Successful pregnancy after high dose chemotherapy and marrow transplantation for treatment of aplastic anemia.

A 29-year-old woman with severe idiopathic aplastic anemia was given immunosuppressive therapy with procarbazine, 37.5 mg/kg, antithymocyte globulin, 36 mg IgG/kg, and cyclophosphamide, 200 mg/kg. This was followed by a marrow transplant from her HLA identical sister, immunosuppressive therapy with intermittent methotrexate for 3 months postgrafting and ultimate restoration of hematopoiesis. Two years after transplantation the patient delivered a healthy male infant. This is the first successful pregnancy after a high dose chemotherapy and marrow transplantation for treatment of aplastic anemia.

Periplasmic expression of part of the major rotavirus capsid protein VP7 containing all the three antigenic regions in Escherichia coli.

Part of the porcine rotavirus outer capsid protein VP7 containing all the three antigenic regions was expressed as a chimeric protein with bacterial alkaline phosphatase (AP) in E. coli. The construct contains an ompF promoter, the DNA encoding the signal sequence and the first 12 amino acids of mature OmpF, part of vp7 and the DNA encoding mature AP. The chimeric protein is stable, retains the biological property of AP and ability to react with polyclonal antiserum against the virus, and can be exported through the bacterial inner membrane into the periplasm.

Synthesis and biological evaluation of sialylmimetics as rotavirus inhibitors.

Rotaviruses cause severe gastroenteritis in infants and are estimated to be responsible for over 600 000 deaths annually, primarily in developing countries. The development of potential inhibitors of this virus is therefore of great interest, particularly since the safety and efficacy of rotaviral vaccines has recently been questioned. This study describes the synthesis of a variety of compounds that can be considered as mimetics of N-acetylneuraminic acid thioglycosides and the subsequent in vitro biological evaluation of these sialylmimetics as inhibitors of rotaviral infection. Our results show that readily accessible carbohydrate-based compounds have the potential to act as inhibitors of rotaviral replication in vitro, presumably through inhibition of the rotaviral adhesion process.

Expression of rotavirus VP7 antigens in fusions with bacterial proteins.

We have cloned antigenic regions of the VP7 gene from rotavirus RV-5 into the lamB gene. The insertions discussed in this paper comprise 250bp of rotavirus DNA and cover the A and B antigenic regions of the protein. The fusion proteins are expressed and are present in the outer membrane: they react with anti-rotavirus antibody. However, as yet we have not been able to demonstrate that the fusion has LamB protein functions with regard to maltodextrins or L phage, and the fusion protein appears to exist in the outer membrane as a monomer rather than as a trimer.

Self-injection with olive oil. A cause of lipoid pneumonia.

A 48-year-old man with unipolar depression and a psychosexual problem concerning his body image was injecting his scrotum repeatedly with olive oil to increase the size of his genitals. He developed respiratory failure following accidental intravenous injection of olive oil and was found to have lipogranulomatous lesions in the lung and the scrotum.

Immunofluorescence in duodenal mucosa of children with acute enteritis due to a new virus.

Electron microscopy od duodenal mucosa from children with acute non-bacterial enteritis has shown virus particles in epithelial cells. Indirect immunofluorescent techniques applied to the same tissue showed virus antigen localized in the cytoplasm of epithelial cells of the villi. Specific IgM antibody was present in sera from infected patients as early as two days after the onset of symptoms. Virus particles from different patients appeared to share a common antigen. The evidence presented supports our belief that this new virus was the cause of acute enteritis in the children studied.

An epidemic of diarrhoea in human neonates involving a reovirus-like agent and 'enteropathogenic' serotypes of Escherichia coli.

