Veterinary Microbiology 183 (2016) 103–109

Different counteracting host immune responses to clade 2.2.1.1 and2.2.1.2 Egyptian H5N1 highly pathogenic avian influenza viruses innaïve and vaccinated chickens

Ahmed A. Samya, Mona I. El-Enbaawyb, Ahmed A. El-Sanousic, Soad A. Nasefa,Mahmoud M. Naguiba, E.M. Abdelwhaba,f, Hirokazu Hikonod,e, Takehiko Saitod,*aReference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Dokki, Giza 12618, EgyptbMicrobiology Department, Faculty of Veterinary Medicine, Cairo University, Giza 12211, EgyptcVirology Department, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egyptd Influenza and Prion Disease Research Centre, National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), Tsukuba,Ibaraki 305-0856, JapaneDepartment of Veterinary Medicine, Iwate University, Morioka, Iwate 020-8550, Japanf The Federal Research Institute for Animal Health, Friedrich-Loeffler Institute, Suedufer 10, D-17493 Greifswald-Insel Riems, Germany

A R T I C L E I N F O

Article history:Received 17 November 2015Received in revised form 4 December 2015Accepted 10 December 2015

Keywords:Avian influenzaH5N1ChickensCytokineVaccineEgypt

A B S T R A C T

In Egypt, two distinct lineages of H5N1 highly pathogenic avian influenza (HPAI) viruses, “classic 2.2.1.2”and “variant 2.2.1.1” strains, have evolved. The underlying host immune responses counteracting theseviruses in chickens remain not well understood. In the present study, the cytokine responses to a classicstrain (C121) and those to a variant strain (V1063) were compared in naïve and vaccinated chickens. Innaïve chickens, the C121 replicated more efficiently than the V1063. Both the C121 and theV1063 increased interferon (IFN)-g and interleukin (IL)-10 gene expression at 48 h post inoculation(hpi) in the lung and spleen but the levels of these cytokines were lower in chickens infected with theC121 than those infected with the V1063. In contrast, in chickens vaccinated with inactivated C121-basedvaccine, the C121 replicated less than the V1063. Both challenge with the C121 and that with theV1063 did not increase IFN-g gene expression at 48 hpi; rather, the C121 increased IL-4 gene expressionin the lung accompanied with lower viral titer and higher HI titers. These results suggested that thepathogenicity of HPAI viruses correlated with IFN-g-producing helper and/or cytotoxic T cell responses innaïve chickens, whereas vaccine efficacy to HPAI viruses correlated with IL-4 producing helper T cellresponses in the lung in vaccinated chickens. It implies that IL-4 in the lung, in addition to the traditionalserum HI titers, could be used to screen novel vaccine strategies, such as strains, adjuvant, prime/boostprotocols, against HPAI in chickens.

ã 2015 Elsevier B.V. All rights reserved.

Contents lists available at ScienceDirect

Veterinary Microbiology

journal homepage: www.else vie r .com/locate /ve tmic

1. Introduction

Highly pathogenic avian influenza (HPAI) is a devastatingdisease of poultry and poses a potential pandemic threat inhumans (Cattoli et al., 2011). The disease has become endemic inpoultry in China, Viet Nam, Indonesia, India, and Egypt, causing adestructive effect on poultry production and posing serious threatsto the economy in those countries (Swayne et al., 2011). Since theintroduction of the H5N1HPAI 2.2.1 virus into poultry in Egypt in2006, over 30 million birds have been killed by the virus or culled

* Corresponding author.E-mail address: taksaito@affrc.go.jp (T. Saito).

http://dx.doi.org/10.1016/j.vetmic.2015.12.0050378-1135/ã 2015 Elsevier B.V. All rights reserved.

to control its spread (Abdelwhab and Hafez, 2011). After the lastwinter (2014–2015), Egypt now considered the country with thehighest human cases worldwide with 114 deaths out of342 infected human cases reported as of 30 April 2015, and mostof the cases had a history of poultry exposure (WHO, 2015).

