Role of Genetic and Molecular Dynamics in the Emergence, Reemergence, and Interspecies Transmission of Equine Influenza Viruses

C H A P T E R

34 Role of Genetic and Molecular Dynamics in the Emergence, Reemergence, and Interspecies Transmission of Equine Influenza Viruses Mohamed Boukharta1, Hamid El Amri1, Fathiah Zakham2 and Moulay Mustapha Ennaji2 1

Genetic Laboratory of Royal Gendarmerie, Rabat, Morocco 2Laboratory of Virology, Microbiology, Quality, Biotechnologies/Eco-Toxicology and Biodiversity, Faculty of Sciences and Techniques, Mohammedia, University Hassan II of Casablanca, Casablanca, Morocco

ABBREVIATIONS M2e NA NEP NP NS PF RBD RBS vRNPs

ectodomain ion-channel protein M2 neuraminidase nuclear export protein nucleoprotein nonstructural fusion peptide RNA-binding domain receptor-binding site ribonucleoprotein viral complex

Emerging and Reemerging Viral Pathogens DOI: https://doi.org/10.1016/B978-0-12-819400-3.00034-X

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© 2020 Elsevier Inc. All rights reserved.

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INTRODUCTION Equine influenza is an infectious and contagious disease of the upper respiratory tract in horses, donkeys, and their crossbred products (Barquero et al., 2007; Gurkirpal, 1997). This disease causes significant economic losses that weigh heavily on the equine value production due to the drop in performance and the persistence of pulmonary sequelae (Lai et al., 2004; Lewis et al., 2011). The causative agent is a virus that belongs to the family Orthomyxoviruses (Orthomyxoviridae) and the genus Influenza A virus. Two distinct viral subtypes of equine influenza virus emerged in 1957 and 1963, respectively, prototypes A/equine/Prague/1/56 (H7N7) and A/equine/Miami/1/63 (H3N8) (Waddell et al., 1963; Sovinova et al., 1958). These two equine influenza viruses, in particular, the H3N8 subtype, have two characteristics. First, their genetic composition changes as they replicate in equids, leading to the emergence of new antigenic variants that are incompatible with the vaccine strains (Oxburgh and Hagstrom, 1999); second, the risk of the emergence of H3N8 influenza viruses of equine origin in other mammalian species, especially dogs. In January 2004, after the outbreaks of acute respiratory symptoms reported in 22 dogs racing in Florida, United States, the two viruses A/ ca/Florida/43/2004 and A/ca/Iowa/13628/2005 were isolated. The genetic analysis of their sequences indicated a great similarity with the strain of equine influenza (H3N8) that had been circulating in the United States since the 1990s (Buonavoglia and Martella, 2007). This suggested a transmission event from the horse to the dog, which is probably facilitated by direct contact between these two species (Payungporn et al., 2008). Also in North America, a retrospective study showed that there were traces of antibodies directed against the equine virus (H3N8) in the serum of certain dogs before the discovery of the first virus in 2004. This serological feature was confirmed by the isolation of strain A/ca/ Florida/242/03 from conserved tissues of a greyhound that died of hemorrhagic bronchopneumonia in 2003 (Kruth et al., 2008). In Canada, Ontario, a seroprevalence study of canine influenza was conducted on 225 samples obtained from dogs and showed a seroprevalence level of 1/225. The only dog that showed evidence of antibodies was a racing greyhound of American origin (Florida) (Songserm et al., 2006). In England a retrospective study showed an acute respiratory illness in 92 foxhounds in 2002, and it was a canine flu. The respiratory tract cells of dead dogs had specific receptors for H3N8 equine viruses (Daly et al., 2008). In China, phylogenetic analysis of two porcine influenza isolates revealed that high homology to equine influenza (H3N8) viruses of European lineage circulated in the 1990s (Tu et al., 2009).

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The abovementioned examples clearly showed the expansion of the equine virus (H3N8) within the animal kingdom and its potential emergence in other mammalian species. We could even solve the potential risk of transmission of these viruses to humans, given the role of gene exchange in pigs between different lineages (avian and mammalian) (Taubenberger and Morens, 2010; Taubenberger and Kash, 2010). The horses no longer played the role of epidemiological Dead end of equine influenza viruses, especially for the subtype (H3N8). In this chapter, we will present the structural and functional characteristics of the influenza A viruses; the pathological particularities of the equine influenza disease; the therapeutic, preventive, and curative means to counteract it; and the virulence determinants involved in the occurrence of the viral emergence and reemergence.

INFLUENZA VIRUS Taxonomy and Classification According to the International Committee on Taxonomy of Viruses, influenza viruses belong to the Orthomyxovirus family (from the Greek word “orthos”: exact and “myxa” refers to mucus), which refers to their ability to infect the upper respiratory tract (Huraux et al., 2003; Zientara, 2001). The term “myxovirus” was first proposed in 1955 by Andrews et al. for the group of viruses with similar physical, chemical, and morphological properties. The prefix “myxovirus” indicates an affinity for some mucins (Rice, 1961). Currently, this family comprises five genera, including three influenza viruses (so-called types): Influenza A virus, Influenza B virus, and Influenza C virus, which are usually responsible for influenza illness in many bird and mammal species. The other two genera Thogotovirus and Isavirus do not cause influenza symptomatology in the infected host but were recently included in the Orthomyxovirus family because of their morphological and molecular similarities to influenza viruses. The Thogotovirus (Dhori and Thogotovirus) viruses are transmitted by ticks, and they infect vertebrates and sometimes human, while the only virus that belongs to the genus Isavirus is transmitted through water and causes anemic infections in fish (salmon) (Calisher, 2009). Influenza A, B, and C viruses were isolated in many birds and mammal species, and they have no cross-immunity. Each type is defined by the antigenic characteristics of its internal proteins: the nucleocapsid (NP) and the matrix (M1) (Castillo Alvarez et al., 2008). Influenza A virus was isolated for the first time in 1931 in a pig by the American, Richard Shope (Taubenberger and Morens, 2010;

