Efficacy of Coxsackievirus A5 Vaccine Candidates in an Actively Immunized Mouse Model.

Coxsackievirus A5 (CV-A5) has recently emerged as a main hand, foot, and mouth disease (HFMD) pathogen. Following a large-scale vaccination campaign against enterovirus 71 (EV-71) in China, the number of HFMD-associated cases with EV-71 was reduced, especially severe and fatal cases. However, the total number of HFMD cases remains high, as HFMD is also caused by other enterovirus serotypes. A multivalent HFMD vaccine containing 4 or 6 antigens of enterovirus serotypes is urgently needed. A formaldehyde-inactivated CV-A5 vaccine derived from Vero cells was used to inoculate newborn Kunming mice on days 3 and 10. The mice were challenged on day 14 with a mouse-adapted CV-A5 strain at a dose that was lethal for 14-day-old suckling mice. Within 14 days postchallenge, groups of mice immunized with three formulations, empty particles (EPs), full particles (FPs), and a mixture of the EP and FP vaccine candidates, all survived, while 100% of the mock-immunized mice died. Neutralizing antibodies (NtAbs) were detected in the sera of immunized mice, and the NtAb levels were correlated with the survival rate of the challenged mice. The virus loads in organs were reduced, and pathological changes and viral protein expression were weak or not observed in the immunized mice compared with those in alum-inoculated control mice. Another interesting finding was the identification of CV-A5 dense particles (DPs), facilitating morphogenesis study. These results demonstrated that the Vero cell-adapted CV-A5 strain is a promising vaccine candidate and could be used as a multivalent HFMD vaccine component in the future.IMPORTANCE The vaccine candidate strain CV-A5 was produced with a high infectivity titer and a high viral particle yield. Three particle forms, empty particles (EPs), full particles (FPs), and dense particles (DPs), were obtained and characterized after purification. The immunogenicities of EP, FP, and the EP and FP mixture were evaluated in mice. Mouse-adapted CV-A5 was generated as a challenge strain to infect 14-day-old mice. An active immunization challenge mouse model was established to evaluate the efficacy of the inactivated vaccine candidate. This animal model mimics vaccination, similar to immune responses of the vaccinated. The animal model also tests protective efficacy in response to the vaccine against the disease. This work is important for the preparation of multivalent vaccines against HFMD caused by different emerging strains.

Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis.

Reverse genetics systems have been established for all major groups of plant DNA and positive-strand RNA viruses, and our understanding of their infection cycles and pathogenesis has benefitted enormously from use of these approaches. However, technical difficulties have heretofore hampered applications of reverse genetics to plant negative-strand RNA (NSR) viruses. Here, we report recovery of infectious virus from cloned cDNAs of a model plant NSR, Sonchus yellow net rhabdovirus (SYNV). The procedure involves Agrobacterium-mediated transcription of full-length SYNV antigenomic RNA and co-expression of the nucleoprotein (N), phosphoprotein (P), large polymerase core proteins and viral suppressors of RNA silencing in Nicotiana benthamiana plants. Optimization of core protein expression resulted in up to 26% recombinant SYNV (rSYNV) infections of agroinfiltrated plants. A reporter virus, rSYNV-GFP, engineered by inserting a green fluorescence protein (GFP) gene between the N and P genes was able to express GFP during systemic infections and after repeated plant-to-plant mechanical passages. Deletion analyses with rSYNV-GFP demonstrated that SYNV cell-to-cell movement requires the sc4 protein and suggested that uncoiled nucleocapsids are infectious movement entities. Deletion analyses also showed that the glycoprotein is not required for systemic infection, although the glycoprotein mutant was defective in virion morphogenesis. Taken together, we have developed a robust reverse genetics system for SYNV that provides key insights into morphogenesis and movement of an enveloped plant virus. Our study also provides a template for developing analogous systems for reverse genetic analysis of other plant NSR viruses.

Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus.

