Establishment of a subgenomic replicon for bovine viral diarrhea virus in Huh-7 cells and modulation of interferon-regulated factor 3-mediated antiviral response

We describe the development of a selectable, bi-cistronic subgenomic replicon for bovine viral diarrhea virus (BVDV) in Huh-7 cells, similar to that established for hepatitis C virus (HCV). The selection marker and reporter (Luc-Ubi-Neo) in the BVDV replicon was fused with the amino-terminal protease N(pro), and expression of the nonstructural proteins (NS3 to NS5B) was driven by an encephalomyocarditis virus internal ribosome entry site. This BVDV replicon allows us to compare RNA replication of these two related viruses in a similar cellular background and to identify antiviral molecules specific for HCV RNA replication. The BVDV replicon showed similar sensitivity as the HCV replicon to interferons (alpha, beta, and gamma) and 2'-beta-C-methyl ribonucleoside inhibitors. Known nonnucleoside inhibitor molecules specific for either HCV or BVDV can be easily distinguished by using the parallel replicon systems. The HCV replicon has been shown to block, via the NS3/4A serine protease, Sendai virus-induced activation of interferon regulatory factor 3 (IRF-3), a key antiviral signaling molecule. Similar suppression of IRF-3-mediated responses was also observed with the Huh-7-BVDV replicon but was independent of NS3/4A protease activity. Instead, the amino-terminal cysteine protease N(pro) of BVDV appears to be, at least partly, responsible for suppressing IRF-3 activation induced by Sendai virus infection. This result suggests that different viruses, including those closely related, may have developed unique mechanisms for evading host antiviral responses. The parallel BVDV and HCV replicon systems provide robust counterscreens to distinguish viral specificity of small-molecule inhibitors of viral replication and to study the interactions of the viral replication machinery with the host cell innate immune system.     

177 Molecular Systematics of the Green Algal Orders Ulotrichales and Ulvales Based on 18S rDNA Sequences

The traditional use of thallus morphology to define lineages within the Ulvophyceae proved problematic because parallelism and convergence obscured natural relationships. The advent of transmission electron microscopy led to a re‐evaluation of groups on the basis of zoid characters. Within TEM‐defined lineages, the Ulotrichales and Ulvales were considered closely related orders, and molecular evidence has substantiated this hypothesis. In our molecular systematic studies of ulvophyceous green algae, we have broadened taxon sampling among these two orders. We show that the Ulvales and Ulotrichales are indeed monophyletic orders. However, the phylogenetic placement of species in the genera Bolbocoleon, Ctenocladus, Phaeophila, Pseudendoclonium and Acroblaste indicates that one or more additional lineages, placed between Ulvales and Ulotrichales in gene sequence trees, must be recognized. These species may constitute a separate order (Ctenocladales) consisting of multiple families. Our findings will be compared with taxonomic concepts based on morphological criteria.     

178 Molecular Systematics of the Ulvellaceae (Ulvales, Ulvophyceae) Inferred from Nuclear and Chloroplast DNA Sequences

The traditional use of thallus morphology to define lineages within the Ulvophyceae proved problematic because parallelism and convergence obscured natural relationships. The advent of transmission electron microscopy led to a re-evaluation of groups on the basis of zoid characters. Within TEM-defined lineages, the Ulotrichales and Ulvales were considered closely related orders, and molecular evidence has substantiated this hypothesis. In our molecular systematic studies of ulvophyceous green algae, we have broadened taxon sampling among these two orders. We show that the Ulvales and Ulotrichales are indeed monophyletic orders. However, the phylogenetic placement of species in the genera Bolbocoleon, Ctenocladus, Phaeophila, Pseudendoclonium and Acroblaste indicates that one or more additional lineages, placed between Ulvales and Ulotrichales in gene sequence trees, must be recognized. These species may constitute a separate order (Ctenocladales) consisting of multiple families. Our findings will be compared with taxonomic concepts based on morphological criteria.     

