Sei sulla pagina 1di 20
Seediscussions,stats,andauthorprofilesforthispublicationat: https://www.researchgate.net/publication/256488773 Avian

Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.researchgate.net/publication/256488773

Article in FutureMicrobiology·September2013

DOI:10.2217/fmb.13.81·Source:PubMed

CITATIONS

10

4authors,including:

54 PUBLICATIONS 440 CITATIONS

26 PUBLICATIONS 1,055 CITATIONS

READS

251

26 PUBLICATIONS 1,055 CITATIONS SEEPROFILE READS 251 StephenAndrewBustin AngliaRuskinUniversity 221 PUBLICATIONS

221 PUBLICATIONS 22,111 CITATIONS

Someoftheauthorsofthispublicationarealsoworkingontheserelatedprojects:

GrapheneoxideBiomedicalapplications Viewproject Viewproject

duckandchicken Viewproject Viewproject

Prevalence,IsolationandmolecularcharacterizationofshigatoxinproducingEscherichiacolifrom

AllcontentfollowingthispagewasuploadedbyStephenAndrewBustinon27February2015.

Theuserhasrequestedenhancementofthedownloadedfile.

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: 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

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

[1].

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
Keywords
LPAI [2, 201].
n
avian influenza n diagnosis
n
real-time PCR n surveillance
n
vaccines n virus isolation
n
waterfowl
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].

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

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

Avian influenza viruses: virology, diagnosis & surveillance

Review

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

Review
Review

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.

Approximate

number of

molecules

per virion

30–60

30–60

30–60

500

1000

100

Localization and features Nascent protein length § in amino acids

757

87

759

716

550

498

454

Virion interior, infected cell

nuclei

Virion interior, infected cell

nuclei

Virion interior, infected cell

nuclei

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.

sequencing.

Ribonucleoprotein;

protein

approaches.

and

RNP:

analysis

genetic

complex;

sequence

and

[194]. protein

by biochemical

nucleotide

taken Polymerase

from

by

Determined

Determined

complex:

§ ‡ Data P

Encoded

protein(s)

production

 

Polymerase

basic 1, 87 kDa

 

Polymerase

basic 2, 96 kDa

Polymerase

 

85.5 kDa

Hemagglutinin,

kDa

homotrimer

 

Nucleoprotein,

Neuraminidase,

kDa

homotetramer

 

description

 

acidic,

220

55 kDa

240

             

Gene

RNA segment (mRNA ) length in nucleotides

segment

number

RNA

PB1

2341 (2320)

1

PB1-F2

PB2

2341 (2320)

2

PA

2233 (2211)

3

HA

1778 (1757)

4

NP

1565 (1540)

5

NA

1413 (1392)

6

Avian influenza viruses: virology, diagnosis & surveillance

Review

 

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

responses

Mediates nuclear export of vRNPs

 

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

Approximate

number of

molecules

per virion

3000

3000

 

Localization and features Nascent protein length §

in amino

acids

252

97

230

121

 

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.

sequencing.

Ribonucleoprotein;

protein

approaches.

and

RNP:

analysis

genetic

complex;

sequence

and

[194]. protein

by biochemical

nucleotide

taken Polymerase

from

by

Determined

Determined

complex:

§ ‡ Data P

Encoded

protein(s)

M1, 28 kDa

M2, 15 kDa

homotetramer

NS1, 25 kDa

dimer

NS2, 14 kDa

     

description

production

Gene

RNA segment (mRNA ) length in nucleotides

segment

number

RNA

 

M1

1027 (1005)

7

M2

NS1

890 (868)

8

NEP/NS2

Review

El Zowalaty, Bustin, Husseiny & Ashour

Hemagglutinin Neuraminidase M2 ion channel RNP
Hemagglutinin
Neuraminidase
M2 ion channel
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

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

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

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
N–
HA0
TM
–C
Cleavage site
Avirulent avian isolate H5
RETR
G
LPAI: proteases localized in
respiratory and intestinal organs
Avirulent avian isolate H7
KXR
G
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
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.