BACKGROUND: Recovery of recombinant negative-stranded RNA viruses from cloned cDNAs is an inefficient process as multiple viral components need to be delivered into cells for reconstitution of infectious entities. Previously studies have shown that authentic viral RNA termini are essential for efficient virus rescue. However, little is known about the activity of viral RNAs processed by different strategies in supporting recovery of plant negative-stranded RNA virus. METHODS: In this study, we used several versions of hammerhead ribozymes and a truncated cauliflower mosaic virus 35S promoter to generate precise 5' termini of sonchus yellow net rhabdovirus (SYNV) antigenomic RNA (agRNA) derivatives. These agRNAs were co-expressed with the SYNV core proteins in Nicotiana benthamiana leaves to evaluate their efficiency in supporting fluorescent reporter gene expression from an SYNV minireplicon (MR) and rescue of full-length virus. RESULTS: Optimization of hammerhead ribozyme cleavage activities led to improved SYNV MR reporter gene expression. Although the MR agRNA processed by the most active hammerhead variants is comparable to the capped, precisely transcribed agRNA in supporting MR activity, efficient recovery of recombinant SYNV was only achieved with capped agRNA. Further studies showed that the capped SYNV agRNA permitted transient expression of the nucleocapsid (N) protein, and an agRNA derivatives unable to express the N protein in cis exhibited dramatically reduced rescue efficiency. CONCLUSION: Our study reveals superior activity of precisely transcribed, capped SYNV agRNAs to uncapped, hammerhead ribozyme-processed agRNAs, and suggests a cis-acting function for the N protein expressed from the capped agRNA during recovery of SYNV from plasmids.

HIV/AIDS epidemic among older adults in China during 2005-2012: results from trend and spatial analysis.

BACKGROUND: Recent studies have indicated an increasing burden of human immunodeficiency virus (HIV)/AIDS among older adults. METHODS: All identified people living with HIV/AIDS (PLWHA) recorded through the Chinese HIV/AIDS CRS during 2005-2012 were included in the study, except for the cases that lacked specific spatial information. Trend tests and spatial analyses were conducted. RESULTS: Information about 73,521 PLWHA (aged ≥50 years) was collected during 2005-2012. Three provinces-Guangxi, Henan, and Yunnan-accounted for 54.4% of the identified cases during the study period. Compared with 2005, the ratio between residents and migrants among the study population decreased to 40.1% in 2012. The ratio of HIV-infected patients to AIDS patients and the ratio of males to females increased gradually among older infected adults. Results of spatial analysis indicate a clustered distribution of HIV/AIDS among older adults throughout the country. Hot spots were observed in 4 provinces (Guangxi, Henan, Yunnan, and Sichuan) and 1 municipality (Chongqing). A trend from central provinces toward southern provinces was also identified. CONCLUSIONS: The number and proportion of HIV/AIDS among older adults have increased in recent years. The hot spots showed movement from central to southern China. A focused intervention strategy targeting the older PLWHA is urgently required in China.

The HIV, syphilis, and HCV epidemics among female sex workers in china: results from a serial cross-sectional study between 2008 and 2012.

BACKGROUND: Studies on the transmission of human immunodeficiency virus (HIV) and sexually transmitted diseases in female sex workers (FSWs) have been limited primarily to inferences drawn by focusing on defined geographical areas. METHODS AND FINDINGS: This serial cross-sectional study was conducted in mainland China from 2008 through 2012. Data for 827 079 participants was analyzed. We classified venues such as karaoke bars and hotels as high tier and venues such as hair salons and barbershops, massage parlors, and other public outdoor venues as low tier based on the participants' socioeconomic status. FSWs who worked at the venues and those who were present on the days of the survey were recruited. The prevalence of HIV decreased from 0.6% in 2008 to 0.3% in 2012, the syphilis prevalence ranged from 2.4% to 3.2% between 2008 and 2012, and hepatitis C virus (HCV) prevalence decreased from 0.9% in 2008 to 0.8% in 2012. Further, we found that HIV, syphilis, and HCV prevalence proportions were high in FSWs from low tiers. CONCLUSIONS: HIV, syphilis, and HCV prevalence among FSWs in our study decreased during the study period. Comprehensive intervention strategies, particularly those that focus on low-tier and older FSWs, are needed in order to decrease the disease burden in this population.

