H5Nx HIGHLY PATHOGENIC AVIAN INFLUENZA.

1. Introduction

Avian influenza viruses are highly contagious, extremely variable viruses that are widespread in birds. Wild birds in aquatic habitats are thought to be their natural reservoir hosts, but domesticated poultry and other birds can also be infected. Most viruses cause only mild disease in poultry and are called low pathogenic avian influenza viruses (LPAIVs). Highly pathogenic avian influenza viruses (HPAIVs) can develop from certain LPAIVs, usually, while they are circulating in poultry flocks(Swayne, 2007). Highly pathogenic avian influenza viruses can kill up to 90-100% of the flock, and cause epidemics that may spread rapidly, devastate the poultry industry and result in severe trade restrictions (OIE, 2014; CDC, 2015). 

Avian influenza viruses (AIVs) can occasionally affect mammals, including humans, usually after close contact with infected poultry. While infections in people are often limited to conjunctivitis or mild respiratory disease, some viruses can cause severe illness. Avian influenza viruses of the subtype H5Nx are often HPAIVs, which were originally discovered in geese in China’s Guangdong province in 1996. Several outbreaks occurred in farmed geese in Sanshui, a small town 50 miles outside the capital of Guangdong with a mortality rate of more than 40%(Wan, 2012). By 1997, the A/goose/Guangdong/1/1996-like viruses spilled over into the live poultry markets in Hong Kong with high rates of mortality. Simultaneously, there were 18 confirmed human cases of HPAIVs infection, 6 of whom died (Bender et al., 1999). There was a large degree of homology between the avian isolates and the viral isolates collected from these human infections indicating that these viruses were being transmitted from birds to human hosts(Bender et al., 1999).

The 1997 outbreak was contained through the culling of “stamping out” of all poultry in Hong Kong (Chan, 2002). However, AIVs continued to circulate in healthy duck populations in surrounding areas. Its re-emergence in 2003 resulted in the infection of 2 human cases caused by novel H5N1 genetic variants that continued to circulate and evolve into 10 phylogenetic clades (0-9)(Li et al., 2004). At the end of 2017, 860 laboratory-confirmed cases of H5N1 influenza virus infection from 16 different countries, resulting in 454 deaths had been reported to the World Health Organization (WHO). Infection of humans with AIVs are rare, but sporadic infections can occur due to direct contact with infected birds or through contaminated environments (WHO, 2017). According to the FAO, China has around 64% of the world’s domesticated ducks and 95% of the domesticated goose population breeding in live poultry markets alongside other poultry and swine. These conditions allow these markets to become breeding grounds for H5Nx influenza virus circulation. Outbreaks caused by AIVs have devastated live poultry markets in Asia and have had a substantial negative impact on the US economy(Guan and Smith, 2013). In this review, H5Nx viruses will be discussed for their replication, infection, evolution, threat and vaccination to the poultry industry, with emphasis on the need for a broadly reactive vaccine to protect the poultry population.

2. Etiology

2.1. Virus classification

Avian influenza viruses are classified in the family Orthomyxoviridae, genus influenza virus A (Perez et al., 2011). In the current classification system, influenza A viruses are categorized into 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes, although recently described bat influenza viruses may increase the number of hemagglutinin subtypes. The viral HA, and to a lesser extent the NA, are major targets for the immune response. There is ordinarily little or no cross-protection between different HA or NA types.

A virus is classified as HPAIVs or LPAIVs by its ability to cause severe disease in intravenously inoculated young chickens in the laboratory, or by its possession of certain genetic features that have been associated with high virulence in HPAIVs (i.e. the sequence at the HA cleavage site) (Swayne, 2008).

In the laboratory, the insertion of genetic sequences from HPAIVs into non-H7, non-H5 viruses has created some viruses that are pathogenic only after intravenous inoculation, and other viruses (containing H2, H4, H8 or H14) that were highly virulent after both intravenous and intranasal inoculation (Veits et al., 2012). In rare cases, an H5 or H7 virus has a genetic signature that classifies it as an HPAIVs, but causes only mild illness in poultry. Such viruses may have been isolated when they were evolving to become more virulent. Their presence triggers the same regulatory responses as fully virulent HPAIVs.

A numerical clade system has been adopted to better relate the evolutionary changes in these related H5Nx isolates over time; a clade is a taxonomic group comprising a single common ancestor and all descendants of that ancestor. For the Eurasian H5Nx hemagglutinin gene, the reference isolate is A/Goose/Guangdong/1/1996 (H5N1). The initial outbreak viruses from Hong Kong from 1997 were included in a single clade with the prototype virus, based on the hemagglutinin sequence. However, since 2003 the viruses have spread to progressively more regions beyond China and have evolved into several independent but related clades. By 2004, 10 distinct first order genetic clades were recognized (0-9), and continued evolution in subsequent years has resulted in a total of 30 additional second, third, and fourth order clades (e.g. 2.1, 2.2, 2.1.3, 2.2.1, 2.1.3.2 and 2.2.1.1, etc.) (Figure 1). This clade nomenclature system readily identifies the genetic linkage of the virus regardless of the geographic location, source of the isolate, or year of the isolate. The strain nomenclature system will continue to be maintained in repositories and be used to identify sequences deposited into databases such as GenBank.

Figure 1: Genetic evolution of the HA gene of H5N1 high-pathogenicity avian influenza viruses (HPAIV), A/goose/Guangdong/1/1996 lineage, since its emergence in 1996. The constant genetic divergence of HA leading to the emergence of new genetic groups (shown in pink shading) is illustrated by phylogenetic trees supporting updated clade classifications proposed by the WHO, OIE, and FAO H5N1 Evolution Working Group. Viruses not detected since 2008 are marked with a red star. From Updated unified nomenclature system for the highly pathogenic H5N1 avian influenza viruses, WHO (adapted and updated). http://www.who.int/influenza/gisrs_laboratory/h5n1_nomenclature/en/.

2.3. Morphology

Virions are typically spherical to pleomorphic (100 nm) but can be filamentous with lengths up to several hundred nm (Figure 2) (Perez et al., 2011; Shaw and Palese, 2013).

The surface is covered by two types of glycoprotein projections (10-14 nm in length and 4-6 nm in diameter): (1) rod‐shaped trimers of HA, and (2) mushroom‐shaped tetramers of NA, and a tetrameric Matrix 2 (M2) protein. Virus buoyant density is 1.19g/cm3 in aqueous sucrose and single virion molecular weight (Mr) is 250 x 106. The nucleocapsid is helical (Perez et al., 2011). The viral genome is composed of eight segments of single‐stranded, negative‐sense RNA that code for a minimum of 10 or up to 17 proteins depending on the strain (Vasin et al., 2014). Eight proteins are constituents of the virus (HA, NA, nucleoprotein [NP], matrix 1 [M1], matrix 2 [M2], polymerase basic protein 1 [PB1], polymerase basic protein 2 [PB2], polymerase acidic protein [PA], and a minor amount of nonstructural protein 2 [NS2]), and a nonstructural protein 1 (NS1) is located in the host cell cytoplasm. Expression of PB1‐F2 is variable, depending on the virus strain. In eight proteins of the virus, HA and NA are remarkable proteins.

Hemagglutinin of HPAIV 

Hemagglutinin is located on the surface of the influenza virus and it facilitates viral entry into the host cell by binding to sialic acid on the host cell surface (Sauter et al., 1989). Avian-adapted strains of influenza virus preferentially bind to N-acetylneuraminic acid with α-2,3- sialic acids (Matrosovich et al., 1997). These sialic acids are located in the gut and the digestive tract of avian species and in the lower respiratory tract of humans (Costa et al., 2012). HA is synthesized as polypeptide chain-encoded domains HA1 and HA2, co-translationally translocated into the lumen of the endoplasmic reticulum and eventually to the surface (Klenk et al., 1975). The HA protein contains a cleavage site between the HA1 and HA2 domains, cleavage is essential for infectivity and allows the HA molecule to undergo an irreversible conformation change in acidic endosomes. This cleavage is performed by cellular proteases to create two subunit HA1 and HA2 domains linked by disulfide bonds (Chen et al., 1998). The cleavage nature of H5 HA proteins is achieved when virions are incubated with trypsin. This results in the conversion of HA to HA1 and HA2. The cleavage of HA can be blocked by a protease inhibitor (Klenk et al., 1975). HA is expressed on the virion as a trimeric protein that is stabilized by residues on the HA2 region. The HA ectodomain is composed of two regions, a stem region and a globular head region (Skehel and Wiley, 2000). Neutralizing antibodies directed to the globular head of HA are critical for reducing viral infection and disease (Fleury et al., 1999). An important factor affecting viral pathogenicity depends upon the sequence of the amino acids in the HA0 cleavage site (Webster and Rott, 1987). HA proteins from HPAIVs contain a multibasic cleavage site that is cleaved by the ubiquitous furin cellular protease (Horimoto and Kawaoka, 1994). In contrast, in LPAIVs, that contain only one basic amino acid, the cleavage of HA is tissue specific, which results in a lower clinical manifestation in poultry. 

The polybasic cleavage site on HA is a strong determinant for high pathogenicity of H5 viruses, however insertion of polybasic sequences into an LPAIVs HA does not always result in a lethal phenotype as tested in chickens (Bogs et al., 2010). Other influenza proteins such as PB2, PB1, and NP may increase pathogenicity of an influenza virus. The pathogenic phenotype of H5 viruses is not HA dependent. Pathogenicity and efficiency of replication can also be dependent on PB2, NP, NA, and M genes. The deleted stalk region of NA found in HPAIVs also confers pathogenicity, where rescue of the NA stalk region leads to a decreased pathogenesis in chickens (Stech et al., 2015). Deletion of the stalk region increases lethality and transmission compared with the wild-type viruses that display a lower lethality. In addition to H5 viruses, this same NA stalk deletion abrogates H2N2 virus replication in ducks, but shifts the virus tropism from the intestinal tract to the respiratory tract in chickens (Sorrell et al., 2010). The presence of the polybasic HA cleavage site was sufficient enough to induce viral neurotropic (Bogs et al., 2010).

The dominant circulating AIV strains that have arisen since 2015 comprise viruses in the clade 2.3.4.4, which includes reassortant viruses in the H5N6, H5N8, and H5N2 subtypes. However, the strains within this clade that have crossed over into the human population are limited to the H5N6 subtype. The ability of H5N6 viruses to spill over into the human population may be associated with mutations in the HA molecule that affect the specific RBS binding preference of HA. These H5N6 viruses preferentially bind to different sialic acids depending on the host from which each virus was isolated (Zhao et al., 2017).

