**2. Evolution of influenza A viruses**

### **2.1 Antigenic drift**

Influenza viruses cause annual recurrent epidemics in humans, which approximately result in 3–5 million cases of severe illness and 250,000–500,000 deaths worldwide [11]. Most of these were occurred by influenza A and B viruses. Especially influenza A viruses change viral surface glycoproteins HA and NA by accumulations of point mutations in HA and NA genes and escape from human immunity called as antigenic drift, which may occur as seasonal influenza epidemic. But antigenic drift does not evolve virulence of influenza A viruses which cause severe disease in humans [12]. Seasonal influenza viral infections are limiting disease and mild, which cause severe disease and death by factors of host immune conditions. This does not mean that there is no necessity of treatment of anti-influenza viral therapy

**97**

polymerase proteins

*Adequate Monitor of Avian Influenza Viral Infections and Foresight About Possibilities of Its…*

for seasonal influenza infections. The adequate diagnosis and treatment for seasonal influenza infection diminish severe illness and subsequent death from seasonal influenza infections and lead to awareness of emerging infectious disease [13].

In 1997 HPAI (H5N1) viruses occurred among wild birds and caused an outbreak in poultry and sporadic human infection in Hong Kong [14]. This was firstly identified as the crossover of avian-human species barrier and resulted in 18 infected individuals and 6 of 18 patients died [15]. Zoonotic infection of avian influenza virus H5N1 has been widely spread from Asia to Europe and Africa, which has been endemic in some countries and caused outbreaks resulting in millions of poultry infections, several hundreds of human infectious cases, and many human deaths since 2003 [16]. Avian influenza virus H5N1 causes a disease which has aggressive symptoms different from a mild disease of seasonal influenza virus infection in humans [9]. HA is a surface viral protein which binds to and uses sialic acid-containing molecules as receptors for viral infection to multiple cells. Avian influenza virus strains circulated in waterfowl contain HAs with preference for binding to sialic acids linked to the rest of the sugar by an α2–3 linkage. In contrast, HAs from human influenza virus strains show enhanced binding to α2–6-linked sialic acids [17]. Seasonal influenza viruses cause symptoms of upper respiratory infection by correlation with an abundance of α2–6-linked sialic acids in the upper respiratory tract of humans [18]. More detailed analysis of H5N1 infection showed an abundance of α2–3-linked sialic acids in the lower respiratory tract of humans, and the explanation of severe disease of the lung was done, as pneumonia and respiratory failure in H5N1 infection [19]. In addition to an abundance of avian influenza viral receptor in lower respiratory tract in humans, the autopsy reports of H5N1-infected patients revealed avian influenza virus infection intra and extra lung concluding the brain, intestine, heart, spleen, lymphoid tissue, kidney, and placenta by applying immunohistochemistry (IHC) with monoclonal antibodies to hemagglutinin (HA) and nucleocapsid protein (NP) and/or in situ hybridization with sense and antisense probes to HA and NP to detect viral antigens and genomic sequences in various organs of H5N1 cases and RT-PCR and nucleic acid sequence-based amplification H5 detection assays [20–25]. HAs with multiple residues at the cleavage site of precursor HAs that can be activated by ubiquitous intracellular proteases may cause systemic infections; on the other hand, HAs with a single arginine residue at the cleavage site of precursor HAs that can only be cleaved by extracellular trypsin-like proteases present in the upper respiratory and gastrointestinal tracts may give rise to local infections [26]. The disease severity in human cases varies from mild to extremely severe; mutations of HA may always be related to the ability of receptor binding but not the virulence of viruses, which imply that there are other factors responsible for the virulence of H5N1 influenza viruses in humans [27]. As the characterization of avian influenza A H5N1, the analysis of H5N1 infections showed the deletion of 19 amino acids of the stalk region in NA protein that cleaves and separates the HA of progeny virions from the sialic acid-containing receptors on the surface of the infected host cells, in which they were generated [14, 28]. This deletion may play a role in the transmission of virus across species [29]. The polymerase complex is composed of three viral

