Evaluation of diagnostic accuracy of developed rapid SARS-COV-2 IgG antibody test kit using novel diluent system.

Immunochromatographic assay kits are used in primary diagnostics which is based on the principle of antigen and antibody interaction. These kits play pivotal role in rapid surveillance of infectious diseases at early stages as well as for the surveillance of the contagious diseases. The immunochromatographic test kits lacks sensitivity and specificity with certain diseases. In this study, our intention was to develop a rapid test kit for SARS-COV-2 with a novel diluent system to enhance the efficacy of antigen-antibody binding and thereby the improvement in the sensitivity outlined. Finally, IgG antibodies against SARS-COV-2 virus peptides were analyzed using 25 positive and 25 negative confirmed clinical samples. The sensitivity of the clinical studies showed 91% sensitivity and 100% specificity. Therefore, the authors propose that this assay will be a potential tool for efficient community or sentinel surveillance of SARS-COV-2 infection and additionally, for effective monitoring of convalescent sera therapy.

A slow, efficient and safe nanoplatform of tailored ZnS QD-mycophenolic acid conjugates for in vitro drug delivery against dengue virus 2 genome replication.

Dengue is a major health concern causing significant mortality, morbidity and economic loss. The development of anti-dengue viral drugs is challenging due to high toxicity, as well as off-target/side effects. We engineered size tuned ZnS QDs as a platform for the efficient delivery of mycophenolic acid (MPA) against dengue virus serotype 2 (DENV2) to evaluate the drug efficacy and toxicity using the DENV2 sub-genomic replicon system in BHK21 cells. The results indicate that the Selectivity Index 50 (SI50) of the ZnS QD-MPA conjugate was two orders higher than that of free MPA with lower cytotoxicity. The effect is attributed to the sustained release of MPA from ZnS QD-MPA. The conjugated MPA caused significant inhibition of the virus at the level of replication and viral protein translation. The study underpins the efficiency of the ZnS QD for the delivery of antiviral drugs against DENV2 with negligible toxicity and side effects.

Semi-quantification of antibody-dependent enhancement (ADE) in the uptake of Adenovirus serotype 5 into THP-1 cells.

Replication defective recombinant Ad5 vectors (rAdV5) are extensively explored for its applications in gene therapy and vaccine delivery. Ad5 enter into monocytes and macrophages through CAR independent route as an immune complex termed as antibody-dependent enhancement (ADE). We developed an effective method for estimating the ADE of rAdV5 encoding GFP (rAdV5-GFP) into THP-1 cells, using fluorimetric semi-quantification of GFP. Initially, twenty numbers of human sera samples were screened in HeLa cells for anti-Ad5 antibody titer using neutralization assay. Uptake of rAdV5-GFP in THP-1 cells was observed only after pre-incubation with the serially diluted human sera which are attributed to ADE. The optimal dilution which showed the maximum GFP expression as per the fluorescence microscopic analysis in THP-1 cells was used for further analysis. Fluorimetric analysis of the THP-1 cell lysate showed a maximum GFP intensity of 17058 RFU, which was equivalent to the 0.397 pmoles of Alexa Fluor 488 under the same experimental condition. Similarly, immunoblot analysis of GFP in THP-1 cell lysate and HeLa cell lysate confirmed the entry of rAdV5-GFP into the cells. The assay can serve as a platform for understanding the molecular events involved in ADE for the uptake of viruses into immune cells.

Mycophenolic acid (MPA) modulates host cellular autophagy progression in sub genomic dengue virus-2 replicon cells.

Cellular autophagy (Macrophagy) is a self-degradative process, executed through the network of autophagy associated genes (ATGs) encoded proteins. Both in vitro and in vivo studies suggest that dengue virus (DENV) induces autophagy and supports the viral genome replication and translation. Therefore, the cellular autophagy induced by dengue virus can be a good target for antiviral drug development. The action of mycophenolic acid (MPA), a specific inhibitor of DENV replication, was investigated in the stable BHK-21/DENV2 replicon cells. The inhibition was mediated by enhanced degradation of autophagic substrates in stable BHK-21/DENV2 replicon cells as evidenced by a decrease in lapidated LC3 (LC3II) and p62 expression in the presence of MPA. In contrast, the results indicated that four gene sets, namely Transmembrane protein 74 (TMEM74), Unc-51-like kinase 2 (ULK2), Cathepsin D (CTSD) and Estrogen receptor 1 (ESR1) were upregulated in stable BHK-21/DENV2 replicon cells, due to the sustained dynamic replication of DENV2 genome. These ATGs involved in the pre-autophagosomal structure (PAS) formation, were suppressed in the presence MPA. Instead, MPA induced the expression of different set of autophagy genes such as ATG4, AKT1, APP, ATG16L1, ATG16L2, B2M and HPRT1. An enzyme involved in the nucleotide salvage pathway, HPRT1, was highly expressed in the presence of MPA. The study shows that DENV2 replication is dependent on PAS formation and is inhibited in the presence of MPA by enhancing the degradation of autophagic substrates and suppression of PAS formation. This study provides impetus in designing MPA analogues to effectively inhibit dengue viral replication.