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,

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

Review

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

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

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

studies

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

Review

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

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

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,

Inc., ME, USA) [163].

Vaccines

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

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

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.

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.

References

Papers of special note have been highlighted as:

n of interest

nn of considerable interest

1. World Organization for Animal Health. Avian

influenza. In: Manual of Diagnostic Tests and

Vaccines for Terrestrial Animals: Mammals,

Birds, and Bees. (6th Edition, Volume 1). Office

International des Epizooties, France, 465–481

(2008).

2. Swayne DE. The global nature of avian

influenza. In: Avian Influenza. Wiley-

Blackwell, NJ, USA, 126–127 (2009).

3. Kurtz J, Manvell RJ, Banks J. Avian influenza

virus isolated from a woman with

conjunctivitis. Lancet 348(9031), 901–902

(1996).

4. Webster RG, Geraci J, Petursson G, Skirnisson

K. Conjunctivitis in human beings caused by

influenza A virus of seals. N. Engl. J. Med.

304(15), 911 (1981).

5. Webster RG, Bean WJ, Gorman OT,

Chambers TM, Kawaoka Y. Evolution and

ecology of influenza A viruses. Microbiol. Mol.

Biol. Rev. 56(1), 152–179 (1992).

6. Kilbourne ED. Influenza immunity: new

insights from old studies. J. Infect. Dis. 193(1),

7–8 (2006).

7. Lindstrom S, Cox N, Klimov A. Genetic analysis of human H2N2 and early H3N2 influenza viruses, 1957–1972: evidence for genetic divergence and multiple reassortment events. Virology 328(1), 101–119

(2004).

8. Palese P Shaw ML Orthomyxoviridae: the viruses and their replication. In: Fields’ Virology. Knipe DM, Howley PM, Griffin DM, Lamb RA, Martin MA (Eds). Lippincott, Williams & Wilkins, PA, USA, 1647–1689 (2007).

9. Capua I, Alexander DJ. Avian influenza and human health. Acta Tropica 83(1), 1–6 (2002).

10. Saif YM. Diseases of Poultry. Iowa State Press,

IA, USA (2003).

11. Arzt J, White W, Thomsen B, Brown C.

Agricultural diseases on the move early in the

third millennium. Vet. Pathol. 47(1), 15–27

(2010).

12. El Zowalaty ME, Abin M, Chander Y, Redig

PT, Goyal SM. Isolation of H5 avian

influenza viruses from waterfowl in the upper

midwest region of the United States. Avian

Dis. 55(2), 259–262 (2011).

13. El Zowalaty ME, Abin M, Raju S et al.

Isolation of avian influenza virus from

polymerase chain reaction-negative cloacal

samples of waterfowl. J. Vet. Diagn. Invest.

23(1), 87–90 (2011).

First comprehensive study in literature on

the isolation of AI viruses from waterfowl

after passage in embryonating chicken

eggs and prior to rapid RT-qPCR to

improve the detection and isolation of live

influenza viruses for further

characterization. It is imperative to

perform virus isolation during AI

surveillance efforts to obtain a clear

picture on the circulating AIV and to

understand that depending solely on

qRT-PCR may yield spurious results.

14. Garten RJ, Davis CT, Russell CA et al. Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science 325(5937), 197–201 (2009).

15. Mounts AW, Kwong H, Izurieta HS et al. Case–control study of risk factors for avian influenza A (H5N1) disease, Hong Kong, 1997. J. Infect. Dis. 180(2), 505–508 (1999).

16. Alexander DJ. Avian influenza: historical aspects. Avian Dis. 47, 4–13 (2003).

17. Lupiani B, Reddy SM. The history of avian influenza. Comp. Immunol. Microbiol. Infect. Dis. 32(4), 311–323 (2009).

18. Nichols JE, Leduc JW. Influenza.

In: Vaccines for Biodefense and Emerging and

Neglected Diseases. Barrett AT, Stanberry LR

(Eds). Academic Press, Waltham, MA, USA,

497-525 (2008).

