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Transmissible spongiform encephalopathies (TSEs or prion diseases) are characterized by a constellation of typical though variable pathological changes in the brain. Deposition of disease-associated abnormal prion protein (PrPSc) is the pathological feature of TSEs most consistent and accessible for quantification. However, the evaluation of PrPSc deposits detected by immunohistochemical techniques has been traditionally based on arbitrarily assigned semiquantitative scores. This approach is limited by its subjectivity and bias, yielding considerable variability. In this study, we used MetaMorph 6.1 image analysis software for quantitative analysis of immunostained PrPSc deposits in the CNS of hamsters infected with the 263K strain of scrapie agent. Computerized morphometric analysis (CMA) allowed unambiguous detection of even minimal amounts of immunostained PrPSc. CMA values for intensity of staining and area stained correlated well with semiquantitative scores, providing reproducible quantitative data and objective criteria for analyzing PrPSc deposition. CMA provides a simple and reliable method for improved and consistent diagnosis of TSEs that may also be used to quantify other immunostained biomarkers.

To determine whether subtypes of HIV-1 and HIV-2 vary in their ability to induce T cell apoptosis in vitro, human peripheral blood mononuclear cells (PBMC) from healthy donors and CEM.NKR-CCR5 cells were infected with a variety of HIV-1 and HIV-2 isolates in vitro. Apoptotic cell levels and chemokine and cytokine production were analyzed. Significant variations in cytopathic effects following in vitro infection with primary isolates of HIV-1 or HIV-2 subtypes were observed in PBMCs. The percent of apoptotic cells from each individual ranged from 2 to 78% after HIV-1 infection and from 0 to 28% after HIV-2 infection (p0.01). We did not observe significant differences in the degree of apoptosis induced among cells infected with different HIV-1 group M subtypes or group O virus, nor among cells infected with different HIV-2 isolates. However, HIV-2 induced significantly lower degree of apoptosis overall in PBMC and CEM.NKR-CC5 cells when compared with HIV-1 subtypes (p0.0001). No significant differences were observed in the production of chemokines, such as RANTES, MIP-1alpha, and MIP-1beta, and cytokines, such as TNF-alpha and TNF-beta when PBMC cultures were infected with different HIV-1 subtype viruses, or HIV-2 isolates. In conclusion, HIV-2 isolates induced significantly lower levels of T cell apoptosis in both PBMC and CEM.NKR-CCR5 cells than HIV-1 isolates. No differences in T cell apoptosis levels were seen between different subtypes of HIV-1 group M or group O isolates. This is consistent with the mild clinical course of infection with HIV-2 that has been reported relative to that observed with HIV-1.

In anticipation of large-scale smallpox vaccination, clinical trials of new vaccine candidates with improved safety profiles, and new vaccinia immune globulin (VIG) products, there is an immediate need to develop new assays to measure vaccinia-specific immune responses. The classical assay to measure vaccinia neutralization, the plaque-reduction neutralization test (PRNT), is slow, labor intensive, and difficult to validate and transfer. Here we describe the development of a novel vaccinia-neutralization assay based on the expression of a reporter gene, beta-galactosidase (beta-Gal). Using a previously constructed vaccinia-beta-Gal recombinant virus, vSC56, we developed a neutralization assay that is rapid, sensitive, and reproducible. The readout is automated. We show that the neutralizing titers, ID(50), for several VIG products measured by our assay were similar to those obtained by PRNTs. A new Food and Drug Administration VIG standard was established for distribution to other laboratories. The new assay will serve as an important tool both for preclinical and clinical trials of new smallpox vaccines and for evaluation of therapeutic agents to treat vaccine-associated adverse reactions.

