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Objectives: To evaluate immediate placement and immediate restoration of a novel implant with a 12°-angled prosthodontic platform, in fresh extraction sockets of the aesthetic zone of the maxilla. Materials and methods: Tapered, roughened surface implants of 4 mm (n=15) or 5 mm (n=13) diameter were placed in 27 participants (mean age: 47.1 years; range: 21–71 years) requiring an immediate replacement of single anterior maxillary teeth. Provisional screw-retained all-ceramic crowns were placed within 4 h following optical impressions. At 8 weeks (baseline), definitive screw-retained all-ceramic crowns were placed in occlusion. Results: Twenty-six of the 28 implants met the inclusion criteria at surgery. Marginal bone levels revealed bone gain between surgery and baseline, and between baseline and 1 year of 0.2 mm (SD 0.75) and 0.78 mm (SD 2.45). Mean mid-buccal mucosal margins showed gains of 0.2 mm (SD 0.44). Prosthodontic maintenance and the aesthetics of the screw-retained implant crowns were facilitated by the external hex 12°-angled prosthodontic platform on the novel implant design, re-orientating the access cavity to the palatal or occlusal surfaces. All-ceramic implant crowns showed a high success rate with low maintenance issues over 1 year. Conclusion: Tapered, roughened-surfaced implants with a novel 12°-angled prosthodontic platform immediately placed in fresh extraction sockets, immediately restored with provisional crowns and subsequent definitive crowns at 8 weeks were successful for 1 year. To cite this article: Brown SDK, Payne AGT. Immediately restored single implants in the aesthetic zone of the maxilla using a novel design: 1-year report. Clin. Oral Impl. Res. 22, 2011; 445–454.

We have used X-ray crystallography to determine the structure of a decay accelerating factor (DAF)-binding, clinic-derived isolate of echovirus 11 (EV11-207). The structures of the capsid proteins closely resemble those of capsid proteins of other picornaviruses. The structure allows us to interpret a series of amino acid changes produced by passaging EV11-207 in different cell lines as highlighting the locations of multiple receptor-binding sites on the virion surface. We suggest that a DAF-binding site is located at the fivefold axes of the virion, while the binding site for a distinct but as yet unidentified receptor is located within the canyon surrounding the virion fivefold axes.

The Beaudette strain of IBV was passaged 16 times in chick kidney cells. Total cellular RNA was analyzed by Northern hybridization and was probed with 32 P-labeled cDNA probes corresponding to the first 2 kb of the 5′ end of the genome, but excluding the leader, and to the last 1.8 kb of the 3′ end of the genome. A new, defective IBV RNA species (CD-91) was detected at passage 6. The defective RNA, present in total cell extract RNA and in oligo-(dT) 30 -selected RNA from passage 15, was amplified by the reverse transcription-polymerase chain reaction (RT-PCR) to give four fragments. The oligonucleotides used were selected such that CD-91 RNA, but not the genomic RNA, would be amplified. Cloning and sequencing of the PCR products showed that CD-91 comprises 9.1 kb and has three regions of the genome. It contains 1133 nucleotides from the 5′ end of the genome, 6322 from gene 1b corresponding to position 12,423 to 18,744 in the IBV genome, and 1626 from the 3′ end of the genome. At position 749 one nucleotide, an adenine residue, was absent from CD-91 RNA. By Northern hybridization CD-91 RNA was detected in virions in higher amounts than the subgenomic mRNAs.

Previous studies in vitro of the processing of cloned polyprotein fragments from the coronavirus infectious bronchitis virus (IBV) large open reading frame (ORF1), confirmed the activity of a predicted 3C-like proteinase (3CLP) domain and suggested that the proteinase is released autocatalytically from the polyprotein in the form of a 35 kDa protein, 3CLpro, capable of further cleavages in trans. In order to identify such cleavages within the ORF1 polyprotein mediated by 3CLpro, the proteinase was expressed in bacteria, purified and used in trans cleavage assays with polyprotein fragments lacking the 3CLP domain as targets. The proteinase was expressed as a polyprotein fragment which was able to process during expression in bacterial cells, releasing mature 3CLpro. A histidine (His6) tag was introduced close to the C-terminus of the proteinase to aid purification. Processing demonstrated by the tagged proteinase was indistinguishable from that of the wild-type enzyme indicating that the site chosen for the tag was permissive. From these studies we were able to demonstrate trans cleavages consistent with the use of most of the previously predicted or identified sites within the open reading frame of gene 1. This tentatively completes the processing map for the ORF1 region with respect to 3CLpro.

