**2. Human infections with avian influenza virus**

The first human transmission of Highly Pathogenic Avian Influenza (HPAI) subtype H5N1 occurred in 1997 in Hong Kong. It became a global public health concern, knowing that pandemic influenza viruses in the past originated from animals [20]. The H5N1 was thus considered a potential pandemic threat [21]. The HPAI H5N1 lineage (A/Goose/Guangdong/1/96) was initially isolated from a goose farm in Guangdong Province, China in 1996. In the following year, outbreaks of highly pathogenic H5N1 were reported in poultry at farms and live bird markets in Hong Kong. Subsequently, contact with poultry and exposure to infected live and/ or dead birds became the medium for human exposure and in Hong Kong there were altogether, 18 cases (6 fatal) reported in the first known instance of human infection with this virus [22].

from LPAIs. Interestingly, the H5N8 (clade 2.3.4.4) though virulent in poultry has remained of low susceptibility in human, but another newly emerged H5N6 first identified in a peafowl arising from reassortment of H5 clade 2.3.4.4 has shown virulence in human and has killed 7 people among 17 that were infected since 2016 [27]. Repeated cases in human have raised concerns that subtype H5N6 virus also has the potential for crossover human infection which if sustained, may also be a candidate for influenza pandemic [28]. The notification of the first human case of novel subtype H7N4 to the Centre for Health Protection in Hong Kong on the 14th of February 2018 is a reminder that avian influenza is continually evolving bird-human transmission [29]. This H7N4 event and previous H7N9 detection first in human before cases in poultry were noticed also shows that humans are fast becoming sentinel for influenza sur-

Preventing Zoonotic Influenza

37

http://dx.doi.org/10.5772/intechopen.76966

Triple reassortant influenza A/H1N1pdm09 originating in swine caused the 1st pandemic of the twenty-first century in 2009. This was at the time of HPAI H5N1 epizootics in Asia, Europe and Africa. The strain and the region thought to be probable epicenter of future pandemic was H5N1 and Asia. The severity and spread of HPAI H5N1 in poultry and subsequent transmission to human, lent credence to scientific speculations that the zoonotic virus might have been a pandemic strain. Unpredictably, while attention was on HPAI H5N1, a pandemic H1N1 influenza virus emerged in Mexico, although the virus was believed to have been circulating in pigs many years before its first detection in human [16, 30]. The 2009 influenza pandemic spread to more than 214 countries and an estimated 151,700–575,400 respiratory and cardiovascular deaths were associated with the infection worldwide [31]. Lessons learnt include the realization that a zoonotic and pandemic virus may emerge from an animal reservoir in an unexpected location and spread rapidly throughout the world within a short time [32]. Also important is the realization that that the 2009 H1N1 pandemic virus was subsequently transmitted from human to pigs in a phenomenon that has been variously described as reverse zoonosis reported in more than 20 countries in America, Europe, Asia and Africa. Interestingly, the swine influenza sequence data available in public gene bank showed that humans transmit far more influenza A virus to swine than pigs have ever transmitted to humans, at least in terms of viruses that are transmitted onward in the new host as against dead end or accidental hosts [14]. The implication is that endemic human-like influenza virus that is enzootic in pigs will continuously pose public health risk in the generation of Influenza variants (combination of human and swine influenza viruses). This has also been reported to

**3. The pandemic H1N1pdm09, zoonosis and reverse zoonosis**

cause human infections in people exposed to pigs especially in America [14].

Virus strains or variants resulting from reassortment of swine influenza A(H3N2) and influenza A/H1N1pdm09 and similar viruses have been detected in swine in many countries. It is therefore of concern that emerging influenza variants could efficiently be transmitted among humans. Over 300 human cases of A(H3N2)v have been described between 2011 and 2012 in the United State alone beside clusters of human-human transmission further demonstrating that variant influenza viruses also pose a public health threat at the human-animal interface. Animals and humans may infect each other in intensive farms, abattoirs and agricultural fairs

veillance at the human-animal interface.

Early symptoms of influenza H5N1 virus usually develop 2 to 4 days after exposure to sick poultry and most patients infected with influenza H5N1 virus presents symptoms of fever, cough, shortness of breath and radiological evidence of pneumonia [21]. The number of human H5N1 cases reported globally was heightened in 2003 and since then, the virus has maintained a steady infection, morbidity and mortality at the animal-human interface. Those primarily at risk are cohorts of poultry farmers, handlers and operators in live bird markets and their immediate family members or contact. Though human to human transmission of H5N1 is not yet efficient, evolving nature of influenza virus in the environment is a reminder of the risk of emergence of a strain adapted for that possibility.

