**2. Avian influenza in humans**

**1. Introduction**

60 Influenza - Therapeutics and Challenges

Influenza viruses belong to the family *Orthomyxoviridae*. These are RNA-containing viruses possessing a negative fragmented genome. To date, there are four types (serotype) of influenza viruses—influenza A, B, C, and D. Influenza A viruses affect humans and a wide range of mammals (horses, pigs, dogs, wild and domestic cats, seals, ferrets) and birds (chickens, wild waterfowl, gulls, etc.). Only influenza A viruses are known as causative agents of severe epidemics and pandemics. The antigenic properties of influenza A viruses are based on two

Wild waterfowl are considered as a natural reservoir of influenza A viruses which is characterized by high divergence. The 16 HA subtypes and nine NA subtypes were detected in migratory waterfowl and poultry [1]. Sometimes, avian influenza viruses overcome the interspecies barrier and infect poultry and mammals. Avian influenza viruses of subtypes H5N1, H7N3, H7N7, H7N9, and H9N2 may become pathogenic for humans and occasionally cause very severe infections. As part of an influenza pandemic preparedness program, the World Health Organization (WHO) analyzes a range of zoonotic and potentially pandemic influenza viruses for the development of appropriate vaccines as seasonal influenza vaccination does

After isolation of the first influenza viruses in 1933–1936, the development of influenza vaccines in England, the United States, Australia, and in the USSR began. The development of active immunization against influenza using live attenuated vaccines was conducted in Russia under the leadership of A.A. Smorodintsev since 1937, and in the USA since 1960, where the group of H.F. Maassab also obtained cold-adapted attenuated variants of influenza viruses A and B. At present, two types of LAIVs are commercially available. The first, based on cold-adapted master donor viruses (MDVs) A/ Leningrad/134/17/57 (H2N2) and B/USSR/60/69 [3–5], was licensed in 1987 for the people 3 years and older as Ultravac (Microgen, Russia). The second, known as FluMist based on cold-adapted MDVs, A/Ann Arbor/6/60ca (H2N2), and B/Ann Arbor/1/66ca, was licensed in 2003 (MedImmune, Inc., USA). FluMist is used for the prevention of influenza in persons younger than 49 and older than 2 years of age [6]. According to World Health Organization (WHO), vaccination prevents influenza in 80–90% of vaccinated people, and the economic effect of influenza vaccinations is 10–20 times higher than the cost of vaccination. In the past 10 years, attention was paid due to the advantages of LAIV that cause the formation of systemic and strong local (secretory) immunity. By contrast, parenteral inactivated influenza vaccines (IIV) stimulate mainly the formation of serum strain-specific antibodies which offer only limited protection against newly emerging viruses [7]. Intranasal implementation of LAIV produces immune response similar to natural infection and therefore induces an earlier, broader, and more long-lasting protection than inactivated vaccines [8]. Besides, the cost of live vaccine is five times less than inactivated vaccine, and the productivity of the biotechnological production pro-

cess is significantly higher which is also important in the event of pandemic.

surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA).

not protect against pandemic avian influenza viruses [2].

Most avian viruses are initially low virulent for birds, causing only transient asymptomatic intestinal infections in wild waterfowl [9]. Viruses of subtypes H5 and H7 can be widespread among poultry, while acquiring the increased pathogenicity. This was observed during outbreaks caused by H5N2 viruses in 1983 or 1994–1995 in North America [10, 11], subtype H7 (H7N7 or H7N2)—in Europe and in Australia [12]. For the first time, "bird plague," a disease caused (as is now known) by highly pathogenic influenza viruses, was described in 1878 during an outbreak among chickens in Italy. The outbreak causative agent was isolated in 1902 (virus A/Chicken/Brescia/1902 (H7N7)). During similar outbreaks, repeatedly observed in Europe and around the world, several other viruses of H7 subtype were isolated. In 1955, those viruses were identified as belonging to a group of influenza viruses [13]. The first of the highly pathogenic (HP) viruses of the H5N3 subtype—the A/Tern/South Africa/61—was isolated in 1961 [14]. HP avian influenza viruses can cause a mass death of chickens in a short time as a result of dissemination of infection in poultry with rapidly progressive neurologic symptoms, diarrhea, and fatal outcome. Until 1997, there was no obvious evidence of direct infection of humans with avian viruses. Nevertheless, serological studies revealed the presence of antibodies against avian viruses of various subtypes in human sera in southern China, Hong Kong, and East Asia, indicating exposure of some people to avian influenza viruses [15].