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Taubenberger and Kash, 2010). A few years later, influenza B and C viruses were found in humans in 1940 and 1947, respectively (Smith, 1952). In addition, sporadic infections with influenza B and C viruses have been reported in horses, swine, dogs, and cattle (Dea et al., 1980). The influenza A and B viruses are phylogenetically similar, their genome is segmented into eight so-called mini-chromosome parts, while the type C viruses have only seven (Hongo et al., 1999). Surface glycoproteins are distinct structures for type A and type B viruses. Thus they are grouped into a single hemagglutinin (HA) esterase for type C viruses. Until now, only infection with types A and B causes morbidity, sometimes mortality in humans and animals (Biere et al., 2010). Influenza B is associated with sporadic and sometimes epidemic influenza, which is of less importance than influenza A. Influenza C is the most often sporadic and asymptomatic virus (Vabret et al., 2010). Influenza A viruses are frequently found in aquatic and wild birds, domestic poultry, and many mammal species (humans, horses, dogs, pigs, etc.) (Fouchier et al., 2003). They have been classified into subtypes according to the antigenic characteristics of two surface glycoproteins [HA and neuraminidase (NA)], which have a determining biological role (Kaverin et al., 2000). The emergence of the two subtypes currently isolated in equines is a result of interspecies transmission from birds to horses.

Morphology and General Structure The viral particles are highly pleomorphic, spherical (80
120 nm in diameter), ovoid, or filamentous (Burnouf et al., 2004; Nayak and Baluda, 1967). Two glycoproteins such as HA and NA are present on the viral surface. The schematic representation of influenza A virus is shown in Fig. 34.1A. Each virion consists of an envelope, genetic material (genome), and proteins. The envelope is the structure that delimits the virus; the genetic material contains the information that codes for the synthesis of new proteins, according to their functions, in the process involved in replication, transcription, and assembly of virions. These proteins are engaged in interactions with cellular proteins involved in cellular immune system (Noah and Krug, 2005). Fig. 34.1B shows the images of influenza viruses observed under an electron microscope with different forms coexisting in the same viral sample (Dea et al., 1980).

Viral Envelope The envelope is attached to the viral surface glycoproteins (HA and NA) and the nucleocapsid segments inside the virion. Its inner face is

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FIGURE 34.1 (A) Schematic representation of influenza A virus (Noah and Krug, 2005, license code 4342390642284). (B) Influenza viruses observed under the electron microscope (Dea et al., 1980; https://www.ncbi.nlm.nih.gov/pmc/about/copyright/). One of the morphological features of influenza viruses is the presence on their surface of about 500 spicules of transmembrane glycoproteins that form an opaque band around the virions (black arrows).

covered by a polymer of a Matrix protein M1. The M2 matrix protein passes through the viral envelope and acts as an ion channel. This viral protein is involved in the early phase of viral infection and the acidification of the interior of the virion, which is a key step in the release of vRNPs (viral ribonucleic complex) into the cytoplasm (Goffard et al., 2006). Proteins embedded in the HA, NA, and M2 envelope are synthesized late in the infection cycle by ribosomes attached to endoplasmic reticulum.

Viral Genome The genome is composed of eight segments of (2)ssRNA (890
2341 nucleotides) with a total molecular weight of 13.6 kb and is protected by a nucleoprotein that confers helical symmetry by polymerization at each segment. Each of the six segments encodes a protein HA, NA, nucleoprotein (NP), three polymerases: PA, PB1, and PB2 (A for acid and B for basic), segment 7 codes for two proteins of the matrix (M1 and M2), and segment codes 8 for both nonstructural (NS1) and (NS2) proteins and [NEP (nuclear export protein)] (Fig. 34.2). For the most influenza viruses, RNA segments encode 10 proteins, although the 11th protein (PB1-F2) can be expressed by some strains recognized as very virulent (Burnouf et al., 2004).

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Taille (aa)

759

757

716

498 454

252

NP

230

1

2

3

4

5

6

7

8

9

NS2

121 NS1

96 M2

M1

NA

HA

PA

PB1

PB2

566

10

FIGURE 34.2 Diagram illustrating the size of the eight RNA segments of influenza A virus and associated proteins.

Viral Proteins According to the location of their synthesis, viral proteins are classified into external and internal proteins. The former corresponds to the viral proteins HA, NA, and the ion channel M2 that are present on the surface of the viral particle and embedded in the envelope. These proteins have been synthesized by ribosomes linked to the endoplasmic reticulum. Then, the Golgi system is used to insert the proteins into the plasmic membrane and therefore called late proteins due to their synthesis latency during the viral cycle (Webster et al., 1992). The so-called internal proteins are synthesized by free ribosomes in the cytoplasm and are the early NP proteins, the three subunits of the polymerase (PB1, PB2, and PA), NS1, and the two late proteins M1 and NEP (Bouvier and Palese, 2008). A detailed description of the HA (HA1, HA2), NA, NS, and NP proteins is necessary for a good understanding of the virulence mechanism of equine influenza viruses.

Hemagglutinin General Structure HA is a glycoprotein with an ability to agglutinate erythrocytes. It is encoded by segment 4 of the viral genome and synthesized in its uncleaved initial form (HA0) (75 kDa, 550 amino acids) by ribosomes bound to endoplasmic reticulum (Louisirirotchanakul et al., 2007). When transported to the cytoplasmic membrane of the infected cell, it

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FIGURE 34.3 Linear representation of HA protein (HA1 and HA2 subunits) in the equine influenza virus PF. HA, Hemagglutinin; PF, fusion peptide.

undergoes glycosylation and additions of palmitate residues (Webster et al., 1992). The combination of three HA0 monomers forms a homotrimer projected as spicules on the surface of the viral particle 135 A long and a triangular cross section of 15
40 A (Wilson et al., 1981). It is cleaved at arginine 329 by intracellular proteases into two polypeptides of 328 amino acids (50 kDa, HA1) and 221 amino acids (25 kDa, HA2), linked together by a disulfide bridge between residues 14 of HA1 and 137 HA2 (Bullough et al., 1994; Chen et al., 1999). Fig. 34.3 shows a schematic linear structure of HA, where ectodomain accounts 90% of the polypeptide chain, while the remaining 10% represents the transmembrane domain embedded in the envelope (Chen et al., 1999). The three-dimensional structure of HA consists of two distinct regions (Fig. 34.4): • A long fibrous region close to the cell membrane containing residues from both the HA1 chain and the HA2 chain is structured as a triplestranded alpha helix. • A globular region containing residues originating solely from the HA1 chain; this distal region of the cell membrane has an eight-sheet beta structure. The HA1 subunit is the preferred site for minor drift mutations, particularly at the five antigenic sites (A, B, C, D, and E). Since 1963 the equine influenza virus (H3N8), especially the HA1 subunit of H3, has evolved into two lineages with a rate of 0.8 amino acid substitutions per year (Myers and Wilson, 2006), while the HA2 subunit generally exhibits a high degree of conservation and plays a key role in fusion and viral penetration (Vareckova et al., 2008). Hemagglutinin 1 Subunit Cellular Receptor-Binding Sites

The cell receptor-binding site “RBS” exists in the globular region (HA1), forming a pouch consisting of distal ends of three monomers.

