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Avian influenza: virology, diagnosis and surveillance

Future Microbiology

Mohamed E El Zowalaty* 1,2,3 , Stephen A Bustin 1 , Mohamed I Husseiny 3,4 & Hossam M Ashour* 5,6

1 Postgraduate Medical Institute, Faculty of Health, Social Care & Education, Anglia Ruskin University, Chelmsford, Essex, UK 2 Faculty of Public Health & Tropical Medicine, Jazan University, Jazan, Saudi Arabia 3 Department of Microbiology & Immunology, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt 4 Beckman Research Institute at City of Hope, Duarte, CA, USA 5 Department of Pharmacy Practice, Eugene Applebaum College of Pharmacy & Health Sciences, Wayne State University, Detroit, MI, USA 6 Department of Microbiology & Immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt„ *Authors for correspondence: n

Avian influenza virus (AIV) is the causative agent of a zoonotic disease that affects

populations worldwide with often devastating economic and health consequences.

Most AIV subtypes cause little or no disease in waterfowl, but outbreaks in poultry


can be associated with high mortality. Although transmission of AIV to humans

occurs rarely and is strain dependent, the virus has the ability to mutate or reassort

into a form that triggers a life-threatening infection. The constant emergence of

new influenza strains makes it particularly challenging to predict the behavior,

spread, virulence or potential for human-to-human transmission. Because it is

difficult to anticipate which viral strain or what location will initiate the next

pandemic, it is difficult to prepare for that event. However, rigorous implementation

of biosecurity, vaccination and education programs can minimize the threat of

AIV. Global surveillance programs help record and identify newly evolving and

potentially pandemic strains harbored by the reservoir host.

Avian influenza (AI) is an acute avian respira-

tory disease caused by AI viruses (AIVs) that

affect many species of domestic and wild birds

and can also infect other animal species. In

poultry, AI is characterized by marked varia-

tion in morbidity and mortality. The notifiable

form of AI (NAI) describes an outbreak of AI

in poultry due to any H5 or H7 subtype of

influenza A virus (IAV) or an infection by any

AIV resulting in an intravenous pathogenicity

index >1.2 or causing at least 75% mortality

HPNAI is a ‘list A’ notifiable disease (B ox 1) of the

World Organization for Animal Health (OIE),

which describes serious transmissible diseases

that can potentially spread very rapidly between

countries and are of major significance for the


has been transmitted to humans (e.g., H1N1 in

1976, 1986 and 1988; H3N2 in 1993; H7N7

in 1996; and H9N2 in 1998, 1999 and 2003)

inducing mild, severe or fatal infections [3,4].

While it has been suggested that aerosolized

droplets or direct contact with virus in poultry

feces can lead to infections in humans [5], poul-

try-to-human transmission of IAVs is unlikely

to cause pandemic influenza. This requires the

emergence of viral subtypes with human-to-

human transmissibility arising from mutations,

genetic rearrangement of viral RNA segments or

genetic reassortment between different strains of

virus [6,7] in a permissive host [8], with pigs being

the probable ‘mixing vessel’ [9].

Several examples of human-adapted IAVs exist

international trade of animals and animal prod- ucts, and so have severe socioeconomic or public
international trade of animals and animal prod-
ucts, and so have severe socioeconomic or public
health costs [1]. LPNAI includes all IAVs of H5
and H7 subtype that are not HPNAI viruses.
Low pathogenicity avian influenza (LPAI) refers
to all infections caused by AIVs that are not NAI
viruses and include H1-H4, H6, and H8-H16
The HPAI viruses are restricted to subtypes
H5 and H7, although not all H5 and H7 viruses
cause HPAI. The risk from AI is generally low
in most people, as AIVs do not usually infect
humans. However, there have been several cases
of conjunctivitis in humans related to LPAI. AIV
that have initiated a number of pandemics in the
last century [10]. The emergence of the Asian-
based HPAI H5N1 virus, in particular, high-
lights the continuing public health concerns over
human fatalities and its potential emergence as a
future pandemic virus. This implicates AI as one
of the most threatening trans-boundary diseases
facing humans [11], highlights the potential risk
IAV poses to human health and economy [12–14]
and strongly emphasizes the urgent requirement
for continued high levels of awareness and sur-
veillance programs to monitor for emerging AIVs
[15]. Accordingly, the WHO has complemented
its human surveillance network with a program
LPAI [2, 201].
avian influenza n diagnosis
real-time PCR n surveillance
vaccines n virus isolation
part of
10.2217/FMB.13.81 © 2013 Future Medicine Ltd
Future Microbiol. (2013) 8(9), 1–19
ISSN 1746-0913


El Zowalaty, Bustin, Husseiny & Ashour

Box 1. Notifiable avian influenza.

List A notifiable diseases of the World Organization for Animal Health are defined as transmissible diseases that have the potential for very serious and rapid spread, irrespective of national borders, are of serious socioeconomic or public health consequence and are of major importance in the international trade of animals and animal products.

Notifiable avian influenza (AI) is defined as an infection of poultry caused by any influenza A virus of the H5 or H7 subtypes or by any AI virus with an intravenous pathogenicity index in chickens of greater than 1.2 (or, as an alternative, at least 75% mortality). AI can be categorized into highly pathogenic notifiable avian influenza, including high-pathogenicity H5 and H7 subtypes, low-pathogenicity avian influenza (LPAI), which includes all H5 and H7 LPAI subtypes, and all other LPAIs, which are not notifiable to the World Organization for Animal Health,

including H1–H4, H6 and H8–H16 LPAIs.

focusing on the ecology and surveillance of

influenza viruses in wild animals [16,17].

Human influenza pandemics

The three worldwide influenza outbreaks (pan-

demics) in the 20th century occurred in 1918,

1957 and 1968 [18]. Each pandemic caused

significant social and economic upheaval. The

rapid spread, combined with high morbidity and

mortality, gave rise to intense levels of anxiety

and fear among the public. The ‘Spanish flu’

(19181919) was caused by direct transmission

of the H1N1 virus from birds [19] and is consid-

ered to be the ‘mother of all pandemics’ [20]. It

spread across the entire world within 3 months,

infecting 50% of the world’s population and

resulting in an estimated 3050 million deaths

[21]. The ‘Asian flu’ (19571958) was caused by

genetic reassortment between human H1N1

and avian H2N2 influenza viruses. This pan-

demic spread across the world in approximately

6 months and, together with a second wave of

infections, affected approximately 4050% of

the world’s population, killing approximately

1 million people [22]. The ‘Hong-Kong flu’

(19681969) pandemic was caused by a H3N2

virus, which resulted from reassortment of cir-

culating human H2N2 and avian H3 virus in

Box 2. Antigenic drift versus antigenic shift.

