End-point Dilution Research Articles

Detection of paraneoplastic anti-neuronal autoantibodies on paraffin-embedded tissues.

Anti-neuron-specific autoantibodies are widely recognised as useful, though non-specific, diagnostic markers of paraneoplastic neurological disorders. However, controversies on the best way to detect these autoantibodies have recently arisen, and the use of different procedures for their detection by different laboratories has made results difficult to compare. The aim of this study was to adapt the existing immunohistochemical techniques used for the detection of anit-neuron autoantibodies to improve their visualisation and to facilitate a wide application of these procedures. Sera and cerebrospinal fluid (CSF) were obtained from 15 patients known to carry paraneoplastic anti-neuronal autoantibodies; in addition, one serum with "atypical" anti-neuron autoantibody and 18 control sera were studied. Paraformaldehyde-fixed, paraffin-embedded rat nervous tissue and formalin-fixed, paraffin-embedded human nervous tissue treated in a microwave oven were used as substrate; the reactions were developed by immunoperoxidase methods. At the dilutions used for diagnostic purposes, all the sera and CSFs showed staining whose intensity and specificity was comparable to that obtained using frozen tissue; the end-point dilutions were, however, reduced. The atypical pattern of staining of one serum was confirmed and better emphasised using these procedures; all control sera and CSFs were negative. The morphology was improved by the use of paraffin-embedded tissues; moreover, the results obtained are permanent because of peroxidase staining, which makes it possible to use them as standards for further investigations and for comparison between different laboratories. The convenience of using paraffin-embedded material could facilitate a wide application of these procedures in clinical neurology.

Hepatitis C virus detection by single-round PCR specific for the terminal 3' noncoding region.

A single-round PCR method with primers specific for the 3' noncoding region (NCR) of hepatitis C virus (HCV) has been developed. Using a double RNAzol-B extraction, a high-temperature reverse-transcription step with SuperScript II reverse transcriptase, and a 40-cycle two-temperature PCR with a TaqStart antibody hot-start procedure, we were able to detect a 92-nucleotide fragment of the recently discovered 98-nucleotide highly conserved sequence at the 3' terminus of the HCV genome. Direct sequencing of the PCR products confirmed the specificity of the PCR and demonstrated conservation in this region. Only one nucleotide change in 14 specimens was found. End point dilution titration of sera with known viral RNA titers showed the sensitivity of the single-round 3' NCR PCR to be comparable to those of the established nested 5' NCR assays (fewer than 25 HCV genome equivalents). To evaluate specificity and sensitivity, a panel of 116 serum samples characterized by nested 5'-end PCR, genotyping, and quantitative assays was tested. A high degree of concordance (96%) between the 3' NCR and 5' NCR PCR results was found. The sequence conservation at the 3' end of the HCV genome among common genotypes and the savings in time, labor, and reagents from a single-round PCR make this assay a useful addition to the detection systems available to identify and monitor HCV infection.

Effects of mold proteases on the biological activity of allergenic pollen extracts

Abstract Objective To determine the effect of mold extracts with high protease activity on the biological activity of allergenic tree, grass, and weed extracts, using in vivo and in vitro methods, in atopic dogs. Animals 15 dogs with history and clinical signs of atopy. All dogs had strong positive reactions (3+ or 4+) to 1 or more preselected allergens and negative reactions (0) to molds. Procedure Mold extracts and saline solution were coincubated separately with tree, grass, and weed pollen extracts at 4 C for 30 and 180 days. Skin end-point titration (30-day incubation) and ELISA inhibition (30- and 180- day incubations) tests were performed on all samples. The biological activity of pollen extracts coincubated with mold extracts was compared with that of pollen extracts coincubated with saline solution. Results In the skin end-point titration test, weed pollen extracts coincubated with a mixed mold extract lost a statistically significant amount of biological activity, compared with saline coincubated controls. In the ELISA inhibition test, grass and weed pollen extracts incubated with a mixed mold extract lost a significant amount of biological activity, compared with saline coincubated controls. A significant correlation in the measurement of biological activity was found between a loss of end-point dilution in the skin end-point titration test and a decrease in relative potency, as measured by the ELISA inhibition test for allergenic grass and weed extracts. Conclusion and Clinical Relevance Mold proteases can decrease the biological activity of certain grass and weed pollen extracts when coincubated in the same vial for 30 days. Separation of mold and pollen extracts, when preparing immunotherapy vaccines, may help prevent loss of pollen extract potency and increase the vaccine’s stability and efficacy. (Am J Vet Res 1996;57:1447-1452)

Testing plasma pools for markers of viral contamination: the UK experience.