(PB1, PB2, and PA) involved in viral RNA synthesis. In the polymerase complex of avian influenza viruses, glutamic acid to lysine substitution at position 627 of PB2 was isolated but not a prerequisite for high virulence in humans [30]. The 1918 pandemic was called as Spanish flu and proposed that the 1918 virus was not a reassorted virus (like those of the 1957 and 1968 pandemics), but more likely an entirely

*DOI: http://dx.doi.org/10.5772/intechopen.86551*

**2.2 Avian influenza A H5N1 viruses**

*Adequate Monitor of Avian Influenza Viral Infections and Foresight About Possibilities of Its… DOI: http://dx.doi.org/10.5772/intechopen.86551*

for seasonal influenza infections. The adequate diagnosis and treatment for seasonal influenza infection diminish severe illness and subsequent death from seasonal influenza infections and lead to awareness of emerging infectious disease [13].

#### **2.2 Avian influenza A H5N1 viruses**

*Viruses and Viral Infections in Developing Countries*

infectious disease of influenza A viruses [9].

Influenza viruses cause annual recurrent epidemics in humans, which approximately result in 3–5 million cases of severe illness and 250,000–500,000 deaths worldwide [11]. Most of these were occurred by influenza A and B viruses. Especially influenza A viruses change viral surface glycoproteins HA and NA by accumulations of point mutations in HA and NA genes and escape from human immunity called as antigenic drift, which may occur as seasonal influenza epidemic. But antigenic drift does not evolve virulence of influenza A viruses which cause severe disease in humans [12]. Seasonal influenza viral infections are limiting disease and mild, which cause severe disease and death by factors of host immune conditions. This does not mean that there is no necessity of treatment of anti-influenza viral therapy

**2. Evolution of influenza A viruses**

**2.1 Antigenic drift**

antigenic subtypes by the combinations of the main viral antigens: hemagglutinin (HA) and neuraminidase (NA) [3]. Recently 18 different HA subtypes and 11 different NA subtypes have been identified [4, 5]. Influenza A viruses are classified from H1N1 to H18N11 and are called as avian influenza virus, swine influenza virus, or other types of animal influenza viruses depending on original infectious host. Humans, swine, and avian are infective host of influenza A virus, but infectious influenza A viral subtypes among intraspecies are restricted by the binding affinity of the HA protein to sialyloligosaccharides (oligosaccharides containing terminal silica acids linked to galactose) on host cell surface glycoprotein or glycolipids [6]. Influenza A viruses cause normally self-limited disease (asymptomatic or mild) and are shared with humans and many different animals. Most subtypes of influenza A viruses are primarily reserved in aquatic birds, are distinct from human influenza A viruses, and do not easily transmit to human due to host species barriers [7]. Zoonotic infection between humans and animals or interspecies of infection of influenza A viruses makes viral evolutions which can help to surmount species barriers [8]. Avian influenza viruses transmit from aquatic birds to poultry and change virus properties and result in change pathogenesis ranging from mild disease to severe disease with high death rates in host species. According to these phenomena, avian influenza viruses cause severe disease and high death rate in host, which are called as highly pathogenic avian influenza (HPAI) viruses, and avian influenza viruses result in mild disease or asymptomatic infection to host, which are called as low pathogenic avian influenza (LPAI) viruses. A virus is defined as HPAI or LPAI by its ability to cause disease and mortality in intravenously inoculated young chickens in the laboratory or by its possession of molecular characteristic features associated with HPAI viruses. Avian, swine, and other zoonotic influenza infections in humans may happen on the condition of the successful transmission with the sufficient contact between donor and recipient species, successful species jumps of influenza viruses, the influenza viral complex adaptations, and achieved sustained transmission in a new species [7]. Those may cause several ranges of disease in humans depending on viral factors and human factors [9]. HPAI virus infections have been monitored from avian to humans and caused the endemic disease of high fatality rate [10]. HPAI viruses may cause pandemic from endemic on the condition of no effective protection for wide shedding of HPAI viruses. Monitoring of disease severity of avian and human influenza A virus infection could prevent the severe damage to the society from the emerging