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FIGURE 34.4 Tertiary and quaternary structure of HA. RCSB/Code: 4UO0. (A) The tertiary structure of each HA subunit is the folding of its helices and strands into a compact structure divided into two domains (globular and fibrous domains). (B) The quaternary structure of HA is stabilized by lateral interactions between the long helices (cylinders) in the fibrous domains of the three subunits, forming a triple-stranded helical twisted rod. Each of the distal globular domains in HA binds sialic acid to the surface of the target cells. HA, Hemagglutinin.

The site is inaccessible to antibodies, which makes the production of protective antibodies very difficult. The amino acids forming the cell RBS are the residues Tyr 98, Trp 153, Thr 155, His 183, Glu 190, and Leu 194. These residues are highly conserved in all HA1 subtypes, while other amino acids on the surface of the molecule have diverged widely in recent years. Other conserved Cys 97, Pro 99, Cys 139, Pro 147, Tyr 195, and Arg 229 residues are located behind the RBS and appear to stabilize the site architecture without being in a position to interact with the receptor site (Al-Majhdi, 2007). The RBS bottom’s formed by the phenolic hydroxyl group of the tyrosine residue 98 and the aromatic ring of the tryptophan residue 153. Glutamine 190 and leucine 194 are projected downward from a small helix to define the back site with histidine 183 and threonine 155. Finally, the residues 228
374 from the left side and residues 134
138 from the right side of this depression. Binding specificity is determined by glycosidic bonds between the terminal sialic acid and the galactose residues of cellular receptors (Temoltzin-Palacios and Thomas, 1994) (Fig. 34.5). Human influenza A viruses bind preferentially to the (2,6) galactose sialic acid, while avian and equine viruses prefer the (2,3) galactose

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FIGURE 34.5 Schematic model of the cell RBS showing the hydrogen bonds established between sialic acid and the different residues forming the RBS (Temoltzin-Palacios and Thomas, 1994; http://creativecommons.org/licenses/by-nc-sa/4.0/). RBS, Receptorbinding site.

(Suzuki et al., 2000; Shen et al., 2009) (Fig. 34.6). A (2,6) galactose sialic acids are predominant in the human respiratory epithelium, whereas those of α (2,3) galactose are predominant on the surface of epithelial cells of the intestinal tract in ducks (Munier et al., 2010). The specificity can be modified with the affinity toward the cellular receptors. Mutations at residues 226 and 228 of the human influenza virus (H3) RBS facilitate its replication in ducks (Meisner et al., 2008; Munier et al., 2010). Also some mutations on antigenic sites, for example, a single amino acid substitution (Ser/205/Tyr) on the antigenic site D, located far from RBS, affected the ability of A/Mem/1/ 71-Bel/42 virus to bind to sialic acids on the erythrocytes (Suzuki et al., 1989). These two examples clearly showed that the mutations produced in the globular region of HA1 could influence the biological properties of the virus, change the tissue tropism of the virus and thus trigger a state of emergence or reemergence.

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FIGURE 34.6 Sialic acid molecule α (2,3) linked to a galactose molecule. For a (2,6)type bond, the carbon number 2 of sialic acid would be linked, via an oxygen atom, to the number 6 carbon of galactose. Hemagglutinin recognizes the type of link between sialic acid and galactose (Munier et al., 2010; Tscherne and Garcı´a-Sastre, 2011). Cleavage Site

The HA0 precursor is inactive and must be cleaved into two HA1 and HA2 chains via proteases (trypsin-like or furin-like proteases). Trypsin-like proteases determine the sites of viral replication; these proteases are common in the respiratory and intestinal mucosa (Roberts, 2008). The cleavage of HA0 occurs at a conserved residue arginine 329 of a 19 residues cleavage site (323
328 of HA1, Q329, and 1
12 of HA2) (Chen et al., 1999) (Fig. 34.7). This is a critical step to viral infection. However, some influenza viruses, such as the virus responsible of the 1918 pandemic (H1N1), are able to replicate in vitro without need for trypsin in the cell medium (Jindrich et al., 2007; Al-Majhdi, 2007). For the less virulent strains limited to the respiratory tract, this site generally consists of a single arginine [e.g., HA1-PEKQIR-GI-HA2 strain A/ equine/Miami/1963(H3N8)/AAA43164]. On the other hand, in highly

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FIGURE 34.7 Three-dimensional structure of HA0 showing cleavage site (thin arrows) (Galloway et al., 2013; https://www.ncbi.nlm.nih.gov/pmc/about/copyright/#copyPMC). HA, Hemagglutinin.

pathogenic avian strains, this cleavage site consists of several basic residues, forming a consensus sequence of the R-X-K/R-R type (e.g., HA1-PQRERRRKKR-GL-HA2) strain [A/chicken/Hong Kong/258/97 (H5N1)/AAC14418]. Different proteases recognize monobasic sequences and cleave HA0 into HA1 and HA2 (Klenk and Garten, 1994). One of them would be tryptase Clara (extracellular protease). The polybasic site HAs would be cleavable by ubiquitous proteases such as furin (intracellular protease present in the Golgi apparatus) and that leads to a much larger tropism of the virus and a serious systemic infection (Stieneke-Grober et al., 1992; Roberts, 2008; Walker et al., 1994; Cross et al., 2009). Antigenic Sites

Three-dimensional analysis of HA, combined with amino acid sequence analysis of different HAs, allowed the localization of five antigenic sites on the surface of this molecule. A substitution on at least one amino acid residue of each of these sites was observed in epidemic/epizootic strains. Generally, at least four mutations are needed for a new epidemic to appear (Martella et al., 2007). HA may also have additional glycosylation sites, which may hide the antigenic sites targeted by potential neutralizing antibodies (Munier et al., 2010). The antigenic sites are studied in detail in human subtype (H3N2) viruses (Wiley and Skehel, 1987). According to Daniels et al.