Neuraminidase of HPAIV

Characterization of LPAIVs or HPAIVs infections in poultry usually refers to the pathogenicity of the virus during infection and whether the virus contains a polybasic cleavage site in its HA molecule (as reviewed above). However other proteins, such as NA, can add to the pathogenic nature of the virus. To date, the newly circulating strains of AIV in China are H5N6, H5N8, and H5N2 of type H5Nx viruses (Lee et al., 2017a). These viral re-assortments can result in a dominant NA molecule that increases the pathogenicity and release of viral particles. Overall, this can increase viral transmission between hosts. The role of non-HA viral gene products and how these proteins contribute to viral tissue tropism and virulence are still not well understood. Multiple passages of H5N3 viruses in poultry result in a mutation in the catalytic site of NA, which increases the virulence of these viruses in poultry (Diederich et al., 2015). Mutations in internal genes, such as PB2, have also been linked to increased viral pathogenicity in H5Nx viruses. Multiple passages of H5N5 viruses in mice resulted in a substitution in amino-acid position 627 from glutamic acid to lysine (E627K) in the HA protein (Yu et al., 2018). This adaptive mutation increased the pathogenicity of these viruses in mice by 1000× and enhanced viral replication in vivo and in vitro (Yu et al., 2018). There were significant structural and functional differences in the NA proteins (N6, N8, and N2) from several viruses associated with the clade 2.3.4.4 (Diederich et al., 2015). The HA/NA interplay may be age dependent: whereas nonfunctional H5 viruses result in the death of day-old chickens, infection with the same virus in week-old chickens showed no signs of clinical illness at all (Hoffmann et al., 2015). This seems to be an H5-specific phenomenon, whereas H7 viruses were less dependent on a functional NA to cause illness. The dominant AIVs that infected humans have been associated with H5N6 viruses from clade 2.3.4.4. Out of the 17 human infections with H5N6 virus, 16 of the viruses contain a NA stalk deletion. Recombinant H5N6 viruses containing a 10 amino acid NA stalk deletion (amino acids 58–68) had an increase of viral replication in mammalian cell lines compared with the intact NA of H5N6 viruses. These viruses containing the NA stalk deletion also showed an increased viral replication in avian CEF cells, whereas H5N2 virus had lower titers in these cells (Yu et al., 2017). This recombinant virus with the NA deletion (∆H5N6) did not infect neural tissue in mice, whereas the full length H5N6 recombinant virus was neurotropic (Yu et al., 2017). Wild-type H5N6 viruses had higher rates of viral transmission and were more lethal to poultry compared with the ∆H5N6 virus. Wild-type H5N6 viruses were 100% lethal to chickens. All birds died within 10 days post infection (DPI), whereas, only 85% of the ∆H5N6 challenged chickens died by day 14 post infection. These data suggest that the NA stalk region in H5N6 viruses plays an important role in pathogenicity in mammalian hosts and displayed a decreased pathogenicity in chicken cells.

2.4. Virus replication

The stages of virus replication have been reported by various investigators in great detail (Shaw and Palese, 2013). In brief, AI virus HA protein attaches to sialic acid found on host glycoproteins, initiating receptor‐mediated endocytosis. The endosomes naturally acidify which triggers a conformational change in the HA2 protein that causes fusion of the viral envelope with the endosome membrane. The HA protein is synthesized as a polypeptide that must be proteolytic ally cleaved into HA1 and HA2 subunits to allow the virus to be infectious. The viral nucleocapsids are transported to the nucleus where viral transcriptase complex synthesizes mRNA. Six monocistronic mRNAs are produced in the nucleus and transported to the cytoplasm for translation into HA, NA, NP, PB1, PB2, and PA proteins. The mRNA of NS and M gene segments undergo splicing with each producing two mRNAs, which are translated into NS1, NS2, M1, and M2 proteins. The HA and NA proteins are glycosylated in the rough endoplasmic reticulum, trimmed in the Golgi, and transported to the surface where they are embedded in the plasma membrane. The eight viral gene segments along with internal viral proteins (NP, PB1, PB2, PA, and M2) assemble and migrate to areas of the plasma membrane containing the integrated HA, NA, and M2 proteins. The M1 protein promotes close association with the plasma membrane and budding of the virions.

2.5 Antigenic shift and drift of influenza A viruses 

Influenza A viruses are very diverse, and two viruses that share a subtype may be only distantly related. Some variability results from the gradual accumulation of mutations, a process called “antigenic drift”. Once the viral HA or NA has changed enough, immune responses generated against its former proteins may no longer be protective. More rapid changes can occur when two different influenza viruses infect the same cell. In this situation, gene segments from both viruses may be packaged into a single, novel virion, a process called genetic reassortment. Genetic reassortment can occur between any two Influenza A viruses, whether they are adapted to circulate in birds or mammals. If genetic reassortment results in the acquisition of a new HA and/or NA protein, this can cause an “antigenic shift” among the viruses circulating in a species. Antigenic shifts may be sufficient for the reassortant virus to completely evade existing immunity. After a subtype has circulated in a species for a while, genetic reassortments and antigenic drift can produce numerous viral variants, which may differ in their virulence for birds and/or mammals.

3. Detection HPAIVs type A/H5 in domestic and wild birds

3.1 H5N1 HPAIVs 

The Asian lineage HPAIV A (H5Nx) was initially diagnosed in humans in Hong Kong in 1997. The virus then re-emerged in 2003 and 2004, and spread from Southeast Asia across Asia to Europe and Africa, and has gained an endemic status in poultry populations in several countries including Egypt and Indonesia where spill-over infections in humans continue to be registered. HPAIV A (H5N1) has caused several hundred human cases and deaths, as well as the destruction of hundreds of millions of poultry worldwide. In 2005, HPAIV A (H5N1) caused massive mortalities among several aquatic wild bird species at Lake Qinghai, north-western China (Chen et al., 2005). During 2005, HPAIV A (H5N1) spread from this location via southern Siberia, Kazakhstan, and Russia to reach Europe in autumn 2005, and Africa in 2006 (Wallace et al., 2007). Epidemic outbreaks among wild birds were recorded in spring 2006 in central and south-eastern Europe and again, in central Europe, during summer 2007 (Globig et al., 2009). Since 2011, a number of countries in Asia have experienced several new virus introductions, particularly of virus clade 2.3.2.1, in which in most cases wild birds were implicated (Nagarajan et al., 2012). These include India, Bangladesh, the Republic of Korea, Laos, Malaysia, Japan, Myanmar, and Nepal. HPAIV A (H5N1) outbreaks in the poultry populations from these countries as well as from China, Vietnam and Indonesia have been continuously reported to date (Figure 5). Bangladesh, India, and Nepal informed also about three events in wild birds (crows and whooper swan) in 2016 and 2017. Since the first introduction to Nigeria in 2006 HPAIV A (H5N1) has circulated and spread in West African countries (Tassoni et al., 2016; Ekong et al., 2017). The disease was confirmed in Burkina Faso, Cameroon, Cote d’Ivoire, Ghana, Niger, Nigeria, and Togo to date. Furthermore, Nigeria reported four wild bird events from the Delta and Kano provinces in 2016. The outbreaks in Israel and the Occupied Palestinian Territories have been most frequently related to the endemic situation of clade 2.2.1.2 in Egypt; however, no links could be set up on the outbreaks in Iraq and Iran as clade 2.3.2.1 was identified. Since November 2015, the European avian lineage of HPAI H5 with three different subtypes such as H5N1, H5N2 and H5N9 emerged in south-western France and caused severe outbreaks in domestic ducks and domestic geese until March 2017 (Briand et al., 2017).

3.2 H5N6 HPAIVs 

A novel reassortant A (H5N6) HPAIV was detected for the first time from sewage in a live poultry market in China in December 2013 (Qi et al., 2014). In subsequent years, a few countries of Southeast Asia had notified infected with this virus. Initially, the detections were largely confined to China, Laos, and Vietnam (Wong et al., 2015; Chu et al., 2016). During the autumn/winter season 2016/2017, the virus caused large epidemics in the Republic of Korea (poultry) and Japan (wild birds) (Figures 6 and 7). In the Republic of Korea, more than 300 outbreaks were confirmed, mostly in ducks and chickens. The virus was also detected in more than 160 cases in dead wild birds (predominantly swans, but also ducks, geese, gulls, cranes, grebes, birds of prey, owls, and others), including birds kept at zoos. The affected wild bird species were similar to those found positive for A (H5N8) virus in Europe in 2016/2017. Single outbreaks were recently also reported from Hong Kong SAR, Chinese Taipei, Myanmar, and Vietnam.

3.3 H5N8 HPAIVs 

Since the first cases of HPAIV A (H5N8) were detected in November 2016 in common coots (Fulica atra) and Eurasian wigeons (Anas penelope) in Egypt and Tunisia as well as on a poultry farm in Nigeria in December 2016, A (H5N8) has been recorded also from Cameroon, the Democratic Republic of the Congo, Niger, South Africa, Uganda, and Zimbabwe. In Egypt, Cameroon and Nigeria several outbreaks of subtype A (H5N8) were detected in domestic birds in 2017 and currently HPAIV A (H5N1) and A (H5N8) viruses are co-circulating in the poultry population of these countries (see also Section 3.3.1.1). Infections of wild birds (e.g. white-winged black tern, Chlidonias leucopterus) were reported in January 2017 from Lake Victoria in Uganda and these spread to poultry farms in the Democratic Republic of Congo and Uganda in the following months. Over the last 3 months, further outbreaks of HPAIV A (H5N8) have been reported from South Africa and Zimbabwe. In addition to poultry farms, the virus was also detected in several wild bird species e.g. Egyptian goose (Alopochen aegyptiaca), southern masked weaver (Ploceus velatus), yellow-billed duck (Anas undulata), spur-winged goose (Plectropterus gambensis), sacred ibis (Threskiornis aethiopicus), house sparrow (Passer domesticus) and African rock pigeon (Columba guinea) in South Africa (Figure 8).

HPAIV A (H5N8) has also been reported in Asia (China, India, Kazakhstan, Republic of Korea, Nepal, Russia) and the Middle East (Iran, Israel, Kuwait). An overview is provided by FAO via the A (H5N8) HPAIV global situation updates (https://www.fao.org/ag/againfo/programmes/en/empres/h5n8/situation_update.html).

4. Susceptible hosts

Avian influenza viruses have been shown to naturally infect a wide variety of wild and domestic birds, especially free‐living birds occupying aquatic habitats. Some avian influenza infections have involved wild terrestrial birds, but these birds do not represent a major source or reservoir of AIVs (Stallknecht, 2003), but potentially play an important role in local transmission of H5 Gs/GD lineage HPAIV (Siengsanan et al., 2009). In brief, AIVs have been isolated from more than 90 species of free‐living birds representing 13 different orders: Anseriformes (ducks, geese, and swans), Charadriiformes (e.g., shorebirds [turnstones and sandpipers], gulls, terns, puffins, and guillemots), Ciconiiformes (herons and ibis), Columbiformes (doves), Falconiformes (raptors), Galliformes (partridge and pheasant), Gaviiformes (loons), Gruiformes (coots and moorhen), Passeriformes (perching birds-e.g., mynahs, finches, and weaverbirds), Pelecaniformes (cormorant), Piciformes (woodpecker), Podicipediformes (grebe), and Procellariiformes (shearwater) (Stallknecht and Brown, 2016). This represents 61% of known avian families, but the actual number of naturally infected species is most likely much greater (Alexander, 1993). 

In man‐made ecosystems (agriculture, caged, hobby flocks, and exhibition systems), infections have been reported in Psittaciformes (parrots, cockatoos, and parakeets), Casuariiformes (emu), Struthioniformes (ostrich), Rheiformes (rhea), and most domesticated Galliformes and Anseriformes. The latter two groups include chickens, turkeys, Japanese quail (Coturnix japonica), helmeted guineafowl (Numida meleagris), Bobwhite quail (Colinus virginianus), pheasants (various species), chukar partridges (Alectoris chukar), geese (Anser anser domesticus), and ducks (mallards [Anas platyrhynchos domesticus] and Muscovy [Cairina moschata domesticus]) (Easterday et al., 1997). Birds of the orders Psittaciformes probably are infected after capture and during mixing with infected birds at holding sites or in quarantine (Easterday et al., 1997). Some infections of free‐living Passeriformes (perching birds— starlings and sparrows) have been associated with outbreaks on poultry farms where they may have acquired infections from close contact with poultry (Morgan and Kelly, 1990). Experimentally, sparrows have been shown to be capable of transmitting infection back to poultry (Gutierrez et al., 2011). 

LPAIVs have caused epidemics or sporadic cases of respiratory disease in mink, seals, whales, and other sea mammals (Kroeze and Kuiken, 2017). H5Nx Gs/GD lineage HPAIV have been reported to cause sporadic infections in donkeys, large felids (tigers, leopards, lions) domestic dogs, house cats, mink, red foxes, Owston’s palm civets, a stone martin, and pigs (Kroeze and Kuiken, 2017). Most of these cases in carnivores involved close contact with or consumption of infected birds. Cases of natural infections by AIVs in humans have been reported and there is serological evidence of more widespread infection. 

In experimental studies, specific strains of AIV have been shown to infect pigs, ferrets, rats, rabbits, guinea pigs, mice, dogs, foxes, cats, mink, nonhuman primates, and humans (Shortridge et al., 1998; Easterday et al., 1997). 

