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Studies on a temperature-sensitive mutant of fowl plague virus having a mutation in gene 7 coding for the M protein.

A fowl plague virus (FPV) temperature-sensitive mutant, ts 303/1 having a ts mutation in gene 7 coding for the matrix (M) protein has been obtained. The mutant induced synthesis of virus-specific RNA and polypeptides as well as ribonuclear protein (RNP) formation in cells under non-permissive conditions; however, haemagglutinin cleavage was reduced, functionally active haemagglutinin and neuraminidase were absent and virions were not formed. In mutant-infected cells at 36 degrees C haemagglutinin cleavage was also reduced and virions formed had an altered NP:M ratio as well as a decreased haemagglutinin content. A population of virions formed under these conditions was heterogeneous both in morphology and in buoyant density. The data obtained suggest that a mutation in the M proteins of orthomyxoviruses can affect processing of the haemagglutinin and impair final stages of virion morphogenesis.

Genes, Viral↗

Differences in the control of virus mRNA splicing during permissive or abortive infection with influenza A (fowl plague) virus.

Spliced transcripts of influenza A (fowl plague) virus (FPV) RNA (vRNA) segments 7 and 8 accumulate to a much greater extent during non-productive infection of mouse L cells, than they do during productive infection in primary chick embryo fibroblasts (CEF). Virus-specific protein synthesis, or a consequent event in virus replication appears necessary to promote splicing of vRNA segment 8-encoded mRNAs in both cell types, and of vRNA segment 7-encoded mRNAs in CEF. In L cells, however, splicing of the segment 7-encoded mRNAs seems to be independent of such virus-specific control. This observation is discussed in relation to the defect in expression of vRNA 7 which has been observed previously in FPV-infected L cells, and which is thought to account for the failure of virus replication.

Animals↗

The structural proteins of chick embryo lethal orphan virus (fowl adenovirus type 1).

Chick embryo lethal orphan (CELO) virus (fowl adenovirus type 1) contains at least 14 structural proteins with polypeptide molecular weights ranging from 100K to about 6K. A nomenclature of the CELO virion polypeptides is presented and the molar proportion of each polypeptide has been estimated. The CELO virus pentons were specifically released from the virion by dialysis against borate-based calcium-magnesium saline. The penton base (polypeptide III, mol. wt. 92K) and two fibres were separated, characterized and their polypeptides were correlated with their morphological positions in the virion. Peptide mapping suggested that the long fibre (polypeptide IV, mol. wt. 65K), and the short fibre (polypeptide VII, mol. wt. 44.5K) were not related in their primary sequences and are therefore probably encoded by separate genes. The time course of synthesis of the CELO virion polypeptides indicated that, like their mammalian adenovirus counterparts, they are synthesized late (after viral DNA replication).

Adenoviridae↗

Studies of fowl plague virus temperature-sensitive mutants with defects in synthesis of virion RNA.

Three different types of impairment in the synthesis of virion RNA (vRNA) were detected in three groups of temperature-sensitive (ts) mutants of fowl plague virus (FPV) having ts mutations in genes 1, 3 and 5 respectively. Normal synthesis of poly-(A+) cRNA, poly(A-) cRNA and vRNA was observed under non-permissive conditions early in infection in cells infected with the ts43 mutant having a ts mutation in gene 1 coding for the PB2 protein. However, 4 h after infection synthesis of vRNA ceased, synthesis of poly(A+) cRNA was reduced drastically, but the rate of poly(A-) cRNA synthesis was the same as that in cells infected with wild-type FPV. In cells infected with the ts 166 mutant having a ts mutation in gene 3, coding for the PA polypeptide, a drastic reduction was observed in poly(A+) cRNA synthesis under non-permissive conditions. Synthesis of poly(A-) cRNA was also reduced and synthesis of vRNA was not detected. The ts 60 mutant, having a ts mutation in gene 5 coding for the NP polypeptide, induced synthesis of all types of virus-specific RNA under non-permissive conditions, but the regulation of synthesis of vRNA and poly(A+) cRNA was affected, there being predominant syntheses of RNA segments 5 and 8 late in infection. In cells infected with mutants ts43 and ts 166 synthesis of virus-specific proteins was impaired, which reflected defects in the synthesis of virus-specific RNAs. The data obtained suggest that the PB2 protein may be contained in an enzyme complex responsible for synthesis of vRNA, that different enzyme complexes may be involved in the synthesis of poly(A-) cRNA and vRNA, and that the NP protein plays a significant role in the regulation of vRNA synthesis.

