New procedure for isolation of Rous sarcoma virus-specific RNA from infected cells.

The use of mercurated "strong stop" complementary DNA (complementary to the 5'-terminal 101 nucleotides of Rous sarcoma virus RNA) in the isolation of virus-specific RNA from infected chicken embryo fibroblasts is described. Strong stop Rous sarcoma virus complementary DNA was mercurated chemically, and, as a result of the low complexity of this DNA, short hybridization times (up to 15 min) and heating in the absence of formamide were found to be adequate conditions for the isolation of virus-specific RNA. The purity of the isolated RNA was demonstrated by analysis of labeled RNase T1-resistant oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. The isolated RNA could be translated in the in vitro protein synthesis system derived from rabbit reticulocytes, and an analysis of polypeptides programmed by isolated RNA before and after immunoprecipitation further demonstrated both the purity of the isolated mRNA and the quantitative nature of the isolation procedure.     

Defective in vitro production of influenza virus-specific cytotoxic T lymphocytes in ataxia-telangiectasia

Peripheral blood mononuclear cells (PBMC) from patients with ataxia-telangiectasia (A-T) were studied for their capacity to proliferate and to generate influenza virus-specific cytotoxic T lymphocytes (CTL) after in vitro stimulation with influenza A/Hong Kong (A/HK (H3N2)) virus. PBMC from 11 patients proliferated poorly to A/HK and 10 of the 11 patients failed to exhibit significant CTL effector activity when tested on influenza A/HK virus-infected autologous target cells. In contrast, PBMC from each of 18 simultaneously studied, unrelated normal individuals proliferated to A/HK and generated influenza-immune CTL. In each of the 10 A-T patients, deficient CTL activity was shown to be due to a lack of generation of CTL and not to target cell resistance to lysis, because the virtually infected target cells of the patients were lysed by parental influenza-immune CTL. Determinations of T cell numbers and existing serum antibody titers to H3N2 influenza virus suggest this nonresponsiveness cannot be simply explained by a lack of T cells or the absence of exposure to type A (H3N2) influenza virus. Studies in which CTL were generated in A-T plasmas and during co-culture of PBMC from an A-T patient and an MHC-matched sibling failed to demonstrate either plasma or cellular suppression as a mechanism for the lack of CTL production in A-T patients. This immune defect in the production of cytotoxic effector T cells may be a cause of the increased frequency of infections and neoplasms observed in A-T patients.     

Preferential HLA usage in the influenza virus-specific CTL response

To study whether individual HLA class I alleles are used preferentially or equally in human virus-specific CTL responses, the contribution of individual HLA-A and -B alleles to the human influenza virus-specific CTL response was investigated. To this end, PBMC were obtained from three groups of HLA-A and -B identical blood donors and stimulated with influenza virus. In the virus-specific CD8(+) T cell population, the proportion of IFN-gamma- and TNF-alpha-producing cells, restricted by individual HLA-A and -B alleles, was determined using virus-infected C1R cells expressing a single HLA-A or -B allele for restimulation of these cells. In HLA-B*2705- and HLA-B*3501-positive individuals, these alleles were preferentially used in the influenza A virus-specific CTL response, while the contribution of HLA-B*0801 and HLA-A*0101 was minor in these donors. The magnitude of the HLA-B*0801-restricted response was even lower in the presence of HLA-B*2705. C1R cells expressing HLA-B*2705, HLA-A*0101, or HLA-A*0201 were preferentially lysed by virus-specific CD8(+) T cells. In contrast, the CTL response to influenza B virus was mainly directed toward HLA-B*0801-restricted epitopes. Thus, the preferential use of HLA alleles depended on the virus studied.     

Isolation of virus-specific double-stranded RNA from cells transformed with Rous sarcoma virus

Several methods were used for isolation of double-stranded (ds) RNA from the cytoplasm of Rous sarcoma virus-transformed chick embryo cells. The dsRNA was shown to have a high melting temperature (82.5 degrees C) in 0.16 M phosphate buffer (pH 6.8), which shifted to more than 90 degrees C after RNase treatment. The size of a single strand was approximately 1300-1600 nucleotides and RNase-resistant fragments were 50-250 nucleotides long. Double-stranded RNA formed hybrids with the labeled genomic RSV RNA RNA so that the major subpopulation of the dsRNA hybridized to 6-10% of RSV RNA and the minor subpopulation -- to 90-94% of RSV RNA. It was suggested that this large subpopulation of dsRNA was abundant in sequences homologous to proviral end fragments as judged by Southern procedure. The data are discussed by considering the analogy between retroviral proviruses and mobile genetic elements.     

Influenza virus-specific antibody-secreting cells in the murine lung during primary influenza virus infection.

An enzyme-linked immunosorbent plaque assay is described which can reliably enumerate influenza virus-specific antibody-secreting cells and exhibits specificity similar to that of the indirect enzyme-linked immunosorbent assay. The assay was used to characterize the development of specific antibody-secreting cells, principally within lung tissue, during primary murine influenza virus infection after intranasal inoculation. Cells secreting influenza virus-specific immunoglobulin M (IgM), IgG, and IgA were detected in greatest numbers in lung tissue, and the data presented indicated that the cells may have originated from specific B-cell precursors in lung tissue which are demonstratable in vitro. At 11 months after infection, cells secreting IgG and IgA were still present in lung tissue. Influenza virus-specific antibody-secreting cells were also detected in spleen tissue and blood. Antibody-secreting cells appeared earlier in spleen than in lung tissue and declined more rapidly in spleen tissue.     

