Immunogenicity and Cross-Protective Efficacy Induced by an Inactivated Recombinant Avian Influenza A/H5N1 (Clade 2.3.4.4b) Vaccine against Co-Circulating Influenza A/H5Nx Viruses.

Controlling avian influenza viruses (AIVs) is mainly based on culling of the infected bird flocks or via the implementation of inactivated vaccines in countries where AIVs are considered to be endemic. Over the last decade, several avian influenza virus subtypes, including highly pathogenic avian influenza (HPAI) H5N1 clade 2.2.1.2, H5N8 clade 2.3.4.4b and the recent H5N1 clade 2.3.4.4b, have been reported among poultry populations in Egypt. This demanded the utilization of a nationwide routine vaccination program in the poultry sector. Antigenic differences between available avian influenza vaccines and the currently circulating H5Nx strains were reported, calling for an updated vaccine for homogenous strains. In this study, three H5Nx vaccines were generated by utilizing the reverse genetic system: rgH5N1_2.3.4.4, rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2. Further, the immunogenicity and the cross-reactivity of the generated inactivated vaccines were assessed in the chicken model against a panel of homologous and heterologous H5Nx HPAIVs. Interestingly, the rgH5N1_2.3.4.4 induced high immunogenicity in specific-pathogen-free (SPF) chicken and could efficiently protect immunized chickens against challenge infection with HPAIV H5N1_2.3.4.4, H5N8_2.3.4.4 and H5N1_2.2.1.2. In parallel, the rgH5N1_2.2.1.2 could partially protect SPF chickens against infection with HPAIV H5N1_2.3.4.4 and H5N8_2.3.4.4. Conversely, the raised antibodies to rgH5N1_2.3.4.4 could provide full protection against HPAIV H5N1_2.3.4.4 and HPAIV H5N8_2.3.4.4, and partial protection (60%) against HPAIV H5N1_2.2.1.2. Compared to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2 vaccines, chickens vaccinated with rgH5N1_2.3.4.4 showed lower viral shedding following challenge infection with the predefined HPAIVs. These data emphasize the superior immunogenicity and cross-protective efficacy of the rgH5N1_2.3.4.4 in comparison to rgH5N8_2.3.4.4 and rgH5N1_2.2.1.2.

Preparation and evaluation of formalized bivalent Newcastle and Salmonella poultry vaccine

This study was conducted to prepare and evaluate the potency of different inactivated vaccine formulations that protect chickens against Salmonella Enteritidis and Newcastle disease virus using Montanide as adjuvant. Protection and the humoral immune response of prepared vaccines against Salmonella Enteritidis and Newcastle disease virus was evaluated and compared to imported vaccine. In this study, different formulae of Salmonella Enteritidis and Newcastle disease vaccines were prepared and compared with the imported one by measuring the antibody titer against Newcastle disease virus by hemagglutination inhibition test and the antibody titer against Salmonella Enteritidis using Enzyme Linked Immunosorbent Assay. On the other hand, the protection percentages against Newcastle disease and Salmonella Enteritidis were recorded to determine the best effective formula. The highest hemagglutination inhibition antibody level against NDV at first week was recorded for the prepared combined Newcastle disease and Salmonella Enteritidis vaccine (4.2 log2) followed by the prepared monovalent Newcastle disease (3.4 log2); the lowest antibody level (3.1 log2) was obtained with the imported vaccine. A gradual increase was observed in all groups to 7.1 log2, 6.8 log2 and 6.4 log2 at fourth week post vaccination, respectively. The antibody titer against Salmonella Enteritidis was 552 for the prepared combined Salmonella Enteritidis and Newcastle disease, followed by the prepared monovalent Salmonella Enteritidis (477) at first week post vaccination; the antibody titer obtained for the imported vaccine was 477. There was a gradual increase to 1456, 1406 and 1130 at fourth week post vaccination, respectively. Prepared combined vaccines gave the highest protection percentage, followed by prepared monovalent types and finally imported vaccines. Vaccination by the prepared combined Salmonella Enteritidis and Newcastle disease vaccine may be a way to increase the resistance of birds to Salmonella and Newcastle and to decrease the shedding rate(AU)