Since the emergence of the epizootic, Egypt has used massvaccination of poultry as part of a national strategy to control HPAI.Late 2007, there are evidences that the use of unsuitableH5 vaccines as part of this strategy in commercial poultry resultedin the emergence of a genetically and antigenically distinctive“variant” strains that clustered in clade 2.2.1.1 (Grund et al., 2011).These variant strains harbor major changes in immunogenicepitopes of the hemagglutinin (HA) protein that enabled the virusto evade the humoral immune responses evoked by H5N2-based

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104 A.A. Samy et al. / Veterinary Microbiology 183 (2016) 103–109

vaccine commonly used in Egypt (Hassan et al., 2012). In addition,viruses isolated from non-vaccinated backyard birds and small-scale commercial poultry and human clustered in a distinguishableclade designated as 2.2.1.2 (Arafa et al., 2015). The differences inpathogenesis between the co-evolving classic and variant strains inchickens are not well understood.

Cytokines have positive and/or negative effects on thepathogenesis of HPAI viruses, but these effects vary among strains,hosts (Kuchipudi et al., 2014) and depend on the stage of theinfection (Nagai et al., 2003). Previous studies using mouse modelssuggested that overactive inflammatory response with cytokinedysregulation is likely to be the cause of the high fatality rate ofinfluenza infection (Tisoncik et al., 2012). In particular, macro-phage infection caused high activation and rapid proliferation ofT-cell and natural killer cells, that in turn caused a cytokine stormtriggering excessive induction of apoptosis. In contrast, anotherstudy reported that H5HPAI virus infection was lethal to micelacking tumor necrosis factor (TNF) and interleukin (IL)-6(Salomon et al., 2007).

HPAI virus infection in chickens is characterized by highmortality up to 100% within 24–48 h post infection (hpi) with no-overt clinical signs of infection until just 2 h before death (Suzukiet al., 2009). The rapid onset of the disease implies that virus–hostinteractions, such as immune responses, may contribute to thepathogenesis of HPAI viruses in chickens. That was probed by(Karpala et al., 2011; Kuribayashi et al., 2013) who described stronginflammatory and T-helper 1 (Th1) cytokine responses followingH5N1HPAI virus infection. Also, the rapid and extensive prolifera-tion of H5N1HPAI viruses was well correlated with excessivecytokine responses that cause fatal multiple organ failure inchickens (Kuribayashi et al., 2013). However, in peracute cases ofH5N1HPAI virus infection, cytokine gene expression was notsignificantly increased in the lung until death (Suzuki et al., 2009).

In the present study, we compared the cytokine responses to anEgyptian H5N1HPAI virus classic strain (C121) with those to avariant strain (V1063) in naïve and vaccinated chickens toinvestigate how cytokine responses counteract infection of andvaccine efficacy to the two distinct co-evolving genotypes inchickens.

2. Material and methods

2.1. Viruses

A variant virus (A/chicken/Egypt/1063/2010; V1063, GenBankaccession number HQ198269) from clade 2.2.1.1 and a classic virus(A/chicken/Egypt/121/2012; C121, GenBank accession numberJQ858483) from clade 2.2.1.2 were obtained from the influenzavirus repository at the Reference Laboratory for Quality Control onPoultry Production (RLQP), Giza, Egypt. The viruses werepropagated in specific pathogen free (SPF) and intravenouspathogenicity index (IVPI) were calculated following OIE estab-lished protocols (OIE, 2014). IVPI of C121 and V1063 were 2.8 and

Table 1Experimental design and sample collection time points.

Group (no. of chickens) Vaccination Challengea

1 (10) Vaccinated C121b

2 (10) Vaccinated V1063c

3 (10) No No4 (10) No C121b

5 (10) No V1063c

Dpc = days post-challenge.a Challenge at 30 days post-vaccination using 106 EID50/bird.b Bird of the classic strain.c Bird of the variant strain Vaccination was done using inactivated C121 based vacci

2.4, respectively. All experiments were performed in biosafetylevel-3 (BSL3) facilities at the National Institute of Animal Health(NIAH), Tsukuba, Japan using approved protocols.

