Emergence of Highly Pathogenic Avian Influenza A Virus (H5N1) of Clade 2.3.4.4b in Egypt, 2021-2022.

Wild migratory birds have the capability to spread avian influenza virus (AIV) over long distances as well as transmit the virus to domestic birds. In this study, swab and tissue samples were obtained from 190 migratory birds during close surveillance in Egypt in response to the recent outbreaks of the highly pathogenic avian influenza (HPAI) H5N1 virus. The collected samples were tested for a variety of AIV subtypes (H5N1, H9N2, H5N8, and H6N2) as well as other pathogens such as NDV, IBV, ILT, IBDV, and WNV. Among all of the tested samples, the HPAI H5N1 virus was found in six samples; the other samples were found to be negative for all of the tested pathogens. The Egyptian HPAI H5N1 strains shared genetic traits with the HPAI H5N1 strains that are currently being reported in Europe, North America, Asia, and Africa in 2021-2022. Whole genome sequencing revealed markers associated with mammalian adaption and virulence traits among different gene segments, similar to those found in HPAI H5N1 strains detected in Europe and Africa. The detection of the HPAI H5N1 strain of clade 2.3.4.4b in wild birds in Egypt underlines the risk of the introduction of this strain into the local poultry population. Hence, there is reason to be vigilant and continue epidemiological and molecular monitoring of the AIV in close proximity to the domestic-wild bird interface.

Molecular Epidemiology and Evolutionary Analysis of Avian Influenza A(H5) Viruses Circulating in Egypt, 2019-2021.

The highly pathogenic avian influenza (HPAI) H5N8 virus was first detected in Egypt in late 2016. Since then, the virus has spread rapidly among different poultry sectors, becoming the dominant HPAI H5 subtype reported in Egypt. Different genotypes of the HPAI H5N8 virus were reported in Egypt; however, the geographic patterns and molecular evolution of the Egyptian HPAI H5N8 viruses are still unclear. Here, extensive epidemiological surveillance was conducted, including more than half a million samples collected from different poultry sectors (farms/backyards/live bird markets) from all governorates in Egypt during 2019-2021. In addition, genetic characterization and evolutionary analyses were performed using 47 selected positive H5N8 isolates obtained during the same period. The result of the conducted surveillance showed that HPAI H5N8 viruses of clade 2.3.4.4b continue to circulate in different locations in Egypt, with an obvious seasonal pattern, and no further detection of the HPAI H5N1 virus of clade 2.2.1.2 was observed in the poultry population during 2019-2021. In addition, phylogenetic and Bayesian analyses revealed that two major genotypes (G5 and G6) of HPAI H5N8 viruses were continually expanding among the poultry sectors in Egypt. Notably, molecular dating analysis suggested that the Egyptian HPAI H5N8 virus is the potential ancestral viruses of the European H5N8 viruses of 2020-2021. In summary, the data of this study highlight the current epidemiology, diversity, and evolution of HPAI H5N8 viruses in Egypt and call for continuous monitoring of the genetic features of the avian influenza viruses in Egypt.

Comparison of genomic and antigenic properties of Newcastle Disease virus genotypes II, XXI and VII from Egypt do not point to antigenic drift as selection marker.

Newcastle disease (ND), caused by avian orthoavulavirus type-1 (NDV), is endemic in poultry in many regions of the world and causes continuing outbreaks in poultry populations. In the Middle East, genotype XXI, used to be present in poultry in Egypt but has been replaced by genotype VII. We investigated whether virus evolution contributed to superseding and focussed on the antigenic sites within the hemagglutinin-neuraminidase (HN) spike protein. Full-length sequences of an NDV genotype VII isolate currently circulating in Egypt was compared to a genotype XXI isolate that was present as co-infection with vaccine-type viruses (II) in a historical virus isolated in 2011. Amino acid differences in the HN glycoprotein for both XXI and VII viruses amounted to 11.7% and 11.9%, respectively, compared to the La Sota vaccine type. However, mutations within the globular head (aa 126-570), bearing relevant antigenic sites, were underrepresented (a divergence of 8.8% and 8.1% compared to 22.4% and 25.6% within the protein domains encompassing cytoplasmic tail, transmembrane part and stalk regions (aa 1-125) for genotypes XXI and VII, respectively). Nevertheless, reaction patterns of HN-specific monoclonal antibodies inhibiting receptor binding revealed differences between vaccine-type viruses and genotype XXI and VII viruses for epitopes located in the head domain. Accordingly, compared to Egyptian vaccine-type isolates and the La Sota vaccine reference strain, single aa substitutions in 6 of 10 described neutralizing epitopes of HN were found. However, the same alterations in neutralization sensitive epitopes were present in old genotype XXI as well as in newly emerged genotype VII isolates. In addition, isolates were indistinguishable by polyclonal chicken sera raised against different genotypes including vaccine viruses. These findings suggest that factors other than antigenic differences within the HN protein account for facilitating the spread of genotype VII versus genotype XXI viruses in Egypt.

Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of Avian Influenza Virus H9N2 HA Gene.

The H9N2 subtype of avian influenza A virus (aIAV) is circulating among birds worldwide, leading to severe economic losses. H9N2 cocirculation with other highly pathogenic aIAVs has the potential to contribute to the rise of new strains with pandemic potential. Therefore, rapid detection of H9 aIAVs infection is crucial to control virus spread. A qualitative reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of aIAV subtype H9N2 was developed. All results were compared to the gold standard (real-time reverse transcription polymerase chain reaction (RT-PCR)). The RT-RPA assay was designed to detect the hemagglutinin (HA) gene of H9N2 by testing three pairs of primers and a probe. A serial concentration between 106 and 100 EID50 (50% embryo infective dose)/mL was applied to calculate the analytical sensitivity. The H9 RT-RPA assay was highly sensitive as the lowest concentration point of a standard range at one EID50/mL was detected after 5 to 8 min. The H9N2 RT-RPA assay was highly specific as nucleic acid extracted from H9 negative samples and from other avian pathogens were not cross detected. The diagnostic sensitivity when testing clinical samples was 100% for RT-RPA and RT-PCR. In conclusion, H9N2 RT-RPA is a rapid sensitive and specific assay that easily operable in a portable device for field diagnosis of aIAV H9N2.

Evaluation of Protective Efficacy of Influenza Virus Like Particles Prepared from H5N1 Virus of Clade 2.2.1.2 in Chickens.

Highly pathogenic Avian Influenza (HPAI) viruses continue to cause severe economic losses in poultry species worldwide. HPAI virus of subtype H5N1 was reported in Egypt in 2006, and despite vaccination efforts, the virus has become endemic. The current study aims to evaluate the efficacy of a virus-like particle (VLP) based vaccine in vivo using specific pathogen-free (SPF) chickens. The vaccine was prepared from the HPAI H5N1 virus of clade 2.2.1.2 using the baculovirus expression system. The VLPs were quantitated and characterized, including electron microscopy. In addition, the protection level of the VLPs was evaluated by using two different regimens, including one dose and two-dose vaccinated groups, which gave up to 70% and 100% protection level, respectively. The results of this study emphasize the potential usefulness of the VLPs-based vaccine as an alternative vaccine candidate for the control of AIV infection in poultry.

Epidemiological surveillance of H9N2 avian influenza virus infection among chickens in farms and backyards in Egypt 2015-2016.

BACKGROUND AND AIM: LPAI H9N2 infection among the poultry population in Egypt constitutes an additional risk factor in the poultry industry. This study aimed to determine the prevalence of H9N2 avian influenza virus (AIV) in commercial and backyard chickens in Egypt. A 2-year survey of H9N2 AIV in chickens in farms and backyards was carried out in 2015 and 2016. MATERIALS AND METHODS: Direct detection of H9N2 AIV was performed by detecting the virus in tracheal and cloacal swabs using real-time polymerase chain reaction assays. A total of 20,421 samples were collected from chickens in farms and backyards in 26 Egyptian governorates. RESULTS: In 2015, cases positive for H9N2 AIV numbered 388 (3.9%) out of 10,016 examined cases. However, in 2016, the total positive cases numbered 447 (4.3%) out of 10,405 examined cases. The prevalence of H9N2 AIV among chickens on commercial farms was 4.6% out of the 16,666 chickens examined. The rates of positive cases in 2015 and 2016 were 4.4% (349/7884) and 4.7% (417/8782), respectively. The prevalence of H9N2 AIV in backyard chickens was 1.8% (69/3755). The rates of positive cases in backyard chickens were 1.8% (39/2132) in 2015 and again 1.8% (30/1623) in 2016. The highest positivity rate of H9N2 in chicken farms was in Beni-Suef (61.5%) (8/13), whereas the highest positivity rate in backyard chickens was in Fayoum (8.2%) (8/97). CONCLUSION: The analysis of H9N2 infections among chicken farms and in backyard chickens in the different governorates of Egypt over 2 years indicated widespread infection throughout the country. Thus, continuous surveillance and implementation of control programs are warranted.