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(1985), human and equine H3 viruses share the same antigenic relationship and have the same antigenic sites. The antigenic sites presented as sites A [A1 (121,126), A2 (131,137), A3 (142,146)], the antigenic site B [B1 (155
163), B2 (186
199)], the site antigen C [C1 (48,55), C2 (273,278)], the antigenic site D [D1 (170,174), D2 (201,218), D3 (242,248)], and the antigenic site E [E1 (62,63) and E2 (78,83)] (Wilson and Cox, 1990; Both et al., 1983; Nakajima et al., 2004; Underwood, 1982; Virmani et al., 2010; Martella et al., 2007). Different studies suggest that at least four amino acid changes in two different antigenic sites of HA are required to have antigenic drift (Wilson and Cox, 1990). However, some have reported that only two amino acid changes might significantly alter virulence (Jin et al., 2005). The determination of the nature of amino acid changes and their location in antigenic sites is a good indicator for predicting the extent of antigenic variation (Lee and Chen, 2004). According to Hay et al. (2001), between 1986 and 1998, a threshold of 10 mutations in the antigenic sites of new virus variant of human influenza (H3N2) was enough to make the change in vaccination strategy. Hemagglutinin 2 Subunit HA2 subunit is generally highly conserved (Vareckova et al., 2008; Nobusawa et al., 1991) and provides the structural
functional basis for fusogenic activity, which is often considered one of the biological properties essential for viral infection (Macosko et al., 1997). The first 185 residues of HA2 are the ectodomain (ED), of which the N-terminal end of 23 amino acids constitutes the “fusion peptide (PF)”. Residues 185
211 represent the transmembrane domain (TM) and finally residues 211
221 form the cytoplasmic domain embedded within the viral particle (CD) (Chen et al., 1999; Smirnova et al., 2009) (Fig. 34.8). The high conservation of the N-terminal region of the HA2 subunit, particularly for the first 11 amino acids “GIFGAIAGFIE,” currently renders this conserved region of HA2 and those of the M2e (ectodomain ion-channel protein M2) and nucleoprotein (NP), which are the basis of research for the development of universal vaccines (Sui et al., 2009; Du et al., 2010) (Fig. 34.9). Biological Role HA is a major determinant of virulence and host specificity. Its ongoing evolution is responsible for epidemics and pandemics. It has two known functions; first, it binds to cell receptors that have sialic acid molecules bound in their surface, and then it allows the membrane

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FIGURE 34.8 Different conformations of HA of the influenza virus. (A) HA1 and HA2 chains of a neutral pH HA monomer. (B) Trimeric structure of HA in the same form at neutral pH. (C) Trimeric structure of HA composed of the HA2 chain (residues 38
175) linked by a disulfide bridge to the HA1 chain (residues 1
27) at acid pH. (D) Superior view of the HA2 conformation (Isin et al., 2002, license code 4342420494733). HA, Hemagglutinin.

fusion of the viral envelope to the host cell by releasing the capsid in cytoplasm (Doms et al., 1985). Generally, three factors determine the infection or cellular tropism of influenza viruses: • Presence of the right kind of glycosidic bonds [α (2,3) and α (2,6)]. • Presence of protease activity to allow cleavage of HA. • Acidification of the endosome to allow the activation of the HA.

Neuraminidase Structure NA (N-acetyl-neuraminyl-hydrolase) is encoded by segment 6 of the viral genome, which is in the form of a homo-tetramer on the surface of the viral particle. It is a tetrameric glycoprotein of 470 amino acids in the form of a fungus whose N-terminus is oriented toward the inside of the virus (Fig. 34.10). It consists of a stem responsible for its anchoring to the viral membrane and a globular portion responsible for its enzymatic activity including antigenic sites. Mutations at this level give rise to new epidemic strains resistant to NA inhibitors.

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FIGURE 34.9 Schematic diagram of conserved and proposed sequences for universal vaccines (Du et al., 2010, license code 4342420756455).

FIGURE 34.10

Homo-tetrameric structure of neuraminidase (Venselaar et al., 2010; https://www.ncbi.nlm.nih.gov/pmc/about/copyright/).

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Specificity of receptor binding (SA)

Specificity of receptor cleavage (SA)

Glycosylation sites

Length of the stem

Sensitivity to pH

Sensitivity to pH

FIGURE 34.11 Diagram showing the joint action of the two surface glycoproteins (HA and NA) that are critical in the process of infection. When adapting to a new host, HA and NA coevolve so that the functional equilibrium between the two proteins (whose major parameters are indicated in the boxes) is preserved. HA, Hemagglutinin; NA, neuraminidase.

Biological Role NA plays an essential role in activating the viral particles of infected cells by cleaving the terminal sialic acid residue of the receptors on the cell surface, thus preventing the aggregation of virions at this level. In addition, NA cleaves sialic acid from the mucins present in the upper respiratory tract that facilitates the access of the virus to epithelial cells (Myers and Wilson, 2006). Therefore this protein is involved in the propagation and dissemination of the viral infection (Fig. 34.11). NA is the target of Oseltamivir (Tamiflu, Roche), Zanamivir (Relenza, GlaxoSmithKline), and analogs of sialic acids commonly used for therapeutic purposes against the influenza virus. Proteins From the Matrix The membrane protein M1, also known as the matrix protein, is the major protein in the viral structure. It is encoded by a small genomic segment, segment 7 composed of 1027 nucleotides, which is its major product. It is an unglycosylated protein that extends across the lipid

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FIGURE 34.12

Diagram showing the opening mechanism of the ion channel M2. When opening the ion channel M2, protons enter the virus. Acidification allows vRNPs to dissociate from other viral components (M1) and to enter the cell, which ends decapsidation (Das et al., 2010; https://www.ncbi.nlm.nih.gov/pmc/about/copyright/).