**96**

In 1997 HPAI (H5N1) viruses occurred among wild birds and caused an outbreak in poultry and sporadic human infection in Hong Kong [14]. This was firstly identified as the crossover of avian-human species barrier and resulted in 18 infected individuals and 6 of 18 patients died [15]. Zoonotic infection of avian influenza virus H5N1 has been widely spread from Asia to Europe and Africa, which has been endemic in some countries and caused outbreaks resulting in millions of poultry infections, several hundreds of human infectious cases, and many human deaths since 2003 [16]. Avian influenza virus H5N1 causes a disease which has aggressive symptoms different from a mild disease of seasonal influenza virus infection in humans [9]. HA is a surface viral protein which binds to and uses sialic acid-containing molecules as receptors for viral infection to multiple cells. Avian influenza virus strains circulated in waterfowl contain HAs with preference for binding to sialic acids linked to the rest of the sugar by an α2–3 linkage. In contrast, HAs from human influenza virus strains show enhanced binding to α2–6-linked sialic acids [17]. Seasonal influenza viruses cause symptoms of upper respiratory infection by correlation with an abundance of α2–6-linked sialic acids in the upper respiratory tract of humans [18]. More detailed analysis of H5N1 infection showed an abundance of α2–3-linked sialic acids in the lower respiratory tract of humans, and the explanation of severe disease of the lung was done, as pneumonia and respiratory failure in H5N1 infection [19]. In addition to an abundance of avian influenza viral receptor in lower respiratory tract in humans, the autopsy reports of H5N1-infected patients revealed avian influenza virus infection intra and extra lung concluding the brain, intestine, heart, spleen, lymphoid tissue, kidney, and placenta by applying immunohistochemistry (IHC) with monoclonal antibodies to hemagglutinin (HA) and nucleocapsid protein (NP) and/or in situ hybridization with sense and antisense probes to HA and NP to detect viral antigens and genomic sequences in various organs of H5N1 cases and RT-PCR and nucleic acid sequence-based amplification H5 detection assays [20–25]. HAs with multiple residues at the cleavage site of precursor HAs that can be activated by ubiquitous intracellular proteases may cause systemic infections; on the other hand, HAs with a single arginine residue at the cleavage site of precursor HAs that can only be cleaved by extracellular trypsin-like proteases present in the upper respiratory and gastrointestinal tracts may give rise to local infections [26]. The disease severity in human cases varies from mild to extremely severe; mutations of HA may always be related to the ability of receptor binding but not the virulence of viruses, which imply that there are other factors responsible for the virulence of H5N1 influenza viruses in humans [27]. As the characterization of avian influenza A H5N1, the analysis of H5N1 infections showed the deletion of 19 amino acids of the stalk region in NA protein that cleaves and separates the HA of progeny virions from the sialic acid-containing receptors on the surface of the infected host cells, in which they were generated [14, 28]. This deletion may play a role in the transmission of virus across species [29]. The polymerase complex is composed of three viral polymerase proteins