19. Taubenberger JK, Reid AH, Krafft AE,

Bijwaard KE, Fanning TG. Initial genetic

characterization of the 1918 ‘Spanish’

influenza virus. Science 275(5307),

1793–1796 (1997).

20. Taubenberger J, Morens D. 1918 Influenza:

the mother of all pandemics. Emerg. Infect.

Dis. 12(1), 15–22 (2006).

21. Cox NJ, Tamblyn SE, Tam T. Influenza

pandemic planning. Vaccine 21(16),

1801–1803 (2003).

22. Hehme N, Engelmann H, Künzel W,

Neumeier E, Sänger R. Pandemic

preparedness: lessons learnt from H2N2 and

H9N2 candidate vaccines. Med. Microbiol.

Immunol. 191(3–4), 203–208 (2002).

23. Newcastle disease. In: Manual of Standards for

Diagnostic Tests and Vaccines. Office

International des Epizooties, France, 161–169

(1996).

24. Garten RJ, Davis CT, Russell CA et al.

Antigenic and genetic characteristics of

swine-origin 2009 A (H1N1) influenza viruses circulating in humans. Science 325(5937), 197–201 (2009).

25. Igarashi M, Ito K, Yoshida R, Tomabechi D, Kida H, Takada A. Predicting the antigenic structure of the pandemic (H1N1) 2009 influenza virus hemagglutinin. PLoS ONE 5(1), e8553 (2010).

26. Smith GJ, Vijaykrishna D, Bahl J et al. Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature 459(7250), 1122–1125

(2009).

27. Zhang W, Qi J, Shi Y et al. Crystal structure of the swine-origin A (H1N1)-2009 influenza

Avian influenza viruses: virology, diagnosis & surveillance

Review

A virus hemagglutinin (HA) reveals similar

antigenicity to that of the 1918 pandemic

virus. Protein Cell 1(5), 459–467 (2010).

28. Perez-Padilla R. Pneumonia and respiratory failure from swine-origin influenza A (H1N1)

in Mexico. N. Engl. J. Med. 361(7), 680

(2009).

29. Smith GJ, Vijaykrishna D, Bahl J et al. Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature 459(7250), 1122–1125

(2009).

30. Centanni ESE. La peste aviaria I & II. Communicazione fatta all’accademia delle

scienze mediche e naturali de Ferrara. (1901).

31. Stubs EL, Biester HE, Devries L. Fowl pest.

In: Diseases of Poultry. Iowa State Press, IA,

USA, 493–502 (1952).

32. Perroncito E. Epizoozia tifoide nei gallinacei.

Ann. Accad. Agric. Torino. 126, 21–87 (1878).

33. Becker WB. The isolation and classification of

tern virus: influenza virus A/tern/South

Africa/1961. J. Hyg. (Lond.) 64(3), 309–320

(1966).

34. Subbarao K, Klimov A, Katz J et al.

Characterization of an avian influenza A

(H5N1) virus isolated from a child with a

fatal respiratory illness. Science 279(5349),

393–396 (1998).

35. Webster RG. Predictions for future human

influenza pandemics. J. Infect. Dis.

176(Suppl. 1), S14–S19 (1997).

36. Guo Y, Xu X, Wan X. [Genetic

characterization of an avian influenza A

(H5N1) virus isolated from a sick goose

in China]. Zhonghua Shi Yan He Lin

Chuang Bing Du Xue Za Zhi 12(4), 322–325

(1998).

37. Claas EC, Osterhaus AD, van Beek R et al.

Human influenza A H5N1 virus related to a

highly pathogenic avian influenza virus.

Lancet 351(9101), 472–477 (1998).

38. Mounts AW, Kwong H, Izurieta HS et al.

Case–control study of risk factors for avian

influenza A (H5N1) disease, Hong Kong,

1997. J. Infect. Dis. 180(2), 505–508 (1999).

39. Peiris JS, Yu WC, Leung CW et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet 363(9409), 617–619 (2004).

40. Banner D, Kelvin AA. The current state of H5N1 vaccines and the use of the ferret model for influenza therapeutic and prophylactic development. J. Infect. Dev. Ctries 6(06), 465–469 (2012).

