RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in Human Enterovirus A-D from Clinical Specimens.

OBJECTIVE: To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases. METHODS: A panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A-C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID 50 per µL and copies per µL, and the newly established methods were tested in clinical specimens collected in recent years. RESULTS: The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID 50 per µL and 10 virus copies per µL, and for the complete VP1 gene was 1 CCID 50 per µL and 100 virus copies per µL, using serially-diluted virus stocks of five serotypes. As a proof-of-concept, 25 serotypes were identified and complete VP1 sequences of 23 serotypes were obtained by this system among 858 clinical specimens positive for HEVs during the past eight surveillance seasons. CONCLUSION: This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A-D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.

[Spatio-Temporal Patterns and Environmental Risk of Endocrine Disrupting Chemicals in the Liuxi River].

This study aimed to investigate the occurrence and spatio-temporal distribution of 4-tert-octylphenol (4-t-OP), 4-nonylphenol (4-NP), triclosan (TCS), estrone (E1), 17ß-estradiol (E2), and bisphenol-A (BPA) as endocrine disrupting chemicals (EDCs) in the water of the Liuxi River and to evaluate the risks for estrogenic activity. The results showed that EDCs had been detected at the 14 monitoring sites and the total concentration ranged from 26.07 ng·L-1 to 7109.5 ng·L-1, with the highest contribution rate coming from 4-NP (78.62%), followed by BPA (11.91%), and the other four EDCs (≤ 4.92%). On a spatial and temporal scale, the EDC contents increased longitudinally from upstream to downstream, especially in the heavily-polluted Baiyun section where the water quality was lower than level Ⅴ. The EDC contents in the tributaries were much higher than those in the main channels. Influenced by the monsoon precipitation, the contents of 4-NP, 4-t-OP, and total EDCs in the rainy season were significantly (P<0.05) higher than those in the dry season, while the seasonal changes of E1 and E2 followed the opposite tendency. A Pearson correlation analysis showed that DO was significantly negatively correlated with all the EDCs, suggesting that the EDCs and reductive organic pollutants might coexist. As TN, TP, NH4+-N, permanganate index, and EC were significantly positively correlated with E1, E2, BPA, and TCS but not obviously correlated with 4-NP (P>0.05), we presumed that the pollution source of E1, E2, BPA, and TCS might be the same with nitrogen and phosphorus nutrition, originating from the point source emission of the domestic sewage, industrial, and agricultural wastewater. In contrast, 4-NP and 4-t-OP more likely originated from the non-point source pollution from agriculture. RDA results showed that the variation of the EDCs contents by season was more obvious than that in space (RDA1 56.14%>RDA2 14.20%), which was much more influenced by 4-NP in the rainy season and by BPA in the dry season. As E1, E2, and TCS were positively correlated with the Cu, Zn, cyanide, and fecal coliform, these three target compounds could be used to indicate the multiple pollution components for water quality. Compared with the worldwide reported EDC contents in waters, 4-NP, BPA, and TCS contents in the middle and lower reaches of the Liuxi River were at higher levels, while E1, E2, and 4-t-OP were at the middle and lower levels. The risk assessment for estrogenic activity showed that the RQ values in the middle and lower reaches of the Liuxi River were all greater than 1, indicating that the downstream river sections were under high risk for estrogenic activity. As a result, appropriate precautions are needed to improve environmental management.

[Patterns of Bacterial Community Through Soil Depth Profiles and Its Influencing Factors Under Betula albosinensis Burkill in the Xinjiashan Forest Region of Qinling Mountains].