Cycloheximide↗

A mutant of fowl plague virus (influenza A) with an enhanced electrophoretic mobility of RNA segment 8.

A temperature-sensitive mutant (ts 1/9) obtained by undiluted passage of fowl plague virus (FPV) at 33 degrees C carried a strong ts defect in RNA segment 6 [neuraminidase (NA) gene] and a weak ts defect in RNA segment 8 [non-structural (NS) protein Although the viral proteins have normal migration rates, the NS gene migrated during polyacrylamide gel electrophoresis (PAGE) significantly faster than the NS gene of wild-type FPV, even after denaturation by glyoxal. Despite this observation, the NS gene of ts 1/9 did not carry a deletion as shown by sequence determination. There were only five base replacements which resulted in three changes in amino acids. Three of the base replacements led to a more compact secondary structure of RNA segment 8, which seems to be responsible for the faster migration rate during PAGE and which seems to resist, at least partially, the treatment with glyoxal.

Amino Acid Sequence↗

Secretion of fowl plague virus haemagglutinin from insect cells requires elimination of both hydrophobic domains.

In the present study we have investigated the role of the hydrophobic domains of the fowl plague virus (FPV) haemagglutinin (HA) on its intracellular transport and maturation in insect cells. To this end processing of full-length HA (A+) has been compared to that of two truncated forms lacking either the cytoplasmic domain and the transmembrane domain (A-) or lacking the entire HA2 subunit, i.e. the transmembrane domain and the fusion peptide (HA2-). All glycosylation sites present on A- and HA2- were glycosylated, indicating that both truncated forms were completely translocated in the endoplasmic reticulum. Unlike A+, A- and HA2- did not form trimers as indicated by cross-linking, gradient centrifugation and studies employing conformation-specific antibodies. Whereas HA2- was efficiently secreted, A- was retained in the cells in an apparently membrane-bound form. The data show that the carboxy-terminal transmembrane region is essential for the formation and stability of the trimers of the FPV HA. These observations also indicate that, under certain conditions, the fusion peptide of the FPV HA can serve as a membrane anchor.

Animals↗

Genomic mapping and sequence analysis of the fowl adenovirus serotype 10 hexon gene.

The gene for the major capsid protein (hexon) of fowl adenovirus serotype 10 (FAV-10) has been identified by the use of the expression vector pGEX and rabbit polyclonal antisera raised against FAV-10. The nucleotide sequence of the entire hexon gene has been determined. Sequence analysis revealed an open reading frame of 2808 bp coding for a putative polypeptide 936 amino acids long with a molecular mass of 105.5 kDa. The translation initiation codon has a local sequence which conforms with the optimal translation start sequence of CC(A/G)CCATGG. The location of the hexon gene in the FAV genome was from 46.85 to 52.81 map units, which is to the left of the hexon gene in the genomes of both bovine and human adenovirus (52.4 to 60.5 map units.). A splice acceptor site was identified 12 bp upstream of the initiation codon by using mRNA and PCR. It had the sequence TAGG which conforms to the consensus sequence of (C/T)AGG. Comparison of the amino acid sequence of the FAV-10 hexon with those of the bovine, human and murine hexon gene products revealed highest levels of identity occurring in the regions corresponding to the pedestals which form the base region of the hexon, and the lowest levels of identity in the regions corresponding to the loops which are exposed to the external environment.

Amino Acid Sequence↗

Sequence and transcriptional analysis of terminal regions of the fowl adenovirus type 8 genome.

