Evaluation of an infectivity standard for real-time quality control of human immunodeficiency virus type 1 quantitative micrococulture assays. Participating Laboratories of The AIDS Clinical Trials Group

Quantitative microculture assays of cryopreserved human immunodeficiency virus type 1-infected cell suspensions and culture supernatants were compared among seven assays sites. There was no significant change in titer during 1 year of storage. The overall standard deviation for infected cell suspensions was approximately 0.8 log10 virus titer. A method for detecting deviant assay results was developed and was used to identify two donor cell preparations (n = 54) that gave consistently low titers.     

The effects of oestrogen and progesterone on insulin sensitivity in female rats

Several reactive oxygen species, including singlet oxygen (1O2) and hydroxyl free radical (.OH), may potentially be involved in the photoinactivation of viruses by agents such as methylene blue (MB) and rose bengal (RB). Both 1O2 and .OH also mediate the formation of 8-oxoguanine (8-oxoGua) in DNA and RNA. Evidence that MB-or RB-induced bacteriophage (R17 or Q beta) inactivation and 8-oxoGua formation in RNA result from 1O2 rather than .OH was obtained utilizing complementary experimental approaches which show that: (i) the rate of phage photoinactivation by MB was unchanged by the presence of iron chelators or by different temperatures in the 13-37 degrees C range; (ii) MB- and RB-mediated rates of 8-oxoGua formation in isolated RNA have very little, if any, temperature dependence, in contrast to a significant temperature dependence of 8-oxoGua formation by a .OH generating system, the ultraviolet light irradiation of H2O2; and (iii) deuterium oxide (D2O) enhanced the RB-mediated rate of phage photoinactivation and 8-oxoGua formation in isolated RNA. The presence of superoxide dismutase in the RB photoinactivation reaction did not alter the rate of phage inactivation. The data suggest that 8-oxoGua serves as a marker that correlates qualitatively with 1O2-mediated lethal lesions in RNA bacteriophages.     

Lymphoid/nonlymphoid compartmentalization of donor leukocyte chimerism in rat recipients of heart allografts, with or without adjunct bone marrow

A spectrum of oxidative lesions was observed in a bacteriophage-based model system that is very sensitive to the photodynamic activity of selected dyes. When suspensions of the intact bacteriophage Q beta were exposed to methylene blue plus light (MB + L), inactivating events, or "hits" occurred that were oxygen-dependent and that were associated with the formation of several specific lesions: (1) carbonyl moieties on proteins, (2) 8-oxo-7,8-dihydroguanine (8-oxoGua), and (3) single-strand breaks (ssb) in the RNA genome and (4) RNA-protein crosslinks. Formation of carbonyl groups associated with protein in the Q beta phage preparation correlated positively with photoinactivation of the phage with increasing doses of either of the sensitizers MB or rose bengal. Strand breaks in the Q beta genomic RNA were observable at high MB concentrations but appeared not to be significant at the lower concentrations of MB, as full-length Q beta RNA was observable well beyond the 99% inactivation point in MB dosage. It was shown that the number of 8-oxoGua lesions were unlikely to be sufficient to account for the number of lethal events. Following exposure to MB + L, crosslink formation between Q beta RNA and protein was observed by virtue of the location of RNA at the interface of phenol-aqueous extractions of phage suspensions. A significant increase over background of RNA-protein complexes (including full-length Q beta RNA) was observed at the lowest concentration of MB tested (0.5 microM), which corresponded roughly to an average of 2 lethal hits per phage or approximately 13% survival compared to the zero MB control (100% survival). Due to its close correlation with Q beta inactivation and its expected lethality, RNA-protein crosslink formation may be important as an inactivating lesion in bacteriophage Q beta following MB + L exposure.     

