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Monitoring SARS-CoV-2 spread and evolution through genome sequencing is essential in handling the COVID-19 pandemic. Here, we sequenced 892 SARS-CoV-2 genomes collected from patients in Saudi Arabia from March to August 2020. We show that two consecutive mutations (R203K/G204R) in the nucleocapsid (N) protein are associated with higher viral loads in COVID-19 patients. Our comparative biochemical analysis reveals that the mutant N protein displays enhanced viral RNA binding and differential interaction with key host proteins. We found increased interaction of GSK3A kinase simultaneously with hyper-phosphorylation of the adjacent serine site (S206) in the mutant N protein. Furthermore, the host cell transcriptome analysis suggests that the mutant N protein produces dysregulated interferon response genes. Here, we provide crucial information in linking the R203K/G204R mutations in the N protein to modulations of host-virus interactions and underline the potential of the nucleocapsid protein as a drug target during infection. In this study, the authors sequence 892 SARS-CoV-2 genomes from Saudi Arabia and describe population dynamics and importations into the country. They identify a nucleocapsid protein mutation associated with increased viral load and host interactions and characterise its role through biochemical analyses.

Background Klebsiella pneumoniae is a major bacterial and opportunistic human pathogen, increasingly recognized as a healthcare burden globally. The convergence of resistance and virulence in K. pneumoniae strains has led to the formation of hypervirulent and multidrug-resistant strains with dual risk, limiting treatment options. K. pneumoniae clones are known to emerge locally and spread globally. Therefore, an understanding of the dynamics and evolution of the emerging strains in hospitals is warranted to prevent future outbreaks. Methods In this study, we conducted an in-depth genomic analysis on a large-scale collection of 328 multidrug-resistant (MDR) K. pneumoniae strains recovered from 239 patients from a single major hospital in the western coastal city of Jeddah in Saudi Arabia from 2014 through 2022. We employed a broad range of phylogenetic and phylodynamic methods to understand the evolution of the predominant clones on epidemiological time scales, virulence and resistance determinants, and their dynamics. We also integrated the genomic data with detailed electronic health record (EHR) data for the patients to understand the clinical implications of the resistance and virulence of different strains. Results We discovered a diverse population underlying the infections, with most strains belonging to Clonal Complex 14 (CC14) exhibiting dominance. Specifically, we observed the emergence and continuous expansion of strains belonging to the dominant ST2096 in the CC14 clade across hospital wards in recent years. These strains acquired resistance mutations against colistin and extended spectrum β-lactamase (ESBL) and carbapenemase genes, namely bla OXA-48 and bla OXA-232 , located on three distinct plasmids, on epidemiological time scales. Strains of ST2096 exhibited a high virulence level with the presence of the siderophore aerobactin ( iuc ) locus situated on the same mosaic plasmid as the ESBL gene. Integration of ST2096 with EHR data confirmed the significant link between colonization by ST2096 and the diagnosis of sepsis and elevated in-hospital mortality ( p- value < 0.05). Conclusions Overall, these results demonstrate the clinical significance of ST2096 clones and illustrate the rapid evolution of an emerging hypervirulent and MDR K. pneumoniae in a clinical setting.

Background The excessive inflammatory responses provoked by SARS-CoV-2 infection are critical factors affecting the severity and mortality of COVID-19. Previous work found that two adjacent co-occurring mutations R203K and G204R (KR) on the nucleocapsid (N) protein correlate with increased disease severity in COVID-19 patients. However, links with the host immune response remain unclear. Methods Here, we grouped nasopharyngeal swab samples of COVID-19 patients into two cohorts based on the presence and absence of SARS-CoV-2 nucleocapsid KR mutations. We performed nasopharyngeal transcriptome analysis of age, gender, and ethnicity-matched COVID-19 patients infected with either SARS-CoV-2 with KR mutations in the N protein (KR patients n  = 39) or with the wild-type N protein (RG patients n  = 39) and compared to healthy controls ( n  = 34). The impact of KR mutation on immune response was further characterized experimentally by transcriptomic and proteomic profiling of virus-like-particle (VLP) incubated cells. Results We observed markedly elevated expression of proinflammatory cytokines, chemokines, and interferon-stimulated (ISGs) genes in the KR patients compared to RG patients. Using nasopharyngeal transcriptome data, we found significantly higher levels of neutrophils and neutrophil-to-lymphocyte (NLR) ratio in KR patients than in the RG patients. Furthermore, transcriptomic and proteomic profiling of VLP incubated cells confirmed a similar hyper-inflammatory response mediated by the KR variant. Conclusions Our data demonstrate an unforeseen connection between nucleocapsid KR mutations and augmented inflammatory immune response in severe COVID-19 patients. These findings provide insights into how mutations in SARS-CoV-2 modulate host immune output and pathogenesis and may contribute to more efficient therapeutics and vaccine development.

The Coronavirus Disease 2019 (COVID-19), caused by SARS-CoV-2, continues to spread globally with significantly high morbidity and mortality rates. Antigen-specific responses are of unquestionable value for clinical management of COVID-19 patients. Here, we investigated the kinetics of IgM, IgG against the spike (S) and nucleoproteins (N) proteins and their neutralizing capabilities in hospitalized COVID-19 patients with different disease presentations (i.e., mild, moderate or severe), need for intensive care units (ICU) admission or outcomes (i.e., survival vs death). We show that SARS-CoV-2 specific IgG, IgM and neutralizing antibodies (nAbs) were readily detectable in almost all COVID-19 patients with various clinical presentations. Interestingly, significantly higher levels of nAbs as well as anti-S1 and -N IgG and IgM antibodies were found in patients with more severe symptoms, patients requiring admission to ICU or those with fatal outcomes. More importantly, early after symptoms onset, we found that the levels of anti-N antibodies correlated strongly with disease severity. Collectively, these findings provide new insights into the kinetics of antibody responses in COVID-19 patients with different disease severity.

