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Journal of Virological Methods 159 (2009) 244–250

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Characterization of an avian influenza virus H5N1 Egyptian isolate

M.M. Bahgata, M.A. Kutkatb, M.H. Nasraaa, A. Mostafaa, R. Webbyc, I.M. Bahgatd, M.A.A. Ali a,∗

a Virology Lab., Infectious Diseases and Immunology Group, the Center of Excellence for Advanced Sciences, the National Research Center, 12311 Dokki, Giza, Egyptb Department of Poultry Diseases, Veterinary Research Division, National Research Center, 12311 Dokki, Giza, Egyptc Department of Infectious Diseases, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105-2794, USAd Department of Biology, Faculty of Education, Seuss Canal University, Port Saied, Egypt

Article history:Received 11 October 2008Received in revised form 10 April 2009Accepted 20 April 2009Available online 3 May 2009

Keywords:Avian influenza virusH5N1Egyptian isolateVaccine

a b s t r a c t

The highly pathogenic influenza virus H5N1 that infected chickens in Egypt in 2006 was characterized atimmunologic and molecular levels. Cloacal swabs from chicken were analyzed by rapid antigen detectionand RT-PCR using H5- and N1-specific primers, which confirmed the presence of an H5N1 influenza virusin infected chickens. Sequencing results revealed 100% homology of both genes with previously publishedsequences of H5N1 isolates from Egypt and the Middle East. The virus was isolated and propagated inMDBK cells in culture. Host cells showed a substantial cytopathic effect within 2 days of infection, whichincreased dramatically by the fourth day. Plaque infectivity titers of virus harvested from cell culturewere initially 105 PFUs/ml and increased to 108 PFUs/ml after two additional passages and ultrafiltration.Formaldehyde treatment completely inactivated the virus, and MDBK cells inoculated with the killed virusshowed no cytopathic effect. Two days after chickens were immunized with the killed virus, their serashowed that the killed Egyptian isolate was highly immunogenic. Western blot analysis showed that serahad antibodies reacting to four viral peptides: hemagglutinin (61.5 kDa), RNA-binding protein (56 kDa),neuraminidase (50 kDa), and 45-kDa protein. In a challenge infection, the vaccine protected immunizedchickens from death and reduced viral shedding.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Historically, influenza viruses have caused multiple pandemicsin the human population, resulting in massive numbers of deaths.The influenza virus continues to pose a significant threat to pub-lic health. Waterfowl and poultry movement in Eurasia, Egypt, andAfrica has caused the spread of H5N1 avian influenza viruses (AIVs),which threatens the health of not only avian species but also mam-malian species, including humans.

During the past decade, several AIVs, including H5N1, H7N7,H9N2, and H7N3, have infected humans (Trampuz et al., 2004).H5N1 HPAIV was first identified in 1996 as the causative agent of thegeese outbreak in the Guangdong Province, China (Xu et al., 1999);in 1997, the same subtype caused outbreaks in chickens, with 18human infections reported in Hong Kong. The 1997 H5N1 outbreakin Hong Kong was controlled by mass slaughter of all poultry infarms and markets (1.5 millions), which prevented transmission ofthe virus (Subbarao et al., 1998; Yuen et al., 1998).

Despite the implementation of active control measures, the virushas since become endemic in Asia, causing outbreaks in poultry(Li et al., 2004). In 2005, H5N1 infection spread to Europe, the

∗ Corresponding author.E-mail address: mohamedahmedali2004@yahoo.com (M.A.A. Ali).

Middle East, and Africa (Chen et al., 2005; Peiris et al., 2007).Human infection is relatively rare but frequently fatal; most humanshave acquired infection by contact with infected poultry, but a fewcases of suspected human-to-human transmission have also beenreported (Horimoto and Kawaoka, 2001; Webster et al., 2006).

The possibility that the highly pathogenic AIVs (HPAIV) H5N1are now endemic to both domestic and migratory birds makes thecontrol of such infections more difficult. In addition, as ducks arenot always killed by H5N1 infection, they are considered a majorcontributor to viral spread (Webster et al., 2006). From 2003 to2008, 6414 H5N1 outbreaks were reported in 61 countries in bothdomestic poultry and wild birds (OIE, 2008). Also, more than 209million poultry have died since January 2004 (FAO/OIE/WHO FAO,2005).

