Crystal Structures of Fusion Cores from CCoV-HuPn-2018 and SADS-CoV.

Cross-species spillover to humans of coronaviruses (CoVs) from wildlife animal reservoirs poses marked and global threats to human and animal health. Recently, sporadic infection of canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018) in hospitalized patients with pneumonia genetically related to canine and feline coronavirus were identified. In addition, swine acute diarrhea syndrome coronavirus (SADS-CoV) had the capability of broad tropism to cultured cells including from humans. Together, the transmission of Alphacoronaviruses that originated in wildlife to humans via intermediate hosts was responsible for the high-impact emerging zoonosis. Entry of CoV is mainly mediated by Spike and formation of a typical six helix bundle (6-HB) structure in the postfusion state of Spike is pivotal. Here, we present the complete fusion core structures of CCoV-HuPn-2018 and SADS-CoV from Alphacoronavirus at 2.10 and 2.59 Å, respectively. The overall structure of the CCoV-HuPn-2018 fusion core is similar to Alphacoronavirus like HCoV-229E, while SADS-CoV is analogous to Betacoronavirus like SARS-CoV-2. Collectively, we provide a structural basis for the development of pan-CoV small molecules and polypeptides based on the HR1-HR2 complex, concerning CCoV-HuPn-2018 and SADS-CoV.

Molecular and functional characterization of an antenna-enriched glutathione S-transferase BminGSTd3 involved in undecanol degradation in the citrus fruit fly, Bactrocera minax (Enderlein) (Diptera Tephritidae).

Bactrocera minax is a disastrous pest of citrus crops in China. Numerous studies focused on the molecular mechanism of odorant perception of B. minax, but the molecular mechanism of odorant degradation remains unclear. Glutathione S-transferases (GSTs) are considered as a class of odorant-degrading enzymes involved in degrading odorant molecules in insects' olfactory system. Here, we identified a delta-class GST gene, BminGSTd3, from B. minax. It was predominantly expressed in adult's olfactory organ antennae. The bacterially expressed recombinant BminGSTd3 was able to catalyze the conjugation of glutathione (GSH) with 2, 4-dinitrochlorobenzene (CDNB). Spectrophotometric analysis showed that undecanol can inhibit catalytic activities of BminGSTd3. Metabolic assays exhibited that undecanol can be depleted by BminGSTd3. Undecanol is believed to be an important B. minax sex pheromone component. The other components of the pheromone remain unclear. To understand how BminGSTd3 specifically recognizes undecanol, a 3D model of BminGSTd3 was constructed by homology modeling. Molecular docking based on this model revealed that E64 and S65 are the key amino acids recognizing undecanol, and this was proven by site-directed mutagenesis and intrinsic fluorescence assays. We suggest that BminGSTd3 is an undecanol metabolizing GST in B.minax, and E64 and S65 may serve as the key binding sites.

Key Amino Acid Residues Involved in Binding Interactions between Bactrocera minax Odorant-Binding Protein 3 (BminOBP3) and Undecanol.

Insect odorant-binding proteins (OBPs) are significant in binding and transporting odorants to specific receptors. Our previous study demonstrated that BminOBP3 exhibited a strong affinity with undecanol. However, the binding mechanism between them remains unknown. Here, using homology modeling and molecular docking, we found that the C-terminus (I116-P122), especially the hydrogenbonds formed by the last three amino acid residues (V120, F121, and P122) of the C-terminus, is essential for BminOBP3's ligand binding. Mutant binding assays showed that the mutant T-OBP3 that lacks C-terminus (I116-P122) displayed a significant decrease in affinity to undecanol (Ki = 19.57 ± 0.45) compared with that of the wild-type protein BminOBP3 (Ki = 11.59 ± 0.51). In the mutant 3D2a that lacks F121 and P122 and the mutant V120A in which V120 was replaced by alanine, the bindings to undecanol were completely abolished. In conclusion, the C-terminus plays a crucial role in the binding interactions between BminOBP3 and undecanol. Based on the results, we discussed the ligand-binding process of BminOBP3.

Potent NTD-Targeting Neutralizing Antibodies against SARS-CoV-2 Selected from a Synthetic Immune System.