In this study, vertical changes in bacterial α-diversity and community composition were investigated at four soil depths(0-10, 10-20, 20-40 and 40-60 cm) in Betula albosinensis Burkill forest of Qinling Mountains by sequencing of the 16S rDNA regions using Illumina MiSeq high-throughput technology. The results showed that the decreases of OTUs, Chao1 and Shannon were numerical but not significant, and the highest values of 1688, 2314 and 8.66 were obtained in 0-10 cm, respectively. At the phylum level, Acidobacteria and Proteobacteria were the most dominant bacteria in four soil layers. At the genus level, Gp4, Gp6 and Gp16 were the most dominant bacteria. The relative abundance of Acidobacteria in 40-60 cm soil depth(62.88%) was higher than those in other soil depths. Proteobacteria in 0-10 cm(23.62%) was more abundant than that in 40-60 cm. The relative abundance of Acidobacteria was significantly correlated with the total N, soil organic carbon, C/N, and soil dissolved organic carbon. Soil water content, soil organic matter and soil dissolved organic carbon were the key factors affecting soil Proteobacteria. RDA sequencing results showed that soil dissolved organic carbon was the key factor contributing to the bacteria community abundance. The results demonstrated that there are plenty of bacterial distribution in all four soil layers, which provides a fundamental basis for vertical soil bacterial community diversity, and possesses very important research value in biogeochemical cycling.

[Vertical distribution of soil active carbon and soil organic carbon storage under different forest types in the Qinling Mountains].

Adopting field investigation and indoor analysis methods, the distribution patterns of soil active carbon and soil carbon storage in the soil profiles of Quercus aliena var. acuteserrata (Matoutan Forest, I), Pinus tabuliformis (II), Pinus armandii (III), pine-oak mixed forest (IV), Picea asperata (V), and Quercus aliena var. acuteserrata (Xinjiashan Forest, VI) of Qinling Mountains were studied in August 2013. The results showed that soil organic carbon (SOC), microbial biomass carbon (MBC), dissolved organic carbon (DOC), and easily oxidizable carbon (EOC) decreased with the increase of soil depth along the different forest soil profiles. The SOC and DOC contents of different depths along the soil profiles of P. asperata and pine-oak mixed forest were higher than in the other studied forest soils, and the order of the mean SOC and DOC along the different soil profiles was V > IV > I > II > III > VI. The contents of soil MBC of the different forest soil profiles were 71.25-710.05 mg x kg(-1), with a content sequence of I > V > N > III > II > VI. The content of EOC along the whole soil profile of pine-oak mixed forest had a largest decline, and the order of the mean EOC was IV > V> I > II > III > VI. The sequence of soil organic carbon storage of the 0-60 cm soil layer was V > I >IV > III > VI > II. The MBC, DOC and EOC contents of the different forest soils were significanty correlated to each other. There was significant positive correlation among soil active carbon and TOC, TN. Meanwhile, there was no significant correlation between soil active carbon and other soil basic physicochemical properties.

[Molecular epidemiology of HFMD-associated pathogen coxsackievirus A6 in Fujian Province, 2011-2013].

In order to characterize the molecular epidemiology of HFMD-associated Coxsackievirus A6 (CVA6) in Fujian Province, a total of 1340 specimens from non-EV71 non-CVA16 HFMD patients were collected during 2011-2013. Isolated virus strains were identified and subtyped. Full-length coding regions for the VP1 gene of the predominant serotype CVA6 isolates were amplified and sequenced. Among the 375 non-EV71 non-CVA16 HFMD cases confirmed by virus isolation and molecular subtyping, 182 (48.5%) were found to be caused by CVA6, accounting for 7.9%, 16.2% and 39.6% HFMD-associated enteroviruses in FujianProvince during 2011, 2012, and 2013, respectively. Compared with general features observed in the HFMD epidemic, no difference in CVA6-specificity or severity rates was observed between geographical origins, gender, or age groups. Nucleotide sequence analyses of VP1 genes revealed high diversity levels of 16.2%-18.6% among CVA6 strains from Fujian Province, in contrast to the prototype CVA6 strain, and showed low levels of diversity in the amino acid sequences (4.3%-6.2%). Phylogenetic analysis also indicated that CVA6 isolates from Fujian Province were distinct from the prototype strain and other isolates from abroad; however, it was homologous to domestic strains, although the Fujian isolates clustered into multiple branches. These results suggested that significant changes in the pathogenic spectrum of HFMD in Fujian Province occurred during 2011-2013, as CVA6 was one of the predominant serotypes of HFMD. CVA6 isolates from Fujian Province were co-circulating and co-evolving with other domestic strains as multiple closely related CVA6 transmission chains were observed in Fujian Province overall and within each prefecture.

