Antigenic and biological criteria of differentiation of the newly isolated strains of respiratory syncytial virus

Respiratory syncytial (RS) virus strains isolated in different years varied by their antigenic and biological properties. The lowest degree of relatedness was found between the "street" virus and the prototype Long strain; the highest occured among the isolates from a given isolation period. Based on the mean indices of efficiency of the virus reproduction in human embryo lung (HEL) cells at 37 degrees C and 39 degrees C as well as on the degree of virus sensitivity to reference antibodies, the isolates from various years could be divided into three groups, namely high, mild and low virulent strains. The incidence of RS virus infections in children depended on the strain characteristic of virus population circulating in a community of children during the long-term observation period of 1976-1979. Cyclic variation was found in isolation rates of RS viruses; the duration of each cycle in different years ranged from 21 to 41 days. The variability of isolation cycles and the frequency of RS virus reinfections were closely related to the biological characteristics of circulating virus strains.     

Dynamics of some genetic marker changes of respiratory-syncytial virus strains circulating among nursery-school children

Respiratory-syncytial (RS) virus strains circulating during several years appeared polymorphic in respect of two genetic markers: the regression coefficient of infectious activity (RCIA39) characterizing the isolates by their reproduction in tissue cultures at supraoptimal temperature (39 degrees C) and the regression coefficient of neutralization indices (RCNI) characterizing the degree of sensitivity of the strains to antibodies. High-yield RS viruses were more often isolated from children frequently afflicted by the disease, moderate-yield viruses from moderately sick children, while low-yield or none-yield (at 39 degrees C) strains were isolated from rarely afflicted children. On the other hand, RS strains of low reactivity with prototype antibodies were mainly found in often or moderately sick children and the high-reactive ones in rarely sick children. The variability of the RS virus population was continuous, which is consistent with the uninterrupted course of the epidemic process in the nursery-school community. A change of the RCIA39 marker was observed in nearly 50% of strains already after 1 1/2 to 2 months, but most frequently within 5-6 months from the end of disease. The changes of growth intensity at 39 degrees C followed the pattern: high----moderate----low----none, however, in the next epidemic season these properties showed reversion in an opposite direction.     

Detection of respiratory syncytial virus in nasopharyngeal secretions by inhibition of enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay developed for the demonstration of respiratory syncytial (RS) virus immunoglobulin G antibodies was used for the detection of RS virus in specimens of nasopharyngeal secretions (NPS) obtained from children with acute respiratory disease. Samples of NPS were incubated with a fixed amount of standard serum (human serum antibodies to RS virus) before being added to the enzyme-linked immunosorbent assay test plate. A decrease in the optical density value determined for this standard serum was seen with majority of NPS specimens from which RS virus had been isolated in tissue culture. The reliability and the specificity of this inhibiton test were supported by experiments with purified RS virus and by tests with NPS specimens containing other respiratory viruses.     

Occurrence of respiratory syncytial virus subtypes A and B strains in Sweden

The subtype characteristics of 22 strains of respiratory syncytial (RS) virus isolated in Sweden were determined by the use of monoclonal antibodies. Eleven antibodies specific for distinct epitopes on five different structural proteins were used in immunofluorescence and radioimmune precipitation assays. One group of 12 isolates were derived from a three-month epidemic during 1984, whereas the other ten virus isolates were recovered during a time period of 13 years (1971-1983). All isolates could be allocated to the previously defined groups of subtype A and B strains of RS virus. During the single epidemic season, five subtype A and seven subtype B strains were found. During the 13-year period a randomly alternating appearance of six subtype A and four subtype B strains was observed. Thus RS virus strains of different subtype characteristics may occur alternately or concomitantly. The possible significance of consecutive infections with RS virus subtypes for immunopathological events deserves further studies.     

Immunoglobulin G antibody avidity in patients with respiratory syncytial virus infection.

