Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro, with affinity to staphylococcal enterotoxin B (SEB) that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APTSEB1, successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide.The bacterium  Staphylococcus aureus, which is estimated to cause 250,000 cases of foodborne illness each year S. aureus secrete a family of small (26–30 kDa) heat-resistant toxins, known as staphylococcal enterotoxins (SEs) Each year, 1 in 6 Americans contract a foodborne disease, and one of the common foodborne bacterial pathogens is Staphylococcus aureus secrete a closely-related family of 23 SEs (SEA – SElV) Strains of To detect and quantify SEs in food matrices, immuno-affinity based methods, such as the bead-based multiplexing immuno-affinity assay (Luminex) However, immuno-affinity based detection of the SEs in food matrices is limited by the availability and quality of antibodies. Antibodies are costly and time consuming to produce and are commonly harvested from mice, sheep and rabbits. The cost of antibody development is reflected in the relatively high cost of commercial immuno-affinity assays. Further, at least two non-classical SEs (SES and SET) have emerged as potent SFP-causing toxins, for which no antibodies or assays are currently available in vitro from a library of nucleic acids  containing ∼1015 individual sequences using a method known as SELEX  An aptamer is a nucleic acid (or peptide) that binds to a target with high affinity and specificity in vitro which allows any target to be used, despite its toxicity to animals. Second, polymerase chain reaction (PCR) can produce a large, highly pure, quantity of a known aptamer at a relatively low cost. Third, nucleic acids may be modified with a number of functional groups with greater ease, and without negative effects (e.g. loss of affinity), than an antibody. Finally, aptamers are inherently more stable over a greater range of conditions than antibodies. Indeed, many immuno-affinity assays have been successfully transferred to aptamer-affinity assays with similar figures of merit Aptamers offer several significant advantages that make them ideal candidates to supplant antibodies for use in toxin detection assays et. al.et. al., discovered a Spiegelmer (an enantiomeric L-DNA) ssDNA aptamer with affinity to SEB Staphylococcal enterotoxins.Recently, aptamers with affinity to toxins and whole-cell pathogens important to the field of food safety have been successfully discovered SEB1 was characterized to bind to SEB with high selectivity amongst other enterotoxins. This protocol will serve the future of the aptamer initiative at the US FDA and be applied to target molecules of interest to food safety such as toxins, allergens and even entire pathogens.This paper serves to outline the general and rapid method that was used to discover an aptamer with affinity to SEB et. al.The SELEX methodology outlined below is adapted from the work of Murphy, LIB) consists of a central string of 40 randomized nucleotides that are flanked by defined primer binding regions necessary for PCR amplification. The forward and reverse primers (with and without a biotin moiety attached to the 5′ nucleotide) were synthesized at 25 nmole scale and then desalted . The APTLIB was synthesized at 1 µmole scale, with machine mixing for bases within the center random sequence domain, and purified by PAGE (IDT). All DNA stock was maintained at 100 µM in 10 mM Tris, pH 8.0, 0.1 M EDTA, and stored at −40°C until use.The DNA sequences used in this work are listed in 8 (3 mg) Dynabeads© M-270 Epoxy using the Dynabeads Co-Immunoprecipitation Kit . Following conjugation and washing, the SEB-coated magnetic beads were suspended at 6.7×105 beads/µl, or 10 µg/µl, in PBS-T . Uncoated beads for counter-selection were produced in a similar manner without a ligand.Thirty micrograms of highly purified staphylococcal enterotoxin B  was bound to 2×107 coated-beads were washed and resuspended in 50 µl of 50 mM ammonium bicarbonate and 9% acetonitrile. One microgram of the protease trypsin was added to the suspended beads and the mixture was allowed to react at 60°C for 4 hours before quenching the proteolysis with 1% acetic acid . The resultant peptides were analyzed by LC-MS  and those data were compared to a reference standard SEB (10 ng/µl in of 50 mM ammonium bicarbonate and 9% acetonitrile) that was treated in the same manner.Mass spectrometry was used to confirm the binding of SEB to the Dynabeads. The detailed protocol is described elsewhere 15 different ssDNA sequences) of the library were diluted 10-fold in PBS-T. The ssDNA was denatured at 95°C for 5 minutes and then left to cool on ice for at least 10 minutes. Fifty microliters (3.4×107) of beads were washed twice in 500 µl PBS-T and then resuspended in 1 ml PBS-T. The ssDNA library and beads were diluted into 48.5 ml PBS-T in a 50 ml centrifuge tube. To limit non-specific interactions, 50 µl of 1 mg/ml BSA (Sigma) and 5 µl of 1 mg/ml poly(deoxyinosinic-deoxycytidylic) acid (Sigma) were also added. The mixture was allowed to incubate, with gentle rotation, for 30 minutes.For the first round of SELEX, 5 nmoles  for 20 minutes to collect the magnetic beads. The majority of the supernatant was aspirated and the multiple reactions were pooled. After aspirating all excess supernatant, the tube was removed from the magnet and the beads were resuspended prior to transferring the mixture to a microcentrifuge tube. The microcentrifuge tube was placed onto a smaller magnet . After removing the supernatant, the beads were washed once with 500 µl of PBS-T, which was immediately removed.All of the beads were transferred to a PCR tube using 22.5 µl of nuclease-free water. To the bead and water mixture, 2.5 µl of 10 µM forward and 5′ biotinylated reverse primer mix and 25 µl of AmpliTaq Gold® Fast PCR Master Mix (Life Technologies) was added. To produce a significant amount of dsDNA while reducing the possibility of incorrectly-sized products, multiple PCRs were carried out in tandem.1) proceeded as follows: 10 minutes at 95°C followed by 15 cycles of 96°C for 3 seconds, 56°C for 3 seconds, and 68°C for 5 seconds. After the final cycle the reaction was held at 72°C for 10 seconds before cooling the PCR1 product to 4°C. The paramagnetic beads were removed via magnet from the PCR1 product.The first PCR (PCR2) proceeded with 4 reaction tubes each using PCR1 product as the template. One microliter of PCR1 product was added to 21.5 µl of nuclease-free water, 2.5 µl forward and 5′ biotinylated reverse primer mix and 25 µl of AmpliTaq Gold® Fast PCR Master Mix. PCR2 proceeded as follows: 10 minutes at 95°C followed by 35 cycles of 96°C for 3 seconds, 56°C for 3 seconds, and 68°C for 5 seconds. After the final cycle the reaction was held at 72°C for 10 seconds before cooling the PCR2 product to 4°C.The second PCR . Twenty microliters of PCR2 product was loaded onto the gel and visualized by ethidium bromide staining. The pooled PCR product (135 µl) was mixed with 34.5 µl of 5 M NaCl and then incubated with 1 mg of Dynabeads® M-270 Streptavidin (Life Technologies) for 10 minutes. To separate the ssDNA aptamer candidates from the complementary strand, the beads were incubated for 5 minutes in 50 µl of freshly prepared (daily from a 1 M NaOH stock stored at 4°C) 100 mM NaOH. To adjust the pH to 7.4, the supernatant was transferred to a microcentrifuge tube containing 850 µl PBS-T and 100 µl sodium phosphate monobasic.At this point, the 4 PCRThe ssDNA was denatured at 95°C for 5 minutes and then cooled on ice before proceeding to the next round. In the counter-selection rounds (4–14), the cooled ssDNA was first incubated with counter-selection beads for 10 minutes. Then, using a magnet, the supernatant was transferred to the appropriate amount of washed selection beads to incubate see .E. coli (Life Technologies) using the rapid chemical transformation protocol. The E. coli (50 µl) was plated and grown overnight on pre-warmed (37°C) LB agar plates containing 100 µg/ml ampicillin. The plates with a few hundred colonies were sent to GENEWIZ  where 50 colonies were randomly selected for Sanger sequencing using the T7 sequencing primer that was incorporated into the TOPO TA vector.After round 14, the PCR product was inserted into the TOPO® TA cloning vector (Life Technologies) according to manufacturer instructions. The vector then was inserted into One Shot® Top10 The sequences were trimmed to remove known plasmid and primer regions, assessed for quality (i.e. proper length and sequence confidence), and then aligned with Geneious 5.5 SEB1, (2) three random 78 base ssDNA molecules, and (3) forward primer were diluted into 200 µl of PBS-T. A sixth tube contained no DNA and served as a negative control. The three random 78 base ssDNA molecules were identified in other SELEX experiments and were not predicted to exhibit affinity towards SEB. The diluted ssDNA was heated to 95°C for 5 minutes and then placed on ice. Meanwhile, 2 mg of Dynabeads M-270 Streptavidin were aliquoted into 6 tubes. The beads were washed twice with 500 µl of PBS-T.In separate tubes, 5 µg of biotinylated (1) APTAfter the final wash was removed, the chilled DNA was added to the streptavidin-coated magnetic beads. The beads and DNA were mixed by rotation at room temperature for 30 minutes. The beads were then washed 3 times with 500 µl PBS-T and then resuspended in 500 µl PBS-T containing 10 µg BSA. Following a thorough resuspension, 1 µg of SEB was added to each of the 5 tubes containing DNA-coated beads and the negative control. The beads were allowed to incubate in the protein mixture for 30 minutes at room temperature with rotation.Afterwards, the beads were again washed 3 times with 500 µl PBS-T. After the final wash was removed, 50 µl of 1X LDS sample buffer (Life Technologies) supplemented with 0.5 M NaCl was added on top of the coated beads, and the mixture was incubated at 50°C with agitation for 10 minutes. For a positive control, standards of BSA and SEB  were diluted into 50 µl of 1X LDS loading buffer (Life