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Simple Summary The spread of citrus Huanglongbing caused significant damage to the world’s citrus industry. Thermotherapy and chemical agents were used to control this disease; however, the effectiveness of these treatments is frequently inconsistent. In addition, CLas cannot be cultured in vitro. Therefore, structure-based virtual screening is a novel method to find compounds that work against CLas. This study used CLas GMPS as a target for high-throughput screening and selected some compounds which have a higher binding affinity to test their inhibition of CLas GMPS. Finally, two molecules were identified as the lead compound to control citrus HLB. Abstract Citrus production is facing an unprecedented problem because of huanglongbing (HLB) disease. Presently, no effective HLB-easing method is available when citrus becomes infected. Guanosine 5′-monophosphate synthetase (GMPS) is a key protein in the de novo synthesis of guanine nucleotides. GMPS is used as an attractive target for developing agents that are effective against the patogen infection. In this research, homology modeling, structure-based virtual screening, and molecular docking were used to discover the new inhibitors against CLas GMPS. Enzyme assay showed that folic acid and AZD1152 showed high inhibition at micromole concentrations, with AZD1152 being the most potent molecule. The inhibition constant (Ki) value of folic acid and AZD1152 was 51.98 µM and 4.05 µM, respectively. These results suggested that folic acid and AZD1152 could be considered as promising candidates for the development of CLas agents.

Citrus huanglongbing (HLB) is a destructive disease caused by Candidatus Liberibacter species and is a serious global concern for the citrus industry. To date, there is no established strategy for control of this disease. Previously, Bacillus amyloliquefaciens GJ1 was screened as the biocontrol agent against HLB. In this study, two-year-old citrus infected by Ca. L. asiaticus were treated with B. amyloliquefaciens GJ1 solution via root irrigation. In these plants, after seven irrigation treatments, the results indicated that the photosynthetic parameters, chlorophyll content, resistance-associated enzyme content and the expression of defense-related genes were significantly higher than for the plants treated with the same volume water. The content of starch and soluble sugar were significantly lower, compared to the control treatment. The parallel reaction monitoring (PRM) results revealed that treatment with B. amyloliquefaciens GJ1 solution, the expression levels of 3 proteins with photosynthetic function were upregulated in citrus leaves. The accumulation of reactive oxygen species (ROS) in citrus leaves treated with B. amyloliquefaciens GJ1 flag22 was significantly higher than untreated plants and induced the defense-related gene expression in citrus. Finally, surfactin was identified from the fermentation broth of B. amyloliquefaciens GJ1 by high-performance liquid chromatography. These results indicate that B. amyloliquefaciens GJ1 may improve the immunity of citrus by increasing the photosynthesis and enhancing the expression of the resistance-related genes.

Background: An ideal animal model to study SARS-coronavirus 2 (SARS-CoV-2) pathogenesis and evaluate therapies and vaccines should reproduce SARS-CoV-2 infection and recapitulate lung disease like those seen in humans. The angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS-CoV-2, but mice are resistant to the infection because their ACE2 is incompatible with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Methods: We generated a mouse-adapted strain SARS-CoV-2 by serial passages in the lung of BALB/c mice. Complete genome deep sequencing of different generations of viruses was performed to characterize the dynamics of the adaptive mutations in SARS-CoV-2. Indirect immunofluorescence analysis and Biolayer interferometry experiments demonstrated that two mutations in RBD significantly increased its binding affinity towards mouse ACE2. Significantly, TLR7/8 agonist Resiquimod block SARS-CoV-2 in vitro and in vivo. Findings: We adapted a wild-type SARS-CoV-2 by serial passages in the lung of BALB/c mice. The mouse-adapted strain WBP-1 showed increased infectivity in BALB/c mice and led to severe interstitial pneumonia. We characterized the dynamics of the adaptive mutations in SARS-CoV-2 and demonstrated that Q493K and Q498H in RBD significantly increased its binding affinity towards mouse ACE2. Additionally, The TLR7/8 agonist Resiquimod was able to protect mice against WBP-1 challenge, demonstrating this mouse-adapted strain is a useful tool to investigate COVID-19 and develop new therapies. Interpretation: We found for the first time that the Q493K and Q498H mutations in the RBD of WBP-1 enhanced its interactive affinities with mACE2. The mouse-adapted SARS-CoV-2 provides a valuable tool for the evaluation of novel antiviral and vaccine strategies, especially in determining the immunopathological consequences of any intervention. This study also verified the antiviral activity of TLR7/8 agonist Resiquimod against SARS-CoV-2 in vitro and in vivo. Funding Statement: This research was funded by Emergency Science and Technology Project of Hubei Province（2020FCA046）and Independent Science and Technology Innovation Fund of Huazhong Agricultural University in 2020 (2662020PY002). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The animal experiments were approved by the Research Ethics Committee, Huazhong Agricultural University, Hubei, China (HZAUMO-2020-0007). All the animal experiments were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals from the Research Ethics Committee, Huazhong Agricultural University, Hubei, China.