Enteractinococcus coprophilus gen. nov., sp. nov., of the family Micrococcaceae, isolated from Panthera tigris amoyensis faeces, and transfer of Yaniella fodinae Dhanjal et al. 2011 to the genus Enteractinococcus as Enteractinococcus fodinae comb. nov.

A novel actinobacterium, designated strain YIM 100590(T), was isolated from Panthera tigris amoyensis faeces collected from Yunnan Wild Animal Park in Yunnan province, south-west China. Phylogenetic analysis based on 16S rRNA gene sequence data showed that strain YIM 100590(T) is a member of the family Micrococcaceae. Cells were coccoid to oval (0.7-1.5 µm in diameter) occurring singly or in clusters. Growth was observed at 10-37 °C (optimum 28 °C) and at pH 7.0-11.0 (optimum pH 8.0). The major fatty acids were iso-C(15:0) (32.22%), anteiso-C(15:0) (31.64%) and iso-C(16:0) (17.38%). The peptidoglycan was of A4α type (L-Lys-Gly-L-Glu). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides, dimannosyl diacylglycerol, an unknown glycolipid and two unknown phospholipids. The quinone system comprised menaquinones MK-7 (91.9%) and MK-8 (8.3%). The DNA G+C content of strain YIM 100590(T) was 56.2 mol%. Chemotaxonomic data indicated that the strain belongs to the family Micrococcaceae. On the basis of morphological and chemotaxonomic data and phylogenetic analysis, strain YIM 100590(T) is considered to represent a novel species of a new genus within the family Micrococcaceae, for which the name Enteractinococcus coprophilus gen. nov., sp. nov. is proposed. The type strain of Enteractinococcus coprophilus is YIM 100590(T) (=DSM 24083(T)=JCM 17352(T)). Yaniella fodinae DSM 22966(T) was transferred to the new genus as Enteractinococcus fodinae comb. nov. (type strain G5(T)=DSM 22966(T)=JCM 17931(T)=MTCC 9846(T)).

Nonomuraea soli sp. nov., an actinomycete isolated from soil.

A straight-chain, spore-forming actinobacterium, strain YIM 120770(T), was isolated from soil. Phylogenetic analysis on the basis of 16S rRNA gene sequence comparisons revealed that the isolate represents a distinct cluster within the clade comprising the genus Nonomuraea and is related most closely to Nonomuraea rhizophila YIM 67092(T) (96.5% similarity). Cells of strain YIM 120770(T) grew in the presence of 0-3% (w/v) NaCl, at 15-37 °C and at pH 7.0-8.0. The diagnostic amino acid was meso-diaminopimelic acid, cell hydrolysates contained madurose, glucose, mannose, ribose and galactose, the predominant cellular fatty acids were 10-methyl C(17:0) and iso-C(16:0), and the DNA G+C content was 66.4 mol%, data consistent with affiliation of strain YIM 120770(T) to the genus Nonomuraea. Strain YIM 120770(T) shared low levels of 16S rRNA gene sequence similarity (<97%) with the type strains of recognized species of the genus Nonomuraea and could be differentiated from its closest phylogenetic relative based on phenotypic characteristics. These results suggested that strain YIM 120770(T) represents a novel species of the genus Nonomuraea, for which the name Nonomuraea soli sp. nov. is proposed. The type strain is YIM 120770(T) (=DSM 45533(T)=JCM 17347(T)).

Methylobacterium soli sp. nov. a methanol-utilizing bacterium isolated from the forest soil.

A Gram-negative, pink-pigmented, non-spore-forming rod shaped, methanol-utilizing bacterium, strain YIM 48816(T), was isolated from forest soil collected from Sichuan province, China. Strain YIM 48816(T) can grow at 4-37 °C, pH 5.0-7.0 and 0% NaCl (w/v). Based on 16S rRNA gene sequence similarity studies, it belonged to the genus Methylobacterium, and formed a phyletic line. The 16S rRNA gene sequence similarities were 96.2% to Methylobacterium mesophilicum DSM 1708(T) and 96.0% to Methylobacterium brachiatum DSM 19569(T), and the phylogenetic similarities to all other Methylobacterium species with validly published names were less than 96.0%. The major menaquinones detected were Q-10 (97.14%) and Q-9 (2.86%). The major fatty acids were C18:1 ω7c (80.84%). The DNA G + C content was 66.2 mol%. It is apparent from the genotypic and phenotypic data that strain YIM 48816(T) belongs to a novel species of the genus Methylobacterium, for which the name Methylobacterium soli sp. nov. is proposed. The type strain is YIM 48816(T) (CCTCC AA 208027(T) = KCTC 22810(T)).