Antigenic drift is defined as the process whereby small changes or mutations accumulate in the virus, mainly in the surface glycoproteins (predominantly hemagglutinin [HA]), which happen continually over time. The new virus strains thus produced have different antigenic profiles and may not be recognized by antibodies against the older strains, thereby making the individual susceptible to infection with the new strains. Antigenic shift is an abrupt, major change in the HA and/or neuraminidase (NA) proteins of the virus that occurs when surface HA and NA segments reassort between different viruses, resulting in the emergence of a new subtype. Alternatively, the new HA and NA combination may have emerged from an animal population that is so different from the same subtype in humans that most people do not have immunity to the new virus.

China, spreading to Hong Kong and the rest of the world, and causing the deaths of a few million individuals [22]. In February 2009, a new swine-origin influ- enza A (H1N1) virus emerged in Mexico from multiple reassortment events involving a unique combination of gene segments from human, swine and avian type A viruses [23–27], result- ing in the first influenza pandemic of the 21st

century [28,29]. By mid-April, it had reached the USA via human-to-human transmission, leading the WHO to raise its pandemic alert successively to phases 4 (27 April), 5 (29 April) and 6 (11

June) [24]. Although now in the post-pandemic

phase, the H1N1 (2009) virus is one of the

seasonal influenza viruses in global circulation

and continues to be a major challenge to public


History of HPAI

In 1878, a contagious disease of poultry asso-

ciated with high mortality was first described

in Italy and named ‘fowl plague’. In 1955, the

causative agent was identified as an IAV, based

on the presence of A type-specific ribonucleo-

protein. This resulted in the term fowl plague

being replaced by the more suitable term HPAI

[17, 30–32]. In 1961, a high proportion of deaths in

common terns (Sterna hirundo) in South Africa

was attributed to the HP A/H5N3 virus, which

was the first demonstration of a HPAI virus in

wild birds before the HP A/H5N1 [33]. The 1997

outbreak of respiratory disease in humans in

Hong Kong caused by H5N1, which was previ-

ously known to infect only avian species, raised

serious concerns about the potential for another

pandemic in humans [34,35]. The H5N1 virus

seems to have emerged in southeast China dur-

ing 1996 [36], where it reassorted with viruses

circulating in other avian species and emerged

as a new H5N1 virus in Hong Kong. After an

initial major outbreak of HPAI in chickens and

other birds, it was unexpectedly transmitted to humans [37], and in May 1997 a 3-year-old boy became the first human to die of a respira- tory illness related to an H5N1 virus infection [38] . An additional 17 cases were diagnosed in November and December of the same year [17]. A few years later, the virus spread to several Asian countries, infecting humans for the second time in February 2003 [39]. Since late 2003 until the present day, the world has witnessed the deadliest outbreak in the history of HPAI in birds. In addition, the transmission of A/H5N1 virus to humans is on the increase in Asia, southeast Asia, Africa and

Avian influenza viruses: virology, diagnosis & surveillance


the Middle East. Between 1997 and April 2013, HPAI A/H5N1 infection has been identified in 628 human cases, with 374 deaths confirmed in 15 countries, resulting in a fatality rate of 59.55%; the highest numbers of cases/deaths were reported in Indonesia, Egypt, Vietnam and China, respectively [202]. Although A/H5N1 originated in China, it is still unclear why the

number of cases is not the highest in its originat- ing location. So far, there has been no evidence of sustained human-to-human transmission of AIV. However, if the virus mutates or rearranges deliberately or spontaneously into a form that is

more easily transmissible between humans, it will

have the potential to kill millions in a pandemic.

The concurrent A/H5N1 virus possesses

three of the four properties necessary to cause

a pandemic: it infects people, most people are

immunologically naive and it is highly lethal.

However, it lacks the capacity for sustained

human-to-human transmission [18,40]. Although

that capacity may require only a single genetic

reassortment or mutation, up until now, most

infected people have been infected through

direct contact with infected poultry [41]. While

there have been extensive efforts by the WHO to

report all cases, the numbers might not reflect the

real picture, especially in developing countries,

since not all cases are being admitted to hospitals

or reported by governments. In developed coun-

tries, the strict implementation of biosecurity

and preventive procedures in poultry farms helps

guard against the spread of AIV. Annual surveil-

lance programs among waterfowl have also been

implemented in the USA and Europe.

In the spring of 2013, another ongoing out-

break of novel avian A/H7N9 virus that origi-

nated from multiple reassortment events has

again emerged in China, where H7N9 has so

far caused infection in 131 laboratory-confirmed

human cases, including 36 deaths [42,43]. The

recent A/H7N9 could potentially produce

a human pandemic since the virus is more

humanized in traits and could increase human- to-human transmission, if transmitted among

poultry or pigs, raising public health concerns of

a looming pandemic influenza in human [44,45]. In addition to AIV, other influenza viruses exist and can cause influenza epizootics among various animal species [5]. In some species, the disease mimics human influenza, while in others, there are no signs of disease. Since the demonstration of swine influenza (Hsw1N1) in 1930 and later in 1955, and with the identifica- tion of influenza viruses in horses and ducks, transmission to other animals has become more

evident and has attracted the attention of influ- enza researchers. Influenza A/H3N2 viruses dis- covered in horses were also found in pigs, cattle, chickens, dogs and other species throughout the

world [46]. An outbreak of severe respiratory dis- ease in a pack of English foxhounds in the UK in September 2002 was caused by an equine A/ H3N8 virus [47]. H3N8 and H3N2 canine influ- enza viruses have been reported in racing canine populations in the USA [48]. Aquatic birds have been revealed as the source of influenza viruses in sea mammals, such as seals (H4N5, H7N7) and whales (H13N2, H13N9,

H1N3) [5]. In this context, it is worth noting

the concerns expressed about the use of ferrets

as a model. Ferrets are considered to be the best

model for IAV because of their high suscepti-

bility and the fact that IAV infection in ferrets

closely resembles that in humans with respect

to clinical signs, pathogenesis and immunity.

Indeed, recent studies in ferrets have shown that

IAV can acquire the capacity for airborne trans-

mission between mammals without recombina-

tion in an intermediate host, and therefore con-

stitute a high risk for human pandemic influenza

[49,50]. The ability to produce such an infection

raises both biosecurity and biosafety concerns

and could even lead to a human pandemic [51].