Priming of influenza virus-specific cytotoxic T lymphocytes vivo by short synthetic peptides.

Influenza virus-specific CTL were primed in vivo by immunization with short synthetic peptides representing major CTL epitopes from the nucleoprotein of type A influenza virus. The resultant CTL after in vitro boosting of primed spleen cells recognized both virus-infected and peptide-pulsed target cells. The requirement of CD4+ T cell activation was investigated in several ways. First the addition of helper epitopes to the CTL epitope did not enhance CTL generation, suggesting that helper activity was either not limiting or not required. However, in vivo depletion of CD4+ T cells completely inhibited the generation of CTL by peptide immunization. The inclusion of anti-CD4 in the in vitro restimulation with peptide also prevented the generation of CTL, whereas in vitro reactivation of virus immune spleen cells with peptide was not inhibited by anti-CD4. Thus there appears to be heterogeneity in the requirement of CD4+ T cell proliferation in CTL generation. One possibility is that virus infected cells can stimulate higher affinity T cells that are less helper T cell dependent.     

A double-stranded RNA-inducible fish gene homologous to the murine influenza virus resistance gene Mx.

We cloned and sequenced a 2.35-kilobase EcoRI fragment of genomic DNA from a local freshwater fish (Perca fluviatilis) that strongly hybridized to probes derived from the murine influenza virus resistance gene Mx. The cloned fish DNA contained blocks of sequences related to Mx gene exons 3 to 8, which appeared to represent exons of a bona fide fish gene because they were separated by intron sequences flanked by consensus splice acceptor and donor sites. Injection of double-stranded RNA into the peritoneal cavity of trouts resulted in 5- to 10-fold elevated levels of two liver mRNAs of about 2.0 to 2.5 kilobases in length that hybridized to the cloned genomic DNA. High sequence similarity between this fish gene and the murine Mx gene, identical exon lengths, and similar inducibilities in vivo by double-stranded RNA indicate that we isolated a fragment of a fish Mx gene.     

Persistence of influenza virus-specific antibody-secreting cells and B-cell memory after primary murine influenza virus infection

Influenza virus-specific antibody-secreting cells (ASCs), enumerated using an ELISA-plaque assay, were found in the lung and spleen up to 18 months after primary murine influenza infection. The number of ASCs generated in stimulated lung and spleen cell cultures increased 50- to 200-fold after influenza infection. Whereas the level of response did not change in spleen cell cultures up to 18 months after infection, there was a gradual reduction in ASCs in lung cell cultures obtained more than 6 months after infection, predominantly due to a reduction in B memory cells. Homotypic re-infection increased ASCs in the lung only, whereas B-cell memory increased in both the lung and spleen. Although ASCs increased in both the lung and spleen after heterotypic challenge, ASCs and B-cell memory specific for the original subtype were not increased.     

Consequences of immunodominant epitope deletion for minor influenza virus-specific CD8+-T-cell responses

The extent to which CD8+ T cells specific for other antigens expand to compensate for the mutational loss of the prominent DbNP366 and DbPA224 epitopes has been investigated using H1N1 and H3N2 influenza A viruses modified by reverse genetics. Significantly increased numbers of CD8+ KbPB1(703)+, CD8+ KbNS2(114)+, and CD8+ DbPB1-F2(62)+ T cells were found in the spleen and in the inflammatory population recovered by bronchoalveolar lavage from mice that were first given the -NP-PA H1N1 virus intraperitoneally and then challenged intranasally with the homologous H3N2 virus. The effect was less consistent when this prime-boost protocol was reversed. Also, though the quality of the response measured by cytokine staining showed some evidence of modification when these minor CD8+-T-cell populations were forced to play a more prominent part, the effects were relatively small and no consistent pattern emerged. The magnitude of the enhanced clonal expansion following secondary challenge suggested that the prime-boost with the -NP-PA viruses gave a response overall that was little different in magnitude from that following comparable exposure to the unmanipulated viruses. This was indeed shown to be the case when the total response was measured by ELISPOT analysis with virus-infected cells as stimulators. More surprisingly, the same effect was seen following primary challenge, though individual analysis of the CD8+ KbPB1(703)+, CD8+ KbNS2(114)+, and CD8+ DbPB1-F2(62)+ sets gave no indication of compensatory expansion. A possible explanation is that novel, as yet undetected epitopes emerge following primary exposure to the -NP-PA deletion viruses. These findings have implications for both natural infections and vaccines.     

Comparison between three rapid methods for direct diagnosis of influenza and the conventional isolation procedure.

Besides the rapid diagnostic tests based on influenza A and B antigens nucleoproteins detection, which are routinely used, the isolation of influenza strains is still required to obtain recent variant isolates for full antigenic characterization, in order to up-date the influenza vaccine composition. To increase the rapidity and the efficacy of the virus growth, we implemented a culture test in 24-well plates by centrifugation of samples on to LLCMK2 cells in the presence of trypsin. This test was routinely applied to 331 nasopharyngeal swabs collected during the influenza A outbreak in the winters 1988-1989 and to 962 in 1989-1990. The centrifugation culture assay has been compared with the direct detection of NP antigens in the clinical samples by immunofluorescence and capture ELISA tests and with the conventional virus isolation by inoculation of the samples to embryonated eggs and to LLCMK2 cell cultures. Compared with the NP antigen detection tests, the centrifugation culture assay closely correlated (r = 0.95) and the sensitivity and specificity were also excellent, 93.4% and 99.6%, respectively. Compared with the conventional culture assays, the centrifugation culture markedly increased the performance (five times) and rapidity (2 days) of influenza virus isolation and identification.