Recombinant human respiratory syncytial virus (RSV) from cDNA and construction of subgroup A and B chimeric RSV

OBJECTIVE: The present study was performed to discriminate between central and peripheral effects of noradrenaline (NA) in normotensive, non-obese, type 2 diabetic patients. METHODS: Study I: In 10 patients and 10 healthy volunteer (HV) cumulative doses of NA were infused intravenously until mean arterial pressure (MAP) rose with 20 mmHg, and subsequently the effects on the forearm blood flow (FBF) was measured. Also, the FBF response to intra-arterial NA (0.025, 0.1, 0.4 micrograms min-1) was measured. Study II: In 13 patients and 14 HV the venous constrictor response to a cumulative local infusion of NA in a dorsal hand vein was determined. RESULTS: In study I the circulating plasma NA concentrations inducing a rise in MAP of 20 mmHg, were lower in the type 2 patients relative to the HV (p < 0.01). The relationship between changes in pressure and changes in heart rate were similar in both groups. Moreover, FBF responsiveness to intra-arterial NA was not different between the two groups. The slopes of the delta MAP/NA regression lines were correlated with basal insulin levels and relative insulin resistance in the healthy volunteers (R = 0.77, p < 0.01, and R = 0.83, p < 0.01), but not in the type 2 diabetic patients. In study II no differences were observed in the dose generating half maximum (ED50) and the maximum (Emax) response to NA between the type 2 patients and the HV. CONCLUSIONS: Non-obese normotensive type 2 patients have an increased pressor response to NA, which is not based upon a defect in skeletal muscle resistance arterioles, peripheral veins, or a defect in the baroreceptor system. Therefore, in type 2 diabetes the noradrenergic responsiveness of other vascular beds, such as the splanchnic or renal, must be enhanced.     

Recombinant respiratory syncytial virus (RSV) bearing a set of mutations from cold-passaged RSV is attenuated in chimpanzees

A set of five missense mutations previously identified by nucleotide sequence analysis of subgroup A cold-passaged (cp) respiratory syncytial virus (RSV) has been introduced into a recombinant wild-type strain of RSV. This recombinant virus, designated rA2cp, appears to replicate less efficiently in the upper and lower respiratory tracts of seronegative chimpanzees than either biologically derived or recombinant wild-type RSV. Infection with rA2cp also resulted in significantly less rhinorrhea and cough than infection with wild-type RSV. These findings confirm the role of the cp mutations in attenuation of RSV and identify their usefulness for inclusion in future live attenuated recombinant RSV vaccine candidates.     

Some virological and pathomorphological aspects of the respiratory system in the experimental infection with respiratory syncytial virus associated with influenza virus, parainfluenza virus type 3 and adenovirus in the mouse

Infections with respiratory syncytial virus Long strain, associated with influenza virus, A/Beijing 353/89 (H3N2) strain, parainfluenza virus type 3, 739-2D strain, and adenovirus type 3, were experimentally induced in white mice, causing histological, histochemical and histoenzymatic lesions at the respiratory system level, the severity of which exceeded the one observed in the controls infected with a single virus. The pathomorphological changes made up an inflammatory, predominantly infiltrative, lymphohistiocytic, then exudative and alterative picture. The severest and most frequent lesion was the diffuse interstitial, often peribronchiolovascular, bronchopneumonia, which might involve large parenchyma areas. Another highly frequent pulmonary lesion was the thickening of interalveolar septa, due to stasis hyperemia, oedema and the predominantly lymphocytic cytoinfiltrate. At the level of the extrapulmonary airways, the lesion present in all experimental models was the denudation of epithelium cilia. In the viral association in which influenza virus was included, an alteration, the hyalinosis of tunica media of the vessels, as well as of the Reisseisen's muscle, was also observed, in addition to the cytoinfiltrate; when the association was achieved with parainfluenza virus type 3, many macrophages and erythrocytes and a few fibroblasts appeared in the cytoinfiltrate, the alteration being the same as in the former model; when the association contained adenovirus, there appeared necrosis, abundant lymphocytes and lysis of the Reisseisen's muscle in the bronchopulmonary block. The associated infections were demonstrated by the presence of homologous serum antibodies and by positive IF reactions in the pulmonary tissue.     