Molecular evolution of the hemagglutinin gene and epidemiological insight into low-pathogenic avian influenza H9N2 viruses in Egypt.

Despite the low pathogenicity of the H9N2 avian influenza viruses, they can induce severe economic losses in various poultry sectors in conjunction with other factors. In Egypt, low-pathogenic avian influenza (LPAI) H9N2 became endemic in 2011 and has undergone continuous genetic evolution since then. The regular monitoring of the evolution of the virus is necessary to control its spread. During 2017-2020, there were 44 positive samples isolated, and these viruses were genetically sequenced to determine the hemagglutinin (HA) gene circulating in Egypt. The molecular analysis revealed at least nine changes in amino acid residues in comparison with the reference Egyptian strain from the original introduction in 2011 (A/qu/Egypt/113413v/2011), with a similarity of 95%-96%. Amino acid residues 180 and 216 are the most important residues in terms of positive selection pressure. Phylogenetically, the new Egyptian H9N2 viruses in 2017-2020 belonged to a new subcluster related to the strains that had been circulating since 2015. Comparative analysis of the HA gene of LPAI H9N2 viruses in Egypt from 2011 to 2020 supports a continuous evolution through the years with persistent markers.

Efficacy of a Turkey Herpesvirus Vectored Newcastle Disease Vaccine against Genotype VII.1.1 Virus: Challenge Route Affects Shedding Pattern.

The control of Newcastle disease (ND) highly relies on vaccination. Immunity provided by a ND vaccine can be characterized by measuring the level of clinical protection and reduction in challenge virus shedding. The extent of shedding depends a lot on the characteristics of vaccine used and the quality of vaccination, but influenced also by the genotype of the challenge virus. We demonstrated that vaccination of SPF chicks with recombinant herpesvirus of turkey expressing the F-gene of genotype I ND virus (rHVT-ND) provided complete clinical protection against heterologous genotype VII.1.1 ND virus strain and reduced challenge virus shedding significantly. 100% of clinical protection was achieved already by 3 weeks of age, irrespective of the challenge route (intra-muscular or intra-nasal) and vaccination blocked cloacal shedding almost completely. Interestingly, oro-nasal shedding was different in the two challenge routes: less efficiently controlled following intra-nasal than intra-muscular challenge. Differences in the shedding pattern between the two challenge routes indicate that rHVT-ND vaccine induces strong systemic immunity, that is capable to control challenge virus dissemination in the body (no cloacal shedding), even when it is a heterologous strain, but less efficiently, although highly significantly (p < 0.001) suppresses the local replication of the challenge virus in the upper respiratory mucosa and consequent oro-nasal shedding.

Isolation of a Novel Reassortant Highly Pathogenic Avian Influenza (H5N2) Virus in Egypt.

Highly pathogenic avian influenza (HPAI) H5N1 and H5N8 have become endemic among domestic poultry in Egypt since 2006 and 2016, respectively. In parallel, the low pathogenic avian influenza H9N2 virus has been endemic since 2010. Despite the continuous circulation of these subtypes for several years, no natural reassortant has been detected so far among the domestic poultry population in Egypt. In this study, the HPAI (H5N2) virus was isolated from a commercial duck farm, giving evidence of the emergence of the first natural reassortment event in domestic poultry in Egypt. The virus was derived as a result of genetic reassortment between avian influenza viruses of H5N8 and H9N2 subtypes circulating in Egypt. The exchange of the neuraminidase segment and high number of acquired mutations might be associated with an alteration in the biological propensities of this virus.

Combined H5ND inactivated vaccine protects chickens against challenge by different clades of highly pathogenic avian influenza viruses subtype H5 and virulent Newcastle disease virus.

