Embedded microcomputer-based force plate system validation when evaluating lameness severity differentiation under an induced synovitis model in lactating dairy cattle.

Bovine lameness has relatively large negative economic and welfare implications on the U.S. dairy industry. Due to the ramifications, early lameness detection will aid in assisting dairy producers to mitigate downstream effects through early treatment. The objective of this study was to determine the minimum standing time required among 2-, 3-, 4-, 5-, and 10 min time intervals to obtain an accurate weight distribution estimate for each leg when attempting to detect lameness. An embedded microcomputer-based force plate system was developed to measure vertical forces from individual cow limb weight distribution to detect bovine lameness when utilizing an induced synovitis lameness model. The force plate has four quadrants, with each load cell quadrant measuring the force placed on it from a single limb. The force plate recorded weight (kg) every second from each load cell quadrant, after which, a 60 s moving average for weight distribution was calculated. A sequential study design was employed to evaluate non-lame and induced lameness to ensure time requirements were consistent. Prior to induction, the force plate system was used to measure weight distribution every second for 15 min. After lameness induction, additional 15 min increments were recorded every 24 h for seven days. Lameness was induced by injecting the left hind distal interphalangeal joint in three cows with amphotericin B, 12 h prior to the start of the study. Data were analyzed using a linear mixed effect that included the fixed effects of day relative to lameness induction, time period, foot and injected foot. Cow within replicate was included as a random effect. Cumulative minutes were assessed up to 15 min by comparing the least square rolling 60 s cumulative means expressed as a percentage of each animal's BW percentage placed on each leg for 2-, 3-, 4-, 5-, and 10 min intervals. Results indicate that the minimum time needed for accurate lameness detection in cows was 2 min.

Effect of dietary fat concentration from condensed corn distillers' solubles, during the growing phase, on beef cattle performance, carcass traits, digestibility, and ruminal metabolism.

The objectives of this study were to determine the effect of fat concentration from corn distillers' solubles (CDS), fed during the growing phase, on DMI, gain, carcass traits, digestibility, ruminal metabolism, and methane emissions of steers. In Exp. 1, 40 steers (age = 136 ± 20 d; BW = 185 ± 11 kg) were randomly allotted to 1 of 5 dietary treatments: 1) a cosrn-based gro\wing diet (CNT), 2) 0% CDS, 3) 10% CDS, 4) 19% CDS, or 5) 27% CDS. Diets 2 through 5 included coproducts (corn gluten feed and soybean hulls) and were formulated to achieve fat concentrations of 3, 5, 7, and 9%, respectively. Diets were fed once daily for 106 d (growing phase). All steers were fed a corn-based diet from d 107 to 196. Contrasts were used to examine 1) the difference between CNT and 10% CDS and 2) linear and quadratic effects of CDS inclusion. During the growing phase, steers fed CNT had increased ( < 0.01) ADG and G:F compared with steers fed 10% CDS. Increasing CDS inclusion increased (linear, ≤ 0.02) ADG and G:F. Overall, steers fed CNT had increased ( < 0.01) ADG compared with steers fed 10% CDS, but increasing CDS inclusion had no effect ( = 0.19) on overall ADG. Overall DMI and G:F were not different ( ≥ 0.16) in any contrast. There was a trend (Linear; = 0.08) for ultrasound marbling at d 196 to increase as CDS inclusion increased; however, there were no effects ( ≥ 0.20) of treatment on carcass marbling or quality grade. In Exp. 2, 5 steers (BW = 335 ± 56 kg) were fed Exp. 1 diets for ad libitum intakes in a 5 × 5 Latin square design. Apparent DM digestibility increased (linear, = 0.02) with increasing dietary CDS inclusion. Steers fed CNT had greater ( = 0.01) DM digestibility than those fed 10% CDS. Fat digestibility increased (linear, < 0.01) in steers with increasing CDS, but NDF and ADF digestibility were not affected ( ≥ 0.17) by treatment. Similarly, ruminal pH and VFA concentrations were not affected ( ≥ 0.13). Also, there was no difference ( ≥ 0.37) in ruminal methane emissions (g/h). In conclusion, feeding corn during the growing phase increased overall ADG compared with 10% CDS coproduct-based diet but did not affect carcass traits or methane production. Increasing dietary fat inclusion from CDS in coproduct-based diets linearly increased DM and fat digestibility and predicted marbling scores via ultrasound but did not affect marbling at slaughter, NDF digestibility, propionate, or methane production.

