Samah Eid

The circular polymerase extension reaction (CPER) has emerged as a high-fidelity, versatile and rapid tool for generating infectious cDNA clones of positive-sense, single-stranded RNA viruses. Here, we implemented this strategy to generate infectious cDNA clones of two closely related members of the family Arteriviridae : equine arteritis virus KY84 strain (EAV KY84) and porcine reproductive and respiratory syndrome virus VR2332 strain (PRRSV VR2332). Overlapping cDNA fragments spanning the entire viral genomes were generated and assembled with a linker sequence incorporating critical expression elements, yielding a circularized, full-length viral cDNA genome ready for direct transfection into permissive cells. Following transfection, infectious rEAV KY84 and rPRRSV VR2332 strains were recovered and characterized. Similarly, we generated reporter viruses by inserting the mCherry gene downstream of the nsp1/nsp2 cleavage junction in EAV KY84 ORF1a and the GFP gene downstream of ORF1b of PRRSV VR2332. All CPER-generated viruses showed high identity to their parental strains by next-generation sequencing. Growth kinetics of CPER-generated non-reporter and rEAV KY84-RFP mCherry viruses were comparable to their parental strains; however, the reporter rPRRSV VR2332-GFP showed a significant reduction in viral titres despite its stability for at least 15 passages. In contrast, mCherry expression from rEAV KY84-mCherry was negligible at passage 15 and associated with the deletion of the reporter gene. Taken together, the CPER-based method can be used as a rapid tool to generate infectious cDNA clones of arteriviruses, allowing the easy generation of mutants or reporter viruses that can facilitate the investigation of viral gene functions and their interactions with the host.

The infectious bronchitis virus (IBV) causes a severe infectious disease in poultry, leading to significant financial losses. The prevention and treatment of this disease are extremely challenging due to the virus’s rapid mutation. Therefore, quick diagnosis of IBV infections is crucial for controlling the disease. This study aimed to develop a real-time reverse transcription recombinase-aided amplification (RT-RAA) method for IBV. The most effective primer combination was selected for further validation. To determine the assay’s analytical sensitivity, a serial dilution from 10⁵ to 10⁰ EID₅₀/mL was used, and the limit of detection was calculated. The assay could detect down to 10² EID₅₀/mL. The limit of detection (95% Confidence Interval) was 67 EID₅₀ per reaction. There was no cross-reaction with common poultry diseases. When analyzing 39 clinical samples, RT-RAA and RT-PCR showed 100% diagnostic sensitivity and specificity. In conclusion, the IBV RT-RAA detection method is rapid, sensitive, and specific. This approach can be used to improve IBV diagnosis at the point of need.

Background and Aim: Foot-and-mouth disease (FMD) virus causes continuous outbreaks, leading to serious economic consequences that affect animal productivity and restrict trade movement. The potential influence of the disease was due to the emergence of new strains or re-emergence of local strains with major antigenic variations due to genetic mutations. This study aims to evaluate circulating virus in samples collected from infected animals during an outbreak using antigenic characterization and identify whether there is an emergence of a new strain or mutation. Materials and Methods: Reverse-transcription polymerase chain reaction (RT-PCR) was used to screen 86 samples. Viral protein 1 (VP1) codon sequencing was performed. The virus was isolated from the samples inoculated on the baby-hamster kidney cell line and Enzyme-linked immunosorbent assay was performed for serotyping and antigen detection. Results: Based on the RT-PCR screening results, 10 positive samples were selected for sequencing. The sequences belonged to the FMD serotype A African topotype originating from the ancestor prototype Sudan/77, with which it shared 98.48% ± 1.2% similarity. The divergence with local isolates from 2020 was 9.3%. In addition, the sequences were 96.84% ± 1.01% and 95.84% ± 0.79% related to Egyptian-Damietta type 2016 and Sudanese-2018, respectively. Divergence with vaccinal strains ranged from 10% to 17%. Amino acid sequence analysis revealed that the isolates had variation in the most prominent antigenic regions (residues 35–75) and the immunogenic determinants of the G-H loop of VP1 (residues 100–146 and 161–175). Conclusion: The current isolates should be included in the locally produced vaccine to provide broader immunogenic coverage against serotype A African topotypes. Keywords: Epicenters, foot and mouth disease virus, foot-and-mouth disease virus Africa type G-IV, mutations, outbreaks, viral protein 1.

