Uninfected Cell Lysates Research Articles

Maternal Immunization with a Single-Cycle Herpes Simplex Virus (HSV) Candidate Vaccine, ΔgD-2, Protects Neonatal Mice from Lethal Viral Challenge

BackgroundPerinatal HSV is associated with ~60% mortality if untreated and with substantial morbidity even with appropriate therapy. We recently engineered a single-cycle virus deleted in glycoprotein-D (ΔgD-2) that induces high-titer antibodies (Abs) that are non-neutralizing but activate the Fc receptor (FcR) to elicit antibody-dependent cellular cytotoxicity (ADCC). Immunization with ΔgD-2 completely protects adult mice from HSV-1 and HSV-2 disease following vaginal, skin, intraocular, or intranasal challenge and prevents the establishment of latency (ELife, 2014, JCI Insight, 2016). Thus we hypothesize that maternal immunization with ΔgD-2 and/or passive transfer of immune serum will protect neonates from HSV.MethodsFour- to 6-week-old C57Bl/6 female mice were primed and boosted at 3-week intervals with ΔgD-2 or an equal volume of uninfected cell lysates (VD60 cells). Two weeks post-boost, mice were mated and pups were challenged with a lethal dose of HSV-1 (Bx31.1) at day 7 of birth. To differentiate the contribution of transplacental vs. colostrum Abs, mothers were switched at birth. Alternatively, 7-day-old mice born to nonimmunized mothers received a single dose of immune serum (400 μg total Ab) intraperitoneally at time of intranasal challenge.ResultsThirty-eight of 47 (81%) of the pups born to and nursed by ΔgD-2-immunized mothers survived, exhibited little or no signs of disease and were protected from latency as measured by quantifying HSV DNA by PCR in neuronal tissue. In contrast, 12/14 (86%) of pups born to control vaccinated and nursed mice developed neurological signs of disease and died (P < 0.0001, Fisher’s exact test). Survival was associated with increased ADCC Abs in the serum of neonatal mice. In contrast, passive transfer of immune serum, which consistently protects adult mice from infection, did not protect neonates. If newborns born to immunized mice suckled with control mice, protection was partially abrogated (11/19, 58% survival), suggesting that both systemic and mucosal Abs are required for complete protection.ConclusionMaternal vaccination with ΔgD-2 provides significant protection against intranasal neonatal challenge but may require exposure to systemic and mucosal Abs.Disclosures W. R. Jacobs Jr., xvax: Scientific Advisor, Research support; B. C. Herold, X-vax: Grant Investigator, Research grant and Research support

Proteoform-Specific Insights into Cellular Proteome Regulation

Knowledge regarding compositions of proteomes at the proteoform level enhances insights into cellular phenotypes. A strategy is described herein for discovery of proteoform-specific information about cellular proteomes. This strategy involved analysis of data obtained by bottom-up mass spectrometry of multiple protein OGE separations on a fraction by fraction basis. The strategy was exemplified using five matched sets of lysates of uninfected and human respiratory syncytial virus-infected A549 cells. Template matching demonstrated that 67.3% of 10475 protein profiles identified focused to narrow pI windows indicative of efficacious focusing. Furthermore, correlation between experimental and theoretical pI gradients indicated reproducible focusing. Based on these observations a proteoform profiling strategy was developed to identify proteoforms, detect proteoform diversity and discover potential proteoform regulation. One component of this strategy involved examination of the focusing profiles for protein groups. A novel concordance analysis facilitated differentiation between proteoforms, including proteoforms generated by alternate splicing and proteolysis. Evaluation of focusing profiles and concordance analysis were applicable to cells from a single and/or multiple biological states. Statistical analyses identified proteoform variation between biological states. Regulation relevant to cellular responses to human respiratory syncytial virus was revealed. Western blotting and Protomap analyses validated the proteoform regulation. Discovery of STAT1, WARS, MX1, and HSPB1 proteoform regulation by human respiratory syncytial virus highlighted the impact of the profiling strategy. Novel truncated proteoforms of MX1 were identified in infected cells and phosphorylation driven regulation of HSPB1 proteoforms was correlated with infection. The proteoform profiling strategy is generally applicable to investigating interactions between viruses and host cells and the analysis of other biological systems.

