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Objective To investigate the epidemiological characteristics of dengue fever in Shenz-hen city in 2014 and to analyze the evolutional characteristics of the epidemic dengue virus(DENV)strains in order to provide scientific guidelines for the prevention and control of dengue fever. Methods Descrip-tive epidemiological analysis was used to analyze the prevalence of dengue fever in Shenzhen city in 2014. Immunochromatography and real-time PCR were performed to detect the specific antibodies(IgM and IgG) and DENV nucleic acids in serum samples collected from suspected cases of dengue fever. Serum samples collected from the patients at early stage of dengue fever were used to infect the C6 / 36 cell line for further isolation of DENV strains. The types of isolated DENV strains were determined by using real-time PCR. E genes of the isolated DENV strains were amplified by RT-PCR and then sequenced. DNAStar and Clustslx (1. 83)softwares were used to analyze the homology between DENV strains isolated in Shenzhen and other areas. A phylogenetic tree based on the sequences of E genes of Shenzhen strains and other sequences of DENV reference strains downloaded from GenBank was constructed for further analysis. Results A total of 454 cases of dengue fever were reported in Shenzhen in 2014 with a male to female ratio of 1. 43 ∶ 1. Local patients accounted for 76. 21% and the rest 23. 79% were imported cases mainly from Southeast Asian and surrounding cities. There were 441 cases reported from September to November,accounting for 97. 14% of all reported cases. Most of the infected subjects were aged 20 to 50,accounting for 76. 73% . Of the 270 samples positive for DENV nucleic acids,strains of DENV-1,DENV-2,DENV-3 and DENV-4 accounted for 87. 41% ,8. 89% ,0. 37% and 2. 22% ,respectively. The phylogenetic tree analysis revealed that the DENV-1 strains belonged to two genotypes,which were genotypeⅠ and genotype Ⅴ. The DENV strains of genotypeⅠ were highly similar to the epidemic strain isolated in Shenzhen in 2010 and the genotype Ⅴstrains were first reported in Shenzhen. The homology analysis of the nucleotides of E genes showed that mi-nor differences in the nucleotide sequences were found between DENV-2 strains. All of the DENV-2 strains belonged to the genotype Ⅳ as indicated by the phylogenic tree. Conclusion There were 454 cases of den-gue fever(including both local and imported cases)reported in Shenzhen city in 2014,reaching an all-time high. DENV-1 was the predominant pathogen in combination with an increased infection of DENV-2. This study indicated that the prevalent DENV strains might be imported from Southeast countries and neighboring cities. Further researches should be conducted to analyze whether dengue fever is endemic in Shenzhen City.
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Objective To analyze the genetic characteristics and molecular variation of human rhi-novirus strains isolated in Shenzhen.Methods RNA samples were extracted from nasopharyngeal swab samples collected from influenza-like subjects in Shenzhen and analyzed by fluorescent RT-PCR.The VP4-VP2 and VP1 gene regions of human rhinovirus strains were amplified by nested RT-PCR.Clustal W and MEGA programs were used to evaluate molecular variation of the human rhinovirus strains.Results Both human rhinovirus A and B were prevalent in Shenzhen during 2012.Human rhinoviruses A was the predomi-nant pathogen, including subtypes A47, A31, A90, A18 and so on.Two recombinant strains of human rhi-noviruses A47 and A31 were detected.The mutations scattered on the VP1 protein and varied in different subtypes.The receptor binding sites ( loop BC, DE and HI) in different subtypes showed polymorphism. Five out of twenty-five drug sensitivity sites ( I121V, L123M, V167I, Y189H and H259G) showed muta-tion.Conclusion Multiple subtypes of human rhinovirus were prevalent in Shenzhen and were in a state of constant recombination and variation.
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Objective To study the epidemic pattern and molecular variation of respiratory syncy-tial virus ( RSV) strains isolated in Shenzhen from year 2012 to 2013.Methods The clinical samples iso-lated from patients with influenza-like illness were analyzed by fluorescent RT-PCR to screen RSV positive strains.The C-terminal variable regions of genes encoding G proteins were amplified by nested RT-PCR. Molecular variation was analyzed by using Clustal W and MEGA softwares.Results RSV strains were wide-ly prevalent in Shenzhen from 2012 to 2013.Two epidemic peaks usually occurred in spring and summer/au-tumn of each year.The RSV isolates were subtyped into group A belonging to genotype NA1 and group B be-longing to genotype BAⅨ.Most of the mutations scattered at the C-terminal region of G protein.A few mu-tations caused the disappearance of certain glycosylation sites.A novel recombinant virus strain containing 24 inserted amino acids was identified in 2013, which was likely to be introduced into our country from abroad. Conclusion RSV strains were widely and continuously prevalent in Shenzhen, characterized by constant evolution and variation.