5. Transmission

5.1 Transmission of AIVs in birds

Avian influenza viruses are shed in the feces and respiratory secretions of birds, although the relative amount of virus can vary with the specific virus, host species and other factors (Swayne, 2008; Fenner et al., 1987). The feces contain large amounts of virus in aquatic birds such as waterfowl, and the fecal oral route is thought to predominate in wild bird reservoirs (Fouchier and Munster, 2009). Fecal-cloacal transmissions might also be possible, but respiratory transmission is ordinarily thought to play little or no role (Fouchier and Munster, 2009). However, there are some exceptions. Some viruses that have adapted to gallinaceous poultry, such as recent isolates of Asian lineage H5Nx HPAIVs, can be found in higher quantities in respiratory secretions than the feces, even in wild waterfowl (Pantin-Jackwood and Suarez, 2013). There are also reports of a few LPAIVs found mainly in respiratory swabs from wild waterfowl (Krauss et al., 2013), and respiratory spread might be important in some wild terrestrial birds (Fouchier and Munster, 2009). 

Once an AIV has entered a poultry flock, it can spread on the farm by both the fecal-oral route and aerosols, due to the close proximity of the birds. Fomites can be important in transmission, and flies may act as mechanical vectors (Swayne 2008). The possibility of windborne transmission of HPAIVs between farms was suggested by one study (Ypma et al., 2013), but has not been conclusively demonstrated. Avian influenza viruses have also been found in the yolk and albumen of eggs from chickens, turkeys and quail infected with HPAIVs (Kilany et al., 2010). Although HPAIVs infected eggs are unlikely to hatch, broken eggs could transmit the virus to other chicks in the incubator. It might be possible for LPAIVs to be shed in eggs, but the current evidence suggests this is very rare, if it occurs at all (Cappucci Jr et al., 1985). 

How long birds remain contagious differs between avian species, and varies with the severity of the infection (chickens and turkeys infected with HPAIVs die very soon after infection). Most chickens usually excrete LPAIVs for a week, and a minority of the flock for up to two weeks, but individual birds of some species, including waterfowl, can shed some LPAIVs or HPAIVs for a few weeks in the laboratory (Van der Goot et al., 2007).

5.2 Transmission of AIVs to mammals 

People and other mammals are usually infected with avian influenza viruses during close contact with infected birds or their tissues, although indirect contact via fomites or other means is also thought to be possible (CDC, 2015). Respiratory transmission is likely to be an important route of exposure, and the eye may also act as an entry point (Belser et al., 2013; Aamir et al., 2009; Bischoff et al., 2011). A few H5Nx HPAIVs infections in animals, and rare cases in humans, have been linked to the ingestion of raw tissues from infected birds (Lipatov et al., 2009; Zhang et al., 2010). Housecats in an animal shelter might have become infected from contaminated avian feces, ingested while grooming (Leschnik et al., 2007). Feeding experiments provide evidence that H5Nx viruses can enter the body by the oral route in cats, pigs, ferrets, mice, hamsters and foxes, and transmission has been confirmed in cats by direct inoculation of the virus into the gastrointestinal tract (Rimmelzwaan et al., 2006; Shinya et al., 2011). In humans, the strongest evidence for oral transmission is that two people became infected with an Asian lineage H5Nx virus after eating uncooked duck blood (Lipatov et al., 2009). There are other human cases where ingestion probably occurred, but additional routes of exposure also existed (Mail, 2009). 

A ferret model suggested that some viruses might be transmitted to the fetus, when there is high viremia during systemic infections (Sweet and Smith, 1980). Viral antigens and nucleic acids were also found in the fetus of a woman who died of an Asian lineage H5Nx infection (Gu et al., 2007). Trans placental transmission seems much less likely with influenza viruses that replicate only in the respiratory tract.

6. Pathogenesis

6.1 Incubation period

The incubation period in poultry can be a few hours to a few days in individual birds, and up to 2 weeks in the flock (Fenner et al., 1987). A 21-day incubation period, which takes into account the transmission dynamics of the virus, is used for an avian population in the context of disease control (Swayne, 2008). The incubation period for AIVs in mammals is also thought to be short, and might be as little as 1-2 days in some cases (Vahlenkamp et al., 2010).

6.2 Pathogenesis of the infectious process

In poultry, the process begins by inhalation or ingestion of infectious LPAIV or HPAIV virions. Because trypsin‐like enzymes in respiratory and intestinal epithelial cells allow cleavage of the surface hemagglutinin, multiple replication cycles occur in respiratory and/or intestinal tracts with either type of virus. In gallinaceous poultry, the nasal cavity is a major site of initial replication. 

With HPAIVs, after initial replication in respiratory epithelium, the virions invade the submucosa, entering capillaries. The virus replicates within endothelial cells and spreads via the vascular or lymphatic systems to infect and replicate in a variety of cell types in visceral organs, brain, and skin. Alternatively, the virus may become systemic before having extensive replication in vascular endothelial cells. The virus is present in the plasma, and red and white blood cell fractions. Macrophages appear to play a role in systemic virus spread. The presence of a HA proteolytic cleavage site that can be cut by ubiquitous furin‐like cellular enzymes is responsible for this pantropic replication. Clinical signs and death are due to multiple organ failure. Damage caused by AIVs is the result of one of four processes: (1) direct virus replication in cells, tissues, and organs; (2) indirect effects from production of cellular mediators such as cytokines; (3) ischemia from vascular thrombosis, and (4) cardiovascular collapse from coagulopathy or disseminated intravascular coagulation. 

For the LPAIVs, replication usually is limited to the respiratory or intestinal tracts. Illness or death is most often from respiratory damage, especially if accompanied by secondary bacterial infections. Sporadically in some species, LPAIVs spread systemically, replicating and causing damage in kidney tubules, pancreatic acinar epithelium, oviduct, and other organs with epithelial cells having trypsin‐like enzymes. Pathogenesis of the infection process is less well understood in non‐gallinaceous birds. 

7. Clinical signs and pathology

7.1 Clinical signs

Clinical signs of disease are extremely variable and depend on other factors including host species, age, sex, concurrent infections, acquired immunity, and environmental factors (Easterday et al., 1997).

In wild and domestic waterfowl, most HPAIVs replicate to a limited degree and produce few clinical signs. The major exception to this rule are some H5Nx Gs/GD lineage HPAIVs which can infect and cause clinical disease including neurological signs, depression, anorexia, and sudden death (Lee et al., 2005). Occasional sporadic, isolated cases of mortality have been reported in wild birds with other HPAIVs. One unusual outbreak in wild birds occurred in 1961 with H5Nx HPAIVs outbreak in common terns in South Africa, which produced sudden death without any other clinical signs, and was localized to the tern population without involvement of gallinaceous birds. 

In domestic chickens, turkeys, and related galliformes, clinical signs reflect virus replication and damage to multiple visceral organs, and cardiovascular and nervous systems. However, clinical manifestations vary depending on the extent of damage to specific organs and tissues (i.e., not all clinical signs are present in every bird). In most cases in chickens and turkeys, the disease is fulminating with some birds being found dead prior to observation of any clinical signs. If the disease is less fulminating and birds survive for 3-7 days, individual birds may exhibit nervous disorders such as tremors of the head and neck, inability to stand, torticollis, opisthotonus, and other unusual positions of head and appendages. The poultry houses may be unusually quiet because of decreased activity and reduction in normal vocalizations of the birds. Listlessness is common as are significant declines in feed and water consumption. Precipitous drops in egg production occur in breeders and layers with typical declines including total cessation of egg production within six days. Respiratory signs are less prominent than with LPAIVs but can include rales, sneezing, and coughing. Other poultry have similar clinical signs but may live longer and have evidence of neurologic disorders such as paresis, paralysis, vestibular degradation (torticollis and nystagmus), and general behavior aberrations (L. Perkins and Swayne, 2001). Corneal opacity has been observed in domestic ducks infected with H5Nx Gs/GD lineage HPAIVs (Yamamoto et al., 2007). 

In ostriches (Struthio camelus), reduced activity and appetite, listlessness, ruffled feathers, sneezing, hemorrhagic diarrhea, and open mouth breathing have been reported (Manvell et al., 2003). In addition, some birds were uncoordinated, exhibited torticollis, and had paralysis of the wings and tremors of the head and neck. However, signs observed depend on the virulence of the virus, for example an H5Nx AIV in South Africa caused subclinical infection in most ostriches (Toffan et al., 2010). Immune status, management, population density, and other causes of stress in ostriches are regarded as the ultimate determinants of the severity of avian influenza in this species (Abolnik et al., 2016).

7.2 Gross lesion

In gallinaceous poultry, HPAIVs produce a variety of edematous, hemorrhagic, and necrotic lesions in visceral organs and the skin. Although, when death is peracute, no gross lesions may be observed. In chickens, swelling of the head, face, upper neck, and feet may be observed which results from subcutaneous edema and may be accompanied by petechial‐to‐ecchymotic hemorrhages (Figures 10, 11, and 12). Periorbital edema may be seen (Figure 12). Necrotic foci, hemorrhage, and cyanosis of the non‐feathered skin have been reported, especially wattles and combs. Lesions in visceral organs vary with virus strain but most consistently are represented by hemorrhages on serosal or mucosal surfaces and foci of necrosis within parenchyma of visceral organs. Especially prominent are hemorrhages on the epicardium (Figure 13), in pectoral muscles, and in mucosa of the proventriculus and ventriculus (Figure 14). With the H5Nx Gs/GD lineage HPAIVs, necrosis and hemorrhage in Peyer’s patches of the small intestine were common as was reported with outbreaks of fowl plague in the early 1900s (Figure 15), and these viruses tend to produce more severe hemorrhage and edema in the lungs than other HPAIVs (Figure 16).

With most HPAIVs, necrotic foci are common in pancreas (Figure 17), spleen, and heart, and occasionally in liver and kidney. The kidney lesions may be accompanied by urate deposits. Lungs have focal ventral‐to‐diffuse interstitial pneumonia with edema. The lungs can be congested or hemorrhagic. The cloacal bursa and thymus are usually atrophic. Splenomegaly is frequent in gallinaceous birds infected with H5Nx Gs/GD lineage HPAIVs. In ostriches, HPAIVs produced edema of head and neck, severe hemorrhagic enteritis, enlarged and firm pancreas, mild‐to‐severe air sacculitis, hepatitis, peritonitis, renomegaly, and splenomegaly. Lesions are more severe and frequent in young birds.

8. Phenotypic characterization

8.1 H5N1 HPAIVs 

HPAIV of the A (H5N1) subtype responsible for the ongoing outbreaks worldwide continue to exhibit high pathogenicity for gallinaceous poultry and moderate to low pathogenicity for waterfowl. Comparative assessment of pathogenicity of A (H5N1) HPAIV belonging to clades 2.3.2.1b, 2.3.2.1c, 2.3.4, and 2.3.4.1 for mallards provided evidence for efficient infection, with the varied clinical course: relatively mild for 2.3.4 and lethal for 2.3.2.1b (Ducatez et al., 2017). Mandarin ducks (Aix galericulata) inoculated with clade 2.3.2.1 A (H5N1) isolated in South Korea in 2010 remained healthy throughout the experiment and shedding of the virus was minimal (Kang et al., 2017). The clade 7.2 A (H5N1) representatives were subject to animal experiments and showed high pathogenicity in chickens (intravenous pathogenicity index (IVPI) of 2.84–2.97) and no evidence of replication in ducks (Liu et al., 2016). Infection of ferrets with selected avian-derived A (H5N1) isolates of clade 2.3.2.1 b and 2.3.2.1 c led to severe disease, systemic replication and death (Pearce et al., 2017).