The HPAI (H5, H7) viruses circulating in terrestrial poultry (Chicken and turkeys) and are transmitted to human, normally emerge from the low pathogenic precursors in waterfowls. This arises by mutations in the gene and occurrence of multiple basic amino acids in the connecting peptide between the HA1 and HA2 domains of the HA0 precursor protein [23]. Trypsin-like proteases found in the respiratory and gastrointestinal tracts may be responsible for this limited enzymatic cleavage hence pathology are usually restricted to these systems. However, when multiple basic amino acids are introduced by insertion or deletion in the HA cleavage site, the HA0 precursor becomes cleavable by a wide range of ubiquitous proteases found in many host tissues [24]. Consequently, the virus is able to replicate in almost all the tissues/organs beside the respiratory and gastrointestinal tracts such as brain (nervous) and the cardiovascular (hematopoietic) system, resulting in fulminant and disseminated disease with high mortality index particularly in turkey and chicken [25].

In a peculiar incident, in February and March 2013, three patients were hospitalized with severe lower respiratory tract disease of unknown cause in China. The causative virus was later identified as novel avian-origin reassortant influenza A (H7N9), and phylogenetic analysis of all genes of the isolate showed that each gene segment was of avian origin [17]. The HA cleavage site possessed only a single basic amino acid R (arginine) as against polybasic, indicating tendency to be of low pathogenicity in poultry. On the contrary, cases in human host were severe, with patients developing severe pneumonia, acute respiratory distress, and eventually death. All the three patients had pre-existing medical conditions, but more importantly two of them had a history of direct contact with poultry [17]. The switch in virulence and pathogenicity were associated with certain mutations in the reassorted virus that may have contributed to severity of human infection and death. Similarly, waves of H5N8 outbreaks, first detected in domestic birds in China in 2010, which later spread from 2014 through 2016 in Europe and North America was heighten in the winter of 2016 and affected a wide range of domestic and wild birds but no human infection was recorded. Experimental studies even showed low virulence in ferret hence risks to human were considered low [26], even though the contemporary HPAI viruses of subtypes H5N2, H5N5, H5N6 and H5N8, all contain genes from 1997 A/Goose/Guangdong H5N1 lineage with acquired internal genes from LPAIs. Interestingly, the H5N8 (clade 2.3.4.4) though virulent in poultry has remained of low susceptibility in human, but another newly emerged H5N6 first identified in a peafowl arising from reassortment of H5 clade 2.3.4.4 has shown virulence in human and has killed 7 people among 17 that were infected since 2016 [27]. Repeated cases in human have raised concerns that subtype H5N6 virus also has the potential for crossover human infection which if sustained, may also be a candidate for influenza pandemic [28]. The notification of the first human case of novel subtype H7N4 to the Centre for Health Protection in Hong Kong on the 14th of February 2018 is a reminder that avian influenza is continually evolving bird-human transmission [29]. This H7N4 event and previous H7N9 detection first in human before cases in poultry were noticed also shows that humans are fast becoming sentinel for influenza surveillance at the human-animal interface.

### **3. The pandemic H1N1pdm09, zoonosis and reverse zoonosis**

year, outbreaks of highly pathogenic H5N1 were reported in poultry at farms and live bird markets in Hong Kong. Subsequently, contact with poultry and exposure to infected live and/ or dead birds became the medium for human exposure and in Hong Kong there were altogether, 18 cases (6 fatal) reported in the first known instance of human infection with this

Early symptoms of influenza H5N1 virus usually develop 2 to 4 days after exposure to sick poultry and most patients infected with influenza H5N1 virus presents symptoms of fever, cough, shortness of breath and radiological evidence of pneumonia [21]. The number of human H5N1 cases reported globally was heightened in 2003 and since then, the virus has maintained a steady infection, morbidity and mortality at the animal-human interface. Those primarily at risk are cohorts of poultry farmers, handlers and operators in live bird markets and their immediate family members or contact. Though human to human transmission of H5N1 is not yet efficient, evolving nature of influenza virus in the environment is a reminder