Nosocomial infections caused by multi-drug resistant are a global public health threat that ought to be promptly identified, reported, and addressed accurately. Many carbapenem-resistant -associated genes have been identified in Saudi Arabia but not the endemic carbapenemases (KPCs), which are encoded by genes. KPCs are known for their exceptional spreading potential. We collected  = 286 multi-drug resistant (MDR) isolates as part of screening for resistant patterns from a tertiary hospital in Saudi Arabia between 2014 and 2018. Antimicrobial susceptibility testing was carried out using both VITEK II and the broth microdilution of all collected isolates. Detection of resistance-conferring genes was carried out using Illumina whole-genome shotgun sequencing and PacBio SMRT sequencing protocols. A Carbapenem-resistant (CRE) subsp. strain was identified as a novel ST-3510 carrying a carbapenemase encoding gene. The isolate, designated as NGKPC-421, was obtained from shotgun Whole Genome Sequencing (WGS) surveillance of 286 MDR . clinical isolates. The NGKPC-421 isolate was collected from a septic patient in late 2017 and was initially misidentified as . The sequencing and assembly of the NGKPC-421 genome resulted in the identification of a putative ~ 39.4 kb IncX6 plasmid harboring a gene, flanked by transposable elements (IS -IS ). This is the first identification of a KPC-2-producing CRE in the Gulf region. The impact on this finding is of major concern to the public health in Saudi Arabia, considering that it is the religious epicenter with a continuous mass influx of pilgrims from across the world. Our study strongly highlights the importance of implementing rapid sequencing-based technologies in clinical microbiology for precise taxonomic classification and monitoring of antimicrobial resistance patterns.

Molecular diagnosis and surveillance of pathogens such as SARS‐CoV‐2 depend on nucleic acid isolation. Pandemics at the scale of COVID‐19 can cause a global shortage of proprietary commercial reagents and BSL‐2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. An open‐source method, magnetic‐nanoparticle‐aided viral RNA isolation from contagious samples (MAVRICS), built upon readily available reagents, and easily assembled in any basically equipped laboratory, is thus developed. The performance of MAVRICS is evaluated using validated pathogen detection assays and real‐world and contrived samples. Unlike conventional methods, MAVRICS works directly in samples inactivated in phenol‐chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities. MAVRICS allows wastewater biomass immobilized on membranes to be directly inactivated and lysed in TRIzol followed by RNA extraction by magnetic nanoparticles, thereby greatly reducing biohazard risk and simplifying processing procedures. Using 39 COVID‐19 patient samples and two wastewater samples, it is shown that MAVRICS rivals commercial kits in detection of SARS‐CoV‐2, influenza viruses, and respiratory syncytial virus. Therefore, MAVRICS is safe, fast, and scalable. It is field‐deployable with minimal equipment requirements and could become an enabling technology for widespread testing and wastewater monitoring of diverse pathogens. One important bottleneck in the diagnosis and surveillance of COVID‐19 is the shortage of kits for RNA extraction. Magnetic‐nanoparticle‐aided viral RNA isolation from contagious samples (MAVRICS) is an open‐source, safe, fast, and scalable method for RNA extraction. MAVRICS rivals commercial kits but requires minimal materials, and thus could become an enabling technology for widespread community testing of diverse pathogens.

The coronavirus disease 2019 (COVID-19) pandemic imposes an urgent and continued need for the development of safe and cost-effective vaccines to induce preventive responses for limiting major outbreaks around the world. To combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we repurposed the VSV∆51M oncolytic virus platform to express the spike receptor-binding domain (RBD) antigen. In this study, we report the development and characterization of the VSV∆51M-RBD vaccine. Our findings demonstrate successful expression of the RBD gene by the VSV∆51M-RBD virus, inducing anti-RBD responses without attenuating the virus. Moreover, the VSV∆51M-RBD vaccine exhibited safety, immunogenicity, and the potential to serve as a safe and effective alternative or complementary platform to current COVID-19 vaccines.

Middle East Respiratory Syndrome coronavirus (MERS-CoV) infects dromedary camels and zoonotically infects humans, causing a respiratory disease with severe pneumonia and death. With no approved antiviral or vaccine interventions for MERS, vaccines are being developed for camels to prevent virus transmission into humans. We have previously developed a chimpanzee adenoviral vector-based vaccine for MERS-CoV (ChAdOx1 MERS) and reported its strong humoral immunogenicity in dromedary camels. Here, we looked back at total RNA isolated from whole blood of three immunised dromedaries pre and post-vaccination during the first day; and performed RNA sequencing and bioinformatic analysis in order to shed light on the molecular immune responses following a ChAdOx1 MERS vaccination. Our finding shows that a number of transcripts were differentially regulated as an effect of the vaccination, including genes that are involved in innate and adaptive immunity, such as type I and II interferon responses. The camel Bcl-3 and Bcl-6 transcripts were significantly upregulated, indicating a strong activation of Tfh cell, B cell, and NF-κB pathways. In conclusion, this study gives an overall view of the first changes in the immune transcriptome of dromedaries after vaccination; it supports the potency of ChAdOx1 MERS as a potential camel vaccine to block transmission and prevent new human cases and outbreaks.