Animal and human health implications of avian influenza infections.
ABSTRACT Avian influenza (AI) is a listed disease of the World Organisation for Animal Health (OIE) that has become a disease of great importance both for animal and human health. Until recent times, AI was considered a disease of birds with zoonotic implications of limited significance. The emergence and spread of the Asian lineage highly pathogenic AI (HPAI) H5N1 virus has dramatically changed this perspective; not only has it been responsible of the death or culling of millions of birds, but this virus has also been able to infect a variety of non-avian hosts including human beings. The implications of such a panzootic reflect themselves in animal health issues, notably in the reduction of a protein source for developing countries and in the management of the pandemic potential. Retrospective studies have shown that avian progenitors play an important role in the generation of pandemic viruses for humans, and therefore these infections in the avian reservoir should be subjected to control measures aiming at eradication of the Asian H5N1 virus from all sectors rather than just eliminating or reducing the impact of the disease in poultry.
Article: Cloning and expression of highly pathogenic avian influenza virus full-length nonstructural gene in Pichia pastoris.[show abstract] [hide abstract]
ABSTRACT: Avian influenza (AI) is a highly contagious and rapidly evolving pathogen of major concern to the poultry industry and human health. Rapid and accurate detection of avian influenza virus is a necessary tool for control of outbreaks and surveillance. The AI virus A/Chicken/Malaysia/5858/2004 (H5N1) was used as a template to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA by PCR amplification of the NS1 region. Products were cloned into pCR2.0 TOPO TA plasmid and subsequently subcloned into pPICZαA vector to construct a recombinant plasmid. Recombinant plasmid designated as pPICZαA-NS1 gene was confirmed by PCR colony screening, restriction enzyme digestion, and nucleotide sequence analysis. The recombinant plasmid was transformed into Pichia pastoris GS115 strain by electroporation, and expressed protein was identified by SDS-PAGE and western blotting. A recombinant protein of approximately ~28 kDa was produced. The expressed protein was able to bind a rabbit polyclonal antibody of nonstructural protein (NS1) avian influenza virus H5N1. The result of the western blotting and solid-phase ELISA assay using H5N1 antibody indicated that the recombinant protein produced retained its antigenicity. This further indicates that Pichia pastoris could be an efficient expression system for a avian influenza virus nonstructural (NS1).Journal of Biomedicine and Biotechnology 01/2011; 2011:414198. · 2.44 Impact Factor
Article: Membrane-based inverse transition cycling: an improved means for purifying plant-derived recombinant protein-elastin-like polypeptide fusions.[show abstract] [hide abstract]
ABSTRACT: Elastin-like peptide (ELP) was fused to two different avian flu H5N1 antigens and expressed in transgenic tobacco plants. The presence of the ELP tag enhanced the accumulation of the heterologous proteins in the tobacco leaves. An effective membrane-based Inverse Transition Cycling was developed to recover the ELPylated antigens and antibodies from plant material. The functionality of both the ELPylated neuraminidase and an ELPylated nanobody was demonstrated.International Journal of Molecular Sciences 01/2011; 12(5):2808-21. · 2.60 Impact Factor
Article: Coexpression of avian influenza virus H5 and N1 by recombinant Newcastle disease virus and the impact on immune response in chickens.[show abstract] [hide abstract]
ABSTRACT: To analyze the contribution of neuraminidase (NA) toward protection against avian influenza virus (AIV) infection, three different recombinant Newcastle disease viruses (NDVs) expressing hemagglutinin (HA) or NA, or both, of highly pathogenic avian influenza virus (HPAIV) were generated. The lentogenic NDV Clone 30 was used as backbone for the insertion of HA of HPAIV strain A/chicken/Vietnam/P41/05 (H5N1) and NA of HPAIV strain A/duck/Vietnam/TG24-01/05 (H5N1). The HA was inserted between the genes encoding NDV phosphoprotein (P) and matrixprotein (M), and the NA was inserted between the fusion (F) and hemagglutinin-neuraminidase protein (HN) genes, resulting in NDVH5VmPMN1FHN. Two additional recombinants were constructed carrying the HA gene between the NDV P and M genes (NDVH5VmPM) or the NA between F and HN (NDVN1FHN). All recombinants replicated well and stably expressed the HA gene, the NA gene, or both. Chickens immunized with NDVH5VmPMN1FHN or NDVH5VmPM were protected against two different HPAIV H5N1 and also against HPAIV H5N2. In contrast, immunization of chickens with NDVN1FHN induced NDV- and AIV N1-specific antibodies but did not protect the animals against a lethal dose of HPAIV H5N1. Furthermore, expression of AIV N1, in addition to AIV H5 by NDV, did not increase protection against HPAIV H5N1.Avian Diseases 09/2011; 55(3):413-21. · 1.46 Impact Factor