Genome & virology of AIV

Influenza viruses are members of the family

Orthomyxoviridae (orthos, Greek for straight;

myxa, Greek for mucus) which belongs to

group V (negative-polarity ssRNA) of the Bal-

timore system of virus classification [52]. There

are five genera: influenza virus A, B and C,

Isavirus and Thogotovirus [53,54]. Each genus

includes only one species of IAV, influenza B

virus and influenza C virus. Influenza A and C

viruses infect multiple species, while influenza B

viruses almost exclusively infect humans [55].

IAVs include avian, swine, equine and canine

influenza viruses, as well as human IAVs. The structure of IAVs has been extensively studied and reported [56]. The genome of IAVs comprises eight negative-sense, viral ssRNA seg- ments that are numbered in order of decreasing length, as summarized in TaBle 1. The two main surface glycoproteins of influenza viruses as shown in Figure 1 are the hemagglutinin (HA) and neuraminidase (NA) proteins, which function in the attachment and release of the virion to and from cells. They are major antigenic determi- nants and so are major targets for the immune response to which neutralizing antibodies are

made [57]. Theoretically, different combinations


El Zowalaty, Bustin, Husseiny & Ashour


Proposed protein function(s)

Heterotrimeric P complex associated with NP and virion

RNA, RNA transcription and the replication component of

RNA polymerase (polymerase subunit); initiation of

transcription, RNA transcriptase, RNA elongation and

endonuclease activity

Proapoptotic activity

Polymerase subunit; RNA transcriptase, host cell mRNA cap

recognition and virulence

Polymerase subunit; RNA transcriptase and protease

Surface glycoprotein, virus binding to sialic acid-containing

receptors on host cell; penetration of virus genome into

host cell cytoplasm by fusion of virus and host cell

(attachment to cell membranes and receptormembranes

binding) and major antigenic determinant

Encasidates RNA; RNA synthesis and RNA nuclear import

interacts with RNAs and polymerase proteins asregulation;

a major component of the nucleocapsid; role in vRNA

replication; role in virus maturation and packaging

Surface glycoprotein; antigenic determinant, sialidase

(neuraminidase)-catalyzing cleavage of terminal sialic acid

residues from glycoconjugates, thereby digesting mucin to

enable the virus to reach its target epithelium and

facilitating release of infectious progeny virus


Table 1. The genomic segments of influenza A virus and their encoded proteins and functions.


number of


per virion







Localization and features Nascent protein length § in amino acids








Virion interior, infected cell


Virion interior, infected cell


Virion interior, infected cell


Globular head bears

antigenic sites and receptor

binding site; stem;

transmembrane span;

cytoplasmic tail

Virion interior, associated

with P complex and viral RNA

Virion envelope, infected cell

surface, cytoplasmic tail;

transmembrane span;

extracellular stalk; globular

head bears antigenic sites

and enzyme active site

Deduced from RNA sequence, excluding poly-A tract. Total RNA nucleotide sequence length is 13,588.

vRNP: Viral ribonucleoprotein.












[194]. protein

by biochemical


taken Polymerase






§ ‡ Data P






basic 1, 87 kDa



basic 2, 96 kDa



85.5 kDa














55 kDa




RNA segment (mRNA ) length in nucleotides





2341 (2320)




2341 (2320)



2233 (2211)



1778 (1757)



1565 (1540)



1413 (1392)


Avian influenza viruses: virology, diagnosis & surveillance



Proposed protein function(s)

Central role in replication and virus assembly, modulating

nuclear transport of vRNP; early infection: bound to vRNP

prior to RNP transport to nucleus; late infection: binds RNP,

signaling RNP transport from nucleus to cell surface,

mediates association of RNP with hemagglutinin and

neuraminidase at the cell membrane, promoting virion

formation and budding

Membrane cation channel activity; virus uncoating and

assembly; infected cell: specifically raises pH of Golgi to

protect pH-sensitive hemagglutinin; virion: may permit

acidification of virus interior during passage through

endosomal pathway in order to dissociate vRNP from M1

Multifunctional protein and viral interferon antagonist

protein; regulation of host gene expression; binds RNA,

thereby inhibiting host mRNA translation, regulating viral

pre-mRNA splicing and translation and viral polymerase

activity, and downregulating dsRNA-induced antiviral


Mediates nuclear export of vRNPs


Table 1. The genomic segments of influenza A virus and their encoded proteins and functions (cont.).


number of


per virion




Localization and features Nascent protein length §

in amino







Beneath lipid bilayer of virion

envelope; associates with

vRNPs in mature virion to

form nucleocapsid

Virion envelope, infected cell

surface (abundant);

extracellular region;

transmembrane span;

cytoplasmic tail; tetramers

form cation-selective channel

Infected cell nuclei

Associated with core

components of virion;

cytoplasm of infected cells

Deduced from RNA sequence, excluding poly-A tract. Total RNA nucleotide sequence length is 13,588.

vRNP: Viral ribonucleoprotein.












[194]. protein

by biochemical


taken Polymerase






§ ‡ Data P



M1, 28 kDa

M2, 15 kDa


NS1, 25 kDa


NS2, 14 kDa





RNA segment (mRNA ) length in nucleotides






1027 (1005)




890 (868)




El Zowalaty, Bustin, Husseiny & Ashour

Hemagglutinin Neuraminidase M2 ion channel RNP
M2 ion channel

Figure 1. Molecular structure representation of influenza A virus showing two surface

glycoproteins (hemagglutinin and neuraminidase), the major antigenic determinants of the

virus. The hemagglutinin protein is responsible for binding to sialic acid receptors on host cells.

Human influenza A viruses preferentially bind to sialic acids in an a2,6 conformation, while those

from avian species bind mostly to sialic acids in an a2,3 conformation.

RNP: Ribonucleoprotein.

Image was reproduced from the Centers for Diseases Control and Prevention (CDC), Atlanta,

Georgia, USA [206] with permission.

from the 17 HA and ten NA subtypes [58,59] can

be found, and each subtype may contain several

subtypes. Mutations can introduce additional

variability, and so the number of AIV strains

is indefinite.