The effect of interferon γ (IFN-γ) and interleukin 6 (IL-6) on infection of macrophages with human immunodeficiency virus type 1 (HIV-1) was investigated. By using a polymerase chain reaction–based viral entry assay and viral infectivity assay, it was demonstrated that IL-6 and IFN-γ augmented susceptibility of monocyte-derived macrophages (MDMs) to infection with T-cell tropic CXCR4-utilizing (X4) HIV-1 strains. Consistent with this finding, IFN-γ and IL-6 augmented fusion of MDMs with T-tropic envelope-expressing cells. The enhanced fusion of cytokine-treated MDMs with T-tropic envelopes was inhibited by the CXCR4 ligand, SDF-1, and by T22 peptide. IFN-γ and IL-6 did not affect expression of surface CXCR4 or SDF-1–induced Ca++ flux in MDMs. In contrast to the effect of IFN-γ on the infection of MDMs with X4 strains, IFN-γ inhibited viral entry and productive infection of MDMs with macrophage-tropic (M-tropic) HIV-1. Consistent with this finding, IFN-γ induced a decrease in fusion with M-tropic envelopes that correlated with a modest reduction in surface CCR5 and CD4 on MDMs. It was further demonstrated that macrophage inflammatory protein (MIP)-1α and MIP-β secreted by cytokine-treated MDMs augmented their fusion with T-tropic–expressing cells and inhibited their fusion with M-tropic envelope-expressing cells. These data indicate that proinflammatory cytokines, which are produced during opportunistic infections or sexually transmitted diseases, may predispose macrophages to infection with X4 strains that, in turn, could accelerate disease progression.

Mice transgenic with the human poliovirus receptor gene develop clinical signs and neuropathology similar to those of human poliomyelitis when neurovirulent polioviruses are inoculated into the central nervous system (CNS). Factors contributing to disease severity and the frequencies of paralysis and mortality include the poliovirus strain, dose, and gender of the mouse inoculated. The more neurovirulent the virus, as defined by monkey challenge results, the higher the rate of paralysis, mortality, and severity of disease. Also, the time to disease onset is shorter for more neurovirulent viruses. Male mice are more susceptible to polioviruses than females. TGM-PRG-3 mice have a 10-fold higher transgene copy number and produce 3-fold more receptor RNA and protein levels in the CNS than TGM-PRG-1 mice. CNS inoculations with type III polioviruses differing in relative neurovirulence show that these mouse lines are similar in disease frequency and severity, demonstrating that differences in receptor gene dosage and concomitant receptor abundance do not affect susceptibility to infection. However, there is a difference in the rate of accumulation of clinical signs. The time to onset of disease is shorter for TGM-PRG-3 than TGM-PRG-1 mice. Thus, receptor dosage affects the rate of appearance of poliomyelitis in these mice.

Two mouse lines transgenic with the human poliovirus receptor gene (PVR), TGM-PRG-1 and TGM-PRG-3, were characterized to determine whether transgene copy number and PVR expression levels influence susceptibility to poliovirus. The mouse lines have been bred for more than 10 generations and the transgene was stably transmitted to progeny as determined by Southern blot hybridization and restriction fragment length polymorphism. The transgene copy number is 10 in the TGM-PRG-3 mouse line and one in the TGM-PRG-1 mouse line. Abundance of PVR RNA is on average three-fold higher in TGM-PRG-3 relative to TGM-PRG-1 tissues, and the abundance of the receptor molecule is three-fold higher in TGM-PRG-3 central nervous system tissues compared to TGM-PRG-1 tissues as determined by Western blot analysis. When TGM-PRG-1 and TGM-PRG-3 mice were inoculated intracranially with a neurovirulent type III poliovirus strain, they developed clinical symptoms and CNS lesions characteristic of human poliomyelitis. These results indicate that the PVR gene is expressed as a functional receptor in the CNS of both mouse lines rendering the mice susceptible to poliovirus infection. Even though the two mouse lines have different copy numbers of the transgene and different levels of PVR RNA and protein, they are similar in their susceptibility to poliovirus.

Transgenic mice susceptible to poliovirus (TgPVR mice) have been used to study poliovirus neurovirulence and attenuation. It was shown recently that mouse line TgPVR21 may be a suitable model to evaluate neurovirulence safety of oral poliovirus vaccine. It was important to determine whether TgPVR21 mice are sensitive enough to discriminate between type-3 reference and ‘marginal’ vaccines, i.e. those that failed the monkey test while containing only slightly increased amounts of neurovirulent revertants at position 472 of the viral genome as measured by a molecular assay MAPREC. Data presented here demonstrate that TgPVR21 mice are not less sensitive than monkeys in the detection of marginal vaccines. In contrast to the monkey neurovirulence test, which is based on histological examination of the CNS, the TgPVR21 mouse neurovirulence test revealed marginal vaccines by simple analysis of clinical signs without requiring a laborious histological examination.