In mid-February 2006, H5N1 infection was reported in Egyptamong domestic poultry in more than 15 governorates, resultingin severe losses for the poultry industry. This sudden increase inH5N1 activity occurred without prior identification of any deathsof wild birds in those regions. The Egyptian government initiateda poultry stamping out program that appeared to limit the 2006outbreak; however, H5N1 viruses reemerged in a series of avianepidemics in several governorates in 2007 and 2008. Poor hygiene,lack of awareness, and uncontrollable, random raising of domesticpoultry, especially in the rural areas of Egypt, increases the chancesof virus transmission.

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From 2006 till date, there have been 1084 outbreaks of H5N1among poultry in Egypt (OIE, 2008). According to data published in2008 by the WHO, 22 of 50 humans infected with H5N1 in Egyptdied: 10 of 18, 9 of 25, and 3 of 7 in 2006, 2007, 2008, respectively.These infected humans were in close contact mainly with domesticpoultry in rural areas.

Because no vaccines have been prepared from local Egyptianisolates, the Egyptian control program for HPAIVs is depen-dent on the use of imported vaccines. The commercial vaccinesavailable currently for H5 AIVs include two oil-emulsion for-mulations: one derived from killed low-pathogenic H5N2 virusA/chicken/Mexico/232/94/CPA and the other from a reassortantH5N1 virus from China. A major problem with using these vac-cines was that they were not evaluated for their protective efficacyagainst a challenge infection with Egyptian isolates before theirintroduction to the market. The absence of this important stepwas probably the reason for the disappointing results of protec-tive efficacy for both vaccines. For vaccines to have a high efficacy,the strains used to produce the vaccine must be sufficiently closelyrelated to the circulating strains to ensure the induction of effec-tive protective immunity against infection (Hoffmann et al., 2002).A recent study that made a phylogenetic comparison between the2006 and 2008 Egyptian isolates of H5N1 (Ali et al., unpublished)has clearly demonstrated that emergence of significant nucleotidechanges, particularly in the HA domains, might result in changes inthe antigenicity of isolates.

Although it has been reported that inactivated oil-emulsion vac-cines can reduce the transmission of H5N1 AIVs (Ellis et al., 2004),there are concerns that these vaccines may not be completely effec-tive practically and may not prevent virus shedding and spread.

Vaccination with high-efficacy vaccines can substantially helpcontrol AIV outbreaks when administered as part of a com-prehensive control program that includes quarantines; animalmovement controls; and improved biosecurity, surveillance, andeducation. Therefore, adequate tools are needed to monitor vac-cination programs, such as detection of serologic conversion anddifferentiation of infection from vaccinated animals (DIVA). Onelimitation of the killed-virus vaccine approach is that vaccinatedand naturally infected birds are difficult to detect by com-monly available diagnostic tests, making the surveillance of AIVsdifficult.

Several DIVA strategies have been proposed to overcome diag-nostic limitations. The first strategy is to use subunit vaccinestargeted to only the hemagglutinin protein. This approach usesserologic detection of antibody responses to other viral proteinsas a diagnostic marker of infection (Ghedin et al., 2005). A sec-ond similar strategy is to vaccinate birds with an inactivated virusthat contains a hemagglutinin homologous to that in the circulatingstrain and a neuraminidase (NA) of a heterologous subtype (Capuaet al., 2004). Serologic surveillance can then be performed for thehomologous NA as evidence of natural infection and for the heterol-ogous NA as evidence of vaccination. A third strategy is to measurethe serologic response to the nonstructural protein 1 (NS1), whichis produced in large quantities in infected cells but is not packagedin the virion. Inactivated influenza vaccines for agricultural use areprimarily made with whole virions, thus allowing the detection of adifferential NS1 antibody response between naturally infected andvaccinated animals (Ghedin et al., 2005; Zhao et al., 2005).

Egypt is part of the African continent and the Middle East, whereseveral outbreaks of AIVs have been recently reported; therefore,characterizing the causative agent of the epidemics in Egypt is ofmajor importance to employ appropriate control strategies in thisentire region. In this study, we have characterized at the molecu-lar and immunologic levels the HPAIV H5N1 that caused the 2006outbreak in Egyptian poultry. We have also assessed the ability ofan oil-emulsion preparation of the killed virus to induce immunity

in chickens and the diagnostic value of the killed virus to monitorantibody titers in vaccinated chickens.

