Factors affecting infection and pathogenesis of avian influenza H5N1 virus
Rabeh Abd El-Haq Mohamed El-Shesheny;
Abstract
Influenza, commonly known as flu, is an infectious disease caused by a group of
influenza viruses which have been the causative agents responsible for epidemics and
pandemics around the world. Influenza viruses belong to the family of Orthomyxoviridae.
In 1996, H5N1 HPAIV was first identified as the causative agent of geese outbreak in
Guangdong Province, China. Since 2003, HPAI H5N1 viruses spread to other countries and
gradually developed into a global issue. Clade 2.2 H5N1 viruses emerged in Egypt in 2006
(Saad et al., 2007). In 2008, H5N1 was declared to be endemic in Egypt and the viruses in
this country were re-classified as clade 2.2.1 (WHO/OIE/FAO, 2009). The endimicity of
H5N1 viruses in Egypt was due to several factors related to vaccines, vaccination strategy,
lax biosecurity measures, and presence of the virus in different poultry sectors
The H5N1 situation in Egypt is alarming. Egypt is among the countries with the
highest number of human infections and fatalities due to zoonotic H5N1 infections (203
cases, 72 deaths, 35.5% (CFR)) (WHO, 2015), also Indonesia has more laboratory-confirmed
human infections reported to WHO until 2013 (194 cases, 162 deaths, 83.5% CFR) (WHO,
2013), globally, From 2003 through January 2015, 718 laboratory-confirmed human cases of
avian influenza A(H5N1) virus infection have been officially reported to WHO from 16
countries. Of these cases, 413 have died (WHO, 2015).
Results in this study were summarized as follows:
1- Characterization of the antigenic variation of 33 isolates H5N1viruses (2006-2011)
has been examined. The HI activity of a panel of anti-H5 MAbs prepared against
different antigenic epitopes of A/Viet Nam/1203/04 and A/bar-headed
goose/QH/1A/05 (H5N1), against our isolates was mentioned. The cartography of the
antigenic map showed that the H5N1 viruses fell into 3 clusters, A, B and C.
2- To analysis cross-reactivity of 33 isolates H5N1viruses (2006-2011), we prepared rat
hyperimmune sera raised against 8 Egyptian H5N1 viruses, assessed to be
antigenically different with the MAbs panel, to evaluate antigenic reactivity.
Cartography of the HI assay results showed extensive overlap, indicating that all
viruses isolates ware shared epitopes.
Summary
- 111 -
3- Full genome sequence and phylogenetic analyses of 45 H5N1 isolated during 2006–
2013 through systematic surveillance in Egypt, and 53 viruses that were previously
sequenced and available in the public domain. Results indicated that H5N1 viruses in
Egypt were continued to evolve and a new distinct cluster has been emerged.
4- Analyze the distribution of amino acid changes in viral proteomes. The cleavage site
motif PQGERRRKKR↓G (↓denotes cleavage site) was found in most sub-clade 2.2.1
isolates (12 of 13 viruses 92.3%), while 1 isolate had a slight variation
PQGEKRRKKR↓G. Isolates from sub-clade 2.2.1.1 had 2 different motifs of cleavage
site, PQGERRRKKR↓G motif (22 of 45 viruses 48.9%) and PQGEGRRKKR↓G with
a glycine substitution (23 of 45 viruses 51.1%). On the other hand, the new cluster
strains had three different multibasic cleavage sites, the most predominant motif was
PQGEKRRKKR↓G (similar to the single isolate from sub-clade 2.2.1) (35 of 40
viruses 87.5%), and PQGGKRRKKR↓G (1 strain isolated from a human case). The
motif PQGERRRKKR↓G, which was dominant in clade 2.2.1 strains, was found in
the new cluster (4 of 40 viruses 10%).
5- Egyptian strains possessed 3 N-linked glycosylation sites at amino acid positions 11,
23, and 286 (H5 numbering). Viruses from sub-clade 2.2.1 and the new cluster had an
additional glycosylation site at position 165 (NNT), whereas sub-clade 2.2.1.1 viruses
were characterized by an additional glycosylation site at position 72 (NVS). In this
sub-clade, viruses had a glycosylation site at position 154 (NNT) (29 of 45 viruses
64.4%), while other viruses had NDT at the same position (7 of 45 viruses 15.5%).
6- Analysis of the NA gene of Egyptian H5N1 viruses revealed that there were a 20-
amino acid deletion in the NA stalk region (positions 49-68). None of these viruses
displayed oseltamivir-resistant markers at positions E119, H275, R293, and N295 (N1
numbering) except for 1 isolate which had a mutation at position N295S.
