Epidemiological and molecular characteristics of emergent dengue virus in Yunnan Province near the China-Myanmar-Laos border, 2013-2015.

BACKGROUND: Yunnan Province is located in southwestern China and neighbors the Southeast Asian countries, all of which are dengue-endemic areas. In 2000-2013, sporadic imported cases of dengue fever (DF) were reported almost annually in Yunnan Province. During 2013-2015, we confirmed that a large-scale indigenous DF outbreak emerged in cities of Yunnan Province near the China-Myanmar-Laos border. METHODS: Epidemiological characteristics of DF in Yunnan Province during 2013-2015 were evaluated by retrospective analysis. A total of 232 dengue virus (DENV)-positive sera were randomly collected for sequence analysis of the capsid/premembrane region of DENV from patients with DF in Yunnan Province. The envelope gene of DENV isolates was also amplified and sequenced. Phylogenetic analyses were performed using the neighbor-joining method with the Tajima-Nei model. RESULTS: Phylogenetically, all DENV-positive samples could be classified into DENV-1 genotype I and DENV-2 Asian I genotype during 2013-2015 and DENV-4 genotype I in 2015 from Ruili City; and DENV-3 genotype II in 2013 and DENV-2 Cosmopolitan genotype in 2015 from Xishuangbanna Prefecture. CONCLUSIONS: Our results indicated that imported DF from patients from Laos and Myanmar was the primary cause of the DF epidemic in Yunnan Province. Additionally, DENV strains of all four serotypes were identified in indigenous cases in Yunnan Province during the same time period, while the dengue epidemic pattern observed in southwestern Yunnan showed characteristics of a hypoendemic nature: circulation of DENV-1 and DENV-2 over consecutive years.

[The current situation and strategies of snake antivenomimmunoglobulins research and development].

Snake antivenomimmunoglobulins are considered to be the most efficient drugs in snake envenomings. Most snake antivenomimmunoglobulins all over the world are still prepared by fragmentation of polyclonal antibodies isolated from hyper-immunized horse serum till now. In this review, we retrospect the history of snake antivenomimmunoglobulins, analysis the present situation and pay the close attention on the key technological links in the process of research and manufacturing, such as properties of IgG and its fragments, selection and preparation of immunogen, optimization of immunization schedule and protein isolation and purification, which can be available for the reference in the research and development of snake antivenom.

Larvicidal activity of essential extract of Rosmarinus officinalis against Culex quinquefasciatus.

Constituents in rosemary (Rosmarinus officinalis) have been shown to have larvicidal activity against invertebrates. In order to explore the properties of crude extract of rosemary further, we studied the chemical composition and its activity against dichlorodiphenyltrichloroethane (DDT)-susceptible, DDT-resistant, and field strains of Culex quinquefasciatus larvae. The major components of R. officinalis were found to be eucalyptol and camphor, with relative percentages of 10.93% and 5.51%, respectively. Minor constituents included limonene, (+)-4-carene, isoborneol, 1-methyl-4-(1-methylethylidene)-cyclohexene, and pinene. The median lethal concentration (LC50) values of the essential oil of R. officinalis against DDT-susceptible, DDT-resistant, and field strains of larvae of Cx. quinquefasciatus were 30.6, 26.4, and 38.3 mg/liter, respectively. The single median lethal dose (LD50) in Kunming mice was 4752 mg/kg. Essential oils from R. officinalis may, therefore, provide an effective natural plant product for use in mosquito prevention and control.

[Genetic evolution analysis of matrix protein 2 gene of avian influenza H5N1 viruses from boundary of Yunnan province].

OBJECTIVE: To elucidate the variation in characterizations and genetic evolution of the matrix protein 2 or ion channel protein(M2) genes of avian influenza subtype H5N1 viruses in the boundary region of Yunnan province from 2008 to 2012. METHODS: A total of swab samples were collected from foreign poultry such as the junction between Yunnan and Vietnam, Laos,myanmar and wild birds in boundary region of Yunnan province from 2008 to 2012 and screened by H5N1 subtype-specific multiplex RT-PCR. The M genes of H5N1 virus from the positive samples were amplified by RT-PCR and cloned into pMD18-T vectors for sequencing. The alignment and phylogenetic analysis of M2 genes were performed with sequences of the known reference strains. RESULTS: A total of 71 positive samples were found out of 1240 samples and the positive rate was 5.72%. A total of 14 different M2 sequences were obtained from 30 positive samples and were divided into 3 distinct clades or sub-clades(1.2.1, 1.2.2 and 2) by phylogenetic analysis, 5, 7 and 2, respectively. The M2 genes and Hemagglutinin(HA) genes of H5N1 viruses from the boundary region of Yunnan province had showed different relationship of genetic evolution. The substitution or mutation of key amino acids sites had been found among the domains of epitope, adamantane-resistance, and poultry or human original viral strains. CONCLUSION: The M2 genes of H5N1 subtype viruses in boundary region of Yunnan province from 2008 to 2012 showed genetic divergence and the virus of clade 1.2.2 had become dominant epidemic strain in this region.