Technologies) supplemented with 0.5 M NaCl.Twenty five microliters each of the samples and standards, as well as 5 µl of the molecular weight ladder , were loaded onto a NuPAGE® 4–12% Bis-Tris pre-cast polyacryamide gel (Life Technologies) with MOPS as the running buffer. Electrophoresis was conducted at 125 V for the initial 5 minutes and then at 200 V for approximately 30 minutes. The proteins were visualized with silver stain (Thermo Fisher Scientific).SEB1 was conjugated to 2 mg of streptavidin Dynabeads, as outlined above. Following several washes with PBS-T, the aptamer coated beads were incubated at room temperature for 30 minutes with one of two mixtures. Mixture 1 contained the following enterotoxins (1 ng/µl): SEA, SEB, SEC1, SEC2, SEC3, SED, and SEE. Mixture 2 contained the same enterotoxins as mixture 1, but without SEB. The total volume of reaction was 1 ml. The coated beads were then washed, and the toxins were eluted and analyzed by PAGE, as described above.In two tubes, 5 µg of biotinylated APTSEB1 was prepared and conjugated to 2 mg streptavidin Dynabeads as described above. The washed APTSEB1-coated beads were incubated for 30 minutes at room temperature with 3 ml of cell-free culture supernatant (CFCS) from the following S. aureus strains: BAA1747 Five micrograms of biotinylated APTIntact, unmodified, natively-folded SEB was directly immobilized to M-270 epoxy Dynabeads using already established conjugation strategies. This method was chosen over various other methods because the epoxy-functionalized surface allows for a generalized conjugation protocol to bind proteinacious ligands to the surface of the bead without prior protein modification (i.e. biotinylation). Uncoated beads were generated using the same protocol, but without a protein ligand. These beads were used in the “counter-selection” steps outlined below in order to remove ssDNA aptamer candidates that may have non-specifically bound to the unfunctionalized regions of the bead surface during SELEX. Mass spectrometry was used to ensure that the conjugation reaction yielded SEB covalently bound to beads prior to SELEX (data not shown). Efforts were not made to calculate the concentration of the bound SEB because one could simply modulate the number of beads used for a round of selection to increase selective pressure.15 unique DNA sequences. The first round of selection was the most liberal with respect to selective pressure. The entire DNA sequence space was exposed to 3.4×107 beads and had the longest incubation time at 30 minutes , selective pressure was increased gradually. Increasing the selective pressure forced the imes see .7 uncoated beads for 10 minutes. Following incubation, the beads were partitioned using a magnet and the supernatant was immediately added to the specified amount of SEB-coated beads and incubated for the specified time  and not to SEB. Heated and cooled ssDNA were washed over 3.4×10time see . RemarkaThe method was rapid enough to allow for the completion of ∼10 rounds of selection per week. In practice, many targets may be screened at once, as an efficient use of time and resources, to produce a suite of aptamers.2 product was inserted into the TOPO TA cloning vector. This cloning strategy was selected because it did not require the use of restriction enzymes as this technology exploits the 3′ adenosine overhangs that result from Taq polymerase chain extension. Thus the PCR product can be directly inserted into the cloning vector.After round 14, the PCRE. coli, the bacteria were plated onto ampicillin selective plates. Fifty positive clonal colonies were sequenced. Of the 49 sequences returned, 48 were identical  with a 5′ biotin moiety to allow for easy attachment to streptavidin coated Dynabeads. Once conjugated, the coated beads were used for an aptamer-precipitation assay to partition SEB from a 10-fold excess solution of BSA. Following incubation, the beads were extensively washed with PBS-T to fully remove non-specifically bound proteins (i.e. BSA). Relative to the negative controls  Staphylococcal enterotoxins. As observed in SEB1 successfully partitioned SEB away from the other classical enterotoxins. Remarkably, even though pair-wise alignment analysis of SEB (GI:15625508) and SEC1 (GI:119625) revealed an identity of 68%, APTSEB1 did not significantly bind to any of the SEC variants.To further demonstrate the selectivity of APTSEB1, an aptamer-precipitation assay was performed on a complex mixture of toxins. Four strains, for which toxin profiles are known , were cultured, and the toxin-rich cell-free culture supernatant (CFCS) was extracted. Strain BAA1747 is known to secrete SEB . The other three strains, BAA1751 , NRS111 , and NRS109  do not secret SEB; however, together they secrete 17 non-SEB toxins. These 4 strains offered sufficient toxin diversity to challenge the selectivity of APTSEB1.Within a complex mixture, affinity and selectivity are two critical requirements of an aptamer if it is to be used in any assay. To further challenge the selectivity of APTSEB1 was incubated with the CFCS from BAA1747, a single protein whose MW is consistent with the SEB standard was isolated (SEB1 coated beads from the toxin-rich CFCS of the other three strains (SEB1 have an affinity to SEB, but it is also highly selective for SEB.The protein profiles of each CFCS were quite complex and many proteins were not adequately resolved when loaded directly on the polyacrylamide gel (isolated , lane 3.There is a need for the rapid development and deployment of aptamers with affinity to toxins and allergens related to food safety. Aptamers and their use in aptamer-affinity assays would serve two roles. First, with this modified protocol, aptamers could be efficiently developed to molecules for which there are no available antibodies and immuno-affinity assays. Second, aptamers could supplant commercial antibodies to generate aptamer-affinity assays that are lower in cost and can be widely distributed.One of the more important advantages of an aptamer over an antibody is the ability to freely distribute the molecule to allow other scientists to immediately use this aptamer to develop an assay that suits their needs. Aptamer-affinity assays could be developed and evaluated in a manner similar to that of immuno-affinity assays. The key difference is that any laboratory with the aptamer sequence could perform the assay.SEB1 marks the beginning of the US FDA's aptamer initiative, with respect to food safety and security. Aptamers possess binding properties similar to receptors or antibodies, without the ethical concerns of animal use, and they may be freely distributed throughout the world, making their use in assays an attractive alternative to immuno-affinity assays. Efforts are underway to develop and optimize assays with APTSEB1 to detect and quantify the presence of SEB in food matrices. Specifically, work developing an assay utilizing the surface plasmon resonance biosensor platform and other aptamer-affinity assays similar to that used in this study are currently in progress.APTS. aureus exotoxins is in progress, and the selected aptamers will be subsequently applied to the developed and optimized assays. With a concerted effort, aptamers could not only reduce the cost of food safety field assays, but also allow for widespread implementation of those assays by local health inspection agencies thereby empowering them with the tools necessary to enhance public health protection.Further, selection for a full range of aptamers corresponding to the family of


To the Editor: Different arboviral diseases have expanded their geographic range in recent times. Of them, Rift Valley fever, West Nile fever, and African horse sickness are of particular concern. They are endemic in sub-Saharan Africa but occasionally spread beyond this area. Trade and transport of animals and animal products, along with wildlife movements, are considered the driving factors in the spread of these pathogens.Camelus dromedarius) are valuable livestock appreciated as a meat source and as a means for transportation of goods. Camels are susceptible to infection by Rift Valley fever virus (RVFV), West Nile virus (WNV), and African horse sickness virus (AHSV), although their epidemiologic role in these diseases is uncertain . No differences in prevalence of RVFV antibodies were found by sex (p = 0.29), but prevalence was higher in Smara  and among camels >6 years of age  .Fifteen of 100 samples were positive for RVFV-specific antibodies by competitive ELISA, all of which were confirmed by virus-neutralization test, with neutralization titers ranging from 40 to 1,280 (geometric mean titer = 229). With regard to WNV antibodies, the ELISA detected 44 positive samples and 1 doubtful sample, of which 29 were confirmed as positive by virus-neutralization test . As for AHSV antibodies, none of the samples was positive by ELISA. Prevalence data were analyzed by generalized linear model with locality (Dakhla or Smara), sex, and age as fixed factors. No differences by origin or sex were found in prevalence for WNV (p>0.14) but antibodies were more prevalent in camels >3 years of age (χ = 0.04) . We also–Antibodies to 2 zoonotic arboviruses, i.e., RVFV and WNV, were present in camels moving to the northwestern part of the Sahara Desert, and antibodies to AHSV were absent in the populations examined. Despite the higher percentage of seropositivity for WNV than for RVFV, the epidemiologic consequence of RVFV-specific antibodies in this population could be higher than that for WNV antibodies. Camels can act as reservoir hosts for RVFV (The results of this study support that camels moving across the Sahara have contact with RVFV and WNV, and frequently the same animals have been infected by both agents. In a particularly dry environment such as the desert, particular attention should be paid to singular wet areas such as oases. The presence of water in these areas results in an abundance of competent mosquitoes and hosts, which in turn makes these viruses likely to cycle and infect domestic animals such as camels coming to drink and rest.


