Phylogenetic and molecular characterization of coxsackievirus A24 variant isolates from a 2010 acute hemorrhagic conjunctivitis outbreak in Guangdong, China

<p>Abstract</p> <p>Background</p> <p>Acute hemorrhagic conjunctivitis is a common disease in China. As a notifiable disease, cases are registered by ophthalmologists on the AHC surveillance system. An AHC outbreak caused by CA24v was observed in Guangdong Province in 2007 by the National Disease Supervision Information Management System. Three years later, a larger outbreak occurred in Guangdong during the August-October period (2010). To characterize the outbreak and compare the genetic diversity of CA24v, which was determined to be the cause of the outbreak, the epidemiology and the molecular characterization of CA24v were analyzed in this study.</p> <p>Results</p> <p>A total of 69,635 cases were reported in the outbreak. 73.5% of index cases originated from students, children in kindergarten and factory workers, with the ≦ 9 age group at the highest risk. The male to female ratio was 1.84:1 among 0-19 years. 56 conjunctival swabs were collected to identify the causative agent from five cities with the AHC outbreak. 30 virus strains were isolated, and two of the genomes had the highest identity values (95.8%) with CA24v genomes. Four CA24v genotypes were identified by phylogenetic analysis for the VP1 and 3C regions. CA24v which caused the outbreak belonged to genotype IV. Furthermore, full nucleotide sequences for four representative isolates in 2010 and 2007 were determined and compared. 20 aa mutations, two nt insertions and one nt deletion were observed in the open reading frame, with 5'- and 3'- UTR respectively between them.</p> <p>Conclusions</p> <p>CA24v was determined to be the pathogen causing the outbreak and belongs to genotype IV. VP1 is more informative than 3C^Profor describing molecular epidemiology and we hypothesize that accumulative mutations may have promoted the outbreak.</p

Single Endemic Genotype of Measles Virus Continuously Circulating in China for at Least 16 Years

The incidence of measles in China from 1991 to 2008 was reviewed, and the nucleotide sequences from 1507 measles viruses (MeV) isolated during 1993 to 2008 were phylogenetically analyzed. The results showed that measles epidemics peaked approximately every 3 to 5 years with the range of measles cases detected between 56,850 and 140,048 per year. The Chinese MeV strains represented three genotypes; 1501 H1, 1 H2 and 5 A. Genotype H1 was the predominant genotype throughout China continuously circulating for at least 16 years. Genotype H1 sequences could be divided into two distinct clusters, H1a and H1b. A 4.2% average nucleotide divergence was found between the H1a and H1b clusters, and the nucleotide sequence and predicted amino acid homologies of H1a viruses were 92.3%–100% and 84.7%–100%, H1b were 97.1%–100% and 95.3%–100%, respectively. Viruses from both clusters were distributed throughout China with no apparent geographic restriction and multiple co-circulating lineages were present in many provinces. Cluster H1a and H1b viruses were co-circulating during 1993 to 2005, while no H1b viruses were detected after 2005 and the transmission of that cluster has presumably been interrupted. Analysis of the nucleotide and predicted amino acid changes in the N proteins of H1a and H1b viruses showed no evidence of selective pressure. This study investigated the genotype and cluster distribution of MeV in China over a 16-year period to establish a genetic baseline before MeV elimination in Western Pacific Region (WPR). Continuous and extensive MeV surveillance and the ability to quickly identify imported cases of measles will become more critical as measles elimination goals are achieved in China in the near future. This is the first report that a single endemic genotype of measles virus has been found to be continuously circulating in one country for at least 16 years

Etiologic Diagnosis of Lower Respiratory Tract Bacterial Infections Using Sputum Samples and Quantitative Loop-Mediated Isothermal Amplification

Etiologic diagnoses of lower respiratory tract infections (LRTI) have been relying primarily on bacterial cultures that often fail to return useful results in time. Although DNA-based assays are more sensitive than bacterial cultures in detecting pathogens, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. Here we report a nationwide cohort study on 2986 suspected LRTI patients across P. R. China. We compared the performance of a DNA-based assay qLAMP (quantitative Loop-mediated isothermal AMPlification) with that of standard bacterial cultures in detecting a panel of eight common respiratory bacterial pathogens from sputum samples. Our qLAMP assay detects the panel of pathogens in 1047(69.28%) patients from 1533 qualified patients at the end. We found that the bacterial titer quantified based on qLAMP is a predictor of probability that the bacterium in the sample can be detected in culture assay. The relatedness of the two assays fits a logistic regression curve. We used a piecewise linear function to define breakpoints where latent pathogen abruptly change its competitive relationship with others in the panel. These breakpoints, where pathogens start to propagate abnormally, are used as cutoffs to eliminate the influence of contaminations from normal flora. With help of the cutoffs derived from statistical analysis, we are able to identify causative pathogens in 750 (48.92%) patients from qualified patients. In conclusion, qLAMP is a reliable method in quantifying bacterial titer. Despite the fact that there are always latent bacteria contaminated in sputum samples, we can identify causative pathogens based on cutoffs derived from statistical analysis of competitive relationship