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Objective To determine the pathogen of a local dengue fever outbreak in Shenzhen city in 2010,and to analyze the molecular characteristics of the epidemic dengue virus strain as well as explore the possible origin.Methods The serum samples collected from the suspect dengue fever cases were detected for IgM, IgG by enzyme-linked immunosorbent assay ( ELISA ),immunochromatography and dengue virus nucleic acid by real-time polymerase chain reaction (PCR).Serum samples from patients with early stage dengue fever were used to isolate virus with C6/36 and BHK-21 cell lines.The type of isolated virus strain was determined by RT-semi-nested-PCR and realtime PCR.E gene of isolated virus strain was amplified by RT-PCR and sequenced.Homology and phylogenetic tree of E gene of Shenzhen dengue virus with the strains isolated from other areas were constructed.Results IgM,IgG and RNA of type 1 dengue virus were detected in serum samples from dengue fever suspected patients.Type 1 dengue virus named DEV1-SZ1029 was successfully isolated from the serum sample.The homology of nucleotide sequence of E gene of SZ1029 strain with standard type 1 dengue virus HAWAII 45,Fj231/04,GD14/97 and GD05/99 were 94.8％,99.6％,97.7％ and 98.5 ％,respectively.The phylogenetic tree indicated that SZ1029 had the greatest similarity with the D1/Malaysia/36000/05 strain,SG(EHI)DED142808 strain and Fj231/04 strain and they lied in the same branch of the phylogenetic tree.The isolated dengue virus type 1 belonged to genetype Ⅰ with GZ/80,Taiwan87,All patients lived in a certain construction site in Shenzhen and had no recent travel history outside the area in one month before infection.Conclusions The virological,serological and molecular features all identify that the local dengue fever outbreak in Shenzhen in 2010 is caused by type 1 dengue virus and SZ1029 strain may be transferred from Southeast Asian region,and there may be a plague focus in Shenzhen.
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Objective To detect infection of Angiostrongylus cantonensis and examine effection of treatment to prepare monoclonal antibodies(McAbs). Methods Six-week-old BALB/c mice were imrnunized by the intraperitoneal injection of e/s antigens of Angiostrongylus cantonensis. Fusion of splecn cells from immunized mice with prepared SP2/0-Ag14 myeloma cells was performed in RPMI 1640. Fused cells were suspended in RPMI 1640 containing 1% HAT and 20% fetal calf serum and dispensed into 96-well cell culture plates. The supernatants of clones were screened by ELISA with sera of patients of angiostrongyliasis.Distribution of cohere antigen of 12D5 and 21B7 monoclonal antibodies was analyzed with immunohistochemistry. Two McAbs ( 12D5 and 21B7) were applied to detect the circulating antigen (CAg) in the sera of rats infected with A. cantonensis and angiostrongyliasis patients respectively by double antibody sandwich ELISA.Results 12D5 McAb was identified as IgG1 and 21 B7 McAb was IgM. Western blot result showed two McAbs could used to identified 55 × 103 protein of adult worms of A. cantonensis. Cohere antigen of 12D5 and 21B7 monoclonal antibodies were distributed on intestine surface of A. cantonensis. The detection rates of CAg in the sera of infected rats 100% (48/48), the detection rates of CAg in the sera of angiostrongyliasis patients was 100% (32/32). No cross-reaction to sera of patients with other infection of parasites, such as clonochiasis, fasiolopsiasis, ancylostomiasis, trichinosis, anisakiasis as well as schsitosomiasis, and health srea did not reacted with 12D5 and 21B7 McAbs,and detaction rate of antibody of angiostrongyliasis patients only reached 75% (24/32) with antigen of A. cantonensis. Conclusion Cohere antigen of 12D5 and 21B7monoclonal antibodies were antigens of enteric epithelium. Sandwich ELISA with 12D5 and 21B7 McAbs showed high specificity act as detecting CAg of A. cantonensis in sera of infection animal and patients. It is apparent that Sandwich ELISA with 12D5 and 21 B7 is not only rapid and simple without requirement of special instrument, but also rather sensitive and specific for the detection of current infection with A. cantonensis.
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To identify the gene differentially expressed in female Anopheles anthropophagus and to analyze its gene sequence, this gene amplified by PCR was identified by real-time PCR and its cDNA was then amplified with rapid amplification of cDNA ends (RACE) technology. It was found that the expression ratio of the female differentially expressed gene in female and male mosquitoes was 267.49 according to the formula F=2~(-⊿⊿CT).The size of mRNA of the gene was 364 bp, and the amino acid sequence deduced from the open reading frame (ORF) was found to be similar to the sequence of tectin protein of Culex quinquefasciatus and proteins of other species. The mRNA sequence of this gene was submitted to NCBI with a accession number of FJ907236. This gene may provide a foundation for further studies on the biological functions of mosquitoes.
