[Preparation of monoclonal antibodies against the envelope protein extracellular domain of Zika virus in mice].

Objective To prepare monoclonal antibodies against the envelope protein extracellular domain (Eecto) of Zika virus (ZIKV) in mice. Methods A prokaryotic expression plasmid, pET28a-ZIKV-Eecto of ZIKV Eecto, was constructed, transformed into Escherichia coli BL21 and induced by isopropyl ß-D-thiogalactoside (IPTG). The recombinant Eecto protein was expressed in the form of inclusion bodies, and purified proteins were obtained through denaturation, renaturation and ultrafiltration. After three rounds of immunization with the Eecto protein, the serum of BALB/c mice was obtained and the titer of polyclonal antibodies in serum was determined. The reactivity of polyclonal antibodies was analyzed with Western blotting and immunofluorescence assay in HEK293T cells expressing the ZIKV prME. Spleen cells from mice with higher antibody titers were prepared and fused with SP2/0 myeloma cells. The hybridoma cells secreting antibodies were screened through the limited dilution method, and the ascites containing antibody were harvested for titer measurement and subclass analysis. The Eecto from the envelope proteins of Japanese encephalitis virus (JEV), Yellow fever virus (YFV), Dengue virus (DENV1-4), and Tick borne encephalitis virus (TBEV) were coated and used to analyze the cross-reactivity of ZIKV monoclonal antibodies by ELISA. Further specificity analysis was conducted on antibodies with high titers and strong specificity. Results The plasmid pET28a-ZIKV-Eecto was successfully constructed. The purified Eecto protein was obtained with good immunogenicity. Four monoclonal antibodies were prepared and screened, namely 1D6, 4F11, 4H7, and 4F8. Among them, 1D6, 4H7, and 4F8 are IgG (K) type antibodies, and 4F11 is an IgM (K) antibody. The ascitic fluid titer of 1D6 was higher than 1:108. Antibodies 1D6 and 4H7 are ZIKV-specific and showed no cross-reactivity with other Flaviviruses. Conclusion The mice monoclonal antibodies against ZIKV-Eecto are produced successfully, which will provide experimental materials for the establishment of ZIKV detection methods and the study of its pathogenesis.

[Preparation of mouse monoclonal antibodies against the ectodomain of Western equine encephalitis virus E2 (E2ecto) protein].

Objective To prepare mouse monoclonal antibodies against the ectodomain of E2 (E2ecto) glycoprotein of Western equine encephalitis virus (WEEV). Methods A prokaryotic expression plasmid pET-28a-WEEV E2ecto was constructed and transformed into BL21 (DE3) competent cells. E2ecto protein was expressed by IPTG induction and presented mainly as inclusion bodies. Then the purified E2ecto protein was prepared by denaturation, renaturation and ultrafiltration. BALB/c mice were immunized with the formulated E2ecto protein using QuickAntibody-Mouse5W as an adjuvant via intramuscular route, boosted once at an interval of 21 days. At 35 days post-immunization, mice with antibody titer above 1×104 were inoculated with E2ecto intraperitoneally, and spleen cells were fused with SP2/0 cells three days later. Hybridoma cells secreting specific monoclonal antibodies were screened by the limited dilution method, and ascites were prepared after intraperitoneal inoculation of hybridoma cells. The subtypes and titers of the antibodies in ascites were assayed by ELISA. The biological activity of the mAb was identified by immunofluorescence assay(IFA) on BHK-21 cells which were transfected with eukaryotic expression plasmid pCAGGS-WEEV-CE3E2E1. The specificity of the antibodies were evaluated with E2ecto proteins from EEEV and VEEV. Results Purified WEEV E2ecto protein was successfully expressed and obtained. Four monoclonal antibodies, 3G6G10, 3D7G2, 3B9E8 and 3D5B7, were prepared, and their subtypes were IgG2c(κ), IgM(κ), IgM(κ) and IgG1(κ), respectively. The titers of ascites antibodies 3G6G10, 3B9E8 and 3D7G2 were 105, and 3D5B7 reached 107. None of the four antibody strains cross-reacted with other encephalitis alphavirus such as VEEV and EEEV. Conclusion Four strains of mouse mAb specifically binding WEEV E2ecto are successfully prepared.

Amide herbicides: Analysis of their environmental fate, combined plant-microorganism soil remediation scheme, and risk prevention and control strategies for sensitive populations.