Animal and Human Health Implications of Avian
Ilaria Capua Æ Æ Dennis J. Alexander
Published online: 29 June 2007
? The Biochemical Society 2007
Health (OIE) that has become a disease of great importance both for animal and human
health. Until recent times, AI was considered a disease of birds with zoonotic
implications of limited significance. The emergence and spread of the Asian lineage
highly pathogenic AI (HPAI) H5N1 virus has dramatically changed this perspective; not
only has it been responsible of the death or culling of millions of birds, but this virus has
also been able to infect a variety of non-avian hosts including human beings. The
implications of such a panzootic reflect themselves in animal health issues, notably in
the reduction of a protein source for developing countries and in the management of the
pandemic potential. Retrospective studies have shown that avian progenitors play an
important role in the generation of pandemic viruses for humans, and therefore these
infections in the avian reservoir should be subjected to control measures aiming at
eradication of the Asian H5N1 virus from all sectors rather than just eliminating or
reducing the impact of the disease in poultry.
Avian influenza (AI) is a listed disease of the World Organisation for Animal
Avian influenza ? Ecology ? Hosts ? Interspecies transmission ?
Avian influenza (AI) represents one of the greatest concerns for public health that has
emerged from the animal reservoir in recent times. AI, in its highly pathogenic form
Dennis J. Alexander—Unaffiliated Consultant Virologist
I. Capua (&)
OIE, FAO and National Reference Laboratory for Newcastle Disease and Avian Influenza, Istituto
Zooprofilattico Sperimentale delle Venezie, Viale dell’Universita ` 10, Legnaro, Padova 35020, Italy
D. J. Alexander
Virology Department, Veterinary Laboratories Agency Weybridge, Surrey, UK
Biosci Rep (2007) 27:359–372
(HPAI), has been known to the veterinary community since the end of the 19th century,
when an Italian scientist, Edoardo Perroncito reported what is believed to be the first
documented evidence of ‘‘fowl plague’’ as a distinct disease. However, for over
100 years, HPAI revealed itself to be a poultry disease of rare occurrence, which in most
cases affected an irrelevant number of birds, and was, generally speaking, either self-
limiting or controlled efficiently through the application of measures involving stamping
out. Approximately at the turn of the millennium however, a sharp increase in the
number of outbreaks of AI in poultry occurred. It has been calculated that the impact of
AI on the poultry industry has increased 100-fold with 23 million birds affected in a 40-
year-period between 1959 and 1998 and over 200 million from 1999 to 2004 (Capua and
Alexander 2004). In addition to this, since 1997, the consequential human health
implications of AI infections of poultry have been identified, especially as a result of the
spread of Asian H5N1 virus. This has dramatically attracted the attention of the
scientific community and AI infections have assumed a completely different profile both
in the veterinary and medical scientific environments.
In recent times some outbreaks have maintained the characteristic of minor
relevance while others, such as the Italian 1999–2000, the Dutch 2003, the Canadian
2004 outbreaks have caused substantial damage to the poultry industry. The Dutch and
the Canadian outbreaks resulted in human infections and caused more general human
health concerns. Although these three outbreaks were believed to be exceptional with
regards to magnitude, costs and human health involvement, these were just a prelude to
the spread of Asian H5N1 virus and the concerns generated about the emergence of a
new pandemic virus for humans via the avian-human link.
