COVID-19 impedimetric biosensor based on polypyrrole nanotubes, nickel hydroxide and VHH antibody fragment: specific, sensitive, and rapid viral detection in saliva samples.

SARS-CoV-2 rapid spread required urgent, accurate, and prompt diagnosis to control the virus dissemination and pandemic management. Several sensors were developed using different biorecognition elements to obtain high specificity and sensitivity. However, the task to achieve these parameters in combination with fast detection, simplicity, and portability to identify the biorecognition element even in low concentration remains a challenge. Therefore, we developed an electrochemical biosensor based on polypyrrole nanotubes coupled via Ni(OH)2 ligation to an engineered antigen-binding fragment of heavy chain-only antibodies (VHH) termed Sb#15. Herein we report Sb#15-His6 expression, purification, and characterization of its interaction with the receptor-binding domain (RBD) of SARS-CoV-2 in addition to the construction and validation of a biosensor. The recombinant Sb#15 is correctly folded and interacts with the RBD with a dissociation constant (KD) of 27.1 ± 6.4 nmol/L. The biosensing platform was developed using polypyrrole nanotubes and Ni(OH)2, which can properly orientate the immobilization of Sb#15-His6 at the electrode surface through His-tag interaction for the sensitive SARS-CoV-2 antigen detection. The quantification limit was determined as 0.01 pg/mL using recombinant RBD, which was expressively lower than commercial monoclonal antibodies. In pre-characterized saliva, both Omicron and Delta SARS-CoV-2 were accurately detected only in positive samples, meeting all the requirements recommended by the World Health Organization for in vitro diagnostics. A low sample volume of saliva is needed to perform the detection, providing results within 15 min without further sample preparations. In summary, a new perspective allying recombinant VHHs with biosensor development and real sample detection was explored, addressing the need for accurate, rapid, and sensitive biosensors.

Single point mutations in the helicase domain of the NS3 protein enhance dengue virus replicative capacity in human monocyte-derived dendritic cells and circumvent the type I interferon response.

Dengue is the most prevalent arboviral disease worldwide. The outcome of the infection is determined by the interplay of viral and host factors. In the present study, we evaluated the cellular response of human monocyte-derived DCs (mdDCs) infected with recombinant dengue virus type 1 (DV1) strains carrying a single point mutation in the NS3hel protein (L435S or L480S). Both mutated viruses infect and replicate more efficiently and produce more viral progeny in infected mdDCs compared with the parental, non-mutated virus (vBACDV1). Additionally, global gene expression analysis using cDNA microarrays revealed that the mutated DVs induce the up-regulation of the interferon (IFN) signalling and pattern recognition receptor (PRR) canonical pathways in mdDCs. Pronounced production of type I IFN were detected specifically in mdDCs infected with DV1-NS3hel-mutated virus compared with mdDCs infected with the parental virus. In addition, we showed that the type I IFN produced by mdDCs is able to reduce DV1 infection rates, suggesting that cytokine function is effective but not sufficient to mediate viral clearance of DV1-NS3hel-mutated strains. Our results demonstrate that single point mutations in subdomain 2 have important implications for adenosine triphosphatase (ATPase) activity of DV1-NS3hel. Although a direct functional connection between the increased ATPase activity and viral replication still requires further studies, these mutations speed up viral RNA replication and are sufficient to enhance viral replicative capacity in human primary cell infection and circumvent type I IFN activity. This information may have particular relevance for attenuated vaccine protocols designed for DV.

Structural characterization of the internal transcribed spacer 2 (ITS2) of the ribosomal DNA (rDNA) cluster in Calyptratae (Diptera: Schizophora) and its implications for molecular phylogenetic analyses.

The internal transcribed spacer 2 (ITS2) of the eukaryotic ribosomal DNA (rDNA) cluster plays an essential role in processing of the ribosomal RNA, which is primarily accomplished by the secondary structures acquired by the molecule after transcription. Two possible structural conformation models have been proposed for the ITS2 region, the "ring model" and the "hairpin model," and the former has been widely used in many molecular phylogenetic analyses incorporating structural information available to date. To evaluate the validity of this model, in vitro transcribed ITS2 molecules from species representing the three superfamilies of the Calyptratae clade (Diptera: Schizophora), namely Cochliomyia hominivorax, Musca domestica, and Glossina morsitans, were submitted to enzymatic digestion with single- and double-stranded specific nucleases (RNases I, A, T1, and V1). The resulting fragments were analyzed by capillary electrophoresis and digestion sites were mapped in the secondary structure models which were obtained by in silico prediction with further refinement by homology comparisons. The pattern of RNA fragments generated by these RNases show a high degree of correlation to most of the predicted helix-loop regions and structural motifs. Discrepancies to the models can be explained by alternative structural conformation dynamics (in M. domestica and G. morsitans) and by higher-order factors (such as tertiary interactions) that may stabilize thermodynamically unfavored structures (in C. hominivorax).