Proteolytic processing of the polyprotein encoded by mRNA 1 is an essential step in coronavirus RNA replication and gene expression. We have previously reported that an open reading frame (ORF) 1a-specific proteinase of the picornavirus 3C proteinase group is involved in processing of the coronavirus infectious bronchitis virus (IBV) 1a/1b polyprotein, leading to the formation of a mature viral protein of 100 kDa. We report here the identification of a novel 10-kDa polypeptide and the involvement of the 3C-like proteinase in processing of the ORF 1a polyprotein to produce the 10-kDa protein species. By using a region-specific antiserum, V47, raised against a bacterial-viral fusion protein containing IBV sequence encoded between nucleotides 11488 and 12600, the 10-kDa polypeptide was detected in lysates from both IBV-infected and plasmid DNA-transfected Vero cells. Coexpression, deletion, and mutagenesis studies showed that this novel polypeptide was encoded by ORF 1a from nucleotide 11545 to 11878 and was cleaved from the 1a polyprotein by the 3C-like proteinase domain. Evidence presented suggested that a previously predicted Q-S (Q3783 S3784) dipeptide bond encoded by ORF 1a between nucleotides 11875 and 11880 was responsible for the release of the C terminus of the 10-kDa polypeptide and that a novel Q-N (Q3672 N3673) dipeptide bond encoded between nucleotides 11542 and 11547 was responsible for the release of the N terminus of the 10-kDa polypeptide.

The construction of a full-length clone of the avian coronavirus infectious bronchitis virus (IBV) defective RNA (D-RNA), CD-91 (9,080 nucleotides [Z. Penzes et al., Virology 203:286-293]), downstream of the bacteriophage T7 promoter is described. Electroporation of in vitro T7-transcribed CD-91 RNA into IBV helper virus-infected primary chick kidney cells resulted in the production of CD-91 RNA as a replicating D-RNA in subsequent passages. Three CD-91 deletion mutants were constructed--CD-44, CD-58, and CD-61--in which 4,639, 3,236, and 2,953 nucleotides, respectively, were removed from CD-91, resulting in the truncation of the CD-91 long open reading frame (ORF) from 6,465 to 1,311, 1,263, or 2,997 nucleotides in CD-44, CD-58, or CD-61, respectively. Electroporation of in vitro T7-transcribed RNA from the three constructs into IBV helper virus-infected cells resulted in the replication and packaging of CD-58 and CD-61 but not CD-44 RNA. The ORF of CD-61 was further truncated by the insertion of stop codons into the CD-61 sequence by PCR mutagenesis, resulting in constructs CD-61T11 (ORF: nucleotides 996 to 1,058, encoding 20 amino acids), CD-61T22 (ORF: nucleotides 996 to 2,294, encoding 432 amino acids), and CD-61T24 (ORF: nucleotides 996 to 2,450, encoding 484 amino acids), all of which were replicated and packaged to the same levels as observed for either CD-61 or CD-91. Analysis of the D-RNAs showed that the CD-91- or CD-61-specific long ORFs had not been restored. Our data indicate that IBV D-RNAs based on the natural D-RNA, CD-91, do not require a long ORF for efficient replication. In addition, a 1.4-kb sequence, corresponding to IBV sequence at the 5' end of the 1b gene, may be involved in the packaging of IBV D-RNAs or form part of a cis-acting replication element.

We have reviewed the results of ^sup 99m^technetium-methylene diphosphonate (Tc-MDP) bone scintigrams performed on patients following total knee arthroplasty. In addition, p 67 gallium (Ga) citrate scintigrams were carried out sequentially on 29 patients. Three groups of patients were identified: those with asymptomatic knees (undergoing scans for other reasons); those with aseptic or septic loosening; and those with pain without radiologic evidence of loosening. There was good correlation between the results of the scans and the final outcome. We conclude that sequential ^sup 99m^Tc-MDP and p 67 Ga citrate scintigrams are useful for demonstrating the presence of aseptic and septic loosening in knee prostheses, and pain with a normal scan appearance is probably not due to loosening or infection.

Nucleotide sequence analysis has shown previously that the genomic-length mRNA (mRNA1) of the coronavirus infectious bronchitis virus (IBV) contains two large open reading frames (ORFs), 1a and 1b, with the potential to encode polyproteins of approximately 441 and 300 kDa, respectively. We have characterized the specificity of a set of region-specific antisera raised against the 5′-portion of ORF 1a by immunoprecipitation of in vitro-synthesized, C-terminally truncated 1a polypeptides and used these antisera to detect virus-specific proteins in IBV-infected Vero cells. Two antisera, which had specificity for IBV sequences from nucleotides 710 to 2079 and 1355 to 2433, respectively, immunoprecipitated a polypeptide of approximately 87 kDa from IBV-infected Veto cells. In vitro translation of ORF 1a sequence terminating at nucleotide 5763 did not produce this protein unless the in vitro translation products were incubated with Vero cell S10 extracts prepared from either IBV-infected or mock-infected Vero cells. However, processing of the 87-kDa protein was also observed when the same region was expressed in Vero cells using the vaccinia virus/T7 expression system. This observation indicates that the 87-kDa polypeptide is encoded within the 5′-most 3000 nucleotides of mRNA 1 and that it might be cleaved from the 1a polyprotein by viral and cellular proteinases.

