Antibody production and characterization of the nucleoprotein of sever fever with thrombocytopenia syndrome virus (SFTSV) for effective diagnosis of SFTSV.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease caused by the Dabie bandavirus, [or SFTS virus (SFTSV)] that has become increasingly widespread since it was first reported in 2009. The SFTSV comprises three essential single-stranded RNA gene segments, with the S segment encoding the nucleocapsid (N) protein. Since the N protein is the most abundant and stable viral protein, it is a useful diagnostic marker of infection. Various SFTSV N-protein-based detection methods have been developed. However, given the limited research on antibodies of an SFTSV N-protein, here we report the characterization of the antibodies against SFTSV N protein especially their mapping results which is essential for more efficient and optimized detection of SFTSV. METHODS: To generate SFTSV-N-protein-specific monoclonal antibodies, recombinant full-length SFTSV N protein was expressed in E. coli, and the purified N protein was immunized to mice. The binding epitope positions of the antibodies generated were identified through binding-domain mapping. An antibody pair test using a lateral flow immunoassay (LFIA) was performed to identify effective diagnostic combinations of paired antibodies. RESULTS: Nine monoclonal antibodies specific for the SFTSV N protein were generated. Antibodies #3(B4E2) and #5(B4D9) were specific for sequential epitopes, while the remainder were specific for conformational epitopes. Antibody #4(C2G1) showed the highest affinity for the SFTSV N protein. The binding domain mapping results indicated the binding regions of the antibodies were divided into three groups. The antibody pair test demonstrated that #3(B4E2)/#4(C2G1) and #4(C2G1)/#5(B4D9) were effective antibody pairs for SFTSV diagnosis. CONCLUSIONS: Effective virus detection requires at least two strong antibodies recognizing separate epitope binding sites of the virus antigen. Here, we generated SFTSV-N-protein-specific monoclonal antibodies and subsequently performed epitope mapping and an antibody pair test to enhance the diagnostic efficiency and accuracy of SFTSV. Confirmation of epitope mappings and their combination immune response to the N protein provide valuable information for effective detection of SFTSV as well as can respond actively to detect a variant SFTSV.

Development of a Rapid Diagnostic Kit for Congestive Heart Failure Using Recombinant NT-proBNP Antigen.

Background and Objectives: In patients with congestive heart failure, brain natriuretic peptide (BNP) and N-terminal prohormone of brain natriuretic peptide (NT-proBNP) are released due to excessive heart muscle expansion; they can be used for the early detection, progress monitoring, and treatment of congestive heart failure. Recently, considerable efforts have been made to develop an NT-proBNP-based biomarker for detecting heart failure. This study attempts to develop a rapid and accurate congestive heart failure diagnostic kit using NT-proBNP. Materials and Methods: A new gene based on NT-proBNP was selected, recombined, and expressed in Escherichia coli strains, and then monoclonal antibodies were produced using the hybridoma technique. Additionally, antigen-antibody reactivity was confirmed using indirect enzyme-linked immunosorbent assay (ELISA). Furthermore, the first pair and full-strip pair tests were conducted to select candidate clones; these were applied to a rapid diagnosis kit based on gold conjugates and compared with other currently available antigens. Results: NT-proBNP-based antigens with high specificity and monoclonal antibodies were produced, and the optimal antigen-antibody reactivity was confirmed using indirect ELISA. The first pair and full-strip pair tests were performed to select the optimal candidate clones, and a rapid diagnosis kit with excellent reactivity was developed by applying these to a rapid diagnosis kit based on gold conjugates. Conclusions: The development of this rapid diagnosis kit with excellent performance in congestive heart failure is expected to improve disease management by providing an early assessment of the risk of heart failure.

Genotypic Heterogeneity of Orientia tsutsugamushi in Scrub Typhus Patients and Thrombocytopenia Syndrome Co-infection, Myanmar.

Serologic and molecular surveillance of serum collected from 152 suspected scrub typhus patients in Myanmar revealed Orientia tsutsugamushi of genotypic heterogeneity. In addition, potential co-infection with severe fever with thrombocytopenia syndrome virus was observed in 5 (3.3%) patients. Both scrub typhus and severe fever with thrombocytopenia syndrome are endemic in Myanmar.

