Influenza A is a highly contagious virus, which can cause dramatic illness, periodic pandemics and outbreaks globally every year. The major source of these onsets per annum is the emergence of an antigenically novel virus which the human population lack protective immunity against. Meanwhile, animals are a source of panzootics which largely contribute to influenza pandemics. Therefore, it is critical to comprehend the epidemiological and molecular mechanisms by which influenza A viruses acquire the capability to cross species barriers.
I. An Introduction to Established and Emerging Zoonotic Diseases
In general, the transfer of viruses to new hosts requires contact between a host and virus and amplification initiated by the infection of an individual which may lead to an outbreak. However, the production of variable viral features with the capacity to effectively extend throughout the new host population is critical. Infections from animal reservoirs are a major source of emerging diseases in humans including Ebola fever, Severe Acute Respiratory Syndrome (SARS) and Nipah virus. It has been estimated that the 13 zoonoses causing the greatest human morbidity and mortality result in the deaths of 2.2 million people every year (Grace et al., 2012).
II. The Relentless Nature of Influenza A Viruses
Influenza pandemics occurred in 1918 (‘Spanish flu’, H1N1), 1957 (‘Asian flu’, H2N2), 1968 (‘Hong Kong flu’, H3N2) (Landolt and Olsen, 2007) and 2009 (reassorted H1N1 virus, ‘swine flu’). The 1918 ‘Spanish flu’ pandemic killed an estimated 40’50 million people worldwide (Landolt and Olsen, 2007) which highlights its devastating effects.
Pandemic Year Virus Strain Approximate No. Of People Infected Estimated No. Of Deaths Worldwide Case Fatality Rate
Spanish Flu 1918 (-1919) A/H1N1 500 million (approximately one third of world population) 50-100 million (3-5% of the world’s population) >2.5%
Asian Flu 1957 (-1958) A/H2N2 Unknown 2 million <0.1% Hong Kong Flu 1968 (-1969) A/H3N2 Unknown 1 million <0.1% Swine Flu 2009 (-2010) H1N1/09 (previous triple re-assortment of bird, swine and human flu viruses combined with Eurasian pig flu virus) Estimated between 43 million and 89 million (> 622,482 lab-confirmed) 14,286 (ECDC)
18,036 (WHO) 0.03%
H5N1 Pandemic Threat 2003- A/H5N1 638 379 59% (some studies show lower values of 14-33%, does not equate to pandemic statistics)
H7N9 Pandemic Threat 2013- A/H7N9 419 (as of April 2014) Unofficially 127 (as of April 2014) 30% (does not equate to pandemic statistics)
Seasonal Flu Every Year Majorly A/H3N2, A/H1N1, B 5’15% (340 million ‘ 1 billion) 250,000’500,000/year <0.1% Table 1. A display of statistics of notable influenza pandemics and threats (adapted from information provided by the WHO, WHO Human Animal Interface, WPRO, CDC, ECDC). The incidence of influenza A virus, IAV, infection in the human population varies significantly from year to year. It can be dependent on the attack rate, virulence of the circulating strain, and on the degree of immunity of individuals in the population (Alexander and Brown, 2000). Regardless, the impact of yearly human influenza epidemics is substantial. In the United States alone the result is on average 114,000 hospitalizations, 36,000 deaths and up to $10 billion in lost income and medical bills (Landolt and Olsen, 2007). Progress has been made concerning the formation of a stable preventative medication but due to the high mutability of the virus it is extremely difficult. Therefore the topic is of major importance and under constant scrutiny. The urgency of its understanding is not only due to the possible cost of human lives but also the threat to livestock. III. The Composition of Influenza A Viruses Influenza A viruses are of the Orthomyxoviridae genera and are all enveloped, negative-sense, single-stranded, segmented RNA viruses. Within the lipid envelope of influenza A is an M2 protein, functioning as an ion channel, NA and HA molecules. The HA, haemagglutinin, serves as the viral receptor-binding protein and mediates fusion of the virus envelope and host cell endosomal membrane (Skehel and Wiley, 2000). It is a trimer composed of a globular head, containing the receptor binding site, and a stalk. The NA, neuraminidase, is a type II integral membrane protein responsible for the cleavage of the a-ketosidic linkage between sialic acid molecules and adjacent sugar residues (Landolt and Olsen, 2007). NA is composed of an enzymatically active head domain and a stalk region which inserts into the viral envelope and aids the release of budding virus particles to further infection within the host (Lamb and Krug, 2006). It is also involved in viral invasion of the respiratory tract promoting cleavage of the a-ketosidic linkage between the terminal SA and adjacent sugar residue in mucus. Both the HA and NA have antigenic determinants and undergo antigenic variation under immune pressure. They can mutate by antigenic shift, a sudden dramatic replacement of the predominant strain with a novel HA leading to pandemics, or drift, a gradual adjustment of amino acids to bring about a