The transmission of viral infections through the use of products derived from blood has emphasised the need for adequate validation of the production process, testing of materials used in production and quality control tests on the final product. Since the late 1980s, as part of its batch release procedures, NIBSC has tested for markers of viral infectivity plasma pools used in production of blood products used in the UK. As a result of testing over 9,000 pools, NIBSC has identified 9 pools contaminated with HBsAg and 2 pools containing antibodies to HIV-1. Since routine screening of plasma pools for anti-HCV was introduced in 1993, 8 pools out of the 4,000 tested have been found to contain antibodies to HCV. In addition, the release of 12 batches of blood products was withheld and it is known that further batches of material produced from the positive pools were not submitted for batch release. Studies involving assays of dilutions of known positive plasma samples indicated that there is considerable variation in the endpoint dilutions of antigen or antibody detected by test kits from different manufactures. The selection and validation of the kits used in such testing is therefore important. The usefulness of standardised low-level external controls in assays of plasma pools for markers of viral infection is discussed.

Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA

The complete nucleotide sequence of the genome of classical swine fever virus (CSFV) strain Alfort/187 was determined from three cDNA libraries constructed by cloning of DNA fragments obtained from independent sets of reverse transcription and PCR. The cDNA fragments were then assembled and inserted downstream of a T7 promoter in a P15A-derived plasmid vector to obtain the full-length cDNA clone pA187-1. The first nucleotide of the CSFV genome was positioned at the transcription start site of the T7 promoter. Cleavage at an SrfI restriction site introduced at the exact 3' end of the cloned viral cDNA allowed the in vitro synthesis of full-length viral RNA by runoff transcription. This RNA proved to be infectious after transfection into porcine kidney cells. Infectivity was not increased after capping of the synthetic RNA. Virus recovered from transfected cells was titrated in porcine kidney cells by endpoint dilution using indirect immunofluorescence and a CSFV-specific monoclonal antibody. RNA transcripts generated from plasmid DNA isolated from bacteria which had been cultured and cloned 10 times remained infectious, indicating that the full-length clone is stable in bacterial cells. A silent point mutation introduced at position 11842 of the genome was retained in the recombinant virus recovered from transfected cells. An infectious chimeric construct was obtained by replacing a 696-bp fragment in pA187-1 with the corresponding cDNA fragment from the CSFV strain CAP. The stably cloned full-length CSFV cDNA allows site-specific mutagenesis of the viral genome and thus will be useful for detailed molecular characterization of the virus as well as for studies of viral pathogenesis.

Quantification of low levels of human immunodeficiency virus (HIV) type 1 RNA in P24 antigen-negative, asymptomatic, HIV-positive patients by PCR

A nested PCR was used to quantify small numbers of human immunodeficiency virus (HIV) type 1 (HIV-1) RNA particles in the serum specimens of 26 p24 antigen-negative, asymptomatic, HIV-positive patients undergoing antiretroviral therapy. Fifteen patients received zidovudine (ZDV) and alpha interferon, and 11 patients received ZDV monotherapy. After PCR, the amounts of RNA were quantified by comparing the endpoint dilutions of serum samples with a standard curve with known amounts of viral particles. Before the beginning of the antiviral therapy, HIV-1 RNA was detected in 92% of the patients. After treatment, a fall in the number of viral particles was detected in patients receiving combination therapy (mean titers +/- standard errors of the means, 3,617 +/- 756 pretherapy versus 1,800 +/- 845 posttherapy; P < 0.05) and in patients receiving monotherapy (3,763 +/- 642 pretherapy versus 1,353 +/- 394 posttherapy; P < 0.05). Our results indicate that PCR with nested primers may be useful for assessing the changes in viremia in HIV-positive patients with low viral load undergoing antiviral therapy.