Costs and benefits of respiratory syncytial virus immunoglobulin to prevent hospitalization for lower respiratory tract illness in very low birth weight infants

BACKGROUND: Respiratory syncytial virus immunoglobulin intravenous (RSV-IGIV) has been shown to reduce the risk of lower respiratory illness (LRI) hospitalization in preterm infants and infants with bronchopulmonary dysplasia (BPD). The purpose of this analysis was to estimate the economic costs and benefits of prophylaxis with RSV-IGIV in these groups. METHODS: The analysis was performed from a payer's perspective and therefore included only costs and cost savings that would be realized by an insurer. Estimates of the direct costs of prophylaxis and the risk and cost of LRI hospitalization were based on data about preterm very low birth weight infants cared for at our medical center. Estimates of the reduction in risk of LRI hospitalization associated with RSV-IGIV were based on data from a randomized trial (the PREVENT Study). RESULTS: The range of cost for a five-dose course of RSV-IGIV was estimated to be $3280 to $8800 for infants weighing 1.2 to 10.0 kg at the time of the initial dose. Risks of LRI hospitalization were estimated to be 12, 17 and 28%, respectively, for preterm infants without BPD, with mild BPD and with moderate to severe BPD. Estimates of duration and per diem cost of LRI hospitalizations were, respectively, 5 days and $971. The estimated net cost of prophylaxis per infant ranged between $5415 for a 6-kg infant without BPD to $1689 for an infant with BPD and age < or =3 months. CONCLUSIONS: The cost of RSV-IGIV typically exceeds the cost of hospitalizations prevented by several thousand dollars. Cost minus benefit is lower for infants with BPD and infants 3 months of age or younger.     

Serological indication for persistence of bovine respiratory syncytial virus in cattle and attempts to detect the virus

To identify putative persistent bovine respiratory syncytial virus (BRSV) infections in cattle, seven cattle that had experienced BRSV infections were treated with corticosteroids for two periods of 5 days. During the 5-day periods and the 3 weeks after treatment, attempts were made to isolate BRSV from lung lavage fluid and nasal swab specimens. Fluorescent antibody tests were used to detect BRSV antigen in lung lavage cells. A BRSV specific polymerase chain reaction (PCR) assay was developed, and was performed on lung lavage samples of all seven cattle as well as on various tissues of five of the cattle. In addition, nasal swabs of 74 over-one-year-old cattle, in a closed dairy herd were also assayed by PCR. The virus or its RNA was not detected in putative carriers, by any of the methods used, whereas all positive controls were positive. After corticosteroid treatment, three of the seven cattle showed a fourfold rise in antibody titre, suggesting induction of virus replication. BRSV-seronegative sentinel calves, that were housed together with each corticosteroid-treated animal, did not develop antibodies to BRSV indicating that BRSV was not shed by corticosteroid-treated cattle, or was shed at a very low level. In addition BRSV was not detected in seropositive cattle in a closed farm in summer. Although we consider the rises in antibody titres against BRSV an indication for persistence of BRSV in cattle, BRSV or its RNA was not detected in infected cattle.     

Replication of bovine respiratory syncytial virus in bovine and ovine peripheral blood lymphocytes and monocytes and monocytic cell lines

The present study compared the replication of bovine respiratory syncytial virus (BRSV) in bovine and ovine peripheral blood mononuclear cells, ovine and bovine monocytic cell lines and ovine alveolar macrophages. Low titres of virus were detected in ovine and bovine lymphocytes and monocytes 24-96 h post-exposure to the virus but there was no apparent replication of the virus in ovine alveolar macrophages during the culture period. The virus replicated to higher but statistically insignificant titres in ovine and bovine peripheral blood monocytes than in lymphocytes, with lymphocytes yielding peak titres significantly earlier. The secondary cell lines obtained from ovine liver and bone marrow also supported the replication of BRSV to high titres. The titres of BRSV in ovine and bovine lymphocytes and monocytes were significantly lower than in secondary cell lines. The addition of human recombinant tumour necrosis factor alpha after exposure to the virus or pre-incubation of ovine or bovine monocytic cells with either human recombinant interleukin 2 or phorbol myristate acetate before exposure to BRSV, did not significantly affect virus titre. Pre-incubation of cells with indomethacin or actinomycin significantly lowered virus titre (p < 0.05).     

Baculovirus expression of the fusion protein gene of bovine respiratory syncytial virus and utility of the recombinant protein in a diagnostic enzyme immunoassay

The fusion (F) protein of bovine respiratory syncytial virus (BRSV) was expressed by using a baculovirus vector. Antigenicity was tested by immunofluorescence analysis with F-specific monoclonal and polyclonal antibodies. Antibodies to recombinant F protein raised in a rabbit neutralized BRSV and human respiratory syncytial virus infectivity when tested in a plaque reduction assay. The recombinant F protein was evaluated as a source of antigen in an enzyme-linked immunosorbent assay (ELISA), and this ELISA was compared with the virus neutralization (VN) test for detecting BRSV antibodies in 10 consecutive serum samples from four calves vaccinated with a live modified BRSV vaccine and from two nonvaccinated control calves. The ELISA with the baculovirus-expressed F protein as an antigen compared favorably with the VN test and is a rapid, sensitive, and specific method for detecting serum antibodies to BRSV.     

