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Influenza viruses, belongs to the family of Orthomyxoviridae, are negative-sense, single stranded, enveloped RNA viruses with a segmented genome.(3, 7) Among the 3 influenza types (Influenza A, B, C), influenza A has been responsible for all known major epidemics and pandemics.(3) Influenza A virus move in more than 18 mammalian species including pigs and humans but the main natural reserviour is acquatic wild fowl, including migratory birds.(2, 3) The influenza virus genome is segmented that encodes 11 proteins in their 8 separate gene segments.(2) Influenza A viruses are classified according to the subtypes of 2 surface proteins, haemagglutinin (HA or H) and neuraminidase (NA or N).(3, 8) Haemagglutinin is a surface glycoprotein which is necessary to bind a virus particles and entry of it to host cells, contain the primary epitope for protective neutralizing antibodies. Neuraminidase is essential for releasing of virus progeny from infected cells and plays a lesser role in protective immunity but may be able to mitigate the severity and duration of disease. Among the 16 known subtypes of HA, only 3 have successfully adapted to human circulation causing pandemics- H1 in 1918, H2 in 1957 and H3 in 1968. Another viral envelope protein M2 which is a transmembrane ion channel causing viral replication by acidification of viral interior.(7-8) Influenza RNA polymerase formed by polymerase basic (PB) protein 1, PB protein 1-F2, PB protein 2 and polymerase acidic protein, all are need for replication and transcription of viral RNAs.(9) Antigenic characteristics of influenza A virus are constantly changing by frequent mutation of these genes occurs as influenza RNA polymerase has no proofreading mechanisms like all RNA viruses. M1 protein, is formed by 3 polymerase proteins with neucleoprotein that helps to transport riboneucleoprotein complexes to the nucleus.(8) Non structural proteins 1 and 2, localized to the nucleus that binds to different RNA molecules and helps to mediate the expression of viral proteins and viral replications, respectively.
The HA protein on the viral envelope binds with the cell surface on target cells through the surface receptors sialic acid (SA). After cellular attachment, classical receptor mediated endocytosis in clathrin-coated vesicles facilitates to entry of virus particles. Then acidification of endosomes occurs that is essential and helps in the following steps of the replication cycle of the virus in two ways: a) conformational changes in the haemagglutinin causing fusion of the viral and cellular membranes which is favoured by low pH , as a result of virus can enter to cell cytoplasm b) uncoating of the virus and allowing the seperation of ribonucleoprotein complexes from the virus particles through the acidification of viral interior, so that they are released into the cytoplasm and transported to cell nucleus. The RNA polymerase complex in the nucleus of the infected cells facilitates the replication and transcription of the viral RNAs and transcribed RNA are exported in the cell cytoplasm by the protein NEP and M2, where viral RNAs are translated. The progeny virion is assembled and exit through the budding at the plasma membrane that has been previously modified with the viral membrane proteins H, NA and M2. For instance, influenza virus HA binds to cell surfaces sialic acid for entry, again binds with sialic acid for exit. But viral neuraminidase cleaves sialic acid from the membrane allowing viral escape. Following replication at the site of entry, virus can remain localized (eg.influenza virus in respiratory epithelium) and infects adjacent cells or may spread to subepithelial tissues to cause systemic infections.(10-11)
Fig- Replication cycles of Influenza A viruses. Virus enters the cell by endocytosis, ribonucleoptotein complexes are released into cytoplasm to enter nucleus where replication and transcription occurs. Messenger RNAs synthesis and released into cytoplasm to form early protein. In the late phase of infection cycles, M1 and NS2 help to exit ribonucleoprotein complexes. The progeny viruses are assembled and released through budding of plasma membrane. Source- Arias et al. Archives of Medical Research. 2009; 40: 643-654