transformation. To date 18 antigenic variants of HA and 11 NA variants have been discovered, including bat viruses, which may combine in any arrangement resulting in the formation of differing subtypes. H1 to H4, H6 and H8 to H16 are all low-pathogenic avian influenza, LPAI, viruses while a number of H5 and H7 are high pathogenic, HPAI, viruses. Viruses of 16 haemagglutinin and nine neuraminidase subtypes have been discovered in wild seabirds and waterfowl. Hence, waterfowl provide a vast global reservoir of influenza viruses in nature from which novel viruses can emerge to infect mammalian species. The H5 and H7 strains undergo adaptation to become highly pathogenic. In the wild the virus may be LPAI but due to the time propagated within domestic species they can be released and establish themselves as highly pathogenic in wild waterfowl again (Landolt and Olsen, 2007). Unlike LPAI viruses, causing mild respiratory disease and infection, HPAI viruses spread systemically often causing rapid death. Formerly the majority of HPAI outbreaks have been caused by a single lineage previously eliminated from the domestic bird population. However, the Asian H5N1 outbreak appeared to follow a different pattern as multiple reassortant viruses were detected in poultry (Guan et al., 2003). The presence of herd immunity, from previous exposure, drives emergence of new antigenic variants which are then able to infect the population again maintaining the virus in circulation. Influenza genetic diversification is thought to be driven by the immune system in humans. Decreasing protection conferred by influenza specific immunoglobulins and increasing genetic divergence of HA is usually accompanied by decreasing cross-protection as the antigenic divergence between the two strains increases (Landolt and Olsen, 2007). A viral strain is a virus type having identical physiological characteristics. Phylogenetic analyses indicate that genetic evolution of human influenza viruses follows a multi-strain population dynamic with 95% of strains remaining in the population for less than one year. An estimated 1% of influenza virus strains become established, able to infect and replicate, in the human population globally (Landolt and Olsen, 2007). Studies by Wolf et al 2006 have indicated that human influenza virus evolution might not be linear as anticipated but undergo rapid fitness change periods whereby new dominant strains replace old lineages. These are interspersed by stasis periods with neutral sequence substitutions. The susceptibility of new hosts is difficult to predict. Recent transfers of H3N8 equine influenza virus to dogs and of avian H5N1 to cats demonstrated transfers may occur between hosts in different vertebrate orders and classes. Viruses of avian origin have been shown to spread to mink, whales, horses, seals and pigs. Human infection with H5N1 influenza viruses most often occurs after the infection of poultry on farms or in live bird markets, allowing viruses of wild birds to gain access to human populations. In 1979-1980 numerous seals died and were found to have a H7N7 subtype influenza virus residing in their lungs and brain. H3N3 and H4N6 have also been isolated in seals (Landolt and Olsen, 2007). Viruses of H13N2, H13N9, and H1N3 subtypes have been detected in the lungs of whales (Landolt and Olsen, 2007). Meanwhile, avian origin H10N4 viruses, causing systemic infection and disease, were isolated from farm-raised mink (Landolt and Olsen, 2007). H5N1 subtypes have been discovered in felines and influenza viruses were detected in a number of greyhounds dying of pneumonia in 2004 closely related to the H3N8 subtype (Landolt and Olsen, 2007). The first clinical recognition of swine influenza was in 1918. After further reassortment this virus caused the 2009 pandemic. The occurrence of pandemics is most commonly accepted to initiate with a virus of a novel subtype being introduced into a human population from an avian reservoir with or without genetic reassortment. Phylogenetic studies have confirmed this showing that the 1918 'Spanish flu' was caused by a virus of entirely avian origin while reassortment of human and avian influenza viruses caused the pandemic strains of 1957 and 1968. Viruses transmitted to new hosts are often assumed to be poorly adapted, replicate insufficiently, and are inefficiently transferred. They need to adapt so require a greater rate of variation. Hence, emerging zoonoses tend to be rapidly evolving RNA viruses. These often partake in cross-species transmission due to their high diversity arising from error-prone replication, lack of proofreading mechanisms, rapid replication, short virus generation times, and large virus populations. In contrast, most DNA viruses are less variable and more often associated with virus-host cospeciation (Parrish et al, 2008). A) 1977 1915 1957 1968 2009 B) 1950 1960 1970 1980 1990 2000 2007 2015 Figure 1. (A) Known human influenza A pandemics. (B) Animal epidemic and pandemic strains, outbreaks, and human transfers in the past 60 years (adapted from Parrish et al. 2008). IV. Transfer 