Detection of Fiji disease virus in sugarcane by ELISA

A direct double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Fiji disease virus (FDV) in sugarcane (Saccharum spp. hybrid) cultivars NCo310 and CP44-101. Optimal results were obtained when Dynatech microplates, coated with 2 �g/mL FDV-specific immuno-gamma globulin (IgG) coupled with alkaline phosphatase-lgG conjugate at a concentration of 1/1000, were used. The test could detect FDV in crude extracts (fresh or frozen) of gall and gall-free infected tissues with an end point dilution of 1/5000 and 1/1000, respectively. The FDV antibody showed a high degree of specificity and failed to react with 3 other field-associated sugarcane viruses. When the distribution of FDV within the plant was studied, the highest titre was found in the basal axillary buds decreasing towards the top of the plant and into the roots. However, the apical meristem produced a virus titre significantly higher than any adjacent axillary buds. The ELISA was well suited for large-scale screening programs and its potential was further extended to quarantine work since it could be used to identify the virus in frozen, non-galled tissues.

Testing Plasma Pools for Markers of Viral Contamination: The UK Experience

Abstract The transmission of viral infections through the use of products derived from blood has emphasised the need for adequate validation of the production process, testing of materials used in production and quality control tests on the final product. Since the late 1980s, as part of its batch release procedures, NIBSC has tested for markers of viral infecti vity plasma pools used in production of blood products used in the UK. As a result of testing over 9,000 pools, NIBSC has identified 9 pools contaminated with HBsAg and 2 pools containing antibodies to HIV-1. Since routine screening of plasma pools for anti-HCV was introduced in 1993, 8 pools out of the 4,000 tested have been found to contain antibodies to HCV In addition, the release of 12 batches of blood products was withheld and it is known that further batches of material produced from the positive pools were not submitted for batch release. Studies involving assays of dilutions of known positive plasma samples indicated that there is considerable variation in the endpoint dilutions of antigen or antibody detected by test kits from different manufacturers. The selection and validation of the kits used in such testing is therefore important. The usefulness of standardised low-level external controls in assays of plasma pools for markers of viral infection is discussed.

Correlation of CD8 lymphocyte activation with cellular viremia and plasma HIV RNA levels in asymptomatic patients infected by human immunodeficiency virus type 1.

The relationship between CD8 lymphocyte phenotypic alterations and virological parameters was studied in 47 asymptomatic subjects with human immunodeficiency virus type 1 (HIV-1) infection and CD4 T cell counts above 400/microliters. CD8 subsets were examined by means of three-color flow cytometry, using an extensive panel of monoclonal antibody combinations. Virological parameters were measured by both end-point dilution culture of peripheral blood mononuclear cells (PBMCs) and plasma and branched-DNA (bDNA) signal amplification of plasma HIV RNA. Whereas HIV-infected patients had a near-normal CD4 cell count (mean, 782 cells/microliter), several subsets of activated CD8 cells were markedly expanded relative to values in 23 HIV-seronegative controls. The PBMC cultures were positive in 38 cases and plasma HIV RNA was detected in 31. The percentage of CD4 cells correlated negatively with both cellular viremia and plasma HIV RNA levels. Conversely, a positive correlation was observed between viral load and the percentage of CD8 cells. Among CD8 lymphocytes, the CD38+CD8 and HLA-DR+CD8 subsets correlated best with viral load. Three-color analysis showed that the subpopulations involved in this relationship were CD38+HLA-DR+, CD38+CD28-, HLA-DR+CD28+, HLA-DR+CD57-, CD38+CD57-, CD38+CD45RO+, and HLA-DR+CD45RO+. Our data provide the first evidence that viral load correlates with subsets of activated CD8 lymphocytes in asymptomatic HIV-infected subjects who have near-normal numbers of CD4 lymphocytes.

Titration of human-bovine rotavirus reassortants using a tetrazolium-based colorimetric end-point dilution assay.