Wild migratory birds have the capability to spread avian influenza virus (AIV) over long distances as well as transmit the virus to domestic birds. In this study, swab and tissue samples were obtained from 190 migratory birds during close surveillance in Egypt in response to the recent outbreaks of the highly pathogenic avian influenza (HPAI) H5N1 virus. The collected samples were tested for a variety of AIV subtypes (H5N1, H9N2, H5N8, and H6N2) as well as other pathogens such as NDV, IBV, ILT, IBDV, and WNV. Among all of the tested samples, the HPAI H5N1 virus was found in six samples; the other samples were found to be negative for all of the tested pathogens. The Egyptian HPAI H5N1 strains shared genetic traits with the HPAI H5N1 strains that are currently being reported in Europe, North America, Asia, and Africa in 2021–2022. Whole genome sequencing revealed markers associated with mammalian adaption and virulence traits among different gene segments, similar to those found in HPAI H5N1 strains detected in Europe and Africa. The detection of the HPAI H5N1 strain of clade 2.3.4.4b in wild birds in Egypt underlines the risk of the introduction of this strain into the local poultry population. Hence, there is reason to be vigilant and continue epidemiological and molecular monitoring of the AIV in close proximity to the domestic–wild bird interface.

The highly pathogenic avian influenza (HPAI) H5N8 virus was first detected in Egypt in late 2016. Since then, the virus has spread rapidly among different poultry sectors, becoming the dominant HPAI H5 subtype reported in Egypt. Different genotypes of the HPAI H5N8 virus were reported in Egypt; however, the geographic patterns and molecular evolution of the Egyptian HPAI H5N8 viruses are still unclear. Here, extensive epidemiological surveillance was conducted, including more than half a million samples collected from different poultry sectors (farms/backyards/live bird markets) from all governorates in Egypt during 2019–2021. In addition, genetic characterization and evolutionary analyses were performed using 47 selected positive H5N8 isolates obtained during the same period. The result of the conducted surveillance showed that HPAI H5N8 viruses of clade 2.3.4.4b continue to circulate in different locations in Egypt, with an obvious seasonal pattern, and no further detection of the HPAI H5N1 virus of clade 2.2.1.2 was observed in the poultry population during 2019–2021. In addition, phylogenetic and Bayesian analyses revealed that two major genotypes (G5 and G6) of HPAI H5N8 viruses were continually expanding among the poultry sectors in Egypt. Notably, molecular dating analysis suggested that the Egyptian HPAI H5N8 virus is the potential ancestral viruses of the European H5N8 viruses of 2020–2021. In summary, the data of this study highlight the current epidemiology, diversity, and evolution of HPAI H5N8 viruses in Egypt and call for continuous monitoring of the genetic features of the avian influenza viruses in Egypt.

Aim: A total of 112 freshly dead ducks aged from 2 to 20 weeks old with a history of respiratory manifestations were investigated for the implication of Pasteurellaceae family members.. Materials and Methods: Isolation and identification to the family level were conducted by conventional bacteriological methods, including microscopic examination and biochemical characterization. Identification to the species level was conducted by polymerase chain reaction (PCR) and analytical profile index (API) 20E kits. Results: Conventional bacteriological isolation and biochemical characterization revealed the infection of 16/112 examined birds with a prevalence rate of 14.3%. PCR confirmed the detection of Pasteurellaceae family conserved genes RpoB and Bootz in 16/16 (100%) isolates. PCR was also used for genus and species identification of the isolated Pasteurellaceae members; the results revealed that 5/16 (31.3%) of isolates were Gallibacterium anatis and 2/16 of isolates (12.5%) were Pasteurella multocida. Riemerella anatipestifer, Mannheimia haemolytica, and Avibacterium paragallinarum were not detected by PCR. Biotyping by API 20E successfully identified 5/16 (31.3%) isolates that could not be typed by PCR and confirmed their belonging to Pasteurella pneumotropica. Neither the available PCR primer sets nor API 20E succeeded for species identification of 4/16 (25%) isolates. Antibiotic susceptibility profiling of isolates revealed that 16/16 (100%) of isolates demonstrated multidrug resistance (MDR) phenotypes. Moreover, 16/16 (100%) of isolates demonstrated a phenotypic resistance pattern to neomycin. Conclusion: Combined genotypic, phenotypic, biotyping, and virulence characterizations are required for laboratory identification of pathogenic Pasteurellaceae. Moreover, P. multocida was not the prevailed member implicated in respiratory problems in ducks as P. pneumotropica, G. anatis, and unidentified strains were involved with higher prevalence. Chloramphenicol and ampicillin demonstrated the highest in vivo effects on the studied Pasteurellaceae. Furthermore, the prevalence of multidrug-resistant isolates signified the demand to implement targeted surveillance in the ducks' production sector, and MDR survey in poultry sectors in Egypt to apply effective control measures.