Involvement of Protein Kinase D in Expression and Trafficking of ATP7B (Copper ATPase)

ATP7B is a P-type ATPase involved in copper transport and homeostasis. In experiments with microsomes isolated from COS-1 cells or HepG2 hepatocytes sustaining ATP7B heterologous expression, we found that ATP7B utilization of ATP includes autophosphorylation of an aspartyl residue serving as ATPase catalytic intermediate as well as phosphorylation of serine residues by protein kinase D (PKD). The latter was abolished by specific PKD inhibition with CID755673. The presence of PKD protein in the microsomal fraction was demonstrated by Western blotting. PKD is a serine/threonine kinase that associates with the trans-Golgi network, regulating fission of transport carriers destined to the cell surface. Parallel studies on cultured cells showed that nascent WT ATP7B transits to the Golgi complex where it undergoes serine phosphorylation by PKD. Misfolded ATP7B protein (especially if subjected to deletions) underwent proteasome-mediated degradation, which provides effective quality control. Inhibition of proteasome-mediated degradation with MG132 yielded additional, but nonfunctional protein. On the other hand, serine phosphorylation protected WT ATP7B from degradation. Protection was enhanced by PKD activation with phorbol esters and limited by PKD inhibition with CID75673. As a final step, phosphorylated ATP7B was transferred from the Golgi complex to cytosolic trafficking vesicles. Phosphorylation and trafficking were completely prevented by mutations of critical copper binding sites, demonstrating copper dependence of both PKD-assisted phosphorylation and trafficking. ATP7B trafficking was markedly reduced by the Ser-478/481/1121/1453 to Ala mutation. We conclude that PKD plays a key role in copper-dependent serine phosphorylation, permitting high levels of ATP7B protein expression and trafficking.

Vaccinia Virus A56/K2 Fusion Regulatory Protein Interacts with the A16 and G9 Subunits of the Entry Fusion Complex

Deletion of the A56R or K2L gene of vaccinia virus (VACV) results in the spontaneous fusion of infected cells to form large multinucleated syncytia. A56 and K2 polypeptides bind to one another (A56/K2) and together are required for interaction with the VACV entry fusion complex (EFC); this association has been proposed to prevent the fusion of infected cells. At least eight viral polypeptides comprise the EFC, but no information has been available regarding their interactions either with each other or with A56/K2. Utilizing a panel of recombinant VACVs designed to repress expression of individual EFC subunits, we demonstrated that A56/K2 interacted with two polypeptides: A16 and G9. Both A16 and G9 were required for the efficient binding of each to A56/K2, suggesting that the two polypeptides interact with each other within the EFC. Such an interaction was established by the copurification of A16 and G9 from infected cells under conditions in which a stable EFC complex failed to assemble and from detergent-treated lysates of uninfected cells that coexpressed A16 and G9. A recombinant VACV that expressed G9 modified with an N-terminal epitope tag induced the formation of syncytia, suggesting partial interference with the functional interaction of A56/K2 with the EFC during infection. These data suggest that A16 and G9 are physically associated within the EFC and that their interaction with A56/K2 suppresses spontaneous syncytium formation and possibly "fuse-back" superinfection of cells.

Experimental Transmission and Induction of Ranaviral Disease in Western Ornate Box Turtles (Terrapene ornata ornata) and Red-Eared Sliders (Trachemys scripta elegans)