8.2 H5N6 HPAIVs 

The phenotype of the recent A (H5N6) viruses from Asia seems similar to that of the 2016-2017 A (H5N8) viruses: highly virulent for chickens, less virulent for domestic ducks, and variable virulence for wild birds. Increased mortality in galliforms, as well as increased mortality and neurological signs (torticollis, ataxia) in domestic ducks, were reported from the Republic of Korea. However, virus isolation from apparently healthy ducks and geese in live bird markets in China was also described (Jiao et al., 2016; Sun et al., 2016). A Korean A (H5N6) isolate was tested in 6-week-old SPF chickens inoculated intravenously and the IVPI value was 2.66 (Si et al., 2017). Another South Korean A (H5N6) isolate, detected in 2016, had the IVPI value of 2.94 and the mean death time (MDT) for embryos was 36 h (Kim et al., 2017). The IVPI value of two avian-origin A (H5N6) viruses isolated from swine in China was 2.8 and 2.99 (Li et al., 2015). All IVPI values meet the criteria for high virulence according to OIE Diagnostic Manual. Experimental infection of 6-week-old chickens infected via the intranasal route (10⁶ median egg infectious dose EID₅₀) with A (H5N6) HPAIV isolated from apparently healthy ducks in southern China resulted in the quick progression of symptoms, including depression, anorexia, and death of 100% birds within 6-7 days post-infection. The virus replicated in a wide range of organs and was successfully transmitted to naive chickens that also died (Jiao et al., 2016). Field observations of clinical manifestation in wild birds are rare. There was detection of A (H5N6) HPAIV in South Korea in three whooper swans (Cygnus cygnus) in 2016, one with neurological signs and two found dead. It is possible that whooper swans brought HPAIV A (H5N6) into Korea. However, it is also possible that whooper swans, which are highly susceptible to HPAIV A (H5N1) infection, may have been exposed locally through direct or indirect contact with other A (H5N6)-infected but asymptomatic migratory species, such as mallards or spot-billed ducks, or perhaps through exposure to infected poultry (Jeong et al., 2017). In winter 2016-2017 in Japan, 230 cases of HPAIV caused by A (H5N6) viruses were reported from wild birds, captive birds and poultry farms throughout the country. The Japanese A (H5N6) isolates differed slightly from that of HPAIVs isolated previously in Japan and China. The virus exhibited high pathogenicity and a high replication capacity in chickens, whereas virus growth was slightly lower in ducks compared with an A (H5N8) HPAIV isolate collected in Japan in 2014 (Hiono et al., 2017). Conversely, the A (H5N6) virus was detected in apparently healthy Northern pintails (Anas acuta) sampled during active surveillance in Hong Kong SAR.

8.3 H5N8 HPAIVs 

Data on the phenotypic characteristics of the current A (H5N8) HPAIV clade 2.3.4.4 (2016/2017) virus from outside Europe is scarce. Conversely, a large amount of data has been published recently on the pathogenicity of the A (H5N8) clade 2.3.4.4 (2014/2015) virus detected in Asia and North America. However, caution is recommended when extrapolating these results on the properties of the recent A (H5N8) viruses, as despite some similarities, the recent A (H5N8) virus seems to evoke higher mortality for certain species, especially wild birds. In general, the A (H5N8) clade 2.3.4.4 (2014/2015) virus seemed to be less virulent for domestic waterfowl than gallinaceous poultry. Despite its high lethality for chickens, its apparent virulence and transmissibility for this species were lower in comparison with A (H5N1) HPAIV (Lee et al., 2016). Intraclade-dependent differences in virulence were also observed (Tanikawa et al., 2016). Higher resistance of some local breeds/lineages of chickens has been reported from South Korea (Lee et al., 2016). Experimental inoculation of Pekin ducks with an A (H5N8) virus from North America resulted in no mortality, lack or only mild clinical signs (conjunctivitis, diarrhea) but virus shedding and transmission to contact-exposed ducks was observed (Pantin-Jackwood et al., 2017). Experimentally infected Muscovy ducks survived infection and were seroconverted (Lee et al., 2016). Mild clinical signs but occasionally nervous symptoms were observed following experimental inoculation of Chinese geese (Agnes cygnoides) (Pantin-Jackwood et al., 2017). The absence of clinical signs but replication of A (H5N8) strains isolated in Korea in 2014 was reported in pigeons. Transmission to contact birds was not observed (Kwon et al., 2017). The A (H5N8) clade 2.3.4.4 (2014/2015) was detected from a variety of wild bird species in Asia/North America but few studies have addressed the pathogenicity of the isolates for wild birds in the experimental setting. In one of these, the absence of clinical signs but efficient replication and transmission to co-housed birds was reported after experimental infection of Mandarin ducks (Aix galericulata) with a South Korean A (H5N8) isolate (Kwon et al., 2017). Mallards (Anas platyrhynchos) inoculated with an A (H5N8) HPAIV from North America exhibited fever, decreased bodyweight, shed low titers of the virus to contact ducks and had moderate lesions at necropsy (Pantin-Jackwood et al., 2016).

9. Genetic characterization

9.1 H5N1 HPAIVs 

Since the first detection of Gs/GD/96 ‘Guangdong’ lineage of A (H5N1) HPAIV in 1996, the HA of the virus has undergone an extensive evolution with the continuous emergence (and disappearance) of multiple genetic clades. The following clades were detected between 2013 and 2017 (Smith and Donis, 2015; Liu et al., 2016; Shittu et al., 2017): 

• 2.1.3.2a (Indonesia) 

• 2.2.1.1 (Egypt) 

• 2.2.1.2 (Egypt) 

• 2.2.1.2a (Egypt, Israel, and Occupied Palestinian Territories) 

• 2.3.2.1.a (Bangladesh, India) 

• 2.3.2.1.b (China and Hong Kong SAR) 

• 2.3.2.1.c (Cambodia, China, Laos, Indonesia, India, Vietnam, Iraq, Iran, Lebanon, Nigeria, Burkina Faso, Niger, Ghana, Ivory Coast, Romania, and Bulgaria) 

• 7.2 (China)

The A (H5N1) HPAIV virus that was detected in North America in December 2014 should be treated separately as it was a reassortant that contained the HA of the Gs/GD/96 lineage (clade 2.3.4.4), but four segments (including NA) from the North American lineage LPAIV (Torchetti et al., 2015). 

A separate event was also associated with the emergent A (H5N1) virus from France (2015/2016) that turned out to belong to the European avian lineage and was clearly distinguishable genetically from the Gs/GD/96-like lineage (Briand et al., 2017). 

Intraclade and interclade reassortants of Gs/Gd/96-like A (H5N1) have been described (Marinova-Petkova et al., 2016a). There is also evidence of the intersubtype reassortments (including A (H5N1)/A (H9N2)) of clades 2.3.2.1a (Marinova-Petkova et al., 2016b) or 7.2 (Liu et al., 2016). 

A (H5N1) viruses circulating worldwide continue to exhibit markers for increased zoonotic potential (Arafa et al., 2015) but so far no definite mutations have occurred that would enable sustained human-to-human transmission. The receptor-binding site of the HA in the A (H5N1) virus from France (2015/2016) was ‘avian-like’ and no major host adaptation or transmission markers indicative of the increased affinity to mammalian species were found (Briand et al., 2017).

9.2 H5N6 HPAIVs 

Phylogenetic analysis

Phylogenetic studies showed that the A (H5N6) viruses have been generated through multiple reassortment events. The primary A (H5N6) virus (detected at the end of 2013 in China) contained the HA gene of the A (H5N1) HPAIV clade 2.3.4.4, the internal genes of A (H5N1) clade 2.3.2.1, and the NA gene from the H6N6 LPAIV (Qi et al., 2014). Since that time, subsequent reports have provided evidence about growing genetic diversity caused by multiple reassortment events, mostly with Eurasian-origin AIV, including local HPAIV and LPAIV strains circulating in wild birds and poultry. However, the vast majority of HA genes still belong to the 2.3.4.4 lineage, although there are reports of an A (H5N6) virus with the HA derived from the 2.3.2 clade (Du et al., 2017). In a recently published paper by Yang et al. (2017), three events have been suggested to explain the generation of novel A (H5N6) reassortants. In the first event, the ‘reassortant A-type’ acquired HA gene segment from H5N2 clade 2.3.4.4 virus, NA gene segment (non-truncated) from H6N6 virus and internal gene segments from A (H5N1) clade 2.3.2.1.c. In the second event, the ‘reassortant B-type’ was generated by the acquisition of HA gene segment from A (H5N8) clade 2.3.4.4 virus, NA gene segment (truncated) from H6N6 virus and internal gene segments from A (H5N1) clade 2.3.2.1.c. The ‘reassortant C-type’ was generated as a result of the reassortment between reassortant B (HA and NA genes) and poultry-adapted A (H9N2) virus (internal genes). Notably, A (H5N6) viruses with an insert of internal A (H9N2)-like genes seemed to prevail in live poultry markets (LPMs) in different regions of China (Chen et al., 2017). Studies carried out in China on 175 A (H5N6) AIVs isolated between 2014 and 2015 in LPMs in Hunan Province provided evidence for the existence of at least six genotypes arising from segment reassortment, including a variant that possessed an HA from A (H5N1) clade 2.3.2 (Du et al., 2017). In the surveillance in LPM in eastern China in 2016, a novel subtype H7N6 was described in chicken. The virus possessed gene segments derived from A (H5N6), A (H9N2) and A (H7N9) viruses (Wu et al., 2017). The A (H5N6) AIVs detected in Japan in November 2016 were classified into the genetic clade 2.3.4.4 c and were genetically closely related to A (H5N6) HPAIVs that had been recently isolated in South Korea and China (Okamatsu et al., 2017). The A (H5N6) viruses found in wild birds and poultry in Korea in 2016 seem to be closely related A (H5N6) viruses circulating in Guangdong province in China. Reassortment events with Eurasian LPAIVs were also detected (Kwon et al., 2017; Lee et al., 2017b). In another study, the A (H5N6) from faecal samples was proved to contain genes derived from H4N2 and H1N1 (Si et al., 2017). Jeong et al. (2017) characterised genetically two novel reassortants A (H5N6) AIVs detected in November 2016 in whopper swans in South Korea and found them to be distinguishable from the A (H5N8) and A (H5N1) HPAIVs previously isolated in Korea. Kim et al. (2017) analyzed five A (H5N6) isolates from fecal wild bird samples in South Korea and found that they were reassortants generated from numerous Eurasian AI virus subtypes, including A (H5N8) highly pathogenic viruses.

Molecular marker analysis

Two predominant HA cleavage site amino acid motifs found in A (H5N6) viruses from Asia are: PLREKRRKRGLF, PLRERRRKRGLF, occasionally also PLKEKRRKRGLF, PQRERRRKRGLF and PLREKRRRRGLF, all consistent with high virulence. The available studies on the genetic markers of virulence and host adaptation show that although most of A (H5N6) viruses exhibit preferential binding to sialic acid receptors joined to sugar through an a-2,3 sialic acid linkage (Kim et al., 2017), a feature typical of avian influenza viruses, a change towards human receptor-binding preference (a-2,6 sialic acid linkage) has also been described (Sun et al., 2016; Guo et al., 2017). For example, the A (H5N6) Chinese isolates from poultry had lost the glycosylation site at residue 158 of HA, bound both a2,6-resialylated and a2,3-resialylated chicken red blood cells (cRBCs), showed extensive binding to human tracheal epithelial and alveolar cells, replicated in the lungs of mice, and were transmissible through direct contact between ferrets (Sun et al., 2016). Two types of A (H5N6) can be distinguished based on the length of NA: with- and without truncated NA stems (deletions at amino acid positions 59–69), a signature of adaptation to terrestrial poultry (Bi et al., 2016; Sun et al., 2016; Yang et al., 2017). The A (H5N6) viruses that acquired the internal gene cassette from A (H9N2) viruses have been shown to carry mutations related to transmissibility and virulence in mammals or Adamantine resistance in PB1, PA, M1 and M2. The A (H5N6) reassortant viruses that derived NS1 from A (H5N1) viruses possess mutations indicating increased virulence in mice (Yang et al., 2017).