Discovery of entry inhibitors for HIV-1 via a new de novo protein design framework

A new (to our knowledge) de novo design framework with a ranking metric based on approximate binding affinity calculations is introduced and applied to the discovery of what we believe are novel HIV-1 entry inhibitors. The framework consists of two stages: a sequence selection stage and a validation stage. The sequence selection stage produces a rank-ordered list of amino-acid sequences by solving an integer programming sequence selection model. The validation stage consists of fold specificity and approximate binding affinity calculations. The designed peptidic inhibitors are 12-amino-acids-long and target the hydrophobic core of gp41. A number of the best-predicted sequences were synthesized and their inhibition of HIV-1 was tested in cell culture. All peptides examined showed inhibitory activity when compared with no drug present, and the novel peptide sequences outperformed the native template sequence used for the design. The best sequence showed micromolar inhibition, which is a 3-15-fold improvement over the native sequence, depending on the donor. In addition, the best sequence equally inhibited wild-type and Enfuvirtide-resistant virus strains.     

Host- plant effects on the behavior of eketmocerus sr nr. Californicus females raised from melon

The behaviors of female Eretmocerus sp. nr. californiens raised from Bemisia argentifolii Bellows & Perring on melon, Cucumis melo L., were analysed on four different host plant species (cotton, melon, sweet potato and Abutilon theophrasti Medic). Comparison with previously published results of similarly treated females reared from sweet potato plants showed performance related differences leading to oviposition. In the present study, the generalized behavioral pathway (walking, host- encounter, antennation, probing and oviposition) did not vary among host plant species for melon- reared parasitoid females. Host assessment by antennation leading to host acceptance for probing varied from 10.5- 12.3 sec among the host plant species with no host stage- related differences in duration. Initial and repeated (multiple probes on the same host nymph) probing events varied from 52.4 to 77.3 sec among host plant species and no stage- related differences were detected. Oviposition occurred under 62 % of the nymphs on cotton, 100 % of the nymphs on melon and 51 % of the nymphs on sweet potato under which the ovipositor was exserted. On cotton, melon and sweet potato proportionally fewer first instar nymphs were encountered than present, a larger proportion of the second instars that were encountered were subsequently antennated, and a larger proportion of second instars on cotton were then subsequently probed. For cotton, melon, and sweet potato, the overall frequency of oviposition was lower than reported in previous studies. Results from these studies showed that the greatest factor in influencing overall parasitism in laboratory experiments was the propensity of females to alight and remain to search for hosts on a particular host plant. The role of “pre- conditioning” parasitoids to be better- suited to a particular host or host plant species is discussed relative to the findings herein.     

Clitoral stimulation and the effect of age, breed, technician, and postpartum interval on pregnancy rate to artificial insemination in beef cattle

An experiment was conducted to assess the effect of clitoral stimulation on pregnancy rate to artificial insemination in 1,856 beef females. For unadjusted data, pregnancy rate to first service in nonstimulated females was 61% for cows and 59% for heifers, and that of females receiving clitoral stimulation was 69% for cows and 58% for heifers. After adjusting the data for the influences of age, breed, technician, and postpartum interval, clitoral stimulation increased first service pregnancy rate in cows (74 +/- 3% vs 59 +/- 3%, P<0.05), but not in heifers (53 +/- 5% vs 57 +/- 5%, P>0.10). Postpartum interval significantly influenced pregnancy rate, regardless of treatment, and clitoral stimulation was beneficial when applied to cows with relatively short postpartum intervals. Variation in the effect of clitoral stimulation on pregnancy rate was observed among technicians. Clitoral stimulation improved pregnancy rate more effectively in 3 to 4 year old cows than in cows 2 years old and cows 5 years of age or older. We concluded that clitoral stimulation at the time of artificial insemination was an effective means of increasing pregnancy rate in cows, but not in heifers.     

