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 Avian influenza: virology, diagnosis

Review
Future Microbiology
and surveillance
Mohamed E El Zowalaty*1,2,3, Stephen A Bustin1, Mohamed I Husseiny3,4
& 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: hossamking@mailcity.com n elzow001@gmail.com

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

of
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

ro
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.
rP
Avian influenza (AI) is an acute avian respira- has been transmitted to humans (e.g., H1N1 in
tory disease caused by AI viruses (AIVs) that 1976, 1986 and 1988; H3N2 in 1993; H7N7
affect many species of domestic and wild birds in 1996; and H9N2 in 1998, 1999 and 2003)
and can also infect other animal species. In inducing mild, severe or fatal infections [3,4] .
ho

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
ut
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-

index >1.2 or causing at least 75% mortality [1] .
HPNAI is a ‘list A’ notifiable disease (Box 1) of the
World Organization for Animal Health (OIE),
which describes serious transmissible diseases
A
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

that can potentially spread very rapidly between
countries and are of major significance for the
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
LPAI [2, 201] .
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
the probable ‘mixing vessel’ [9] .
Several examples of human-adapted IAVs exist
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 Keywords
facing humans [11] , highlights the potential risk n avian influenza n diagnosis
IAV poses to human health and economy [12–14] n real-time PCR n surveillance
and strongly emphasizes the urgent requirement n vaccines n virus isolation
n waterfowl
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 part of

10.2217/FMB.13.81 © 2013 Future Medicine Ltd Future Microbiol. (2013) 8(9), 1–19 ISSN 1746-0913 1
 Review 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] .
of
Human influenza pandemics
The three worldwide influenza outbreaks (pan-
demics) in the 20th century occurred in 1918,
ro
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’
rP
(1918–1919) 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
ho
resulting in an estimated 30–50 million deaths
[21] . The ‘Asian flu’ (1957–1958) 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
ut
infections, affected approximately 40–50% of
the world’s population, killing approximately
1 million people [22] . The ‘Hong-Kong flu’
(1968–1969) pandemic was caused by a H3N2
A
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.
2 Future Microbiol. (2013) 8(9)
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
health.
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
future science group

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
rP
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-
ho
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-
ut
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
A
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
future science group
Avian influenza viruses: virology, diagnosis & surveillance Review
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
of
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.
ro
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
www.futuremedicine.com 3
4
Table 1. The genomic segments of influenza A virus and their encoded proteins and functions.
RNA RNA segment Gene Encoded Localization and features Nascent Approximate Proposed protein function(s)
segment (mRNA†) production protein(s)‡ protein number of
Review

number length in description length§ molecules

nucleotides in amino per virion
acids
1 2341 (2320) PB1 Polymerase Virion interior, infected cell 757 30–60 Heterotrimeric P complex associated with NP and virion
basic 1, 87 kDa nuclei RNA, RNA transcription and the replication component of
RNA polymerase (polymerase subunit); initiation of
transcription, RNA transcriptase, RNA elongation and
endonuclease activity
PB1-F2
A 87 Proapoptotic activity
2 2341 (2320) PB2 Polymerase Virion interior, infected cell 759 30–60 Polymerase subunit; RNA transcriptase, host cell mRNA cap
basic 2, 96 kDa nuclei recognition and virulence
3 2233 (2211) PA Polymerase Virion interior, infected cell 716 30–60 Polymerase subunit; RNA transcriptase and protease
acidic, nuclei
ut
85.5 kDa
El Zowalaty, Bustin, Husseiny & Ashour

4 1778 (1757)
5 1565 (1540)
HA Hemagglutinin, Globular head bears 550 500 Surface glycoprotein, virus binding to sialic acid-containing
220 kDa antigenic sites and receptor receptors on host cell; penetration of virus genome into
homotrimer binding site; stem; host cell cytoplasm by fusion of virus and host cell
transmembrane span; membranes (attachment to cell membranes and receptor
ho
cytoplasmic tail binding) and major antigenic determinant
NP Nucleoprotein, Virion interior, associated 498 1000 Encasidates RNA; RNA synthesis and RNA nuclear import
55 kDa with P complex and viral RNA regulation; interacts with RNAs and polymerase proteins as
a major component of the nucleocapsid; role in vRNA
replication; role in virus maturation and packaging

Future Microbiol. (2013) 8(9)

rP
6 1413 (1392) NA Neuraminidase, Virion envelope, infected cell 454 100 Surface glycoprotein; antigenic determinant, sialidase
240 kDa surface, cytoplasmic tail; (neuraminidase)-catalyzing cleavage of terminal sialic acid
homotetramer transmembrane span; residues from glycoconjugates, thereby digesting mucin to
extracellular stalk; globular enable the virus to reach its target epithelium and
head bears antigenic sites facilitating release of infectious progeny virus
ro
and enzyme active site
†
Deduced from RNA sequence, excluding poly-A tract. Total RNA nucleotide sequence length is 13,588.
‡
Determined by biochemical and genetic approaches.
§
Determined by nucleotide sequence analysis and protein sequencing.
P complex: Polymerase protein complex; RNP: Ribonucleoprotein; vRNP: Viral ribonucleoprotein.
Data taken from [194].
of