2.2. Sequencing of the HA gene

Viral RNA was extracted from the allantoic fluid using theQIAamp viral RNA mini kit (QIAGEN, Germany). The HA genes ofthe viruses were amplified using OneStep RT-PCR kit (QIAGEN)with the primers published by Hoffmann et al. (2001). The PCRproducts were subjected to electrophoresis in 1.5% agarose gel, andthe specific amplicons of DNA were excised, purified from the gelusing a QIAquick Gel Extraction kit (QIAGEN). Sequences wereobtained using BigDye Terminator Kit 3.1 cycle sequencing kit(Applied Biosystems, USA) on a 3130 Genetic Analyzer (AppliedBiosystems). Nucleotide and amino acid sequences were analyzedusing BioEdit software v7.0 (Hall, 1999). A maximum likelihoodphylogenetic tree was constructed using IQ-tree software version1.1.3 (Nguyen et al., 2015). The phylogenetic tree was viewed andedited using FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/) and Inkscape 0.48. The deduced amino acid sequencesof the HA were compared with those of an ancestral H5N1 virusesof clade 2.2 (A/Bar-headed-Goose/Qinghai-65/05; GenBank acces-sion no. DQ095622) and an H5N1 virus first isolated in Egypt(A/chicken/Egypt/2253-1-2006; GenBank accession no.CY020645). Homology models of the HA protein of H5N1 strainC121 and V1063 were created using SWISS-Model server (Biasiniet al., 2014) with A/chicken/Egypt/2253-1/2006 (H5N1) as atemplate. Amino acid substitution mutations were viewed usingRasTop version 2.2 (http://www.geneinfinity.org/rastop/).

2.3. Vaccine preparation

Propagated viruses (C121 and V1063) were inactivated by theaddition of beta-propiolactone to the harvested allantoic fluid in afinal concentration of 1/1000 for 4 h at room temperature and thenincubated at 4 �C overnight for hydrolysis of beta-propiolactone.Moreover, complete virus inactivation was confirmed by twopassages of the inactivated virus in five SPF eggs. Failure to recoverthe virus was confirmed by hemagglutination test. The inactivatedvirus was then purified from the allantoic fluid using standardsucrose density-gradient centrifugation (Kida and Yanagawa,1979).

2.4. Experimental design

Experimental design is summarized in Table 1. Four-week-oldSPF White Leghorn chickens (Gallus gallus domesticus) werepurchased from Nisseiken Co. (Japan) and housed inBSL3 animal facilities with continuous access to food and water.All chickens were serologically tested prior to vaccination usinghemagglutination inhibition (HI) test for antibodies againstH5N1 viruses. All experiments were performed in BSL3 facilities

Organs and sampling time

Lung, spleen, tracheal and cloacal swabs and serum at 2,6 and 14 dpc

Lung, spleen, tracheal and cloacal swabs at 2 dpi

ne.

http://tree.bio.ed.ac.uk/software/figtree/http://tree.bio.ed.ac.uk/software/figtree/http://www.geneinfinity.org/rastop/

A.A. Samy et al. / Veterinary Microbiology 183 (2016) 103–109 105

according to the biosafety manual of the National Institute ofAnimal Health (NIAH), Tsukuba, Japan.

Fifty chickens were allocated into five groups (10 chickenseach); group 1 was vaccinated with the inactivated classic strainand then challenged with the classic strain, group 2 was vaccinatedwith the inactivated classic strain and then challenged with thevariant strain, group 3 was un-vaccinated and unchallenged, group4 was unvaccinated but challenged with the classic strain, andgroup 5 was unvaccinated and challenged with the variant strain.

