Magdy F. El-Kady

S1 gene sequence analysis of a nephropathogenic strain of avian infectious bronchitis virus in Egypt

BackgroundInfectious bronchitis is highly contagious and constitutes one of the most common and difficult poultry diseases to control. IBV is endemic in probably all countries that raise chickens. It exists as dozens of serotypes/genotypes. Only a few amino acid differences in the S1 protein of vaccine and challenge strains of IBV may result in poor protection. Tropism of IBV includes the respiratory tract tissues, proventriculus and caecal tonsils of the alimentary tract, the oviduct and the kidney.ResultsInfectious bronchitis virus (IBV) strain closely related to Massachusetts (Mass) serotype was isolated from broiler chickens suffering from severe renal and respiratory distresses. The isolate was serologically identified by Dot-ELISA and further characterized by RT-PCR then genotyped using S1 gene sequence analysis. Alignment of the S1 sequence of the isolate with 16 IBV strains revealed high homology to isolates related to Mass serotype. Inoculation with the strain reproduced the disease in experimental 1-day-old chickens and resulted in 20% mortality, severe renal and moderate respiratory distresses. Marked histopathological changes in both kidney and trachea were observed in experimentally infected chickens. A protection study using the H120 live attenuated vaccine showed low protection rate in spite of high S1 sequence homology (97%). Protection based criteria were: virus re-isolation attempts from trachea, tracheal and renal histopathology as well as IBV antigens detection by immunofluorescent antibody technique in kidney sections.ConclusionPeriodical evaluation of cross-protective capabilities of IBV vaccine(s) versus recently recovered field isolates should be performed to ensure optimum control of IBV.

Sequence diversity of the haemagglutinin open reading frame of recent highly pathogenic avian influenza H5N1 isolates from Egypt

The sequences encoding the haemagglutinin (HA) of twelve H5N1 isolates obtained in 2006 and 2007 from different avian species in backyard holdings and poultry farms in Egypt revealed amino acid variations across the polypeptide and also in the polybasic cleavage motif of three of the isolates from backyard poultry with one, so far, unique mutation in an isolate from a chicken. The HAs of two isolates (A/goose/Egypt/R4/2007, A/chicken/Egypt/R3/2007) collected on the same day in the same village from two neighbouring houses were found to differ from each other. Five out of the seven nucleotide exchanges in these two isolates were translationally silent, and two resulted in amino acid substitutions: one in the polybasic cleavage motif and the other in the signal peptide. Circulation of different H5N1 strains possessing considerable variations in backyard poultry, particularly domestic waterfowl, draws attention to the evolution of H5N1 subtypes in Egypt.

Isolation and mutation trend analysis of influenza A virus subtype H9N2 in Egypt

BackgroundAvian influenza virus H9N2 is a panzootic pathogen that affects poultry causing mild to moderate respiratory distress but has been associated with high morbidity and considerable mortality. Interspecies transmission of H9N2 from avian species to mammalian hosts does occur. The virus possesses human virus-like receptor specificity and it can infect humans producing flu-like illness.MethodsRecently, mild influenza like symptoms were detected in H5N1 vaccinated flocks. Influenza A subtype H9N2 was isolated from the infected flock. The virus evolution was investigated by sequencing the viral genes to screen the possible virus recombination. The viral amino acid sequences from the isolated H9N2 strains were compared to other related sequences from the flu data base that were used to assess the robustness of the mutation trend. Changes in the species-associated amino acid residues or those that enabled virulence to mammals were allocated.ResultsPhylogenetic analyses of haemagglutinin and neuraminidase genes showed that the recently isolated Egyptian strain belonged to the H9N2 sub-lineage that prevails in Israel. The six internal segments of the isolated virus were found to be derived from the same sub-lineage with no new evidence of reassortment. The results demonstrated conserved genetic and biological constitution of H9N2 viruses in the Middle East. The recently isolated H9N2 virus from chicken in Egypt possessed amino acids that could enable the virus to replicate in mammals and caused severe disease in domestic chickens.ConclusionThe study highlights the importance of continuous monitoring of the mutations evolved in avian influenza viruses and its impact on virulence to avian species in addition to its importance in the emergence of new strains with the capacity to be a pandemic candidate.