In 1972, the WHO recommended a new sys-

tem for influenza nomenclature and classifica-

tion of influenza viruses into genetically distinct subtypes based on their NA and HA antigens [60,61]. The nomenclature consists of two parts:

a strain designation and a description of the NA and HA antigens [60,62] . To date, only three HA (H1, H2 and H3) and two NA (N1 and N2) sub- types have caused human epidemics, as defined by sustained and widespread person-to-person transmission [8]. Nevertheless, the recent discov- ery of the highly divergent IAV subtype H17N10 in bats from Guatemala in South America rein- forces the importance of surveillance for moni- toring the evolution of influenza viruses among different animal populations. Thus far, this new

distinct H17N10 virus is the only subtype that is

not found in an avian species [58,59].

Epidemiology of AI

The evolutionary success of IAV is a prototypic

example of the ability of microbes to adapt to

their many hosts. Influenza is fundamentally a

recurring background disease that re-emerges slightly differently each year due to the con- tinuously evolving nature of their surface gly- coproteins, referred to as antigenic drift [63]. At unpredictable time periods, influenza presents a newly emerging disease caused by viruses with completely different surface antigens, termed antigenic shift (Box 2), infecting humans with little or no immunity against these strains [57]. This antigenic variability of AIVs is due to their highly error-prone replication process [64] that,

together with viral genome assortment, allows influenza viruses to adapt to their host, thus acquiring a pandemic potential [65].

Avian influenza viruses: virology, diagnosis & surveillance


The ecology and epidemiology of AI have changed substantially in the last two decades [66]. AI infections due to LP A/H9N2 subtype have become widespread in Asia, whereas the HP A/H5N1 subtype has been the causative agent of widespread infections in poultry across other areas of the world, resulting in a modified eco- epidemiology and zoonotic potential [67]. As shown in Figure 2, the primary difference between LPAI and HPAI virus is local versus

systemic replication, respectively. One of the key determinants of virulence is the ability of the host to proteolytically cleave the HA precursor

HA0 into HA1 and HA2 subunits, an essen-

tial requirement for binding to the cell surface

receptors and fusion of the viral envelope with

the endosomal membrane. An analysis of HA

subtypes that circulate in aquatic birds, as well

as HAs representative of the subtypes that have

infected the human population, indicates that

the cleavage efficiency can vary significantly for

different HA subtypes and some display strin-

gent selectivity for specific proteases [68]. The

HA of mammalian viruses contains only a sin-

gle arginine and, rarely, a single lysine, at the

cleavage site and is cleaved extracellularly, limit-

ing their spread in mammalian hosts to tissues

that express the appropriate proteases. Similarly,

LPAI viruses have an HA cleavage site with one basic arginine or lysine residue that is cleaved only by proteases with monobasic specificity. As a result, LPAI infection in birds is restricted to the digestive or respiratory tracts and results in milder illness or no disease at all. By contrast, HPAI viruses are invariably HA subtypes H5 or H7 with a polybasic cleavage site, introduced

either as a result of insertion or substitution [69–71]. This polybasic cleavage site is susceptible to the protease furin, which is ubiquitous in cells [72]. The factors that cause mutation from LPAI to HPAI, which occurs only in birds, are not

known, but it seems that the wider the circula-

tion of LPAI in poultry, the higher the chance

that mutation into HPAI will occur [73]. It is

important to remember, however, that acquisi-

tion of a polybasic HA cleavage site is the only

necessary step for the evolution of LPAI strains

into HPAI viruses, since not all H5 or H7 sub-

types are hypervirulent. There are additional

virulence determinants within the HA itself

[74,75] and in other viral proteins, suggesting that

virulence is under polygenic control [73,76,77].

Persistence of AIV in the environment

LPAI viruses are ubiquitous [16] and approxi-

mately 90 species from some 12 of the 50 orders

N– HA0 TM –C Cleavage site Avirulent avian isolate H5 RETR G LPAI: proteases localized
Cleavage site
Avirulent avian isolate H5
LPAI: proteases localized in
respiratory and intestinal organs
Avirulent avian isolate H7
Virulent avian isolate H5
HPAI: ubiquitous proteases
Virulent avian isolate H7
Avian strains isolated from human H5N1 (1997 HK)
Avian strains isolated from human H9N2 (1999 HK)
Avian strains isolated from human H7N7 (2003 NL)
Avian strains isolated from human H5N1 (2004 Asia)

Figure 2. Schematic representation of the protease cleavage site in influenza A viruses hemagglutinin. HA0 is cleaved into disulfide linked subunits HA1 and HA2 at a specific cleavage site shown in red (single letter amino acid code is used to identify the amino acid sequence at the hemagglutinin cleavage site). The hemagglutinins of LPAI viruses are cleaved by proteases that are localized in respiratory and intestinal organs, resulting in mild localized infections, whereas the hemagglutinins of HAI viruses have multiple basic amino acids, which are cleaved by ubiquitous proteases in a wide range of organs, resulting in lethal systemic infection. Most human isolates of avian strains also possess polybasic cleavage sites. The TM domain is shown in orange. HK: Hong Kong; HPAI: High-pathogenicity avian influenza; LPAI: Low-pathogenicity avian influenza; NL: The Netherlands; TM: Transmembrane.


El Zowalaty, Bustin, Husseiny & Ashour

of birds carry all strains of influenza, with birds inhabiting wetland and aquatic environments showing the highest rates of influenza infection [78]. Mixed infections with different influenza subtypes among waterfowl are also common

[12,13,79]. Carriers are mainly of the orders Anseri- formes (especially the families Anatidae [ducks, geese, and swans], Charadriiformes [terns and waders] and Procellariiformes [shorebirds, gulls and seabirds]) and thus act as a reservoir for the virus [80]. The migratory nature of many of these species and the persistence of influenza in these populations facilitates the dissemination

of influenza viruses worldwide [81]. Influenza

viruses infect different species and have become

very successful parasites in their avian hosts,

causing mostly silent or asymptomatic infections

while creating the opportunity to infect other

immunologically naive hosts [5].