The HPAI (H5, H7) viruses circulating in terrestrial poultry (Chicken and turkeys) and are transmitted to human, normally emerge from the low pathogenic precursors in waterfowls. This arises by mutations in the gene and occurrence of multiple basic amino acids in the connecting peptide between the HA1 and HA2 domains of the HA0 precursor protein [23]. Trypsin-like proteases found in the respiratory and gastrointestinal tracts may be responsible for this limited enzymatic cleavage hence pathology are usually restricted to these systems. However, when multiple basic amino acids are introduced by insertion or deletion in the HA cleavage site, the HA0 precursor becomes cleavable by a wide range of ubiquitous proteases found in many host tissues [24]. Consequently, the virus is able to replicate in almost all the tissues/organs beside the respiratory and gastrointestinal tracts such as brain (nervous) and the cardiovascular (hematopoietic) system, resulting in fulminant and disseminated disease

In a peculiar incident, in February and March 2013, three patients were hospitalized with severe lower respiratory tract disease of unknown cause in China. The causative virus was later identified as novel avian-origin reassortant influenza A (H7N9), and phylogenetic analysis of all genes of the isolate showed that each gene segment was of avian origin [17]. The HA cleavage site possessed only a single basic amino acid R (arginine) as against polybasic, indicating tendency to be of low pathogenicity in poultry. On the contrary, cases in human host were severe, with patients developing severe pneumonia, acute respiratory distress, and eventually death. All the three patients had pre-existing medical conditions, but more importantly two of them had a history of direct contact with poultry [17]. The switch in virulence and pathogenicity were associated with certain mutations in the reassorted virus that may have contributed to severity of human infection and death. Similarly, waves of H5N8 outbreaks, first detected in domestic birds in China in 2010, which later spread from 2014 through 2016 in Europe and North America was heighten in the winter of 2016 and affected a wide range of domestic and wild birds but no human infection was recorded. Experimental studies even showed low virulence in ferret hence risks to human were considered low [26], even though the contemporary HPAI viruses of subtypes H5N2, H5N5, H5N6 and H5N8, all contain genes from 1997 A/Goose/Guangdong H5N1 lineage with acquired internal genes

of the risk of emergence of a strain adapted for that possibility.

with high mortality index particularly in turkey and chicken [25].

virus [22].

36 Influenza - Therapeutics and Challenges

Triple reassortant influenza A/H1N1pdm09 originating in swine caused the 1st pandemic of the twenty-first century in 2009. This was at the time of HPAI H5N1 epizootics in Asia, Europe and Africa. The strain and the region thought to be probable epicenter of future pandemic was H5N1 and Asia. The severity and spread of HPAI H5N1 in poultry and subsequent transmission to human, lent credence to scientific speculations that the zoonotic virus might have been a pandemic strain. Unpredictably, while attention was on HPAI H5N1, a pandemic H1N1 influenza virus emerged in Mexico, although the virus was believed to have been circulating in pigs many years before its first detection in human [16, 30]. The 2009 influenza pandemic spread to more than 214 countries and an estimated 151,700–575,400 respiratory and cardiovascular deaths were associated with the infection worldwide [31]. Lessons learnt include the realization that a zoonotic and pandemic virus may emerge from an animal reservoir in an unexpected location and spread rapidly throughout the world within a short time [32]. Also important is the realization that that the 2009 H1N1 pandemic virus was subsequently transmitted from human to pigs in a phenomenon that has been variously described as reverse zoonosis reported in more than 20 countries in America, Europe, Asia and Africa. Interestingly, the swine influenza sequence data available in public gene bank showed that humans transmit far more influenza A virus to swine than pigs have ever transmitted to humans, at least in terms of viruses that are transmitted onward in the new host as against dead end or accidental hosts [14]. The implication is that endemic human-like influenza virus that is enzootic in pigs will continuously pose public health risk in the generation of Influenza variants (combination of human and swine influenza viruses). This has also been reported to cause human infections in people exposed to pigs especially in America [14].