bilayer and contributes to the stiffness of the viral envelope. The M1 protein can interact with the envelope and the nucleocapsid (Ito et al., 1999). The M2 protein (96 amino acids) is also encoded in segment 7 and present in small numbers in the viral envelope (20
60 molecules). It is a transmembrane homotetrameric protein connected by two disulfide bridges with three domains: a 54 amino acid intracellular portion, 19 amino acid transmembrane portion, and a 23 amino acid extracellular portion. The four helices form a channel in the lipid bilayer that serves as a protonic canal that allows acidifying the interior of the virus. This facilitates the dissociation of the RNPv complex from other viral components by completing decapsidation (Castrucci and Kawaoka, 1995). Fig. 34.12 shows the function of the M2 protein as an ion channel, and its role in the dissociation of M1 protein (RNPv) complexes. In addition, the M2 protein is the target of one of two classes of available antivirals against influenza. It is also involved in the antiviral resistance phenomenon (Wang et al., 1993). Nonstructural Proteins NS proteins are not incorporated in the structure of virions but participate in the replication of virions. These are NS1, NS2, and PB1-F2 proteins. The presence of double-stranded RNA (dsRNA) in the target cell is a signal of viral infection, triggering the activation of cellular immune system, in particular the alpha/beta IFN proteins resulting from the activation of several transcription factors including IRF-3, IRF7, NF-kB, and c-Jun/ATF2 (Peirong et al., 2008). To inhibit these cellular defense agents, influenza viruses have evolved a strategy to counteract these inhibitory factors. NS1 protein is widely regarded as the main antagonist of the immune response of the host cell (Hale et al., 2008). It is a protein of 230 residues and 27 kDa. It is coded in the smallest

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segment of the viral genome (segment 8) that also codes for NS2 protein or NEP. The transcription is performed by a splicing of the NS gene the microRNA (mRNA) resulting from the transcription of the NS gene (Wang et al., 2008) (Fig. 34.13). The NS1 protein is composed of two domains linked by an unstructured region of about 10 amino acids. The first N-terminal third of the protein is the RNA-binding domain (RBD), consisting of three helices: Asn4-Asp24 (helix 1), Pro31-Leu50 (helix 2), and Ile54-Lys70 (helix 3) (Dongzi et al., 2007). It is always in the form of a homodimer in which each of the twopeptide chains provides three α helices. The two helices (α2 and α20 ) occupying antiparallel positions bind to dsRNA molecules through several basic amino acids including arginine 38 and lysine 41 (Marc, 2012). The distal two-thirds forming the effector domain (ED) are in the form of a homodimer, where the monomer is composed of three helices α and seven strands β; these later are organized into a large sheet of antiparallel β strands enveloping the long α5 helix (Dundon and Capua, 2009; Dongzi et al., 2007; Marc, 2012) (Fig. 34.14).

FIGURE 34.13

Schematic representation of alternative splicing at the NS gene level.

NS, Nonstructural.

FIGURE 34.14

Left to right image: Three-dimensional structure of a RBD monomer and that of the NS1 protein ED. ED, Effector domain; NS, nonstructural; RBD, RNAbinding domain.

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The NS1 protein binds to the small U6 and U6atac nuclear RNAs involved in the splicing mechanism by its RBD (1
73) (Marc, 2012). At the effector domain (ED) (residues 74
230), specific binding sites exist for cleavage and polyadenylation specific factor proteins (residues 175
210), proteins [(PABPII) poly(A)-binding protein-II] (residues 218
225), and those of the p85-β regulatory subunit of phosphatidylinositol 3-kinase (residues 89
93, 137
142, and 164
167) (Darapaneni et al., 2009). Other specific binding sites for cellular proteins are recognized by the NS1 protein: (PABPI), importin α, nucleolin, NS1-BP, eIF4GI, hStaufen, NS1-I, PKR, PACT, Crk/CrkL, PDZ, viral polymerase, and finally the cellular components of the nuclear export of mRNA (E1B-AP5, p15, NXF1, and Rae1) (Hale et al., 2008) (Fig. 34.15). The nucleocytoplasmic distribution of this protein is related to the coexistence in the peptide sequence of NS1, nuclear localization signal (NLS) (NLS1: residues 34
38, NLS2: residues 211
216), and a nuclear export signal (residues 132
141) (Darapaneni et al., 2009). The main NLS consists of the basic amino acid series of the RNAbinding α2 helix (Arg35, Arg38, and Lys41). This basic amino acid sequence is recognized by host α-importins that bind to NS1 and transport it to the nucleus (Marc, 2012).

Importin-α RIG-I

R38, K41

NLS1

1

p85β

Polymerase?

p85β

Y89

123–127

P164, P167

NES

73

RNA-binding domain

1–81

PABPI

Nucleolin

Importin-α

PKR

dsRNA

E1B-AP5

CrkL

PDZ

212–217 227–230

207

NoLS NLS2

230

Disordered “tail”?

Effector domain

103, 106

144–188

223–230

CPSF30

CPSF30

PABPII

81–113 elF4GI 73–230

hStaufen

p15

FIGURE 34.15 Diagram of different binding domains to the NS1 protein (Hale et al., 2008; https://creativecommons.org/licenses/by/3.0/). NS, Nonstructural.