Distribution of HIV type 1 (HIV-1) in blood components: detection and significance of high levels of HIV-1 associated with platelets

BACKGROUND: Although inactivation of enveloped viruses transmitted by plasma derivatives has been successful, no methods for virus inactivation or removal have been established for platelet concentrates or red cell (RBC) components. Relatively little is known regarding the extent or significance of virus interactions with the cellular constituents in these components. STUDY DESIGN AND METHODS: Units of whole blood were collected from six HIV type 1 (HIV-1)-positive, asymptomatic individuals and separated into peripheral blood mononuclear cells (PBMNCs), cell-free plasma, white cell-reduced platelet concentrate, and white cell-reduced RBCs. DNA and RNA polymerase chain reaction and virus culture methods were used to study the compartmentalization of HIV-1 immediately after component preparation and after storage. RESULTS: As expected, HIV DNA and infectious virus were detected in fresh blood and in PBMNCs, and virion-associated RNA was detected in fresh plasma from all six donors. The levels of viral nucleic acids in these preparations remained relatively stable with 4 degrees C storage, whereas infectivity of PBMNCs was rapidly lost. Washed RBCs tested negative for HIV in all assays at all time points. Platelets retained high levels of HIV RNA (but not infectivity) after extensive washing, as well as after storage at 4 and 22 degrees C. High-level platelet-associated HIV-1 was also demonstrated in samples collected during early seroconversion. Periseroconversion and postseroconversion levels of platelet-associated HIV-1 correlated with the level of plasma viremia and with the rate of progression to AIDS. Cell-free virus from donor plasma and tissue culture fluid rapidly and firmly attached to platelets from noninfected donors. Infectivity of tissue culture virus bound to platelets was demonstrated in vitro. CONCLUSION: Significant levels of HIV-1 are associated with platelets during all stages of infection. Platelet-associated HIV could either mediate virus clearance or facilitate virus dissemination and expanded tropism. Finally, virus inactivation research must address virus associations with platelets.     

Cumulative viral titer reduction demonstrated by sequential challenge of a tangential flow membrane filtration system and a direct flow pleated filter cartridge

The efficiency of sequential incorporation of two approaches to virus removal, i.e., tangential flow ultrafiltration using the Omega 300K VR Maximate System, and, direct flow microfiltration using the Ultipor VF grade DV50 virus removal filter, was evaluated using bacteriophage phi 6 in Dulbecco's modified Eagle medium (MEM) as the carrier fluid. A combined log titer reduction value of > 10 was demonstrated, with each step, individually, providing > 4.5 log reduction. Significant improvement in filter life was also demonstrated when the combined system was used. The data suggest that this system may have applications in the manufacture of biologicals and biopharmaceuticals, where there is a requirement for multiple robust methods of virus removal to ensure freedom from endogenous or adventitious viral contamination.     

Photochemical inactivation of duck hepatitis B virus in human platelet concentrates: a model of surrogate human hepatitis B virus infectivity

BACKGROUND: Photochemical decontamination of platelet concentrates (PCs) has been demonstrated by the use of 8-methoxypsoralen and ultraviolet A light. Systems for studying the inactivation of blood-borne viruses facilitate the evaluation of photochemical decontamination protocols. STUDY DESIGN AND METHODS: Duck hepatitis B virus (HBV), a model for human HBV, was adapted for the study of hepadnavirus inactivation. A highly specific in vitro infectivity assay used primary duck hepatocyte cultures and was followed by the detection of replicated duck HBV sequences. RESULTS: Duck HBV-infected primary duck hepatocyte cultures produced authentic infectious virus. High-titer (> 10(9) virus genome equivalents/mL) duck HBV-infected sera were completely inactivated in serum or PCs by the use of 100 micrograms per mL of 8-methoxypsoralen and 70 J per cm2 of ultraviolet A light. Intracellular duck HBV (> 4.2 log10) in PCs was also inactivated. Culture results were confirmed by a sensitive duckling infectivity assay that indicated that 6.3 log10 of infectious duck HBV had been inactivated by photochemical decontamination. CONCLUSION: The sensitivity of the culture assay was comparable to that of the duckling assay using polymerase chain reaction gene amplification to detect duck HBV. Duck HBV inactivation in PCs was dependent on the dose of ultraviolet A light and independent of 8-methoxypsoralen concentrations of 100 to 300 micrograms per mL: 100 micrograms per mL 8-methoxypsoralen inactivated 4 to 5 log10 of virus in conjunction with 20 to 40 J per cm2 of ultraviolet A light. The polymerase chain reaction-enhanced duck HBV culture system has utility in optimizing photochemical decontamination protocols.     