Construction of a Sonchus Yellow Net Virus minireplicon: a step toward reverse genetic analysis of plant negative-strand RNA viruses.

Reverse genetic analyses of negative-strand RNA (NSR) viruses have provided enormous advances in our understanding of animal viruses over the past 20 years, but technical difficulties have hampered application to plant NSR viruses. To develop a reverse genetic approach for analysis of plant NSR viruses, we have engineered Sonchus yellow net nucleorhabdovirus (SYNV) minireplicon (MR) reporter cassettes for Agrobacterium tumefaciens expression in Nicotiana benthamiana leaves. Fluorescent reporter genes substituted for the SYNV N and P protein open reading frames (ORFs) exhibited intense single-cell foci throughout regions of infiltrated leaves expressing the SYNV MR derivatives and the SYNV nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins. Genomic RNA and mRNA transcription was detected for reporter genes substituted for both the SYNV N and P ORFs. These activities required expression of the N, P, and L core proteins in trans and were enhanced by codelivery of viral suppressor proteins that interfere with host RNA silencing. As is the case with other members of the Mononegavirales, we detected polar expression of fluorescent proteins and chloramphenicol acetyltransferase substitutions for the N and P protein ORFs. We also demonstrated the utility of the SYNV MR system for functional analysis of SYNV core proteins in trans and the cis-acting leader and trailer sequence requirements for transcription and replication. This work provides a platform for construction of more complex SYNV reverse genetic derivatives and presents a general strategy for reverse genetic applications with other plant NSR viruses.

Analysis of miRNAs involved in mouse brain injury upon Coxsackievirus A6 infection.

Introduction: Coxsackievirus A6 (CV-A6) has emerged as the predominant epidemic strain responsible for hand, foot and mouth disease (HFMD). CV-A6 infection can result in severe clinical manifestations, including encephalitis, meningitis, and potentially life-threatening central nervous system disorders. Our previous research findings demonstrated that neonatal mice infected with CV-A6 exhibited limb weakness, paralysis, and ultimately succumbed to death. However, the underlying mechanism of CV-A6-induced nervous system injury remains elusive. Numerous reports have highlighted the pivotal role of miRNAs in various viral infections. Methods: Separately established infection and control groups of mice were used to create miRNA profiles of the brain tissues before and after CV-A6 transfection, followed by experimental verification, prediction, and analysis of the results. Results: At 2 days post-infection (dpi), 4 dpi, and 2dpi vs 4dpi, we identified 175, 198 and 78 significantly differentially expressed miRNAs respectively using qRT-PCR for validation purposes. Subsequently, we predicted target genes of these differentially expressed miRNAs and determined their potential targets through GO (Gene Ontology) enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis. Finally, we verified the miRNA-mRNA pairing via double luciferase experiments while confirming functional enrichment of target genes through Western Blotting analyses. Discussion: The results from this study suggest that transcriptional regulation, neuronal necrosis, pro-inflammatory cytokine release, and antiviral immunity are all implicated in the pathogenesis of central nervous system injury in mice infected with CV-A6. Brain injury resulting from CV-A6 infection may involve multiple pathways, including glial cell activation, neuronal necrosis, synaptic destruction, degenerative diseases of the nervous system. It can even encompass destruction of the blood-brain barrier, leading to central nervous system injury. The dysregulated miRNAs and signaling pathways discovered in this study provide valuable insights for further investigations into the pathogenesis of CV-A6.

Identification of Critical Amino Acids of Coxsackievirus A10 Associated with Cell Tropism and Viral RNA Release during Uncoating.