Influenza A viruses uses different mechanisms constantly such as point mutation, recombination and reassortment for emergence of recurrent epidemics and pandemics.(10) The most important is the absence of proofreading activity of the viral RNA polymerase during replication of the influenza genomic segments which is responsible for high level of point mutation, leading to "antigenic drift". Seasonal epidemics and reduce the effectiveness of the previous seasonal vaccine are largely affected by antigenic drift.(11-12) The rate of mutation during replication of the influenza genome is about 1 nucleotide change for every copied genome. Moreover, the highest evolutionary rates occurs in proteins, such as haemagglutinin and neuraminidase which are target of neutralizing antibodies and for unknown reasons the M2 protein as well.(11) The mutation frequency in the H and N amino acid sequences during 1-year period is estimated to be less than 1% and it takes 2-5 years to achieve differences which can be detected in serological tests such as haemagglutination inhibition assay.(13) The permanent antigenic drift between HA and NA proteins necessitates constant surveillance to review the antigenicity of circulating strains that helps to prepare the effective yearly vaccine. However, the current diversity of HA and NA subtypes in influenza A viruses caused by antigenic drift that has been happening for thousands of years.(11) So, the main mechanisms of influenza virus evolution is antigenic variation through drift and shift of HA and NA proteins by which the virus can avoid host immune responses. "Antigenic shift" or "genomic reassortment" due to the segmented nature of its genome which allows the development of new progeny viruses having novel combination of segments when two or more different virus subtypes follows a new niche to enter.(10-11) By this process, a new protein introduce in circulating viral populations that can drastically change in biological properties of the virus. Antigenic shift is commonly associated with emergence of pandemic influenza viruses; such as the acquisition of new PB1, H and/or N proteins to which human population is not immune, played a major role in the 1957 and 1968 pandemics. The highly pathogenic "Spanish" influenza was an avian-like H1N1 virus and H1N1 viruses were dominant in human populations from 1918 till about 1956. In 1957, a new human pandemic H2N2 "Asian" strain was appeared through reassortment of H1N1 with avian influenza strains. In 1968, antigenic shift resulted in the appearance of a pandemic H3N2 "Hong kong" strain. It was reported, H1N1 strains reappeared beginning in 1976, when a novel strain causing an influenza outbreak among military personnel at Forx Dix, New Jersy. Human H3N2 and H1N1 strains have remained in co-circulation as seasonal influenza till today.(14) However, the emergence of current human pandemic H1N1 2009 strain spreading in the United States and around the world caused by a "quadruple reassortant" virus, originating from further reassortment of the previous swine triple-reassortant virus containing gene segments from human H3N2 influenza A strain, avian and swine lineages with gene segments of Eurasian swine.(3, 14-15) Recombination by template switching is another mechanism which is also associated with evolution of Influenza A virus. In this type of recombination, genetic materials may be come from more than one origin like ribosomal RNA and viral RNA or from two different viral segments and this non homologus recombination could be associated with changes in viral pathogenecity.(11)
Viral tropism and receptor specificity: Influenza A virus can infect a wide variety of animals. But the main natural reserviour for all influenza A subtypes is aquatic wildfowl with certain subtypes circulate in more than 18 mammalian species including pigs and humans.(3) The initial step of influenza virus cell infection is the attachment of viral HA with SA molecules on the host cell surface. SAs are nine carbon monosaccharides, bound to galactose via Î± 2,3 or Î± 2,6 linkages which act as a major determinant for the tropism of influenza viruses and the type of linkage given the high specificity of viral strains. Thus, human influenza viruses bind to receptors on epithelial cells of respiratory tract through SA Î± 2,6 linkages, whereas avian viruses prefer to bind to SA Î± 2,3configuration on epithelial cells within the intestinal tract of waterfowl. But pig epithelial cells of intestinal tract express both receptors and the virus can infect pigs through the recognition of both types of receptors, making this animal a "mixing vessels" for co-infection with influenza subtypes and generated a reassortant new strains.(2, 11,15) After attaching the virus with respiratory columnar epithelial cells, it prevents host cell protein synthesis that induces apoptotic death of the host cells. But virions are released before cell death and enter nearby healthy cells and, consequently, the pathological changes such as necrotizing bronchitis, intra-alveolar haemorrhage and oedema ensues.(2) These sites of epithelial damage might be aggravated by bacterial infection. Host immune response initiate after initial illness through pre-existing memory T cell activation.