The persistence of genetic homogeneity among Trypanosoma brucei rhodesiense isolates from patients in north-west Tanzania

The decrease in titer of PRV antibodies in serum was evaluated at 10, 37, 67, 109 and 173 days of age in 16 non-vaccinated pigs and 43 pigs vaccinated at 3, 67 and 80 days of age with a modified live TK/gIII gene deleted pseudorabies virus (PRV) vaccine. Serum samples were analyzed for antibodies to PRV by the serum-virus neutralization test (SN), a commercial competitive ELISA (CELISA), and the CELISA OMNIMARK PRV differential (OMD) diagnostic kit. At 10 days of age, all pigs had SN titers > or = 1:4 and were CELISA+/OMD+, indicating circulating antibodies to field strains of PRV. At 109 days, all non-vaccinated pigs had SN titers < 1:4. Forty-five percent of vaccinated pigs had SN titers > or = 1:4, 56% were CELISA positive and most were CELISA+/OMD-, indicating antibodies due to vaccination. At 24 weeks of age, all pigs had SN titers > or = 1:4 and were CELISA+/OMD+ due to exposure to field strains. Although circulating maternal antibodies interfere with the development of active immunity, vaccination at 3 days of age resulted in detectable antibodies by 67 days of age, and a limited immune response could be measured at 109 days of age.     

Inactivation of glycoprotein gE and thymidine kinase or the US3-encoded protein kinase synergistically decreases in vivo replication of pseudorabies virus and the induction of protective immunity

To evaluate the contribution of glycoprotein E (gE), thymidine kinase (TK), and the US3-encoded protein kinase (PK) in the induction of protective immunity to pseudorabies virus (PRV), we intranasally inoculated pigs, the natural host of this virus, with mutant PRV strains in which the genes encoding these proteins were inactivated. Both single and double mutants were constructed. Of these proteins, gE has previously been demonstrated to induce antibodies (in mice and pigs), which require complement to neutralize the virus, and helper T cell responses (in mice). PK and TK have thus far not been reported to induce B or T cell responses. All mutants had a strongly reduced virulence for pigs in comparison with wild-type (wt) PRV. After primary infection, most virus was excreted by wt PRV-inoculated animals. Animals inoculated with gE-PK- and gE-TK- double mutants excreted less virus than animals inoculated with gE-, PK-, and TK- single mutants. After challenge infection with the virulent PRV strain NIA-3, no virus was excreted by wt PRV- and PK- mutant-immunized animals, indicating complete protective immunity. Only one of seven gE- and two of seven TK- mutant-immunized animals excreted virus after the challenge inoculation. In contrast, most animals immunized with the gE-PK- or gE-TK- double mutants excreted virus after the challenge inoculation. Daily mean virus excretion after challenge infection was inversely correlated with daily mean virus excretion after primary infection. In most animals, lack of virus excretion was associated with lack of secondary antibody responses, probably attributable to inadequate stimulation of memory B cells as a consequence of early elimination of viral antigen. Thus, inactivation of gE, TK, and PK all affected the immunogenicity of PRV and the effect of gE and TK and gE and PK inactivation appeared synergistic. We found no simple correlation between in vitro growth properties of the mutants and their immunogenic capacity. Strains lacking PK reached lower end titers in vitro than the other mutants. The most likely explanation for the lower protective capacity of some of the mutants appears their reduced replicative capacity in some cells or tissues in vivo, rather than a loss of particular epitopes.     