2. Materials and methods

2.1. Cloacal swabs

To isolate the virus causing the outbreak, cloacal swabs from25 chickens were randomly taken at a poultry farm in the Qalu-bia Governorate (one of the suburban governorates that borderswith Cairo), which reported a marked increase in death (as manyas 1170 deaths per day). Swabs were placed in Dulbecco’s mini-mum essential medium (DMEM; Bio Whittaker, Combrex Company,Belgium) containing an antibiotic–antimycotic mixture and trans-ported to the laboratory on ice. All the collected samples weretested individually for the presence of the viral antigen by usingthe rapid antigen-detection kit for influenza viruses type A andspecifically for H5 hemagglutinin antigen (Animal Genetics, Inc.,Korea).

2.2. Oligonucleotides, cDNA synthesis, and RT-PCR amplification

Viral RNA was extracted from a 200-�l sample of cloacal mate-rial by using a Biozol RNA extraction reagent, according to themanufacturer’s instructions (BioFlux, China). Reverse transcrip-tion was performed by heating a mixture of 9 �l viral RNA, 1 �l(250 ng) pd(N)6 random hexamer (Amersham Pharmacia Biotech,Cat No. 32970), and 4 �l DEPC-treated water at 65 ◦C for 5 min.The tubes were chilled on ice for 2 min. A mixture of 2 �l dNTPs(Finzyme; 2.5 mM each), 1 �l (40 U) RNasin (Promega), 2 �l 10×reverse transcriptase (RT) buffer (Finzyme), and 1 �l AMV RT(20 U, Finzyme) was then added. For cDNA synthesis, the tubeswere incubated in the thermal cycler (GeneAmp® PCR system9600; Applied Biosystems) at 42 ◦C for 30 min linked to a heat-ing step at 70 ◦C for 10 min to inactivate the RT. Amplificationsof both H5- and neuraminidase (N1)-specific partial sequences ofthe virus were performed in 50 �l PCR reaction mixtures usingspecific primers for both genes (Wright et al., 1995; Yuen etal., 1998). Each PCR reaction contained 5 �l cDNA, 5 �l 10× PCRbuffer, 1 �l MgCl2 (25 mM), 0.5 �l GoTaq-DNA polymerase (5 U/�l;Promega), 1 �l forward primer (200 nM/�l) and 1 �l reverse primer(200 nM/�l), and 5 �l dNTPs (2.5 mM each). The primers used forH5 amplifications were H5-F (5′-GCCATTCCACAACATACACCC-3′)and reverse H5-R (5′-CAAATTCTCTATCCTCCTTTCCAA-3′) with anexpected product size of 358 bp (Yuen et al., 1998). The primers usedfor N1 amplifications were N1-F (5′-TTGCTTGGTCAGCAAGTGCA-3′) and reverse N1-R (5′-TCTGTCCATCCATTA GGATCC-3′), with anexpected product size of 615 bp (Wright et al., 1995). Condi-tions for partial H5 gene amplification included an initial 3-mindenaturation step at 94 ◦C and 40 cycles of denaturation at94 ◦C for 1 min, annealing at 55 ◦C for 1 min, and extension at72 ◦C for 1 min. An additional extension step at 72 ◦C for 7 minwas done to complete the amplification. The amplification ofN1 differed only in annealing time (2 min) and extension time(3 min). Amplified RT-PCR products were electrophoresed on a 1.5%agarose gel.

2.3. DNA sequencing and sequence analysis

The full-length amplified H5 gene was sequenced using dideoxy-nucleotide cycle sequencing and products were run on anautomated sequencer. The resulting sequence was analyzed by mul-tiple alignment with the published H5 sequences of H5N1 isolatesfrom Egypt in the GenBank (ncbi.nlm.nih.gov/nucleotides), usingthe basic nucleotide blast “NCBI/BLAST/blastn suite.”

