Regulation of TNF-related apoptosis-inducing ligand-mediated death-signal pathway in human beta cells by Fas-associated death domain and nuclear factor kappaB.

Transfectants of human CM and NES2Y beta cell lines and primary islets transfected by FADD-DN (dominant-negative form of Fas-associated death domain), a mutant of FADD and/or a superrepressor of nuclear factor kappaB (NF-kappaB) (AdIkappaB(SA)2), were examined for their susceptibility to the TRAIL (TNF-related apoptosis-inducing ligand)-induced death signal pathway, compared with controls, wild-type cells, and vector transfectants in caspase fluorescence, Western blot, electrophoretic mobility shift, apoptosis, and cytotoxicity assays. FADD-DN inhibited caspase-8 activation induced by TRAIL in the transfectants of CM and NES2Y cells. TRAIL-induced apoptosis and cytotoxicity to the FADD-DN transfectants were decreased in comparison to those responses in controls (CM, p < 0.01 and p < 0.01; NES2Y, p < 0.05, and p < 0.02, respectively). When CM, NES2Y, and primary islet cells were transfected by AdIkappaB(SA)2, TRAIL-induced IkappaB degradation and nuclear translocation of NF-kappaB p50/p65 were blocked. TRAIL-induced apoptosis and cytotoxicity to AdIkappaB(SA)2 transfectants of these cells were also reduced (CM, p < 0.02 and p < 0.02; NES2Y, p < 0.01 and p < 0.01, respectively, and islet p < 0.01 for cytotoxicity). Finally, cytotoxicity induced by TRAIL in CM and NES2Y cells transfected with both FADD-DN and AdIkappaB(SA)2 was reduced, compared with that observed in these cells transfected with either FADD-DN alone or AdIkappaB(SA)2 alone, suggesting that FADD and NF-kappaB have synergistic proapoptotic regulatory effects on the susceptibility of beta cell lines and islet cells to TRAIL-induced destruction.

A genetically engineered attenuated coxsackievirus B3 strain protects mice against lethal infection.

Coxsackievirus B3 (CVB3) is a common human pathogen that is endemic throughout the world. There is currently no vaccine available, although the virus is known to be highly lethal to newborns and has been associated with heart disease and pancreatitis in older children and adults. Previously, we showed that the virulence of CVB3 is reduced by a lysine-to-arginine substitution in the capsid protein VP2 (K2168R) or a glutamic acid-to-glycine substitution in VP3 (E3060G). In this report, we show that the double mutant virus CVB3(KR/EG) displays additional attenuation, particularly for the pancreas, in A/J mice. In addition, two other attenuating mutations have been identified in the capsid protein VP1. When either the aspartic acid residue D1155 was replaced with glutamic acid or the proline residue P1126 was replaced with methionine, the resulting mutant also possessed an attenuated phenotype. Moreover, when either of these mutations was incorporated into CVB3(KR/EG), the resulting triple mutant viruses, CVB3(KR/EG/DE) and CVB3(KR/EG/PM), were completely noncardiovirulent and caused only small foci of damage to the pancreas, even at a high dose. Both triple mutants were found to be immunogenic, and a single injection of young A/J mice with either was found to protect them from a subsequent lethal challenge with wild-type CVB3. These findings indicate that the triple mutants could be exploited for the development of a live attenuated vaccine against CVB3.

Attenuating mutations in coxsackievirus B3 map to a conformational epitope that comprises the puff region of VP2 and the knob of VP3.

Ten antibody escape mutants of coxsackievirus B3 (CVB3) were used to identify nucleotide substitutions that determine viral virulence for the heart and pancreas. The P1 region, encoding the structural genes of each mutant, was sequenced to identify mutations associated with the lack of neutralization. Eight mutants were found to have a lysine-to arginine mutation in the puff region of VP2, while two had a glutamate-to-glycine substitution in the knob of VP3. Two mutants, EM1 and EM10, representing each of these mutations, were further analyzed, initially by determining their entire sequence. In addition to the mutations in P1, EM1 was found to have two mutations in the 3D polymerase, while EM10 had a mutation in stem-loop II of the 5' nontranslated region (5'NTR). The pathogenesis of the mutants relative to that of CVB3 strain RK [CVB3(RK)] then was examined in A/J mice. Both mutants were found to be less cardiotropic than the parental strain, with a 40-fold (EM1) or a 100- to 1,000-fold (EM10) reduction in viral titers in the heart relative to the titers of CVB3(RK). The mutations in VP2, VP3, and the 5'NTR were introduced independently into the RK infectious clone, and the phenotypes of the progeny viruses were determined. The results substantiated that the VP2 and VP3 mutations reduced cardiovirulence, while the 5'NTR mutation in EM10 was associated with a more virulent phenotype when expressed on its own. Stereographic imaging of the two mutations in the capsomer showed that they lie in close proximity on either side of a narrow cleft between the puff and the knob, forming a conformational epitope that is part of the putative binding site for coreceptor DAF.

