Background
The incursion of highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 of phlyogenetic clade 2.2, subclade 2.2.1 [
1], into Egypt in 2005/2006 caused severe economic losses in the commercial (previous total annual production of 850 million birds) and backyard sectors (250 million birds) of poultry production in this country [
2]. The virus also possesses considerable zoonotic potential. Human cases of HPAIV H5N1 infection, characterized by a high fatality rate, started to occur due to virus exposure of humans at the poultry-human interface which is highly fissured in Egypt [
3]. In order to restore poultry production capacities and to mitigate risks of an emergence of new virus variants with increased pandemic potential in the human population, efforts to control HPAIV H5N1 were given a high priority [
4].
Despite intense control measures including blanket vaccination, surveillance and depopulation of infected poultry holdings, HPAI H5N1 has gained endemic status in Egyptian poultry populations [
5] and continuous, year-round circulation of HPAI H5N1 virus has been reported [
6‐
8]. This is at least in part due to the highly divergent evolution of H5N1 viruses in Egypt which seems to be accelerated and shaped by vaccine-induced selection pressure leading to the emergence of genetically and antigenically distinct viruses [
9]. Strains of the parent subclade 2.2.1 proper (2.2.1p) are reported to circulate nationwide mainly in unvaccinated birds, particularly waterfowl from backyard holdings [
7]. The vast majority of human infections (34 fatalities out of 109 infected cases until 22
nd June 2010 [
10]) is attributable to viruses of this group. Since 2007, viruses of a variant sublineage emerged from clade 2.2.1. These viruses, which will here be referred to as lineage 2.2.1v, originated from and circulate predominantly in vaccinated commercial chickens [
8]. These antigenically drifted strains were shown to escape immunity induced by standard H5 vaccination and are prevalent mainly in Lower Egypt, particularly in the Nile Delta [
7,
8,
11,
12]. Knowledge of the epidemiology, especially the transmission pathways, of those two lineages between commercial farms, backyard birds, feral birds and humans is incomplete but urgently required to improve control measures. So far, assignment of viruses to either lineage requires virus isolation and antigenic characterization by hemagglutination inhibition or sequencing and molecular analysis. No rapid typing tools are currently available.
A number of RT-qPCR assays for diagnosis and characterization with respect to subtype and pathogenicity of HPAIV H5N1 have been published. These assays target the matrix gene [
13,
14], the nucleoprotein gene [
15,
16], the neuraminidase and the hemagglutinin [
17‐
23]. Egypt's surveillance program embarked on the H5-specific RT-qPCR assay [
23], which is recommended by the World Organization of Animal Health (OIE). The assay was initially highly successful in detecting H5N1 infections in Egypt [
5,
7]. Since 2007, however, an increasing number of strains in Egypt escaping detection by this assay was reported [
24]. Therefore, the aim of this study was to develop a sensitive multiplex RT-qPCR able to detect all HPAIV H5N1 variants of clade 2.2 currently circulating in Egypt and, simultaneously, to distinguish between conventional 2.2.1p strains and the 2.2.1v lineage of vaccine-driven escape variants.
Discussion
We report here the development of a multiplex RT-qPCR assay for detection and differentiation of Egyptian H5N1 HPAI viruses of clade 2.2.1 proper (p) and the emerging 2.2.1 lineage of viruses which escape standard vaccine-induced immunity (designated here 2.2.1v). The assay was shown to have a detection limit of 2-5 cRNA copies per reaction. Based on a phylogenetic analysis of 50 isolates tested [
7,
8], the assay is fully specific with regard to assigning the Egyptian H5N1 isolates to either phylogenetic cluster. No unspecific reactivity with either non-H5 AIV or other avian pathogens was evident. However, the assay can not be used for generic detection of subtype H5 viruses as a large percentage of non-Egyptian H5 subtype strains could not be detected.
