Background: qPCR is a widely used technique in scientific research as a basic tool in gene expression analysis. Classically, the quantitative endpoint of qPCR is the threshold cycle (C_T) that ignores differences in amplification efficiency among many other drawbacks. While other methods have been developed to analyze qPCR results, none has statistically proven to perform better than the C_T method. Therefore, we aimed to develop a new qPCR analysis method that overcomes the limitations of the C_T method. Our f₀% [eff naught percent] method depends on a modified flexible sigmoid function to fit the amplification curve with a linear part to subtract the background noise. Then, the initial fluorescence is estimated and reported as a percentage of the predicted maximum fluorescence (f₀%). Results: The performance of the new f₀% method was compared against the C_T method along with another two outstanding methods—LinRegPCR and Cy₀. The comparison regarded absolute and relative quantifications and used 20 dilution curves obtained from 7 different datasets that utilize different DNA-binding dyes. In the case of absolute quantification, f₀% reduced CV%, variance, and absolute relative error by 1.66, 2.78, and 1.8 folds relative to C_T; and by 1.65, 2.61, and 1.71 folds relative to LinRegPCR, respectively. While, regarding relative quantification, f₀% reduced CV% by 1.76, 1.55, and 1.25 folds and variance by 3.13, 2.31, and 1.57 folds regarding C_T, LinRegPCR, and Cy₀, respectively. Finally, f₀% reduced the absolute relative error caused by LinRegPCR by 1.83 folds. Conclusions: We recommend using the f₀% method to analyze and report qPCR results based on its reported advantages. Finally, to simplify the usage of the f₀% method, it was implemented in a macro-enabled Excel file with a user manual located on https://github.com/Mahmoud0Gamal/F0-perc/releases.

The outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) signifies a serious worldwide concern to public health. Both transcriptome and proteome of SARS-CoV-2-infected cells synergize the progression of infection in host, which may exacerbate symptoms and/or progression of other chronic diseases such as Parkinson’s disease (PD). Oxidative stress is a well-known cause of endoplasmic reticulum (ER) stress observed in both SARS-CoV-2 and PD. In the current study, we aimed to explore the influence of PKR-like ER kinase (PERK) stress pathway under SARS-CoV-2-mediated infection and in human cell model of PD. Furthermore, we investigated whether they are interconnected and if the ER stress inhibitors could inhibit cell death and provide cellular protection. To achieve this aim, we have incorporated in silico analysis obtained from gene set enrichment analysis (GSEA), a literature review and laboratory data. The neurotoxin, 6-hydroxy dopamine (6OHDA), was used to mimic the biochemical and neuropathological characteristics of PD by inducing oxidative stress in dopamine-containing neurons differentiated from ReNVM cell line (dDCNs). Furthermore, we explored if ER stress influences activation of caspases-2, -4 and -8 in SARS-CoV-2 and in stressed dDCNs. Our laboratory data using Western blot, immunocytochemistry and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) analyses indicated that 6OHDA-induced toxicity triggered activation of caspases-2, -4 and -8 in dDCNs. Under SARS-CoV-2 infection of different cell types, GSEA revealed cell-specific sensitivities to oxidative and ER stresses. Cardiomyocytes and type II alveolar epithelial-like cells were more vulnerable to oxidative stress than neural cells. On the other side, only cardiomyocytes activated the unfolded protein response, however, the PERK pathway was operative in both cardiomyocytes and neural cells. In addition, caspase-4 activation by a SARS-CoV-2 was observed via in silico analyses. These results demonstrate that the ER stress pathway under oxidative stress in SARS-CoV-2 and PD are interconnected using diverse pathways. Furthermore, our results using the ER stress inhibitor and caspase specific inhibitors provided cellular protection suggesting that the use of specific inhibitors can provide effective therapeutic approaches for the treatment of COVID-19 and PD.

