New genotype invasion of dengue virus serotype 1 drove massive outbreak in Guangzhou, China.

BACKGROUND: Dengue fever is a mosquito-borne infectious disease that has caused major health problems. Variations in dengue virus (DENV) genes are important features of epidemic outbreaks. However, the associations of DENV genes with epidemic potential have not been extensively examined. Here, we assessed new genotype invasion of DENV-1 isolated from Guangzhou in China to evaluate associations with epidemic outbreaks. METHODOLOGY/PRINCIPAL FINDINGS: We used DENV-1 strains isolated from sera of dengue cases from 2002 to 2016 in Guangzhou for complete genome sequencing. A neighbor-joining phylogenetic tree was constructed to elucidate the genotype characteristics and determine if new genotype invasion was correlated with major outbreaks. In our study, a new genotype invasion event was observed during each significant outbreak period in 2002-2003, 2006-2007, and 2013-2014. Genotype II was the main epidemic genotype in 2003 and before. Invasion of genotype I in 2006 caused an unusual outbreak with 765 cases (relative risk [RR] = 16.24, 95% confidence interval [CI] 12.41-21.25). At the middle and late stages of the 2013 outbreak, genotype III was introduced to Guangzhou as a new genotype invasion responsible for 37,340 cases with RR 541.73 (95% CI 417.78-702.45), after which genotypes I and III began co-circulating. Base mutations occurred after new genotype invasion, and the gene sequence of NS3 protein had the lowest average similarity ratio (99.82%), followed by the gene sequence of E protein (99.86%), as compared to the 2013 strain. CONCLUSIONS/SIGNIFICANCE: Genotype replacement and co-circulation of multiple DENV-1 genotypes were observed. New genotype invasion was highly correlated with local unusual outbreaks. In addition to DENV-1 genotype I in the unprecedented outbreak in 2014, new genotype invasion by DENV-1 genotype III occurred in Guangzhou.

Characteristics of and variation in airborne ARGs among urban hospitals and adjacent urban and suburban communities: A metagenomic approach.

Environmental antibiotic resistance genes (ARGs) have received much attention, while the characteristics of ARGs carried by particulate matter (PM) as a function of urban functional region are almost unknown. In this study, ARGs carried by PM2.5 and PM10 in an urban hospital, a nearby urban community and the nearest suburban community were detected using metagenomics. In total, 643 ARG subtypes belonging to 22 different ARG types were identified. The chloramphenicol exporter gene, sul1, bacA, and lnuA were the most abundant ARG subtypes in all air samples. The hospital exhibited higher ARG abundance and richness than the nearby communities. ARG profiles depended on functional region: hospital and suburban samples clustered separately, and samples from the nearby urban community interspersed among them. The representation of multidrug and quinolone resistance genes decayed with distance from the hospital to the urban community to the suburban community, indicating that hospital PM may be a hotspot for ARGs encoding proteins conferring multidrug and quinolone resistance. Airborne ARGs carried by PM in the hospital environment were more closely associated with clinically important pathogens than were those in nearby communities. In particular, carbapenemase genes, including blaNDM,blaKPC,blaIMP,blaVIM,and blaOXA-48, were discovered in hospital PM. In the suburban community, crAssphage, a human host-specific bacteriophage, was applied to predict ARG abundance and found to be enriched due to anthropogenic pollution but showed no clear evidence for ARG selection. In the hospital and the nearby urban community, the drivers of ARGs were complex. Our results highlighted that PM ARGs were closely related to human activities and revealed a potential hotspot, which could provide new evidence for further research and consequently mitigate the formation of airborne ARGs and transfer risks.

Rapid Detection of Bacillus cereus Using Cross-Priming Amplification.

Bacillus cereus is a spore-forming gastrointestinal pathogen that can cause life-threatening diseases. Here, a simple and effective assay to detect B. cereus was developed, using cross-priming amplification (CPA). Amplicons were detected using disposable cartridges that contained nucleic acid detection strips. The sensitivity of CPA assay for B. cereus was assessed using serial dilutions of genomic DNA, which indicated a detection limit of 3.6 × 101 CFU/mL. No cross-reactions were detected when genomic DNA extracted from 12 different B. cereus strains and 20 other bacterial foodborne strains were tested, suggesting that the assay is highly specific. Finally, we evaluated the practical applications of the CPA assay for the detection of B. cereus in 150 food samples and found that its sensitivity and specificity, compared with real-time PCR, were approximately 98.18 and 100%, respectively. In conclusion, CPA combined with nucleic acid detection strips is easy to perform, requires simple equipment, and offers highly specific and sensitive B. cereus detection.