Abstract. Two poliovirus-susceptible transgenic mouse (Tg PVR) strains, Tg1 and Tg21, were compared with the monkey test for their sensitivity to neurovirulence of live oral poliovirus vaccine (OPV). Intracerebral (i.c.) and intraspinal (i.s.) routes of inoculation were investigated to determine the most suitable combination of mouse strain and route. Evaluation of the mouse tests was performed using several indicators; clinical score and failure time were selected as the most efficient. Tg1 and Tg21 mice inoculated i.s. with type 2, and Tg21 mice inoculated i.s. with type 3 OPV were determined to be the most appropriate systems, whereas they are shown not to be suitable for type 1 OPV. The sensitivity of each of the two mouse models was at least equal to that of the monkey test, suggesting that these mouse systems might be considered as a potential replacement for the monkey test of OPV. However, more data are needed to establish regulatory criteria of acceptability for vaccine lots tested in Tg PVR mice. This is the first study conducted with Tg PVR mice with all three types of poliovirus vaccine preparations.

Mutations that consistently accumulated in the attenuated Sabin 2 strain of poliovirus during propagation in cell cultures were identified by sequence heterogeneity assay and quantified by mutant analysis by PCR and restriction enzyme cleavage (MAPREC). Eight additional sites previously identified in stool isolates were also examined by MAPREC in the virus passages. The pattern of selectable mutations and the rate of their accumulation depended on the type and confluence of the cell culture and the temperature of virus growth. Five unstable genomic sites were identified in Sabin 2 virus passaged 10 times at 34 degrees in African green monkey kidney (AGMK) cells, with the mutations accumulating in the range 1 to 24%. Accumulation of these mutations did not appear to result in a loss of attenuated phenotype since the virus passaged under these conditions passed the monkey neurovirulence test (MNVT). The content of the 481-G revertant known to be related to neurovirulence in monkeys did not increase. Thus, our results suggest that upon growth of Sabin 2 virus in AGMK cells at 34 degrees, the key determinant(s) of attenuation remained stable, and the mutations that occurred did not affect monkey neurovirulence. In virus passaged 10 times at 37 degrees in AGMK cells, 4 unstable genomic sites were identified, in some of them accumulating up to 12% of the mutants. This virus sample severely failed the MNVT. Virus passaged in Vero cells at 34 and 37 degrees accumulated mutants at 7 and 14 genomic sites, respectively, including 481-G in both cases, with almost complete substitution of the original nucleotides at some of the sites. We tested 44 commercial monopools of Type 2 OPV and found out that all of them contained 481-G revertants in the range 0.4-1.1%. An increase in the 481-G revertants in passaged viruses to the level of 4% and above correlated with failure of these samples by the MNVT. Since the pattern of selectable mutations differed in viruses grown in the two cell cultures used in this study, specific mutation profiles should be determined for each cell substrate used for vaccine production to assess manufacturing consistency.

We have previously found that upon passaging type 3 oral poliovirus vaccine (OPV) in cell cultures the proportion of revertants at nucleotide 472 rapidly increases [Chumakov et al.: Proceedings of the National Academy of Sciences of the United States of America 88:199–203 1991]. Systematic study on the accumulation of these revertants showed that it was dependent on the multiplicity of infection and the temperature at which virus was grown. Revertants at position 472 of type 3 OPV accumulated faster in vaccines derived from Sabin Original (SO) substrain than from RNA-plaque purified (RSO) substrain. The rate of accumulation of 472-C revertants differed among cell lines and was higher in overgrown cell cultures suggesting that host factors are involved in the selection of mutants. We also found that accumulation of mutants occurred in vitro at position 480 in type 1 and position 481 in type 2 OPV, making the selection for revertants in domain F of the 5′-noncoding region a general phenomenon for all three Sabin strains. Assessment of the abundance of these mutants may be used for evaluation of the quality of OPV lots. © 1994 Wiiey-Liss, Inc.

Quantitation of virus revertants by PCR and restriction enzyme cleavage may give nonlinear results and, in some cases, produce artifacts caused by nucleotide misincorporation and heteroduplex formation, occurring during PCR. Modifications of the procedure allowed us to overcome these problems and develop a highly sensitive and reliable method of mutant quantitation. This procedure can be used to assess the quality of live vaccines and to study heterogeneity of viral and bacterial populations.

