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Molecular characterization for non structural proteins of Avian Influenza Virus

Master's Thesis 2015 149 Pages

Biology - Micro- and Molecular Biology

Excerpt

CONTENTS

1. Introduction

2. Review of literature

3. Material and methods

4. Results

5. Discussion

6. Summary and Conclusion

7. References

Arabic summary

List of Tables

1 Clinical data of chicken and duck flocks suspected to be affected with AIV. 40

2 Sequences of the oligonucleotide primers and probe used in the study. 46

3 Results of viral isolation, HA, RT-PCR, and IHC of HPAIV H5N1 from infected chicken and duck flocks.

4 Distribution of viral antigen NP in IHC stained tissues and cells of HPAIV H5N1 infected chickens.

List of Figures

Figure 1: Structure of IAV virion showing encoded viral structural and non structural proteins.

Figure 2: Binding sites of cellular proteins on the domain of the NS1 protein.

Figure 3: Arrangement of the NS1 and nuclear export protein (NEP) mRNAs of the IAV.

Figure 4: Topology diagram (A) and hypothetical model (B) of the C-terminus monomer of the NS1 protein.

Figure 5 (A-F): Clinical picture of chickens and ducks suspected to be infected with HPAIV H5N1.

Figure 6 (A-B): Evidence of IAV in inoculated Embryonated Chicken Eggs (ECEs).

Figure 7 (A-B): Detection of IAV using rapid HA.

Figure 8 (A-B): Identification and subtyping of IAV using Reverse Transcriptase - Polymerase Chain Reaction (RT-PCR).

Figure 9: Phylogenetic tree on basis of nucleotide sequences of complete coding region of NS gene of HPAIV H5N1.

Figure 10: Phylogenetic tree of H gene nucleotide sequences at the cleavage site of HPAIV H5N1.

Figure 11: Detection of IAV in tissue specimens and serum samples using real- time RT-PCR.

Figure 12 (A-C): Detection of viral antigen nucleoprotein (NP) by IHC in trachea, brain, and lung.

Figure 13 (A-F): Detection of nucleoprotein (NP) viral antigen by IHC in pancreas, proventriculus, spleen, bursa, liver, and testis.

LIST OF ABBREVIATIONS

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List of abbreviations and codes of Amino acids (Rules, 1969)

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Thanks are continued to members of department of Virology for helpful advices and support.

Many thanks to Prof. Dr. Mohamed Azawy for kindly providing clinical samples that were used in this study. Thanks for Mohammed Afifi for helpful statistical analysis.

Thanks are also extended to the Egyptian Ministry of Higher Education (ParOwn Grant members), Egypt for funding my 6 months training grant in USA at which most of laboratory experiments were done.

It is my pleasure also, to thank Prof. Dr. Zheng Xing, associate professor of Virology and Immunology, Department of Veterinary Medical Sciences, University of Minnesota, USA for hosting me in his laboratory during the practical work of my thesis. He helped me in conceiving the project, performing experiments, analysis of data, and writing as well.

Special thanks to Jan Shivers, chief of Immunohistochemistry (IHC) laboratory, Veterinary Diagnostic laboratory, University of Minnesota, USA for her help in performing cross section Immunohistochemistry experiments. Thanks also for Dr. Rob Porter for photographing of slides of IHC.

I would like to express my deepest gratitude and thankful greetings to PLOS ONE Editorial Team for accepting publishing data from this thesis under title of “Pathogenicity of Highly Pathogenic Avian Virus H5N1 in Naturally Infected Poultry in Egypt”.

DEDICATION

To

“ MY MOTHER, MY FATHER

ALL MEMEBERS OF MY FAMILY

AND

THOSE WHO I LEARNED FROM

THEM EVERYWHERE ”

1. INTRODUCTION

Avian Influenza (AI) is contagious endemic and epidemic viral infection affecting wide variety of avian and mammalian hosts (Irvine et al., 2007). Influenza A virus (IAV) is negative- stranded, segmented RNA virus, classified within the genus Influenza A viruses in the family Orthomyxoviridae (Cox and Subbarao, 2000). IAV is classified into various subtypes according to their hemagglutinin and neuraminidase surface glycoprotiens and highly pathogenic (HP) or low pathogenic (LP) viruses based on their virulence (Suarez and SchultzCherry, 2000).

The segmented genome of IAV consists of 8 segments that code 10 or 11 proteins depending on whether the 11th protein, PB1-F2, is present or not (Chen et al., 2001). These proteins are divided into three main categories: A) surface proteins (hemagglutinin; H, neuraminidase; N and matrix 2; M2), B) internal proteins (polymerase subunits; PB2, PB1, PA, nucleoprotein; NP, matrix1; M1 and nuclear export protein; NEP), and C) non-structural proteins (NS1 and PB1-F2) (Webster et al., 1992; Cheung and Poon, 2008). Novel extra protein products have been recently identified as PB1-N40, PA- X, PA-N155, PA-N182, and M3 increasing the number of the IAVs encoded proteins to 15 or 16 (Muramoto et al., 2013).

The non structural protein (NS1), considered a virulence factor, is thought to play an important role in viral replication and pathogenicity during infection by antagonizing the host interferon defense mechanism (Garcia-Sastre et al., 1998; Bergmann et al., 2000). It has been previously reported that mutations or deletions within the NS1 protein significantly hampered replication of influenza viruses, both in vitro and in vivo due to an increased interferon (IFN) response and rapid elimination of the virus (Dankar et al., 2011; Petersen et al., 2013).

The HPAI H5N1 viruses produce systemic infections, morbidity and mortality as high as 100% (Spickler et al., 2008; Swayne, 2007), cause severe agricultural and economic burden, and pose a serious public health threat. They were transmitted to Africa with reported outbreaks in Nigeria, Egypt, Cameroon, and other African countries in 2006 (Enserink, 2006; Aly et al., 2008). Since then, they become endemic only in Egypt, spreading from farms to farms, causing several economic losses to poultry industry, and infecting human, even under H5 vaccine induced immune pressure, that potentially lead to continuous viral evolution and mutations (Abdelwhab et al., 2010).

Strikingly, it has been recently reported that the Egyptian HPAIV H5N1 viruses possess the greatest pandemic risk due to their unique genomic fingerprints including the mutations at H154-156, where a glycosylation site is missing, and PB2627K (Neumann et al., 2012). Accordingly, providing more information about the Egyptian HPAIV H5N1 recent outbreaks with supplementary genetic, antigenic, and pathogencity data is of great global interest, which is the main objective of this study.

The objectives of this study are:

1. Isolation, identification, and subtyping of IAV caused outbreaks in commercial chickens and backyard ducks, Sharkia, Egypt, 2013.
2. Sequencing of Non Structural (NS) and Hemagglutinin (H) genes to characterize molecular pathogenicity determinants of HPAI H5N1 viruses.
3. Investigation of distribution and spread of the HPAI H5N1 viruses in different tissues of naturally infected chickens and ducks by RT-qPCR and Immunohistochemistry (IHC).

2. REVIEW OF LITERATURE

2.1. Avian Influenza Virus (AIV) infection: nature and economic importance:

Avian influenza viruses can infect and causes in a large variety of birds and mammals worldwide (Alexander, 2000). Infections in birds can give rise to a wide variety of clinical signs that may vary according to the host, strain of virus, the host's immune status, ranging from respiratory manifestation and high mortalities as high as 100% (Spickler et al., 2008; Swayne, 2007). The on-going epidemic of highly pathogenic avian influenza (HPAI) H5N1 virus infections in poultry continues to cause severe economic problems and threatens human health worldwide, particularly where the infections became endemic (Capua and Alexander, 2006; Peiris et al., 2007).

2.2. History of AIV:

AIV was firstly reported as fowl plaque or fowl pest in 1878 in Italy (Perroncito, 1878). In 1901, Centainii and Savunozzi determined that the cause was a filterable agent. By then it was already shown that human influenza viruses, identified as a virus in 1933, (Smith et al., 1933). It was recorded that avian and human influenza virus counterparts many biological properties including the ability to grow in chick embryos and agglutinate red blood cells (Hirst, 1941). Low pathogenic avian influenza virus LPAI or midly pathogenic avian influenza was reported in the middle of the 20th century and the oldest outbreak was winter strain of Germany isolates in chicken in 1949 (H10N7). It was demonstrated that fowl plague was an avian influenza virus whose genomic composition was virtually identical to the one found in the human influenza virus (Schafer, 1955).

The first report of HPAIV outbreak caused by H5N1 was occurred in Scotlandin 1959. Alexander listed five substantiated outbreaks since 1975; this occurred in many areas in the world as USA, Australia, England and others (Alexander and Gough, 1986). By the early part of the 20th century, the disease was reported in other areas all over the world including Egypt. The terminology “highly pathogenic avian influenza” was officially adopted in 1981 at the First International Symposium on Avian Influenza to designate the highly virulent forms of avian influenza. The Office International des Epizooties (OIE) that codifies sanitary and health standards, has included HPAIV as a List A reportable disease. HPAIV has been recognized for more than 100 years it was endemic in the first third of the 20th century in some European countries, USA and occurred regular in others, further more the involved isolates were classified as H7N1 and H7N7, HPAIV outbreaks have been reported about 26 times since 1955 till 2004 (Capua and Alexander, 2004).

Since end of 2003, simultaneous HPAIV outbreaks were recorded in domestic and wild birds in at least 48 countries in the Middle East, Africa and European countries in addition to East Asia. On January 2006, an outbreak of Highly Pathogenic Avian Influenza Virus (HPAIV) was recorded in Nigeria for the first time (Fasina et al., 2009).

In 2006 the AIV was reported in ten African countries including Egypt, Toga, Sudan, Benin, Niger, Nigeria, Ghana, Cot d’Ivoir, Djibouti, Caeroon (OIE, 2008). Even without these H5N1 outbreaks, the period 1995 to 2008 will be considered significant in the history of HPAIV because of the vast numbers of birds that died or were culled in three of the other ten epizootics during this time (Belshe, 2005 and Alexander, 2008).

Egypt was the second African country, after Nigeria, to declare infection of poultry with HPAIV H5N1 on 16 February 2006 (Aly et al., 2008). More than 30 million birds were culled in the first wave of the outbreak in 2006 (Meleigy, 2007; Aly et al., 2008) and 52 human fatalities out of 150 infected persons have been reported until 6, July 2011 (WHO, 2011).

Full hemagglutinin gene sequencing was performed and the data revealed that all Egyptian strains were very closely related and belonging to subclade 2.2 of the H5N1 virus of Eurasian origin, the same one circulating in the Middle East region and introduced into Africa at the beginning of 2006 (Aly et al., 2008). Re-emerging of H5N1 severe outbreaks in vaccinated chickens at Sharkia Province in Egypt was observed in October 2007 (Hussein et al., 2009). Despite intense attempts to eradicate the virus, endemic status is reported in Egypt. Continuous viral circulation is likely increases risks of sporadic human infections.

In 2008, the Egyptian Government declared that H5N1 has become endemic in Egypt (Taha et al., 2010). Since that time, active, passive and targeted surveillances were established to elucidate the spread of H5N1 usually in poultry sectors and rarely in other feral birds or farm animals. More information on surveillance, diagnosis and control activities mobilized to confront H5N1 virus in Egypt and the major challenges hampering the containment of the disease has been reviewed in details by (Abdelwhab and Hafez, 2011). Recently, it was recorded that the rate of HPAIV H5N1 in commercial poultry was significantly lower than that in backyards and live bird markets (El-Zoghby et al., 2013).

Two subclades of H5N1 are circulating in Egypt (Arafa et al., 2010): ‘‘Classic’’ 2.2.1 strains are present mainly in backyard birds and have caused the majority of human cases (Abdelwhab et al., 2010). ‘‘Variant’’ 2.2.1 strains circulated mainly in vaccinated commercial farms since late 2007 (Arafa et al., 2010; Hafez et al., 2010). Viruses of this lineage represent antigenic drift variants and limit the efficiency of the currently used vaccines (Grund et al., 2011).

Vaccination of backyard birds using inactivated H5 vaccines was provided by the government free of charge while the commercial sector adopted their pertained vaccination practices with widely varying standards (Hafez et al., 2010). However, vaccination coverage was 1-50% and increase risk of human infection due to silent circulation of the virus in vaccinated backyard incited the government to cesses vaccination of birds in the backyard sector (Peyre et al., 2009; Abdelwhab and Hafez, 2011). On the contrary, several types of inactivated vaccines based on H5N1 and H5N2 strains are supplied by a number of vaccine manufacturers and are permanently applied in the commercial sector (Abdelwhab et al., 2009).

2.3. Taxonomy and classification of AIV:

Influenza virus A is a member of the Family Orthomyxoviridae, this family composed of five genera, influenza virus A, B and C viruses, Thogoto viruses and Isa virus es. Type designation A, B and C is based on the antigenic character of the matrix protein located in the virus envelope and the nucleoprotein within the virus particle. The name influenza comes from the Italian: influenza, meaning "influence", (Latin: influentia) (Eccles, 2005).

2.4. Structure and morphology of AIV:

Influenza A viruses are enveloped, small (80 to 120 nm in diameter), pleomorphic particles. The virions are typically spherical to pleomorphic but can be filamentous virions (20 nm in diameter and 200 to 300 nm long). Its genome consists of 8 segments of linear, negative sense, single-stranded RNA that encodes 10th or 11th proteins (Fig. 1) depending on whether the 11th protein, PB1- F2, is present or not (Chen et al., 2001). These proteins are divided into three main categories: A) surface proteins (hemagglutinin; H, neuraminidase; N and matrix 2; M2), B) internal proteins (polymerase subunits; PB2, PB1, PA, nucleoprotein; NP, matrix1; M1 and nuclear export protein; NEP) and C) non-structural proteins (NS1 and PB1-F2) (Webster et al., 1992; Cheung and Poon, 2008). Novel extra protein products have been recently identified as PB1-N40, PA- X, PA-N155, PA-N182, and M3 increasing the number of IAV encoded proteins to 15 or 16 (Muramoto et al., 2013).