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Ribonucleoprotein Viral Complex The NP gene (segment 5) plays a critical role in the interspecies barrier and adaptation within the host of influenza A viruses (Xu et al., 2009; Gorman et al., 1990). The nucleoprotein comprises a sequence of 498 amino acids that polymerize around genomic RNA associated with the polymerase complex (PA, PB1, and PB2). The ensemble forms the ribonucleoprotein complex (vRNP) (B250 kDa), which is essential for the transcription and replication steps of the genome in the cell nucleus (Klaus et al., 1997; Voeten et al., 2000). The nucleoprotein (NP) sequence is highly conserved (Boukharta et al., 2012) and contains a viral RNA binding region at its N-terminus (residues 1
181), two domains responsible for NP
NP autointeraction (residues 189
358 and 371
465) (Albo et al., 1995; Elton et al., 1999; Kobayashi et al., 1994) and three NP regions [aa (1
161), aa (255
340), and aa (340
465)] that interreact independently with PB2 (polymerase basic protein 2) (Ka-Leung et al., 2012; Li et al., 2009). Influenza viruses that are responsible for severe infections are able to escape neutralizing antibodies following the accumulation of “antigenic drift” mutations in their surface glycoproteins (HA and NA) and the introduction of mutations to conserved epitopes of viral proteins including NP and M1 (Epstein, 2003). The nucleoprotein contains the most conserved epitopes of influenza and appears to be the major target of the cytotoxic T lymphocyte (CTL) response, which limits the replication of the influenza virus (Varich et al., 2009; Wahl et al., 2009). In humans the antigenic epitopes of the nucleoprotein are essential for binding to the major histocompatibility complex class I and specific CTLs (Voeten et al., 2000). Four epitopes of influenza A virus NP have been analyzed and identified by several studies. They are in the form of short sequences recognized by CTLs: E1 (aa 265
273), E2 (aa 338
347), E3 (aa 380
388), and E4 (aa 383
391) (Voeten et al., 2000; Huet et al., 1990; DiBrino et al., 1993). Phylogenetic analysis of NP gene sequences of influenza A viruses indicates that the NP gene is highly conserved within the lineage (Thippamom et al., 2010). Eight lineages have been identified, including the human, porcine, avian, and equine (Xu et al., 2009) (Fig. 34.16). The hetero-tri-complex polymerase (vRNP) (B250 kD) consists of three proteins: PA, PB1, and PB2. It plays an essential role in the viral cycle and the synthesis of viral RNA (Yingfang et al., 2009).

Replicative Cycle of the Virus Knowing the roles and functions of various viral proteins listed above, the different puzzles of the replicative cycle of influenza become

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34. ROLE OF GENETIC AND MOLECULAR DYNAMICS IN THE EMERGENCE

FIGURE 34.16 Phylogenetic tree of influenza A viral NP genes. Eight lineages are denoted: Human, pandemics H1N1 2009, Classical swine, Eurasian avian, Eurasian swine, North American avian, Oceanian avian, and equine (Xu et al., 2011).

obvious. The replicative cycle phases are briefly described and the mechanism of action of some involved key proteins. Fig. 34.17 illustrates the different stages of the viral cycle of influenza. Fixation, Endocytosis, and Release of vRNPs Complexes The infection cycle begins with the specific binding of the viral particle to specific receptors (glycoproteins, gangliosides, glycolipids) located on the surface of the cell membrane, which carry sialic acids at their ends. Once the virus is attached to the host cell due to many HA/sialic acid interactions, the next step is the cleavage of HA by cellular proteases. This allows exposure of the viral PF and facilitates an entry of the virus into the host cell by endocytosis (Beby-Defaux et al., 2003).

EMERGING AND REEMERGING VIRAL PATHOGENS

FIGURE 34.17

Schematic representation of different viral cycle stages of influenza A virus (Capitanio and Wozniak, 2012).

766

34. ROLE OF GENETIC AND MOLECULAR DYNAMICS IN THE EMERGENCE

The interior of the endosome becomes acidic due to cellular proton pumps, leading to a three-dimensional modification of the HA that allows fusion of the viral and endosomal membranes (Fig. 34.17). This intraendosomal acidification activates the proton channels of the virion and opens the channel formed by the viral proteins M2. Thus the interior of the virion becomes acidified, causing the dissociation of the vRNPs from the M1 matrix protein layer that lines the inner surface of the viral membrane. The fusion of the viral and endosomal membrane following the decrease in pH and dissociation M1 of the vRNPs allow for release of the eight genomic segments in the cytoplasm (Lakadamyali et al., 2004). Active Import of the Viral Genome Into the Cell Nucleus vRNPs in the cytoplasm of the host cell must gain access to the nucleus to be transcribed and replicated in the early phase of infection. The nucleoprotein (NP) has been shown to be important for the import of the viral RNA into the nucleus and has been proposed to contain at least three different NLSs (Cros et al., 2005). Viral Transcription and Replication in the Kernel The viral particle enters in the eclipse period that is characterized by two phases: messenger RNA transcription and genome replication. The viral RNA has a negative polarity [RNA(2)] and must be transcribed into (1)RNA to enable ribosomal transcription. The host cell does not have an enzyme capable of performing this, thus the viral genome encodes its own transcriptase. This is composed of the heterotri-complex polymerase (PA, PB1, and PB2) of the ribonucleoprotein complex. Thus the PB1 subunit of the polymerase binds to m7Gppp caps and cleaves about 10 base cell mRNAs after the cap; these RNA fragments serve as primers for the initiation of viral mRNA synthesis (Plotch et al., 1981). Viral mRNAs are 30 polyadenylated (like most cellular mRNAs) due to a uridine-rich sequence near the 50 end of the template vRNA. The vRNAs and cRNA (replicative intermediate of positive polarity) are covered with nucleoproteins, but the viral mRNA is not covered. There is a temporal regulation of the viral transcription, as the products of viral mRNA synthesis vary in proportion during the infection. The replication of the genome also takes place in the nucleus, and the transcription begins for each of the eight segments. It does not require the presence of a primer. The vRNAs are transcribed into eight cRNA that will serve as a template for the synthesis of new viral genomes (Samji, 2009).

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EMERGENCE AND EVOLUTION OF EQUINE INFLUENZA VIRUSES

767

Assembly and Budding The assembly of the viruses takes place in the cytoplasm, and the cell membrane is rearranged by the insertion of the viral glycoproteins HA and NA and by affixing on the internal face of the proteins, M1 and M2, which will constitute the matrix. In the nucleus, NP protein associates with formed vRNAs to constitute the hetero-tri-complex vRNPs of various segments. These segments then migrate through the nuclear pore via the chromatin maintenance protein 1
specific export system. Two late viral proteins, NEP and M1 seem to play a key role in this stage (Nayak et al., 2009). The budding of the virus is not immediately lethal for the cell that remains normal in appearance, but it becomes exhausted and eventually dies. However, the destruction of the cells is mainly due to the cytotoxic immune response. The new viral particles remain attached to the membrane of the infected cell, because of the binding between the viral HA and the cellular sialic acid. NA proteins break this link, allowing new viruses to break away and infect new cells (Samji, 2009).

