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1.
J Oral Rehabil ; 45(4): 334-343, 2018 Apr.
Article in English | MEDLINE | ID: mdl-29314144

ABSTRACT

Long-standing loss of natural teeth in the mandible can lead to severe jaw atrophy and even mandibular fracture. There is no consensus on the best pre-prosthetic surgical treatment to reconstruct the atrophic edentulous mandible. The purpose of this review was to provide an overview of the existing literature and to give an evidence-based recommendation for bone grafting and future research. This systematic review was conducted according to the PRISMA statement. A literature search was performed in online databases Pubmed and Cochrane library for articles published between January 1980 and September 2017. The search was conducted using Medical Subject Heading terms: alveolar ridge augmentation; mouth, edentulous and mandible. Eligible articles were included according to in- and exclusion criteria and assessed on quality. Dental implant survival and bone stability were the primary outcomes. Secondary outcomes were complications. Twenty-four text articles matched the criteria and were included. Eleven articles were assessed to be of adequate quality for analysis. Graft stability seems to be higher in vertical distraction and tent-pole grafting, but as the dental implant survival is high (91.7% or higher) regardless of the procedure used for bone augmentation, this is of no clinical relevance. The survival rate of dental implants is high, regardless of the bone augmentation procedure used. High-quality clinical trials are needed to support the current evidence and guidelines on pre-implant bone grafting. Reporting of future research should include proper baseline characteristics and treatment description, as well as uniform outcome rendering.


Subject(s)
Alveolar Ridge Augmentation/methods , Dental Implantation, Endosseous/methods , Dental Prosthesis, Implant-Supported , Mandible/surgery , Mouth, Edentulous/surgery , Alveolar Bone Loss , Dental Implants , Humans , Mouth, Edentulous/physiopathology
2.
Ned Tijdschr Geneeskd ; 152(46): 2485-8, 2008 Nov 15.
Article in Dutch | MEDLINE | ID: mdl-19055252

ABSTRACT

A 28-year-old pregnant woman was given local anaesthesia in the perineum with a Monoject disposable needle in preparation for an episiotomy. The needle was bent manually in order to facilitate infiltration. During infiltration the needle broke off and disappeared. The delivery was uneventful. Radio diagnostic examination located the needle deep in the patient's left buttock. It was removed a week later via a small incision. Breakage of the needle was probably caused by the bending in combination with a sudden movement by the patient at the moment of infiltration. In obstetrics episiotomy is a common surgical procedure. For infiltration anaesthesia of the perineum a long, flexible, reusable Unimed needle is recommended instead of a short, inflexible, disposable Monoject needle.


Subject(s)
Anesthesia, Local/instrumentation , Anesthesia, Obstetrical/instrumentation , Episiotomy/instrumentation , Foreign-Body Migration/surgery , Needles , Adult , Anesthesia, Local/methods , Anesthesia, Obstetrical/methods , Episiotomy/adverse effects , Episiotomy/methods , Female , Humans , Perineum , Pregnancy
4.
J Virol ; 80(5): 2326-36, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16474139

ABSTRACT

The severe acute respiratory syndrome coronavirus (SARS-CoV) open reading frame 3a protein has recently been shown to be a structural protein. The protein is encoded by one of the so-called group-specific genes and has no sequence homology with any of the known structural or group-specific proteins of coronaviruses. It does, however, have several similarities to the coronavirus M proteins; (i) they are triple membrane spanning with the same topology, (ii) they have similar intracellular localizations (predominantly Golgi), (iii) both are viral structural proteins, and (iv) they appear to interact with the E and S proteins, as well as with each other. The M protein plays a crucial role in coronavirus assembly and is glycosylated in all coronaviruses, either by N-linked or by O-linked oligosaccharides. The conserved glycosylation of the coronavirus M proteins and the resemblance of the 3a protein to them led us to investigate the glycosylation of these two SARS-CoV membrane proteins. The proteins were expressed separately using the vaccinia virus T7 expression system, followed by metabolic labeling. Pulse-chase analysis showed that both proteins were modified, although in different ways. While the M protein acquired cotranslationally oligosaccharides that could be removed by PNGaseF, the 3a protein acquired its modifications posttranslationally, and they were not sensitive to the N-glycosidase enzyme. The SARS-CoV 3a protein, however, was demonstrated to contain sialic acids, indicating the presence of oligosaccharides. O-glycosylation of the 3a protein was indeed confirmed using an in situ O-glycosylation assay of endoplasmic reticulum-retained mutants. In addition, we showed that substitution of serine and threonine residues in the ectodomain of the 3a protein abolished the addition of the O-linked sugars. Thus, the SARS-CoV 3a protein is an O-glycosylated glycoprotein, like the group 2 coronavirus M proteins but unlike the SARS-CoV M protein, which is N glycosylated.