0f Influenza A Viruses Predominantly, transmission of viruses between differing species is dependent on epidemiological factors as well as host and viral factors being impeded by a requirement for multiple, complex adaptive virus changes. Donor and recipient contact is a precondition affected by geographical, ecological, and behavioural separation. Factors influencing geographical distribution of host species, such as wildlife trade and introduction of domestic species, or decreasing behavioural separation, bush meat hunting, tend to promote viral emergence. Human population expansion and travel, developing immunosuppression such as in HIV patients, intravenous drug use, sexual practices and contacts, and farming practices, deforestation and agricultural expansion promote transfer (Parrish et al, 2008). Influenza virus may be transmitted among humans in three ways: by direct contact with infected individuals; by contact with fomites and by inhalation of virus-laden aerosols. (Parrish et al, 2008). Direct contact with infectious waterfowl or poultry can transmit influenza A viruses to domesticated birds. Viral replication occurs in avian intestinal tracts preferentially, being shed in high concentrations in the faeces and contaminating the lakes, ponds, fields or nests that birds visit increasing their transmission (Landolt and Olsen, 2007). Infected birds also shed the virus in their nasal secretions and saliva. Food and water can become infected so if healthy birds eat with infected individuals or carriers they may contract the virus also. Further fomites include furniture, soap or clothes though virus transmission is dependent on the nature of the contaminated clothing (Ikeda et al., 2015). With heavy environmental contamination influenza A viruses generally survive for less than 8-12 hr on cloth, paper, and tissues but can persist for 24-48 hr on hard, nonporous surfaces such as stainless steel (Bean et al., 1982). There is evidence of A/H5N1 virus transmitting to humans from cleaning or plucking infected birds. There is also speculation that some people have become infected after swimming in water contaminated by infected bird faeces. Some infections have occurred in people who handle fighting cocks too. Influenza A viruses infect a wide variety of species but a number display only partial restriction of their host range meaning many viruses are rarely detected anywhere other than their natural host but some transmit amongst other species (Landolt and Olsen, 2007). V. Immune Factors Determining Virulence Replication potential, host density or immune statuses of hosts may be restricted at different levels including receptor binding, entry or fusion, trafficking within the cell, genome replication, and gene expression. These restrictions may be by accident, aspects the virus is incapable of coping with, or design whereby the cell is set up to prevent viral ability. The production and shedding of infectious virus can also be host specific. Innate antiviral responses (interferon- and cytokine-induced responses), and intrinsic mechanisms also counteract viral infection and produce host range restrictions. Some inhibitors include apolipoprotein B-editing catalytic polypeptide (APOBEC) proteins, restricting retrovirus activity specifically, and tripartite motif (TRIM5??) protein. Concerning influenza, three interferon induced transmembrane proteins, IFITM1, 2 and 3, possess antiviral qualities limiting IAV infection in cultured cells. Murine IFITM3 controls influenza virus in vivo and human IFITM3 polymorphisms correlate with seasonal and highly pathogenic IAV severity (Bailey et al, 2014). Interferon-induced Mx proteins also possess a strong antiviral activity against the viruses. Avian strains are sensitive to MxA and murine Mx1. Human strains have a reduced susceptibility to MxA, a resistance facilitated by changes in the nucleoprotein, NP. A combination of isoleucine-100, proline-283, and tyrosine-313 mutations is essential for reduced Mx sensitivity in cell culture and in vivo. The human IAV resistance is due to the nucleoprotein, the Mx functional target, containing particular amino acids. The first example of an avian IAV naturally less sensitive to Mx-mediated inhibition is the H7N9 virus with an NP that overcomes the human MxA restrictions. This may clarify why H7N9 viruses transmitted efficiently to humans (Riegger et al, 2015). Mammalian defences include host factors that bind to virion components to prevent infection. Glycans or lectins bind and eliminate incoming viruses. For example, in porcine plasma, sialylated ??