Fowl adenovirus (FAdV) type 1, CELO strain has no homologies to mastadenovirus E1A, E1B, E3 and E4, which regulate virus gene expression, DNA replication and virus-host interaction. Similarly, the right 5 kb and left 15 kb ends of CELO virus DNA are non-homologous to mastadenoviruses. To compare CELO virus with another FAdV, 7.5 kb of the left and 17 kb of the right ends of FAdV type 8 (strain A-2A) were sequenced and nine and 17 open reading frames (ORFs), respectively, were found. This FAdV-8 genome was similar to CELO virus in that (1) the central region contained the major structural protein genes including the fibre, pVIII, 100K, late 33K and pIVa2 genes, which were in the same order as in mastadenoviruses, (2) no homologues of mastadenovirus E1A, E1B, E3 and E4 were found in the ends, and (3) the left 6 kb and the right 13 kb ends showed no homology to mastadenoviruses. Several genomic features were unique to FAdV-8 compared to CELO virus. FAdV-8 contained one fibre gene in contrast to two in CELO virus. Three of eight unassigned ORFs in the left and five of 13 unassigned ORFs in the right ends were unique compared to CELO virus. Two sets of tandem repeats, one with five identical 33 bp repeats and the other with more than ten identical 135 bp repeats, mapped between 4.5 and 7.5 kb from the right terminus. No virus-associated RNA gene was found. Fifteen of 16 unique FAdV-8 ORFs tested were, as determined by RT-PCR, transcribed early.

Aviadenovirus↗

The complete nucleotide sequence of fowl adenovirus type 8.

The fowl adenovirus type 8 (FAdV-8) genome was sequenced and found to be 45063 nucleotides in length, the longest adenovirus (AdV) genome for which the complete nucleotide sequence has been determined so far. No regions homologous to early regions 1, 3 and 4 (E1, E3 and E4) of mastadenoviruses were recognized. Gene homologues for early region 2 (E2) proteins, intermediate protein IVa2 and late proteins were found by their similarities to protein sequences from other AdVs. However, sequences homologous to intermediate protein IX and late protein V could not be identified. Sequences for virus-associated RNA could also not be recognized. Two regions of repeated sequences were found on the FAdV-8 genome. The shorter repeat region contained five identical and contiguous direct repeats that were each 33 bp long, while the longer repeat region was made of 13 identical and contiguous, 135 bp long repeated subunits.

Aviadenovirus↗

Deletion of the non-essential UL0 gene of infectious laryngotracheitis (ILT) virus leads to attenuation in chickens, and UL0 mutants expressing influenza virus haemagglutinin (H7) protect against ILT and fowl plague.

Infectious laryngotracheitis virus (ILTV), a member of the Alphaherpesvirinae, possesses several unique genes. One of them, UL0, encodes an abundantly expressed protein that accumulates in the nuclei of ILTV-infected cells. This study demonstrates that this protein is dispensable for in vitro virus replication and that UL0 deletion mutants exhibit only minor growth defects in cultured cells. The UL0 gene locus of ILTV was also used for insertion of foreign DNA sequences encoding enhanced GFP or haemagglutinin (HA), subtype H7, of a highly pathogenic avian influenza virus under the control of the human cytomegalovirus immediate-early gene promoter. Expression of foreign proteins was shown by (immuno)fluorescence tests and Western blot analyses. After experimental infection of chickens, UL0 deletion mutants proved to be attenuated when compared to both parental wild-type ILTV and an UL0 rescue mutant. Nevertheless, all animals immunized with UL0-negative ILTV were protected from clinical disease after subsequent infection with virulent ILTV. Furthermore, all animals immunized with HA-expressing ILTV survived a lethal challenge with H7 subtype avian influenza virus with minimal clinical signs. Thus, an UL0-negative and HA-expressing ILTV recombinant may be used as a bivalent live virus vaccine against ILT and fowl plague. Unlike inactivated influenza virus vaccines, HA-expressing ILTV recombinants should be suitable for mass application and would also permit serological discrimination between vaccinated and virus-infected animals in the field.

