A new modified live equine influenza virus vaccine: phenotypic stability, restricted spread and efficacy against heterologous virus challenge.

Flu Avert IN vaccine is a new, live attenuated virus vaccine for equine influenza. We tested this vaccine in vivo to ascertain 1) its safety and stability when subjected to serial horse to horse passage, 2) whether it spread spontaneously from horse to horse and 3) its ability to protect against heterologous equine influenza challenge viruses of epidemiological relevance. For the stability study, the vaccine was administered to 5 ponies. Nasal swabs were collected and pooled fluids administered directly to 4 successive groups of naïve ponies by intranasal inoculation. Viruses isolated from the last group retained the vaccine's full attenuation phenotype, with no reversion to the wild-type virus phenotype or production of clinical influenza disease. The vaccine virus spread spontaneously to only 1 of 13 nonvaccinated horses/ponies when these were comingled with 39 vaccinates in the same field. For the heterologous protection study, a challenge model system was utilised in which vaccinated or naïve control horses and ponies were exposed to the challenge virus by inhalation of virus-containing aerosols. Challenge viruses included influenza A/equine-2/Kentucky/98, a recent representative of the 'American' lineage of equine-2 influenza viruses; and A/equine-2/Saskatoon/90, representative of the 'Eurasian' lineage. Clinical signs among challenged animals were recorded daily using a standardised scoring protocol. With both challenge viruses, control animals reliably contracted clinical signs of influenza, whereas vaccinated animals were reliably protected from clinical disease. These results demonstrate that Flu Avert IN vaccine is safe and phenotypically stable, has low spontaneous transmissibility and is effective in protecting horses against challenge viruses representative of those in circulation worldwide.

Cloning of a family of serine protease genes from the cat flea Ctenocephalides felis.

Serine protease gene fragments approximately 480 nucleotides in length were amplified from Ctenocephalides felis larval and adult cDNA libraries using degenerate oligonucleotide PCR primers. Partial clones of thirty-eight distinct serine protease encoding sequences were isolated, and nineteen different full-length cDNAs encoding mature serine proteases were subsequently cloned and sequenced. All of the mature proteases contained the histidine, aspartic acid and serine amino acids of the catalytic triad characteristic of serine proteases. The mature C. felis serine proteases had amino acid sequences that were at most 29-53% identical to those known insect and arachnid serine proteases. Two of the C. felis gene sequences had similarity with the Drosophila melanogaster developmental genes snake and stubble. mRNA expression of selected serine protease genes was examined in different life stages, tissues, genders, and in response to bloodfeeding.

Southern blot and polymerase chain reaction exon analyses of HPRT- mutations induced by radon and radon progeny.

A linear dose response was observed for radon-induced mutations at the CHO-hprt locus with an induction frequency of 1.4 x 10(-4) mutants per viable cell per gray. Mutants isolated after two levels of radon exposure were evaluated using Southern blot techniques and polymerase chain reaction (PCR) exon amplification. No significant differences in mutational spectra were detected at these two exposure levels. Of 52 radon-induced mutations, 48% sustained a gene deletion, 23% underwent a rearrangement of the banding patterns or loss of one or more exons, and 29% showed no change from the parental line. These mutants were compared with mutants produced after X irradiation (3 Gy) and with spontaneous mutants from untreated cells. The spectra of mutation types in cells treated with radon and X rays were not significantly different. In contrast, 31 spontaneous mutations exhibited a low percentage of gene deletion events (16%); most spontaneous mutants showed no change (74%); the remaining 10% were classified as alterations. In conclusion, the principal lesion seen at the CHO-hprt locus after radiation exposure is gene deletion, while the predominant class of spontaneous mutations is composed of smaller events not detectable by Southern blot or PCR exon analysis.

Brain fibronectin expression in prenatally irradiated mice.