Subject(s)
Protein Modification, Translational , Protein Processing, Post-Translational , Severe acute respiratory syndrome-related coronavirus/chemistry , Viral Matrix Proteins/metabolism , Viral Proteins/metabolism , Amino Acid Sequence , Coronavirus M Proteins , Gene Expression , Glycosylation , Molecular Sequence Data , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism , Radioisotopes , Sialic Acids/analysis , Staining and Labeling , Viral Envelope Proteins , Viral Matrix Proteins/chemistry , Viral Proteins/chemistry , Viroporin Proteins
5.
J Virol ; 79(24): 15054-63, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16306576

ABSTRACT

Group 2 coronaviruses encode an accessory envelope glycoprotein species, the hemagglutinin esterase (HE), which possesses sialate-O-acetylesterase activity and which, presumably, promotes virus spread and entry in vivo by facilitating reversible virion attachment to O-acetylated sialic acids. While HE may provide a strong selective advantage during natural infection, many laboratory strains of mouse hepatitis virus (MHV) fail to produce the protein. Apparently, their HE genes were inactivated during cell culture adaptation. For this report, we have studied the molecular basis of this phenomenon. By using targeted RNA recombination, we generated isogenic recombinant MHVs which differ exclusively in their expression of HE and produce either the wild-type protein (HE+), an enzymatically inactive HE protein (HE0), or no HE at all. HE expression or the lack thereof did not lead to gross differences in in vitro growth properties. Yet the expression of HE was rapidly lost during serial cell culture passaging. Competition experiments with mixed infections revealed that this was not due to the enzymatic activity: MHVs expressing HE+ or HE0 propagated with equal efficiencies. During the propagation of recombinant MHV-HE+, two types of spontaneous mutants accumulated. One produced an anchorless HE, while the other had a Gly-to-Trp substitution at the predicted C-terminal residue of the HE signal peptide. Neither mutant incorporated HE into virion particles, suggesting that wild-type HE reduces the in vitro propagation efficiency, either at the assembly stage or at a postassembly level. Our findings demonstrate that the expression of "luxury" proteins may come at a fitness penalty. Apparently, under natural conditions the costs of maintaining HE are outweighed by the benefits.


Subject(s)
Hemagglutinins, Viral/metabolism , Murine hepatitis virus/enzymology , Viral Fusion Proteins/metabolism , Viral Proteins/metabolism , Animals , Gene Expression , Hemagglutinins, Viral/genetics , Hemagglutinins, Viral/immunology , Mice , Molecular Sequence Data , Murine hepatitis virus/genetics , Murine hepatitis virus/physiology , RNA, Viral/analysis , Vaccinia virus/genetics , Viral Fusion Proteins/genetics , Viral Fusion Proteins/immunology
6.
J Virol ; 77(17): 9567-77, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12915570