#### **2.1. H5N1 influenza viruses**

For the first time, attention to H5 avian influenza viruses as possible pandemic agents was brought in May 1997 in Hong Kong during a mass outbreak among chickens when the avian virus H5N1 was isolated from a child who died from viral pneumonia [16]. To the end of 1997, an infection with the virus H5N1 similar to poultry viruses identified in the region was confirmed in another 17 people, five of whom died [17].

It is possible that before the appearance of the virus H5N1 in humans, a series of reassortments during the circulation of a number of precursor viruses in birds have occurred. Thus, HA of H5N1 viruses isolated from humans were almost identical to those of the A/Goose/ Guandong/1/96 (H5N1) [18], and NA may have been acquired from the virus H6N1 [24]. It is assumed that the internal genes were borrowed from the same H6N1 virus or H9N2 A/Qail/ Hong Kong/G1/97 (H9N2) influenza viruses during transmission from waterfowl to quails and chickens [19].

The mechanisms of avian influenza viruses "step-by-step" adaptation to new hosts are well characterized [20]. The change in host cell specificity and the increase in the pathogenicity of influenza viruses can be influenced either by amino acid substitutions in the receptor binding site of HA or by substitutions affecting the conformation and steric availability of this center. In particular, this can be influenced by changes in the number of glycosylation sites or their localization. High pathogenicity of avian influenza viruses in mammals is polygenic in nature. The HA of H5 or H7 HP viruses with a polybasic cleavage site is known as a primary virulence factor, although the unusual severity of clinical manifestations during human infection with influenza H5N1 viruses can also be associated with mutations in internal proteins (PB1-F2, PB2) and non-structural (NS) proteins.

2013, there were 1566 cases of avian influenza H7N9 in the world, of which 613 (39%) were fatal [28]. At the same time, 88% of the infected developed severe pneumonia, 68% was hospi-

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Experts believe that the virus H7N9 is not likely transmitted from person to person, but can spread with prolonged contact, especially when people care for sick family members. Moreover, the reassortment of several viruses is also not excluded. Genome analysis of human-isolated H7N9 viruses has shown adaptive evolution and convergent changes in eight viral genes, including sites in the PB2 gene (Q591K, E627K, and D701N), in HA (R156K, V202A, and L244Q), and in NA (R289K). These substitutions are known as playing a role in

The H9N2 influenza viruses readily transmit from birds to animals and humans due to the easy appearance of variants that have an affinity for sialic receptors in mammals [30]. Sero-epidemiological studies revealed antibodies to viruses H9N2 among 15% of poultry workers in China [31]. Viruses H9N2 were isolated from people with symptoms of respiratory infection in Hong Kong and China from 1997 to 2009 [32] belonging mainly to the antigenic G1 line, unlike other H9N2 viruses isolated from swine and poultry belonging to the antigenic variety G9 [33]. Phylogenetic analysis showed that after 1994, Eurasian H9N2 after complex genetic reassortment of G1 and G9 viruses circulating among wild and domestic birds formed several antigenic lines [34]. The H9N2 viruses, which caused human cases, were not HP as they did not possess highly cleavable HA and were not highly virulent for poultry, although molecular analysis demonstrated similarity of genes for internal proteins with HP H5N1 viruses, which caused an outbreak among people in 1997 [35]. Due to the fact that the avian influenza viruses of the H9 subtype are transmitted to humans, have genetic similarity to the H5N1 viruses, and are widespread in Asia, Europe, and the Middle East, the WHO has included H9N2 vaccine development in the

Serological studies in Southern China revealed that 13% of people from different provinces have antibodies to the influenza virus of H6 subtype [37]. Phylogenic analysis of influenza A viruses indicates that the closely related genes coding the internal proteins could be found in influenza A viruses of different subtypes and that the reassortment between the avian and human influenza viruses is possible [38]. It was also shown that some of the fragments of the NP and NA genes of highly pathogenic H5N1 viruses originated from the H6 virus of wild ducks [18, 39]. Therefore, the avian influenza viruses of H6N1 subtype may represent a

Thus, various avian influenza viruses can pose a threat to humans that necessitates the development of a corresponding vaccine strain for the protection of humans from possible infection. As part of an influenza pandemic preparedness program, the WHO monitors the

talized in the intensive care unit. Mortality in different years ranged from 31 to 39%.

crossing species barriers from avian to human [29].

overall plan for pre-pandemic training [36].