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Figure 1: Structure of Influenza A virus showing encoded viral structural and non structural proteins.

Hemagglutinin (H) surface protein:

Hemagglutinin (H) is one of surface proteins that is responsible for attachment and fusion of the virus with the host cell receptors. The H is synthesized as a precursor polypeptide, H0 then cleaved by ubiquitous proteolytic enzymes into H1 and H2 (Steinhauer, 1999). The H monomer consists of a globular head region mainly of H1 connected to a fibrous stalk domain formed by the two polypeptide segments of H1 and H2. Several structures are present on the H1 protein namely receptor binding domain (RBD), proteolytic cleavage site (PCS), N-Linked glycosylated carbohydrate (GS), antigenic sites and immunogenic epitopes (Chen et al., 1998; Brown, 2000), while the transmembrane domain and fusion peptide are associated with the H2 protein (Armstrong et al., 2000).

The proximity of the globular head region harbors the receptor-binding pocket of the virus which usually binds to α2-3 linkage sialosides abundant in the intestinal tract of birds in case of avian influenza viruses (AIV) while human-adapted viruses are specific for the α2-6 linkage mainly in the respiratory tract (Parrish and Kawaoka, 2005). A switch from the α2-3 linkage to the α2-6 linkage receptor specificity is a prerequisite for emergence of avian viruses with pandemic potential (Stevens et al., 2006). All influenza A viruses have PCS of an arginine (R) residue adjacent to a conserved glycine (G) amino acid, the later becomes the N-terminus of H2 protein (Garten and Klenk, 1983). Avian influenza of low pathogencity phenotypes has monobasic amino acid, arginine or lysine (K) residues, in the cleavage site while the existence of multibasic amino acids with an R-X-K/R-R motif is a feature of high pathogenic subtypes (Klenk and Garten, 1994).

Two different classes of proteases are responsible for cleavage-activation of influenza viruses, and the distribution of these proteases in the host appears to be the prime determinant of tropism and pathogenicity (Klenk and Garten, 1994; Steinhauer, 1999). The proteases that cleave non pathogenic viruses are encountered in a limited number of cell or tissue types, so these viruses normally cause localized infections in, for example, the respiratory tract of mammals or the intestinal tract of wild birds.

On the contrary, proteases that activate pathogenic influenza viruses are ubiquitously expressed, allowing for the systemic spread of the virus in infected hosts (Munch et al., 2001). Five immunogenic epitopes (denoted A - E) of recent H5N1 hemagglutinin were mapped (Kaverin et al., 2007; Duvvuri et al., 2009). The repertoire of immunocompetent antibody-producing cells is directed almost against the upper surface of the H5 H molecule (Kaverin et al., 2007). Therefore, most of the positively selected sites were found to be within or adjacent to the immunogenic epitopes with a higher evolution rate which could help the virus to circumvent the host immune response (Lee et al., 2004; Duvvuri et al., 2009).

Non structural protein 1 (NS1):

The NS1 protein is a multifunctional protein that participates in both protein-protein and protein-RNA interactions. It binds non-specifically to double-stranded RNA (dsRNA) and to specific protein targets. Multifunctional proteins usually show a modular organization, with different domains responsible for different functions. Two important domains have been described in this 26 kDa NS1 protein accomplishing its multiple functions (Fig. 2). The N-terminal structural domain (RNA-binding domain, RBD), which protects the virus against the antiviral state induced by IFN-α/β, primarily by blocking the activation of the 2'-5'-oligo(A) synthetase/ RNase L pathway; and the C-terminal structural domain (effector domain), which inhibits the maturation and exportation of the host cellular antiviral mRNAs by binding cleavage and polyadenylation specificity factor (CPSF) and inhibiting poly(A)-binding protein (PAB II) function. The effector domain is crucial for the function of the RBD (Krug et al., 2003; Wang et al., 2002).

The influenza A virus RNA segment 8, which contains 890 nucleotides, directs the synthesis of two mRNAs in infected cells. One is colinear with the viral RNA segment and encodes for NS1 protein of 230 amino acids; the other is derived by alternative splicing from the NS1 mRNA and translated into nuclear export protein (NEP) of 121 amino acids. (Bullido et al., 2001) (Figs. 2 and 3).

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Figure 2: Binding sites of cellular proteins on the domains of the NS1 protein (Dongzi LIN et al. 2007).

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Figure 3: Arrangement of the NS1 and nuclear export protein (NEP) mRNAs of the influenza A virus (Dongzi LIN et al. 2007).

Structure and function of N-terminus of the NS1 Protein:

The dsRNA-binding domain of the NS1 protein is located at its N-terminal end. An N terminal structural domain, which comprises the first 73 amino acids of the intact protein NS1 (1−73), possesses all of the dsRNA binding activities of the full- length protein (Qian et al., 1995). The function of the dsRNA- binding activity of the NS1 protein during influenza A virus infection has not been elucidated yet. Some of the previously held theories have been disproved by new findings. In the early studies, NS1 protein inhibited the activation of protein kinase RNA-regulated (PKR) by sequestering dsRNA through the dsRNA binding domain of the NS1 protein (Bergmann et al., 2000; Lu et al., 1995; Hatada et al., 1999).

However, it was reported recently that the inhibition was realized by the direct binding of NS1 protein and the N-terminal 230 amino acid region of PKR, for which the dsRNA-binding domain is not responsible (Li et al., 2006). In another aspect, previous studies reported that high levels of IFN-α/β and its mRNA were produced in cells infected with a recombinant influenza A/Wisconsin/33 (A/WSN/33) virus expressing an NS1 protein with a mutated RNA-binding domain (Donelan et al., 2003; Wang et al., 2000).

However, a new study has shown that this mutant WSN NS1 protein is located in the cytoplasm, rather than the nucleus of infected cells and the phenotype of this mutant WSN virus is due to the mislocalization of the mutant NS1A protein rather than to the loss of NS1 dsRNA-binding activity. Mutant NS1 protein expressed by recombinant A/Udorn/72 virus could localize in the nucleus of the infected cells for the second nuclear localization signal. The experiment using this recombinant A/ Udorn/72 virus revealed that the RNA-binding activity of the NS1A protein does not have a role in inhibiting the influenza A virus-induced synthesis of IFN-β mRNA, but is required for the protection of influenza A virus against the antiviral state induced by IFN-β. This protection primarily involves inhibiting the IFN- α/β-induced 2'-5'-oligo(A) synthetase/RNase L pathway (Min et al., 2006).

Besides type I IFN, the NS1 protein is also involved in the inhibition of other pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin 6 (IL6), macrophage inflammatory protein-1 alpha (MIP-1α), IL1β and IL18. NS1 protein regulates the production of pro-inflammatory cytokines in infected macrophages through the function of both N and C-terminal domains. Moreover, the N-terminal part of the NS1 protein appeared to be crucial for the inhibition of IL1βand IL18 production, whereas the C-terminal part was important for the regulation of IFN-β, tumor necrosis factor-α, IL6 and MIP-1α production in influenza A virus-infected human macrophages (Stasakova et al., 2005).

The dsRNA binding domain of the NS1 protein can also bind to the 5'untranslated region of viral mRNAs and poly(A) binding protein 1 (PABP1). The eukaryotic initiation factor 4GI (eIF4GI) binding domain is located in the middle of the NS1A protein, a region close to PABP1 interacting domain. Accordingly, it is reasonable to infer that the NS1 interactions with eIF4GI and PABP1, as well as with viral mRNAs, could promote the specific recruitment of the viral mRNA translation initiation complexes, thus enhancing the translation of the viral mRNA (Burgui et al., 2003).

Structure and function of C-terminus of the NS1 Protein:

The C-terminus of the NS1 protein mainly contains three functional domains: eIF4GI, the 30 kDa subunit of CPSF (CPSF30), and the PAB II binding domain. The biophysical study (Bornholdt et al., 2006) on the NS1 effector domain showed that each monomer consists of seven β-strands and three α-helixes. Six of the β-strands form an antiparallel twisted β- sheet, but not the last one (Fig. 4). Six of the β-strands surround a central long α-helix, which is held in place through an extensive network of hydrophobic interactions between the twisted β-sheet and the α-helix. The CPSF30 binding domain is at the base of the largest α-helix. Asp92, whose mutation to glutamate is linked to increased virulence and cytokine resistance in certain H5N1 strains, is located in the bottom of a structurally dynamic cleft and is involved in strong hydrogen- bonding interactions with Ser195 and Thr197, shown in (Figure 4).As described previously, there are binding sites for eIF4GI, CPSF30 and PAB II in the C-terminus of the NS1 protein, and the interaction between eIF4GI and NS1 protein is associated with enhancement of the translation of the viral mRNA. It is also mentioned that the dsRNA binding activity of the NS1 protein is not related to the inhibition of the synthesis of IFN-β mRNA. Nevertheless, the level of the IFN-β does decrease in virus- infected cells. Why? It has already been identified that NS1 protein binds and inhibits the function of two cellular proteins that are essential for the 3'-end processing of cellular pre- mRNAs, CPSF30 and PAB II by way of its effector domain, thereby inhibiting the production of mature cellular mRNAs, including IFN-β mRNA (Noah et al., 2003).

The binding to CPSF30 and the resulting inhibition of 3'- end processing of cellular premRNAs is mediated by amino acid 144 of the NS1 protein, as well as by amino acids 184 to 188 (the 186 region). These two regions interact with the CPSF30. Amino acids 215 to 237 of the NS1 protein have been identified as the binding site for PABII. Binding of NS1 and PABII, which facilitates the elongation of oligo(A) tails during the generation of the 3' poly(A) ends of mRNAs, prevents PAB II from properly extending the poly-A tail of pre-mRNA within the host cell nucleus, and blocks these pre-mRNAs exporting from the nucleus (Chen et al., 1999). It was also reported that another role of the C-terminal of the NS1 protein in vivo is to stabilize and/or facilitate formation of NS1 dimers and therefore, to promote the RNA binding function of the NS1 N-terminal domain (Wang et al., 2002).

The cytokine resistance conferred by the D92E mutation might be due to the increased affinity for dsRNA with this mutation (Li et al., 2004). Because of the proximity of Asp92 to the dimeric interface, this mutation might alter the stability or orientation of the RBD to affect its dsRNA binding affinity. However, the mutation D92E might lower the efficiency of NS1 phosphorylation. It is known that NS1 phosphorylation is required for the induction of apoptosis that allows viral ribonucleoprotein (vRNP) exporting from the nucleus. This mutation results in a virulent phenotype by prolonging the viral life cycle (Bornholdt et al., 2006).

The deletion of residues 80−84 found in recent H5N1 strains could increase cytokine resistance by altering either the orientation or the stability of the RBD, or both, as these residues are parts of a flexible linker between the RBD and the effector domain (Bornholdt et al., 2006).

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Figure 4: Topology diagram (A) and hypothetical model (B) of the C-terminus monomer of the NS1 protein. The cleavage and polyadenylation specificity factor binding site is shown in orange, purple shows the nuclear export signal, and yellow indicates Asp92, Ser195 and Thr197. The β-strands (blue) are numbered 1−7, and the α-helices (red) are marked a, b and c. The N-terminus and C-terminus are also shown. (Reproduced from Bornholdt and Prasad) (Bornholdt et al., 2006).

Prospects of NS1 proteins:

The NS1 protein has various functions during IAV infection through both its RNA-binding domain and effector domain, such as protecting influenza A virus against the antiviral state, inhibiting several kinds of pro-inflammatory cytokines, and blocking the maturation and exportation of the host cellular antiviral mRNAs. The crystal structures of the NS1 RNA binding domain and effector domain indicate that NS1 protein functions as a dimer. In this dimer, the NS1 RNA binding domain and effector domain form a six-helical chain fold and an α-helix β-crescent fold, respectively, which is unique. Together with the hereditary conservation, the NS1 protein is regarded as an appealing specific target against influenza A virus. At present, the vaccines and antiviral drugs used to aim directly at the haemagglutinin (HA) and neuraminidase (NA) of influenza A virus have rendered prevention and treatment less predictably effective because of the viral antigenic mutation. Based on the above-mentioned data, it is feasible to develop live attenuated viral vaccines using the NS1-mutational viruses (Falcon et al., 2005), and design effective antiviral drugs to directly target some of the functional sites, such as the CPSF30 binding site (Twu et al., 2006). It is also possible to explore the assisting function in tumor therapy using the recombinant virus expressing truncated NS1 protein (Efferson et al., 2006).

2.5. Physical and chemical properties of AIV:

Family Orthomyxoviridae possesses enveloped viruses that are sensitive to acid pH values, although their retention of infectivity is dependent on the degree of acidity that is obtained and the virus strain (Puri et al., 1990). Furthermore, they can be inactivated through ionizing radiation and UV rays that have a potential application in the laboratory for sterilizing tools and for biological reagent manufacturing. The primary target by which radiation brings about virus inactivation is viral RNA rather than viral proteins and the radiation dose necessary for inactivation tends to be correlated to the genome size (House et al., 1990).