ABSTRACT

Toroviruses (family Coronaviridae, order Nidovirales) are enveloped, positive-stranded RNA viruses that have been implicated in enteric disease in cattle and possibly in humans. Despite their potential veterinary and clinical relevance, little is known about torovirus epidemiology and molecular genetics. Here, we present the first study into the diversity among toroviruses currently present in European swine and cattle herds. Comparative sequence analysis was performed focusing on the genes for the structural proteins S, M, HE, and N, with fecal specimens serving as sources of viral RNA. Sequence data published for animal and human torovirus variants were included. Four genotypes, displaying 30 to 40% divergence, were readily distinguished, exemplified by bovine torovirus (BToV) Breda, porcine torovirus (PToV) Markelo, equine torovirus Berne, and the putative human torovirus. The ungulate toroviruses apparently display host species preference. In phylogenetic analyses, all PToV variants clustered, while the recent European BToVs mostly resembled the New World BToV variant Breda, identified 19 years ago. However, we found ample evidence for recurring intertypic recombination. All newly characterized BToV variants seem to have arisen from a genetic exchange, during which the 3' end of the HE gene, the N gene, and the 3' nontranslated region of a Breda virus-like parent had been swapped for those of PToV. Moreover, some PToV and BToV variants carried chimeric HE genes, which apparently resulted from recombination events involving hitherto unknown toroviruses. From these observations, the existence of two additional torovirus genotypes can be inferred. Toroviruses may be even more promiscuous than their closest relatives, the coronaviruses and arteriviruses.


Subject(s)
Torovirus/classification , Torovirus/genetics , Animals , Base Sequence , Cattle , Cattle Diseases/epidemiology , Cattle Diseases/virology , DNA, Viral/genetics , Europe/epidemiology , Evolution, Molecular , Genetic Variation , Humans , Microscopy, Electron , Models, Genetic , Molecular Epidemiology , Molecular Sequence Data , Phylogeny , Recombination, Genetic , Sequence Homology, Nucleic Acid , Sus scrofa , Swine Diseases/epidemiology , Swine Diseases/virology , Torovirus/isolation & purification , Torovirus Infections/epidemiology , Torovirus Infections/veterinary , Torovirus Infections/virology
7.
EMBO J ; 21(23): 6571-80, 2002 Dec 02.
Article in English | MEDLINE | ID: mdl-12456663

ABSTRACT

Arteri-, corona-, toro- and roniviruses are evolutionarily related positive-strand RNA viruses, united in the order Nidovirales. The best studied nidoviruses, the corona- and arteriviruses, employ a unique transcription mechanism, which involves discontinuous RNA synthesis, a process resembling similarity-assisted copy-choice RNA recombination. During infection, multiple subgenomic (sg) mRNAs are transcribed from a mirror set of sg negative-strand RNA templates. The sg mRNAs all possess a short 5' common leader sequence, derived from the 5' end of the genomic RNA. The joining of the non-contiguous 'leader' and 'body' sequences presumably occurs during minus-strand synthesis. To study whether toroviruses use a similar transcription mechanism, we characterized the 5' termini of the genome and the four sg mRNAs of Berne virus (BEV). We show that BEV mRNAs 3-5 lack a leader sequence. Surprisingly, however, RNA 2 does contain a leader, identical to the 5'-terminal 18 residues of the genome. Apparently, BEV combines discontinuous and non-discontinuous RNA synthesis to produce its sg mRNAs. Our findings have important implications for the understanding of the mechanism and evolution of nidovirus transcription.


Subject(s)
RNA Caps , RNA, Messenger , Torovirus/genetics , Transcription, Genetic , Base Sequence , Molecular Sequence Data , RNA Splicing
8.
Vet Microbiol ; 69(1-2): 89-91, 1999 Sep 01.
Article in English | MEDLINE | ID: mdl-10515275

ABSTRACT

The biosynthesis and function of the gE-gI complex of feline herpesvirus (FHV) was studied by heterologous expression of the cloned genes in the vaccinia virus-based vTF7-3 expression system and by analysis of FHV recombinants. This work has led to the identification of domains in gI involved in complex formation with gE, and to a model for the disulfide-bonded structure of gI. The effects of mutations in gI on gE-gI-dependent cell-to-cell transmission are discussed.