The following Brief Report was written by residents. A discussion by a member of the residents' faculty follows. We invite any resident to submit such articles, together with commentary by a faculty member.

A group of 48 patients, consecutive apart from exclusions, from a 1-year series of 60 cases of aneurysmal subarachnoid haemorrhage (SAH), was reviewed, in respect of clinical and radiological features, surgical management, clinical outcome, psychological distress and psychometric status, the neuropsychological assessment being compared with a closely-matched group of controls, the postoperative assessment being accompanied by a single positron emission computed tomogram (SPECT) scan. A review of those features which might have been expected to have a bearing on cognitive outcome (CT abnormalities at the outset, angiographic vasospasm, operative aneurysmal leakage, temporary vessel occlusion) failed to show a significant difference on cognitive tests. There was, however, a cognitive deficit shown by the patient group as a whole, when compared with the controls. Thus, SAH itself, the initial insult, would appear to be the essential factor in the production of persistent cognitive deficits.

We present here evidence demonstrating that four previously predicted Q-S(G) cleavage sites, encoded by the IBV sequences from nucleotide 15,129 to 15,134, 16,929 to 16,934, 18,492 to 18,497, and 19,506 to 19,511, respectively, can be recognised and transcleaved by the 3C-like proteinase. Five mature products with sizes of approximately 100 kDa, 65 kDa, 63 kDa, 42 kDa and 35 kDa are released from the ORF1b polyprotein by the 3C-like proteinase-mediated cleavage at these positions. Meanwhile, expression of plasmids containing only the ORF1b region showed no autocleavage of the polyprotein encoded, suggesting that the 3C-like proteinase may be the sole proteinase involved in processing of the 1b polyprotein. These data may therefore represent a complete processing map of the polyprotein encoded by ORF1b of mRNA1.

Deletion mutagenesis has been used to identify essential regions for rescue of coronavirus defective RNAs (D-RNAs). Using this technique on a cloned IBV D-RNA CD-91, we have identified a region potentially important in its rescue. Comparing the sequence of D-RNAs rescued with those not rescued we have deduced that a 72 base region corresponding to base number 13824 to 13896 in the viral genome is required for rescue. This may be an IBV D-RNA packaging signal or a cis-acting element involved in replication. Further experiments and modification of our techniques will be required to differentiate between the two processes.

A region of the infectious bronchitis virus (IBV) genome between nucleotide positions 8693 and 10927 which encodes the predicted 3C-like proteinase (3CLP) domain and several potential cleavage sites has been clones into a T7 transcription vector. In vitro translation of synthetic transcripts generated from this plasmid was not accompanied by detectable processing activity of the nascent polypeptide unless the translation was carried out in the presence of microsomal membrane preparations. The processed products so obtained closely resembled in size those expected from cleavage at predicted glutamine-serine (Q/S) dipeptides and included a protein with a size of 35 kDa (p35) that corresponds to the predicted size of 3CLP. Efficient processing was dependent on the presence of membranes during translation; processing was found to occur when microsomes were added posttranslationally, but only after extended periods of incubation. C-terminal deletion analysis of the encoded polyprotein fragment revealed that cleavage activity was dependent on the presence of most but not all of the downstream and adjacent hydrophobic region MP2. Dysfunctional mutagenesis of the putative active-site cysteine residue of 3CLP to either serine or alanine resulted in polypeptides that were impaired for processing, while mutagenesis at the predicted Q/S release sites implicated them in the release of the p35 protein. Processed products of the wild-type protein were active in trans cleavage assays, which were used to demonstrate that the IBV 3CLP is sensitive to inhibition by both serine and cysteine protease class-specific inhibitors. These data reveal the identity of the IBV 3C-like proteinase, which exhibits characteristics in common with the 3C proteinases of picornaviruses.

In order to investigate the mechanisms involved in the processing of infectious bronchitis virus polyproteins, several candidate regions of the genome have been cloned and expressed in vitro. During these studies it was observed that the translation product encoded by one of these clones (pKT205) was poorly expressed. Biochemical and genetic analyses revealed that the basis for the poor expression was a post-translational event involving ubiquitination of the protein and degradation by an ATP-dependent system operating in the reticulocyte lysate used for the in vitro expression. Two independently acting regions which conferred instability were identified, one of which mapped to the predicted 3C protease domain, contained within the 5′ end of the clone, while the other, more C-terminal region, was effective in conferring instability upon a heterologous protein to which it had been transferred. These regions may influence the stability of the authentic viral protein(s) in vivo and hence allow for the control of their expression and/or function at the level of proteolysis by cellular protease(s).