AIM: The aim of the current study was to evaluate the efficacy of a trivalent-inactivated oil-emulsion vaccine against challenge by different clades highly pathogenic avian influenza (HPAI) viruses including HPAI-H5N8 and the virulent genotype VII Newcastle disease virus (NDV) (vNDV). MATERIALS AND METHODS: The vaccine studied herein is composed of reassortant AI viruses rgA/Chicken/Egypt/ME1010/2016 (clade 2.2.1.1), H5N1 rgA/Chicken/Egypt/RG-173CAL/2017 (clade 2.2.1.2), and "NDV" (LaSota NDV/CK/Egypt/11478AF/11); all used at a concentration of 108 EID50/bird and mixed with Montanide-ISA70 oil adjuvant. Two-week-old specific pathogen free (SPF) chickens were immunized subcutaneously with 0.5 ml of the vaccine, and hemagglutination inhibition (HI) antibody titers were monitored weekly. The intranasal challenge was conducted 4 weeks post-vaccination (PV) using 106 EID50/0.1 ml of the different virulent HPAI-H5N1 viruses representing clades 2.2.1, 2.2.1.1, 2.2.1.2, 2.3.4.4b-H5N8, and the vNDV. RESULTS: The vaccine induced HI antibody titers of >6log2 against both H5N1 and NDV viruses at 2 weeks PV. Clinical protection against all HPAI H5N1 viruses and vNDV was 100%, except for HPAI H5N1 clade-2.2.1 and HPAI H5N8 clade-2.3.4.4b viruses that showed 93.3% protection. Challenged SPF chickens showed significant decreases in the virus shedding titers up to <3log10 compared to challenge control chickens. No virus shedding was detected 6 "days post-challenge" in all vaccinated challenged groups. CONCLUSION: Our results indicate that the trivalent H5ND vaccine provides significant clinical protection against different clades of the HPAI viruses including the newly emerging H5N8 HPAI virus. Availability of such potent multivalent oil-emulsion vaccine offers an effective tool against HPAI control in endemic countries and promises simpler vaccination programs.

Full genome sequences of chicken anemia virus demonstrate mutations associated with pathogenicity in two different field isolates in Egypt.

Chicken anemia virus (CAV) is an important pathogen associated with immunosuppression in chicken. In this study, out of samples collected from 115 commercial poultry farms, 12 samples were CAV positive by PCR. Partial sequence and phylogenetic analysis of VP1 gene revealed that the detected viruses were clustered to genotype I (n = 3) and genotype II (n = 9). Motifs of both low (E144) and high pathogenic strains (T89, I125, Q141) were found in the three viruses of genotype I. Whereas genotype II viruses demonstrated the characteristic motifs of highly pathogenic strains (I75, T89, I125, Q141, and Q144). Three isolates representative of both genotypes (CAV/CA1, CAV/GZ1 and CAV/SK4) were selected for full genome sequencing and results revealed that the VP2 gene had two substitutions at V153 and E 175, while VP3 gene had only one substitution at C118. To evaluate virus pathogenicity, two isolates from each genotype (CAV/SK4 of genotype I and CAV/CA1 of genotype II) were intramuscularly inoculated in two groups of one-day-old specific pathogen free chicks. Eighteen days post inoculation, PCR detected CAV in 75 and 90% of chicks in group I and II; respectively. Mortalities in inoculated chicks were 5 and 20% and packed cell volume values were 0.21 and 0.19; respectively. CAV/CA1 and CAV/SK4 isolates showed pathogenic evidences at the level of genetic (Q141 and 394Q) with variable degree of virulence. In conclusion, the study reports the circulation of at least two genotypes of CAV among chicken population with mutation associated with pathogenicity.

Potential Biological and Climatic Factors That Influence the Incidence and Persistence of Highly Pathogenic H5N1 Avian Influenza Virus in Egypt.

