Emergence of equine influenza virus H3Nx Florida clade 2 in Arabian racehorses in Egypt.

BACKGROUND: Equine influenza is an important cause of respiratory disease in equids. The causative virus; EIV, is highly variable and can evolve by accumulation of mutations, particularly in the haemagglutinin (HA) gene. Currently, H3N8 is the sole subtype circulating worldwide with Florida clade 1 (FC1) is most prevalent in the Americas and FC2 in Asia and Europe. In Egypt, EIV was detected in two occasions: subtype H7N7 in 1989 and subtype H3N8 (FC1) in 2008. No data is available on the circulation pattern of EIV during the last decade despite frequent observation of suspected cases. METHODS: Twenty-two nasal swabs were collected from vaccinated and non-vaccinated horses showing respiratory signs suggestive of EIV infection in 2017-18. Three additional swabs were retrieved during a national race event in January 2018 from Arabian mares with high fever, gait stiffness and dry cough. Samples were screened by RT-qPCR and HA1 domain of the hemagglutinin gene was amplified and sequenced for sequence and phylogenetic analysis. RESULTS: RT-qPCR screening revealed that only the 3 samples from the race were positive with cycle thresholds ranging from 16 to 21 indicating high viral load. Isolation attempts in hen's eggs were unsuccessful. Sequence analysis of the HA1 domain gene has revealed two identical nucleotide sequences, while the third contained 3 synonymous mutations. Phylogenetic analysis clustered study sequences with recent FC2 sequences from Europe. Amino acid alignments revealed 14 and 13 amino acid differences in the study sequences compared to A/equine/Egypt/6066NANRU-VSVRI/08 (H3N8) and A/equine/Kentucky/1997 (H3N8), respectively, available as EIV vaccines in Egypt. Nine amino acids were different from A/equine/Richmond/1/2007 (H3N8), the recommended FC2 vaccine strain by the world organization of animal health expert surveillance panel (OIE-ESP), two of which were unique to the Egyptian sequences while the remaining 7 changes were shared with the FC2-144V subgroup detected in the United Kingdom from late 2015 to 2016. CONCLUSIONS: The study represents the first reported detection of FC2-144V related EIV from Arabian mares in Egypt, and probably from the entire middle east region. The presented information about EIV epidemiology and spread may require reconsideration of the vaccine strains used in the national vaccination programs.

Suitcase Lab for Rapid Detection of SARS-CoV-2 Based on Recombinase Polymerase Amplification Assay.

In March 2020, the SARS-CoV-2 virus outbreak was declared as a world pandemic by the World Health Organization (WHO). The only measures for controlling the outbreak are testing and isolation of infected cases. Molecular real-time polymerase chain reaction (PCR) assays are very sensitive but require highly equipped laboratories and well-trained personnel. In this study, a rapid point-of-need detection method was developed to detect the RNA-dependent RNA polymerase (RdRP), envelope protein (E), and nucleocapsid protein (N) genes of SARS-CoV-2 based on the reverse transcription recombinase polymerase amplification (RT-RPA) assay. RdRP, E, and N RT-RPA assays required approximately 15 min to amplify 2, 15, and 15 RNA molecules of molecular standard/reaction, respectively. RdRP and E RT-RPA assays detected SARS-CoV-1 and 2 genomic RNA, whereas the N RT-RPA assay identified only SARS-CoV-2 RNA. All established assays did not cross-react with nucleic acids of other respiratory pathogens. The RT-RPA assay's clinical sensitivity and specificity in comparison to real-time RT-PCR (n = 36) were 94 and 100% for RdRP; 65 and 77% for E; and 83 and 94% for the N RT-RPA assay. The assays were deployed to the field, where the RdRP RT-RPA assays confirmed to produce the most accurate results in three different laboratories in Africa (n = 89). The RPA assays were run in a mobile suitcase laboratory to facilitate the deployment at point of need. The assays can contribute to speed up the control measures as well as assist in the detection of COVID-19 cases in low-resource settings.

Glycosylation is a novel TGFß1-independent post-translational modification of Smad2.