Transgenic mice susceptible to poliovirus were recently produced by two groups of investigators. In this study, we compared the sensitivity of PVR Tg-1 transgenic mice and rhesus monkeys to poliovirus type 3. We found that intracerebrally inoculated Tg-1 mice are able to differentiate wild-type strain from attenuated strains and from a vaccine revertant. However, this mouse system can not discriminate between live poliovirus vaccine lots which passed the intraspinal (i.s.) monkey neurovirulence safety test (WHO) and those that failed. Unlike the monkey test which can detect as failed those vaccine lots which possess above 1% revertants at the 472 (U-->C) position, the test in Tg-1 mice inoculated intracerebrally (i.c.) did not recognize virus preparations containing even three percent revertants. Thus, the PVR Tg-1 i.c. mouse model is suitable for epidemiological and other virological studies, but it does not appear to be useful for neurovirulence testing of live poliovirus vaccines. A solution to the latter may be found in the use of a more sensitive i.s. route of inoculation of PVR Tg mice.

The hypothesis was tested that it is possible to influence cellular responses of intact cells using synthetic peptide substrates, pseudosubstrates, and inhibitors of protein kinases. Using cytotoxic T-cells (CTL), we demonstrate here that some basic amino acid-containing synthetic peptide substrates of protein kinases [e.g., of cGMP-dependent protein kinase (peptide PKG-S), synthetic peptide inhibitor of cGMP-dependent protein kinase (peptide PKG-I), and peptide corresponding to the tyrosine phosphorylation site in pp60src (peptide RR-src)] were strongly inhibitory in T-cell receptor (TCR) and T-cell growth factor, interleukin 2 (IL-2)-triggered proliferation of CTL. These peptides also inhibited other cellular responses of CTL. Peptides which contain basic amino acids, but do not have substrate specificity determinants for protein kinase, were not inhibitory. The inhibition with peptides is not due to their toxicity, since no cell death was observed by the trypan blue exclusion test and by lactate dehydrogenase release. Use of the granule exocytosis assay provided opportunities to clarify the mechanism of the peptide action. Tested peptides inhibited not only cell-surface ligand-induced CTL activation, but also affected cell-surface receptor-independent CTL activation (granule exocytosis and gamma-interferon secretion) induced by the synergistic action of the protein kinase C activator (PMA) and ionophore A23187. It was found that minor changes in amino acid composition or amino acid position in the synthetic peptides dramatically change their ability to affect lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurovirulence of several mumps virus strains was assessed in a prototype rat neurovirulence test and compared to results obtained in the monkey neurovirulence test. The relative human neurovirulence of these strains was proportional to the severity of hydrocephalus in rats but not to lesion scores in the monkeys.

Addition of ATP or ATP analog to the incubation media is shown to result in cell death in experiments with different cultured cell lines as evidenced by the results of several independent assays, both in the absence or presence of extracellular Ca2+. Cytolytic T-lymphocyte (CTL) clone itself was not only resistant to cytolytic effects of ATP, but was able to "rescue" antigen-nonbearing 51Cr-labeled cells from lytic effects of extracellular ATP (but not from lytic effects of adenosine 5'-thiotriphosphate) when present during assay. To test whether the resistance of CTL to ATP is due to a high activity of ecto-ATPase, four independent assays of ATPase activity were utilized to demonstrate the presence and relatively high activity of the ecto-ATPase(s) on CTL surface. Studies of substrate specificity of CTL ecto-ATPase suggest that there is more than one nucleoside 5'-triphosphatase on the surface of CTL. The enzyme(s) activity is Ca2+- and Mg2+-dependent and in this respect is similar to recently described hepatic cells ecto-ATPase. We tested effects of known ATP-binding site-specific reagents fluorescein 5'-isothiocyanate (FITC) and 5'-fluorosulfonylbenzoyladenosine (FSBA) to find covalent modification procedures to be used in studies of functional role of ecto-ATPase. FSBA, but not FITC, inhibits lymphocyte ecto-ATPase but addition of ATP together with FSBA protects ecto-ATPase activity. Inactivation of CTL ecto-ATPase by pretreatment with FSBA makes CTL susceptible to lytic effects of extracellular ATP, as was hypothesized for the functional role of this enzyme in CTL.