-2-macroglobulins can bind human influenza viruses. Viruses lacking efficient esterase or neuraminidase activity for new host glycans can be bound and inactivated. Galactosyl(??1-3)galactose is a glycan not found in humans but present on some intestinal bacteria. Hence, it enlists a response from antibodies intrinsic to the human body. Virions produced in hosts which have galactosyl(??1-3)galactose-modified proteins are rapidly recognized and inactivated by antibodies when they enter humans, preventing infection (Parrish et al, 2008). A variant virus, H3N2v, was discovered in swine and fears arose it may combine with H1N1 to produce a novel reassortant and cause a pandemic. The reassorted virus was produced and although it successfully infected swine and ferrets it was attenuated in human cells. This is a feature due to IFN-beta and IFN-lambda gene expression in human cells which is not evident in swine (Powell et al, 2014). VI. The alpha-2,3-Gal, alpha-2,6-Gal Dilemma Both HA and NA molecules of IAVs act on cell sialic acid receptors. Complementarity between virus binding, HA, and cleavage, NA, activities is necessary for optimal binding to and discharge from cells expressing different glycan receptors (Parrish et al, 2008). Two features determine the binding of influenza virus to its cellular receptor; the SA species, N-acetylneuraminic (NeuAc) or N-glycolylneuraminic (NeuGc) acid, and the NeuAc or NeuGc linkage to galactose residues (e.g. ??2,6Gal or ??2,3Gal) on the cell membrane. Avian viruses prefer binding to ??2,3Gal linked SA in NeuGc or NeuAc forms while human lineage viruses tend to bind to NeuAca2,6Gal-linked receptors (Landolt and Olsen, 2007). Avian intestinal cells principally express ??2,3-linked receptors. Meanwhile, ??2,6-linked receptors are primarily expressed by human tracheal epithelial cells (Landolt and Olsen, 2007). Avian influenza viruses with a preference for ??2,3'SA attach to cells of the lower respiratory tract, type II pneumocytes and non'ciliated cells of birds. Comparatively, viruses transmitting between mammals with a preference for ??2,6'SA were proven to bind primarily to ciliated cells of the upper respiratory tract (van Riel et al, 2013). However, Wan and Perez, 2006, discovered that Japanese quail express both ??2,3 and ??2,6 forms in their trachea. In humans' ??2,3Gal-linked SA are also expressed by a small subset of human tracheal respiratory epithelial cells. Hence, the viruses with the avian receptor can establish themselves and infect the host. Another difference occurs when the methyl group of the N-acetyl chain is inserted into the hydrophobic pocket of the virus-binding site. Rotation around the glycosidic bond allows the galactose molecule to adopt either a cis or a trans position and integrate different haemagglutinin molecules. Avian HA molecules are commonly bound in a trans conformation, while human receptors tend to be cis. Accordingly, glycans possessing ??2,6-linked sialic acid have greater conformational flexibility producing umbrella-like shapes glycans while those containing ??2,3-linked sialic acid have impeded conformational freedom and form a cone-like glycan structure. There have been suggestions that inefficient viral attachment to respiratory tissues, low levels of replication in these tissues and poor virus release and aerosolization of virus particles may explain why influenza viruses with ??2,3'SA preference are unsuccessfully transmitted via the airborne route between mammals (Sorrell et al, 2011). As avian viruses do not replicate efficiently in human hosts just as human viruses do not replicate well in birds an important intermediate host is indicated. Both receptor types reside in the porcine respiratory tract. The virus must adapt to bind a particular type preferentially so the adaptive change could occur within the pig. The avian influenza virus could infect the pig binding SA??2,3GaI receptors but eventually adapt to preferentially bind NeuAca2,6Gal receptors and move between birds and humans. Another possibility is that pigs can act as 'mixing vessels' becoming infected by both avian and human influenza strains and encouraging genetic reassortment as RNA segments are exchanged at replication and novel viral variants are produced. This has occurred in pigs in Europe between human-like H3N2 and avian-like H1N1 viruses (Landolt and Olsen, 2007). Reassortant H3N2 and H1N2 viruses have also been isolated from humans in Europe and Hong Kong (Landolt and Olsen, 2007). Further evidence for this 'mixing vessel' theory is provided by 2-way, human/swine, and 3-way, avian/human/swine, reassortant viruses of H3N2, H1N2, H1N1, and H3N1 subtypes that have emerged in pigs since 1998 (Landolt and Olsen, 2007). In Italy studies were performed on workers in close contact with turkey populations where H7N3 viral strains were circulated. They indicated that zoonotic transmission had occurred. However, it is known that human and swine lineages are unable to effectively replicate in avian hosts suggesting pigs are the likely intermediate host (Landolt and Olsen, 2007). Direct swine-human infections occur and have been noted in N. America and Asia. Concurrently, H5N1 and H9N2 viruses isolated from land-based poultry were found to have lower affinity for SA??2,3Gal than those acquired from aquatic birds (Landolt and Olsen, 2007). Therefore poultry could be another likely intermediate host supporting adaptation from 2,3 to 2,6. VII. Further Mutational Adaptations of Influenza A Viruses NS1 protein has been shown to have various effects in infected cells, including regulation of the