The titer and avidity of respiratory syncytial virus-specific antibodies were measured in 196 serum specimens from 93 children with an acute, laboratory-confirmed respiratory syncytial virus infection. An enzyme immunoassay method based on the ability of urea to dissociate the bound antibodies with low avidity from the antigen was used. Three patterns of immune responses were observed. Children less than 6 months of age usually had low titers of antibodies with high avidity in their acute-phase serum samples. These antibodies were concluded to be of maternal origin, since their reaction pattern was similar to that of healthy adults. During the next few weeks, a slight increase in titers with a concurrent decrease in antibody avidity was observed. All children 6 to 24 months of age had low-avidity antibodies in their acute-phase serum samples, which matured to high avidity during the follow-up. On the contrary, about half of the children greater than 24 months of age had high-avidity antibodies already in the acute-phase serum samples. We conclude that the former children were experiencing primary infections with respiratory syncytial virus and the latter were experiencing reinfections. All adults with remote immunity had antibodies with high avidity.     

Rapid diagnosis of respiratory syncytial virus infection in children by the immunofluorescent technique

The immunofluorescent examination of nasopharyngeal secretions has been compared with conventional cell culture for the diagnosis of respiratory syncytial (RS) virus infection in children under the age of 2 years in Newcastle upon Tyne and Manchester. Two hundred and sixty-eight identifications of RS virus were made by the fluorescent method, of which 258 (96%) were confirmed culturally. In 267 children from whom virus was isolated in cell culture a positive diagnosis was made by the fluorescent method in 258 (97%). In 238 infants with bronchiolitis a diagnosis of RS virus infection was made on the basis of the fluorescent test in 185 (78%) of which 180 (97%) were subsequently confirmed in cell culture. The reasons for false results are briefly discussed. It is concluded that the method is reliable for the early diagnosis of respiratory syncytial virus infection.     

Cellular and antibody response to respiratory syncytial (rs) virus in human colostrum,maternal blood,and cord blood

Samples of colostrum, maternal blood, and cord blood from a group of 21 women were examined for the presence of cellular reactivity to respiratory syncytial (RS) virus using a transformation assay and for the level of specific IgA, IgG, and IgM antibodies to RS virus by membrane immunofluo-rescence. Six of the 18 colostral cell cultures and six of the 16 maternal blood cultures gave a significant proliferative response to RS virus antigen, although a positive response in both local and systemic cell cultures was found in only one mother. In addition, one of 18 samples of cord blood gave a proliferative response to RS virus antigen. Detectable titres of IgA antibodies to RS virus were found in 15 of the 20 samples of colostral whey and in 13 of the 17 samples of maternal plasma examined. RS virus-specific IgG antibodies were detected in 10 of 20 colostral whey samples and in all samples of maternal cord plasma. In this study, it was not possible to demonstrate a relationship between a positive proliferative response of colostral cell cultures to RS virus and the level of specific IgA or IgG antibodies in colostral whey. Similarly, the proliferative response of maternal blood cultures was unrelated to the titre of specific IgA or lgG antibodies in maternal plasma. The relevance of the local cellular proliferative response to RS virus in colostral cell cultures to the protection afforded by breast-feeding is discussed.     

Kinetic and chemical analyses of the biologic significance of lipoteichoic acids in mediating adherence of serotype III group B streptococci.

The mechanism(s) involved in the binding of lipoteichoic acid (LTA), isolated from virulent, asymptomatic, or avirulent serotype III strains of group B streptococci, to human embryonic epithelial cells (HEC), human fetal epithelial cells (HFC), and human adult buccal epithelial cells was investigated. It was determined that the binding of purified [3H]LTA to human adult buccal epithelial cells differed from the binding to HEC and HFC. LTA from all group B streptococcus strains bound to human adult buccal epithelial cells in a similar manner and was enhanced by the lipid portion of the polymer; in contrast, [3H]LTA binding to HEC and HFC was mediated by hydrophobic as well as specific interactions due to the glycerolphosphate backbone of LTA. Binding avidity of the LTAs to HEC and HFC varied depending on the bacterial strain. Polymers from asymptomatic and avirulent strains were easily dissociated from cell surfaces with unlabeled virulent LTA through competitive interactions; however, 10-fold greater levels of the same material were required to displace virulent [3H]LTA from HEC and HFC surfaces. These observed differences in binding avidity were shown to be due to longer LTA chains (30 to 35 glycerolphosphate units) in virulent strains when compared with LTA chains (10 to 12 glycerolphosphate units) of asymptomatic and avirulent strains. Thus, LTA appears to enhance the ability of virulent group B streptococci to bind to HEC and HFC with stronger avidity by virtue of the increased length of the cell-associated polymers synthesized by these strains. Mild enzymatic treatment of HEC and HFC with trypsin or periodate abolished LTA binding, which suggests the presence of a certain glycoprotein receptor(s) for LTA which does not appear to be present on human adult buccal epithelial cells. These data may therefore partially explain the increased susceptibility of newborn infants to group B streptococcal infections.     