Influenza A viruses are sensitive to temperature. Recent studies on the effect of microwave and autoclave treatments on Influenza A viruses demonstrated that for a human influenza A virus, A/New Caledonia E 4020 (H1N1), from an initial titre of 105 EID50/ml on swabs, no virus could be detected after microwave treatment for 5 s, and autoclave treatment for 20 min was sufficient to inactivate the virus (Elhafi et al., 2004). The effect of heat treatment on HPAIV (A/chicken/Korea/ES/2003, H5N1 subtype) in chicken meat has also been investigated (Swayne, 2006). Thigh and breast chicken meat, from experimentally infected birds, was examined for virus infectivity after exposure at 30, 40, 50, 60 and 70° C and after treatment at 70 °C for 1, 5, 10, 30 and 60 s, using the heating block of a thermocycler as the inactivation method. The initial titres of infected thigh and breast meat with the H5N1 strain were 106.8 and 105.6 EID50/g, respectively. After exposure at 30, 40 and 50° C, the titre in both types of meat sample remained unchanged. Complete inactivation was only reached after exposure at 70° C (1 s) and at 70° C for 5 s in the breast and thigh meat, respectively (Swayne, 2006).

On the basis of their resistance to chemical agents, viruses can be divided into three categories (A, B, C) according to the classification proposed by Noll and Youngner (1959). This classification is based on the presence/ absence of lipids on the virus and on the virus size, which appear to be the characteristics that most influence resistance to chemical agents. Avian influenza viruses belong to category A, which includes all enveloped viruses of intermediate to large size. Many authors have reached the same conclusion regarding the susceptibility of viruses to chemical agents, i.e. the presence of lipids is associated with a high susceptibility to all disinfectants (Maris, 1990).

2.6. Antigenic and genetic properties of AIV:

Influenza A viruses are classified into subtypes based on HA and NA. Previously, Influenza A viruses are classified into various subtypes; sixteen subtypes of H gene and nine subtypes N gene have been identified (Fouchier et al., 2005; Alexander, 2007). After the recent discovery of a new virus genome subtype identified from bat, H17N10 (Tong et al., 2012), there are currently 17 H subtypes (H1 to H17) and 10 N subtypes (N1 to N10) known. More recently, there are 18 H (H1 - H18) and 11 N (N1 - N11) subtypes, the newly extra subtypes were isolated from bats (Tong et al., 2013).

All subtypes of influenza A virus are prevalent in wild and domesticated birds (Webster et al., 1992). Three H subtypes (H1, H2 and H3) and two N subtypes (N1 and N2) are usually infecting humans. However, and recently, human infections by the previously avian-restricted subtypes H5, H7 and H9 have been frequently reported (Perdue and Swayne, 2005). Likewise, swine and horses are infected with a much narrower range of AIV subtypes (Alexander, 2000).

Avian influenza viruses are classified according to the pathogenicity for poultry into two main categories; low pathogenic strains (LPAIV) result in mild or asymptomatic infections and HPAIV causing up to 100% morbidity and mortality (Swayne, 2009). To date, only H5 or H7 subtypes fulfilled the defined criteria of high pathogenicity. Meanwhile, the existence of H5 and H7 viruses of low pathogenicity were also documented and these strains can potentially evolve into high path subtypes (Garcia et al., 1996; Halvorson, 2002).

All H5 and H7 viruses have been listed as a “notifiable disease” by the OIE which mandates all member countries to report the OIE within 24 hours of confirming AIV infections (Pearson, 2003). Therefore, the OIE defined the HPAIV as: (1) viruses cause 75% mortality of 8 susceptible 4- to 8-week-old chickens within a 10 days observation period or (2) viruses have an intravenous pathogenicity index (IVPI) of greater than 1.2 upon inoculation of 10 susceptible 4- to 8-week-old chickens or (3) H5 or H7 AIV with PCS amino acid sequence similar to any of those that have been previously observed in HPAI viruses. Moreover, nonpathogenic H5 and H7 in chickens that do not posses PCS similar to any of those that have been observed in HPAI viruses are designated as notifiable LPAI viruses while other non-H5 or non-H7 AIV that are not virulent for chickens are identified as LPAI viruses (OIE, 2009).

In the European Union (EU), the Council directive 2005/94/EC defined HPAIV infection in poultry or other captive birds as: (1) infection with any influenza A virus of the subtypes H5 or H7; or with any influenza having an IVPI >1.2 in 6-week old chickens and/or (2) infection with H5 or H7 AIV subtypes that have multiple basic amino acids at the PCS (cleavage site) of the HA similar to that observed for HPAI viruses. AIV of subtypes that do not comply with the previously mentioned criteria were defined as LPAIV (EC, 2005).

Constant genetic and antigenic variation of AIV is an intriguing feature for continuous evolution of the virus in nature (Brown, 2000). Gradual antigenic variation via incremental acquisition of point mutations is defined as “antigenic drift” which is commonly regarded as the driving mechanism for influenza virus epidemics from one year to another. However, possible “antigenic shift” of influenza virus occurs by exchange genes from different subtypes of influenza “reassortment” leading to a complete alteration in the antigenic structure and emergence of new viruses with novel gene constellations (Brown, 2000). This unpredictable process is relatively infrequent, however it results in severe pandemics since the human population has no prior immunity to these de-novo surface proteins (Ferguson et al., 2003).

The H5N1 H gene has evolved into ten phylogenetically distinct clades (designated as clade 0 - 9) (WHO/OIE/FAO, 2009). Two major phylogenetic clades are wide-spread: Clade 1 viruses in Cambodia, Thailand, and Vietnam and clade 2 viruses spread from China and Indonesia to the Europe, Middle East and Africa. To date, six distinct subclades of clade 2 have been identified (WHO/OIE/FAO, 2009).

In a previous study, genome analysis of viruses collected from Europe, Northern Africa and the Middle East from late 2005 to 2006 in addition to Asian H5N1 revealed emergence of a new European-Middle Eastern-African (EMA) lineage which further was diversified into 3 distinct independently evolving clades, designated as EMA clade 1, EMA clade 2 and EMA clade 3. The early Egyptian strains in 2006 clustered within EMA clade 1 (Salzberg et al., 2007). In another study, African strains were classified into 3 sublineages denominated A - C, where the early Egyptian strains clustered within the sublineage B along with isolates from Southwest Nigeria and Djibouti (Ducatez et al., 2007).

Later on, H5N1 viruses isolated from Egypt, Israel, the Gaza Strip, Nigeria, and Europe in 2006 and 2007 were classified as clade 2.2.1, within this clade the Egyptian viruses further diversified to several subclades or groups (WHO/OIE/FAO, 2009). The most recent WHO classification allocated the Egyptian strains from human and backyard origin within the 2.2.1/C group, meanwhile viruses from vaccinated chickens belong to the 2.2.1/F group (WHO, 2011a).

2.8. Pathogenesis of AIV:

The mechanisms by which virulent strains of AI cause disease and death of their hosts is not clear. Particularly, the specific cells involved in viral replication and the mechanisms by which these viruses injure these cells have not been defined (Van-Campen et al., 1989).

AIV is normally transmitted by direct contact between infected and susceptible birds or indirect contact through aerosol droplets or exposure to virus contaminated fomites (Easterday et al., 1997). The H protein of avian influenza viruses initiate infection by binding sialic acid (SA)-containing glycoproteins on cells (Rogers and Paulson, 1983). Hemagglutinin cleavability is dependent on its primary structure at the site where cleavage occurs and the presence of the right proteases in target tissues that can carry out that cleavage. In epithelial cells lining the respiratory and intestinal tracts, the hemagglutinin of all incoming avian influenza viruses is cleaved by host proteases, thereby activating its fusion activity and allowing its entry; however, in other tissues, only the hemagglutinin of virulent viruses is cleaved, leading to systemic disease and death. This phenomenon accounts not only for viral strain differences but also for the susceptibility or resistance of different avian species (Murphy et al., 1999).

The cleavage of the H precursor molecule H0 is required to activate virus infectivity, and the distribution of activating proteases in the host is one of the determinants of tropism and, as such, pathogenicity. The H proteins of mammalian and nonpathogenic avian viruses are cleaved extracellularly, which limits their spread in hosts to tissues where the appropriate proteases are encountered. On the other hand, the H proteins of pathogenic viruses are cleaved intracellularly by ubiquitously occurring proteases and therefore have the capacity to infect various cell types and cause systemic infections (Steinhauer, 1999).

Influenza virus enters its host cell by endocytosis. The low pH inside the endosome triggers conformational changes in the major viral membrane protein, hemagglutinin, leading to fusion of the viral with the endosomal membrane (Günther- Ausborn et al., 2000). M2 protein plays a key role in the triggering process because it is an integral membrane protein that allows H+ ions to enter into the virion, which causes a conformational change of the H at the lower pH to allow the fusion domain to become active (Pinto and Lamb, 2007). The viral nucleocapsids are transported to the nucleus where viral transcriptase complex synthesizes mRNA. Transcription is initiated with 10--13 nucleotide RNA fragments generated from host heterogenous nuclear RNA via viral endonuclease activity of PB2. Six monocistronic mRNAs are produced in the nucleus and transported to the cytoplasm for translation into H, N, NP, PB1, PB2, and PA proteins. The mRNA of NS and M gene segments undergo splicing with each producing two mRNAs which are translated into NS1, NS2, MI. and M2 proteins. The H and N proteins are glycosylated in the rough endoplasmic reticulum, trimmed in the Golgi and transported to the surface where they are embedded in the plasma membrane. The eight viral gene segments along with internal viral proteins (NP, PBI, PB2, PA and M2) assemble and migrate to areas of the plasma membrane containing the integrated H, N, and M2 proteins. The M1 protein promotes close association with the plasma membrane and budding of the virions (Saif et al., 2008).

HPAIV H5N1 isolate caused systemic infections in chickens and quail and killed all of the birds within 2 and 4 days of intranasal inoculation, respectively. This isolate also replicated in multiple organs and tissues of ducks and caused some mortality. However, lower virus titers were observed in all corresponding tissues of ducks than in chicken and quail tissues, and the histological lesions were restricted to the respiratory tract (Lee et al., 2005). HPAI viruses, including HPAIV H5N1, cause severe systemic disease in galliform species as well as in anseriform species and bird species of other orders (Kuiken et al., 2010).

Following IV inoculation of AI virus leads to demonstration of intranuclear and intracytoplasmic influenza nucleoprotein in kidney tubule epithelium verifies the kidney as a primary site of influenza virus replication and confirms the nephropathogenicity of influenza virus. Furthermore, the presence of diffuse, severe renal tubule necrosis in chickens that died suggests acute renal failure, with associated blood electrolyte and nitrogenous waste abnormalities as the cause of death (Swayne and Slemons, 1994).

In contrast to systemic infection following IV inoculation, IT and IN inoculation of influenza viruses resulted in influenza virus replication and lesions in only the local area of exposure, i.e., the respiratory tract. The absence of mortality, the lack of kidney lesions, and the failure to isolate influenza virus from kidney tissue (Slemons and Swayne 1990) following IN and IT inoculation suggested that under defined experimental conditions an innate barrier exists in the respiratory and immune systems that prevents low-virulence avian-origin influenza viruses from entering the blood stream and producing viremia and systemic lesions. However, in chickens this innate barrier has been abated in some experimental studies by specific test conditions or laboratory manipulations. The predominant and severe endothelial cell tropism or lymphocytic cellular tropism of high- pathogenic avian influenza viruses in chickens obscured the nephrotropic and/or nephropathogenic properties (Olander et al., 1991 and Van Campen et al., 1989).

In addition, some avian-origin high-virulence influenza viruses have no or minimal nephrotropism and nephropathogenic properties (Acland et al., 1984). Finally, Swayne and Slemons, (1994) indicated that low-virulence avian-origin influenza viruses were nephrotropic during simulated systemic infection (IV inoculation) and pneumotropic during simulated local infection (IT and IN inoculation).

AIV antigen was located in the cerebrum, brain stem, and pancreas, mainly in association with histological lesions. Intranuclear and intracytoplasmic staining was seen in neurons and glial cells of the cerebral gray matter and brain stem in 80% of infected ducks. In the pancreas, immunolabeling was detected in the nucleus and cytoplasm of necrotic acinar cells of 20% of ducks (Vascellari et al., 2007). They found that through the application of IHC, the localization of viral antigen was closely correlated to clinical manifestations of disease and the histologic lesions detected. In some samples, viral demonstration in necrotic pancreatic foci was not possible, presumably due to the extensive necrosis of affected cells. In contrast, both IHC and ISH were able to reveal viral infection in individual cells before the development of histologic lesions. ISH was more sensitive than IHC, revealing a small amount of viral RNA in some samples where viral nucleoprotein has not been detected by IHC. They concluded that AI virus showed high pathogenicity, associated with marked CNS and pancreatic damage. IHC and ISH detected virus spread even in cells and tissues where histologic lesions were not present, showing the strong viral neurotropism in ducks.

Bröjer et al ., (2009) found that high number of ducks with encephalitis, in association with high levels of virus as detected by IHC, suggests that the virus is highly neurotropic, as previous studies showed by (Brown et al., 2006; Keawcharoen et al ., 2008). Signs of neurologic disturbance were, in fact, the main observed clinical signs in infected birds. It is likely that the encephalitis, in combination with an inability to feed or drink, was the ultimate cause of death in most of the birds. Neurotropism of the virus was also observed in the peripheral nervous system, with detection of virus in the submucosal and myenteric plexa of the intestine and in ganglion cells.

