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Chapter 2.

Review of Literature

Avian influenza (AI) is an infectious highly contagious disease caused by Influenza virus
A genus of the Orthomyxoviridae family. The causative agent is negative-strand,
segmented RNA virus. Influenza A viruses can be divided into subtypes on the basis of
the possession of one of 15 antigenically distinct haemagglutinin (HA) antigens (H1 to
H15) and one of nine neuraminidase (NA) antigens (N1 to N9) (Alexander, 2000). LPAI
viruses cause respiratory and gastrointestinal infections without infecting the meat. By
contrast, HPAI viruses produce infection of respiratory and gastrointestinal tracts,
produce a viremia and virus is present in the meat and internal contents of eggs during the
acute stages of the infection (Swayne, 2004). Virtually all HA and NA combinations have
been isolated from birds. Wild waterfowl and shorebirds are considered the reservoir of
influenza A viruses because these species harbor all 15 hemagglutinin (HA) subtypes
(Alexander, 1993; Alexander, 2000; Condoberry and Slemons, 1992; Stallknecht, 1992;
Webster, 1992; Webster, 1978).

HPAI was considered a rare disease until recent times in domestic poultry with only 17
episodes being reported worldwide in the 40-year period 1959 to 1998. However, further
outbreaks have occurred since 1999, resulting in eight episodes involving 12 countries in
the 7 years covering 1997 to March 2004. Recently, there also appears to have been a
marked increase in the number of LPAI outbreaks caused by H5 and H7 viruses
(Alexander, 2001).

It was revealed that only type A influenza viruses are known to cause natural infections
in birds, but viruses of all 15 haemagglutinin and all nine neuraminidase influenza A
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Chapter 2. Review of Literature

subtypes in the majority of possible combinations have been isolated from avian species.
The very virulent viruses causing highly pathogenic avian influenza (HPAI) have been
restricted to subtypes H5 and H7, although not all viruses of these subtypes cause HPAI
(Alexander,, 2000).

During the spring of 2002, a low pathogenic avian influenza (LPAI) A (H7N2) virus
caused a major outbreak in commercial poultry in Virginia and adjacent states. The virus
primarily affected turkey flocks causing respiratory distress and decreased egg
production. Turkeys were more susceptible than chickens to H7N2 virus infection
(Tumpey,, 2004).

AI viruses were reported in chickens 200 km southwest of Islamabad, Pakistan. The

affected population was layers, broiler breeders and broilers with approximately 75% of
the flocks experiencing mortality ranging between 20 and 85%. H9N2 and H7N3 AI and
Newcastle disease viruses were isolated in various combinations. Both the HPAI and
LPAI forms of the H7N3 virus were identified (Swayne & Suarez 2001).

An outbreak of HPAI caused by a virus of H7N3 subtype was reported in 1995 affecting
northern Pakistan, causing the death of 3.2 million birds, primarily broiler breeders and
broilers. A vaccination campaign with a homologous vaccine was implemented, coupled
to an upgrading of biosecurity measures, and this led to the eradication of HPAI (Naeem,

During this outbreak HPAI viruses with three different HA0 cleavage site amino acid
sequences were detected i.e. PETPKRKRKRGLF, PETPKRRKRGLF and
PETPKRRNRGLF (Banks, 2000a).

Low pathogenic avian influenza (LPAI) H5N2 virus, which mutated to a HPAI virus with
the HA0 cleavage site sequence RKRKTRGLF and caused an outbreak of HPAI in
Mexico in 1994/95, continued to circulate in chickens, and the virus has been isolated
each year from 1997 (Senne, 1998).

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Chapter 2. Review of Literature

The H5N2 viruses, genetically similar to the virus circulating in Mexico, were isolated in
the Central American countries of Guatemala in 2000 and El Salvador in 2001. These
were the first reports of AI virus in Central America. It was concluded that movement of
contaminated poultry products and equipment between Mexico and Guatemala were the
most probable means of virus introduction (Senne, 2003).

In November 1997 outbreak of HPAI occurred near Tamworth, New South Wales. The
viruses isolated from chickens on two commercial farms were identified as HPAI viruses
of H7N4 subtype, with HA0 cleavage site amino acid sequences of RKRKRG and
intravenous pathogenicity index (IVPI) values of 2.52 and 2.90. The outbreak resulted in
the destruction of a total of 310565 chickens and 1232074 eggs, at a cost of a $ 4445000
(Selleck, 2003).

It was reported that between October 1997 and January 1998 eight outbreaks of HPAI
due to infections with virus of H5N2 subtype were recorded in the Veneto and Friuli-
Venezia Giulia regions of NE Italy. IVPI values of 2.98 to 3.00 were recorded for the
viruses isolated and the HA0 cleavage site was PQRRRKKRGLF (Capua, 1999).

A LPAI H5N9 virus from a flock of 2000 chickens in Emilia Romagna Italy in 1998 was
isolated. The IVPI was 0.00 and the cleavage site motif PQKETRGLF and reported
outbreaks due to H9N2 LPAI occurred in chickens in Italy in 1994 and 1996 (Fioretti, 1999; 1998).

Between 18 March and 17 April 1998, 29 outbreaks of LPAI, due to a virus of H7N7
subtype, were recorded in the Republic of Ireland; 27 commercial turkey, one turkey
breeder and one broiler breeder farm were affected (Campbell & De Geus 1999).

Three outbreaks, one in meat turkeys, one in broiler breeders and one in turkey breeders,
of H7N7 LPAI occurred in Northern Ireland in March to April 1998, apparently as a
result of spread from the Republic of Ireland (Graham, 1999).

LPAI virus of H5N2 subtype was isolated in Belgium in 1999 from a backyard flock of
100 chickens with 10% mortality and diarrhea and respiratory signs. The IVPI value

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Chapter 2. Review of Literature

obtained for the virus was 0.00 and the cleavage site amino acid motif PQKETRGLF.
Despite the LPAI nature of the virus, a stamping-out policy was employed (Meulemans, 2000).

In March 1999, first isolation of a LPAI virus of H7N1 subtype was reported. The isolate
had an IVPI value of 0.00 and HA0 cleavage site PEIPKGRGLF. From March to
December 1999, 199 farms were shown to be infected with the virus (Capua and
ALexender, 2004).

In December 1999, HPAI outbreak in a meat turkey flock in Italy was reported showing
high mortality was characterized as H7N1 with an IVPI value of 3.0 and a deduced HA0
cleavage site amino acid sequence of PEIPKGSRVRRGLF (Capua, 2000).

Between December 1996 and April 1998, over 2.5 million layer chickens on 24 premises
and 47 flocks in Pennsylvania were infected with a LPAI H7N2 virus with HA0 cleavage
site sequence PENPKTRGLF. Approximately 25% of birds exhibited clinical signs of
respiratory disease and a temporary decrease in egg production (Davison, (2003),
Henzler, (2003) and Senne, (2003).

LPAI virus of H7N2 subtype continued to circulate in the live bird market system in the
US, and the virus again spilled over into the industrial poultry population of the
Shanandoah valley in Virginia in 2002. In all, 197 outbreaks were diagnosed (primarily
in turkeys) and a total of about 5 million birds was stamped out at a cost of about $149
000 000 (Spackman & Suarez, 2003).

In March 2003, two outbreaks of LPAI H7N2 virus were confirmed in New London
County, Connecticut, affecting 2 900 000 table-egg layer hens in two commercial
operations managed by the same enterprise. In February 2004 two farms in Delaware
were confirmed as infected with LPAI of H7N2 subtype. One was a small-scale quasi-
backyard type farm with approximately 11000 chickens of various types, which supplied
live-bird markets. At the beginning of March 2004 a flock in Maryland was shown to be
infected with H7N2 virus as a result of surveillance in the Delmarva Peninsula following
the Delaware outbreaks (Capua and Alexander, 2004).
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Chapter 2. Review of Literature

In 2000 a flock of turkeys in Ontario, with slightly elevated mortality and egg production
problems, was shown to be infected with a LPAI virus of H7N1 subtype, which had an
IVPI of 0 and a HA cleavage site sequence of PENPKTRGLF (Pasick, 2003).