9.3 H5N8 HPAIVs 

Phylogenetic analysis 

In May, 2016, a novel reassortant A (H5N8) HPAIV belonging to clade 2.3.4.4 was identified in the Tyva Republic in Russia near the border with Mongolia and in Qinghai Lake in China. Phylogenetic analysis showed that three genes (HA, NA and NS) of novel Russian and Chinese isolates were derived from clade 2.3.4.4B, whereas the remaining segments (PB2, PB1, PA, NP, and M) clustered with LPAIV detected in wild birds in Mongolia, China, and Vietnam (Lee et al., 2017b; Li et al., 2017). Since then, further A (H5N8) clade 2.3.4.4 B reassortants have been identified across Europe and Asia. In October 2016, two slightly different genotypes of A (H5N8) viruses were detected at two zoos in India, with most of the gene segments closely related to the A (H5N8) sequences from Tyva Republic, Qinghai Lake, and Uvs-Nuur Lake. However, the NP and PA genes (first genotype) or only NP gene (second genotype) showed the highest similarities to the Eurasian LPAIVs sequences (Nagarajan et al., 2017). Similar observations were also made for the Korean A (H5N8) isolates obtained in December 2016, which were proved to be reassortants generated from A (H5N8) clade 2.3.4.4B and Eurasian LPAIVs (Kim et al., 2017). Novel reassortants were also detected in December 2016 in Egypt (Kandeil et al., 2017) The analysis of partial sequences of HA genes from outbreaks in Iran in November 2016 also showed that they belonged to clade 2.3.4.4B (Ghafouri et al., 2017). The data suggests that multiple genotypes were generated in the summer of 2016 in central Asia, and then were disseminated to the Far East, Middle East, Africa, and Europe. 

Molecular analysis 

Most of A (H5N8) isolates identified in Asia and Africa show the avian-like receptor specificity as indicated by the presence of glutamine (Q) at amino acid position 226 of HA protein. However, Marchenko et al. (2017) reported N94S and T123P substitutions in the HA protein, associated with increased interactions with human-type sialic acid receptors. All available studies indicate that analyzed isolates are susceptible to amantadine and neuraminidase inhibitors. The most prominent mutations responsible for increased pathogenicity for mammals such as PB2 E627K and D701N were not detected. However, markers of mammalian host specificity were observed in other genes, e.g. PB1 L13P in isolates from India and Egypt (Kandeil et al., 2017; Nagarajan et al., 2017). Variability in PB1- F2 protein length was also observed, isolates from India possessed truncated PB1-F2 protein (11 amino acids), whereas in isolates from South Korea this protein was of full length.

10. Diagnosis

10.1 Different diagnosis

Clinical diagnosis is at best presumptive and only used during epizootics, because of the extreme variability in the clinical signs accompanying influenza virus infections in birds. At the flock level, mortality, egg production, and body weight charts are often early indicators of nonspecific infectious and noninfectious illness, including avian influenza virus infection. Because of the broad spectrum of signs and lesions reported with infections by AIVs in several species, a definitive diagnosis must be made by virologic and serologic methods. For HPAIVs, other causes of high mortality must be excluded such as velogenic Newcastle disease, septicemic fowl cholera, heat exhaustion, water deprivation, and some toxins. For LPAIVs, other causes of respiratory disease and drops in egg production must be investigated such as lentogenic NDV, avian metapneumovirus, and other paramyxoviruses, infectious laryngotracheitis, infectious bronchitis, chlamydia, mycoplasma, and various bacteria. Concurrent infections with other viruses or other bacteria have been commonly observed (Easterday et al., 1997).

10.2 Laboratory diagnosis

Laboratory-based testing typically involves real-time (quantitative) RT-PCR (RT-qPCR) assay to detect the matrix protein (M) gene, as this is highly conserved in all avian and mammalian influenza viruses. Samples positive by this assay then are tested for specific H5 and H7 genes by RT-qPCR. If samples are H5 or H7 positive by RT-qPCR, sequence analysis is undertaken to determine the properties of the cleavage site. H5/H7 negative samples can be sequenced to determine the HA subtype. If several basic amino acids are detected at the cleavage site, then regulatory action is taken to eliminate the focus of infection. Virus isolation is used to obtain viruses for antigenic analyses and for in vivo pathogenicity tests; isolations are also performed for non-H5 or -H7 viruses, especially if there is any mortality associated with the sampled premise. Virus is best isolated from cloacal swabs (wild birds and aquatic poultry) and tracheal swabs (terrestrial poultry). Specimens are inoculated into the allantoic cavity of 10 11-day-old embryonating eggs, or on to MDCK cells, and the presence of virus is indicated by hemagglutinating activity using chorioallantoic or cell culture fluids and chicken or turkey red blood cells. Isolates are routinely characterized by gene-specific RT-qPCR assays or with monospecific antisera using hemagglutination-inhibition (HI) tests. Infection of flocks can also be assessed using serologic tests such as agar gel immunodiffusion, ELISA tests, and hemagglutination-inhibition tests to detect antibodies to influenza antigens. The initial screening is with a broad serological test for influenza viruses (such as agar gel immunodiffusion or ELISA), followed by 16 different hemagglutinin- and nine neuraminidase-specific tests for subtyping.

11. Morbidity and mortality

Avian influenza differs in severity, depending on the species of bird as well as the virus. LPAIVs usually cause mild illnesses or asymptomatic infections in birds, including chickens and ducks, but outbreaks can be more severe when there are concurrent infections or other exacerbating factors (Swayne, 2008). High mortality is occasionally seen in young ostriches infected with either LPAIVs or HPAIVs, although adult birds seem to be only mildly affected by both (Swayne, 2007). 

HPAIVs usually cause high and rapidly escalating mortality in chicken and turkey flocks, with cumulative morbidity and mortality rates that may approach 90-100% (Swayne, 2008). Some reports suggest that Asian linage H5N8 viruses might spread somewhat more slowly through chicken flocks than H5N1 viruses, and that the clinical presentation may be somewhat less severe (Lee et al., 2016). Any birds that survive an HPAIVs outbreak are usually in poor condition and do not begin laying again for several weeks. Morbidity and mortality rates can sometimes approach 100% in other domesticated and wild birds, as well; however, susceptibility varies greatly, and certain species such as waterfowl tend not to be severely affected (Desvaux et al., 2009).

Some Asian lineage H5Nx viruses cause severe illness even in waterfowl, and the introduction of these viruses may be heralded by unusual deaths among wild birds (e.g., swans in Europe and recently crows in Pakistan) (Desvaux et al., 2009). Thousands of wild birds were killed in some outbreaks, such as one at Qinghai Lake, China in 2005 (Chen et al., 2006). Wild bird deaths have also been associated with some Asian lineage H5 reassortants, such as H5N8 viruses, in Asia (Kim et al., 2015).

12. Treatment

There is no specific treatment for influenza virus infections in animals. Poultry flocks infected with HPAIVs are depopulated (this is generally mandatory in HPAIV-free countries), while the disposition of infected LPAIVs flocks may differ, depending on the specific virus and the country.

13. Control and prevention

13.1 Control

Control of avian influenza virus infections of domestic poultry is reliant on biosecurity, surveillance, and depopulation whenever HPAIVs are detected. Biosecurity is critical to prevent potentially catastrophic economic loss as a result of epizootics of HPAIVs infections and to prevent the evolution of H5 and H7 LPAIVs to HPAIVs by segregating domestic poultry from wild birds. 

A quick response is vital for containing avian influenza outbreaks, and in some cases, for minimizing the risk of zoonotic transmission. In addition to national notification requirements, HPAIVs and LPAIVs that contain H5 or H7 must be reported to the OIE by member nations (Epizooties, 2014). Veterinarians who encounter or suspect a reportable disease should follow their country-specific guidelines for informing the proper authorities (state or federal veterinary authorities in the U.S. for diseases in animals). Unusual mortality among wild birds should also be reported. Control of AIV infections of domestic poultry is reliant on biosecurity, surveillance, and depopulation whenever HPAIVs are detected. Biosecurity is critical to prevent potentially catastrophic economic loss as a result of epizootics of HPAIVs infections, and to prevent the evolution of H5 and H7 LPAIVs to HPAIVs by segregating domestic poultry from wild birds.

13.2 Prevention

The risk of introducing a virus to poultry or other birds can be reduced by good biosecurity and hygiene, which includes preventing any contact with other domesticated or wild birds, mechanical vectors and fomites including water sources. All-in/ all-out flock management is helpful in poultry flocks, and birds should not be returned to the farm from live bird markets or other slaughter channels. To help prevent reassortment between human and avian influenza viruses, people are encouraged to avoid contact with birds while suffering flu symptoms (Reid and Taubenberger, 2003). 

Avian influenza vaccines include both traditional inactivated whole virus vaccines and newer recombinant vectored vaccines (Bouma et al., 2007; Chen, 2009). Most vaccines are produced for chickens, although they may be validated for use in turkeys, and their effectiveness can differ in other species (Van der Goot et al., 2007; Koch et al., 2009). In addition to suppressing clinical signs, some vaccines are capable of increasing resistance to infection, and decreasing virus excretion and transmission (van der Goot et al., 2007; Maas et al., 2009). However, clinical protection is not necessarily correlated with reduced virus shedding, and some birds can become infected even in the best-case scenario (Lee et al., 2004). Thus, vaccination can mask infections if good surveillance programs are not used simultaneously (Capua and Marangon, 2006; Swayne, 2008). Vaccination can also place selection pressures on influenza viruses, which may encourage the emergence of vaccine resistant isolates (Lee et al., 2004). In different countries, vaccines may either be used routinely to protect poultry flocks, as an adjunct control measure during an outbreak, or to protect valuable species such as zoo birds from highly virulent viruses such as H5N1(Capua and Marangon, 2006).

During outbreaks, HPAIVs are normally eradicated by depopulation of infected flocks, combined with other measures such as movement controls, quarantines, and perhaps vaccination. Insect and rodent control, disposal of contaminated material, and thorough cleaning and disinfection are also important. 

For mammals, prevention involves avoiding close contact with infected birds or their tissues. Keeping susceptible animals indoors may be helpful during outbreaks in birds.

14. Vaccination

Until recently, AIVs infections caused by viruses of the H5 and H7 subtype occurred rarely, and vaccination was not considered because stamping out was the recommended control option. Primarily, for this reason, vaccinology for AI has not grown at the same rate as for other infectious diseases of animals. 

Guidelines on disease prevention and control have been issued as joint recommendations of the OIE, FAO, and WHO. These recommendations, however, need to be put into practice in a variety of different field situations; the applicability of 1 system rather than another in a given situation must be evaluated, weighing the benefits of a successful result against the drawbacks of failure. 

Vaccination can be a powerful tool to support eradication programs if used in conjunction with other control methods. Vaccination has been shown to increase resistance to field challenge, reduce shedding levels in vaccinated birds, and reduce transmission (Capua et al., 2004; Van Der Goot et al., 2005). All these effects of vaccination contribute to controlling AI; however, experience has shown that to be successful in controlling and ultimately in eradicating the infection, vaccination programs must be part of a wider control strategy that includes biosecurity and monitoring the evolution of infection.

An encouraging system, based on the detection of anti-NS1 antibodies, has been recently developed and can be used with all inactivated vaccines, provided they have the same hemagglutinin subtype as the field virus (Tumpey et al., 2005). This system is based on the fact that the NS1 protein is synthesized only during active viral replication and, therefore, is rarely present in inactivated vaccines. Birds vaccinated with such vaccines will develop antibodies to NS1 only after field exposure. 

To date, the only system that enables detection of field exposure in a vaccinated population and that has resulted in eradication is based on heterologous vaccination and known as “DIVA” (differentiating infected from vaccinated animals). This system was developed to support the eradication programs in the presence of several introductions of LPAIVs of the H7 subtype (Capua et al., 2004). Briefly, a vaccine is used that contains a virus possessing the same HA, but different NA, as the field virus. This vaccination strategy enables detection of antibodies to the neuraminidase antigen of the field virus.