In this study, we predicted the environmental fate of amide herbicides (AHs) using the EQC (EQuilibrium Criterion) model. We found that the soil phase is the main reservoir of AHs in the environment. Second, a toxicokinetic prediction indicated that butachlor have a low human health risk, while the alachlor, acetochlor, metolachlor, napropamide, and propanil are all uncertain. To address the environmental and human-health-related threats posed by AHs, 27 new proteins/enzymes that easily absorb, degrade, and mineralize AHs were designed. Compared with the target protein/enzyme, the comprehensive evaluation value of the new proteins/enzymes increased significantly: the absorption protein increased by 20.29-113.49%; the degradation enzyme increased by 151.26-425.22%; and the mineralization enzyme increased by 23.70-52.16%. Further experiments revealed that the remediating effect of 13 new proteins/enzymes could be significantly enhanced to facilitate their applicability under real environmental conditions. The hydrophobic interactions, van der Waals forces, and polar solvation are the key factors influencing plant-microorganism remediation. Finally, the simulations revealed that appropriate consumption of kiwifruit or simultaneous consumption of ginseng, carrot, and spinach, and avoiding the simultaneous consumption of maize and carrot/spinach are the most effective means reduce the risk of exhibiting AH-linked toxicity.

[Preparation of mouse monoclonal antibodies against human adenovirus 55 Hexon (HAdV55 Hexon) protein].

Objective To prepare specific mouse monoclonal antibody (mAb) against human adenovirus type 55 Hexon protein (HAdV55 Hexon). Methods The Hexon genes of HAdV55, 3, 4, 7, 16 and 21 were chemically synthesized as templates for PCR amplification. The prokaryotic expression plasmids pET28a-HAdV55 Hexon and eukaryotic expression plasmids pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon were constructed respectively. The pET28a-HAdV55 Hexon plasmid was transformed into E. coli competent cell BL21 (DE3) and was induced by IPTG. After the purified inclusion body was denatured and renatured, Hexon55 protein was purified by tangential flow filtration system. pCAGGS-HAdV55 Hexon was used to immunize BALB/c mice by cupping, and HAdV55 Hexon protein was used to booster immunization. The anti-HAdV55 Hexon mAb was prepared by hybridoma technique and the titer and subclass were determined. The specificity of antibody was identified by Western blot using HEK293T cells transfected with pCAGGS-HAdV55 Hexon and by immunofluorescence assay (IFA) using BHK cells transfected with pCAGGS-HAdV55 Hexon. Both clones with high titer were selected, and the cross-reactivity of pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon transfected cells were analyzed by Western blot analysis and IFA. Results PET28a-HAdV55 Hexon and pCAGGS-HAdV55 Hexon, 3, 4, 7, 16 and 21 expression plasmids were successfully constructed. BL21 transformed with pET28a-HAdV55 Hexon was induced by IPTG. The HAdV55 Hexon protein was mainly expressed in the form of inclusion body. After denaturation and renaturation, the purified HAdV55 Hexon protein was obtained by ultrafiltration. Six hybridoma cell lines secreting HAdV55 Hexon mAb were obtained. The antibody subclass analysis showed that 2 strains were IgG2a subtypes and 4 strains were IgG2b. Two specific HAdV55 Hexon antibodies with high titer were obtained, and there was no cross-reactivity with HAdV3, 4, 7, 16, 21 Hexon. Conclusion The specific mice mAb against HAdV55 Hexon provides an experimental basis for establishing its antigen detection method.

Molecular Mechanisms Underlying Metabolic Resistance to Cyflumetofen and Bifenthrin in Tetranychus urticae Koch on Cowpea.

Tetranychus urticae Koch (T. urticae) is one of the most tremendous herbivores due to its polyphagous characteristics, and is resistant to most acaricides. In this study, enzyme-linked immunosorbent assay (ELISA), transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) were carried out to analyze the mechanisms of T. urticae metabolic resistance to cyflumetofen and bifenthrin on cowpea. The enzyme activity of UDP-glucuronosyltransferases (UGTs) and carboxylesterases (CarEs) in the cyflumetofen-resistant (R_cfm) strain significantly decreased, while that of cytochrome P450 monooxygenases (P450s) significantly increased. Meanwhile, the activities of glutathione-S-transferases (GSTs), CarEs and P450s in the bifenthrin-resistant (R_bft) strain were significantly higher than those in the susceptible strain (Lab_SS). According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses, in the R_cfm mite strain, two carboxyl/cholinesterase (CCE) genes and two P450 genes were upregulated and one gene was downregulated, namely CYP392E7; in the R_bft mite strain, eleven CCE, nine UGT, two P450, four GST and three ABC genes were upregulated, while four CCE and three P450 genes were downregulated. Additionally, 94 differentially expressed genes (DEGs) were common to the two resistant groups. Specifically, TuCCE46 and TuCCE70 were upregulated in both resistant groups. Furthermore, the qRT-PCR validation data were consistent with those from the transcriptome sequencing analysis. Specifically, TuCCE46 (3.37-fold) was significantly upregulated in the R_cfm strain, while in the R_bft strain, TeturUGT22 (5.29-fold), teturUGT58p (1.74-fold), CYP392A11 (2.89-fold) and TuGSTd15 (5.12-fold) were significantly upregulated and TuCCE01 (0.13-fold) and CYP392A2p (0.07-fold) were significantly downregulated. Our study indicates that TuCCE46 might play the most important role in resistance to cyflumetofen, and TuCCE01, teturUGT58p, teturUGT22, CYP392A11, TuGSTd15, TuGSTm09 and TuABCG-13 were prominent in the resistance to bifenthrin. These findings provide further insight into the critical genes involved in the metabolic resistance of T. urticae to cyflumetofen and bifenthrin.