Animals↗

Genome sequence analysis of the avian retrovirus causing so-called fowl glioma and the promoter activity of the long terminal repeat.

So-called fowl glioma is a retroviral infectious disease caused by avian leukosis virus subgroup A (ALV-A). We determined the complete nucleotide sequence of the virus genome. The full-length sequence was consistent with a genetic organization typical of a replication-competent type C retrovirus lacking viral oncogenes. The coding sequences were well conserved with those of replication-competent viruses, but the 3' noncoding regions including LTR were most related to those of replication-defective sarcoma viruses. The U3 region of the LTR had a few deletions and several point mutations compared to that of other ALVs. The promoter activities of the LTRs of glioma-inducing ALV and ALV-A standard strain, RAV-1, were equivalent in chick embryo fibroblasts (CEF), while that of glioma-inducing ALV was significantly lower than that of RAV-1 in human astrocytic cells. These subtle differences of the promoter activity of the LTR may be related to the induction of glial neoplasm.

Alpharetrovirus↗

A fourth allele in the plasma esterase-1 (Es-1) system of the domestic fowl.

Plasma samples of fowl were analysed by horizontal polyacrylamide gel electrophoresis (pH 9.0). Evidence was presented for the subdivision of an earlier reported esterase-1 allele (Es-1A) into two alleles designated Es-1A1 and Es-1A2. Family data were consistent with the hypothesis that the Es-1 phenotypes were controlled by four codominant, autosomal alleles Es-1C, Es-1A1, Es-1A2 and Es-1B). The White Leghorn samples showed high frequency of Es-1A1 (about 0.7) and also had considerable frequency of Es-1A2 (0.2) and of Es-1B (0.1). The three meat-type breeds studied (White Plymouth Rock, Rhode Island Red and New Hampshire) showed a very high frequency of Es-1B (0.8-1.0).

Alleles↗

The connection between the seminiferous tubules and the rete testis in the domestic fowl (Gallus domesticus). Morphological study.

The tubules connecting the seminiferous tubules proper to the rete testis in the fowl were studied with the aid of light and electron microscopy. The material examined ultrastructurally was fixed by vascular perfusion through the thoracic aorta. The seminiferous tubules were joined to the rete testis in three different ways; they were either linked by a terminal segment and a tubulus rectus, by a terminal segment only, or opened directly into the rete cavities. The terminal segment of the seminiferous tubules was lined with columnar cells (modified Sertoli cells). These cells were characterized by having an indented nucleus with a prominent nucleolus, many mitochondria, a sizable Golgi apparatus, electron dense bodies and many cytoplasmic protrusions into the lumen. Intraepithelial lymphocytes as well as macrophages in the lumina of the terminal segment, the tubuli recti and the rete testis were also observed. Myoid cells were found in the boundary tissue of the terminal segment.

Animals↗

Specialized cell contacts and the blood-testis barrier in the seminiferous tubules of the domestic fowl (Gallus domesticus).

The ultrastructure of Sertoli-Sertoli and Sertoli-germ cell surface specializations in the domestic fowl was studied in material fixed by vascular perfusion through the thoracic aorta. Three main types of surface specializations were found between adjacent Sertoli cells. These are focal tight junctions, desmosome-like devices, and a specialization characterized by the presence of long and dilated subsurface cisternae of rough endoplasmic reticulum. Typical inter-Sertoli cell junctions similar to those of mammals were absent. Germ cells were attached to Sertoli cells mainly by desmosome-like devices of varying appearance. The junctions between Sertoli cells and elongating or elongated spermatids, "the mantle", consisted of only slight condensations of filamentous material in the Sertoli cell. The tight junctions between adjacent Sertoli cells were efficient in preventing lanthanum from passing towards the lumen beyond the level of the spermatogonia.

Animals↗

Development of the blood-testis barrier in the domestic fowl (Gallus domesticus).