41. Bartlett JG. Planning for avian influenza. Ann. Intern. Med. 145(2), 141–144 (2006).

42. Li Q, Zhou L, Zhou M et al. Preliminary report. epidemiology of the avian influenza A

(H7N9) outbreak in China. N. Engl. J. Med. doi:10.1056/NEJMoa1304617 (2013) (Epub ahead of print).

43. Liu D, Shi W, Shi Y et al. Origin and diversity of novel avian influenza A H7N9 viruses causing human infection:

phylogenetic, structural, and coalescent analyses. Lancet 381(9881), 1926–1932

(2013).

44. Zhu H WD, Kelvin Dj, Li L et al. Infectivity, transmission, and pathology of human H7N9 influenza in ferrets and pigs. Science 341(6142), 183–186 (2013).

nn„

Recent outbreaks caused by avian H7N9

influenza viruses could be the beginning

of a human avain influenza pandemic with

possible human-to-human spread. The

finding that efficient transmissibility of

H7N9 viruses in ferrets by direct contact

raises grave public health concerns.

45. Uyeki TM, Cox NJ. Global concerns

regarding novel influenza A (H7N9) virus

infections. N. Engl. J. Med. 368(20),

1862–1864 (2013).

46. Easterday BC. Animals in the influenza

world. Phil. Trans. R. Soc. Lond. 288(1029),

433–437 (1980).

47. Daly JM, Blunden AS, Macrae S et al.

Transmission of equine influenza virus to

English foxhounds. Emerg. Infect. Dis. 14(3),

461–464 (2008).

48. Song D, Kang B, Lee C et al. Transmission of

avian influenza virus (H3N2) to dogs. Emerg.

Infect. Dis. 14(5), 741–746 (2008).

49. Herfst S, Schrauwen EJ, Linster M et al.

Airborne transmission of influenza A/H5N1

virus between ferrets. Science 336(6088),

1534–1541 (2012).

50. Imai M, Watanabe T, Hatta M et al.

Experimental adaptation of an influenza H5

HA confers respiratory droplet transmission

to a reassortant H5 HA/H1N1 virus in

ferrets. Nature 486(7403), 420–428 (2012).

51. Berns KI, Casadevall A, Cohen ML et al.

Adaptations of avian flu virus are a cause for

concern. Science 335(6069), 660–661 (2012).

52. Baltimore D. Expression of animal virus genomes. Bacteriol. Rev. 35(3), 235–241

(1971).

53. Virology Division International Union of Microbiologival Societies. Virus Taxonomy. Eighth Report of the International Committee on the Taxonomy of Viruses. Fauquet CM, Mayo MA, Maniloff J, Desselberger, Ball LA (Eds). Academic Press, MA, USA (2005).

54. MacLachlan NJ, Dubovi EJ. Orthomyxoviridae. In: Fenner’s Veterinary Virology (4th Edition). Academic Press, MA, USA, 353–370 (2011).

55. Hay AJ, Gregory V, Douglas AR, Lin YP. The evolution of human influenza viruses. Phil. Trans. R. Soc. Lond. 356(1416), 1861– 1870 (2001).

56. Bouvier NM, Palese P. The biology of influenza viruses. Vaccine 26, D49–D53

(2008).

57. Steinhauer D, Skehel J. Genetics of influenza viruses. Annu. Rev. Genet. 36, 305–332

(2002).

58. García-Sastre A. The neuraminidase of bat influenza viruses is not a neuraminidase. Proc. Natl Acad. Sci. USA 109(46), 18635– 18636 (2012).

59. Tong S, Li Y, Rivailler P et al. A distinct

lineage of influenza A virus from bats. Proc.

Natl Acad. Sci. USA 109(11), 4269–4274

(2012).

60. A revision of the system of nomenclature for

influenza viruses: a WHO memorandum.

Bull. World Health Organ. 58(4), 585–591

(1980).