Antinociceptive effects of heterotopic electroacupuncture in formalin-induced pain.

This study examined the antinociceptive effect of electroacupuncture (EA) to heterotopic acupoints on formalin-induced pain in rats. EA (2 ms, 10 Hz, and 3 mA) was delivered to heterotopic acupoints HE(7) and PE(7), or non-acupoints at the right fore limb, for 30 min and was immediately followed by subcutaneous formalin injection into the left hind paw, respectively. The quantified pain score, electromyogram (EMG) response of the C-fiber reflex, and cFos immunoreactivity were assessed, respectively. EA to heterotopic acupoints significantly reduced both early- and late-phase pain-like behaviors and significantly decreased the EMG responses of the C-fiber reflex after formalin injection. By contrast, EA to non-acupoints had no significant effects on pain-like behavior or the EMG response. In addition, EA to heterotopic acupoints decreased cFos immunoreactivity in the lumbar spinal dorsal horn. Therefore, EA induced pre-emptive antinociception via the extra-segmental inhibition of the formalin-induced pain, suggesting that EA to heterotopic acupoints is a useful treatment for inflammatory pain.

Moxibustion at ST36 alleviates pain in complete Freund's adjuvant-induced arthritic rats.

This study was to investigate the antinociceptive effects of moxibustion in a complete Freund's adjuvant (CFA)-induced arthritic rat model, and the effects of moxibustion on immunohistochemical changes at the spinal cord level. Moxibustion was applied to the ipsilateral (right) Zusanli (ST36) acupoint to the lesion side for 9 days to CFA-induced arthritic rats. The stepping force was measured as a behavioral test, c-Fos immunohistochemistry, NO production and nNOS Western blots were examined to evaluate antinociceptive effects. Moxibustion at ST36 significantly improved the stepping force in the affected hind limb in CFA-induced arthritis. Moreover, moxibustion at ST36 suppressed the production of NO and the protein expression of c-Fos and nNOS induced by arthritis. These results suggest that moxibustion at ST36 has a potent antinociceptive effect in an arthritic rat model, and modulates neuronal excitability and endogenous NO production by suppressing c-Fos and nNOS protein expression.

Asymmetric activation of extracellular signal-regulated kinase 1/2 in rat vestibular nuclei by unilateral labyrinthectomy.

The expression of phosphorylated extracellular signal-regulated kinase 1/2 (pERK 1/2) was evaluated in the vestibular nuclei (VN) of rats following unilateral labyrinthectomy (UL). Immunohistochemistry revealed a significant asymmetrical increase in pERK 1/2 expression in the VN, 5 min after UL, after which pERK 1/2 immunoreactivity decreased rapidly and was undetectable by 90 min after UL. These results suggest that unilateral deafferentation of the vestibular system triggers intracellular signal pathways that activate ERK 1/2 in the VN.

Immunohistochemical detection of phosphorylated form of extracellular signal-regulated kinase 1/2 in rat vestibular nuclei following hemorrhagic hypotension.

The purpose of this study was to evaluate the expression of the phosphorylated form of extracellular-regulated protein kinase 1/2 (pERK1/2), which is one of the major regulatory factors for transcription of the c-fos oncogene in neurons, within the vestibular nuclei (VN) of rats following acute arterial hypotension. Following acute arterial hypotension induced by rapid hemorrhage, a significant number of pERK1/2-immunoreactive neurons appeared bilaterally in the caudal aspect of the medial and inferior VN. No labeling of pERK1/2 was observed in the lateral VN. The peak expression of pERK1/2-immunoreactive neurons in these nuclei occurred within 5 min after hemorrhage. In bilaterally labyrinthectomized rats, the appearance of pERK1/2-immunoreactive neurons was eliminated in the VN. These results suggest that, following acute hypotension, afferent signals from the peripheral vestibular receptors are required for activation of extracellular signal-regulated kinase 1/2 in the VN.