Biology of Avian Influenza Viruses
Influenza viruses have segmented, negative sense, single strand RNA genomes and are
placed in the family Orthomyxoviridae. At present the Orthomyxoviridae family consists
of five genera, only viruses of the Influenzavirus A genus are known to infect birds.
Influenza A viruses are further divided into subtypes based on the antigenic
relationships in the surface glycoproteins, haemagglutinin (HA) and neuraminidase
(NA). At present 16 HA subtypes have been recognised (H1–H16) and nine NA
subtypes (N1–N9). Each virus has one HA and one NA antigen, apparently in any
combination. All influenza A subtypes in the majority of possible combinations have
been isolated from avian species. To date only viruses of H5 and H7 subtype have been
shown to cause HPAI in susceptible species, but not all H5 and H7 viruses are virulent.
For allinfluenza Avirusesthe haemagglutininglycoprotein isproduced asa precursor,
HA0, which requires post translational cleavage by host proteases before it is functional
and virus particles are infectious (Rott 1992). The HA0 precursor proteins of AI viruses
of low virulence for poultry (LPAI viruses) have a single arginine at the cleavage site and
another basic amino acid at position –3 or –4 from the cleavage site. These viruses are
limited to cleavage by extracellular host proteases such as trypsin-like enzymes and thus
restricted to replication at sites in the host where such enzymes are found, i.e. the
respiratory and intestinal tracts. HPAI viruses possess multiple basic amino acids
(arginine and lysine) at their HA0 cleavage sites either as a result of apparent insertion or
apparent substitution (Senne et al. 1996; Vey et al. 1992; Wood et al. 1993) and appear to
360Biosci Rep (2007) 27:359–372
processing subtilisin-related endoproteases of which furin is the leading candidate
(Stieneke-Grober et al. 1992). HPAI viruses are able to replicate throughout the bird,
damaging vital organs and tissues, which results in disease and death.
To date only viruses of the H5 and H7 subtypes have been shown to cause HPAI. It
appears that HPAI viruses arise by mutation after a LPAI precursor has been
introduced into poultry. It follows that all HPAI viruses should have a LPAI progenitor,
although the latter have only been identified in a limited number of cases, and these do
not include the Asian H5N1 virus which has now spread to three continents. Several
mechanisms appear to be responsible for this mutation. Most HPAI viruses appear to
have arisen as result of spontaneous duplication of purine triplets which results in the
insertion of basic amino acids at the HA0 cleavage site and that this occurs due to a
transcription fault by the polymerase complex (Perdue et al. 1997). However, as pointed
out by Perdue et al. (1997) this is clearly not the only mechanism by which HPAI viruses
arise as some appear to result from nucleotide substitution rather than insertion while
others have insertions without repeating nucleotides. The Chile 2002 (Suarez et al. 2004)
and the Canada 2004 (Pasick et al. 2005) H7N3 HPAI viruses show distinct and unusual
cleavage site amino acid sequences. These viruses appear to have arisen as a result of
recombination with other genes (nucleoprotein gene and matrix gene respectively)
resulting in an insertion at the cleavage site of 11 amino acids for the Chile virus and 7
amino acids for the Canadian virus.
The factors that bring about mutation from LPAI to HPAI are not known. In some
instances mutation seems to have taken place rapidly (at the primary site) after
introduction from the wild bird reservoir, in others the LPAI virus has circulated in
poultry for months before mutating. Therefore, it is impossible to predict if and when
this mutation will occur. However, it can be reasonably assumed that the wider the
circulation of LPAI in poultry, the higher the chance that mutation to HPAI will occur.