Subject(s)
Herpesviridae/pathogenicity , Membrane Proteins/physiology , Viral Envelope Proteins/physiology , Animals , Cats , Cell Communication , Membrane Proteins/genetics , Molecular Weight , Structure-Activity Relationship , Viral Envelope Proteins/genetics
9.
J Gen Virol ; 80 ( Pt 7): 1799-1805, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10423149

ABSTRACT

The alphaherpesvirus glycoproteins gE and gI form a hetero-oligomeric complex involved in cell-to-cell transmission. The gI-deficient recombinant feline herpesvirus (FHV), FHVdeltagI-LZ, produces plaques that are only 15% the size of those of wild-type FHV. Here, we have complemented FHV(delta)gI-LZ allotopically by expressing intact gI and C-terminally truncated gI derivatives from the thymidine kinase locus. The effect on gE-gI-mediated cell-to-cell spread was assessed by plaque assay employing computer-assisted image analysis (software available at http://www.androclus.vet.uu.nl/spotter/spotter.htm+ ++). Allotopic complementation with intact gI fully restored plaque size. Deletion of the C-terminal 11 residues of gI did not affect cell-to-cell spread, whereas deletion of the complete cytoplasmic tail reduced plaque size by only 35%. Mutants expressing gI166, roughly corresponding to the N-terminal half of the ectodomain, displayed a small-plaque phenotype. Nevertheless, their plaques were reproducibly larger than those of matched gI-deficient controls, indicating that the gE-gI166 hetero-oligomer, though crippled, is still able to mediate cell-to-cell spread. Our data demonstrate that plaque analysis provides a reliable and convenient tool to measure and quantitate gE-gI function in vitro.


Subject(s)
Alphaherpesvirinae/genetics , DNA, Recombinant , Viral Envelope Proteins/genetics , Alphaherpesvirinae/pathogenicity , Animals , Cats , Gene Deletion , Gene Expression Regulation, Viral , Virulence/genetics , Virus Replication/genetics
10.
J Virol ; 72(9): 7245-54, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9696819

ABSTRACT

Alphaherpesvirus glycoproteins E and I (gE and gI, respectively) assemble into a hetero-oligomeric complex which promotes cell-to-cell transmission, a determining factor of virulence. Focusing on gI of feline herpesvirus (FHV), we examined the role of disulfide bonds during its biosynthesis, its interaction with gE, and gE-gI-mediated spread of the infection in vitro. The protein's disulfide linkage pattern was determined by single and pairwise substitutions for the four conserved cysteine residues in the ectodomain. The resulting mutants were coexpressed with gE in the vaccinia virus-based vTF7-3 system, and the formation and endoplasmic reticulum (ER)-to-Golgi transport of the hetero-oligomeric complex were monitored. The results were corroborated biochemically by performing an endoproteinase Lys-C digestion of a [35S]Cys-labeled secretory recombinant form of gI followed by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the peptides under reducing and nonreducing conditions. We found that (i) gI derivatives lacking Cys79 (C1) and/or Cys223 (C4) still assemble with gE into transport-competent complexes, (ii) mutant proteins lacking Cys91 (C2) and/or Cys102 (C3) bind to gE but are retained in the ER, (iii) radiolabeled endoproteinase Lys-C-generated peptide species containing C1 and C4 are linked through disulfide bonds, and (iv) peptides containing both C2 and C3 are not disulfide linked to any other peptide. From these findings emerges a model in which C1 and C4 as well as C2 and C3 form intramolecular disulfide bridges. Since the cysteines in the ectodomain have been conserved during alphaherpesvirus divergence, we postulate that the model applies for all gI proteins. Analysis of an FHV recombinant with a C1-->S substitution confirmed that the C1-C4 disulfide bond is not essential for the formation of a transport-competent gE-gI complex. The mutation affected the posttranslational modification of gI and caused a slight cold-sensitivity defect in the assembly or the intracellular transport of the gE-gI complex but did not affect plaque size. Thus, C1 and the C1-C4 bond are not essential for gE-gI-mediated cell-to-cell spread, at least not in vitro.