A colorimetric end-point dilution assay was developed for the titration of rotavirus-containing samples that uses commercially available tetrazolium dyes as an indicator of virus infection. This assay offers several advantages over both plaque assays and traditional end-point dilution methods. The latter assays require manual counting of plaques or the scoring of wells for the presence of virus based on observed cytopathic effects. The colorimetric end-point dilution assay enables the scoring of wells based upon absorbance readings alone, thereby eliminating time-consuming and subjective manual screenings. This method also has the potential for automating the analysis of large numbers of samples. Virus titers of human-bovine rotavirus reassortants obtained using this method are comparable to those determined by plaque assay. The scoring of wells based on absorbance readings was also found to agree with manual scoring of cytopathic effects and with the production of viral antigen.

Biological Characterization and Molecular Cloning of Murine C-type Retroviruses Derived from the TSZ Complex from Mainland China

Characterization of the SRS murine retrovirus complex, derived from the TSZ system of murine leukemia developed in China, was carried out. The initial stock contained XC+, NB-tropic virus (and possibly other viruses), and induced several neoplastic diseases in neonatally inoculated NIH Swiss mice: erythroid leukemia, myeloid leukemia (acute myeloblastic leukemia), and lymphoblastic lymphoma (both B- and T-lymphoid). In addition, approximately 30% of inoculated animals developed central nervous system disease—hindlimb paralysis or semilateral paralysis. Rescue of virus from the spleen of an animal with combined erythroid/myeloid leukemia, followed by endpoint dilution gave two stocks: 19-6 (XC+, NB-tropic) and 19-7 (XC-, NB-tropic). Both stocks induced erythroid and myeloid leukemia, and 19-6 induced CNS symptoms as well. Southern blot analysis indicated that the predominant viruses from the 19-6 and the 19-7 cultures were related, but different in the env region. An infectious molecular clone of provirus from 19-6 cells was obtained. The resulting cloned virus [SRS 19-6 murine leukemia virus (MuLV)] induced four kinds of leukemia: erythroid, myeloid, B-lymphoma, and T-lymphoma; in many cases, more than one tumor type was identified in the same animal. Such a broad spectrum of leukemias induced by a cloned MuLV is unusual. Flaccid hindlimb paralysis induced by SRS 19-6 MuLV could be attributed to meningeal B-lymphoma. Immunofluorescent staining with a panel of env-specific monoclonal antibodies confirmed that the 19-6 and 19-7 viral stocks contained different viruses, which differed from previously characterized MuLVs. The viruses of the SRS complex may provide interesting reagents for investigations of MuLV-induced disease.

Investigation of proviral load in individuals infected with human T-lymphotropic virus type II.

Human T-lymphotrophic virus type II (HTLV-II) has not yet been associated with any disease. Little is known about the proviral loads of HTLV-II in vivo and its relationship, if any, to lack of pathogenicity. We determined the HTLV-II proviral copy number in peripheral blood lymphocyte (PBL) samples from 49 HTLV-II-infected individuals, of whom 25 were coinfected with human immunodeficiency virus type 1 (HIV-1). The HTLV-II copy numbers were determined by polymerase chain reaction (PCR) amplification of end-point dilutions of PBL lysates, followed by hybridization to a 32P-labeled HTLV-II-specific probe. The proviral copy number for the 49 samples ranged from < 0.02 to 200 per 1000 PBLs; 6% had < 0.02, 16% had 0.02, 20% had 0.2, 18% had 2, 31% had 20, and 8% had 200 copies per 1000 PBLs. The distributions of HTLV-II copy numbers in the coinfected and singly infected subgroups were not significantly different (Wilcoxon rank sum, p = 0.24). In the coinfected subgroup, there was no significant correlation between the HTLV-II proviral load and the counts of CD4-positive lymphocytes or CD8-positive lymphocytes (Spearman Coefficient = 0.26, p = 0.20; = 0.091, p = 0.67, respectively). Our data demonstrate the presence of a wide range of viral loads in HTLV-II-infected individuals. The high viral loads (> or = 20 copies/1000 lymphocytes) detected in 39% of our samples suggest that the low pathogenicity of HTLV-II is not related to the presence of low viral loads in the infected subjects. Our data from the HIV-1 coinfected individuals show no apparent effect of HIV-1 on HTLV-II proviral loads.

Epstein-Barr virus DNA in peripheral blood leukocytes of patients with posttransplant lymphoproliferative disease.