Virus strains or variants resulting from reassortment of swine influenza A(H3N2) and influenza A/H1N1pdm09 and similar viruses have been detected in swine in many countries. It is therefore of concern that emerging influenza variants could efficiently be transmitted among humans. Over 300 human cases of A(H3N2)v have been described between 2011 and 2012 in the United State alone beside clusters of human-human transmission further demonstrating that variant influenza viruses also pose a public health threat at the human-animal interface. Animals and humans may infect each other in intensive farms, abattoirs and agricultural fairs when in close proximity [33, 34]. Our ability to predict and prevent outbreaks of zoonotic pathogens like influenza therefore requires an understanding of their ecology and evolution in reservoir hosts [35]. This is important because Influenza A viruses from animals including reassortant, novel and variants are considered of significant threat in the emergence of the next pandemic due to the abundance of permanent animal reservoirs harboring viruses that are now frequently spilling over into human.

### **4. Mutation, reassortment and variants influenza virus**

Over the past 100 years, the IAVs have caused several pandemics including the one that has been described as "the greatest medical holocaust in history" [36]. Mutation and reassortment are two well established factors that have contributed in zoonotic influenza viruses gaining the ability to adapt to humans, leading to pandemics and thereafter sustained human-to-human transmissions. The accumulation of mutations and genome reassortments have been the driving force for most of the IAV adaptability in humans as the IAV RNA genome replication lacks the exonuclease proofreading capability, thus giving rise to high nucleotide mutation rates [37]. Antigenic drift and shift are the two major phenomena in influenza viruses that lead to antigenically variant influenza viruses [1, 38, 39]. The antigenic drift refers to point mutations in the HA and/or NA while the antigenic shift leads to the formation of a new virus subtype with a novel combination of HA and NA from different subtypes. While the antigenic drift is responsible for yearly epidemics, the antigenic shift has been responsible for some of the devastating pandemics in influenza history claiming many lives, including the 1918-Spanish flu. A list of zoonotic influenza outbreaks have been summarized in **Table 1**.

The human influenza viruses have limited subtypes of HA and NA (H1, H2, H3 and N1, N2) whereas the avian influenza viruses infecting the poultry may harbor almost all the subtypes of HA and NA [40], thus giving rise to multiple recombination of HA and NA in avian species. Since 1996, the HPAI-H5N1virus have claimed several lives resulting in high mortality rate while the recently identified LPAI-H7N9 in East China region had a mortality rate of 40% [41]. The H7N9 virus isolates have the capability of binding to both avian and human influenza virus receptors due to presence of a leucine at amino acid position 217 [42]. A relatively limited number of mutations in the zoonotic IAV genome can lead to production of new viral progenies with capability of efficient transmission among mammals and studies have also demonstrated that amino acid substitutions in the HA protein can change the preference of binding receptors of influenza viruses. For example, the G186 V mutation in HA protein of H7N9 virus has been identified as the potential adaptation of the virus to human-type receptors [43]. A recent study conducted on a non-laboratory-adapted virus A/Vietnam/1203/2004 (H5N1) with an HA2-K58I point mutation (K to I at position 58) showed that a decrease in the HA activation pH (pH 5.5) influenced the viral properties as compared to the wild type virus (HA activation pH 6.0) in mammalian hosts [44]. The mutation increased the viral load in ferret's nasal cavity while it reduced the viral load in lungs thus supporting the fact that a single mutation could lead to an increased viral growth in mammalian upper respiratory tract [44].

Several studies in ferrets have shown that the viruses such as H5N1 [45], H7N9 [46] and H7N1 [47] could transmit through respiratory droplets after acquiring mutations in their genomes. Another study on A/Anhui/1/13 (H7N9) virus showed that substitutions at G219S and K58I resulted in high affinity for α2,6-linked sialic acid receptor and acid and temperature stability [48]. The increased polymerase activity due to mutation in the viral PB2 has also been linked to enhanced viral replication. The PB2 subunit from all avian viruses generally contains polymerases with glutamic acid at amino acid position 627 (E627) while the PB2 from human viral isolates almost exclusively have lysine at 627 (K627). Mehle et al. have shown that E627K mutation of PB2 conferred a high level of polymerase activity in human and porcine cells thus increasing the viral replication [49]. Another study showed that a basic amino acid at position 591 of the PB2 subunit compensated for the lack of PB2-627 K in HPAI-H5N1 and pandemic H1N1viruses markedly increased the replication of these viruses in mammalian species [50]. The PB2 gene mutation in duck H7N9 also enhanced the polymerase activity and thus viral replication in human cells [51]. The H1N1 influenza virus that caused the 1918 pandemic and

**Year (Country) Influenza** 

**subtype**

1997/2003-present (Asia, Europe and Africa) H5N1 660 N224 K (HA)

