A novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) and continues to be a global health challenge. To understand viral disease biology, we have carried out proteo-genomic analysis using next-generation sequencing (NGS) and mass spectrometry on nasopharyngeal swabs of COVID-19 patients to examine the clinical genome and proteome. Our study confirms the mutability of SARS-CoV-2 showing multiple single-nucleotide polymorphisms. NGS analysis detected 27 mutations, of which 14 are synonymous, 11 are missense, and 2 are extragenic in nature. Phylogenetic analysis of SARS-CoV-2 isolates indicated their close relation to a Bangladesh isolate and multiple origins of isolates within the country. Our proteomic analysis, for the first time, identified 13 different SARS-CoV-2 proteins from the clinical swabs. Of the total 41 peptides captured by high-resolution mass spectrometry, 8 matched to nucleocapsid protein, 2 to ORF9b, and 1 to spike glycoprotein and ORF3a, with remaining peptides mapping to ORF1ab polyprotein. Additionally, host proteome analysis revealed several key host proteins to be uniquely expressed in COVID-19 patients. Pathway analysis of these proteins points toward modulation in immune response, especially involving neutrophil and IL-12-mediated signaling. Besides revealing the aspects of host-virus pathogenesis, our study opens new avenues to develop better diagnostic markers and therapeutic approaches.

A novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19 and continues to be a global health challenge. To understand viral disease biology, we have carried out proteo-genomic analysis using next generation sequencing (NGS) and mass-spectrometry on nasopharyngeal swabs of COVID-19 patients to examine clinical genome and proteome. Our study confirms the hyper mutability of SARS-CoV-2 showing multiple SNPs. NGS analysis detected 27 mutations of which 14 are synonymous, 11 are missense and 2 are extragenic in nature. Phylogenetic analysis of SARS-CoV-2 isolates indicated their close relation to Bangladesh isolate and multiple origins of isolates within a country. Our proteomic analysis, for the first time identified 13 different SARS-CoV-2 proteins from the clinical swabs. Of the total 41 peptides captured by HRMS, 8 matched to nucleocapsid protein, 2 to ORF9b, 1 to spike glycoprotein and ORF3a, with remaining mapping to ORF1ab polyprotein. Additionally, host proteome analysis revealed several key host proteins to be uniquely expressed in COVID-19 patients. Pathway analysis of these proteins points towards modulation in immune response, especially involving neutrophil and IL-12 mediated signaling. Besides revealing the aspects of host-virus pathogenesis, our study opens new avenues to develop better diagnostic markers and therapeutics.

Hsp90 gene of G. lamblia has a split nature comprising two ORFs separated by 777 kb on chromosome 5. The ORFs of the split gene on chromosome 5 undergo transcription to generate independent pre-mRNAs that join by a unique trans-splicing reaction that remains partially understood. The canonical cis-acting nucleotide elements such as 5’SS-GU, 3’SS-AG, polypyrimidine tract and branch point adenine are present in the independent pre-mRNAs and therefore trans-splicing of Hsp90 must be assisted by spliceosomes in vivo. Using an approach of RNA-protein pull down, we showed that an RNA helicase selectively interacts with HspN pre-mRNA. Our experiments involving high resolution chromosome conformation capture technology as well as DNA FISH show that the trans-spliced genes of Giardia are in three-dimensional spatial proximity in the nucleus. Altogether our study provides a glimpse into the in vivo mechanisms involving protein factors as well as chromatin structure to facilitate the unique inter-molecular post-transcriptional stitching of split genes in G. lamblia.