The isolation of a LPAI virus of H7N7 from a small mixed free-range flock consisting of
60 layer-type chickens, 10 adult broilers, two bronze turkeys, 30 geese and 25 ducks in
Southern Germany was reported. The virus had an IVPI of 0.03 and a HA0 cleavage site
motif of PEIPKGRGLF (Werner, 2003).

Between the end of April and the first week of May 2002, Samples were collected from
the premises and processed in the laboratory, resulting in the isolation of a LPAI virus of
H7N3 subtype. On 23 May following an official inspection, HPAI was suspected. The
laboratory confirmed HPAI with the isolation of a virus, of H7N3 subtype, which proved
to be highly virulent for chickens (Rojas, 2002).

During August 2002, serological testing at the abattoir detected antibodies to H7 virus in
three meat-type turkey flocks were examined. Intensive surveillance in the whole area did
not reveal any additional outbreaks. In October 2002, haemagglutination inhibition tests
on serum samples from meat turkeys in the Brescia province were again found to be
positive for antibodies to the H7 subtype of AI. Virus isolation yielded a LPAI virus of
H7N3 subtype and HA0 cleavage site sequence PEIPKGRGLF (Capua and Alexander,

At the end of February 2003, HPAI was suspected in layer farms located in the Gelder
Valley province in The Netherlands. The outbreak was confirmed with the isolation of a
HPAI virus of H7N7 subtype. In the middle of April the infection spread to Belgium and
Germany. On 20 January 2004 Taiwan confirmed the infection of two farms, with a LPAI
virus of H5N2 subtype. A second farm 3 km from the first outbreak was confirmed as
infected with HPAI on 12 March 2004.In February 2004 HPAI caused by a virus of
H5N2 subtype was confirmed in a broiler flock of 6608 birds in Gonzales County, Texas.
A further outbreak in a flock of 12 000 birds was reported at the beginning of March.
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Chapter 2. Review of Literature

LPAI of H7N3 subtype was detected on a broiler breeder farm in British Columbia
Canada in February 2004 as a result of a routine monitoring programme (Capua and
Alexander, 2004).

Outbreaks due to H9N2 LPAI occurred in domestic ducks, chickens and turkeys in
Germany during 1995 to 1998 (Werner, 1999; 1998). While in pheasants in Ireland in
1997 (Campbell, 1998).

Outbreaks due to H9N2 LPAI occurred in turkeys in the US in 1995 and 1996 (Halvorson, 1998), while in ostriches in South Africa in 1995 (Banks, 2000b).

Infections of chickens with H9N2 viruses in Korea were first detected in 1996 and
reported to be widespread (Mo, 1998; Lee, 2000).

Widespread infections have been reported in China and Hong Kong, due to H9N2 viruses
(Liu, 2003; Guan, 2000).

Since 1998, an epidemic of avian influenza has occurred in the Iranian poultry industry.
The agent was pathotyped as non-highly pathogenic and subtyped as an H9N2 avian
influenza virus (Nili and Asasi, 2002).

An epidemic of avian influenza (AI) (H9N2) occurred in broiler chicken farms in Iran
during 1998-2001. Mortality between 20% and 60% was commonly observed on the
affected farms (Nili and Asasi, 2003).

Recently seven isolates of avian influenza virus (AIV) serotype H9N2 recovered from an
outbreak of AI were analyzed on the basis of their biological and molecular
characteristics. All the isolates belonged to the low-pathogenicity group of AIV (Bano, 2003).

LPAI H9N2 infections have been reported in the Middle East since 1998 and have also
caused widespread outbreaks in commercial chickens in Pakistan (Naeem, 2003;

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Chapter 2. Review of Literature

There were three instances on record of an isolation of AI viruses from humans up to

1996. The first was an HPAI virus of H7N7 subtype obtained from a patient with
hepatitis in 1959. The second related to a laboratory worker in Australia who developed
conjunctivitis after accidental exposure directly in the eye with a HPAI virus of H7N7
subtype. The third was of conjunctivitis associated with an avian LPAI virus, again of
H7N7 subtype, which spread to an animal handler from an infected seal. (Webster,
1981, Taylor & Turner, 1977 and Campbell, 1970).

It was investigated that aquatic birds provide the natural reservoir for influenza A viruses,
but in general, avian influenza is asymptomatic in feral birds. Occasionally, however,
highly pathogenic strains of influenza cause serious systemic infections in domestic
poultry (Zambon, 2001).

An avian H5N1 influenza A virus obtained from a tracheal aspirate of a 3-year-old child
in Hong Kong with a fatal illness consistent with influenza. The virus caused 87.5 to 100
percent mortality in experimentally inoculated White Plymouth Rock and White Leghorn
chickens. These results may have implications for global influenza surveillance and
planning for pandemic influenza (Subbarao,, 1998).

Natural infections with influenza A viruses in a variety of animal species including

humans, pigs, horses, sea mammals, mustelids and birds were observed. Occasionally
devastating pandemics occur in humans. Although viruses of relatively few HA and NA
subtype combinations have been isolated from mammalian species, all 15 HA subtypes
and all 9 NA subtypes, in most combinations, have been isolated from birds (Capua and
Alexander, 2002).

Chickens were inoculated with one of five H5N2 Mexican-origin avian influenza virus
(AIV) isolates to determine their pathogenicity for chickens. In laboratory infections,
three AIVs, H5/94, M5/94, and J12/94, produced sporadic illness and death and were
categorized as mildly pathogenic. Q1/95 produced illness and death in all inoculated
chickens and was categorized as highly lethal and highly pathogenic (HP). P11/94B
commonly produced clinical illness, but deaths were infrequent (Swayne,, 1997).
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Chapter 2. Review of Literature

Genes from animal influenza viruses were captured by the human virus via reassortment,
which occurred when a host has been simultaneously infected by both virus types. Such
an antigenic shift appears to present the human immune system with a novel antigenic
experience, usually resulting in high morbidity and mortality. In addition to antigenic
shift, slight changes in the surface proteins caused by point mutations (antigenic drift)
also allow the viruses to evade the human system, but it may take 3–5 years for a virus of
a given subtype to accumulate enough point mutations to cause disease when it reinfects
a previously exposed person (Peter and Adolfo, 2002).

In 1997 an avian H5N1 influenza virus was directly transmitted from birds to humans in
Hong Kong and resulted in 18 confirmed infections, thus strengthening the pandemic
threat posed by avian influenza (AI). Indeed, reassortant viruses, harboring a combination
of avian and human viral genomes, have been responsible for major pandemics of human
influenza (Tollis and Trani, 2002).

In 2003, highly pathogenic strains of avian influenza virus, including the H5N1 and
H7N7 subtypes, again crossed from birds to humans and caused fatal disease. Direct
avian-to-human influenza transmission was unknown before 1997 (Webby and Webster,

The occurrence of avian influenza viruses (AIV) infections in southern Pakistan was
evaluated. A second investigation was undertaken in selected broiler-breeder, broiler and
layer flocks. In this investigation, nine H9N2 AIV isolates were recovered (Naeem,,

Avian influenza (AI) outbreak occurred in meat-type chickens in central Pennsylvania

from December 2001 to January 2002. Avian influenza virus (AIV), H7N2 subtype, was
isolated. The H7N2 isolates were characterized as a low pathogenic strain at the National
Veterinary Services Laboratories based on molecular sequencing of the virus
hemagglutinin cleavage site and virus challenge studies in specific-pathogen-free leghorn
chickens (Lu, 2004).

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Chapter 2. Review of Literature

An outbreak of low-pathogenicity H7N2 avian influenza virus (AIV) in the Shenandoah

Valley of Virginia during the spring and summer of 2002 was reported affecting 197
farms and resulted in the destruction of over 4.7 million birds. The outbreak affected
primarily turkey farms (28 breeders, 125 grow out) with some spillover into chicken
farms (29 breeders, 13 grow out, 2 table-egg layers) (Akey, 2003).