In terms of diagnostic performance regarding the HPAIV H5N1 strains currently circulating in Egypt, the multiplex assay was at least equal to standard RT-qPCRs targeting the M gene of AIV and superior to a generic H5 RT-qPCR [
23] when examining swabs which originated from experimental infections or from field samples of poultry holdings in Egypt. The generic H5-specific RT-qPCR assay described by Slomka et al. [
23] apparently missed five Egyptian field samples, possibly due to mismatches in binding regions of primers and/or probes; one of the missed samples was assigned to lineage 2.2.1v while four of them belonged to 2.2.1p. In three field samples the multiplex assay detected both clades with almost similar Ct values (Additional file
4, Table S4, #9, #17, #18). The field samples analysed were derived from pooled swabs of five birds of each holding; as such it can not be excluded that infections with both clades occurred simultaneously at these holdings. Work is in progress to clarify these cases by sequencing clones of HA gene fragments. A similar situation was also encountered with two isolates (Additional file
1, Table S1, #3, #18); alignment of primer and probe sequences with the published sequences of these isolates, however, did not yield any hint for an unspecific reactivity. A contamination of these isolates can only be excluded by sequencing clones of HA gene fragments.
Reducing the amount of circulating HPAIV H5N1 virus by concerted actions of rapid and specific testing, culling and vaccination of poultry is the key to mitigate the risk of human infections and fatalities in Egypt. Controlling the endemic HPAIV H5N1 situation in Egypt is particularly painstaking because of [
1] the concentration of the majority of commercial and backyard poultry business in a very small part of the whole country (Nile valley and, particularly, Nile delta), [
2] the integration/contacts of backyard birds within small commercial poultry farms (farms with 5.000-20.000 birds represent circa 75% of the poultry production), [
3] the marketing system (random uncontrolled movement of birds to/from live bird markets), and [
4] day labourers at commercial farms usually raise backyard birds in their houses. In addition, continuing viral evolution which is even further accelerated and skewed by vaccination pressure remains a daily challenge for diagnostic measures which are at the root of all efforts to control the situation. Characterization of currently circulating strains and, if required, adaptation of amplification-based diagnostic tools, such as introduced here, is essential to improve the situation.
Conclusions
The necessity to update commercial and generic H5-specific RT-qPCRs for the Egyptian situation has been stressed recently [
24]. The current assay provides this update. The assay is tailored to suite the special Egyptian situation. Therefore, the multiplex assay is not recommended for use elsewhere, particularly in areas where non-clade 2.2 HPAIV H5N1 are prevalent. Also, should new lineages of HPAIV H5N1 be introduced into Egypt, such as the 2.3.2 subclade viruses which already escaped from Central and South-eastern Asia to South-eastern Europe earlier in 2010, the current assay will need updating again. In addition to detection of clade 2.2.1p H5 HPAIV the multiplex assay also allows the positive identification of the 2.2.1v lineage of vaccine escape mutants. This lineage probably evolved in commercial chicken farms where vaccination using standard LPAIV H5 strains was practiced. Recent studies have shown that new vaccines might be required to efficiently induce protective immunity against lineage 2.2.1v viruses in poultry [
11]. The multiplex assay therefore may also be instrumental in decision-making regarding the type of vaccine to be used for the specific outbreak situation.
Methods
Reference viruses and bacteria
A panel of 50 HPAIV H5N1 strains isolated in SPF-chicken eggs in the National Laboratory for Quality Control on Poultry Production (NLQP) in Egypt was used for determination of the analytical specificity and sensitivity of the PCR assays (Additional file
1, Table S1). In addition 42 further avian influenza viruses of subtypes H1 [
4], H2 [
5], H5 [
22], H6 [
5], H7 [
3], and H9 [
3] from the repository of German National Reference Laboratory for Avian Influenza, Friedrich-Loeffler Institute, were analysed (Additional file
2, Table S2). Non-orthomyxoviruses and several bacterial species were used to further determine the specificity of the assay.
Primer/probe design
A collection of 316 near full length H5 gene segment sequences of H5N1 viruses circulating in Egypt between 2006 and 2010 was retrieved from the public GenBank data base. Sequences were aligned using MUSCLE [
25] and manually edited. Primers and probes design were selected from a variable region of the HA2 gene for detection of an 85 bp fragment and a more conserved region in the HA1 gene region for detection of a 106 bp fragment (Table
1, Figure
1). Primers P1FW-Standard-EGY, P1RV-EGY and probe PRO1a-Standard-EGY were used for detection of the clade 2.2.1p strains (HEX channel). Primers P1FW-Variant-EGY, P1RV-EGY and probe PRO-Variant-EGY (FAM channel) were used for detection of the clade 2.2.1v variant strains. Primers P2FW-Standard-EGY, P2FW-Variant-EGY, P2RV-Standard-EGY and P2RV-Variant-EGY and probe PRO2-EGY were used for detection of both lineages via the ROX channel.