Subject(s)
Alphaherpesvirinae/chemistry , Disulfides , Glycoproteins/chemistry , Protein Conformation , Viral Proteins/chemistry , Animals , Cats , Cell Line , Electrophoresis, Polyacrylamide Gel , Glycoproteins/metabolism , Kinetics , Oxidation-Reduction , Protein Folding , Rabbits , Sodium Dodecyl Sulfate , Viral Proteins/metabolism
11.
Vet Rec ; 142(25): 683-6, 1998 Jun 20.
Article in English | MEDLINE | ID: mdl-9670455

ABSTRACT

A putative bovine respiratory torovirus (BRTV) was propagated in bovine fetal diploid lung and human colonic tumour cells, and fringed pleomorphic particles were detected in the culture supernatants by electron microscopy. Antisera directed against a bovine (Breda strain) and equine (Berne strain) torovirus failed to react with BRTV-infected cells in immunofluorescence assays and did not neutralise BRTV. No toroviral RNA was found in the supernatants of infected cells by means of a reverse transcriptase-polymerase chain reaction with torovirus-specific primers. On the other hand, bovine coronavirus-specific antisera and monoclonal antibodies did neutralise the cytopathic effects, and coronaviral antigen was detected in the cultures by immunofluorescence. Furthermore, bovine coronavirus RNA was detected in the supernatants of BRTV-infected cells after nucleic acid amplification. It is concluded that the cytopathic BRTV isolate is a coronavirus.


Subject(s)
Cattle Diseases/virology , Coronavirus, Bovine/classification , Torovirus/classification , Animals , Cattle , Cell Culture Techniques/methods , Coronavirus, Bovine/isolation & purification , Fluorescent Antibody Technique, Direct , Humans , Polymerase Chain Reaction , RNA, Viral/analysis
12.
J Virol ; 72(5): 4508-14, 1998 May.
Article in English | MEDLINE | ID: mdl-9557750

ABSTRACT

Recent evidence suggests that the type II feline coronavirus (FCoV) strains 79-1146 and 79-1683 have arisen from a homologous RNA recombination event between FCoV type I and canine coronavirus (CCV). In both cases, the template switch apparently took place between the S and M genes, giving rise to recombinant viruses which encode a CCV-like S protein and the M, N, 7a, and 7b proteins of FCoV type I (K. Motowaka, T. Hohdatsu, H. Hashimoto, and H. Koyama, Microbiol. Immunol. 40:425-433, 1996; H. Vennema, A. Poland, K. Floyd Hawkins, and N. C. Pedersen, Feline Pract. 23:40-44, 1995). In the present study, we have looked for additional FCoV-CCV recombination sites. Four regions in the pol gene were selected for comparative sequence analysis of the type II FCoV strains 79-1683 and 79-1146, the type I FCoV strains TN406 and UCD1, the CCV strain K378, and the TGEV strain Purdue. Our data show that the type II FCoVs have arisen from double recombination events: additional crossover sites were mapped in the ORF1ab frameshifting region of strain 79-1683 and in the 5' half of ORF1b of strain 79-1146.


Subject(s)
Coronavirus, Canine/genetics , Coronavirus/genetics , Recombination, Genetic , Animals , Base Sequence , Cats , Cell Line , DNA, Viral , Dogs , Molecular Sequence Data
13.
J Virol ; 72(5): 3507-11, 1998 May.
Article in English | MEDLINE | ID: mdl-9557628

ABSTRACT

A porcine torovirus (PoTV) was identified and characterized; it is a novel member of the genus Torovirus (family Coronaviridae, order Nidovirales), closely related to but clearly distinct from the already recognized equine torovirus (ETV) and bovine torovirus (BoTV) representatives. Immunoelectron microscopy of feces from piglets revealed elongated, 120- by 55-nm particles which were recognized by a torovirus-specific antiserum. Amplification by reverse transcriptase (RT) PCR with primers designed to detect conserved regions (on the basis of the genomes of BoTV strain Breda and ETV strain Berne) resulted in the identification of the 489-bp nucleocapsid gene, encoding a 18.7-kDa protein. The sequence identity in this region between PoTV and both ETV and BoTV was only about 68%, whereas the latter two show 81% identity. Neutralizing antibodies directed against ETV were found in sera of adult and young pigs. In all 10 herds sampled, seropositive animals were present, and 81% of randomly selected adult sows possessed antibodies. A longitudinal study with RT PCR showed that piglets shed virus in the feces for 1 or more days, starting 4 to 14 days after weaning.