Two antigenically and genetically distinct groups of Avian influenza A H9N2 viruses
were reported to widespread among domestic poultry and were recently isolated from
humans with respiratory illness (Xiuhua,, 2001).

Avian influenza A H9N2 viruses are circulating in domestic poultry worldwide. Although
this avian subtype is generally not highly pathogenic for avian species, these viruses have
recently been transmitted to mammalian species, including humans. In Hong Kong, H9N2
viruses were isolated from domestic pigs in 1998 and one year later were isolated from
two children with uncomplicated febrile respiratory illnesses (Alexander, 2001).

In Cambodia a new outbreak was reported in chickens. More than 600 chickens fell ill
and started dying on 19/09/04 on a farm at Kien Svay District in Kandal Province, near
the Cambodian capital. The farm had 4,500 one-month old chickens. All chicks were
bought from Takhmao District in the same province. About 2,300 chickens died, and the
remaining 2,200 birds were culled. Testing by the Pasteur Institute in Phnom Penh
confirmed that the strain was H5N1. It was suspected that the farm's owner used water
from the pond for the chickens. In Indonesia deaths of 350 chickens was reported. Tests
on dead birds confirmed H5N1 strain on 29/09/04 (FAO 2004).

Recent outbreaks in China, Viet Nam, Cambodia, Malaysia and Thailand show that the
virus continues to circulate in the region and will not probably be eradicated in the near
future. Need of more and urgent research was felt as the role of wildlife, domestic ducks
and pigs in transmitting the virus among animals is still not fully understood (FAO 2004).

It was reported that the Australia had experienced outbreaks of HPAI in 1976 (H7N7),
1985 (H7N7), 1992 (H7N3) and 1994 (H7N3) (Westbury, 1998).

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Chapter 2. Review of Literature


Influenza A viruses infecting poultry can be divided into two distinct groups on the basis
of their ability to cause disease. The very virulent viruses cause highly pathogenic avian
influenza (HPAI), which may result in flock mortality as high as 100%. These viruses
have been restricted to subtypes H5 and H7, although not all viruses of these subtypes
cause HPAI. All other viruses cause a much milder disease consisting primarily of mild
respiratory disease, depression and egg production problems in laying birds (low
pathogenicity avian influenza) LPAI (Alexander, 2000).

The surface proteins of H7N3 viruses was isolated from wild ducks in Italy in 2001 and
compared with H7N3 strains that circulated in Italian turkeys in 2002-2003. It was
observed that the wild and domestic avian strains appeared strictly related at both
phenotypic and genetic level. Homology percentages in seven of their genes were
comprised between 99.8% (for PB2) and 99.1% (for M), and their NA genes differed
mainly because of a 23-aminoacid deletion in the NA stalk (Campitelli, 2004).

LPAI H7N1 virus was isolated during the 1999-2001 epizootic in Italy. The
epidemiologic relationships with hemagglutinin (HA) of H7N3 viruses showed 97.8%
nucleotide similarity with H7N1, and NP, M, and PB2 gene similarities were 93.6%,
98.2%, and 96.2%, respectively. Phylogenetic analyses of HA, PB2, and M genes showed
that H7N3 and H7N1 viruses were closely related (Di Trani, 2004).

H5N2 and H7N2 subtypes are low pathogenic; they did undergo mutations and acquired
an additional basic amino acid at the H cleavage site. The minimum number of basic
amino acids in correct sequence (B-X-B-R, where B = basic amino acid, X = need not be
basic amino acid, and R = arginine) required for high pathogenicity was lacking. It was
observed that a low pathogenic H5 or H7 subtype might become highly pathogenic by
acquiring additional basic amino acids at the H cleavage site (Panigrahy, 2002).

Two H9N2 viruses were isolated, for the first time, from humans in Hong Kong in 1999.
Sequence analysis of the HA genes revealed that HA of A/Hong Kong/1073/99 (H9N2)

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Chapter 2. Review of Literature

is most closely related to that of A/quail/HK/G1/97 (H9N2) that contains the internal
genes similar to those of Hong Kong/97 (H5N1) viruses. The present study suggested that
isolation of the H9 influenza viruses from humans requires precaution against the
emergence of a novel human influenza (Saito, 2001).

Outbreaks of avian influenza due to an H7N1 virus of low pathogenicity occurred in

domestic poultry in northern Italy in1999 when a highly pathogenic avian influenza
(HPAI) virus emerged. After Nucleotide sequencing for the HA1 and the stalk region of
the neuraminidase (NA), It was suggested that HPAI viruses arose from low
pathogenicity viruses and that they were most closely related to a wild bird isolate.
Additional glycosylation sites were present at amino acid position 149 of the HA for two
separate lineages, and at position 123 for all HPAI and some low pathogenicity viruses
(Banks, 2001).

Banks, (2000a) obtained a 945 nucleotide region (bases 76-1020) of the HA1 part of the
HA gene for 31 influenza viruses of H7 subtype isolated primarily from Europe, Asia and
Australia over the last 20 years. These were analyzed phylogenetically and compared
with sequences of the same region from 23 H7 subtype viruses available in Genbank. The
overall results showed two geographically distinct lineages of North American and
Eurasian viruses with major sublineages of Australian, historical European and equine

AI hemagglutinin appears to be unique in its capacity to accept basic amino acids at its
proteolytic cleavage site (PCS). We have previously noted that stable RNA secondary
structure near the PCS is related to the acquisition of virulence and have proposed that
the secondary structure may promote the insertion of basic amino acids (Perdue,,

Sequences of the hemagglutinin (HA) gene of five Korean H9N2 AIV were analysed. It
was observed that these viruses were closely related and possibly came from the same
source. Phylogenetic analysis of the HA1 subunit of H9 subtype isolates revealed that
Korean AIV isolates were different from isolates from the poultry markets in Hong Kong
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Chapter 2. Review of Literature

in 1997. None of the Korean AIVs had multiple basic amino acids at the HA cleavage
site that confer high pathogenicity to some H5 and H7 AIVs. Phylogenetic analysis of the
nucleoprotein and matrix gene demonstrated that Korean isolates cluster with Eurasian
origin AIVs (Lee, 2000).

Low-pathogenic H7 AIV in 1994 was detected. The first isolate from 1994 had the amino
acid threonine at the 2 position of the hemagglutinin cleavage site, which was the most
commonly observed amino acid at that site for North American H7 AIVs. In January
1995 a new genotype with a proline at the 2 position was detected. A third viral genotype,
detected in November 1996, had an eight-amino-acid deletion within the putative
receptor-binding site. Although isolates with proline at the 2 position without the deletion
were still observed in viruses from the last sampling date (David, 1999).

Artificial mixtures of the two hemoagglutinin variants in birds was inoculated. It was
observed that as few as 1 in 1000 particles containing the glycosylation site was sufficient
to exhibit 100% lethality in birds. It was proposed that presence of carbohydrate near the
receptor site on the H7 avian influenza virus hemagglutinin might influence virulence
(Perdue et al. 1995).

Three North American isolates were reported, which were found to contain two
electrophoretic variants occurring within populations as few as one embryo passage away
from the original clinical specimen. Pulse-chase assays indicated none of the variant HA
molecules were cleavable in chick embryo fibroblasts. In the highly pathogenic
Australian isolate, both HA variants are cleavable in fibroblasts, without added trypsin,
and the differences are localized within the HA1 region. With all the strains tested, the
slower migrating HA variant was associated with a consistently higher hemagglutinin
titer in embryos. It was observed that H7 isolates from imported birds exhibit similar
variants. It was indicating that their occurrence was not limited to domestic poultry. That
consistent presence of two distinct electrophoretic variants in several avian H7 isolates
suggested multiple allelic forms of the H7 hemagglutinin (Perdue,, 1994).

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Chapter 2. Review of Literature

Phylogenetic analysis of the N8 neuraminidase (NA) genes from 18 influenza A viruses,

representing equine and avian hosts in different geographic locations, revealed three
major lineages: (i) currently circulating equine 2 viruses; (ii) avian viruses isolated in the
Eurasian region and (iii) avian viruses isolated in North America. Comparison of
mutation rates indicated that avian N8 genes have evolved more slowly than their equine
counterparts. It was observed that in both avian lineages, 72% of the nucleotide changes
were silent in the terminal branches of the phylogenetic tree, whereas in equine 2 viruses,
59% of the nucleotide changes were silent (Saito, 1993).