During December 1974, an epidemic of diarrhoea occurred in the Royal Children's Hospital, Melbourne, in a ward caring for neonates with acute or chronic medical and surgical problems. Electron microscopy of diarrhoeal faeces revealed a reovirus-like particle ('duovirus' or 'rotavirus') known to cause acute enteritis in older children. This virus is considered to have been primarily involved in the aetiology of the epidemic. In addition, three 'enteropathogenic' serotypes of Escherichia coli were isolated from babies during the epidemic, but none produced enterotoxin when tested in ligated ileal loops of rabbits or in monolayers of Y1 adrenal cells. Further epidemics of neonatal diarrhoea must be studied using culture and electron microscopy of faeces to determine the relative importance of this virus and of E. coli in the aetiology of diarrhoea in this age-group.

Development of rotavirus molecular epidemiology: electropherotyping.

Early in the era of rotavirology it was realized that the characteristic patterns of bands produced in polyacrylamide gels following electrophoresis of genomic dsRNA were useful for checking the identity of rotavirus isolates. However it was Romilio Espejo who first proposed the use of this technique for epidemiology, although most others did not take the suggestion seriously because the technique was then rather specialized and RNA staining methods were not very sensitive. Using samples collected by Ruth Bishop in Melbourne following the original identification of human rotaviruses, Sue Rodger recorded the "electropherotypes" of all samples available to 1979 and painstakingly compared them, side by side (since minor variations in conditions, especially temperature, alter the relative migration distances of dsRNA bands). These efforts produced the first longitudinal, extensive study of human rotavirus strain variation. Since then, technical improvements have greatly increased the sensitivity of the procedures, and electropherotyping has been recognized as a powerful and economical method for epidemiological studies of rotaviruses.

An improved enzyme-linked immunosorbent assay for the detection of rotavirus in faeces of neonates.

A direct enzyme-linked immunosorbent assay (EIA) for the detection of rotavirus in neonatal stools was developed. Rabbit antiserum against SA 11 rotavirus was incorporated as both coating and detector antibody, and rotavirus-negative rabbit serum was applied as a coating antibody control to eliminate false positive results. Pretreatment of stools with EDTA was found to increase both the sensitivity and specificity of the assay. This effect was greatest when 0.25 M EDTA (tetrasodium salt) was included in homogenized stool suspensions before the removal of solid debris by centrifugation. By electron microscopy, this EDTA pretreatment appeared to partly uncoat human rotavirus particles in faeces. Potentially suitable solid phase supports and horseradish peroxidase substrates were evaluated in the development of the assay. Screening of stool samples revealed that repeated freezing and thawing of stools eliminated positive EIA reactions. The SA 11 coating antibody compared favourably with a reference coating antiserum prepared against human faecal rotavirus strains. This EIA showed greater sensitivity for rotavirus detection than electron microscopy of stool concentrates prepared by ultracentrifugation, on testing 143 stools from 99 neonates and children. The assay has been applied successfully to detection of rotavirus in stools of neonates containing meconium, smaller amounts of viral antigen than in older children, and lacteal antirotaviral antibody. It is likely to be particularly useful for cross-infection studies in hospital wards and neonatal nurseries.

Selection of rotavirus VP4 cell receptor binding domains for MA104 cells using a phage display library.

Rotavirus infection of host cells, like other viruses, is a complex process that has not been fully elucidated, and much attention has been focused on the regions of the viral attachment protein, VP4, that are involved in binding to the cellular receptor. In this study, phage display technology was employed to generate a g3p VP4 gene-targeted phage display peptide library using the porcine rotavirus strain CRW8, and a method was optimised for panning this library on adherent MA104 cells to identify receptor binding domains. Recombinant phage that displayed expressed peptides from both the rotavirus VP4 trypsin cleavage products VP8* and VP5* were selected, and while some of the phage clones contained insert sequences from regions of VP4 implicated previously in cell binding and infection, new domains were also identified. In all, four regions within VP8* and six regions of VP5* were selected by panning. To our knowledge, this paper is the first description of using a gene-targeted phage display library to identify receptor binding domains on viral proteins.

The avian rotavirus Ty-1 Vp7 nucleotide and deduced amino acid sequences differ significantly from those of Ch-2 rotavirus.

