Randomly primed,strand-switching,MinION-based sequencing for the detection and characterization of cultured RNA viruses

RNA viruses rapidly mutate, which can result in increased virulence, increased escape from vaccine protection, and false-negative detection results. Targeted detection methods have a limited ability to detect unknown viruses and often provide insufficient data to detect coinfections or identify antigenic variants. Random, deep sequencing is a method that can more fully detect and characterize RNA viruses and is often coupled with molecular techniques or culture methods for viral enrichment. We tested viral culture coupled with third-generation sequencing for the ability to detect and characterize RNA viruses. Cultures of bovine viral diarrhea virus, canine distemper virus (CDV), epizootic hemorrhagic disease virus, infectious bronchitis virus, 2 influenza A viruses, and porcine respiratory and reproductive syndrome virus were sequenced on the MinION platform using a random, reverse primer in a strand-switching reaction, coupled with PCR-based barcoding. Reads were taxonomically classified and used for reference-based sequence building using a stock personal computer. This method accurately detected and identified complete coding sequence genomes with a minimum of 20× coverage depth for all 7 viruses, including a sample containing 2 viruses. Each lineage-typing region had at least 26× coverage depth for all viruses. Furthermore, analyzing the CDV sample through a pipeline devoid of CDV reference sequences modeled the ability of this protocol to detect unknown viruses. Our results show the ability of this technique to detect and characterize dsRNA, negative- and positive-sense ssRNA, and nonsegmented and segmented RNA viruses.     

Detection of infectious bursal disease virus in experimentally infected chickens by in situ hybridization

In situ hybridization was used in a pathogenesis study of three vaccine pathotypes (Delaware variant A, D78, and BursaVac) of infectious bursal disease virus (IBDV). Tissues were excised (bursa, thymus, spleen, proventriculus, and cecal tonsils), fixed in formalin, and paraffin embedded at 12, 24, 48, 72, and 120 hr postinoculation (HPI). With an antisense VP2 gene probe, viral nucleic acid was detected in bursas from both D78- and BursaVac-infected chickens at 24, 48, 72, and 120 HPI. However, viral RNA was detected only in the Delaware variant A-infected birds at 72 HPI. Thymus and spleen were positive in the D78-infected birds at 48 HPI and in the BursaVac-inoculated group at 72 HPI. Viral nucleic acid was not present in detectable levels among any of the tissues tested at 12 HPI. However, by 24 hr, scattered positive lymphoid cells were visualized in the bursal follicles of chickens infected with D78 and BursaVac. In addition, low levels of viral nucleic acids were detected in the thymus and spleen among the D78- and BursaVac-infected birds. The sites of viral replication were consistent between the two vaccine-infected groups (D78 and BursaVac), whereas the chickens infected with Delaware variant A had limited IBDV replication in the bursa.     

Stereodependent bioactivation of R(+)-ethyl S-propyl methylphosphonothioate: Is the S-oxide the active metabolite?

R(+)-Ethyl S-propyl methylphosphonothioate is bioactivated both in vivo and when perfused through isolated liver to give a product which is much more active as an inhibitor of acetylcholinesterase than the parent compound. The bioactivation does not occur in hepatectomised animals. Acetylcholinesterase inhibited by the active metabolite is not reactivated by pyridine-2-aldoxime methanesulphonate (P2S), whereas enzyme inhibited by the parent compound and its S(?) enantiomer is reactivatable. Attempts to identify the active metabolite were unsuccessful and experiments to explore its stability were inconclusive. Extensive in vitro studies of the inhibition of acetylcholinesterase by the enantiomers of ethyl S-propyl methylphosphonothioate and ethyl S-diisopropylaminoethyl methylphosphonothioates and subsequent reactivation of the enzyme by P2S showed that (a) there are large differences between the rates of inhibition of the R and S enantiomers of both compounds, (b) reactivation profiles are critically dependent on reaction conditions, and (c) the reactivation profiles of the R and S enantiomers of the former compound are indistinguishable under all conditions whereas differences are observed under some conditions for the latter pair of enantiomers. The results are discussed in terms of the possibility that the S-oxide of R(+)-ethyl S-propyl methylphosphonothioate is the active metabolite and it is concluded that this is unlikely.     

Amazon deforestation and climate change

A coupled numerical model of the global atmosphere and biosphere has been used to assess the effects of Amazon deforestation on the regional and global climate. When the tropical forests in the model were replaced by degraded grass (pasture), there was a significant increase in surface temperature and a decrease in evapotranspiration and precipitation over Amazonia. In the simulation, the length of the dry season also increased; such an increase could make reestablishment of the tropical forests after massive deforestation particularly difficult.     

Aminopyralid soil residues affect rotational vegetable crops in Florida

BACKGROUND: Bahiagrass (Paspalum notatum Flueggé) is a poor host of several soilborne pests of vegetable crops; therefore vegetable crops are commonly grown in a rotation with bahiagrass pastures in Florida. The herbicide aminopyralid provides foliar and soil residual weed control and increases forage production in bahiagrass pastures; however, the soil residual activity of aminopyralid makes carryover injury likely in subsequent sensitive vegetable crops. Field research was conducted to determine the sensitivity of five vegetable crops to soil residues of aminopyralid. RESULTS: At an aminopyralid soil concentration of 0.2 µg kg^?1 (the limit of quantitation for aminopyralid in this research), crop injury ratings were 48% (bell pepper), 67% (eggplant), 71% (tomato), 3% (muskmelon) and 3% (watermelon), and fruit yield losses (relative to the untreated control) at that concentration were 61, 64, 95, 8 and 14% in those respective crops. CONCLUSIONS: The crops included in this research were negatively affected by aminopyralid at soil concentrations less than the limit of quantitation (0.2 µg kg^?1). Therefore, it was concluded that a field bioassay must be used to determine whether carryover injury will occur when these crops are planted on a site where aminopyralid has been previously applied. Copyright © 2011 Society of Chemical Industry     

Equine colonic motility: interactions among wall motion, propulsion, and fluid flow

Transmission of intestinal content with respect to wall position and intraluminal pressure was studied using implanted catheters in portions of the haustrated left ventral colon and nonhaustrated pelvic flexure and left dorsal colon in 3 conscious, standing, and feeding ponies. Wall position and content movement was studied in 1 noncatheterized conscious pony that was standing and eating. When coordinated wall movements involving greater than or equal to 30 cm of adjacent colon were seen, point-to-point content movement accompanied intraluminal pressure peaks occurring in the same direction. Near the pelvic flexure, intraluminal pressure peak maximums coincided and reduced colonic diameters and reduced intercatheter distances. Qualitative observation of 3 related aspects of intestinal motility: wall motion, propulsion, and fluid movement can be studied in the conscious feeding pony.     

Long distance transport of foot-and-mouth disease virus over the sea

The conditions required for the transport of foot-and-mouth disease (FMD) virus in the atmosphere over long distances and in sufficient concentrations to cause infection in exposed animals are described. Using these factors a series of 23 outbreaks of FMD in Europe, where the original outbreaks were separated from later outbreaks by sea passage, have been investigated. The findings obtained support the hypothesis that under certain conditions the airborne transmission of FMD over a long sea passage is possible.