Chickens were vaccinated intramuscularly with 104 haemag-glutination units of the purified inactivated virus. Serum sampleswere taken on 30 day post-vaccination to measure antibody titersby using HI tests using antigens prepared from C121 andV1063 strains. Naïve and vaccinated chickens were challengedintranasally with 106 EID50 of the virus on 30 day post-vaccination. Oropharyngeal swabs, lungs, and spleens were takento measure virus titers on 2, 6, and 14 day post-challenge (dpc).Parts of the lungs and spleens were kept in RNA later solutionaccording to manufacturer instruction (Ambion, USA) and storedat �20 �C until RNA extraction was done to measure cytokine geneexpression.

2.5. Cytokine gene expression and viral RNA quantification

Total RNA was isolated from collected tissues and swabs usingthe RNeasy mini RNA Purification kit (QIAGEN) with DNasetreatment (QIAGEN). The concentration of RNA in each sample wasdetected using a NanoDrop1 Spectrophotometer ND-1000(Thermo Scientific, USA). Quantitative real-time PCR was per-formed using Quantitect probe RT-PCR (QIAGEN). Primers andprobes were designed for IL-1b, IL-4, IL-6, IL-8, IL-10, IL-18, 28Sribosomal RNA (rRNA), interferon (IFN)-a, IFN-g and HA detectionand quantification (sequences will be made available uponrequest). The cytokine gene amplification data were normalizedagainst 28S rRNA gene expression. Real-time RT-PCR wasperformed using a 7500 Real-time PCR System (Applied Bio-systems). PCR conditions were the same for each targeted gene ofdifferent cytokines as follows: 30 min at 50 �C, 95 �C for 15 min,followed by 40 cycles of 95 �C for 15 sec and 60 �C for 1 min. For HA,30 min at 50 �C, 95 �C for 15 min, followed by 40 cycles of 95 �C for10 s, 54 �C for 30 s and 72 �C for 30 s.

2.6. Statistical analysis

Gene expression fold change was determined by the DDCtmethod (Livak and Schmittgen, 2001) using 28S rRNA as theendogenous reference gene to normalize the level of target geneexpression and then calibrated against a control group (non-infected and non-vaccinated group). Logarithmic transformationwas performed on fold change values before being analyzed byStudent’s t-test. The t-test was used to determine significantdifferences between fold change values of chickens challenged

Table 2Receptor binding sites, Glycosylation sites and cleavage site profile of the HA of H5N1 viruclade 2.2.1 A/Bar-headed-Goose/Qinghai-65/05 and the ancestor of H5N1 virus in Egyp

Virus Receptor binding Antigenic sites

Ca Sa S

222 224 140 141 154 156 1

A/Bar-headed-Goose/Qinghai-65/05 Q G R S N A AA/chicken/Egypt/2253-1-2006 – – – – – – –A/chicken/Egypt/121/2012 – – – A – – –A/chicken/Egypt/1063/2010 – – G – – T E

with H5N1HPAI virus classic and variant strains and the controlgroup. Standard errors were calculated using the fold changevalues of three chickens for each gene measured.

3. Results

3.1. Sequences analysis of the HA gene

Phylogenetic analysis indicated that the C121 and V1063 virusesbelonged to clade 2.2.1.2 and clade 2.2.1.1, respectively (Fig. S1). HAcoded protein of both viruses revealed a multiple basic cleavagesite of PQGEKRRKKR/GLF and PQGEGRRKKR/GLF for the classic andvariant virus, respectively, with only one amino acid substitution(K341G; H5 numbering). Both viruses showed markers of avianreceptor specific binding: Q222 and G224. The HA sequence ofC121 possessed six potential glycosylation sites (PGS) at positions11, 23, 165, 286, 484, and 543, which is typical of the ancestralstrain introduced to Egypt in 2006. While, V1063 possessed4 additional PGS at positions 72, 154, 236, and 273; and lacked thePGS at position 165. Furthermore, amino acid substitutions weredetected in antigenic sites Sa, Sb, Cb and Ca of the variant strain.This is noteworthy given that only one mutation was detected inthe antigenic site Ca of the classical strain compared to A/chicken/Egypt/2253-1-2006 (Table 2). Most of these mutations are locatedat the exposed surface of HA protein (Fig. 1).