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

The Application of Space Vector in Calculation of Plinth Elevation Angle

The plinth elevation angle was related to the upstream slope and face slab shape of CFRD, as well as to the dam axis, which was very important in plinth design. Based on the space vector algorithm, the function established consists of plinth elevation angle and three-dimensional coordinate of key points of plinth line, which was taken as the unique input variable, and then the plinth elevation angle was directly obtained. The improved vector method with characteristics of high efficiency, simple input parameters and clear spatial concept, is obviously superior to the traditional geometric method which was deduce by doing the auxiliary line, and it has been widely applied and well verified in engineering

Effects of One-Year Simulated Nitrogen and Acid Deposition on Soil Respiration in a Subtropical Plantation in China

Atmospheric nitrogen (N) and acid deposition have become global environmental issues and are likely to alter soil respiration (Rs); the largest CO2 source is from soil to the atmosphere. However, to date, much less attention has been focused on the interactive effects and underlying mechanisms of N and acid deposition on Rs, especially for ecosystems that are simultaneously subjected to elevated levels of deposition of both N and acid. Here, to examine the effects of N addition, acid addition, and their interactions with Rs, we conducted a two-way factorial N addition (control, CK; 60 kg N ha−1 a−1, LN; 120 kg N ha−1 a−1, HN) and acid addition (control, CK; pH 4.5, LA; pH 2.5, HA) field experiment in a subtropical plantation in China. Our results showed the following: (1) During the one-year observation period, the seasonal dynamics of Rs presented a single peak curve model, which was closely related to the surface soil temperature. (2) The simulated N deposition and acid deposition significantly decreased the Rs in the subtropical plantation. Compared to the CK plots, the LN and HN treatments reduced the annual mean values of Rs by 41% and 56%, and the annual mean values of Rs were inhibited by 26% and 31% in the LA and HA plots. The inhibition of N application on Rs was stronger than that of the simulated acid deposition. (3) Significant interactions between N addition and acid addition on Rs were detected, and Rs was significantly inhibited under four co-addition treatments. (4) The underlying mechanism and main reason for the responses of Rs to simulated N and acid deposition in this study might be the inhibition of soil microbial biomass and soil enzyme activity due to soil acidification under increased N and acid input

The Effects of Biochar on Microbial Community Composition in and Beneath Biological Soil Crusts in a <i>Pinus massoniana</i> Lamb. Plantation

Biological soil crusts (BSCs) hold promise for reducing soil erosion in subtropical forest plantations, and microorganisms profoundly affect the formation and development of BSCs. The effects of biochar as a soil conditioner on the diversity and structure of soil microbial communities in BSCs are largely unknown. Therefore, our aim was to determine how biochar might improve microbial community composition and BSC function. Herein, a field experiment was conducted in a P. massoniana plantation; the addition of biochar was the treatment, and no biochar addition was the control (CK). Soil microbial communities associated with moss BSCs (in and beneath BSCs) with and without the addition of biochar were analyzed by Illumina sequencing technology. The results showed that Acidobacteria (28.35%), Proteobacteria (22.53%), Actinobacteria (17.41%), and Chloroflexi (16.74%) were the dominant bacterial phyla, whereas Basidiomycota (70.00%) and Ascomycota (22.76%) were the dominant fungal phyla in BSCs. The soil bacterial and fungal OTU number and richness in BSCs were higher than those beneath BSCs. The relative abundances of Acidobacteria, Chloroflexi, and Basidiomycota were higher in BSCs than beneath BSCs, whereas the relative abundances of Actinobacteria, Firmicutes, Ascomycota, and Chytridiomycota showed the opposite trend. Beneath BSCs, biochar addition increased the soil bacterial OTU number and richness (ACE index and Chao1) but decreased the soil fungal OTU number and richness. Biochar had little effect on soil microbial community structures in BSCs; however, beneath BSCs, it significantly increased the relative abundances of Acidobacteria, Chloroflexi, and Basidiomycota and significantly decreased the relative abundances of Actinobacteria, Firmicutes, Ascomycota, and Chytridiomycota. Biochar-induced changes in soil microbial communities were related to soil environmental factors, especially urease activity, organic matter content, pH, total nitrogen content, and sucrase activity. We demonstrated the different effects of biochar on soil microbial communities in and beneath the BSCs of subtropical forest plantations; these findings provided new insights into soil stabilization with BSCs below the forest canopy in subtropical regions