Interspecies barriers and the host species speci-

ficity mechanisms, such as receptor preference

and host factors interacting with HA, NA, viral

polymerase and other internal genes, are impor-

tant molecular constraints and determinants for

their transmissibility of AIVs among different

species [82,83]. IAV particles bind their target cells

through interaction between their HA molecules

and the sialic acid-containing cell-surface recep-

tors on the host cells. Human influenza viruses

bind preferentially to N-acetylneuraminic acid

(sialic acid), which is attached to a galactose

molecule by an a2,6 linkage (SAa2,6Gal),

while AIVs mostly bind to silalic acid with an

a2,3 linkage [84]. In human tracheal epithelial

cells, a2,6 linkages predominate, while a2,3-

linkages are more common in duck gut epi-

thelial cells [83]. Recently, it was indicated that

epithelial cells of the human lower respiratory

tract contain both SAa2,3Gal and SAa2,6Gal

but with different distributions [85]. In humans,

a2,3 linkages are also present in respiratory epi-

thelial cells, but their presence is less abundant

than a2,6 linkages [86]. This explains the low infectivity but high pathogenicity of some AI strains in mammalian species [56]. AIVs do not generally replicate well in humans and vice versa [87,88], so it is possible that reassortment took place in an intermediate host. Pig populations have been proposed as a potential mixing vessel where reassortment takes place, since both avian and human strains can infect and replicate in pigs [89]. This is attributed to the fact that tra- cheal cells of pigs, where influenza replication occurs, contain a2,3-linked sialic acid receptors preferred by avian viruses, as well as those with the a2,6-linkage favored by human strains [89].

The mechanisms by which influenza viruses pass from one bird to another and cause infec- tion are not fully understood, but may be due to combinations of factors that include strain of virus, species of bird and environmental factors. In wild ducks, for example, influenza viruses rep-

licate preferentially in the cells lining the intesti- nal tract and are excreted in high concentrations in the feces. Contamination of water supplies by infective feces and fomites that contain concen- trations as high as 10 7 infectious particles/g [5] is one obvious route of infection [5,90]. This has led to the assessment of ducks being the ‘Trojan

horses’ of H5N1 in their surreptitious spread of

viruses because they do not show symptoms after

HP H5N1 infection [91]. Influenza viruses have

coevolved with ducks over a very long period of

time, allowing the establishment of equilibria

between hosts and viruses so that neither suffers

a significant loss of biological fitness. Hence, it is

important to determine whether antigenic diver-

sity is driven naturally in ducks or whether it is it

the consequence of vaccine usage. Furthermore,

it will be necessary to determine the genomic

characteristics of ducks that are associated with

natural resistance in some species and ascertain

what dose of vaccine antigen is required to pre-

vent transmissible levels of virus excretion by

ducks of different species [91].

Clearly, the natural reservoir of IAVs could

be the source of the next human influenza pan-

demic [92] and so the routine surveillance and

early detection of these viruses [93,94], their hosts

and their migratory patterns [95], as well as the

study of the impact of environmental and social

factors on their interactions [97], are key to the

control, management and eradication of the

disease [97]. One consequence of this has been

the formulation in the USA of an interagency

strategic plan by a group of federal and state

resource, as well as science agencies, to conduct

surveillance in wild birds in order to determine

the possible pathways of entry [98]. In addition to carrying mixed populations of AIVs, waterfowl can host additional viruses, especially the avian paramyxoviruses (APMVs) [99,100]. Several studies have demonstrated the circulation of influenza virus with APMV-1 (also known as Newcastle disease virus [NDV]), APMV-2, APMV-4 and APMV-6 [101–105]. The coexistence of NDV with AIV in field samples might present a diagnostic problem when it is desired to isolate and characterize only AIVs for surveillance and epidemiological purposes. The overwhelming growth of NDV may inhibit AIV growth, which in turn will decrease the chance

Avian influenza viruses: virology, diagnosis & surveillance


of detection of AI subtypes. In several AIV surveillance studies, if a sample is found to be positive for NDV, it is not processed for AIV detection; only if a sample is found to be negative for NDV it is screened for AIV [106]. In a recent report, it was found that treatment of the sample with NDV polyclonal antiserum facilitates the isolation of AI from samples containing both AIVs and NDV [102].

Diagnosis of AI

An effective strategy for understanding the ecology and epidemiology of the virus, and

thus controlling the spread of influenza viruses,

depends on the availability of reliable laboratory

techniques permitting accurate and sensitive

detection of AIVs in waterfowl [107,108]. Clinical

diagnosis of AIV in birds is performed by the

isolation and characterization of the virus, which

varies with species and type of infection [109] and

can be either presumptive or definitive. Clinical

symptoms can be a valuable tool for presump-

tive diagnosis of HPAI, whereas LPAI infection

is asymptomatic [110]. Definitive diagnosis of

AIV depends on specific laboratory methods,

including the indirect evidence of infection by

serological methods, which detect anti-influenza

antibodies, and direct detection methods for live

virus, viral antigen or viral nucleic acid [111].

Specimen collection

Clinical specimens for AIV diagnosis in birds

can be obtained from either live or dead birds.

Wild birds in surveillance programs of migrating

waterfowl are usually trapped using night light-

ing, rocket netting and hunter harvesting tech-

niques [203,204]. Samples from live birds should

include both tracheal and cloacal swabs, since

it has been shown that the analysis of both oro-

pharyngeal and cloacal swabs provides an accu-

rate snapshot of AIV status in birds [79,112–114]. If

this is not possible, the collection of fresh feces

may also serve as an alternative [115]. Samples should be placed in an isotonic phos- phate-buffered saline or viral transfer medium consisting of brain–heart infusion medium con- taining several antibiotics to eliminate any bacte- ria that may interfere with subsequent diagnostic procedures [116]. Although diagnostic samples can be stored temporarily at 40°C, it is recom- mended that all samples be kept at -800°C until tested, especially if prolonged storage is antici-

pated [117]. In addition, field samples should be maintained under strict cold chain conditions from the moment of acquisition and should not be subjected to frequent freeze–thaw cycles, as

this results in significantly decreased virus titers [79]. In addition, technical details such as the use of dry or wet swabs, the pH of viral transfer medium and the time taken to transport sam- ples to the testing laboratory play a crucial role in the successful isolation of AIVs from field samples [114].

Serological methods

Serological methods for the diagnosis of AI in birds play an important role in monitoring the disease in populations and can provide an accurate method for the detection of influenza

infection [118]. Serological diagnosis is important

in case of LPAI to ensure freedom of infection

for commercial purposes. Conversely, in cases

of HPAI, serological methods are of little value

since birds die before producing antibodies [111].