An experimental transmission study was designed to determine whether a causal relationship exists between a Ranavirus (BSTRV) isolated from a Burmese star tortoise that died and the lesions observed in that tortoise. A pilot study was performed with 3 box turtles (Terrapene ornata ornata) and 3 red-eared sliders (RESs; Trachemys scripta elegans) to assess their suitability in a larger study. Based on the outcome of this study, RESs were selected, and 2 groups of 4 RESs received either an oral (PO) or intramuscular (IM) inoculum containing10(5) 50% Tissue Culture Infecting Dose (TCID(50)) of a BSTRV-infected cell lysate. One turtle each was mock inoculated PO or IM with the same volume of uninfected cell lysate. Three of four IM-inoculated RESs developed clinical signs (nasal and ocular discharge [3 of 3], oral plaques [1 of 3], conjunctivitis and hyphema [1 of 3] and extreme lethargy [3 of 3]). A Ranavirus was isolated from kidney homogenates of 3 euthanatized turtles; DNA sequences of a portion of the major capsid protein gene were amplified by polymerase chain reaction. Consistent histologic lesions were observed only in IM-inoculated turtles and included fibrinoid vasculitis centered on splenic ellipsoids, multifocal hepatic necrosis, and multicentric fibrin thrombi in a variety of locations, including hepatic sinusoids, glomerular capillary loops, and pulmonary capillaries. Virions compatible with Ranavirus were observed within necrotic cells of the spleen of 1 IM-inoculated turtle using transmission electron microscopy. This study fulfills Koch's postulates, confirming a causal relationship between BSTRV and the clinical and histologic changes in chelonians infected with this virus.

The Putative Terminase Subunit of Herpes Simplex Virus 1 Encoded by U L 28 Is Necessary and Sufficient To Mediate Interaction between pU L 15 and pU L 33

Viral terminases play essential roles as components of molecular motors that package viral DNA into capsids. Previous results indicated that the putative terminase subunits of herpes simplex virus 1 (HSV-1) encoded by U(L)15 and U(L)28 (designated pU(L)15 and pU(L)28, respectively) coimmunoprecipitate with the U(L)33 protein from lysates of infected cells. All three proteins are among six required for HSV-1 DNA packaging but dispensable for assembly of immature capsids. The current results show that in both infected- and uninfected-cell lysates, pU(L)28 coimmunoprecipitates with either pU(L)33 or pU(L)15, whereas pU(L)15 and pU(L)33 do not coimmunoprecipitate unless pU(L)28 is present. The U(L)28 protein was sufficient to stabilize pU(L)33 from proteasomal degradation in an engineered cell line and was necessary to stabilize pU(L)33 in infected cells, whereas pU(L)15 had no such effects. The presence of pU(L)33 was dispensable for the pU(L)15/pU(L)28 interaction in lysates of both infected and uninfected cells but augmented the tendency for pU(L)15 and pU(L)28 to coimmunoprecipitate. These data suggest that pU(L)28 and pU(L)33 interact directly and that pU(L)15 interacts directly with pU(L)28 but only indirectly with pU(L)33. It is logical to propose that the indirect interaction of pU(L)15 and pU(L)33 is mediated through the interaction of both proteins with pU(L)28. The data also suggest that one function of pU(L)33 is to optimize the pU(L)15/pU(L)28 interaction.

Development of an enzyme-linked immunosorbent assay for the detection of antibodies against malignant catarrhal fever viruses in cattle serum

Malignant catarrhal fever (MCF) is a sporadic but fatal lymphoproliferative viral disease of cattle, deer and other ruminants. The causative agents are highly-cell-associated herpesviruses of the subfamily gammaherpesvirinae. In this study, an ELISA (WC11-ELISA) was developed to detect antibody to malignant catarrhal fever virus (MCFV) in cattle serum and compared to the commercially produced competitive-inhibition ELISA (CI-ELISA). Crude lysate antigen from alcelaphine herpesvirus-1 strain WC11 was bound to 96-well microplates and used to capture antibodies to MCFV. Dilutions of test sera were added to wells containing bound MCF antigen and control wells containing uninfected cell lysates. A horseradish peroxidase-labelled rabbit-anti-bovine IgG conjugate detected antibodies to MCF, and the results were expressed as absorbance readings at 450 nm. Samples were selected blind from cattle sera which had been sent to the laboratory for diagnostic testing for MCFV antibodies and were tested in both the WC11-ELISA and the CI-ELISA. Good agreement between the WC11-ELISA and CI-ELISA test ( k = 0.86, n = 95) results was found.

