Coexistence of Different Genotypes in the Same Bat and Serological Characterization of Rousettus Bat Coronavirus HKU9 Belonging to a Novel Betacoronavirus Subgroup

Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9), a recently identified coronavirus of novel Betacoronavirus subgroup D, from Leschenault''s rousette, was previously found to display marked sequence polymorphism among genomes of four strains. Among 10 bats with complete RNA-dependent RNA polymerase (RdRp), spike (S), and nucleocapsid (N) genes sequenced, three and two sequence clades for all three genes were codetected in two and five bats, respectively, suggesting the coexistence of two or three distinct genotypes of Ro-BatCoV HKU9 in the same bat. Complete genome sequencing of the distinct genotypes from two bats, using degenerate/genome-specific primers with overlapping sequences confirmed by specific PCR, supported the coexistence of at least two distinct genomes in each bat. Recombination analysis using eight Ro-BatCoV HKU9 genomes showed possible recombination events between strains from different bat individuals, which may have allowed for the generation of different genotypes. Western blot assays using recombinant N proteins of Ro-BatCoV HKU9, Betacoronavirus subgroup A (HCoV-HKU1), subgroup B (SARSr-Rh-BatCoV), and subgroup C (Ty-BatCoV HKU4 and Pi-BatCoV HKU5) coronaviruses were subgroup specific, supporting their classification as separate subgroups under Betacoronavirus. Antibodies were detected in 75 (43%) of 175 and 224 (64%) of 350 tested serum samples from Leschenault''s rousette bats by Ro-BatCoV HKU9 N-protein-based Western blot and enzyme immunoassays, respectively. This is the first report describing coinfection of different coronavirus genotypes in bats and coronavirus genotypes of diverse nucleotide variation in the same host. Such unique phenomena, and the unusual instability of ORF7a, are likely due to recombination which may have been facilitated by the dense roosting behavior and long foraging range of Leschenault''s rousette.Coronaviruses infect a wide variety of animals in which they can cause respiratory, enteric, hepatic, and neurological diseases of various severities. Based on genotypic and serological characterization, coronaviruses were traditionally classified into three distinct groups, groups 1, 2, and 3 (3, 27, 59). Recently, the Coronavirus Study Group of the International Committee for Taxonomy of Viruses has renamed the traditional group 1, 2, and 3 coronaviruses as Alphacoronavirus, Betacoronavirus, and Gammacoronavirus, respectively (http://talk.ictvonline.org/media/p/1230.aspx). Coronaviruses are known to have a high frequency of recombination as a result of their unique mechanism of viral replication (27). Such tendency for recombination and high mutation rates may allow them to adapt to new hosts and ecological niches (24, 47, 52).The severe acute respiratory syndrome (SARS) epidemic has boosted interest in the study of coronaviruses in humans and animals (21, 34, 38, 41, 54). In the past few years, there has been a dramatic increase in the number of newly described human and animal coronaviruses (2, 4, 5, 8-10, 15-20, 23, 25, 28, 30, 32, 35, 36, 39, 43, 45, 50, 51, 53, 56, 58). Two novel human coronaviruses, human coronavirus NL63 (HCoV-NL63) and human coronavirus HKU1 (HCoV-HKU1), belonging to Alphacoronavirus and Betacoronavirus, respectively, have been discovered, in addition to the human coronavirus OC43 (HCoV-OC43), human coronavirus 229E (HCoV-229E), and SARS coronavirus (SARS-CoV) (17, 29, 45, 53, 55). We have also previously described the discovery of a diversity of novel coronaviruses in wild bats and birds in China, including SARSr-Rh-BatCoV, belonging to Betacoronavirus subgroup B, from Chinese horseshoe bats (30, 48, 56). Among these novel coronaviruses, three avian coronaviruses were found to belong to a novel subgroup of Gammacoronavirus (Gammacoronavirus subgroup C), while three bat coronaviruses were found to belong to two novel subgroups of Betacoronavirus (Betacoronavirus subgroups C and D) (48, 50). Based on the presence of the huge diversity of coronaviruses in Alphacoronavirus and Betacoronavirus among various bat species, bats are likely the reservoir for the ancestor of these two coronavirus genera (47).During our genome analysis of these novel coronaviruses, one of them, Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9), belonging to Betacoronavirus subgroup D, which was identified in Leschenault''s rousette bats, was found to display marked nucleotide and amino acid sequence polymorphism among the four strains with complete genome sequences (50). In our study on HCoV-HKU1, it has been shown that such sequence polymorphisms may indicate the presence of different genotypes (52). By complete genome sequence analysis of the potentially different genotypes of HCoV-HKU1, we have demonstrated for the first time natural recombination in a human coronavirus, resulting in the generation of at least three genotypes (52). We have also recently shown that recombination between different strains of SARSr-Rh-BatCoV from different regions of China may have given rise to the emergence of civet SARSr-CoV (31). To investigate the presence of different genotypes of Ro-BatCoV HKU9, the complete RNA-dependent RNA polymerase (RdRp) (corresponding to nsp12), spike (S), and nucleocapsid (N) gene sequences of Ro-BatCoV HKU9 from 10 additional bats were determined. Since sequence analysis showed the possible coexistence of different genotypes in seven bat individuals, complete genome sequencing of these distinct genotypes from two bats was carried out to investigate for possible recombination events among the different genotypes. In addition, serological characterization of Ro-BatCoV HKU9 was also performed by Western blot and enzyme immunoassays using recombinant Ro-BatCoV HKU9 nucleocapsid proteins and recombinant nucleocapsid proteins of Betacoronavirus subgroup A, B, and C coronaviruses to determine possible cross-reactivity among the different Betacoronavirus subgroups and the seroepidemiology of Ro-BatCoV HKU9 in Leschenault''s rousette bats.     