Lack of islet neogenesis plays a key role in beta-cell depletion in mice infected with a diabetogenic variant of coxsackievirus B4.

Group B coxsackieviruses (CVBs) have a well-established association with type 1 diabetes but the mechanism of depletion of beta-cell mass following infection has not yet been defined. In this report we show that the major difference in pathogenesis between the E2 diabetogenic strain of CVB4 and the prototypic JVB strain in SJL mice is not in tropism for islet cells but in the degree of damage inflicted on the exocrine pancreas and the resulting capacity for regeneration of both acinar and islet tissue by the host. Both strains replicated to a high titre in acinar tissue up to day 3 post-infection (p.i.), while the islets of Langerhans were largely spared. However, the pancreas in the JVB-infected animals then regenerated and many small islets were seen throughout the tissue by day 10 p.i. In contrast, the acinar tissue in E2-infected mice became increasingly necrotic until all that remained by day 21 p.i. were large islets containing varying numbers of dead cells, caught up in strands of connective tissue. Surviving beta cells were found to synthesize little insulin, although islet amyloid polypeptide was detected and glucagon synthesis in alpha cells appeared normal or enhanced. Our results suggest that the key to CVB-E2-induced damage lies in the exocrine tissue and prevention of islet neogenesis rather than from direct effects on existing islets.

Mapping of genetic determinants of rubella virus associated with growth in joint tissue.

Rubella virus (RV) strains vary in their abilities to replicate and persist in cell cultures derived from human joint tissue (synovial cells [SC]), and this arthrotropism appears to be linked to their association with joint symptoms in vivo. In order to map the genetic determinants of arthrotropism, an infectious clone of the Cendehill vaccine strain of RV was constructed, as well as two chimeric clones containing cDNAs from both Cendehill and Therien (wild-type) strains. Replacement of the entire structural gene region of Therien in the infectious clone pROBO302 with the corresponding region of Cendehill did not affect growth in SC. A further observation that Cendehill bound equally well to SC and the permissive Vero cell line indicated that restriction was not at the level of receptor binding, a function of the envelope proteins. Mutations that affected growth in joint cells were mapped to two locations in the nonstructural gene region. The first of these (nucleotides 2803 and 6416) resulted in a 10-fold decrease in yield of progeny virus from SC. This region contained five mutations, at nucleotides 2829, 3060, 3164, and 3528 (near the carboxy terminus of P150 where the protease domain is located) and at nucleotide 4350 in p90. Further substitution of the sequence representing nucleotides 1 to 2803 to give a complete Cendehill infectious clone restricted growth in SC by a further 100-fold to less than 10 PFU/ml. This region contains three mutations, at nucleotides 34, 37, and 55, within the 5' stem-loop structure. In conclusion, the Cendehill-specific mutations believed to be determinants of joint cell growth are located in two regions, the 5' nontranslated region and in a sequence that encodes the carboxy-terminal region of p150 extending into the helicase domain of p90.

Characterization of a highly conserved baculovirus structural protein that is specific for occlusion-derived virions.