**2.4. H6N1 influenza viruses**

potential danger for humans.

**2.3. H9N2 influenza viruses**

From 1997 to 2001, the HA of H5N1 viruses remained antigenically conserved, although, since 2003, there has been an unusually high level of H5N1 viruses evolution. The HP H5N1 viruses isolated from poultry and humans separated into three branches that differ antigenically and genetically [21]. During the outbreak in 2005 on Lake Qinghai, a number of HP H5N1 viruses were isolated from wild waterfowl [22]. This may indicate a reverse drift of similar viruses from poultry to wild birds, which was not observed previously. Along with H5 HA evolution, the extensive reassortment of avian influenza viruses in birds in China resulted in new H5 viruses possessing different NA subtypes (H5N2, H5N5, H5N6, and H5N8) and internal protein genes. In 2014, HA gene segments of H5N1, H5N2, H5N5, H5N6, and H5N8 were designated as clade 2.3.4.4., which were detected in birds in 40 countries in Africa, Asia, and Europe [23].

WHO has consistently recorded cases of human infection caused by HP influenza H5N1 virus, many of which had fatal outcomes. The clinical features of human infection caused highly pathogenic H5N1 viruses are characterized not only by primary viral pneumonia but also by complications with acute distress syndrome and poly-organ lesions [24].

At present, cases of human infection with the avian influenza H5N1 virus were decreased compared to the early 2000s. From 2003 to 2009, 468 cases of this disease were registered in 16 countries, mainly in Vietnam, China, Indonesia, Thailand, and Egypt. In 2010–2014, the number of cases was two times decreased (233 people). In 2016, the virus continued to infect people in only one country—Egypt (10 cases, three of them with a fatal outcome). In 2017, again in Egypt three cases were recorded, one of which was fatal. Thus, even when the absolute number of cases was decreased, mortality remains extremely high. In total, according to WHO data, by mid-2017, 859 people were infected with influenza H5N1, 453 (53%) from which died [25].

During the outbreak in Hong Kong in 1997, there was no direct evidence of a sustained human-to-human transmission of H5N1 viruses, although antibodies against H5 viruses were detected in 3.7% of physicians who had contact with H5-infected patients [26]. In 2008, transmission of an infection with avian influenza H5N1 from a son to his father was registered in China [27]. Under conditions of the continuous appearance HP H5N1 viruses in the humans, there is a risk of such a transmission during close contacts.

#### **2.2. H7N9 influenza viruses**

On March 31, 2013, the first three cases of human infection with the avian influenza H7N9 virus were registered in China. In all three cases, an infection of the respiratory tract was complicated with severe pneumonia. Two patients died, the third was in a critical condition for a long time, but recovered. Since then, the number of laboratory-confirmed cases in China has increased every day. In addition to severe and lethal cases, the sero-diagnostics methods have proved the asymptomatic course of the disease in workers of poultry farms. From March 2013, there were 1566 cases of avian influenza H7N9 in the world, of which 613 (39%) were fatal [28]. At the same time, 88% of the infected developed severe pneumonia, 68% was hospitalized in the intensive care unit. Mortality in different years ranged from 31 to 39%.

Experts believe that the virus H7N9 is not likely transmitted from person to person, but can spread with prolonged contact, especially when people care for sick family members. Moreover, the reassortment of several viruses is also not excluded. Genome analysis of human-isolated H7N9 viruses has shown adaptive evolution and convergent changes in eight viral genes, including sites in the PB2 gene (Q591K, E627K, and D701N), in HA (R156K, V202A, and L244Q), and in NA (R289K). These substitutions are known as playing a role in crossing species barriers from avian to human [29].