7- Characterization of the new cluster based on amino acid identity and genetic
distances. Virus isolates of the new cluster carried several mutations that were novel
or rare in the previously circulating sub-clades. To classify a new clade or sub-clade,
the average between-groups divergence should be ≥1.5% while within-group
divergences should be ≤1.5% by pairwise analysis. Our results showed that the new
cluster have strains from late 2010 till 2013 with high divergence (3, 2.1, and 4.2%)
Summary
- 112 -
when compared to sub-clades 2.2, 2.2.1, and 2.2.1.1 respectively. Viruses within the
new cluster had 0.9% divergence.
8- Diversity and selection pressure of Egyptian H5N1 viruses, the H5N1viral genes were
under purifying selective pressure ranging from ω= 0.064 (for the PB2 gene) to 0.890
(for the M2 gene) using both the SLAC and FEL methods. Only the PB1-F2 had
positive selective pressure (ω=8.476, SLAC method). The diversity of nucleotides of
each gene segment was calculated on the basis of Kimura distances ranging from
1.5% (M1) to 2.2% (PB2). Amino acid divergence in each gene segment ranged from
0.8% (PB1 and M1) to 3.5% (HA)
9- The reverse genetics technology was applied to determine the gene that have vital role
in pathogenicity of influenza H5N1 virus infection. Gene segments can be exchanged
between influenza viruses and hence their impact on viral properties was also
determined. The reassortment of the NP, NA, and NS genes showed the biggest
differences in replication in embryonated chicken eggs for
A/chicken/Egypt/Q1995D/2010 and A/chicken/EgyptM2583A/2010 viruses.
10- To determine the pathogenicity of H5N1 avian influenza viruses that have mutation in
the C-terminal of NS1 protein. We generated viruses with an ESKV motif and a
mutant virus with an NS1 protein containing a C-terminal ESEV motif by reverse
genetics in backbone seven genes from A/Puerto Rico/8/1934 (PR8). The growth
kinetics of the two generated viruses was determined in both cell culture A549 and
MDCK cell lines. Our result of site direct mutagenesis showed that the mutant virus
did not have a significantly increased ability to grow in MDCK cells compared to
PR8-NS2006 at 12 and 24 hpi. However, at the 48 hpi time point PR8-NS2006 virus
grew higher than PR8-NS2006M. In contrast, growth of the PR8-NS2006 in A549
cells was higher than PR8-NS2006M at 12, 24, and 48 hpi.
11- Based on the results obtained in this study, the next step is to investigate the role of
individual amino acids in each of the genes shown to affect H5N1 pathogenicity, as
well as to continue exploring the effect of certain gene combinations. Also in light of
our findings that H5N1 in Egypt continues to evolve, surveillance and molecular
studies need to be sustained.
REFERENCE
influenza viruses which have been the causative agents responsible for epidemics and
pandemics around the world. Influenza viruses belong to the family of Orthomyxoviridae.
In 1996, H5N1 HPAIV was first identified as the causative agent of geese outbreak in
Guangdong Province, China. Since 2003, HPAI H5N1 viruses spread to other countries and
gradually developed into a global issue. Clade 2.2 H5N1 viruses emerged in Egypt in 2006
(Saad et al., 2007). In 2008, H5N1 was declared to be endemic in Egypt and the viruses in
this country were re-classified as clade 2.2.1 (WHO/OIE/FAO, 2009). The endimicity of
H5N1 viruses in Egypt was due to several factors related to vaccines, vaccination strategy,
lax biosecurity measures, and presence of the virus in different poultry sectors
The H5N1 situation in Egypt is alarming. Egypt is among the countries with the
highest number of human infections and fatalities due to zoonotic H5N1 infections (203
cases, 72 deaths, 35.5% (CFR)) (WHO, 2015), also Indonesia has more laboratory-confirmed
human infections reported to WHO until 2013 (194 cases, 162 deaths, 83.5% CFR) (WHO,
2013), globally, From 2003 through January 2015, 718 laboratory-confirmed human cases of
avian influenza A(H5N1) virus infection have been officially reported to WHO from 16
countries. Of these cases, 413 have died (WHO, 2015).
Results in this study were summarized as follows:
1- Characterization of the antigenic variation of 33 isolates H5N1viruses (2006-2011)
has been examined. The HI activity of a panel of anti-H5 MAbs prepared against
different antigenic epitopes of A/Viet Nam/1203/04 and A/bar-headed
goose/QH/1A/05 (H5N1), against our isolates was mentioned. The cartography of the
antigenic map showed that the H5N1 viruses fell into 3 clusters, A, B and C.
2- To analysis cross-reactivity of 33 isolates H5N1viruses (2006-2011), we prepared rat
hyperimmune sera raised against 8 Egyptian H5N1 viruses, assessed to be
antigenically different with the MAbs panel, to evaluate antigenic reactivity.
Cartography of the HI assay results showed extensive overlap, indicating that all
viruses isolates ware shared epitopes.
Summary
- 111 -
3- Full genome sequence and phylogenetic analyses of 45 H5N1 isolated during 2006–
2013 through systematic surveillance in Egypt, and 53 viruses that were previously
sequenced and available in the public domain. Results indicated that H5N1 viruses in
Egypt were continued to evolve and a new distinct cluster has been emerged.
