Identifying SARS-CoV-2-related coronaviruses in Malayan pangolins.

The ongoing outbreak of viral pneumonia in China and across the world is associated with a new coronavirus, SARS-CoV-21. This outbreak has been tentatively associated with a seafood market in Wuhan, China, where the sale of wild animals may be the source of zoonotic infection2. Although bats are probable reservoir hosts for SARS-CoV-2, the identity of any intermediate host that may have facilitated transfer to humans is unknown. Here we report the identification of SARS-CoV-2-related coronaviruses in Malayan pangolins (Manis javanica) seized in anti-smuggling operations in southern China. Metagenomic sequencing identified pangolin-associated coronaviruses that belong to two sub-lineages of SARS-CoV-2-related coronaviruses, including one that exhibits strong similarity in the receptor-binding domain to SARS-CoV-2. The discovery of multiple lineages of pangolin coronavirus and their similarity to SARS-CoV-2 suggests that pangolins should be considered as possible hosts in the emergence of new coronaviruses and should be removed from wet markets to prevent zoonotic transmission.

[Diagnosis of the first imported case of Plasmodium ovale infection at Guangdong Port].

The first imported case of Plasmodium ovale infection in Guangdong Province was identified. The patient worked in Myanmar for one week and had a fever when he arrived at Guangzhou Baiyun International Airport. Epidemiological information and blood sample were collected. The detection was conducted by microscopy, right VIEW rapid malaria test (RDTs) and real-time PCR with Plasmodium genus-specific and species-specific primers and probes. The case showed weak positive RDT result, and was confirmed as P. ovale infection by microscopy and real-time PCR. After treatment with artemether, his symptoms improved.

Cu-Based Organic-Inorganic Composite Materials for Electrochemical CO2 Reduction.

Electrochemical CO2 reduction reaction (CO2 RR) is an attractive pathway to convert CO2 into value-added chemicals and fuels. Copper (Cu) is the most effective monometallic catalyst for converting CO2 into multi-carbon products, but suffers from high overpotentials and poor selectivity. Therefore, it is essential to design efficient Cu-based catalyst to improve the selectivity of specific products. Due to the combination of advantages of organic and inorganic composite materials, organic-inorganic composites exhibit high catalytic performance towards CO2 RR, and have been extensively studied. In this review, the research advances of various Cu-based organic-inorganic composite materials in CO2 RR, i. e., organic molecular modified-metal Cu composites, Cu-based molecular catalyst/carbon carrier composites, Cu-based metal organic framework (MOF) composites, and Cu-based covalent organic framework (COF) composites are systematically summarized. Particularly, the synthesis strategies of Cu-based composites, structure-performance relationship, and catalytic mechanisms are discussed. Finally, the opportunities and challenges of Cu-based organic-inorganic composite materials in CO2 RR are proposed.

[Nested PCR for malaria detection and Plasmodium species identification].

According to the sequences of small subunit ribosomal RNA (SSU rRNA) gene of Plasmodium spp., universal and species-specific primers were designed to detect malaria and identify species. 60 blood samples were detected by the established nested PCR method. The results were compared with those of microscopic examination. 40 blood samples were Plasmodium-positive by nested PCR with 22 samples of P. falciparum, 13 of P. vivas, 3 with P. falciparum and P. vivax mixed infection, 1 of P. ovale and 1 of unclassified malaria infection. Altogether, the coincidence between the results of nested PCR and microscopy stood for 76.7% (46/60), including 18 of P. falciparum, 11 of P. vivax and 17 negatives. Further sequence analysis and real-time PCR were performed to detect blood samples with discrepancy, results of which were the same as that of nested PCR. The amplified product of P. ovale was sequenced and showed 100% homology to the corresponding part of P. ovale SSU rRNA gene sequence (GenBank No. DQ845247), which confirmed that the case was imported ovale malaria.