Airbags have become an increasingly accepted automobile safety feature that can reduce the morbidity and mortality associated with motor vehicle collisions. We present the following case report of an unusual fatality with multiple internal injuries from a minor mechanism motor vehicle collision. The cause of injuries was determined to be secondary to the deployment of a driver's side airbag without the concomitant use of a lap-shoulder belt.

The gene encoding the spike (S) protein from two geographically distinct strains (American and British) of canine coronavirus (CCV) was cloned and sequenced. The nucleotide sequence revealed open reading frames of 1443 or 1453 amino acids, respectively. Structural features include an N-terminal hydrophobic signal sequence, a hydrophilic cysteine-rich cluster near the C-terminus, two heptad repeats and 29 or 33 potential N-glycosylation sites. Pairwise comparisons of S amino acid sequences from these isolates with other CCV strains (Insavc1 and K378) revealed that heterogeneity, found mostly in the form of conservative substitutions, is distributed throughout the canine sequences. However, 5 variable regions could be identified. Similar analysis with feline, porcine, murine, chicken and human coronavirus sequences revealed that the canine sequences are much more closely related to the feline S protein sequence than to the porcine S protein sequences even though they are all from the same antigenic group. Moreover, the sequence similarity between CCV isolates and the feline coronavirus, feline infectious peritonitis virus (FIPV) was comparable. Expression of the CCV or the transmissible gastroenteritis virus (TGEV) S gene using the vaccinia virus system produced a protein of the expected size which could induce extensive syncytia formation in infected canine A72 cells.

Intracerebral inoculation of the neurotropic murine picornavirus, Theiler's murine encephalomyelitis virus (TMEV), results either in an acute encephalitis (GDVII strain) or in the establishment of a persistent infection with the development of demyelinating lesions (BeAn strain). In this article, the expression of the viral RNA polymerase was studied in the central nervous system of both acutely and persistently infected mice and in infected cells in tissue culture. Similar numbers of acutely infected glial cells (80–85%) expressed both viral polymerase and structural proteins in vitro while a much smaller proportion of persistently infected glial cells (0·6–0·9%) expressed these proteins. Following infection of mice with GDVII, many cells in the brain were found to express polymerase. However, in the spinal cord of mice persistently infected with BeAn, very few cells were found to express the polymerase while many more cells showed the presence of viral structural proteins. This suggests that a restriction in viral replication, possibly at the level of polymerase expression, may be a feature of the persistent infection. However, enough polymerase was expressed to maintain a polymerase-specific antibody response in a number of infected animals as late as 21 months post-infection. Mechanisms that may be involved in the establishment and maintainance of TMEV persistence are discussed with reference to these findings.

Avian infectious bronchitis virus (IBV) is the prototype species of the Coronaviridae, a family of enveloped viruses with large positive-stranded RNA genomes. The genomic RNA of IBV is 27.6 kilobases (kb) in length and contains at least 10 distinct open reading frames (ORFs) (Boursnell et al., 1987). Available evidence suggests that five subgenomic mRNA species are produced in virus-infected cells. These mRNAs (mRNAs 2–6) together with the genome-length mRNA (mRNAl) range in length from about 2 kb to 27.6 kb, and have been shown to share a common 3’ -terminus and to form a nested set structure (Stern and Kennedy, 1980ab). Three of these, mRNAs 2,4 and 6, encode the major virion structural proteins spike (S), membrane (M), and nucleocapsid (N), respectively (Stern and Sefton, 1984). Two of the other mRNAs, mRNA 3 and mRNA 5, have recently been shown to encode three and two viral proteins respectively (Smith et al., 1990; Liu et al., 1991; Liu and Inglis, 1992).