[Primary clinical result of digital template as navigation to supper cervical pedicle instrumentation].

OBJECTIVE: To observe the primary clinical result of digital template as navigation to the upper cervical pedicle instrumentation. METHODS: CT scan of upper cervical vertebrae was performed. 3-D model of upper cervical vertebrae was reconstructed by software Amira 3.1 and was preserved in STL format. Then 3-D model was run in software UG Imageware 12.0, the best pedicle channel was extracted according to the reverse engineering principle. A virtual navigational template was established according to he lamina anatomic trait, and the best pedicle channel. The virtual vertebrae and navigational template were manufactured using rapid prototyping. The navigational template was sterilized and used intra operative to assist with the placement of pedicle screw. The Accuracy of screw placement was confirmed with postoperative X-ray and CT scanning. RESULTS: The digital navigational template had been established and used in the 3 cases, the good trajectory of cervical pedicle had been showed by the CT scan of post operation. There were not complications of related pedicle screw insertion. CONCLUSIONS: A novel method of upper cervical pedicle location using Reverse Engineering and rapid prototyping has been developed; the navigational template is found to be highly accuracy and has great expectation.

Effects of rhBMP-2 gene transfection to periodontal ligament cells on osteogenesis.

The present study aims to investigate the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the osteogenesis of periodontal ligament (PDL) cells. The expression vector of rhBMP-2 (pcDNA3.1-rhBMP-2) was established. PDL cells were obtained through the enzymatic digestion and tissue explant methods and verified by immunohistochemistry. Cells were classified into experimental (cells transfected with pcDNA3.1/rhBMP-2-EGFP), blank (cells with no transfection) and control group (cells transfected with empty plasmid). rhBMP-2 expression was assessed via Western blotting analysis. The mineralization ability, alkaline phosphatase (ALP) activity and level of related osteogenic biomarkers were detected to evaluate the osteogenic characteristics of PDL cells. The rhBMP-2 expression vector (pcDNA3.1-rhBMP-2) was successfully established. Primary PDL cells displayed a star or long, spindle shape. The cultured cells were long, spindle-shaped, had a plump cell body and homogeneous cytoplasm and the ellipse nucleus contained two or three nucleoli. Cells displayed a radial, sheaf-like or eddy-like arrangement after adherence growth. Immunohistochemical staining confirmed that cells originated from mesenchymal opposed to epithelium. The experimental group exhibited an enhanced mineralization ability, higher ALP activity and increased expression of rhBMP-2 and osteogenic biomarkers (Runx2, collagen type I and osteocalcin) than the blank and control group. The present study demonstrated that rhBMP-2 transfection enhances the osteogenesis of PDL cells and provides a possibility for the application of rhBMP-2 expression products in dental disease treatment.

IL-7 suppresses osteogenic differentiation of periodontal ligament stem cells through inactivation of mitogen-activated protein kinase pathway.

Periodontal ligament stem cells (PDLSCs) are tissue-specific mesenchymal stem cells (MSCs), having an important role in regenerative therapy for teeth loss. Interleukin-7 (IL-7) is a key cytokine produced by stromal cells including MSCs, and exhibits specific roles for B and T cell development and osteoblasts differentiation of multiple myeloma. However, the effect of IL-7 on osteogenic differentiation of PDLSCs remains unclear. Therefore, in the present study we determined whether IL-7 affects the proliferation and osteogenic differentiation of PDLSCs in vitro and explored the associated signaling pathways for IL-7-mediated cell differentiation. The results demonstrated that the isolated human PDLSCs possessed MSCs features, highly expressing CD90, CD44, CD105, CD29 and CD73, and almost did not expressed CD34, CD45, CD11b, CD14 and CD117. IL-7 could not significantly affect the proliferation of PDLSCs, but it decreased their osteogenic differentiation and inhibited alkaline phosphatase (ALP) activity. The results of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting exhibited that the expression levels of Runx-2, SP7 and osteocalcin (OCN) were significantly reduced by IL-7. Further studies indicated that IL-7 did not significantly change JNK, ERK1/2 and p38 protein production, but markedly suppressed their phosphorylation levels. These data suggest that IL-7 inhibits the osteogenic differentiation of PDLSCs probably via inactivation of mitogen-activated protein kinase (MAPK) signaling pathway.

Tormentic acid inhibits LPS-induced inflammatory response in human gingival fibroblasts via inhibition of TLR4-mediated NF-κB and MAPK signalling pathway.

