Basic Informations

C.V

Master Title

Studies on H5N1 avian influenza in household poultry in Beni-Suef governorate

Master Abstract

Six AI strains were isolated during the 2006-2007 outbreaks from backyard holdings in Beni-Suef governorate. The clinical data of the diseased birds were highly suggestive for HPAI. Cloacal swab suspensions from diseased birds were inoculated in the allantoic cavity of 9-11 days old SPF-ECE. Haemagglutinating fluids from dead and surviving embryos were screened for the presence of group A and H5 AIV antigens using rapid chromatographic strip test. All isolates were positive for AIV group antigen but only R1 isolate was positive for H5 antigen. Isolates were further subjected to molecular characterization using RRT-PCR for matrix, H5 and N1 genes and exclusion of subtype H7 gene using specific probes and primer sets. All of the six isolate were found positive for Matrix, H5 and N1 genes and negative for H7 gene. H5 gene full length amplification and N1 gene partial amplification were performed using RT-PCR and the products were sequenced and found to be related to clade 2.2. Three isolates showed mutation at the HA cleavage site. Cloning of short PCR fragments flanking the cleavage site from such isolates confirmed the finding of direct sequence analysis. Sequence analysis revealed that Egyptian strains are closely related to each other based on (H, N) sequences however considerable variability among circulating strains of H5N1 were recorded and mutations at various positions of the H gene including potential glycosilation sites and the proteolytic cleavage site were detected even between two isolates collected from two neighboring houses on the same day. All strains retain most of H5N1 AIV sequence properties relevant to virus virulence. CLUSTAL W multisequence analyses were performed and considerable similarity of the H and N genes and amino acid to A/Gs/Gd/96 [the donor of H5 and N1 genes of the strain A/Harbin/Re-1 (the seed virus strain of H5N1 inactivated AI vaccine)] were observed. On the other hand, lower identity was observed between the Egyptian A/chicken/Mexico/232/94 H5 genes and amino acids that used as the seed virus strain in at least 5 different types of H5N2 inactivated vaccines used for vaccination of poultry in Egypt. N1 genes of all isolated strains showed high homology to each others and have no mutations in the amino acids responsible for Oseltamivir resistance. Continuous surveillance of AIVs is very crucial in the evolutionary pathway especially from backyard holdings where mixed species (including ducks) and ages which kept in close contact with human pose a special challenge. H5N1 vaccine immune responsiveness in different native, crossbred and foreign chicken breeds were evaluated. The possible beneficiary effect of boostering at different intervals was also screened. Haemagglutination inhibition assay using formalin inactivated antigen from recent Egyptian strain was used as the parameter of the immune responsiveness evaluation. Considerable breed variations were detected after single vaccination at 1day of age. The immune responsiveness reached its peak at 5 wks PV in all breed except Balady one (peaked at 3 wks PV).The HI titers began to decline 2 wks after reaching the peak. Balady and Fayoumy breeds showed higher HI titers followed by Rhode Island. Neither Golden nor Manrarah breed reached an acceptable protective HI titer (theoretically). Two vaccination shots were found to be superior to single vaccination scheme where antibody titers reached its conceivable level earlier than single vaccination with subsequent possible disease prevention from early exposure. We tested boostering at different intervals (1, 2, 3 or 4 wks after the initial dose). Double doses at 1 day and 1 wk of age showed the best combination. This is followed by 1 day and 2 wks program. The other combination showed high titers but delay in reaching acceptable titers was recorded. These findings denote that two vaccination shots is better than single vaccination for rapid elevation of the immune response and double doses at 1 and 7 days of age considered the best combination.

PHD Title

Further Studies on the Current Situation of Avian Influenza in Egypt

PHD Abstract

Poultry industry in Egypt is stricken by two avian influenza virus subtypes; highly pathogenic H5N1 and low pathogenic H9N2 viruses. The viruses circulate in all poultry sectors and they are endemic in the country. Great effort was applied to keep the disease under control but all trials failed and this failure in controlling AI in Egypt is a final result of retarded biosecurity, evolution of escape mutant viruses in poultry, shortage of vaccine coverage and the mismatch between circulating and vaccinal strains. Consequently, there was an urgent need to understand the genetic and antigenic properties of the circulating viruses. Moreover, the development of vaccines that are well-matched, antigenically, to currently circulating viruses is one of the main tools for AIV control. In this study, we collected and genetically subtyped twenty AIVs from naturally infected field cases. The viruses were propagated in SPF-ECE and viral antigens were detected using rapid chromatographic strips. HA and NA genes were sutyped by RT-PCR and full length HA and NA gene sequencing was done. Cross antigenic analysis of selected H5N1 and H9N2 strains was done using sera prepared against them in SPF turkey. Three reassortant viruses were constructed using HA of recent Egyptian strains (two H5N1 and one H9N2) in the genetic background of A/Wisconsin/H1N1/33. The immunogenicity of oil emulsion inactivated vaccine prepared from the H9N1 (WSN/K3) and one H5N1 strain (WSN/F27) was evaluated in commercial chickens after single and booster vaccination regimens. Finally, the protection efficacy of single dose of H5N1 vaccine was evaluated in SPF chicks. Although all isolates were obtained from diseased flocks, virus pathogenicity even within subtype varied greatly. H5N1 viruses isolated from rural poultry sectors induced high mortality rates in chickens and variable mortality rates in ducks. Isolates obtained from commercial flocks being naturally infected with H5N1 AIV showed low mortality rates. Previous vaccination in one flock and mixed infection with the co-circulating H9N2 AIV probably resulted in such low mortality in those flocks. H9N2 AIVs were found more prevalent in 2012 than did H5N1 viruses. The virus was associated with variable mortality rates in chickens. The disease also, induced variable disease signs and lesions and in most cases it was associated with respiratory tract infection. Genetically, HA and NA genes of each subtype were closely related to each other. Moreover, isolates from the same year were found more related. The cleavage site of HA of H5 viruses contained polybasic amino acids motif: a marker of pathogenicity. Variations in the signal peptide, proposed antigenic sites and receptor binding sites of the HA genes were observed. Also, significant variation between the HA gene of Egyptian H5N1 strains obtained in the current study and the commercial vaccine seed virus strains was observed. On the other hand the HA gene of Egyptian H9N2 strains showed lower rate of mutations and the collected strains were both genetically and antigenically related. The amino acids playing a functional and structural role in the NA active site were found conserved in both N1 and N2 NA. Recent Egyptian H5N1 NA showed absence of mutations associated with to resistance to oseltamivir. Antigenically, Egyptian H5N1 isolates crossly reacted in-between but to a lower degree than did the H9N2 isolates. Plasmid based reverse genetics was used to develop reassortant H5N1 and H9N1 candidate vaccine strains. The pathogenicity associated polybasic amino acid sequence in the cleavage site of H5N1 HA was removed producing low pathogenic virus. Single and double dose vaccination regimen using cell culture vaccines prepared from the reassortant viruses were found more or less immunogenic in commercial chickens as did the commercially available vaccines. The vaccines produced acceptable HI titers at 3 or 4 weeks post vaccination. Double dose vaccination program significantly improved the immune response to all of tested vaccines. At 21 days post vaccinataion, a single dose of WSN/F27 vaccine containing 128 HA units per dose protected 50% of the vaccinated SPF chicks from clinical disease and mortality after challenge with 106 EID50 from A/chicken/Egypt/128s/2012 H5N1 AIV until the 10th day post infection.