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2.4. Infection of mammalian cells in culture

Madin–Darby bovine kidney (MDBK) cells were grown in DMEMsupplemented with 10% fetal calf serum (FCS; Biochrome KGBerlin, Germany), 1% penicillin/streptomycin mixture (BiochromeKG), and 250 mg/l Fungizone (Gibco-BRL Life Technologies, GrandIsland, NY). Prior to infection, the DMEM was discarded from theadherent cell layer and cells were treated briefly with 0.5 �g/mltrypsin–EDTA (Sigma, Deisenhofen, Germany). Swab samples wereclarified by low-speed centrifugation and decontaminated by theantibiotic–antimycotic mixture. Trypsin-treated cells were inocu-lated with the decontaminated samples and then incubated at 36 ◦Cfor 1 h for virus adsorption. Maintenance medium was then added,and samples were incubated at 37 ◦C in 5% CO2. Cells were observeddaily under a light microscope to assess the presence of cytopathiceffects.

2.5. Production of cell culture-based inactivated vaccine

Isolated virus was reinoculated onto MDBK cells to prepare 1 lof virus stock. Cell supernatants were harvested and viral titerswere determined by using plaque infectivity assays. Briefly, in a6-well sterile plate, MDBK monolayers were grown and inoculatedwith serial dilutions of the virus containing 0.5 �g/ml trypsin andincubated at 37 ◦C in 5% CO2 for 1 h. A 3-ml 2× agarose overlayerwas then added to each well and left to solidify at room temper-ature. Plates were incubated and observed for plaque formation(Rashad and Ali, 2006). To concentrate viral stocks, infected tis-sue culture supernatants were purified by ultrafiltration througha polysulfone hollow fiber cartridge (50-kDa cut-off) (Amersham).This resulted in a 10-fold increase in viral titers, as measured bythe plaque assay. Virus was inactivated by formaldehyde and thentreated with sodium thiosulfate solution to remove the residualformaldehyde. Vaccine was prepared by mixing the concentratedvirus with Negilla sativa oil, which induces cellular and humoralimmunity when used as an adjuvant (Haq et al., 1999). A representa-tive volume of the virus stock was inoculated onto cell cultures; theabsence of cytopathic effect after 1 week of observation indicatedsuccessful inactivation of the stock.

2.6. Vaccination of chickens

A total of five 3-day-old chicks were immunized intramuscularlywith a 0.2-ml solution containing 107 PFUs of inactivated virus. Toensure the specificity of the immune response, 20 control chickswere immunized with lysates of untreated MDBK host cells and20 were not immunized. Chickens received a booster dose of thevaccine 3 weeks after the first immunization. Serum samples werecollected from the three groups before immunization and thenweekly post-immunization.

2.7. Detection of IgG response by ELISA

Influenza-specific antibodies were detected by ELISA accordingto the method of Bahgat et al. (2006), with minor modifica-tions. Briefly, the ELISA plate was coated (100 �l/well) with killedvirus (108 PFUs/ml) diluted 1:1 in coating buffer (1 M Na2CO3; 1 MNaHCO3, pH 9.6) and then incubated at 37 ◦C for 3 h. Plates werewashed thrice with PBS–0.05% Tween 20 (PBST). Nonspecific bind-ing was blocked by incubating the plates with PBST–1% bovineserum albumin (PBST–BSA; 200 �l/well) at 37 ◦C for 2 h. After wash-ing thrice with PBST, wells were loaded with diluted chicken serain PBST–FCS (1:50; 100 �l/well) and plates were incubated at 37 ◦Cfor 2 h. Plates were washed thrice and then incubated with 1:2000(100 �l/well) anti-chicken IgG conjugated to horseradish peroxi-dase (Kirkgaard & Perry Laboratories; Gaithersburg, Germany) at

37 ◦C for 2 h. Plates were again washed thrice with PBST. For colordevelopment, 100 �l/well OPD substrate (Sigma) was diluted intosubstrate buffer (0.1 M anhydrous citric acid and 0.2 M dibasicsodium phosphate, pH 5.0) containing 0.06% H2O2. Plates were leftfor 10 min at room temperature until color developed. The enzy-matic reaction was stopped with 40 �l/well of 2 M HCl, and thechange in optical density (OD) was recorded at a �max of 490 nmusing a multiwell plate reader (TECAN; SUNRISE, Austria, GmbH).