Van Riel et al., (2009) found in the severely edematous wattle skin, most endothelial cells contained virus antigen, while in all other tissues virus antigen was only detected in a few endothelial cells. Viral antigen IHC showed that H7N7 virus attached to more endothelial cells in wattle skin than in other vascular beds. This might explain, at least partly, the tropism of the virus and the associated severity of lesions in this tissue. Also they found that AI antigen often associated with histologic lesions, although virus antigen was also present in areas without detectable lesions. Viral antigen was most commonly observed in endothelial and mononuclear cells in all tissues. A remarkable finding was that viral antigen was detected in many endothelial cells in the wattle, while in all other tissues viral antigen was only detected in a few, individual endothelial cells. Parenchymal cells of the heart (cardiomyocytes), kidney (tubular epithelial and glomerular cells), lung (epithelial cells), pancreas (acinar cells), and trachea (epithelial cells) also contained viral antigen. Although there was hepatocellular necrosis, virus antigen was not detected in hepatocytes. In the wattle, keratinocytes of the skin contained viral antigen in 1 focus, and a few cells in the feather pulp of 1 feather follicle contained viral antigen.

Destruction of lymphoid tissues by A/turkey/Ont/7732/66 (H5N9) (Ty/Ont) is a characteristic of infection with this highly virulent avian influenza virus and not of other virulent avian H5 viruses, A/tern/South Africa/1961 (H5N3) (Tern/S.A.) or A/chicken/Pennsylvania/1370/83 (H5N2) (Ck/Penn). These three strains vary in the cell type(s) in which viral antigen is found, indicating that they infect and replicate in different cell types (Van Campen et al., 1989). The striking feature of infection with A/turkey/Ont/7732/66 (H5N9) (Ty/Ont) is the destruction of lymphoid tissues. This could occur by virus infection that results in killing lymphocytes and macrophages present in large numbers in the spleen; however, processes other than viral replication might be involved. In investigating this possibility, they found that Ty/Ont affected the in vitro response of avian lymphocytes to mitogen in a dose-dependent manner. Possible explanations for the enhanced response with low doses of Ty/Ont include the release of lymphocyte-activating factors by macrophages, direct activation of lymphocytes or a direct mitogenic effect of influenza virus on lymphocytes (Van Campen et al., 1989).

HPAIV H5N1 nucleoprotein was detected by IHC in the nucleus and cytoplasm of neutrophils in the placental blood of a pregnant woman. Viral RNA was detected in neutrophils by in situ hybridization and enhanced real time polymerase chain reaction. Therefore, neutrophils may serve as a vehicle for viral replication and transportation in avian influenza (Zhao et al., 2008).

2.10. Diagnosis of AIV:

The confirmation of AIV should be carried out with appropriate laboratory tests following the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (OIE, 2009). This includes samples collection, and in the primary outbreak in a given country virus isolation and identification and assessment of the pathogencity.

Isolation of AIV:

Embryonated chicken eggs (ECEs) obtained from specific pathogen free or specific antibody negative fowls are the method of choice for isolation and propagation of AIV (Woolcock, 2008; OIE, 2009). Inoculation of 9 -11 days old ECE via the allantoic sac has been used for decades as a superior route for growing of AIV. Occasionally, yolk sac or the chorioallantoic membrane routes might be useful in isolation of non-chicken originated AIV (Woolcock, 2008).

Allantoic fluid collected from inoculated ECE which have hemagglutinating activity when mixed with chicken erythrocytes could indicate presence of an AIV; however other hemagglutinating viruses (e.g. paramyxoviruses) and contaminating bacteria should be ruled out. Typically, an HPAIV kills the embryo within 24-48 hours after inoculation of ECE but further passages are required to propagate viruses of low pathogenicity (Woolcock, 2008; OIE, 2009). High cost, availability, less specificity and sensitivity are the main disadvantages of ECE for AIV isolation (Suarez, 2008; Woolcock, 2008). On the other hand, cell cultures and cell lines were found to be as sensitive as egg inoculation in terms of virus isolation, titration, selection and pathogenicity. Madin-Darby canine kidney (MDCK), primary chicken embryo kidney (CEK), primary chicken embryo fibroblast (CEF) cell cultures and baby hamster kidney (BHK-21) cell lines are efficient systems for growth of AIV. However, MDCK, CEK, and CEF were found useful and cost-effective to process a higher volume of samples (Moresco et al., 2010).

In case of LPAIV propagation in tissue cultures, trypsin must be added. Nevertheless, chicken kidney cells produce trypsin-like proteases which could allow replication of LPAIV without prior addition of trypsin (Suarez, 2008). General speaking, virus isolation remains the only tool for providing a live virus for further investigation (Charlton et al., 2009). Yet, confirmation and subtyping of AIV after primary isolation is usually done by HI, agar gel immunodiffusion assay (AGID), commercial immunoassay kits or RT-PCR (Spackman et al., 2008; Woolcock, 2008; OIE, 2009).

Detection of nucleic acid by RT-PCR:

Several types of RT-PCR methods have been developed since the early 2000s for diagnosis and differentiation of AIV which are widely employed in surveillance, monitoring of outbreaks, and research activities. Among those methods, the RT-qPCR was described to be of high sensitivity, high specificity, rapid, cheap, quantitative and cost-effective method (Spackman and Suarez, 2008). A number of RT-qPCR assays for diagnosis and characterization of AIV have been published. These assays target the matrix gene (Spackman et al., 2002), the nucleoprotein gene, the neuraminidase or the hemagglutinin gene (Hoffman et al., 2001). Using specific primers and probes, amplification of a conserved region within the matrix gene among all AIV subtypes followed by or simultaneously with HA and NA subtype-specific RT-qPCR is the common used approach (Spackman et al., 2002).

Detection of viral antigen by Immunohistochemistry (IHC):

Reverse Transcriptase Real-time Polymerase Chain Reaction (RT-qPCR) assays and IHC staining have been recently developed for rapid and accurate diagnosis of the HPAIV H5N1 infections worldwide (Cattoli et al., 2004; Tsukamoto et al., 2010; Nuovo, 2006).

IHC staining is a method to detect and locate the target viral antigen in tissue sections (Nuovo, 2006). It has been used to detect avian influenza virus nucleoprotein (NP) antigen. IHC is suitable technique for a routine avian influenza diagnostic laboratory because it does not need any sophisticated equipment or skills (Chamnanpood et al., 2011).

Sequencing of AIV genome:

Identification of AIV genome sequence data is very important to develop novel influenza vaccines, therapies and diagnostics and increase our understanding for molecular evolution, virulence-associated genetic markers and hostpathogen interaction (Spackman et al., 2008). Genome sequence of AIV has become relatively less expensive due to the recent advancement in the field of automated sequencing technology (Spackman et al., 2008).

In contrast to the standard tests for assessment of AIV pathogenicity which is time-consuming, laborious and logistically complex, sequencing of the PCS motif of the H for rapid assessment of the virulence potential of AIV could be generated easily within 24 hours and has been considered by the OIE as a criteria for notifiable HPAIV (Spackman et al., 2008; OIE, 2009). Furthermore, subtyping of AIV is achievable by direct sequencing of whole or partially amplified H and N gene segments (Spackman et al., 2008). In addition to rapid pathotyping and subtyping of the AIV, sequence analysis was applied successfully in molecular epidemiology to likely identify the possible source of infection, spectrum of susceptible species, ecological niche and geographic range (Shi et al., 2010).

2.11. Control and prophylactic against AIV:

Although enforcement of biosecurity measures and an eradication strategy of an infected flock should be the basic line in any control against H5N1 virus infections (Capua and Marangon 2007); Vaccination as a “tailored synergy” has been implemented as a main tool to confront the disease in many of developing countries and to mitigate the impact of the unbearable pre-emptive culling of infected birds (Swayne, 2009). Several types of H5 vaccines are available to protect birds against H5N1 virus infection. Conventional inactivated heterologous LPAIV (H5N2, H5N3, H5N9) or homologous whole HPAIV H5N1 virus after removal of the PCS by means of reverse genetics are commonly used vaccines in the field (Swayne, 2009). Furthermore, vaccines include recombinant viral vectors (e.g.: adenovirus, fowl poxvirus, Newcastle disease virus, baculovirus, turkey herpes virus and infectious laryngotracheitis virus) with an inserted AI H5 gene are a recently developed promising approach (Beard et al., 1991).

Prevention of the clinical signs, mortality, reduced shedding of the virus in the environment, increased the resistance of birds to an infection, decreased bird-to-bird transmission and limited decrease in the egg production are the main advantages of the AI vaccines (Capua and Marangon 2007; Swayne, 2009). Yet, the virus is still able to infect vaccinated birds and subsequent silent spread usually occurs (van der Goot et al., 2007). It is worth pointing out that continuous circulation of AIV under immune pressure in vaccinated populations for extended period favour the antigenic drift of the field virus away from the vaccine strain as reported in the H5N2 epidemic in Mexico (Lee et al., 2004) and the endemic H5N1 in China (Tian et al., 2010) as well as in Egypt (Peyre et al., 2009).

Generally, the immunity induced by vaccination is of short duration and it is necessary to apply the vaccine several times during one rearing period. There are little or no data available about the frequency of vaccinations required for keeping the breeder and layer flocks protected during the entire production period (Hafez, 2008). Furthermore, there are several factors which could affect the vaccine and vaccination against HPAIV such as: subtype of the vaccinal strain, heterogeneity of the vaccine and circulating virus, potency of the vaccine, dose, antigen mass, adjuvant, surfactant, age of birds, species and the breed of birds (Philippa et al., 2007).

Inappropriate storage, handling and improper administration are further factors for vaccination failure. The quality of the vaccine application is crucial since all non injected chickens are not protected, and improperly injected chicks will be poorly protected. Using post-vaccination necropsy (residue of oil at the site of injection) or serological testing demonstrated that it is not uncommon to see as much as 20 - 30% or even more of chickens that were not injected (Gardin, 2007).

Finally, continuous antigenic and genetic drift of AIV, differentiating vaccinated from field exposed birds and inevitable circulation of the virus in vaccinated birds “silent infection” are considered major challenges of any AIV vaccine (Capua and Marangon, 2007). Therefore, vaccination alone is inadequate to eliminate H5N1 virus in endemic countries. Thus, it is essential to incorporate a sustainable awareness campaign and education programs about the virus and modes of transmission for veterinarians and para-veterinarians involved in the poultry production chain (Hafez, 2008).

2.12. Public health significance of AIV:

The world’s first cases of human infection with the H5N1strain were documented in 1997 in Hong Kong. For the first time, evidence showed that the H5N1 strain can infect humans directly without prior adaptation in a mammalian host. A striking feature of this outbreak was the presence of primary viral pneumonia in severe cases. Usually, pneumonia that occurs in patients with influenza is a secondary bacterial infection. In these cases, however, pneumonia was caused directly by the virus, it did not respond to antibiotics, and it frequently was rapidly fatal. The outbreak, which involved 18 cases, six of which were fatal, coincided with outbreaks of infection of H5N1 in domestic poultry on farms and in live markets (WHO, 2005).

Although no sustained human-to-human transmission of the H5N1 virus has occurred so far and no evidence of genetic reassortment between human and avian influenza virus genes has been found, the epizootic outbreak in Asia poses an important public health risk. If the H5N1 viruses develop the ability for efficient and sustained transmission between humans, an influenza pandemic likely would result, with high rates of illness and death (Ligon, 2005).

3. MATERIAL AND METHODS

3.1. Material:

3.1.1. Specimens:

Sampling was carried out from chickens and ducks flocks suspected to be infected with AIV in Sharkia Province, Egypt, 2013. Specimens from tissues including (trachea, brain, lung, pancreas, proventriculus, spleen, bursa, liver, intestine, and testis) and sera were collected from infected birds.

Specimens were collected from 7 flocks; chicken broilers (3), chicken layers (2), and backyard ducks (2) (Table 1). The clinical picture of the examined birds included sudden deaths, mortalities up to 40%, ecchymoses on the shanks and feet, cyanosis of the comb and wattles, subcutaneous edema of head and neck, and ecchymotic haemorrages on sterum bone for chickens, and nervous signs (torticollis), for ducks (Figure 5).

3.1.2. Reference HPAIV H5N1:

Highly Pathogenic Avian Influenza Virus H5N1 (A/chicken/Egypt/SHAH-1403/2011, GenBank accession number JQ927216) was kindly provided by Dr. Reham El Bakery, Department of Avian and Rabbit Medicine, Faculty of Veterinary Medicine, Zagazig University, Egypt. The virus suspension was passage number 4 with titer of 5.7 log 10 EID50/ml.

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3.1.3. Phosphate buffer saline (PBS), pH 7.4:

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The pH was adjusted by Hcl or Sodium bicarbonate to be 7.4; it was sterilized by autoclaving and kept at 4oC till used. It was used for preparation of samples.

3.1.4. Emryonated chicken eggs (ECE):

A total number of 150 ECE of 9-11 days were used for isolation and propagation of HPAIV H5N1 via allantoic route.

3.1.5. Chicken erythrocytes (RBCs):

Chicken blood was collected in Na citrate 3.8 % by a volume 1:4 and centrifuged in ordinary centrifuge at 1500 rpm/15 minutes. Chicken RBCs were collected from the sediment and washed twice using PBS and then prepared as 10% suspensions in PBS for rapid hemagglutination test.

3.1.6. Antibiotic mixture:

Pen Strept: (Gibco, Invitrogen, Code, 4512)

- Penicillin 10.000 IU/ml
- Streptomycin 10.000 µg/ml

The antibiotic mixture was added to sample supernatants to get a final concentration of 1000 IU/ml Penicillin and 1000 µg/ml Streptomycin.