The development of monoclonal antibodies to respiratory syncytial virus and their use in diagnosis by indirect immunofluorescence

Twelve clones of murine hybridoma cells secreting antibody specific for respiratory syncytial (RS) virus were classified into four groups on the basis of their pattern of staining of unfixed RS virus-infected HEp-2 cells in an indirect immunofluorescence test. Three of the groups reacted with virus antigens present on the membrane of the cells, whilst the fourth group failed to stain most live cells, suggesting specificity for an antigen expressed internally. Representative monoclonals from the membrane antigen staining groups immunoprecipitated the 86K glycoprotein (G), 50K plus 19K glycoprotein (F1,2) and a 23K non-glycosylated protein (VP23). A representative monoclonal from the fourth group that appeared to stain an internally expressed protein immunoprecipitated the virion 34K phospho-protein (P). All four monoclonals stained acetone-fixed tissue culture cells infected with either the Long strain of RS virus or with strains isolated in Newcastle during the 1965, 1972, and 1983 winter epidemics. The anti-fusion protein antibody stained acetone-fixed cells from all of 26 nasopharyngeal secretions from infants with RS virus infection. The anti-G glycoprotein antibody and the anti-VP23 antibody stained cells from secretions poorly or not at all, whilst the anti-P protein antibody stained cells in half the secretions tested but reacted with only a small proportion of cells in comparison with the anti-F or polyclonal antibodies. A pool of all four monoclonals produced more intense staining than the anti-F monoclonal alone and gave a more clearly defined staining reaction than the polyclonal antiserum used for routine diagnosis in over half the secretions. These results indicate that monoclonal antibodies will be of value in the diagnosis of RS virus by indirect immunofluorescence if care is taken in the selection of a suitable pool.     

Viremia in respiratory syncytial virus infection]

Viremia was demonstrated to occur in experimental respiratory syncytial (RS) virus infection in suckling cotton rats and in natural infection in children. RS virus was isolated from the whole blood of the animals in 3 out of 6 experiments at 2, 5, 6, 7 and 15 days after inoculation, the maximum infectious titer being more than 10(4) TCPD50/0.1 ml. RS virus was also isolated from the blood of 7 out of 15 examined children presenting the typical clinical picture of RS virus disease during the epidemic season of RS virus infection. In 6 patients RS virus was isolated from one blood specimen at 1, 6 and 7 days after the onset, in one patient from 3 blood specimens at 6, 9 and 22 days after the onset. The demonstrated long-term persistence of virus in the blood suggests the possibility of existence of chronic RS virus infection.     

Differences in virulence of Naegleria fowleri.

All pathogenic Naegleria fowleri isolated from the environment were highly virulent to mice when instilled intranasally. Axenic cultivation gradually decreased virulence of highly virulent strains. This decrease was most pronounced in environmental isolates and of minor importance in N. fowleri isolated from human cerebrospinal fluid. The low virulent strains obtained by continuous axenic cultivation appeared after clonation to consist of individuals with different virulence. Virulence could be enhanced in low virulent strains by brain passage and passages in Vero cell cultures, but could not be induced by these methods in nonvirulent strains isolated from the environment. Different mice strains showed different sensitivities to infection with pathogenic Naegleria. In addition, older mice were less sensitive than younger animals to low virulent strains.     

Comparison of enzyme-linked immunosorbent assay and neutralization techniques for measurement of antibody to respiratory syncytial virus: implications for parenteral immunization with live virus vaccine.