Planosporangium mesophilum sp. nov., isolated from rhizosphere soil of Bletilla striata.

A Gram-stain-positive, non-motile actinomycete, designated strain YIM 48875(T), was isolated from rhizosphere soil of Bletilla striata and its taxonomic position was established by using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequence data showed that strain YIM 48875(T) belonged to the genus Planosporangium, supported by a bootstrap value of 100 %. Cells of strain YIM 48875(T) showed two kinds of sporangia, which also supported its classification in the genus Planosporangium. Strain YIM 48875(T) grew optimally at 28 °C, at pH 6.0-8.0 and in the presence of 2 % (w/v) NaCl. The level of 16S rRNA gene sequence similarity between strain YIM 48875(T) and Planosporangium flavigriseum YIM 46034(T) was 98.6 %. Strain YIM 48875(T) exhibited a quinone system with menaquinones MK-9(H(4)), MK-9(H(6)) and MK-9(H(8)) as the predominant compounds, a polar lipid profile comprising diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol mannoside and the major fatty acids iso-C(15 : 0) and iso-C(16 : 0); these data were markedly different from those for P. flavigriseum YIM 46034(T). The level of DNA-DNA relatedness between strain YIM 48875(T) and P. flavigriseum YIM 46034(T) was 45.5 %. It is apparent from the genotypic and phenotypic data that strain YIM 48875(T) represents a novel species of the genus Planosporangium, for which the name Planosporangium mesophilum sp. nov. is proposed. The type strain is YIM 48875(T) ( = CCTCC AA 209049(T)  = KCTC 19779(T)).

Rubellimicrobium roseum sp. nov., a Gram-negative bacterium isolated from the forest soil sample.

A novel pink-coloured, non-spore-forming, non-motile, Gram-negative bacterium, designated YIM 48858(T), is described by using a polyphasic approach. The strain can grow at pH 6.5-9 (optimum at pH 7) and 25-30 degrees C (optimum at 28 degrees C). NaCl is not required for its growth. Positive for oxidase and catalase. Urease activity, nitrate reduction, starch and Tween 80 tests are negative reaction. 16S rRNA gene sequence similarity studies showed that strain YIM 48858(T) is a member of the genus Rubellimicrobium, with similarities of 96.3, 95.7 and 95.5% to Rubellimicrobium mesophilum MSL-20(T), Rubellimicrobium aerolatum 5715S-9(T) and Rubellimicrobium thermophilum DSM 16684(T), respectively. Q-10 was the predominant respiratory ubiquinone as in the other members of the genus Rubellimicrobium. The major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphoglycolipid, glycolipid and the major fatty acids were C18:1 omega7c, C16:0 and C10:0 3-OH, which are very different from the valid published species. The DNA G + C content was 67.7 mol%. Both phylogenetic and chemotaxonomic evidence supports that YIM 48858(T) is a novel species of the genus Rubellimicrobium, for which the name Rubellimicrobium roseum sp. nov. is proposed. The type strain is YIM 48858(T) (=CCTCC AA 208029(T) =KCTC 23202(T)).

[Effects of furadan on soil enzyme activity].

By using simulation method, this paper studied the effects of furadan on the activities of soil urease, invertase and alkaline phosphatase, with the affecting factors investigated. The results showed that after adding furadan into soil, soil urease activity was decreased first but increased then, indicating that the ecotoxicity of furadan was reduced gradually. When the concentration of furadan was less than 0.3%, soil urease, invertase and alkaline phosophatase were all activated, and in some soil samples, a significant positive correlation was observed between soil invertase activity and furadan concentration, suggesting that soil invertase activity could be used as an indicator for the soil pollution caused by furadan. It was concluded that the quality of soil and ecological environment would be kept safe under low concentrations of furadan (< or = 0.3%).

Single and joint effects of pesticides and mercury on soil urease.