Smad2 is a crucial component of intracellular signaling by transforming growth factor-ß (TGFß). Here we describe that Smad2 is glycosylated, which is a novel for Smad2 post-translational modification. We showed that the Smad2 glycosylation was inhibited upon treatment of cells with 17ß-estradiol, and was enhanced in cells in a dense culture as compared to cells in a sparse culture. The Smad2 glycosylation was not dependent on the C-terminal phosphorylation of Smad2, and was not affected by TGFß1 treatment of the cells. Smad2 was glycosylated at multiple sites, and one of the predicted sites is Serine110. Thus, Smad2 is glycosylated, and this post-translational modification was modulated by 17ß-estradiol but not by TGFß1.

Evaluating two approaches for using positive control in standardizing the avian influenza H5 reverse transcription recombinase polymerase amplification assay.

Highly pathogenic avian influenza H5N1 virus causes heavy losses in poultry farms worldwide. Molecular diagnostic techniques like RT-PCR and real-time RT-PCR are considered the gold standard for identification of H5 influenza viruses in clinical samples. These techniques are hampered by the need of well-equipped laboratories, large space requirement, and relatively long time-to-result. Recombinase polymerase amplification (RPA) assay represents an excellent alternative to PCR since it is more simple, rapid, economic, and portable. Reverse transcription RPA (RT-RPA) assay was recently developed for sensitive and specific detection of H5N1 virus in 6-10 min. To ensure the accuracy of the developed assay, two approaches for using a positive control were evaluated in this study. These approaches included: 1) all-in-one (internal positive control; IPC), 2) two-tubes-per-one-sample (external positive control; EPC). Sigma virus (SIGV) RNA and turkey mitochondrial DNA were tested as positive controls in both approaches. For all-in-one approach, both targets (H5 and IPC) were strongly inhibited. In contrast, very good amplification signals were obtained for the two types of EPC with no effect on the analytical sensitivity and specificity of H5 RT-RPA assay in two-tubes-per-one-sample approach. The performance of EPC-based H5 RT-RPA was further validated using 13 tracheal swabs. The results were compared to real-time RT-PCR and proved superior specificity in detecting H5N1 but not H5N8 viruses. Inclusion of EPC did not affect the aptitude of both assays in terms of sensitivity, specificity and reproducibility. In conclusion, the two-tubes-per-one-sample approach was more reliable to control the false negative results in H5 RT-RPA assay.

A Path towards Timely VAP Diagnosis: Proof-of-Concept Study on Pyocyanin Sensing with Cu-Mg Doped Graphene Oxide.

In response to the urgent requirement for rapid, precise, and cost-effective detection in intensive care units (ICUs) for ventilated patients, as well as the need to overcome the limitations of traditional detection methods, researchers have turned their attention towards advancing novel technologies. Among these, biosensors have emerged as a reliable platform for achieving accurate and early diagnoses. In this study, we explore the possibility of using Pyocyanin analysis for early detection of pathogens in ventilator-associated pneumonia (VAP) and lower respiratory tract infections in ventilated patients. To achieve this, we developed an electrochemical sensor utilizing a graphene oxide-copper oxide-doped MgO (GO - Cu - Mgo) (GCM) catalyst for Pyocyanin detection. Pyocyanin is a virulence factor in the phenazine group that is produced by Pseudomonas aeruginosa strains, leading to infections such as pneumonia, urinary tract infections, and cystic fibrosis. We additionally investigated the use of DNA aptamers for detecting Pyocyanin as a biomarker of Pseudomonas aeruginosa, a common causative agent of VAP. The results of this study indicated that electrochemical detection of Pyocyanin using a GCM catalyst shows promising potential for various applications, including clinical diagnostics and drug discovery.

Detection of Equid Alphaherpesvirus 1 from Arabian Horses with different clinical presentations between 2016-2019 in Egypt.

Equid alphaherpesvirus 1 (EHV-1) is an important virus causing pathological disorders in horses. This highly contagious pathogen causes persistent outbreaks of upper respiratory tract infection, ocular affections, abortion, and neurological disorders with high mortality in Arabian horses in Egypt. The quick and accurate diagnosis is important to broaden our understanding about EHV-1 in the field, and to implicate stronger preventive, and control measures. Sixty-six Arabian horses from Cairo and Giza governorates were sampled from respiratory, abortigenic and neurological outbreaks over a period of 4 years. EHV-1 was diagnosed in these cases by immunohistochemistry using monoclonal antibody against EHV-1 glycoprotein B and molecular detection using gB, ORF33 specific real-time PCR. EHV-1 was detected in 25 cases, mostly from abortigenic outbreaks (14 abortions, 3 stillbirths, and two early neonatal deaths), in addition to 5 respiratory affections and single EHV-1 myeloencephalopathy. Molecular characterization revealed that the ORF33 sequences from this study were almost identical and closely related to the European EHV-1 strains. Furthermore, no difference in the amino acid sequences compared to previously published EHV-1 sequences from Egypt. The data in this study provides some insights about the prevalance of EHV-1 infection in Arabian horses, discusses EHV-1 diagnostic approaches, highlights the importance of accurate diagnosis and the importance of pregnant mare vaccination, and adds to the previous knowledge about EHV-1 in Egypt which may help in better controlling EHV-1 infections in the future.