3.1.7. Reagents used for conventional RT-PCR:

3.1.7.1 Reagents for RNA extraction:

Blood/Liquid sample Total RNA Rapid Extraction Kit (Spin-Column) (Bioteke Corporation, China) (Cat. #: RP4001) were used for total RNA extraction from 250 µl of HA-positive allantoic fluids according to manufacturer's instructions.

3.1.7.2. Reagents for synthesis of cDNA and PCR reactions:
3.1.7.2.1. RT-PCR (cDNA synthesis) Kit:

The cDNA Diastar™ RT Kit with RNase inhibitor (Cat.#. DR22-R10k, Solegent Co. Itd., Korea) was used for synthesis of cDNA stand using random primer.

3.1.7.2.2. Master Mix:

The 2X Taq PCR master Mix (Bioteke Corporation, China) was used in PCR.

3.1.7.2.3. Primers:

Two sets of primer (Table 2) were used in PCR reaction for subtyping of AIV isolates (H5 and N1 forward and reverse primers) to yield bands of ~317 and ~245 bp for H and N genes respectively.

3.1.7.3. Reagents for agarose gel electrophoresis:
3.1.7.3.1. Tris-Acetate EDTA (TAE) buffer:

It is 50X stock solution (Fermentas). It was used as 1x buffer solution for preparation of agarose and for gel electrophoresis.

3.1.7.3.2. Agarose (Molecular Biology Grade):

It was used in concentration of 1.5% in TAE

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It was heated in microwave and used for agarose gel electrophoresis of PCR products.

3.1.7.3.3. Ethidium Bromide:

A stock solution of ethidium bromide (Fluka) was prepared as the following:

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It was used for staining the agarose gel electrophoresis DNA by adding 50µl from stock solution to 50 ml 1.5% melted agarose to give a final concentration of 0.5 µg/ ml.

3.1.7.3.4. Molecular weight marker: GeneRulerTM, 100 bp plus DNA Ladder, Ready-to-use (Fermentas)

It is composed of fourteen chromatography-purified individual DNA fragments (in base pairs): 3000, 2000, 1500, 1200, 1000, 900, 800, 700, 600, 500, 400, 300, 200, and 100 bp.

It contains two reference bands (1000 and 500 bp) for easy orientation. It was added in volume of 5µl per lane.

3.1.8. Reagents for cloning and sequencing of NS gene:

3.1.8.1 Reagents for TA cloning:
3.1.8.1.1. RT-PCR (cDNA synthesis) Kit:

SuperScriptTM III RT-Kit (InvitrogenTM, USA) (Cat.#:18080-093) was used for reverse transcription of the extracted RNA according to manufacturer's instructions using uni-12 primer (Table 2).

3.1.8.1.2. Amplification kit:

Taq. DNA polymerase enzyme (Takara Bio Inc., Japan), was used for ampliphication of (cDNA) using specific sets of primers for both ends of segment eight of H5N1 influenza virus (NS segment) (Table 2).

3.1.8.1.3. PCR purification combo kits:

Specific bands of NS segment at the expected size (890+29 bp) on gel were excised and purified using PureLinkTM quick gel extraction and PCR purification combo kit (InvitrogenTM, USA)(Cat#: K2200-01) following manufacturer's instructions.

3.1.8.1.4. TA cloning® kits:

TA cloning® kit, with pCRTM 2.1 vector without competent cells (Cat.#: K2020-20), subcloning efficiency TM DH5α TM competent cells (Cat.# 18265-017) and PureLinkTM quick plasmid miniprep kit (Cat.# K2100-10) were used For cloning of NS (segment 8) following manufacturer's instructions.

3.1.8.2. Reagents for sequencing of NS gene:

Reagents for sequencing were performed by ACGT sequencing company, Ilionos, Chicago, USA.

3.1.9. Reagents for purification and sequencing of amplified H gene:

3.1.9.1. Reagents for purification of PCR product (DNA)

The gene JETTM Gel extraction Kit (Cat.# K0691, Fermentus) was used according to manufacturer’s instructions.

3.1.9.2. Reagents for sequencing of H gene:

Reagents for sequencing were performed by Sigma sequencing company, Cairo, Egypt.

3.1.10. Reagents for real-time RT-PCR:

3.1.10.1. Reagents for RNA extraction:

Blood/Liquid sample Total RNA Rapid Extraction Kit (Spin-Column) (Bioteke Corporation, China) (Cat. #: RP4001) were used for total RNA extraction different tissue specimens according to manufacturer's instructions.

3.1.10.2. Real-time RT-PCR kit:

Ambion AgPath-IDTM one step RT-RealTime PCR kit (Applied Biosystems®, USA) was used for TaqMan-based real- time RT-PCR (Applied Biosystems) to measure viral M gene transcripts in these tissues according to manufacturer's instructions using sets of M gene specific primers and probe (Table 2).

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3.1.11. Reagents for Immunohistochemistry (IHC):

3.1.11.1. Neutral buffered formalin 10%:

Ten percent neutral buffered formalin was used for fixation of tissues as a preparatory step for (IHC). It was prepared by adding 10 ml formalin to 90 ml phosphate buffer saline.

3.1.11.2. Citrisolv clearing agent (deparaffinizing solutions):

Different series of ethanol's solutions (100% ethanol: 95% Ethanol; 70% Ethanol; 50% Ethanol) and distilled water were used.

3.1.11.3. Target retrieval solution (Dako; cat. # S1699)
3.1.11.4. Hydrogen peroxide (H2O2), 3%
3.1.11.5. 0.05 M Tris Buffered Saline, PH 7.6 with 0.05% Tween 20
3.1.11.6. Normal goat serum, 1:10 (Sigma)

Goat serum was used as concentration 1:10 in Tris buffered saline (TBS) as blocking solution.

3.1.11.7. Primary antibody:

Primary rabbit anti-influenza A nucleoprotein (NP) polyclonal antibody (Cat.#: 9382; LifeSpan Biosciences, Inc.USA) was used for detection of influenza A virus in tissues of chickens and ducks.

3.1.11.8. Secondary antibody:

The EnVision+/HRP goat anti-rabbit IgG, (Dako; Ready-to-use; cat.#: K3469).

3.1.11.9. Substrate:

Chromomgen-3-Amino-9-Ethylecarbazole+; AEC+ (Dako; Ready-to-use; cat. #: K3469) was used in IHC staining.

3.1.11.10. Counter stain

Mayer's Hematoxylin stain was used as counter stain.

3.1.11.11. Aqueous mounting medium (Riedel-deHäen)
3.1.11.12. Positive and negative controls:

Positive control was known IAV positive tissue while negative control was rabbit immunoglobulin fraction (Dako; cat. # X0903).

3.2. Methods:

3.2.1. Preparation of collected samples:

3.2.1.1. Preparation of tissue samples for virus isolation:

Tissue homogenates 10% suspension was prepared by mixing 0.5g of tissue to 5ml of sterile PBS then centrifuged at 2000rpm for 20 minutes at 4oC. Antibiotics were added to the supernatant fluids to get a final concentration of 1000IU penicillin and 1000µg streptomycin/ml, and then left for one hour at refrigerator. The supernatant was aliquoted into sterile 1.5ml eppendorffs, labeled and used for inoculation of SPF-ECE for virus isolation.

3.2.1.2. Preparation of serum samples:

Blood samples were collected from chickens and ducks and allowed to clot at room temperature then centrifuged at 2000rpm for 10 minutes for serum separation. The supernatant sera were aspirated into small cryovials aliquots and stored at - 20°C until used for RNA extraction.

3.2.2. Isolation of AIV using ECE:

The embryonated chicken eggs of 9-11 days old were inoculated via allantoic cavity route with 0.2ml of sample (trachea and lung tissue homogenates pooled together). Each sample was inoculated into 3 eggs. The inoculated eggs were sealed melted wax. Additionally, three fertile eggs were inoculated with reference AIV subtype H5N1 virus and another three eggs were kept without inoculation as negative control.

These eggs were incubated at 37°C for 5 days, embryonic death was monitored twice daily. The removed ECEs were chilled at 4˚C for 4 hours and then examined. The eggs were cleaned by cotton piece soaked in 70% ethanol. At least three successive embryo passages were applied for each sample to be negative. Allantoic fluids were collected and preserved at (-20C) until tested for hemagglutination activity using washed chicken RBCs 10% (OIE, 2012).

3.2.3. Detection of AIV using rapid HA:

Using clean glass slides, 50 µL of allantoic fluid from each sample were mixed with 50 µL of washed chicken RBCs 10% and incubated at room temperature for 3-5 minutes. Tested allantoic fluids which showed agglutination in form of aggregation of RBCs were considered HA positive and subjected to RT-PCR for subtyping AI viruses.

3.2.4. Detection, identification and subtyping of AIV isolates using RT-PCR:

3.2.4.1. Extraction of RNA:

Viral RNA was extracted directly from 250µl of HA- positive allantoic fluids, positive and negative controls were included as well. RNA extraction step were done using Blood/Liquid sample Total RNA Rapid Extraction Kit (Bioteke Corporation, China) according to Manufacturer’s instructions. A 750µl of Lysis buffer were added to 250µl allantoic fluid in one microcentrifuge tube followed by vortexing for 2 minutes. Microcentrifuge tube was incubated for 10 minutes at room temperature followed by adding 150µl chloroform and shaking for 15 seconds, and then incubation for 3 minutes at room temperature. After then, Samples were centrifuged at 12,000 rpm/10 minutes where the mixture was separated into 3 phases. The upper aqueous phase was transferred to a fresh tube where we added 500μl 70% ethanol. The alcohol-aqueous mixture was transferred to the spin-column followed by centrifugation at 10,000 rpm and two steps of washing using washing buffers. Finally, the spin column was placed into RNase-free centrifuge tube and 60µl was added to the center of the column to elute extracted RNA from silica membrane of spin-column.

3.2.4.2. Synthesis of cDNA:

The extracted viral RNAs from allantoic fluids were reverse transcribed to cDNA using cDNA DiaStarTM RT Kit (Solgent Co. ltd. Korea) according to Manufacturer’s instructions. In a PCR tube, 5µl of extracted RNA was mixed with 1µl of random primer. The mixture was heated to 56oC for 5 minutes and cooled immediately on ice. This was followed by preparation of a mixture with the following condition:

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The mixture was mixed properly, incubated at 50oC for 60 minutes. The reaction was inactivated by heating at 95oC to stop the action of RT enzyme. Pure cDNA was produced and ready for amplification.

3.2.4.3. PCR reaction using H and N primers:

A total volume of 25µl in a sterile 0.2 ml RNase free PCR tube using 2X power PCR master Mix (Bioteke Corporation, China). The solution phase PCRs contain the following contents:

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The optimized PCR cyclic reaction conditions for H gene were performed in MWG-Biotech thermal cycler as described as followings:

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The optimized PCR cyclic reaction conditions for N gene were performed in Biotech thermal cycler as described as followings:

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3.2.4.4. Agarose gel electrophoresis of RT-PCR products of both H and N genes:

Fifty ml from 1.5% agarose was prepared in 1x TAE buffer by heating and melting in microwave. The melted agarose left till cool to about 45oC then 50µl from ethidium bromide (stock=0.5 mg/ml) was added to give a final concentration of 0.5 µg/ml. The gel was poured, left for solidification, and the comb was removed then 1X TAE buffer was added. Five µl of the PCR products and 5 µl molecular weight marker were added into the marked wells formed in gel. Electrophoresis was done at 100 volts for 40 min then the gel was viewed and photographed on the UV transilluminator.

3.2.5. Cloning and sequencing of NS gene (Segment 8)

3.2.5.1. Synthesis of cDNA:

The extracted RNA from Allantoic fluid was reverse transcribed to cDNA using SuperScriptTM III RT-Kit (InvitrogenTM, USA)(Cat.#:18080-093) according to manufacturer's instructions. Seven μl of extracted RNA, 0.5μl uni-12 primer and 2.5μl RNase free water were added together in one PCR tube and incubated at 70°C for 5 minutes, this was called part #1. In another PCR tube, 1μl dNTPs (Takara 10 mM), 4μl Invitrogen 5X 1st strain buffer, 1μl Invitrogen M-MLV polymerase, 1μl RNase inhibitor, 2μl of 0.1mM DTT and 1μl of H2O were mixed together in one PCR tube, this was called part #2. Part #1 was added to part #2 and transferred to thermocycler under optimized cyclic reaction as followings: (25°C/15 minutes, 42°C/1.5 hour, 75°C/10 minutes and 4°C forever). The Reverse Transcription product (cDNA) was used as template for the amplification reaction.

3.2.5.2. Amplification reaction:

One microliter of (uni-12) RT-product was used as a template for PCR using 2.5μl (10X) buffer, 1 μl dNTP, 0.5 μl Taq. DNA polymerase enzyme (Takara Bio Inc., Japan), 19 μl Nuclease free water and 0.5 μl from both forward and reverse primer for NS segment.

The optimized PCR cyclic reaction conditions for NS gene were as described as followings:

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3.2.5.3. Agarose gel electrophoresis of RT-PCR product of NS:

Agar gel electrophoresis was prepared as previously described in H and N genes.

3.2.5.4. Purification of specific PCR amplicons from agarose gel:

The PCR amplicons (~890 +29bp) were considered specific bands for non structural (NS) segment of HPAIV H5N1 (Hoffman et al., 2001). Specific bands were excised using gel cutter. About 300-450µl of gel solublizing buffer (InvitrogenTM, USA) were added to excised gel slice in one eppendorff, incubated in water bath 45°C until the gel slice was solubilized. The solubilized solution was transferred to silica column followed by two steps of washing according to manufacturer's instructions (PureLinkTM quick gel extraction and PCR purification combo kit, InvitrogenTM, USA). Finally, 40µl TrisHCL was used as ellution buffer.