Orthohepadnavirus and represents a new species.During an analysis of the virome of bats from Myanmar, a large number of reads were annotated to orthohepadnaviruses. We present the full genome sequence and a morphological analysis of an orthohepadnavirus circulating in bats. This virus is substantially different from currently known members of the genus  Hepadnaviridae comprises 2 genera (Orthohepadnavirus and Avihepadnavirus), and viruses classified within these genera have a narrow host range. The genus Orthohepadnavirus consists of pathogens that infect mammals, and it currently contains 4 species: hepatitis B virus, woodchuck hepatitis virus, ground squirrel hepatitis virus, and woolly monkey hepatitis B virus. The genus Avihepadnavirus contains 2 avian species: duck hepatitis B virus and heron hepatitis B virus , Hipposideros armiger (n = 8), Rhinolophus ferrumequinum (n = 176), Myotis chinensis (n = 11), Megaderma lyra (n = 6), and Hipposideros fulvus (n = 12). All bat tissue samples were subjected to viral metagenomic analysis (unpublished data). The sampling of bats for this study was approved by the Administrative Committee on Animal Welfare of the Institute of Military Veterinary, Academy of Military Medical Sciences, China.We purchased 853 freshly killed insectivorous bats in Sedon and Wutao Counties in southeastern Kachin State, Myanmar; the counties are adjacent to Yunnan Province, People’s Republic of China. The bats covered 6 species: Miniopterus fuliginosus). Of these bats, 2.19% (7/320) were from Sedon County and 4.69% (15/320) from Wutao County; the viruses they harbored shared >98% nt identity. No other species had positive amplification results, indicating that M. fuliginosus was the most likely species to harbor orthohepadnaviruses.We used PCR to further study the prevalence of orthohepadnavirus in the 6 bat species; the condition of the samples made serologic assay and pathology impracticable. Viral DNA was extracted from liver tissue of each of the 853 bats by using the QIAamp DNA Mini Kit . To detect virus in the samples, we conducted PCR by using the TaKaRa PCR Kit  with a pair of degenerate pan-orthohepadnavirus primers (sequences available upon request). The PCR reaction was as follows: 45 cycles of denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 40 s, and a final extension at 72°C for 7 min. Positive results were obtained for 22 long-fingered bats  and 4 pairs of specific primers (sequences available upon request). Four overlapping amplicons were obtained, sequenced in both directions, and assembled into the full genomic sequence by using SeqMan, version 7.1.0 . All 3 full genomes (GenBank accession nos. JX941466– JX941468) were 3,230 nt in length, which is close to the size of primate hepatitis viruses  but smaller than rodent hepatitis viruses . We analyzed the genome structure by using Vector NTI Advance 10 . The results showed that the bat hepatitis viruses (BtHVs) contained the same circular and compact genomic structure as other orthohepadnaviruses, comprising 4 open reading frames encoding the multifunctional Pol, preS1/preS2/S, preC/C, and X proteins in the same direction  were constructed with ClustalW version 2.0  and MEGA5 , followed by clarification by low-speed centrifugation to remove cell debris. We then passed the pooled sample through a 0.22-μm syringe filter . Polyethylene glycol 6000 was added, and the resulting precipitate was sedimented at 12,000 × g in a desktop centrifuge  for 40 min at 4°C. The pellet was resuspended and examined after negative staining in a JEM-1200 EXII transmission electron microscope . Numerous spherical particles of ≈20 nm diameter were observed  or to a narrow host range of the virus. Further study is required to determine the tropism and prevalence of BtHVs in other bat species.Our observations provide strong evidence for the circulation of orthohepadnaviruses in at least 1 species of bats,


In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications. A range of subcloning methodologies has been developed for the synthesis and manipulation of genetic constructs, each with an unique dependence on endonuclease The immobilization and hybridization of DNA onto paramagnetic beads or other solid-phases have shown to be invaluable in several applications including next generation DNA sequencing Here we show a subcloning platform in which target genes are inserted into bead-immobilized acceptor vectors via solid-phase annealing and ligation. To demonstrate the utility of the platform, we have cloned human gene fragments, coding for potential cancer therapeutic targets, into bacterial, fungal, and mammalian expression vectors. We also demonstrate scale-up and automation of the platform and use it to transfer 95 target genes from a donor vector into a mammalian expression vector.A schematic view of the solid-phase cloning platform is shown in We constructed a panel of solid-phase acceptor vectors that can be used to express genes in several hosts  were excised from the donor vector and ligated to two separate, immobilized acceptor vectors, the ormation . Alternaormation .The solution-to-solid phase subcloning gave ligation efficiencies (∼50 cfu/ng vector DNA) much lower than a fully solution-based protocol . Notably, ligation efficiencies were not dependent on gene insert or vector size . We alsoWe hypothesized that ligation efficiency at higher bead loadings could be improved if both vector and gene DNA were immobilized onto beads; here the paramagnetic beads could be brought into very close contact with a magnet. This bead-bead subcloning process is depicted in We sought to characterize the critical bead-bead ligation step using both confocal microscopy and flow cytometry. We attached red and green fluorophores to gene- and vector beads, respectively. After ligation, the beads were examined qualitatively with a confocal microscope or quantitatively using a flow cytometer.Representative microscopy images are shown in Flow cytometry allowed quantification of the success of the bead-bead ligation reaction. Multi-bead constructs have a dual-color signature, as well as increased forward- and side-scatter. We expected gene-vector ligation efficiency to be greatly reduced in the solid phase reaction, partially due to the slow motion of the beads. To facilitate bead-bead contact we performed the ligation in the presence of a magnet, which brings the paramagnetic insert and acceptor-beads into close contact. To test the robustness of the procedure, we also investigated the effect of gene and vector length on ligation efficiency.S. carnosus surface display vector pSCEM2, the ligation efficiency was reduced 8-fold when the magnet was not used during ligation. We also observed a reduced number of successful clones, indicating vector re-ligation. The reduction in ligation efficiency in the absence of magnet is consistent with the change in the number of bead-bead constructs observed via flow cytometry.The ligation efficiency and accuracy were not dependent on the length of the gene or vector DNA. The presence of magnet had a strong positive effect on ligation efficiency. For example, in the ligation of TNF to the Notably, bead-bead ligation efficiency was reduced at higher DNA loadings. Increasing the vector DNA loading from 0.5 ng to 2 ng DNA/µg bead reduced the pSCEM2-TNF and pSCEM2-EGFR bead-bead ligation efficiencies by 2.4 fold and 1.8 fold, respectively. This is consistent with the observed decrease in ligation with bead loading in the solution-to-solid ligations .E.coli in a 96-well format. The total subcloning process, excluding transformation, was completed in approximately 3 h.We envisioned using the solid-phase cloning platform to rapidly switch gene cassettes between vectors in a large-scale manner. We programmed the optimized bead-bead protocol to a Magnatrix 8000 robotic liquids handler and used it to transfer 95 genes from the vector pAff8c to the mammalian expression vector pLentiHAp in a 96-well format. The genes ranged in size from 90 bp to 3300 bp .To demonstrate the potential of the solid-phase subcloning platform in aiding protein-expression studies, we chose three genes from our collection of clinically relevant genes  and subcloned them from the donor vector pAff8c into three expression vectors . DNA sequencing of successful clones verified correct in-frame ligation of the target gene into the expression vector. Of the three genes, only TNF was successfully expressed in S. carnosus surface-expression of the target genes from the pSCEM2 vector as determined by flow cytometry. TNF, CA9 ectodomain and PSMA ectodomain are expressed . Solid-phase vector construction could be used in tandem with a high-throughput protein expression and purification platform, such as one recently developed in our laboratory pastoris , althougE. coli. However, linear DNA could be used directly in transformations of hosts capable of linear-DNA uptake. Alternatively, whole bead-DNA constructs could be used for transfections We have developed a subcloning platform based upon the immobilization of DNA onto paramagnetic beads. The protocol can be used with PCR products or, in cases where additional PCR is not desirable, with inserts excised from donor vectors. We have circularized DNA after elution from beads for transformation into intra-bead bridging between proximal vector strands via ligation of compatible sticky-ends may hinder addition of the gene insert. This problem would be exacerbated at high vector densities, which is consistent with the observed reduced ligation efficiencies at high bead loading . This is consistent with previous solid-phase DNA manipulation studies, which demonstrated both efficient enzyme cleavage and strand DNA hybridization at sites >20 nt from the biotin immobilization site The ability to quickly pull out bead constructs via magnetic separation eliminated the need for gel extractions and spin-columns and simplified automation of the protocol. The sublconing process could be fully automated, with bead capture, enzymatic digestion, and DNA washing performed by a robot. Solid-phase cloning could be adapted to recently-developed microfluidics devices which are designed to manipluate magnetic microparticles with high precision An automated platform would also greatly aid high-throughput expression and function studies. We envisioned a scenario in which several expression hosts would be tested for the optimal expression of a panel of proteins. Using our optimized protocol, in which restriction digests were performed simultaneously and reagents were combined and stored as stock solutions, we were able to transfer 95 target genes from a donor vector to a mammalian expression vector in 3 h. A preliminary test of several of these proteins in different expression vectors showed interesting discrepancies in terms of secreted titer and glycosylation state.An additional direction of the bead-based cloning method is assembly of multicomponent constructs via sequential addition of genetic elements In conclusion, the approach shown here has demonstrated the power of magnetic beads for standardized highly automated subcloning of large number of fragments into expression vectors for protein expression screening and we point out a direction for use of magnetic beads in broader terms for generation of longer multicomponent genetic assemblies, relevant for metabolic engineering.All enzymes were from Fermentas UAB  and were FastDigest. We found the use of these FastDigest enzymes to be critical for simultaneous restriction digestions.solution-to-bead and bead-to-bead ligations were from an E. coli “donor” vector pAff8c Genes for Acceptor beads were prepared in bulk