Differentiation of Capripox Viruses by Nanopore Sequencing.

The genus capripoxvirus (CaPV), family Poxviridae, includes three virus species: goatpox virus (GPV), sheeppox virus (SPV) and lumpy skin disease virus (LSDV). CaPV causes disease outbreaks with consequent economic losses in Africa and the Middle East. LSDV has recently spread to Southeast Europe. As CaPVs share 96-97% genetic similarity along the length of the entire genome and are difficult to distinguish using serological assays, simple, reliable and fast methods for diagnosis and species differentiation are crucial in cases of disease outbreak. The present study aimed to develop a field-applicable CaPV differentiation method. Nanopore technology was used for whole genome sequencing. A local database of complete CaPV genomes and partial sequences of three genes (RPO30, P32 and GPCR) was established for offline Basic Local Alignment Search Tool (BLAST). Specificities of 98.04% in whole genome and 97.86% in RPO30 gene runs were obtained among the three virus species, while other databases were less specific. The total run time was shortened to approximately 2 h. Functionality of the developed procedure was proved by samples with high host background sequences. Reliable differentiation options for the quality and capacity of hardware, and sample quality of suspected cases, were derived from these findings. The whole workflow can be performed rapidly with a mobile suitcase laboratory and mini-computer, allowing application at the point-of-need with limited resource settings.

A novel bivalent Pasteurellosis-RHD vaccine candidate adjuvanted with Montanide ISA70 protects rabbits from lethal challenge.

In the present study, a bivalent vaccine against Pasteurella multocida and rabbit hemorrhagic disease virus (RHDV) was formulated with Montanide™ ISA70 oil adjuvant (Seppic, Paris, France). Its efficacy was evaluated and compared to similar monovalent preparations and commercially available monovalent vaccines. White new Zeeland rabbit groups (n = 10) received 2 successive doses of the tested vaccines and were challenged 2 weeks after 2nd dose with Pasteurella multocida and RHDV or either pathogens according to their vaccination schedule. Challenged not-vaccinated group of rabbits (n = 10) was included as a control. The bivalent and monovalent ISA70 preparations were found stable, safe, sterile, pure and of low viscosity. Group 3 (GP3) which received bivalent vaccine showed the highest antibody geometric mean titers against Pasteurella multocida and RHDV evaluated by ELISA and hemagglutination inhibition (HI) respectively. Following virulent challenge; Gp3 rabbits were 90% protected from challenge over other groups that showed 80% protection. Detection of either pathogen in the livers of dead and euthanized rabbits had failed except for non-vaccinated controls. The bivalent vaccine candidate was fully protective. Immunization against both pathogens can be achieved by single vaccination.

Acquisition of Invasiveness by Breast Adenocarcinoma Cells Engages Established Hallmarks and Novel Regulatory Mechanisms.

BACKGROUND/AIM: Proteomics of invasiveness opens a window on the complexity of the metastasis-engaged mechanisms. The extend and types of this complexity require elucidation. MATERIALS AND METHODS: Proteomics, immunohistochemistry, immunoblotting, network analysis and systems cancer biology were used to analyse acquisition of invasiveness by human breast adenocarcinoma cells. RESULTS: We report here that invasiveness network highlighted the involvement of hallmarks such as cell proliferation, migration, cell death, genome stability, immune system regulation and metabolism. Identified involvement of cell-virus interaction and gene silencing are potentially novel cancer mechanisms. Identified 6,113 nodes with 11,055 edges affecting 1,085 biological processes show extensive re-arrangements in cell physiology. These high numbers are in line with a similar broadness of networks built with diagnostic signatures approved for clinical use. CONCLUSION: Our data emphasize a broad systemic regulation of invasiveness, and describe the network of this regulation.