The Vp7 gene of the avian strain Ty-1, which is classified as G7 serotype, was sequenced and the amino acid sequence deduced. The gene is 1065 nucleotides long with a long open reading frame of 987 nucleotides producing a protein 329 amino acids in length. The amino acid homology of the Ty-1 Vp7 protein to that of the avian Ch-2 Vp7 was 70%. The A, B, and C variable epitope regions of Ty-1 were unique compared to those of Ch-2 and other strains representing the 14G serotypes. The low 53% homology of the A and C regions of Ty-1 and Ch-2 would suggest that Ty-1 may be of a different serotype to the G7 reference strain Ch-2.

VP4 relationships between porcine and other rotavirus serotypes.

VP4 relationship of Australian porcine rotaviruses were identified using genetic reassortants and MAbs. All porcine virus isolates except BEN-144 appeared to share VP4 antigenicity with OSU virus. VP4 and BEN-144 virus (Gottfried-like virus) showed some antigenic relationships with the human neonatal viruses ST-3 and RV-3. In addition, VP4 of porcine CRW-8 showed antigenic relationships with simian SA-11. RRV and also canine K9 viruses, while that of porcine TFR-41 showed at least one way VP4 antigenic relatedness with UK bovine rotavirus. Furthermore, BMI-1 virus which is antigenically similar to an American virus SB1-A (a naturally occurring reassortant) may have arisen similarly by gene reassortment in nature in Australia.

Attachment of SA-11 rotavirus to erythrocyte receptors.

Treatment of human group O and sheep erythrocytes with receptor-destroying enzyme rendered them inagglutinable by simian rotavirus SA-11. The erythrocyte receptors were also removed by periodate oxidation and markedly reduced by incubation with a high concentration of trypsin, but they were not altered by infectivity-enhancing concentrations of trypsin, p-hydroxymercuribenzoate, or sodium sulfite (Na2SO3). Hemagglutinating activity of the virus particles was destroyed by periodate oxidation at 37 degrees C, p-hydroxymercuribenzoate, and a high concentration of trypsin and decreased by Na2SOa but was not altered by incubation with receptor-destroying enzyme, infectivity-enhancing concentrations of trypsin, or periodate oxidation at 4 degrees C. These results indicate that neuraminic acid-containing receptor substances are involved in the interaction of the virus with human and sheep erythrocytes, and suggest that SA-11-erythrocyte union involves carbohydrate on the surface of erythrocytes but not on the virion. Sensitivities of the SA-11 hemagglutinin to alcohols and repeated freeze-thaw cycles were also investigated.

Coproantibody response to rotavirus infection.

During three months after a family outbreak of diarrhoeal disease, rotavirus-specific immunoglobulins of the IgA, IgG and IgM classes were measured by enzyme-linked immunosorbent assay in faecal extracts from the four people involved. Shortly afterwards, sequential extracts were obtained from another infant after a proven rotavirus infection. Rotavirus infection was diagnosed by electron microscopy in three of the patients from whom acute-phase faecal samples were obtained, and all five patients developed a transient specific-antibody response. Antirotaviral IgA, IgM and IgG all reached peak titres between two and four weeks after infection, then dropped back to undetectable levels after two months. If these findings are confirmed in larger numbers of cases, they will provide the basis for simple diagnosis of recent rotavirus infections, without the need of even a single sample of serum.

Transfer of antirotaviral antibodies from mothers to their infants.

Levels of rotavirus-specific immunoglobulin G (IgG), IgA, IgM, and secretory immunoglobulin in maternal and cord serum, colostrum and milk, and infants' stools were measured by enzyme-linked immunosorbent assay in 92 mothers and their infants. Although antirotaviral IgG, IgA, and secretory immunoglobulin were present in most maternal sera, only IgG crossed the placenta. All samples of colostrum and milk tested contained antirotaviral secretory immunoglobulin and IgA except those of two women in whom IgA deficiency was subsequently described. Specific IgM and IgG were also detected in many colostral samples. Antirotaviral IgA was detected in many colostral samples. Antirotaviral IgA was detected in stools of breast-fed but not bottle-fed neonates. Apparently the human infant receives rotaviral antibodies both transplacentally and via maternal colostrum and milk.