Multiple nucleic acid-based techniques (NAT) have been implemented for testing blood and plasma donors for HIV-1 RNA which may be detected at an earlier stage of infection when HIV antigen or antibody is absent or below the limit of detection of current assays. The available NAT assays are based on different technologies. In order to evaluate the performance of nucleic acid-based techniques (NAT assays) and to allow accurate comparisons of results from different assays, it is essential to have well characterized specimens with known copy numbers as a standard. For this purpose, a comprehensive study was conducted to develop two HIV-1 RNA reference panels. The first (Panel 1) was prepared using a single specimen from the HIV-1 group M subtype B and consists of panel members with a wide range of HIV-1 RNA copy numbers. Panel 2 consists of 26 members representing HIV-1 group M subtypes A, C, D, E, F, G and groups O and N. For accurate determination of HIV-1 RNA copy numbers of each member of Panel 2, they were analyzed using various testing platforms/technologies available through the cooperation of five independent laboratories participating in the study. A consensus value for HIV RNA copy number was assigned to each member of Panel 2 based on statistical analysis of the data provided by the participants. Both panels could serve as reference panels to be used by manufacturers of HIV NAT tests to evaluate the sensitivity limits of their assays.

The viral and transmissible spongiform encephalopathy (TSE) safety of therapeutics of biological origin (biologicals) is greatly influenced by the nature and degree of variability of the source material and by the mode of purification. Plasma-derived and recombinant DNA products currently have good viral safety records, but challenges remain. In general, large enveloped viruses are easier to remove from biologicals than small 'naked' viruses. Monoclonal antibodies and recombinant DNA biopharmaceuticals are derived from relatively homogeneous source materials and purified by multistep schemes that are robust and amenable to scientific analysis and engineering improvement. Viral clearance is more challenging for blood and cell products, as they are complex and labile. Source selection (e.g. country of origin, deferral for CJD risk factors) currently occupies the front line for ensuring that biologicals are free of TSE agents, but robust methods for their clearance from products are under development.

Similar to other segmented RNA viruses, influenza viruses can exchange genome segments and form a wide variety of reassortant strains upon coreplication within a host cell. Therefore, the mapping of genome segments of influenza viruses is essential for understanding their phenotypes. In this work, we have developed an oligonucleotide microarray hybridization method for simultaneous genotyping of all genomic segments of two highly homologous strains of influenza B virus. A few strain-specific oligonucleotide probes matching each of the eight segments of the viral genomes of the B/Beijing/184/93 and B/Shangdong/7/97 strains were hybridized with PCR-amplified fluorescently labeled single-stranded DNA. Even though there were a few mismatches among the genomes of the studied virus strains, microarray hybridization showed highly significant and reproducible discrimination ability and allowed us to determine the origins of individual genomic segments in a series of reassortant strains prepared as vaccine candidates. Additionally, we were able to detect the presence of at least 5% of mixed genotypes in virus stocks even when conventional sequencing methods failed, for example, for the NS segment. Thus, the proposed microarray method can be used for (i) rapid and reliable genome mapping of highly homologous influenza B viruses and (ii) extensive monitoring of influenza B virus reassortants and the mixed genotypes. The array can be expanded by adding new oligoprobes and using more quantitative assays to determine the origin of individual genomic segments in series of reassortant strains prepared as vaccine candidates or in mixed virus populations.

The only US FDA licensed smallpox vaccine, Dryvax, was associated with rare but serious neurological adverse events. After smallpox was eradicated in the United States, mass vaccination ceased in 1971. As counter-bioterrorism/biowarfare measures, new smallpox vaccines are now being investigated. However, there are no established pre-clinical neurotoxicity assays with which to evaluate these new vaccines prior to licensure. Here we report the development and initial characterization of a small animal neurotoxicity assay for vaccinia-based smallpox vaccines using Dryvax virus as a reference vaccine strain and the neuroadapted Western Reserve (WR) strain as a neurotoxic positive control. In neonatally inoculated mice, the WR strain produced significantly greater and more rapid onset of mortality than the Dryvax vaccine reference. Expression of virus antigen in neural cells and infectious virus replication in the brain was also significantly different between the two strains. In addition, the appearance of high titer virus antibody correlated with the clearance of virus from brain. With further validation, this assay incorporating a licensed vaccine reference standard and positive control strain may provide important pre-clinical neurotoxicity data on new vaccinia-based smallpox vaccine strains.

Transgenic (Tg) mice expressing the human poliovirus receptor (PVR) were vaccinated with inactivated poliovirus vaccine (IPV) and evaluated for induced immunity against type 3 poliomyelitis. One injection of monovalent type 3 IPV elicited protective immunity against wild-type poliovirus. In contrast, 2 injections of trivalent IPV were required for protection. Neutralizing antibody response and protection were vaccine dose-dependent. Administration of polio-immune mouse plasma protected unimmunized mice, demonstrating that neutralizing antibody was sufficient for immunity. IPV heated to remove its D antigen component did not induce protection in Tg PVR mice. IPV derived from a wild-type poliovirus strain gave better protection against wild-type viral challenge than IPV derived from an attenuated poliovirus strain. The newly developed Tg PVR mouse-protection test may be useful in evaluating existing IPV potency tests and for attempts to improve formulations of trivalent IPV or combined vaccines for childhood immunization schedules.

