The present article reports on a simple, economical, and green preparative strategy toward water-soluble, fluorescent carbon nanoparticles (CPs) with a quantum yield of approximately 6.9% by hydrothermal process using low cost wastes of pomelo peel as a carbon source for the first time. We further explore the use of such CPs as probes for a fluorescent Hg(2+) detection application, which is based on Hg(2+)-induced fluorescence quenching of CPs. This sensing system exhibits excellent sensitivity and selectivity toward Hg(2+), and a detection limit as low as 0.23 nM is achieved. The practical use of this system for Hg(2+) determination in lake water samples is also demonstrated successfully.

In this contribution, we demonstrate a green, cost-effective, one-pot preparative route toward Ag nanoparticles-graphene (AgNPs-G) nanocomposites in aqueous solution with the use of tannic acid (TA), an environmentally friendly and water-soluble polyphenol, as a reducing agent. Such AgNPs-G nanocomposites were synthesized through one-pot reduction of AgNO 3 and GO by TA. We investigated surface enhanced Raman scattering (SERS) and electrochemical properties of the resultant AgNPs-G nanocomposites. It is found that such AgNPs-G nanocomposites show excellent SERS activity as SERS substrates and exhibit notable catalytic performance toward the reduction of H 2 O 2 . This enzymeless H 2 O 2 sensor has a fast amperometric response time of less than 2 s. The linear range is estimated to be from 1 6 10 24 M to 0.01 M (r = 0.999) and the detection limit is estimated to be 7 6 10 26 M at a signal-to-noise ratio of 3. A glucose biosensor was further fabricated by immobilizing glucose oxidase (GOD) into chitosan-AgNPs-G nanocomposite film on the surface of a glassy carbon electrode (GCE). This sensor exhibits good response to glucose, and the linear response range is estimated to be from 2 to 10 mM (R = 0.996) at 20.5 V. The detection limit of 100 mM was achieved at a signal-to-noise ratio of 3. More importantly, we demonstrate successfully its application for glucose detection in human blood serum.

Recently, a new coronavirus was isolated from the lung tissue of autopsy sample and nasal/throat swabs of the patients with Severe Acute Respiratory Syndrome (SARS) and the causative association with SARS was determined. To reveal further the characteristics of the virus and to provide insight about the molecular mechanism of SARS etiology, a proteomic strategy was utilized to identify the structural proteins of SARS coronavirus (SARS-CoV) isolated from Vero E6 cells infected with the BJ-01 strain of the virus. At first, Western blotting with the convalescent sera from SARS patients demonstrated that there were various structural proteins of SARS-CoV in the cultured supernatant of virus infected-Vero E6 cells and that nucleocaspid (N) protein had a prominent immunogenicity to the convalescent sera from the patients with SARS, while the immune response of spike (S) protein probably binding with membrane (M) glycoprotein was much weaker. Then, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the complex protein constituents, and the strategy of continuous slicing from loading well to the bottom of the gels was utilized to search thoroughly the structural proteins of the virus. The proteins in sliced slots were trypsinized in-gel and identified by mass spectrometry. Three structural proteins named S, N and M proteins of SARS-CoV were uncovered with the sequence coverage of 38.9, 93.1 and 28.1% respectively. Glycosylation modification in S protein was also analyzed and four glycosylation sites were discovered by comparing the mass spectra before and after deglycosylation of the peptides with PNGase F digestion. Matrix-assisted laser desorption/ionization-mass spectrometry determination showed that relative molecular weight of intact N protein is 45 929 Da, which is very close to its theoretically calculated molecular weight 45 935 Da based on the amino acid sequence deduced from the genome with the first amino acid methionine at the N-terminus depleted and second, serine, acetylated, indicating that phosphorylation does not happen at all in the predicted phosphorylation sites within infected cells nor in virus particles. Intriguingly, a series of shorter isoforms of N protein was observed by SDS-PAGE and identified by mass spectrometry characterization. For further confirmation of this phenomenon and its related mechanism, recombinant N protein of SARS-CoV was cleaved in vitro by caspase-3 and -6 respectively. The results demonstrated that these shorter isoforms could be the products from cleavage of caspase-3 rather than that of caspase-6. Further, the relationship between the caspase cleavage and the viral infection to the host cell is discussed.