T cells of the human intestinal lamina propria are high producers of interleukin-10

BACKGROUND: The transmission of hepatitis A virus (HAV) has been associated with the use of a number of solvent/detergent-treated factor VIII concentrates and possibly a factor IX concentrate. These reports have emphasized the necessity of using virus-inactivation methods for plasma products that are capable of inactivating nonenveloped viruses such as HAV. STUDY DESIGN AND METHODS: A simple, highly accurate titration procedure for HAV, which allows extensive kinetic investigations of virus-inactivation procedures, has been developed. This system has now been used to evaluate the efficacy of vapor heating in inactivating HAV after the addition of the virus to a range of human plasma products. RESULTS: It was demonstrated that HAV was significantly more thermostable than other picornaviruses, which reinforced the fact that such viruses cannot be used as model viruses for HAV-inactivation studies. A one-step vapor-heating procedure was demonstrated to inactivate between 5.9 and > 6.3 log10 of HAV in different products. A two-step vapor-heating procedure had the capacity to inactivate between > 8.7 and > 10.4 log10 of HAV. Both procedures were more effective in inactivating HAV than was the pasteurization procedure used for virus inactivation in human albumin solutions. CONCLUSION: These data demonstrate the efficacy of vapor heating in inactivating high-titer HAV after the spiking of plasma products with virus. This study confirms and explains the results of controlled clinical trials and long-term clinical usage with respect to the lack of HAV transmission by such vapor-heated products.     

Efflux of cyclic GMP from activated human platelets

Activation of human platelets is associated with an increased level of cGMP, when total cGMP in individual samples is measured. However, by discriminating between intracellular and extracellular cGMP we were able to demonstrate that cGMP accumulates in the extracellular space only, whereas the level of intraplatelet cGMP actually decreases. Therefore, during the first minutes of platelet aggregation cGMP is released from the cell, and it thereby escapes hydrolysis by intracellular phosphodiesterases. In contrast, during direct activation of soluble guanylyl cyclase by nitrovasodilators, such as sodium nitroprusside, the newly synthesized cGMP remains mainly inside the cells. Elevation of intracellular calcium and activation of protein kinase C are likely to be involved in promoting cGMP efflux. Our results are discussed in contrast to the general hypothesis that the cGMP increase associated with platelet aggregation may represent a feedback mechanism designed to terminate early events of activating signal transduction. According to our data the apparent cGMP increase results from cGMP release from thrombocytes, rather than soluble guanylyl cyclase activation. This cGMP efflux provides a mechanism of decreasing the intracellular cGMP level upon stimulation with platelet agonists and thus favors platelet activation.     

Impact of various red cell concentrate preparation methods on the efficiency of prestorage white cell filtration and on red cells during storage for 42 days

BACKGROUND: White cell (WBC) reduction prior to storage of red cell (RBC) concentrates may reduce the incidence of HLA alloimmunization and may improve the quality of stored RBCs. STUDY DESIGN AND METHODS: An integrated WBC-reduction filter system was tested after various RBC preparation procedures (from whole blood), and the influence of filtration on RBCs during storage for 42 days was investigated. Four additive system RBC preparation protocols were used. Units prepared from conventional triple blood bags were held for 4 to 6 hours at 22 degrees C, and then the RBCs were separated via a hard spin and filtration, performed immediately (Group 1) or after 18 hours' storage at 4 degrees C (Group 2). Units prepared from a top-and-bottom collection system were held at 22 degrees C for 4 to 6 or 22 to 24 hours; the centrifuged RBCs were filtered immediately after preparation (Groups 3 and 4, respectively, by holding time). WBC reduction and filtration time were analyzed. The impact of WBC filtration on pH, hemolysis rate, hemoglobin content, ATP, potassium glucose, and lactate was investigated weekly during storage for 42 days. RESULTS: Filtration reduced the mean WBC count by 3 to 4 log10, to 0.19 +/- 0.25 x 10(6), regardless of the RBC preparation method. Mean filtration times differed significantly between the groups and were longest for Group 1. Besides hemolysis and pH values, which were greater in all filtered units, no major differences were found in filtered and unfiltered RBCs during the storage interval. CONCLUSION: The efficiency of prestorage WBC filtration of RBCs was unaffected by the preparation procedure. However, the filtration time for RBCs freshly prepared in the conventional triple blood bag system without buffy-coat depletion was unacceptable. No major metabolic differences between filtered and unfiltered RBCs during 42 days of storage were found.     

