A novel conserved B-cell epitope in pB602L of African swine fever virus.

African swine fever virus (ASFV) is a complex DNA virus and the only member of the Asfarviridae family. It causes high mortality and severe economic losses in pigs. The ASFV pB602L protein plays a key role in virus assembly and functions as a molecular chaperone of the major capsid protein p72. In addition, pB602L is an important target for the development of diagnostic tools for African swine fever (ASF) because it is a highly immunogenic antigen against ASFV. In this study, we expressed and purified ASFV pB602L and validated its immunogenicity in serum from naturally infected pigs with ASFV. Furthermore, we successfully generated an IgG2a κ subclass monoclonal antibody (mAb 7E7) against pB602L using hybridoma technology. Using western blot and immunofluorescence assays, mAb 7E7 specifically recognized the ASFV Pig/HLJ/2018/strain and eukaryotic recombinant ASFV pB602L protein in vitro. The 474SKENLTPDE482 epitope in the ASFV pB602L C-terminus was identified as the minimal linear epitope for mAb 7E7 binding, with dozens of truncated pB602l fragments characterized by western blot assay. We also showed that this antigenic epitope sequence has a high conservation and antigenic index. Our study contributes to improved vaccine and antiviral development and provides new insights into the serologic diagnosis of ASF. KEY POINTS: • We developed a monoclonal antibody against ASFV pB602L, which can specifically recognize the ASFV Pig/HLJ/2018/ strain. • This study found one novel conserved B-cell epitope 474SKENLTPDE482. • In the 3D structure, 474SKENLTPDE482 is exposed on the surface of ASFV pB602L, forming a curved linear structure.

A candidate nanoparticle vaccine comprised of multiple epitopes of the African swine fever virus elicits a robust immune response.

The African swine fever (ASF) pandemics pose a significant threat to the global swine industry, and the development of safe and effective vaccines is a daunting but necessary challenge. The level and persistence of immunity are very important for the effectiveness of the vaccine. Targeting antigens to antigen presenting cells (APCs) can greatly enhance immunogenicity. In this study, we developed a self-assembled nano-ASFV vaccine candidate (NanoFVax) targeting DCs, by covalently coupling the self-assembled 24-mer ferritin with the dominant B and T cell epitopes of the highly immunogenic ASFV antigen (p72, CD2v, pB602L and p30) and fused with the chemokine receptor XCL1 (a DC targeting molecule) through the SpyTag/SpyCatcher protein ligase system. Compared to monomeric protein, the nanoparticle vaccines can induce a more robust T-cell response, and the high-level antibody response against ASFV can last for more than 231 days. Therefore, the NanoFVax is a novel and promising vaccine candidate for ASFV.

Sphingolipids: Regulators of azole drug resistance and fungal pathogenicity.

In recent years, the role of sphingolipids in pathogenic fungi, in terms of pathogenicity and resistance to azole drugs, has been a rapidly growing field. This review describes evidence about the roles of sphingolipids in azole resistance and fungal virulence. Sphingolipids can serve as signaling molecules that contribute to azole resistance through modulation of the expression of drug efflux pumps. They also contribute to azole resistance by participating in various microbial pathways such as the unfolded protein response (UPR), pH-responsive Rim pathway, and pleiotropic drug resistance (PDR) pathway. In addition, sphingolipid signaling and eisosomes also coordinately regulate sphingolipid biosynthesis in response to azole-induced membrane stress. Sphingolipids are important for fungal virulence, playing roles during growth in hosts under stressful conditions, maintenance of cell wall integrity, biofilm formation, and production of various virulence factors. Finally, we discuss the possibility of exploiting fungal sphingolipids for the development of new therapeutic strategies to treat infections caused by pathogenic fungi.

The OxrA Protein of Aspergillus fumigatus Is Required for the Oxidative Stress Response and Fungal Pathogenesis.