Cyanobacterial blooms have a range of social, environmental and economic impacts due to their content of secondary metabolites involving toxins. Microcystins (MCs) is one of the cyanotoxins found in freshwater ecosystems. This study was aimed to adopt a rapid technique for determining the microcystin in blooms and freshwater by the detection of phycocyanin operon of cyanobacteria using PCβF and PCαR primers as well as mcyE gene encoding to microcystin/ nodularin using HEP primers. The molecular results showed that phycocyanin operon and mcyE gene were disclosed in the freshwater and bloom of samples at the studied sites. In conclusion, The PCR assay used in this study was helpful and rapid, in particular when the target organism concentration in the freshwater sample is very low.

Background An ideal animal model to study SARS-coronavirus 2 (SARS-CoV-2) pathogenesis and evaluate therapies and vaccines should reproduce SARS-CoV-2 infection and recapitulate lung disease like those seen in humans. The angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS-CoV-2, but mice are resistant to the infection because their ACE2 is incompatible with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein . Methods SARS-CoV-2 was passaged in BALB/c mice to obtain mouse-adapted virus strain. Complete genome deep sequencing of different generations of viruses was performed to characterize the dynamics of the adaptive mutations in SARS-CoV-2. Indirect immunofluorescence analysis and Biolayer interferometry experiments determined the binding affinity of mouse-adapted SARS-CoV-2 WBP-1 RBD to mouse ACE2 and human ACE2. Finally, we tested whether TLR7/8 agonist Resiquimod (R848) could also inhibit the replication of WBP-1 in the mouse model. Findings The mouse-adapted strain WBP-1 showed increased infectivity in BALB/c mice and led to severe interstitial pneumonia. We characterized the dynamics of the adaptive mutations in SARS-CoV-2 and demonstrated that Q493K and Q498H in RBD significantly increased its binding affinity towards mouse ACE2. Additionally, the study tentatively found that the TLR7/8 agonist Resiquimod was able to protect mice against WBP-1 challenge. Therefore, this mouse-adapted strain is a useful tool to investigate COVID-19 and develop new therapies. Interpretation We found for the first time that the Q493K and Q498H mutations in the RBD of WBP-1 enhanced its interactive affinities with mACE2. The mouse-adapted SARS-CoV-2 provides a valuable tool for the evaluation of novel antiviral and vaccine strategies. This study also tentatively verified the antiviral activity of TLR7/8 agonist Resiquimod against SARS-CoV-2 in vitro and in vivo. Funding This research was funded by the National Key Research and Development Program of China (2020YFC0845600) and Emergency Science and Technology Project of Hubei Province (2020FCA046) and Robert A. Welch Foundation (C-1565).

Citrus huanglongbing (HLB) is a destructive disease that causes significant damage to many citrus producing areas worldwide. To date, no strategy against this disease has been established. Inosine 5&prime, monophosphate dehydrogenase (IMPDH) plays crucial roles in the de novo synthesis of guanine nucleotides. This enzyme is used as a potential target to treat bacterial infection. In this study, the crystal structure of a deletion mutant of CLas IMPDH&Delta, 98-201 in the apo form was determined. Eight known bioactive compounds were used as ligands for molecular docking. The results showed that bronopol and disulfiram bound to CLas IMPDH&Delta, 98-201 with high affinity. These compounds were tested for their inhibition against CLas IMPDH&Delta, 98-201 activity. Bronopol and disulfiram showed high inhibition at nanomolar concentrations, and bronopol was found to be the most potent molecule (Ki = 234 nM). The Ki value of disulfiram was 616 nM. These results suggest that bronopol and disulfiram can be considered potential candidate agents for the development of CLas inhibitors.