Quantitative Analysis of Viral RNA in the Murine Heart and Pancreas with Different Concentration of Coxsackievirus B3

Objectives: We investigated the clinical features, pathologic changes, and viral RNA kinetics in the course of acute and subacute experimental coxsackievirus B3 (CVB3) infection in a murine model. Methods: Five-week-old A/J inbred male mice were divided into 5 groups. Four of those groups were inoculated intraperitoneally with 5 × 10⁴ (group 1), 1 × 10⁵ (group 2), 5 × 10⁵ (group 3), or 1 × 10⁶ (group 4) PFU of CVB3. Control mice were inoculated with uninfected Vero cell lysate in DMEM. Mice from each group were sacrificed on days 7 or 14 after inoculation. Results: Bloody diarrhea, earlier weight loss, perianal swelling, and death were correlated with higher viral load. One of ten mice in group 3 and 5 of 10 mice in group 4 died spontaneously between days 4 and 12 after inoculation. All of the remaining 34 mice of infected groups demonstrated extensive pancreatic inflammation. Focal myocarditis developed in only 4 (11.8%) of those 34 subjects. Amylase and creatine kinase activities in the serum were increased in the mice of infected groups. CVB3 RNA was detected in the heart and pancreatic tissue in all subjects. The CVB3 RNA copy number in pancreatic tissue was not correlated with the severity of inflammation. Conclusions: In the murine model, viral loading dose determines the clinical features of CVB3-induced infection, and the severity of pancreatitis is not correlated with the viral loading dose or tissue level of viral RNA.

Cellular immune responses to feline immunodeficiency virus (FIV) induced by dual-subtype FIV vaccine

Vaccine-induced T cell responses to FIV were assessed by measuring FIV-specific cytokine and cytotoxic-effector molecule production. A total of 22 cats at 10–12 weeks of age received either dual-subtype FIV vaccine ( n = 12), uninfected cell lysate ( n = 5) consisting of cells used to produce vaccine viruses, or no immunization ( n = 5). Significant increases in mRNA and protein production of T-helper 1 (Th1) cytokines (IL-2, IFNγ), mRNA production of a cytotoxic-effector molecule (perforin), and lymphoproliferation response were observed in peripheral blood mononuclear cells (PBMC) from dual-subtype FIV-vaccinated cats after in vitro stimulation with inactivated FIV. In contrast, no statistically significant increase in FIV-stimulated mRNA production of Th2 cytokines (IL-4, IL-6) or other cytotoxic-effector molecules (TNFα, FasL) was observed in the PBMC from dual-subtype vaccinated cats. Moreover, no FIV-specific increases in the IFNγ, IL-2, and perforin mRNA productions and in the IFNγ bioactivity and lymphoproliferation responses were observed in the PBMC from cell-immunized cats. These observations suggest that IFNγ induction, lymphoproliferation, and significant portion of IL-2 and perforin productions in the PBMC from dual-subtype vaccinated cats are clearly specific for viral antigens. Overall, dual-subtype FIV vaccine elicited strong Th1 response (IFN(, IL-2), which may contribute to the vaccine protection by enhancing the perforin-mediated cytotoxic-cell activity against FIV.

Salivary production of IgA and IgG to human herpes virus 8 latent and lytic antigens by patients in whom Kaposi's sarcoma has regressed.

IgG and IgA antibodies with specificities to a latent and a lytic antigen of human herpes virus 8 (HHV-8) were detectable in the saliva and serum of eight patients whose Kaposi's sarcoma had regressed, seven of whom were HIV-1 infected. The measurement of antibody-specific activity and secretion rate, and the detection of secretory IgA all indicate anti-HHV-8 antibody activity in saliva. The specific humoral responses possibly influence mucosal replication of HHV-8, and in turn, that of HIV.