Subject(s)
Swine Diseases/virology , Torovirus Infections/veterinary , Torovirus/classification , Amino Acid Sequence , Animals , Cattle , Cell Line , Feces/virology , Horses , Molecular Sequence Data , Sequence Homology, Amino Acid , Swine , Swine Diseases/blood , Torovirus/genetics , Torovirus/isolation & purification , Torovirus/ultrastructure , Torovirus Infections/blood , Torovirus Infections/virology
14.
J Virol ; 72(1): 862-7, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9420301

ABSTRACT

Simian hemorrhagic fever virus (SHFV) was recently reclassified and assigned to the new virus family Arteriviridae. During replication, arteriviruses produce a 3' coterminal, nested set of subgenomic mRNAs (sgRNAs). These sgRNAs arise by discontinuous transcription, and each contains a 5' leader sequence which is joined to the body of the mRNA through a conserved junction sequence. Only the 5'-most open reading frame (ORF) is believed to be transcribed from each sgRNA. The SHFV genome encodes nine ORFs that are presumed to be expressed from sgRNAs. However, reverse transcription-PCR analysis with leader- and ORF-specific primers identified only eight sgRNA species. The consensus sequence 5'-UCNUUAACC-3' was identified as the junction motif. Our data suggest that sgRNA 2 may be bicistronic, expressing both ORF 2a and ORF 2b. SHFV encodes three more ORFs on its genome than the other arteriviruses. Comparative sequence analysis suggested that SHFV ORFs 2a, 2b, and 3 are related to ORFs 2 through 4 of the other arteriviruses. Evidence which suggests that SHFV ORFs 4 through 6 are related to ORFs 2a through 3 and may have resulted from a recombination event during virus evolution is presented.


Subject(s)
Arterivirus/genetics , Evolution, Molecular , Genome, Viral , Multigene Family , RNA, Messenger/genetics , RNA, Viral/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Line , DNA Primers/genetics , Molecular Sequence Data , Open Reading Frames , Polymerase Chain Reaction , Sequence Homology, Amino Acid
15.
J Virol ; 71(11): 8397-404, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9343196

ABSTRACT

Alphaherpesvirus glycoproteins gE and gI form a noncovalently associated hetero-oligomeric complex, which is involved in cell-to-cell spread. In the absence of gI, feline herpesvirus (FHV) gE is transport incompetent and fully retained in the endoplasmic reticulum. Here, we assess the effect of progressive C-terminal truncations of FHV gI on the biosynthesis, intracellular transport, and function of the gE-gI complex. The truncated gI proteins were coexpressed with gE in the vaccinia virus-based vTF7-3 expression system. The results were corroborated and extended by studying FHV recombinants expressing truncated gI derivatives. The following conclusions can be drawn. (i) Deletion of the cytoplasmic tail, the transmembrane region plus the C-terminal half of the ectodomain of gI, does not affect intracellular transport of gE. Apparently, the N-terminal 166 residues of gI constitute a domain involved in gE-gI interaction. (ii) A region mediating stable association with gE is located within the N-terminal 93 residues of gI. (iii) The cytoplasmic domain of gI is not essential for gE-gI-mediated cell-to-cell transmission of FHV, as judged from plaque morphology. Deletion of the cytoplasmic tail of gI reduced plaque size by only 35%. (iv) Recombinants expressing the N-terminal 166 residues of gI display a small-plaque phenotype but produce larger plaques than recombinants with a disrupted gI gene. Thus, a complex consisting of gE and the N-terminal half of the gI ectodomain may retain residual biological activity. The implications of these findings for gE-gI interaction and function are discussed.