An efficient reactive oxygen species (ROS) detoxification system is vital for the survival of the pathogenic fungus Aspergillus fumigatus within the host high-ROS environment of the host. Therefore, identifying and targeting factors essential for oxidative stress response is one approach to developing novel treatments for fungal infections. The oxidation resistance 1 (Oxr1) protein is essential for protection against oxidative stress in mammals, but its functions in pathogenic fungi remain unknown. The present study aimed to characterize the role of an Oxr1 homolog in A. fumigatus. The results indicated that the OxrA protein plays an important role in oxidative stress resistance by regulating the catalase function in A. fumigatus, and overexpression of catalase can rescue the phenotype associated with OxrA deficiency. Importantly, the deficiency of oxrA decreased the virulence of A. fumigatus and altered the host immune response. Using the Aspergillus-induced lung infection model, we demonstrated that the ΔoxrA mutant strain induced less tissue damage along with decreased levels of lactate dehydrogenase (LDH) and albumin release. Additionally, the ΔoxrA mutant caused inflammation at a lower degree, along with a markedly reduced influx of neutrophils to the lungs and a decreased secretion of cytokine usually associated with recruitment of neutrophils in mice. These results characterize the role of OxrA in A. fumigatus as a core regulator of oxidative stress resistance and fungal pathogenesis. IMPORTANCE Knowledge of ROS detoxification in fungal pathogens is useful in the design of new antifungal drugs and could aid in the study of oxidative stress resistance mechanisms. In this study, we demonstrate that OxrA protein localizes to the mitochondria and functions to protect against oxidative damage. We demonstrate that OxrA contributes to oxidative stress resistance by regulating catalase function, and overexpression of catalase (CatA or CatB) can rescue the phenotype that is associated with OxrA deficiency. Remarkably, a loss of OxrA attenuated the fungal virulence in a mouse model of invasive pulmonary aspergillosis and altered the host immune response. Therefore, our finding indicates that inhibition of OxrA might be an effective approach for alleviating A. fumigatus infection. The present study is, to the best of our knowledge, a pioneer in reporting the vital role of Oxr1 protein in pathogenic fungi.

Contribution of traditional Chinese medicine combined with conventional western medicine treatment for the novel coronavirus disease (COVID-19), current evidence with systematic review and meta-analysis.

This study provides current evidence for efficacy and safety of treating COVID-19 with combined traditional Chinese medicine (TCM) and conventional western medicine (CWM). Six databases were searched from January 1 to December 31, 2020. Randomized controlled trials (RCTs), case-control studies (CCTs), and cohort studies on TCM or TCM combined with CWM treatment for COVID-19 were included. The quality of included RCTs was assessed by Cochrane risk of bias tool, and the Newcastle-Ottawa Scale (NOS) was used to assess the quality of cohort studies and CCTs. Review Manager 5.4 software was used to perform meta-analysis. The quality of evidence was assessed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. A total of 35 studies (3,808 patients) composing 19 RCTs and 16 observational studies were included. The results of meta-analysis revealed that comparing with CWM alone, integrated TCM and CWM had significant improvement in total effective rate, improvement rate of chest CT, the rate of disease progression, as well as improvement of fever, fatigue and cough. The overall quality of evidence was very low to moderate. In conclusion, TCM combined with CWM was a potential treatment option for increasing clinical effective rate, improving the clinical symptoms, and preventing disease progression in COVID-19 patients. High-quality clinical trials are required in the further.

A sphingolipid synthesis-related protein OrmA in Aspergillus fumigatus is responsible for azole susceptibility and virulence.

Previous studies identified that the budding yeast Saccharomyces cerevisiae have two sphingolipid synthesis-related proteins, Orm1p and Orm2p, that negatively regulate the activities of SPT, which is a key rate-limiting enzyme in sphingolipid synthesis. However, little is known about whether sphingolipids in the cell membrane, which are closely related to ergosterols, could affect the efficacy of azole drugs, which target to the ergosterol biosynthesis. In this study, through genome-wide homologue search analysis, we found that the Aspergillus fumigatus genome only contains one Orm homologue, referred to as OrmA for which the protein expression could be induced by azole antifungals in a dose-dependent manner. Deletion of ormA caused hypersensitivity to azoles, and adding the sphingolipid synthesis inhibitor myriocin rescued the azole susceptibility induced by lack of ormA. In contrast, overexpression of OrmA resulted in azole resistance, indicating that OrmA is a positive azole-response regulator. Further mechanism analysis verified that OrmA is related to drug susceptibility by affecting endoplasmic reticulum stress responses in an unfolded protein response pathway-HacA-dependent manner. Lack of ormA led to an abnormal profile of sphingolipid ceramide components accompanied by hypersensitivity to low temperatures. Furthermore, deletion of OrmA significantly reduced virulence in an immunosuppressed mouse model. The findings in this study collectively suggest that the sphingolipid metabolism pathway in A. fumigatus plays a critical role in azole susceptibility and fungal virulence.