Evolutionary and phylodynamic analyses of Dengue virus serotype I in Guangdong Province, China, between 1985 and 2015.

To determine the evolutionary and phylodynamic history of DENV-1 in Guangdong, the strains detected between 1985 and 2015 were determined with phylogenetic and Bayesian analyses of the E gene. Three DENV-1 genotypes (I, V, and VI) were circulating in Guangdong, and genotype I was detected most frequently. The evolutionary rate of DENV-1 was estimated to be 1.03 × 10-3 nucleotide substitutions/site/year. The most recent ancestor of the viruses existed approximately 141 years ago. The observed epidemiological dynamics correlated with similar fluctuations in diversity, and the epidemiological dynamics of DENV-1 transmission reflect dramatic changes in the viral population sizes. Two recombination events were identified in those strains. The selection pressures were estimated and revealed an abundance of negatively selected sites but few positively selected sites. These data improve our understanding of the evolution and molecular epidemiology of DENV-1 and provide insights that will facilitate the surveillance and control of DENV-1.

Fluorescence resonance energy transfer between bovine serum albumin and fluoresceinamine.

Physical binding-mediated organic dye direct-labelling of proteins could be a promising technology for bio-nanomedical applications. Upon binding, it was found that fluorescence resonance energy transfer (FRET) occurred between donor bovine serum albumin (BSA; an amphiphilic protein) and acceptor fluoresceinamine (FA; a hydrophobic fluorophore), which could explain fluorescence quenching found for BSA. FRET efficiency and the distance between FA and BSA tryptophan residues were determined to 17% and 2.29 nm, respectively. Using a spectroscopic superimposition method, the saturated number of FAs that bound to BSA was determined as eight to give a complex formula of FA8-BSA. Finally, molecular docking between BSA and FA was conducted, and conformational change that occurred in BSA upon binding to FA molecules was also studied by three-dimensional fluorescence microscopy.

Circulation of Coxsackievirus A6 in hand-foot-mouth disease in Guangzhou, 2010-2012.

BACKGROUND: Hand, foot and mouth disease (HFMD) is usually caused by Enterovirus 71(EV71), and Coxsackievirus A16 (CV-A16) in Guangzhou, the biggest city of South China. However, Coxsackievirus A6 (CV-A6) were observed increased dramatically from 2010-2012. METHODS: In order to understand and to describe the epidemiologic and genetic characteristics of CV-A6, specimens of 5482 suspected HFMD cases were collected and examined by real-time fluorescence PCR. All samples positive for enteroviruses were analyzed by descriptive statistics. Phylogenetic analysis of CV-A6 based on the VP1 sequences was performed to investigate molecular and evolutionary characteristics. RESULTS: Coxsackievirus A6 increased dramatically from 9.04% in 2010 to 23.21% in 2012 and became one of the main causative agents of HFMD in Guangzhou. CV-A6 attack rates were highest in one to two year olds (33.14%). Typical clinic symptoms of CV-A6 HFMD include fever (589/720, 81.81%), maculopopular rash and vesicular exanthema around the perioral area (408/720, 56.66%), intraoral (545/720, 75.69%), the buttock (395/720, 54.86%), the trunk (244/720, 33.89%), the knee (188/720, 26.11%), and the dorsal aspects of hands (437/720, 60.69%). Phylogenetic analysis showed the CV-A6 isolates in this study belonged to Cluster A1 and were similar to those found in Shanghai in 2011 and 2012 (JX495148, KC414735), Shenzhen in 2011 (JX473394), Japan in 2011 (AB649243, AB649246), France in 2010(HE572928), Thailand in 2012(JX556564) and Israel in 2012 and 2013(.KF991010, KF991012).

High-spin states and level structure in stable nucleus strontium-84.