(PB1, PB2, and PA) involved in viral RNA synthesis. In the polymerase complex of avian influenza viruses, glutamic acid to lysine substitution at position 627 of PB2 was isolated but not a prerequisite for high virulence in humans [30]. The 1918 pandemic was called as Spanish flu and proposed that the 1918 virus was not a reassorted virus (like those of the 1957 and 1968 pandemics), but more likely an entirely avian-like virus that has adapted to humans. But the 1918 influenza virus was analyzed as 10 amino acids change the polymerase complex including the lysine residue at position 627 [31]. These mutations require more investigation for the precursors of a new influenza pandemic. PB1-F2 is a small mitochondrial protein that is encoded on an open reading frame of PB1 [32]. This open reading frame is highly conserved in avian influenza isolates [33]. The reconstructed 1918 influenza virus linked to the PB1-F2 protein and recombinant virus with a single mutation in the PB1-F2 protein [serine (S) instead of asparagine (N) at position 66] of H5N1 (Hk/97) increase viral pathogenicity [34, 35]. PB1-F2 may induce apoptosis of immune cells and an insufficient adaptive immune response [35]. The NS proteins (NS1 and NS2) are viral proteins, and NS1 has multiple functions including splicing and nuclear export of cellular mRNA as well as stimulation of translation [36]. The NS1 protein is crucial for evading the innate immune response of the host by inhibiting the antiviral response mediated by type I interferons [37]. The presence of glutamic acid at position 92 of NS1 has been investigated to be a prerequisite for the resistance to antiviral cytokines considering the results of severe disease induction by inoculation of reassortant influenza virus (H1N1) bearing the NS gene of the H5N1/97 virus to pigs and the lack of glutamic acid at position of the NS1 protein in the recent human and avian isolates [38–41]. Two PDZ ligand (PL) sequence motifs (a Glu-Pro-Glu-Val (EPEV), a Glu-Ser-Glu-Val (ESEV)) were detected in viruses isolated during 1997 but not in viruses after 1997 as a potential codominants of virulence disrupting several PDZ-domain protein-mediated pathways for cell signaling [33].

M1 and M2 are matrix proteins, and M2 is a 97-residue single-pass membrane protein with its N-terminus directed toward the outside of the virus. It determines proton selectivity and unidirectional conductance of the channel by mutagenesis studies [42]. A serine to asparagine substitution at residue 31 of M2 protein is associated with resistance to adamantanes which were used for therapy of influenza A virus infections [43]. On the other hand, M2 protein is suggested to be a candidate for the adaptation of the virus to a new host in addition to PB1-F2 from a study of Thai and Indonesian isolates [43].

#### **2.3 Avian influenza A H7N9 viruses**

Avian influenza viruses sometimes infect poultry as LPAI, and there is no concern to LPAI, but even LPAI suddenly caused the sporadic infection among humans which started on February 19, 2013, in Shanghai [44]. There was no epidemic of severe disease in poultry infections with H7N9 before human infections with H7N9. In April 2013, the number of human cases of H7N9 virus infections increased significantly, reaching 125 confirmed cases in China, which was suggested to be derived from contacting with poultry and/or contaminated environments in farmers' markets with live birds [45]. After closure of live birds' markets, the number of new human cases infected with H7N9 declined rapidly. But the second wave of human infections with H7N9 virus started again owing to the lower fall temperature and reopening of poultry markets in the fall of 2013. This wave is characterized by the extensive geographic spread from the province of Eastern China to Southern China and a large number of human infections with H7N9 (440 human infections and 122 associated death of 440 as of May 16, 2014) [46]. For the protection of widespread of H7H9, two strategies composed of suspending farmers' markets with live birds and stopping the transport for trading of live birds carrying the virus were applied by provincial and Chinese authorities [47, 48]. These measures controlled the more spread of H7N9 virus infections in China, but the potential benefits of public health measures should be balanced against the potentially significant societal and economic costs [49]. The lack of protective antibodies in human against

**99**

occurred in human infections [51].

**2.4 Avian influenza A H7N7 viruses**

Outbreaks of HPAI virus infections mostly originate from endemic LPAI viral strains of H5 and H7 subtypes among wild waterfowl. These LPAI virus strains can transmit from waterfowl to poultry and change virulence to HPA virus among poultry [58]. In the Netherlands, an epidemic of HPAI virus H7N7 infection occurred in 2003 that resulted in 255 affected flocks and culling (killing and removing of infected poultry flocks) of 30 million birds [59]. Control measures

*Adequate Monitor of Avian Influenza Viral Infections and Foresight About Possibilities of Its…*