2.8. Western blot analysis of viral antigens

Concentrated killed virus was analyzed by SDS-PAGE (sodiumdodecyl sulfate polyacrylamide gel electrophoresis) (Laemmli,1970) through 4% stacking and 16% resolving gels in 0.75-mmthick vertical slab gels. Inactivated virus (108 PFUs/ml) diluted 1:1in sample buffer (0.125 M Tris base, 4% SDS, 20% glycerol, 10%mercaptoethanol, and 0.1% bromophenol blue as a tracking dye)was heated in a boiling water bath for 5 min and loaded ontothe gel. Low-range-molecular-weight marker (Mr) proteins from14.5 to 97 kDa (Bio-Rad Laboratories) were also run on the samegel. After electrophoresis, proteins were electrically transferred(Towbin et al., 1979) from the gel to a BA85 nitrocellulose sheet(pore size, 0.45 mm; Schleicher and Schuell, Dassel, Germany) at6 V/cm and 250 mA overnight at 4 ◦C in the transfer buffer (0.025 MTris, 0.186 M glycine, and 25% methanol). The strip carrying theMr was cut, stained with Amido Black (Sigma), and destainedwith 60% methanol to visualize the bands. Immunorecognitionwas performed on cut membrane strips carrying chicken sera(dilution 1:50). Incubation and washing conditions were used asdescribed previously (Ruppel et al., 1985), the only modificationbeing that 1% BSA (Serva, Heidelberg, Germany) in PBS-0.3% T wasused to block the protein-free binding sites on the nitrocellulosemembrane. Immune detection was carried out with peroxidase-conjugated goat anti-chicken IgG (Kirkgaard & Perry Laboratories;Gaithersburg, Germany) diluted 1:2000 in PBS-0.3% T. Immunecomplexes on the nitrocellulose membrane were visualized bydeveloping the strips with 0.01 M PBS (100 ml, pH 7.4) contain-ing 50 mg 3,3′-diaminobenzidine (Sigma) and 100 ml of 3% H2O2.Molecular weights of the immunogenic viral peptides were deter-mined by comparing their migration fronts to those of the Mr.

2.9. Challenge infection

Four weeks after initial infection, vaccinated chickens and con-trol chickens were challenged intramuscularly with 107 PFUs ofhomologous virus particles. Viability of the chickens was monitoredby daily visual inspection. Viral antigen shedding in their feces wasmonitored daily by using a rapid H5N1 antigen-detection kit. Allexperiments involving the handling of live virus, culturing, con-centration, and challenge infection were carried out in biosafetycontained conditions and all precautions were taken to avoid spreadof the virus.

2.10. Statistical analysis

Statistical analysis, including means and standard deviations,was performed by the Student’s t-test, using the GraphPad InStatsoftware (GraphPad Instat, San Diego, CA).

3. Results

3.1. Symptoms in infected birds and propagation of the virus

The signs of severe AIV infection in chickens included severedepression, inappetence, facial edema, swollen cyanotic comb andwattles, and diffused hemorrhage in the areas between the hocks

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Fig. 1. RT-PCR analysis of the H5N1 antigen-positive swabs, using specific primersfor partial sequences of the H5 and N1 genes. The amplification products for H5and N1 appeared at the expected molecular weights of 358 bp (lane 2) and 615 bp(lane 3), respectively. The molecular weights were verified by including a 100-bpmolecular weight ladder (lane 1) on the same gel.

and feet. Subcutaneous edema in the head and neck regions, severecongestion of the musculature, and pinpoint petechial hemorrhageon the inside of both the keel and peritoneum were also observed.Cloacal swabs were taken from birds that appeared to have AIVinfection and analyzed by rapid antigen detection. Samples thatwere positive for H5 were confirmed by RT-PCR using specificprimers for partial sequences of both H5 and N1 genes. The amplifi-cation products were identified at the expected molecular weightsof 358 bp for H5 and 615 bp for N1 (Fig. 1).

Virus was isolated from RT-PCR-confirmed samples and thenpropagated in MDBK cells. Two days after inoculation, cell culturesshowed a marked cytopathic effect, which increased dramaticallyby Day 4 (Fig. 2A). Plaque infectivity titers of the virus harvestedfrom infected cells at Day 4 showed approximately 105 PFUs/ml.

Table 1IgG responses in sera from vaccinated chickens.