Duplex TaqMan real-time PCR assay for simultaneous detection and quantification of Anaplasma capra and Anaplasma phagocytophilum infection.

Anaplasma capra and A. phagocytophilum, two species of the family Anaplasmataceae, are zoonotic tick-borne obligate intracellular bacteria affecting wild and domestic ruminants, dogs, cats, horses and humans. A. capra and A. phagocytophilum infections have been steadily increasing in both number and geographic distribution, and the accurate diagnosis of these infections is challenging. This study aimed to develop a rapid, sensitive and reliable duplex real-time PCR assay for the specific detection and differentiation of these Anaplasma species. We designed primers and probes against the conserved regions of A. capra groEL and A. phagocytophilum 16S rRNA genes. A range of PCR-related parameters were evaluated such as the dosage of primers and probes, and annealing temperature. The specificity, sensitivity and repeatability of this assay were evaluated. Assay performance was further evaluated using samples collected from 124 goats in four regions of Henan, China. This set of samples was also tested using conventional PCR under conditions previously described. The developed duplex real-time PCR assay allowed the simultaneous detection of A. capra and A. phagocytophilum in a reasonably short time at levels as small as 102 copies/µL, respectively, with optimal specificity and reproducibility. In addition, this duplex real-time PCR assay is the first DNA-based method designed to detect A. capra and A. phagocytophilum, and will be valuable for timely diagnosis and treatment of these infections.

Palmitoylation of the Cysteine Residue in the DHHC Motif of a Palmitoyl Transferase Mediates Ca2+ Homeostasis in Aspergillus.

Finely tuned changes in cytosolic free calcium ([Ca2+]c) mediate numerous intracellular functions resulting in the activation or inactivation of a series of target proteins. Palmitoylation is a reversible post-translational modification involved in membrane protein trafficking between membranes and in their functional modulation. However, studies on the relationship between palmitoylation and calcium signaling have been limited. Here, we demonstrate that the yeast palmitoyl transferase ScAkr1p homolog, AkrA in Aspergillus nidulans, regulates [Ca2+]c homeostasis. Deletion of akrA showed marked defects in hyphal growth and conidiation under low calcium conditions which were similar to the effects of deleting components of the high-affinity calcium uptake system (HACS). The [Ca2+]c dynamics in living cells expressing the calcium reporter aequorin in different akrA mutant backgrounds were defective in their [Ca2+]c responses to high extracellular Ca2+ stress or drugs that cause ER or plasma membrane stress. All of these effects on the [Ca2+]c responses mediated by AkrA were closely associated with the cysteine residue of the AkrA DHHC motif, which is required for palmitoylation by AkrA. Using the acyl-biotin exchange chemistry assay combined with proteomic mass spectrometry, we identified protein substrates palmitoylated by AkrA including two new putative P-type ATPases (Pmc1 and Spf1 homologs), a putative proton V-type proton ATPase (Vma5 homolog) and three putative proteins in A. nidulans, the transcripts of which have previously been shown to be induced by extracellular calcium stress in a CrzA-dependent manner. Thus, our findings provide strong evidence that the AkrA protein regulates [Ca2+]c homeostasis by palmitoylating these protein candidates and give new insights the role of palmitoylation in the regulation of calcium-mediated responses to extracellular, ER or plasma membrane stress.

Damage resistance protein (Dap) contributes to azole resistance in a sterol-regulatory-element-binding protein SrbA-dependent way.