Subject(s)
Alphaherpesvirinae/physiology , Viral Envelope Proteins/physiology , Alphaherpesvirinae/growth & development , Alphaherpesvirinae/pathogenicity , Animals , Biological Transport , Cats , Cell Compartmentation , Cells, Cultured , Protein Binding , Sequence Deletion , Structure-Activity Relationship
16.
Virology ; 234(2): 349-63, 1997 Aug 04.
Article in English | MEDLINE | ID: mdl-9268167

ABSTRACT

Feline coronavirus (FCoV) persistence and evolution were studied in a closed cat-breeding facility with an endemic serotype I FCoV infection. Viral RNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in the feces and/or plasma of 36 of 42 cats (86%) tested. Of 5 cats, identified as FCoV shedders during the initial survey, 4 had detectable viral RNA in the feces when tested 111 days later. To determine whether this was due to continuous reinfection or to viral persistence, 2 cats were placed in strict isolation and virus shedding in the feces was monitored every 2-4 days. In 1 of the cats, virus shedding continued for up to 7 months. The other animal was sacrificed after 124 days of continuous virus shedding in order to identify the sites of viral replication. Viral mRNA was detected only in the ileum, colon, and rectum. Also in these tissues, FCoV-infected cells were identified by immunohistochemistry. These findings provide the first formal evidence that FCoV causes chronic enteric infections. To assess FCoV heterogeneity in the breeding facility and to study viral evolution during chronic infection, FCoV quasispecies sampled from individual cats were characterized by RT-PCR amplification of selected regions of the viral genome followed by sequence analysis. Phylogenetic comparison of nucleotides 7-146 of ORF7b to corresponding sequences obtained for independent European and American isolates indicated that the viruses in the breeding facility form a clade and are likely to have originated from a single founder infection. Comparative consensus sequence analysis of the more variable region formed by residues 79-478 of the S gene revealed that each cat harbored a distinct FCoV quasispecies. Moreover, FCoV appeared to be subject to immune selection during chronic infection. The combined data support a model in which the endemic infection is maintained by chronically infected carriers. Virtually every cat born to the breeding facility becomes infected, indicating that FCoV is spread very efficiently. FCoV-infected cats, however, appear to resist superinfection by closely related FCoVs.


Subject(s)
Coronavirus, Feline/genetics , Feline Infectious Peritonitis/virology , Genetic Variation , Genome, Viral , Amino Acid Sequence , Animals , Base Sequence , Breeding , Cats , Evolution, Molecular , Molecular Sequence Data
17.
J Virol ; 71(7): 5277-86, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9188596

ABSTRACT

We have characterized the 3'-most 3 kb of the genome of bovine torovirus (BoTV) strain Breda. A novel 1.2-kb gene, located between the genes for the membrane and nucleocapsid proteins, was identified. This gene, the 3'-most 0.5 kb of which is also present in the genome of the equine torovirus isolate Berne virus (BEV), codes for a class I membrane protein displaying 30% sequence identity with the hemagglutinin-esterases (HEs) of coronaviruses and influenza C viruses. Heterologous expression of the BoTV HE gene yielded a 65,000-molecular weight N-glycosylated protein displaying acetylesterase activity. Serologic evidence indicates that the HE homolog is expressed during the natural infection and represents a prominent antigen. By using an antiserum raised against residues 13 to 130 of HE, the HE protein was detected in radioiodinated, sucrose gradient-purified BoTV preparations. Formal evidence that HE is a structural protein was provided by immunoelectron microscopy. In addition to the large, 17- to 20-nm spikes, BoTV virions possess shorter surface projections (6 nm on average). We postulate that these surface projections, which are absent from the BEV virion, are composed of the BoTV HE homolog. The HE gene, which has now been demonstrated in three different virus genera, is a showpiece example of modular evolution.