HPAI viruses are not necessarily virulent for all species of birds and the clinical
severity seen in any host appears to vary with both bird species and virus strain
(Alexander et al. 1978, 1986). In particular, ducks rarely show clinical signs as a result of
HPAI infections although there are reports that some of the Asian H5N1 viruses have
caused disease (Sturm-Ramirez et al. 2005) and the HPAI viruses A/duck/Italy/2000
H7N1 and A/chicken/Germany/34 (H7N1) have been reported to cause disease and
death in naturally and experimentally infected waterfowl (Alexander et al. 1978; Capua
and Mutinelli 2001b).
Avian influenza viruses have been shown to infect birds and mammals. Generally
speaking the former are infected more readily and efficiently than the latter, and the
inter and intraspecies transmission within the Class Aves occurs to a greater extent than
in the Class Mammalia. One of the main factors that influence susceptibility to infection
appears to be the receptor conformation on the host cells. Avian influenza viruses bind
preferably to sialic acid (SA)-a2,3-Gal terminated saccharides, which are prominent in
avian cells. Human influenza viruses, in contrast, bind preferentially to SA-a2,6-Gal
terminated saccharides, well represented in human epithelial cells. This different
binding preference is believed to be one of the major factors that impede the crossing of
the species barrier. However, the fact that avian influenza viruses do occasionally infect
people indicates that this barrier is not insurmountable.
Biosci Rep (2007) 27:359–372 361
Influenza viruses have been shown to infect a great variety of birds (Alexander 2000,
2001; EFSA 2005; Hinshaw et al. 1981; Lvov 1978), including free-living birds, captive
caged birds, domestic ducks, chickens, turkeys and other domestic poultry.
HPAI viruses cause a severe systemic disease in galliform birds, which results in
mortality rates approaching 100%. Extensive viral replication in vital organs determines
nervous and enteric symptoms which in many species brings about the death of the bird.
In contrast, LPAI viruses cause a mild disease in domesticated birds, which may be
inapparent and is often self limiting. Turkeys appear to develop a more serious clinical
condition under certain circumstances (Capua and Mutinelli 2001a). The marked clinical
difference between HPAI and LPAI infections does not occur in ostriches (Struthio
camelus), the reason for this still to be determined (Capua and Mutinelli 2001a).
It was not until the mid-1970s that any systematic investigations of influenza in feral
birds were undertaken. These investigations revealed enormous pools of influenza
viruses to be present in the wild bird population (EFSA 2005; Olsen et al. 2006;
Stallnecht 1998; Stallnecht and Shane 1988) especially in waterfowl, Family Anatidae,
Order Anseriformes. In the surveys listed by Stallknecht and Shane (1988) a total of
21,318 samples from all species resulted in the isolation of 2,317 (10.9%) viruses.
However, 14,303 of these samples were from birds of the Order Anseriformes which
yielded 2,173 (15.2%) isolates. The next highest isolation rates were 2.9% and 2.2%
from the Passeriformes and Charadriiformes, respectively; but these compare with an
overall isolation rate of 2.1% from all birds other than ducks and geese. However,
studies by Sharp et al. (1993), suggest that waterfowl do not act as a reservoir for all
avian influenza viruses. It seems likely that part of the influenza gene pool is maintained
in shorebirds and gulls, from which the predominant number of isolated influenza
viruses are of a different subtype to those isolated from ducks (Kawaoka et al. 1988).
Prior to the ongoing H5N1 epizootic, HPAI had only once affected wild birds
significantly. This outbreak occurred in South Africa in 1961 and caused the death of
approximately 1,300commonterns(Becker 1966). Itappeared,therefore,thatHPAI was
a disease of domesticated birds and that wild birds usually only harboured the low
pathogenic form of these viruses. The unprecedented situation occurring in Asia has
resulted in the spill-over of infection to naı ¨ve populations of wild birds. Although to date
all the wild birds from which Asian HPAI H5N1 virus has been isolated were either dead
or dying, the incubation period of this disease in Asian migratory birds is unknown, and
at the moment the scientific community only has an indication of the species that may be
infected and succumb to the virus. Knowledge and information on all species that are
susceptible to infection including the incubation period for those birds that do develop a
clinical condition, their ability to fly significant distances if infected and data on the route,
duration and titre of viral shedding are unavailable. At this stage only hypotheses can be
formulated on the eco-epidemiological consequences of this spill-over.