Virulence and antigenic determinants: Most of the viral proteins play a significant role to determine the virulence of influenza A viruses, including the adaptation of infection and circulation into new host species, the ability to replicate properly and the capacity to modulate the host immune responses. The HA, PB1, PB2 and NS1 proteins are more pathogenic and two other proteins such as NA and M2 are associated with the development of antiviral resistance. It has been shown that the exchange of complete genomic segments or by single mutation can influence the virulence of influenza viruses directly. In addition, it is not necessary to have a direct correlation between virulence markers and virulence because highly virulent strains not always contain all identified virulence factors, on the other hand some low virulence strains might have some of them. So, it is very difficult to reach a conclusion, which mutation are more relevant virulence factors for mammals, especially in case of human infections. In spite of that, several available data suggest that proteolytic cleavage of multibasic sequences at the HA and the presence of a lysine at 627 position of PB2 seems to be universal determinants of viral pathogenesis.(11) Influenza virus enters the host cell through the haemagglutinin (HA) surface receptor that binds with target cell receptor SA. HA mediates fusion as well as the tissue tropism of influenza viruses to specific host. The amino acid in the active site of HA plays a critical role in receptor binding and specificity. For instance, H2 and H3 subtype of HA can recognize avian SA receptors due to the presence of glutamine and glycine at amino acid residues 226 and 228 in HA, whereas leucine and serine residues at the same position allows binding to human SA receptors (Î±2,6). Furthermore, for HA of the H1 subtype, glutamic acid and glycine residues at position 190 and 225 are responsible for the interaction with avian SA receptors and the presence of aspartic acid at the same position provide binding specificity to human SA receptors. So, the ability of influenza A (H1N1) 2009 strains infect human host can be explained by the presence of aspartic acid at amino acid residues 190 and 225. Therefore, haemagglutinin also helps to mediate the fusion of viral and cellular attachment and it is the main target of neutralizing antibodies. HA is a trimeric rod shaped protein with a globular head and a transmembrane domain that inserted into the viral membrane. The globular head form a spike by three identical domains, projecting away from the viral surfaces, assuming it has at least five antigenic sites to surround the receptor binding site. The post translational proteolytic cleavage of HA protein into HA1 and HA2 domain is essential for viral replication as low pH of endocytic vesicles causes conformational changes of HA, exposing a hydrophobic fusion peptide located at the amino terminus of the HA2 domain. This fusion peptide is necessary to exit the viral ribonucleoprotein complex into the cytoplasm through the fusion of viral and host cell membrane. So, the HA protein and its cleavage is a critical determinants of virulence and pathogenesis of influenza virus. Highly virulent strains of avian influenza (H5 and H7) causing systemic spread of the virus due to the presence of multiple basic amino acid residues at the cleavage site of HA protein that are recognized by ubiquitous proteases, but low virulent strain possess only one arginine residue at the cleavage site that are cleaved by tissue specific proteases resulting infections are localized and mild. For instance, the influenza A (H1N1) 2009 strains that cause mild localized infection due to the presence of only one arginine at the HA cleavage site.(16,17) PB1- F2 is a small protein encodes the PB1 polymerase gene that act as an apoptosis inducing factor. In a mouse model, it contributes the pathogenecity of the viral strains by inducing apoptosis but this protein is not expressed in all influenza strains. PB1-F2 also exacerbated the frequency and severity of secondary bacterial infections by its pro-inflammatory responses during primary viral infection in mice. But the mechanism is still unknown. It has been shown that various mutations in PB2 like amino acid substitution and host body temperature as well have been associated with diverse virulence strains including transmission and host range specificity. The substitution of glutamic acid at amino acid residue 627 that is present in most avian viruses, converts an H5N1 virus from non pathogenic to pathogenic in mice. The temperature at human upper respiratory tract is 330c which is favourable for those viruses that have lysine at position 627 compared with 410c found in birds. So, lysine at position 627 is now identified as a determinant of viral pathogenecity in several mammalian species.(11, 17) Other mutation in PB2 like Asp70Asn enhances binding of this protein to cellular nuclear import factor Î±1 in mouse cells that can explain the better replication in mammalian cells. So, it can be concluded, the molecular markers 627Lys and 701Asn in PB2 are responsible for efficient transmission among human. It has been reported, the current pandemic virus influenza A (H1N1) 2009 having glutamic acid and aspartic acid at position 627 and 701, respectively.(16,17) The non structural protein NS1 of influenza A virus which is expressed at very high levels in infected cells, although it is not a viral particles. It has a ability to bind with double-stranded RNA (ds RNA). So, this protein can modulate the viral pathogenecity by preventing the activation of PKR which is a dsRNA - dependent protein kinase and also inhibit the cellular interferon response by preventing the activation of interferon-B stimulated gene products. The exact mechanism of action of this protein is still unknown. So, the virulence and pathogenecity are also associated with mutation of NS1 protein and mutant strain can modify the host responses. For instances, the NS1 protein of highly pathogenic H5N1 strain and the 1918 H1N1 pandemic strain contain a four residue motif at the carboxy-terminal end of the protein that forms a potential PDZ domain which is a protein-protein recognition module, present in many cellular proteins that stimulate a different cell signalling cascades associated with high virulence. But PDZ binding domain is absent in low virulent strain. Interestingly, the influenza A (H1N1) 2009 strains have amino acid at position 92 and does not have PDZ domain due to the deletion of 11 carboxy-terminal amino acid. On the other hand, the presence of glutamic acid rather than aspartic acid at position 92 correlates with high pathogenecity in avian strain (H5N1).