Objective: By investigating the genotype and evolutionary variation of hantavirus (HV) in Tiantai county, a national surveillance site for hemorrhagic fever with renal syndrome (HFRS) was set in Zhejiang province, from 2011 to 2018, to reveal the molecular epidemiological characteristics of hantavirus (HV) in Tiantai. Methods: Total RNA was extracted from ultrasound treated HV antigen- positive rat lung samples in Tiantai from 2011 to 2018. After cDNA was prepared, nested PCR was used to amplify partial sequence of M fragments by using specific primers of HV. The sequences of HV in Tiantai from 2011 to 2018 were compared with other known HV sequences in order to identify the genotype and analyze the evolution and variation of the virus. Results: In 67 HV antigen-positive lung specimens, 31 were positive in nested PCR amplification with type-specific primers, including 30 Hantaan virus (HTNV) positive samples, 1 Seoul virus (SEOV) positive sample, and all the 31 samples were from Apodemus agrarius. The phylogenetic tree based on partial M segment was divided into monophyletic group, 30 strains were distributed in HTNV group and 1 was in SEOV group. The HTNV strain Tiantai T2018-130 was independently in one branch, sharing 84.8%-87.9% homology with other strains both at home and abroad, including 29 strains in HTNV group in Tiantai. The other 29 HTNV strains in Tiantai showed closer relationship. The SEOV strain T2016-31 from Apodemus agrarius showed closer relationship with previous strains of SEOV, Tiantai ZT71, ZT10 and Z37 strains of Wenzhou, Zhejiang province. Conclusions: HTNV, the main genotype of HV in Tiantai of Zhejiang province, showed obvious geographic clustering, but the strain T2018-130 was distinct from the others in Tiantai. Meanwhile, by sequence analysis, we confirmed that The SEOV strain T2016-31 existed in in Apodemus agrarius, indicating there was a phenomenon of "spillover" between virus and host in SEOV evolution.目的： 研究2011－2018年肾综合征出血热（HFRS）国家监测点浙江省天台县汉坦病毒（HV）的基因型别和进化变异情况，了解天台县HV分子流行病学特点。 方法： 将天台县2011－2018年HV抗原阳性的鼠肺标本超声后提取核酸，利用HV型特异性引物，应用巢式PCR对部分M片段进行扩增分型和序列测定，将天台县2011－2018年的HV序列与国内外其他已知的HV序列进行比较，以明确该地区的基因型别，分析病毒的进化变异情况。 结果： HV抗原阳性的67份鼠肺标本经型特异性引物巢式PCR扩增后31份标本为阳性，其中30份为汉滩病毒（HTNV）、1份为汉城病毒（SEOV），31份PCR阳性鼠肺均来自黑线姬鼠。31份巢式PCR阳性产物的部分M片段核苷酸序列有30株分布在HTNV单元群，1株分布在SEOV单元群。HTNV单元群中的天台T2018-130株与天台其余29株及国内外其他株同源性为84.8%~87.9%，差异较大，天台其余29株亲缘关系较近；SEOV发生群中的T2016-31株来自黑线姬鼠的鼠肺标本，与SEOV天台以往分离株ZT71株、ZT10株和浙江温州分离株Z37株在系统发生树上分布于同一或临近分支。 结论： 浙江省天台县HV流行的主要型别HTNV基因表现出明显的地理聚集现象，但也存在着基因差异较大的变异株T2018-130株；同时从病毒基因序列分析证实SEOV T2016-31株存在于黑线姬鼠中，可能意味着在SEOV进化过程中病毒在宿主间发生了"溢出"现象。.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that caused a global outbreak of coronavirus disease 2019 (COVID-19) pandemic. To elucidate the mechanism of SARS-CoV-2 replication and immunogenicity, we performed a comparative transcriptome profile of mRNA and long non-coding RNAs (lncRNAs) in human lung epithelial cells infected with the SARS-CoV-2 wild-type strain (8X) and the variant with a 12-bp deletion in the E gene (F8). In total, 3,966 differentially expressed genes (DEGs) and 110 differentially expressed lncRNA (DE-lncRNA) candidates were identified. Of these, 94 DEGs and 32 DE-lncRNAs were found between samples infected with F8 and 8X. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyzes revealed that pathways such as the TNF signaling pathway and viral protein interaction with cytokine and cytokine receptor were involved. Furthermore, we constructed a lncRNA-protein-coding gene co-expression interaction network. The KEGG analysis of the co-expressed genes showed that these differentially expressed lncRNAs were enriched in pathways related to the immune response, which might explain the different replication and immunogenicity properties of the 8X and F8 strains. These results provide a useful resource for studying the pathogenesis of SARS-CoV-2 variants.