Hemagglutinin of influenza virus H7 was altered at its multibasic cleavage site by site-
directed mutagenesis and assayed for proteolytic activation after expression in CV-1
cells. Studies on plaque variants of influenza virus H7N7 showed that alteration of the
consensus sequence resulted in a loss of pathogenicity for chickens (Vey,, 1992).

The probability of different and independent rates of evolution of HA and NA was

speculated, reflecting the greater selective pressure of HA antibodies, which forces the
more rapid emergence of HA escape mutants. The slower antigenic change found for NA
further supports the potential for NA-specific infection-permissive immunization as a
useful stratagem against influenza (Kilbourne,, 1990).


The sensitivity and specificity of the RRT-PCR assay were compared to those of VI
(Virus Isolation) in embryonating eggs with 1,550 clinical swab samples from New York
and New Jersey live-bird markets. Of the swab samples tested by both RRT-PCR and VI,
1,183 were negative by both assays and 202 were positive by both assays. Overall the
results of the two assays agreed on 1,385 samples (89%) and disagreed on 165 samples
(11%). Of the 165 samples on which results for the assays differed, 101 samples were
positive by VI and negative by RRT-PCR; RRT-PCR detected 202, or 66.6%, of the 303
samples that were positive by VI. Sixty-four samples were positive by RRT-PCR but
negative by VI; VI detected 75.6% of the samples, which were RRT-PCR positive
(Spackman, 2004).
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Chapter 2. Review of Literature

Universal AIV RT-PCR including a PCR-enzyme-linked immunosorbent assay (ELISA)

procedure was compared directed against nucleoprotein (NP) (NP RT-PCR-ELISA) and
the subtype specific RT-PCR for H5 and H7. Using inoculation in SPF fowl eggs as
standard the sensitivity of the NP RT-PCR-ELISA and the RT-PCR for H5 or H7 was
91% and 94%, and the corresponding specificity 98% and 96%. In comparison with
inoculation into eggs an additional of 9 samples were positive by NP RT-PCR-ELISA
and 13 samples were positive by RT-PCR for one of the HA subtypes (Dybkaer,

Direct comparison of H5-H7 multiplex reverse transcriptase-polymerase chain reaction

RRT-PCR with hemagglutination inhibition (HI) was performed with 83 AI RRT-PCR
and virus isolation positive tracheal and cloacal swab samples obtained from various
avian species and environmental swabs from live-bird markets in New York and New
Jersey. Both multiplex RRT-PCR and HI agreed on the subtype determination of 79
(95.2%) of the 83 samples, of which 77 were positive for H7 and two were determined to
be non-H5/non-H7 subtypes. No samples were determined to be the H5 subtype by either
assay (Spackman, 2003).

ELISA test, using a monoclonal antibody (MAb) specific for the H7 influenza surface
glycoproteins was developed. It was concluded that the H7 ELISA had a 99%
concordance of results with the classical hemagglutination inhibition (HI) test (Sala,

Nucleic acid sequence-based amplification (NASBA) technique has been developed that
allows the detection of avian influenza A subtype H5 from allantoic fluid harvested from
inoculated chick embryos. The amplified viral RNA is detected by
electrochemiluminescence. The NASBA technique is rapid and specific for the
identification of influenza A subtype H5 viruses of the Eurasian lineage. It can be used to
distinguish highly pathogenic and low pathogenic strains of the H5 subtype (Collins, 2002).

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Chapter 2. Review of Literature

Sensitivity and specificity of the real-time PCR assay were directly compared with those
of the current standard for detection of influenza virus: virus isolation (VI) in
embryonated chicken eggs and hemagglutinin subtyping by hemagglutination inhibition
(HI) assay. The comparison was performed with 1,550 tracheal and cloacal swabs from
various avian species and environmental swabs obtained from live-bird markets in New
York and New Jersey. Influenza virus-specific RRT-PCR results correlated with VI
results for 89% of the samples. The remaining samples were positive with only one
detection method. Overall the sensitivity and specificity of the H7- and H5-specific RRT-
PCR were similar to those of VI and HI (Spackman, 2002).

Reverse transcription (RT)-PCR heteroduplex mobility assay (HMA) was designed To

detect and partially characterize influenza A viruses from different animal species, This
M gene RT-PCR was shown to be sensitive and specific for the detection of human,
avian, and swine influenza A viruses. PCR amplicons from human, avian, and swine
viruses of 15 different subtypes, with between 1.9 and 21.4% nucleotide divergence, were
differentiated by HMA. The avian H9N2 virus, which crossed the species barrier to
humans, was screened against the reference panel. It was considered that the RT-PCR
HMA method described to offer a rapid and sensitive means for screening for novel or
unusual influenza viruses (Ellis, 2001).

Performance of the C-ELISA was evaluated by testing 756 chickens, 1123 turkeys, 707
emus, and 1261 ostriches, for a total of 3847 serum samples. Relative to the agar gel
immunodiffusion (AGID) test, the C-ELISA had a sensitivity of 100% for all four
species. The C-ELISA's sensitivity relative to the hemagglutination-inhibition (HI) test
results was 100% for chicken, turkey, and emu and 96.2% for the ostrich serum samples.
More than 90% of the AGID-negative/ C-ELISA-positive serum samples were found
positive by HI for at least one influenza serotype. It was concluded that the C-ELISA
was more sensitive and more specific than the AGID test and as sensitive and as specific
as the HI test. It was suggested that C-ELISA has the potential to replace the AGID test
for screening sera from avian species, including ratites, for detection of antibodies to type
A influenza virus (Zhou, 1998).

Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 21
Chapter 2. Review of Literature

AI AGID and CELISA tests were compared for sensitivity and specificity using 1651
experimental and reference antisera. Samples discrepant in AGID and CELISA test
results were further evaluated by the AI indirect fluorescent antibody (IFA),
hemagglutination-inhibition (HI), and neuraminidase-inhibition (NI) tests. The results
demonstrated a high degree of correlation between the AGID and CELISA test results;
with the IFA, HI, and NI tests offering additional support of CELISA test specificity. It
was concluded the CELISA is a rapid, economical, sensitive, and specific serodiagnostic
method for screening large numbers of avian sera for antibodies to avian influenza virus
Shafer, 1998).

Indirect double antibody sandwich DAS-ELISA was evaluated for its ability to detect the
AIV antigen in tracheal and cloacal specimens from turkeys inoculated with AIV. Results
of indirect DAS-ELISA were compared with those of conventional virus isolation.
Percentage agreement between indirect DAS-ELISA and virus isolation in AIV-positive
samples was found to be 76.1% and, in AIV-negative samples, it was found to be 82.1%.
It was concluded that the DAS-ELISA might be a viable alternative to virus isolation
because of its rapidity, compared with virus isolation (Kodihalli, 1993).

Sequential serological tests using AGID, HI, and ELISA were performed for measuring
antibodies to AIV up to 4 weeks postvaccination, when birds were challenged
intranasally using H4N2 live AIV. The ELISA was 25 to 1600 times more sensitive than
the HI test and was able to detect antibody production earlier than the HI test. All turkeys
with an ELISA titer of greater than or equal to 800 were protected against homologous
challenge, as measured by virus recovery 3 days postchallenge. Four turkeys out of 20
serologically negative by AGID and HI tests but ELISA-positive were protected
(Abraham, 1988).

Individual variations in serological response were demonstrated to avian influenza virus

infection after experimental infection of specific-pathogen-free chickens with H6N2
influenza virus. Homologous antibodies were detected from the 6th to the 157th day after
infection using HI or ELISA and from the 11th to the 157th day by AGID (Meulemans, 1987).
Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 22
Chapter 2. Review of Literature

An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibody

to type A avian influenza (AI) virus. The sensitivity and group specificity of the AI-
ELISA were compared with those of the agar-gel-precipitin test (AGPT) and the
hemagglutination-inhibition (HI) test under conditions of both controlled and field
exposure. It was observed that AI-ELISA was able to detect specific AI antibody as early
as 8 days post-inoculation (PI), and it measured rising levels of antibody through 35 days
PI, at which time the chickens were re-exposed to AI virus. Conversely, AGP tests were
negative through 35 days PI, and HI tests began to detect low levels of AI antibody only
at 21 days PI (Snyder, 1985).