2003 (USA) H7N2 1 Not determined 2003 (Hong Kong) H9N2 1 Q226L (HA)

2003 (The Netherlands) H7N7 89 E627K (PB2) 2004 (Egypt) H10N7 2 Not determined 2004 (Canada) H7N3 2 Not determined 2007 (UK) H7N2 4 Not determined 2008–2009 (Hong Kong) H9N2 2 Not determined 2012 (Mexico) H7N3 2 Not determined 2013 (China) H10N8 3 Not determined 2013 (China, Taiwan, Hong Kong) H7N9 137 Q226L (HA)

2013 (Taiwan) H6N1 1 P186L (HA) Since 2014 (China) H5N6 16 G540A (NS) 2018 (China) H7N4 1 Not determined

**Table 1.** Zoonotic influenza A viruses and identified adaptations (reviewed in [53] with modification).

**Confirmed cases Adaptation in segment**

http://dx.doi.org/10.5772/intechopen.76966

N158D (HA) T160A (HA) E627K (PB2)

Preventing Zoonotic Influenza

39

G228S (HA) T212A (HA)

E627K (PB2)


when in close proximity [33, 34]. Our ability to predict and prevent outbreaks of zoonotic pathogens like influenza therefore requires an understanding of their ecology and evolution in reservoir hosts [35]. This is important because Influenza A viruses from animals including reassortant, novel and variants are considered of significant threat in the emergence of the next pandemic due to the abundance of permanent animal reservoirs harboring viruses that

Over the past 100 years, the IAVs have caused several pandemics including the one that has been described as "the greatest medical holocaust in history" [36]. Mutation and reassortment are two well established factors that have contributed in zoonotic influenza viruses gaining the ability to adapt to humans, leading to pandemics and thereafter sustained human-to-human transmissions. The accumulation of mutations and genome reassortments have been the driving force for most of the IAV adaptability in humans as the IAV RNA genome replication lacks the exonuclease proofreading capability, thus giving rise to high nucleotide mutation rates [37]. Antigenic drift and shift are the two major phenomena in influenza viruses that lead to antigenically variant influenza viruses [1, 38, 39]. The antigenic drift refers to point mutations in the HA and/or NA while the antigenic shift leads to the formation of a new virus subtype with a novel combination of HA and NA from different subtypes. While the antigenic drift is responsible for yearly epidemics, the antigenic shift has been responsible for some of the devastating pandemics in influenza history claiming many lives, including the 1918-Spanish flu. A list of zoonotic influenza outbreaks have been

The human influenza viruses have limited subtypes of HA and NA (H1, H2, H3 and N1, N2) whereas the avian influenza viruses infecting the poultry may harbor almost all the subtypes of HA and NA [40], thus giving rise to multiple recombination of HA and NA in avian species. Since 1996, the HPAI-H5N1virus have claimed several lives resulting in high mortality rate while the recently identified LPAI-H7N9 in East China region had a mortality rate of 40% [41]. The H7N9 virus isolates have the capability of binding to both avian and human influenza virus receptors due to presence of a leucine at amino acid position 217 [42]. A relatively limited number of mutations in the zoonotic IAV genome can lead to production of new viral progenies with capability of efficient transmission among mammals and studies have also demonstrated that amino acid substitutions in the HA protein can change the preference of binding receptors of influenza viruses. For example, the G186 V mutation in HA protein of H7N9 virus has been identified as the potential adaptation of the virus to human-type receptors [43]. A recent study conducted on a non-laboratory-adapted virus A/Vietnam/1203/2004 (H5N1) with an HA2-K58I point mutation (K to I at position 58) showed that a decrease in the HA activation pH (pH 5.5) influenced the viral properties as compared to the wild type virus (HA activation pH 6.0) in mammalian hosts [44]. The mutation increased the viral load in ferret's nasal cavity while it reduced the viral load in lungs thus supporting the fact that a single mutation could lead to an increased viral growth in mammalian upper respiratory tract [44].

are now frequently spilling over into human.

38 Influenza - Therapeutics and Challenges

summarized in **Table 1**.