Role of the fowlpox virus thymidine kinase gene for the growth of FPV recombinants in cell culture

Fowlpox virus (FPV) insertion plasmids were constructed that, upon integration into the viral genome via in-vivo recombination, inactivate the viral thymidine kinase (tk) gene. Using this approach, no wild-type virus-free stocks of recombinant virus could be obtained. In contrast, either integration of foreign genes into the intergenic region of the intact FPV tk gene and the open reading frame located downstream, or the functional substitution of the inactivated FPV tk gene by an intact vaccinia virus tk gene resulted in the predicted stable recombinants that were free of wild-type virus. Our results suggest that in already highly attenuated poxvirus strains an intact tk gene is essential for efficient growth of the virus in cell culture.     

Safety and immunogenicity of a respiratory syncytial virus subunit vaccine (PFP-2) in the institutionalized elderly

The safety and immunogenicity of purified fusion protein (PFP-2) respiratory syncytial virus (RSV) vaccine was evaluated in an open label study in 37 frail institutionalized persons over age 65. Vaccination was well tolerated without significant side-effects. Thirty-six of 37 volunteers completed the study. Nineteen of 36 (53%) vaccinees had a greater than or equal to fourfold increase in IgG to F protein at 4 weeks and 17 (47%) had a greater than or equal to fourfold rise in neutralizing titers to either group A or B virus. Although response rate to PFP-2 vaccine in the frail elderly was somewhat diminished compared to results in the healthy elderly, the vaccine was well tolerated and relatively immunogenic.     

Polymerase chain reaction versus culture for detection of Ureaplasma urealyticum and Mycoplasma hominis in the urogenital tract of adults and the respiratory tract of newborns

The efficiency of the polymerase chain reaction (PCR) was compared with that of culture for detection of Ureaplasma urealyticum and Mycoplasma hominis in 726 clinical specimens comprising 189 gynecological samples, 362 urological samples, and 175 samples from newborn infants. The sensitivity of PCR versus culture was 95% for both organisms, while the sensitivity of culture versus PCR was 91% for Ureaplasma urealyticum and 84% for Mycoplasma hominis. Furthermore, PCR tests were faster than culture tests, allowing the time to diagnosis to be reduced from two to five days to 24 h.     

Role of bovine respiratory syncytial virus in the aetiology of viruspneumonia in calves and yearlings ("pinkengriep") (author's transl)

The clinical picture of "pinkengriep", an enzootic form of bronchopneumonia in young cattle, is described. In addition to cough, conjunctivitis and a soporous state, accelerated respiration initially is an outstanding clinical symptom. In some cases, symptoms of fog fever appear during the second stage of the disease. In the autumn of 1973 and that of 1974, a total number of 292 animals with "pinkengriep" were serologically examined for known respiratory virus infections and Chlamydia. Complement fixation tests showed that there was significant increase in antibodies to respiratory syncytial virus in 76 per cent of the animals studied. In the case of para-influenza virus 3, virus diarrhoea virus, the adenoviruses of the antigen groups 1, 2 and 3, those of the antigen groups 4 to 10 inclusive and Chlamydia, these proportions were 48, 13, 12, 11 en 10 per cent respectively. On the other hand, symptoms of infection with infectious bovine rhinotracheitis virus were absent in the herds studied. These findings suggest a possible role of bovine respiratory syncytial virus in "pinkengriep".     

A peptide mimic of a protective epitope of respiratory syncytial virus selected from a combinatorial library induces virus-neutralizing antibodies and reduces viral load in vivo

Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and pneumonia in infants and young children worldwide. As yet, there is no effective vaccine against RSV infection, and previous attempts to develop a formalin-inactivated vaccine resulted in exacerbated disease in recipients subsequently exposed to the virus. In the work described here, a combinatorial solid-phase peptide library was screened with a protective monoclonal antibody (MAb 19) to identify peptide mimics (mimotopes) of a conserved and conformationally-determined epitope of RSV fusion (F) protein. Two sequences identified (S1 [HWYISKPQ] and S2 [HWYDAEVL]) reacted specifically with MAb 19 when they were presented as solid-phase peptides. Furthermore, after amino acid substitution analyses, three sequences derived from S1 (S1S [HWSISKPQ], S1K [KWYISKPQ], and S1P [HPYISKPQ]), presented as multiple antigen peptides (MAPs), also showed strong reactivity with MAb 19. The affinity constants of the binding of MAb 19, determined by surface plasmon resonance analyses, were 1.19 x 10(9) and 4.93 x 10(9) M(-1) for S1 and S1S, respectively. Immunization of BALB/c mice with these mimotopes, presented as MAPs, resulted in the induction of anti-peptide antibodies that inhibited the binding of MAb 19 to RSV and neutralized viral infection in vitro, with titers equivalent to those in sera from RSV-infected animals. Following RSV challenge of S1S mimotope-immunized mice, a 98.7% reduction in the titer of virus in the lungs was observed. Furthermore, there was a greatly reduced cell infiltration in the lungs of immunized mice compared to that in controls. These results indicate the potential of peptide mimotopes to protect against RSV infection without exacerbating pulmonary pathology.