Rapid immunoassay and clinical evaluation of the SARS-CoV-2 antibody assay on the real express-6 analyzer.

We developed a rapid and simple magnetic chemiluminescence enzyme immunoassay on the Real Express-6 analyzer, which could simultaneously detect immunoglobulin G and immunoglobulin M antibodies against SARS-CoV-2 virus in human blood within 18 min, and which could be used to detect clinical studies to verify its clinical efficacy. We selected blood samples from 185 COVID-19 patients confirmed by polymerase chain reaction and 271 negative patients to determine the clinical detection sensitivity, specificity, stability, and precision of this method. Meanwhile, we also surveyed the dynamic variance of viral antibodies during SARS-CoV-2 infection. This rapid immunoassay test has huge potential benefits for rapid screening of SARS-CoV-2 infection and may help clinical drug and vaccine development.

Fiber1, but not fiber2, is the essential fiber gene for fowl adenovirus 4 (FAdV-4).

Fibre is the viral protein that mediates the attachment and infection of adenovirus to the host cell. Fowl adenovirus 4 (FAdV-4) possesses two different fibre trimers on each penton capsomere, and roles of the separate fibres remain elusive. Here, we attempted to investigate the function of FAdV-4 fibres by using reverse genetics approaches. Adenoviral plasmids carrying fiber1 or fiber2 mutant genes were constructed and used to transfect chicken LMH cells. Fiber1-mutated recombinant virus could not be rescued. Such defective phenotype was complemented when a fiber1-bearing helper plasmid was included for co-transfection. The infection of fiber-intact FAdV-4 (FAdV4-GFP) to LMH cells could be blocked with purified fiber1 knob protein in a dose-dependent manner, while purifed fiber2 knob had no such function. On the contrary, fiber2-mutated FAdV-4, FAdV4XF2-GFP, was successfully rescued. The results of one-step growth curves showed that proliferative capacity of FAdV4XF2-GFP was 10 times lower than that of the control FAdV4-GFP. FAdV4XF2-GFP also caused fewer deaths of infected chicken embryos than FAdV4-GFP did, which resulted from poorer virus replication in vivo. These data illustrated that fiber1 mediated virus adsorption and was essential for FAdV-4, while fiber2 was dispensable although it significantly contributed to the virulence.

CXCR3.1 and CXCR3.2 Differentially Contribute to Macrophage Polarization in Teleost Fish.

The study of multiple copies of chemokine receptor genes in various teleosts has long appealed to investigators seeking to understand the evolution of the immune system. The CXCR CXCR3 gene has two isoforms, CXCR3.1 and CXCR3.2, which are both expressed in macrophages. The distinct roles of teleost CXCR3s have not been identified previously. In this article, we found that CXCR3.1 and CXCR3.2 differentially contributed to macrophage polarization in the teleosts: ayu (Plecoglossus altivelis), grass carp (Ctenopharyngodon idella), and spotted green pufferfish (Tetraodon nigroviridis). In ayu macrophages, the P. altivelis CXCR3.1 (PaCXCR3.1) gene was constitutively expressed, whereas the P. altivelis CXCR3.2 (PaCXCR3.2) gene was induced postinfection with Escherichia coli Upon E. coli infection, PaCXCR3.1+ and PaCXCR3.2+ macrophages showed an M1 and an M2 phenotype, respectively. CXCL9-11-like proteins mediated M1 and M2 polarization by interacting with the PaCXCR3.1 and PaCXCR3.2 proteins on macrophages, respectively. The transcription factors P. altivelis STAT1 and P. altivelis STAT3 were activated in PaCXCR3.1+ and PaCXCR3.2+ macrophages, respectively. Furthermore, the prognosis of septic ayu adoptively transferred with PaCXCR3.2+ macrophages was improved. Our data reveal a previously unknown mechanism for macrophage polarization, suggesting that redundant genes may regulate crucial functions in the teleost immune system.

LECT2 drives haematopoietic stem cell expansion and mobilization via regulating the macrophages and osteolineage cells.