[Influence of accessory protein P19 from Bacillus thuringiensis on insecticidal crystal protein Cry11Aa].

p19 gene, cry11Aa gene and p20 gene from Bacillus thuringienesis subsp. israelensis are organized as a single operon. It is reported that P20 polypeptide is not required for high-level expression of Cry11Aa and crystal formation in B. thuringiensis. It is deduced that P19 might relate to Cry11Aa crystallization. In this study, two recombinant plasmids pHcy1 and pHcy3 containing cryllAa gene were constructed, the latter absent from p19 gene encoding a possible accessory protein between cry11Aa promoter and cry11Aa gene. The recombinant plasmids were introduced into an acrystalliferous mutant 4Q7 of B. thuringiensis subsp. israelensis. SDS-PAGE showed that Cry11Aa protein per unit of culture medium had a higher expression level in 4Q7(pHcy1) with p19 and cry11Aa genes than in 4Q7(pHcy3) with only cry11Aa gene. Both two B. thuringiensis strains formed Cry11Aa crystals in a similar size and shape during sporulation. Toxicity bioassay showed 4Q7 (pHcy1) and 4Q7 (pHcy3) exhibited a comparable mosquito-larvicidal activity against 3rd-instar Culex quinquefasciatus. It indicated that accessory protein P19 did not have an effect on cry11Aa crystallization and high mosquitocidal toxicity. However, it could enhance Cry11Aa expression amount to a certain extent.

[Effects of helper protein P20 from Bacillus thuringiensis on Vip3A expression].

Insecticidal crystal proteins (ICPs) produced in Bacillus thuringiensis accumulate as crystalline inclusions that represent up to 30% of total dry weight the cell produces. The mechanisms of in vivo crystallization of these insecticidal proteins remain interests, yet unclear. A 20-kDa protein (P20), the product of the third open reading frame of cry11A operon in B. thuringiensis subsp. israelensis has been defined to be an important molecular chaperone (helper protein) for forming Cyt1A crystal and enhancing Cry11A expression. The novel vegetative insecticidal proteins (VIPs) are secreted outside the cell of B. thuringiensis during mid-logarithmic growth. VIP3A shows activity against many lepidopteran insect larvae in a different mechanism from that of ICPs. To investigate the influence of helper protein P20 on Vip3A production and its insecticidal activity, P20 was coexpressed with Vip3A protein in B. thuringiensis and the yields and insecticidal toxicity of Vip3A were also analyzed. The recombinant plasmid pHVP20 was constructed by inserting a 5.4kb foreign fragment containing both vip3A gene and p20 gene into the shuttle vector pHT3101. The plasmid pHPT3 only containing vip3A gene was used as control. pHVP20 and pHPT3 were transformed into the B. thuringiensis acrystalliferous strain CryB not containing vip3A gene by electroporation. The obtained B. thuringiensis transformants were CryB(pHVP20) and CryB(pHPT3) respectively. Western blot showed that Vip3A protein reached its maximum yield after 48h of CryB (pHVP20) growth and remained high expression level during the sporulation. The maximum yield of Vip3A protein in CryB (pHVP20) was about 1.5 fold as compared with that in CryB(pHPT3) by the mean of ImageMaster VDS software. It is considered that P20 might combine with the native Vip3A protein during the sporulation, stabilize Vip3A and protect Vip3A from unspecific full proteolysis. Bioassay showed that the cell pellets of CryB (pHVP20) and CryB(pHPT3) performed high insecticidal toxicity against the first instar larvae of Spodoptera litura. Their LC50s of were 48.79 microg/mL and 78.00 microg/mL respectively and were not significantly different. Cell supernatants of two strains containing small amounts of secreted Vip3A were not toxic to the tested insect. It suggests that p20 can enhance the expression of Vip3A, but not improve its insecticidal toxicity remarkably.

[Construction of a eukaryotic expression plasmid containing gp120 gene of HIV-1 subtype B and its expression in HepG2 cells].

OBJECTIVE: To construct an eukaryotic expression plasmid containing gp120 gene of HIV-1 subtype B and obtain gp120 gene expression in HepG2 cells. METHODS: According to the published gp120 gene sequence in Genbank, a pair of primers was designed and synthesized. The PCR amplification product of gp120 gene was cloned into pMD-18T vector using TA cloning followed by BamHI and XhoI digestion and sequence analysis. The target gene was then subcloned into a highly efficient eukaryotic expression vector pcDNA3.1 (+). The recombinant plasmid was sequenced and identified by restrictive endonuclease digestion, and transfected into HepG2 cells via liposome. The expression of gp120 gene was analyzed by RT-PCR and Western blotting, respectively. RESULTS: Restriction endonuclease digestion and sequence analysis verified successful construction of the recombinant vector pcDNA3.1(+)/gp120. The target fragment gp120 was identical with U26942 in Genbank, and the expression of gp120 gene was detected in the lysate of the transfected HepG2 cells by RT-PCR and Western blotting. CONCLUSION: The eukaryotic expression plasmid for gp120 has been constructed successfully, which is capable of stable expression in HepG2 cells.

