Genetic Characterization of a Novel HIV-1 CRF07_BC/CRF55_01B Recombinant Form Identified in Jiangmen, China.

New recombinant variants are a predominant challenge for preventing the spread of the HIV-1 epidemic. In this study, we confirmed a novel HIV-1 CRF07_BC/CRF55_01B recombinant form for the first time, which was isolated from a male patient in Jiangmen, China. The genomic sequence of the variant with four CRF55_01B segments inserted into the CRF07_BC backbone is 8,510 bp in length, extending from nucleotides 669 to 9,293 according to the HXB2 genome. Specifically, the recombinant strain contains site mutations associated with drug resistance.

Multi-locus sequence typing of Laribacter hongkongensis isolates from freshwater animals, environment and diarrhea patients in southern China.

Laribacter hongkongensis is a novel emerging bacterium associated with gastroenteritis and invasive bacteremic infections. Freshwater fish and edible frogs have been identified as major reservoirs of L. hongkongensis. Currently one of the main challenges in L. hongkongensis research is to identify their sources and possible transmission routes. The aim of this study was to determine the genetic diversity and relatedness of these L. hongkongensis isolates to their hosts in the hope of shedding light on these issues. In this study, multi-locus sequence typing (MLST) was used to determine the genetic characteristics of 114 L. hongkongensis strains, including 113 isolated from humans, fish, frogs, Amazonian snails and water sample in Guangzhou and Jiangmen, Southern China, and one reference isolate HZ242, recovered from a diarrhea patient in Hangzhou. The relationships among the STs and the relatedness among the isolates were assessed by phylogenetic and eBURST analysis. A total of 72 different sequence types (STs) from 114 isolates of L. hongkongensis were identified by MLST analysis, and ST99-ST161were novel. Significant difference of the prevalence of different STs between fish isolates (41.8%, 23/55) and frog isolates (82.4%, 42/51) was revealed (p=0.000). The most frequent ST (ST45) was identified 28 times and only found in fish isolates. In addition, 10 groups were identified by eBURST in this study. Combined the MLST data from Hong Kong and the present study, there were eight eBRUST lineages (group A-H) included the isolates (49.2%, 128/260) from either numerous hosts or multiple geographic origins, which contained 33.1% (53/160) of all the STs. Group A (n=57, STs=20) consisted exclusively of isolates from fish and 92.9% (39/42) of isolates in group B (n=42, STs=16) were only from fish. Group C-F (n=22, STs=14) were found to be associated with human, apart from other hosts. In this study, extensive genetic heterogeneity among the L. hongkongensis isolates from various hosts was observed. Specifically, there is higher genetic diversity of L. hongkongensis isolates of frog-origin than those of fish-origin. This study indicated some isolates exhibited a preference for specific hosts or geographic areas. ST45 was revealed to be the most frequent ST, which was only found in the fish isolates in Southern China, but might be irrelative to human infection. This MLST study further revealed that frog was likely to be another major source for human infection with L. hongkongensis apart from fish.

[Current prevalence of Clonorchis sinensis infection in Jiangmen, Guangdong Province].

OBJECTIVE: To investigate the prevalence of clonorchiasis in Jiangmen City. METHODS: From May to December 2011, each town was randomly chosen from east, south, west, north and central area of 7 cities/districts of Jiangmen City. Four or five villages were randomly selected from each town. In each village, the residents above 3-year-old in 10% randomly sampled families were treated as research objects. Total of 14,000 fecal boxes were issued and 12,661 ones back. Eggs in stool were examined by modified Kato-Katz thick smear method (three slides per specimen). RESULTS: A total of 1316 clonorchiasis cases were found from 12,661 pepople in 140 villages with a prevalence of 10.39% (1316/12,661). The average egg density was 98.3 eggs per gram (EPG) feces. Among 7 cities/districts, the prevalence in Pengjiang District (26.68%, 402/1507) was the highest, and that of Taishan City (0.93%, 19/2049) was the lowest. The egg density in Heshan City was the highest (225.4 EPG) and the lowest one was found in Taishan City (5.13 EPG). The prevalence was negatively related with the distance to major rivers (r=-0.61, P<0.01). The prevalence and the egg density in males and females was 13.20% (807/6112) and 80.9 EPG, and 7.77% (509/6549) and 39.4 EPG, respectively. The prevalence and intensity of infection increased obviously in the groups of above 20-year-old. The people with a higher prevalence was the group of 60-69 year-old, and the people above 70 years showed heavier infection (153.8 EPG). Light, moderate and heavy infection occupied 99.91%, 0.09%, and 0. CONCLUSION: Clonorchiasis is endemic in seven districts of Jiangmen City with different epidemic degrees. There are significant differences in the prevalence and intensity of infection among different areas. The villages with higher prevalence distribute along the middle and lower sections of the two major rivers.