61. Fouchier RA, Munster V, Wallensten A et al.

Characterization of a novel influenza A virus

hemagglutinin subtype (H16) obtained from

black-headed gulls. J. Virol. 79(5),

2814–2822 (2005).

62. [A revised system of nomenclature for

influenza A viruses: WHO report]. Bull.

World Health Organ. 57(4), 611–617 (1979).

63. Fauci AS. Emerging and re-emerging

infectious diseases: influenza as a prototype

of the host–pathogen balancing act. Cell

124(4), 665–670 (2006).

64. Zambon MC. Epidemiology and

pathogenesis of influenza. J. Antimicrob.

Chemother. 44(Suppl. B), 3–9 (1999).

65. Desselberger U, Nakajima K, Alfino P et al.

Biochemical evidence that new influenza

virus strains in nature may arise by

recombination (reassortment). Proc. Natl

Acad. Sci. USA 75(7), 3341–3345 (1978).

66. Olsen B, Munster VJ, Wallensten A,

Waldenström J, Osterhaus AD, Fouchier

RA. Global patterns of influenza A virus in

wild birds. Science 312(5772), 384–388

(2006).

67. Capua I, Alexander DJ. Avian influenza infections in birds a moving target. Influenza Other Respi. Viruses 1(1), 11–18 (2007).

68. Galloway SE, Reed ML, Russell CJ, Steinhauer DA. Influenza HA subtypes demonstrate divergent phenotypes for cleavage activation and pH of fusion:

implications for host range and adaptation. PLoS Pathog. 9(2), e1003151 (2013).

69. Senne DA, Panigrahy B, Kawaoka Y et al. Survey of the hemagglutinin (HA) cleavage site sequence of H5 and H7 avian influenza viruses: amino acid sequence at the HA

Review

El Zowalaty, Bustin, Husseiny & Ashour

cleavage site as a marker of pathogenicity potential. Avian Dis. 40(2), 425–437

(1996).

70. Vey M, Orlich M, Adler S, Klenk HD, Rott R, Garten W. Hemagglutinin activation of pathogenic avian influenza viruses of serotype H7 requires the protease recognition motif R-X-K/R-R. Virology 188(1), 408–413 (1992).

71. Wood GW, McCauley JW, Bashiruddin JB, Alexander DJ. Deduced amino acid sequences at the haemagglutinin cleavage site of avian influenza A viruses of H 5 and H 7 subtypes. Arch. Virol. 130(1–2), 209–217 (1993).

72. Stieneke-Gröber A, Vey M, Angliker H.

Influenza virus hemagglutinin with

multibasic cleavage site is activated by furin,

a subtilisin-like endoprotease. EMBO J. 11(7),

2407–2414 (1992).

73. Alexander DJ. An overview of the

epidemiology of avian influenza. Vaccine

25(30), 5637–5644 (2007).

74. Bogs J, Veits J, Gohrbandt S et al. Highly

pathogenic H5N1 influenza viruses carry

virulence determinants beyond the polybasic

hemagglutinin cleavage site. PLoS ONE 5(7),

e11826 (2010).

75. Gohrbandt S, Veits J, Hundt J et al. Amino

acids adjacent to the haemagglutinin cleavage

site are relevant for virulence of avian

influenza viruses of subtype H5. J. Gen. Virol.

92(1), 51–59 (2011).

76. Hatta M, Kawaoka Y. The NB protein of

influenza B virus is not necessary for virus

replication in vitro. J. Virol. 77(10),

6050–6054 (2003).

77. Seo SH, Hoffmann E, Webster RG. Lethal

H5N1 influenza viruses escape host anti-viral

cytokine responses. Nat. Med. 8(9), 950–954

(2002).

78. Hinshaw VS, Webster RG, Turner B. The

perpetuation of orthomyxoviruses and

paramyxoviruses in Canadian waterfowl.

Can. J. Microbiol. 26(5), 622–629 (1980).

79. El Zowalaty ME, Abin M, Chander Y, Redig

PT, Goyal SM. Improved method for the isolation and sub-typing of avian influenza viruses from oropharyngeal samples of ducks. Avian Dis. 55(3), 439–442 (2011).