Source of sera IgG reactivitya (±SD) P-value†

Chicks with maternal immuneresponse (n = 11)

1.51 (0.97) 0.006

Chicks vaccinated with killed EgyptianH5N1 vaccine (n = 5)

0.84 (0.34) 0.006

Chicks vaccinated with commercialH5N2 vaccine (n = 31)

1.47 (0.59) <0.0001

a IgG reactivity was determined by measuring OD at 490 nm.† P-values were calculated by comparing the IgG reactivity from experimental

groups with that from control animals (n = 5) vaccinated with lysate from uninfectedMDBK cells (mean, 0.074 ± 0.039).

After two further passages on MDBK cells, the viral titer increased toapproximately 107 PFUs/ml. After concentration by ultrafiltration,a viral titer of 108 PFUs/ml was achieved (Fig. 2B).

When products of the amplified full-length H5 gene were ana-lyzed by automated sequencing followed by blasting the obtainednucleotide sequence with the available sequences till 2006 onGenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi.), the sequenceshowed 100% homology with previously published H5 sequencesof H5N1 isolates from Egypt and the Middle East (data not shown).The sequence has been submitted to GenBank, with the accessionnumber FJ472343.

The purified virus stock was completely inactivated by treat-ment with formaldehyde, as shown by absence of the cytopathiceffect when a representative volume of the inactivated virus wasincubated for 10 days on MDBK cells (data not shown).

3.2. IgG responses in vaccinated chicken sera

To determine the IgG response of chickens vaccinated with theinactivated virus, ELISA was performed on serum samples takenat various points before and after vaccination. The killed Egyp-tian H5N1 isolate was used to detect antibodies to viral proteinsin sera from chickens vaccinated with either the homologous anti-gen or a commercially available oil-emulsion killed H5N2 vaccine(Fig. 3). Interestingly, serum samples taken from unimmunized 3-day-old chicks had detectable H5-specific IgG titers (Fig. 3A andTable 1), which dropped to background levels 1 week thereafter.These IgG responses were most likely because of maternal IgGantibodies transmitted from vaccinated mothers. Three-day-oldchicks vaccinated with the killed Egyptian H5N1 isolate had nodetectable antibodies after boosting, whereas those that receivedtheir first vaccination at 10 days of age showed marked serocon-version upon boosting (Fig. 3A and Table 1). These data suggest

Fig. 2. Propagation of the H5N1 Egyptian isolate in MDBK cells in culture. (A) The cytopathic effect was recorded 2 days postinfection and dramatically increased on thefourth day. (B) Plaque infectivity titration assay of the virus harvested from cell culture showed approximately 105 PFUs/ml, which increased to approximately 107 PFUs/mlafter two passages in MDBK cells (I) and to approximately 108 PFUs/ml after concentration by ultrafiltration (II). Uninfected MDBK cells were included as a negative control(dish C in I) in which no plaques were observed after crystal violet staining.

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Fig. 3. Enzyme-linked immunosorbent assays of IgG responses in chicken sera. (A)IgG reactivity was measured in sera from 3-day-old chicks prior to immunizationor from 10-day-old chicks that were vaccinated with killed H5N1 Egyptian isolate, acommercial killed H5N2 vaccine, or untreated MDBK lysate (control). IgG reactivitywas high in the untreated 3-day-old chicks, indicating transmission of maternal IgG(triangles) from vaccinated mothers. The killed Egyptian isolate was highly antigenic.The IgG response of chicks vaccinated with the killed Egyptian H5N1 isolate (closedcircles) was much higher than that in controls (open circles). Sera from chicks thatreceived three vaccinations of commercial H5N2 vaccine showed the highest IgGlevels (squares). (B) Chickens that received multiple vaccinations of the commer-cial vaccine could be classified into three groups according to their IgG reactivities:strong reactors (triangles; OD490 > 1.5), moderate reactors (squares; OD490 < 1.5 or>1.0), and poor reactors (circles; OD490 < 1.00).

that maternal antibodies can interfere with the vaccination ofnewborns.

Sera from chickens (n = 30) that received three doses of the com-mercial H5N2 vaccine showed the highest IgG levels. Vaccinatedchickens in this group were classified into three distinct groups onthe basis of their IgG reactivity: (1) high good reactors (OD490 > 1.5),(2) moderate reactors (OD490 < 1.5 or >1.0), and (3) poor reactors(OD490 < 1.0) (Fig. 3B).