Serological methods are thus useful epidemio-

logically for strain surveillance, but cannot be

used for rapid diagnosis, which would allow

therapeutic intervention [119]. Common serologic

tests for AIV include hemagglutination inhibi-

tion (HI), NA inhibition, agar gel precipitation

(also known as agar gel immunodiffusion),

virus neutralization, complement fixation (CF),

enzyme immunoassay and indirect immunofluo-

rescence [120,121]. These tests are based on the

presence of influenza-specific antibodies that

first appear approximately 2 weeks after initial

infection and peak at 47 weeks after initial

infection [121]. HI and NA inhibition assays are

labor-intensive and time-consuming and require

several controls for standardization. However,

they are inexpensive, use readily available rea-

gents and display high specificity in identifying

strains [122]. HI is more specific in differentiating

between HA subtypes [123]. The agar gel immu-

nodiffusion test detects IgM and some IgG and

is type-specific [124]. Although CF tests have

been used for the identification of influenza iso-

lates [119], the Canadian Public Health Labora-

tories stopped offering this test in 2009 because influenza CF serology does not provide a reliable indicator of immunity or response to vaccination and is not recommended for the diagnosis of acute influenza infection [205].

Virus isolation

Influenza virus was first isolated in 1933, by inoculation of specimens into the amniotic cav- ity of 10–12-day-old embryonating chicken eggs (ECEs) [125], where permissive infection occurs in the cells lining the amniotic and allantoic cavities [119]. This method, using either spe- cific pathogen-free chicken eggs or specific


El Zowalaty, Bustin, Husseiny & Ashour

antibody-negative eggs [126], continues to be the gold standard technique for AIV diagnosis and remains the most sensitive method for generat-

ing very high titers of all but one AIV [120]. The exception is the recently discovered bat virus A/ H17N10; to date, all attempts to propagate this virus in cell cultures or chicken embryos have failed [59]. Although commercial non-specific pathogen-free eggs have been used for AIV propagation and isolation, their vaccination history should be carefully checked to confirm the absence of other pathogens that might affect AIV isolation [13].

Allantoic fluids negative for virus can be

passaged into ECEs for up to three passages

and retested for HA before being recorded

as negative. However, AIV isolation in ECEs

requires a readily available continuous supply

of fertilized chicken eggs and special incuba-

tors [127]. It can take days to weeks to achieve

high titers, the method is labor- and resource-

intensive and there is a mandatory requirement

of a high level of biosecurity facilities (generally

biosafety level 3) if HPAI is suspected. Hence,

it is not widely used for the routine diagnosis

of influenza infection. On the other hand, egg

isolation provides high quantities of live infec-

tious virus and reference laboratories therefore

utilize this culture system to ensure high sensi-

tivity and for biological characterization, full-

length sequence analysis and antiviral resistance


production of virus stocks for epidemiological

monitoring and vaccine updating, which is a

critical requirement for the diagnosis of AI in

the index cases.

The technique of conventional culture of

influenza viruses in a cell culture was introduced

in 1940s [121] and provides a useful method for

the primary isolation of some human or swine

influenza isolates that do not grow well in ECEs

[118,129]. Since viral culture amplifies the inocu-

lum, it is more sensitive than direct methods of detection [13]. Cell cultures can be monitored for the development of cytopathic effects, the manifestation of hemadsorption after the addi- tion of erythrocytes or for the presence of influ- enza antigens [121]. Various mammalian and bird cell lines have been used to isolate influ- enza viruses. The most commonly used cells are: primary rhesus monkey kidney and rhe- sus monkey kidney (LLC MK2) cells, African green monkey kidney cells, mink lung epithelial cell line (Mv1Lu), buffalo green monkey kid- ney, Madin–Darby canine kidney, green mon- key continuous cell line (Vero), human lung

[111,128]. This method also enables the

embryonating cells (MRC-5), CACO-2 cell lines, CCL-141 (duck embryo) cells, CCL-169 (goose embryonic kidney) cells, duck embryo fibroblast cells and chicken embryo fibroblast

cells [130–135].

Alternatively, since viruses are released slowly from the cell surface of virus-infected cells, hemadsorption results in erythrocytes adher- ing directly to these infected cells, which can

be observed microscopically [119]. In addition to ECEs, Madin–Darby canine kidney cells are now considered a valuable system for the isola- tion of influenza viruses. However, since AIVs

have different host and in vitro growth proper-

ties depending on the strain [136,137] and not all

host cells are universally permissive to all AIV

subtypes, virus isolation is effective only when

the cell culture is sensitive to the inoculated virus

[138]. In addition, this method requires the rapid

transport of specimens to the laboratory, since

delays may lead to inactivation of the virus and

hence failure to isolate any infectious agent [139].

Although conventional cell culture takes up

to 2 weeks to generate results, it is very sensi-

tive. Cytopathic effects such as intracytoplasmic

basophilic inclusion bodies are observed. The

presence of influenza virus can be ascertained

using either hemadsorption with guinea pig red

blood cells [108] or immunofluorescence on cul-

tured cells. Immunofluorescence has the twin

advantages of higher sensitivity in the detection

of positive cultures and can also be used to type

the isolated virus.

Another useful technique for the culture of

AIVs is the rapid shell vial culture, which uses

single or mixed cell lines using monolayers of

two different cell types in a single vial [121].

This technique has the advantage of enhancing

sensitivity and shortening the time to detection

through enhancing the viral infectivity of the

cells by centrifugation (time to diagnosis: 24 h

vs 13 days postinoculation) [140,141]. Sensitivity

is equivalent to that of conventional tube cul- tures and greater than that of direct fluorescent- antibody tests [142].

Molecular methods

Molecular methods, also known as nucleic acid tests, detect viral nucleic acid (i.e., RNA) and promise to shorten the turnaround time for the laboratory diagnosis of AIV. There is a range of different types of molecular diagnostic tests. Until recently, the most commonly used was reverse transcription PCR (RT-PCR), where purified RNA of the virus, isolated from chicken eggs, cell cultures or from clinical specimens, is

Avian influenza viruses: virology, diagnosis & surveillance


reverse transcribed into cDNA, which is then exponentially amplified using AIV-specific oli- gonucleotide primers [143–148]. Conventional RT-PCR is an end point assay, where the PCR product is analyzed on agarose gel after eth- idium bromide staining to separate the PCR product according to molecular weight, which

allows presumptive identification [128,146,149]. Its main disadvantage is the ease with which the assay can be contaminated and the tedi- ous amount of work involved in running and visualizing gels. Other nucleic acid test methods include the

use of RT-PCR with detection by ELISA [150],

nucleic acid sequence-based amplification [151]

and H5 RT-loop-mediated isothermal amplifica-

tion [152]. However, none of these can compete

with quantitative real-time PCR (RT-qPCR),

which is a homogeneous assay requiring minimal

hands-on time and no post-PCR processing [153].