Infectivity of nanovirus DNAs: induction of disease by cloned genome components of Faba bean necrotic yellows virus.

Circumstantial evidence suggests that the genome of Faba bean necrotic yellows virus (FBNYV), a nanovirus, consists of eight distinct, circular, single-stranded DNAs, each of about 1 kb and encoding only one protein. Here, the use of cloned full-length FBNYV DNAs for reproducing FBNYV-like symptoms in Vicia faba, the principal natural host of FBNYV, is reported. Characteristic symptoms of FBNYV infection were obtained in faba bean plants following biolistic DNA delivery or agroinoculation with all eight FBNYV DNAs. Although the eight different DNAs have been invariably detected in field samples infected with the various geographical FBNYV isolates, experimental infection with different combinations of fewer than eight DNAs also led to typical FBNYV symptoms. Even only five genome components, DNA-R, DNA-S, DNA-M, DNA-U1 and DNA-U2, were sufficient for inducing disease symptoms in V. faba upon agroinoculation. Symptomatic plants agroinoculated or bombarded with eight DNAs contained typical FBNYV virions; however, the virus was not transmitted by Aphis craccivora or Acyrthosiphon pisum, two efficient aphid vectors of FBNYV.

RNA-dependent activation of primer RNA production by influenza virus polymerase: different regions of the same protein subunit constitute the two required RNA-binding sites.

The capped RNA primers required for the initiation of influenza virus mRNA synthesis are produced by the viral polymerase itself, which consists of three proteins PB1, PB2 and PA. Production of primers is activated only when the 5'- and 3'-terminal sequences of virion RNA (vRNA) bind sequentially to the polymerase, indicating that vRNA molecules function not only as templates for mRNA synthesis but also as essential cofactors which activate catalytic functions. Using thio U-substituted RNA and UV crosslinking, we demonstrate that the 5' and 3' sequences of vRNA bind to different amino acid sequences in the same protein subunit, the PB1 protein. Mutagenesis experiments proved that these two amino acid sequences constitute the functional RNA-binding sites. The 5' sequence of vRNA binds to an amino acid sequence centered around two arginine residues at positions 571 and 572, causing an allosteric alteration which activates two new functions of the polymerase complex. In addition to the PB2 protein subunit acquiring the ability to bind 5'-capped ends of RNAs, the PB1 protein itself acquires the ability to bind the 3' sequence of vRNA, via a ribonucleoprotein 1 (RNP1)-like motif, amino acids 249-256, which contains two phenylalanine residues required for binding. Binding to this site induces a second allosteric alteration which results in the activation of the endonuclease that produces the capped RNA primers needed for mRNA synthesis. Hence, the PB1 protein plays a central role in the catalytic activity of the viral polymerase, not only in the catalysis of RNA-chain elongation but also in the activation of the enzyme activities that produce capped RNA primers.

Characterization of the in vitro activity of the RNA-dependent RNA polymerase associated with the ribonucleoproteins of rice hoja blanca tenuivirus.

An RNA-dependent RNA polymerase (RdRp) activity associated with the ribonucleoproteins of rice hoja blanca tenuivirus (RHBV) was detected and analyzed. Conditions for in vitro RNA synthesis and for coupled RNA synthesis-translation of RHBV were established. In both cases, synthesis of the viral and viral complementary genomic and subgenomic RNA3 and RNA4 were observed, demonstrating that both transcription and replication occurred. Though coupling of RNA synthesis to translation allowed efficient translation of the newly synthesized subgenomic RNAs, studies of the effect of various inhibitors of protein synthesis revealed that RNA synthesis was independent of translation. Primer extension experiments demonstrated that in the presence of capped exogenous RNAs, a stretch of 10 to 16 nonviral nucleotides was added to the 5' end of a population of newly synthesized viral complementary RNA4. It appears that in addition to RdRp activity, RHBV-associated protein(s) also possessed cap-snatching capacity.

Comparison of Colombian and Costa Rican strains of rice hoja blanca tenuivirus.

The sequences of RNA-3 and RNA-4 of rice hoja blanca tenuivirus isolates from Colombia and from Costa Rica were determined and analyzed. These isolates were 98.9% and 98.6% identical in the coding and non-coding regions of RNA-3, and 96.9 and 91.5% identical in the coding and non-coding regions of RNA-4, and are therefore strains of the same virus. There is about three times as much variation between isolates (based on consensus sequences) as there is within isolates (based on sequences of individual clones). There is also considerably more variation for RNA-4 (both between and within isolates) than there is for RNA-3, even though between tenivirus species RNA-3 has diverged more than RNA-4, implying that the evolution of the tenuivirus RNAs is not necessarily dependent on the amount of variation found for these RNAs.