OBJECTIVE: Periodontal disease is one of the most prevalent oral diseases, which is associated with inflammation of the tooth-supporting tissues. Tormentic acid (TA), a triterpene isolated from Rosa rugosa, has been reported to exert anti-inflammatory effects. The aim of this study was to investigate the anti-inflammatory effects of TA on lipopolysaccharide (LPS)-stimulated human gingival fibroblasts (HGFs). METHODS: The levels of inflammatory cytokines such as interleukin (IL)-6 and chemokines such as IL-8 were detected by enzyme-linked immunosorbent assay (ELISA). The expression of Toll-like receptor 4 (TLR4), nuclear factor kappa B (NF-κB), IκBα, p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) was determined by Western blotting. RESULTS: The results showed that Porphyromonas gingivalis LPS significantly upregulated the expression of IL-6 and IL-8. TA inhibited the LPS-induced production of IL-6 and IL-8 in a dose-dependent manner. Furthermore, TA inhibited LPS-induced TLR4 expression; NF-κB activation; IκBα degradation; and phosphorylation of ERK, JNK, and P38. CONCLUSION: TA inhibits the LPS-induced inflammatory response in HGFs by suppressing the TLR4-mediated NF-κB and mitogen-activated protein kinase (MAPK) signalling pathway.

Repeated pancreatitis-induced splenic vein thrombosis leads to intractable gastric variceal bleeding: A case report and review.

Gastric varices (GV) are one of the most common complications for patients with portal hypertension. Currently, histoacryl injection is recommended as the initial treatment for bleeding of GV, and this injection has been confirmed to be highly effective for most patients in many studies. However, this treatment might be ineffective for some types of GV, such as splenic vein thrombosis-related localized portal hypertension (also called left-sided, sinistral, or regional portal hypertension). Herein, we report a case of repeated pancreatitis-induced complete splenic vein thrombosis that led to intractable gastric variceal bleeding, which was treated by splenectomy. We present detailed radiological and pathological data and blood rheology analysis (the splenic artery - after a short gastric vein or stomach vein - gastric coronary vein - portal vein). The pathophysiology can be explained by the abnormal direction of blood flow in this patient. To our knowledge, this is the first reported case for which detailed pathology and blood rheology data are available.

[Research methods in protein-protein and protein-nucleic acid interactions and application in the study of human enterovirus A71].

Methods for analyses of protein-protein interactions include: yeast two hybrid (Y2H), phage dis- play (PD), co-immunoprecipitation (Co-IP), glutathione S-transferase pull-down (GST pull-down), cellular co-localization, far-western blotting, virus overlay protein binding assay (VOPBA), surface plasmon resonance (SPR), and fluorescence resonance energy transfer (FRET). Technologies for the detection of protein-nucleic acid interactions include: yeast one hybrid (Y1H), chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), Southwestern blotting, reporter gene, Co-IP, GST pull-down, and PD. These methods are often used in the study of the human enterovirus A71 (EV-A71) by our research team. Reviews in the Chinese literature in this field are lacking, so we reviewed applications of these methods in the study of EV-A71. This review may impart important knowledge in the research of other viruses with regard to protein-protein and protein-nucleic acid interactions.

Purification, characterization and gene cloning of Da-36, a novel serine protease from Deinagkistrodon acutus venom.

A serine protease termed Da-36 was isolated from crude venom of Deinagkistrodon acutus. The enzyme was a single chain protein with an apparent molecular weight of 36,000 on SDS-PAGE with an isoelectric point of 6.59. Da-36 could clot human plasma by cleaving the Aα, Bß and γ chains of fibrinogen and also exhibited arginine esterase activity. The proteolytic activity of Da-36 toward TAME was strongly inhibited by PMSF and moderately affected by benzamidine and aprotinin, indicating that it was a serine protease. Meanwhile, Da-36 showed stability with wide temperature (20-50 °C) and pH value ranges (pH 6-10). Divalent metal ions of Ca(2+), Mg(2+), and Mn(2+) had no effects but Zn(2+) and Cu(2+) inhibited the arginine esterase activity of Da-36. Total DNA was extracted directly from the lyophilized crude venom and the gene (5.5 kbp) coding for Da-36 had been successfully cloned. Sequence analysis revealed that the Da-36 gene contained five exons and four introns. The mature Da-36 was encoded by four separate exons. The deduced mature amino acid sequence of Da-36 was in good agreement with the determined N-terminal sequence of the purified protein and shared high homology with other serine proteases isolated from different snake venoms. Blast search using amino acid sequence of Da-36 against public database revealed that Da-36 showed a maximal identity of 90% with both Dav-X (Swiss-Prot: Q9I8W9.1) and thrombin-like protein 1 (GenBank: AAW56608.1) from the same snake species, indicating that Da-36 is a novel serine protease.

[Isolation and characterization of rotavirus from bat].