Contributions of Antibody and T Cell Subsets to Protection Elicited by Immunization with a Replication-Defective Mutant of Herpes Simplex Virus Type 1

Replication-defective mutants of herpes simplex virus 1 (HSV-1) elicit immune responses in mice that reduce acute and latent infection after corneal challenge and are protective against development of disease. To understand the basis for the protective immunity induced by this new form of immunization, we investigated the contribution of various components of the immune response to protection against corneal infection and disease. Passive transfer of sera from mice immunized with the replication-defective mutant virus,d301, its parental HSV-1 strain, or uninfected cell lysate was used to examine the role of antibody. Despite posttransfer neutralizing antibody titers equivalent to those in control mice directly immunized with mutant virus, recipients of immune serum showed no reductions in primary replication in the eye, keratitis, or latent infection of the nervous system. However, immune serum protected mice from encephalitis and death. To examine the contribution of T cell subsets to protection, mice were immunized once with mutant virus and then were depletedin vivoof CD4+or CD8+T cells prior to corneal challenge. CD4 depletion resulted in higher titers of challenge virus in the eye at 3 to 4 days after challenge compared to control mice. Latent infection of the nervous system was increased by depletion of CD4+T cells but not by depletion of CD8+T cells. Keratitis developed only in a portion of the CD8+T cell-depleted mice, suggesting that an immunopathologic potential of CD4+T cells is held in check when immune CD8+T cells are also present. Taken together, these data support a role for antibody induced by immunization with a replication-defective virus principally in protecting the central nervous system from disease, roles for CD4+T cells in reducing primary replication in the eye and protecting against latent infection of the nervous system, and a role for CD8+T cells in regulating the immunopathologic activity of CD4+T cells.

Histones as a Target for Influenza Virus Matrix Protein M1

Matrix protein M1 purified from influenza A and B viruses has been analyzed for its ability to specifically interact with cellular proteins by immune coprecipitation and by anin vitrobinding assay on nitrocellulose on PVDF membranes. When M1 was mixed with lysates of uninfected cells there was selective binding of histones H2A, H2B, H3, and H4. Week binding of H1 was also observed. The binding specificity of M1 was confirmed by using purified histones. The M1–histone complexes were dependent on pH and ionic strength, indicating electrostatic interactions. Chemical cleavage of M1 by formic acid into an N-terminal 9-kDa fragment and a C-terminal 18-kDa fragment did not abolish interaction with histones. However, after treatment with 1Msodium chloride cleaved M1 no longer bound to histones, whereas uncleaved M1 showed an increased binding activity after salt treatment. These findings suggest that both N- and C-terminal domains of M1 are involved in histone binding and that conformation of M is an important factor in this interaction. The data support the notion that there is specific interaction of M1 with nucleosomes during the nuclear phase of influenza virus replication.

Herpes simplex virus vector system: analysis of its in vivo and in vitro cytopathic effects

With its natural propensity to infect and establish life-long latency in neurons, herpes simplex virus type 1 (HSV-1) has been successfully employed by various laboratories as vectors for gene transfer into neurons. However, analysis of its cytopathic effects in vivo and in vitro has been limited. In this study, we examined the cytopathic effects of 2 HSV-1 α4 mutants (ts756 and d120) on adult rat hippocampus and striatum and of d120 on hippocampal neurons in culture. We assessed damage by stringent counting of surviving neurons after infection and demonstrated that while neither ts756 nor d120 infection resulted in any gross anatomical or behavioral changes of the animals, ts756, but not d120, produced a significant amount of damage in the CA4 cell field and dentate gyrus of the hippocampus. Thus, since crude examination is insufficient to detect subtle but significant degrees of neuron loss, the cytopathic effects of HSV or any vector system must be carefully analyzed. Furthermore, we also observed that uninfected cell lysates damaged neurons, both in vivo and in vitro. This cytotoxicity occurred within the first 24 h post-inoculation and probably arose through the activation of glutamate receptors. For the preparation of HSV vectors, purification of the virus from soluble cellular components by a simple pelleting step can significantly decrease such acute toxicity.