We tested the hypotheses that Epstein-Barr virus (EBV) DNA levels in peripheral blood leukocytes (PBL) of transplant recipients with posttransplant lymphoproliferative disease (PTLD) (1) exceed those of patients without PTLD, (2) rise with or before clinical detection of the disease, and (3) fall with effective therapy. Using the polymerase chain reaction (PCR) and an endpoint dilution technique, we compared EBV DNA levels in sequential specimens from 5 patients with PTLD, 16 solid organ transplant recipients without PTLD, and 5 young adults with primary infectious mononucleosis (IM), and in single specimens from 21 healthy seropositive subjects. EBV DNA levels in the first two groups rose with induction of immunosuppression despite prophylactic acyclovir. Markedly elevated levels of EBV DNA were seen in 4 of 5 patients with PTLD at or before clinical diagnosis. The peak levels in these patients exceeded those of transplant recipients without PTLD (P = 0.02) and healthy adults with IM (P = 0.02). EBV DNA levels fell dramatically with effective therapy. Four of 21 healthy seropositive subjects demonstrated low levels of EBV DNA, similar to levels seen late in the course of patients with IM. We conclude that a semiquantitative PCR assay for EBV DNA in PBL can assist in the detection of PTLD and in monitoring the effect of therapy.

UP-REGULATION OF A TUMOR-ASSOCIATED ANTIGEN (TAG-72) BY INTERFERON-ALPHA AND INTERFERON-GAMMA IN PATIENTS WITH CUTANEOUS BREAST-CANCER RECURRENCES

Up-regulation of a tumor-associated antigen (TAA) TAG-72 was investigated immunohistochemically in skin recurrences of 10 breast cancer patients treated with intralesional injections of natural interferons (nIFNs) alpha and gamma. Up-regulation was assessed by comparing the immunoreactivity of pre-IFN and post-IFN specimens stained with monoclonal antibodies (MoAbs) B72.3 and CC49 at near end-point dilutions. Varying degrees of enhanced immunoreactivity were detected in IFN-injected and non-IFN-injected lesions except in 1 patient with high constitutive expression of TAG-72 and another patient not expressing TAG-72. Thus, IFN-mediated up-regulation of TAG-72 can occur in patients whose breast cancer cells express this TAA, independently of the initial levels of antigenic expression.

Correlation between viral DNA load and serum anti p19 antibody concentration in symptomless human T-lymphotropic virus type-I (HTLV-I)-infected individuals.

In order to determine whether serum anti-human T-cell lymphotropic virus type I (HTLV-I) antibody concentration is correlated with cellular viral DNA load, these 2 biological parameters were established in 22 symptomless HTLV-I carriers. The proviral copy (PVC) number was determined through quantificative polymerase chain reaction. Specific antibody titers were determined by Western blot with the end-point dilution method; the quantification of each antibody was performed through ScanBlot by determination of the peak height of each Western-blot band. A positive correlation was observed between the PVC number and the titer of total antibodies. When the association between the peak height of each antibody and the PVC number was studied, a significant positive correlation was observed only with anti-p 19. Further evaluation through follow-up studies of symptomless HTLV-I individuals is needed to clarify the value of anti-HTLV-I antibody titer as a predictor of disease progression.

Identification in the Calcineurin A Subunit of the Domain That Binds the Regulatory B Subunit

Calcineurin (CaN) is the serine/threonine protein phosphatase (phosphatase 2B) that is activated by binding of Ca2+ to its B subunit and to calmodulin (CaM). This paper identifies residues between the catalytic region and the CaM-binding domain of the A subunit as the domain that binds the regulatory B subunit. A purified fusion protein containing residues 328-390 of the A subunit 1) binds CaN B subunit, and 2) inhibits (IC50 = 0.1 microM) the in vitro stimulation of CaN A phosphatase activity by purified CaN B subunit. A synthetic peptide corresponding to residues 341-360 blocked the binding of CaN B to residues 328-390 in the fusion protein, so 4 hydrophobic residues within this region (Val349-Phe350 and Phe356-Val357) were mutated to either Glu (E mutant) or Gln (Q mutant). The wild-type and mutant A subunits were expressed individually or coexpressed with B subunit in Sf9 cells, purified and characterized. The mutant A subunits were similar to wild-type A subunit in terms of basal phosphatase activity (1-3 nmol/min/mg) and activation by Mn2+/CaM. Addition of purified B subunit to purified wild-type A subunit at a 1:1 molar ratio gave a 40-fold increase in phosphatase activity whereas addition of B subunit to either of the mutant A subunits had no effect on phosphatase activity, even at a 3:1 molar excess of B subunit. Furthermore, when wild-type or mutant A subunits were coexpressed with B subunit and purified on CaM-Sepharose, the B subunit co-eluted with the wild-type A subunit but not with either mutant A subunit. These results demonstrate that residues 328-390 in the A subunit bind B subunit and that the mutated hydrophobic residues are essential.