Background: Avian metapneumovirus (aMPV) is an economically significant respiratory pathogen of poultry that affects performance, egg production, and fertility of breeder flocks. Despite its impact, aMPV continues to be ill-defined in Egypt with no integrated surveillance studies conducted in breeder flocks. Therefore, the current study was designed to provide the first integrated molecular and serological evaluation of aMPV circulation in broiler breeders in Egypt. Methods: Between 2024 and 2025, a total of 6,000 serum samples and 800 tracheal swabs were collected from 60 unvaccinated broiler breeder flocks across twelve Egyptian governorates. Sera were obtained at 16 weeks (rearing phase) and 35 weeks (production phase) of age and screened for aMPV subtypes A and B by ELISA. Tracheal swabs collected during the production phase were pooled into 80 composite samples and tested for the viral RNA by reverse transcription quantitative real time PCR (RT-qPCR). Results: Serological analysis revealed widespread aMPV exposure, with governorate-level seroprevalence ranging between 64.4% (Luxor) and 89% (Giza). Antibody titers increased between 16 and 35 weeks of age, reflecting cumulative viral exposure. Molecular testing detected aMPV RNA in 67 (83.75%) of pooled swab samples. Subtype B was the predominant genotype detected solely in 65 (81.25%) sample pools and in co-detection with subtype A in 2 pools (2.5%). Serological and molecular findings were generally aligned, with flocks positive for aMPV RNA often exhibiting higher antibody titers. Conclusion: These findings indicate that aMPV, particularly subtype B, is likely endemic across the Egyptian broiler breeder flocks. The study highlights critical knowledge gaps and emphasizes the need for viral isolation, sequencing, and controlled evaluation of biosecurity and vaccination strategies.

Avian metapneumovirus (aMPV) is a major respiratory pathogen of poultry with a significant economic impact; however, its epidemiology at the wildlife–poultry interface remains poorly understood, particularly within Afro–Eurasian migratory systems. This cross-sectional study (December 2024–April 2026) investigated aMPV occurrence in wild birds across eleven Egyptian governorates representing key ecological zones along major migratory flyways. A total of 1280 samples were collected from 800 wild birds representing migratory waterfowl and synanthropic species, including 800 oropharyngeal swabs tested by real-time RT-qPCR for aMPV subtypes A and B and 480 serum samples analyzed using indirect ELISA. aMPV RNA was detected in 28/800 samples (3.5%), with all positives identified as subtype B and confined to the Nile Delta, Middle Egypt, and Canal Region. In contrast, serological analysis revealed a high seroprevalence of 58.3% (280/480), indicating widespread prior exposure with significant spatial and species-level variation (p < 0.05). The marked disparity between low molecular detection and high seroprevalence supports transient infection with cumulative exposure. The exclusive detection of subtype B may reflect epidemiological connectivity between poultry and wild bird populations within shared ecological interfaces; however, the directionality of transmission and the possibility of independent wildlife maintenance could not be determined within the scope of the present cross-sectional study. Future studies incorporating whole-genome sequencing, longitudinal surveillance, and broader flyway-scale sampling are needed to resolve transmission pathways and distinguish field strains from potential vaccine-derived viruses within wildlife–poultry interfaces.

Avian metapneumovirus (aMPV) is an important respiratory pathogen of poultry associated with respiratory disease, reproductive losses, secondary bacterial complications, and economic losses in commercial poultry systems worldwide. This narrative review summarizes current evidence regarding the epidemiology, molecular diversity, transmission ecology, diagnosis, vaccination, and wildlife–poultry interactions of aMPV across Africa and the Middle East. This literature was identified through structured searches of PubMed, Scopus, Web of Science, Google Scholar, and ScienceDirect using predefined keywords related to epidemiology, molecular characterization, surveillance, wild birds, diagnostics, and vaccination. Available evidence demonstrates widespread endemic circulation of aMPV, with subtype B predominating across most investigated poultry systems. However, major heterogeneity exists among studies regarding surveillance intensity, diagnostic methodologies, molecular characterization, and reporting standards. Current evidence suggests possible involvement of wild birds in viral dissemination, although definitive reservoir competence and transmission directionality remain insufficiently established. Important surveillance gaps persist across several African and Middle Eastern countries, emphasizing the need for harmonized surveillance, subtype-matched vaccination strategies, and integrated regional monitoring.