and used in solution-to-solid and solid-to-solid ligations. Briefly, acceptor plasmids were linearized (NotI/AscI) and a biotinylated linker containing a complementary AscI restriction site was attached to one end. The plasmids were then incubated with DynaBeads M270 streptavidin-coated beads  to create acceptor beads. Typical bead loadings were 0.5 ng plasmid DNA/µg beads, corresponding to ca. 1 500 DNA/bead. Details on acceptor vectors, including primer sequences, see Supplemental Information Vector beads were first treated with NotI (37 C 1 hr), which produced a free NotI end, and then washed twice in Tris buffer pH 8. Genes from the pAff8c clone library were prepared as NotI/AscI-digested, purified fragments, or as NotI/AscI-digested PCR products. The ligation reaction volume was 50 µL and included 5 µg of acceptor beads (loaded at 0.5 ng DNA/µg beads), a 10∶1 molar excess of insert DNA, 5 µL of T4 DNA ligase buffer, 5% v/v PEG4000 and 1 µL T4 DNA ligase. The ligation reaction proceeded at room temperature for 45 min under shaking. The beads were removed via magnetic separation and washed. The DNA was removed from beads with AscI treatment . The new vector constructs were circularized with addition of T4 DNA ligase and 0.5 mM ATP (22°C 10 min) and 3 µL used for bacterial transformation.Solution-to-bead ligation of the fluorescent capping oligo to M270-pHISZ was performed similarly but used 10 pmol of an 18 bp oligo containing a BamHI site and a 5′-AlexaFluor 488 . For the positive control, the pHISZ vector was capped with the BamHI oligo before bead loading, ensuring near 100% ligation and maximal fluorescence at each loading.Solid-to-solid bead ligations were performed with acceptor beads and donor beads. Donor beads were prepared by incubating streptavidin-coated M270 beads with biotinylated genes from the pAff8c clone library. Biotinylated genes were either from PCR with biotinylated primers or constructed via excision from the pAff8c vector followed by addition of a biotinylated oligo. For details of the preparation of donor beads see SI Methods.We performed the solid-to-solid ligation of acceptor and donor beads in 1.5 mL eppendorf tubes. For ligations in the presence of magnet we used a magnetic separation stand. In each case the ligation volume was 50 µL, included 1 µg of M270-acceptor beads (∼0.5 ng acceptor plasmid) and 10 µg of M270-donor beads (∼5 ng donor DNA), 5 µL of T4 DNA ligase buffer, 5% v/v PEG4000 and 1 µL T4 DNA ligase. The ligation reaction proceeded for 60 min, with pipette stirring every 10 min. DNA was removed from beads with AscI treatment, circularized and used for transformation as described above.Fluorophores AlexaFluor 488 and AlexaFluor 647 succinimidyl esters were from Invitrogen. Acceptor and donor-loaded beads were modified at pH 9 with AlexaFluor 647 and AlexaFluor 488 respectively, according to the supplier's protocol (50 µg donor beads and 50 µg acceptor beads were incubated with 50 µg of fluorophore for 1 h at 22°C). In this reaction the streptavidin is covalently modified at its amine groups. The beads were washed 3 times and re-suspended in Tris buffer pH 8. The donor-beads were loaded at 0.5 ng DNA/µg bead with ITGAL2b cut (NotI AscI) from the vector pAff8c and the vector beads were loaded with pLentiHAp at 0.5 ng DNA/µg bead. Donor-acceptor ligations were performed with the labeled beads according to the above protocols  in the presence and absence of magnet.The ligation mixture was analyzed with a Beckman-Coulter Gallios flow cytometer using FL-6 (647 nm) and FL-1 (488 nm) excitation and detection. Before analysis the ligation mixture was diluted to 300 µL in Tris buffer pH 8. Fifty thousand events were recorded for each sample.The subcloning protocol was programmed onto the Magnatrix 8000 robot  using the manufacturer's software. The pAff8c-donor library was used in 96-well format. Enzymes, M270 beads and acceptor beads were prepared as reagents and stored at 4°C during the procedure. Details of the protocol can be found in Supplemental Methods S1.E. coli, P. pastoris, S. carnosus and Chinese Hamster Ovary Cells .The target proteins TNF, CA9 and PSMA were expressed in E. coli RR1ΔM15 was transformed with pAff8c-target vector by heat shock and subjected to Km selection. Colonies were PCR screened with primers N2 and U5 and sequenced verified. Picked colonies were used to inoculate 10 mL of TSB medium (+Km) and grown overnight at 37°C. A culture aliquot (100 µL) was used to inoculate 50 mL of TSB (+Km) which grew to OD600 = 0.8 prior to induction with 0.5 mM IPTG. The cultures were moved to 25°C. After 6 hours the cells were pelleted, lysed with 5 mL of 7 M GdHCl (37°C 3 h). Lysis supernatant (5 µL) was run on SDS-PAGE gel.Staphylococcus carnosus was transformed with pSCEM-target vector via electroporation according to a published protocol Pichia pastoris strain SMD1168H and the P. pastoris expression vector pPICZα-C (Invitrogen) were a kind gift from Dr. Harry Brumer. P. pastoris was transformed with 0.5 µg pPICZαCp-target vector via electroporation according to the protocol of Wu PmeI before electroporation. Transformation mixtures were subjected to Zeomycin selection (125 µg/µL) and grown at 30°C. Colonies appeared after ca. 3 days. Colonies were lysed with Lyticase (Sigma) and PCR-screened Pichia pastoris colonies were cultivated in 50 mL solutions BMGY and BMMY at 25°C according to the Invitrogen protocol, with daily addition of MeOH to 0.5% v/v. After 96 hrs the cells were pelleted and lysed. Lysis supernatants were analyzed via Western blot, with anti-His6 antibodies (Sigma) and HRP detection (Sigma).Successfully transformed Mammalian cells CHO-S (ATCC) were transfected via lentiviral delivery, as described in detail in Supplemental Information. Transfected cells were selected via puromycin resistance. Cells were cultured for one week after transfection at a maintained 70–80% confluence then harvested and sonicated. Lysates were filtered and analyzed via Western blot, with anti-His6 antibodies  and HRP detection .Table S1Vectors used in this study.(DOC)Click here for additional data file.Table S2Oligos used in this study.(DOC)Click here for additional data file.Table S3Antigens in the pAff8c donor library.(DOC)Click here for additional data file.Figure S1Detailed bead-based subcloning strategy.(TIF)Click here for additional data file.Figure S2Size distribution of pAff8c donor library.(TIF)Click here for additional data file.Figure S3Detailed schematic of donor-bead construction and characterization.(TIF)Click here for additional data file.Methods S1Preparation of acceptor plasmids. Preparation of acceptor beads from acceptor plasmids. Preparation of pAff8c library. Preparation of donor beads from pAff8c library. Automation of bead-to-bead protocol. Transfection of CHO-S and mammalian cell culture.(DOC)Click here for additional data file.


Escherichia coli O157 antibodies and magnetic beads of two different sizes (1 μm and 2.6 to 2.8 μm) that contained a streptavidin coating, activated carboxyl groups or tosylated surfaces. The synthesized IMB, together with a commercially available IMB, were used to capture different strains of E. coli O157:H7 and E. coli O157:NM. The E. coli capture was measured by the time resolved fluorescence (TRF) intensity using a sandwich assay which we have previously demonstrated of having a sensitivity of 1 CFU/g after 4.5 hour enrichment [E. coli O157:H7, streptavidin-coated IMB elicited the highest response, on average. Streptavidin-coated IMB also provided an economic benefit, costing less than $0.50 per assay. The results could be used to guide the proper choice of IMB for applications in developing detection processes for E. coli O157:H7.Immunomagnetic beads (IMB) were synthesized using anti- This IMS also has the advantage of being very versatile. Many methods of detection can be used to quantify and/or identify the captured bacteria. IMB can be plated directly onto a selective medium  or used -3 to 10-6 s). This long decay time coupled with a large Stokes' shift (>200 nm) and narrow emission peak allow the chelate label to be read after nonspecific background has already decayed. This allows for increased sensitivity and enhanced signal-to-noise ratio. IMS combined with TRF has been successful in the detection of 10 cfu/mL of E. coli O157:H7 in apple cider [E. coli O157:H7 in ground beef [Salmonella and E. coli O157 from germinated alfalfa sprouts [In recent years, time-resolved fluorescence (TRF) has also been used quite extensively as a means of pathogen detection. TRF utilizes lanthanide chelate labels which produce intense fluorescent signals with a long half-life  were also used for comparison. Information about these beads is proprietary. However, the size and density were estimated by microscopic examination and discontinuous sucrose density gradient centrifugation, respectively [Two sizes of IMB consisting of anti- O157:H7 . IMB froectively . In addi2.2.E. coli O157:H7 antibodies  using a PolyLink Protein Coupling Kit (Bangs) following the manufacturer's instructions. Water soluble carbodiimide was used to activate the carboxyl groups on the surface of carboxylated beads. This reaction creates an active ester, which binds to the primary amine groups of the antibody. Biotinylated antibodies were conjugated onto beads containing a layer of streptavidin covalently attached to the bead surface. The affinity of streptavidin for biotin represents a strong noncovalent interaction, having a dissociation constant of ∼10-15 M, rivaling that of covalent bonds. Biotinylated goat anti-E. coli O157:H7 antibodies (KPL) were conjugated to both IMB-S and IMB-S1 at concentrations of 6 μg/mg and 20 μg/mg, respectively, as follows: suspensions of 100 μL of beads were mixed with 900 μL phosphate buffered saline . Six or twenty microliters of biotinylated goat anti-E. coli O157:H7 antibody  were added and allowed to bind at room temperature for 30 minutes with gentle rocking on a Specimix . After conjugation, the beads were washed 4 times with a solution of 20 mM PBS, 150 mM NaCl, 2 mM EDTA, and 0.5% BSA. After the final washing, the beads were resuspended in 1 mL of the same solution and stored at 2–8°C. Goat anti-E. coli O157:H7 antibody (KPL) was conjugated to IMB-T and IMB-T1 at concentrations of 20 μg/mg and 40 μg/mg, respectively, following the manufacturer's guidelines. Hydroxy groups on tosylactivated beads were treated with p-toluensulphonyl chloride, resulting in a sulphonyl ester which binds to amino or sulfhydryl groups of the antibody. The optional addition of 0.1% BSA after the first 10 minutes of conjugation was applied. The amount of conjugated antibody and linking chemistry of the IMB-D are considered proprietary by the manufacturer.Both sizes of carboxylated beads were conjugated with 16 μg/mg of goat anti-2.3.The amount of antibody bound to the IMB was determined by absorbance at 280 nm using a Cary 50 spectrometer . Standard curves of absorbance vs. concentration for each type of antibody were generated by measuring the absorbance of various antibody concentrations at 280 nm using a quartz cuvette. The extinction coefficients at 280 nm derived from the standard curves were used for estimating free antibody concentrations after conjugation. The amounts of antibody bound to beads were calculated from the differences between total-applied prior to and free-remained after the conjugation. The calculated concentrations of antibody bound to the beads are shown in 2.4.E. coli O157:H7 strains B1409 , SEA 13B 88 , and 380-94  along with E. coli O157:NM strain MF 13180-NM (FSIS) and non-O157 strain K12 (source unknown) were grown overnight in 25 mL mEC