Validation of SYTO 9/Propidium Iodide Uptake for Rapid Detection of Viable but Noncultivable <Emphasis Type="Italic">Legionella pneumophila</Emphasis>

Legionella pneumophila is an ubiquitous environmental microorganism that can cause Legionnaires’ disease or Pontiac fever. As a waterborne pathogen, it has been found to be resistant to chlorine disinfection and survive in drinking water systems, leading to potential outbreaks of waterborne disease. In this work, the effect of different concentrations of free chlorine was studied (0.2, 0.7, and 1.2 mg l^?1), the cultivability of cells assessed by standard culture techniques (buffered charcoal yeast extract agar plates) and viability using the SYTO 9/propidium iodide fluorochrome uptake assay (LIVE/DEAD® BacLight?). Results demonstrate that L. pneumophila loses cultivability after exposure for 30 min to 0.7 mg l^?1 of free chlorine and in 10 min when the concentration is increased to 1.2 mg l^?1. However, the viability of the cells was only slightly affected even after 30 min exposure to the highest concentration of chlorine; good correlation was obtained between the rapid SYTO 9/propidium iodide fluorochrome uptake assay and a longer cocultivation with Acanthamoeba polyphaga assay, confirming that these cells could still recover their cultivability. These results raise new concerns about the assessment of drinking water disinfection efficiency and indicate the necessity of further developing new validated rapid methods, such as the SYTO 9/propidium iodide uptake assay, to assess viable but noncultivable L. pneumophila cells in the environment.     

Methicillin-resistant Staphylococcus epidermidis carrying biofilm formation genes: detection of clinical isolates by multiplex PCR

Staphylococcus epidermidis is the most prevalent coagulase-negative Staphylococcus (CNS) and is a major cause of hospital bacteremia. Based on 18 reference strains and 149 Staphylococcus clinical strains, used in a novel multiplex PCR method, the aim of this study was to identify S. epidermidis with respect to the sequence of three genes: recN, which encodes a recombination/repair protein, mecA (methicillin resistance), and icaAB, which is involved in biofilm formation. Amplicons of 219 bp (S. epidermidis-recN gene), 154 bp (mecA gene), and 546 bp (icaAB genes) were obtained. Reliable results were achieved for 100% of the evaluated strains, suggesting that this new multiplex-PCR approach could be useful for the accurate identification of methicillin-resistant S. epidermidis with the potential to produce biofilm.     

Physical mapping of stable RNA genes in Bacillus subtilis using polymerase chain reaction amplification from a yeast artificial chromosome library.

A new approach for mapping genes which utilizes yeast artificial chromosome clones carrying parts of the Bacillus subtilis genome and the polymerase chain reaction technique is described. This approach was used to physically map stable RNA genes of B. subtilis. Results from over 400 polymerase chain reactions carried out with the yeast artificial chromosome clone library, using primers specific for the genes of interest and designed from published sequences, were collected. The locations of 10 known rRNA gene regions (rrnO, rrnA, rrnE, rrnD, rrnB, rrnJ-rrnW, and rrnI-rrnH-rrnG) have been determined by this method, and these results correlate with those observed by standard genetic mapping. All rRNA operons, except rrnB, are found between 0 and 90 degrees, while rrnB has been placed in the area of 270 degrees on the chromosome map. Also localized were the tRNA gene clusters associated with the following ribosomal operons: rrnB (21 tRNAs), rrnJ (9 tRNAs), rrnD (16 tRNAs), and rrnO and rrnA (2 internal tRNAs). A previously unmapped four-tRNA gene cluster, trnY, a tRNA gene region that is not associated with a ribosomal operon, was found near the origin of replication. The P-RNA gene, important for processing of tRNAs, was found between map locations 197 and 204 degrees.     