3.2.5.5. Ligation of purified specific PCR amplicons into cloning vector:

Purified PCR fragments were ligated into pCRTM 2.1 vector by adding 3µl from purified fragment to 0.5µl ligation buffer, 0.5µl ligase and 1.0µl pCR 2.1 plasmid vector in one eppendorf, then, incubated at 14°C/overnight.

3.2.5.6. Transformation of ligated plasmids into bacteria:

Transformation process was done using chemically competent E. coli that has modified cell membrane and can accept entry of plasmid vector (Subcloning efficiency TM DH5α TM competent cells, InvitrogenTM, USA). This process was held through the following successive steps:

Ice incubation: 3µl ligated plasmid were added to 35µl competent bacteria followed by good gentle mixing and ice incubation for about 40 minutes.

Heat shock: Sudden moving to water bath (42°C) for one minute.

Recovery of bacteria: adding 150µl pre-warmed antibiotic free LB medium to the eppendorf that contains 3µl ligated plasmid and 35µl competent bacteria.

Incubation with shaking: Incubating recovered bacteria in shaker incubator (37°C/250rpm) for 30 minutes and not more than 60 minutes.

Staining of transformed bacteria by X-gal substrate: Adding 20-40μl X-gal substrate of blue color to the transformed competent bacteria.

Plating on petry dishes: Streaking of the X-gal stained transformed bacteria into LB semi-solid agar that contain Ampicillin followed by incubation of plates in inverted direction.

Picking up successively transformed bacteria: Only white bacterial colonies were picked up and transferred to eppendorfs that contain LB fluid media. Only white colonies were picked up because white color means that these bacterial colonies have our cloned NS gene because this gene will stimulate beta- galactosidase enzyme to change color of X-gal from blue to white color. Picked up white colonies were subjected to hybrid cloned plasmid extraction step.

3.2.5.7. Extraction of hybrid cloned plasmids from transformed bacteria:

This step was done by adding 1.5ml of the bacterial culture to 2ml eppedorfs followed by centrifugation at 4000rpm for 2 minutes. Supernatants were discarded and 100µl of resuspension buffer were added followed by vortexing to resuspend bacteria. For lysis of bacterial cell wall, 200µl of lysis buffer were added to resuspended bacteria. For neutralization of basic pH that caused by lysis buffer, 150µl of neutralizing buffer were added followed by vigorous mixing and centrifugation at maximum speed for 2 minutes. Then, the harvested supernatants that contain protein and nucleic acids were transferred to clean eppendorf followed by addition of 200µl phenol and 200µl chloroform and vigorous shaking to obtain nucleic acids free in supernatants and get rid of proteins after centrifugation at maximum speed for 2 minutes. 1ml of Ethyl alcohol was added to the harvested supernatants followed by centrifugation at maximum speed for 2 minutes. Finally, extracted plasmid pellets were left to dry on bench until next step (plasmid digestion) was started.

3.2.5.8. Purification of hybrid cloned plasmids:

Purification of white plasmid pellets was an essential step before sending plasmids for sequencing. It was done using PureLinkTM quick plasmid miniprep kits following manfacturer's instruction. The steps were similar to that previously mentioned for purification of PCR amplicons from gel slices.

3.2.6. Full length sequencing of NS gene (segment 8):

Purified hybrid cloned plasmids were sent for sequencing (ACGT sequencing company, Ilionos, Chicago, USA). Sequences were obtained using an ABI Big Dye Terminator v.1.1 sequencing kit and run on a 3730 XL DNA Analyzer (Applied Biosystems, Foster City, CA). The NS nucleotide sequences of our isolates are available on GenBank database under the accession numbers (KJ192204, KJ192205 and KJ192206).

3.2.7. Partial sequencing of H gene:

The PCR products of the predicted molecular size (~345) were purified using GeneJETTM Gel Extraction Kit (Fermentus) as recommended by the manufacturer. Purified PCR products were sent for sequencing (Sigma sequencing company, Cairo, Egypt). The H nucleotide sequences of our isolates are available on GenBank database under the accession numbers (KP311329 and KP311330).

3.2.8. Phylogenetic analysis of sequences of NS and H genes:

Phylogenetic analysis of the NS and H genes was based on nucleotides 39-704 (666 bases) of NS and 791-1100 (309 bases) of H genes. All gene sequence data of known H5N1 strains were collected from the National Center for Biotechnology Information (NCBI) flu database. Multiple alignments were constructed using ClustalW Multiple alignment using the MegAlign module of DNAStar software (Lasergene version 7.2 (DNASTAR, Madison, WI, USA). The neighbour- joining method with Kimura two-parameter distances was used for constructing the phylogenetic tree using the Mega 4.1 (Kimura, 1980). The tree was rooted to the A/goose/Guangdong/1/1996 virus sequence. The reliability of the internal branches was assessed by the p-distance substitution model and 1000 bootstrap replications. The NS and H genotypes were determined using the Influenza A Virus FluGenome web server, (http://www.flugenome.org/) (Lu et al, 2007).

3.2.9. Deduced amino acid sequence analysis of NS and H genes:

The amino acid sequences of NS1, NS2 and H cleavage site were deduced from the nucleotide sequences. The multisequence alignment tool available in the flu database was used to compare the deduced amino acid sequences of the Egyptian H5N1 strain under study with other H5N1 lineages circulated in Egypt, Middle East and worldwide in order to screen amino acid residues that were identified as pathogenic determinants of highly pathogenic avian influenza viruses.

3.2.10. Detection of AIV in tissue specimens and serum samples using real-time RT-PCR:

3.2.10.1. Extraction of RNA:

Tissue homogenates from birds previously screened as RT-PCR H5N1 positive were subjected to RNA extraction using Blood/Liquid sample Total RNA Rapid Extraction Kit (Bioteke Corporation, China) according to Manufacturer’s instructions. Positive and negative controls were included as well.

3.2.10.2. Real time RT-PCR reaction:

The extracted viral RNA from different tissues was used for TaqMan-based real-time RT-PCR to measure viral M gene transcripts in tissues using sets of M gene specific primers and probe (Table 2). Reaction conditions were optimized in accordance with national veterinary service laboratory (Spackman, 2005, USDA, USA) using the Ambion AgPath-IDTM one step RT-PCR kit (Applied Biosystem®, USA). The experiments were held in three successive times and positive and negative controls were included along with the tested samples as well.

The solution phase PCRs contain the following contents:

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The Thermal Cycler Profile for M gene was performed using the following machine (Applied Biosystems 7500 RealTime PCR System) as described as followings:

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3.2.11. Detection of AIV Nucleoprotein (NP) antigen in different tissues using IHC (Key et al., 2006):

The collected tissues were fixed in 10% neutral buffered formalin, routinely processed. Paraffin blocks were sectioned and immunohistochemically stained.

3.2.11.1. Tissue deparaffization and rehydration:

The slides were placed at slides rack and then placed in oven (58-62 °C) for 30 minutes to warm and melt paraffin in formalin-fixed, paraffin-embedded tissues sections.

Deparaffinization were done through 4 changes of citrissolv clearing agent (3 minutes/each solution) inside a fume hood. Tissues were rehydrated through a graded ethanol series (100%, 95%, 70%, and 50%) and distilled water (3 minutes/each) inside a fume hood.

3.2.11.2. Antigen retrieval:

The bottom of decloaker was firstly filled with 500 ml of distilled water. Plastic slide racks were filled with antigen retrieval solution and then were set inside decloaker. Plastic racks with blank slides were set inside decloaker as well. Decloaker was tightly closed and set to start heating/pressurizing process up to 120°C/17-24 psi. Once temp/pressure was reached, slides were held at that point for 30 seconds, after then, stop button was pushed. Once decloaker reaches 85°C/10 seconds and pressure was zero psi, decloaker lid was opened. Slide racks were cooled immediately by placing them in two changes of 0.05 MTBS, PH 7.6, within tween 20 added (0.05% per volume) for 5 minutes/each.

3.2.11.3. IHC staining:

Endogenous peroxidase was firstly blocked with 3% H2O2 for 15 minutes followed by rinsing slides in TBS/Tween 20 for 5 minutes. Non specific binding sites were blocked with normal goat serum, 1:10 in TBS, for 15 minutes. Slides were incubated with primary antibody overnight at 4°C followed by rinsing in TBS/Tween 20 for 5 minutes and incubation with EnVision+/HRP goat anti-rabbit IgG for 60 minutes. After rinsing slides in TBS/Tween 20 for 5 minutes, immunoreactivity was detected by adding substrate (3-Amino-9-Ethylocarbazole). Positive staining development time was 10 minutes, after which slides were rinsed with distilled water and placed in a glass dish for running tap water for 5 minutes.

Finally, slides were counterstained with Mayer's Hematoxylin for 5 minutes then rinsed in water followed by covering slide by cover slip using aqueous mounting medium. Two unstained sections per case block, one to be incubated with primary antibody and one to be used as negative control (incubated with negative control rabbit IgG fraction). All previous steps were done at room temperature except the primary antibody step, which was done at 4°C overnight.

3.2.12. Statistical Analysis

Data were collected and continuous variables were analyzed using one-way analysis of variance (ANOVA), then comparison of means was carried out with Duncan’s multiple range tests (DMRT) and summarized as mean ± standard deviation.

4. RESULTS

4.1. Isolation of IAV using ECE:

Inoculated embryos died within 48-72 hours post inoculations with diffuse hemorrhages after three successive passages were considered as positive samples (Fig. 6B).

4.2. Detection of IAV using rapid HA:

Allantoic fluids were tested for hemagglutination reactivity using rapid hemagglutination assay (HA) and positive reactions were detected (Fig. 7B). Hemagglutinating viruses were detected in 34 birds out of 46 birds with a percentage of 73.9 % with 30 of 42 (71.4%) in chickens and 4 of 4 (100%) in ducks, respectively (Table 3).

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Figure 5: Clinical picture of chickens and ducks suspected to be infected with HPAIV H5N1.A. Chicken Broilers: Ecchymosis on shanks and feet; B. Chicken Broilers: cyanosis of comb and wattles; C. Chicken Broilers: fascial oedema (arrow); D. Chicken Layers: Hemorrhages on sternum bone (arrow); E. Backyard Ducks: Nervous signs, torticollus (arrow); F. Backyard Ducks: Liver necrotic lesions (arrow).

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Figure 6: Evidence of IAV in inoculated ECEs. (A) Normal control negative chicken embryo inoculated with PBS with antibiotic mixture. (B) Chicken embryo inoculated with supernatants of homogenized testicular tissue of naturally infected chicken layers showing dead embryos within 48h with severe congestion and hemorrhages after the first passage (arrow).

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Figure 7: Detection of IAV using rapid HA. (A) Normal control negative washed chicken RBCs (10%). (B) Positive reactions of tested allantoic fluids by rapid HA using 10% washed chicken RBCs in form of aggregation or agglutination (arrow).

4.3. Detection, identification and subtyping of IAV using RT-PCR:

Allantoic fluids which showed positive reactions with rapid HA assay were submitted to RT-PCR for subtyping of viral isolates using specific primers for H and N genes. Reference AIV subtype H5N1 and positive samples produced bands at ~345 bp (Fig. 7A) and ~245 bp (Fig. 7B) specific to AIV subtype H5N1using H5 and N1 primers respectively. AIV H5N1 was detected in 34 birds out of 46 sampled birds with a percentage of 73.9 % that distributed between chickens and ducks by 30 of 42 (71.4%) and 4 of 4 (100%) respectively (Table 3).

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Figure 8: Detection, identification and subtyping of IAV isolates using RT-PCR. (A) PCR amplification for H gene showing band size of ~345 bp (arrow), First lane: Molecular marker of 100 bp, Lane 1-7: Positive samples, Ctrl +ve: Positive AIV subtype H5N1, Ctrl -ve: Negative control (allantoic fluid of non inoculated ECE). (B) PCR amplification for N gene showing band size of ~245 bp (arrow), First lane: Molecular marker of 100bp, Lane 1-5: Positive samples, Ctrl +ve: Positive AIV subtype H5N1, Ctrl -ve: Negative control (allantoic fluid of non inoculated ECE).

4.3. Cloning and sequencing of NS gene (segment 8):

The NS genomic segments (segment 8) of the Egyptian H5N1 isolates in this study were completely sequenced. The results showed that the lengths of the RNA region coding NS1 and NS2 proteins were 822 bp. All NS1 and NS2/NEP genes shared the first 30 nucleotides of the coding region. The percent of identity of nucleotide sequences of the NS segments of our isolates was 99 %. The sequences showed homology of 99% with those of the HPAIV H5N1 viruses circulating in Egypt at the time of this investigation, and confirmed that our isolates belongs to genotype (NS1E) upon using the Influenza A Virus FluGenome web server, (http://www.flugenome.org/) (Lu et al, 2007).

4.4. Partial sequencing of H gene:

The H gene (segment 4) of our isolates was also partially sequenced at the coding region of amino acids motifs at H cleavage site. The results showed that the percent of identity of nucleotide sequences of H gene of our isolates was 99 %. The sequences showed identity of 98-99% with those of HPAIV H5N1 viruses circulating in Egypt at the time of this investigation, and confirmed that our isolates belongs to genotype (5J), using the same IAV FluGenome web server (Lu et al, 2007). Interestingly, we noticed that the surface H protein of the HPAIV H5N1 currently circulating in Egypt belongs to genotype (5J), a different genotype from the genotypes of the viral strains used in some available commercial vaccines (Fig. 9)

4.5. Phylogenetic analysis of sequences of NS and H genes:

Several nucleotide sequences of NS gene (segment 8) of known HPAIV H5N1 strains were collected from NCBI Influenza Data base and were used for phylogenetic analysis. The phylogenetic analysis, based on complete coding region of NS gene, showed that our isolates formed a uniform cluster, together with highly pathogenic H5N1 viruses isolated from Egypt in (2010, 2011, 2012 and 2013), however, this cluster was far from other viruses isolated from Egypt in 2006, 2007, 2008 (Fig. 8).