**4. Mutation, reassortment and variants influenza virus**

**Table 1.** Zoonotic influenza A viruses and identified adaptations (reviewed in [53] with modification).

Several studies in ferrets have shown that the viruses such as H5N1 [45], H7N9 [46] and H7N1 [47] could transmit through respiratory droplets after acquiring mutations in their genomes. Another study on A/Anhui/1/13 (H7N9) virus showed that substitutions at G219S and K58I resulted in high affinity for α2,6-linked sialic acid receptor and acid and temperature stability [48]. The increased polymerase activity due to mutation in the viral PB2 has also been linked to enhanced viral replication. The PB2 subunit from all avian viruses generally contains polymerases with glutamic acid at amino acid position 627 (E627) while the PB2 from human viral isolates almost exclusively have lysine at 627 (K627). Mehle et al. have shown that E627K mutation of PB2 conferred a high level of polymerase activity in human and porcine cells thus increasing the viral replication [49]. Another study showed that a basic amino acid at position 591 of the PB2 subunit compensated for the lack of PB2-627 K in HPAI-H5N1 and pandemic H1N1viruses markedly increased the replication of these viruses in mammalian species [50]. The PB2 gene mutation in duck H7N9 also enhanced the polymerase activity and thus viral replication in human cells [51]. The H1N1 influenza virus that caused the 1918 pandemic and the H5N1 avian influenza virus isolated in 1997 (Hong Kong) both harbors the N66S mutation in PB1-F2 which drastically enhanced the pathogenicity of these viruses [52].

According to the Centers for Disease Control and Prevention (CDC), when an influenza virus that normally circulates in swine (not in humans) is detected in humans, it is referred to as variant influenza viruses. The human infections with H1N1, H3N2 and H1N2 variant viruses have been reported from United States [13, 65]. Although the variant influenza viruses rarely show sustained human-to-human transmission, yet there have been few strains that overcame this barrier. All the cases reported in US were of swine origin rather than avian origin. In 2009, triple reassortant variant influenza virus was detected throughout the world and caused the first pandemic of twenty-first century. This variant virus had genes from avian, human, and swine influenza viruses claiming more than 12,500 lives in the US alone and about 575,400 globally [31, 66]. Later H3N2 variant viruses which had similarity with triplereassortant viruses were detected in US swine but had acquired the matrix gene from highly transmissible influenza A H1N1-2009 viruses which contributed in efficiency of transmission of the variant virus [67]. A recent study has also identified two distinct variants of H3N2 influenza virus that grows in cell culture [68]. Both the variants differed in just one single mutation at amino acid 151 of NA. The D151 viral variant could efficiently grow in cell culture while the G151 viral variant showed extremely poor growth in cell culture system [68]. More in-depth studies are still needed to better understand the viral properties of variant influenza

Preventing Zoonotic Influenza

41

http://dx.doi.org/10.5772/intechopen.76966

The isolation of influenza A/H1N1 in 1933 quickly ushered the development of the first generation of live-attenuated influenza vaccines (LAIV). The initially developed inactivated influenza vaccine (IIV) only targeted a single influenza strain (influenza A). Then, in 1942, a vaccine targeting both influenza A and B viruses were produced soon after the discovery of influenza B. Subsequently, scientists discovered that influenza viruses mutated, leading to antigenic changes (antigenic drift and antigenic shift). Since 1973, the World Health Organization (WHO) has been providing yearly recommendations for the composition of the influenza vaccine, based on results of the virological surveillance conducted by the WHO's Global Influenza Surveillance and Response System (GISRS). Later in 1978, the trivalent influenza vaccine was developed that included two influenza A strains and one influenza B strain. Currently, two influenza B lineages are circulating (Yamagata and Victoria) therefore, since 2013, the WHO recommendations suggested a second B strain to be added to make a quadrivalent influenza vaccine (QIV) [69]. Influenza vaccines protect against infection and can reduce illness and severity of infection especially in groups at risk for flu complications such as children, the elderly, pregnant women, and individuals with underlying medical conditions like asthma, HIV/AIDS, and chronic heart or lung diseases [70]. Frequent influenza infections at the human-animal interface may also warrant occupational vaccination for veterinarians, researchers, health care providers, farmers and animal traders who are more

For over half a century now, WHO has been collaborating with scientists, epidemiologists, and policymakers to create an integrated approach to manufacture, test, and approve

viruses as they continue to pose threat to human lives.

**5. Immunity and challenges of vaccination**

likely to be exposed to zoonotic influenza virus [71].