Effects of antibodies, trypsin, and trypsin inhibitors on susceptibility of neonates to rotavirus infection.

Levels of antirotaviral secretory immunoglobulin A were measured by enzyme-linked immunosorbent assay in colostrum and milk samples collected daily for the first 5 days postpartum from 49 mothers breast-feeding their infants. The trypsin-inhibitory capacity of these lacteal secretion samples was assessed by their ability to inhibit the hydrolysis of alpha-N-benzoyl-DL-arginine-p-nitroanilide by trypsin. Stools passed by these breast-fed infants and by an additional 43 bottle-fed infants were pooled by individual and examined by electron microscopy for rotavirus. Stool trypsin levels were estimated with the gelatin hydrolysis test. Breast-fed infants were significantly less likely to become infected with rotavirus and showed significantly lower stool tryptic activity than did bottle-fed infants. Breast-fed infants who did not excrete rotavirus over the 5-day period received milk of significantly higher antirotaviral secretory immunoglobulin A or trypsin-inhibitory capacity or both than breast-fed infants who were infected with rotavirus. A case of probable maternal rotavirus infection during pregnancy, producing greatly elevated lacteal antirotaviral secretory immunoglobulin A levels lasting for 2 years, was detected. Results of this study suggest that both antibodies and trypsin inhibitors in human milk can be associated with the protection of neonates against rotavirus infection in the first 5 days of life.

Direct serotyping of porcine rotaviruses using VP7-specific monoclonal antibodies by an enzyme immunoassay.

Employing a serotyping EIA test using MAbs both cell culture adapted and faecal porcine rotaviruses were classified into serotypes G3, G3/5, G4, and G5. The MAbs have confirmed and extended the serotyping results obtained using polyclonal antisera. These MAbs are therefore potential reagents for serotyping of porcine rotaviruses. Using subgroup specific MAbs serotypes G3, G3/5, and G5 were found to contain subgroup I antigens while G4 rotaviruses contained either subgroup II or subgroup I antigens.

Morphology and development of bovine ephemeral fever virus.

The development of the virus of bovine ephemeral fever in mouse brain has been studied by electron microscopy. The virus particles are bullet-shaped, 70 by 145 nm, and slightly tapered toward the rounded end. The outer envelope is closely apposed to an electron-dense shell, about 12 nm thick, but no other internal structure is visible. The virus strains studied bud from the marginal membranes of neurones, but intracytoplasmic development, possibly aberrant, was also observed with one strain. Ephemeral fever virus is thus obviously a rhabdovirus, with points of resemblance to vesicular stomatitis, Flanders-Hart Park, and Kern Canyon viruses on the one hand, and to rabies virus on the other, but is structurally distinct from any of these.

Protein synthesis in Bunyamwera virus-infected cells.

In Vero cells infected with Bunyamwera virus there is a rapid inhibition of cell RNA and protein synthesis to levels of 30 and 3% respectively of the control rate, both the rate of inhibition and the time lag before its initiation being multiplicity dependent. Using u.v.-irradiated virus, investigation of the mechanism of inhibition of host cell protein synthesis indicates that synthesis of new virus components is required for inhibition to occur. Quantitative comparison of the proteins synthesized in infected cells shows that at higher m.o.i. synthesis of virus, as well as cellular proteins, is inhibited. Bunyamwera virus-infected Vero cells synthesized three virus-specific proteins identified as the structural virion proteins. Nucleoprotein is synthesized predominantly early in infection while the major envelope glycoprotein and the minor glycoprotein are synthesized predominantly late in the infection cycle.