Public veterinary medicine: public health. Serologic evaluation of vesicular stomatitis virus exposure in horses and cattle in 1996

OBJECTIVE: To determine potential risk factors for vesicular stomatitis (VS) in Colorado livestock in 1995 and evaluate VS virus (VSV) exposure of Colorado livestock in 1996. DESIGN: Retrospective case-control study of VS risk factors and seroprevalence evaluation. SAMPLE POPULATION: Premises included 52 that had VS-positive animals and 33 that did not have VS-positive animals during the 1995 epidemic, and 8 in the vicinity of premises that had VS-positive animals during the 1995 epidemic. PROCEDURE: Layout and management data for premises were collected during site visits in 1996. Signalment and management data were collected for animals from which samples were obtained, and samples were tested by serologic examination and virus isolation. The VSV seroprevalence rate was estimated for Colorado, using serum obtained for equine infectious anemia testing and from the Market Cattle Identification program in Colorado. RESULTS: At least 1 animal was seropositive for VSV on 35 of 52 (67%) premises, and 71 of 228 (31%) animals tested were seropositive for VSV. Seroprevalence was 63 of 170 (37%) for horses and 8 of 54 (15%) for cattle. Seroprevalence of VSV in animals from non-study premises in Colorado in 1996 was estimated to be 1.1% in cattle and 0.8% in horses. CLINICAL IMPLICATIONS: Overall VSV seroprevalence in Colorado livestock was less than seroprevalence in epidemic areas, and seroprevalence rates in epidemic areas were greater for horses than cattle. Results may indicate that some animals had subclinical VSV infection during epidemics and that animals may be exposed to VSV between epidemics.     

Study on splitting of sucrose glycoside bonds by beta-fructosidase of Aspergillus niger VKM F-801

Functional groups of beta-fructosidase of Aspergillus niger VKM F-801 were identified. Based on the pH dependence of the enzyme, the calculated heat of ionization, the photoinactivation of the enzyme in the presence of methylene blue photosensitizer, and also the enzyme inactivation by diethyl pyrocarbonate, the catalytic center of the beta-fructosidase was found to include carboxyl and imidazole groups of amino acid residues. The glycoside bond in the sucrose molecule is apparently cleaved by the catalytically active carboxylate--imidazole pair.     

Bioaccumulation of manganese and its toxicity in feral pigeons (Columba livia) exposed to manganese oxide dust (Mn3O4)

BACKGROUND: Studies were conducted to determine the capability of a hydrogen peroxide gas plasma sterilization process to inactivate several types of viruses. Six test agents were used: HIV type 1, human hepatitis A virus, respiratory syncytial virus, vaccinia, herpes simplex virus type 1, and poliovirus type 2. METHODS: The test viruses were suspended in cell culture medium and dried on the bottom of sterile glass petri dishes. The inoculated dishes were processed in the hydrogen peroxide gas plasma system for half the normal sterilization cycle time. Four inoculated carriers for each virus were used in two separate half cycles. Infectivity of the test viruses and cytotoxicity to the indicator cell lines were assayed. RESULTS: The hydrogen peroxide gas plasma sterilization process produced inactivation of the six viral test agents under these experimental conditions. The reduction in viral titers ranged from 2.5 log10 to 5.5 log10, a 99.68% to 99.999% decrease. CONCLUSIONS: These results clearly demonstrate the virucidal effectiveness of the hydrogen peroxide gas plasma sterilization process against both lipid and nonlipid viruses.     