Coxsackievirus A10 (CV-A10) is a prevailing causative agent of hand-foot-mouth disease, necessitating the isolation and adaptation of appropriate strains in cells allowed for human vaccine development. In this study, amino acid sequences of CV-A10 strains with different cell tropism on RD and Vero cells were compared. Various amino acids on the structural and non-structural proteins related to cell tropism were identified. The reverse genetic systems of several CV-A10 strains with RD+/Vero- and RD+/Vero+ cell tropism were developed, and a set of CV-A10 recombinants were produced. The binding, entry, uncoating, and proliferation steps in the life cycle of these viruses were evaluated. P1 replacement of CV-A10 strains with different cell tropism revealed the pivotal role of the structural proteins in cell tropism. Further, seven amino acid substitutions in VP2 and VP1 were introduced to further investigate their roles played in cell tropism. These mutations cooperated in the growth of CV-A10 in Vero cells. Particularly, the valine to isoleucine mutation at the position VP1-236 (V1236I) was found to significantly restrict viral uncoating in Vero cells. Co-immunoprecipitation assays showed that the release of viral RNA from the KREMEN1 receptor-binding virions was restricted in r0195-V1236I compared with the parental strain r0195 (a RD+/Vero+ strain). Overall, this study highlights the dominant effect of structural proteins in CV-A10 adaption in Vero cells and the importance of V1236 in viral uncoating, providing a foundation for the mechanism study of CV-A10 cell tropism, and facilitating the development of vaccine candidates.

Humoral and cellular immunogenicity and efficacy of a coxsackievirus A10 vaccine in mice.

Coxsackievirus A10 (CV-A10) has become one of the major pathogens of hand, foot and mouth disease (HFMD), and studies on the vaccine and animal model of CV-A10 are still far from complete. Our study used a mouse-adapted CV-A10 strain, which was lethal for 14-day-old mice, to develop an infected mouse model. Then this model was employed to establish an actively immunized-challenged mouse model to evaluate the efficacy of a formaldehyde-inactivated CV-A10 vaccine, which was prepared from a Vero cell-adapted strain. CV-A10 vaccine at a dose of 0.5 or 2.0 µg was inoculated intraperitoneally in neonatal Kunming mice on the third and ninth day. Then the mice were challenged on day 14. The survival rate of mice immunized with 0.5 or 2.0 µg vaccine were 90% and 100%, respectively, while all Alum-inoculated mice died. Compared to those in the two vaccinated groups, the Alum-inoculated mice showed severe pathological damage, strong viral protein expression and high viral loads. The antisera from vaccinated mice showed high level of neutralizing antibodies against CV-A10. Meanwhile, three potential T cell epitopes located at the carboxyl-terminal regions of the VP1 and VP3 were identified and exhibited CV-A10 serotype-specific. The humoral and cellular immunogenicity analysis showed that immunization with two doses of the vaccine elicited CV-A10 specific neutralizing antibody and T cell response in BALB/c mice. Collectively, these findings indicated that this actively immunized-challenged mouse model will be invaluable in future studies on CV-A10 pathogenesis and evaluation of vaccine candidates.

Identification of a Conserved, Linear Epitope on VP3 of Enterovirus A Species Recognized by a Broad-Spectrum Monoclonal Antibody.

Outbreaks of hand, foot and mouth disease (HFMD) have occurred frequently in the Asian-Pacific region over the last two decades, caused mainly by the serotypes in Enterovirus A species. High-quality monoclonal antibodies (mAbs) are needed to improve the accuracy and efficiency of the diagnosis of enteroviruses associated HFMD. In this study, a mAb 1A11 was generated using full particles of CV-A5 as an immunogen. In indirect immunofluorescence and Western blotting assays, 1A11 bound to the viral proteins of CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, and EV-A71 of the Enterovirus A and targeted VP3. It has no cross-reactivity to strains of Enterovirus B and C. By mapping with over-lapped and truncated peptides, a minimal and linear epitope 23PILPGF28 was identified, located at the N-terminus of the VP3. A BLAST sequence search of the epitope in the NCBI genus Enterovirus (taxid: 12059) protein database indicates that the epitope sequence is highly conserved among the Enterovirus A species, but not among the other enterovirus species, first reported by us. By mutagenesis analysis, critical residues for 1A11 binding were identified for most serotypes of Enterovirus A. It may be useful for the development of a cost-effective and pan-Enterovirus A antigen detection for surveillance, early diagnosis and differentiation of infections caused by the Enterovirus A species.

[Association study on the microRNA-1 target gene polymorphism and the risk of premature coronary artery disease].