Calcium regulation of calcineurin phosphatase activity by its B subunit and calmodulin. Role of the autoinhibitory domain.

Calcineurin (CaN) contains an autoinhibitory element (residues 457-482) 43 residues COOH-terminal of the calmodulin-binding domain (Hashimoto, Y., Perrino, B. A., and Soderling, T. R. (1990) J. Biol. Chem. 265, 1924-1927) that regulates the Ca(2+)-dependent activation of its phosphatase activity. Substitution of Arg476 and Arg477 or Asp467 to Ala in the autoinhibitory peptide 457-482 significantly decreased its inhibitory potency. CaN A subunits with these residues mutated to Ala were coexpressed with the Ca(2+)-binding B subunit using the baculovirus/Sf9 cell system. Kinetic analysis showed that although the purified mutants had no activity in the absence of calcium, they were less dependent than the wild-type enzyme on calcium and calmodulin for activity. To determine if additional autoinhibitory motifs were present in the COOH terminus of calcineurin, the A subunit was truncated at residues 457 or 420 and co-expressed with B subunit. The Vmax values of both truncation mutants with or without Ca2+ were increased relative to wild-type calcineurin. The increased Ca(2+)-independent activity of CaN420 relative to CaN457 indicates the presence of additional autoinhibitory element(s) within residues 420-457. CaN420 had similar high Vmax values with or without Ca2+, but the Km value for peptide substrate was increased 5-fold to 125 microM in the absence of Ca2+. The Km values of all the expressed calcineurin species were increased in the absence of Ca2+. The CaN A or CaN A420 subunits alone have low Vmax and high Km (115 microM) values even in the presence of Ca2+. These results indicate that 1) there are several autoinhibitory motifs between the CaM-binding domain and the COOH terminus that are relieved by Ca2+ binding to CaM and the B subunit, 2) Ca2+ binding to the B subunit also regulates enzyme activity by lowering the Km of the catalytic subunit for substrate, 3) binding of the B subunit is required for high Vmax values even after removal of the autoinhibitory domain. These results are consistent with synergistic activation of calcineurin by Ca2+ acting through both CaM and the B subunit.

Conventional virus detection in cell substrates for the production of biopharmaceuticals

Cocultivation of SIPC-2 myeloma cells with clone A31, BALB/3T3 cells results in the continuous production of a C-type virus by the A31 cells that has been designated S/A-1. S/A-1 virus possessed the essential properties of a murine retrovirus, including a buoyant density of 1.16 g/cm3, C-type morphology, high molecular weight (70 S) RNA, and an RNA-dependent DNA polymerase. After cloning by three endpoint dilution passages in A31, and SIRC cells, the virus was shown to possess ecotropic as well as xenotropic activity. For example, the S/A-1 virus producibility infected mouse cells, including BALB/3T3, NIH/3T3, SC-1, 3T3 FL, and F1 cells as well as non-mouse cells, including normal rat kidney, rabbit corneal (SIRC), Chinese hamster peritoneal, mink lung, Pekin duck embryo, human rhabdomyosarcoma, and guinea pig embryo cells. The S/A-1 virus infected A31, cells do not induce XC-syncytia and the virus does not induce foci in mink lung cells. Thus, the data suggest that it is an amphotropic virus whose ecotropism is different (i.e., NB-tropic) from the previously reported amphotropic viruses (i.e., N-tropic). In addition, S/A-1 virus is different from the mink cell focus-inducing (MCF) viruses (J. W. Hartley, N. K. Wolford, L. J. Old, and W. P. Rowe 1977, Proc. Nat. Acad. Sci. USA74, 789–792) in terms of its lack of focus-inducing activity on mink lung cells, B-tropism, and ability to replicate in human rhabdomyosarcoma and F1 mouse cells. The structural proteins of cloned S/A-1 virus were compared to those of 1504-A virus, a previously described amphotropic virus isolated from feral mice (J. W. Hartley and W. P. Rowe 1976, J. Virol.19 19–26; S. Rasheed, M. B. Gardner, and E. Chan 1976, J. Virol.19, 13–18), by electrophoresis on a linear 7–20% acrylamide slab gel and staining with Coomassie blue. S/A-1 differed from 1504-A in terms of (a) the mobility of a protein with a molecular weight of approximately 12,000 daltons (pl5) and (b) the fact that it has a “double” p30.