Measurement of yeast intracellular pH by image processing and the change it undergoes during growth phase

The intracellular pH of the yeast Saccharomyces cerevisiae was determined by a fluorescence microscopic image processing technique. Image processing was carried out using a modification of the ratio imaging method for measurement of yeast intracellular pH. Care was necessary when taking fluorescence images in order to obtain accurate measurement of yeast intracellular pH. Until now it has been difficult to measure the intracellular pH of cells in actual cultivation conditions. This method enabled us not only to measure the intracellular pH of dilute cell suspensions, but also to obtain two-dimensional information. In the case of resting cells, the intracellular pH was dependent upon the extracellular pH, and this value was constant when the extracellular pH was constant. On the other hand, in the case of actively growing cells, intracellular pH was found to change, even if the extracellular pH was constant: the values observed were intracellular pH 5.7 during lag phase, intracellular pH 6.8 during exponential phase and intracellular pH 5.5 during stationary phase. These results for intracellular pH indicate that the yeast proton pump was activated during growth from the point of view of pH in vivo.     

Effect of hydration on the water content of human erythrocytes

An ideal, hydrated, nondilute pseudobinary salt-protein-water solution model of the RBC intracellular solution has been developed to describe the osmotic behavior of human erythrocytes during freezing and thawing. Because of the hydration of intracellular solutes (mostly cell proteins), our analytical results predict that at least 16.65% of the isotonic cell water content will be retained within RBCs placed in hypertonic solutions. These findings are consistent not only with the experimental measurements of the amount of isotonic cell water retained within RBCs subjected to nonisotonic extracellular solutions (20-32%) but also with the experimental evidence that all of the water within RBCs is solvent water. By modeling the RBC intracellular solution as a hydrated salt-protein-water solution, no anomalous osmotic behavior is apparent.     

Duck hepatitis B virus inactivation and 8-methoxypsoralen photoadduct formation in human platelet concentrates

Photochemical inactivation (PCI) of virus and bacteria in platelet concentrates (PC) has been demonstrated using 8-methoxypsoralen (8-MOP) and long-wavelength UV light (UVA). To study inactivation of blood-borne virus, we have employed duck hepatitis B virus (DHBV), a model for human hepatitis B virus. A specific hepatocyte culture infectivity assay, with PCR detection, could measure 5-6 log10 virus kill. The DHBV inactivation in PC was dependent on UVA dose, was enhanced when plasma was reduced from 100% to 20% and was limited by 8-MOP solubility in the reduced-plasma medium. Optimum conditions for PCI were 100 micrograms/mL 8-MOP in 20% plasma and 80% synthetic platelet storage medium. A radiolabeling assay for 8-MOP photoadducts in hepatocytes seeded into PC confirmed that DHBV inactivation reflected DNA modification and indicated that adduct formation was insensitive to minor variations in conditions. Kinetic modeling indicated that optimum adduct formation was a compromise between 8-MOP dark binding and optical transmittance and that plasma proteins competed for 8-MOP binding. The PCI results in various media correlated with corresponding DNA modification densities and were compared to statistical models incorporating DHBV characteristics and predictions of 8-MOP crosslink formation between DNA strands. Behavior was consistent with one or a small number of lethal modifications per DNA strand, including monoadducts, but probably not crosslinks alone. A minor subpopulation of DHBV was found to be somewhat more difficult to inactivate, consistent with three-fold lower modification, due possibly to single-stranded DNA character or host repair of photoadducts.     

Interferon induction as a quasispecies marker of vesicular stomatitis virus populations