EMERGENCE AND EVOLUTION OF EQUINE INFLUENZA VIRUSES Equine influenza viruses are RNA dependent, and RNA polymerase is recognized by its poor replication. As a result, point mutations occur continuously on the two surface glycoproteins HA and NA: the main carriers of viral virulence. This so-called minor mechanism of antigenic variation is largely responsible for the evolution of lineages and sublineages, particularly subtype viruses (H3N8). For the subtype virus (H7N7), and according to several authors, it had cocirculated together with the H3N8 viruses until 1979. Others presumed that it circulated until the year 1989. Since 1980, the declared epizootics are exclusively due to subtype viruses (H3N8) (Murcia et al., 2011).

Equine Influenza Virus (H7N7) Isolation and Viral Evolution The subtype (H7N7) was first described in 1956 in the former Czechoslovak Republic following the isolation of A/equine virus/ Prague/1/56 (H7N7) (Sovinova et al., 1958), which was responsible for the equine flu in Europe for more than 20 years. The strain reached North America in 1960 and India in 1964 (Murcia et al., 2011). This equine virus was respectively identified in Chile in 1977 (A/equine-1/ Santiago/77) (Muller et al., 2005), Yugoslavia in 1978 (Quinlivan et al.,

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34. ROLE OF GENETIC AND MOLECULAR DYNAMICS IN THE EMERGENCE

2005), Italy in 1979, India in 1987 with the isolation of strain A/equine/ Ludhiana/5/H7N7, and finally in Egypt in 1989 (Gurkirpal, 1997; Paillot et al., 2005). Insidious Circulation Since 1989, no viral isolation has been observed; however, serological surveys have revealed the circulation of this virus in Central Asia and Eastern Europe in 1991 (Gibbs and Anderson, 2010). Similarly in Africa, traces of antibodies have been reported in Mali (2002), Algeria (2006), and Morocco (2010) (Sidibe´ et al., 2002; Bererhi et al., 2009; Boukharta et al., 2012). On this basis, two hypotheses are currently retained; either the virus continues to circulate silently or it has disappeared after being supplanted by subtype viruses (H3N8) (Zientara, 2001). In addition, the Expert Committee on Equine Influenza Surveillance, headed by the Office International des Epizooties (OIE), recommends each year that vaccine manufacturers do not incorporate subtype viruses (H7N7) into the vaccine composition and to perform rigorous surveillance of these viruses to identify any recurrence (Boliar et al., 2006).

Equine Influenza Virus (H3N8) Viral Isolation In 1963 a major epizootic in the United States allowed isolation and identification of the equine (H3N8) virus for the first time, probably of avian origin (Fig. 34.18), which was named A/equine/Miami/1/1963 (Waddell et al., 1963). Second, due to international horse movements, this virus spread widely in South America and Europe, leading to significant epizootics in 1964/1965 (Myers and Wilson, 2006). Antigen Evolution In Japan a major epizootic of equine influenza occurred in 1971/72 following the importation of infected horses from Europe. As a result, more than 6782 horses were infected in Tokyo, resulting in the suspension of horse racing for 10 weeks (Sugiura et al., 2001). Between 1978 and 1981, several epizootics were notified in the United States and Europe, despite rigorous use of the vaccine. In 1986 and 1987 the H3N8 virus infected the immunologically naive equine population of South Africa through imported asymptomatic and vaccinated horses that were carriers of the virus. This epizootic led to the suspension of the races for 5 months (Kawaoka and Webster, 1989). The same scenario occurred in India in 1987 (Ilobi et al., 1994). The year 1989 documented the beginning of genetic divergence of this virus. Severe epizootics with high morbidity have occurred in

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769

FIGURE 34.18 Hypothetical origin of strain A/equine/Miami/1/(H3N8). An ancestral influenza virus, possibly avian, would have given rise to a series of antigenically linked animal viruses including a virus isolated from horses in Miami in 1963 and a virus isolated from ducks during the same year in Ukraine. Another virus related to this family would have recombined 5 years later with the Asian strain H2N2, then present in humans, giving birth to the strain Hong Kong (H3N2) (Dea et al., 1980; https://www.ncbi.nlm.nih.gov/ pmc/about/copyright/).

Europe and America and mainly attributed to point drift mutations of the gene encoding HA and particularly to the antigenic sites of the subunit (HA1). The new strains were partially recognizable by preexisting antibodies (Barbic et al., 2009), and the vaccines used at the time became obsolete. At the same time, several studies have reported that, first, H3N8 equine viruses evolved into a single lineage (Kawaoka et al., 1998) and then split into two distinct lineages from 1990 (Martella et al., 2007): European [e.g., A/equine/Suffolk/89 (H3N8)] and American [e.g., A/equine/Newmarket/1/93 (H3N8)] (Borchers et al., 2005). The American lineage has subsequently diverged into three sublineages: South America, Florida, and Kentucky (Lai et al., 2004). It is important to note that viruses from these two lineages, favored by international horse movements and their circulation worldwide without geographical barriers (Ito et al., 2008). According to several authors, the old strains (H3N8) circulating in the 1960s [A/equine/Miami/1/63 (H3N8)] and in 1970/80

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34. ROLE OF GENETIC AND MOLECULAR DYNAMICS IN THE EMERGENCE

[A/equine/Fontainebleau/76(H3N8)] apparently disappeared (Martella et al., 2007). In China, two major epizootics occurred in 1989
90 and 1993
94. The generated economic loss was of 20,000 infected equine and at least 400 deaths (2%) for the first epizootic, and 2255,000 infected equine and 24,600 deaths (1%) for the second one. Genetic analysis of isolated viruses revealed great homology to European lineage viruses (Timoney, 1996). In 1989 a severe epizootic occurred again in China. More than 13,000 horses were infected with a morbidity rate that reached 80% and a mortality rate of 20% (Murcia et al., 2011). Genetic analysis showed that six of the eight genomic segments of this virus [named A/equine/Jilin/1/89 (H3N8)] were highly similar to the segments of an avian strain (Guo et al., 1995). This epizootic limited out rapidly testifying the possibility of the direct transmission of an avian (H3N8) virus to horses (Paillot et al., 2007). In 1995, two strains of European lineage were isolated from an epizootic in the Netherlands. WHO and OIE had recommended for the first time the incorporation of two representative vaccine strains of European and American lineage into vaccines. Other minor epizootics were notified, in Hong Kong in 1992; Dubai and South Africa in 1993; Morocco in 1997/2004; Tunisia in 1998; Philippines in 1997; Egypt in 2000; the United Kingdom and South Africa in 2003; and Argentina, Canada, Croatia, Denmark, France, Germany, Greece, Hungary, Ireland, Italy, Sweden, the United Kingdom, and United States in 2004. The viruses responsible for the 2004 outbreaks in Europe and America belong to the American lineage. The annual substitution rate of HA is of the order of 0.8 amino acid (Myers and Wilson, 2006). These data reinforce the need for international monitoring of equine (H3N8) viruses, particularly in the countries from which most of the movement of horses takes place (sales, racing, competition, etc.) (Powell, 1985). Current World Situation According to the last meeting of OIE Expert Group on monitoring and the composition of equine influenza vaccines, held on March 22, 2017, several outbreaks have been reported in the United States, Ireland, and the United Kingdom. Gene sequencing (HA1) of isolated viruses had been shown to belong to the American lineage (Florida sublineage). The viruses identified in the United States are clade 1, while those identified in Ireland and the United Kingdom are clade 2. Compared to strains isolated in 2016 that share the same sublineage with those notified in 2015, several amino acid substitutions at the level of HA had been observed on viruses of both clades, which

