Until vaccines and effective therapeutics become available, the practical solution to transit safely out of the current coronavirus disease 19 (CoVID-19) lockdown may include the implementation of an effective testing, tracing and tracking system. However, this requires a reliable and clinically validated diagnostic platform for the sensitive and specific identification of SARS-CoV-2. Here, we report on the development of a de novo, high-resolution and comparative genomics guided reverse-transcribed loop-mediated isothermal amplification (LAMP) assay. To further enhance the assay performance and to remove any subjectivity associated with operator interpretation of results, we engineered a novel hand-held smart diagnostic device. The robust diagnostic device was further furnished with automated image acquisition and processing algorithms and the collated data was processed through artificial intelligence (AI) pipelines to further reduce the assay run time and the subjectivity of the colorimetric LAMP detection. This advanced AI algorithm-implemented LAMP (ai-LAMP) assay, targeting the RNA-dependent RNA polymerase gene, showed high analytical sensitivity and specificity for SARS-CoV-2. A total of ~200 coronavirus disease (CoVID-19)-suspected NHS patient samples were tested using the platform and it was shown to be reliable, highly specific and significantly more sensitive than the current gold standard qRT-PCR. Therefore, this system could provide an efficient and cost-effective platform to detect SARS-CoV-2 in resource-limited laboratories.

Avian coronaviruses (ACoVs) are continuously evolving and causing serious economic consequences in the poultry industry and around the globe. Owing to their extensive genetic diversity and high mutation rates, controlling ACoVs has become a challenge. In this context, the potential contribution of wild birds in the disease dynamics, especially in domesticated birds, remains largely unknown. In the present study, five hundred fifty-seven (n = 557) cloacal/fecal swabs were collected from four different wild bird species from eight Egyptian governorates during 2016 and a total of fourteen positive isolates were used for phylodynamics and evolutionary analysis. Genetic relatedness based on spike (S1) gene demonstrated the clustering of majority of these isolates where nine isolates grouped within Egy/variant 2 (IS/885 genotype) and five isolates clustered within Egy/variant 1 (IS/1494/06 genotype). Interestingly, these isolates showed noticeable genetic diversity and were clustered distal to the previously characterized Egy/variant 1 and Egy/variant 2 in Egyptian commercial poultry. The S1 gene based comparison of nucleotide identity percentages revealed that all fourteen isolates reported in this study were genetically related to the variant GI-23 lineage with 92–100% identity. Taken together, our results demonstrate that ACoVs are circulating in Egyptian wild birds and highlight their possible contributions in the disease dynamics. The study also proposes that regular monitoring of the ACoVs in wild birds is required to effectively assess the role of wild birds in disease spread, and the emergence of ACoVs strains in the country.

No abstract

The addition of a methyl group to the N6-position of adenosine (m6A) is considered one of the most prevalent internal post-transcriptional modifications and is attributed to virus replication and cell biology. Viral epitranscriptome sequencing analysis has revealed that hemagglutinin (HA) mRNA of H1N1 carry eight m6A sites which are primarily enriched in 5′-DRACH-3′ sequence motif. Herein, a large-scale comparative m6A analysis was conducted to investigate the conservation patterns of the DRACH motifs that corresponding to the reference m6A sites among influenza A viruses. A total of 70,030 complete HA sequences that comprise all known HA subtypes (H1–18) collected over several years, countries, and affected host species were analysed on both mRNA and vRNA strands. The bioinformatic analysis revealed the highest degree of DRACHs conservation among all H1 sequences that clustered largely in the middle and in the vicinity to 3′ end with at least four DRACH motifs were conserved in all mRNA sequences. The major HA-containing subtypes displayed a modest DRACH motif conservation located either in the middle region of HA transcript (H3) or at the 3′ end (H5) or were distributed across the length of HA sequence (H9). The lowest conservation was demonstrated in HA subtypes that infect mostly the wild type avian species and bats. Interestingly, the total number and the conserved DRACH motifs in the vRNA were found to be much lower than those observed in the mRNA. Collectively, the identification of putative m6A topology provides a foundation for the future intervention of influenza infection, replication, and pathobiology in susceptible hosts.

The addition of a methyl group to the N6 position of adenosine (m6A) is the most common posttranscriptional RNA modification, and it regulates most steps of RNA metabolism including splicing, stability, translation, nuclear-export, and RNA structures. Besides cellular RNA, m6A modifications have also been detected on viral RNA. A range of recent studies have demonstrated the crucial roles of m6A in the virushost interactions; however, m6A cellular machineries are only characterized in limited mammalian species. Herein, we aim to present comprehensive evolutionary insights into major m6A writers, erasers, and readers and draw a comparative structural analysis between avian and mammalian m6A-associated machineries. The comparative collinearity on the chromosomal scale revealed that the majority of m6A-related genes were found less syntenic even among avian species. Genetic analysis of avian m6A erasers revealed a distinct phylogenetic clustering compared to mammalian orthologs and shared a weak percent (55%) identity with mammalian species with low identity percentage (55%). The overall comparative three-dimensional (3D) structure analyses among different mammalian species were maintained through synonymous structural mutations. Unlike erasers, the putative 3D structures in the active sites as for the aromatic cage in YTH-domain of YTHDC1 and two pivotal loops in MTD-domains in METTL3 exhibited structural alterations in chicken. In conjunction with in silico investigations, influenza viruses significantly downregulated gene the transcription of m6A writers and erasers, whereas m6A readers were moderately regulated in chicken fibroblasts. In light of these findings, future detailed biochemical and crystallographic studies are warranted to define the roles of m6A machinery in regulating both viral and cellular RNA metabolism in avian species.