SARS-CoV-2 viral protein Nsp2 stimulates translation under normal and hypoxic conditions.

When viruses like SARS-CoV-2 infect cells, they reprogram the repertoire of cellular and viral transcripts that are being translated to optimize their strategy of replication, often targeting host translation initiation factors, particularly eIF4F complex consisting of eIF4E, eIF4G and eIF4A. A proteomic analysis of SARS-CoV-2/human proteins interaction revealed viral Nsp2 and initiation factor eIF4E2, but a role of Nsp2 in regulating translation is still controversial. HEK293T cells stably expressing Nsp2 were tested for protein synthesis rates of synthetic and endogenous mRNAs known to be translated via cap- or IRES-dependent mechanism under normal and hypoxic conditions. Both cap- and IRES-dependent translation were increased in Nsp2-expressing cells under normal and hypoxic conditions, especially mRNAs that require high levels of eIF4F. This could be exploited by the virus to maintain high translation rates of both viral and cellular proteins, particularly in hypoxic conditions as may arise in SARS-CoV-2 patients with poor lung functioning.

Non-structural protein 1 and hematology parameters as predictors of dengue virus infection severity in Indonesia.

Dengue virus infection (DVI) remains a significant health challenge, and diagnosis must still be considered. Non-structural protein 1 (NS1) is a potential marker of the dengue virus that can help diagnose DVI. The study aimed to assess the role of NS1 as a predictor of the severity of DVI. We utilized Dengue PCR-confirmed samples and employed semi-quantitative NS1Ag ELISA for NS1 examination, adhering to the World Health Organization South-East Asia Region (WHO-SEARO) 2011 criteria for DVI. We included DVI patients from Indonesia aged 1-65 years. Secondary infections had more severe clinical conditions than primary infections. Leukocyte and platelet levels had a more significant effect on NS1 positivity (6.19 (1.9-30.2); p<0.001; 190 (11-417); p=0.015; respectively). Multivariate analysis revealed leukocytes as a more significant predictor of NS1 values than platelets, with an odds ratio of 5.38 contributing to 30.5% of the NS1 value variation. The NS1 value could distinguish undifferentiated fever and dengue fever in the children group with a sensitivity of 76.0% and specificity of 87.5% (p=0.015). The number of NS1(-) in the severe dengue hemorrhagic fever (DHF) group was higher than NS1(+). DENV-4 type and primary infection were dominant in this study, although they did not significantly differ from the NS1 value. NS1 value can be used as a predictor to determine the severity of DVI in children but not in the adult group. The levels of leukocytes and platelets influenced the NS1 value.

Developing a robust method integrating with selective membrane-based preconcentration and signal amplification for field virus detection.

Infectious diseases caused by viruses have attracted global concern owing to their rapid spread and catastrophic consequences. Therefore, developing fast and reliable on-site virus detection methods is essential for the prevention and treatment of virus-related diseases. In this study, immunoassays on a membrane, combining virus preconcentration with nanoparticle-based signal amplification, were used to realize the rapid and accurate visual detection of viruses. The biotin-streptavidin scaffolds for target virus preconcentration were established on a membrane, and subsequently a Zika aptamer (Apt) immobilized on the membrane recognized and captured the nonstructural protein 1 of Zika virus (ZIKV-NS1). The probe for detection was synthesized by conjugating the Zika Apt with a high level of horseradish peroxidase on gold nanoparticles. The ZIKV-loaded membrane was incubated with the probes, and the viral signal was amplified as the signal of horseradish peroxidase. In the presence of 3,3,5',5'-tetramethyl benzidine and hydrogen peroxide, the green color of the probe-coated membrane indicated the level of ZIKV-NS1. Our developed method could reach a detection limit of 5 ng mL-1, and the whole procedure could be completed within 1 h. Analyses of rabbit serum and environmental water samples demonstrated that an immunoassay-based approach on the membrane could accurately determine the level of ZIKV-NS1 against the complicated matrix. Our results suggest that this virus detection method has a high potential for application in clinical and environmental settings.

Development of Monoclonal Antibodies to Detect for SARS-CoV-2 Proteins.

The COVID-19 pandemic caused by SARS-CoV-2 infection has impacted the world economy and healthcare infrastructure. Key reagents with high specificity to SARS-CoV-2 proteins are currently lacking, which limits our ability to understand the pathophysiology of SARS-CoV-2 infections. To address this need, we initiated a series of studies to generate and develop highly specific antibodies against proteins from SARS-CoV-2 using an antibody engineering platform. These efforts resulted in 18 monoclonal antibodies against nine SARS-CoV-2 proteins. Here we report the characterization of several antibodies, including those that recognize Nsp1, Nsp8, Nsp12, and Orf3b viral proteins. Our validation studies included evaluation for use of antibodies in ELISA, western blots, and immunofluorescence assays (IFA). We expect that availability of these antibodies will enhance our ability to further characterize host-viral interactions, including specific roles played by viral proteins during infection, to acquire a better understanding of the pathophysiology of SARS-CoV-2 infections.