Molecular cloning and characterization of an immunosuppressive and weakly oncogenic variant of Friend murine leukemia virus, FIS-2.

The FIS variant is a weakly leukemogenic, relatively strong immunosuppressive murine retrovirus which was isolated from the T helper cells of adult NMRI mice infected with Friend murine leukemia virus (F-MuLV) complex (FV). Unlike FV, it does not induce acute erythroleukemia but retains the immunosuppressive property of FV and induces suppression of the primary antibody response rapidly and persistently in adult mice. A previous study showed that the FIS variant contains two viral components, a replication-competent virus and a defective virus. In this study, we have biologically purified the FIS variant by end point dilution and we show that the replication-competent virus FIS-2 alone can induce immunosuppression as the parental FIS variant. Most newborn mice infected with FIS-2 developed erythroleukemia, but with an increased latency period compared with that of F-MuLV clone 57. In contrast, FIS-2 induced suppression of the primary antibody response and disease more rapidly than F-MuLV clone 57 in immunocompetent, adult mice. FIS-2 was further molecularly cloned and characterized. Restriction mapping and nucleotide sequence analysis of FIS-2 showed a high degree of homology between FIS-2 and F-MuLV clone 57, suggesting that FIS-2 is a variant of F-MuLV. The striking difference is the deletion of one of the tandem repeats in the FIS-2 long terminal repeat and the single point mutation in the binding sites for core-binding protein and FVa compared with the long terminal repeat of F-MuLV clone 57. Two single point mutations led to the appearance of two extra potential N glycosylation sites in the FIS-2 gag-encoded glycoprotein. Together, the results suggest that FIS-2 represents an interesting murine model to study retrovirus-induced immunosuppression on the basis of its unique combined property of low leukemogenicity and relatively strong and persistent immunosuppressive activity in adult mice.

Sequence analysis of proviral HIV RT amplified directly by a semi-quantitative technique from AZT treated patients.

To minimise possible arbitrary selective effects of culturing HIV, proviral RT DNA was isolated directly from PBMCs of four patients treated for 6-14 months with AZT. RT DNA was amplified by PCR and sequenced directly without further in vitro manipulation. Eighteen changes additional to those 4 or 5 changes previously shown by genetic reconstruction experiments [Kellam et al.: Proceedings of the National Academy of Sciences of the United States of America 89:1934-1938, 1992] were found in the 14 different sequences analysed. Substitutions clustered in two defined areas of the RT, from amino acids 60 to 70 and from 180 to 220. Mutations were observed at each of the two areas independently or at both sites simultaneously. Amino acid changes in RT from patients harbouring resistant strains of HIV-1 were found in positions 60 (Val), 62 (Ala), 93 (Gly), 100 (Phe), 161 (Pro), 173 (Asn), 177 (Glu), 180 (Ile), 181 (Tyr), 182 (Leu), 186 (Asp), 194 (Gln), 196 (Glu), 200 (Ile), 209 (Val), 210 (Trp), 211 (Lys), and 214 (Phe) in addition to those described previously. It was anticipated that multiple proviral DNAs would be present in a single clinical sample. Therefore end point dilution PCR methodology was used, which allowed sequence analysis of separate proviral DNA molecules from the patients' proviral DNA. Even in patients who had received AZT for more than 10 months wild-type "AZT-sensitive" RTs co-existed with mutated "AZT-resistant" RTs in the same patient sample.