Genome Exchange and Integration of Influenza A Virus

*Small changes in the NA coding gene segment

Similar to the HA protein, the NA protein also undergoes minor changes during its activity. Studies have shown that amino acid changes occur on the surface of the NA protein, where there are sialic acid binding sites (positions 222, 329, and 344 on the N2 gene) [92, 118]. Similar to the HA surface antigen, the NA antigen also has the ability to generate protective antibodies. Mutations and changes in the NA protein (especially in the conserved region) affect the antigenicity of NA during the immune response, influenza viruses change to "evade" the effects of anti-NA antibodies, creating a new antibody lineage that is different from the previously acquired antibody lineage when the mutation did not appear [92]. However, only acquired immunity when interacting with the conserved part of NA is protective.

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*Small changes in the M gene

The study of the evolution of the M gene showed that M1 had 26.4% amino acid changes and M2 had 48.5% amino acid changes. This difference is due to the fact that the M2 gene is under higher selective pressure from protective antibodies than M1. However, the large changes in the M2 gene were only observed in human and swine influenza viruses, and almost never occurred in avian influenza strains. Observing the M1 gene, most of the changes in the gene did not lead to amino acid changes. In addition, comparing the mutation frequency per year on M1 and M2 and the remaining genes of the virus showed that this frequency was lower than the mutation frequency of the remaining genes, so the M gene can be considered a highly conserved gene [29].

Major genetic changes associated with antigenic variation

Laboratory studies comparing viruses isolated from the 1918, 1947-1956, 1957-1967, and 1968 epidemics showed that these epidemics had the same cause, influenza A subtype H1N1, but had major variations in genetics and antigenic properties (no or low titer cross-reactions when performing hemagglutination inhibition reactions between newly emerged viruses and residual antibodies produced by old virus subtypes).

Thus, although they have the same virus subtype name (H1N1), the nature of the virus has completely changed, so specific immune responses will not result in cross-protective responses between known virus subtypes of the same name.

The emergence of a new virus without a viable antibody in the population would result in a worldwide pandemic (the 1918, 1977, 2009 influenza pandemics with influenza A subtype H1N1) [51, 70, 97, 98]. The emergence of major mutations leading to a complete change in the antigenic properties of both the HA and NA gene segments of influenza viruses is explained by the hypothesis of exchange and integration between different virus subtypes.

1.1.4. Exchange and integration in the genome of influenza A virus

Scientists have shown that, with such a segmented genome, the rearrangement of genetic material between influenza virus strains is likely to occur when there is a co-infection of two or more influenza A viruses of different subtypes on a host. The result is the creation of a new virus whose genome is the mixing and rearrangement of gene segments of the co-infecting influenza viruses. In fact, when analyzing the family tree of influenza A/H1N1 virus strains circulating from 1918 to the present, it was found that the virus has undergone many exchanges and integrations of genes of each gene segment. The gene segments encoding PB1, NA and M that appeared in the early 1940s are still circulating in the viruses of 1947, especially the gene segment encoding HA of the 1947 epidemic virus is completely different from the HA of the virus strains circulating in 1943-1945 [12, 30, 33]. In 2009, the pandemic H1N1 strain had an exchange and integration between human influenza, avian influenza and swine influenza viruses, in which the gene segments encoding HA, NP, NS were integrated from the swine influenza strain, NA, M from the Eurasian swine influenza virus strain, especially PA and PB2 are 2 genes with a third level of integration exchange (these 2 genes are the exchange of all 3 influenza virus strains from humans, poultry and pigs) [5, 27]. This influenza virus strain carries a genome that has an exchange and integration with the genome originating from the virus that causes disease in humans, so the new virus strain is fully capable of replicating in human respiratory epithelial cells and easily spreading in the human population over a large range. However, the surface proteins of

The new virus is completely different from previous influenza virus strains that have circulated in humans and the host is not protected by residual antibodies. The newly emerged H7N9 influenza virus also carries a highly integrated and exchanged genome, in which the HA gene segment is similar to the avian H7 gene, the NA gene segment is similar to the N9 gene of the A/H11N9 virus, and the remaining genes are similar to the A/H9N2 virus [63]. In addition, through this exchange and integration, the new influenza virus can carry drug resistance genes from other influenza virus strains, typically the M gene carrying the amantadine resistance mutation of the H1N1pdm09 virus and the NA gene segment carrying the oseltamivir resistance mutation of the H7N9 virus [30, 63]. Thus, the new virus strain could be the cause of a global pandemic, threatening the safety of countries and the health of people around the world [33, 34].