Electropolymerized-molecularly imprinted polymers (E-MIPS) as sensing elements for the detection of dengue infection.

A straightforward in situ detection method for dengue infection was demonstrated through the molecular imprinting of a dengue nonstructural protein 1 (NS1) epitope into an electropolymerized molecularly imprinted polyterthiophene (E-MIP) film sensor. The key enabling step in the sensor fabrication is based on an epitope imprinting strategy, in which short peptide sequences derived from the original target molecules were employed as the main template for detection and analysis. The formation of the E-MIP sensor films was facilitated using cyclic voltammetry (CV) and monitored in situ by electrochemical quartz crystal microbalance (EC-QCM). Surface properties were analyzed using different techniques including atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and polarization modulation-infrared reflection-adsorption (PM-IRRAS). The standard calibration curve (R = 0.9830) was generated for the detection of the epitope, Ac-VHTWTEQYKFQ-NH2, with a linear range of 0.2 to 30 µg/mL and detection limit of 0.073 µg/mL. A separate calibration curve (R = 0.9786) was obtained using spiked buffered solutions of dengue NS1 protein, which resulted in a linear range of 0.2 to 10 µg/mL and a detection limit of 0.056 µg/mL. The fabricated E-MIP sensor exhibited long-term stability, high sensitivity, and good selectivity towards the targeted molecules. These results indicated that the formation of the exact and stable cavity imprints in terms of size, shape, and functionalities was successful. In our future work, we aim to use our E-MIP sensors for NS1 detection in real-life samples such as serum and blood.

Localization of SARS-CoV-2 Capping Enzymes Revealed by an Antibody against the nsp10 Subunit.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 pandemic. One of the key components of the coronavirus replication complex are the RNA methyltransferases (MTases), RNA-modifying enzymes crucial for RNA cap formation. Recently, the structure of the 2'-O MTase has become available; however, its biological characterization within the infected cells remains largely elusive. Here, we report a novel monoclonal antibody directed against the SARS-CoV-2 non-structural protein nsp10, a subunit of both the 2'-O RNA and N7 MTase protein complexes. Using this antibody, we investigated the subcellular localization of the SARS-CoV-2 MTases in cells infected with the SARS-CoV-2.

Levels of Circulating NS1 Impact West Nile Virus Spread to the Brain.

Dengue virus (DENV) and West Nile virus (WNV) are arthropod-transmitted flaviviruses that cause systemic vascular leakage and encephalitis syndromes, respectively, in humans. However, the viral factors contributing to these specific clinical disorders are not completely understood. Flavivirus nonstructural protein 1 (NS1) is required for replication, expressed on the cell surface, and secreted as a soluble glycoprotein, reaching high levels in the blood of infected individuals. Extracellular DENV NS1 and WNV NS1 interact with host proteins and cells, have immune evasion functions, and promote endothelial dysfunction in a tissue-specific manner. To characterize how differences in DENV NS1 and WNV NS1 might function in pathogenesis, we generated WNV NS1 variants with substitutions corresponding to residues found in DENV NS1. We discovered that the substitution NS1-P101K led to reduced WNV infectivity in the brain and attenuated lethality in infected mice, although the virus replicated efficiently in cell culture and peripheral organs and bound at wild-type levels to brain endothelial cells and complement components. The P101K substitution resulted in reduced NS1 antigenemia in mice, and this was associated with reduced WNV spread to the brain. Because exogenous administration of NS1 protein rescued WNV brain infectivity in mice, we conclude that circulating WNV NS1 facilitates viral dissemination into the central nervous system and impacts disease outcomes. IMPORTANCE Flavivirus NS1 serves as an essential scaffolding molecule during virus replication but also is expressed on the cell surface and is secreted as a soluble glycoprotein that circulates in the blood of infected individuals. Although extracellular forms of NS1 are implicated in immune modulation and in promoting endothelial dysfunction at blood-tissue barriers, it has been challenging to study specific effects of NS1 on pathogenesis without disrupting its key role in virus replication. Here, we assessed WNV NS1 variants that do not affect virus replication and evaluated their effects on pathogenesis in mice. Our characterization of WNV NS1-P101K suggests that the levels of NS1 in the circulation facilitate WNV dissemination to the brain and affect disease outcomes. Our findings facilitate understanding of the role of NS1 during flavivirus infection and support antiviral strategies for targeting circulating forms of NS1.

Improved detection of flaviviruses in Australian mosquito populations via replicative intermediates.