broth  at 37°C with shaking at 160 rpm . After overnight enrichment, bacteria in 1 mL of each culture were pelleted by centrifugation  and resuspended in PBS. The bacteria were then diluted 1:100 in PBS and enumerated using a Petroff-Hausser counting chamber (Hausser Scientific). Six microliters of the 1:100 diluted culture were placed onto the counting chamber slide. The slide consists of 25 0.2 mm×0.2 mm squares. The bacteria in five random squares were counted in duplicate, and cell concentration in cells/mL calculated. Serial dilutions of each E. coli strain were prepared in PBS from 1×108 to 1×102 cells/mL following enumeration. Two hundred microliters of the diluted suspensions were subjected to the TRF immunoassay as described in section 2.6.2.5.E. coli O157:H7 in the ground beef.For the ground beef experiments, a suspension of 1 mL PBS containing 25 cells was prepared and used to inoculate 25 g of ground beef in a stomacher bag with mesh filter . The inoculum was manually massaged into the ground beef followed by the addition of 225 mL of mEC broth. The sample was then mixed in a stomacher  on low for 30 s, followed by enrichment for 24 h at 37°C with shaking at 160 rpm. After enrichment, aliquots of 200 μL from the side of the mesh filter without ground beef were withdrawn for IMS capture and TRF assay as described in section 2.6. A second aliquot of ground beef was inoculated with 1mL PBS to serve as a blank. The blank was run alongside the sample to gauge the growth of background organisms. The post-enriched sample was also plated onto Sorbitol MacConkey agar supplemented with cefixime and tellurite  to enumerate the growth of O157:H7 in ground beef. The post-enrichment blank was plated on both plate count agar  and CT-SMAC to enumerate background organisms and ensure no indigenous 2.6.E. coli cells or enriched ground beef samples with a volume of 200 μL were added to 20 μL of beads. To rows B and D, 200 μL of washing buffer diluted 1:25 from wash concentrate  supplemented with 0.5% Tween 20  was added. Goat anti-E. coli O157:H7 (KPL) was labeled with europium using a DELFIA® Eu-Labeling Kit  following the manufacturer's specifications. The labeled stock was stored frozen at -20°C. Prior to the assay, the europium-labeled detection antibody was diluted in assay buffer  supplemented with 0.1% Tween 20 to a concentration of 1 μg/mL, filtered through a 0.45 μm syringe filter , and 200 μL added to each well of row C. Lastly, 200 μL of enhancement solution  was added to row E. The samples were processed in the KingFisher, which magnetically transfers the beads from row to row. After initial binding of bacteria to beads in row A for 15 min, the bead/bacteria complexes were washed for 1 min in row B. The washed complexes were then transferred to row C and allowed to bind with the europium-labeled detection antibody for 1 h. After another 1 min wash in row D, the complexes were transferred into the enhancement solution in row E. Proprietary chelators in the enhancement solution extracted the europium to form an Eu-chelator that emitted strong fluorescent signal at 615 nm. The fluorescence intensity displayed as counts per second (CPS) was measured using a VICTOR2 1420 multilabel counter .Immunomagnetic separation (IMS) was carried out using the KingFisher magnetic particle processor . The KingFisher automatically transfers beads between binding and washing steps. The entire assay was performed in black 96-well microtiter plates . To row A, suspensions of cultured 2.7.Data were replicated at least three times, with averages and percent error reported. The percent error was calculated by dividing the standard deviation among replicates by the average signal obtained by the TRF assay and multiplying by 100. Microsoft Excel was applied to determine the statistical parameters of variance and standard deviation using Microsoft Excel spreadsheet. Normalized responses per number of applied beads data were calculated by dividing the TRF response by the number of beads used in the assay for further evaluation on the interaction of the bacteria with the beads.2.8.E. coli antibody costs $400/1 mL. In the labeling procedure, 100 μL of IMB are labeled with 6 μL antibody. Therefore, the beads are $8.99 for 100 μL, and the antibody is $0.40 per μL or $2.40 per 6 μL. Adding the cost of the beads and antibody together, the total cost for preparing 1mL of IMB-S is $11.39. Since 20 μL of beads are used per assay, the cost per assay is $0.23.The cost of each IMB per assay was estimated by taking into consideration the cost of the beads, antibody, and coupling kit (for carboxylated beads only) used in the conjugation of each IMB. For example, IMB-S costs $899/10mL. Biotinylated goat anti-3.3.1.Additionally, a cost analysis was done to give an approximate cost of each IMB per assay (20 μL), taking into account the amount of antibody applied, amount of beads used in the labeling process, and coupling kits where applicable. Common reagents/solutions were not included in the cost analysis since they were made in-house from lab grade chemicals. Cost analysis results are also shown in 3.2.E. coli O157:H7 strains B1409, 380-94, and SEA 13B 88 along with E. coli O157:NM were diluted in PBS and assayed with each IMB. E. coli K12 was also tested with each IMB for cross-reactivity. No detectable signal was observed for K12 (data not shown). 7 cells/mL of E. coli O157. The top graph, A, shows the capture of bacteria as measured by the fluorescence intensity (CPS) for each strain tested with each IMB. For all strains, IMB-D provided the highest response, followed by IMB-C or IMB-T. IMB-S provided the lowest response for all strains except O157:NM. The data in the bottom graph, B, has been normalized to the number of beads contained in the 20 μL assay volume . However, when the data is normalized to the number of beads used in the assay, it becomes clear that three IMB have advantages over the others. IMB-S, IMB-C, and IMB-T all elicit higher signals per bead than the others. This is consistent with the observations in suspension that the larger beads capture more bacteria and thus elicit a higher signal. In addition, the normalized data is also consistent with results of IMB-D as seen in suspension. Therefore, it can be said with confidence that neither background flora present in ground beef nor the matrix of ground beef itself interferes with the capture and detection of E. coli O157:H7.The total aerobic plate count for background microflora revealed 2.0×104.E. coli O157 react differently to the same beads. This could be due to antigenic differences between the strains. For all strains tested with the 1 μm beads, the streptavidin-coated beads elicited the highest response. For the larger beads, IMB-S also provided the highest normalized signal for strains B1409 and O157:NM. All IMB except IMB-D produced similar signals with strain 380-94. Strain SEA 13B 88 was the only strain which saw IMB-S produce the lowest signal. Again, different antigenic features of this strain are the most probable explanation as to the lower capture with the IMB-S. While each type of antibody linkage tested favorably, especially in a real world sample, the streptavidin-coated beads may hold a slight advantage. On the whole, streptavidin-coated particles performed very well in the capture of E. coli O157:H7 and O157:NM. Perhaps the orientation of the antibodies on the surface of the streptavidin-coated beads allows for enhanced capture. In addition, streptavidin-coated IMB also provide an economic benefit, costing under $0.50 per assay.We have shown in this report and others that larger size IMB are more effective in the capture of a target of interest. While the surface antibody concentration of IMB-S and IMB-T was less than that of the smaller beads, they still elicited a higher response. This is due to the ability of the larger beads to interact with target organism in larger volumes as proposed in our previous study , 19. IMB


West Nile virus RNA was detected in brain tissue from a horse that died in June 2003 in Nuevo Leon State, Mexico. Nucleotide sequencing and phylogenetic analysis of the premembrane and envelope genes showed that the virus was most closely related to West Nile virus isolates collected in Texas in 2002. Flavivirus (family Flaviviridae), was first recognized in the Western Hemisphere during an outbreak in New York in 1999 (West Nile virus (WNV), a mosquitoborne virus in the genus  in 1999 , 7 Canad in 1999 , and sev in 1999 –7.A phylogenetic analysis of the prototype New York strain (WN-NY99-flamingo382-99), isolated from a dead flamingo from the Bronx Zoo in 1999, showed that this strain was most similar to an Israeli isolate from 1998 . HoweverMore recently, Estrada-Franco et al.  reportedCerebellar tissue was taken from a dead 12-year-old stallion from a privately owned ranch in the municipality of Juarez in Nuevo Leon State, Mexico, approximately 240 km south of the Texas border. The horse was first observed with neurologic symptoms on June 20, 2003, and it was euthanized 7 days later. The horse had never been outside the state of Nuevo Leon and had not been vaccinated against WNV. The tissue sample was immediately placed on dry ice and transported to the biosafety-level-3 facilities at Colorado State University for processing. Although we were unable to isolate virus from the sample by passing brain homogenate in Vero cells, we successfully amplified viral RNA.Total RNA was extracted from approximately 100 µg of cerebellar tissue with Trizol reagent , according to the manufacturer's instructions. The prM-E genes were amplified as two fragments by reverse transcription-polymerase chain reaction (RT-PCR) by using primers designed from the nucleotide sequence of the prototype WN-NY99 strain (GenBank accession no. AF196835). PCR amplifications were performed by using Ex Taq DNA polymerase , which has 3´ → 5´ exonuclease activity. Amplification products were separated by agarose gel electrophoresis, visualized with crystal violet, and extracted by using the rapid gel extraction system . The resulting DNA fragments were reamplified by PCR because of the low RNA copy number in the original material and purified by using the QIAquick PCR purification kit . Purified DNAs were sequenced on both strands with an ABI 377 DNA sequencer  and eight pairs of WNV-specific primers.The nucleotide sequence of the prM-E genes of the WNV from Nuevo Leon State, Mexico (designated MexNL-03) was submitted to GenBank (GenBank accession no. AY426741). This region comprises 2004 nucleotides and corresponds to nucleotides 466 to 2469 of the genomic RNA of the WN-NY99 strain . AlignmeThe nucleotide sequence of MexNL-03 differed from that of the WN-NY99 strain (GenBank accession no. AF196835) in six positions . The bootstrap support for this topologic arrangement ranged from 58% to 73%. Similar findings were also reported by Estrada-Franco et al. . AdditioThe data presented here indicate that WNV was introduced into Nuevo Leon State, Mexico, from inland Texas. A likely mode of introduction was by infected birds traveling for short distances . Earlier