Several nucleotide sequences of H gene (segment 4) of known HPAIV H5N1 strains were also retrieved from NCBI Influenza Data base and were aligned for further use in construction of phylogenetic tree. The phylogenetic analysis, based on partial coding region of H gene, showed that our isolates formed a uniform cluster, together with the HPAI H5N1 viruses from Egypt isolated in 2009, 2010, 2011, 2012, and 2013; however, this cluster was not identical to the HPAIV H5 strains used for commercial vaccine development in Egypt, and was also phylogenetically distant from the viruses isolated from Egypt in 2006, 2007, and 2008 (Fig. 9).

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Figure 9: Phylogenetic tree on basis of nucleotide sequences of complete coding region of NS gene of HPAIV H5N1. The tree was constructed using Neighborhood joining method with bootstrap values calculated for 1,000 replicates and cut off value 50%. Sequences from this study are marked with solid triangle.

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Figure 10: Phylogenetic tree of the H gene nucleotide sequences at the cleavage site of HPAIV H5N1. Vaccine H5 strains were included in the tree and marked with solid circles. The tree was constructed with multiple alignment of a 309 base- nucleotide sequence of HA genes using the Neighborhood- joining method in MEGA4. The tree topology was evaluated by 1,000 bootstrap analyses.

4.5. Deduced Amino Acid Sequences Analysis of NS and H genes:

The percent of identities of amino acid sequences among NS1 and NS2 proteins of our isolates were more than 99%. The NS1 proteins of the two Egyptian H5N1 isolates did not differ from each other, but differed significantly when compared with those of the low pathogenic avian influenza (LPAIV) isolates retrieved from Gene bank (NCBI). In the current study, the molecular determinants of HPAIV strains were identified within NS1 protein of our H5N1 isolates, including 225 amino acids in length, deleted80 TMASV84 motif, Glutamate at position 92 (92E), and C-terminus E-S-E-V motif. The following residues (T5, P31, D34, R38, K41, G45, R46 and T49) were identified in the RNA binding domain of NS1 protein. The NS2 protein of our H5N1 isolates contained 121 amino acids residues with tryptophan at position 78. The nuclear export signal (NES) motif was identified in its N-terminus region to be 12 ILVRMSKMQL21.

The percent of H amino acid sequence identities of our sequenced isolates was 99%. We found that the HA of our isolates encodes a multibasic amino acid motif, 321- PQGERRRKKR*GLF-333, at the H cleavage site, which is a characteristic feature of all HPAIV H5N1 strains. Interestingly, we found that one of our isolates has a substitution of amino acid (R325K) at this cleavage site, to make it PQGEKRRKKR*GLF.

4.6. Detection of IAV in tissue specimens and serum samples using real-time RT-PCR:

Chicken and duck tissues including trachea, lung, liver, spleen, intestine, brain, testis, and serum were collected from birds that showed positive results by viral isolation, HA assay, and RT-PCR. These tissues were subjected to total RNA extraction and real-time RT-PCR with primers specific to M gene to examine viral RNA in each organ type.

By real-time RT-PCR, viral RNA of the HPAIV H5N1 M gene was detected in all tissues tested both in chickens and ducks, with an exception to testis which was only positive for chicken layers (Fig.10). Higher levels of viral RNA were in general detected in tissues of chicken broilers and layers, including trachea, lung, spleen, intestine, brain, and serum, than in those of ducks (Fig.10). In chicken broilers, higher viral RNA levels appeared in brain, trachea, and serum samples with significance difference with those in chicken layers (Fig.10). No significant differences in viral RNA levels were observed in various tissues of ducks, except for higher levels detected in trachea, lung, and liver tissues that were significant different from those of chickens (Fig.10). However, these samples from different birds cannot be emphatically compared because they came from natural outbreaks with uncertain timing of the course of infection.

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Figure 11: Detection of IAV in tissue specimens and serum samples using real-time RT-PCR. The tests were performed in triplicates for each sample and the C t numbers are average with 3times ± SD (error bar), (* P<0.05).Results were expressed as C t values. C t values lower than (35) were considered positive while values greater than (35) were considered negative. Strong positive tissues included C t values between 15 and 25, moderate positive tissues included C t values between 25 and 30, and weak positive tissues included C t values between 30 and 35.

4.7. Detection of IAV antigen (Nucleoprotein) in different tissues using IHC:

Chickens and ducks tissues included trachea, lung, brain, spleen, bursa, pancreas, liver, proventriculus, and testis were collected from birds that showed positive results by real-time RT-PCR. These tissues were prepared for cross-section IHC using specific antiserum against influenza A virus nucleoprotein (NP) to evaluate viral tissue tropism in tissues of chickens and ducks naturally infected with HPAI H5N1 virus. The viral NP antigen was observed in all tested tissues included trachea, lung, brain, spleen and bursa, pancreas, proventriculus, liver, and testis (Figs. 11 and 12).

Nucleoprotein viral antigen was clearly detected in endothelial and epithelial cells of trachea (Fig. 11A), Neurons, glial cells of Perkinji cell layer, and endothelial cells of brain (Fig. 11B), mononuclear cells of lung (Fig. 11C). acinar epithelium of pancreas (Fig. 12A), glandular epithelium of proventriculus (Fig. 12B), lymphocytes and mononuclear cells of spleen (Fig. 12C), lymphocytes of follicular layer of bursa (Fig. 12D), and VanKuppfer cells of liver in between liver sinusoids (Fig. 12E).

Strikingly, viral NP antigen was detected between seminephrous tubules of testicular tissue and even sticking to heads of sperms inside these tubules (Fig. 12F)

Staining of most tissues shared a common characteristic feature which was detection of viral antigen in their endothelial and mononuclear cells, which suggest that viral pathogenesis of the HPAIV H5N1 may be associated with endothelial invasion, and that the virus could be carried by infected monocytes.

A summary of viral antigen staining in various tissues that were examined is shown in (Table 4).

It worse mention, that we prepared samples from duck tissues and performed the same IHC staining on duck tissues as chicken tissues. However, we could not detect viral NP in all tissues examined (data not shown). This could be attributed to the preparation of samples, since they were collected from dead ducks in the field. Although viral RNA was still present and live virus isolated and subtyped by RT-PCR successfully (Figs. 6, 7, and 10), no sufficient viral antigen existed in the tissues due to prolonged exposure at ambient temperature and/or prolonged preservation in formalin before doing IHC staining.

Table 3: Results of viral isolation, HA, RT-PCR, and IHC of HPAIV H5N1 from infected chicken and duck flocks.

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Table 4: Distribution of viral antigen NP in IHC stained tissues and cells of HPAIV H5N1 infected chickens.

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IHC scoring system: (+) few viral antigen distribution; (++) moderate viral antigen distribution; (+++) strong viral antigen distribution.

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Figure 12 (A-C): Detection of viral antigen nucleoprotein (NP) in HPAIV H5N1 infected birds by IHC. (A) Trachea; (A-1) Trachea of control HPAIV H5N1 non infected birds (X=50μm); (A-2) Trachea of control HPAI H5N1 none infected birds (X=20μm); (A-3) Viral antigen in endothelial cells of trachea (arrow) (X=20μm); (A-4) Viral antigen in epithelial cells of trachea (arrow) (X=20μm). (B) Brain; (B-1) Brain of control HPAIV H5N1 none infected birds (X=50μm); (B-2) Brain of control HPAIV H5N1 none infected birds (X=20μm); (B-3) Viral antigen in endothelial cells, neurons, and glia cells of brain (arrow) (X=20μm), (B-4) Viral antigen in endothelial cells of brain (arrow) (X=20μm). (C) Lung; (C-1) Lung of control HPAIV H5N1 none infected birds (X=50μm); (C-2) Viral antigen in mononuclear cells of lung (arrow) (X=50μm), (C-3) Viral antigen in mononuclear cells of lung (arrow) (X=20μm).

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Figure 13 (A-F): Detection of nucleoprotein (NP) viral antigen in HPAIV H5N1 infected birds by IHC. (A) Pancreas; (A-1) Viral antigen in acinar epithelium of pancreas (arrow) (X=50μm); (A-2) Viral antigen in acinar epithelium of pancreas (arrow) (X=20μm). (B) Proventriculus; (B-1) Viral antigen in glandular epithelium of proventriculus (X=50μm); (B-2) Viral antigen in glandular epithelium of proventriculus (arrow) (X=20μm). (C) Spleen; (C-1) Viral antigen in lymphocytes of spleen (X=50μm); (C-2) Viral antigen in lymphocytes of spleen (arrow) (X=20μm). (D) Bursa; (D-1) Viral antigen in lymphocytes of follicular layer of bursa (X=50μm); (D-2) Viral antigen in lymphocytes of follicular layer of bursa (arrow) (X=20μm). (E) Liver; Viral antigen in VanKupffer cells of liver (arrow) (X=50μm). (F) Testis; Viral antigen in-between seminephrous tubules of testicular tissue (arrow) (X=20μm).

5. DISCUSSION

HPAIV H5N1 is still circulating and causing outbreaks with significant mortality to both commercial chickens and domestic backyard ducks in Egypt. Our data showed that vaccination with H5N1 and H5N2 vaccines may provide partial protection, especially to layers which were vaccinated twice. For both broilers and layers in commercial farms, they appeared to be protected with even one-time vaccination when compared to a mortality of over 70% in non-vaccinated chickens (Grund et al., 2011).Unfortunately, we did not perform serological evaluation of the immunological status or vaccine efficacy in chickens, therefore are unable to assess how well the mortality is correlated to the vaccination. Therefore, we cannot conclude emphatically that the much lower mortality in the layers, which were vaccinated twice, was absolutely attributed to the boosted immunity.

Vaccine efficacy may best be assessed in the setting of natural outbreaks, which apparently differs from that designed experimentally. In naturally infected free-living birds, the clinical and pathologic manifestations of an HPAIV infection may be influenced by multiple factors including the age of the bird, the dosage of virus and routes of viral exposure, the presence of concomitant infections, and the levels of immunity acquired from vaccination or during previous exposure to influenza viruses (Keawcharoen et al., 2008, Bröjer et al., 2009), which could be significantly different from trials with experimental challenges. Data about vaccine protection and efficacy from natural outbreaks may be more valuable when case studies are carefully planned and serological survey is thoroughly performed and assessed. Even though the mortalities were lowered, the pathogenicity of the infection appeared to be severe, and the virus was highly virulent in sick chickens as shown in Fig. 5 and Table 1.

Laboratory diagnosis of Influenza A virus infections is performed by viral isolation and identification followed by subtyping using hemagglutination-inhibtion test and/or RT-PCR (OIE, 2005). In this study, using viral isolation (Fig. 6), RTPCR (Fig. 7), and sequencing, we identified that the IAV that caused an outbreak in commercial chickens and backyard ducks in Sharkia province, Egypt, was of H5N1 subtype.

Complete genotype nomenclature is essential to describe gene segment reassortment (Lu et al, 2007). The reassortment of NS gene segments between different AI viruses, particularly the H5N1 subtype has been previously reported (Munir et al., 2013). Nucleotide sequences analysis of NS and H genes of our isolates showed homology of 98-99% with those of the H5N1 viruses circulating in Egypt at the time of this investigation. Genotyping tool (Lu et al, 2007), confirmed that our isolates belongs to genotypes NS1E and 5J for NS and H genes respectively which indicates that the current circulating H5N1 viruses in Egypt did not undergo reassortment of NS gene segments till time of investigation. Moreover, they harbor a different H genotype (5J) from the commercial vaccinal strains genotypes (Fig. 9), which may illustrate why vaccination failure commonly occurs, but confirming this hypothesis need further studies. Phylogenetic analysis of NS and H genes, showed that our isolates were phylogenetically distant from the H5N1 viruses isolated from Egypt in 2006, 2007, 2008, indicating the Egyptian H5N1 strain has dramatically evolved from the parental strain that hit Egypt in 2006 (Figs. 8 and 9).The phylogenetic analysis of H gene, showed that our isolates formed a uniform cluster that was not identical to the HPAIV H5 strains used for commercial vaccine development in Egypt, which may also elucidate why vaccination failure occurs, but further studies are crucial to confirm this assumption (Fig. 9).

AIV pathogenicity can be determined by calculating the intravenous pathogenicity index or by characterizing the molecular pathogenicity markers such as multiple basic amino acids located at the cleavage site of the H protein (OIE manual, 2005). Analysis of H gene amino acids sequences at cleavage site revealed that our isolates encodes a multibasic amino acid motif, 321-PQGERRRKKR*GLF-333, at the H protein cleavage site, which is a characteristic feature of all HPAIV H5N1 strains. Interestingly, we found that one of our isolates has a substitution of amino acid (R325K) at this cleavage site, to make it PQGEKRRKKR*GLF, whether this mutation could or could not have a role in the increased pathogenicity of the isolated strains, this may need further studies.