Activation of gene transcription by radiation has been recently demonstrated in vitro. However, little is known on the specificity of these alterations on gene transcription. Prenatal irradiation is a known teratogen that affects the developing mammalian central nervous system (CNS). Altered neuronal migration has been suggested as a mechanism for abnormal development of prenatally irradiated brains. Fibronectin (FN), an extracellular glycoprotein, is essential for neural crest cell migration and neural cell growth. In addition, elevated levels of FN have been found in the extracellular matrix of irradiated lung. To test whether brain FN is affected by radiation, either FN level in insoluble matrix fraction or expression of FN mRNA was examined pre- and postnatally after irradiation. Mice (CD1), at 13 d of gestation (DG), served either as controls or were irradiated with gamma rays at 0.5 or 1 Gy. Control and irradiated animals were killed either at 13 DG, 14 DG, 17 DG, or 5, 6, or 14 d postnatal. Brain and liver were collected from offspring and analyzed for either total FN protein levels or relative mRNAs for FN and tubulin. Results of prenatal irradiation on reduction of postnatal brain weight relative to whole body weight and morphological reduction in cerebral cortex regions of postnatal brains are comparable to that reported by others. Insoluble matrix fraction (IMF) per gram of brain, liver, lung, and heart weight was not significantly different either between control and irradiated groups or between postnatal stages, suggesting that radiation did not affect the IMF. However, total amounts of FN in brain IMF at 17 DG were significantly different (p < .02) between normal (1.66 +/- 0.80 micrograms) and irradiated brains (0.58 +/- 0.22 microgram). FN mRNA was detectable at 13, 14, and 17 DG, but was not detectable at 6 and 14 d postnatal, indicating that FN mRNA is developmentally regulated. After 0.5 Gy of irradiation, expression of FN mRNA was reduced to 36% +/- 22% (1 h), 52% +/- 10% (1 d), and 76% +/- 10% (4 d) of the control level. After 1 Gy of irradiation, relative FN mRNA was 62% +/- 28% (1 h) and 75% +/- 3% (4 days) to the control level, respectively. This reduction was comparable to that reported by others for the cytoskeletal protein beta-actin. In contrast, mRNA for tubulin, another cytoskeletal protein, increased at 1 h after irradiation but then approached normal postnatally. The longer lasting alteration of FN may be more directly related to neural development, particularly if the reduction in FN is nonuniform.

A highly repetitive DNA sequence possibly unique to canids.

A short interspersed nucleotide (nt) element (SINE) was cloned from the genomic DNA of the domestic dog, Canis familiaris. Southern-blot analysis of canine DNA digested with four restriction endonucleases indicated that the SINE is widely dispersed throughout the genome. Hybridizations also indicated that the element may be unique to canids and is absent in a variety of other mammals, including members of four closely-related carnivore families. Three examples of the SINE have been located and sequenced. The 130-bp SINE contains putative RNA polymerase III transcriptional control sequences. The SINE is flanked at the 3' end by a (TC)8-repeat region followed by a poly(A) tract of 35-65 nt. Computer database searches located two homologous sequences with approx. 80% identity to the SINE. These sequences were located in untranslated regions of the canine genes encoding interferon-omega and clotting factor IX.

Physical mapping of the Coxiella burnetii genome.

Coxiella burnetii isolates from different genomic groups contain restriction fragment polymorphisms that were easily distinguishable using pulsed field gradient electrophoresis (PFGE). Conversely, isolates that belong to the same genomic group yield identical patterns indicating that PFGE can be used to identify the genomic grouping of new C. burnetii isolates. Intact C. burnetii cells were embedded in agarose and lysed in situ. The genomic DNA was digested with low-frequency cutting restriction endonucleases, and subjected to PFGE analysis. NotI and SfiI cut C. burnetii DNA least often and produced the largest fragments. ApaI, MluI, SalI, XbaI or XhoI produced only small DNA fragments (+/- 50 kbp). When PFGE was used to analyse C. burnetii genomes for the presence of plasmid-related sequences, all the plasmid sequences in Nine Mile and Priscilla were associated with their 36 kbp or 39 kbp plasmid bands, respectively. If these isolates contained plasmid sequences which had integrated into their chromosomes those sequences would have been visible as additional bands. These same studies also showed that plasmid sequences in the plasmidless-Ko isolate were completely contained within two NotI fragments, indicating that the integrated plasmid is localized to a concise region of the C. burnetii genome. Since it is difficult to conduct genetic analyses of obligate intracellular parasites using standard techniques, a physical map is being developed using PFGE. In addition to providing a means for determining gene loci, the physical maps provide a means for comparing genetic organization among the different strains of C. burnetii.

Use of pulsed field gel electrophoresis to differentiate Coxiella burnetii strains.