The interferon (IFN)-inducing capacity of different isolates of vesicular stomatitis virus (VSV) of the Indiana (IN) and New Jersey (NJ) serotypes were measured to assess the extent of variability of this phenotype. Over 200 preparations of wild-type field isolates, laboratory strains, and plaque-derived subpopulations were examined. Marked heterogeneity was found in the ability of these viruses to induce IFN, covering a 10,000-fold range. A good fit to a normal distribution for the log of the IFN yields suggests a continuum of incremental changes in the viral genome may govern the IFN-inducing capacity of consensus populations derived from independently arising infections. A broad range in the magnitude of these changes, skewed towards inducers of high IFN yields, is consistent with a comparable series of ribonucleotide changes in the VSV genome, a sine qua non of a quasispecies population. Plaque- or vesicle-derived populations displayed standard deviations less than the mean IFN yields, though skewed to higher yielders, whereas populations from field and laboratory samples which differed widely in time and origin of isolation gave standard deviations greater than the means. The plaque isolation of IFN-inducing particles of VSV-IN, normally masked in populations by the predominance of non-IFN-inducing particles that suppress IFN induction, and the isolation of potent wild-type IFN-inducing VSV-IN from cows during an outbreak of vesicular stomatitis in a region that had yielded only virus expressing the non-IFN-inducing phenotype in prior and subsequent years, supports the view that genetic bottlenecks are operative in the natural transmission of this disease.     

Stability of hemagglutinating activity of extracellular and intracellular forms of Japanese encephalitis virus exposed to acidic pH

The hemagglutinating (HA) activity of extracellular and intracellular forms of Japanese encephalitis (JE) virus was comparatively titrated by exposure to acidic pH below 7.0. A pH-dependent irreversible loss in titer was observed with the virus grown in both C6/36 and BHK 21 (BHK) cells maintained in the pH range of 5.8 to 7.0 for 10 min at 37 C. The HA activity of intracellular virus was relatively more stable than that of extracellular virus in the pH range of 5.8 to 6.4. Virion structural components, envelope glycoprotein (E), capsid (C), and membrane (M) proteins in extracellular virus and E, C, and the precursor form of M (prM) proteins in intracellular virus were detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. A panel of monoclonal antibody (mAb) directed for nine antigenic epitopes on the JE virus E protein molecule was used for the analysis of antigenic reactivity of E protein after treatment at pH 6.0. The reaction between the extracellular virus and three HA-inhibiting (HI) mAbs was significantly reduced after acid treatment; however, the antigenic reactivity of intracellular virus was much more stable with a 100- to 1,000-fold difference. Infectivity titers of extracellular and intracellular viruses in Vero cells were reduced by 1/24,100 and 1/21,666 after acidic treatment at pH 6.0. In contrast, the infectivity of intracellular viruses was more stable, with residual infectivity of 1/182 and 1/340 for BHK and C6/36 cell-grown virus, respectively. Acidic treatment of JE virus not only resulted in the irreversible loss of its HA activity but also affected the antigenic reactivity of HI epitopes on its E protein molecule.     

Anatomical variation of cerebral venous drainage: the theoretical effect on jugular bulb blood samples

A workshop was convened to discuss safety issues for traditional-approach HIV vaccines, especially inactivated vaccines. The topics included issues pertaining to (1) cell substrates used for production and (2) vaccine virus inactivation. The use of cell substrates such as tumor-derived continuous cell lines (TCLs) or virus-transformed. CLs may be the most feasible approach to provide commercial-scale virus yields. However, especially because of concerns about tumorigenicity, TCLs have not been used to produce preventive vaccines for human trials with healthy subjects in the United States. Residual TCL material (e.g., DNA, cellular proteins, viruses) may not be removed during purification of intact HIV virions to the same extent achievable for a recombinant protein. Manufacturing processes, e.g., physicochemical methods of destroying DNA, could decrease tumorigenicity risk. Methods to assess potential for tumorigenicity may need further development. Another potential substrate for viral production that merits further study is human peripheral blood mononuclear cells (PBMCs). Regardless of the cell substrate used, extensive testing for adventitious agents (including non-HIV retroviruses) is needed. Vaccine virus inactivation can be considered in statistical terms, i.e., the probability of a surviving infectious particle. One formula to determine a "safety margin" (SM) is reduction of titer in log10 for all inactivation steps minus initial viral infectivity in log10. Calculations for appropriate SMs should include all sources of variability (e.g., lot-to-lot differences). Ensuring a specified SM, as the lower bound of the 95% confidence interval, for production lots was discussed. Sensitivity and specificity of infectivity assays may present limitations.