Mosquito-borne flaviviruses are significant contributors to the arboviral disease burdens both in Australia and globally. While routine arbovirus surveillance remains a vital exercise to identify known flaviviruses in mosquito populations, novel or divergent and emerging species can be missed by these traditional methods. The MAVRIC (monoclonal antibodies to viral RNA intermediates in cells) system is an ELISA-based method for broad-spectrum isolation of positive-sense and double-stranded RNA (dsRNA) viruses based on detection of dsRNA in infected cells. While the MAVRIC ELISA has successfully been used to detect known and novel flaviviruses in Australian mosquitoes, we previously reported that dsRNA could not be detected in dengue virus-infected cells using this method. In this study we identified additional flaviviruses which evade detection of dsRNA by the MAVRIC ELISA. Utilising chimeric flaviviruses we demonstrated that this outcome may be dictated by the non-structural proteins and/or untranslated regions of the flaviviral genome. In addition, we report a modified fixation method that enables improved detection of flavivirus dsRNA and inactivation of non-enveloped viruses from mosquito populations using the MAVRIC system. This study demonstrates the utility of anti-dsRNA monoclonal antibodies for identifying viral replication in insect and vertebrate cell systems and highlights a unique characteristic of flavivirus replication.

Clinical and epidemiologic characteristics associated with dengue fever in 2011-2016 in Bang Phae district, Ratchaburi province, Thailand.

BACKGROUND: Dengue is a major public health problem in Thailand, but data are often focused on certain dengue-endemic areas. Methods: To better understand dengue epidemiology and clinical characteristics in Thailand, a fever surveillance study was conducted among patients aged 1-55 years, who presented with non-localized febrile illness at Bang Phae Community Hospital in Ratchaburi province, Thailand from October 2011 to September 2016. RESULTS: Among 951 febrile episodes, 130 were dengue-confirmed. Individuals aged 10-14 years were mostly affected, followed by those 15-19 years-of-age, with about 15% of dengue-confirmed cases from adults 25 years and older. There were annual peaks of dengue occurrence between June-November. Most prevalent serotype in circulation was DENV-2 in 2012, DENV-3 in 2014, and DENV-4 & -3 in 2015. Among dengue cases, 65% were accurately detected using the dengue NS1 RDT. Detection rate was similar between secondary and primary dengue cases where 66% of secondary vs. 60% of primary dengue cases had positive results on the NS1 RDT. Among dengue cases, 66% were clinically diagnosed with suspected dengue or DHF, prior to lab confirmation. Dengue was positively associated with rash, headache, hematemesis and alterations to consciousness, when compared to non-dengue. Dengue patients were 10.6 times more likely to be hospitalized, compared to non-dengue cases. Among dengue cases, 95 were secondary and 35 were primary infections. There were 8 suspected DHF cases and all were identified to be secondary dengue. Secondary dengue cases were 3.5 times more likely to be hospitalized compared to primary dengue cases. Although the majority of our dengue-positive patients were secondary dengue cases, with few patients showing manifestations of DHF, our dengue cases were mostly mild disease. Even among children < 10 years-of-age, 61% had secondary infection and the rate of secondary infection increased with age. CONCLUSION: While the majority of dengue-confirmed cases were children, almost three-quarters of dengue-confirmed cases in this study were secondary dengue. Our study results consistent with previous data from the country confirm the hyperendemic transmission of DENV in Thailand, even in the non-epidemic years. With various interventions becoming available for dengue prevention and control, including dengue vaccines, decision-making on future implementation strategies should be based on such burden of disease data.

Automatic and sensitive detection of West Nile virus non-structural protein 1 with a portable SERS-LFIA detector.

A portable surface-enhanced Raman scattering (SERS)-lateral flow immunoassay (LFIA) detector has been developed for the automatic and highly sensitive detection of West Nile virus (WNV) non-structural protein 1 (NS1) and actual WNV samples. Au@Ag nanoparticles (Au@Ag NPs) labeled with double-layer Raman molecules were used as SERS tags to prepare WNV-specific SERS-LFIA strips. On this platform, the WNV-specific antigen NS1 protein was quantitatively and sensitively detected. The detection limit for the WNV NS1 protein was 0.1 ng/mL, which was 100-fold more sensitive than visual signals. The detection limit for inactivated WNV virions was 0.2 × 102 copies/µL. The sensitivity of the SERS-LFIA detector was comparable to that of the fluorescence quantitative reverse transcription-polymerase chain reaction assay. The prepared SERS-LFIA strips exhibited high sensitivity and good specificity for WNV. Thus, the strips developed herein have clinical application value. Moreover, the portable SERS-LFIA detector enabled automatic and rapid detection of the SERS-LFIA strips. The platform established herein is expected to make a substantial contribution to the diagnosis and control of outbreaks of emerging infectious diseases, including WNV.