Soluble antigen fluorescent test was studied as a tool for serological investigation of
influenza type A infection in wild birds using an artificial matrix of cellulose acetate disc
as a substrate for antigen and the test results are scanned and recorded by a fluorometer.
It was suggested the soluble antigen fluorescent antibody test was a sensitive technique
for the detection of specific influenza A antibodies in several avian species, and could be
adapted for use in large scale surveys (Al-Attar, 1981).

Inactivated AI vaccine H7N2 (CP/97) virus significantly reduced viral shedding from
vaccinated turkeys in comparison with sham controls but did not prevent infection. A
second dose of vaccine increased antiviral serum immunoglobulin G and
hemagglutination inhibition (HI) reactivity titers in tow different turkey age groups.
Serum from CP/97 vaccinated turkeys reacted equally well to CP/97 TV/02 antigens by
HI and ELISA. It was concluded that antigenically related 1997 H7N2 virus, as a
vaccine candidate for protection in poultry against a H7N2 virus will be beneficial
(Tumpey, 2004).

It was indicated that H7N2 subtype contained minimal pathogenicity for chickens. Seven-
day-old SPF chickens were not infected when inoculated with 10(0.7-2.0) mean embryo
lethal dose (ELD50) of the H7N2 virus per bird. At this dose level, the immune response

Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 23
Chapter 2. Review of Literature

to this virus was not detected by the hemagglutination-inhibition (HI) test. The H7N2
subtype of AIV induced a measurable immune response in all birds within 2 wk after
10(4.7-5.7) ELD50 doses per bird of infectious virus exposure. Antibody titers were
associated with AIV infectious doses and age of exposure of birds (Lu and Castro, 2004).

Three monoclonal antibodies (mAbs) recognizing HA of H9 subtype of AIV were

obtained, which may provide a useful tool for the antigenic analysis, the serological
diagnosis, the epidemiological survey and the evaluation of AIV vaccine (Zeng,

Protection against influenza is thought to be mainly conferred by serum antibodies,

although mucosal IgA antibodies and cell-mediated immune responses almost certainly
contribute. About one to two weeks after a primary infection neutralizing,
haemagglutination inhibiting (HAI) antibodies as well as antibodies to neuraminidase
occur in serum, peaking at approximately three to four weeks. After reinfection, the
antibody response is more rapid. Secretory IgA antibodies peak approximately 14 days
after infection and can be detected in saliva, nasal secretions, sputum and in tracheal
washings. Preceding the occurrence of antibody-producing cells, cytotoxic T
lymphocytes with specificity for influenza appear, and serve to limit the infection. Also,
mononuclear cells infiltrate infected airways providing antibody dependent cell-mediated
cytotoxicity against influenza-infected cells (WHO, 2002).

The delivery of H1N1 subtype influenza viral antigens as immunostimulating complexes

(ISCOM) induced broad cross-protection in mice against challenge with various
influenza virus subtypes, including the avian H9 and the H5 strains that were recently
responsible for deaths in humans. The ISCOM delivery system induced high and long-
lived serum antiviral antibodies and class I-restricted cytotoxic T-lymphocytes (CTL). It
was suggested that exogenous delivery of influenza antigens as ISCOM could influence
their antigen processing and presentation, their ability to induce/recall CTL specificities,
and their capacity to mediate broad cross-protection against influenza virus variants
(Sambhara, 2001).

Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 24
Chapter 2. Review of Literature

Live attenuated vaccines were administered directly to the respiratory tract providing
more effective immunity against influenza than subunit or split inactivated vaccines. It
was observed that immunological responses relevant to both the prevention of and
recovery from influenza are best induced by natural infection (Wareing and Tannock,

The use of Escherichia coli heat-labile toxin was investigated as adjuvant, complexed
with lecithin vesicles improved the immunogenicity of the trivalent inactivated influenza
virus vaccine. It was considered that such “virosomal” preparations could be given
intranasally and have been found to elicit a protective immune response, including an
influenza-specific cell-mediated immunity (Glueck, 2001).

The development of a fowlpox virus recombinant expressing the haemagglutinin

molecule from a highly virulent avian influenza virus was reported. On immunization
chickens and turkeys with the recombinant, protection was afforded against a lethal
challenge with either the homologous or a heterologous influenza virus strain. It was
indicated that fowlpox virus provided a suitable candidate for the development of a
species-specific recombinant viral vector (Taylor, 1998).

Embryonating 18-day-old white rock (WR) and white leghorn (WL) eggs was inoculated
at 1.5 inches depth with various Inactivated oil-emulsion avian influenza vaccine
volumes and antigen concentrations. Serum hemagglutination-inhibition (HI) titers were
first detected in chickens at 2 wk post hatch. Protection against morbidity and mortality
was demonstrated in all of 10 chickens vaccinated as embryos and challenged with highly
pathogenic AI virus at 34 days of age where as all of five unvaccinated control chickens
died. In pooled groups from successive hatches, the hatchability of WR or WL embryos
injected with 100 microliters of vaccine was not significantly different (P > 0.05) from
unvaccinated hatchmate controls when needle gauges of 22, 20, and 18 were used.
Seroconversion rates of chickens vaccinated as embryos ranged from 85% to 100%.
Geometric mean HI titers of chickens ranged from 11 to 733, and in pooled groups, the
range was 49 to 531. Titers for AI vaccinated groups ranged from 156 to 1178. It was

Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 25
Chapter 2. Review of Literature

demonstrated that acceptable hatchability, seroconversion rates, and protective immunity

could be attained with inovo inoculation of AI oil emulsion vaccines if the vaccines are
prepared with sufficient antigen and administered properly (Stone, 1997).

An innovative approach to influenza vaccination took advantage of the infection-

permissive nature of immunization with NA as the minor surface antigen. Immunization
of BALB/c mice with purified N1- and N2-subtype NAs demonstrated no antigenic
competition in primary or secondary response. However, when homotypic or heterotypic
infection followed immunization, cross-reactive antibodies between N1 and N2 were
found and "reverse antigen competition" occurred with initial NA priming suppressing
response to HA following infection with virus containing homologous NA. It was
concluded that these studies of antigen mixtures had implications for the use of combined
and chimeric vaccines for diseases other than influenza (Johansson and Kilbourne, 1994).

Mice were immunized with either inactivated whole virus influenza A (H3N2) virus
(WV) vaccine or with purified N2 neuraminidase (NA) vaccine, after challenge with
mouse-adapted homologous infective virus at intervals of 1-141 days later, the optimal
vaccine-infection interval for induction of resistance to subsequent infection was
observed (Johansson and Kilbourne, 1991).

Chickens were immunized against the highly virulent H5N9 virus and then challenged
with a lethal dose of the virus. Based on the ELISA results, a majority (5/6) of the
chickens produced antibodies to three of the five neutralizing epitopes on the viral HA.
After challenge, two of six immunized chickens shed virus and died. It was indicated that
chickens immunized against highly virulent influenza viruses might excrete virulent
variants following challenge with live virus (Hinshaw, 1990).

De, (1988) cloned cDNA copy of the haemagglutinin (HA) gene of H5N1 influenza
virus and expressed in vaccinia virus. Challenges of immunized and non-immunized
adult chickens with virulent H5N1 influenza virus showed that the immunized animals
were highly protected while the non-immunized controls died.

Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 26
Chapter 2. Review of Literature

Role of the neuraminidase in eliciting protection against a lethal influenza A virus

(H5N2) infection in chickens was investigated. Isolated N2 neuraminidase administered
in adjuvant did not prevent infection but did prevent systemic spread of virus and death
of chickens. It was observed that chickens vaccinated with isolated N2 in adjuvant or
with inactivated H5N2 influenza virus were protected from clinical signs and death after
challenge with virulent influenza virus. However, these animals were completely
susceptible and died of infection with a heterologous subtype (H7N7) of influenza virus
(Webster, 1988).