OBJECTIVE: To investigate the association between the genetic variant of miRNA-1 target gene COG6 rs9548934 C→T and the risk of premature coronary artery disease (pCAD). METHODS: This study included 226 pACD patients and 275 gender and age matched pCAD-free controls hospitalized in our hospital, diagnosis was made based on coronary angiography (CAG) results. The genotypes of miRNA-1 target gene COG6 rs9548934 C→T were detected by PCR-RFLP. RESULTS: Compared with the wide genotype CC, subjects with the variant genotypes CT of rs9548934 C→T was associated with a 45% lower risk of pACD (adjusted OR = 0.55, 95%CI = 0.36 - 0.82, P = 0.003), and the subjects with CT/TT genotypes were also associated with a significantly lower risk of pACD (adjusted OR = 0.64, 95%CI = 0.44 - 0.92, P = 0.015). Using the median serum TG level (1.20 mmol/L) in control group as the cutoff value, subjects with higher serum TG levels were associated with increased risk of pACD after adjustment for age, gender and BMI (adjusted OR = 2.32, 95%CI = 1.57 - 3.41, P < 0.001). In addition, subjects with higher HDL-C levels were associated with significantly lower risk of pACD (adjusted OR = 0.48, 95%CI = 0.31 - 0.75, P = 0.001). Stratified analyses showed that the risk reduction for pCAD in CT/TT genotypes carriers was more significant in the female subjects (adjusted OR = 0.54, 95%CI = 0.30 - 0.97, P = 0.040), and in subjects with lower TG, TC, HDL-C and LDL-C levels (adjusted OR = 0.62, 95%CI = 0.39 - 0.98, P = 0.040; adjusted OR = 0.55, 95%CI = 0.35 - 0.85, P = 0.008; adjusted OR = 0.43, 95%CI = 0.22 - 0.87, P = 0.018; adjusted OR = 0.49, 95%CI = 0.32 - 0.75, P = 0.001, respectively). CONCLUSION: The polymorphism of miRNA-1 target gene COG6 rs9548934C→T is associated with lower risk of pCAD, especially in female subjects and subjects with lower serum lipid levels.

Associations between dietary fiber intake and cardiovascular risk factors: An umbrella review of meta-analyses of randomized controlled trials.

Although several meta-analyses have revealed the beneficial effects of dietary fiber intake on human health, some have reported inconsistent findings. The purpose of this work was to perform an umbrella meta-analysis to evaluate the relevant evidence and elucidate the effect of dietary fiber intake on glycemic control, lipid profiles, systematic inflammation, and blood pressure. Eligible studies were searched in several electronic databases, including Web of Science, PubMed, Scopus, and the Cochrane Library, up to March 2022. A total of 52 meta-analyses involving 47,197 subjects were identified to assess the pooled effect size. Overall, higher dietary fiber intake was significantly associated with reductions in parameters involving glycemic control, including fasting plasma glucose (ES = -0.55, 95% CI: -0.73, -0.38, P < 0.001), fasting plasma insulin (ES = -1.22, 95% CI: -1.63, -0.82, P < 0.001), homeostasis model assessment of insulin resistance (HOMA-IR) (ES = -0.43, 95% CI: -0.60, -0.27, P < 0.001), and glycosylated hemoglobin (HbA1c) (ES = -0.38, 95% CI: -0.50, -0.26, P < 0.001). In terms of lipid profiles, higher dietary fiber intake was associated with significant reductions in the serum level of total cholesterol (ES = -0.28, 95% CI: -0.39, -0.16, P < 0.001) and low-density lipoprotein cholesterol (ES = -0.25, 95% CI: -0.34, -0.16, P < 0.001), but not triglycerides (ES = -0.001, 95% CI: -0.006, 0.004, P = 0.759) and high-density lipoprotein cholesterol (ES = -0.002, 95% CI: -0.004, 0.000, P = 0.087). Higher dietary fiber intake was also significantly associated with improved tumor necrosis factor-alpha serum levels (ES = -0.78, 95% CI: -1.39, -0.16, P = 0.013), while no significant effect was observed for C-reactive protein (ES = -0.14, 95% CI: -0.33, 0.05, P = 0.156). Finally, blood pressure was also significantly improved following higher dietary fiber intake (systolic blood pressure: ES = -1.72, 95% CI: -2.13, -1.30, P < 0.001; diastolic blood pressure: ES = -0.67, 95% CI: -0.96, -0.37, P < 0.001). Subgroup analysis revealed that the study population and type of dietary fiber could be partial sources of heterogeneity. In conclusion, the present umbrella meta-analysis provides evidence for the role of dietary fiber supplementation in the improvement of established cardiovascular risk factors.

