Viral metagenomics analysis of poultry faeces in live poultry market, Haikou, China

Objective:To conduct in-depth study of the distribution and diversity of viruses in poultry is of great importance in monitoring the emergence of interspecies transmission of novel viruses that may cause epidemics with public health significance. Poultry is an economically important source of meat, eggs and feathers which plays an important role as natural reservoirs of many pathogenic viruses. Compared with wild animals, poultry have more frequent interactions and therefore opportunities to transmit their viruses to human.Methods:To study the viromes of different types of poultry in Hainan, China, we used metagenomics for deep viral nucleic acid sequencing of the faecal samples collected from chickens, ducks and pigeons from a live poultry market in Haikou.Result:The poultry viromes were identified by sequence similarity comparisons of viral reads (BLASTxE score, <5) against viral reference database. A total of 15 309 viral reads were obtained, approximately 13 063, 1 370 and 876 viral reads were generated from the chicken, duck, pigeon faecal samples, respectively. The majority of the sequences were homologous to the animal virus of Adenoviridae, Herpeaviridae, Picobirnaviridae, Reoviridae, Retroviridae, Circoviridae, Paramyxoviridae, Astroviridae, Caliciviridae, Coronaviridae, Picornaviridae, and Orthomyxoviridae. The VP4 and VP7 segments of a pigeon rotavirus, similar to fox rotavirus in group A, were sequenced and phylogenetically analyzed. The near full genome of a pigeon circovirus was also analyzed.Conclusion:The major types of poultry in a Haikou harbor many different families of viruses in their feces which may have the potential for interspecies transmissions. Further studies should be conducted to identify the most prevalent and important viruses among a larger number of poultry in Haikou and other areas in Hainan.

Complete genome sequence of a rodent torque teno virus in Hainan Island, China

Objective:Torque teno virus(TTV), are reported in a wide range of mammals. In this study, we sequenced and analyzed the complete genome of a genetic variant of Rodent TTV, RoTTV3-HMU1 (Hainan Medical University). The virus was harbored by a Rattus norvegicus in the residential areas of Hainan Island, China.Methods:Torque Teno virus (TTV) was found widely distributed throughout the world infecting an extensively wide range of mammals .We extracted the viral DNA from a Rattus norvegicus liver which was caught from the residential areas of Hainan Island. Purifying the amplicons in the range of 250-500 bp. Then Five hundred nanograms DNA was subjected to high-throughput sequencing. The contigs were compared with the NCBI nucleiotide database, designed the primers to cover the genome by PCR amplification and amplicons of each PCR which have been cloned and sequenced. Finally the genome was annotated by using NCBI ORF finder and FGENESV0. Phylogenetic analysis was implemented by the neighbor-joining method in the MEGA6 software package.Results:We sequenced the complete genome of a genetic variant of Rodent TTV, RoTTV3- HMU1. The genomic sequence of RoTTV3-HMU1 has been deposited in GenBank under accession number MF688246.1. The complete genome of RoTTV3-HMU1 is 2 570 nucleotides (nt) in length with a G+C content of 46.93%. RoTTV3-HMU1 encoded 3 unidirectional overlapping open reading frames (ORF). Sequence analysis indicated that the genome of RoTTV3-HMU1 virus was most closely related to RN_2_15 (GenBank accession no. KM668486.1). Phylogenetic analysis based on both ORF1 and the total genome sequence placed RoTTV3-HMU1 in to the clad RoTTV3 of the RoTTV.Conclusions:Hainan Island faces mainland across the sea, however, the same genotype of RoTTV was identified in both Hainan Island and the other part of China. The detection of RoTTV3-HMU1 contributed to a better understanding about the origin and evolution of RoTTV.