Because bats are associated with emerging zoonoses, identification and characterization of novel viruses from bats is needed. Using a modified rapid determination system for viral RNA/DNA sequences, we identified a novel bat betaherpesvirus 2 not detected by herpesvirus consensus PCR. This modified system is useful for detecting unknown viruses. Since the 1990s, bats have been associated with several emerging zoonotic agents, including Hendra, Nipah, Ebola, lyssa, and severe acute respiratory syndrome coronavirus-like viruses  was noted at third passage. The collected supernatant was injected into fresh primary kidney cells and caused apparent cytopathic effect at first passage.During June–August, 2008, with the permission of the governor of Wakayama Prefecture, Japan, we caught 8 insectivorous vespertilionid bats, Before using the RDV method, we had attempted to detect herpesvirus by nested PCR with the consensus primer sets DFA, ILK, KG1, TGV, and IYG, which were designed according to the consensus-degenerate hybrid oligonucleotide primers program  search. In a BLASTx search, 1 cDNA fragment (deduced sequence of 29 aa) was homologous to the glycoprotein B (gB) amino acid sequence of the tupaiid herpesvirus 1 (TuHV-1) (79% identity), which belongs to subfamily Betaherpesvirinae. We designed new consensus-degenerate hybrid oligonucleotide primers (http://blocks.fhcrc.org/codehop.html) selective for the betaherpesvirus gB and DNA polymerase (DPOL) genes, and we determined the complete gB sequence and the partial DPOL sequence of the isolated virus . BLAST search indicated that the complete gB sequence was novel and most similar to that of TuHV-1 (59% aa sequence identity)  . We nameBetaherpesvirinae consists of the genera Cytomegalovirus, Muromegalovirus, and Roseolovirus. TuHV-2 and caviid herpesvirus 2 are species unassigned to any genus in the subfamily Betaherpesvirinae.We constructed a phylogenetic tree by using the neighbor-joining method with the gB amino acid sequence and the available sequences of known herpesviruses . The phyM. fuliginosus, from the same location for an epizootologic study . The variety of virus sequences and gene mutations often prevents successful amplification of virus genes. RDV, however, can detect viral cDNA fragments independent of virus species and thus is useful as a first-choice tool for identifying emerging known and unknown viruses in animals and humans.,,BLAST search showed that the complete gB sequence of the isolated virus was novel and most similar to that of TuHV-1. Recently, bats have been described as hosts for herpesviruses in several countries in Europe, America, Africa, and Asia (Our epizootologic study found relatively high (8%) prevalence of BatBHV-2 in insectivorous bats. Although the virus genome was detected in a few parenchymal organs by nested PCR, no amplification was possible for serum, intestine, or urinary bladder samples, which may exclude apparent virus shedding by the bats. In addition, all 50 bats collected appeared clinically healthy. To understand the life cycle of this virus, the possibility of a latent infection in these insectivorous bats must be explored.Phylogenetic tree based on the deduced amino acid sequences of complete glycoprotein B. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test  is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The tree was rooted to herpes simplex virus type 1 (X14112). The evolutionary distances were computed by using the Poisson correction method and are in units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated from the dataset. The final dataset included a total of 698 positions. Phylogenetic analyses were conducted in MEGA4 (System for Rapid Determination of Viral RNA Sequences, Version 3.1.Epizootologic Study.


Purpose. To evaluate sex-related differences in the prevalence of and cardiovascular risk factors related to gallstone disease (GSD) in an elderly agricultural and fishing population of Taipei, Taiwan. Methods. The study sample consisted of 6511 healthy elderly participants (3971 men and 2540 women) who were voluntarily admitted to a teaching hospital for a physical checkup in 2010. The participants' blood samples and real-time ultrasound fatty liver results were collected. Results. The prevalence of GSD in the study population was 13.2%, which increased significantly with population age (P < .0001). Women were associated with significantly higher GSD prevalence than men . In a multiple logistic regression analysis, female sex, older age, and metabolic syndrome (MetS) were significantly associated with GSD. Multiple logistic regression analysis also revealed that obesity (odds ratio (OR) = 1.26, 95% confidence interval (CI): 1.09–1.44) and metabolic factors  were significantly associated with GSD in women but not in men. Conclusion. In the study population, female sex, older age, and MetS were associated with higher GSD prevalence. The population exhibited other sex-related differences. Gallstone disease (GSD) is the most common disease of the biliary system and most cases are asymptomatic . It is aGSD fulfills the Wilson screening criteria, which specify that the disease is a critical health problem and are listed as follows: the disease natural history should be understood; there should be a recognizable latent or early symptomatic stage; there should be a test that is easy to perform and interpret, acceptable, accurate, reliable, sensitive, and specific; there should be an accepted treatment recognized for the disease; treatment should be more effective if started early; there should be a policy on who should be treated; diagnosis and treatment should be cost effective; and case finding should be a continual process . In agriThe sample for this cross-sectional study consisted of 6511 elderly adults (3971 men and 2540 women) with agricultural and fishing occupations who were voluntarily admitted to a teaching hospital in Northern Taiwan for an annual physical checkup between January 2010 and December 2010. Blood samples and ultrasound fatty liver results were collected. The participants completed a questionnaire on their demographic data, personal disease history, medications, and family history. Fasting blood samples were collected by clinical nurses using venipuncture. Overnight-fasting serum and plasma samples (from whole blood preserved with EDTA and NaF) were stored at −20°C until analysis. All procedures were performed according to the guidelines of the hospital institutional ethics committee and adhered to the tenets of the Declaration of Helsinki. All patient information was anonymous.In this study, the age ranges of study participants were stratified as follows: 60–64 yrs, 65–74 yrs, 75–84 yrs, and ≥85 yrs. The medical histories and measurements of the participants were obtained by well-trained nurses. Personal and family histories of hypertension, type 2 diabetes, cardiovascular diseases, and other chronic diseases were obtained by a structured health interview questionnaire. The study participants were asked to take off the shoes and any other belongings that could possibly add extra weight when they were weighed. Heights and weights were evaluated according to body mass index (BMI). Also the waist circumference was measured at the level of the iliac processes and the umbilicus with a soft tape measure to estimate abdominal obesity. Blood pressure for each subject was measured twice in the sitting position with an interval of 15 minutes between the measurements, by means of standard sphygmomanometers of appropriate width, after a rest period for 30 minutes. Those taking antihypertensive therapy were considered to have known hypertension .2; hypercholesterolemia, cholesterol ≥200 mg/dL; high BUN, BUN ≥20 mg/dL; high creatinine, creatinine ≥1.4 mg/dL; and hyperuricemia, uric acid ≥7 mg/dL in men and ≥6 mg/dL in women. Serum ALT or AST levels ≥40 U/L were classified as elevated [The definitions of the relevant diseases or conditions were as follows: obesity, body mass index (BMI) ≥25 Kg/melevated .GSD was diagnosed by a panel of radiologists using real-time ultrasound . The abdominal regions were examined after the patients had fasted for at least 8 hours, and GSD was identified based on the presence of movable hyperechoic foci with acoustic shadows. GSD was classified as single GSD, multiple GSD, or cholecystectomy, excluding gallbladder polyps. Any type of GSD was considered a case in the study population .To ensure consistent GSD diagnosis between the 2 specialists, the weighted Kappa statistic was used to assess the agreement of interobserver reliability. A pilot study was performed using 50 randomly selected nonstudy participants and included 4 states of GSD. For interobserver reliability, the weighted Kappa value for the diagnosis of GSD between the 2 specialists was 0.78 : 0.70–0.87). For intraobserver reliability, the weighted Kappa value for the diagnosis of GSD was 0.80 (95% CI: 0.75–0.87) for one radiologist and 0.82 (95% CI: 0.74–0.91) for the other radiologist.P  value < .05 was considered to represent a statistically significant difference between 2 test populations.Statistical analysis was performed using SAS for Windows . Crude and adjusted odds ratios (ORs) (adjusted for sex and age) were estimated, and 95% CIs were applied. Multiple logistic regression was also performed to investigate the independence of the factors associated with the prevalence of GSD. A P = .003). According to a Cochran-Armitage trend test, the prevalence of each type of GSD increased with age (P < .0001). The participants aged ≥85 years (82/323 = 25.4%) were associated with a more than 2-fold higher risk of GSD compared with the participants aged 60–64 years (225/2140 = 10.5%).As shown in We evaluated the effects of independent associated risk factors for GSD by using a multiple logistic regression model. As shown in Taiwan has experienced rapid economic development and industrialization, accompanied by changes in traditional diets and increasingly sedentary lifestyles. One of the crucial benefits of early screening for GSD by using ultrasonography is the detection of asymptomatic cases, which can enable the early treatment of GSD and the prevention of serious outcomes such as acute GSD pancreatitis and gallbladder cancer , 13. FewIn this study, we applied the methods for GSD assessment used in previous studies , 8, obsePatients with MetS  are associated with higher risk of cardiovascular disease irrespective of previous history of cardiovascular events . PreviouObesity is a major risk factor for GSD development because it is associated with increased hepatic secretion of cholesterol , 18. TheOne of the major limitations of this study was the evaluation of GSD prevalence and GSD-related risk factors through the screening of an elderly population residing in a single area. However, our study results retained sufficient statistical power for evaluating any sex differences among the various risk factors for GSD because of the considerably large sample size. Second, we evaluated only elderly participants, who might have characteristics that differ from those of the general population. However, this subpopulation is known to be more susceptible to GSD compared with other populations of Taiwan. Third, nonrespondents who did not return for physical examination may have more prevalent GSD or cholecystectomy and the prevalence of GSD may be underestimated. Fourth, we did not evaluate the postcholecystectomy syndrome in this study. This syndrome not only can represent either the continuation of symptoms thought to be caused by the gallbladder or the development of new symptoms normally attributed to the gallbladder but also includes the development of symptoms caused by removal of the gallbladder . Should Our study results indicate that female sex, older age, and MetS are associated with higher prevalence of GSD in the elderly occupational population of Taiwan. In addition, we observed several sex-related differences in the study population. Further investigation is required to elucidate the temporal sequence of events leading to GSD development and to determine the underlying mechanisms of the sex-related differences in GSD prevalence in the elderly population.