80. Stallknecht DE. Ecology and epidemiology of avian influenza viruses in wild bird populations: waterfowl, shorebirds, pelicans, cormorants, etc. Avian Dis. 47(Special issue), 61–69 (2003).

81. Charlton B, Crossley B, Hietala S. Conventional and future diagnostics for avian influenza. Comp. Immunol. Microbiol. Infect. Dis. 32(4), 341–350 (2009).

82. Peiris JM, de Jong MD, Guan Y. Avian influenza virus (H5N1): a threat to human

health. Clin. Microbiol. Rev. 20(2), 243–267

(2007).

83. Webby R, Hoffmann E, Webster R. Molecular constraints to interspecies transmission of viral pathogens. Nat. Med. 10, S77–S81 (2004).

84. Couceiro J, Paulson JC, Baum LG. Influenza virus strains selectively recognize sialyloligosaccharides on human respiratory epithelium; the role of the host cell in selection of hemagglutinin receptor specificity. Virus Res. 29(2), 155–165 (1993).

85. Shinya K, Ebina M, Yamada S, Ono M, Kasai N, Kawaoka Y. Avian flu: influenza virus

receptors in the human airway. Nature

440(7083), 435–436 (2006).

86. Matrosovich M, Zhou N, Kawaoka Y, Webster

R. The surface glycoproteins of H5 influenza

viruses isolated from humans, chickens, and

wild aquatic birds have distinguishable

properties. J. Virol. 73(2), 1146–1155 (1999).

87. Beare AS. Replication of avian influenza

viruses in humans. Arch. Virol. 119(1–2), 37

(1991).

88. Hinshaw VS, Webster RG, Naeve CW,

Murphy BR. Altered tissue tropism of

human–avian reassortant influenza viruses.

Virology 128(1), 260–263 (1983).

89. Ito T, Couceiro JN, Kelm S et al. Molecular

basis for the generation in pigs of influenza A

viruses with pandemic potential. J. Virol.

72(9), 7367–7373 (1998).

90. Slemons RD, Easterday BC. Virus replication

in the digestive tract of ducks exposed by

aerosol to type-A influenza. Avian Dis. 22(3),

367–377 (1978).

91. Kim JK, Negovetich NJ, Forrest HL, Webster

RG. Ducks: the trojan horses of H5N1

influenza. Influenza Other Respi. Viruses 3(4),

121–128 (2009).

92. Janies D, Hill AW, Guralnick R, Habib F,

Waltari E, Wheeler WC. Genomic analysis

and geographic visualization of the spread of

avian influenza (H5N1). Syst. Biol. 56(2),

321–329 (2007).

93. Ollier L, Caramella A, Giordanengo V, Lefebvre JC. High permissivity of human HepG2 hepatoma cells for influenza viruses. J. Clin. Microbiol. 42(12), 5861–5865 (2004).

94. Perez DR, Sorrell EM, Donis RO. Avian influenza: an omnipresent pandemic threat. Pediatr. Infect. Dis. J. 24(11 Suppl.), S208–S216 (2005).

95. Ferro PJ, El-Attrache J, Fang X et al. Avian influenza surveillance in hunter-harvested waterfowl from the gulf coast of Texas (November 2005–January 2006). J. Wildl. Dis. 44(2), 434–439 (2008).

96. Furuse Y, Suzuki A, Kamigaki T, Oshitani H. Evolution of the M gene of the influenza A

virus in different host species: large-scale sequence analysis. Virol. J. 6, 67 (2009).

97. Munster VJ, Fouchier RA. Avian influenza virus: of virus and bird ecology. Vaccine 27(45), 6340–6344 (2009).

98. Pedersen K, Swafford SR, Deliberto TJ. Low pathogenicity avian influenza subtypes isolated from wild birds in the United States, 2006–2008. Avian Dis. 54(1 Suppl.), 405–410 (2010).

99. Hua YP, Chai HL, Yang SY, Zeng XW, Sun

Y. Primary survey of avian influenza virus and

Newcastle disease virus infection in wild birds in some areas of Heilongjiang Province,

China. J. Vet. Sci. 6(4), 311–315 (2005).