Este estudio se llevó a cabo para preparar y evaluar la potencia de diferentes formulaciones de vacunas inactivadas que protegen a los pollos contra Salmonella Enteritidis y el virus de la enfermedad de Newcastle utilizando Montanide como adyuvante. Se evaluó la protección y la respuesta inmune humoral de las vacunas preparadas contra Salmonella Enteritidis y el virus de la enfermedad de Newcastle y se comparó con la vacuna importada. En este estudio se prepararon diferentes fórmulas de vacunas contra Salmonella Enteritidis y la enfermedad de Newcastle y se compararon con la importada midiendo el título de anticuerpos contra el virus de la enfermedad de Newcastle mediante la prueba de inhibición de la hemaglutinación y el título de anticuerpos contra Salmonella Enteritidis mediante ELISA. Por otra parte, se registraron los porcentajes de protección contra la enfermedad de Newcastle y Salmonella Enteritidis para determinar la fórmula más eficaz. El mayor nivel de anticuerpos inhibidores de la hemaglutinación contra el virus de la enfermedad de Newcastle, en la primera semana, se registró con la vacuna combinada preparada contra la enfermedad de Newcastle y Salmonella Enteritidis (4,2 log2), seguida de la vacuna monovalente preparada contra la enfermedad de Newcastle (3,4 log2); el menor nivel de anticuerpos (3,1 log2) se obtuvo con la vacuna importada. Se observó un aumento gradual en todos los grupos hasta alcanzar 7,1 log2, 6,8 log2 y 6,4 log2 en la cuarta semana tras la vacunación, respectivamente. El título de anticuerpos contra Salmonella Enteritidis fue de 552 para la vacuna combinada preparada contra la Salmonella Enteritidis y enfermedad de Newcastle, seguida por la vacuna monovalente preparada contra Salmonella Enteritidis (477) en la primera semana después de la vacunación; el título de anticuerpos obtenido con la vacuna importada fue de 477. Hubo un aumento gradual hasta 1456, 1406 y 1130 en la cuarta semana después de la vacunación, respectivamente. Las vacunas combinadas preparadas dieron el mayor porcentaje de protección, seguidas por los tipos monovalentes preparados y, por último, por las vacunas importadas. La vacunación con la vacuna combinada preparada contra la Salmonella Enteritidis y la enfermedad de Newcastle puede ser una forma de aumentar la resistencia de las aves a la Salmonella y Newcastle y de disminuir la tasa de excreción(AU)

Cardenolides and pentacyclic triterpenes isolated from Acokanthera oblongifolia leaves: their biological activities with molecular docking study.

Pentacyclic triterpenes and cardenolides were isolated from Acokanthera oblongifolia leaves. Their chemical structures were determined based on comprehensive 1D and 2D NMR spectroscopy. Their MIC was determined against 12 microorganisms. Their exerted cytotoxicity on the immortalized normal cells, hTERT-RPE1 was assessed by the sulforhodamine-B assay. The viral inhibitory effects of compounds against Newcastle disease virus (NDV) and H5N1 influenza virus IV were evaluated. Four in vitro antioxidant assays were performed in comparison with BHT and trolox and a weak activity was exhibited. Acovenoside A was with potent against H5N1-IV and NDV with IC50 ≤ 3.2 and ≤ 2.1 µg/ml and SI values of 93.75 and 95.23%, respectively, in comparison to ribavirin. Its CC50 record on Vero cells was > 400 and 200 µg/ml, respectively. Acobioside A was the most active compound against a broad range of microbes while Pseudomonas aeruginosa was the most sensitive. Its MIC (0.07 µg/ml) was 1/100-fold of the recorded CC50 (7.1 µg/ml/72 h) against hTERT-RPE1. The molecular docking of compounds on human DNA topoisomerase I (Top1-DNA) and IV glycoprotein hemagglutinin were studied using MOE program. This study has introduced the cardenolides rather than triterpenoids with the best docking score and binding interaction with the active site of the studied proteins.

Development of an effective contemporary trivalent avian influenza vaccine against circulating H5N1, H5N8, and H9N2 in Egypt.

Low pathogenicity avian influenza (LPAI) H9N2, highly pathogenic avian influenza (HPAI) H5N1, and H5N8 circulate in Egyptian poultry and cause veterinary and public health burdens. In response, AIV vaccines are commonly used. The main objective of this study was to develop a broad, cross-protective, trivalent vaccine based on circulating AIVs in Egypt. We generated highly replicating avirulent AIVs, H5N1, and H5N8, to be used in combination with H9N2 strain for the generation of an inactivated vaccine. Immunogenicity and protective efficacy of this vaccine were tested. Results showed that a single immunization dose enhanced humoral immune responses giving full protection against challenges with LPAI H9N2, HPAI H5N1, and H5N8 viruses. This efficacious vaccine will reduce the cost of vaccination for poultry growers and is expected to be effective in the field as it is based on contemporary viruses currently in circulation among Egyptian poultry.

Antiviral activity of Aloe hijazensis against some haemagglutinating viruses infection and its phytoconstituents.

Evaluation of the antiviral activities of flowers, flower-peduncles, leaves, and roots of Aloe hijazensis against haemagglutinating viruses of avian paramyxovirus type-1 (APMV-1), avian influenza virus type A (AI-H5N1), Newcastle disease virus (NDV), and egg-drop syndrome virus (EDSV) in specific pathogen free (SPF) chicken embryos were carried out. Extract of the flowers and leaves showed relatively higher activity than the extracts of other plant parts. Thirteen compounds were isolated from both the flowers and flower-peduncles of A. hijazensis. The isolated compounds were classified into: five anthraquinones; ziganein, ziganein-5-methyl ether, aloesaponarin I, chrysophanol, aloe-emodin, one dihydroisocoumarin; feralolide, four flavonoids; homoplantaginin, isoorientin, luteolin 7-glucuronopyranoside, isovitexin, one phenolic acid; p-coumaric acid, the anthrone; barbaloin together with aloenin. Eleven compounds were attributed to the flowers and seven to the flower-peduncles. Homoplantaginin and luteolin 7-glucuronopyranoside are reported here for the first time from Aloe spp. To the best of our knowledge, this is the first report on the chemical composition and biological activity of those plant parts.