Bats are considered as important animal reservoirs for many pathogenic viruses to humans. The viral metagenomic analysis was performed to study gut and lung tissues of 30 insectivorous bats collected in Yunnan Province and 26 reads were noted to group A rotavirus (RVA). Further RT-PCR screening on bat samples and in vitro viral isolation on cell cultures confirmed the presence of a novel RVA, named as RVA/Bat-tc/MYAS33/2013/G3P[10], in one of 30 Stoliczka's trident bats. The VP7 gene of this strain MYAS33 was closely related to that of an equine RVA strain from Argentina and the nucleotide sequence similarity was 93%, while its VP4 gene was a rare P[10] type and obtained the maximum sequence identity (94.8%) with that of a human strain from Thailand. The present study highlights the potential role of bats as reservoirs for RVAs.

[Genetic evolution of non-structural gene among avian influenza H5N1 viruses isolated from the boundary of Yunnan province].

OBJECTIVE: To elucidate the characteristics of variation and the genetic evolution of non-structural protein (NS1, NS2) genes related to avian influenza subtype H5N1 viruses isolated from the boundary region of Yunnan province. METHODS: Swab samples were collected from foreign poultry and wild birds in the boundary regions of Yunnan province and screened by H5/N1 subtype-specific multiplex RT-PCR. The NS segment of H5N1 virus from the positive samples were amplified by RT-PCR and cloned into pMD18-T vectors for sequencing. The alignment and phylogenetic analysis on those available NS1, NS2 genes were performed with sequences of the known reference strains. RESULTS: 71 positive samples were identified from 1240 samples, with the positive rate as 5.72%. Fourteen different NS segment sequences were obtained from 30 representative positive samples and could be divided into 3 distinct clades or sub-clades (I-1, I-2 and II), by phylogenetic analysis. The NS1/NS2 genes and Hemagglutinin (HA) genes of H5N1 viruses from the boundary regions of Yunnan province showed different relationships regarding the characteristics on genetic evolution. The substitution or mutation of key amino acids sites had been noticed in the nuclear location signal domains, effect domain, and other pathogenicity markers. CONCLUSION: NS genes of H5N1 subtype viruses in boundary region of Yunnan province showed genetic divergence and the virus of clade I-2 and II had become dominant epidemic strains in this region since 2010.

[Genetic diversification of avian influenza H5N1 virus in boundary areas of Yunnan province].

OBJECTIVE: To elucidate the genetic diversifications of avian influenza subtype H5N1 viruses in the boundary regions of Yunnan province during 2009 to July, 2011. METHODS: Swab samples were collected from foreign poultry and wild birds in boundary regions of Yunnan province during 2009 to July, 2011 and tested by H5/N1 subtype-specific multiplex RT-PCR. The HA genes of H5N1 virus from the positive samples were amplified by RT-PCR and cloned into pMD18-T vectors for sequencing. Both alignment and phylogenetic analysis were performed with sequences of the known reference strains. RESULTS: Fifteen different HA sequences were obtained from 36 representative positive samples and could be divided into 2 distinct Clades (2.3.2 and 2.3.4). Through phylogenetic analysis, Clade 2.3.2 and 2.3.4 could then be further divided into 3 (II-1 to II-3) and 2 smaller clades (I-1 and I-2), respectively. The viruses of Clade 2.3.2 II-1 and II-2 were new variant strains of H5N1 virus. The cleavage sites of HA from positive samples all possessed molecular characterization of highly pathogenic avian influenza virus. Mutation of key amino acids had been found among receptor binding sites, potential glycosylation sites, neutralizing epitopes and others. CONCLUSION: It seemed evident that the H5N1 subtype viruses showed genetic diversifications and had undergone the evolution progress of multi-clade (2.3.2, 2.3.4) to single calde (2.3.2) in the boundary regions of Yunnan province, during 2009 to July, 2011.

[Cloning of full-length coding sequence of tree shrew CD3E and prediction of its molecular characteristics].

The use of tree shrews (Tupaia belangeri) in human disease studies demands essential research tools, in particular cellular markers and their monoclonal antibodies for immunological studies. Here we cloned the full-length cDNAs encoding CD3E from total RNA of the spleen, liver and peripheral blood of tree shrews and analyzed their structural characteristics in comparison with other mammals by Discovery Studio software. The results showed that the open reading frame sequence of tree shrew CD3E was 582 bp, encoding 194 amino acids. The overall structure of tree shrew CD3E protein was similar to its counterparts of other mammals, intracellular and transmembrane domain highly conserved. However, detailed analysis revealed two potential glycosylation sites and different surface charges in the extracellular domain. Availability of the entire open-reading-frame and related sequence information would therefore facilitate the preparation of monoclonal antibodies against tree shrew CD3 and further studies for its function.