High genetic differentiation of Indo-Pacific humpback dolphins (Sousa chinensis) along the Asian Coast of the Pacific Ocean.

The Indo-Pacific humpback dolphin (Sousa chinensis) is a vulnerable marine mammal species that inhabits shallow, coastal waters from Southeast China, southward throughout Southeast Asia, and westward around the Bay of Bengal to eastern India. Polymorphic microsatellites are useful for elucidating ecological and population genetics-related questions. Here, 18 new polymorphic microsatellites were developed from S. chinensis genomic DNA by Illumina MiSeq sequencing. Population genetic analyses were conducted on 42 S. chinensis individuals from three geographic locations, including the Xiamen Bay of China, the Western Gulf of Thailand, and Andaman Sea. Our microsatellite data revealed a strong and significant population structure among the three sampling regions (overall F ST = 0.371, p = .001). Pairwise mutual information index also demonstrated high levels of genetic differentiation between different region pairs (values range from 0.272 to 0.339, p < .001). Moreover, Structure analysis inferred three genetic clusters, with the high assignment probabilities of 95.92%, 99.47%, and 99.68%, respectively. Principal coordinate analysis plots of individuals divided entire genotypes into three clusters, indicating high level of genetic differentiation. Our results indicated the strong genetic structure in S. chinensis populations is a result of geographic distances. Other factors such as environmental variables, anthropogenic interference, and social behavior may also have contributed to population differentiation.

The prevalence and potential implications of microplastic contamination in marine fishes from Xiamen Bay, China.

The wide presence of microplastics (MPs) in the ocean leads their exposure on marine fish. MP contamination was reported for the gastrointestinal tracts and gills of 117 marine fishes attributed to nine species from Xiamen Bay, a special economic zone in China. Among species, MP abundance ranged from 1.07 items individual-1 to 8.00 items individual -1. Fibers dominated MP shapes, accounting for 59.03% of all MPs. Polymer composition was dominated by polyamide (26.97%) and rayon (17.56%). MPs were most commonly (55.22%) transparent, and most (77.61%) were < 1 mm in size. Our report represents the first of MP contamination in wild marine fish from Xiamen Bay, which we determine to be at an intermediate to slightly higher level compared with levels reported elsewhere, and provides further insights into potential risks of MPs pose to fish and human health.

Identification of mercury-containing nanoparticles in the liver and muscle of cetaceans.

Natural mercury-containing nanoparticles (Hg-NPs) have been found in the environment, but the information for Hg-NPs in organisms was still limited. Clarifying the unique roles of Hg-NPs in organisms is crucial to fully understand the health risks of Hg. Herein, liver and muscle tissues of cetaceans were collected to identify the presence and characteristics of Hg-NPs. We found that methylmercury (MeHg) was the dominant species of Hg in muscles, while inorganic Hg (IHg) accounted for 84.4-99.0% (average 94.0%) of Hg in livers. By using transmission electron microscopy (TEM), size exclusion chromatography coupled with inductively coupled plasma mass spectrometry (SEC-ICPMS) and single particle ICPMS (sp-ICPMS), large amounts (9-161 µg/g) of Hg-NPs in livers and small amounts (0.1-0.4 µg/g) in muscles were observed, indicating that Hg-NPs was an important form of Hg in livers. Both small sized (5-40 nm) and large sized (>100 nm) Hg-NPs were identified, which were mainly complexed with selenium (Se) and sulfur (S) as well as a few cadmium (Cd), lead (Pb) and silver (Ag). This study provided direct evidence of Hg-NPs in marine mammals as well as their chemical form and size distribution, which are helpful for further understanding the biogeochemical cycle and health risk of Hg.

