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 Inactivated Avian Influenza Virus and Newcastle Disease Virus Bivalent Vaccination Program Against Newly Circulated H5N8 and NDV Strains

Avian influenza virus (AIV) and Newcastle disease virus (NDV) are respiratory illness syndromes that have recently been detected in vaccinated flocks and are causing major financial losses in the chicken farming industry. The objective was to evaluate the efficacy of Valley Vac H5Plus NDVg7 vaccine in protecting chickens against the H5N8 and NDV strains that have recently been circulating in comparison with the efficacy of the commercially available bivalent H5+ND7 vaccine. In contrast to the H5+ND7 vaccine, which was made of genetically distinct H5N8/2018 clade 2.3.4.4b genotype group (G5), H9N2/2016, H5N1/2017, and genetically comparable NDV genotype VII 1.1/2019 of the recently circulating challenge viruses, the Valley Vac H5Plus NDVg7 vaccine consisted of the recently isolated (RG HPAI H5N1 AIV/2015 Clade 2.2.1.2, RG HPAIV H5N8/2020 Clade 2.3.4.4b genotype group 6 (G6), and NDV genotype VII 1.1/2012) which were genetically similar to challenged strains. To determine the effectiveness of the Valley Vac H5Plus NDVg7 vaccine, a total of 70-day-old commercial chicks were divided into 7 groups of 10 birds each. Groups (G1 and G4) received Valley Vac H5Plus NDVg7 vaccine. Groups (G2 and G5) groups received commercial H5+ND7 vaccine. While groups (G3 and G6) were kept nonvaccinated, and group (G7) was kept as a nonchallenged and nonvaccinated. After 3-wk post vaccination (WPV), groups G1, G2, and G3 were challenged with A/Duck/ Egypt/SMG4/2019(H5N8) genotype G6. On the other hand, groups G4, G5, G6 were challenged with NDV/EGYPT/18629F/2018 genotype VII 1.1 with an intranasal injection of 0.1 mL. Antibody titer was calculated at the first 3 wk after vaccination, and the viral shedding titer was calculated at 3-, 5-, and 7-days post challenge. Mortality and morbidity rates were monitored daily during the experiment, and for the first 10 d after the challenge, to provide an estimate of the protection rate. The results showed that a single dosage of 0.5 mL per bird of Valley Vac H5Plus NDVg7 vaccine provides 80% protection against both H5N8 and NDV, compared to the bivalent H5+ND7 vaccine, which provided 20 and 80% protection against H5N8 and NDV, respectively. In addition, 0.5 mL per bird of Valley Vac H5Plus NDVg7 vaccine produced a greater immune response against both viruses than commercial vaccination at 1 to 3 WPV with a significant difference at 1 WPV for H5N8 and a comparatively higher immune response for NDV. Furthermore, it reduced virus shedding of H5N8 on the third, fifth, seventh, and tenth days lower than H5+ND7 vaccine with a significant difference on the third day for H5N8 and relatively lower than bivalent H5+ND7 vaccine for NDV with a significant difference on the fifth day. The Valley vaccinated group demonstrated more tissue intactness compared to the commercially vaccinated group against the H5N8 challenge, however the bivalent commercially vaccinated group showed the similar level of tissue integrity against NDV. In conclusion, Valley Vac H5Plus NDVg7 that contains the genetically similar strain to recently circulating challenged virus (H5N8 genotype G6) provided better protection with greater immune response and decreased the amount of virus shed against H5N8 genotype G6 and showed less histopathological alteration than the commercial bivalent H5+ND7 vaccine that contain genetically distinct (H5N8 genotype G5). However the Valley Vac H5Plus NDVg7 provided the same protection with relatively high immune response and relatively decreased the amount of virus shed and showed equal tissue integrity than the commercial bivalent H5+ND7 vaccine against NDV genotype VII 1.1 that contain the same genotype of NDV genotype VII 1.1

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

Molecular Characterization and Phylogenetic Analysis of Fowl Adenoviruses Isolated from Broiler Chicken Flocks

FADV has caused high economic losses in poultry industry in Egypt in the last few years. The study aimed to detect and genetically characterize the fowl adenovirus (FAdV) species prevalent in Egyptian commercial broiler chicken flocks during 2023. The 63 suspected samples were collected from Egyptian broiler chickens from 5 governorates during 2023. The molecular characterization was performed by using polymerase chain reaction (PCR) and the positive samples was isolated in primary chicken embryo liver (CEL) cells. The genetic characterization of 8 selected samples represented different governorates by sequencing of loop 1 (L1) of the hexon gene. Clinically, the poultry suffered from depression, watery diarrhea, and ascites and decreased body weight with a mortality rate of 10–30%. The post-mortem inspection showed liver was pale, enlarged with petechial haemorrhage. 27 out of 63 samples (42.8%) were positive by PCR. The molecular charctersation of the L1 hexon gene’s revealed that the FADV (from Eg-ANY1-2023 to EG-ANY4-2023) genetically charcterized as FADV-D 2/11 strains, the FADV-EG-ANY5-2023 to FADV-EG-ANY8-2023 genetically characterized as FADV E/8a and FADV E/8b. By mutation analysis, the strains in our study related to FADV-E/8a (FADV-EG-ANY5, ANY6) had R171K in the HVR4 and strain related to 8b (FADV-EG-ANY7, ANY8) had S95N in the HVR2 and A91T between HVR1 and HVR2 compared to other reference strains. Thus, these findings demonstrate that many mutated virus genotypes are circulating in commercial chicken flocks. Further research is needed to study the pathogencity of these strains and implement control measures and vaccine production to prevent economic loss in the poultry industry