RT-qPCR typically uses a fluorescently labeled

probe to detect the increase in PCR product

while the test is being performed and the results

are reported in real-time. All these methods have

the potential to provide rapid and sensitive diag-

nostic results.

RNA is most commonly extracted from AIVs

using phenol-guanidinium thiocyanate [154] or

one of the recently developed commercially avail-

able kits that depend on the adsorption of RNA

to silica gel-based columns. These methods for

the detection of influenza are highly sensitive,

specific and versatile [155], with the column-based

methods having the advantage in terms of the

reliable generation of intact RNA [156]. Once the

viral RNA is extracted, it can be used not only

to identify the isolate as AIV, but also to fur-

ther determine the subtype and even the strain

by sequence analysis utilizing an array of several

influenza-specific primers. The viral genotype

can be determined by sequencing some or all

of the AIV genes, although some level of virus

amplification in ECEs or cell culture is required for full-length sequence analysis [121]. Molecular methods are rapid, of acceptable sensitivity, and provide results within a relatively short time for targeted treatment and limitation of the spread of infection. This is crucial in situations in which rapid diagnosis is necessary, such as in epidemics and in patients with specific medical conditions [120,157]. There are several concerns with molecular diagnostic methods. Foremost is the techni- cal problem associated with the viral sequence that gives rise to both genetic drift and shift. The exquisite specificity of PCR can suddenly

become a liability if the sequence change or reassortment includes primer binding sites and the assay no longer amplifies the altered target sequences. Consequently, nucleic acid-based testing requires routine updating of the primers [13]. Another is the potential for obtaining false- negative results that may occur due to the pres- ence of inhibitors (which are concentrated along with pathogens during sample processing), low virus titers, degradation of target RNA before amplification and errors in setting up a reaction [158,159]. The problem of RNA quality, in par-

ticular, is a critical issue, as these tests require

RNA samples to be of good quality and the

use of degraded RNA or inefficient PCR reac-

tions can make these methods unreliable and

may yield inaccurate results [13,107]. Hence, it is

important to follow a rigid quality assessment

program and, taking the example of RT-qPCR,

there is a requirement to report the protocols

accurately and comprehensively using the mini-

mum information for publication of quantitative

real-time PCR experiments (MIQE) guidelines

[160]. Even then, the conversion of mRNA to

cDNA is highly variable, and RT-qPCR results

can vary considerably with the choice of reverse


mRNA molecules are not linear molecules,

but instead form secondary structures through

extensive intrastrand base pairing. This results in

numerous stem-loop structures consisting of sin-

gle-stranded loops and double-stranded hairpin

structures of varying length. The efficiency of

the RT step depends heavily on the primers used

for cDNA synthesis not targeting the hairpin

structures, since the intermolecular (i.e., primer

to mRNA) hybridization kinetics are infavour-

able compared with intramolecular (i.e., mRNA

to itself) hybridization. Hence, it is important to

choose suitable RT primers that target the loops,

rather than the stems. Similarly, it is important

to ensure that the amplicon itself is free from

secondary structures at the PCR primer binding sites, as extensive secondary structures there can give rise to significantly different results. It must be noted that assays only determine total patho- gen number and do not provide information as to whether a pathogen has the ability to establish an infection or not. Hence, a positive result may

not necessarily pose a public health threat, and so there is a requirement for additional assays that can determine viability. None of the diagnostic tests discussed so far are easily carried out in the field, and none can provide virtually instant results. Rapid point- of-care diagnosis is critical for the control and


El Zowalaty, Bustin, Husseiny & Ashour

management of influenza infection in humans, as it enables the fast administration of appro- priate antiviral therapy within the first 2 days

of illness [161], reduces the length of in-patient hospital stay and minimizes the unnecessary use of antibiotics [162]. Rapid antigen capture immu- noassay tests can provide results within 30 min or less and are easy to perform [163]. They are based on an immunochromatographic reaction that uses a monoclonal antibody targeted to a specific antigen of influenza, usually the nucleo- protein. They can therefore detect any AIV [164– 167] and include tests such as the Directigen™

Flu A and Directigen Flu A and B tests (Becton

Dickinson Diagnostic Systems, MD, USA) and

the BinaxNow ® influenza A and B test (Binax,

Inc., ME, USA) [163].


Vaccination in poultry can minimize the threat

of AI. Vaccines against AI are important tools

for protecting the poultry industry and humans,

since vaccines help increase resistance to infec-

tion, prevent illness and death, reduce virus

replication and reduce viral transmission to

birds and mammals, including humans. DNA

vaccination could provide protective immunity

in animal models against different infectious

diseases. The basic principle of DNA vaccina-

tion is the induction of an immune response by

intramuscular injection or the use of a ‘gene gun’

of naked DNA (plasmid) encoding the targeted

gene into the host cells [168]. The restrictions of

DNA vaccination are vaccine cost, problems

with delivery into birds, low efficiency in birds

and its requirement of large amounts of puri-

fied plasmids [169]. The reason DNA vaccines

are not suitable in the poultry industry include

their time-consuming application, expenses and

undue stress to the chickens.

DNA delivery technologies are effective and

essential for inducing a strong and long-lasting

immune response that is required to induce high and continued levels of antigen production at proper target sites. A variety of intracellular bac- teria have been utilized as live carriers for effi- cient delivery of either DNA vaccine constructs or vaccine antigens [170] directly into professional APCs [171]. This strategy can elicit humoral and cellular responses against the pathogens from which the target genes are derived [172–175]. Live oral attenuated Salmonella typhimurium is a well-known, useful carrier for the expres- sion and delivery of heterologous antigens to mucosal lymphoid tissue, including the HA of

AIV [176]. The viable bacteria multiply inside the

Salmonella-containing vacuole and deliver the recombinant antigen into the host cell cytosol [177,178]. DNA vaccines delivered by attenuated S. typhimurium SL7207 and boosted with a con- ventional killed vaccine confer protection on chickens against infection with the H9 subtype of AIV [179]. Another approach is the utiliza- tion of SPI2-encoded type III secretion system

[180] for antigen expression and delivery into the cytoplasm of APCs, which increases safety and efficacy. Such vaccines have been shown to be very effective in eliciting both CD8 + and CD4 + T-cell-mediated immune responses in models of

infectious diseases and cancer [178,181–184].