During 2009/10 a major measles epidemic caused by genotype B3 occurred in South Africa. Measles inclusion body encephalitis (MIBE) was diagnosed in a number of highly immuno-compromised HIV patients. The diagnosis was based on typical clinical and MRI findings and positive measles virus PCR in brain or CSF.To characterize the brain virus, nucleoprotein, matrix, fusion and haemagglutinin genes from 4 cases was compared with virus from acutely infected patients.cDNA was synthesized using random primers and viral genes were amplified by nested RT-PCR. PCR products were sequenced in the forward and reverse direction and a contig of each gene was created. Sequences were aligned with reference sequences from GenBank and other local sequences.Brain virus was very similar to the South African epidemic virus. Features characteristic of persistent measles virus in the brain were absent. Mutation frequency in brain virus was similar to epidemic virus and had the same substitution preference (U to C and C to U). The virus of 2 patients had the same L454W mutation in the fusion protein.The brain virus was very similar to the epidemic strain. The relatively few mutations probably reflect the short time from infection to brain disease in these highly immuno-compromised patients. During 2009 and 2010 a widespread measles virus epidemic occurred in South Africa. More than 18 000 cases were laboratory confirmed. The majority of infections were in young people. One third were infants less than one year of age and the rest were between one and 40 years  were allThere are three neurological complications following acute measles infection. Within 2 weeks of the onset of the rash, an acute demyelinating encephalomyelitis (ADEM) may develop. This is an auto-immune phenomenon as measles virus is not present in the brain . MeaslesComplete sequence data for N, M, F and H genes was obtained for brain virus of 2 patients (1 and 3) and epidemic virus from 6 patients (GenBank accession numbers KC305651-KC305689]. Incomplete sequence data was obtained from brain virus of a further 2 patients (2 and 4). Partial sequence was due to limited clinical material available for PCR amplification. For patient 4, most of the H gene sequence was available (nucleotides 246–1874) and partial sequence for the F gene (nucleotide 1–1690) and for patient 2, the complete N, M and F genes were sequenced.Phylogenetic analysis of the nucleoprotein and haemagglutinin genes confirmed that the South African epidemic in 2009/10 was caused by genotype B3. . A concatenated phylogenetic tree of the full N, M, F and H genes confirmed a close relationship of the South African measles viruses to B3 viruses from North Africa .Sequences from the acute epidemic virus were aligned and used to create a South African consensus sequence to which the brain virus of patients 2, 3 and 4 were compared. The brain virus from patient 1 was compared with her own acute blood virus from 3 months earlier. In all patients the brain virus was very similar to the epidemic virus. Typical features of measles virus of SSPE/MIBE cases were not present, namely hyper mutation of the matrix and fusion proteins or truncation of C terminal end of fusion protein gene . Each MIU to C and C to U mutations were the most common mutations present in the epidemic virus and were also the most frequent in the brain virus. The calculated mutation frequency in brain virus was 0.87 per 1000 nucleotides. This was not significantly different from the mutation frequency of the epidemic virus which was 0.56 per 1000 nucleotides (p=0.19). Interestingly, of the 10 substitutions that occurred in coding regions in the brain virus, 8 were non-synonymous. The most variable region was the non coding region of the fusion gene.Patients 1 and 3 had an identical U to G mutation at position 1944 in the F gene which gave rise to a L454W substitution in the fusion protein. Of note, the L454W substitution was not present in the blood virus of patient 1 collected during acute measles infection 3 months earlier.The measles virus outbreak in South Africa was due to genotype B3 a known African genotype. It was probably introduced into South Africa from the north where ongoing circulation of measles virus of this genotype has been documented in various African countries for many years ,11. InfeWhile invasion of the brain during acute measles may occur , MIBE isOn the whole, the brain virus was very similar to the acute epidemic virus. This is in contrast to what is found with SSPE ,15 and aet al. .None of the authors have any competing interests.DH: conceived the study, analysed the results and wrote the manuscript. JH and CA: identified the cases of MIBE and helped to write the manuscript. HS: designed primers to amplify measles virus genes; assembled contigs of the genes; performed phylogenetic analysis and wrote part of the manuscript. All authors read and approved the final maunscript.Primer sequences used in amplification of measles virus nucleocapsid, matrix, fusion and haemaglutinin genes.Click here for file


A surprising portion of both mammalian and Drosophila genomes are transcriptionally paused, undergoing initiation without elongation. We tested the hypothesis that transcriptional pausing is an obligate transition state between definitive activation and silencing as human embryonic stem cells (hESCs) change state from pluripotency to mesoderm. Chromatin immunoprecipitation for trimethyl lysine 4 on histone H3 (ChIP-Chip) was used to analyze transcriptional initiation, and 3′ transcript arrays were used to determine transcript elongation. Pluripotent and mesodermal cells had equivalent fractions of the genome in active and paused transcriptional states (∼48% each), with ∼4% definitively silenced (neither initiation nor elongation). Differentiation to mesoderm changed the transcriptional state of 12% of the genome, with roughly equal numbers of genes moving toward activation or silencing. Interestingly, almost all loci (98–99%) changing transcriptional state do so either by entering or exiting the paused state. A majority of these transitions involve either loss of initiation, as genes specifying alternate lineages are archived, or gain of initiation, in anticipation of future full-length expression. The addition of chromatin dynamics permitted much earlier predictions of final cell fate compared to sole use of conventional transcript arrays. These findings indicate that the paused state may be the major transition state for genes changing expression during differentiation, and implicate control of transcriptional elongation as a key checkpoint in lineage specification. Initially thought to occur only in rare instances such as Drosophila heat shock genes  and beating cardiomyocyte cultures were collected on day 14 BRACHYURY T but not SOX1 transcription at day 2 were subjected to further analysis.Undifferentiated H7 (WiCell) human embryonic stem cells were maintained on Matrigel-coated plates in mouse embryo fibroblast (MEF) conditioned medium as previously described g for 5 minutes) and washed twice with cold PBS and stored at −80°C. Thawed pellets were lysed with IP buffer  supplemented with Complete™ Protease Inhibitors Cocktail Tablets (Roche). The nuclear pellet was collected after centrifugation , resuspended in IP buffer and sonicated for 6 rounds of 10 one-second pulses with half-second pauses at a power level of 5 (Misonix 3000 sonicator with a micro tip Cat#S3000). Sheared chromatin was distributed such that approximately 2×106 cells-worth was combined with an antibody recognizing tri-methyl lysine 4 on histone H3 (H3K4me3) or pan-histone H3. Protein-A sepharose beads (Amersham Cat#17-5280-01) were used to collect precipitated nucleosomes. After extensive washes with IP buffer, 100 µL of 10% w/v Chelex® 100 Molecular Biology Grade Resin (Bio-Rad Cat#142-1253) beads were added to each sample, and the samples were boiled for 10 minutes after a brief vortex. Proteinase K  was added and samples were digested 30 minutes at 55°C at 1200 RPM in a shaking heat block, and then boiled for an additional 10 minutes.Samples were prepared and analyzed as recently described by Nelson et al. Immunoprecipitated H3K4me3 DNA and control pan-histone H3 immunoprecipitated DNA were purified by treatment with RNAse A, proteinase K and two successive phenol∶chloroform∶isoamyl alcohol extractions. Purified DNA ends were blunted using T4 DNA polymerase, and were then ligated to linker oligonucleotides. The resulting DNA was amplified using a two-stage ligation-mediated PCR (LMPCR) protocol. Amplified DNA was labeled with either Cy5-dUTP (H3K4me3) or Cy3-dUTP (histone H3) using Bioprime random primer labeling kits (Invitrogen) and purified using spin columns. Labeled DNA was then mixed (5–6 µg each of immunoenriched H3K4me3 DNA and control histone H3 DNA) and hybridized to arrays in Agilent hybridization chambers for up to 40 hours at 65°C. Arrays were then washed and scanned in an Agilent DNA microarray scanner BA using the extended dynamic range setting. Complete details of the entire ChIP-chip procedure are described in www.agilent.com). The array consists of 2 slides each containing ∼244,000 60mer oligos designed to cover regions between approximately −4 kb and +4 kb relative to the transcription start sites of ∼18,464 genes at a density of about 1 oligo per 250 bp.The human promoter array (244K Human Promoter Array) was purchased from Agilent Technology . Total RNA was purified the MirVana RNA purification kit . Sample amplification, labeling and hybridization on Illumina Human WG6 Sentrix BeadChips were performed for all arrays in this study according to the manufacturer's instructions  using an Illumina BeadStation in the Burnham Institute's Microarray Core Facility. Oligo dT priming ensured that only full-length transcription was measured. Raw data extraction was performed in the BeadStudio software suite  and probes with a detection score less than 0.99, a measure of confidence that the signal observed is above background fluorescence, in all of the samples were discarded. The resulting probes were then quantile-normalized to correct for between-sample variation Each sample was prepared from approximately 1×10p-value for a given transcript was less than 10−3  we considered the locus to be producing full-length transcript.If the detection H3K4me3 data were normalized and stored in metagene format as log2 over panH3. If any of the following are true, then the locus is determined to be H3K4me3 bound :Any point over 1.5 log2 over panH3.Any point over 0.8 log2 over panH3 within −1250 to 750 bp relative to the transcription start siteA customized, Python-based program was written to assign loci to specific transcription states and characterize the transitions between these states. For each cell population, if a locus is called as having both full-length transcript and H3K4me3, it is placed into a list of actively transcribed loci. If the locus instead is only called as being H3K4me3 bound, it is placed in the paused list. If the locus is neither called for having full-length transcript nor H3K4me3, it is added in the silent list .For two cell populations and three possible transcriptional states, there are nine potential transitions. Using the lists of transcriptionally active, paused or silent loci for the pluripotent and mesodermal cell populations generated, we employed the following logic . If a loFor gene ontology analysis, we used NCBI's capture of the gene ontology database in the gene2go.gz file, to gather the list of loci annotated as being within a