100. Stallknecht DE, Shane SM, Zwank PJ, Senne

DA, Kearney MT. Avian influenza viruses

from migratory and resident ducks of coastal

Louisiana. Avian Dis. 34(2), 398–405 (1990).

101. Douglas KO, Lavoie MC, Kim LM, Afonso

CL, Suarez DL. Isolation and genetic

characterization of avian influenza viruses and

a Newcastle disease virus from wild birds in

Barbados: 2003–2004. Avian Dis. 51(3),

781–787 (2007).

102. El Zowalaty ME, Chander Y, Redig PT,

El Latif HKA, El Sayed MA, Goyal SM.

Selective isolation of Avian influenza virus

(AIV) from cloacal samples containing AIV

and Newcastle disease virus. J. Vet. Diagn.

Invest. 23(2), 330–332 (2011).

103. Hinshaw VS, Wood JM, Webster RG, Deibel

R, Turner B. Circulation of influenza viruses

and paramyxoviruses in waterfowl originating

from two different areas of North America.

Bull. World Health Organ. 63(4), 711–719

(1985).

104. Jahangir A, Ruenphet S, Ueda S et al. Avian

influenza and Newcastle disease viruses from

northern pintail in Japan: isolation,

characterization and inter-annual

comparisons during 2006–2008. Virus Res.

143(1), 44–52 (2009).

105. Slemons RD, Johnson DC, Osborn JS, Hayes

F. Type-A influenza viruses isolated from wild

free-flying ducks in California. Avian Dis.

18(1), 119–124 (1974).

106. Slemons RD, Shieldcastle MC, Heyman LD, Bednarik KE, Senne DA. Type A influenza viruses in waterfowl in Ohio and implications for domestic turkeys. Avian Dis. 35(1), 165–173 (1991).

107. Lira J, Moresco KA, Stallknecht DE, Swayne DE, Fisher DS. Single and combination diagnostic test efficiency and cost analysis for detection and isolation of avian influenza virus from wild bird cloacal swabs. Avian Dis. 54(Suppl. 1), 606–612 (2010).

108. Weinberg A, Mettenbrink CJ, Ye D, Yang CF. Sensitivity of diagnostic tests for influenza

Avian influenza viruses: virology, diagnosis & surveillance

Review

varies with the circulating strains. J. Clin. Virol. 33(2), 172–175 (2005).

109. Philippa JD. Avian influenza. In: Zoo and Wild Animal Medicine Current Therapy (6th Edition). Fowler ME (Ed.). Saunders, PA, USA, 79–87 (2008).

110. Swayne DE, Suarez DL. Highly pathogenic avian influenza. Rev. Sci. Tech. 19(2), 463–482 (2000).

111. Suarez DL, Das A, Ellis E. Review of rapid molecular diagnostic tools for avian influenza virus. Avian Dis. 51(1 Suppl.), 201–208

(2007).

112. Ellström P, Latorre-Margalef N,

Griekspoor P et al. Sampling for low-

pathogenic avian influenza A virus in wild

mallard ducks: oropharyngeal versus cloacal

swabbing. Vaccine 26(35), 4414–4416

(2008).

113. Jindal N, De Abin M, Primus AE et al.

Comparison of cloacal and oropharyngeal

samples for the detection of avian influenza

virus in wild birds. Avian Dis. 54(1), 115–119

(2010).

114. Roelandt S, Outtrim L, Browning C,

Alexander DJ, Brown IH, Irvine RM.

Evaluation of two different swab transport

systems in the detection of avian influenza

virus excretion from infected pekin ducks

Anas platyrhynchos. J. Virol. Methods

184(1–2), 8–14 (2012).

115. WHO. Laboratory procedures. In: WHO

Manual on Animal Influenza Diagnosis and

Surveillance. WHO Press, Switzerland, 98,

15–19 (2002).