Subject(s)
Hemagglutinins, Viral/metabolism , Torovirus/enzymology , Viral Fusion Proteins , Viral Proteins/metabolism , Viral Structural Proteins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cattle , Cell Line , Cricetinae , DNA, Complementary , Genome, Viral , Hemagglutinins, Viral/biosynthesis , Hemagglutinins, Viral/chemistry , Hemagglutinins, Viral/genetics , Molecular Sequence Data , Sequence Homology, Amino Acid , Torovirus/genetics , Viral Proteins/biosynthesis , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Structural Proteins/biosynthesis , Viral Structural Proteins/chemistry , Viral Structural Proteins/genetics
18.
J Virol ; 70(8): 5466-75, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8764058

ABSTRACT

The biosynthesis of glycoproteins E and I of feline herpesvirus was studied by using the vaccinia virus vTF7-3 expression system. gE and gI were synthesized as N-glycosylated, endoglycosidase H (EndoH)-sensitive precursors with Mrs of 83,000 and 67,000, respectively. When coexpressed, gE and gI formed sodium dodecyl sulfate-sensitive hetero-oligomeric complexes that were readily transported from the endoplasmic reticulum (ER). Concomitantly, the glycoproteins acquired extensive posttranslational modifications, including O glycosylation, leading to an increase in their apparent molecular weights to 95,000 and 80,000 to 100,000 for gE and gI, respectively. In the absence of gE, most gI remained EndoH sensitive. Only a minor population became EndoH resistant, but these molecules were processed aberrantly as indicated by their Mrs (100,000 to 120,000). By immunofluorescence microscopy, gI was detected primarily in the ER but also at the plasma membrane. gE, when expressed by itself, remained EndoH sensitive and was found only in the ER and the nuclear envelope. These results were corroborated by studying the biosynthesis of gE in feline herpesvirus (FHV)-infected cells. In cells infected with wild-type FHV, gE acquired the same co- and posttranslational modifications as during vTF7-3-driven expression. However, an FHV mutant lacking gI failed to produce mature gE. We conclude that gE is retained in the ER, presumably by associating with molecular chaperones, and becomes transport competent only when in a complex with gI.


Subject(s)
Gene Expression Regulation, Viral , Simplexvirus/physiology , Viral Envelope Proteins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cats , Cloning, Molecular , Molecular Sequence Data , Viral Envelope Proteins/genetics , Virus Assembly , Virus Integration
19.
Virology ; 212(2): 622-31, 1995 Oct 01.
Article in English | MEDLINE | ID: mdl-7571432

ABSTRACT

Feline coronaviruses (FCoVs) have been subdivided into feline enteric coronaviruses (FECVs) and feline infectious peritonitis viruses (FIPVs) on the basis of pathogenic properties. Serologically, a distinction has been made between type I and II FCoVs, the latter of which more closely resemble canine coronavirus (CCV). To gain more insight into the genetic relationships between different FCoV biotypes, we determined the nucleotide sequences of the ORF7a/7b transcription unit of nine strains. The following observations were made: (i) The sequences are 87-100% identical. In this part of the genome, type I and II FCoVs are more closely related to each other than to CCV. To explain the genetic and antigenic differences between the spike genes of type I and II FCoVs, we postulate that type II FcoVs have arisen by an RNA recombination event between a type I FCoV and CCV. (ii) The avirulent "FECV" strains UCD and 79-1683 are more similar to virulent "FIPV" strains than to each other. Our findings thus support the notion that FECV and FIPV are not different species but merely virulence variants. (iii) In contrast to FECV 79-1683, FECV UCD contains an intact ORF7b, indicating that ORF7b deletions are not a universal distinguishing property of FECVs. (iv) ORF7b deletions readily occur in vitro, correlating with loss of virulence. By reverse transcription-polymerase chain reaction analysis, we show that in naturally occurring FCoVs ORF7b is maintained. Thus, ORF7b seems to provide a distinct selective advantage during natural infection.


Subject(s)
Coronavirus, Feline/genetics , Coronavirus/genetics , Open Reading Frames/genetics , Sequence Analysis, DNA , Amino Acid Sequence , Animals , Base Sequence , Cats , Coronavirus/pathogenicity , Coronavirus, Canine/genetics , Coronavirus, Feline/pathogenicity , Dogs , Evolution, Molecular , Genetic Variation/genetics , Molecular Sequence Data , RNA, Viral/analysis , RNA, Viral/genetics , Sequence Deletion/genetics , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Transcription, Genetic , Virulence/genetics
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