Highly pathogenic H5N1 avian influenza virus (A/H5N1) of clade 2.2.1 is endemic in poultry in Egypt where the highest number of human infections worldwide was reported. During the last 12 years the Egyptian A/H5N1 evolved into several genotypes. In 2007-2014 vaccinated poultry suffered from antigenic drift variants of clade 2.2.1.1 and in 2014/2015 an unprecedented upsurge of A/H5N1 clade 2.2.1.2 occurred in poultry and humans. Factors contributing to the endemicity or re-emergence of A/H5N1 in poultry in Egypt remain unclear. Here, three potential factors were studied: climatic factors (temperature, relative humidity, and wind speed), biological fitness in vitro, and pathogenicity in domestic Pekin and Muscovy ducks. Statistical analyses using negative binomial regression models indicated that ambient temperature in winter months influenced the spread of A/H5N1 in different geographic areas analyzed in this study. In vitro, at 4 and 56°C 2.2.1.1 and recent 2.2.1.2 viruses were more stable than other viruses used in this study. Further, Pekin ducks were more resistant than Muscovy ducks and the viruses were excreted for up to 2 weeks post-infection assuming a strong role as a reservoir. Taken together, ambient temperature in winter months potentially contributes to increasing outbreaks in some regions in Egypt. Heat stability of clade 2.2.1.1 and recent 2.2.1.2 viruses probably favors their persistence at elevated temperatures. Importantly, asymptomatically infected Pekin ducks may play an important role in the spread of avian and human-like A/H5N1 in Egypt. Therefore, control measures including targeted surveillance and culling of silently infected Pekin ducks should be considered.

Multiple introductions of reassorted highly pathogenic avian influenza viruses (H5N8) clade 2.3.4.4b causing outbreaks in wild birds and poultry in Egypt.

Recently, an increased incidence of outbreaks of highly pathogenic avian influenza (HPAI) H5N8 in poultry linked to infected migratory birds has been reported from different European, Asian and African countries. In Egypt, incursion of HPAI H5N8 virus of clade 2.3.4.4b has been recently registered. Full genomic characterization of 3 virus isolates from wild birds and poultry (backyard and commercial farm sectors) showed high nucleotide similarity among the HA, NA, M, and NS gene segments of the three Egyptian HPAI H5N8 viruses, indicating that they are descendants of a common ancestral virus. However, the analyzed Egyptian H5N8 viruses revealed distinct genotypes involving different origins of the PB2, PB1, PA and/or NP segments. In genotype-1 represented by strain A/common-coot/Egypt/CA285/2016 the PB2 and NP segments showed closest relationship to H5N6 and H6N2 viruses, recently detected in Italy. The second is replacement of PB1 and NP genes A novel reassortant, represented by strain A/duck/Egypt/SS19/2017, showed an exchange of PB1 and NP genes which might have originated from H6N8 or H1N1 and H6N2 viruses. Finally, replacement of PA and NP genes characterized strain A/duck/Egypt/F446/2017. Bayesian phylogeographic analyses revealed that Egyptian H5N8 viruses are highly likely derived from Russian 2016 HPAI H5N8 virus (A/great_crested_grebe/Uvs-Nuur_Lake/341/2016 (H5N8)) and the reassortment likely occurred before incursion to Egypt.

Preparation and immunological evaluation of inactivated avian influenza virus vaccine encapsulated in chitosan nanoparticles.

Efficacy maximization of inactivated avian influenza vaccine using safe adjuvants was investigated. Chitosan nanoparticles were prepared by ionic gelation method with average size of 150 nm and their Zeta potential was 11.5 mV. After encapsulation of avian influenza vaccine, the average size was 397 nm and Zeta potential was 4.29 mV. The highest HI antibody titer results were shown in chicken group vaccinated with inactivated avian influenza virus AIV-chitosan followed by the group vaccinated with inactivated AIV-chitosan nanoparticles then the group vaccinated with oil inactivated AIV vaccine, on using chicken antigen at 2 weeks post second vaccination. Upon using duck antigen, the highest HI antibody titers were shown in chicken group vaccinated with inactivated AIV oil emulsion vaccine followed by chicken group vaccinated with AIV-chitosan nanoparticles then the group vaccinated with AIV-chitosan. Chicken in the group vaccinated with AIV-chitosan nanoparticles induced the best results of lymphocyte proliferation assay. The results of phagocytic activity percentage and phagocytic index of AIV-chitosan nanoparticles and AIV-chitosan groups at 3 days post first vaccination were increased significantly in comparison with other groups, whereas at 14 days post first vaccination, group vaccinated with AIV-chitosan nanoparticles showed significant increase in phagocytic activity percentage and phagocytic index.

Reactivity and sensitivity of commercially available influenza rapid diagnostic tests in Japan.