The influence of two pesticides including chlorimuron-ethyl and furadan and mercury (Hg) on urease activity in 4 soils (meadow burozem and phaeozem) was investigated. The soils were exposed to various concentrations of the two pesticides and Hg individually and simultaneously. Results showed that there was a close relationship between urease activity and organic matter content in soil. Chlorimuron-ethyl and furadan could both activate urease in the 4 soils. The maximum increment of urease activity by chlorimuronethyl was up to 14%-18%. There was almost an equal increase (up to 13%-21%) in the urease activity by furadan. On the contrary, Hg markedly inhibited soil urease activity. A logarithmic equation was used to describe the relationship (P<0.05) between the concentration of Hg and the activity of soil urease in the 4 tested soils. Semi-effect dose (ED50) values by the stress of Hg based on the inhibition of soil urease in the 4 soils were 88, 5.5, 24 and 20 mg/kg, respectively, according to the calculation of the corresponding equations. The interactive effect of chlorimuron-ethyl or furadan with metal Hg on soil urease was mainly synergic at the highest tested concentrations.

[Effects of Hg on soil enzyme activity].

With simulation test, this paper studied the effects of Hg on the activities of urease, invertase and neutral phosphotase in four soils. The results showed that Hg inhibited soil urease and invertase activities markedly, but its inhibitory effect differed with test soils. There was a significant logarithmic correlation between the concentration of HgCl2 and the activities of these two enzymes (P < 0.05). In test soils, the ED50 of urease activity was 87.99, 5.47, 24.05 and 19.88 mg x kg(-1), and that of invertase activity was 76.68, 727.49, 236.52 and 316.59 mg x kg(-1), respectively. Urease was more sensitive than invertase to Hg contamination, while organic matter had a protective effect on soil enzymes. Soil neutral phosphatase was not sensitive to Hg contamination, except that it was significantly activated by Hg in the meadow brown soil applied with plenty of organic fertilizer.

[Effect of temperature on kinetic of soil urease inhibited by Hg].

Urease kinetics inhibited by Hg in two meadow burozem, fertilized by different ways and two phaeozem soils with different organic matter content was investigated at different temperatures. The results showed that the urease activity and kinetic parameter V(max) and V(max)/K(m) were higher in soils with high organic matter content than that in soils with low organic matter among the same soil type. It indicated that organic matter had great adsorption capacity to urease. Soil urease V(max) and V(max)/K(m) in phaeozem were generally higher than that in meadow organic matter had great adsorption capacity to urease. Soil urease V(max) and V(max)/K(m) in phaeozem were generally higher than that in meadow burozem, but urease activity and K(m) were not comparable among different soil types. K(m) depended on not only the organic matter content of soils, but also fertilization ways. As incubating temperature increased, urease activity, V(max) and V(max)/K(m) value enhanced under the optical catalysis temperature. Hg behaved as uncompetitive inhibitor to soil urease in this experiment. The negative effect that Hg inhibited urease activity, K(m), V(max) and V(max)/K(m) increased with temperature increasing, indicated that the protective capacity of soil on urease decreased with the temperature increasing.

Relationships between humic substance-bound mercury contents and soil properties in subtropical zone.

The bioavailability of humic substance-bound mercury (HS-Hg) has been established, while the distribution of HS-Hg in soils in relation to soil properties remains obscure. Path analysis and principal component analysis were employed in present study to investigate how soil factors influence the contents of HS-Hg in soils. Results showed that HS-Hg ranged from 0.0192 to 0.2051 mg/kg in soils. The two fractions existed in soils as humic acid-bound mercury (HA-Hg) > fulvic acid-bound mercury (FA-Hg) and the ratio of HA-Hg/FA-Hg was 1.61 on the average. Soil organic carbon (OC) and HS favorably determined soil HS-Hg and the two fractions. The mercury source forming HS-Hg derived from soil total mercury and HS-Hg. FA-Hg and HA-Hg served as mercury source for each other. In acidic soils, FA-Hg and HA-Hg consistently rose with the increase of OC, and generally HA-Hg increased more dramatically. Soils with lower pH and lighter texture contained more HS-Hg, particularly fraction of FA-Hg. Among all influencing factors, organic material source showed the strongest effect, followed by other soil properties and soil mercury source.