We tested the ability of inactivated SARS-CoV vaccine to induce neutralizing antibodies in BALB/c mice. The inactivated vaccine was prepared by SARS-CoV virus propagation in Vero cells, with subsequent beta-propiolactone inactivation and Sepharose 4FF column chromatography purification. One hundred forty BALB/c female mice were divided into seven groups of 20 mice each. Of the seven groups, three groups were inoculated with 0.1, 1, and 3 microg of the vaccine without adjuvant while three other groups were inoculated at the same three dosages of vaccine with aluminum hydroxide as adjuvant, respectively. The remaining group was set up as a blank control. Each mouse was inoculated twice at an interval of 3 weeks. One week after the second immunization, mice sera were collected to detect serum neutralizing antibodies. An assay for determining neutralizing antibody titers was developed. The results can be summarized as follows: (1) higher dosages of vaccine induced higher levels of neutralizing antibody titer; (2) the level of neutralizing antibodies induced by the inoculation with aluminum hydroxide adjuvant was slightly higher than that without adjuvant, but the difference was not statistically significant.

The purified inactivated SARS vaccine could induce high levels of neutralizing antibody, and protect the monkeys from the challenge of SARS-CoV. The SARS vaccine prepared in the study appeared to be safe in monkeys.

Severe acute respiratory syndrome (SARS) is a zoonotic disease caused by SARS-related coronavirus (SARS-CoV) that emerged in 2002 to become a global health concern. Although the original outbreak was controlled by classical public health measures, there is a real risk that another SARS-CoV could re-emerge from its natural reservoir, either in its original form or as a more virulent or pathogenic strain; in which case, the virus would be difficult to control in the absence of any effective antiviral drugs or vaccines. Using the well-studied SARS-CoV isolate HKU-39849, we developed a vaccinia virus-based SARS-CoV reverse genetic system that is both robust and biosafe. The SARS-CoV genome was cloned in separate vaccinia virus vectors, (vSARS-CoV-5prime and vSARS-CoV-3prime) as two cDNAs that were subsequently ligated to create a genome-length SARS-CoV cDNA template for in vitro transcription of SARS-CoV infectious RNA transcripts. Transfection of the RNA transcripts into permissive cells led to the recovery of infectious virus (recSARS-CoV). Characterization of the plaques produced by recSARS-CoV showed that they were similar in size to the parental SARS-CoV isolate HKU-39849 but smaller than the SARS-CoV isolate Frankfurt-1. Comparative analysis of replication kinetics showed that the kinetics of recSARS-CoV replication are similar to those of SARS-CoV Frankfurt-1, although the titers of virus released into the culture supernatant are approximately 10-fold less. The reverse genetic system was finally used to generate a recSARS-CoV reporter virus expressing Renilla luciferase in order to facilitate the analysis of SARS-CoV gene expression in human dendritic cells (hDCs). In parallel, a Renilla luciferase gene was also inserted into the genome of human coronavirus 229E (HCoV-229E). Using this approach, we demonstrate that, in contrast to HCoV-229E, SARS-CoV is not able to mediate efficient heterologous gene expression in hDCs.

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The genome sequence of the Severe Acute Respiratory Syndrome (SARS)-associated virus provides essential information for the identification of pathogen(s), exploration of etiology and evolution, interpretation of transmission and pathogenesis, development of diagnostics, prevention by future vaccination, and treatment by developing new drugs. We report the complete genome sequence and comparative analysis of an isolate (RIOI ) of the coronavirus that has been recognized as a pathogen for SARS. The genome is 29725 nt in size and has 11 ORFs (Open Reading Frames). It is composed of a stable region encoding an RNA-dependent RNA polymerase (composed of 2 ORFs) and a variable region representing 4 CDSs (coding sequences) for viral structural genes (the S, E, M, N proteins) and 5 PUPs (putative uncharacterized proteins). Its gene order is identical to that of other known coronaviruses. The sequence alignment with all known RNA viruses places this virus as a member in the family of Coronaviridae. Thirty putative substitutions have been identified by comparative analysis of the 5 SARSassociated virus genome sequences in GenBank. Fifteen of them lead to possible amino acid changes (non-synonymous mutations) in the proteins. Three amino acid changes, with predicted alteration of physical and chemical features, have been detected in the S protein that is postulated to be involved in the immunoreactions between the virus and its host. Two amino acid changes have been detected in the M protein, which could be related to viral envelope formation. Phylogenetic analysis suggests the possibility of non-human origin of the SARS-associated viruses but provides no evidence that they are man-made. Further efforts should focus on identifying the etiology of the SARS-associated virus and ruling out conclusively the existence of other possible SARS-related pathogen(s).