interferon-induced signalling and effector mechanisms. This is evident in certain NS1 variants of avian H5N1 influenza viruses displaying an enhanced virulence for pigs (Parrish et al, 2008). Meanwhile, when individual components of the eight RNA segments of the influenza genome were reassorted into the background of a virus from an alternative host, the viral replication rate was majorly reduced. Fitness trade-offs are also prominent. Mutations assisting replication and establishment in a new host likely reduce viral viability in donor species and partially adapted viruses quickly go extinct. PA, PB1, and PB2, the replication proteins of influenza A virus, act as a heterotrimeric complex. Altering their combinations through reassortment of genomic segments can reduce replication efficiency. It may also produce a need for subsequent adaptation to the combinations of proteins from different strains and animal sources. Evidence exists indicating that all eight gene segments of the influenza virus play a role in the host range. However, examination of each individual proteins contribution has a number of complications. Firstly, the mutations may not only appear in one but multiple gene segments during adaptation to a new host. Consequent sequence analyses of six human H5N1 isolates have revealed that the virus has acquired a variety of amino acid substitutions. These affect not only the HA, but also the internal proteins, PB2, PB1, PA, NP, M, and NS (Landolt and Olsen, 2007). Specific constellations of gene segments may also be involved in controlling influenza virus species specificity with HA being identified as a key player in determination of specificity. The N linked oligosaccharides may also determine species specificity. A glycosylation site at position 63, commonly found in H3 viruses of human origin, is absent in avian-lineage H3 viruses illustrating their importance (Landolt and Olsen, 2007). Competitive inhibitors found in many species serums can restrict host ranges. A2-macroglobulin molecules in guinea pig and horse serum inhibit infection by human H3 viruses with SAa2,6Gal receptor specificity, but do not affect equine and avian H3 virus infection (Landolt and Olsen, 2007). Efficient growth of the influenza virus is dependent on balanced action between the HA receptor binding affinity and the NA receptor-destroying activity (Landolt and Olsen, 2007). Hence, NA's substrate specificity and cleavage ability often change to aid HA's alterations. For example, following the introduction of the 1957 pandemic strain into the human population, the a2,6Gal cleavage activity of the avian NA increased (Landolt and Olsen, 2007). This indicates that the neuraminidase was altered to match the HA's a2,6 preference. NA stalk length and amino acid sequence vary amongst viruses. NA's with shorter stalks are released less efficiently as the enzymatic domain in the head cannot reach the substrate effectively. Conversely, shortening of the NA stalk is associated with the adaptation of duck viruses to land based poultry (Landolt and Olsen, 2007). pH also appears to have an effect on viral efficiency as a lowering of the optimal fusion pH for avian H5 HA viruses culminates in enhanced replication and more efficient contact transmission in ferret upper respiratory tracts (Shelton et al, 2013). Aside from a switch in receptor specificity to facilitate infection of cells in the upper respiratory tract, increased virus production in the URT and efficient release of virus particles to yield airborne viruses may also be required (Herfst et al, 2012). The polymerase complex is necessary to consider as it has been hypothesised that the temperature at which replication occurs is important in selecting the host tropism of the virus (Landolt and Olsen, 2007). Avian viruses primarily replicate at temperatures around 41??C, the temperature in their intestinal tract. However, for replication in humans the viruses need to adapt to function at 33??C, the temperature of the human URT (Herfst et al, 2012). The PB2 gene of the polymerase has achieved the most evidence to link it to a role in determining species specificity. Clements and co-workers demonstrated that a human-avian reassortment could replicate within birds containing only an avian PB2 gene but not replicate efficiently in mammals. This is believed to depend on a single amino acid transition at position 627. It is also position 627 of PB2 which maintains the cold sensitivity of the avian viruses in mammalian cells encouraging the temperature dependence theory. Avian PB2 is composed of glutamate at position 627 while human viruses contain lysine. Lysine enhances pathogenicity, viral replication and severity of H5N1 and H7N7 in mammalian hosts, however, glutamate does not. An H7N7 HPAI virus isolated from a patient in The Netherlands contained a Lys substitution at position 627 (Landolt and Olsen, 2007) as did several of the H5N1 strains isolated from humans in Asia (Landolt and Olsen, 2007). Hatta and co-workers further indicated that the same Gly-to-Lys substitution at position 627 in the PB2 protein influenced