The targeting of stress-response regulators has emerged as a powerful strategy to enhance azole drug efficacy and to abrogate azole drug resistance. Previously, we reported that a damage resistance protein (Dap) family, composed of DapA, DapB, and DapC, could respond to azole stress stimuli in Aspergillus fumigatus, although the exact response mechanisms remain unknown. In this study, RNA-seq analysis found that a total of 180 genes are induced by azole in a dapA-dependent manner. These genes are involved in oxidation-reduction, metabolic processes, and transmembrane transport. Following azole stress stimuli, DapA and DapC consistently show a stable endoplasmic reticulum (ER)-localization pattern. In comparison, the sterol-regulatory element-binding protein SrbA is capable of nuclear translocation from the ER after azole-stress stimuli, suggesting that SrbA, but not Daps, can directly sense azole stress. Moreover, we found that SrbA is required for the normal expression of DapA and DapC but not of DapB. In addition, in the absence of SrbA, the enhanced expression of DapA induced by azole-itraconazole is blocked, indicating that SrbA is required for the DapA response to azole stress. Double mutants together with overexpression experiments suggest that DapA might act downstream of SrbA to respond to azole stress stimuli. Compared with the ΔsrbA strain, no additional increase in sensitivity was observed in the double mutants ΔsrbAΔdapB and ΔsrbAΔdapC, indicating that DapA might be of central importance in the response to azole drugs. Thus, our findings demonstrate that Dap proteins indirectly sense azole stress and link the function of the azole stress-regulator SrbA with the role of Daps in azole susceptibility.

A putative mitochondrial calcium uniporter in A. fumigatus contributes to mitochondrial Ca(2+) homeostasis and stress responses.

Ca(2+) uptake into mitochondria plays a central role in cell physiology by stimulating ATP production, shaping cytosolic Ca(2+) transients and regulating cell survival or death. Although this system has been studied extensively in mammalian cells, the physiological implications of Ca(2+) uptake into mitochondria in fungal cells are still unknown. In this study, a bi-directional best-hit BLASTP search revealed that the genome of Aspergillus fumigatus encodes a homolog of a putative mitochondrial Ca(2+) uniporter (MCU) and a mitochondrial carrier protein AGC1/MICU1 homolog. Both putative homologs are mitochondrially localized and required for the response to azole and oxidative stress such that the loss of either McuA or AgcA results in reduced susceptibility to azole and oxidative stress, suggesting a role in environmental stress adaptation. Overexpressing mcuA restores the azole-resistance phenotype of the ΔagcA strain to wild-type levels, but not vice versa, indicating McuA plays a dominant role during these stress responses. Using a mitochondrially targeted version of the calcium-sensitive photoprotein aequorin, we found that only mcuA deletion leads to dysfunctional [Ca(2+)]mt and [Ca(2+)]c homeostasis, suggesting that McuA, but not AgcA, contributes to Ca(2+) uptake into mitochondria. Further point-mutation experiments combined with extracellular Ca(2+) chelator treatment verified that two predicted Ca(2+)-binding sites in McuA are required for Ca(2+) uptake into mitochondria and stress responses through the regulation of [Ca(2+)]c homeostasis.

The newly nonsporulated characterization of an Aspergillus fumigatus isolate from an immunocompetent patient and its clinic indication.

Aspergillus fumigatus (A. fumigatus) commonly produces abundant and heavily melanized infectious conidia, which are the primary agents that cause invasive aspergillosis (IA) in immunocompromised patients. We isolated a white nonsporulating A. fumigatus strain (A1j) from an immunocompetent patient. It was identified by histopathological examination and morphological observation, and subsequently confirmed by DNA sequencing of internal transcribed spacer (ITS) regions and partial ß-tubulin genes. Neither a long waiting time nor passage on various medium types could stimulate the formation of spores and pigment. No significant relative difference was found in sensitivity to antifungal agents or cell wall destabilizing reagents, as compared to wild-type A. fumigatus Af293. Nevertheless, A1j was hypovirulent in the immunosuppressed mice model, consistent with the good result in our patient. RNA deep-sequencing analysis (RNA-seq) revealed that hundreds of transcripts were significantly dysregulated, including those related to pigmentation and sporulation. qRT-PCR confirmed the anergic state of key regulator brlA for sporulation under the induction of conidiation conditions, but without mutation. To the best of our knowledge, this is the first report of a white, nonsporulating A. fumigatus strain infection in an immunocompetent patient. In our opinion, A1j may represent a mutant of typical A. fumigatus, providing a new clue for identification of clinical A. fumigatus isolates. Furthermore, the good prognosis of our patient and the reduced virulence in the mice model infected with A1j highlight the potential of sporulation inhibitors as a new generation of antifungal agents.