1.1.5. Pathogenicity of influenza virus

Influenza is an acute respiratory infection caused by influenza virus. The main transmission route of influenza virus is through small droplets of saliva carrying the virus when the patient comes into direct contact with healthy people. The average incubation period is 2 days, usually lasting from 1 to 4 days [43], after which the patient shows symptoms such as fever, fatigue, muscle pain mainly in the back and symptoms of runny nose, watery eyes... The patient may have no fever after 5 to 7 days with fatigue and cough, and recover after about 10 days if there are no complications of secondary infection [4].

Since the beginning of the 20th century, the world has recorded 4 influenza pandemics, starting in Spain in 1918-1919 caused by the A/H1N1 influenza virus, which is one of the influenza pandemics with a high mortality rate, with an estimated 40 million people dying from influenza infection [43]. The 1957-1958 Asian flu pandemic originated in Singapore caused by the A/H2N2 influenza virus with about 69,800 deaths. The 1968-1969 influenza pandemic in Hong Kong was caused by the A/H3N2 influenza virus [84]. The 2009 A/H1N1pdm09 influenza pandemic started in Mexico, then quickly spread to the United States, Canada, Europe and Asia. At the end of May 2009, Vietnam recorded

The first case of pandemic A/H1N1 influenza. The number of cases increased rapidly, by December 2009, more than ten thousand samples suspected of being infected with A/H1N1pdm09 influenza were sent to the Influenza Laboratory, Central Institute of Hygiene and Epidemiology [2]. The first avian influenza epidemic appeared in Hong Kong in 1997-2002, caused by the H5N1 subtype A influenza virus. The virus was transmitted from poultry to humans, killing 6/18 cases [95]. In 2003, avian influenza appeared in Vietnam, to date there have been 125 confirmed cases of A/H5N1 influenza, including 62 deaths [115]. In March 2013, the World Health Organization (WHO) received notice of the first cases of A/H7N9 influenza infection in China, the virus was determined to be transmitted from poultry to humans with a total of 133 infected people and 34 deaths [116].

Based on laboratory surveillance data since 2001 and the national influenza surveillance program in Vietnam (National Institute of Hygiene and Epidemiology in collaboration with US-CDC since 2006), influenza in Vietnam occurs year-round, with two distinct peaks in winter and spring with the circulation of influenza B virus (February - March) and summer with the circulation of strains belonging to influenza subtype A (July - August) [5].

1.1.6. Evolution of influenza A virus

The evolution of influenza viruses was first recognized as genetic evolution with small changes, large changes in genes. Small changes that occur frequently may not create antigenic changes but create diversity in the genotype of influenza virus strains. Large changes that affect antigenic properties, although occurring at low frequency, are important changes in the evolution of influenza viruses. A characteristic feature of influenza virus evolution is the exchange and integration of gene segments to create new influenza virus subtypes. The effects of this evolution are changes in antigenic properties, changes in hosts, increased virulence, increased transmission, drug resistance, etc.

The evolution of influenza A virus is confirmed to be adaptive evolution, occurring only sporadically in the influenza virus population, under the influence of a number of phenomena such as:

co-infection in the same host, or the emergence of a virus variant from elsewhere [83]. The two gene segments encoding the surface glycoproteins HA and NA are under great pressure from the neutralizing ability of antibodies produced during infection. The remaining gene segments, although not under selective pressure from the immune response, are thought to have undergone selection to adapt to each host species, typically the gene segment encoding PB2 [57].

The impact of humans on the evolution of influenza A viruses is not yet clearly assessed, but activities such as poultry use, vaccination, and the use of antiviral drugs are also likely to influence evolution. In addition, with the use of antiviral drugs, drug resistance has emerged, so the virus has begun to change to adapt to the environment, and these changes will be the next evolutionary steps of influenza viruses.

1.2. Prevention and treatment of influenza A virus

1.2.1. Flu vaccine

Influenza vaccines have been used since 1945 with the aim of reducing the incidence of influenza and the burden of disease caused by influenza viruses. The vaccines used are produced in embryonated chicken eggs with antigens including two A strains (one A/H3N2 strain and one A/H1N1pdm09 strain) and one influenza B strain, selected from more than 100 national influenza centers in more than 80 countries around the world. The selection of virus strains is carried out by WHO every February for countries in the Northern Hemisphere and every September for countries in the Southern Hemisphere [117]. To be effective in disease prevention, vaccines must be administered to the community every year, due to the rapid change of the virus in the HA and NA gene segments. However, in Vietnam, influenza vaccines have not been widely used in the entire population, so the effectiveness of disease prevention is still limited.