The sensitivity of an enzyme-linked immunosorbent assay (ELISA) to detect low levels of antibody to respiratory syncytial (RS) virus was compared with a tube dilution neutralization test (NEUT) on sera obtained from children who received a parenteral live RS virus vaccine. Among the children who developed antibody in response to live RS virus vaccine. ELISA was as sensitive as NEUT at detecting antibody increases. Some children who did not have detectable prevaccine ELISA antibody possessed NEUT antibody; these children were generally less than 12 months old, suggesting that they had low levels of maternal antibody. Low levels of NEUT or ELISA antibody were associated with the absence of antibody increases after injection of live RS virus vaccine. The quantity of antibody stimulated by this live RS virus vaccine was small compared with that which was stimulated by naturally acquired RS virus infection. We concluded that ELISA is a satisfactory test for determining antibody to RS virus in vaccine field trials, given the understanding that low levels of preexisting antibody are not detected in some instances.     

Monoclonal antibodies demonstrate heterogeneity in the G glycoprotein of prototype strains and clinical isolates of respiratory syncytial virus

In order to study variation among prototype strains and clinical isolates of respiratory syncytial (RS) virus, four prototype strains (Long, A2, CH18537, 9320) were used to produce monoclonal antibodies to this virus. The majority of monoclonals reacted with all four prototype strains by fluorescent antibody staining. Among the non-cross-reacting monoclonals, five additional patterns of reactivity with the prototype strains were recognized. Fourteen monoclonals, including ones representative of each of the patterns of reactivity with the prototype strains, were selected to use for typing prototype strains and community isolates. All 14 were found by immunoprecipitation to recognize the RS virus G glycoprotein. These monoclonals could uniquely identify each of the prototype strains. In addition to the antigenic differences among the prototype strains detected by the monoclonals, differences were also detected in the migration of the G glycoprotein of the prototype strains in polyacrylamide gel electrophoresis. Fluorescent antibody staining with panels of monoclonals distinguished two antigenic types among 114 isolates of RS virus recovered from children in St. Louis during the period 1981-86. The predominant type (80% of isolates) had a pattern of reactivity that resembled but differed from that of either the Long or A2 strains. The second type had a pattern of reactivity identical with that of 9320. The possible significance of this heterogeneity must be considered in developing diagnostic tests as well as active or passive immunotherapy for infections caused by RS virus.     

Evaluation of the Antibiotic Removal Device.

A new system called an Antibiotic Removal Device (ARD), used for the removal of residual antibiotics in blood specimens, was studied in 300 patients diagnosed as clinically septic despite antimicrobial therapy. Blood specimens from these patients were processed with and without the ARD into aerobic and anaerobic media. There were 53 patients who had one or more positive blood culture bottles, for a total of 109 positive blood culture bottles. Of these 109 bottles, 33.9% were positive with ARD-processed blood in aerobic media, 24.8% with ARD-processed blood in anaerobic media, 22.9% with conventionally processed blood in aerobic media, and 18.3% with conventionally processed specimens in anaerobic media. After 6 h of incubation, 14 bottles with blood processed by the ARD and 9 bottles processed conventionally were positive; after 24 h, 48 bottles with blood processed by the ARD and 31 bottles processed conventionally were positive. The same organisms were isolated from both ARD- and conventionally processed blood, with four exceptions. The ARD system, when compared with a conventional system of blood specimen processing, significantly increased the detection of bacteremia and decreased the time required for its detection in patients clinically septic despite ongoing antimicrobial therapy.     

Association of hemolysin production, hemagglutination of human erythrocytes, and virulence for chicken embryos of extraintestinal Escherichia coli isolates.