Human Herpesvirus 6 Inhibits the Proliferative Responses of Human Peripheral Blood Mononuclear Cells

Human herpesvirus 6 (HHV-6) was recently isolated from the blood of immunosuppressed individuals and children with exanthem subitum. In this study, the effect of HHV-6 on the proliferative response of human peripheral blood lymphocytes was tested. T cell lines or human peripheral blood lymphocytes were infected with HHV-6 variant A, strain GS, or variant B, strain Z29. Infected and uninfected cell lysates were UV inactivated and sonicated; when lysates were added to peripheral blood mononuclear cells from HHV-6-seropositive adults, lymphoproliferative responses to antigens (tuberculin purified protein derivative or mumps) and mitogens (phytohemagglutinin or interleukin-2) were inhibited in a dose-dependent manner. This inhibition was reversed by a rabbit anti-HHV-6(GS) antiserum, and inhibition was not due to cell lysis induced by viral infection, since cell death was not observed. This in vitro suppression of cellular immune responses by HHV-6 proteins may be relevant to the in vivo pathogenesis of HHV-6 and its relationship to human diseases.

Characterization of the in Vitro System for the Synthesis of mRNA from Human Respiratory Syncytial Virus

An in vitro transcription system for human respiratory syncytial virus (RSV) is described. Purified viral nucleocapsid (RNP) isolated from virus-infected cells was shown to support transcription of all 10 genes encoded by the virus as determined by Northern blot hybridization. The mRNAs synthesized were polyadenylated and comigrated with the corresponding mRNAs synthesized in virus-infected cells when analyzed in agarose-urea gel electrophoresis. The in vitro-synthesized mRNAs are functional as determined by their capacity to synthesize protein in vitro. The transcriptional reaction was significantly stimulated by the uninfected host cell lysate, indicating a requirement of host factor(s) in mRNA synthesis. Preliminary results suggest that cellular actin is involved in this process.

Production and Characterization of a Monoclonal Antibody Directed Against HTLV-1 pl9: Use in a Specific Capture Enzyme Immunoassay

An enzyme immunoassay (EIA) was developed for detection of Human T-cell Leukemia Virus antigen in culture supernatants and cell lysates. The assay used a mouse monoclonal antibody against HTLV-I p19 major core protein as capture antibody. It has a sensitivity of 1 microgram/ml of HTLV-I protein, 250 pg/ml of purified recombinant p19 and detected p19 in an 10(-2) diluted supernatant of MT2 infected cell and in a 100 MT2 cells lysate (10(6) cells taken at day 7 of culture). The assay enable us to discriminate between HTLV-I and HTLV-II antigens and is reproducibly negative for supernatants and cell lysates of uninfected cells and of HIV-1 infected cells. The assay was found to be more specific and 10 times more sensitive than the reverse transcriptase (RT) assay, and the EIA test became positive three days earlier than RT assay for the HTLV-I cell lines supernatants.

Poliovirus infection results in structural alteration of a microtubule-associated protein.

Poliovirus infection results in profound changes in cellular metabolism and architecture. To identify alterations in cellular proteins following poliovirus infection which might account for these changes, monoclonal antibodies were prepared by screening for differences in antigen pattern in infected and uninfected cell lysates. Further characterization of the antigen of one such antibody (25 C C1) is described in this report. The 25 C C1 antigen is a cytoskeleton-associated protein which decreases in size 4 to 5 h postinfection. It copurifies with some of the protein synthesis initiation factors but not with eucaryotic initiation factor (eIF)-4F, the p220 subunit of which is cleaved following infection (D. Etchison, S. C. Milburn, I. Edery, N. Sonenberg, and J. W. B. Hershey, J. Biol. Chem. 257:14806-14810, 1982). Unlike alteration of p220, alteration of the 25 C C1 antigen is not due to a protease which can be detected by cell lysate mixing experiments. Alteration of the antigen occurs during purification, suggesting progressive proteolysis, but the alteration is more extensive in preparations from infected cells than in those from uninfected cells. A recombinant phage expressing the antigenic determinant was isolated from a human fibroblast cDNA library, and the sequence of the cDNA insert was found to be entirely contained within the established sequence of microtubule-associated protein (MAP) 4 (R. R. West, K. M. Tenbarge, and J. B. Olmsted, J. Biol. Chem. 266:21886-21896, 1991). The antigen distribution, as detected by indirect immunofluorescence, was similar to, but more diffuse than, the distribution of tubulin. The antibody recognized the largest abundant HeLa cell MAP, which copurified with tubulin after three cycles of polymerization-depolymerization, thus confirming the identity of the antigen as MAP 4. These results indicate that poliovirus infection of HeLa cells affects the structural integrity of a cytoskeletal protein, MAP 4.