Pharmacodynamic study on insomnia-curing effects of Shuangxia Decoction in Drosophila melanogaster / 中国天然药物

The present study aimed to establish a pharmacodynamic method using the pySolo software to explore the influence of freeze-dried powders of Shuangxia Decoction (SXD) on the sleep of normal Drosophila melanogaster and the Drosophila melanogaster whose sleep was divested by light. The dose-effect and the time-effect relationships of SXD on sleep were examined. The effect-onset concentration of SXD was 0.25%, the plateau appeared at the concentration of 2.5% and the total sleep time showed a downtrend when the concentration was greater than 2.5%. The sleep time was the longest on the fourth day after SXD was given. The fruit fly sleep deprivation model was repeated by light stimulation at night. The middle dosage group (2.5%) had the best insomnia-curing effect. In conclusion, using the pySolo software, an approach for the pharmacodynamics study was established with Drosophila melanogaster as a model organism to determine the insomnia-curing effects of the traditional Chinese medicine (TCM). Our results demonstrated the reliability of this method. The freeze-dried powders of SXD could effectively improve the sleep quality of Drosophila melanogaster.

Prokaryotic expression of DNA recombinase FLP and its purification with enzymatic activity / 生物工程学报

DNA recombinase FLP gene exists on the 2 micro plasmid of Saccharomyces cerevisiae. Recombinase FLP could recognize an FRT site composed of 34bp and function the sequences for exchange, recombination, deletion and reversion between the two orientated FRT sites. These functions are highly recognized by molecular biologists and biotechnology engineers for theoretic and applicable technology studies. This work constructed a prokaryotic over-expressed vector harboring FLP gene nominated as pQE30-flpe and established its over-expression culture system in which recombinase FLP could be efficiently expressed in E. coli strain M15. Purification procedures for high purity and active FLP are established through combination of ammonium sulfate precipitation with a 0.5-1.0 mL micro-column technique of Ni affinity chromatography with gradient elution. To verify the recombinase activity of purified FLP, substrate vectors, sequence donor vector (pUC18-FRT-gfp-FRT) and sequence accepting vector (pET30a-FRT) are constructed with various number, orientation of FRTs harboring the GFP gene for the expression of visible assay of the functions of recombination, exchange and deletion. Results showed that the system not only over expressed recombinase FLP in prokaryotic E. coli, but also efficiently purified the enzyme with a higher activity of the function of recombination, exchange and deletion. The system and the method are easily implemented and feasibly manipulated for theoretic study and biotechnology application.

A clinical study of abnormal bone metabolism in patients with HBV liver cirrhosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the pathogenesis of abnormal bone metabolism in patients with HBV liver cirrhosis.</p><p><b>METHODS</b>NM-300 signal-energy X-ray absorptiometry system was used to measure the bone mineral density (BMD) in 61 liver cirrhosis patients and 30 age-matched healthy controls. The serum levels of 1,25(OH)2D3, parathyroid hormone (PTH), calcitonin (CT), bone gamma-carboxyglutamic acid-containing protein (BGP), IL-1beta, IL-6, tumor necrosis factor (TNF)alpha and urine crosslaps were also detected in these patients.</p><p><b>RESULTS</b>BMD in patients with HBV liver cirrhosis was lower than those of the controls. The serum levels of 1,25(OH)2D3 and BGP in cirrhosis patients were lower than those in the controls, and they were much lower in the osteoporosis (OP) group than in the non-osteoporosis (NOP) group. The PTH and CT were higher significantly in the patients than in the controls. The changes of serum 1,25(OH)2D3 and BGP were correlated with the changes of BMD. The serum levels of IL-1beta, IL-6, TNFalpha and urine crosslaps in cirrhosis patients were higher than those of the controls, and they were much higher in the OP group than in the NOP group. We also found that the serum levels of IL-1beta, IL-6, TNFalpha and urine crosslaps had a negative correlation with BMD.</p><p><b>CONCLUSIONS</b>These data suggest that bone formation is weakened and bone resorption is increased in patients with HBV liver cirrhosis, 1,25(OH)2D3 plays an important role in abnormal bone formation. Elevation of serum IL-1beta, IL-6, TNFalpha can accelerate bone resorption and cause hepatic bone disease (HBD). Taking 1,25(OH)2D3 and reducing the level of IL-1beta, IL-6, TNFalpha may be very important in preventing and treating HBD.</p>