H7N9 virus leads to the result of influenza pandemic in humans, nevertheless of zoonotic infection of poultry to humans [50]. During October 1, 2016–August 7, 2017, the largest fifth epidemic of Asian H7N9 infections since 2013 was reported as 759 human infections including 281 deaths [51]. The total of human infections has reached to 1557 human infections and at least 605 deaths (39%) during March 31, 2013–August 7, 2017 [51]. Fourteen clusters of two or three persons with H7N9 happened during the fifth epidemic Asian H7N9 that was explained in association with wider geographic spread and higher prevalence of Asian virus among poultry and not as a result of any increased incidence of poultry to humans or human-to-human spread [51]. In the genesis of H7N9, H7 of H7N9 is derived from H7N3 viruses isolated among ducks in Eastern China in 2010–2011, and N9 of H7N9 is derived from avian H2N9 and/or H11N9 viruses isolated among wild migratory birds along the East Asian flyway [50, 52]. Both of them are Eurasian lineage of avian viruses, and reassortment of gene (so-called as antigenic shift) may happen and produce the possible precursor of H7N9 virus. Six viral genes except H7 and N9 of H7N9 virus likely originated from chicken H9N2 sub-lineages 1 and 2 circulating in Eastern China [50, 52]. Antigenic shift is suggested to form H7N9 virus during the generation and evolution of viruses. In the analysis of H7N9, viral sequences revealed that H7N9 virus possesses leucine or isoleucine at position 226 of HA which binds to both sialic acid-α2,3-galactose (avian-type receptor) and sialic acid-α2,6-galactose (human -type receptor) [53]. This may mean as the avian virus infection to humans in addition to expression of sialic acid-α2,6-galactose in the upper respiratory tract and sialic acid-α2,3-galactose in the lower respiratory tract in humans [19, 21–25]. According to PB2 protein of H7N9, sequence analysis revealed that many isolated H7N9 viruses encode lysine at position 627 of PB2 [54]. This substitution of glutamic acid (E) to lysine (K) of position 627 of PB2 (E627K mutation) may enable avian viruses to adapt to efficient replication at 33 and 37°C of human temperature lower than original avian host temperature (41°C) [55, 56]. Other mutations of position 627 of PB2 in these viruses have been reported and investigated [56]. The resistant H7N9 variants encoding the R294K mutation in NA that confers resistance to oseltamivir have been detected in patients treated with neuraminidase inhibitors (NAIs). R294K substitution was revealed as multidrug resistance with extreme oseltamivir resistance by the study of using protein- and virus-based assays [57]. On the other hand, K294 mutation of H7N9 infections was found. Compared with H7N9 viruses containing K294 which leads to mild resistance to NAIs, R294K appeared in clinical course under the selective pressure of oseltamivir treatment and conferred not only multidrug resistance to NAIs but also decreases NA activity and impaired virus replication. These phenomena may explain the failure for predominance of R294K-mutated strains of H7N9 in this outbreak. But the drug-resistant mutations should be monitored vigilantly in order to help the most effective drug administration and the prevention of unnecessary loss of human lives in clinical course [57]. Recently LPAI H7N9 viruses have evolved to HPAI viruses and caused the increased morbidity and mortality in poultry, and the same phenomenon has more likely

*DOI: http://dx.doi.org/10.5772/intechopen.86551*

#### *Adequate Monitor of Avian Influenza Viral Infections and Foresight About Possibilities of Its… DOI: http://dx.doi.org/10.5772/intechopen.86551*