Supplementary material related to this article found, in theonline version, at http://dx.doi.org/10.1016/j.vetmic.2015.12.005.

3.2. Pathogenicity of the viruses in naïve chickens

All naïve chickens challenged with C121 died within 48 hpi,whereas one of naïve chickens challenged with V1063 died within48 hpi and the remaining chickens died by 72 hpi with typicalclinical signs beginning to appear at 48 hpi. Thus, the IVPI ofC121 and V1063 were 2.8 and 2.4, respectively. The viral titers inlung and spleen of birds infected with C121 were higher than thoseinfected with V1063 at 48 hpi: 8 and 8.1 log10 EID50/ml for C121 and6.77 and 5.9 log10 EID50/ml for V1026, respectively.

3.3. Cytokine responses in naïve chickens

In the lung at 48 hpi (Fig. 2A), both C121 and V1063 increasedgene expression of IL-1b and IFN-g, but the level of IFN-g inducedby C121 was lower than that induced by V1063. C121 increasedIL-6, whereas V1063 increased IL-10 expression. In contrast, C121,but not V1063, decreased IFN-a gene expression.

In the spleen at 48 hpi (Fig. 2B), both C121 and V1063 increasedgene expression of IL-10 and IFN-g but the levels of these cytokinesinduced by C121 were lower than those induced by V1063. V1063,but not C121, increased IL-1b and IL-8. Both C121 andV1063 decreased IL-4 gene expression, where C121 strain wasmore potent than V1063 to downregulate IL-4.

ses used in the current study in comparison with one of the ancestor H5N1 viruses oft A/chicken/Egypt/2253-1-2006(H5 numbering).

Glycosylation siteat position

Cleavage site

b Cb

84 71 11 23 72 154 165 236 273 286 484 543

L + + – – + – – + + + PQGERRRKKR/GLF – + + – – + – – + + + PQGERRRKKR/GLF – + + – – + – – + + + PQGEKRRKKR/GLF P + + + + – + + + + + PQGEGRRKKR/GLF

http://dx.doi.org/10.1016/j.vetmic.2015.12.005

Fig. 1. Predicted tertiary structures of the HA monomer of A/chicken/Egypt/121/2012 (a) and A/chicken/Egypt/1063/2010 (b) (For interpretation of the references to color inthis figure legend, the reader is referred to the web version of this article.).Molecular models were obtained using A/chicken/Egypt/2253-1/2006(H5N1) as a template. Blue depicts amino acid changes distinguishing each virus from the template. TheHA monomers of selected sequences were generated using SWISS modell (Biasini et al., 2014) and the 3D molecular structures of the protein were viewed using RasTop.

Fig. 2. Cytokine gene expression in naïve chickens after inoculation with HPAI H5N1 A/chicken/Egypt/121/2012 (classic 2.2.1.2) or A/chicken/Egypt/1063/2010 (variant2.2.1.1).Cytokine gene expression levels after inoculation with a classic strain (black column) or a variant strain (gray column) in lung (A) and spleen (B) at 48 h post-inoculation.Significance statistical differences between the experimental and control groups were indicated by asterisk: *(P < 0.05) and ** (P < 0.01).

106 A.A. Samy et al. / Veterinary Microbiology 183 (2016) 103–109

Table 3Virus titers in chickens that were vaccinated with a classic strain (C121) and thenchallenged with the classic strain (C121) or the variant strain (V1063).

Time point Virus titera

Oropharyngeal swab Lung Spleen

C121 V1063 C121 V1063 C121 V1063

2 dpc 2.1 4.1 1.76 2.01 1.45 2.306 dpc nd 3.1 nd 3.34 nd 2.4714 dpc nd nd nd nd nd nd

dpc, Days post-challenge; nd, not detected.a Mean virus titer (log10 EID50/ml).