Prevalence of avian respiratory viruses in broiler flocks in Egypt

Abstract In this study, respiratory viral pathogens were screened using real-time RT-PCR in 86 broiler chicken flocks suffering from respiratory diseases problems in 4 Egyptian governorates between January 2012 and February 2014. The mortality rates in the investigated flocks ranged from 1 to 47%. Results showed that mixed infection represented 66.3% of the examined flocks. Mixed infectious bronchitis (IBV) and avian influenza (AI)-H9N2 viruses were the most common infection (41.7%). Lack of AI-H9N2 vaccination and high rates of mixed infections in which AI-H9N2 is involved indicate an early AI-H9N2 infection with a potential immunosuppressive effect that predisposes for other viral infections. High pathogenic AI-H5N1 and virulent Newcastle disease virus (vNDV) infections were also detected (26.7% and 8.1%, respectively). Interestingly, co-infection of AI-H9N2 with either AIV-H5N1 or vNDV rarely resulted in high mortality. Partial cell-mediated immunity against similar internal AI genes, as well as virus interference between AI and vNDV, could be an explanation for this. Highly prevalent IBV and AI-H9N2 were isolated and were molecularly characterized based on S1 gene hypervariable region 3 (HVR3) and hemagglutinin gene (HA) sequences, respectively. IBV strains were related to the variant group of IBV with multiple mutations in HVR3. Though AI-H9N2 viruses showed low rate of evolution in comparison to recent strains, few amino acid substitutions indicative of antibody selection pressure were observed in the HA gene. In conclusion, mixed viral infections, especially with IBV and AI-H9N2 viruses, are the predominant etiology of respiratory disease problems in broiler chickens in Egypt. Further investigations of the role of AI, IBV, and ND viruses’ co-infections and interference in terms of altering the severity of clinical signs and lesions and/or generating novel reassortants within each virus are needed.

Erratum to: Serological surveillance reveals widespread influenza A H7 and H9 subtypes among chicken flocks in Egypt

Multiple avian influenza viruses’ subtypes are circulating worldwide possessing serious threat to human populations and considered key contributors to the emergence of human influenza pandemics. This study aimed to identify the potential existence of H7 and H9 avian influenza infections circulating among chicken flocks in Egypt. Serum samples were collected from chicken flocks that experienced respiratory distresses and/or variable mortality rates. H7 and H9 virus infections were screened by haemagglutination inhibition assay using chicken erythrocytes. Serum samples were collected from 9 broiler, 12 breeder and 18 layer flocks. Out of 1,225 examined sera, 417 (34 %) from 14 flocks and 605 (49.4 %) from 21 flocks were found positive for H7 and H9, respectively. Prevalence of both H7 and H9 antibodies were higher in layer followed by breeder then broiler flocks. Special consideration should be paid to control influenza viruses in Egypt, as pandemic influenza strains may develop unnoticed given the presence of subclinical infections, and the possibility of re-assortment with the prevailing endemic H5N1 virus strains in Egypt do exist.

Evolutionary trajectories and diagnostic challenges of potentially zoonotic avian influenza viruses H5N1 and H9N2 co-circulating in Egypt.