Efficacy of a coxsackievirus A6 vaccine candidate in an actively immunized mouse model.

Coxsackievirus A6 (CV-A6) has been emerging as a major pathogen of hand, foot and mouth disease (HFMD). Study on the pathogenesis of CV-A6 infection and development of vaccines is hindered by a lack of appropriate animal models. Here, we report an actively immunized-challenged mouse model to evaluate the efficacy of a Vero-cell-based, inactivated CV-A6 vaccine candidate. The neonatal Kunming mice were inoculated with a purified, formaldehyde-inactivated CV-A6 vaccine on days 3 and 9, followed by challenging on day 14 with a naturally selected virulent strain at a lethal dose. Within 14 days postchallenge, all mice in the immunized groups survived, while 100% of the Alum-only inoculated mice died. Neutralizing antibodies (NtAbs) were detected in the serum of immunized suckling mice, and the NtAb levels correlated with the survival rate of the challenged mice. The virus loads in organs were reduced, and pathological changes and viral protein expression were weak in the immunized mice compared with those in Alum-only inoculated control mice. Elevated levels of interleukin-4, 6, interferon γ and tumour necrosis factor α were also observed in Alum-only control mice compared with immunized mice. Importantly, the virulent CV-A6 challenge strain was selected quickly and conveniently from a RD cell virus stock characterized with the natural multi-genotypes. The virulent determinants were mapped to V124M and I242 V at VP1. Together, our results indicated that this actively immunized mouse model is invaluable for future studies to develop multivalent vaccines containing the major component of CV-A6 against HFMD.

Emergence of a novel recombinant of CV-A5 in HFMD epidemics in Xiangyang, China.

BACKGROUND: Hand, foot and mouth disease (HFMD) is caused by a variety of enterovirus serotypes and the etiological spectrum worldwide has changed since a large scale of outbreaks occurred in 1997. METHODS: A large number of clinical specimens of HFMD patients were collected in Xiangyang and genotyping was performed by qRT-PCR, conventional PCR amplification and sequencing. Among the 146 CV-A5 detected cases, the complete genome sequences of representative strains were determined for genotyping and for recombination analysis. RESULTS: It was found that CV-A5 was one of the six major serotypes that caused the epidemic from October 2016 to December 2017. Phylogenetic analyses based on the VP1 sequences showed that these CV-A5 belonged to the genotype D which dominantly circulated in China. Recombination occurred between the CV-A5 and CV-A2 strains with a breakpoint in the 2A region at the nucleotide 3791. CONCLUSIONS: The result may explain the emergence of CV-A5 as one of the major pathogens of HFMD. A multivalent vaccine against HFMD is urgently needed to control the disease and to prevent emerging and spreading of new recombinants.

Determination and analysis of harmful components in synthetic running tracks from Chinese primary and middle schools.