Identification of a novel linear B-cell epitope on the p30 protein of African swine fever virus using monoclonal antibodies.

The outbreak of African Swine Fever (ASF) has caused huge economic losses to the pig industry. There are no safe and effective vaccines or diagnostics available. The p30 protein serves as a key target for the detection of ASFV antibodies and is an essential antigenic protein for early serological diagnosis. Here, the p30 protein was purified after being expressed in E. coli and its immunogenicity was verified in sera from pigs naturally infected with ASFV. Furthermore, a monoclonal antibody (McAb) designated as McAb 1B4G2-4 (subtype IgG1/kappa-type) was produced and it was verified to specifically recognize the ASFV Pig/HLJ/2018/strain and eukaryotic recombinant ASFV p30 protein. The epitope identified by McAb 1B4G2-4, defining the unique B-cell epitope 164HNFIQTI170, was located using peptide scanning. Comparing amino acid (aa) sequence revealed that this epitope is conserved in all reference ASFV strains from different regions of China, including the highly pathogenic strain Georgia 2007/1 (NC_044959.2) that is widely distributed. It is also exposed to the surface of the p30 protein, suggesting that it could be an important B-cell epitope. Our study may serve as a basis for the development of serological diagnostic methods and subunit vaccines.

Recent Advances of Photoactive Near-Infrared Carbon Dots in Cancer Photodynamic Therapy.

Photodynamic therapy (PDT) is a treatment that employs exogenously produced reactive oxygen species (ROS) to kill cancer cells. ROS are generated from the interaction of excited-state photosensitizers (PSs) or photosensitizing agents with molecular oxygen. Novel PSs with high ROS generation efficiency is essential and highly required for cancer photodynamic therapy. Carbon dots (CDs), the rising star of carbon-based nanomaterial family, have shown great potential in cancer PDT benefiting from their excellent photoactivity, luminescence properties, low price, and biocompatibility. In recent years, photoactive near-infrared CDs (PNCDs) have attracted increasing interest in this field due to their deep therapeutic tissue penetration, superior imaging performance, excellent photoactivity, and photostability. In this review, we review recent progress in the designs, fabrication, and applications of PNCDs in cancer PDT. We also provide insights of future directions in accelerating the clinical progress of PNCDs.

Unlocking Genetic Profiles with a Programmable DNA-Powered Decoding Circuit.

Human genetic architecture provides remarkable insights into disease risk prediction and personalized medication. Advances in genomics have boosted the fine-mapping of disease-associated genetic variants across human genome. In healthcare practice, interpreting intricate genetic profiles into actionable medical decisions can improve health outcomes but remains challenging. Here an intelligent genetic decoder is engineered with programmable DNA computation to automate clinical analyses and interpretations. The DNA-based decoder recognizes multiplex genetic information by one-pot ligase-dependent reactions and interprets implicit genetic profiles into explicit decision reports. It is shown that the DNA decoder implements intended computation on genetic profiles and outputs a corresponding answer within hours. Effectiveness in 30 human genomic samples is validated and it is shown that it achieves desirable performance on the interpretation of CYP2C19 genetic profiles into drug responses, with accuracy equivalent to that of Sanger sequencing. Circuit modules of the DNA decoder can also be readily reprogrammed to interpret another pharmacogenetics genes, provide drug dosing recommendations, and implement reliable molecular calculation of polygenic risk score (PRS) and PRS-informed cancer risk assessment. The DNA-powered intelligent decoder provides a general solution to the translation of complex genetic profiles into actionable healthcare decisions and will facilitate personalized healthcare in primary care.