Differential polymerase chain reaction for detection of wild-type and a vaccine strain of Aujeszky's disease (pseudorabies) virus

A polymerase chain reaction (PCR) strategy for differentiating between a vaccine mutant strain and wild-type (WT) strains of Aujeszky's disease (pseudorabies) virus (ADV) was evaluated. With this approach, a single virus or a concurrent WT and vaccine virus infection could be distinguished by targeting the genomic alteration within the vaccine strain. PCR primers were designed for a recombinant vaccine virus that has almost all of the WT gX gene replaced by the lacZ gene. One primer, corresponding to a conserved sequence upstream of the altered region, was selected for common use. The differentiating primers were chosen from the unique WT gX and vaccine lacZ gene sequences. The sensitivity of the differential PCR was analyzed using extracted viral DNA and in vitro infected cell lysates. Approximately 10 and between 10 to 100 molecules of WT and vaccine viral DNAs, respectively, could be detected, regardless of the presence or absence of uninfected cell lysates. Detection of viral DNA from in vitro infected cell cultures approximated this level of sensitivity. The specificity of the amplifications was verified by restriction endonuclease analysis and Southern hybridization. Although the vaccine primer pair target was amplified to a lesser degree as compared to the WT primer pair product, utility of the differential PCR was demonstrated using trigeminal nerve ganglia from swine infected with vaccine virus and WT virus. Both viral targets were detected only by their specific primer pair, in either the single or dual infection.

Alzheimer beta/A4-amyloid precursor protein: evidence for putative amyloidogenic fragment.

Recombinant baculovirus was used to overexpress human Alzheimer beta/A4-amyloid precursor protein (APP) in Spodoptera frugiperda (Sf9) cells. Lysates of these cells were then analyzed for the presence of carboxyl-terminal fragments of APP by an immunoblotting assay using either an antibody against the APP cytoplasmic domain (rabbit anti-human 695APP645-694) or an antibody against the amino terminus of beta/A4-amyloid (rabbit anti-human 695APP586-606). Anti-human 695APP645-694 identified APP holoprotein, a 25-kDa species, and a prominent group of carboxyl-terminal fragments of 17, 16, and 14 kDa, whereas anti-human 695APP586-606 identified APP holoprotein and a single prominent low-molecular-mass protein species comigrating with the 17-kDa carboxyl-terminal fragment identified by anti-human 695APP645-694. No immunoreactive species was detected at these molecular mass positions when either antibody was used for analysis of lysates of either uninfected Sf9 cells or Sf9 cells infected with wild-type Autographa californica baculovirus. For each antibody, specific immunoreactivity was abolished by preabsorption with the corresponding peptide immunogen. The incorporation of a beta/A4-amyloid amino-terminal epitope into a 17-kDa fragment of APP suggests that, in the baculoviral overexpression system, the electrophoretic microheterogeneity of APP carboxyl-terminal fragments is due, at least in part, to alternative proteolysis of APP. If such carboxyl-terminal fragments of APP containing an intact beta/A4-amyloid domain are produced in human brain, then they may represent intermediates in the conversion of APP to deposited beta/A4-amyloid.(ABSTRACT TRUNCATED AT 250 WORDS)

A monoclonal antibody capture ELISA to detect antibody to border disease virus in sheep serum

An enzyme-linked immunosorbent assay (ELISA) has been developed to detect antibody to border disease virus (BDV) in sheep serum. A monoclonal antibody bound to 96-well microplates was used to capture antigen from detergent-solubilised BDV-infected cells. Single dilutions of test sera were then added to wells containing bound BDV antigen and control wells containing uninfected cell lysates. Specific antibody to BDV was detected by an anti-ovine IgG antiserum conjugated with horseradish peroxidase and the results expressed as ELISA units with reference to a standard curve. Sequential sera from 16 experimentally infected sheep and single sera from 103 sheep involved in a field outbreak were tested in the ELISA and for neutralising antibody. There was good qualitative correlation between the two tests.