Seasonal influenza virus routinely causes epidemic infections throughout the world. Sporadic infections by H5N1, H5N6, and H7N9 viruses are also reported. To treat patients suffering from such viral infections, broadly reactive and highly sensitive influenza rapid diagnostic tests (IRDTs) are required. Here, we examined the reactivity and sensitivity of 25 IRDTs available in Japan for the detection of seasonal H1N1pdm09, H3N2, and type B viruses, as well as highly pathogenic H5 and H7 viruses. All of the IRDTs tested detected the seasonal viruses and H5 and H7 viruses albeit with different sensitivities. Several IRDTs detected the H5 and H7 viruses and the seasonal viruses with similar (high) sensitivity.

Insights into genetic diversity and biological propensities of potentially zoonotic avian influenza H9N2 viruses circulating in Egypt.

Low pathogenic avian influenza (LPAI) H9N2 viruses have established endemic status in Egyptian poultry populations since 2012. Recently, four cases of human H9N2 virus infections in Egypt demonstrated the zoonotic potential of these viruses. Egyptian H9N2 viruses obtained from 2011 to 2014 phylogenetically grouped into three clusters (1-3) within subclade B of the G1 lineage. Antigenically, a close clustering of the Egyptian H9N2 viruses with other recent G1-B like H9N2 strains and a significant antigenic distance from viruses outside the G1-B lineage was evident. Recent Egyptian LPAIV H9N2 showed a tendency to increased binding with erythrocytes expressing α 2,6-linked sialic acid which correlated with the Q226L amino acid substitution at the receptor binding unit of the hemagglutinin (Q234L, H9 numbering). Sequence analyses of the N2 neuraminidase (NA) revealed substitutions in the NA hemadsorption site similar to the N2 of prepandemic H3N2/1968, but no distinct antigenic or functional characteristics of the H9N2 NA associated with increased zoonotic potential could be identified.

Heterologous post-infection immunity against Egyptian avian influenza virus (AIV) H9N2 modulates the course of subsequent infection by highly pathogenic AIV H5N1, but vaccination immunity does not.

In Egypt, zoonotic A/goose/Guangdong/1/96 (gs/GD-like) highly pathogenic avian influenza virus (HPAIV) H5N1 of clade 2.2.1.2 is entrenched in poultry populations and has co-circulated with low-pathogenic avian influenza virus H9N2 of the G1 lineage since 2010. Here, the impact of H9N2 infection or vaccination on the course of consecutive infection with a lethal Egyptian HPAIV H5N1 is studied. Three-week-old chickens were infected with H9N2 or vaccinated with inactivated H9N2 or H5N1 antigens and challenged three weeks later by an HPAIV H5N1. Interestingly, pre-infection of chickens with H9N2 decreased the oral excretion of H5N1 to levels that were comparable to those of H5N1-immunized chickens, but vaccination with inactivated H9N2 did not. H9N2 pre-infection modulated but did not conceal clinical disease by HPAIV H5N1. By contrast, homologous H5 vaccination abolished clinical syndromic surveillance, although vaccinated clinical healthy birds were capable of spreading the virus.

Highly Pathogenic Avian Influenza Virus (H5N8) Clade 2.3.4.4 Infection in Migratory Birds, Egypt.

We isolated highly pathogenic avian influenza virus (H5N8) of clade 2.3.4.4 from the common coot (Fulica atra) in Egypt, documenting its introduction into Africa through migratory birds. This virus has a close genetic relationship with subtype H5N8 viruses circulating in Europe. Enhanced surveillance to detect newly emerging viruses is warranted.

New real time and conventional RT-PCRs for updated molecular diagnosis of infectious bronchitis virus infection (IBV) in chickens in Egypt associated with frequent co-infections with avian influenza and Newcastle Disease viruses.

In Egypt, currently two geographically restricted genotypes of the infectious bronchitis coronavirus (IBV) are circulating with detrimental effects for poultry industry. A sensitive real-time RT-PCR assay targeting the IBV nucleocapsid gene (N) was developed to screen clinical samples for presence of IBV. Conventional RT-PCRs amplifying hypervariable regions (HVRs 1-2 and 3) of the IBV S1 gene were developed and amplificates used for nucleotide sequence-based typing of IBV field strains in Egyptian chickens directly from clinical samples.