Application of CuCoMnO (x) coat by sol gel technique on aluminum and copper substrates for solar absorber application

Solar thermal heaters are used widely in domestic and industrial applications. The main part of solar thermal heaters is the absorber surface which must have a maximum absorptivity (alpha) and minimum emissivity (epsilon) of solar radiation. This is achieved by application of selective coating on the absorber surface. In the present work, solar selective CuCoMnO (x) spinel films are deposited by sol gel technique using a dip-coating technique on copper and aluminum sheets. The precursor's ratio Co:Cu:Mn applied is 1:3:3. Different precursor molar ratios were combined with a fixed amount of solvents for the coating process. Process parameters such as withdrawal rate, heat treatment, and substrate materials on the coat characteristics and optical properties were studied. The coated metallic samples were heat treated at 450A degrees C for 30 min in the case of aluminum and at 200A degrees C at different times in the case of copper. Optical properties of the coatings, namely absorptivity (alpha) and emissivity (epsilon) were measured and the deposition process parameters were optimized in order to produce the maximum selectivity (alpha/epsilon) values. The deposition parameters were found to influence both the thickness and surface roughness of the coatings. As the coating thickness decreases, the absorptivity increases while the emissivity decreases irrespective of the substrate material. It was also observed from the results that when applying the coat on aluminum substrates, a maximum selectivity value of (alpha/epsilon) = 31 was realized while for the copper substrates a maximum value of (alpha/epsilon) = 81.8 was obtained. The deposited coatings were analyzed using SEM, XRD, and AFM

Genetic variability of the Avian leukosis virus subgroup J gp85 gene in layer flocks in Lower Egypt

Aim: This study aimed to determine the prevalence of layer flock tumor disease in Lower Egypt during the period of 2018- 2019 and to undertake molecular characterization and determine the genetic diversity of all identified viruses. Materials and Methods: Forty samples were collected from layer chicken located in six governorates of Lower Egypt during the period of 2018-2019. Samples were taken from tumors in different organs. Tumor tissues were identified by histopathological sectioning and then further confirmed by a reverse-transcription polymerase chain reaction. Finally, genetic evolution of Avian leukosis virus (ALV-J) gp85 gene was studied. Results: All the study samples were negative for Marek's disease virus, reticuloendotheliosis virus A,B,C and D and 20 samples were positive for ALV-J in backyard in six governrates. Sequencing of ALV-J gp85 gene was performed for six representative samples (one from each governorate), and they were found to be genetically related to prototype virus HPRS-1003 (identity percentage: 91.2-91.8%), but they were from a different group that was similar to the AF88-USA strain (first detected in 2000) with specific mutations, and they differed from a strain that was previously isolated in Egypt in 2005, forming two different subgroups (I and II) that had mutations in the hr1domain (V128F, R136A) and hr2 domain (S197G, E202K). Conclusion: The ALV-J virus was the main cause of neoplastic disease in layer chickens from Lower Egypt in the period of 2018-2019. We found that the genetic evolution of ALV-J gp85 gene was related to prototype virus HPRS-1003 but in a different group with a specific mutation. Further studies are needed to evaluate the antigenicity and pathogenicity of recently detected ALV-J strains

Genetic evolution of Marek's disease virus in vaccinated poultry farms

Background and Aim: The Marek's disease virus (MDV) is a neoplastic disease causing serious economic losses in poultry production. This study aimed to investigate MDV occurrence in poultry flocks in the Lower Egypt during the 2020 breakout and genetically characterized Meq, gL, and ICP4 genes in field strains of MDV. Materials and Methods: Forty samples were collected from different breeds from eight Egyptian governorates in 2020. All flocks had received a bivalent vaccine (herpesvirus of turkey FC-126 + Rispens CVI988). However, weight loss, emaciation, reduced egg production, paralysis, and rough/raised feather follicles occurred. Samples were collected from feather follicles, liver, spleen, and nerve tissue for diagnosis by polymerase chain reaction. MDV genetic characterization was then performed by sequencing the Meq, gL, and ICP4 genes of five positive samples representing different governorates and breeds. Results: A total of 28 samples were positive for MDV field strains, while two were related to MDV vaccinal strains. All samples tested negative for ALV (A, B, C, D, and J) and REV. Phylogenetic analysis of the Meq gene of sequenced samples revealed that all MDVs were related to the highly virulent European viruses (Gallid herpesvirus 2 ATE and PC12/30) with high amino acid (A.A.) identity 99.2-100%. Alternatively, there was low A.A. identity with the vaccine strains CVI988 and 3004 (up to 82.5%). These results indicate that further investigation of the efficacy of current Egyptian vaccines is required. The Egyptian strains also harbor a specific mutation, allowing clustering into two subgroups (A and B). By mutation analysis of the Meq gene, the Egyptian viruses in our study had R101K, P217A, and E263D mutations present in all Egyptian viruses. Furthermore, R176A and T180A mutations specific to our strains contributed to the high virulence of highly virulent strains. There were no mutations of the gL or ICP4 genes. Conclusion: Further studies should evaluate the protection contributed by current vaccines used 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