H7N9 virus leads to the result of influenza pandemic in humans, nevertheless of zoonotic infection of poultry to humans [50]. During October 1, 2016–August 7, 2017, the largest fifth epidemic of Asian H7N9 infections since 2013 was reported as 759 human infections including 281 deaths [51]. The total of human infections has reached to 1557 human infections and at least 605 deaths (39%) during March 31, 2013–August 7, 2017 [51]. Fourteen clusters of two or three persons with H7N9 happened during the fifth epidemic Asian H7N9 that was explained in association with wider geographic spread and higher prevalence of Asian virus among poultry and not as a result of any increased incidence of poultry to humans or human-to-human spread [51]. In the genesis of H7N9, H7 of H7N9 is derived from H7N3 viruses isolated among ducks in Eastern China in 2010–2011, and N9 of H7N9 is derived from avian H2N9 and/or H11N9 viruses isolated among wild migratory birds along the East Asian flyway [50, 52]. Both of them are Eurasian lineage of avian viruses, and reassortment of gene (so-called as antigenic shift) may happen and produce the possible precursor of H7N9 virus. Six viral genes except H7 and N9 of H7N9 virus likely originated from chicken H9N2 sub-lineages 1 and 2 circulating in Eastern China [50, 52]. Antigenic shift is suggested to form H7N9 virus during the generation and evolution of viruses. In the analysis of H7N9, viral sequences revealed that H7N9 virus possesses leucine or isoleucine at position 226 of HA which binds to both sialic acid-α2,3-galactose (avian-type receptor) and sialic acid-α2,6-galactose (human -type receptor) [53]. This may mean as the avian virus infection to humans in addition to expression of sialic acid-α2,6-galactose in the upper respiratory tract and sialic acid-α2,3-galactose in the lower respiratory tract in humans [19, 21–25]. According to PB2 protein of H7N9, sequence analysis revealed that many isolated H7N9 viruses encode lysine at position 627 of PB2 [54]. This substitution of glutamic acid (E) to lysine (K) of position 627 of PB2 (E627K mutation) may enable avian viruses to adapt to efficient replication at 33 and 37°C of human temperature lower than original avian host temperature (41°C) [55, 56]. Other mutations of position 627 of PB2 in these viruses have been reported and investigated [56]. The resistant H7N9 variants encoding the R294K mutation in NA that confers resistance to oseltamivir have been detected in patients treated with neuraminidase inhibitors (NAIs). R294K substitution was revealed as multidrug resistance with extreme oseltamivir resistance by the study of using protein- and virus-based assays [57]. On the other hand, K294 mutation of H7N9 infections was found. Compared with H7N9 viruses containing K294 which leads to mild resistance to NAIs, R294K appeared in clinical course under the selective pressure of oseltamivir treatment and conferred not only multidrug resistance to NAIs but also decreases NA activity and impaired virus replication. These phenomena may explain the failure for predominance of R294K-mutated strains of H7N9 in this outbreak. But the drug-resistant mutations should be monitored vigilantly in order to help the most effective drug administration and the prevention of unnecessary loss of human lives in clinical course [57]. Recently LPAI H7N9 viruses have evolved to HPAI viruses and caused the increased morbidity and mortality in poultry, and the same phenomenon has more likely occurred in human infections [51].

#### **2.4 Avian influenza A H7N7 viruses**

Outbreaks of HPAI virus infections mostly originate from endemic LPAI viral strains of H5 and H7 subtypes among wild waterfowl. These LPAI virus strains can transmit from waterfowl to poultry and change virulence to HPA virus among poultry [58]. In the Netherlands, an epidemic of HPAI virus H7N7 infection occurred in 2003 that resulted in 255 affected flocks and culling (killing and removing of infected poultry flocks) of 30 million birds [59]. Control measures

*Viruses and Viral Infections in Developing Countries*

Thai and Indonesian isolates [43].

**2.3 Avian influenza A H7N9 viruses**

avian-like virus that has adapted to humans. But the 1918 influenza virus was analyzed as 10 amino acids change the polymerase complex including the lysine residue at position 627 [31]. These mutations require more investigation for the precursors of a new influenza pandemic. PB1-F2 is a small mitochondrial protein that is encoded on an open reading frame of PB1 [32]. This open reading frame is highly conserved in avian influenza isolates [33]. The reconstructed 1918 influenza virus linked to the PB1-F2 protein and recombinant virus with a single mutation in the PB1-F2 protein [serine (S) instead of asparagine (N) at position 66] of H5N1 (Hk/97) increase viral pathogenicity [34, 35]. PB1-F2 may induce apoptosis of immune cells and an insufficient adaptive immune response [35]. The NS proteins (NS1 and NS2) are viral proteins, and NS1 has multiple functions including splicing and nuclear export of cellular mRNA as well as stimulation of translation [36]. The NS1 protein is crucial for evading the innate immune response of the host by inhibiting the antiviral response mediated by type I interferons [37]. The presence of glutamic acid at position 92 of NS1 has been investigated to be a prerequisite for the resistance to antiviral cytokines considering the results of severe disease induction by inoculation of reassortant influenza virus (H1N1) bearing the NS gene of the H5N1/97 virus to pigs and the lack of glutamic acid at position of the NS1 protein in the recent human and avian isolates [38–41]. Two PDZ ligand (PL) sequence motifs (a Glu-Pro-Glu-Val (EPEV), a Glu-Ser-Glu-Val (ESEV)) were detected in viruses isolated during 1997 but not in viruses after 1997 as a potential codominants of virulence disrupting