pathogenicity in highly pathogenic H5N1 virus in 1997. Apart from temperature sensitivity, it is possible that amino acid substitutions in the polymerase proteins influence host range by altering interactions of the polymerase complex with host cell factors. The nucleoprotein itself selectively regulates NA expression and glycoprotein functional balance (Brooke et al, 2014). During virus replication, the influenza NP is modified by host-cell-derived phosphokinases (Lamb and Krug, 2006). The phosphorylation pattern of the NP protein appears to determine the extent to which a particular cell line supports virus growth demonstrating its relevance. Both the M and NP segments were associated with reduced replication of human-avian reassortants in the respiratory tract of squirrel monkeys (Landolt and Olsen, 2007). With the importance displayed through numerous experiments it is conceivable that the quality of these protein-to-protein interactions may have an impact on influenza species specificity. However, to what extent such protein'protein interactions determine host range has yet to be determined and more than changes in the polymerase complex are required to traverse species barriers. Influenza viruses could utilise a host of methods to overcome these difficulties. Intermediate and amplifier hosts may play a critical role as they unite animal viruses, often having had little contact with alternative hosts, into close contact with recipient hosts. This is true of many diseases. For example, the fruit bat resides in orchards and transfers Nipah virus to pigs which may then transmit to farmers. After transmitting to a new host recombination and reassortment are important for incremental host adaptation. This is evident as in 1968 a human H3N2 virus emerged containing HA and PB1 gene segments imported from avian viruses into the backbone of 1918-descended H1N1 viruses. This secondary reassortment aided further survival and transmission. Other examples include the H2N2 pandemic in 1957 and the H3N2 influenza strain in 2003 displaying interclade reassortment. Certain individuals within populations may play a greater role in the emergence and transmission of infection possibly due to several factors, for example some secrete larger quantities of virus and infect larger ranges of secondary contacts (superspreading) (Parrish et al, 2008). As of yet there is no evidence available to support a genetic susceptibility towards H5N1 virus but proof exists to reinforce a theory of H1N1 susceptibility to pneumonia in various mammals (Janke BH, 2014; Kwon et al., 2014; Pigott et al., 2014). As mentioned earlier it has been postulated that intermediate hosts are key to effective viral spread and infection between hosts. It has been proposed that pigs support two processes that support pandemic potential, adaptation and genetic reassortment. Virtually all HA subtypes, H1-13, can successfully replicate within the pig suggesting it may be the intermediate in which viral adaptation occurs (Landolt and Olsen, 2007). In 1979 a wholly avian H1N1 influenza virus crossed the species barrier to infect pigs in Europe (Landolt and Olsen, 2007). Avian viruses H1N1, H3N2, H5N1, and H9N2 have transmitted to pigs in China and Hong Kong (Landolt and Olsen, 2007) as have H4N6, H1N1, and H3N3 viruses in Canada (Landolt and Olsen, 2007). Swine IAVs include the subtypes H1N1, H1N2 and H3N2 with H1N1 and H3N2 having been present in humans also. One swine IAV has been detected possessing all eight gene segments of the human seasonal H3N2 virus. Often only IAVs transmitting in swine for over a year are considered successfully adapted. Rates of nucleotide substitution for the H1, H3, and N2 segments were consistently lower in human seasonal viruses than in closely related viruses in swine (Nelson et al, 2014). Avian influenza viruses have not yet acquired the ability to pass between humans by aerosol or droplet transmission but studies have proven that ferrets are capable of hosting both human and avian A/H5N1 viruses and have the ability to transmit human influenza viruses within their species. It had been previously demonstrated that several amino acid substitutions in the receptor binding sites of the HA surface glycoprotein of A/Indonesia/5/2005 change the binding preference from the avian ??-2,3'linked SA receptors to the human ??