One hundred forty-two strains of Escherichia coli isolated from extraintestinal infections were examined for colicin V (ColV) and hemolysin (Hly) production. For comparison, 20 strains isolated from the feces of normal individuals and 12 enteropathogenic strains of E. coli were tested for these properties. Thirty-five to 59% of extraintestinal isolates were Hly+, but only one fecal strain was Hly+. Colicin V biosynthesis was found for 12% of blood culture isolates, 7% of urine culture isolates and 16% of the strains from other extraintestinal infections. None of the fecal isolates was ColV+. Selected strains were tested for virulence in 13-day-old chicken embryos; these same strains were tested for their ability to hemagglutinate chicken or human erythrocytes. Of 22 extraintestinal isolates, 13 (59%) killed greater than or equal to 60% of the embryos within 72 h. Only one of eight normal fecal isolates and two of three enteropathogenic strains tested were virulent. About 80% of the virulent strains were Hly+. The most striking finding, however, was the hemagglutination of human erythrocytes by virulent extraintestinal isolates. It seems possible that the hemagglutination property reflects a specific common adherence factor.     

Deoxyoligonucleotide probes that differentiate antigenic and pathogenetic variants of the tick-borne encephalitis virus]

Experiments on molecular hybridization were carried out using a panel of 11 deoxyoligonucleotide probes complementary to different parts of tick-borne encephalitis (TBE) virus, strain Sophyin, genome. Under study were the TBE virus strains differing by 3 criteria: (1) source of isolation (patients with acute and chronic TBE, Ixodes persulcatus and D. nuttalli ticks, small mammals); (2) serotype (eastern and Siberian Aina/1448), (3) virulence for Syrian hamsters. RNA of all the strains was hybridized with kDNA, 90% of strains with probe Sh5 complementary to protein E gene, nucleotide positions 1285-1311. The highest differentiating capacity was observed with probes P131 and Sh3 complementary to genes of proteins ns2b and M. These probes reacted with RNA of 100% of highly virulent strains of the eastern serotype and only with 20-30% of strains of the Aina/1448 serotype of lower virulence. A certain differentiating capacity was demonstrated by probes Sh2 and P10 complementary to genes of prm and C proteins: they hybridized with RNA of 80% of eastern serotype strains highly virulent for hamsters and with only 20% of Aina/1448 serotype strains of low virulence. The panel of probes used revealed no significant differences among strains in relation to their isolation source, with the exception of a strain isolated from D. nuttalli ticks which reacted only with kDNA and probe P2 complementary to nsI protein gene, but not with other probes. The TBE virus strains isolated from patients with chronic TBE were shown to represent a genetically heterogeneous group.     

Maturation of immunoglobulin G avidity after rubella vaccination studied by an enzyme linked immunosorbent assay (Avidity-ELISA) and by haemolysis typing

Two tests were introduced recently for assessment of the avidity of rubella immunoglobulin antibodies. In the quantitative test—avidity—enzyme linked immunosorbent assay (ELISA)—IgG antibodies obtained from individuals shortly after primary infection with rubella virus are distinguished from those with past immunity by their antigen-elution characteristics. This method uses agents that disrupt hydrophobic bonds in proteins [Kamoun PP (1988): Denaturation of globular proteins by urea: Breakdown of hydrophobic bonds? Trends in Biological Sciences 13:424–425.]. In the semiquantitative, presumptive test—haemolysis typing—the lowavidity rubella-IgG antibodies are distinguished from the high-avidity antibodies by the quality of their haemolytic zones in a radial haemolysis test. In the present study, both tests were applied to sera taken before and after vaccination with two different strains (Cendehill or RA 27/3) of live attenuated rubella virus. It was found that after vaccination of previously nonimmune subjects, IgG synthesized during the first 2 months had a very low avidity; IgG avidity increased dramatically during the subsequent 4 months and less markedly between 6 and 12 months after vaccination. On the contrary, the initially high IgG avidity of previous immune vaccinees remained at an elevated level postvaccination. These results provide a basis for identification of recent primary rubella virus infections, or vaccination reactions, by the avidity of specific IgG and also for their separation from rubella reinfections.     

Comparison of monoclonal antibody time-resolved fluoroimmunoassay with monoclonal antibody capture-biotinylated detector enzyme immunoassay for respiratory syncytial virus and parainfluenza virus antigen detection.