#### **2.3. H9N2 influenza viruses**

virulence factor, although the unusual severity of clinical manifestations during human infection with influenza H5N1 viruses can also be associated with mutations in internal proteins

From 1997 to 2001, the HA of H5N1 viruses remained antigenically conserved, although, since 2003, there has been an unusually high level of H5N1 viruses evolution. The HP H5N1 viruses isolated from poultry and humans separated into three branches that differ antigenically and genetically [21]. During the outbreak in 2005 on Lake Qinghai, a number of HP H5N1 viruses were isolated from wild waterfowl [22]. This may indicate a reverse drift of similar viruses from poultry to wild birds, which was not observed previously. Along with H5 HA evolution, the extensive reassortment of avian influenza viruses in birds in China resulted in new H5 viruses possessing different NA subtypes (H5N2, H5N5, H5N6, and H5N8) and internal protein genes. In 2014, HA gene segments of H5N1, H5N2, H5N5, H5N6, and H5N8 were designated as clade 2.3.4.4., which were detected in birds in 40 countries in

WHO has consistently recorded cases of human infection caused by HP influenza H5N1 virus, many of which had fatal outcomes. The clinical features of human infection caused highly pathogenic H5N1 viruses are characterized not only by primary viral pneumonia but

At present, cases of human infection with the avian influenza H5N1 virus were decreased compared to the early 2000s. From 2003 to 2009, 468 cases of this disease were registered in 16 countries, mainly in Vietnam, China, Indonesia, Thailand, and Egypt. In 2010–2014, the number of cases was two times decreased (233 people). In 2016, the virus continued to infect people in only one country—Egypt (10 cases, three of them with a fatal outcome). In 2017, again in Egypt three cases were recorded, one of which was fatal. Thus, even when the absolute number of cases was decreased, mortality remains extremely high. In total, according to WHO data, by mid-2017, 859 people were infected with influenza H5N1, 453 (53%) from

During the outbreak in Hong Kong in 1997, there was no direct evidence of a sustained human-to-human transmission of H5N1 viruses, although antibodies against H5 viruses were detected in 3.7% of physicians who had contact with H5-infected patients [26]. In 2008, transmission of an infection with avian influenza H5N1 from a son to his father was registered in China [27]. Under conditions of the continuous appearance HP H5N1 viruses in the humans,

On March 31, 2013, the first three cases of human infection with the avian influenza H7N9 virus were registered in China. In all three cases, an infection of the respiratory tract was complicated with severe pneumonia. Two patients died, the third was in a critical condition for a long time, but recovered. Since then, the number of laboratory-confirmed cases in China has increased every day. In addition to severe and lethal cases, the sero-diagnostics methods have proved the asymptomatic course of the disease in workers of poultry farms. From March

there is a risk of such a transmission during close contacts.

also by complications with acute distress syndrome and poly-organ lesions [24].

(PB1-F2, PB2) and non-structural (NS) proteins.

Africa, Asia, and Europe [23].

62 Influenza - Therapeutics and Challenges

which died [25].

**2.2. H7N9 influenza viruses**

The H9N2 influenza viruses readily transmit from birds to animals and humans due to the easy appearance of variants that have an affinity for sialic receptors in mammals [30]. Sero-epidemiological studies revealed antibodies to viruses H9N2 among 15% of poultry workers in China [31]. Viruses H9N2 were isolated from people with symptoms of respiratory infection in Hong Kong and China from 1997 to 2009 [32] belonging mainly to the antigenic G1 line, unlike other H9N2 viruses isolated from swine and poultry belonging to the antigenic variety G9 [33]. Phylogenetic analysis showed that after 1994, Eurasian H9N2 after complex genetic reassortment of G1 and G9 viruses circulating among wild and domestic birds formed several antigenic lines [34]. The H9N2 viruses, which caused human cases, were not HP as they did not possess highly cleavable HA and were not highly virulent for poultry, although molecular analysis demonstrated similarity of genes for internal proteins with HP H5N1 viruses, which caused an outbreak among people in 1997 [35]. Due to the fact that the avian influenza viruses of the H9 subtype are transmitted to humans, have genetic similarity to the H5N1 viruses, and are widespread in Asia, Europe, and the Middle East, the WHO has included H9N2 vaccine development in the overall plan for pre-pandemic training [36].