Genetic reassortment in influenza viruses is yet another vital event that leads to sudden outbreaks of influenza. Influenza viruses have a segmented genome and thus, simultaneous infection with other IAVs results in reassortment event leading to formation of new viral progenies containing gene segments of mixed parental origin. Several pandemics have emerged in the past [54] and appears to be more frequent now than previously thought [55]. The reassortment event can be a result of errors during the replication of viral RNA polymerase, the host environment, the immune or evolutionary pressure [56]. The pandemics of 1957 and 1968 were caused by reassortant viral strains [57]. The HA, NA, and PB1 genes of the H2N2 1957 pandemic strain and the HA and PB1 fragments of the H3N2 1968 pandemic strain were both derived from avian influenza virus strains [57]. The HPAI subtype H5N1 isolated from geese in Guangdong province in 1996 evolved to produce H5N8 clades 2.3.4.4 Gs/GD HPAIV. A recent study on the evolution and pathogenicity of H5N2 avian influenza viruses isolated in H5N1 endemic areas in China revealed that these viral isolates were derived from reassortment events in which few isolates had the HA and NS derived from H5N1 while few had the HA derived from endemic H9N2 viruses [58]. A similar study from South Korea reported the emergence of novel reassortant H5N8 viruses in 2014 in ducks raised in breeder farms [59]. Since its first appearance, lineage of the HPAI H5N1 continues to circulate with lots of diversification of the HA gene into multiple genetic clades. The H5 clade 2.3.4.4 of the H5N8 subtype was subsequently detected in several countries of Europe by the end of 2014 and in summer of 2016, it was detected again in wild aquatic birds sampled in western Siberia [60]. A recent study has also shown that the reassortment event between the Gs/GD lineage H5N8 virus and North American origin viruses further resulted in the emergence of H5N1 and H5N2 viruses in the US [61].

Experimental observations have further revealed that reassortment between zoonotic and seasonal IAVs can result in production of airborne-transmissible viruses in mammals [62–65]. A study showed that a reassortant virus, comprising of the H5 hemagglutinin having 4 mutations from H5N1 avian virus and remaining seven segments from the 2009 pandemic H1N1 virus lead to reassortant H5 HA/H1N1 virus that gained the capacity of droplet transmission in ferret model [62]. Another experiment further showed that the avian H5N1 subtype viruses do have the potential to attain mammalian transmissibility by genetic reassortment [63]. The authors utilized reverse genetics to create several reassortant viruses between duck H5N1 (HA gene) and human-infective H1N1 virus to show that the new reassortant viruses could efficiently infect and sustained droplet transmission in guinea pigs without mortality [63]. Similar study reported that the avian-human H9N2 reassortant virus harboring the surface proteins of avian H9N2 in a human H3N2 backbone gained the ability of transmission through the respiratory droplets and caused clinical infection in ferrets similar to human influenza infections [64]. A recent study performed in a novel transfection-based inoculation system generated a reassortant H9N1 virus by transfecting the plasmids containing genes from H9N2 virus and pandemic H1N1 (pH1N1) virus into HEK 293 T cells. The resulting transfections gave rise to two reassortant viruses (H9N1) that had the capability of droplettransmissibility [65].

According to the Centers for Disease Control and Prevention (CDC), when an influenza virus that normally circulates in swine (not in humans) is detected in humans, it is referred to as variant influenza viruses. The human infections with H1N1, H3N2 and H1N2 variant viruses have been reported from United States [13, 65]. Although the variant influenza viruses rarely show sustained human-to-human transmission, yet there have been few strains that overcame this barrier. All the cases reported in US were of swine origin rather than avian origin. In 2009, triple reassortant variant influenza virus was detected throughout the world and caused the first pandemic of twenty-first century. This variant virus had genes from avian, human, and swine influenza viruses claiming more than 12,500 lives in the US alone and about 575,400 globally [31, 66]. Later H3N2 variant viruses which had similarity with triplereassortant viruses were detected in US swine but had acquired the matrix gene from highly transmissible influenza A H1N1-2009 viruses which contributed in efficiency of transmission of the variant virus [67]. A recent study has also identified two distinct variants of H3N2 influenza virus that grows in cell culture [68]. Both the variants differed in just one single mutation at amino acid 151 of NA. The D151 viral variant could efficiently grow in cell culture while the G151 viral variant showed extremely poor growth in cell culture system [68]. More in-depth studies are still needed to better understand the viral properties of variant influenza viruses as they continue to pose threat to human lives.