Haematopoietic stem cells (HSCs) can differentiate into cells of all lineages in the blood. However, the mechanisms by which cytokines in the blood affect HSC homeostasis remain largely unknown. Here we show that leukocyte cell-derived chemotaxin 2 (LECT2), a multifunctional cytokine, induces HSC expansion and mobilization. Recombinant LECT2 administration results in HSC expansion in the bone marrow and mobilization to the blood via CD209a. The effect of LECT2 on HSCs is reduced after specific depletion of macrophages or reduction of osteolineage cells. LECT2 treatment reduces the tumour necrosis factor (TNF) expression in macrophages and osteolineage cells. In TNF knockout mice, the effect of LECT2 on HSCs is reduced. Moreover, LECT2 induces HSC mobilization in irradiated mice, while granulocyte colony-stimulating factor does not. Our results illustrate that LECT2 is an extramedullar cytokine that contributes to HSC homeostasis and may be useful to induce HSC mobilization.

Mobilisation and dysfunction of haematopoietic stem/progenitor cells after Listonella anguillarum infection in ayu, Plecoglossus altivelis.

Haematopoietic stem/progenitor cells (HSPCs) can mobilise into blood and produce immune cell lineages following stress. However, the homeostasis and function of HSPCs after infection in teleosts are less well known. Here, we report that Listonella anguillarum infection enhances HSPC mobilisation and reduces their differentiation into myeloid cells in ayu (Plecoglossus altivelis), an aquacultured teleost in East Asia. We established a colony-forming unit culture (CFU-C) assay to measure HSPCs using conditioned medium from peripheral blood mononuclear cells stimulated with phytohaemagglutinin. The number of CFU-Cs decreased in the head kidney and increased in the blood and spleen of ayu infected with L. anguillarum. HSPC mobilisation after L. anguillarum infection was mediated by norepinephrine. Furthermore, HSPCs from ayu treated with L. anguillarum lipopolysaccharide (LPS) showed defective myeloid differentiation and could no longer rescue L. anguillarum-infected ayu. HSPC expansion was suppressed after L. anguillarum infection or its LPS treatment in vitro. These results reveal a link between HSPC regulation and pathogen infection in teleosts.

Molecular Characterization of E-Type Prostanoid Receptor 4 (EP4) from Ayu (Plecoglossus altivelis) and Its Functional Analysis in the Monocytes/Macrophages.

Prostaglandin E2 (PGE2) plays an important role in a broad spectrum of physiological and pathological processes by interacting with E-type prostanoid receptors (EPs). EP4 is one of four EP subtypes known to mediate the immune response in mammalian monocytes/macrophages. However, the precise function and characteristics of EP4 in fish remain unclear. In this study, we characterized a novel EP4-like (PaEP4L) gene from ayu, Plecoglossus altivelis. The cDNA sequence of PaEP4L is 2781 nucleotides (nts) in length, encoding a polypeptide of 459 amino acid residues with a calculated molecular weight of 51.17 kDa. Sequence comparison and phylogenetic tree analysis showed that PaEP4L shared 76% amino acid identity with that of the Atlantic salmon (Salmo salar). PaEP4L mRNA was detected by real-time quantitative PCR (QPCR) in all tested tissues and head kidney-derived monocytes/macrophages (MO/MФ). It varied greatly in liver, spleen and MO/MФ upon Vibrio anguillarum infection. Western blot analysis revealed a significant increase of PaEP4L in cell homogenates from ayu MO/MФ upon V. anguillarum infection. Moreover, anti-PaEP4L IgG reversed the down-regulation of interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) mRNA expression as well as phagocytosis in ayu MO/MФ caused by PGE2. There were no significant differences in the respiratory burst response between PGE2 treated and untreated cells. We further found that cAMP mediated PGE2/PaEP4L signal in ayu MO/MФ. In conclusion, our results indicate that PaEP4L mediates PGE2 effects on ayu MO/MФ function, revealing that EP4 also plays a role in the modulation of cells of the fish's innate immune system.