The genome-length mRNA (mRNA 1) of the coronavirus infectious bronchitis virus (IBV) contains two large open reading frames (ORFs), 1a and 1b, with the potential to encode polypeptides of 441 and 300 kDa, respectively. The downstream ORF, ORF 1b, is expressed by a ribosomal frameshifting mechanism. In an effort to detect viral polypeptides encoded by ORF 1b in virus-infected cells, immunoprecipitations were carried out with a panel of region-specific antisera. A polypeptide of approximately 100 kDa was precipitated from IBV-infected, but not mock-infected, Vero cells by one of these antisera (V58). Antiserum V58 was raised against a bacterially expressed fusion protein containing polypeptide sequences encoded by ORF 1b nucleotides 14492 to 15520; it recognizes specifically the corresponding in vitro-synthesized target protein. A polypeptide comigrating with the 100,000-molecular-weight protein (100K protein) identified in infected cells was also detected when the IBV sequence from nucleotides 8693 to 16980 was expressed in Vero cells by using a vaccinia virus-T7 expression system. Deletion analysis revealed that the sequence encoding the C terminus of the 100K polypeptide lies close to nucleotide 15120; it may therefore be generated by proteolysis at a potential QS cleavage site encoded by nucleotides 15129 to 15135. In contrast, expression of IBV sequences from nucleotides 10752 to 16980 generated two polypeptides of approximately 62 and 235 kDa, which represent the ORF 1a stop product and the 1a-1b fused product generated by a frameshifting mechanism, respectively, but no processed products were observed. Since the putative picornavirus 3C-like proteinase domain is located in ORF 1a between nucleotides 8937 and 9357, this observation suggests that deletion of the picornavirus 3C-like proteinase domain and surrounding regions abolishes processing of the 1b polyprotein. In addition, the in vitro translation and in vivo transfection studies also indicate that the ORF 1a region between nucleotides 8763 and 10720 contains elements that down-regulate the expression of ORF 1b.

Canine coronavirus (CCV), a causative agent of enteritis in newborn dogs, was first identified in 19711. The disease is characterized by infection of the absorptive epithelium of the villi and the onset of diarrhoea followed by villus atrophy2. The CCV virion is known to contain at least three protein species viz. the 204 kDa spike glycoprotein, S, the 32 kDa membrane glycoprotein, M, and the 50 kDa nucleocapsid protein, N3.

We conducted two field studies using a behavioral consultation approach to reduce children's problem behaviors in public school settings. The first study consisted of a descriptive analysis in which the students and their teachers were observed during naturally occurring classroom activities. The results of the descriptive analysis provided hypotheses regarding the operant function of the students' problem behaviors. The hypotheses were tested in the second experiment directly through a modified experimental analysis and indirectly through an evaluation of the treatment effects. The interventions were designed to disrupt the inappropriate response-reinforcer relation by discontinuing contingent reinforcement (i.e., extinction), providing the reinforcer contingent on appropriate play behaviors, and teaching the students verbal skills functionally equivalent to the inappropriate response. The classroom teachers were trained to implement the interventions and conduct the experimental analyses during classroom activities in which the problem behaviors occurred most frequently. The interventions were effective in decreasing the students' problem behaviors while concurrently increasing their appropriate verbal skills.

The development of an osteonecrotic (avascular necrosis) hip joint in a patient with symptomatic CDC Stage 1V C2 disease is reported. The diagnosis was aided by radiological assessment and confirmed by radioisotope bone scan. This case serves as yet another musculoskeletal manifestation in association with HIV infection.

Nucleotide sequences from the third open reading frame of mRNA D (D3) of infectious bronchitis virus (IBV) were expressed in bacteria as part of a fusion protein with beta-galactosidase. Antiserum raised in rabbits against this fusion protein immunoprecipitated from IBV-infected chick kidney or Vero cells a polypeptide of 12.4K, the size expected for a D3-encoded product. The D3 polypeptide is apparently non-glycosylated, and appears to be associated with the membrane fraction of infected cells, as judged by cell fractionation and immunofluorescence.

The avian Coronavirus infectious bronchitis virus (IBV) has a single-stranded, polyadenylated, positive-sense RNA genome some 27.6 Kb in length (Boursnell et al., 1987). IBV replication almost certainly proceeds by the same mechanism that occurs in the more intensively studied Coronavirus, mouse hepatitis virus (MHV). Genomic RNA is first translated to produce the viral RNA-dependent RNA polymerase, which transcribes the genomic RNA to generate a negative-stranded full-length copy of the virion RNA. This copy RNA then serves as a template for the production of a nested set of 3′ -coterminal transcripts from which virus polypeptides are translated (reviewed in Spaan et al., 1988). These transcripts include a genomic-sized RNA (mRNA 1) and five subgenomic RNAs (mRNAs 2–6) of which mRNA 6 is the smallest (Stern and Kennedy, 1980). Almost all detailed analysis of IBV gene function to date has concentrated on the products of mRNAs 2, 4 and 6 which encode the viral structural proteins spike, membrane and nucleocapsid respectively (see Figure 1). Together, these mRNAs account for less than 30% of the genomic coding capacity; most of the remaining information is in two large, overlapping open-reading frames (ORFs) la and lb, encoded by mRNA 1. These ORFs could potentially produce some 740 kD of protein and must encode the RNA polymerase (Boursnell et al., 1987). A number of associated activities predicted to be involved in viral replication are also suspected to be encoded by mRNA 1 (Gorbalenya et al., 1989). To date, however, the only product of this RNA that has been detected in coronavirus-infected cells is a 28 kD MHV polypeptide (p28) of unknown function (Denison and Perlman, 1987) which is derived from the 5′ -end of the MHV genome (Soe et al., 1987).