The formation of the blood-testis barrier (BTB) in the domestic fowl was studied at the electronmicroscopic level employing lanthanum as a tracer. No effective barrier could be demonstrated in testes before puberty, although several components of the Sertoli junctional complex such as focal tight junctions and desmosomes were already existent. The time of onset of meiosis after hatching showed great individual variation and meiosis did not occur synchronously in the tubules of a given testis. An effective barrier could first be detected in tubules containing early spermatids, and in which spermatogonia and primary spermatocytes at the leptotene stage were still within the open compartment. Thus, barrier formation was correlated with the occurrence of haploid germ cells. Complete compartmentation of seminiferous tubules, leaving only spermatogonia within the open compartment, was attained in tubules containing elongated spermatids of the maturation phase. In these tubules, primary spermatocytes at the leptotene stage were situated in an intermediate compartment.

Animals↗

The major oligosaccharides in the large subunit of the hemagglutinin from fowl plague virus, strain Dutch. Structure elucidation by one-dimensional and two-dimensional 1H nuclear magnetic resonance and by methylation analysis.

The N-glycosidically linked glycans in the large subunit (HA1) of the hemagglutinin from fowl plague virus, strain Dutch (containing about 15%, w/w, of carbohydrates), were liberated by alkaline hydrolysis, and were filtrated through Bio-Gel as the re-N-acetylated oligosaccharide alditols. One major fraction (90%, mol/mol) was obtained. It was subfractionated by concanavalin A affinity chromatography and was analyzed by methylation/capillary gas chromatography/mass fragmentography and especially by one-dimensional and two-dimensional 1H nuclear magnetic resonance. The major HA1 glycans, which are not sialylated, were thus found to comprise about 40%, 30% and 20% (mol/mol), respectively, of biantennary intersected, biantennary, and triantennary N-acetyllactosaminic ('complex') oligosaccharides. About two thirds of the internal GlcNAc residues in these glycans are substituted by Fuc(alpha 1----6), all the triantennary species carry the third Gal(beta 1----4)GlcNAc(beta 1----unit at the Man(alpha 1----6)-branch, and roughly one fourth of the N-acetyllactosamine units in the non-intersected biantennary oligosaccharides are incomplete.

Amino Acids↗

Ultrastructural changes in nervous elements associated with the granular cells of the common carotid artery of the domestic fowl after distal vagal ganglionectomy.

Intramural granular cells of the left and right common carotid artery of the domestic fowl were examined with the electron microscope after left distal vagal ganglionectomy. The vast majority of the nerve fibers and endings associated with the granular cells of the left common carotid artery degenerated rapidly after ipsilateral ganglionectomy, indicating that they are derived from the left vagus. On the other hand, left distal vagal ganglionectomy resulted in transformation of the intramural granular cells of the right common carotid artery and the few nervous elements associated with them, into a typical chemoreceptor tissue. This tissue was made up of small granule (60-120 nm) containing cells associated with many nerve fibers, nerve endings and fenestrated capillaries. The nerve endings were of two types; presumptive afferent endings and adrenergic efferent endings. Both types of endings made synaptic junctions with the granular cells.

Animals↗

The structure of the harderian gland of the guinea fowl at embryonic and post embryonic stages.

The Harderian gland of the guinea fowl is a bright, pink and relatively large orbital organ situated at the ventromedial aspect of the orbit. It possesses a single duct that has its exit on the convex medial surface. The outline is irregular with its caudal half narrower than the rostral half, and possessing a slight constriction about the mid point. Histologically, the gland had been outlined with the existence of a large contorted lumen by day 18 of incubation. The surface epithelial lining showed two layers of cells all through and lymphocytes were also present. By day 19 of incubation, certain zones of the surface epithelium had become pseudostratified and some of these cells contained vacuoles indicative of secretory materials within their cytoplasm. By day 21, few definitive acini with lumina had been observed and at day 23, the epithelium had assumed only a single layer of cells that were tall columnar cells, except at the junctions of the folds. By day 24, the tunica propria seemed to have completely disappeared with the acini occupying every available space. Plasma cells were seen three days after hatching.

Animals↗