Currently it is unclear whether or not Asian HPAI H5N1 virus is truly endemic in any
part of the Eurasian wild bird population or merely limited to spill-over events from
domestic birds. If the latter is true, then provided the domestic source of infection is
eliminated, and the infections are responsible for the death of the wild avian hosts,
presumably the prevalence of infection will gradually be reduced to zero. In contrast if
HPAI infection does not bring about the death of the wild bird host and becomes
362 Biosci Rep (2007) 27:359–372
compatible with normal behavioural patterns and migration in at least some species, this
will result in the development of an endemic cycle in wild birds, mimicking the well-
known LPAI ecology. The consequences of such a situation are unpredictable.
Avian Influenza Infections of Mammals
Ostensibly pigs play a crucial role in influenza ecology and epidemiology, primarily
because of their dual susceptibility to human and avian viruses. They possess both SA-
a2,3-Gal terminated saccharides and SA-a2,6-Gal terminated saccharides, and are
therefore considered a potential ‘‘mixing vessel’’ for influenza viruses, from which
reassortants may emerge.
Kida et al. (1994) demonstrated experimentally that pigs were susceptible to
infection by at least one virus representative of each of the subtypes H1–H13.
The introduction of classical swine H1N1 influenza viruses to turkeys from infected
pigs has been reported to occur regularly in the USA, and in some cases, influenza-like
illness in pigs has been followed immediately by disease signs in turkeys (Halvorson
et al. 1992; Mohan et al. 1981; Pomeroy 1982). Genetic studies of H1N1 viruses from
turkeys in the USA has revealed a high degree of genetic exchange and reassortment of
influenza A viruses from turkeys and pigs in the former species (Wright et al. 1992). In
Europe, avian H1N1 viruses were transmitted to pigs, became established, and were
subsequently reintroduced to turkeys from pigs (Ludwig et al. 1994; Wood et al. 1997).
An independent introduction of H1N1 virus from birds to pigs occurred in Europe in
1979 (Pensaert et al. 1981). A similar introduction occurred in Asia in the early 1990s;
and these latter viruses are genetically distinct from the viruses in Europe (Guan et al.
1996). H9N2 viruses were introduced into pigs in South-East Asia (Peiris et al. 2001).
Serological evidence has been obtained of infections of pigs with viruses of H4, H5 and
H9 subtypes (Ninomiya et al. 2002). During the HPAI H7N7 epidemic in The
Netherlands in 2003, 13 pig herds on farms with infected poultry were shown to have
antibodies to H7 subtype, though no virus was detected (Loeffen et al. 2003, 2004). In
Canada, however, avian viruses of H3N3 and H4N6 subtypes have been isolated from
pigs (Karasin et al. 2000, 2004). Clearly the introduction of avian influenza viruses to
pigs is not an uncommon occurrence. But despite this, the only subtypes to have become
truly established in pig populations and readily transmissible from pig-to-pig and herd-
to-herd are H1N1, H3N2, and the reassortant H1N2, although genotype analysis of
isolates of these subtypes suggests that they can be the result or reassortment of viruses
from different progenitor host species (pig, human and avian).
There have been sporadic unpublished reports of natural infection of H5N1 in pigs
(Van Reeth 2007), and three experimental infections have been carried out with a total
of 8 H5N1 viruses. Six of these replicated in pigs, although clinical signs were mild or
unapparent and shedding levels were not high. In none of the experiments was pig-to-
pig transmission observed (Choi et al. 2005; Isoda et al. 2006; Shortridge et al. 1998).
Although there have been isolated reports of evidence of infection of horses with viruses
of subtypes H1N1, H2N2 and H3N2 (Tumova 1980), influenza infections of horses have
been restricted essentially to H7N7 and H3N8 subtypes of influenza A and these viruses
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