The word “evolution” usually evokes thoughts of events which occurred in the distant past, but it is self-evidently an ongoing process. The application of intense selective pressure, for example by the wide-spread use of vaccination, and also the transport of infected animals over large distances may have contributed to what may be considered “short-term” evolution. Papers relevant to putative Coronavirus evolution in both recent and distant time-scales are presented in this volume.

A cDNA clone of the infectious bronchitis virus (IBV) spike protein gene has been recombined into vaccinia virus. Cells infected with the recombinant virus synthesized IBV spike antigen which was recognized by antibody raised against purified spike protein. Immunofluorescence showed that the IBV spike antigen was transported to the infected cell surface membrane and immunoprecipitation showed the presence of the glycosylated 180K mol. wt. polypeptide precursor of the two spike subunits S1 and S2 that comigrated with this antigen from IBV-infected cells. Vaccinated mice produced antibody that recognized the IBV spike antigen by ELISA and which neutralized IBV infectivity as shown by ciliostasis tests on tracheal organ cultures.

cDNA clones prepared from genomic RNA of coronavirus IBV have been sequenced. The nucleotide sequence for the complete 5' region of mRNA C, which is not present in mRNAs A and B, has been determined. A sequence of 1224 bases is presented which contains a long open reading frame predicting a polypeptide of molecular weight 25 443. This is in agreement with the molecular weight of 23 000 reported for the unglycosylated form of the membrane polypeptide.

The polymerase-encoding region of the genomic RNA of the coronavirus infectious bronchitis virus (IBV) contains two very large, briefly overlapping open reading frames (ORF), F1 and F2, and it has been suggested on the basis of sequence analysis that expression of the downstream ORF, F2, might be mediated through ribosomal frame-shifting. To examine this possibility a cDNA fragment containing the F1/F2 overlap region was cloned within a marker gene and placed under the control of the bacteriophage SP6 promoter in a recombinant plasmid. Messenger RNA transcribed from this plasmid, when translated in cell-free systems, specified the synthesis of polypeptides whose size was entirely consistent with the products predicted by an efficient ribosomal frame-shifting event within the overlap region. The nature of the products was confirmed by their reactivity with antisera raised against defined portions of the flanking marker gene. This is the first non-retroviral example of ribosomal frame-shifting in higher eukaryotes.

Two patients with osteoporosis circumscripta of the skull are presented who have each been treated with a 3-month course of intravenous infusions of (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD) (total dose of 185 and 375 mg), which in both cases was associated with rapid and dramatic remineralization of the porotic bone and marked clinical and biochemical remission. However, osteolytic disease reappeared (after 9 and 18 months, respectively) in both cases as an osteolytic ring showing high uptake of radiolabelled bisphosphonate on bone scan, in formerly unaffected bone just distal to the original leading edge of osteolysis. Further treatment was associated with healing of these new lesions. These cases suggest that the diseased osteoclasts entering previously normal bone are protected against the drug probably because normal bone matrix takes up relatively little APD, and that APD taken up by diseased bone behind this front confers long-term resistance to further resorption. In assessing treatment strategies with bisphosphonates for Paget's disease, the response at the leading edge of osteoporosis circumscripta may be particularly informative.

RNA sequences encoding the surface projection (spike) of the coronavirus infectious bronchitis virus, strain Beaudette, have been cloned into pBR322 using cDNA primed with a specific oligonucleotide. A 5.3 kilobase viral insert in the clone pMB179 has been identified. The region of this clone coding for the spike gene has been sequenced by the chain termination method, and we present here the first report of DNA sequence data for a coronavirus spike protein, the protein which forms the characteristic 'corona' after which the group is named. The amino acid sequence of the primary translation product, deduced from the DNA sequence, predicts a polypeptide of 1162 amino acids with a molecular weight of 127 006. This has many interesting features which confirm and extend our knowledge of this recently characterized membrane glycoprotein. The polypeptide is subsequently cleaved to S1 and S2, and partial amino acid analysis of the amino-terminus of the S1 polypeptide has been employed to locate the position of this terminus of S1 within the large open reading frame. The amino acid analysis also reveals the presence of an 18 amino acid putative signal sequence on the primary translation product which is not present on the mature S1 polypeptide.