A.A. Samy et al. / Veterinary Microbiology 183 (2016) 103–109 107

3.4. Pathogenicity of the viruses in vaccinated chickens

In chickens vaccinated with the inactivated C121, no deaths orclinical signs were detected after the homologous challenge withC121. In contrast, 3 chickens showed clinical signs at 5 dpc include(depression, ruffled feather) after heterologous challenge withV1063 with no recorded deaths. The viral titers of C121 were lower

Fig. 3. Cytokine gene expression in vaccinated chickens after inoculation with HPAI H5N2.2.1.1).Chickens were vaccinated with a classic strain-based vaccine then challenge with the holevels in the lung (A) and spleen (B) 48 h, 6 day and 14 day post-challenge were mexperimental and control groups were indicated by asterisk: *(P < 0.05) and **(P < 0.01). Bgrey bars refer to chickens vaccinated with C121 and challenged with V1063.

than those of V1063 in the oropharyngeal swab, lung, and spleenon 2 and 6 dpc (Table 3). C121 and V1063 were not detected in anyorgans tested at 14 dpc.

3.5. Cytokine responses in vaccinated chickens

In the lung at 48 hpc (Fig. 3Aa), both C121 and V1063 decreasedgene expression of IFN-g and IL-10. C121 decreased 1L-8 and IL-18,whereas V1063 decreased IFN-a, in contrast C121 inducedsignificantly higher expression of IL4 comparing to V1063. At6 dpc (Fig. 3Ab), both C121 and V1063 increased gene expression ofIFN-a. C121 increased gene expression of IL-4, whereasV1063 increased IL-6, IFN-g, and IL-10� At 14 dpc (Fig. 3Ac), bothC121 and V1063 almost have the same gene expression levels ofnon-vaccinated and non-challenged chickens.

In the spleen at 48 hpc (Fig. 3Ba), both C121 andV1063 decreased gene expression of IFN-a that significantly wasmore predominant in birds challenged with V1063. C121 decreasedIL-6 and IL-8, whereas V1063 decreased IL-4. At 6 dpc (Fig. 3Ab),

1 A/chicken/Egypt/121/2012 (classic 2.2.1.2) or A/chicken/Egypt/1063/2010 (variant

mologous classic strain or with the heterologous variant strain. Cytokine expressioneasured using Real Time RT-PCR. Significance statistical differences between thelack bars refer to chickens vaccinated with C121 and challenged with C121 whereas

Table 4Serum antibody titers in chickens that were vaccinated with a classic strain (C121) and then challenged with a classic strain (C121) or a variantstrain (V1063).

Time Point(dpc)

Antibody titera

C121-vaccinated/C121-challenged C121-vaccinated/V1063-challengedHI titer to C121 HI titer to V1063

0 7.5# 5.32 4.33*,# 3.336 7.0*,# 5.33

14 8.0* 9.0

dpc, Days post-challenge.a geometric mean HI titers (log2).* Statistically significant (P < 0.05) against HI titer on 0 dpc.# Statistically significant (P < 0.05) against HI titer to V1063.

108 A.A. Samy et al. / Veterinary Microbiology 183 (2016) 103–109

C121 decreased gene expression of IL-8 and IL-18, whereasV1063 increased IL-18, IFN-g and IL-10� At 14 dpc (Fig. 3Ac), bothC121 and V1063 increased gene expression of IL-6 and IL-4, but thelevels of IL-4 induced by C121 were higher than these induced byV1063. Also, both C121 and V1063 decreased gene expression ofIL-8 and IL-18.

3.6. Antibody responses in vaccinated chickens

Vaccination with the inactivated C121 induced antibodiesreactive to C121 as well as V1063 at day 30 post vaccination(Table 4). The HI antibody titers against C121 increased from 7.5 log2at the day of challenge to 8.0 log2 at 14 dpc and from 5.3 to 9.0 log2against V1063. At day 2 post inoculation, transient decrease in HItiters was observed which were elevated at 6 dpc (Table 4).