Types of vaccines

Inactivated vaccine

This vaccine is produced from virus strains grown in the allantois of embryonated chicken eggs, which are then inactivated by formaldehyde or β-propiolactone and purified by ultracentrifugation. This vaccine includes whole and partial vaccines. The whole vaccine is safe and well tolerated with a protective efficacy of 60-90% in adults and children. The partial vaccine consists of only two components, the HA and NA proteins, and other components of the virus have been removed. This vaccine causes few side effects, but it is thought that the vaccine is less effective in the presence of new virus strains [50].

Live attenuated vaccine

This vaccine is used to enhance the body's humoral and local immune responses (in the respiratory tract) to influenza viruses. Unlike inactivated vaccines, the components of live attenuated vaccines are virus strains adapted to temperatures lower than body temperature (25 o C) so that when introduced through the nose at body temperature, the viruses will be inactivated to stimulate reproduction.

antibodies in the upper and lower respiratory tracts and also enhances the cellular immune response. This vaccine was introduced in the United States in 2003 and in previous years in the former Soviet Union with a fairly high safety profile [50].

New generation vaccines

The production of vaccines containing influenza viruses cultured on MDCK (Madin-Darby Canine Kidney) cells or Vero cells, which can replace vaccines produced on embryonated chicken eggs, is of interest to vaccine manufacturers not only because of the potential for increased productivity but also because the side effects of this type of vaccine are expected to be lower than those currently used.

Vaccines produced based on the application of genetic technology (vaccines using reverse genetic technology, DNA vaccines, protein vaccines) are also being researched with the hope of being widely applied in the future.

Vaccines produced based on changes in the virus's genetic structure that reduce the ability to replicate (M2 and NS2 genes are removed) and lose the ability to synthesize cellular interferon inhibitors (NS1 gene is removed) are capable of inducing an immune response, not following the current vaccine production process [50].

1.2.2. Drugs for treating influenza A virus

Antiviral drugs inhibit entry into infected cells

Amantadine and rimantadine are derivatives of adamantane, used since the 1960s (Figure 1.6). The mechanism of action of these substances is to inhibit the M2 transmembrane ion exchange channel of influenza A virus, H + ions cannot enter the virus, the pH in the virus does not change, limiting the fusion process of the virus membrane with the host cell, preventing the virus from "undressing" to penetrate the cell.

host cells (Figure 1.6). Amatadine is effective against all previous influenza A subtypes that have infected humans (H1N1, H2N2, H3N2) but not against influenza B viruses because the M2 protein is only found in influenza A viruses [31]. Resistance often occurs with influenza A/H3N2 strains due to the appearance of mutations in the M gene (M2 part) leading to changes in amino acids at five positions 26, 27, 30, 31 and 34, changing the interaction site of amantadine with M2. In which, position 31 changes from Serine (Ser-S) to Arginine (Asn-N), which is a common position in virus strains in studies around the world [9, 42, 89].

Figure 1.6. Structure and mechanism of action of amantadine and rimantadine (Source: http://umanitoba.ca and Drugs discovery approaches – Wiley 2007)

Neuraminidase inhibitor antiviral drugs

Oseltamivir and zanamivir are two selective inhibitors of the surface enzyme neuraminidase of influenza virus (Figure 1.7). Neuraminidase cleaves sialic acid on the host cell surface from the HA glycoprotein of newly budding virus particles at specific α 2,3Gal-sialic or α 2,6 Gal-sialic bridges. Therefore, neuraminidase helps release virus particles, creating conditions for new virus particles to be able to penetrate other epithelial cells, enhancing virus dissemination. By inhibiting this process, the virus is only able to infect and multiply in infected cells but cannot spread to invade other healthy cells, preventing the virus from causing disease (Figure 1.8).

Neuraminidase is an enzyme on the surface of the virus that is quite conserved in influenza virus strains, scientists predict that the possibility of virus resistance to oseltamivir is lower than amantadine [21]. However, recent domestic and international studies have shown the emergence of virus strains resistant to oseltamivir mainly on the A/H1N1 subtype and especially an avian influenza virus strain.

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