The production of interleukin-1 (IL-1) by cultured neonatal rat microglia was studied using the D10 cell assay. The results show that IL-1 was secreted in response to lipopolysaccharide (LPS) in a dose- and time-dependent fashion. IL-1 production was specific to microglia and was not induced in astrocytes. Indomethacin, which is known to modulate the release of IL-1 from monocytes, had no effect on LPS-stimulated microglia. Aging of the microglia from two weeks to 4 weeks in culture, however, reduced the release of IL-1 in response to LPS. Our data indicate that microglia are a major source of IL-1 and that the release of IL-1 depends on the presence of inflammatory mediators such as LPS and the age of the culture.

We hypothesized that cytolytic T lymphocytes (CTL) may utilize extracellular ATP (ATPo) during the effector phase of the CTL-target cell interactions and that CTL could be the source of ATPo. It is demonstrated here that incubation of CTL with activating ligands [Con A or monoclonal antibody (mAb) to the T-cell antigen receptor (TCR)] results in the extracellular Ca2(+)-independent accumulation of the ATPo. The addition of the ATP-degrading enzymes into the mixture of CTL and target cells results in a strong inhibition of the CTL-mediated, TCR-triggered lethal-hit delivery to the target cell. In a parallel control experiment, the employed enzymes did not affect target cell-induced, TCR-triggered exocytosis of granules from CTL. Thus, the removal of ATPo with enzymes does not interfere with the activation of CTL by the target cell but does block lytic events. Cloned helper T lymphocytes also accumulate ATPo after incubation with anti-TCR mAb or Con A, suggesting the possibility that ATPo, which acts in concert with ectoprotein kinases and/or purinergic receptors, may be of general use as a messenger in cellular interactions of T lymphocytes.

In a recent clinical trial, dietary supplementation with a single dose of oral iodized oil (OIO) improved the survival of infants in a population at risk for mild-to-severe iodine deficiency disorders. The study was designed so that OIO was administered at the first visit for the recommended childhood vaccination schedule of the WHO Expanded Programme on Immunization (EPI). Placebo or OIO was administered immediately following trivalent oral poliovirus vaccine (OPV). An important concern was that OIO might have a deleterious effect on OPV, since polioviruses are inactivated by iodine, or might otherwise reduce immune responses to the vaccine. Serum samples were collected upon enrollment of infants at 6-10 weeks of age and again at the final follow-up evaluation at 6 months of age. Pre-immune and post-immune sera from 478 infants were tested for virus-neutralizing antibody activity against each of the three poliovirus serotypes. Iodine supplementation did not reduce immune responses to OPV. These results indicate that supplementation with OIO may be combined with delivery of the first dose of OPV in the EPI schedule.

Potency Test of Inactivated Poliovirus Vaccine in Transgenic Mice Expressing Human Poliovirus Receptor. • 772

In studies with polioviruses (PV) isolated from infected children, Sabin, seeking suitable strains to attenuate for live oral PV vaccine (OPV), derived substrains with exceptionally low virulence for monkeys inoculated intraspinally-the species and route of inoculation most sensitive to residual neurovirulence. Because OPV strains tend to undergo partial genetic reversion, it is important to verify that live vaccine preparations retain all markers of attenuation, especially lack of neurovirulence. For many years US federal regulations have required that each monovalent virus pool be tested for neurovirulence by injection into the spinal cords of monkeys before incorporation into trivalent OPV. Monkey neurovirulence testing (MNVT) is expensive and uses very large numbers of animals-testing for a single lot of trivalent OPV requiring 88 monkeys or more. We have studied three tests that might eventually reduce the reliance on MNVT if not replace it: 1) A molecular assay to detect increased content in PV vaccine of those virions with revertant mutations at sites in the viral genome thought to be responsible for neurovirulence (“mutant analysis by polymerase chain reaction and restriction enzyme cleavage”/MAPREC). 2) Intraspinal inoculation of transgenic (Tg) mice expressing the human PV-receptor gene and susceptible to infection with PV. 3) Increased replication of neurovirulent strains of type-3 PV in cultures of interferon-treated human neuroblastoma cells. MAPREC and Tg-mouse tests successfully detected preparations of type-3 OPV (the least stable type of OPV most often incriminated in vaccine-associated paralytic poliomyelitis) that had failed MNVT. The neuroblastoma test recently showed promise in detecting revertant type-3 OPV. Both MAPREC and Tg-mouse tests are currently subjects of multi-center studies coordinated through the World Health Organization.