Summary The subgenomic mRNAs of the fowl coronavirus infectious bronchitis virus (IBV) form a 3′ co-terminal or ‘nested’ set. The RNase T1 oligonucleotide mapping data which demonstrated this form of sequence organization provided no evidence for the presence of non-contiguous (leader) sequences fused to the 5′ termini of the mRNAs. However, we have been able to demonstrate, by extension of an end-labelled synthetic oligonucleotide primer, the presence of a leader sequence on IBV mRNA A. Sequencing by the chemical degradation method of the extended product has yielded the almost complete sequence of the 60 base leader and its point of fusion to the sequence of the virus genomic RNA.

An efficient method for the generation of gene banks from large RNA viruses is described using infectious bronchitis virus as an example. Randomly primed clones have been characterized and found to be representative of the viral genome including 5' leader sequences.

The nucleotide sequence determination of the genome of the Beaudette strain of the coronavirus avian infectious bronchitis virus (IBV) has been completed. The complete sequence has been obtained from 17 overlapping cDNA clones, the 5'-most of which contains the leader sequence (as determined by direct sequencing of the genome) and the 3'-most of which contains the poly(A) tail. Approximately 8 kilobases at the 3' end of this sequence have already been published. These contain the sequences of mRNAs A to E within which are the genes for the spike, the membrane and the nucleocapsid polypeptides: the main structural components of the virion. The remainder of the sequence, equivalent to the 'unique' region of mRNA F, is some 20 kilobases in length and is thought to code for a polymerase or polymerases which are involved in the replication of the genome and the production of the subgenomic messenger RNAs. This sequence contains two large open reading frames, potentially coding for polypeptides of molecular weights 441,000 and 300,000. Unlike other large open reading frames in the virus, the 300,000 open reading frame appears to have no subgenomic RNA associated with it which would allow it to be at the 5' end of an mRNA species. Because of this, and because of the characteristics of the sequence in the region immediately upstream of its start codon, other mechanisms of translation, such as ribosome slippage, must be postulated.

Summary EBNA-containing chromatin from Raji cells was solubilized by treatment with high concentrations of urea and salt and fractionated by hydroxyapatite chromatography. Fractions eluting at different phosphate concentrations were analysed for the presence of EBNA by means of an anti-EBNA-specific 125I-labelled IgG absorption assay. The antigen was found in fractions containing non-histone chromatin proteins.

cDNAs prepared from viral genomic RNA purified from two strains of infectious bronchitis virus (IBV) (Beaudette and M41) have been cloned into pBR322. Three of these clones, which contain the complete sequences of mRNA A for both strains, except for the leader sequences which are only present on the subgenomic messenger RNAs, have been sequenced using the dideoxy method. The sequences are similar for both strains, each containing a single long open reading frame of 1227 bases which predicts a polypeptide of molecular weight approximately 45 000. The genome position and size of this predicted polypeptide are consistent with it being the gene for the nucleocapsid protein. The amino acid sequence shows considerable homology with those of the nucleocapsids of murine hepatitis virus strains A59 and JHM. The major difference between the sequences determined for the two IBV strains is that the 3' non-coding region of the Beaudette strain contains a 184 base segment which is not present in the M41 strain.

Circulating red blood cell volumes were determined by the carbon monoxide method, and plasma volumes were calculated in four men 20, 29, 71, and 75 years old, and two women 29 years of age before, during, and after exposure to an altitude of 3800 m. In the four youngest subjects there were early increases in hemoglobin concentration during the first days at the stated altitude attributed to decreases in plasma volume. At the same time, hemoglobin concentration decreased and plasma volume increased in the oldest subject. Red cell volumes were slow to change, and it was concluded that 3 weeks or more of exposure to this altitude are required to affect significantly the red cell volume in man.

Blood volume was determined with both T-1824 (Evans blue) and carbon monoxide (CO) for 2 young women, 2 young men, and 2 men ages 70 and 75. In the CO method a precise volume of CO was administered from a closed system, and the CO concentrations in blood were determined on the Van Slyke-Neill apparatus before and at the end of a 10-min. rebreathing period. The manometric technic is presented in detail and possible sources of errors are discussed. Comparisons were made during the spring in Indiana, in the hot desert, and at an altitude of 3800 m. Blood volume was taken as the sum of plasma volume (by T-1824 method) and red cell volume (by CO method); from these values body hematocrit was calculated. The ratios of body hematocrit to observed hematocrit ranged from 0.89 to 1.05 in 16 observations, with a median value of 0.93. In 12 of the 16, they ranged from 0.89 to 0.95, also with a median value of 0.93. Blood volumes determined by the CO method, using the 0.93 ratio, were within -1 to +4% of the blood volumes as determined above in 12 of 16 subjects. The other four differed by -7 to -13%.