Magnetic resonance of a dextran-coated magnetic fluid intravenously administered in mice

Magnetic resonance was used to investigate the kinetic disposition of magnetite nanoparticles (9.4 nm core diameter) from the blood circulation after intravenous injection of magnetite-based dextran-coated magnetic fluid in female Swiss mice. In the first 60 min the time-decay of the nanoparticle concentration in the blood circulation follows the one-exponential (one-compartment) model with a half-life of (6.9 +/- 0.7) min. The X-band spectra show a broad single line at g approximately 2, typical of nanomagnetic particles suspended in a nonmagnetic matrix. The resonance field shifts toward higher values as the particle concentration reduces, following two distinct regimes. At the higher concentration regime (above 10(14) cm(-3)) the particle-particle interaction responds for the nonlinear behavior, while at the lower concentration regime (below 10(14) cm(-3)) the particle-particle interaction is ruled out and the system recovers the linearity due to the demagnetizing field effect alone.     

Propensity of adult lymphoid progenitors to progress to DN2/3 stage thymocytes with Notch receptor ligation

Notch family receptors control critical events in the production and replenishment of specialized cells in the immune system. However, it is unclear whether Notch signaling regulates abrupt binary lineage choices in homogeneous progenitors or has more gradual influence over multiple aspects of the process. A recently developed coculture system with Delta 1-transduced stromal cells is being extensively used to address such fundamental questions. Different from fetal progenitors, multiple types of adult marrow cells expanded indefinitely in murine Delta-like 1-transduced OP9 cell cocultures, progressed to a DN2/DN3 thymocyte stage, and slowly produced TCR(+) and NK cells. Long-term cultured cells of this kind retained some potential for T lymphopoiesis in vivo. Adult marrow progressed through double-positive and single-positive stages only when IL-7 concentrations were low and passages were infrequent. Lin(-)c-Kit(low)GFP(+)IL-7Ralpha(+/-) prolymphocytes were the most efficient of adult bone marrow cells in short-term cultures, but the assay does not necessarily reflect cells normally responsible for replenishing the adult thymus. Although marrow-derived progenitors with Ig D(H)-J(H) rearrangements acquired T lineage characteristics in this model, that was not the case for more B committed cells with V(H)-D(H)J(H) rearrangement products.     

A new species of Microlicia (Melastomataceae) from Brazil

A new species of the genusMicrolicia,M. flava, from the highland “campo rupestre” vegetation of Serra da Canastra National Park, São Roque de Minas, Minas Gerais, Brazil, is described and illustrated.     

Continuous aqueous two-phase extraction of human antibodies using a packed column

The performance of a pilot scale packed differential contactor was evaluated for the continuous counter-current aqueous two-phase extraction (ATPE) of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cells supernatant (CS) enriched with pure protein. Preliminary studies have been firstly performed in order to select the dispersed phase (phosphate-rich or polyethylene glycol 3350 Da (PEG)-rich phase) and the column packing material. The PEG-rich phase has been selected as the dispersed phase and the stainless steel as the preferred material for the column packing bed since it was not wetted preferentially by the selected dispersed phase. Hydrodynamic studies have been also performed, and the experimental results were successfully adjusted to the Richardson-Zaki and Mísek equations, typically used for the conventional organic-aqueous two-phase systems. An experimental set-up combining the packed column with a pump mixer-settler stage showed to have the best performance and to be advantageous when compared to the IgG batch extraction. An IgG recovery yield of 85% could be obtained with about 50% of total contaminants and more than 85% of contaminant proteins removal. Mass transfer studies have revealed that the mass transfer was controlled by the PEG-rich phase. A higher efficiency could be obtained when using an extra pump mixer-settler stage and higher flow rates.     

Whole-genome sequence of Corynebacterium pseudotuberculosis PAT10 strain isolated from sheep in Patagonia, Argentina

In this work, we report the complete genome sequence of a Corynebacterium pseudotuberculosis PAT10 isolate, collected from a lung abscess in an Argentine sheep in Patagonia, whose pathogen also required an investigation of its pathogenesis. Thus, the analysis of the genome sequence offers a means to better understanding of the molecular and genetic basis of virulence of this bacterium.     

Adjustments in the Time,Distance and Direction of Foraging in Dinoponera quadriceps Workers

We measured individual decisions regarding the adjustments of time, distance and direction of foraging in Dinoponera quadriceps. We observed two colonies in an area of secondary Atlantic Forest, FLONA-ICMBio, in Northeastern Brazil. The workers were individually marked. We recorded the displacement of workers, their returns to the nest with and without food, the time spent searching for food, maximum and total distance, inter-trip latency and direction of trips. The time spent searching for food, maximum distance and transport velocity did not vary with food size. The previous trip success reduced the latency between foraging trips and increased the percentage of success on the next trip. However, this previous success did not demonstrate a significant variation relative to the time spent searching on the next trip or direction of search. The workers maintained an individual directional fidelity during foraging. The adjustments of these foraging variables under individual control contributed to the efficiency at the colony level. D. quadriceps is compatible with the central place theory and risk sensitivity model of behavior.