The majority of neutralizing antibodies (NAbs) against SARS-CoV-2 recognize the receptor-binding domain (RBD) of the spike (S) protein. As an escaping strategy, the RBD of the virus is highly variable, evolving mutations to thwart a natural immune response or vaccination. Targeting non-RBD regions of the S protein thus provides a viable alternative to generating potential, robust NAbs. Using a pre-pandemic combinatorial antibody library of 1011, through an alternate negative and positive screening strategy, 11 non-RBD-targeting antibodies are identified. Amongst one NAb that binds specifically to the N-terminal domain of the S protein, SA3, shows mutually non-exclusive binding of the angiotensin-converting enzyme 2 receptor with the S protein. SA3 appears to be insensitive to the conformational change and to interact with both the "open" and "closed" configurations of the trimeric S protein. SA3 shows compatible neutralization as S-E6, an RBD-targeting NAb, against the wild type and variant of concern (VOC) B.1.351 (Beta) of the SARS-CoV-2 pseudo virus. More importantly, the combination of SA3 with S-E6 is synergistic and recovers from the 10-fold loss in neutralization efficacy against the VOC B.1.351 pseudo virus.

Molecular identification and lipid mobilization role of adipokinetic hormone receptor in Spodoptera litura (F.).

Energy homeostasis is essential for organisms to maintain fluctuation in energy accumulation, mobilization. Lipids as the main energy reserve in insects, their metabolism is under the control of many physiological program. This study aimed to determine whether the adipokinetic hormone receptor (AKHR) was involved in the lipid mobilization in the Spodoptera litura. A full-length cDNA encoding AKHR was isolated from S. litura. The SlAKHR protein has a conserved seven-transmembrane domain which is the character of a putative G protein receptor. Expression profile investigation revealed that SlAKHR mRNA was highly expressed in immatural stage and abundant in fat body in newly emerged female adults. Knockdown of SlAKHR expression was achieved through RNAi by injecting double-stranded RNA (dsRNA) into the 6th instar larvae. The content of triacylgycerol (TAG) in the fat body increased significantly after the SlAKHR gene was knockdown. And decrease of TAG releasing to hemolymph with increase of free fatty acid (FFA) in hemolymph were observed when the SlAKHR gene was knowned-down. In addition, lipid droplets increased in fat body was also found. These results suggested that SlAKHR is critical for insects to regulate lipids metabolism.

Neutralizing Antibodies to SARS-CoV-2 Selected from a Human Antibody Library Constructed Decades Ago.

Combinatorial antibody libraries not only effectively reduce antibody discovery to a numbers game, but enable documentation of the history of antibody responses in an individual. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has prompted a wider application of this technology to meet the public health challenge of pandemic threats in the modern era. Herein, a combinatorial human antibody library constructed 20 years before the coronavirus disease 2019 (COVID-19) pandemic is used to discover three highly potent antibodies that selectively bind SARS-CoV-2 spike protein and neutralize authentic SARS-CoV-2 virus. Compared to neutralizing antibodies from COVID-19 patients with generally low somatic hypermutation (SHM), these three antibodies contain over 13-22 SHMs, many of which are involved in specific interactions in their crystal structures with SARS-CoV-2 spike receptor binding domain. The identification of these somatically mutated antibodies in a pre-pandemic library raises intriguing questions about the origin and evolution of these antibodies with respect to their reactivity with SARS-CoV-2.

Functional Characterization of Two Antenna-Enriched Odorant-Binding Proteins From Bactrocera minax (Diptera: Tephritidae).

Olfaction is of great significance for insect mate-seeking and host-locating behaviors. Insect odorant-binding proteins (OBPs), especially those antenna-enriched OBPs, are thought to discriminate, capture and transport odorant molecules to olfactory receptors, but this has not been fully clarified in Bactrocera minax (Enderlein), an economically important pest of citrus crops. Our previous studies showed that seven OBP genes (BminOBP1-7) were identified from B. minax adults via a head transcriptome analysis, of which only BminOBP3 and 6 were highly expressed in antennae, suggesting an olfactory role. To confirm their functions, here, BminOBP3 and 6 were cloned, expressed in Escherichia coli cells. Binding properties of the recombinant BminOBPs with 13 volatiles, most of which can elicit a significant behavioral response from B. minax adults, were determined by fluorescent competitive binding assays. The results showed that Both BminOBP3 and 6 exhibited a remarkable selectivity towards the 13 ligands tested. BminOBP3 displayed strong binding affinity only with undecanol. BminOBP6 demonstrated strong binding affinity with undecanol and limonene among 13 ligands tested. Undecanol is believed to be main sex pheromone component of B. minax. Limonene is an important volatile compound enriched in citrus fruits. Taken together, we concluded that BminOBP3 and 6 may play a prominent role in the process of B. minax mate-seeking and host-locating behaviors through recognizing and transporting these volatiles. It is conceivable that this study will increase our molecular understanding of B. minax olfaction, facilitating the development of OBP-based behavioral interference that is potentially useful for the integrated management of B. minax.