Identification of a linear B-cell epitope on the African swine fever virus CD2v protein.

African swine fever virus (ASFV) poses a serious threat to domestic pigs and wild boars, which is responsible for substantial production and economic losses. A dominant ASFV specific linear B cell epitope that reacted with the convalescent serum was explored and identified with the help of immune informatics techniques. It is essential in understanding the host immunity and in developing diagnostic technical guidelines and vaccine design. The confirmation of dominant epitopes with a positive serological matrix is feasible. To improve the immunogenicity of the epitope, we designed the dominant epitope of CD2v in the form of 2 branch Multiple-Antigen peptide (MAPs-2), CD2v-MAPs-2. Notably, CD2v peptide can be taken up by dendritic cells (DCs) to activate T lymphocytes and induce highly effective valence antibodies in BALB/c mice. The specific CD8+ T cell response were observed. The dominant epitope peptide identified in this study was able to effectively activate humoral and cellular immunity in mice model.

Establishment of a Dual-Antigen Indirect ELISA Based on p30 and pB602L to Detect Antibodies against African Swine Fever Virus.

African swine fever (ASF) is an acute, virulent, and highly fatal infectious disease caused by the African swine fever virus (ASFV). There is no effective vaccine or diagnostic method to prevent and control this disease currently, which highlights the significance of ASF early detection. In this study, we chose an early antigen and a late-expressed antigen to co-detect the target antibody, which not only helps in early detection but also improves accuracy and sensitivity. CP204L and B602L were successfully expressed as soluble proteins in an Escherichia coli vector system. By optimizing various conditions, a dual-antigen indirect ELISA for ASFV antibodies was established. The assay was non-cross-reactive with antibodies against the porcine reproductive and respiratory syndrome virus, classical swine fever virus, porcine circovirus type 2, and pseudorabies virus. The maximum serum dilution for detection of ASFV-positive sera was 1:1600. The intra-batch reproducibility coefficient of variation was <5% and the inter-batch reproducibility coefficient of variation was <10%. Compared with commercial kits, the dual-antigen indirect ELISA had good detection performance. In conclusion, we established a detection method with low cost, streamlined production process, and fewer instruments. It provides a new method for the serological diagnosis of ASF.

The Hole Sealing Technology of Solid-Liquid Materials with Three Pluggings and Two Injections for Gas Extraction Hole in the Coal Mine.

The sealing quality of the gas extraction holes determines the extracted gas concentration. Based on this, the paper reveals the basic principle of hole sealing by analyzing the gas leakage mechanism of the borehole. The hole sealing technology of solid-liquid materials with three pluggings and two injections for the gas extraction hole is proposed, and the hole sealing device and material are developed. Through testing the granularity distribution of the solid material, as well as the surface tension and contact angle of the slurry, the hole sealing material that can meet the requirements of accessible, sticky, and anti-deformation is selected. The sealing material enters microcracks and bonds coal rock more easily. First, the solid material is injected for hole sealing. Second, the liquid material can be injected repeatedly to maintain a high concentration for holes with poor sealing and gas concentration attenuation in the late stage of gas extraction. Field tests show that the gas concentration of solid material is 1.3 times that of the conventional material after 30 days of sealing. The liquid material injected after the concentration decline enables the gas extraction concentration to be recovered at 85%.

Acidic/Alkaline Stress Mediates Responses to Azole Drugs and Oxidative Stress in Aspergillus fumigatus.