#### **2.4. H6N1 influenza viruses**

Serological studies in Southern China revealed that 13% of people from different provinces have antibodies to the influenza virus of H6 subtype [37]. Phylogenic analysis of influenza A viruses indicates that the closely related genes coding the internal proteins could be found in influenza A viruses of different subtypes and that the reassortment between the avian and human influenza viruses is possible [38]. It was also shown that some of the fragments of the NP and NA genes of highly pathogenic H5N1 viruses originated from the H6 virus of wild ducks [18, 39]. Therefore, the avian influenza viruses of H6N1 subtype may represent a potential danger for humans.

Thus, various avian influenza viruses can pose a threat to humans that necessitates the development of a corresponding vaccine strain for the protection of humans from possible infection. As part of an influenza pandemic preparedness program, the WHO monitors the number of zoonotic and potentially pandemic influenza viruses to schedule candidates for the development of appropriate vaccines [40].

acid residues (lysine and arginine) in the proteolytic cleavage site [47], which causes effective cleavage of HA by intracellular proteases expressed in most organs and tissues of birds and mammals. Unlike HP avian influenza viruses, non-pathogenic viruses contain a single arginine residue (R) in the cleavage site [44]. For non-pathogenic viruses proteolytic activation, the presence of trypsin-like enzymes is required, which is expressed by a limited range of cells and is

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The reassortant vaccine strains were prepared in the Virology Department, Institute of Experimental Medicine, using classical genetic reassortment in CE as previously described [48]. The H5N2 reassortant virus inherited only the HA gene from the H5N2 parent virus, and the remaining seven genes from the Len/17 MDV (7,1 genome composition) [49]. The reassortants of subtypes H7N3, H9N2, and H6N1 inherited the HA and NA from parental avian influenza viruses (6,2 genome composition). All the reassortant strains were studied for temperaturesensitive (*ts-*) and cold-adapted (*ca-*) phenotype [49–52]. For those purposes, the reassortant viruses were propagated in CE for 2 days at 25, 34, and 40°C. The yield of "wild-type" avian influenza viruses at 40°C was the same or greater than at 34°C. Only when the temperature was increased to 41°C, the reproduction of these strains was partially limited. Thus, the high degree of temperature resistance of all the above viruses was demonstrated. In contrast to parental avian viruses, all vaccine candidates poorly reproduced at 40°C in titers not exceeded 1.5–1.8 log10 EID50/ml. At the same time, these reassortant strains grew well at low temperatures. Thus, all obtained reassortants acquired the genes of internal and nonstructural proteins from the A/Leningrad/134/17/57 (H2N2) MDV inherited the *ts-* and *ca-*phenotype. The pronounced difference in optimal reproductive conditions between the temperature-resistant viruses of avian influenza and the cold-adapted attenuation donor is due to the properties of viral polymerases [53]. This difference in the temperature optimum of the parental viruses may facilitate the isolation of the reassortant viruses possessing the desired gene composition

The ability of LAIV to induce antibodies not only to the homologous variant subtype but also to cross-reacting antibodies to antigenically different variants including HP variants was

Among all vaccine candidates based on non-pathogenic avian influenza viruses, the H6N1 LAIV was characterized by the highest HI titers in mice after a single administration (GMT = 17.4). The LAIV of H7N3 subtype raised serum antibodies not only against the homologous virus but also against H7N9, which possessed the difference of 3% in the HA amino acid sequence. In the sera from mice double-vaccinated with H7N3 LAIV, serum HI titers against H7N9 were 20–40 times higher than against H7N3 (P < 0.05) [56]. At the same time, local IgA levels were higher against homologous H7N3 compared with H7N9 after vaccination with LAIV. The H5N2 LAIV induced detectable HI and neutralizing antibody titers only against the homologous H5N2 virus, perhaps due to the genetic differences between H5N2 vaccine strain and infectious viruses H5N1 isolated in 1997, 2003, and 2005 (10–12%

found in the airways.

after selective passages at a lower temperature.

shown in several mouse studies [50–52, 54–56].

differences of the HA1 amino acid sequence).

**3.2. Immunogenicity and cross-protection in mice**