The discovery of new anticoccidial drug targets is amongst the necessary efforts needed to control chicken coccidiosis caused by Eimeria species.In this study, the fragment coding for the putative Eimeria tenella glycogen synthase kinase-3 (GSK-3) was amplified from the cDNA of E. tenella.Homology search showed that generated E. tenella GSK-3 sequence has high similarities with GSK-3 sequences from other organisms.The conserved domains of GSK-3 and residues important for the GSK-3 activity were also predicted within the E. tenella GSK-3.Secondary structure analysis and homology modelling predicted that the protein structure is divided into a beta strand domain at the N terminal and an alpha helix domain at terminal C, which are characteristics of GSK-3 enzymes.These results supported the E. tenella GSK-3 codes for the GSK-3 protein in E. tenella.Although the degree of conservation is high, significant differences were observed between GSK-3 of E. tenella and its host.The Ser 9 residue reported to be important for the inhibition of the GSK-3 activity was not conserved within the E. tenella GSK-3.Considering that Ser 9 is a phosphorylation site in GSK-3β of vertebrates, the absence of this residue in the E. tenella GSK-3 sequence suggests that the regulation of E. tenella GSK-3 involves a different phosphorylation site and mechanism.Phylogenetic analysis suggests that E. tenella GSK-3 has a closer relationship to plant Superposition analysis between E. tenella GSK-3 and Homo sapiens GSK-3β predicted that E. tenella GSK-3 is able to interact with a GSK-3 inhibitor.Taken together, these results suggested that the E. tenella GSK-3 has the potential to be developed into an anticoccidial drug target.
Cermet coatings deposited using high-velocity oxy-fuel (HVOF) are widely used due to their excellent wear and corrosion resistance. The new agglomeration-rapid sintering method is an excellent candidate for the preparation of WC-Co-Cr feedstock powders. In this study, four different WC-10Co-4Cr feedstock powders containing WC particles of different sizes were prepared by the new agglomeration-rapid sintering method and deposited on steel substrates using the HVOF technique. The microstructures and mechanical properties of the coatings were investigated using scanning electron microscopy, X-ray diffraction, nanoindentation, and Vickers indentation. The through-thickness residual stress profiles of the coatings and substrate materials were determined using neutron diffraction. We found that the microstructures and mechanical properties of the coatings were strongly dependent on the WC particle size. Decarburization and anisotropic mechanical behaviors were exhibited in the coatings, especially in the nanostructured coating. The coatings containing nano- and medium-sized WC particles were dense and uniform, with a high Young's modulus and hardness and the highest fracture toughness among the four coatings. As the WC particle size increased, the compressive stress in the coating increased considerably. Knowledge of these relationships enables the optimization of feedstock powder design to achieve superior mechanical performance of coatings in the future.
Objective To establish a TaqMan based real-time reverse transeription-polymerase chain reaction (RT-PCR) assay for the detection of Japanese encephalitis virus. Methods The gene sequences of Japanese encephalitis virus downloaded from the GenBank was aligned, using the biologic software. Specific primers and probes were designed in the conserved region of the C gene for Japanese encephalitis virus. The real-time RT-PCR reactive condition was optimized and the sensitivity, specificity and the stability of the assay were evaluated. Mosquitoes collected from Zhejiang province were detected by this assay. Results Mg2+, primer and probe were optimized at 5 mmol/L, 0.2 μmol/L and 0.1 μmol/L respectively. The specificity of the assay was high and there were no cross reactions with dengue virus, rabies virus, seoul virus or hantan virus. The detection limits of the assay was 0.1 TCID50. Results from preliminary application showed that TaqMan RT-PCR for Japanese encephalitis virus was sensitive, easier and faster to perform the process of traditional virus isolation and identification. It took only three hours to extract viral RNA and perform the real-time RT-PCR. Conclusion This TaqMan-based one-step RT-PCR assay was a quick, sensitive and specific tool for molecular diagnosis of Japanese encephalitis virus.