A highly conserved baculovirus late gene called odvp-6e was shown to be a structural protein that is specific for occlusion-derived virus (ODV) envelopes. The complete sequence of this gene is presented for both Orgyia pseudotsugata nuclear polyhedrosis virus (OpMNPV) and Cydia pomonella granulosis virus (CpGV). The predicted sizes of the OpMNPV and CpGV ODVP-6E are 40, 241, and 38,655 respectively. The OpMNPV odvp-6e gene was transcriptionally mapped and was shown to initiate from a consensus late gene motif, TTAAG, and is expressed from 18-120 hr postinfection. Polyclonal antiserum was generated against a bacterial fusion protein and used to analyze the cellular steady-state levels of ODVP-6E and to determine if this protein was a component of either budded virus (BV) or ODV. Western blots showed that ODVP-6E is a component of the ODV but not BV. This was confirmed by immunoelectron microscopy of ODV from Autographa californica NPV (AcMNPV) which localized ODVP-6E to the ODV envelope. The sequences of the odvp-6e gene from the baculoviruses Choristoneura fumiferana NPV (CfMNPV), AcMNPV, and Helicoverpa zea NPV (HzSNPV) were obtained from GenBank. Comparisons of the predicted amino acid sequences of OpMNPV, CpGV, AcMNPV, CfMNPV, and HzSNPV show that there are two possible membrane-spanning domains and a cysteine-rich domain that are conserved in all of the proteins.

Selective infection of astrocytes in human glial cell cultures by rubella virus.

BACKGROUND: Rubella virus (RV) can cause a wide variety of neurologic symptoms, particularly when infection occurs in utero. However, little is known about the pathogenesis of these infections and the cell types in human brain susceptible to infection have not been characterized. EXPERIMENTAL DESIGN: Cell cultures derived from human brain tissue were examined for susceptibility to two wild-type and two vaccine strains of RV. Cell types expressing viral antigen were identified by double-label immunofluorescence with monoclonal antibodies to specific cell markers and a polyclonal anti-RV antibody. Viral yield was determined by plaque assay. RESULTS: All four RV strains replicated in the brain cultures, although the titers obtained in the case of the vaccine strains were more than 100-fold lower than those found for wild-type isolates. Astrocytes appeared to be the main cell type infected, expressing large amounts of viral antigen. In contrast, oligodendrocytes were rarely productively infected, even when surrounded by infected astrocytes. Occasional neurons expressing viral antigen were also seen. CONCLUSIONS: The main cell type permissive to RV infection in developing brain tissue is the astrocyte. Although not highly cytolytic, the virus may disrupt the functioning of these cells, resulting in neurologic deficits. The restricted replication of this virus in oligodendrocytes correlates with the lack of demyelination generally reported as being typical of RV neurologic disease.

Characterization of rubella virus strain differences associated with attenuation.

A comparison of the phenotypic properties of three rubella vaccines (HPV77/DE5, RA27/3 and Cendehill) and four wild-type (wt+) isolates (M33, Therien, Thomas and IB2) has been carried out. Differences in growth characteristics, plaque morphology and temperature sensitivity were identified. In addition differential reactivity of the strains to polyclonal and a monoclonal anti-E1 antibody were found in immunoperoxidase-staining reactions. The ability of the wt+ and vaccine strains to infect lymphoreticular cells and chondrocytes, also varied in that the RA27/3 and Cendehill strains were highly restricted in both these cell types while the wt+ strains and HPV77/DE5 vaccine grew to higher titer. This biological variation was associated with differences in E1 and E2 glycoproteins detected on immunoblots.

Characterization of a strain of murine cytomegalovirus which fails to grow in the salivary glands of mice.

Characterization of a tissue culture-adapted strain of murine cytomegalovirus (MCMV), the Vancouver strain, which demonstrated altered tissue tropism in mice was undertaken to help understand the mechanism of pathogenesis of cytomegaloviruses. The Vancouver strain grew to a limited extent in the spleen but failed to grow in the salivary glands of inoculated mice. This mutation probably arose during multiple in vitro passaging of the parental Smith strain. The Vancouver strain replicated more quickly and produced a greater yield of virus per cycle than the Smith strain in vitro, resulting in a larger plaque size. In addition to these phenotypic differences, the Vancouver strain was found to have a 9.4 kb deletion spanning the XbaI I/L junction of the parental Smith strain (0.960 to 0.995 map units), and a 0.9 kb insertion which mapped to the EcoRI K fragment (0.37 to 0.47 map units). Analysis of virus-induced proteins at various times post-infection identified only one major change in Vancouver strain-infected cells, the absence of a 42K protein found in Smith-infected cells at early and late times.

Non-permissiveness of synovial membrane cells to human parvovirus B19 in vitro.

The ability of cultured human synovial cells derived from synovial membrane and cartilage to support the replication of human parvovirus B19 was assessed. No viral DNA synthesis nor viral antigens were detected suggesting that B19 virus is not capable of replicating in synovial cells. The significance of this finding in relationship to the pathogenesis of parvovirus arthritis is discussed.