(11) The integral membrane protein M2 is the main target for antiviral drugs of adamantanes class. Resistance to this class of antiviral drugs has been increasing throughout the world; nowadays most seasonal H1N1 and H3N2 strains of human and swine origin including the influenza A (H1N1) 2009 strain showing resistance to this antiviral drugs due to mutation in M2 such as single amino acid substitution at position 26,27,30,31 and 34.(11) However, some animal study shows that few protective antibodies generated after influenza infection against M2.(7) Another class of antiviral such as NA inhibitor target NA. Although most strain of influenza A virus show sensitive to this drug but some reports reflects the emergence of NA inhibitor resistance influenza A (H1N1) 2009 in Japan, Denmark, Holland and the United States.(11,14) In 1978-79 season the Influenza A virus subtype H1N1 was isolated. Three strains - A (H1N1), A (H2N2), A (H3N2) was pandemic in 1918, 1957 and 1968, respectively in this century. These strains were originated by genetic reassortment which contained gene segments from human, avian and swine lineages.(18) As many as 50 million people were killed globally by the 1918 pandemic and approximately half of all influenza related fatalities occurred in young adults in the age group between 20-40 years.(3) In 197/78 the virus reappeared and current human seasonal H1 protein is from the same lineages. These viruses contained HA, NA and PB1 of human origin; NP, M and NS genes of classical swine and PB2 and PA genes which was North American avian virus origin. Epidemiological data show that the current outbreak of influenza-like respiratory illness commenced in Mexico, in February 2009.(12) 41 to 84 million 2009 cases were identified as of January 16, 2010 in the United States since April 2009, with an estimation of 8,330 to 17,160 death. However, most of the infections were in mild severity and did not require to be hospitalised, only small fraction of the confirmed cases had been hospitalised who were known to have underlying medical conditions such as cardiac diseases, asthma, diabetes, pregnancy and immunosuppressive therapy. According to the most recent updates by WHO, total of 2,67,105 reported cases of swine influenza were confirmed affecting 175 countries, with a total of 2,692 casualties.(15) In Australia, around 38,000 laboratory confirmed cases of H1N1 2009 influenza were identified with 13% requiring hospitalization and 14% of these needing intensive care support. In Australia, 191 deaths were attributed to H1N1 2009 influenza.(19) Even though seasonal influenza is typically a disease affecting the extreme of age; 90% of the approximately 36,000 influenza caused deaths in the United States each year occur in individuals who are 65 years and older, most of the children and younger adults were affected by the 2009 influenza pandemic and was significantly milder in comparison to the 1918 pandemic. The morbidity and mortality caused by the 2009 H1N1 pandemic have been modest globally in comparison to many seasonal influenza years. More than 90% cases and 87% casualties related to the 2009 H1N1 pandemic was found to be in the population younger than 65 years and one study revealed the median age of the patients died was 26 years.(3) Potential explanations for the increased frequency of disease in the young people compared to older people (in contrast to seasonal influenza) may be attributed to the presence of pre-existing cross protective antibodies against the current pandemic H1N1 2009 strain which can be found in individuals over the age of 59 years. This probably reflects exposure to related H1N1 strain circulating in the 1950s earlier in life.(14, 20) The 2009 pandemic influenza virus spread very fast from individual to individual worldwide. It started spreading early in the southern hemispheres during the winter flu season (May to Sep) making it predominant circulating influenza virus in most of the southern hemispheric locations by mid season.(3) All up, we would mention, the H1N1/2009 is significantly milder than seasonal flu or the 1918 pandemic but some of the characteristics made it virulent in the early phase of the disease of pandemic like quick transmission from human to human that spread internationally with exceptional momentum, lack of pre-existing immunity against the 2009 variants and setback in vaccine availability. The pandemic H1N1/09 influenza virus is presently the dominant strain in the majority of areas of the world. The bulk of 2009 H1N1 patients still suffers from mild symptoms and recover completely within 1 week. However, the H1N1/09 pandemic still remains one of the questions and concern; it possibly will persist in the future influenza seasons and substitute the currently circulating seasonal influenza A virus. On top of experiencing antigenic shift, the pandemic H1N1/09 may possibly further adapt to humans by the means of point mutation in a virus instigating more severe diseases. The present H1N1/09 pandemic strongly implies the necessity to have effective preparedness strategies and co-ordinated activities at a global level, like, upgrading the influenza vaccine development along with yearly review and distribution, use of precise personal protective equipment, social distancing intervention to halt transmission and timely and early access to effective antiviral therapy. Furthermore, continuing research needs to be carried out to ensure the control of both swine and avian influenza virus infection.