3.3. Killed H5 vaccines induce immunity to four viral antigens

The specificity of the antibodies raised in response to vaccina-tion was determined by Western blot analyses. Serum samples fromvaccinated birds were used to probe blots containing antigens of thekilled Egyptian H5N1 isolate (Fig. 4). The sera groups included serafrom chickens (1) with high maternal antibody titers, (2) vaccinatedwith two doses of the killed Egyptian H5N1 isolate, and (3) vacci-nated with three doses of the commercial oil-emulsion killed H5N2vaccine. All serum samples contained antibodies that bound to fourviral peptides: hemagglutinin (61.5 kDa), the major surface glyco-protein; RNA-binding protein (56 kDa); neuraminidase (50 kDa), asurface glycoprotein; and a 45-kDa unidentified protein that mightrepresent HA1 (Andrea et al., 1992). These results indicate thatimmunization with the killed vaccines induced immunity to multi-ple viral antigens. None of the peptides were detected by antibodiesin sera from control animals (Fig. 4).

Fig. 4. Western blot analysis of H5N1 antigens in sera from vaccinated chickens. Serawas collected from 3-day-old chicks that had high maternal IgG (lane 1) and 10-day-old chicks vaccinated with killed H5N1 Egyptian isolate (lane 2) or multiple doses ofthe commercial oil-emulsion killed H5N2 vaccine (lane 3). Sera from all three groupsstrongly reacted to four H5N1 peptides: hemagglutinin (61.5 kDa), RNA-binding pro-tein (56 kDa), neuraminidase (50 kDa), and an unidentified 45-kDa protein. None ofthe peptides were detected by the sera from control chickens immunized with thelysates of untreated MDBK cells (lane 4).

3.4. Killed Egyptian H5N1 isolate effectively protects againstchallenge infection

To determine the level of protection from subsequent viralinfection provided by the killed H5N1 vaccine, chickens initiallyvaccinated with the killed Egyptian isolate or untreated MDBK celllysates (control) were challenged with 107 PFUs/ml of homologouswild-type virus 4 weeks after the initial vaccination. All control ani-mals succumbed within 48 h of the challenge infection. In contrast,none of the five birds that were initially immunized with the killedvirus died after the challenge. After the challenge was administered,animals that had been previously vaccinated with the killed Egyp-tian H5N1 vaccine still shed virus, but with a lower intensity thancontrol animals did before they died (data not shown).

4. Discussion

The evolution of influenza is a continuing process that involvesviral and host factors. The increasing frequency with which highlypathogenic H5N1 that can be potentially transmitted to humans isemerging is of great concern to both veterinary and human publichealth officials. The important question is how soon the next pan-demic will emerge. A combination of factors, including populationdensities of poultry, pigs, humans, and other possible hosts, possi-

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bly affects the evolution of the virus. Highly concentrated poultryand pig farming, in conjunction with traditional live animal or “wet”markets, provides optimal conditions for increased mutation, reas-sortment, and recombination of influenza viruses.

In poultry, neutralizing antibodies to both HA and NA canprovide primary protection against disease, and the cytotoxicT-lymphocyte response can reduce viral shedding in mildlypathogenic avian influenza viruses but provides questionable pro-tection against HPAI; however, the specific contribution of cellularimmune responses in protection remains unknown (Suarez andSchultz-Cherry, 2000). As the continuity of the avian influenza virusto exist relies on escaping host immunity through antigenic alter-ations that arise through genetic drift point mutations of the HAgene or through genetic shift by reassortment exchange of the HAligand with that of viruses retained in avian or nonavian species, thebattle seems to be ongoing as the host tries to update the specificityof its humoral response to infection by the most recent viruses thathave altered antigenic specificities (Hilleman, 2002).

Since their emergence in several countries in 2003, H5N1 viruseshave continued to circulate and evolve into multiple lineages. It istherefore imperative to characterize these viruses at the regionallevel. In this study, we characterized the growth and immunologicproperties of the H5N1 AIV responsible for a lethal outbreak inchickens in Egypt in 2006 in order to assess whether the inactivatedstrain could be used as an effective vaccine against subsequentoutbreaks. We also tested the utility of the rapid antigen detec-tion assay for monitoring AIV infection in a field setting. Using thismethod, we efficiently identified the H5 antigen from naturally andexperimentally infected chickens. H5-specific RT-PCR amplified anamplicon of the expected size, indicating the nature of the virus(Wright et al., 1995; Yuen et al., 1998), which was later confirmed byfull-length sequencing of H5. The rapid antigen detection methodfor primary field screening of influenza type A virus and specificallythe H5 antigen was successful to monitor avian influenza infection.