Promising results have also been obtained with vaccines generated by reverse genetics (Tian et al., 2005). These vaccines are expected to perform like conventional inactivated vaccines; however, data are not yet available as to their efficacy under field conditions. Recombinant fowl pox vaccines that express the hemagglutinin protein of the field virus have also been reported to be efficacious for reducing shedding levels and providing clinical protection (Swayne et al., 2000a). They enable the detection of field exposure because vaccinated unexposed animals do not have antibodies to any of the other viral proteins. However, the performance of these vaccines in relation to the immune status of the host to the vector virus is unclear (Swayne et al., 2000b). Recent encouraging studies indicate that vaccination of day-old chicks with maternal antibodies against fowl pox has been successful. Data are lacking on the performances of such vaccines in a population that has been field exposed to fowl pox. These vaccines are likely to induce protective immunity only in birds that are susceptible to infection with the vector virus.

Regardless of the vaccine and companion test used, mapping the occurrence of infection within the vaccinated population is imperative, primarily to monitor the evolution of infection and to appropriately manage field exposed flocks. Field exposure represents a means by which infectious viruses may continue to circulate in the immune population.

Inadequate biosecurity or vaccination practices can lead to transmission between flocks and the selection of variants that exhibit antigenic drift. The antigenic drift of H5N2 viruses belonging to the Mexico lineage, resulting in lower identity (less similarity) to the vaccine strain, has been described (Lee et al., 2004). 

The international scientific community is debating how the vaccination of poultry would affect human health. On one hand, vaccinated birds shed less virus; on the other, they do not show any clinical signs of disease and could therefore act as silent carriers. Several factors contribute to the development of infection in humans: insufficient hygienic standards, the characteristics of the strain, and the presence of a viral dose sufficient to infect a human being. With reference to the H5N1 crisis, several countries are using vaccination to support control efforts. Vietnam implemented a nationwide vaccination campaign, which was completed in early 2006. The campaign’s main achievement is that despite 61 cases of human infection between January and November 2005, no human cases of AI have been reported in Vietnam after December 2005 (WHO, 2009).

15. References

Aamir, U. B., Naeem, K., Ahmed, Z., Obert, C. A., Franks, J., Krauss, S., ... & Webster, R. G. (2009). Zoonotic potential of highly pathogenic avian H7N3 influenza viruses from Pakistan. Virology, 390(2), 212-220.

Abolnik, C., Olivier, A., Reynolds, C., Henry, D., Cumming, G., Rauff, D., ... & Falch, C. (2016). Susceptibility and status of avian influenza in ostriches. Avian diseases, 60(1s), 286-295.

Alexander, D. J. (1993). Orthomyxovirus infection. Virus infections of birds, 287-287.

Belser, J. A., Gustin, K. M., Pearce, M. B., Maines, T. R., Zeng, H., Pappas, C., ... & Tumpey, T. M. (2013). Pathogenesis and transmission of avian influenza A (H7N9) virus in ferrets and mice. Nature, 501(7468), 556-559.

Bender, C., Hall, H., Huang, J., Klimov, A., Cox, N., Hay, A., ... & Subbarao, K. (1999). Characterization of the surface proteins of influenza A (H5N1) viruses isolated from humans in 1997–1998. Virology, 254(1), 115-123.

Bi, Y., Liu, H., Xiong, C., Liu, D., Shi, W., Li, M., ... & Gao, G. F. (2016). Novel avian influenza A (H5N6) viruses isolated in migratory waterfowl before the first human case reported in China, 2014. Scientific reports, 6(1), 1-10.

Bischoff, W. E., Reid, T., Russell, G. B., & Peters, T. R. (2011). Transocular entry of seasonal influenza–attenuated virus aerosols and the efficacy of N95 respirators, surgical masks, and eye protection in humans. Journal of Infectious Diseases, 204(2), 193-199.

Bogs, J., Veits, J., Gohrbandt, S., Hundt, J., Stech, O., Breithaupt, A., ... & Stech, J. (2010). Highly pathogenic H5N1 influenza viruses carry virulence determinants beyond the polybasic hemagglutinin cleavage site. PloS one, 5(7), e11826.

Bouma, A., Chen, H., Erasmus, B., Jones, P., Marangon, S., & Domenech, J. (2007, March). OIE Ad Hoc Group on AI Vaccination Guidelines. In Vaccination: a tool for the control of avian influenza. Proceedings of a meeting (pp. 3-167).

Briand, F. X., Schmitz, A., Ogor, K., Le Prioux, A., Guillou-Cloarec, C., Guillemoto, C., ... & Niqueux, E. (2017). Emerging highly pathogenic H5 avian influenza viruses in France during winter 2015/16: phylogenetic analyses and markers for zoonotic potential. Eurosurveillance, 22(9), 30473.

Cappucci Jr, D. T., Johnson, D. C., Brugh, M., Smith, T. M., Jackson, C. F., Pearson, J. E., & Senne, D. A. (1985). Isolation of avian influenza virus (subtype H5N2) from chicken eggs during a natural outbreak. Avian Diseases, 1195-1200.

Capua, I., & Mutinelli, F. (2001). colour atlas and text on avian influenza=. Papi editore.

Capua, I., & Marangon, S. (2006). Control of avian influenza in poultry. Emerging Infectious Diseases, 12(9), 1319.

Capua, I., Cattoli, G., & Marangon, S. (2004). DIVA--a vaccination strategy enabling the detection of field exposure to avian influenza. Developments in biologicals, 119, 229-233.

Chan, P. K. (2002). Outbreak of avian influenza A (H5N1) virus infection in Hong Kong in 1997. Clinical Infectious Diseases, 34(Supplement_2), S58-S64.

Chen, H. (2009). Avian influenza vaccination: the experience in China. Revue scientifique et technique (International Office of Epizootics), 28(1), 267-274.

Chen, H., Li, Y., Li, Z., Shi, J., Shinya, K., Deng, G., ... & Kawaoka, Y. (2006). Properties and dissemination of H5N1 viruses isolated during an influenza outbreak in migratory waterfowl in western China. Journal of virology, 80(12), 5976-5983.

Chen, H., Smith, G. J. D., Zhang, S. Y., Qin, K., Wang, J., Li, K. S., ... & Guan, Y. (2005). H5N1 virus outbreak in migratory waterfowl. Nature, 436(7048), 191-192.

Chen, J., Lee, K. H., Steinhauer, D. A., Stevens, D. J., Skehel, J. J., & Wiley, D. C. (1998). Structure of the hemagglutinin precursor cleavage site, a determinant of influenza pathogenicity and the origin of the labile conformation. Cell, 95(3), 409-417.

Chen, L. J., Lin, X. D., Tian, J. H., Liao, Y., Ying, X. H., Shao, J. W., ... & Zhang, Y. Z. (2017). Diversity, evolution and population dynamics of avian influenza viruses circulating in the live poultry markets in China. Virology, 505, 33-41.

Chu, D. H., Okamatsu, M., Matsuno, K., Hiono, T., Ogasawara, K., Nguyen, L. T., ... & Sakoda, Y. (2016). Genetic and antigenic characterization of H5, H6 and H9 avian influenza viruses circulating in live bird markets with intervention in the center part of Vietnam. Veterinary Microbiology, 192, 194-203.

Control, C. f. D. and Prevention (2015). "Centers for Disease Control and Prevention-CDC."

Costa, T., Chaves, A. J., Valle, R., Darji, A., van Riel, D., Kuiken, T., ... & Ramis, A. (2012). Distribution patterns of influenza virus receptors and viral attachment patterns in the respiratory and intestinal tracts of seven avian species. Veterinary research, 43(1), 1-13.

Desvaux, S., Marx, N., Ong, S., Gaidet, N., Hunt, M., Manuguerra, J. C., ... & Reynes, J. M. (2009). Highly pathogenic avian influenza virus (H5N1) outbreak in captive wild birds and cats, Cambodia. Emerging infectious diseases, 15(3), 475.

Diederich, S., Berhane, Y., Embury-Hyatt, C., Hisanaga, T., Handel, K., Cottam-Birt, C., ... & Pasick, J. (2015). Hemagglutinin-neuraminidase balance influences the virulence phenotype of a recombinant H5N3 influenza A virus possessing a polybasic HA0 cleavage site. Journal of virology, 89(21), 10724-10734.

Du, Y., Chen, M., Yang, J., Jia, Y., Han, S., Holmes, E. C., & Cui, J. (2017). Molecular evolution and emergence of H5N6 avian influenza virus in central China. Journal of virology, 91(12), e00143-17.

Ducatez, M., Sonnberg, S., Crumpton, J. C., Rubrum, A., Phommachanh, P., Douangngeun, B., ... & Webby, R. (2017). Highly pathogenic avian influenza H5N1 clade 2.3. 2.1 and clade 2.3. 4 viruses do not induce a clade-specific phenotype in mallard ducks. The Journal of general virology, 98(6), 1232.

Easterday, B. C., Hinshaw, V. S., & Halvorson, D. A. (1997). Influenza. Disease of Poultry. Iowa State University Press, Ames: IA, USA, 583-605.

Ekong, P. S., Fountain‐Jones, N. M., & Alkhamis, M. A. (2018). Spatiotemporal evolutionary epidemiology of H5N1 highly pathogenic avian influenza in West Africa and Nigeria, 2006–2015. Transboundary and emerging diseases, 65(1), e70-e82.

Fenner, F., Bachmann, P. A., Gibbs, E. P. J., Murphy, F. A., Studdert, M. J., & White, D. O. (1987). Veterinary virology, Orthomyxoviridae.

Fleury, D., Barrère, B., Bizebard, T., Daniels, R. S., Skehel, J. J., & Knossow, M. (1999). A complex of influenza hemagglutinin with a neutralizing antibody that binds outside the virus receptor binding site. Nature structural biology, 6(6), 530-534.

Fouchier, R. A. M., & Munster, V. J. (2009). Epidemiology of low pathogenic avian influenza viruses in wild birds. Revue scientifique et technique, 28(1), 49.

Ghafouri, S. A., Langeroudi, A. G., Maghsoudloo, H., Tehrani, F., Khaltabadifarahani, R., Abdollahi, H., & Fallah, M. H. (2017). Phylogenetic study-based hemagglutinin (HA) gene of highly pathogenic avian influenza virus (H5N1) detected from backyard chickens in Iran, 2015. Virus genes, 53(1), 117-120.

Globig, A., Staubach, C., Beer, M., Köppen, U., Fiedler, W., Nieburg, M., ... & Harder, T. C. (2009). Epidemiological and ornithological aspects of outbreaks of highly pathogenic avian influenza virus H5N1 of Asian lineage in wild birds in Germany, 2006 and 2007. Transboundary and emerging diseases, 56(3), 57-72.

Grant, E. J., Chen, L., Quiñones-Parra, S., Pang, K., Kedzierska, K., & Chen, W. (2014). T-cell immunity to influenza A viruses. Critical Reviews™ in Immunology, 34(1).

Gu, J., Xie, Z., Gao, Z., Liu, J., Korteweg, C., Ye, J., ... & Lipkin, W. I. (2007). H5N1 infection of the respiratory tract and beyond: a molecular pathology study. The Lancet, 370(9593), 1137-1145.

Guan, Y., & Smith, G. J. (2013). The emergence and diversification of panzootic H5N1 influenza viruses. Virus research, 178(1), 35-43.

Guo, H., de Vries, E., McBride, R., Dekkers, J., Peng, W., Bouwman, K. M., ... & de Haan, C. A. (2017). Highly pathogenic influenza A (H5Nx) viruses with altered H5 receptor-binding specificity. Emerging infectious diseases, 23(2), 220.

Gutierrez, R. A., Sorn, S., Nicholls, J. M., & Buchy, P. (2011). Eurasian tree sparrows, risk for H5N1 virus spread and human contamination through Buddhist ritual: an experimental approach. PLoS One, 6(12), e28609.