A recent report indicated that T200 molecules interact with elements of the cytoskeleton in BW5147 T lymphoma cells. We have confirmed the cytoskeletal association of T200 by examining nonionic detergent-soluble and detergent-insoluble fractions of murine T cell tumor cell lines, cloned cytotoxic T lymphocyte lines, and thymocytes. Concanavalin A (Con A)-treated and untreated cells were extracted with 0.5% Triton X-100 and the remaining insoluble material was extracted under conditions allowing actin depolymerization. In the absence of Con A treatment, little T200 could be recovered from the depolymerized insoluble fraction. However, in T cells treated with capping concentrations of Con A, a considerable amount of T200 was rendered insoluble in nonionic detergent, and T200 could be recovered from the insoluble fraction by a buffer which dissociates actin polymers. A lesser, but still significant, amount of T200 associated with the detergent-insoluble fraction of thymocytes treated with concentrations of Con A and succinyl Con A, which are mitogenic for T cells. We also found that in T cells treated with mitogenic concentrations of succinyl Con A, more T200 associated with cytoskeleton than did H-2 or LFA-1 molecules. Because T200 is such a predominant molecule on the surface of T cells, such translocations of the molecule may have a major impact on the physiology of the cell, especially if T200 functions as a protein tyrosine phosphatase as recent evidence by others suggests.

Screening for sequence heterogeneities in Sabin Type 3 strains of attenuated poliovirus demonstrated mutations that consistently accumulate to significant levels following 10 passages in cultures of primary African green monkey kidney (AGMK) cells or continuous cultures of Vero cells. Fourteen newly identified mutations were quantified by mutant analysis by PCR and restriction enzyme cleavage in passages and in batches of commercial vaccines made in AGMK and Vero cells from the Sabin original (SO) seed virus and from a seed virus rederived by RNA plaque purification (RSO or "Pfizer" seed). Nine of the 14 mutations were reproducibly observed in more than one series of passages. Although 5 other mutations were observed in only one set of passages each, their content gradually increased to a high percentage, suggesting that all the mutations that we found accumulated consistently. SO-derived samples accumulated more mutations than did RSO-derived ones, and the number of mutations and the rates of their accumulation were higher in Vero than in AGMK cells. While the rates of accumulation of most mutations were higher when passaging was performed at 37 degrees, a U--C transition at nucleotide 5832 occurred faster at 34 degrees, the temperature used for vaccine production. Analysis of Type 3 oral poliovirus vaccine (OPV) monopools made by six manufacturers found only 5 of these newly identified mutations in vaccine batches (nucleotides 3956, 4935, 5357, 5788, and 5832). Some of the mutations were found in trace amounts (less than 0.1%) while others were present at up to 1.8% levels. The pattern of these mutations was characteristic for the type of seed virus and the cell substrate but demonstrated no correlation with results of the monkey neurovirulence test. Therefore the only mutation occurring in Type 3 OPV which contributed to neurovirulence in monkeys was the previously described reversion at nucleotide 472. Quantitation of reversion at nucleotide 472 can be utilized for assessment of acceptability of vaccine lots, while other mutations can be used for monitoring the consistency of vaccine production.

Analysis of dead versus live cells is shown to be possible using Calcoflour White M2R (CFW), a fluorescent brightener. Comparison of CFW with both propidium iodide (PI) and fluorescein diacetate (FDA) was performed on a FACS 440 dual laser flow cytometer on several populations of cultured rat and mouse cell lines, peripheral leukocytes, splenocytes, diatoms, and plant protoplasts. As a measure of cell viability, staining results with CFW were strongly associated with PI (correlation coefficient of 0.9886) and FDA (inverse correlation coefficient of 0.9647). With plant and algal cells, controls are necessary as CFW does stain live cells to some extent. CFW (excitation: UV, emission max: 435 nm) can be used in conjunction with two-color immunofluorescence analysis using fluorochromes excited at 488 nm with no interference.