As several studies have demonstrated the efficacy of oil-emulsion vaccines to protect against influenza (Abraham et al.,1988; van der Goot et al., 2005; Ellis et al., 2006; Cardona et al.,2006; Suarez et al., 2006), we too conducted pilot studies to assessthe ability of the Egyptian isolate to grow in MDBK cells and gener-ate an immune response after being formulated as a killed vaccine.The isolate grew without adaptation to reasonable titers, and thetiters after subsequent passages increased by 10-fold. Vaccinationof chickens with two doses of the oil-emulsion vaccine produceddetectable antibodies to four influenza antigens, which could pro-tect animals from subsequent challenge.

We used ELISA to quantify the IgG response to vaccination as ithas been previously reported to be more sensitive than agar gel pre-cipitation and hemagglutination inhibition when used to measureantibodies raised in vaccinated turkeys against a killed oil-emulsionA/Duck/613/MN/79 (H4N2) vaccine (Abraham et al., 1988). ELISAwas able to detect the antibody response in four turkeys that wereserologically negative by the other two tests, and turkeys with highELISA titers were protected against the challenge.

In this study, a substantial IgG response was detected by ELISAin sera from 3-day-old chicks prior to immunization. This find-ing was unexpected, but can be explained by the presence ofmaternal antibodies from vaccinated mothers. Subsequent mon-itoring of the IgG response in untreated chicks showed that thematernal immune response decreased to undetectable levels by10 days of age. Maternal antibodies in hatchlings might interferewith vaccination strategies; for example, chicks vaccinated whenmaternal antibody levels are still high might fail to mount an IgGresponse to the immunization. These results highlight the needto include assessment of maternal antibodies in influenza vacci-nation strategies and also to develop alternative mechanisms forvaccination.

The variability in IgG titers observed in chickens immunizedwith our vaccine preparation or the commercial vaccine is con-sistent with that seen in previous studies. Cardona et al. (2006)reported highly variable antibody responses in flocks immu-nized with a killed H6N2 vaccine. Variable levels of vaccineprotection and antibody response after administration of a killedoil-emulsion A/Duck/613/MN/79 (H4N2) vaccine have also beenreported (Abraham et al., 1988).

It is considered that such individual variations in the antibodyresponse in vaccinated chickens may be due to the presence of vari-able residual titers of maternal IgG in their sera, which differentiallyblock the vaccines from equally inducing immune response. Alter-natively, unknown genetic factors in chicken populations may causeindividual variations in their ability to mount equal IgG responses.The individual variations in the IgG response reflect that vaccinatedchickens in the field might be not equally protected, as a result ofwhich some can be reinfected.

The mean OD490nm value of the IgG response in sera from birdsthat received the commercial vaccine was higher than that of birdsthat received our vaccine. This can be because of the difference indosing (three doses of the commercial formulation versus only twodoses of our vaccine). Also, not all birds who received the com-mercial vaccine had higher IgG levels than birds who received ourvaccine.

A disadvantage of our vaccine or the commercial H5N2 vaccineis that their antigen contents have not been standardized (Websterand Hulse, 2004). Yet the titer of the inactivated virus (107 PFUs)that we used to vaccinate individual chickens was higher than thedose used in other studies.

Results of Western blotting showed that the killed Egyp-tian H5N1 virus vaccine was specific for multiple influenzaantigens. The absence of a detectable antibody reaction to the low-molecular-weight nonstructural protein 1, which is produced inlarge quantities during infection (Ghedin et al., 2005; Zhao et al.,2005), confirmed that the antibody response which was detectedwas due to vaccination and not infection.

ELISA and Western blotting analyses using the killed H5N1 virusas an antigen represent useful tools for monitoring the protectiveimmune response in vaccinated poultry. Although the killed H5N1vaccine protected chickens from a lethal challenge, the high vari-ability in the induced IgG response clearly underlines the needfor improved vaccines and vaccination strategies to control H5N1under field conditions, which is paramount to reduce the risks topublic health and minimize trade restrictions.

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