In China, incidences involving pupils suffering health problems caused by synthetic running tracks have attracted the public's attention. However, the existence of known and unknown harmful chemicals in the tracks have not yet been explored. Here, the levels of 16 known harmful ingredients were firstly analyzed in 167 school running tracks. In all samples, the recognized toxic solvents and additives, such as the benzene series, soluble mercury, 3,3'-dichloro-4,4'-diaminodiphenylmethane (MOCA) and toluene diisocyanate monomer (TDI) were under the limits of detection. In contrast, polycyclic aromatic hydrocarbons (PAHs), phthalates, Short chain chlorinated paraffins (SCCPs) soluble lead, cadmium and chromium were found in 86%, 88%, 46%, 81%, 43% and 83% of the specimens, respectively. The levels, toxicology and distribution of these known chemicals were evaluated. Then, a static-headspace gas chromatography-mass spectrometer (GC-MS) method in full scan mode was employed to screen for unknown volatile chemicals. Three groups of chemicals reflecting different kinds of pollution sources were discovered: new solvents, such as N, N-Dimethylformamide, new additives, such as 2-ethylhexanoic acid, and by-products, such as carbon disulfide. In summary, the existence of potential risk factors in school plastic tracks was revealed through exhaustive testing. Moreover, most of the hazardous components detected have been recently included in a new national standard to improve the safety performance of synthetic running tracks.

Detection and quantification analysis of chemical migrants in plastic food contact products.

Plastic food contact materials (FCM)-based products were widely used in everyday life. These products were normally imposed to strict regulations in order to pass the enforcement tests of compliance as a prefix condition. However, even in these "qualified" materials, unknown chemical substances, not involving in legislation lists, could migrate from FCM. In this perspective, the present work aims to thoroughly analyze by means of Gas Chromatography-Mass Spectrometry (GC-MS) the different substances/migrants in 120 qualified FCM plastic products. Unexpectedly, among the identified compounds (nearly 100), only 13% was included in the permitted list of Commission Regulation EU No 10/2011. All the identified compounds were classified into 11 categories according to their chemical structure and the FCM type, whereas toxicology data were in addition analyzed. Each plastic type exhibited different preferences of chemical migrants. Fortunately, most of the compounds identified were of low toxicity, and only 4 chemicals were included in priority lists and previous literature reports as potential risk factors. Subsequently, the accurate amount of these 4 chemicals was determined. The amount of Bis(2-ethylhexyl) adipate (DEHA) and Bis(2-ethylhexyl) phthalate (DEHP) were lower than the SML in Commission Regulation EU No 10/2011, and that of stearamide was under the recommended use quantity. The 2,4-di-tert-butylphenol (2,4-DTBP) was widely exist in the investigated FCM products. Among them, the highest level is obtained in polypropylene/low density polyethylene (BOPP/LDPE) materials, up to 45.568±31.513 mg/kg. In summary, a panel of unlisted chemical migrants were discovered and identified by GS-MS screening. The results implied that plastic FCMs were not so "inert" as they usually considered, and further safety evaluation should be performed toward the complete identification of new substances in FCM products.

Two Lysin-Motif Receptor Kinases, Gh-LYK1 and Gh-LYK2, Contribute to Resistance against Verticillium wilt in Upland Cotton.

Lysin-motif (LysM) receptor kinases (LYKs) play essential roles in recognition of chitin and activation of defense responses against pathogenic fungi in the model plants Arabidopsis and rice. The function of LYKs in non-model plants, however, remains elusive. In the present work, we found that the transcription of two LYK-encoding genes from cotton, Gh-LYK1 and Gh-LYK2, was induced after Verticillium dahliae infection. Virus-induced gene silencing (VIGS) of Gh-LYK1 and Gh-LYK2 in cotton plants compromises resistance to V. dahliae. As putative pattern recognition receptors (PRRs), both Gh-LYK1 and Gh-LYK2 are membrane-localized, and all three LysM domains of Gh-LYK1 and Gh-LYK2 are required for their chitin-binding ability. However, since Gh-LYK2, but not Gh-LYK1, is a pseudo-kinase and, on the other hand, the ectodomain (ED) of Gh-LYK2 can induce reactive oxygen species (ROS) burst in planta, Gh-LYK2 and Gh-LYK1 may contribute differently to cotton defense. Taken together, our results establish that both Gh-LYK1 and Gh-LYK12 are required for defense against V. dahliae in cotton, possibly through different mechanisms.

[Establishment of a One-Step Real-Time RT-PCR Method for the Detection of Venezuelan Equine Encephalitis Virus].