Key words:
Japanese encephalitis virus; Real-time reverse transcription-polymerase chain reaction; Detection
Objective: To explore the genetic characteristics and evolution of hantavirus carried by rodents in port area of Ningde in Fujian province in the summer of 2020. Methods: Rodents were captured in the port area of Ningde, the RNA was extracted from rodent lung tissues and detected by using specific kit. The positive samples were used for whole-genome sequencing of the virus. Bioinformatics software was used for the analysis on the similarity and genetic variation of the sequences. Results: A total of 112 rodents were captured, including 5 Rattus norvegicus and 2 Rattus flavipectus, the positive rate of hantavirus was 6.25% (7/112). By virus gene sequencing, two hantavirus complete genome sequences were obtained (named as FJ35 and FJ36, GenBank accession numbers: MW449188-MW449193). The genetic analysis results showed that the hantavirus detected in positive samples were SEOV and shared 99% nucleotide similarity with hantavirus strains LZSF21 and JX20140581 isolated from Shandong province. Phylogenetic analysis using the maximum likelihood method showed that the hantavirus detected in positive samples belonged to S3 subtype, sharing the same subtype with hantavirus strains Z37 from Zhejiang province, LZSF21 from Shandong province, and zy27 and Gongzhuling 415 from northeastern China. Compared with FJ372, the amino acid variation of N259S was observed at sites 251-264 of nucleoprotein, which might be related to antigenicity. Another variation of Q81R was observed in glycoprotein compared with SEOV 80-39 segment of coded amino acid of international reference strain, which might also cause the change in antigenicity. Conclusion: The high positive rate of hantavirus in rodents in the port area of Ningde- would increase the risk of natural human infection and epidemic in local area. The hantavirus positive rodents in this focus might be from an endemic area in Shandong. It is necessary to strengthen the imported rodent control in the port area of Ningde. The virus detected in 2 positive samples belonged to SEOV subtype Ⅲ and shared high homologies of nucleotides and amino acid sequences with the hantavirus strains in surrounding area. However, some slight variations occurred in glycoprotein and nucleoprotein amino acid sequences, which might cause changes in its antigeniity.目的： 分析2020年夏季福建省宁德港地区（宁德港）鼠形动物携带汉坦病毒基因特征及遗传进化关系。 方法： 夹夜法采集宁德地区鼠类，提取鼠形动物肺组织RNA，用汉坦病毒检测试剂盒对样本进行检测，阳性样本进行全基因组测序，对序列进行相似性、遗传与变异等生物信息学分析。 结果： 共捕获鼠形动物112只，汉坦病毒阳性率为6.25%（7/112），其中包括褐家鼠5只，黄胸鼠2只；对阳性样本测序，获得2条汉坦病毒的基因全序列（分别命名为：FJ35和FJ36，GenBank登录号：MW449188~MW449193），全基因组序列均来自雄性褐家鼠；序列比对发现该样本所携带病毒属于汉坦病毒基因型汉城病毒（SEOV），与山东省汉坦病毒分离株JX20140581和LZSF21之间核苷酸相似性较高为99%；采用最大似然法构建系统发育树发现该阳性样本所携带病毒属于S3亚型，与浙江省、山东省以及东北地区汉坦病毒分离株Z37、LZSF21和zy27、Gongzhuling415等属于同一亚型；氨基酸序列分析发现，与国际标准株SEOV80-39 M片段编码的氨基酸相比，FJ35和FJ36第81位谷氨酰胺（Q）变为精氨酸（R）；与FJ372核蛋白氨基酸相比发现N259S变异。 结论： 2020年夏季宁德港鼠形动物汉坦病毒阳性率较高，存在人群自然感染和流行的风险；形成该疫源地的病毒来自山东省某疫区的可能性比较大，需要做好港口鼠形动物的输入性防控；2株阳性样本所携带病毒属于S3亚型，与周围地区病毒株核苷酸及氨基酸同源性较高，但糖蛋白及核蛋白氨基酸存在一些微小的变异，可能引起相关抗原性的改变。.
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