The role of the different antigenic determinants of the N2 NA in chickens by passive

transfer of monoclonal antibodies was also investigated in the same study. Antibodies to
antigenic determinants rimming the enzyme active center reduced disease signs in
approximately half of the birds but did not significantly reduce virus levels. It was
observed that the antibodies to one of the two independent antigenic determinants that are
distant from the enzyme active center were most effective at reducing virus replication
and disease signs (Webster, 1988).

An avian influenza (AI) oil-emulsion vaccine was formulated with one part of inactivated
H5N9 AI virus emulsified in 4 parts oil. Broilers were vaccinated subcutaneously (SC)
either at 1 or 3 days old or at 4 or 5 wks old. Commercial white leghorn (WL) layers were
vaccinated SC at 12 and 20 wks old. HI titers postvaccination (PV) were 1:86-1:320 for
broilers, 1:597 for twice-vaccinated layers, and 1:422 for once-vaccinated layers. Ninety
to 100% of vaccinated broilers were protected against death and morbidity when
challenged with highly pathogenic H5N2 AI virus 4 weeks PV, and all were protected
when challenged 8 wks PV (Stone, 1987).

The ability of old mice to mount a T-cell response to influenza virus was reported. Both
primary and secondary cytotoxic T-lymphocyte (CTL) responses following in vivo
immunization with influenza virus were minimal in old mice at the time point of peak
response in young mice (Effros and Walford, 1983).

Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 27
Chapter 2. Review of Literature

The use of vaccines to control AI is gaining acceptance by veterinary health agencies as a
tool in eradication programmes. The choice of vaccines available includes the traditional
whole-virus inactivated vaccines, purified subunit vaccines and genetically modified
vaccines. The use of inactivated vaccines has been used successfully in many countries to
stop the spread of avian influenza in the poultry industry. The fowlpox-vectored vaccine
TROVAC AI H5™ has been used to vaccinate broiler chickens in Mexico for five years.
For more than 30 years inactivated whole-virus avian influenza vaccines have been the
only product available to control the spread of the disease from infected to susceptible
birds (European Commission, 2000).

It was investigated that inactivated AI vaccine, prepared from

A/Chicken/Pennslyvania/21342/97 (CP/97) virus, significantly reduced viral shedding
from vaccinated turkeys in comparison to sham controls, but did not prevent infection.
The protective effect of vaccination correlated with the level of virus-specific antibody as
a second dose of vaccine increased antiviral serum IgG and hemagglutination inhibition
(HI) reactivity titers in two different turkey age groups (Tumpey,, 2004).

Vaccines have been used to manage economic losses from LPAI or, in some instances,
have been used as a tool in LPAI or HPAI eradication strategies. AI vaccines can prevent
clinical signs and death in poultry, increase resistance of birds to infection, and decrease
the amount of virus shed in the environment (Swayne, 2004).

The HA gene from A/Turkey/Ireland/83 strain was inserted into a non-essential location
in the poxvirus genome resulting in a virus that protect poultry from two infectious
diseases, fowlpox and AI. The vaccine provides excellent protection against a wide range
of highly pathogenic subtype-H5 viruses (Swayne et al. 2000).

Inactivated whole avian influenza (AI) virus vaccines, baculovirus-derived AI

haemagglutinin vaccine and recombinant fowl poxvirus-AI heamagglutinin vaccine were
tested for the ability to protect chickens against multiple highly pathogenic (HP) H5 AI

Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 28
Chapter 2. Review of Literature

viruses. The vaccines protected against clinical signs and death, and reduced the number
of chickens shedding virus and the titer of the virus shed following a HP H5 AI virus
challenge (Swayne,, 2000).

Controlling of AI infections of poultry with H5 and H7 subtype viruses by homologous

and recombinant vaccines without monitoring field exposure and the application of
stringent control measures on field exposed farms (regardless of the state of vaccination)
have been unsuccessful in eradicating the infections in Mexico (H5N2) and Pakistan
(H7N3) (European Commission, 2000).

Broiler breeders (BB) and white leghorn (WL) pullets were vaccinated with a control
fowl poxvirus vaccine (FP-C) and/or a recombinant fowl poxvirus vaccine containing an
H5 hemagglutinin gene insert (FP-HA) were challenged with a HP H5N2 AIV isolated
from chickens in Mexico. When used alone, the FP-HA vaccine protected BB and WL
chickens from lethal challenge, but when given as a secondary vaccine after a primary
FP-C immunization, protection against a HP AIV challenge was inconsistent. Both
vaccines protected against virulent fowl pox challenge (Swayne, 2000).

A universal vaccine generation against influenza A virus was attempted by fusing the
extracellular domain of the conserved M2 protein of an influenza A virus to the hepatitis
B virus core protein. It was found that intraperitoneal or intranasal administration in mice
provided a high degree of protection against viral challenge, but it remains to be seen
whether an immune response against a minor surface antigen like the M2 protein will be
sufficient to provide full protection against influenza in the human population at large
(Neirynck,, 1999).
The possibility to construct a “generic” HA was reported that could be used as an
immunogen in vaccines, based on a framework of conserved amino acids (Bush,,

The influenza virus vaccines formulated in either adjuvant were far superior to the non-
adjuvanted aqueous vaccine in eliciting antibody and T-cell responses in mice,
particularly at lower doses of antigen. In addition, the vaccines containing adjuvant were
superior in eliciting protective immunity (Deliyannis, 1998).
Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 29
Chapter 2. Review of Literature

Six commercial inactivated water in oil H5N2 vaccines were compared with standardized
vaccines to assess their efficacy. Each of the six commercially available vaccines
prevented disease signs, and half of the vaccines significantly reduced viral shedding
from vaccinated birds. There is a need for standardization of AI virus vaccine, and the
antigen content should be increased in some of the commercially available AI vaccines in
Mexico (Garcia,, s1998).

Delivery of a single injection of DNA encoding the influenza HA provided immunity for
the life of the chicken. The method for DNA injection depends on the use of the gold
beads to carry the antigen into the tissue of the animal (Kodihalli and Webster, 1997).

Susceptible chickens were vaccinated with the parent fowlpox vaccine virus or
recombinant viruses and challenged 4 weeks later with highly pathogenic avian influenza
virus, all birds vaccinated by the wing-web method were protected by both recombinants,
while 50% and 70% mortality occurred in the two groups of birds vaccinated by comb
scarification. Birds vaccinated with the unaltered parent fowlpox vaccine virus or
unvaccinated controls experienced 90% and 100% mortality, respectively, following
challenge 9 Beard,, 1991).

The cold-adapted master strains were temperature-sensitive and were well suited for use
as live vaccines because their pathogenicity, in animals as well as humans, was strongly
attenuated. It was suggested that the annually updated vaccine formulations could be
generated by making “6:2 reassortants” in which the two genes encoding major viral
surface antigens (HA and NA) reflect the sequence found in the current strains, whereas
the remaining six genes derive from the cold-adapted master strains 9 Maassab,

Chickens and turkeys were vaccinated with inactivated virus oil-emulsion vaccines
containing different concentrations of either 1 (monovalent) or 4 (polyvalent) strains of
avian influenza virus (AIV) were challenged-exposed with virulent AIV. Four of 6
vaccines protected completely against postexposure mortality. It was indicated that
Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 30
Chapter 2. Review of Literature

control of virulent AIV in chickens and turkeys by vaccination with inactivated vaccines
might be feasible (Brugh,, 1979).

The hemagglutinin concentration of beta-propiolactone-inactivated influenza vaccine

containing H5N2 virus was measured by single-radial-immunodiffusion (SRD) test. After
administration of vaccine to chickens in Freund's complete adjuvant, vaccine efficacy
was assessed by challenge with lethal H5N2 virus. SRD potency values correlated with
post-vaccination antibody levels and protection against infection (Wood, 1985).