In Egypt, since 2006, descendants of the highly pathogenic avian influenza virus (HP AIV) H5N1 of clade 2.2 continue to cause sharp losses in poultry production and seriously threaten public health. Potentially zoonotic H9N2 viruses established an endemic status in poultry in Egypt as well and co-circulate with HP AIV H5N1 rising concerns of reassortments between H9N2 and H5N1 viruses along with an increase of mixed infections of poultry. Nucleotide sequences of whole genomes of 15 different isolates (H5N1: 7; H9N2: 8), and of the hemagglutinin (HA) and neuraminidase (NA) encoding segments of nine further clinical samples (H5N1: 2; H9N2: 7) from 2013 and 2014 were generated and analysed. The HA of H5N1 viruses clustered with clade 2.2.1 while the H9 HA formed three distinguishable subgroups within cluster B viruses. BEAST analysis revealed that H9N2 viruses are likely present in Egypt since 2009. Several previously undescribed substituting mutations putatively associated with host tropism and virulence modulation were detected in different proteins of the analysed H9N2 and H5N1 viruses. Reassortment between HP AIV H5N1 and H9N2 is anticipated in Egypt, and timely detection of such events is of public health concern. As a rapid tool for detection of such reassortants discriminative SYBR-Green reverse transcription real-time PCR assays (SG-RT-qPCR), targeting the internal genes of the Egyptian H5N1 and H9N2 viruses were developed for the rapid screening of viral RNAs from both virus isolates and clinical samples. However, in accordance to Sanger sequencing, no reassortants were found by SG-RT-qPCR. Nevertheless, the complex epidemiology of avian influenza in poultry in Egypt will require sustained close observation. Further development and continuing adaptation of rapid and cost-effective screening assays such as the SG-RT-qPCR protocol developed here are at the basis of efforts for improvement the currently critical situation.

Genetic drift evolution under vaccination pressure among H5N1 Egyptian isolates

BackgroundThe highly pathogenic H5N1 is a major avian pathogen that intensively affects the poultry industry in Egypt even in spite of the adoption of vaccination strategy. Antigenic drift is among the strategies the influenza virus uses to escape the immune system that might develop due to the pressure of extensive vaccination. H5N1 mutates in an intensified manner and is considered a potential candidate for the possible next pandemic with all the catastrophic consequences such an eventuality will entail.MethodsH5N1 was isolated from the pooled organ samples of four different affected flocks in specific pathogen free embryonated chicken eggs (SPF-ECE). A reverse transcriptase polymerase chain reaction (RT-PCR) was performed to the haemagglutingin and neuraminidase. Sequencing of the full length haemagglutingin was performed. Sequence analyses of the isolated strains were performed and compared to all available H5N1 from Egyptian human and avian strains in the flu database. Changes in the different amino acid that may be related to virus virulence, receptor affinity and epitope configuration were assigned and matched with all available Egyptian strains in the flu database.ResultsOne out of the four strains was found to be related to the B2 Egyptian lineage, 2 were related to A1 lineage and the 4th was related to A2 lineage. Comparing data obtained from the current study by other available Egyptian H5N1 sequences remarkably demonstrates that amino acid changes in the immune escape variants are remarkably restricted to a limited number of locations on the HA molecule during antigenic drift. Molecular diversity in the HA gene, in relevance to different epitopes, were not found to follow a regular trend, suggesting abrupt cumulative sequence mutations. However a number of amino acids were found to be subjected to high mutation pressure.ConclusionThe current data provides a comprehensive view of HA gene evolution among H5N1 subtype viruses in Egypt. Egyptian H5N1-AIVs are constantly undergoing genetic changes and reveal a complex pattern of drifts. These findings raise the concerns about the value of using influenza vaccines in correlation with the development of antigenic drift in influenza epidemics.

Experimental co-infection of infectious bronchitis and low pathogenic avian influenza H9N2 viruses in commercial broiler chickens

Abstract In this study, commercial broilers were experimentally infected with single (classical IBV, variant IBV or AIV-H9N2) or mixed AIV-H9N2 with classical, variant or vaccine strains of IBV. Birds were monitored for clinical and pathological outcomes and virus shedding for 10days post infection (DPI). Clinical signs were limited to the respiratory tract in all challenged groups and varied from mild to moderate mouth breathing to severe respiratory signs with snorting sound and extended head. Mortalities were only recorded in mixed AIV-H9N2/variant IBV challenge group. AIV-H9N2 challenge caused tracheal petechial hemorrhage that progressed to tracheal congestion and caseation. In mixed AIV-H9N2/IBV vaccine challenge, severe tracheitis with bronchial cast formation was observed. In mixed AIV-H9N2/variant IBV challenge severe congestion of the tracheal mucosa and excessive exudates with a tendency to form tubular casts were observed. Kidney ureate deposition was only observed in variant IBV challenge group. Histopathologically, tracheal congestion, severe degeneration, and deciliation were noticed in all groups of mixed infection. Interestingly, hemorrhage and atrophy were observed in thymus gland of birds challenged with single AIV-H9N2 or mixed AIV-H9N2/IBV. There was no difference in the tracheal shedding level of variant IBV between single and mixed infected groups while classical IBV shedding increased in mixed infection group. Interestingly, the AIV-H9N2 showed constantly high shedding titers till 7DPI with variant or vaccine IBV co-infection. In conclusion, co-infection of IBV and AIV-H9N2 induced severe clinical outcome and high mortality. Also, IBV co-infection increased the shedding of AIV-H9N2 in experimentally infected birds.