Excessive Nitrogen Fertilization Favors the Colonization, Survival, and Development of Sogatella furcifera via Bottom-Up Effects.

Fertilization can trigger bottom-up effects on crop plant-insect pest interactions. The intensive use of nitrogen fertilizer has been a common practice in rice production, while the yield has long been challenged by the white-backed planthopper, Sogatella furcifera (Horváth). High nitrogen fertilization can facilitate S. furcifera infestation, however, how nitrogen fertilizer leads to high S. furcifera infestation and the nutritional interactions between rice and S. furcifera are poorly understood. Here, we evaluated the effects of various levels of nitrogen fertilizer application (0-350 kg/ha) on rice, and subsequently on S. furcifera performance. We found that higher nitrogen fertilizer application: (1) increases the preference of infestation behaviors (feeding and oviposition), (2) extends infestation time (adult lifespan), and (3) shortens generation reproduction time (nymph, pre-oviposition, and egg period), which explain the high S. furcifera infestation ratio on rice paddies under high nitrogen conditions. Moreover, high nitrogen fertilizer application increased all tested rice physical indexes (plant height, leaf area, and leaf width) and physiological indexes (chlorophyll content, water content, dry matter mass, and soluble protein content), except for leaf thickness, which was reduced. Correlation analysis indicated that the specific rice physical and/or physiological indexes were conducive to the increased infestation behavior preference, extended infestation time, and shortened generation reproduction time of S. furcifera. The results suggested that nitrogen fertilizer triggers bottom-up effects on rice and increases S. furcifera populations. The present study provides an insight into how excess nitrogen fertilization shapes rice-planthopper interactions and the consequent positive effect on S. furcifera infestation.

Diversity, Tissue Localization, and Infection Pattern of Bacterial Symbionts of the White-Backed Planthopper, Sogatella furcifera (Hemiptera: Delphacidae).

The white-backed planthopper (WBPH), Sogatella furcifera (Horváth), is a destructive pest of rice. Bacterial symbionts play an important role in insect hosts, especially hemipteran hosts. This study was designed to examine the bacterial symbionts of the WBPH using 16S rDNA high-throughput sequencing. A total of 63 and 177 operational taxonomic units (OTUs) were identified in females and males of three WBPH populations, respectively. These OTUs included bacteria of 75 genera from 11 phyla, where Wolbachia, Cardinium, and Asaia were the dominant genera, accounting for over 97.99% of all the symbiotic bacteria. Fluorescence in situ hybridization detected Wolbachia, Cardinium, and Asaia in the salivary glands, guts, testes, and eggs of the WBPH, indicating the potential for both horizontal and vertical transmission. Moreover, the infection pattern of the three dominant bacterial symbionts was detected in six WBPH populations. The frequencies of Wolbachia infection of females and Cardinium infection of both sexes were over 96.7%. Wolbachia infection of males ranged between 46.7 and 63.3%, which was significantly lower than that observed for females. Asaia infection of both sexes varied substantially among the populations. These results indicate that the complex host-symbiotic bacteria interaction is influenced by host sex and geographical origin and potentially by the transmission modes of the symbionts.

Effects of Water Immersion and Soil Moisture Content on Larval and Pupal Survival of Bactrocera minax (Diptera: Tephritidae).