High-spin states of (84)Sr are populated through the reaction (70)Zn ((18)O, 4n) (84)Sr at the beam energy of 75 MeV. The measurements of excitation functions, γ-γ coincidences, directional correlations of oriented states (DCO) ratios and γ-transition intensities are performed using eight anticompton HPGe detectors and one planar HPGe detector. Based on the experimental results, we establish a new level scheme of (84)Sr, in which 12 new states and nearly 30 new γ-transitions are identified in the present work. The positive-parity yrast band is extended to spin I(π) = 24(+), while one negative-parity band is extended to spin I(π) = 19(-) and it is found that the even-spin and odd-spin members in high-spin states show the nature of signature staggering. The deformation of (84)Sr is studied by calculating the total-Routhian-surfaces (TRS) of positive-parity yrast states in the cranked shell model formalism.

Complete genome sequence of dengue virus serotype 4 from guangzhou, china.

In 2010, the first complete genome sequence of a dengue virus serotype 4 genotype II strain was reported in Guangzhou, China. Here, we report another isolated strain belonging to the genotype II. Our results will offer help to dengue virus control and precautions.

Molecular characterization of the envelope gene of dengue virus type 3 newly isolated in Guangzhou, China, during 2009-2010.

BACKGROUND: After an absence of 29 years, dengue virus type 3 (DENV-3) re-emerged in Guangzhou in 2009 and again in 2010. However, the geographical route by which the virus entered the city, and how it has changed genetically, remain unclear. Therefore, we carried out a comprehensive investigation into the molecular characteristics of the DENV-3 involved. METHODS: The envelope (E) genes of viruses isolated from dengue patients during the 2009-2010 epidemics were sequenced and compared with previously published E gene sequences of global representative DENV-3 strains available in GenBank, including isolates circulating in other provinces of China. RESULTS: A total of 13 isolates (seven from 2009 and six from 2010) were obtained from human serum samples. Phylogenetic analysis revealed that the isolates were grouped into three genotypes (I, III, and V) and then two clades within genotype III (genotype I from Indonesia, genotype III clade A from Côte d'Ivoire, genotype III clade B from Tanzania, and genotype V from Philippines). In addition, there were 1.3-9.0% and 0.5-3.9% differences in the nucleic and deduced amino acid sequences between the 2009 and 2010 strains, respectively. CONCLUSIONS: The DENV-3 viruses from the period 2009-2010 were not from the continuous spread of an epidemic strain or the re-emergence of the 2009 strains in the 2-year period. The introduction of different DENV-3 genotypes following more than one geographical route was an important contributing factor to the 2009-2010 dengue epidemics in Guangzhou.

Complete genome sequence of dengue virus serotype 3 from guangzhou, china.

In 2009, dengue virus serotype 3 (DENV-3) was first detected in Guangzhou, China. In this study, we identified another isolated strain belonging to genotype II. Phylogenetic analysis shows that the GZ/10476/2012 strain has a close relationship with the DENV-3 genotype II from Southeast Asian strains.

Re-emergence of dengue virus type 3 in Canton, China, 2009-2010, associated with multiple introductions through different geographical routes.

BACKGROUND: Endemic dengue virus type 3 (DENV-3) infections have not been reported in Canton, China, since 1980. In March 2009, DENV-3 was isolated for the second time, occurring about 30 years after the previous circulation. In August, 3 other cases emerged. One much larger outbreak occurred again in 2010. To address the origin and particularly to determine whether the outbreaks were caused by the same viral genotype, we investigated the epidemiological and molecular characteristics of the introduction, spread and genetic microevolution of DENV-3 involved. METHODOLOGY/PRINCIPAL FINDINGS: Three imported cases (index-1,2,3) separately traveled back from Vietnam, India and Tanzania, resulted in 1, 3 and 60 secondary autochthonous cases, respectively. In autochthonous cases, 64.6% positive in IgM anti-DENV and 18.6% in IgG from a total of 48 submitted serum samples, accompanied by 7 DENV-3 isolates. With 99.8%, 99.7%, and 100% envelope gene nucleotidic identity, 09/GZ/1081 from index-1 and endemic strain (09/GZ/1483) belonged to genotype V; 09/GZ/10616 from index-2 and endemic strains (09/GZ/11144 and 09/GZ/11194) belonged to genotype III Clade-A; and 10/GZ/4898 from index-3 and all four 2010 endemic DENV-3 strains belonged to genotype III Clade-B, respectively. CONCLUSIONS/SIGNIFICANCE: Both epidemiological and phylogenetic analyses showed that the 2010 outbreak of dengue was not a reemergence of the 2009 strain. Introductions of different genotypes following more than one route were important contributory factors for the 2009-2010 dengue epidemics/outbreaks in Canton. These findings underscore the importance of early detection and case management of imported case in preventing large-scale dengue epidemics among indigenous peoples of Canton.