Genetic structure of the endangered Irrawaddy dolphin (Orcaella brevirostris) in the Gulf of Thailand.

The Irrawaddy dolphin (Orcaella brevirostris) is an endangered, small cetacean species which is widely distributed in rivers, estuaries, and coastal waters throughout the tropical and subtropical Indo-Pacific. Despite the extensive distribution of this species, little is known of individual movements or genetic exchange among regions in Thailand. Here, we evaluate the genetic diversity and genetic structure of O. brevirostris in the eastern, northern and western Gulf of Thailand, and Andaman Sea. Although phylogenetic relationships and network analysis based on 15 haplotypes obtained from 32 individuals reveal no obvious divergence, significant genetic differentiation in mitochondrial DNA (overall FST = 0.226, P < 0.001; ΦST = 0.252, P < 0.001) is apparent among regions. Of 18 tested microsatellite loci, 10 are polymorphic and successfully characterized in 28 individuals, revealing significant genetic differentiation (overall FST = 0.077, P < 0.05) among the four sampling sites. Structure analysis reveals two inferred genetic clusters. Additionally, Mantel analysis demonstrates individual-by-individual genetic distances and geographic distances follow an isolation-by-distance model. We speculate that the significant genetic structure of O. brevirostris in Thailand is associated with a combination of geographical distribution patterns, environmental and anthropogenic factors, and local adaptations.

Early divergence and differential population histories of the Indo-Pacific humpback dolphin in the Pacific and Indian Oceans.

The currently recognized Indo-Pacific humpback dolphin occurs in estuaries and surrounding shallow waters from the South China Sea to the Asian coast of the Indian Ocean. However, a recent study suggested that the humpback dolphin from the Bay of Bengal may represent a distinct phylogenetic species. In this study, we sequenced 915-bp mtDNA segments from five geographic populations in both Chinese and Thai waters; together with previously published sequences, these data revealed that the ancestral Indo-Pacific humpback dolphin might have split during the transition from the Oligocene to Miocene (23.45 Mya, 95% HPD: 16.65-26.55 Mya), and then dispersed along the Pacific and Indian Ocean coasts of Asia. Genetic differentiation was detected between most of the examined populations, except for only a few pairwise populations in the northern South China Sea. Genetic differentiation/distance between the humpback dolphins from the northern and southern South China Sea met the sub-species threshold value proposed for marine mammals, whereas that between the humpback dolphins in the Pacific and the Indian Ocean was above the species threshold. Bayesian inference of historic gene flow indicated low but constant northward gene flow along the Indian Ocean coast; however, there was a recent abrupt increase in gene flow in the Pacific region, likely due to the shortening coastline at the low stand of sea level. Our results revealed that the current taxonomic classification of Indo-Pacific humpback dolphins may not reflect their phylogeography.

Predictors of Gull-billed tern (Gelochelidon nilotica) nest survival in artificial coastal saltpans, Bohai Bay, China.

BACKGROUND: Coastal saltpans are a common supratidal human-modified wetland habitat found within many coastal landscape mosaics. Commercial salt production and aquaculture practices often result in the creation of exposed coastal substrates that could provide suitable breeding habitat for waterbird populations; however, few studies have quantified waterbird breeding success in these artificial wetlands. METHODS: Here we examine the nesting behavior of the Gull-billed tern (Gelochelidon nilotica) breeding in the Nanpu coastal saltpans of Bohai Bay, Yellow Sea, China over three consecutive nesting seasons (2017-2019) by using nest survival model in Program MARK. RESULTS: The results revealed that nest survival of Gull-billed terns in coastal saltpans (0.697) was higher than previously published estimates from other regions, with an estimated daily survival rate (DSR) of 0.982 ± 0.001 (±95% CI). High nest survival was mainly attributed to low levels of human disturbances and low predation rates, while exposure to strong winds, flooding and silting were the main factors causing nest failure. Model-averaged estimates revealed that eggs laid in nests located on 'habitat islands' with feather or clam shell substrates were most likely to hatch. Initiation date, nest age, clutch size and quadratic effects of nearest-neighbor distance, nearest distance to road and nearest distance to water were all significant predictors of nest success, but the nest survival declined overall from 2017 to 2019 due to the degradation and loss of breeding habitat anthropogenically caused by rising water levels. DISCUSSION: Coastal saltpans represent an alternative breeding habitat for the Gull-billed tern populations in Bohai Bay, but conservation management should prioritize flood prevention to improve the extent and quality of breeding habitat, concurrent with efforts to create further 'habitat islands' with suitable nesting substrate.