future science group

 Table 1. The genomic segments of influenza A virus and their encoded proteins and functions (cont.).
RNA RNA segment Gene Encoded Localization and features Nascent Approximate Proposed protein function(s)
segment (mRNA†) production protein(s)‡ protein number of
number length in description length§ molecules
nucleotides in amino per virion

future science group

7 1027 (1005)
8 890 (868)
acids
M1 M1, 28 kDa Beneath lipid bilayer of virion 252 3000 Central role in replication and virus assembly, modulating
envelope; associates with nuclear transport of vRNP; early infection: bound to vRNP
vRNPs in mature virion to prior to RNP transport to nucleus; late infection: binds RNP,
A form nucleocapsid 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
M2 M2, 15 kDa Virion envelope, infected cell 97 3000 Membrane cation channel activity; virus uncoating and
homotetramer surface (abundant); assembly; infected cell: specifically raises pH of Golgi to
ut
extracellular region; protect pH-sensitive hemagglutinin; virion: may permit
transmembrane span; acidification of virus interior during passage through
cytoplasmic tail; tetramers endosomal pathway in order to dissociate vRNP from M1
form cation-selective channel
ho
NS1 NS1, 25 kDa Infected cell nuclei 230 Multifunctional protein and viral interferon antagonist
dimer 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

www.futuremedicine.com
responses
rP
NEP/NS2 NS2, 14 kDa Associated with core 121 Mediates nuclear export of vRNPs
components of virion;
cytoplasm of infected cells
†
Deduced from RNA sequence, excluding poly-A tract. Total RNA nucleotide sequence length is 13,588.
‡
Determined by biochemical and genetic approaches.
§
Determined by nucleotide sequence analysis and protein sequencing.
ro
P complex: Polymerase protein complex; RNP: Ribonucleoprotein; vRNP: Viral ribonucleoprotein.
Data taken from [194].
of
Avian influenza viruses: virology, diagnosis & surveillance
Review

5
 Review El Zowalaty, Bustin, Husseiny & Ashour

Hemagglutinin

Neuraminidase

of
M2 ion channel

ro
rP
RNP

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
ho

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 distinct H17N10 virus is the only subtype that is
ut

be found, and each subtype may contain several
subtypes. Mutations can introduce additional
variability, and so the number of AIV strains
is indefinite.
A
not found in an avian species [58,59] .
Epidemiology of AI
The evolutionary success of IAV is a prototypic

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
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] .

6 Future Microbiol. (2013) 8(9) future science group

 Avian influenza viruses: virology, diagnosis & surveillance Review

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
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

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
of
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-

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
rP
cleavage site and is cleaved extracellularly, limit-
ing their spread in mammalian hosts to tissues
that express the appropriate proteases. Similarly,
ro
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
ho

Cleavage site

Avirulent avian isolate H5 RETR G LPAI: proteases localized in

Avirulent avian isolate H7 KXR G respiratory and intestinal organs
ut

Virulent avian isolate H5 RKRKKR G HPAI: ubiquitous proteases

Virulent avian isolate H7 KKRKKR G

Avian strains isolated from human H5N1 (1997 HK) RERRRKKR G

Avian strains isolated from human H9N2 (1999 HK) RSSR G
A

Avian strains isolated from human H7N7 (2003 NL) KRRRR G

Avian strains isolated from human H5N1 (2004 Asia) RRKKR G

N– HA1 –C N– HA2 TM –C

S–S

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.

future science group www.futuremedicine.com 7

 Review 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,
of
causing mostly silent or asymptomatic infections
while creating the opportunity to infect other
immunologically naive hosts [5] .
Interspecies barriers and the host species speci-
ro
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
rP
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
ho
(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-
ut
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,
A
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] .
8 Future Microbiol. (2013) 8(9)
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 107 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
future science group

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,
rP
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] .
ho
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-
ut
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-
A
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
future science group
Avian influenza viruses: virology, diagnosis & surveillance Review
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
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
ro
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 4–7 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
www.futuremedicine.com 9
 Review 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
of
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
ro
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
rP
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-
ho
length sequence analysis and antiviral resistance
studies [111,128] . This method also enables the
production of virus stocks for epidemiological
monitoring and vaccine updating, which is a
critical requirement for the diagnosis of AI in
the index cases.
ut
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
A
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
10 Future Microbiol. (2013) 8(9)
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
future science group

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-
rP
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
ho
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
ut
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
A
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
future science group
Avian influenza viruses: virology, diagnosis & surveillance Review
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
of
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
ro
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
transcriptase.
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
www.futuremedicine.com 11
 Review 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,
of
Inc., ME, USA) [163] .
Vaccines
Vaccination in poultry can minimize the threat
ro
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
rP
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
ho
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-
ut
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.
A
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
12 Future Microbiol. (2013) 8(9)
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
future science group
 Avian influenza viruses: virology, diagnosis & surveillance Review

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
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-

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
of
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-

ro
vaccine failure in poultry making the available nosis of AI in humans and birds are not similar
AI vaccines are not efficient to elicit protective and involve different techniques. Thus, AIV
Executive summary
Background & history
rP
„„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.

ho

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.
ut

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.
A

„„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.

future science group www.futuremedicine.com 13

 Review 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.

of
10. Saif YM. Diseases of Poultry. Iowa State Press, 18. Nichols JE, Leduc JW. Influenza.
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