[Expression of SARS spike gene in Shizomycete pombe].

The viral spike protein is the main surface antigen of the coronavirus, and it could be useful in the research of clinical diagnosis, SARS vaccine and the structure biology.According to the analysis of the main antigen of the SARS spike protein, 5 fragments of the whole spike gene were cloned, and ligated to the vector pNMT1. Through electroporation transformantion to TCP1, the recombinant S. pombe strains capable of expressing the 5 fragments were constructed. SDS-PAGE or Western blot analysis of the induced expression products demonstrated that the 5 recombinant proteins were expressed in the fission yeast respectively.

Toxicity of Bacillus sphaericus LP1-G against susceptible and resistant Culex quinquefasciatus and the cloning of the mosquitocidal toxin gene.

Bacillus sphaericus LP1-G, belonging to flagellar serotype H3, has been found to have moderate toxicity against two resistant Culex quinquefasciatus colonies (RLCq1 and RLCq2) and the susceptible contrast (SLCq). With an aim of screening mosquitocidal acting factor, a partial genome library was prepared from a partial HindIII digest of the total DNA from Bacillus sphaericus LP1-G. Two thousand twenty Escherichia coli clones were screened for toxicity against susceptible SLCq, and a toxic clone, designated E-UL68, was chosen for further study. The recombinant E-UL68 performed toxicity against both susceptible and two resistant colonies, having the same level of toxicity as that of wide-type strain LP1-G. Sequence analysis revealed that the inserted fragment was composed of 3876 nucleotides and contained a complete gene, whose sequence was identical to that of the mtx gene from B. sphaericus SSII-1. Because the binary toxin produced during sporulation of strain LP1-G has no activity against the target mosquitoes, this indicates that the Mtx toxin or other active factors might perhaps be responsible for the toxicity of LP1-G against different colonies of mosquito larvae.

[Cloning and expression product of vip3A gene from Bacillus thuringiensis and analysis of inseceicidal activity].

The vip3 A gene in a size of 2.3 kb amplified from wild-type Bacillus thuringiensis strain S184 by PCR was cloned into pGEM-T Easy vector and its sequence was analysized by DNASTAR. The plasmid pOTP was constructed by inserting vip3A-S184 gene into the expression vector pQE30 and then was transformed into E. coli M15. E. coli M15 cells harbouring the plasmid pOTP were induced with 1 mmol/L IPTG to express 89 kD protein which was confirmed to be Vip3A-S184 by Western blot. Experiments showed that about 19% of Vip3A-S184 proteins were soluble, and others were insoluble proteins and formed inclusion bodies observed by transmission electron microscopy(TEM). The target protein was purified under the native condition and the polyclonal antibody was prepared by immunizing rabbits. The polyclonal antibody was used to detect Vip3A proteins expressed in Bacillus thuringiensis. Bioassay showed that Vip3A-S184 showed a high toxicity against 3 tested insect larvae including Spodoptera exigua, Spodoptera litura and Helicoverpa armigera.

[Bacillus thuringiensis helper protein P20 affects the formation of Cry1Ab].

The Cry1Ab differs most significantly from the other related ICPs by its absence of a carboxyl terminus of 28 amino acids including four cysteines; consequently it is less stable. We report that the helper protein P20 plays a role in the expression and crystallization of Cry1Ab. Three Cry1Ab expression plasmids pT1B, pP1B, and pDP1B, were constructed based on the shuttle vector pHT3101. The vector pT1B does not contain the p20 gene, pP1B carries p20, and pDP1B contains p20 with cry1A(c) promoter. Transformants were obtained by electroporating the plasmids into Bacillus thuringiensis acrystalliferous mutant CryB. Western blot demonstrated that crylAb was expressed as a 130 kD protein in all the transformants, and some of the protein was partially degraded into a 60 kD peptide. Quantitative protein analysis indicated that the amount of the 130 kD protein varied in the transformants and was in the ratio of 1:1.4:1.5 for PT1B, pP1B and pDP1B respectively. For the 60 kD proteins, the ratio was 1:1.1:1.6. Microscopic examination revealed that the size of the typical pyramidal crystals in the three transformants was in the order of T1B < P1B < DP1B. Bioassay showed that T1B, P1B and DP1B were all toxic to the larvae of Helicoverpa armigera with similar LC50. This study suggested that P20 plays a role in the expression and crystallization of Cry1Ab.