-2,6'linked SA receptors. After subsequent mutations the A/H5N1 virus became airborne and transmissible between the ferrets. The mutations were consistently an identical set of four amino acid substitutions in the host receptor-binding protein haemagglutinin, and one in the polymerase complex protein basic polymerase 2, PB2. The essential changes required to alter the virus from an ??2-3'linked sialic acid specificity ??2-6'linked sialic acid are two receptor-binding amino acid substitutions, Q222L and G224S in HA. The others include T156A in HA, which interrupts the N-linked glycosylation sequon; H103Y in the HA trimer-interface; and E627K in the PB2 (Herfst et al, 2012). Further to these experiments it has been shown that the A/H7N1 virus has a greater transmission between cohoused ferrets than mammals of only airborne contact as consequent infection only appears after approximately 6 days (Sutton et al, 2014). However, it has been discovered from surveillance data that two of the substitutions are common in A/H5N1 viruses, hence, some viruses may only require an additional three substitutions to become transmissible via respiratory droplets between mammals (Russell et al, 2012). This provides evidence that avian influenza viruses can become transmissible between mammals without reassortment within an intermediate host. The H5N1 virus, though, was not as efficiently spread as the H1N1 virus of the 2009 outbreak which also showed successful viral shedding in na??ve test subjects after just 1 to 2 days (Herfst et al, 2012). The mutations required for airborne transmission between mammals could be developed by a number of studied methods; random mutation, positive selection as E627K substitutions have been shown to increase within-host fitness and longevity as the longer the host is infected the greater the proportion of mutations produced (Russell et al, 2012). Mutations may also aid viral binding and efficiency. 138'Ser has been shown to enhance binding to ??2,6'SA of pig H5N1 viruses, while 226'Leu in H7 avian influenza viruses improves binding to ??2,6'SA and 186'Val influences receptor binding of H7 (Gambaryan et al, 2012; Yang et al, 2014). The binding site that accommodates the SA receptor is a shallow pocket, formed by amino acid residues which are fairly highly conserved among virtually all subtypes and strains (Landolt and Olsen, 2007). Analysis of the HA gene of the 1968 pandemic strain by Bean and co-workers (Bean et al., 1992) revealed that fewer than six amino acid residues in the HA of the 1968 Hong Kong pandemic virus were altered in the process of avian-to-human transmission. Many alterations cause many different changes. Leucine-226 was found to confer SAa2,6Gal specificity in human H2 and H3, but not H1, viruses (Skehel and Wiley, 2000), whereas glutamine at position 226 correlates with SAa2,3Gal preference in avian and equine H3 viruses (Landolt and Olsen, 2007). Receptor-binding specificity is also influenced by the number and position of N-linked oligosaccharides at or around the receptor-binding site (Landolt and Olsen, 2007). Variation of glycosylation around the receptor-binding is made evident at adaptation to new hosts as seen when the virus infects eggs or mice. VIII. Vaccines, Treatment and Further Areas of Research Recent transmission is evident between a range of species. In March 2013 a novel H7N9 influenza A virus was detected in 130 human infections in China. The virus appears to have been derived from H7 viruses reasserting with enzootic H9N2 viruses (Lam et al, 2013). This idea of the coexistence of H7N9 and H9N2 subtypes in chickens is supported by metagenomic sequencing (Yu et al, 2014). In 2004 H3N8 viruses transmitted from horses to dogs then spread across America and throughout Europe. The viruses infecting canines and equines were compared and found to have a distinct difference in their HA. Via antigenic drift four lineages of equine virus were produced and the 'Florida clade 2' line contained an anomalous a-helix A. Meanwhile an amino acid substitution, HA1 Trp-22 to Leu, distinguished canine HA from the equine strands. The same is true of the canine H3N2 virus in S. Korea and China differing from its anticipated avian origin. H3N2 IAV has also infected canines but with minimal transmission, often concentrated in dog shelters, failing to spread to the numerous susceptibles due to limited sufficient contact and overly heterogeneous contact networks (Parrish et al, 2014). Understanding these processes is key to unlocking an effective remedy to this disease. Human travel and trade patterns are studied to highlight and predict likely pathways of the future H5N1 IAV through bird migration and trade. Influenza A viruses are widely distributed geographically as they are transported long distances by migratory birds. Virus transfer is inferred by comparing emergent viruses extracted from recipient hosts with viral ancestors in donor hosts. Any early recognition of viruses transmitting ineffectively throughout a new host can provide opportunities for epidemic control. For example, during the SARS CoV outbreak the first virus that emerged was inefficiently transmitted by most infected people. Its early detection upon outbreak and the subsequent institution of active control measures, particularly quarantine, allowed the epidemic to be stopped before the virus could become fully established in humans. Conventional infection control procedures, including smaller tasks like health monitoring, can substantially reduce contact between reservoir and recipient hosts, preventing outbreaks or terminating them after host transfer while they are still limited in size (Parrish et al, 2008). Even with major funding and research the evolution of viruses to allow adaptation to new hosts is still not well understood. Not only are some aspects still incompletely