several PDZ-domain protein-mediated pathways for cell signaling [33].

M1 and M2 are matrix proteins, and M2 is a 97-residue single-pass membrane protein with its N-terminus directed toward the outside of the virus. It determines proton selectivity and unidirectional conductance of the channel by mutagenesis studies [42]. A serine to asparagine substitution at residue 31 of M2 protein is associated with resistance to adamantanes which were used for therapy of influenza A virus infections [43]. On the other hand, M2 protein is suggested to be a candidate for the adaptation of the virus to a new host in addition to PB1-F2 from a study of

Avian influenza viruses sometimes infect poultry as LPAI, and there is no concern to LPAI, but even LPAI suddenly caused the sporadic infection among humans which started on February 19, 2013, in Shanghai [44]. There was no epidemic of severe disease in poultry infections with H7N9 before human infections with H7N9. In April 2013, the number of human cases of H7N9 virus infections increased significantly, reaching 125 confirmed cases in China, which was suggested to be derived from contacting with poultry and/or contaminated environments in farmers' markets with live birds [45]. After closure of live birds' markets, the number of new human cases infected with H7N9 declined rapidly. But the second wave of human infections with H7N9 virus started again owing to the lower fall temperature and reopening of poultry markets in the fall of 2013. This wave is characterized by the extensive geographic spread from the province of Eastern China to Southern China and a large number of human infections with H7N9 (440 human infections and 122 associated death of 440 as of May 16, 2014) [46]. For the protection of widespread of H7H9, two strategies composed of suspending farmers' markets with live birds and stopping the transport for trading of live birds carrying the virus were applied by provincial and Chinese authorities [47, 48]. These measures controlled the more spread of H7N9 virus infections in China, but the potential benefits of public health measures should be balanced against the potentially significant societal and economic costs [49]. The lack of protective antibodies in human against

**98**

were applied to HPAI virus infection in poultry leading to the potential start of human influenza pandemic, such as culling and banning the movement of infected flocks and tracing and screening of the infection which were implemented, followed by preemptive culling of flocks in a 1-km zone around the infected flock. In this HPAI viral epidemic in poultry, 86 poultry workers and 3 household contacts were identified to be affected with H7N7 by RT-PCR, and the main symptom of this infection was conjunctivitis (83/89 persons) and one fatal case happened [60, 61]. Since 2006–2011 LPAI virus infection in the Netherlands (2006) and in Germany (2011) and HPAI virus infection in England (2008) and in Spain (2009), H7N7 viral infections were reported. These show the difficulty in the elimination of epidemic avian influenza viral infection from the endemic avian influenza virus infection among wild waterfowls. As additional measures, vaccination for poultry in outbreaks of H7N7 significantly reduced the excretion of viruses, which may reduce virus spread in infected areas and the risk of human exposure to viruses, in Italy (2000) [62]. Compared with the H7N1 and H7N3 episodes leading no bird to human transmission in Italy, in the genetic analysis of H7N7, HA gene of H7N7 is quite similar to HA genes of H7N1 and H7N3. So other mutations in NA or internal genes of H7N7 virus including E627K in PB2 except HA may determine the capacity to replicate and cause disease in humans. Although H7N7 virus may replicate well in the human cells near the eyes, it may not well replicate in the human upper respiratory tract [61].