4. Discussion

Pathogenesis of HPAI viruses is not only virus intrinsic, it largelydepends on host factors such as immune responses includingcytokines expression. In Egypt, two genetically and antigenicallydifferent clades of H5N1HPAI viruses, so-called 2.2.1.2 classicstrains and 2.2.1.1 variant strains, have evolved. In this study, theclassic C121 virus replicated more efficiently than the variantV1063 in naïve chickens, whereas C121 replicated less efficientlythan V1063 in C121-vaccinated chickens. Therefore, comparison ofthe cytokine responses to these viruses in naïve and vaccinatedchickens provided a good model to investigate the relationshipbetween cytokine responses and the pathogenesis of/vaccineefficacy to HPAI viruses.

Our current genetic analysis showed that V1063 strainspossessed mutation at residues 74, 140, 144 and 162 that inaddition to mutation at residue 141 were enough to induceantigenic vaccine drift observed for the 2008 field strain (Cattoliet al., 2011). Moreover, V1063 had a mutation at S129L, thatconfirmed to be included in the antigenic drift from homologousH5N1 virus induced antibodies (Li et al., 2013). In addition, itpossesses different glycosylation pattern, as it has deglycosylationat position 165 and glycosylation residues at 72, 154, 236 and 273.These characteristic molecular patterns of variant strains illustrat-ed the ability of these viruses to evade the antibody responseinduced by H5N2 vaccine (Hassan et al., 2012). Our current resultsin HI tests showed that antibodies induced by vaccination withC121 were less cross-reacting with V1063 antigen (day 0 postchallenge). It remains to be assessed how badly V1063 evadedantibody responses induced by vaccination with C121.

Cytokine gene analysis conducted in this study showed thatboth C121 and V1063 increased IFN-g in the lung and spleen innaïve chickens, however the level of IFN-g induced by C121 wassignificantly lower than that induced by V1063. IFN-g is a typicalcytokine produced by type 1 helper T cells and cytotoxic T cells.

These effector T cells play important roles in the protection toinfluenza virus infection (Hikono et al., 2006; Strutt et al., 2013).Therefore, these results suggest that the higher virulence ofC121 correlates with the weaker induction of IFN-g. This resultcoincided with higher expression of IL18 withV1063 in lung andspleen, that is a strong inducing factor of IFN-g (Kaiser et al., 2001).The lower expression of IFNg, IL18 and IL10 could be due to theefficient replication of C121 in cytokines producing cells that agreewith results of CKYM7-HPAI infection (Suzuki et al., 2009). It isworth mentioning that cytokines expression for C121 in this studyand for CKYM7 virus infection was estimated just before theperacute death of the naïve chickens. In context, other studyrecorded that the highest cytokines gene expression at 2 dpi in thebrain, while the onset of the death two days later with higher viralload and lower cytokines expression (Kuribayashi et al., 2013).Altogether, cytokines response related to viral load, the nature ofcells composing infected tissue as well as the severity of infection(Kuribayashi et al., 2013; Moulin et al., 2011) and probably theonset of death. On the other hand, the significantly lowerexpression of IFNa, probably in part, couldn't counteractC121 virus and protect chickens from death which in accordancewith the results obtained by (Daviet et al., 2009).

