Streptococcus suis serotype 2 (SS2) is an emerging human pathogen worldwide. A large outbreak occurred in the summer of 2005 in China. Serum samples from this outbreak revealed that levels of the main proinflammatory cytokines were significantly higher in patients with streptococcal toxic-shock-like syndrome (STSLS) than in patients with meningitis only. However, the mechanism underlying the cytokine storm in STSLS caused by SS2 remained unclear. In this study, we found that suilysin (SLY) is the main protein inflammatory stimulus of SS2 and that native SLY (nSLY) stimulated cytokines independently of its haemolytic ability. Interestingly, a small amount of SLY (Å Mol/L) induced strong, long-term TNF-α release from human PBMCs. We also found that nSLY stimulated TNF-α in wild-type macrophages but not in macrophages from mice that carried a spontaneous mutation in TLR4 (P712H). We demonstrated for the first time that SLY stimulates immune cells through TLR4. In addition, the Myd88 adaptor-p38-MAPK pathway was involved in this process. The present study suggested that the TLR4-dependent inflammatory responses induced by SLY in host might contribute to the STSLS caused by SS2 and that p38-MAPK could be used as a target to control the release of excess TNF-α induced by SS2.

Uropathogenic Escherichia coli (UPEC) is the most common pathogenic bacteria associated with urinary tract infection (UTI). UPEC can cause UTI by adhering to and invading uroepithelial cells. Fimbriae is the most important virulence factor of UPEC, and a potentially promising target in developing novel antibacterial treatments. In this study, the antibacterial properties and effects of the compound dictamnine, extracted from the traditional Chinese medicine Cortex Dictamni, on the bacterial morphology, cell adhesion, and invasion of UPEC were studied. Dictamnine exhibited no obvious antibacterial activity against UPEC, but significantly impeded the ability of UPEC to adhere to and invade uroepithelial cells. RT-qPCR analysis showed that treatment downregulated the expression of type 1 fimbriae, P fimbriae, and curli fimbriae adhesion genes, and also downregulated adhesion-related receptor genes of uroepithelial cells. Transmission electron microscopy showed that dictamnine destroyed the structure of the fimbriae and the surface of the bacteria became smooth. These results suggest that dictamnine may help to prevent UTI by simultaneously targeting UPEC fimbriae and urothelial adhesin receptors, and may have a potential use as a new anti-UPEC drug.

Serious diseases caused by Streptococcus suis serotype 2 (S. suis 2) include septicaemia and meningitis, which are associated with high morbidity and mortality. Proliferation in the blood can result in a breach of the blood–brain barrier (BBB) and provide entry into the cerebrospinal fluid (CSF), where bacteria cause inflammation of the meningeal membranes resulting in meningitis. The molecular mechanisms of how this pathogen crosses the BBB remain unclear. Suilysin (SLY) has been identified as an important secreted virulence factor of S. suis 2 and may play a vital role in provoking meningitis. In this investigation, we demonstrate that SLY can increase the paracellular permeability of BBB, both in vivo and in vitro, via the activation of group III secretory phospholipase A2 (PLA2G3). Our results indicate that at lower, sublytic concentrations, the toxin can stimulate cerebral microvascular endothelial cells to release TNF-α, thereby inducing high level expressions of PLA2G3. Abnormal elevations of PLA2G3 might further injure tissues through direct cytolytic effectors or other responses.

Objective: Influenza B virus (IBV) is highly contagious, spreads rapidly, and causes seasonal epidemic respiratory disease in the human population, especially in immunocompromised people and young children. Clinical manifestations in this high-risk population are often more severe than in immunocompetent hosts and sometimes atypical. Therefore, rapid, and accurate detection of IBV is important.Methods: An amplified luminescent proximity homogeneous assay linked immunosorbent assay (AlphaLISA) was developed for detection of IBV by optimizing the ratio of IBV antibody-labeled receptor beads, streptavidin-conjugated donor beads and biotinylated IBV antibody, as well as the optimal temperature and time conditions for incubation. Assay sensitivity, specificity and reproducibility were evaluated. A total of 228 throat swab samples and inactivated influenza B virus were tested by AlphaLISA and lateral flow colloidal gold-based immunoassay (LFIA).Results: AlphaLISA produced the best results for detection of inactivated influenza B virus when IBV antibody-labeled acceptor beads were 50 μg/ mL, streptavidin-conjugated donor beads were 40 μg/mL, and biotinylated IBV antibody was 0.5 μg/mL at 37°C for 15–10 min. Under these conditions, AlphaLISA had a limit of detection of 0.24 ng/mL for the detection of influenza B nucleoprotein, did not cross react with other common respiratory viruses, and showed good reproducibility with inter-assay coefficient of variation (CV) and intra-assay CV < 5%. The results of 228 clinical throat swab samples showed good agreement between AlphaLISA and LFIA (Kappa = 0.982), and AlphaLISA showed better sensitivity than LFIA for detecting inactivated influenza B virus.Conclusion: AlphaLISA showed higher sensitivity and throughput in the detection of IBV and can be used for IBV diagnosis and epidemic control.

Rotavirus is the main pathogen causing acute viral gastroenteritis. Accurate and rapid diagnosis of rotavirus infection is important to determine appropriate treatment, prevention of unnecessary antibiotics use and control of infection spread. In this study, we established a rapid, accurate, and sensitive amplified luminescent proximity homogeneous assay linked immunosorbent assay (AlphaLISA) for detecting rotavirus and evaluated its efficacy in human stool samples. Our results demonstrated that the sensitivity of AlphaLISA (5⁻⁸) significantly exceeded that of the immunochromatographic assay (ICA, 5⁻⁴) for rotavirus antigen detection. The intra-assay and inter-assay coefficients of variation were 2.99–3.85% and 5.27–6.51%, respectively. Furthermore, AlphaLISA was specific for rotavirus and did not cross-react with other common diarrhea viruses. AlphaLISA and real-time reverse transcription polymerase chain reaction (RT-qPCR, which is considered a gold standard for detecting diarrhea viruses) tests showed consistent results on 235 stool samples, with an overall consistency rate of 97.87% and a kappa value of 0.894 (P < 0.001). The overall consistency rate of ICA compared with RT-qPCR was 95.74%. AlphaLISA showed better consistency with RT-qPCR than the routinely used ICA for rotavirus detection in stool samples. The AlphaLISA method can be used in clinical practice for the rapid, accurate, and sensitive detection of rotavirus infection.

Nipah virus (NiV) is an emerging zoonotic pathogen whose surface glycoprotein (G)-mediated host cell invasion mechanism leads to fatal encephalitis in infected patients (case fatality rate 40–75%). Given the limitations of existing diagnostic technologies, such as low sensitivity and prolonged processing times, we prepared an anti-NiV-G monoclonal antibody to establish a novel Amplified Luminescent Proximity Homogeneous Assay (AlphaLISA) detection system. Firstly, five high-affinity anti-NiV-G monoclonal antibodies were screened from the spleens of immunized mice by flow cytometry-single-cell cloning technology. The reaction system was further optimized, and the optimal dilution ratio of antibody-conjugated receptor microspheres, biotinylated antibodies, and donor microspheres was screened, and the AlphaLISA detection platform was successfully constructed. The detection sensitivity of NiV-G protein was 0.024 ng/mL (41.7 times higher than that of conventional ELISA), the coefficient of variation was <9.5%, and the repetition was good. It showed good specificity in the detection of 5 zoonotic viruses, including Japanese encephalitis virus and Zika virus. At the same time, this method is less disturbed by human serum, and the detection time is less than 30 min, showing a good clinical application prospect.