A quantitative assay for EBNA in human lymphoblastiod cell lines has been developed. The assay employs EBNA-positive and -negative 125-I-IgG preparations as reagents and can be used in a direct or indirect manner. EBNA specificity has been demonstrated in a number of ways. The antigenic relationship between EBNA present in Raji cells and in a number of other human lymphoblastiod cell lines of diverse origins and between the cell-associated antigen present in FT Raji cells and that present in isolated Raji nuclei has been studied. The possibility of carrying out blocking titrations for anti-EBNA determination has been demonstrated and the effect of glutaraldehyde and formaldehyde upon antigenic activity in FT Raji cells has been studied.

Summary: Mutants of Escherichia coli k12 have been isolated that grow on media containing pyruvate or proline as sole carbon sources despite the presence of 10 or 50 mm-sodium fluoroacetate. Such mutants lack either acetate kinase [ATP:acetate phosphotransferase; EC 2.7.2.1] or phosphotransacetylase [acetyl-CoA:orthophosphate acetyltransferase; EC 2.3.1.8] activity. Unlike wild-type E. coli, phosphotransacetylase mutants do not excrete acetate when growing aerobically or anaerobically on glucose; their anaerobic growth on this sugar is slow. The genes that specify acetate kinase (ack) and phosphotransacetylase (pta) activities are cotransducible with each other and with purF and are thus located at about min 50 on the E. coli linkage map. Although Pta- and Ack- mutants are greatly impaired in their growth on acetate, they incorporate [2-14C]acetate added to cultures growing on glycerol, but not on glucose. An inducible acetyl-CoA synthetase [acetate:CoA ligase (AMP-forming); EC 6.2.1.1] effects this uptake of acetate.

The elution at low pH of human EBV-positive 125I-IgG bound to preparations of EBV-carrying non-producer lymphoblastoid cell lines has been investigated. Specific binding was demonstrated. A number of approaches were used to confirm the specificity. The stoichiometry of binding and elution was studied. The sub-cellular location of the EBV-associated antigen (s) detected by this method was shown to be predominantly nuclear. This confirms other studies demonstrating the presence of a nuclear antigen in non-producer lymphoblastoid cell lines carrying the EBV genome.

Summary The solubilisation of Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) by treatment with various molarities of NaCl was investigated using the 125 I—IgG absorption assay. Ninety percent of the antigenic activity detected using the 125 I—IgG absorption assay was insoluble at 0.15 M NaCl. It could be rendered soluble by treatment with 2.0 M NaCl, but reprecipitated upon return to 0.15 M NaCl. EBNA was partially extracted from Raji chromatin by treatment with 0.35 M NaCl. The efficiency of extraction was increased by homogenisation in 2.0 M NaCl followed by dialysis to 0.35 M NaCl. The data demonstrate the close association of EBNA with Raji chromatin and suggest that it may be a chromatin-associated non-histone protein.

The coronavirus Infectious Bronchitis Virus (IBV), a pathogen of chickens, has a genome structure typical of a coronavirus. The virus particle contains a single-stranded RNA molecule of approximately 27kb with positive polarity, and in infected cells six species of virus-specific mRNA are produced (1), designated RNA A to RNA F in order of increasing size (RNA F corresponding to the size of the genomic RNA). These RNAs form a nested set (2), in which the sequences in each of the subgenomic RNAs are also represented in all the larger RNAs; these RNA species each have a short ‘leader’ sequence corresponding to the 5′ end of the genomic RNA (3) followed by progressively larger amounts of information derived from its 3′ -end. For each mRNA, the region of sequence which is not contained in the next smallest RNA is assumed to be used for translation of viral proteins (2). Polypeptide products have been assigned for RNAs A, C and E (nucleoprotein, membrane protein and the spike protein precursor respectively) (4), but the coding function of the other RNAs has not so far been established, although it has generally been assumed that RNA F codes for the viral RNA-dependent-RNA-polymerase.

SUMMARY The subgenomic RNAs of the fowl coronavirus infectious bronchitis virus (IBV) form a 3' co-terminal or 'nested' set. The presence of non-contiguous (leader) sequences fused to the 5' termini of murine hepatitis virus mRNAs has been demonstrated using RNase T 1 oligonucleotide mapping and sequencing. The presence of a leader sequence on IBV mRNA A has been demonstrated previously. In this paper the presence of a leader identical to that present on the 5' terminus of IBV mRNA A is demonstrated to be present on the 5' terminus of IBV genomic RNA. This has been achieved by sequencing of primer extension products and cDNA clones containing the genomic leader. Analysis of these clones has revealed the presence of a sequence at the leader/genome-length RNA junction which is closely related to regions of homology identified previously within the genomic RNA sequence at the leader/body junctions of subgenomic RNAs. The implications of this finding for mechanisms of coronavirus RNA synthesis are discussed.