[Pilot observation on the effect of integrated control measures against clonorchiasis in Pengjiang District of Jiangmen City].

From June to December in 2008, five villages were randomly chosen from Pengjiang District of Jiangmen city and about five hundred residents from each village were examined for clonorchiasis by Kato-Katz method (three slides per specimen). Fifty residents from each village were re-examined one month after treatment. One year later 50 treated residents were chosen from Dalin village and Sanya village for fecal examination. Questionnairing was conducted to determine the knowledge rate on clonorchiasis prevention among residents. The percentage and usage of sanitary toilets were investigated. The average infection rate of clonorchiasis from five villages was 21.5%(537/2501). 86.6%(465/537) of clonorchiasis received treatment voluntarily. One month after treatment the infection rate in four villages declined significantly. The positive rate showed no significant difference between one month and one year after treatment in Dalin and Sanya villages (P>0.05) . Questionairing indicated that 41.2%(170/413) of the clonorchiasis cases ate raw fish frequently, which was significantly higher than those non-infected people [4.2%, 8/192] (P<0.05). After health education, the knowledge awareness rate raised from 23.1% (135/584) to 84.5% (349/413) (P<0.05). The dissemination and usage of sanitary toilets were 93.2% (38 068/40 848) and 100%, respectively.

Cloning and expression of mitochondrial malate dehydrogenase of Clonorchis sinensis.

The NAD-dependent mitochondrial malate dehydrogenase (mMDH, EC1.1.1.37) plays pivotal roles in tricarboxylic acid and is crucial for the survival and pathogenecity of parasites. A cDNA, which was identified by high throughput sequencing from the cDNA library constructed from adult Clonorchis sinensis, encoded a putative peptide of 341 amino acids with more than 50% identity with mMDHs from other organisms. The mMDH was expressed in Escherichia coli as the recombinant protein with a GST tag and purified by glutathione-Sepharose 4B column. The recombinant mMDH showed MDH activity of 63.6 U/mg, without lactate dehydrogenase activity and NADPH selectivity. The kinetic constants of recombinant mMDH were determined.

[Cloning and expression of the gene encoding rat IgE-dependent histamine-releasing factor and effect study on the recombinant protein].

AIM: To clone and express the gene encoding rat IgE-dependent histamine-releasing factor (rHRF) and to study the effect of recombinant rHRF to induce histamine release from rat sensitized mast cells. METHODS: The complete coding region of rHRF was cloned and expressed in BL21 cells. The soluble recombinant rHRF was purified. Aliquots of the mast cells obtained from the lungs of OVA-immunized rats were separately stimulated by recombinant rHRF at different concentration. The released histamine was measured by the OPT spectrofluorometric procedure. RESULTS: The cloned DNA fragment showed 97% nucleotide sequence identity and 95% deduced amino acid sequence identity with the mRNA of rat IgE-dependent histamine-releasing factor (or translationally controlled tumor protein) in GenBank. The recombinant rHRF showed a relative molecular mass of 24 000 in SDS-PAGE. The purified rHRF which was independent of the allergen induced histamine releasing from the sensitized mast cells in a dose-dependent manner. CONCLUSION: The recombinant rHRF, which showed the effect of inducing histamine release from sensitized mast cells, would play an important role in the allergen diseases.

Enzymatic and physico-chemical characteristics of recombinant cMDH and mMDH of Clonorchis sinensis.