Characterization of cassava common mosaic virus and a defective RNA species.

The genome of cassava common mosaic potexvirus (CsCMV) has been sequenced from cDNA clones and consists of 6376 nucleotides (nt). A 76 nt untranslated region (UTR) at the 5' terminus was followed by ORF1 which potentially encodes a protein of 1449 amino acids (aa). ORFs 2, 3, and 4 were predicted to encode proteins of 231, 112 and 97 aa, respectively. ORF5 potentially encodes a 229 aa protein of 25 kDa that is similar to the coat proteins of other potexviruses. The 3'-terminal UTR of 114 nt was followed by a poly(A) tail. The genomic organization of the CsCMV genome is similar to that of other potexviruses. A cDNA clone that was apparently obtained from a defective RNA species contained both the 5' and 3' UTRs and an ORF that potentially encodes the first 263 aa of ORF1 and the last 33 aa of the coat protein. Defective RNA species were found both in purified preparations of the virus and in total nucleic acid isolated from CsCMV-infected plants.

Capped nonviral sequences at the 5' end of the mRNAs of rice hoja blanca virus RNA4.

Subgenomic RNAs of both polarities corresponding to rice hoja blanca virus (RHBV) ambisense RNA4 were detected in RHBV-infected rice tissues. Total RNA extracted from RHBV-infected and noninfected rice tissues and RNA4 purified from RHBV ribonucleoprotein particles were used as templates for primer extension studies. The RNAs extracted from RHBV-infected tissues contain a population of RNA molecules with 10 to 17 nonviral nucleotides at their 5' end. The RNA-cDNA hybrids resulting from primer extension of such RNA molecules were specifically immunoselected with anti-cap antibodies, indicating that the subgenomic RNAs are capped and probably serve as mRNAs and that the additional nucleotides at their 5' end possibly derive from host mRNAs via a cap-snatching mechanism.

Complete nucleotide sequence and coding strategy of rice hoja blanca virus RNA4.

The complete sequence of rice hoja blanca virus (RHBV) RNA4 has been determined, based on the sequence of the corresponding cDNA clones. RNA4 consists of 1991 nucleotides with two open reading frames (ORFs). One putative ORF is located in the 5'-proximal region of the viral RNA4; it encodes a protein of predicted M(r) 20076 which corresponds to the major non-structural protein that accumulates in RHBV-infected rice plants, and which bears limited sequence identity with the helper component of tobacco vein mottling potyvirus. The other ORF is located in the 5'-proximal region of the viral complementary RNA4 and encodes a protein of predicted M(r) 32,469. Between the two ORFs is an intergenic region of 524 nucleotides, part of which can theoretically adopt a stable stem-loop structure; the 5' and 3' ends can potentially base-pair over 16 nucleotides, producing a pan-handle configuration. These characteristics are in favour of an ambisense coding strategy for RHBV RNA4.

Rice hoja blanca virus genome characterization and expression in vitro.

No information exists on the organization and mechanisms of expression of the genome of rice hoja blanca virus (RHBV), a member of the tenuivirus group, but here we describe the first steps in its characterization. RHBV contains four ssRNA and three dsRNA species, the sizes of which were estimated by native and denaturing gel electrophoresis. Hybridization analyses using 32P-labelled riboprobes of viral and viral complementary polarities showed that unequal amounts of the two polarities of at least the smallest RNA are present in the virion, and indicated that the dsRNA species contain the same information as the ssRNA species of corresponding size. Total RHBV RNA directs the synthesis of two major proteins of 23K and 21K in vitro. RNA3 directs the synthesis of a 23K protein designated NS3, and RNA4 of a 21K protein designated NS4. The NS4 protein corresponds to the non-structural protein that accumulates in RHBV-infected rice tissue. The nuclecocapsid protein is not translated from either total RHBV RNA or any individual RHBV RNA in vitro.

Phylogeny of transfer RNA.

Phylogenetic trees of transfer RNA specific for phenylalanine, methionine initiator glycine and valine are constructed. Although the exact relationships between taxa cannot be obtained from the mere analysis of the sequences some conclusions can be drawn about the evolution of this molecule.