Synthesis, antimicrobial, and antiviral activities of some new 5-sulphonamido-8-hydroxyquinoline derivatives.

A series of fused pyranopyrazole and pyranoimidazole, namely 5-(3,6-diamino-4-aryl-5-carbonitrile-pyrano(2,3-c)pyrazol-2-yl)sulphonyl-8-hydroxyquinolines (5a-e), 5-(6-amino-4-aryl-5-carbonitrile-pyrano(2,3-c)pyrazol-3-yl)sulphonamido-8-hydroxyquinolines (6a-e), 5-(2-thioxo-4-aryl-5-carbonitrile-6-amino-pyrano(2,3-d)imidazol-2-yl)sulphonyl-8-hydroxyquinolines (10a-e), and 5-(2-oxo-4-aryl-5-carbonitrile-6-amino-pyrano(2,3-d)imidazol-2-yl) sulphonyl-8-hydroxyquinolines (11a-e), have been prepared via condensation of some arylidine malononitriles with 5-sulphonamido-8-hydroxyquinoline derivatives 3, 4, 8 and 9. All the synthesized compounds were screened for their antimicrobial activities, and most of the tested compounds showed potent inhibition growth activity towards Escherichia coli, Pseudomonas aeruginosa (Gramnegative bacteria). Furthermore, six selected compounds were tested for their antiviral activity against avian paramyxovirus type1 (APMV-1) and laryngotracheitis virus (LTV), and the results showed that a concentration range of 3-4 µg per mL of compounds 2, 3, and 4 showed marked viral inhibitory activity for APMV-1 of 5000 tissue culture infected dose fifty (TCID(50)) and LTV of 500 TCID(50) in Vero cell cultures based on their cytopathic effect. Chicken embryo experiments show that compounds 2, 3, and 4 possess high antiviral activity in vitro with an inhibitory concentration fifty (IC(50)) range of 3-4 µg per egg against avian APMV-1 and LTV and their toxic concentration fifty (CC(50)) of 200-300 µg per egg.

Chemical composition and biological activity of ripe pumpkin fruits (Cucurbita pepo L.) cultivated in Egyptian habitats.

The chemical composition and biological activity of three parts (rind, flesh and seeds) of pumpkin fruits (Cucurbita pepo L.) cultivated in Egypt were studied. Chemical analysis of fibre, protein, ß-carotene, carbohydrates, minerals and fatty acids present in the rind, flesh, seeds and defatted seeds meal was conducted. Chemical, GC-MS and biological assays of organic extracts of the main fruit parts, rind and flesh established their unique constituents. Chromatographic purification of the extracts afforded triglyceride fatty acid mixture (1), tetrahydro-thiophene (2), linoleic acid (3), calotropoleanly ester (4), cholesterol (5) and 13(18)-oleanen-3-ol (6). GC-MS analysis of the extract's unpolar fraction revealed the existence of dodecane and tetradecane. Structures of the isolated compounds (1-6) were confirmed by NMR and EI-MS spectrometry. Antimicrobial, antiviral and antitumour activities of the fruit parts were discussed. The promising combined extract of rind and flesh was biologically studied for microbial and cytotoxic activities in comparison with the whole isolated components.

Phytoconstituents of Jatropha curcas L. leaves and their immunomodulatory activity on humoral and cell-mediated immune response in chicks.

A novel biflavone di-C-glucoside, 6,6"-di-C-beta-D-glucopyranoside-methylene-(8,8")-biapigenin (1), was isolated from the leaves of Jatropha curcas L. (Euphorbiaceae), together with six known compounds; apigenin 7-O-beta-D-neohesperidoside (2), apigenin 7-O-beta-D-galactoside (3), orientin (4), vitexin (5), vicenin II (6), and apigenin (7). Their structures were determined on the basis of extensive chemical and spectroscopic analyses (UV, NMR and HRESI-MS). The immunomodulatory effect of an 80% aqueous methanol extract (AME) and compounds 1-5 (0.25 mg/kg body wt) to one-day-old specific pathogen-free (SPF) chicks was determined. Stimulation of both humoral and cell-mediated seroresponse was observed, especially those of AME and compound 1. Remarkable effective increases of the antibody titers, lymphocyte and macrophage cells, in blood were recorded. SPF chicks treated with the tested samples exhibited protection against Newcastle disease challenge virus after being vaccinated.