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The immune effect of two recombinant protein fragments of spike protein in severe acute respiratory syndrome coronavirus (SARS CoV) was investigated in Balb/c mice. Two partial spike gene fragments S1 (322-1464 bp) and S2 (2170-2814 bp) of SARS coronavirus were amplified by RT-PCR, and cloned into pET-23a prokaryotic expression vector, then transformed into competent Escherichia E.coli BL21 (DE3)(pLysS) respectively. Recombinant proteins were expressed and purified by Ni2+ immobilized metal ion affinity chromatography. The purified proteins mixed with complete Freund adjuvant were injected into Balb/c mice three times at a two-week interval. High titer antibody was detected in the serum of immunized Balb/c mice, and mice immunized with S1 protein produced high titer IgG1, IgG2a, IgG2b and IgG3, while those immunized with S2 protein produced high titer IgG1, IgG2a, but lower titer IgG2b and IgG3. Serum IFN-γ concentration was increased significantly but the concentrations of IL-2, IL-4 and IL-10 had no significant change. And a marked increase was observed in the number of spleen CD8+ T cells. The results showed that recombinant proteins of SARS coronavirns spike protein induced hormonal and cellular immune response in Balb/c mice.
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The immune effect of two recombinant protein fragments of spike protein in severe acute respiratory syndrome coronavirus (SARS CoV) was investigated in Balb/c mice. Two partial spike gene fragments S1 (322 1464 bp) and S2 (2170 2814 bp) of SARS coronavirus were amplified by RT-PCR, and cloned into pET-23a prokaryotic expression vector, then transformed into competent Escherichia E. coli BL21 (DE3)(pLysS) respectively. Recombinant proteins were expressed and purified by Ni2+ immobilized metal ion affinity chromatography. The purified proteins mixed with complete Freund adjuvant were injected into Balb/c mice three times at a two-week interval. High titer antibody was detected in the serum of immunized Balb/c mice, and mice immunized with S1 protein produced high titer IgG1, IgG2a, IgG2b and IgG3, while those immunized with S2 protein produced high titer IgG1, IgG2a, but lower titer IgG2b and IgG3. Serum IFN-concentration was increased significantly but the concentrations of Il-2, IL-4 and IL-10 had no significant change. And a marked increase was observed in the number of spleen CD8+ T cells. The results showed that recombinant proteins of SARS coronavirus spike protein induced hormonal and cellular immune response in Balb/c mice.
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Objective Rab11 GTPases play an essential role in regulating membrane trafficking pathways in eukaryotic cells. Nonetheless, there has been little work done on characterizing the transport machinery of Trichomonas. The aim of this study is to clone and characterize a Rab11 gene of Trichomonas vaginalis.Methods A cDNA expression library was constructed with T. vaginalis total RNA. A cDNA clone, which showed a high degree of homology with Rab proteins of different species, was isolated and sequenced. Sequence analysis was performed using BLASTP, RPS-BLAST and ClustalW programs. The genomic DNA corresponding to the cDNA sequence was amplified using PCR techniques and following by sequencing. Results cDNA with a length of 710 base pairs and an open reading frame of 636 bp was obtained. The deduced amino acid sequence from the open reading frame was found to possess 211 residuals. Sequence analysis demonstrated that this cDNA clone was homologous to the Rab11 subfamily of different species (60% identity and 79% similarity with Arabidopsis thaliana Rab11c, 58% identity and 78% similarity with human Rab11b), and that the amino acid sequence contains all the well known conserved sequence elements of Rab family. Specific Rab motifs were also detected in the deduced amino acid sequence. Phylogenetic analysis showed that its closest homologues are Rab11 proteins from other species. Sequencing of the PCR product of genomic DNA revealed that the genomic DNA sequence encompassing the putative 5'-ATG and 3'-stop codon is identical to the cDNA sequence.Conclusion A cDNA clone corresponding to the T. vaginalis Rab11 gene was obtained.The function of this gene in regulating membrane trafficking pathways of the parasitic protist is still under investigation.
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Objective To construct a prokaryotic expression system containing the dense granule protein 4(GRA4) of Toxoplasma gondii,purify the expressed protein and detect its immunogenicity.Methods The specific fragment of GRA4 gene was amplified by PCR.After subcloning the prokaryotic expression recombinant pET,GRA4,the expressed product was purified with His?BindTM affinity chromatography and analyzed by Western blot.BALB/c mice were immunized with the GRA4 recombinant protein,and the antibody IgG titer was detected by ELISA.Results The pET,GRA4 prokaryotic expression system was obtained.The MW of the expressed protein was Mr 40 000 and formed in inclusion body.After purification,the recombinant protein could be specifically recognized by the T.gondii infected rabbit serum.Mice immunized with the purified recombinant protein elicited high titer of IgG antibody.Conclusion The pET,GRA4 recombinant protein was successfully expressed and purified,which shows the immunogenicity.