Advances in the use of nucleic acid probes in diagnosis of viral diseases of man. Brief review.

A variety of methods are now available for the preparation and labelling of viral nucleic acids for use as probes in diagnostic virology. Some of these are assessed including the use of synthetic oligonucleotides in place of molecularly cloned nucleic acids, and alternatives to labelling with radioactive isotopes such as biotin, enzymes and fluorochromes. Dot blot, sandwich, indirect sandwich and in situ hybridization are covered, and examples given of the current use of nucleic acid probes in detection of human viral infections. The potential and limitations of nucleic acid hybridization are discussed in the light of these new methods.

Molecular cloning of complementary DNA to Newcastle disease virus, and nucleotide sequence analysis of the junction between the genes encoding the haemagglutinin-neuraminidase and the large protein.

Complementary DNA clones to 90% of the Newcastle disease virus (NDV) genome have been produced and mapped. These clones cover the entire HN, F and M genes, most if not all of the L gene and parts of the NP and P genes. The map of overlapping clones gives the gene order 3'-NP-P-M-F-HN-L-5' for NDV, identical to the gene order of Sendai virus, on the assumption that the NP gene of NDV is at the 3' end of the genome as previously suggested by inactivation of NDV transcription by u.v. light. The nucleotide sequence of 453 bases covering the junction between the HN and L genes has been determined. There is nucleotide sequence homology to the consensus polyadenylation and mRNA start sites of Sendai virus and vesicular stomatitis virus. The deduced amino acid sequence of the C terminus of the HN protein of NDV shows homology to the C-terminal amino acid sequences of the HN proteins of simian virus 5 and Sendai virus. An explanation for the presence of HN0, the precursor to HN in some strains of NDV, is suggested by the presence of a long non-coding region at the 3' terminus of the mRNA encoding the HN protein of NDV that could, by mutation, allow synthesis of a larger polypeptide.

Sequence structures of a mouse major urinary protein gene and pseudogene compared.

Laboratory mouse strains carry approximately 35 major urinary protein (MUP) genes per haploid genome, tightly clustered together on chromosome 4. Most belong to two main groups (Groups 1 and 2). The available evidence strongly suggests that the Group 1 genes are active while the Group 2 genes are pseudogenes. Here we present the complete sequence of a Group 1 gene and a Group 2 gene and 700 bp of flanking sequence. The sequence of the Group 1 gene is consistent with its being active. The Group 2 gene contains two stop codons and a frame-shift mutation in the reading frame defined by the Group 1 gene, and would code for a signal peptide 25 rather than 19 amino acids long. The Group 2 gene differs from the Group 1 gene in other ways: a deletion upstream of the TATA box and another in intron 3, a base change in the TATA box itself, a 2 bp duplication at the splice acceptor boundary of intron 6, an altered poly(A) addition signal and a 1-base deletion 5' to the initiation codon. Some of these differences may explain the 10- to 20-fold higher level of Group 1 mRNA in mouse liver, and the fact that Group 1 and Group 2 transcripts are mainly spliced differently. The presence of the stop codon means that the Group 2 gene is a pseudogene in the context of the Group 1 gene. However, there is some evidence that the mature hexapeptide that it would code for may have biological activity. The 12 acceptor splice sites of the two genes all contain the identical sequence ACAG at the exon boundary.(ABSTRACT TRUNCATED AT 250 WORDS)

Spirochaetes in the equine caecum.

Two morphological types of spirochaete were found in the horse caecum measuring 4 to 6 micron by 0.3 to 0.4 micron and 6 to 8 micron by 0.1 to 0.2 micron. Attempts were made to culture the organisms but none survived subculture beyond the primary isolate. Electron microscopy revealed that many of the organisms were infected by bacteriophages.

Studies on the structure of a coronavirus-avian infectious bronchitis virus.

When avian infectious bronchitis virus (IBV) is fixed in formaldehyde, negative stain is able to penetrate the particle and an internal component is visualized. This component is seen as a tongue or flask shaped structure attached at one point to the outer virus membrane. A model yielding transmission patterns similar to the virus has been made. Gradient centrifugation studies on IBV reveal that the RNP is associated with the internal sac.

Electron microscope observations on the entry of avian infectious bronchitis virus into susceptible cells.

Infectious bronchitis virus was observed to enter cells of chicken chorioallantoic membrane by viropexis. There was no support for the suggestion that entry took place by fusion of viral and plasma membranes. The results of electron microscopy showed that virus attachment occurred both at 4 degrees and at 37 degrees C. Viropexis was not observed until the preparations were warmed. Similar results were obtained using chicken kidney cells. Quantitative data obtained from a plaque counting system employing chicken kidney cells indicated that attachment was the same at both temperatures and that some virus particles were taken up at 4 degrees C. Virus uptake was triggered by attachment of the virus to the cell membrane and the subsequent process of virus entry visualised by E. M. appeared to proceed without the involvement of lysosomal enzymes. No intracellular virus was located by electron microscopy in warmed preparations when virus was treated with specific antiserum, either before or after adsorption to the cells.

Haemagglutination by avian infectious bronchitis virus-a coronavirus.

The haemagglutinating ability of three strains of IBV was investigated. It was shown that whereas strain Beaudette had no detectable haemagglutinin, both Connecticut and Massachusetts agglutinated red cells of various species. The haemagglutinin of Connecticut was detectable after sucrose gradient purification whereas that of Massachusetts required both the purification step and incubation with the enzyme phospholipase C to reveal it. The agglutination could be inhibited by specific antisera. Some studies on the nature of the red cell receptor, and the possible presence of a receptor destroying enzyme, are reported.

The polypeptide composition of avian infectious bronchitis virus.

Avian infectious bronchitis virus grown in ovo was purified by differential centrifugation and isopycnic sedimentation in density gradients. The purified virus was analysed by SDS polyacrylamide gel electrophoresis and found to comprise up to sixteen polypeptides, four of which were glycopeptides. Bromelain treatment of the particles removed three polypeptides and two glycopeptides.

Studies on the biosynthesis of mitochondrial malate dehydrogenase and the location of its synthesis in the liver cell of the rat.

A method is described for the isolation of mitochondrial malate dehydrogenase from either the whole tissue homogenate or from the microsomal fraction of rat liver. The procedure involves the treatment of the tissue extract with detergent followed by gel filtration and chromatography on Amberlite CG-50 and DEAE-cellulose. The resulting enzyme was homogeneous by the criterion of gel electrophoresis. Incubation of the microsomal fraction from rat liver under the usual conditions for protein synthesis in the presence of [(3)H]leucine resulted in the incorporation of (3)H into the mitochondrial malate dehydrogenase when purified as described. The results are taken to indicate that the mitochondrial enzyme is synthesized by the cytoplasmic ribosomes. Possible ways in which the cytoplasmic and mitochondrial forms of malate dehydrogenase reach their final locations in the cell are discussed.