Interestingly, the development of an immune

response to HA glycoprotein is important for the

protection of chickens against challenge with the

AIV infection [185]. Accordingly, a wide variety

of vaccines against AIV has been developed and

tested in experimental conditions, but only inac-

tivated whole AIV vaccines and live recombinant

fowlpox virus vectors expressing HA vaccines

have been licensed and widely used in various

countries [186].

Future perspective

The recent outbreak in humans of a novel

influenza A/H7N9 in China in March 2013

shed light on the importance of surveillance

efforts to alleviate public health concerns of

AI. Extensive global surveillance programs are

urgently required to help predict the circulating

AI strains that may produce potential pandem-

ics in humans. Vaccines and molecular immu-

nology approaches are currently the subject of

extensive research efforts aimed at minimiz-

ing the threat of AI. These are complemented

by surveillance programs that are essential for

monitoring the AI dynamics in their recognized

reservoirs. The outcome of active surveillance for

AIVs in different parts of the world, including

Malaysia, will fill the gap in knowledge regard-

ing the true disease status in Malaysia, Egypt and several countries in southeast Asia. Similar to many countries in southeast Asia, Malaysia is endemic for NDV, but its current AI dis- ease status is unknown. Outbreaks of HP A/ H5N1 were reported in 2004, 2006 and 2007 in poultry, although none have been reported since. The country borders Indonesia, which is endemic for A/H5N1, and Thailand, which has also had outbreaks of HP A/H5N1 AI. In addi- tion, in countries such as Thailand, Indonesia, and China where pigs are not fully protected from acquiring infection with any possible avian influenza viruses, the generation of pandemic

Avian influenza viruses: virology, diagnosis & surveillance


influenza strains is a possibility. It was recently reported that the unprotected transport of pigs and the breach of biosecurity measures could facilitate the mixed infection of humans and pigs with avian viruses and the generation of newer strains of greater pandemic potential [187, 188]. This could also explain the emergence of influ- enza A strains of pandemic potential in China and southeast Asia. Because of the lack of active surveillance, particularly in duck populations, the true disease status in such regions remains undetermined. Although the use of influenza vaccines to

protect the poultry industry and humans is an

effective procedure to control the disease, the

current use of vaccination encounters several

challenges [189]. A major hurdle is the increased

chance of having asymptomatically infected

poultry spreading the virus. In addition, poor

administration is the most common cause of

vaccine failure in poultry making the available

AI vaccines are not efficient to elicit protective

immune responses, thus, search for new AI vac- cine delivery mechanisms should be advocated such as nanovaccines. Another drawback of cur- rently applied vaccines used in poultry is the low antigen stability in the vaccines and the short duration of immunity. Worryingly, it has been recently reported that independent recombina- tion events between distinct attenuated infec- tious laryngotracheitis virus vaccine strains can

result in virulent recombinant viruses [190]. This prospect might arise in AIVs as well. Although the use of nanotechnology to deliver AI vaccines may improve vaccine efficacy and overcome the

recombination challenges [191,192], for now, we

advocate strict biosecurity procedures coupled

with the implementation of worldwide intera-

gency surveillance efforts modeled on those of

the USA and Europe in high-risk regions, such

as in developing countries, to identify potentially

pandemic strains [193–195]. Surveillance and diag-

nosis of AI in humans and birds are not similar

and involve different techniques. Thus, AIV

Executive summary

Background & history

Avian influenza virus (AIV), which is closely related to human influenza virus, appeared nearly 150 years go and has recently re-

emerged, causing grave concern among certain populations.

Several pandemics of AIV have afflicted human populations with devastating impact.

The recently emerged viruses, mainly A/H5N1 and A/H7N9, arose from several reassortment events in different hosts and pose a

significant threat to human public health.

Genome & virology of AIV

Although the structure of AIV has been fully elucidated, it remains difficult to predict when the next pandemic will occur.

Hemagglutinin and neuraminidase are the major antigenic determinants of influenza A viruses.

Acquisition of a polybasic hemagglutinin cleavage site is not the only necessary step for the evolution of low-pathogenicity avian

influenza strains into high-pathogenicity avian influenza viruses. There are other factors that are also required.

Epidemiology of AIV

AIVs are natural inhabitants of waterfowl, mainly migratory ducks.

Several interspecies barriers determine the transmissibility of AIVs among different species and, finally, to humans.

Surveillance is of importance in order to monitor the current status of avian influenza in populations and to update vaccine databases.

Global programmed surveillance programs in Asia, a potential origin of the next pandemic, should be promptly initiated.

Diagnosis of AIV

Diagnosis of avian influenza infection in waterfowl is different from diagnosis in humans. Suitable and reliable diagnostic techniques in real time will guard against severe infection in humans and allow monitoring of AIVs spreading in their natural reservoirs. Virus isolation in embryonating chicken eggs should be advocated as the cornerstone diagnostic technique of isolation of live AIVs for subsequent characterization and updating of vaccine databases. Nucleic acid-based tests are useful techniques for the screening of AIVs during surveillance studies. The specificity of quantitative PCR is subject to several factors, such as the primers, RNA extraction method, RNA integrity and presence of inhibitors, among others. Virologic and serological techniques, along with rapid molecular methods (e.g., quantitative PCR), will provide information and suitable intervention strategies to limit the pandemic potential of avian influenza in humans and poultry.

Vaccines Vaccines are useful tools to minimize the threat of avian influenza in poultry, but a concern that is associated with the use of vaccines in poultry is that vaccines could increase the chance of having asymptomatically infected poultry spreading the virus. DNA vaccines and nanovaccines are promising tools to improve currently applied avian influenza vaccines in poultry.


El Zowalaty, Bustin, Husseiny & Ashour

surveillance in AIV’s natural reservoirs of wild birds is of high importance in order to monitor

the silent circulating strains and protect poultry from being infected with LPAI strains, as well as HPAI. Biosecurity measures, environmental pro- tection and handling of poultry in at-risk farms or locations of endemic infections will limit and control the spread of the disease in humans and will minimize poultry-to-human transmission. Virologic and serological monitoring, along with the use of rapid molecular methods (e.g., quantitative PCR), will provide information in real time in order to enable suitable intervention

strategies to limit the AI pandemic and disease

potential in humans and poultry. Finally, it is

advisable to use complementary methods for the identification of AIV, since this approach is most likely to yield a reliable and comprehensive evaluation of circulating viruses [13,107].

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a

financial interest in or financial conflict with the sub- ject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production

of this manuscript.


Papers of special note have been highlighted as:

n of interest

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