the gestation period.[51-53] Adamantanes are only used in those cases where Neuraminidase inhibitors based drugs fail to show any viable potency against the infection. Sometimes, the combination of adamantanes and Neuraminidase inhibitors is more efficient due to their synergistic effects as displayed by various mice-based models.[54-56] Early cases of oseltamivir resistance were reported back in 2017 (Figure 2) in Myanmar when H1N1 infected patients in a local com-munity outbreak were analyzed. [57]

Vaccine Mediated Therapies
The most recent development in the field of vaccines is the use of the AS03 (Adjuvant System 03) type adjuvant system that exploits α-tocopherol oil and water-based emulsion of a very precise ratio. A study based on randomized and controlled trials in adults has shown that by utilizing the AS03 type adjuvant systems of vaccine administration, the negative effects of conven- , overall and stratified according to age, virus subtype or lineage, and vaccine type. [75] tional vaccination procedures can be significantly reduced with a relatively higher persistence rate. This produces both humoral and cell-mediated immune responses following the induction of CD8 T-cells (Cluster of Differentiation 8 Thymus cells). [59][60] Furthermore, antigen selection and cross-protection process can be improved by using a nomenclature system that is easy to comprehend and is based on the phylogeny of Hemagglutinin based lineages. A total of 7,070 H1 sequences were used for the development of the nomenclature annotation tool that yields 99% accurate results most of the time. [61][62] Modern technological development has made it possible to study the interactions of vaccines by using computational simulation analysis and constructing various Mathematical models. These computationally optimized vaccines have shown their activity over a broad range of Influenza A viruses isolated from human and swine subjects. [63][64][65][66][67] Recently developed COBRA (Computationally Optimized Broadly Reactive Antigen) vaccines with HA (Influenza Hemagglutinin) antigen are optimized in such a way that most zoonotic infections were prevented in farmworkers as shown in a study by Morris and colleagues. [68][69][70] miRNA (micro Ribonucleic Acid) alteration technologies are a kind of new approach to deal with the virus with a unique strategy. It has been observed that especially developed miRNA such as miR-let-7b-MRE enhances the biosafety for developing more operative and stable vaccines by decreasing the replication and virulence of the virus. [71][72][73][74] Neutralization can be achieved by using monoclonal antibodies that have performed well in experimental assays to study antigen-antibody interactions (Figure  3). Such an example is murine-based high-affinity MA2077 neutralizing monoclonal antibody that inhibits the 2009 strain of Influenza A in a specifically developed experimental assay. The specificity of this monoclonal antibody lies with the 'Sa' site in an in vitro medium. [76] Chicken eggs are generally used as a basic medium to reproduce viruses and then to produce vaccines along with, but a new type of approach utilizes attenuated metabolically active E. coli to produce an enormous amount of antibodies before primary immunization in a short span of about three weeks. This method can be used as an efficient way to study vaccines without applying any rigorous purification procedures for deriving antigen. On the other hand, this method is much faster than other lengthy conventional methods because it only employs E. Coli based harvested Hemagglutinin. This Hemagglutinin produces a detectable amount of antibodies when observed in MDCK (Madin-Darby Canine Kidney) cells. Furthermore, this immunization technique doesn't require any exogenous adjuvant as a booster dose. [77][78][79][80]

Modern Research and Progress
There is a reinforcement problem that exists in the cases of co-infection such as Influenza A Virus in a combination with the hRSV (Human Respiratory Syncytial Virus). It has been observed that during normal seasonal peaks of both viruses, the ferrets with co-infection show an increase in the levels of pro-inflammatory cy-tokines. This happened when the inoculum dose was increased more than normal with the same ratio of hRSV. This phenomenon is mostly termed as viral interference that increases the challenges while dealing with a subject of co-infection. Immune mediators with other chemokines were also released upon increasing the dosage that poses the possibility of antigenindependent mechanisms that drive the interference process. [81] Later, it was found that autophagy is a process that defines the interaction of dendritic cells with several strains of influenza viruses as observed in mice models. [82] Although the seasonal viral spread can be explained with a reverse zoonosis strategy that promotes the development of new lineages, viral interference is not perfectly understood yet. [83][84] A recent study in hosts other than birds and swine showed that host jumping of several strains of Influenza A is a major threat in control of any future pandemics. Canines and Equines usually have a very stable lineage of Influenza A viruses. The samples that were taken from different southern China (2013-15), upon sequencing have shown that the virus is slowly mutating when the interaction of these species with major reservoirs of Influenza A virus occurs. It means that host jumping can contribute to the development of new lineages in minor hosts as well. [85][86][87] Similarly, experimental studies also support the transmission of the virus from different avian species into mammals. The virus adapts to the host body by exploiting specific HA-receptors. [88] Modern methods of characterization mostly depend upon the genetics of the agent that is being examined. One of the profound examples of using genetic methods was the characterization of a strain of Influenza A present in native pigs of Ontario, CA. The 99% triple re-assortment based identity match of subtypes like H3N2 with the original strain of 2009 confirmed their origin from pigs. Furthermore, the Ontario based strain posed significant resistance to adamantanes but was immensely responsive to Neuraminidase inhibitors based drugs. [89][90][91][92] Other methods still work effectively as shown in an experiment (mice and ferret models) of combined factor vaccines (both Hemagglutinin as well as Neuraminidase) that were utilized to produce a significant amount of immune responses in research subjects. [93][94] Song and colleagues conducted a pathogenic study on the corresponding lineages of Chinese swine and humans (NS, PA, PB2, NP, M, NA, and PB1) by using several techniques and methods to differentiate strains in the laboratory. The study concluded that the viral strain in the Chinese swine sample was homologous with the strain that was previously circulating in North American regions. On the other hand, there were several variations in the pathogenicity when compared to other mammal-infecting strains. Mice models were used for histopathological examinations that showed that the viruses (AV1522, AV1523) progress over time due to the re-assortment in Chinese swine. It ultimately affects the respiratory as well as the nervous system of the infected individuals. [95][96][97][98] Furthermore, the continual resistance of the virus against modern antivirals drugs and vaccines makes it more challenging for researchers to develop new treatment strategies. The new anti-influenza agents that are still evolving and are in rigorous development include RNA (Ribonucleic Acid) inhibitors, RdRp (RNA-dependent RNA polymerase) inhibitors, V-ATPase (Vacuolar-type Adenosine Tri-Phosphatase) inhibitors as well as some species of modified antioxidants. [99][100][101][102][103]

Conclusion
Although the present-day scenario is far more different from the circumstances during the pandemic of 2009, we should always be ready to deal with the unforeseen circumstances. The complications related to the infection caused by the different strains of the virus are now under control due to the rapidly evolved technology that enabled us to develop more effective treatments and advanced therapeutic methodologies. These developments helped people on a global scale in recent decades. [104] If we discover a new strain of the virus with a pandemic causing potential, the management strategies would be less complex because of our far more developed global management system. Overall, we are optimistic that we shall find a cure for this infection as we always have.