Development of a Real-Time Recombinase-Aided Amplification Method for the Rapid Detection of Streptococcus equi subsp. equi.

Streptococcus equi subspecies equi (S. equi) is the causative pathogen of strangles in horses, donkeys, and other equine animals. Strangles has spread globally and causes significant losses to the horse industry. In response to the urgent need for effective disease control, this study introduces a novel nucleic acid diagnostic method known as a real-time recombinase-assisted amplification (RAA) assay, developed based on the eqbE gene, for the rapid detection of S. equi nucleic acid. The real-time RAA method employs specifically designed probes and primers targeting the eqbE gene, enhancing the overall specificity and sensitivity of the detection. After efficiency optimization, this real-time RAA method can detect 10 or more copies of nucleic acid within 20 min. The method demonstrates high specificity for S. equi and does not cross-react with other clinically relevant pathogens. Real-time RAA diagnostic performance was evaluated using 98 nasal swab samples collected from horses and compared with the real-time PCR detection method. Results revealed that 64 and 65 samples tested positive for S. equi using real-time RAA and real-time PCR, respectively. The overall agreement between the two assays was 96.94% (95/98), with a kappa value of 0.931 (p < 0.001). Further linear regression analysis indicated a significant correlation in the detection results between the two methods (R2 = 0.9012, p < 0.0001), suggesting that the real-time RAA assay exhibits a detection performance comparable to that of real-time PCR. In conclusion, the real-time RAA assay developed here serves as a highly specific and reliable diagnostic tool for the detection of S. equi in equine samples, offering a potential alternative to real-time PCR methods. In conclusion, the real-time RAA nucleic acid diagnostic method, based on the eqbE gene, offers rapid and accurate diagnosis of S. equi, with the added advantage of minimal equipment requirements, thus contributing to the efficient detection of strangles in horses.

A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC.

The quadrilateral reassortant IAV A/(H1N1) pdm09 is the pathogen responsible for the first influenza pandemic of the 21st century. The virus spread rapidly among hosts causing high mortality within human population. Efficient accumulation of virions is known to be important for the rapid transmission of virus. However, the mechanism by which A/(H1N1) pdm09 promotes its rapid replication has not been fully studied. Here, we found the NS1 of A/(H1N1) pdm09 mediated complete macroautophagy/autophagy, and then facilitated self-replication, which may be associated with the more rapid spread of this virus compared with H1N1WSN and H3N8JL89. We found that the promotion of self-replication could be mainly attributed to NS1pdm09 strongly antagonizing the inhibitory effect of LRPPRC on autophagy. The interaction between NS1pdm09 and LRPPRC competitively blocked the interaction of LRPPRC with BECN1/Beclin1, resulting in increased recruitment of BECN1 for PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type 3) and induction of the initiation of autophagy. In conclusion, we uncover the unique molecular mechanism by which A/(H1N1) pdm09 utilizes autophagy to promote self-replication, and we provide theoretical basics for the analysis of the etiological characteristics of the A/(H1N1) pdm09 pandemic and the development of anti-influenza drugs and vaccines.Abbreviations: 293T: human embryonic kidney 293 cells; 293T_LRPPRC: stable LRPPRC expression 293T cells; 3-MA: 3-methyladenine; A549 cells: human non-small cell lung cancer cells; AA: amino acid; ACTB: actin beta; BECN1: beclin 1; BECN1 KO: BECN1 knockout 293T cells; Cal: calyculin A; Co-IP: co-immunoprecipitation; CQ: chloroquine; DC: dendritic cell; Eug: eugenol; GFP: green fluorescent protein; HA: hemagglutinin; HIV: human immunodeficiency virus; IAVs: Influenza A viruses; IFN: interferon; JL89: A/equine/Jilin/1/1989 (H3N8); LAMP2: lysosomal associated membrane protein 2; LRPPRC: leucine rich pentatriicopeptide repeat containing; LRPPRC KO: LRPPRC knockout 293T cells; M2: matrix 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MDCK: Madin-Darby canine kidney cells; MOI: multiplicity of infection; MS: mass spectrometry; NP: nucleoprotein; NS1: non-structural protein 1; NS1JL89: non-structural protein 1 of A/equine/Jilin/1/1989 (H3N8); NS1pdm09: non-structural protein 1 of A/(H1N1) pdm09; NS1SC09: non-structural protein 1 of A/Sichuan/2009 (H1N1); NS1WSN: non-structural protein 1 of A/WSN/1933 (H1N1); PB1: polymerase basic protein 1; PB1-F2: alternate reading frame discovered in PB1 gene segment; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PR8: A/PR/8/34 (H1N1); Rapa: rapamycin; RFP: red fluorescent protein; SC09: A/Sichuan/2009 (H1N1); SQSTM1/p62: sequestosome 1; STK4/MST1: serine/threonine kinase 4; TEM: transmission electron microscopy; TOMM20: translocase of outer mitochondrial membrane 20; WHO: World Health Organization; WSN: A/WSN/1933 (H1N1); WSN-NS1JL89: WSN recombinant strain in which NS1 was replaced with that of JL89; WSN-NS1SC09: WSN recombinant strain in which NS1 was replaced with that of SC09.

M.neoaurum infection increased the inhibitory function of Tregs and the death rate associated with Salmonella coinfection.

Mycobacterium neoaurum belongs to the nontuberculous mycobacteria (NTM) and is ubiquitously present in the environment. However, the changes in Treg percentages and suppressive properties in mice infected with M. neoaurum are still not elucidated. In this study, mice were intraperitoneally injected with M. neoaurum. The change in the CD4+CD25+ Treg cell percentage in the spleen was analyzed using flow cytometry. There was a significant increase in the number of CD4+CD25+ cells by week 6 postinfection, with a peak proportion of approximately 2%. The Foxp3 and IL-10 mRNA expression in CD4+CD25+ cells from the spleens of M.neoaurum-infected mice was higher than that in CD4+CD25+ cells from the spleens of noninfected controls. Proliferation suppression assay results indicated that CD4+CD25+ cells suppressed the proliferation of CD4+CD25- cells at week 6 after M.neoaurum infection, and the suppression rate reached 89.8%. However, CD4+CD25+ cells from the noninfected control group did not suppress the proliferation of CD4+CD25- cells. Based on the above results, mice were subjected to oral administration of S. Typhimurium at 6 weeks postinfection with M. neoaurum, and we found that the mortality of the M.neoaurum-S. Typhimurium infection group was higher than that of the S. Typhimurium infection group. In addition, serious pathological changes appeared in the liver and cecum of the M.neoaurum-S.Typhimurium infection group compared with those of the S. Typhimurium infection group. M. neoaurum increased Treg percentages and suppressed spleen function in mice. These results revealed the possibility that persistent M.neoaurum infection could increase the occurrence of secondary infection.