116. Docherty DE, Slota PG. Use of muscovy

duck embryo fibroblasts for the isolation of

viruses from wild birds. Methods Cell Sci.

11(3), 165–170 (1988).

117. Taubenberger JK, Layne SP. Diagnosis of

influenza virus: coming to grips with the

molecular era. Mol. Diagn. 6(4), 291–305

(2001).

118. Potter C, Zuckerman AJ, Banatvala JE,

Pattison JR. Influenza. In: Principles and

Practice of Clinical Virology. Wiley, NY, USA, 199–223 (1987).

119. Newton DW, Treanor JJ, Menegus MA. Clinical and laboratory diagnosis of influenza virus infections. Am. J. Manag. Care 6(Suppl. 5), S265–S275 (2000).

120. Petric M, Comanor L, Petti CA. Role of the laboratory in diagnosis of influenza during seasonal epidemics and potential pandemics. J. Infect. Dis. 194(Suppl. 2), S98–S110

(2006).

121. Prince HE, Leber AL. Comparison of complement fixation and hemagglutination inhibition assays for detecting antibody responses following influenza virus

vaccination. Clin. Vaccine Immunol. 10(3), 481–482 (2003).

122. Julkunen I, Pyhala R, Hovi T. Enzyme immunoassay, complement fixation and hemagglutination inhibition tests in the diagnosis of influenza A and B virus infections. Purified hemagglutinin in subtype-specific diagnosis. J. Virol. Methods 10(1), 75–84 (1985).

123. Pearson JE, Senne DA. Diagnostic procedures for avian influenza. Avian Dis. 47, 222–227

(2003).

124. Smith W, Andrewes CH, Laidlaw PP. A virus from influenza patients. Lancet 2, 66 (1933).

125. Swayne DE, Senne DA, Suarez DL. Avian

influenza. In: A Laboratory Manual for the

Isolation, Identification, and Characterization

of Avian Pathogens. Dufour-Zavala L (Ed.).

American Association of Avian Pathologists,

GA, USA, 128–134 (2008).

Diagnsotic principles followed in the

identification and characcterization of

avian influenza viruses as well as other

avian pathogens.

126. Cattoli G, Drago A, Maniero S et al.

Comparison of three rapid detection systems

for type A influenza virus on tracheal swabs of

experimentally and naturally infected birds.

Avian Pathol. 33(4), 432–437 (2004).

127. Cattoli G, Capua I. Molecular diagnosis of

avian influenza during an outbreak. Dev. Biol.

(Basel) 124, 99–105 (2006).

128. Carman S, Stansfield C, Weber J, Bildfell R,

Van Dreumel T. H3N2 influenza A virus

recovered from a neonatal pig in Ontario –

1997. Can. Vet. J. 40(12), 889–890 (1999).

129. Clavijo A, Tresnan DB, Jolie R, Zhou EM.

Comparison of embryonated chicken eggs

with MDCK cell culture for the isolation of

swine influenza virus. Can. Vet. J. 66(2),

117–121 (2002).

130. Lee CW, Jung K, Jadhao S, Suarez D.

Evaluation of chicken-origin (DF-1) and

quail-origin (QT-6) fibroblast cell lines for

replication of avian influenza viruses. J. Virol.

Methods 153(1), 22–28 (2008).

131. Moresco KA, Stallknecht DE, Swayne DE. Evaluation and attempted optimization of avian embryos and cell culture methods for efficient isolation and propagation of low pathogenicity avian influenza viruses. Avian Dis. 54(1 Suppl.), 622–626 (2010).

132. Munster VJ, Baas C, Lexmond P et al. Practical considerations for high-throughput influenza A virus surveillance studies of wild birds by use of molecular diagnostic tests. J. Clin. Microbiol. 47(3), 666–673 (2009).

133. Reina J, Fernandez-Baca V, Blanco I, Munar M. Comparison of Madin–Darby canine kidney cells (MDCK) with a green monkey

continuous cell line (Vero) and human lung embryonated cells (MRC-5) in the isolation of influenza A virus from nasopharyngeal aspirates by shell vial culture. J. Clin. Microbiol.