Real time RT-PCR optimization
The concentration of primers and probes was optimized in separate PCRs (two primers, one probe) and re-adjusted when combined in the multiplex RT-qPCR to increase the efficiency of amplification. Likewise, different annealing temperatures ranging from 50 to 60°C and different chemistries (SuperScript III One-Step RT-PCR system with Platinum Taq DNA polymerase [Invitrogen]; Quantitect One step kit; [Qiagen]) were evaluated. Reactions were carried out in a 25-μl volume on an MX3005P real time PCR machine (Stratagene).
Quantitative analysis
For preparation of standard controls, the cloned H5 gene segment from A/chicken/Egypt/0879-NLQP/2008 (clade 2.2.1v) and A/chicken/Egypt/NLQP-0918Q/2009 (clade 2.2.1p) was used for generating cRNA in vitro by run-off transcriptions performed as previously described [
18]. Detection limit of the RT-qPCR was determined using 10-fold serial dilutions (10
1-10
7 copies) of cRNA.
Samples from experimentally infected chickens
All animal experiments were conducted following official German animal welfare regulations (LALLF M-V/TSD/7221.3-2.1-031/09). Six weeks old SPF chickens (n = 10, each) were infected by the oculo-nasal route with a dose of 10
6.0 TCID
50 of A/chicken/Egypt/0879-NLQP/2008 (clade 2.2.1v) or A/chicken/Egypt/NLQP-0918Q/2009 (clade 2.2.1p) (Grund et al., unpublished). RNA was extracted from mixed oropharyngeal and cloacal swabs collected from individual birds 2 or 7 days post infection (dpi). RNA was used in quantitative RT-qPCRs described in this study and compared to M and H5 based RT-qPCR assays as previously described [
14,
23].
Field samples
Tracheal and cloacal swabs were collected both from poultry at commercial farms (n = 11) and backyard holdings (n = 39) in the frame of the national surveillance scheme in poultry sectors in Egypt from 2008 to 2010. Oropharyngeal and cloacal swabs from five birds were pooled for RNA extraction and PCR analysis.
Extraction of RNA from 140 μl of allantoic fluid (RNA viral isolates) or swab fluid (field samples, experimental infections) was carried out using the QIAamp viral RNA mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The RNA was eluted from the columns with 50 μl of DEPC-treated water and used immediately or after storage at -80°C. Likewise, DNA was extracted from bacterial species and DNA viruses (Additional file
2, Table S2) using the QIAamp DNA mini kit (Qiagen, Hilden, Germany).
Statistics
In addition to the descriptive evaluation of the test results, the technical sensitivity of the new multiplex RT-qPCR was investigated by comparing positive Ct-values with those of the standard H5-specific RT-qPCR test recommended by Slomka et al. [
23]. For this purpose, Fisher's exact tests were applied considering the one-sided hypothesis of achieving lower Ct-values by the multiplex RT-qPCR than by the standard test. All statistical calculations have been performed using R, Version 2.8.1 (2008-12-22) [
26].
Acknowledgements
We are grateful to our co-workers and colleagues for excellent technical work in the FLI, Germany, and the NLQP, Egypt. El-Sayed is funded by a grant from the German Academic Exchange Service to the Free University of Berlin. Work by the authors on Egyptian HPAIV H5N1 is co-funded in the frame of an O.I.E. Twinning project between the Animal Health Research Institute, Giza, and the FLI, Riems.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
ESAW carried out part of the development studies, the analytical and the diagnostic evaluation; he performed the sequence alignments and helped to draft the manuscript. AME and ASA carried out parts of the analytical and diagnostic evaluation using Egyptian samples. MZ carried out the statistical tests. CG, MMA and HMH provided samples for analysis, participated in the design of the study and helped to draft the manuscript. TCH conceived and coordinated the study, and drafted the manuscript. All authors read and approved the final manuscript.