Mutations in the SARS-Coronavirus (SARS-CoV) can alter its clinical presentation, and the study of its mutation patterns in human populations can facilitate contact tracing. Here, we describe the development and validation of an oligonucleotide resequencing array for interrogating the entire 30-kb SARS-CoV genome in a rapid, cost-effective fashion. Using this platform, we sequenced SARS-CoV genomes from Vero cell culture isolates of 12 patients and directly from four patient tissues. The sequence obtained from the array is highly reproducible, accurate (>99.99% accuracy) and capable of identifying known and novel variants of SARS-CoV. Notably, we applied this technology to a field specimen of probable SARS and rapidly deduced its infectious source. We demonstrate that array-based resequencing-by-hybridization is a fast, reliable, and economical alternative to capillary sequencing for obtaining SARS-CoV genomic sequence on a population scale, making this an ideal platform for the global monitoring of SARS-CoV and other small-genome pathogens.[Supplemental material is available online at www.genome.org and http://www.gis.a-star.edu.sg/homepage/toolssup.jsp.]In April 2003, the SARS coronavirus (SARS-CoV), a singlestranded RNA virus, was identified as the pathogen responsible for Severe Acute Respiratory Syndrome (Drosten et al. 2003;Fouchier et al. 2003). Soon after, the consensus genome sequence for SARS-CoV was published (Marra et al. 2003;Ruan et al. 2003). Our studies have shown that the SARS-CoV mutates at a significant rate. In addition to single nucleotide changes, we observed small deletions of 5-6 nucleotides in some SARS-CoV isolates. Also, we identified several recurrent sequence variants capable of distinguishing genotypes linked to strains originating in different geographical regions (Ruan et al. 2003). As mutations can alter virulence and therapeutic response of viral pathogens, it is important to develop methods for the rapid monitoring of genetic changes in the SARS-CoV in human populations. Moreover, the rapid detection of genetic variants in newly confirmed SARS cases could also provide important clues to the geographic origins of infection, thus facilitating contact tracing.Sequencing of viral genomes has historically used standard dye termination technologies. Recently, sequencing by hybridization to oligonucleotides has been described (Drmanac et al. 1992;Maskos and Southern 1992a;Southern et al. 1992;Schena et al. 1995) and commercialized (Pease et al. 1994;Nuwaysir et al. 2002). These methods have been adapted for studies of genetic diversity in disease, genes, and between species (Drmanac et al. 1998;Hacia et al. 1998;Wang et al. 1998;Hacia 1999;Vahey et al. 1999;Brenner et al. 2000;Drmanac and Drmanac 2001;Fan et al. 2002). Despite these advances, several limitations temper the enthusiasm for sequencing by hybridization, including difficulties in detection of deletions and cost considerations that significantly restrict array modifications and reformatting for optimization (Cutler et al. 2...

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ST283 is a zoonotic GBS clone associated with farmed freshwater fish, capable of causing severe disease in humans. It caused a large foodborne outbreak in Singapore and poses both a regional and potentially more widespread threat.

In this study, we examined the hypothesis that early pulmonary metastases form within the vasculature. We introduced primary tumors in immunocompromised mice by subcutaneous injection of murine breast carcinoma cells (4T1) expressing green fluorescent protein. Isolated ventilated and perfused lungs from these mice were examined at various times after tumor formation by fluorescent microscopy. The vasculature was visualized by counterstaining with 1,1-dioctadecyl-3,3,3 ,3 -tetramethylindocarbocyanine (DiI)-acetylated low-density lipoprotein. These experiments showed that metastatic cells derived by spontaneous metastases were intravascular, and that early colony formation was intravascular. The location of the tumor cells was confirmed by deconvolution analysis. This work extends our previous study 1 We recently reported direct observations of the sequence of events in pulmonary metastasis after injection of fluorescent tumor cells into the venous circulation using fluorescent microscopy of isolated perfused lungs. 1 Because endothelium has receptors for oxidized or acetylated low-density lipoprotein (LDL), the pulmonary endothelium can be precisely visualized in these preparations through infusion of DiI-acetylated LDL. Because the lung is translucent, these methods allow high-resolution imaging of the microvasculature of the lungs at up to 100-m depth beneath the pleural surface and enable the precise localization of the tumor cells. We used these methods to show in a mouse experimental metastasis model that tumor cells attach to the pulmonary endothelium and proliferate intravascularly.We have also demonstrated that differences in apoptosis of tumor cells in vivo correlated to differences in metastatic potential. 1-3 Based on these observations, we proposed a new model for pulmonary metastasis in which endothelium-attached tumor cells that survived the initial apoptotic stimuli proliferate intravascularly. A principal tenet of this new model is that extravasation of tumor cells is not a prerequisite for metastatic foci formation. [1][2][3] Because the initial experiments were based on intravenous injection of tumor cells, it was possible that the results were an artifact of this mode of introduction of the tumor cells or of the relatively large numbers of cells infused simultaneously. To determine whether our observations in an experimental model of metastasis were valid for cells that metastasize spontaneously from a primary tumor, we have tested our hypothesis using a spontaneous metastasis model. Our previous results were obtained using fibrosarcoma cells, so we sought to extend this hypothesis to carcinoma cells by using the breast carcinoma cell line 4T1. 4 The results indeed indicate that extravasation is also rare after spontaneous metastasis. These observations confirmed that metastasizing tumor cells proliferate intravascularly within the lung. This suggests that drug discovery efforts could be directed to blocking the metastatic cascade at the steps of the initial Supported by the Susan G. Komen Bre...

Oseltamivir ring chemoprophylaxis, together with prompt identification and isolation of infected personnel, was effective in reducing the impact of outbreaks of 2009 H1N1 influenza in semiclosed settings.