A study of the association of iNOS and eNOS gene polymorphism with portal hypertension in liver cirrhosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study whether polymorphism of iNOS and eNOS genes is associated with portal hypertension in liver cirrhosis.</p><p><b>METHODS</b>A case control study of 106 patients with liver cirrhosis due to HBV was performed in comparison with 108 controls using PCR-RFLP to detect iNOS promoter -969G --> C and eNOS exon7 894G --> T polymorphism.</p><p><b>RESULTS</b>The frequency of the C allele and GC genotype in the iNOS promoter -969G --> C was significantly higher in the portal hypertension group than in the control group. (P < 0.05). The frequency of the T allele and GT genotype in eNOS exon7 894G --> T was dramatically higher in the portal hypertension group than in the control group (P < 0.05). Multivariate logistic regression analysis revealed that iNOS polymorphism in iNOS promoter -969G --> C and eNOS exon7 894G --> T was an independent new risk factor for portal hypertension. We discovered that iNOS promoter -969G --> C led to an increase in its functional activity.</p><p><b>CONCLUSIONS</b>The polymorphism of iNOS promoter -969G --> C and eNOS exon7 894G --> T is associated with portal hypertension of liver cirrhosis, which is a new independent risk factor found related to the occurrence of portal hypertension. The polymorphism of iNOS promoter -969G --> C results in functional activity increase of the iNOS promoter.</p>

Polymorphism of NOS2A promoter -969(G>C) is associated with portal hypertension of liver cirrhosis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine whether polymorphism of NOS2A promoter -969G>C is associated with the portal hypertension of liver cirrhosis.</p><p><b>METHODS</b>A case control study covering 106 patients with liver cirrhosis due to hepatitis B virus(HBV) in comparison with 108 controls was performed using PCR-restriction fragment length polymorphism. The NOS2A mRNA and protein expression in liver cirrhosis tissues were detected by reverse transcription-PCR and Western blot. The recombinant plasmids of NOS2A promoter luciferase reporter gene were constructed and were transfected transiently into HepG2 cells for analyzing the functional activity of the promoter.</p><p><b>RESULTS</b>The frequencies of the C allele and GC genotype at NOS2A promoter -969G>C were significantly higher in portal hypertension group (16.9%, 33.8%) than in control group(8.8%, 17.6%)(P<0.05), and positive correlation (r=0.18) and association (OR=2.42) were noted. There was no significant difference in frequency distribution between single liver cirrhosis group and control group(P>0.05). The expressions of NOS2A mRNA and protein in liver cirrhosis tissues were more increased in C allele carriers with liver cirrhosis than in G allele carriers with liver cirrhosis, which led to higher functional activity of the promoter. Multivariate logistic regression analysis revealed that NOS2A polymorphism at promoter -969G>C is an independent novel risk factor for the occurrence of portal hypertension in patients with liver cirrhosis.</p><p><b>CONCLUSION</b>The polymorphism of NOS2A promoter -969(G>C) is associated with portal hypertension of liver cirrhosis, which results in functional activity increase of NOS2A promoter and is an independent risk factor for portal hypertension.</p>

Relationship of endothelin-1 (ET-1) TaqI and tumor necrosis factor (TNF) a gene polymorphism with portal hypertension in liver cirrhosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study whether liver cirrhosis and portal hypertension are associated with ET-1 TaqI polymorphism and TNFa promoter-308G to A polymorphism.</p><p><b>METHODS</b>A case control study of 106 patients with liver cirrhosis following HBV C infection was performed in comparison with 108 controls by PCR-RFLP.</p><p><b>RESULTS</b>The frequency of C allele and CC+TC genotype in TaqI polymorphism of ET-1 gene in the portal hypertension group (LC+) was significantly higher than that in the healthy controls, and the frequency of TNF2/1 genotype in TNFa promoter -308 G to A polymorphism in LC+ group was significantly higher than that in the control group. The results by stratification analysis showed that TCF2 genotype frequency was higher in the LC+ group than in the control group. ET-1 TaqI polymorphism and TNFa polymorphism were risk factors for the occurrence of portal hypertension by Logistic regression analysis.</p><p><b>CONCLUSION</b>ET-1 TaqI polymorphism and TNFa polymorphism are associated with portal hypertension, and are new risk factors for the occurrence of portal hypertension. TCF2 genotype may be a susceptible gene of portal hypertension.</p>