A human host exploits stresses such as acidic/alkaline pH, antifungal drugs, and reactive oxygen species to kill microbial pathogens such as the fungus Aspergillus fumigatus. However, A. fumigatus is resistant to these stresses in vitro. Therefore, what accounts for the potent antifungal activity of the human host? In this observation, we show that simultaneous exposure to acidic pH and oxidative stresses is much more potent than the individual stresses themselves and that this combinatorial stress kills A. fumigatus synergistically in vitro. Interestingly, A. fumigatus is resistant to the combination of alkaline pH and oxidative stress. Quantitative real-time PCR analyses showed that acidic/alkaline pH stress can mediate oxidative stress responses in A. fumigatus by regulating the expression of catalase-encoding genes. We further show that A. fumigatus is sensitive to the combination of acidic/alkaline stress and azole drug stress. Transcriptome analysis revealed that the sensitivity of A. fumigatus to azole drugs under acidic/alkaline conditions may be related to changes in genetic stability, sphingolipid metabolism, lipid metabolism, and amino acid metabolism. Collectively, our findings suggest that combinatorial stress represents a powerful fungicidal mechanism employed by hosts against pathogens, which suggests novel approaches to potentiate antifungal therapy. IMPORTANCE The human host combats fungal infections via phagocytic cells that recognize and kill fungal pathogens. Immune cells combat Aspergillus fumigatus infections with a potent mixture of chemicals, including reactive oxygen species, acidic/alkaline stress, and antifungal drugs. However, A. fumigatus is relatively resistant to these stresses in vitro. In this observation, we show that it is the combination of acidic/alkaline pH and oxidative or azole stress that kills A. fumigatus so effectively, and we define the molecular mechanisms that underlie this potency. Our findings suggest that combinatorial stress is a powerful fungicidal mechanism employed by hosts, which suggests novel approaches to potentiate antifungal therapy. This study provides a platform for future studies that will address the combinatorial impacts of various environmental stresses on A. fumigatus and other pathogenic microbes.

Development of an Effective Double Antigen Sandwich ELISA Based on p30 Protein to Detect Antibodies against African Swine Fever Virus.

African swine fever (ASF), the highly lethal swine infectious disease caused by the African swine fever virus (ASFV), is a great threat to the swine industry. There is no effective vaccine or diagnostic method to prevent and control this disease currently. The p30 protein of ASFV is an important target for serological diagnosis, expressed in the early stage of viral replication and has high immunogenicity and sequence conservatism. Here, the CP204L gene was cloned into the expression vector pET-30a (+), and the soluble p30 protein was successfully expressed in the E. coli prokaryotic expression system and then labeled with horseradish peroxidase (HRP) to be the enzyme-labeled antigen. Using the purified recombinant p30 protein, a double-antigen sandwich ELISA for ASFV antibody detection was developed. This method exhibits excellent specificity, sensitivity and reproducibility in clinical sample detection with lower cost and shorter production cycles. Taken together, this study provides technical support for antibody detection for ASFV.

Development of an indirect ELISA for the identification of African swine fever virus wild-type strains and CD2v-deleted strains.

African swine fever (ASF) is a potent infectious disease with detrimental effects on the global swine industry and no currently vaccine available. The emergence of low-virulence CD2v-deleted mutants manifested as non-hemadsorption (non-HAD) strains represents a significant challenge to the prevention and control of ASF. In this study, we aimed to establish an indirect ELISA (IELISA) method for the identification of ASFV wild-type and CD2v-deleted strains. We integrated the CD2v protein extracellular domain sequence (CD2v-Ex, 1-588 bp) of the highly pathogenic strain China/2018/AnhuiXCGQ into the genome of suspension culture-adapted Chinese hamster Ovary-S (CHO-S) cells using lentivirus vectors (LVs). By screening, we identified a monoclonal CHO-S cell line that stably expressed secretory CD2v-Ex Protein. We then used the purified CD2v-Ex Protein as the detection antigen to establish an indirect ELISA method (CD2v-IELISA) for identification of the ASFV wild-type and CD2v-Deleted (CD2v-) strains. The CD2v-IELISA method showed excellent specificity with no cross-reaction with serum samples infected with ASFV (CD2v-), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), porcine circovirus (PCV), porcine pseudorabies virus (PRV), swine foot and mouth disease virus (FMDV) and porcine epidemic diarrhea virus (PEDV). Furthermore, this method showed high sensitivity, allowing identification of ASFV-infected clinical serum samples up to a dilution of 1:2,560. The coefficient of variation both in and between batches was <10% with good reproducibility and a high compliance rate of 99.4%. This CD2v-IELISA method developed here is of great significance for the prevention, control and purification of ASFV.