Pulsed field gradient gel electrophoresis (PFGE) provides a powerful technique for the analysis of bacterial genomic DNA by allowing the resolution of DNA fragments as large as 9000 kilobase pairs (kbp). When isolates of Coxiella burnetii were examined using this method, the restriction enzymes Not I and Sfi I gave the fewest and most easily resolved fragments. Sfi I cuts the genome of the Priscilla isolate of C. burnetii into 15 DNA fragments ranging in size from 320 to 18 kbp, and Not I cuts the DNA of this isolate into 20 fragments from 293 to 10 kbp in size. Analysis of the undigested DNA and summing of the Sfi I restriction fragments both indicate that the C. burnetii DNA contains approximately 1600 kbp, or is about one-third the size of the DNA in Escherichia coli. Comparisons of isolates revealed that the numbers and patterns of DNA fragments observed correlate with proposed strain designations. Because PFGE allows the reproducible separation of restriction endonuclease-digested C. burnetii DNA fragments into precise bands, it greatly facilitates the selection of large DNA fragments for cloning. Bands harvested from the gel can be cloned. Clone banks are invaluable for identifying the location of specific genes and landmarks and providing material for future experiments, including DNA sequencing. Yeast artificial chromosome (YAC) cloning vectors can accept fragments as large as 500 kbp. The fragmentation patterns of C. burnetii that we have obtained with infrequent-cutting enzymes are small enough to be cloned into YAC vectors. Using a PFGE selection method means that only small libraries would have to be created and screened. Thus, the results of these experiments also demonstrate the applicability of PFGE for deriving a physical map of C. burnetii chromosomal DNA. Development of such a macrorestriction map will facilitate genetic and molecular studies with C. burnetii.

Nine introns with conserved boundary sequences in the Euglena gracilis chloroplast ribulose-1,5-bisphosphate carboxylase gene.

The single, chloroplast encoded gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) from Euglena gracilis is found to contain nine intervening sequences. The intervening sequences were identified by heteroduplex analysis between Euglena rbcL and the non-intron-containing rbcL from Spinacea oleracea, by electron microscopy of Euglena rbcL DNA-mRNA hybrids, and by cloning, restriction endonuclease analysis, and partial DNA sequencing. The identification, locus, and coding properties for six of ten exons was confirmed by partial DNA sequence analysis. Each of the nine introns in the approximately 6.5 kb rbcL locus is approximately 0.5 kb in length. The DNA sequence of five 3'-intron/5'-exon and four 3'-exon/5'-intron boundaries are highly conserved. A proposed consensus sequence is (formula; see text) These conserved sequences could play a role in an mRNA splicing mechanism in chloroplasts analogous to that in eucaryotic nuclei.

Detection of multiple, unspliced precursor mRNA transcripts for the Mr 32,000 thylakoid membrane protein from Euglena gracilis chloroplasts.

The psbA gene is the coding locus for a polypeptide of 32 kilodaltons that is involved in electron transport through photosystem II. The 4.9 kilobasepair (kbp) EcoRI restriction endonuclease fragment EcoI from the 145 kbp Euglena gracilis chloroplast DNA was shown to encode psbA. Five transcripts of size 3.1, 2.8, 2.3, 1.8, and 1.2 kilobases were detected by hybridization of psbA probes to nitrocellulose filter blots of electrophoretically separated RNAs. This same pattern was observed when the hybridization probe consisted of only exon sequences from this split gene. A synthetic, intron specific probe hybridized to all RNA precursors except the 1.2 kb mature RNA. These results and psbA DNA sequence data lead to the conclusion that the four higher molecular weight transcripts are unprocessed precursors of the 1.2 kilobase RNA, some of which contain unspliced intervening sequences. There is an increase in psbA transcripts during light induced maturation of the chloroplasts.

Location of the single gene for elongation factor Tu on the Euglena gracilis chloroplast chromosome.

The structural gene for elongation factor Tu (EF-Tu) has been mapped by heterologous hybridization to a 2900 base pair sequence of Euglena gracilis Klebs Strain Z Pringsheim chloroplast DNA within the EcoRI fragment, Eco N. The hybridization probes were obtained from a HhaI restriction fragment containing internal sequences to Escherichia coli EF-Tu, located in the tufA gene locus, and from an EcoRI restriction fragment of chloroplast DNA from the eukaryotic algae Chlamydomonas reinhardii, containing the 3' end of the chloroplast EF-Tu gene. This is the second identified protein gene locus to be mapped on the E. gracilis chloroplast genome, and the first using prokaryotic DNA as a probe.

The gene for the large subunit of ribulose-1,5-bisphosphate carboxylase in Euglena gracilis chloroplast DNA: location, polarity, cloning, and evidence for an intervening sequence.

The gene for the large subunit (LS) of ribulose-1,5,-bisphosphate carboxylase of Euglena gracilis Z chloroplast DNA has been mapped by heterologous hybridization with DNA restriction fragments containing internal sequences from the Zea mays and Chlamydomonas reinhardii LS genes. The Euglena LS gene which has the same polarity as the Euglena rRNA genes has been located with respect to Pst I, Pvu I, and HindIII sites within the Eco RI fragment Eco A. The region of Euglena chloroplast DNA complementary to an 887 bp internal fragment from the Chlamydomonas chloroplast LS gene is interrupted by a 0.5-1.1 kbp non-complementary sequence. This is the first chloroplast protein gene located on the Euglena genome, and the first evidence for an intervening sequence within any chloroplast protein gene.