Vaccination of chickens was evaluated with a commercially available killed H5N2
vaccine as an additional tool to enhanced biosecurity measures and intensive surveillance
for control of highly pathogenic avian influenza subtype H5N1 disease in Hong Kong in
2002 (Ellis, 2004).

It was further reported that to quarantine, depopulation of the affected sheds and
increased biosecurity, vaccination of the unaffected sheds and surrounding unvaccinated
farms was undertaken on three farms. In at least two farms, infection spread to the
recently vaccinated sheds with low rates of H5N1 mortality in sheds when the chickens
were between 9 and 18 days post-vaccination. However, after 18 days post-vaccination
no more deaths from H5N1 avian influenza occurred and intensive monitoring by virus

culture on these farms showed no evidence of asymptomatic shedding of the virus. It was
concluded that H5 vaccine could interrupt virus transmission in a field setting (Ellis,

To control the LPAI virus and to develop a novel control strategy, a "DIVA"
(Differentiating Infected from Vaccinated Animals) strategy was developed. The "DIVA"
strategy was based on the use of an inactivated oil emulsion vaccine containing the same
haemagglutinin (H) sub-type as the field virus, but a different neuraminidase (N). It was
suggested that the combination of a "DIVA" control strategy with a territorial monitoring
Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 31
Chapter 2. Review of Literature

system under official control could represent an effective tool for the control of avian
influenza infections in poultry (Capua and Marangon.2003).

Inactivated whole avian influenza virus (AIV) vaccine provided protection against
homologous haemagglutinin (HA) subtype virus, but poor protection against a
heterologous HA virus. Moreover, it induced chickens to produce antibodies to cross-
reactive antigens, especially nucleoprotein, which was limits AIV serological
surveillance. A recombinant fowl pox virus co-expressing HA (H5 subtype) and NA (NI
subtype) genes of AIV was evaluated for its ability to protect chickens against
intramuscular challenge with a lethal dose of HP AIV. Susceptible chickens were also
vaccinated by wing-web puncture with the parent fowlpox vaccine virus. Following
challenge 4 weeks later with HPAIV, all chickens vaccinated with recombinant virus
were protected, while the chickens vaccinated with either the unaltered parent fowlpox
vaccine virus or unvaccinated controls experienced 100% mortality following challenge
(Qiao, 2003).

A heterologous H7N3 vaccine induced clinical protection levels of 93% in each of the
vaccination schemes used, and prevented viraemia in SPF birds when challenged with the
HPAI H7N1. It was observed that the number of bird shedding infectious virus was
reduced in the vaccinated bird groups when compared to the vaccinated controls. Boyle, (2000) vaccinated chickens at 7 days of age with fowlpox H7 avian influenza virus
recombinants and challenged 21 days later. The vaccine induced protective immune
responses. Few clinical signs of infection developed. In contrast, unvaccinated or
chickens vaccinated with a non-recombinant fowlpox or a fowlpox expressing the H1
haemagglutinin of human influenza were highly susceptible to avian influenza (Cupua, 2002).

It was reported that traditionally, inactivated oil emulsion vaccines have been used
worldwide to control low pathogenic avian influenza virus (LPAI) infections in poultry
and more recently, HPAI in Mexico and Pakistan (Swayne and Suarez, 2000; Garcia and
Alvrez, 1999; Halvorson, 1995; Pomeroy, 1995 and Naeem, 1998).

Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 32
Chapter 2. Review of Literature

Groups of 4-week-old chickens were immunized with inactivated vaccines containing

one of 10 haemagglutinin subtype H5 AI viruses, one heterologous H7 AI virus or
normal allantoic fluid (sham). All 10 H5 vaccines provided good protection from clinical
signs and death, and produced positive serological reactions on agar gel immunodiffusion
and haemagglutination inhibition tests. As the quantity of AI antigen in the vaccines
increased, all parameters of protection improved (Swayne,, 1999).

proteins were expressed as either intact (H7) or slightly truncated versions (H5). these
proteins were more effective when administered subcutaneously in a water-in-oil
emulsion. Vaccination of three-week-old chickens with 1.0 g of protein per bird
generated a more consistent serum antibody response with an average geometric mean
titer (GMT) of 121 (H5) and 293 (H7) at 21 days postvaccination (Crawford,,

One-day-old chickens were immunized with a recombinant fowl poxvirus vaccine

containing a hemagglutinin gene insert (Vector-HA) from an H5 AIV. Vector-HA and
negative control (vector-control) vaccinated chicks were challenged with a HP H5N2
AIV isolated from chickens. All immunized chickens were antibody negative on the agar
gel precipitin test. However, in the hemagglutinin-inhibition test, a few immunized
chickens (8%) had low serologic titers. Protection against illness (90-100%) and death
(90-100%) was provided by the vector-HA vaccine from 3 wk of age to the end of the 20-
wk study (Swayne,, 1997).

The versatility of liposomes was investigated in the incorporation of
hydrophilic/hydrophobic components, their non-toxic nature, biodegradability,
biocompatibility, adjuvanticity, induction of cellular immunity, property of sustained
release and prompt uptake by macrophages, makes them attractive candidates for the
delivery of antigens (Verma, 2004).

Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 33
Chapter 2. Review of Literature

Three monovalent aluminum-adjuvanted whole virus formulations were compared with

different antigen concentrations (1.9, 3.75 and 7.5 microg HA per dose) to a split virus
vaccine (15 microg HA per dose) without aluminum adjuvantation. The sera were tested
for hemagglutination inhibition (HI) antibodies, neuraminidase inhibition (NI) antibodies
and virus neutralizing (VN) antibodies. HI titers reached protective levels (geometric
mean titer (GMT) >1:40) after two vaccine doses. In the group immunized with the
lowest antigen dose the seroprotection rate was 82%. Significant increases in HI, NI and
VN titers were observed, however, no significant local immune response was detected
(Hehme, 2004).

The development of novel and increasingly safer vaccines frequently utilizes well-
characterized antigens, in particular highly purified proteins or synthetic peptides. In spite
of some achievements, this approach is frequently impeded by the fact that such antigens
are often poor immunogens when administered alone. This fact has necessitated the
development of suitable adjuvants that possess the ability to enhance the immunogenicity
of a given antigen, preferably with little or no side effects (Moser, 2003).

Vaccination with influenza virosomes elicits high titers of influenza-specific antibodies,

indicating that HA and NA reconstituted into a membranous environment exhibit strong
immunogenicity. Moreover, virosomes can be used as presentation systems for unrelated
antigens bound to the virosome surface. Because of their intrinsic adjuvant activity,
virosomes support antibody formation and induction of T-helper cell responses against
such surface-associated antigens. Virosomes can also be used to elicit cytotoxic T-cell
responses against encapsulated antigens (Huckriede, et. Al., 2003).

Evaluation of liposomes as vehicle for oral vaccines and characterization, the stability of
polymerized and non-polymerized liposomes was examined. It was suggested that
polymerized and non-polymerized liposomes would serve effectively as an oral delivery
vehicle Alonso-Romanowski, 2003).

It was studied in response that naked DNA vaccines can be degraded by nucleases in situ,
are unable to target antigen-presenting cells (APCs), and exhibit poor performance when
Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 34
Chapter 2. Review of Literature

administered by routes other than the intramuscular. It was shown through animal
experiments that immunization by the intramuscular or the subcutaneous route with
liposome-entrapped plasmid DNA encoding the hepatitis B surface antigen leads to much
greater humoral (IgG subclasses) and cell mediated (splenic IFN-gamma) immune
responses than with naked DNA (Gregoriadis, 2002).

Liposomes are lipid vesicles. The external liposome membrane is composed of the same
lipids as the cell membranes. This is very important, since the fact that the molecules
used are not foreign to the host prevents the induction of immune rejection, and the same
liposome formulation can be used for repeated vaccinations. Iscom are solid particles
generated by combining an antigen with a biocompatible detergent and the adjuvant Quil-
A, thus giving rise to minute structures (35 nm, Fig. 3). These particles can only be used
with antigens that can be mixed with lipids and with Quil-A (normally proteins) (FAO,

Two doses of MF59-adjuvanted and non-adjuvanted influenza H5N3 surface-antigen

vaccine were compared. It was found that non-adjuvanted vaccine was poorly
immunogenic. It was observed that MF59 significantly increased geometric mean titres
(GMTs) of antibody when compared to non-adjuvanted vaccine (P<0.001) (Stephenson, 2002).