4- Analyze the distribution of amino acid changes in viral proteomes. The cleavage site
motif PQGERRRKKR↓G (↓denotes cleavage site) was found in most sub-clade 2.2.1
isolates (12 of 13 viruses 92.3%), while 1 isolate had a slight variation
PQGEKRRKKR↓G. Isolates from sub-clade 2.2.1.1 had 2 different motifs of cleavage
site, PQGERRRKKR↓G motif (22 of 45 viruses 48.9%) and PQGEGRRKKR↓G with
a glycine substitution (23 of 45 viruses 51.1%). On the other hand, the new cluster
strains had three different multibasic cleavage sites, the most predominant motif was
PQGEKRRKKR↓G (similar to the single isolate from sub-clade 2.2.1) (35 of 40
viruses 87.5%), and PQGGKRRKKR↓G (1 strain isolated from a human case). The
motif PQGERRRKKR↓G, which was dominant in clade 2.2.1 strains, was found in
the new cluster (4 of 40 viruses 10%).
5- Egyptian strains possessed 3 N-linked glycosylation sites at amino acid positions 11,
23, and 286 (H5 numbering). Viruses from sub-clade 2.2.1 and the new cluster had an
additional glycosylation site at position 165 (NNT), whereas sub-clade 2.2.1.1 viruses
were characterized by an additional glycosylation site at position 72 (NVS). In this
sub-clade, viruses had a glycosylation site at position 154 (NNT) (29 of 45 viruses
64.4%), while other viruses had NDT at the same position (7 of 45 viruses 15.5%).
6- Analysis of the NA gene of Egyptian H5N1 viruses revealed that there were a 20-
amino acid deletion in the NA stalk region (positions 49-68). None of these viruses
displayed oseltamivir-resistant markers at positions E119, H275, R293, and N295 (N1
numbering) except for 1 isolate which had a mutation at position N295S.
7- Characterization of the new cluster based on amino acid identity and genetic
distances. Virus isolates of the new cluster carried several mutations that were novel
or rare in the previously circulating sub-clades. To classify a new clade or sub-clade,
the average between-groups divergence should be ≥1.5% while within-group
divergences should be ≤1.5% by pairwise analysis. Our results showed that the new
cluster have strains from late 2010 till 2013 with high divergence (3, 2.1, and 4.2%)
Summary
- 112 -
when compared to sub-clades 2.2, 2.2.1, and 2.2.1.1 respectively. Viruses within the
new cluster had 0.9% divergence.
8- Diversity and selection pressure of Egyptian H5N1 viruses, the H5N1viral genes were
under purifying selective pressure ranging from ω= 0.064 (for the PB2 gene) to 0.890
(for the M2 gene) using both the SLAC and FEL methods. Only the PB1-F2 had
positive selective pressure (ω=8.476, SLAC method). The diversity of nucleotides of
each gene segment was calculated on the basis of Kimura distances ranging from
1.5% (M1) to 2.2% (PB2). Amino acid divergence in each gene segment ranged from
0.8% (PB1 and M1) to 3.5% (HA)
9- The reverse genetics technology was applied to determine the gene that have vital role
in pathogenicity of influenza H5N1 virus infection. Gene segments can be exchanged
between influenza viruses and hence their impact on viral properties was also
determined. The reassortment of the NP, NA, and NS genes showed the biggest
differences in replication in embryonated chicken eggs for
A/chicken/Egypt/Q1995D/2010 and A/chicken/EgyptM2583A/2010 viruses.
10- To determine the pathogenicity of H5N1 avian influenza viruses that have mutation in
the C-terminal of NS1 protein. We generated viruses with an ESKV motif and a
mutant virus with an NS1 protein containing a C-terminal ESEV motif by reverse
genetics in backbone seven genes from A/Puerto Rico/8/1934 (PR8). The growth
kinetics of the two generated viruses was determined in both cell culture A549 and
MDCK cell lines. Our result of site direct mutagenesis showed that the mutant virus
did not have a significantly increased ability to grow in MDCK cells compared to
PR8-NS2006 at 12 and 24 hpi. However, at the 48 hpi time point PR8-NS2006 virus
grew higher than PR8-NS2006M. In contrast, growth of the PR8-NS2006 in A549
cells was higher than PR8-NS2006M at 12, 24, and 48 hpi.
11- Based on the results obtained in this study, the next step is to investigate the role of
individual amino acids in each of the genes shown to affect H5N1 pathogenicity, as
well as to continue exploring the effect of certain gene combinations. Also in light of
our findings that H5N1 in Egypt continues to evolve, surveillance and molecular
studies need to be sustained.
REFERENCE
Other data
| Title | Factors affecting infection and pathogenesis of avian influenza H5N1 virus | Authors | Rabeh Abd El-Haq Mohamed El-Shesheny | Issue Date | 2015 |
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