interpreted, such as the development of drug resistance or re-assortment patterns, many are difficult to study due to complex environmental and viral factors or biosecurity issues. Pre-emptive strategies including improved surveillance targeted to regions of high likelihood for disease emergence, improved detection of pathogens in reservoirs or early in outbreaks, broadly based research clarifying necessary steps to favour emergence, and modified forms of classical quarantine should be initiated. Human disease surveillance, longitudinal veterinary and wild-animal infection surveillance must all be associated as the virus and infections span all species (Parrish et al, 2008). Possible vaccines and antiviral drugs with the potential ability to control and prevent airborne transmission of influenza viruses could be further researched as avian viruses have been demonstrated to possess the ability to transmit between mammals and may one day result in another severe pandemic. Antiviral drugs could be used where available, although cost, logistical issues and side effects may label these difficult to utilise in a large-scale outbreak. They would likely work only in the context of other control measures (Parrish et al, 2008). Nonetheless, an H3N8 equine virus could become transmissible to humans as EIVs have spread between horses and canines, pigs and camels and previous experimental inoculations to humans have produced effective infections. A live, attenuated eq/GA/81 ca vaccine has been developed that, when introduced intranasally to mice and ferrets, induced antibodies and conferred protection against the wild type virus. This vaccine could be administered to humans upon further testing in case of an H3N8 pandemic (Baz et al, 2014). However, different approaches must also be applied to different countries. While in developed countries outbreaks of equine influenza can be prevented by vaccines or recovery aided by resting of the animals the same is not true of less economically developed countries where horses and donkeys are a primary resource for work and the disease can have detrimental effects socio-economically. Further investigation could be performed concerning the amino acid substitutions also. If the order with which they are substituted is an important factor it could significantly decrease the rate at which the transmission ability can arise. Greater surveillance in high risk areas where mutations arise at a high level could also provide insight and data on the nature of the virus. As time passes, more experiments are performed, more research conducted and more conclusions drawn. However, many of these are simply deductions from experimental and laboratory settings and no one can be sure that the models and theories produced are supported in real life situations as wildlife are difficult to track and open viral transmissions are impossible to study. Also, a number of studies have been performed on the basis of previous human pandemic analyses. This can be an issue, though, as only four such pandemic analyses are available for use and all are involved a composite virus of both human and avian counterparts. Hence, they do not give much indication of the possibilities of pandemics triggered by whole viruses, for example, of avian origin. Perhaps more research should be concentrated on this area as it has been shown to be possible in mammals (Herfst et al, 2012). Although the studies could be improved they still remain critical to comprehension of influenza. Understanding and predicting the host tropism of influenza proteins lay an important foundation for future work in constructing computation models capable of directly predicting interspecies transmission of influenza viruses (Eng et al, 2014). IV. Concluding Remarks The viral genome maintains a high plasticity and Influenza A holds the qualities necessary for a virus to cause a new emerging disease. This causes the study of influenza A viruses to be a highly topical and ongoing process. Many advances have been made in understanding how it can, and has, transferred between different species including enlightenment on how adaptation can occur, knowledge of the role of intermediates, understanding the molecular process of binding and transmittance and the mutations required. However, there is much more to discover whether it be the order in which mutations occur, successful stages for drug and vaccine interventions or patterns of global transfer to interrupt. It is still not possible to predict which infections may pass between specific species or the severity of their actions. Further studies could highlight common pathways and suggest preventive strategies. Although dangerous and costly, cross species transmission of IAVs has not yet reached the disastrous potential it holds over the natural world. It is extremely likely that the virus will emanate from its animal reservoir and cause extensive disease in mammalian hosts which, whether looked at economically or in terms of mortality, will have detrimental effects on society.
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