given ontology. For each of these loci, we determine which of the nine possible transitions the locus is undergoing using the genome-wide lists previously generated.In order to assess the relative contribution of the paused state to early human development, we used a recently established system of directing the differentiation of human embryonic stem cells to early mesoderm We used the histone H3-lysine-4 trimethyl modification (H3K4me3) as a chromatin marker of transcriptional initiation We first focused on three developmentally regulated loci with known functions in lineage commitment to see that they behaved as expected in our differentiation system: BRACHYURY T (a transcription factor required for mesoderm differentiation)Having verified that genes with well-established biological functions behave as expected in our system, we next computationally sorted all detectable protein-coding loci in the human genome into one of the three transcriptional states  based on their association with H3K4me3 and full-length transcript abundance . We thenWe next sought to determine if genes changing expression transition directly from an active to a silent state (or vice-versa), or if they instead pass through a paused intermediate. Strikingly, we found an overwhelming 98.0 to 98.9% of genes changing expression are transitioning into or out from a paused intermediate . These dTo better understand the physiological significance of these transcriptional changes from embryonic stem cells to mesoderm, the Gene Ontology database We hypothesized that, by focusing on genes that were transcriptionally paused in embryonic stem cells and changed state during commitment to mesoderm, we could predict the later cardiomyocyte fate of the population . ConsistThe paused state of transcription seems to play a central role in the change of gene expression during the differentiation of pluripotent human embryonic stem cells . By usinSeveral studies have recently shed light into the molecular regulation of transcriptional pausing. The negative elongation factor, NELF, is a four-subunit complex that cooperates with the two-subunit DRB-sensitivity inducing factor, DSIF, to bind PolII and repress elongation The widespread use of transcriptional pausing as a means to control mRNA expression differs significantly from translational regulation during ESC differentiation. We recently performed translation state array analysis on differentiating murine ESCs, and we found that the majority of mRNA transcripts synthesized are loaded directly onto ribosomes The induction of mesoderm requires coordinated signaling through members of the TGF-beta superfamily and the Wnt/β-catenin pathways While our study has focused on differentiation of hESCs, we hypothesize that the checkpoint of paused transcription is a more general phenomenon as cells undergo other changes of state. For example, activation of endothelium by cytokines, conversion of blood monocytes to macrophages or differentiation of skeletal myoblasts to myotubes all are accompanied by changes in mRNA transcription. It will be of interest to determine whether paused transcription serves as an obligate checkpoint for transcriptional regulation as these cells change state in response to environmental cues.Figure S1Computational analysis of data. (A) To call if a locus is associated with tri-methylation on lysine 4 of histone H3 (H3K4me3), we used the genome-wide chromatin immunoprecipitation data in metagene format, normalized to the transcription start site of each protein coding locus and as a log2 over the pan-histone H3 signal at the equivalent probe. The locus was declared H3K4me3 bound if any point within the locus's metagene was above 1.5 log2 or over pan-histone H3, over 0.8 log2 over pan-histone H3 from −1250 to +750 from the transcription start site. (B) The logic flowchart to call each locus as being active (A), paused (P), or silent (S) based on the prior calls for H3K4me3 and full-length transcription for each locus. (C) The logic flowchart to determine the transition of each locus from pluripotency to mesendoderm.(TIF)Click here for additional data file.Figure S2During cardiomyocyte differentiation, ontologies of genes involved in cardiac differentiation activate, those involved in ectodermal derivatives silence. For each ontology, the percentage of changing loci that are transcriptionally archiving (from initiating to silenced) or activating (from initiating to elongating) is depicted for our differentiation towards cardiomyocytes. The loci of genes in the ectodermal ontologies of neurotransmitter receptor activity (GO:0030594), keratinization (GO:0031424) and brain development (GO:0007420) starting as initiating tend to archive and lose initiation. The loci of genes in the cardiac mesoderm ontologies of heart development (GO:0007507), blood vessel development (GO:0001568), heart looping (GO:0001947) and regulation of heart contraction (GO:0008016) tend to activate and start elongating.(TIF)Click here for additional data file.


In vivo, the virus predominantly infects lymphocytes and mammary epithelial cells. High level infection of mammary epithelial cells ensures efficient passage of virus to the next generation. It also results in mammary tumor induction, since the MMTV provirus integrates into the mammary epithelial cell genome during viral replication and activates cellular oncogene expression. Thus, mammary tumor induction is a by-product of the infection cycle. A number of important oncogenes have been discovered by carrying out MMTV integration site analysis, some of which may play a role in human breast cancer.Mouse mammary tumor virus (MMTV), which was discovered as a milk-transmitted, infectious cancer-inducing agent in the 1930s, has been used since that time as an animal model for the study of human breast cancer. Like other complex retroviruses, MMTV encodes a number of accessory proteins that both facilitate infection and affect host immune response.  More reentified ,74; howeentified –78. Moreentified ,16,79. T5.in vivo virus infection using MMTV and mice. This is in a large part due to our ability to study the natural route of milk-borne transmission and to the use of both inbred and genetically altered transgenic and knockout mice in the analysis of infection and oncogenesis. Because MMTV has existed as an infectious virus in mice for millions of years, it has evolved to take advantage of its host’s biology, using host genes from transcription factors to immune regulatory molecules, to establish infection. Although it causes mammary tumors, this does not occur until relatively late in life and thus the virus has persisted, since infected mothers are able to transmit virus to offspring. The lack of acute MMTV-induced pathogenesis is most likely due to different host means of limiting virus infection, including factors that operate at the cellular level like intrinsic restriction factors and immune response genes. As additional host-anti-viral genes are discovered, MMTV will continue to serve as an important model for testing the ability of these factors to function in vivo.Much has been learned about Wnt family that are known to play roles in different human cancers. Second, ITAM-mediated signaling in mammary epithelial cells may represent a novel mechanism of transformation; indeed, a novel oncogene associated with invasive breast cancer in humans has been recently shown to transform cells through ITAM-mediated signaling [In addition to serving as an important means for studying virus infection, MMTV has provided a number of critical models for understanding human breast cancer. First, the analysis of MMTV CIS has led to the identification of oncogenes/pathways such as members of the ignaling . Finally


Neotoma leucodon) captured in a region of Mexico in which woodrats are food for humans. Analyses of nucleotide and amino acid sequence data indicated that the woodrats were infected with a novel Tacaribe serocomplex virus, proposed name Real de Catorce virus.We isolated arenavirus RNA from white-toothed woodrats ( Arenaviridae, genus Arenavirus) comprises 7 North American viruses  in southern Texas is the principal host of Catarina virus (Sigmodon hispidus) in southern Florida is the principal host of Tamiami virus (The Tacaribe serocomplex (family  viruses  and 15 SN. leucodon) captured in Mexico . We report the determination of the nucleotide sequence of a 3352-nt fragment of the small (S) genomic segment of arenavirus AV H0030026 from RNA isolated from a white-toothed woodrat captured in 2005 in northeastern Mexico.A recent study found antibody to a Tacaribe serocomplex virus in white-toothed woodrats , and an antibody-positive Merriam’s kangaroo rat (Dipodomys merriami) were tested for arenavirus by cultivation in monolayers of Vero E6 cells (http://paup.csit.fsu.edu). The blood samples were tested by ELISA (Onychomys leucogaster), 19 deer mice (Peromyscus spp.), 11 harvest mice (Reithrodontomys spp.), 13 Nelson’s pocket mice, 45 Merriam’s kangaroo rats, and 16 other kangaroo rats (Dipodomys spp.) were captured during 800 trap-nights; overall trap success rate was 116/800 (14.5%). IgG against Whitewater Arroyo virus was found in 2 (22.2%) of the white-toothed woodrats, 2 (15.4%) of the Nelson’s pocket mice, 1 (2.2%) of the Merriam’s kangaroo rats, and none of the 49 other rodents. Arenavirus was isolated from none of the 12 animals included in the virus assays.Nine white-toothed woodrats, 3 northern grasshopper mice  was determined from RNA isolated from white-toothed woodrat TK133448. The 3,352-nt fragment included the complete glycoprotein precursor (GPC) gene and complete nucleocapsid (N) protein gene of AV H0030026. Nonidentities between the amino acid sequences of the GPC and N protein of AV H0030026 and the amino acid sequences of the GPC and N proteins of the 10 other North American viruses included in the analyses of amino acid sequence data ranged from 28.3% to 35.2% and from 11.6% to 21.0%, respectively . The resThe nucleotide sequence of a 687-nt fragment of the S genomic segment of arenavirus AV H0030028 was determined from RNA isolated from a kidney of white-toothed woodrat TK133451. The sequence of this fragment (GenBank accession no. GU137350) was 99.9% identical to the sequence of the homologous region of the N protein gene AV H0030026. Results of assays for arenavirus RNA in the kidneys of the antibody-positive Nelson’s pocket mice and the antibody-positive kangaroo rat were negative.N. albigula), large-eared woodrats (N. macrotis), Mexican woodrats (N. mexicana), southern plains woodrats (N. micropus), and a bushy-tailed woodrat (N. cinerea) captured in the western United States (N. leucodon) and that Tacaribe serocomplex viruses are enzootic to Mexico.Tacaribe serocomplex viruses have been isolated from white-throated woodrats  and Merriam’s kangaroo rat (D. merriami) are members of the rodent family Heteromyidae. No accounts of arenavirus infections in these or other heteromyid rodents have been published. The anti-arenavirus antibody in the pocket mice and kangaroo rat in this study may be a consequence of infection with an arenavirus other than Real de Catorce virus. Alternatively, the results of the ELISA on these rodents are inaccurate .Nelson’s pocket mouse , Mexican deer mice (Peromyscus mexicanus), and other cricetid rodents are consumed by the Tzeltzal Indians in Chiapas (Human consumption of woodrats is common in rural regions in the highlands of Mexico. For example, white-throated woodrats are consumed by humans in the Potosí-Zacatecan Mexican Plateau (