Hiono, T., Okamatsu, M., Matsuno, K., Haga, A., Iwata, R., Nguyen, L. T., ... & Sakoda, Y. (2017). Characterization of H5N6 highly pathogenic avian influenza viruses isolated from wild and captive birds in the winter season of 2016–2017 in Northern Japan. Microbiology and immunology, 61(9), 387-397.

Hoffmann, D., Röhrs, S., Rahn, J., Stech, J., & Beer, M. (2015). Pathogenicity evaluation of neuraminidase-negative H5 and H7 viruses in day-old chicks and adult chicken. Vaccine, 33(49), 6997-7001.

Horimoto, T., & Kawaoka, Y. (1994). Reverse genetics provides direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza A virus. Journal of virology, 68(5), 3120-3128.

Jeong, J., Woo, C., Ip, H. S., An, I., Kim, Y., Lee, K., ... & Jheong, W. (2017). Identification of two novel reassortant avian influenza a (H5N6) viruses in whooper swans in Korea, 2016. Virology journal, 14(1), 1-4.

Jiao, P., Cui, J., Song, Y., Song, H., Zhao, Z., Wu, S., ... & Liao, M. (2016). New reassortant H5N6 highly pathogenic avian influenza viruses in Southern China, 2014. Frontiers in Microbiology, 7, 754.

Kandeil, A., Kayed, A., Moatasim, Y., Webby, R. J., McKenzie, P. P., Kayali, G., & Ali, M. A. (2017). Genetic characterization of highly pathogenic avian influenza A H5N8 viruses isolated from wild birds in Egypt. The Journal of general virology, 98(7), 1573.

Kang, H. M., Lee, E. K., Song, B. M., Heo, G. B., Jung, J., Jang, I., ... & Lee, Y. J. (2017). Experimental infection of mandarin duck with highly pathogenic avian influenza A (H5N8 and H5N1) viruses. Veterinary microbiology, 198, 59-63.

Kilany, W. H., Arafa, A., Erfan, A. M., Ahmed, M. S., Nawar, A. A., Selim, A. A., ... & Abdelwhab, E. M. (2010). Isolation of highly pathogenic avian influenza H5N1 from table eggs after vaccinal break in commercial layer flock. Avian diseases, 54(3), 1115-1119.

Kim, H. R., Kwon, Y. K., Jang, I., Lee, Y. J., Kang, H. M., Lee, E. K., ... & Bae, Y. C. (2015). Pathologic changes in wild birds infected with highly pathogenic avian influenza A (H5N8) viruses, South Korea, 2014. Emerging infectious diseases, 21(5), 775.

Kim, Y. I., Park, S. J., Kwon, H. I., Kim, E. H., Si, Y. J., Jeong, J. H., ... & Choi, Y. K. (2017). Genetic and phylogenetic characterizations of a novel genotype of highly pathogenic avian influenza (HPAI) H5N8 viruses in 2016/2017 in South Korea. Infection, Genetics and Evolution, 53, 56-67.

Klenk, H. D., Rott, R., Orlich, M., & Blödorn, J. (1975). Activation of influenza A viruses by trypsin treatment. Virology, 68(2), 426-439.

Koch, G., Steensels, M., & Van Den Berg, T. (2009). Vaccination of birds other than chickens and turkeys against avian influenza. Revue scientifique et technique (International Office of Epizootics), 28(1), 307-318.

Krauss, S., Pryor, S. P., Raven, G., Danner, A., Kayali, G., Webby, R. J., & Webster, R. G. (2013). Respiratory tract versus cloacal sampling of migratory ducks for influenza A viruses: are both ends relevant?. Influenza and other respiratory viruses, 7(1), 93-96.

Kwon, J. H., Noh, Y. K., Lee, D. H., Yuk, S. S., Erdene-Ochir, T. O., Noh, J. Y., ... & Nahm, S. S. (2017). Experimental infection with highly pathogenic H5N8 avian influenza viruses in the Mandarin duck (Aix galericulata) and domestic pigeon (Columba livia domestica). Veterinary microbiology, 203, 95-102.

L. Perkins, L. E., & Swayne, D. E. (2001). Pathobiology of A/chicken/Hong Kong/220/97 (H5N1) avian influenza virus in seven gallinaceous species. Veterinary pathology, 38(2), 149-164.

Lee, C. W., Senne, D. A., & Suarez, D. L. (2004). Effect of vaccine use in the evolution of Mexican lineage H5N2 avian influenza virus. Journal of virology, 78(15), 8372-8381.

Lee, C. W., Suarez, D. L., Tumpey, T. M., Sung, H. W., Kwon, Y. K., Lee, Y. J., ... & Kim, J. H. (2005). Characterization of highly pathogenic H5N1 avian influenza A viruses isolated from South Korea. Journal of Virology, 79(6), 3692-3702.

Lee, D. H., Kwon, J. H., Noh, J. Y., Park, J. K., Yuk, S. S., Erdene-Ochir, T. O., ... & Song, C. S. (2016a). Pathogenicity of the Korean H5N8 highly pathogenic avian influenza virus in commercial domestic poultry species. Avian Pathology, 45(2), 208-211.

Lee, D. H., Sharshov, K., Swayne, D. E., Kurskaya, O., Sobolev, I., Kabilov, M., ... & Shestopalov, A. (2017b). Novel reassortant clade 2.3. 4.4 avian influenza A (H5N8) virus in wild aquatic birds, Russia, 2016. Emerging infectious diseases, 23(2), 359.

Lee, D. H., Bertran, K., Kwon, J. H., & Swayne, D. E. (2017a). Evolution, global spread, and pathogenicity of highly pathogenic avian influenza H5Nx clade 2.3. 4.4. Journal of Veterinary Science, 18(S1), 269-280.

Leschnik, M., Weikel, J., Möstl, K., Revilla-Fernández, S., Wodak, E., Bagó, Z., ... & Thalhammer, J. G. (2007). Subclinical Infection with Avian Influenza A H5N1 Virus in Cats. Emerging Infectious Diseases, 13(2), 243.

Li, K. S., Guan, Y., Wang, J., Smith, G. J. D., Xu, K. M., Duan, L., ... & Peiris, J. S. M. (2004). Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia. Nature, 430(6996), 209-213.

Li, M., Liu, H., Bi, Y., Sun, J., Wong, G., Liu, D., ... & Chen, J. (2017). Highly pathogenic avian influenza A (H5N8) virus in wild migratory birds, Qinghai Lake, China. Emerging infectious diseases, 23(4), 637.

Li, X., Fu, Y., Yang, J., Guo, J., He, J., Guo, J., ... & Chen, H. (2015). Genetic and biological characterization of two novel reassortant H5N6 swine influenza viruses in mice and chickens. Infection, Genetics and Evolution, 36, 462-466.

Lipatov, A. S., Kwon, Y. K., Pantin-Jackwood, M. J., & Swayne, D. E. (2009). Pathogenesis of H5N1 influenza virus infections in mice and ferret models differs according to respiratory tract or digestive system exposure. The Journal of infectious diseases, 199(5), 717-725.

Liu, L., Zeng, X., Chen, P., Deng, G., Li, Y., Shi, J., ... & Chen, H. (2016). Characterization of clade 7.2 H5 avian influenza viruses that continue to circulate in chickens in China. Journal of virology, 90(21), 9797-9805.

Maas, R., Tacken, M., van Zoelen, D., & Oei, H. (2009). Dose response effects of avian influenza (H7N7) vaccination of chickens: serology, clinical protection and reduction of virus excretion. Vaccine, 27(27), 3592-3597.

Mail, P. (2009). PRO/AH/EDR> Avian influenza, human-Thailand (06). Sept. 9, 2004. Archive Number 20040909.2513.

Manvell, R. J., English, C., Jorgensen, P. H., & Brown, I. H. (2003). Pathogenesis of H7 influenza A viruses isolated from ostriches in the homologous host infected experimentally. Avian diseases, 47(s3), 1150-1153.

Marchenko, V. Y., Susloparov, I. M., Komissarov, A. B., Fadeev, A., Goncharova, N. I., Shipovalov, A. V., ... & Ryzhikov, A. B. (2017). Reintroduction of highly pathogenic avian influenza A/H5N8 virus of clade 2.3. 4.4. in Russia. Archives of virology, 162(5), 1381-1385.

Marinova-Petkova, A., Franks, J., Tenzin, S., Dahal, N., Dukpa, K., Dorjee, J., ... & Webster, R. G. (2016a). Highly pathogenic reassortant avian influenza a (h5n1) virus clade 2.3. 2.1 an in poultry, bhutan. Emerging infectious diseases, 22(12), 2137.

Marinova-Petkova, A., Shanmuganatham, K., Feeroz, M. M., Jones-Engel, L., Hasan, M. K., Akhtar, S., ... & Webster, R. G. (2016b). The continuing evolution of H5N1 and H9N2 influenza viruses in Bangladesh between 2013 and 2014. Avian diseases, 60(1s), 108-117.

Matrosovich, M. N., Gambaryan, A. S., Teneberg, S., Piskarev, V. E., Yamnikova, S. S., Lvov, D. K., ... & Karlsson, K. A. (1997). Avian influenza A viruses differ from human viruses by recognition of sialyloligosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology, 233(1), 224-234.

Morgan, I. R., & Kelly, A. P. (1990). Epidemiology of an avian influenza outbreak in Victoria in 1985. Australian veterinary journal, 67(4), 125-128.

Nagarajan, S., Kumar, M., Murugkar, H. V., Tripathi, S., Shukla, S., Agarwal, S., ... & Tosh, C. (2017). Novel reassortant highly pathogenic avian influenza (H5N8) virus in zoos, India. Emerging Infectious Diseases, 23(4), 717.

Nagarajan, S., Tosh, C., Smith, D. K., Peiris, J. S. M., Murugkar, H. V., Sridevi, R., ... & Dubey, S. C. (2012). Avian influenza (H5N1) virus of clade 2.3. 2 in domestic poultry in India. PLoS One, 7(2), e31844.

Office International des Epizooties. (2014). Terrestrial animal health code. World Organisation for Animal Health.

Okamatsu, M., Ozawa, M., Soda, K., Takakuwa, H., Haga, A., Hiono, T., ... & Kida, H. (2017). Characterization of highly pathogenic avian influenza virus A (H5N6), Japan, November 2016. Emerging infectious diseases, 23(4), 691.

Organization, W. H. (2014). WHO risk assessment human infections with avian influenza A (H7N9) virus. 02 October, 2014.

Pantin-Jackwood, M. J., & Suarez, D. L. (2013). Vaccination of domestic ducks against H5N1 HPAI: a review. Virus research, 178(1), 21-34.

Pantin-Jackwood, M. J., Costa-Hurtado, M., Bertran, K., DeJesus, E., Smith, D., & Swayne, D. E. (2017). Infectivity, transmission and pathogenicity of H5 highly pathogenic avian influenza clade 2.3. 4.4 (H5N8 and H5N2) United States index viruses in Pekin ducks and Chinese geese. Veterinary Research, 48(1), 1-14.

Pantin-Jackwood, M. J., Costa-Hurtado, M., Shepherd, E., DeJesus, E., Smith, D., Spackman, E., ... & Swayne, D. E. (2016). Pathogenicity and transmission of H5 and H7 highly pathogenic avian influenza viruses in mallards. Journal of virology, 90(21), 9967-9982.

Pearce, M. B., Pappas, C., Gustin, K. M., Davis, C. T., Pantin-Jackwood, M. J., Swayne, D. E., ... & Tumpey, T. M. (2017). Enhanced virulence of clade 2.3. 2.1 highly pathogenic avian influenza A H5N1 viruses in ferrets. Virology, 502, 114-122.

Perez, D., Rimstad, E., Smith, G., Kochs, G., McCauley, J. W., Kaverin, N. V., ... & Hongo, S. (2011). Orthomyxoviridae. Virus Taxonomy. Oxford: Elsevier, 749-762.