Molecular epidemiological and virological study of dengue virus infections in Guangzhou, China, during 2001-2010.

BACKGROUND: Dengue virus (DENV) infection is the most prevalent arthropod-borne viral infection in tropical and subtropical regions worldwide. Guangzhou has the ideal environment for DENV transmission and DENV epidemics have been reported in this region for more than 30 years. METHODS: Information for DENV infection cases in Guangzhou from 2001 to 2010 were collected and analyzed. The DENV strains were cultured and isolated from patients' sera. Viral RNA was extracted from cell culture supernatants. cDNA was synthesized by reverse transcription PCR. Phylogenetic trees of four DENV serotypes were constructed respectively. RESULTS: In total, 2478 DENV infection cases were reported; 2143 of these (86.43%) occurred during 3 months of the year: August, September and October. Of these, 2398 were local cases (96.77%) and 80 were imported cases (3.23%). Among the imported cases, 69 (86.25%) were from Southeast Asian countries. From the 90 isolated strains, 66.67%, 3.33%, 14.44%, and 15.56% belonged to DENV serotypes 1, 2, 3, and 4, respectively. DENV-1 was predominant in most of the years, including during 2 outbreaks in 2002 and 2006; however, none of the strains or genotypes identified in this study were found to be predominant. Interestingly, DENV strains from different years had different origins. Moreover, the strains from each year belonged to different serotypes and/or genotypes. CONCLUSIONS: Southeast Asia countries were found to be the possible source of DENV in Guangzhou. These findings suggest that there is increasing diversity in DENV strains in Guangzhou, which could increase the risk of DENV outbreaks in the near future.

Novel virostatic agents against bluetongue virus.

Bluetongue virus (BTV), a member in the family Reoviridae, is a re-emerging animal disease infecting cattle and sheep. With its recent outbreaks in Europe, there is a pressing need for efficacious antivirals. We presented here the identification and characterization of a novel virostatic molecule against BTV, an aminothiophenecarboxylic acid derivative named compound 003 (C003). The virostatic efficacy of C003 could be improved via chemical modification, leading to a de novo synthesized compound 052 (C052). The 50% effective concentrations (EC(50)) of C003 and C052 were determined at 1.76 ± 0.73 µM and 0.27 ± 0.12 µM, respectively. The 50% cytotoxicity concentration (CC(50)) of C003 was over 100 µM and the CC(50) of C052 was at 82.69 µM. Accordingly, the 50% selective index (SI(50)) of C003 and C052 against BTV was over 57 and 306, respectively. The inhibitory effect of C003/C052 on BTV-induced apoptosis was also confirmed via the inhibition of caspase-3/-7 activation post BTV infection. C003/C052 could inhibit BTV induced CPE even when added as late as 24 h.p.i., indicating that they might act at late stage of viral life-cycle. C003/C052 could reduce over two-logs of both the progeny virus production and the number of genomic viral RNA copies. Interestingly, both the activation of host autophagy and viral protein expression were inhibited post BTV infection when cells were treated with C003 and C052, suggesting that C003/C052 might act as virostatic agents via inhibiting host autophagy activation. Although further investigations might be needed to pin down the exact mechanism of C003/C052, our finding suggested that these compounds might be potent lead compounds with potential novel mechanism of action against BTV.

Defective activities, but not secretions, resulting from gene point mutations of human mannan-binding lectin.

Human mannan-binding lectin (MBL) plays a pivotal role in innate immunity. Substantial literature supports the belief that three point mutations, CGT52TGT, GGC54GAC and GGA57GAA, in the collagen-like region (CLR) of the human MBL gene, are associated with increased susceptibility to infection, autoimmunity and carcinogenesis. To investigate the mechanisms of MBL deficiency, human wild-type and three variant MBL genes were expressed in COS-7 and Chinese hamster ovary (CHO) cells. Results showed that no apparent differences were found among the levels of gene transcription and protein secretion of four forms of MBL. However, the degree of oligomerization of variant forms of MBL was found to be much lower than that of recombinant human wild-type MBL. The ability of variant MBL proteins to bind mannan was much weaker than that of the wild-type MBL protein, and the MBL variants failed to effectively activate the complement lectin pathway. These data suggested that a lower order oligomer, but not decreased plasma levels of MBL, may be the main result of MBL gene mutations and may be associated with immunodeficiency.