Bactrocera minax, one of the most devastating citrus pests in Asia, has two developmental stages (mature larva and pupa) that complete their life cycle in the soil. Currently, southern China has a climate with abundant autumn rains, and soil moisture can be a major factor affecting the survival of larvae and pupae of B. minax. In the present study, we evaluated the effects of water immersion and high soil moisture content on the development of mature larvae and pupae of B. minax. When immersed in water for 1 d, 100% of mature larvae of B. minax were knocked out. When larvae were immersed for less than 6 d, however, more than 92% of knocked-out larvae recovered within 24 h. The days of water immersion with 50% and 90% recovery ratios (indicated as RD50 and RD90) were 10.3 d and 6.4 d, respectively. When larvae were immersed less than 6 d, the mortality ratios of larvae were not significantly different from those that were not immersed at all. The days of immersion causing 50% and 90% mortality of larvae (MD50 and MD90, respectively) were 7.6 d and 11.1 d, respectively. The pupation ratios of larvae were also observed to be not significantly different compared to non-immersion, and the days of immersion causing 50% and 90% pupation (PD50 and PD90, respectively) were 6.6 d and 0.8 d, respectively. Larval respiration rates were reduced after water immersion as a strategy for larval survival. High water content was not detrimental to pupae of B. minax. Adult emergence did not significantly decrease in soil with high water content, even though pupae were under those conditions for 161-175 d. The respiration rates of pupae were lower in soil with different moisture levels and were not significantly different, which ensured the survival of pupae in high water content. Reduced respiration rate is a strategy for survival of larvae and pupae, and remarkable tolerance to high moisture conditions could explain the high rate of spread and geographical distribution of B. minax. The results of this study provide a reference for the occurrence and control of B. minax.

Characterization and Levels of Expression of Sensory Neuron Membrane Proteins in the Adult Citrus Fruit Fly (Diptera: Tephritidae).

Sensory neuron membrane proteins (SNMPs) play an important role in insect chemoreception; however, the SNMPs for Bactrocera minax (Enderlein) (Diptera: Tephritidae), an economically important pest of citrus, remain uncharacterized. Here, we report on the molecular characterization of SNMPs (BminSNMP1 and BminSNMP2) from adult B. minax. The open-reading frames of BminSNMP1 and BminSNMP2 were 1,608 and 1,647 nucleotides, encoding proteins of 535 and 557 amino acid residues, respectively. Phylogenetic analysis showed that the two BminSNMPs belonged to two distinct subgroups, indicating the possibility of their contrasting function in insect chemoreception. Real-time PCR results showed that BminSNMP1 was expressed primarily in the antennae of males and females, where levels of expression were similar at different developmental stages of females, but lower in 1- and 5-d-old males than in 15- and 20-d-old males. In both sexes, BminSNMP2 was expressed at high levels in antennae and in nonolfactory tissues, especially in legs, where levels were higher than in other nonolfactory tissues. We found highest levels of expression of BminSNMP2 in antennae of both sexes in 30-d-old adults, while in legs of both sexes, highest levels of expression were detected in 1- and 30-d-old adults. We discuss the possible physiological functions of BminSNMPs based on our findings.

Influence of Supplemental Protein on the Life Expectancy and Reproduction of the Chinese Citrus Fruit Fly, Bactrocera minax (Enderlein) (Tetradacus minax) (Diptera: Tephritidae).

Bactrocera minax (Enderlein) (Diptera: Tephritidae) is a major citrus pest in China, whose artificial rearing technology of the adult is not well documented to date. In this study, we tried to determine if supplementing proteins to the adult diet could result in the enhancement of some fitness parameters of B. minax. Four feeds with varying protein source were provided as F0 (water), F1 (sucrose), F2 (sucrose + yeast), and F3 (sucrose + peptone). F0 and F1 being the control, F2 and F3 were protein food types. The results showed that adults fed by F2 and F3 lived longer with 40.1 d and 32.8 d, respectively, had reduced death rates (death peaks were delayed for 5.6 d and 4.1 d, respectively), increased mating frequencies (8.1 and 5.3 per females, 4.7 and 7.3 per males, respectively), and longer mating durations (with 42 d and 34 d). In addition, females recorded an increased adult ovary development, more egg load (with 94.8 and 77.3 brood eggs per ovary) and to greater oviposition rates of 63.2 eggs/female and 19.3 eggs/female. Based on our results, protein supplements enhanced B. minax survival, mating, and fecundity. This study does not only provide basic knowledge to implement artificial rearing of B. minax, but also deepens our understanding on its physiology that could be used to enhance the management of the pest.