Venezuelan equine encephalitis (VEE) is a zoonotic disease caused by the Venezuelan equine encephalitis virus (VEEV) complex. This disease has not yet been reported in China, and it is therefore essential to establish a rapid and accurate method for detection of the virus in order to prevent and control this disease. In this study, a one-step real-time quantitative RT-PCR method was developed for the detection of the VEEV complex. A pair of specific primers and a Taqman probe were designed corresponding to a conserved region of the VEEV gene nspl, allowing the detection of all known strains of different sub- types of the virus. Using RNA synthesized by in vitro transcription as template, the sensitivity of this method was measured at 3.27 x 10(2) copies/microL. No signal was generated in response to RNA from Chikungunya virus (CHIKV), nor to RNA encoding the nsp1 fragment of Eastern equine encephalitis virus (EE-EV) or Western equine encephalitis virus (WEEV), all of which belong to the same genus as VEEV. This indicates that the method has excellent specificity. These results show that this one-step real-time quantitative RT-PCR method may provide an effective tool for the detection of VEEV in China.

Diversity of HIV/AIDS epidemic in China: a result from hierarchical clustering analysis and spatial autocorrelation analysis.

OBJECTIVE: To find out the diversity of HIV/AIDS epidemic among different areas in China according to their varied epidemic characteristics. DESIGN AND METHODS: Seventeen provincial variables, generated from original HIV/AIDS epidemic data and socioeconomic indicators to indicate HIV/AIDS epidemic characteristics, were introduced to hierarchical clustering analysis to form subepidemic areas. Then spatial autocorrelation analysis was applied to show the clustering distribution of cases from different most-at-risk populations. RESULTS: Three HIV/AIDS subepidemic areas (A, B, C) were formed, each of which was further divided into two clusters, showing the diversity of HIV/AIDS epidemic in China. A1 was the earliest and severest HIV/AIDS epidemic area and occupied 37% hotspot counties. The epidemic in A1 was driven by IDU in its early period and heterosexual transmission later. Henan, the only province in A2, characterized by its HIV/AIDS epidemic among former plasma donors during the early 1990s, presented strong spatial clustering of blood/plasma transmission occupying 80% blood/plasma hotspots. The epidemic within B3, located in southwest China, was driven by IDU and heterosexual populations, and recently by MSM. The epidemic within B4, covering all four municipalities, had been largely spread among MSM since 2005. B3 and B4 occupied 76% MSM hotspots. For C5 and C6, only sporadic HIV/AIDS infections occurred in the last years among former plasma donors and heterosexual populations, whereas the prevalence among MSM had been increasing. CONCLUSION: China's different HIV/AIDS subepidemic areas had obvious diversity of affected populations, which should be considered when determining prevention policies.

[Spatial analysis on the HIV/AIDS infection status among injecting drug users in China, from 2005 to 2011].

OBJECTIVE: To analyze the spatial distribution of HIV/AIDS epidemics among injecting drug users(IDUs) in China from 2005 to 2011 and to understand its changing trend. METHODS: Using data on people living with HIV and AIDS through injecting drug between 2005 and 2011 to analyze the demographic characteristics of injecting drug users. Analysis on spatial correlation (provincial level and country level) and median center of hot spots(country level)were conducted by Arcgis software. RESULTS: Sex ratio (male/female) and registered place ratio(province/other provinces)reduced as time went by, with the ratios in 2011 as 6.75 and 7.01 respectively. Tape ratio of the disease between HIV and AIDS showed an upward trend (Z = 26.880, P < 0.01). Since 2005, the identified numbers of HIV/AIDS and the spatial correlation and hot spots in provincial level had reduced, the numbers of provincial hot spots were two from 2009 to 2011(Sichuan and Yunnan provinces)at the national level. However, the spatial correlation and hot spots at the provincial level had an increasing trend. Between 2005 and 2011, the Western Median Centers of hot spots was located in Xinjiang province while the Southwestern Median Center of hot spots tended to move towards the north. CONCLUSION: The demographics changes of HIV/AIDS infection among injecting drug users seemed to be regular from 2005 to 2011. Spatial correlation at the provincial level was reducing. However, the spatial correlation and the numbers of hot spots at the country level increased, with hot spots at the country level tended to move from the border areas towards inland.