PHYLOGENETIC POSITION OF BOLBOCOLEON PILIFERUM (ULVOPHYCEAE,CHLOROPHYTA): EVIDENCE FROM REPRODUCTION,ZOOSPORE AND GAMETE ULTRASTRUCTURE,AND SMALL SUBUNIT RRNA GENE SEQUENCES1

Aspects of the reproduction of Bolbocoleon piliferum N. Pringsheim, a common, small, filamentous, endophytic marine green alga, were examined by LM and TEM. These observations were combined with phylogenetic analysis of nuclear‐encoded small subunit rRNA gene sequences to assess the phylogenetic position of B. piliferum. Quadriflagellate zoospores and planozygotes derived from fusion of isogametes yielded plants with identical morphology. Zoosporangia and gametangia divided by sequential cleavages. Plugs at the apices of zoosporangia and gametangia formed during development; tubes were found at zoosporangial and gametangial apices after swarmer release. Flagellar apparatuses of zoospores and gametes were similar to those of algae in the Ulvales (Ulvophyceae), except that terminal caps were entire rather than bilobed and rhizoplasts and “stacked” microtubular root configurations were absent. Structures associated with planozygotes were identical to those observed in other algae currently assigned to Ulotrichales and Ulvales. Molecular phylogenetic analyses placed B. piliferum within the Ulvophyceae, at the base of a clade that contains representatives of the families Ulvaceae, Ulvellaceae, and Kornmanniaceae. The results support an earlier hypothesis that B. piliferum constitutes a distinct lineage. Analyses including Kornmanniaceae recover monophyletic Ulotrichales and Ulvales, whereas analyses omitting the Kornmanniaceae indicate that Ulotrichales is paraphyletic. The structures associated with gamete fusion are conserved within Ulotrichales and Ulvales and perhaps more widely within Chlorophyta.     

Effects of host and parasitoid age on search behaviour and oviposition rates inLariophagus distinguendus Förster (Hymenoptera: Pteromalidae)

Oviposition rates and related behaviours were quantified for Lariophagus distinguendusFörster attacking Calosobruchus chinensis (L.) and C. maculatus (F.). Oviposition rates varied with parasitoid age; parasitoids aged 1–7 days laid approximately twice as many eggs per day as those aged 8–14 days. Similar differences were noted in search rates and handling times; younger parasitoids had higher attack rates and lower handling times than older parasitoids. Search rates and handling times also varied with the host stage available for attack. Search rates were higher and handling times were lower on larger stages. The results are discussed with reference to their impact on the dynamical behavior of insect parasitoid-host populations.     

Multiple linear and nonlinear regression analyses of factors causing calving difficulty

Calving difficulty (CD) was analyzed as the dependent variable from observations on 592 first-calf, 2-year-old heifers that were either Angus X Hereford (AH), Charolais X AH or Simmental X AH. Independent variables were birth weight (BW), pelvic area (PA), BW.(33), PA.(5), cow weight (CW), cow condition score (CS) and sex of calf (S). CD was analyzed as score (1 = no difficulty through 4 = extreme difficulty) CD-I, % CD (1 vs 2 + 3 + 4 , CD-II) or % high CD (1 + 2 vs 3 + 4 , CD-III). R(2) values from CD-II and CD-III multiple correlation (R) analyses were consistently lower (.10 to .13) than from the CD-I analysis. When only BW and PA were included in the model, R(2) values for CD-I were .36, .39 and .39 for the linear, quadratic and cubic analyses, respectively. When BW, PA, CW, CS and S were included in the model, the R(2) values for CD-I were .40 and .42 for the linear and quadratic analyses (cubic effects were not tested). In the linear analysis with BW.(33) and PA.(5), the R(2) for CD-I was .36. In all analyses, the order of importance of the factors studied was BW and PA, with a much lower contribution from CW and S. CS did not account for a significant increase in R(2) in any analysis. CD remained below 50% whenever the PA BW ratio exceeded 8.4. This ratio is a convenient practical method to relate PA and BW to CD. Conclusions are that the linear effects of BW and PA account for the majority of the identified variation in CD (R(2)) and that small increases in R(2) can be obtained by adding CW, CS and S to the model and/or by expanding the model to include quadratic effects.