Molecular epidemiology of dengue viruses in southern China from 1978 to 2006.

To investigate molecular epidemiology of dengue viruses (DENV) in southern China, a total of 14 dengue isolates were collected in southern China during each epidemic year between 1978 and 2006 and their full-length genome sequences were obtained by using RT-PCR method. The E gene sequences from additional 6 dengue fever patients in Guangzhou in 2006 were also obtained by using RT-PCR method. Combined with DENVs sequences published in GenBank, phylogenetic analysis and recombination analysis were performed. One hundred and twenty-five E gene sequences and 60 complete genome sequences published in the GenBank were also involved. Phylogenetic analysis showed that there was a wide genetic diversity of DENVs isolated in southern China. DENV-1 strains exist in almost all of the clades of genotype I and IV except the Asia 1 clade of genotype I; DENV-2 stains are grouped into four of the five genotypes except American genotype. DENV-4 strains are grouped into 2 genotypes (I and II). Phylogenetic analysis also showed that all DENV-4 isolates and two DENV-2 isolates were closely related to the prior isolates from neighboring Southeast Asia countries. The DENV-1 strain isolated during the 2006 epidemic is highly homologous to the strains isolated during the 2001 epidemic.Recombination analysis showed no inter-serotype recombination, but 22 intra-serotype recombination events were found across the 32 complete genomes of all Chinese isolates. The study suggested that dengue fever epidemic in Southern China over the past 30 years presented two important modes, 1) imported-cases-induced endemic prevalence; 2) endogenous epidemic outbreak with natural epidemic focus. Recombination may play an important role in dengue virus evolution and adaptation.

The intrinsically high pitting corrosion resistance of mechanically polished nitinol in simulated physiological solutions.

Nitinol wires have been widely used in many biomedical applications, such as cardiovascular stent due to their superelasticity and shape memory effect. However, their corrosion properties and the related biocompatibility are not well understood, and the reported results are controversial. In this study, we evaluate the pitting corrosion property of nitinol, titanium, nickel, and 316L stainless steel (316LSS) wires with different surface roughnesses in a saline solution at 37 °C. The cyclic potentiodynamic polarization results show that mechanically polished nitinol and Ti wires are highly resistant to pitting corrosion, while Ni and 316LSS wires are susceptible to pitting corrosion. Electrochemical impedance spectroscopy is used to study the interface of oxide film/solution and all mechanically polished nitinol wires are covered by 2-3 nm thick films formed under open circuit potential. Furthermore, the electronic structures and semiconducting properties of passive films on nitinol, Ti and Ni wires are studied by Mott-Schottky analysis. Passive films formed on nitinol and Ti exhibit n-type semiconducting characteristics, whereas films on Ni show p-type semiconducting characteristics. Scanning Kelvin Microscopy is used to measure the surface potential difference between common inclusions from the nitinol matrix and the results indicate that the inclusions are more electrochemically noble than the nitinol matrix. Band energy theory is used to model the electrochemical interface between the passive films of nitinol and the solution under different applied potential conditions. A mechanism for the strong pitting corrosion resistance of nitinol in saline solution is proposed.

[Molecular epidemiologic analysis on new emerged type 3 dengue virus in Guangzhou in 2009].

OBJECTIVE: To analyze and trace the infection source the envelope (E) gene of the new emerged type 3 dengue virus in Guangzhou in 2009. METHODS: Sera were collected from patients infected with local dengue fever. Dengue virus was cultured and isolated by C6/36 cells. The whole length E gene was amplified from the positive specimen by RT-PCR, thereby sequenced and phylogenetic tree drawn by neighbor-joining method. Both data on epidemiologic and molecular studies were processed and analysed. RESULTS: 7 strains of type 3 dengue virus were isolated from samples of the 19 patients. E gene of these strains was amplified. The complete E genes of 7 strains belonged to 1479 nucleotides in length, encoding a polyprotein of 493 amino acids. Data from the phylogenetic analysis showed that 09/GZ/1081, 09/GZ/1483 and 09/GZ/10806 strains fell within the Southeast Asia/South Pacific group. 09/GZ/10616, 09/GZ/11144, 09/GZ/11194 while 09/GZ/13105 strains fell within the India group. CONCLUSION: The type 3 dengue virus identified in Guangzhou area in 2009 was imported and could be decided into two genotypes.