Humoral antibody responses to different H5N1 and H5N2 vaccination regimes: Implications for the development of autogenously based vaccines

Whereas H5N1 vaccine and several H5N2 vaccines are commercially available and are used to control H5N1 outbreaks in some endemic countries, infections hit many vaccinated flocks. The following study was conducted to compare the efficacy of such vaccines and to assess their potential induction of antibodies against the haemagglutinin of local H5N1 isolate after single vaccination. The possible beneficiary effect of booster dose at different intervals was screened for both H5N1 vaccine as well as a selected H5N2 candidate. Differences in the serological immune response among native and cross breeds were also screened. No significant variations were detected between available commercial H5N1 and H5N2 vaccines after single vaccination. Two vaccination shots using H5N1 but not H5N2 vaccine were found to be superior to a single vaccination scheme, where chicks developed more conceivable antibody titers than in single vaccination program. There was considerable variation among chicken lines in the immune response to H5N1 vaccine: native breeds possessed the highest antibody titers as compared to other breeds.

Genotyping and pathotyping of diversified strains of infectious bronchitis viruses circulating in Egypt

AIM To characterize the circulating infectious bronchitis virus (IBV) strains in Egypt depending on the sequence of the spike-1 (S1) gene [hypervariable region-3 (HVR-3)] and to study the pathotypic features of these strains. METHODS In this work, twenty flocks were sampled for IBV detection using RRT-PCR and isolation of IBV in specific pathogen free (SPF) chicks during the period from 2010 to 2015. Partial sequencing and phylogenetic analysis of 400 bp representing the HVR-3 of the S1 gene was conducted. Pathotypic characterization of one selected virus from each group (Egy/Var-I, Egy/Var-II and classic) was evaluated in one day old SPF chicks. The chicks were divided into 4 groups 10 birds each including the negative control group. Birds were inoculated at one day by intranasal instillation of 10(5)EID50/100 μL of IBV viruses [IBV-EG/1212B-2012 (Egy/Var-II), IBV/EG/IBV1-2011 (Egy/Var-I) and IBV-EG/11539F-2011 (classic)], while the remaining negative control group was kept uninfected. The birds were observed for clinical signs, gross lesions and virus pathogenicity. The real-time rRT-PCR test was performed for virus detection in the tissues. Histopathological examinations were evaluated in both trachea and kidneys. RESULTS The results revealed that these viruses were separated into two distinct groups; variant (GI-23) and classic (GI-1), where 16 viruses belonged to a variant group, including 2 subdivisions [Egy/Var-I (6 isolates) and Egy/Var-II (10 isolates)] and 4 viruses clustered to the classic group (Mass-like). IBV isolates in the variant group were grouped with other IBV strains from the Middle East. The variant subgroup (Egy/Var-I) was likely resembling the original Egyptian variant strain (Egypt/Beni-Suif/01) and the Israeli strain (IS/1494/2006). The second subgroup (Egy/Var-II) included the viruses circulating in the Middle East (Ck/EG/BSU-2 and Ck/EG/BSU-3/2011) and the Israeli strain (IS/885/00). The two variant subgroups (Egy/Var-I and Egy/Var-II) found to be highly pathogenic to SPF chicks with mortalities up to 50% than those of the classic group which was of low virulence (10% mortality). Pathogenicity indices were 25 (Egy/Var-II), 24 (Egy/Var-I) and 8 (classic); with clinical scores 3, 2 and 1 respectively. CONCLUSION These findings indicated that the recent circulating Egyptian IBVs have multiple heterogeneous origins in marked diversifying nature of their spread, with high pathotype in specific pathogen free chicks.