The nonstructural protein 1 (NS1), considered the main modulator of host immunity and a virulence factor, is a multifunctional protein that protect IAV against the antiviral state (Falcon et al., 2005). In this study, we found that NS1 protein of our isolates carries molecular pathogenicity determinants of HPAIV H5N1 strains including deleted 80 TMASV84 motif, Glutamate at position 92 (D92E), and C- terminus E-S-E-V motif (Seo et al., 2002, Obenauer et al, 2006) which provide another evidence that our isolates were of high pathogenicity. Glutamate at position 92 (D92E) is associated with Interferons (IFNs) down regulation and cytokine resistance (Seo et al., 2002). The NS1 protein shares in both protein-protein and protein-RNA interactions by two important domains, the N-terminal structural domain (RNA-binding domain, RBD) and the C-terminal structural domain (effector domain). In the RNA binding domain of NS1, the bases responsible for its functions were characterized as following; Thr5, Pro31, Asp34, Arg35, Arg38, Lys41, Gly45, Arg46, Thr49 (Wang et al., 1999). For keeping the RNA-binding activity of the NS1 protein Arg38 and Lys41 are necessary. Locations of Arg38 and Lys41 are highly preservative characteristic for the avian influenza virus strains. Our isolates possess arginin and lysine at positions 38 and 41 respectively. The NS2/NEP protein function depends on the nuclear export signal (NES) motif in its N terminus region. The amino acid sequence in this region is highly conserved. The amino acid sequence of NES and the NS2/NEP sequence in the A/WSN/33 viruses have previously been identified to be12 ILMRMSKMQL21 (Iwatsuki Horimoto et al, 2004). The NES sequence of the NS2/NEP H5N1 samples in this study was identified to be 12 ILVRMSKMQL21. Tryptophan at position 78 (Trp78) is necessary for export of ribonucleoprotein complexes from nucleus (Akarsu et al., 2003). In our studied strains tryptophan was located in position 78. Taken together, full deduced amino acids analysis of NS1 and NS2 proteins of the Egyptian HPAI H5N1 viruses isolated in 2013 indicated that no substitutions, deletion and/ or insertion have been yet occurred at the mostly important amino acid residues of these two proteins.

Pathogenicity of infection by HPAIV H5N1 has been studied experimentally in chickens and domestic ducks. Vietnam HPAIV H5N1 caused high mortality in two- and four-week-old SPF White Leghorn chickens (G. gallusdomesticus) (48/48, 100%) with mean death times (MDT) from 36 to 48 hrs, and two- and five-week-old Pekinwhite ducks (Anasplatyrhynchus) (63/64, 98.4%) with MDTs from 2.7 to 4.4 days (Pfeiffer et al., 2009). Pathogenicity of the Egyptian HPAIV H5N1 have been tested experimentally only in domestic ducks (Wasilenko et al., 2011). While A/ck/Egypt/08 killed 8/8 (100%) with an MDT of 4.1 days, A/ck/Egypt/07 killed 4/8 (50%) with an MDT of 7 days, indicating that HPAIV H5N1 isolates differ in their virulence. Although these two isolates are considered to have evolved from the same origin (Wasilenko et al., 2011), they are far apart within the clade 2.2 in the phylogenetic tree and have evolved different pathogenicity since the HPAIV H5N1 of clade 2.2 was introduced into Egypt. On the other hand, pathogenicity observed in these experimental challenges cannot be directly compared with that in natural outbreaks. Apparent differences exist between SPF birds with a certain age group infected experimentally by the intranasal route (IN) and poultry of various ages in commercial farms by natural exposure. The mortality rates in our report were lower in duck and even much lower in once- and twice-vaccinated chickens during the outbreaks. However, sick birds expressed severe systemic symptoms included nervous disorders in both chickens and ducks, and infection was confirmed by detection of IAV viral antigen NP.

The isolated HPAI H5N1 viruses, A/chicken/Egypt/IT- 1/2013 and A/chicken/Egypt/IT-2/2013, clearly demonstrate their pantropism in tissues of H5N1 infected chickens. The viral RNA and NP antigen were detected in multiple tissues, including trachea, lung, brain, liver, spleen, pancreas, intestines, proventriculus, bursa of fabricius, and testis in infected chickens, similar to those observed in chickens infected with the Vietnamese H5N1 virus (Pfeiffer et al., 2009). We could conclude that even though vaccination may lower mortality rates, it does not change pantropism of HPAIV H5N1 in sick birds.

Our study showed that high levels of viral RNA were detected in the brains of infected chickens (Fig. 10) and that viral NP antigen was observed in the nuclei of neurons and glial cells of the brain (Fig. 11B), clear signs of virus replication in brain, in agreement with (Brown et al., 2009; Pantin-Jackwood et al., 2009; Tang et al., 2009; Goletic et al., 2010) who previously described neurotropism of HPAIV H5N1 strains. Different pathways have been proposed by which the HPAI H5N1 virus infects the central nervous system (CNS) in chickens. It has been hypothesized that the virus could reach the CNS through the olfactory nerves (Majde et al., 2007), the peripheral nervous system (Tanaka et al., 2003; Matsuda et al., 2004), or even the bloodstream (Mori et al., 1995). Interestingly, we observed the viral NP antigen in endothelial cells of brain, which provides direct evidence that the HPAIV H5N1 likely invades the CNS by replicating in blood vessels in the brain, and contributes to the development of severe nervous symptoms. Based on our evidence, we consider this to be one of the routes for HPAIV H5N1 to invade CNS. Severe CNS disorders in birds are probably one of the main causes for mortality when neurons are infected; massive edema due to virus infection-induced altered vascular permeability and multi-organ failure are commonly blamed for high mortality in HPAIV infected birds.

Mechanisms for viral penetration of the blood-brain barrier in the brain have been investigated previously. The virus may invade neurons through the opening of endothelial cell junctional complexes (para-cellular route) (Lossinsky & Shivers, 2004), or through vesiculo-tubular structures (trans- cellular route) (Liu et al., 2002). It could reach the vessels in the brain through the bloodstream, or via a “Trojan horse mechanism” where viral particles are transported through infection of leukocytes and/or mononuclear cells (Verma et al., 2009). In our study, viral RNA or antigen was detected in blood or sera and infected macrophages and monocytes, which suggests that the endothelial cells may play a crucial role in viral penetration of the blood-brain barrier, leading to severe necrotizing encephalopathy and death. Our finding that endothelial cells of the cerebellum were also strongly positive in viral NP antigen supports this hypothesis (Fig. 11B4).

The viral antigen was strongly expressed in the acinar epithelium of pancreas, giving rise to the possibility of a potential role of the pancreas in viral pathogenesis. Evidence showed that the virus also replicated in lymphocytes of follicular layer of the bursa, which may be significant in inducing immunity against the virus, a key process for recovery of sick birds from infection. A remarkable isolation of virus from testicular tissue samples explained the severe embryonic hemorrhages, congestion, and deaths within 48hrs post-infection (Fig. 6A). Moreover, high viral RNA levels were detected from testicular tissue (Fig. 10), with viral NP antigen expressed in between or inside seminephrous tubules or even sticking to sperms (Fig. 12F). However, sexual transmission for apparently healthy cocks to spread HPAIV H5N1 during the incubation period is probably a scenario of low or unlikely probability. It is likely that sperm collected from infected cocks with healthy appearance could disseminate virus to both uninfected birds, and farm handlers and workers either via natural insemination or during application of artificial insemination.

In conclusion, this study, firstly, identified that the outbreak that appeared in commercial chickens and backyard ducks in Sharkia province, Egypt, 2013 was attributed to HPAIV H5N1infection. Secondly, genetic and amino acid analysis of H gene at cleavage site indicated that they carry molecular determinants of HPAIV strains. Moreover, H protein of our isolates belongs to genotype (5J) which is different genotype from those strains used in some available commercial vaccines currently used in Egypt. Thirdly, genetic and amino acid analysis NS gene of viral isolates indicated that they belong to genotype NS1E with no reassortment between H5N1 subtype and other subtypes currently circulating in Egypt. The amino acids residues of NS-1 and NS-2 proteins of our strain did not show progressive evolution as we did not detect amino acids substitution, deletion and or insertion at the most important motifs of the NS1 protein. Fourthly, by IHC we confirmed the pan-tropism of the Egyptian HPAIV H5N1 in naturally infected chickens where endothelial cells, mononuclear cells, and testicular tissues expressed obvious viral antigen. Detection of viral antigen in endothelial and mononuclear cells reflects that the virus may have disseminated in all birds tissues via these cells. Moreover, expression of viral antigen in brain tissues suggests that severe necrotizing encephalopathy may be at least one of the possible causes of death of birds, if not the only cause.

By end of this study, we recommend further studies on continued subtyping and full genome characterization of IA viruses currently circulating in the Egyptian poultry field and also, functional characterization of NS1protien to identify its specific role in virulence of HPAI viruses. Vaccine efficacy studies, possibility of sexual transmission of IAV viruses, and pathogenicity of HPAIV in naturally infected ducks are also necessary studies.

6-SUMMARY

Highly pathogenic avian influenza virus (HPAIV) H5N1 has been endemic in Egypt since 2006 and raised concern recently for its potential to evolve and be of highly transmissible among humans. Infection of HPAIV H5N1 has been described in experimentally challenged birds. However, pathogenicity of HPAIV H5N1, isolated in Egypt, has not been reported in naturally infected chickens and ducks, which could be unique due to distinct transmission routes and dosage of infection. Here we report a recent outbreak of HPAIV H5N1 in 2013, in commercial poultry farms in Sharkia Province, Egypt. The main symptoms were ecchymoses on the shanks and feet, cyanosis of the comb and wattles and subcutaneous edema of head and neck for chickens, and nervous signs (torticollis) for ducks. Within 48-72 hrs of the onset of illness, the average mortality rates were 22.8-30% and 28.5-40% in vaccinated chickens and non- vaccinated ducks, respectively. Tissue samples of chickens and ducks were collected for cross-section immunohistochemistry and realtime RT-PCR for specific viral RNA transcripts. Higher viral RNA transcripts were detected in tissues of chicken broilers and layers, including trachea, lung, spleen, intestine, brain, and serum, than those of ducks which have only viral RNA transcripts in trachea, lung, and liver tissues. In chickens, the highest viral RNA levels appeared to be in brain, trachea, and serum with significant differences detected between chicken broilers and layers in theses tissues in particular. Significant differences of the viral RNA were observed in trachea, lung, and liver tissues of ducks than those of chickens, indicating that HPAI H5N1 replicates with distinct tissue tropism between chickens and ducks. However, these samples from different birds cannot be compared because they came from natural outbreaks with uncertain timing of the course of infection. While the viral RNA was nearly detected in all tissues and serum collected indicating viral pan-tropism, the viral antigen was detected almost ubiquitously accordingly in all tissues including testicular tissues. Interestingly, viral antigen was also observed in endothelial cells of the most organs, and seen clearly in trachea and brain in particular as well as in mononuclear cells of various tissues particularly lungs. We performed phylogenetic analyses and compared the genomic sequences of the surface hemagglutinin (H) and non structural protein 1(NS1) among the isolated viruses, the HPAI H5N1 viruses circulated in Egypt in the past and currently, and some available commercial vaccinal strains. Analysis of deduced amino acids of both HA and NS1 revealed that our isolates carry molecular determinants of HPAI viruses, including the multibasic amino acids at the cleavage site in HA and glutamate at position 92 (D92E), C - terminus E-S-E- V motif, and the deletion at position 80-84 in NS1 protein. Taken together, this is the first study about pathogenicity of the HPAIV H5N1 strain, currently circulating in Egypt, from naturally infected poultry, which provides unique understanding of the viral pathogenesis in HPAIV H5N1 infected chickens and ducks.

In conclusion, this study, firstly, identified that the outbreak that appeared in commercial chickens and backyard ducks in Sharkia province, Egypt, 2013 was attributed to HPAIV H5N1infection. Secondly, genetic and amino acid analysis of H gene at cleavage site indicated that they carry molecular determinants of HPAIV strains. Moreover, H protein of our isolates belongs to genotype (5J) which is different genotype from those strains used in some available commercial vaccines currently used in Egypt. Thirdly, genetic and amino acid analysis NS gene of viral isolates indicated that they belong to genotype NS1E with no reassortment between H5N1 subtype and other subtypes currently circulating in Egypt. The amino acids residues of NS-1 and NS-2 proteins of our strain did not show progressive evolution as we did not detect amino acids substitution, deletion and or insertion at the most important motifs of the NS1 protein. Fourthly, by IHC we confirmed the pan-tropism of the Egyptian HPAIV H5N1 in naturally infected chickens where endothelial cells, mononuclear cells, and testicular tissues expressed obvious viral antigen. Detection of viral antigen in endothelial and mononuclear cells reflects that the virus may have disseminated in all birds tissues via these cells. Moreover, expression of viral antigen in brain tissues suggests that severe necrotizing encephalopathy may be at least one of the possible causes of death of birds, if not the only cause.

Last but not least, this study gives insights into pathogenesis of HPAIV in naturally infected birds which may be different from that obtained from experimentally infected birds due to distinct viral dosage and route of infection, distinct age and immunity of birds, and possibility of presence of contaminant infection. Thus it can serve as an augmentation to and in comparison with experimental studies. This study is also important to the veterinarians to perform accurate diagnosis on the actual field samples.

By end of this study, we recommend further studies on continued subtyping and full genome characterization of IA viruses currently circulating in the Egyptian poultry field and also, functional characterization of NS1protien to identify its specific role in virulence of HPAI viruses. Vaccine efficacy studies, possibility of sexual transmission of IAV viruses, and pathogenicity of HPAIV in naturally infected ducks are also necessary studies.

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Pages
149
Year
2015
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9783668042186
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Language
English
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v301947
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molecular avian influenza virus

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Title: Molecular characterization for non structural proteins of Avian Influenza Virus