In a previous study, a variant strain based vaccine conferedclinical protection against challenge with a variant (homologous)or a classical strain, but with lower virus shedding with ahomologous challenge (Grund et al., 2011). In the present work, thechickens vaccinated with inactivated C121 based vaccine andchallenged with the homologous C121 virus were protected betterthan that challenged with V1063. It is likely that such higherprotection to C121 was due to the higher reactivity of serumantibodies to C121 (Table 4). Regarding cytokines gene expressionresults showed that, in contrast to the primary immune responsesin naïve chickens, both challenge with C121 or V1063 did notincrease IFN-g gene expression at 48 hpc; rather, C121 increasedIL-4 gene expression, especially in the lung. These resultssuggested that IL-4 responses in the lung had a critical role inprotection against HPAI in vaccinated chickens. IL-4 is a typicalcytokine produced by type 2 helper T cells, in murine model itinduced proliferation and differentiation of B-cell precursors,antibodies production (Hofman et al., 1988), and essential forantibodies class switching (Paul, 1987). It is still not well identifiedwhether IL-4 in chickens functionally resembles IL-4 in mammals,however it was shown that IL-4 plasmid injection induced higherspecific antibodies response in chickens after vaccination withanticoccidial (Annamalai and Selvaraj, 2012) or Newcastle diseasevirus (NDV) vaccine (Sawant et al., 2011). Concerning influenzainfection, Th2 cytokines responses found in the lung of mice wereable to clear the influenza virus from the lung (Baumgarth et al.,1994; Sarawar et al., 1992). Similarly, our findings clearly indicatedthat humoral immunity represented by IL4 and/or HI titer has apivotal role in protection against HPAI comparing to CMI (IFNg)

A.A. Samy et al. / Veterinary Microbiology 183 (2016) 103–109 109

which reflected by the decrease of viral shedding. Regardless thepathogenicity and/or the efficiency of the viral replication, thehomologous vaccination could provoke efficient higher humoralimmunity (IL4 and HI titer) against C121. In contrast, and despite ofthe lower ability of V1063 to proliferate in naïve chicken, itreplicated efficiently in vaccinated birds. It is worth mentioningthat the challenge with V1063 in vaccinated chickens inducedpredominant Th1 immune response (IL18 and IFNg) and higherlevel of IL10 at 6 dpc. This resembles the same pattern afterinfection of naive chickens with the same strain at 2dpi, whichprobably contributed to the onset of clinical signs.

In conclusion, the current study described variable cytokinesgene expression profiles induced by two genetically distinctEgyptian H5N1 viruses after the inoculation of naïve andvaccinated chickens. Results suggested that in naïve chickens,the pathogenicity of the two HPAI viruses correlated with IFN-g-producing helper and/or cytotoxic T cell responses. In vaccinatedchickens, in addition to the traditional HI titers, protectioncorrelated with the level of IL-4 in the lung, which may be usefulto assess vaccine efficiency against HPAI infection/vaccination inchickens. Moreover, to achieve full protection of chickens againstthe infection with HPAI H5N1 viruses, homologous vaccines arehighly recommended.

Acknowledgments

The authors are grateful to the members of Influenza and PrionDisease Research Centre, National Institute of Animal Health, Japanfor their excellent technical assistance, with special thanks to Dr.Ryota Tsunekuni for his excellent technical support during thestudy. The authors are also thankful to colleagues in RLQP. Wegratefully acknowledge Trevon Fuller, Center for Tropical Research,Institute of the Environment and Sustainability, University ofCalifornia, Los Angeles, USA for his comments on the manuscript.Ahmed A. Samy was supported by a ParOwn scholarship fordoctoral studies to the National Institute of Animal Health,Tsukuba, Ibaraki, Japan where he conducted this research.

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Different counteracting host immune responses to clade 2.2.1.1 and 2.2.1.2 Egyptian H5N1 highly pathogenic avian influenza...1 Introduction2 Material and methods2.1 Viruses2.2 Sequencing of the HA gene2.3 Vaccine preparation2.4 Experimental design2.5 Cytokine gene expression and viral RNA quantification2.6 Statistical analysis

3 Results3.1 Sequences analysis of the HA gene3.2 Pathogenicity of the viruses in naïve chickens3.3 Cytokine responses in naïve chickens3.4 Pathogenicity of the viruses in vaccinated chickens3.5 Cytokine responses in vaccinated chickens3.6 Antibody responses in vaccinated chickens

4 DiscussionAcknowledgmentsReferences