Liposome- vesicles have been introduced as drug delivery vehicles due to their structural
flexibility in size, composition and bilayer fluidity as well as their ability to incorporate a
large variety of both hydrophilic and hydrophobic compounds. With time the liposome
formulations have been perfected so as to serve certain purposes and this lead to the
design of "intelligent" liposomes, which can stand specifically induced modifications of
the bilayers or can be surfaced with different ligands that guide them to the specific target
sites (Voinea and Simionescu, 2002).

Bangham et al. (1965) created first the concept of the liposome as a microparticulate
lipoidal vesicle separated from its aqueous environment by one or more lipid bilayers.

Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 35
Chapter 2. Review of Literature

Later Gregoriadis and Ryman (1972) suggested using liposomes as drug carrier systems
(Budai M and Szogyi M, 2001).

Liposomes offer an excellent opportunity to selective targeting of drugs which is

expected to optimize the pharmacokinetical parameters, the pharmacological effect and to
reduce the toxicity of the encapsulated drugs (Budai and Szogyi, 2001).

Non-adjuvanted A/Duck/Singapore/97 (H5N3) vaccines were poorly immunogenic and

doses of 7.5-30 microg haemagglutinin alone were unlikely to give protection from
A/Hong Kong/97 (H5N1) virus. Addition of MF59 to A/Duck/Singapore/97 vaccines
boosted the antibody response to protection levels (Nicholson, 2001).

Several adjuvants were evaluated in order to enhance the immune response to influenza
vaccines. Among these, oil in water adjuvant emulsion containing squalene, MF59, has
been combined with subunit influenza antigens and tested in clinical trials in comparison
with non-adjuvanted conventional vaccines. Immunogenicity analyses demonstrated a
consistently higher immune response with statistically significant increases of
postimmunisation geometric mean titres, and of seroconversion and seroprotection rates
compared to nosn-adjuvanted subunit and split influenza vaccines (Podda, 2001).

Inactivated vaccine with the copolymer adjuvant was administered in mice infected with
influenza virus or naive aged mice. It was observed that the serum HI antibody response
substantially enhanced to inactivated influenza vaccine and significantly reduced lung
virus titers following subsequent challenge with live virus compared with mice
administered vaccine alone (Katz, 2000).

MF59 adjuvanted influenza vaccine was compared with a conventional non-adjuvanted

influenza vaccine in elderly ambulatory patients. The adjuvanted vaccine induced more
local reactions than the conventional vaccine; however, the reactions were normally mild
and limited to the first 2-3 days after immunization. No statistically significant difference
between groups in systemic postimmunization reactions was reported except for a mild,
transient malaise after the first immunization. Compared with the first immunization, no

Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 36
Chapter 2. Review of Literature

increase in postimmunization reactions was seen after the second and third
immunizations (Minutello, 1999).

Influenza virus vaccines formulated in either adjuvant were far superior to the non-
adjuvanted aqueous vaccine in eliciting antibody and T-cell responses in mice,
particularly at lower doses of antigen. In addition, the vaccines containing adjuvant were
superior in eliciting protective immunity. After one dose of either adjuvanted vaccine,
strong proliferative responses were achieved (Deliyannis, 1998).

Detergent-disrupted influenza virus vaccines, formulated as Iscoms, or oil-in-water (o/w)

emulsions was administered parenterally to mice and evaluated for immunogenicity and
protective efficacy. Both formulations enhanced both primary and secondary serum
antibody responses. The magnitude of these responses with o/w emulsions was further
enhanced by the addition of the non-ionic block copolymer L121 in the emulsion
(Coulter, 1998).

The addition of 100 micrograms PCPP enhanced the HAI antibody response 10-fold over
the levels elicited by the vaccine alone.. Immunization of aged mice (22 months old) with
trivalent influenza vaccine alone did not sero-convert these mice as measured by HAI or
ELISA whereas significant sero-conversion was achieved when mice were immunized
with PCPP-formulated trivalent vaccine (Payne, 1998).

Syntex adjuvant in its micro fluidized form (SAF-m) was equal to or superior to Freund's
complete adjuvant in stimulating an enhanced hemagglutination inhibition (HI) antibody
response in mice to trivalent influenza virus vaccine (TIV). There was an average 16-fold
increase in HI titer for the three components of the vaccine with no significant differences
among strains (Hjorth, 1997).

The mice injected subcutaneously three times at 14-day intervals with Dehydration-
rehydration liposome vesicles DRVs containing 15 micrograms of rgp120 plus
interleukin 6 (IL-6) or interferon gamma (IFN-gamma) produced significantly greater
DTH responses than mice injected with DRVs containing rgp120 alone. It was concluded

Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 37
Chapter 2. Review of Literature

that cytokine-containing liposomes might be an effective adjuvant for the induction of a

DTH response to envelope-antigen subunit vaccines (Lachman, 1995).

The sterically stabilized liposomes (SSL-IL-2) activity was assessed in vitro and in mice
in comparison with soluble IL-2, IL-2 in conventional liposomes (non-SSL-IL-2), and
pegilated IL-2 (PEG-IL-2). The main observations were as follows: (a) SSLs were far
better carriers than conventional liposomes with regard to encapsulation efficiency and
pharmacokinetics; (b) > 85% of IL-2 biological activity was consistently encapsulated in
SSLs (Kedar, 1994).

Acceptable oil-emulsion vaccines were sought to replace mineral oil-emulsion vaccines.

Tissue reaction from injection of animal-oil and vegetable oil vaccines was less than that
induced by mineral-oil vaccines. It was suggested that an inactivated avian influenza
vaccine formulated from peanut oil induced protection against morbidity and death when
vaccinated chickens were challenged with a virulent isolate of avian influenza virus
(Stone, 1993).

Trivalent avian influenza (AIV) antigens (H4N8, H5N2 and H7N3), mixed with
positively charged, negatively charged and neutral avridine-containing liposomes, and
oil-emulsion were administered subcutaneously to 6-week-old turkeys. Charged
liposomal avridine adjuvant, either positive or negative, produced a better antibody
response than uncharged liposomal avridine or oil-emulsion adjuvants when used in a
trivalent avian influenza vaccine. It was concluded that the antibody response to the
different antigens was significantly greater to the positively charged adjuvanted vaccine

compared with the negatively or neutral charged or oil-emulsion adjuvanted vaccines. It

was suggested that the positively charged liposomal avridine plays a significant role as
adjuvant to the AIV antigens (Fatunmbi, 1992).

It was studied that low dose formalin-inactivated influenza A virus vaccine was
protective in young mice, but not in aged mice, while a higher dose was protective in
both. Administration of low dose vaccine with IL-2 liposomes conferred protection
comparable to the high dose in aged mice (Mbawuike, 1990).
Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 38
Chapter 2. Review of Literature

Inactivated Newcastle disease virus (NDV), avian influenza virus (AIV), and infectious
bronchitis virus (IBV) antigens were evaluated for immunological efficacy in monovalent
and polyvalent vaccines. Avian influenza HI titers were significantly (P less than 0.05)
lower than those of the control monovalent groups when highly concentrated NDV was
part of the polyvalent vaccine (Xie and Stone, 1990).

Avian influenza (AI) oil-emulsion vaccine was formulated with one part inactivated
H5N9 AI virus emulsified in 4 parts oil. Maximum geometric mean hemagglutination-
inhibition titers postvaccination (PV) were 1:86 to1: 320 for broilers, 1:597 for twice-
vaccinated layers, and 1:422 for once-vaccinated layers. Ninety to 100% of vaccinated
broilers were protected against death and morbidity when challenged with highly
pathogenic H5N2 AI virus 4 weeks PV, and all were protected when challenged 8 wks
PV (Stone, 1987).

Comparative immunological studies on commercial oil based and liposomal vaccines of avian influenza H7 39