EMERGING AND REEMERGING VIRAL PATHOGENS

PATHOPHYSIOLOGY

771

FIGURE 34.19 Diagram of genetic evolution of equine influenza viruses (H3N8). As of 2010, strains isolated in America and Europe are clades 1 and 2 of the Florida sublineage (gray boxes).

further widened the sequential divergence (http://www.oie.int/fr/ notre-expertise-scientifique/information-specific-and-recommendations/ grippe-equine/) (Fig. 34.19).

PATHOPHYSIOLOGY Following the inhalation of contaminated aerosols, influenza virus infects epithelial cells of the upper respiratory tract. The mechanism of infection is reported in the chapter dealing with influenza viruses. After an incubation period of 1
3 days the first clinical sign appears. It includes a rise in body temperature (39.1 C
41.7 C) for 4
5 days (Myers and Wilson, 2006). The clinical picture is characterized by a strong, quintessential, dry, and painful cough associated with a nasal discharge that releases viral particles, laryngitis, tracheitis, bronchitis, bronchiolitis, and interstitial pneumonitis often associated with congestion and alveolar edema. Cases of myalgia, orchitis, encephalitis, and myocarditis are also reported. The severity of the disease depends mainly on the circulating strain and the immune status of the infected animal (Heldens et al., 2002). Clinical signs decline in 2 weeks postinfection, unless there are complications due to bacterial superinfections. In some rare cases, especially in old horses, the virus can penetrate the basement membrane of the infected respiratory epithelium. This phenomenon accounts for exceptional cases of myositis, myocarditis, edema, encephalitis, and abortion observed after influenza infection. Equine influenza causes high morbidity, sometimes mortality among foals, unhealthy horses,

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34. ROLE OF GENETIC AND MOLECULAR DYNAMICS IN THE EMERGENCE

and donkeys. In adults, mortality is usually associated with bacterial superinfection leading to pleurisy, pneumonia, or hemorrhagic purpura (Lai et al., 2004).

GENETIC DYNAMISM OF INFLUENZA VIRUSES Interspecies Barrier Currently, all influenza subtypes have been reported in avian species, particularly in wild waterfowl, which participate through their biological migration in global viral circulation. However, the concept of influenza virus species barrier to their hosts is relative and is a subject to many transgressions. Thus during the last decades, several contaminations by avian viruses affecting humans and some species of mammals (dog, cat, etc.) have been reported and to a lesser degree between certain mammals (e.g., between horse and dog). This transmission of interspecies barrier is correlated with genetic variations. First, point mutations mainly affect antigenic sites, then recombination or reassortment of new viruses following coinfection by viruses of different lineages in the presence of a species serving for material genetics exchange “for example, pork,” and finally the direct transfer of a virus unchanged from one species to another. The direct transmission of unchanged influenza viruses was responsible for several epidemics and epizootics. Thus in humans, the highly pathogenic avian (H5N1) virus had crossed the species barrier several times since 1997 (Fig. 34.20).

Antigenic Variations They are known to have two distinct types of antigenic variation: antigenic drift and antigenic shift (Roberts, 2008). The “antigenic drift” variations constantly cause minor and progressive changes in the structure of the HA and NA proteins of the influenza virus strains. These variations are attributed to genome instability, and the lack of mechanisms for repairing RNA-dependent RNA polymerase errors occurs during replication in infected cells. These minor changes give rise to seasonal epidemics, usually in winter, where the responsible strains are partially recognized by the immune system. As a consequence of these minor changes, many attenuated or nonviable influenza genotypes are produced, but the selection pressures (T lymphocytes, antibodies, host receptors, etc.) exerted by the immune system of the host organism lead to the expansion of some variant only (Munier et al., 2010). Antigenic shift or reassortment variations occur only in influenza A viruses, which are sudden and complete changes in one or both surface

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CONCLUSION

773

FIGURE 34.20 Interspecies transmission pattern of influenza A virus (Jindrich et al., 2007, license code 4342450371625).

antigens. This results completely in new viruses having surface antigens different from the antigens of the old viruses circulating in the population.

CONCLUSION Currently, equine influenza is one of the most important fields of research on equine veterinary medicine. This importance is closely related to the specificity of the disease, which is recognized by its high contagiousness, severity of respiratory symptoms, and its responsibility about two-thirds of equine respiratory infections. Second, the characteristics of influenza type A viruses are regularly responsible of epidemics and epizootics in many mammal species (man, pig, horse, dog, etc.),

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wild aquatic birds, and domestic poultry. The genetic dynamism of equine influenza viruses, especially those of the subtype H3N8, and their transmission into other mammalian species clearly shows the health risk of the unpredictable emergence or reemergence of these viruses in other mammalian species, including humans.

Acknowledgments This chapter was funded by the financial support of Ministry of Higher Education, University Hassan II of Casablanca, Faculty of Sciences and Techniques, Mohammedia and also Royal Gendarmery of Morrocco. The authors thank all the research staff at the Laboratory of Virology, Microbiology, Quality and Biotechnologies/Ecotoxicology & Biodiversity and Genetics Laboratory of Royal Gendarmeries for their support and technical help.

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