Aim: This study aimed to investigate the prevalence and implication of extended-spectrum beta-lactamase (ESBL) producing and Class 1 integrons (int1) gene carriers Escherichia coli isolates that demonstrated multidrug resistance (MDR) phenotypes and was isolated from turkeys that suffered from respiratory manifestation. Materials and Methods: A total of 120 freshly dead turkey poults that suffered from respiratory manifestations, with a history of treatment failure at Hefna, Belbis, Sharqia (Egypt) were sampled. From each bird lung and liver were aseptically collected and transported for laboratory investigations. Results: Examination of samples collected from 120 freshly dead turkey poults revealed the isolation of coagulase-positive staphylococci, coagulase-negative staphylococci, Campylobacter spp., Salmonella spp., Proteus spp., Pseudomonas spp., Klebsiella spp., and E. coli with the prevalence rates of 12/120 (10%), 30/120 (35%), 17/120 (14.2%), 5/120 (4.1%), 17/120 (14.2%), 6/120 (5%), 7/120 (5.8%), and 18/120 (15%), respectively. E. coli isolates were subjected for serotyping and characterization, while the rest of isolates were preserved to be investigated later in further studies. Serogrouping of E. coli isolates revealed the identification of O119, O6, O8, and O169, while 1/18 isolate was untypable. Studying phenotypic antibiotic susceptibility profiles of isolates revealed that 18/18 (100%) of isolates demonstrated resistance against cefuroxime, tetracycline, and trimethoprim, 16/18 (88.9%) of isolates demonstrated resistance to amoxicillin/ clavulanic acid, enrofloxacin, and norfloxacin, 14/18 (77.8%) of isolates demonstrated resistance to doxycycline and ciprofloxacin, and 9/18 (50%) of isolates showed resistance to gentamycin. Double disk synergy test showed that 6/18 (33.3%), 8/18 (44.4%), and 13/18 (72.2%) of isolates demonstrated the phenotypic pattern of ESBL producers with cefepime, cefotaxime, and ceftriaxone, respectively. Genotypic attributes for beta-lactamase TEM gene and int1 gene were studied by reverse transcriptase-polymerase chain reaction and revealed that 17/18 (94.4%) of isolates were positive for both genes. Embryo lethality test indicated that the 18 studied E. coli isolates were considered primary pathogens. Conclusion: The results revealed that 18/18 (100%) of E. coli isolates demonstrated MDR against three or more antibiotic groups, 9/18 (50%) of isolates showed extensive resistance against the nine tested chemotherapeutic agents from seven antibiotic groups. It is recommended to monitor the circulation of MDR and ESBL-producing pathogens in poultry production in a one health approach, as a preventive measure to mitigate the risk imposed on public health.

This study assesses the presence of one hundred and fourteen E. colistrains recovered from five hundred chicken examined samples including five hundred heart, five hundred livers and five hundred cloacal swabs with a percentage of 12.8 %. E. coli is one of the most common isolates in avian diseases, which causes colibacillosis, or act as a major factor in development of acute respiratory disease causing high losses especially between chickens. In addition to the conventional methods used for isolation and identification of E. coli, PCR is required as rapid, accurate and specific tool for detection of pathogenic E. coli and their virulence genes.