Qi, X., Cui, L., Yu, H., Ge, Y., & Tang, F. (2014). Whole-genome sequence of a reassortant H5N6 avian influenza virus isolated from a live poultry market in China, 2013. Genome announcements, 2(5), e00706-14.

Reid, A. H., & Taubenberger, J. K. (2003). The origin of the 1918 pandemic influenza virus: a continuing enigma. Journal of General Virology, 84(9), 2285-2292.

Rimmelzwaan, G. F., van Riel, D., Baars, M., Bestebroer, T. M., van Amerongen, G., Fouchier, R. A., ... & Kuiken, T. (2006). Influenza A virus (H5N1) infection in cats causes systemic disease with potential novel routes of virus spread within and between hosts. The American journal of pathology, 168(1), 176-183.

Sauter, N. K., Bednarski, M. D., Wurzburg, B. A., Hanson, J. E., Whitesides, G. M., Skehel, J. J., & Wiley, D. C. (1989). Hemagglutinins from two influenza virus variants bind to sialic acid derivatives with millimolar dissociation constants: a 500-MHz proton nuclear magnetic resonance study. Biochemistry, 28(21), 8388-8396.

Shaw, M. and P. Palese (2013). Orthomyxoviridae, p 1151–1185. Fields virology, Lippincott Williams & Wilkins, Philadelphia, PA.

Shinya, K., Makino, A., Tanaka, H., Hatta, M., Watanabe, T., Le, M. Q., ... & Kawaoka, Y. (2011). Systemic dissemination of H5N1 influenza A viruses in ferrets and hamsters after direct intragastric inoculation. Journal of virology, 85(10), 4673-4678.

Shittu, I., Meseko, C. A., Gado, D. A., Olawuyi, A. K., Chinyere, C. N., Anefu, E., ... & Joannis, T. M. (2017). Highly pathogenic avian influenza (H5N1) in Nigeria in 2015: evidence of widespread circulation of WA2 clade 2.3. 2.1 c. Archives of virology, 162(3), 841-847.

Shortridge, K. F., Zhou, N. N., Guan, Y., Gao, P., Ito, T., Kawaoka, Y., ... & Webster, R. G. (1998). Characterization of avian H5N1 influenza viruses from poultry in Hong Kong. Virology, 252(2), 331-342.

Si, Y. J., Lee, I. W., Kim, E. H., Kim, Y. I., Kwon, H. I., Park, S. J., ... & Choi, Y. K. (2017). Genetic characterisation of novel, highly pathogenic avian influenza (HPAI) H5N6 viruses isolated in birds, South Korea, November 2016. Eurosurveillance, 22(1), 30434.

Siengsanan, J., Chaichoune, K., Phonaknguen, R., Sariya, L., Prompiram, P., Kocharin, W., ... & Ratanakorn, P. (2009). Comparison of outbreaks of H5N1 highly pathogenic avian influenza in wild birds and poultry in Thailand. Journal of wildlife diseases, 45(3), 740-747.

Skehel, J. J., & Wiley, D. C. (2000). Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annual review of biochemistry, 69(1), 531-569.

Smith, G. J., & Donis, R. O. (2015). World Organisation for Animal Health/Food and Agriculture Organization (WHO/OIE/FAO) H5 Evolution Working Group. Nomenclature updates resulting from the evolution of avian influenza A (H5) virus clades 2.1. 3.2 a, 2.2. 1, and 2.3. 4 during 2013–2014. Influenza Other Respir Viruses, 9(5), 271-6.

Sorrell, E. M., Song, H., Pena, L., & Perez, D. R. (2010). A 27-amino-acid deletion in the neuraminidase stalk supports replication of an avian H2N2 influenza A virus in the respiratory tract of chickens. Journal of virology, 84(22), 11831-11840.

Stallknecht, D. E., & Brown, J. D. (2016). Wild bird infections and the ecology of avian influenza viruses. Animal Influenza, 153-176.

Stallknecht, D. E. (2003). Ecology and epidemiology of avian influenza viruses in wild bird populations: waterfowl, shorebirds, pelicans, cormorants, etc. Avian diseases, 47, 61-69.

Stech, O., Veits, J., Abdelwhab, E. S. M., Wessels, U., Mettenleiter, T. C., & Stech, J. (2015). The neuraminidase stalk deletion serves as major virulence determinant of H5N1 highly pathogenic avian influenza viruses in chicken. Scientific reports, 5(1), 1-8.

Sun, H., Pu, J., Wei, Y., Sun, Y., Hu, J., Liu, L., ... & Liu, J. (2016). Highly pathogenic avian influenza H5N6 viruses exhibit enhanced affinity for human type sialic acid receptor and in-contact transmission in model ferrets. Journal of virology, 90(14), 6235-6243.

Swayne, D. (2008). "Avian Influenza. In Foreign Animal Diseases 7th ed. United States Animal Health Association."

Swayne, D. E. (2007). Understanding the complex pathobiology of high pathogenicity avian influenza viruses in birds. Avian diseases, 51(s1), 242-249.

Swayne, D. E., Beck, J. R., & Kinney, N. (2000b). Failure of a recombinant fowl poxvirus vaccine containing an avian influenza hemagglutinin gene to provide consistent protection against influenza in chickens preimmunized with a fowl pox vaccine. Avian diseases, 132-137.

Swayne, D. E., Garcia, M., Beck, J. R., Kinney, N., & Suarez, D. L. (2000a). Protection against diverse highly pathogenic H5 avian influenza viruses in chickens immunized with a recombinant fowlpox vaccine containing an H5 avian influenza hemagglutinin gene insert. Vaccine, 18(11-12), 1088-1095.

Sweet, C., & Smith, H. (1980). Pathogenicity of influenza virus. Microbiological reviews, 44(2), 303-330.

Tanikawa, T., Kanehira, K., Tsunekuni, R., Uchida, Y., Takemae, N., & Saito, T. (2016). Pathogenicity of H5N8 highly pathogenic avian influenza viruses isolated from a wild bird fecal specimen and a chicken in Japan in 2014. Microbiology and immunology, 60(4), 243-252.

Tassoni, L., Fusaro, A., Milani, A., Lemey, P., Awuni, J. A., Sedor, V. B., ... & Monne, I. (2016). Genetically different highly pathogenic avian influenza A (H5N1) viruses in West Africa, 2015. Emerging infectious diseases, 22(12), 2132.

Tian, G., Zhang, S., Li, Y., Bu, Z., Liu, P., Zhou, J., ... & Chen, H. (2005). Protective efficacy in chickens, geese and ducks of an H5N1-inactivated vaccine developed by reverse genetics. Virology, 341(1), 153-162.

Toffan, A., Olivier, A., Mancin, M., Tuttoilmondo, V., Facco, D., Capua, I., & Terregino, C. (2010). Evaluation of different serological tests for the detection of antibodies against highly pathogenic avian influenza in experimentally infected ostriches (Struthio camelus). Avian pathology, 39(1), 11-15.

Torchetti, M. K., Killian, M. L., Dusek, R. J., Pedersen, J. C., Hines, N., Bodenstein, B., ... & Ip, H. S. (2015). Novel H5 clade 2.3. 4.4 reassortant (H5N1) virus from a green-winged teal in Washington, USA. Genome announcements, 3(2), e00195-15.

Tumpey, T. M., Alvarez, R., Swayne, D. E., & Suarez, D. L. (2005). Diagnostic approach for differentiating infected from vaccinated poultry on the basis of antibodies to NS1, the nonstructural protein of influenza A virus. Journal of clinical microbiology, 43(2), 676-683.

Vahlenkamp, T. W., Teifke, J. P., Harder, T. C., Beer, M., & Mettenleiter, T. C. (2010). Systemic influenza virus H5N1 infection in cats after gastrointestinal exposure. Influenza and other respiratory viruses, 4(6), 379-386.

Van der Goot, J. A., van Boven, M., Koch, G., & de Jong, M. C. (2007). Variable effect of vaccination against highly pathogenic avian influenza (H7N7) virus on disease and transmission in pheasants and teals. Vaccine, 25(49), 8318-8325.

Van der Goot, J. A., Koch, G. D., De Jong, M. C. M., & Van Boven, M. (2005). Quantification of the effect of vaccination on transmission of avian influenza (H7N7) in chickens. Proceedings of the National Academy of Sciences, 102(50), 18141-18146.

Vasin, A. V., Temkina, O. A., Egorov, V. V., Klotchenko, S. A., Plotnikova, M. A., & Kiselev, O. I. (2014). Molecular mechanisms enhancing the proteome of influenza A viruses: an overview of recently discovered proteins. Virus research, 185, 53-63.

Veits, J., Weber, S., Stech, O., Breithaupt, A., Gräber, M., Gohrbandt, S., ... & Stech, J. (2012). Avian influenza virus hemagglutinins H2, H4, H8, and H14 support a highly pathogenic phenotype. Proceedings of the National Academy of Sciences, 109(7), 2579-2584.

Wan, X. F. (2012). Lessons from emergence of a/goose/guangdong/1996‐like h5n1 highly pathogenic avian influenza viruses and recent influenza surveillance efforts in southern china. Zoonoses and public health, 59, 32-42.

Webster, R. G. and R. J. C. Rott (1987). "Influenza virus A pathogenicity: the pivotal role of hemagglutinin." 50(5): 665-666.

Wong, F. Y., Phommachanh, P., Kalpravidh, W., Chanthavisouk, C., Gilbert, J., Bingham, J., ... & Morzaria, S. (2015). Reassortant highly pathogenic influenza A (H5N6) virus in Laos. Emerging infectious diseases, 21(3), 511.

World Health Organization. (2009). Cumulative number of confirmed human cases of avian influenza A/(H5N1) reported to WHO. http://www. who. int/csr/di-sease/avianinfluenza/country/en/.

Wu, H., Lu, R., Peng, X., Peng, X., Chen, B., Cheng, L., & Wu, N. (2017). Molecular characterization of a novel reassortant H7N6 subtype avian influenza virus from poultry in Eastern China, in 2016. Archives of virology, 162(5), 1341-1347.

Yamamoto, Y., Nakamura, K., Kitagawa, K., Ikenaga, N., Yamada, M., Mase, M., & Narita, M. (2007). Severe nonpurulent encephalitis with mortality and feather lesions in call ducks (Anas platyrhyncha var. domestica) inoculated intravenously with H5N1 highly pathogenic avian influenza virus. Avian diseases, 51(1), 52-57.

Yang, L., Zhu, W., Li, X., Bo, H., Zhang, Y., Zou, S., ... & Shu, Y. (2017). Genesis and dissemination of highly pathogenic H5N6 avian influenza viruses. Journal of Virology, 91(5), e02199-16.

Ypma, R. J., Jonges, M., Bataille, A., Stegeman, A., Koch, G., Van Boven, M., ... & Wallinga, J. (2013). Genetic data provide evidence for wind-mediated transmission of highly pathogenic avian influenza. The Journal of infectious diseases, 207(5), 730-735.

Yu, Y., Zhang, Z., Li, H., Wang, X., Li, B., Ren, X., ... & Liao, M. (2017). Biological characterizations of H5Nx avian influenza viruses embodying different neuraminidases. Frontiers in microbiology, 8, 1084.

Yu, Z., Cheng, K., Sun, W., Zhang, X., Xia, X., & Gao, Y. (2018). PB2 and HA mutations increase the virulence of highly pathogenic H5N5 clade 2.3. 4.4 avian influenza virus in mice. Archives of virology, 163(2), 401-410.

Zhang, J., Geng, X., Ma, Y., Ruan, S., Xu, S., Liu, L., ... & Li, Z. (2010). Fatal avian influenza (H5N1) infection in Human, China. Emerging infectious diseases, 16(11), 1799.

Zhao, Z., Guo, Z., Zhang, C., Liu, L., Chen, L., Zhang, C., ... & Liu, L. (2017). Avian influenza H5N6 viruses exhibit differing pathogenicities and transmissibilities in mammals. Scientific reports, 7(1), 1-6.