Proteomic analysis reveals the enhancement of human serum apolipoprotein A-1(APO A-1) in individuals infected with multiple dengue virus serotypes.

OBJECTIVES: Human serum protein profiling of the individual infected with multiple dengue virus serotypes for identifying the potential biomarkers and to investigate the cause for the severity of dengue virus infection. METHODS: Dengue virus NS1-positive serum samples were pooled into two groups (S2 and S3) based on the molecular serotyping and number of heterotypic infections. The pooled serum samples were subjected to two-dimensional gel electrophoresis (2DGE) to identify the differentially expressed proteins. The peptide masses of upregulated protein were detected by matrix-assisted laser desorption-ionisation time-of-flight MALDI-TOF mass spectrometry and analysed by MASCOT search engine. The results were compared with the control group (S1). The commonly upregulated protein was validated by quantitative ELISA and compared with control as well as single serotypic infected samples. RESULTS: Based on 2DGE, total thirteen proteins were differentially upregulated in S2 and S3 groups as compared to control. Some of the upregulated proteins were involved in mediating the complement activation of immune response. The apolipoprotein A-1 (APO A-1) was upregulated in S2 and S3 groups. Upon validation, APO A-1 levels were increased in line with the number of heterotypic infection of dengue viruses. CONCLUSION: Heterotypic infection of dengue viruses upregulate the serum proteins involved in the complement pathway in the early phase of infection. There was a significant increase in the level of APO A-1 in three different serotypic infections of dengue virus as compared to control. Further, the role of APO-A1 can be explored in elucidating the mechanism of dengue pathogenesis.

Occurrence of concurrent infections with multiple serotypes of dengue viruses during 2013-2015 in northern Kerala, India.

BACKGROUND: Dengue is a global human public health threat, causing severe morbidity and mortality. The occurrence of sequential infection by more than one serotype of dengue virus (DENV) is a major contributing factor for the induction of Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS), two major medical conditions caused by DENV infection. However, there is no specific drug or vaccine available against dengue infection. There are reports indicating the increased incidence of concurrent infection of dengue in several tropical and subtropical regions. Recently, increasing number of DHF and DSS cases were reported in India indicating potential enhancement of concurrent DENV infections. Therefore, accurate determination of the occurrence of DENV serotype co-infections needs to be conducted in various DENV prone parts of India. In this context, the present study was conducted to analyse the magnitude of concurrent infection in northern Kerala, a southwest state of India, during three consecutive years from 2013 to 2015. METHODS: A total of 120 serum samples were collected from the suspected dengue patients. The serum samples were diagnosed for the presence of dengue NS1 antigen followed by the isolation of dengue genome from NS1 positive samples. The isolated dengue genome was further subjected to RTPCR based molecular serotyping. The phylogenetic tree was constructed based on the sequence of PCR amplified products. RESULTS: Out of the total number of samples collected, 100 samples were positive for dengue specific antigen (NS1) and 26 of them contained the dengue genome. The RTPCR based molecular serotyping of the dengue genome revealed the presence of all four serotypes with different combinations. However, serotypes 1 and 3 were predominant combinations of concurrent infection. Interestingly, there were two samples with all four serotypes concurrently infected in 2013. DISCUSSION: All samples containing dengue genome showed the presence of more than one serotype, indicating 100% concurrent infection. However, the combination of serotypes 1 and 3 was predominant. To the best of our knowledge, this is the first report indicating the concurrent infection of dengue in the northern Kerala, India. The phylogenetic analysis of dengue serotype 1 identified in this study shows a close relationship with the strain isolated in Delhi and South Korea during the 2006 and 2015 epidemics respectively. Similarly this study indicates that the phylogeny of dengue serotype 3 of northern Kerala is more closely related to dengue isolate of Rajasthan state, India. The geographical and climatic conditions of Kerala favours the breeding of both the mosquito vectors of dengue (Aedes albopictus and Aedes aegypti), which may enhance the severity of dengue in the future. Therefore, the study provides an alarming message for the urgent need of an antiviral strategy or other health management systems to curb the spread of dengue infection.