Identification of amino acids within norovirus polymerase involved in RNA binding and viral replication.

Until recently, molecular studies on human norovirus (HuNoV), a major causative agent of gastroenteritis, have been hampered by the lack of an efficient cell culture system. Murine norovirus-1 (MNV-1) has served as a surrogate model system for norovirus research, due to the availability of robust cell culture systems and reverse genetics. To identify amino acids involved in RNA synthesis by the viral RNA-dependent RNA polymerase (NS7), we constructed NS7 mutants in which basic amino acids surrounding the catalytic site were substituted with alanine. Electrophoretic mobility shift assay revealed that these residues are important for RNA binding, particularly R396. Furthermore, in vitro RNA synthesis and reverse genetics were used to identify conserved amino acids essential for RNA synthesis and viral replication. These results provide additional functional insights into highly conserved amino acids in NS7 and provide potential methods of rational attenuation of norovirus replication.This study was supported by grants from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs (A085119) and Basic Science Research Programs through NRF funded by the Ministry of Education (NRF-2013R1A1A2064940, L.J.-H. and NRF-2016R1A6A3A01012238, K.R.H.). BA was supported by funding from Qassim University, Saudi Arabia, and the work in the lab of IG is supported by the Wellcome Trust (097997/Z/11/Z). K.R.H. was a recipient of postdoctoral fellowship from the BK21+ program. IG is a Wellcome Senior Fellow

Pharmacoinformatics and Breed-Based De Novo Hybridization Studies to Develop New Neuraminidase Inhibitors as Potential Anti-Influenza Agents

Influenza represents a profoundly transmissible viral ailment primarily afflicting the respiratory system. Neuraminidase inhibitors constitute a class of antiviral therapeutics employed in the management of influenza. These inhibitors impede the liberation of the viral neuraminidase protein, thereby impeding viral dissemination from the infected cell to host cells. As such, neuraminidase has emerged as a pivotal target for mitigating influenza and its associated complications. Here, we apply a de novo hybridization approach based on a breed-centric methodology to elucidate novel neuraminidase inhibitors. The breed technique amalgamates established ligand frameworks with the shared target, neuraminidase, resulting in innovative inhibitor constructs. Molecular docking analysis revealed that the seven synthesized breed molecules (designated Breeds 1–7) formed more robust complexes with the neuraminidase receptor than conventional clinical neuraminidase inhibitors such as zanamivir, oseltamivir, and peramivir. Pharmacokinetic evaluations of the seven breed molecules (Breeds 1–7) demonstrated favorable bioavailability and optimal permeability, all falling within the specified parameters for human application. Molecular dynamics simulations spanning 100 nanoseconds corroborated the stability of these breed molecules within the active site of neuraminidase, shedding light on their structural dynamics. Binding energy assessments, which were conducted through MM-PBSA analysis, substantiated the enduring complexes formed by the seven types of molecules and the neuraminidase receptor. Last, the investigation employed a reaction-based enumeration technique to ascertain the synthetic pathways for the synthesis of the seven breed molecules

Liposome-Mediated Delivery of MERS Antigen Induces Potent Humoral and Cell-Mediated Immune Response in Mice

The advancements in the field of nanotechnology have provided a great platform for the development of effective antiviral vaccines. Liposome-mediated delivery of antigens has been shown to induce the antigen-specific stimulation of the humoral and cell-mediated immune responses. Here, we prepared dried, reconstituted vesicles (DRVs) from DPPC liposomes and used them as the vaccine carrier system for the Middle East respiratory syndrome coronavirus papain-like protease (DRVs-MERS-CoV PLpro). MERS-CoV PLpro emulsified in the Incomplete Freund’s Adjuvant (IFA-MERS-CoV PLpro) was used as a control. Immunization of mice with DRVs-MERS-CoV PLpro did not induce any notable toxicity, as revealed by the levels of the serum alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN) and lactate dehydrogenase (LDH) in the blood of immunized mice. Immunization with DRVs-MERS-CoV PLpro induced greater antigen-specific antibody titer and switching of IgG1 isotyping to IgG2a as compared to immunization with IFA-MERS-CoV PLpro. Moreover, splenocytes from mice immunized with DRVs-MERS-CoV PLpro exhibited greater proliferation in response to antigen stimulation. Moreover, splenocytes from DRVs-MERS-CoV PLpro-immunized mice secreted significantly higher IFN-γ as compared to splenocytes from IFA-MERS-CoV PLpro mice. In summary, DRVs-MERS-CoV PLpro may prove to be an effective prophylactic formulation to prevent MERS-CoV infection

Amplicon and Metagenomic Analysis of Middle East Respiratory Syndrome (MERS) Coronavirus and the Microbiome in Patients with Severe MERS.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic infection that emerged in the Middle East in 2012. Symptoms range from mild to severe and include both respiratory and gastrointestinal illnesses. The virus is mainly present in camel populations with occasional zoonotic spill over into humans. The severity of infection in humans is influenced by numerous factors, and similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underlying health complications can play a major role. Currently, MERS-CoV and SARS-CoV-2 are coincident in the Middle East and thus a rapid way of sequencing MERS-CoV to derive genotype information for molecular epidemiology is needed. Additionally, complicating factors in MERS-CoV infections are coinfections that require clinical management. The ability to rapidly characterize these infections would be advantageous. To rapidly sequence MERS-CoV, an amplicon-based approach was developed and coupled to Oxford Nanopore long read length sequencing. This and a metagenomic approach were evaluated with clinical samples from patients with MERS. The data illustrated that whole-genome or near-whole-genome information on MERS-CoV could be rapidly obtained. This approach provided data on both consensus genomes and the presence of minor variants, including deletion mutants. The metagenomic analysis provided information of the background microbiome. The advantage of this approach is that insertions and deletions can be identified, which are the major drivers of genotype change in coronaviruses. IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in late 2012 in Saudi Arabia. The virus is a serious threat to people not only in the Middle East but also in the world and has been detected in over 27 countries. MERS-CoV is spreading in the Middle East and neighboring countries, and approximately 35% of reported patients with this virus have died. This is the most severe coronavirus infection so far described. Saudi Arabia is a destination for many millions of people in the world who visit for religious purposes (Umrah and Hajj), and so it is a very vulnerable area, which imposes unique challenges for effective control of this epidemic. The significance of our study is that clinical samples from patients with MERS were used for rapid in-depth sequencing and metagenomic analysis using long read length sequencing