The cytosol and mitochondrial malate dehydrogenases (MDHs, EC 1.1.1.37) of Clonorchis sinensis were expressed in Escherichia coli as a fusion protein with a 6xHis and GST tag, respectively. The cytosol MDH of Clonorchis sinensis (Cs-cMDH) has higher resistibility to acid than mitochondrial MDH (Cs-mMDH). The Cs-cMDH also has higher heat resistibility and thermal stability than Cs-mMDH. Although there is only 22.8% identity between the amino acid sequences of Cs-cMDH and Cs-mMDH, they share several conserved residues. There are some differences between the circular dichroism spectra of Cs-cMDH and Cs-mMDH, but they have approximate percentages of helix. 4,4'-Bisdimethylamino diphenylcarbinol can decrease the Cs-mMDH activity but not the Cs-cMDH activity. Paraziquantel, metronidazole and albendazole did not inhibit the enzymes' activity, but adenosine 5'-monophosphate showed competitive inhibition to enzyme, with the Ki for Cs-cMDH and Cs-mMDH being 2.81 and 0.49 mM, respectively.

Molecular cloning and characterization of a novel adenylate kinase 3 gene from Clonorchis sinensis.

Adenylate kinase (AK) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in living cells. AK catalyzes reversible high energy phosphoryl transfer reactions between ATP (or GTP) and AMP to generate ADP (or GDP). From a Clonorchis sinensis adult worm cDNA library, we isolated a cDNA clone encoding a novel AK3 isozyme. The 956 bp cDNA encodes a putative protein of 228 amino acids with a predicted molecular mass of 26.2 kDa. The recombinant CsAK3 protein produced in Escherichia coli can be refolded into a functional protein with AK3 activity. The optimum pH and temperature for the enzyme are 8.5 and 40 degrees C, respectively. The calculated activation energy is 56.04 kJ mol-1. The Km of the CsAK3 for AMP and GTP are 118 microM and 359 microM, respectively. CsAK3 is inhibited by Ap5A (>70% inhibition by 2.0 mM AP5A). Ap5A may be a potential lead compound acting on C. sinensis in which AK3 as a drug target.

Clonorchis sinensis: molecular cloning and functional expression of novel cytosolic malate dehydrogenase.

The NAD-dependent cytosolic malate dehydrogenase (cMDH, EC 1.1.1.37) plays a pivotal role in the malate-aspartate shuttle pathway that operates in a metabolic coordination between cytosol and mitochondria, and thus is crucial for the survival and pathogenicity of the parasite. In the high throughput sequencing of the cDNA library constructed from the adult stage of Clonorchis sinensis, a cDNA clone containing 1152bp insert was identified to encode a putative peptide of 329 amino acids possessing more than 50% amino acid sequence identities with the cMDHs from other organisms such as fish, plant, and mammal. But low sequence similarities have been found between this cMDH and mitochondrial malate dehydrogenase as well as glyoxysomal malate dehydrogenase from other organisms. Northern blot analysis showed the size of the C. sinensis cMDH mRNA was 1.2 kb. The cMDH was expressed in Escherichia coli M15 as a His-tag fusion protein and purified by BD TALON metal affinity column. The recombinant cMDH showed high MDH activity of 241 U mg(-1), without lactate dehydrogenase and NADP(H) selectivity. It provides a model for the structure, function analysis, and drug screening on cMDH.

Molecular cloning and characterization of cDNA encoding a ubiquitin-conjugating enzyme from Clonorchis sinensis.

The ubiquitin-proteasome system is an essential mechanism for protein degradation in eukaryotes. Protein ubiquitination is composed of a series of enzymatic reactions. The ubiquitin-conjugating enzyme (E2) is one of the important enzymes involved in the process. A cDNA encoding an E2 enzyme was cloned from a Clonorchis sinensis cDNA library by large-scale sequencing. This new cDNA contains 862 bp with a putative open reading frame of 156 amino acids. The deduced amino acid sequence is 77% identical to the human E2, HHR6A and HHR6B. The coding region of this cDNA was expressed in E. coli as a GST-tagged protein, and was purified to electrophoretic homogeneity. Enzymatic assays showed that this E2 had the capacity to form a thiolester linkage, and could conjugate ubiquitin to histone H2A in an E3-independent manner in vitro, which indicated that the expressed protein was functionally active. The nucleotide sequence reported in this paper has been submitted to the Genbank Database with accession number AY632078.