An all-monoclonal antibody, time-resolved fluoroimmunoassay was compared with several enzyme immunoassays for the detection of respiratory syncytial virus and parainfluenza virus type 1, 2, and 3 antigens in clinical specimens. The most sensitive enzyme immunoassay for parainfluenza virus type 1 was an all-monoclonal antibody assay with biotin-labeled detector antibody and streptavidin-peroxidase conjugate, but for respiratory syncytial virus and parainfluenza virus types 2 and 3 the most sensitive assay was a polyclonal antibody assay with horse capture antibodies and bovine or rabbit detector antibodies with anti-species peroxidase. All tests were evaluated with nasopharyngeal aspirate specimens from respiratory illnesses and with cell culture harvests of multiple strains of each virus isolated over many years. The time-resolved fluoroimmunoassay detected respiratory syncytial virus antigen in 92% of the specimens positive by culture, which was a decidedly higher sensitivity than either the monoclonal or polyclonal antibody enzyme immunoassay format (62 and 76%, respectively). For the parainfluenza viruses the time-resolved fluoroimmunoassay detected type-specific antigen in 94 to 100% of culture-positive specimens and again was more sensitive than the all-monoclonal antibody enzyme immunoassays (75 to 89%) or all-polyclonal antibody enzyme immunoassays (66 to 95%). Combined with results from a previously reported adenovirus time-resolved fluoroimmunoassay, these tests identified respiratory antigens in large numbers of clinical specimens.     

Monoclonal antibody-based capture enzyme immunoassays for specific serum immunoglobulin G (IgG), IgA, and IgM antibodies to respiratory syncytial virus

Monoclonal antibodies (MAbs) to the fusion protein (F), attachment protein (G), and nucleoprotein (N) of respiratory syncytial (RS) virus were evaluated for use as detector antibodies in immunoglobulin G (IgG), IgA, and IgM capture enzyme immunoassays. MAb assays were tested against assays using polyclonal antibodies (PAbs) with serum specimens from patients with and without evidence of recent RS virus infection. Assays developed with N MAbs were comparable to or better than PAb assays for detecting specific IgG and IgM antibodies but were somewhat less sensitive for IgA. F MAb assays were less sensitive for IgG and IgM antibodies but identified specific IgA in some specimens negative by N MAb assay. G MAb assays were insensitive for IgG and IgM antibodies but did detect about 50% of the IgA antibodies identified by the PAb assay. The basis for the low sensitivity of the G MAb assays is unclear, since many of these specimens were positive for IgG antibodies to G by Western immunoblot. The sensitivity of MAb assays varied with patient age: N MAb assays detected specific antibody responses to RS virus in all immunoglobulin classes in both adults and infants less than 1 year of age, F MAb assays detected specific IgG responses in adults and IgA responses in both adults and infants, and G MAb assays only detected IgA responses in adults. A mixture of N and F MAbs was complementary overall, identifying 54 of 55 (IgG), 51 of 52 (IgA), and 16 of 17 (IgM) serum specimens positive by PAb assay. These MAb assays were also specific with specimens tested from persons without a history of recent RS virus infection. The availability of these MAb-based assays offers other laboratories the opportunity to have long-term, standardized reagents and tests for serological diagnosis of RS virus infection.     

The immune response to influenza virus: III. Changes in the avidity and specificity of early IgM and IgG antibodies

Serum samples taken from rabbits 5 days after vaccination with SW influenza virus by the intravenous route contained high levels of IgM antibodies. IgG antibodies were either not detected or were present at very low levels. By the 10th day after vaccination both IgM and IgG antibodies were present in the serum. The early IgM antibodies were of high avidity while the early IgG antibodies were of very low avidity. The presence of low avidity IgG antibodies in whole serum caused a decrease in the average avidity of the antibodies in whole serum from the 5th to the 10th day post vaccination. The avidity of the IgM antibodies remained fairly constant for the first 20 days of the immune response but a slight increase was detected after secondary vaccination. The avidity of the early IgG antibodies increased during the test period of 20 days. The early IgM and IgG antibodies were heterogeneous with respect to avidity.

The highly avid IgM antibodies showed high cross-reactivity with related influenza viruses, i.e. they were of low specificity. The early IgG antibodies that were of low avidity cross-reacted with only one other influenza virus out of the four tested, i.e. they were more specific; as the avidity of the IgG antibodies increased so did their cross-reactivity.