Differential ability of wild-type and vaccine strains of rubella virus to replicate and persist in human joint tissue.

Natural rubella has been reported to be associated with a higher incidence of arthropathy than immunisation with rubella vaccine. In addition, the different vaccines (HPV77/DE5, RA27/3, Cendehill) have been shown to vary in their association with joint symptoms in clinical trials. To investigate possible reasons for these differences in arthritogenicity, the susceptibility of human joint tissue to five rubella virus strains (three vaccines and two wt+) has been examined. Human joint tissue in either organ or dispersed cell-culture was infected in vitro and the degree of replication and persistence of each rubella strain compared. The wt+ strains (M33 and Therien) replicated to high titre in both cell and organ cultures and persisted for over 2 months. The HPV77/DE5 strain (Meruvax I) showed a very similar pattern. In contrast, the replication of RA27/3 (Meruvax II) and Cendehill (Cendevax) was highly restricted in joint cells and both of these strains showed very limited ability to penetrate and persist in the organ cultures. These results concur with the differences in arthritogenicity observed between the strains in vivo, suggesting that local viral replication may play a role in the pathogenesis of rubella-associated arthritis.

Susceptibility of normal human joint tissue to viruses.

A model system has been developed to investigate the comparative ability of different viruses to replicate and persist intraarticularly. The viruses chosen for study were rubella, mumps, Coxsackie B4, adenovirus and varicella zoster, a selection of viruses with different degrees of association with joint symptoms in clinical studies. Our results showed that these viruses demonstrated a range of abilities to infect and persist in human joint tissue cultured in vitro. The most arthritogenic viruses, rubella, and to a lesser extent mumps, replicated and penetrated deeply into the synovial membrane. In contrast, the other 3 viruses were much less arthrotropic, and may only induce arthritis by immunopathological mechanisms.

Restricted mumps virus infection of cells derived from normal human joint tissue.

Mumps virus (MuV) is known to be associated with acute arthritis and may also have a role in chronic inflammatory joint disease. The mechanism of induction of joint inflammation is not known but may be associated with direct invasion of joint tissue. To investigate the possibility of persistent intra-articular infection, the interaction of MuV with primary cells from normal human joint tissue was examined. These mixed cultures of synovial membrane cells and chondrocytes were found to be semi-permissive to the virus; only a small proportion of cells (5 to 20%) were infected and produced low titres of progeny virions. In addition, little viral antigen was detected on the cell surface relative to that found on Vero cells. This restricted infection of synovial membrane cells was related to a severely decreased synthesis of the viral glycoproteins, fusion and haemagglutinin-neuraminidase, and the membrane protein in comparison to the levels found in Vero cells. Persistent infections were readily established and could be maintained for 2 to 3 months. During the first month, the infection remained highly focal and supernatant viral titres were low. Thereafter both the percentage of infected cells and viral titres increased until finally the cultures were killed. No evidence was obtained for the generation of temperature-sensitive mutants or defective interfering particles during long-term infection, but the persistent virus derived from the cultures gave cloudy plaques and induced no fusion in Vero cells until passaged. This study has shown that human synovial tissue cells have the intrinsic ability to support MuV replication and persistence which may be important in the pathogenesis of mumps arthritis.

Congenital rubella syndrome associated with calcific epiphyseal stippling and peroxisomal dysfunction.

An infant girl had the clinical and immunologic findings of congenital rubella syndrome but also had arthrogryposis multiplex and calcific epiphyseal stippling. Spastic quadriparesis developed, and both physical and behavioral development were slow. Increased spasticity of the legs at 5 1/2 years was related not to progressive rubella encephalomyelopathy but to spinal cord compression by abnormal cartilaginous tissue. The presence of a peroxisomal disorder was demonstrated by a greatly increased level of phytanic acid and slightly increased levels of hexacosanoate in serum and by reduced activity of peroxisomal dihydroxyacetone phosphate acyltransferase and a slightly increased ratio of cytosolic to peroxisomal catalase activity in cultured fibroblasts. A reduction in the number and size of peroxisomes was demonstrated in cultured fibroblasts, and a needle biopsy specimen of the liver also showed the peroxisomes to have a smaller diameter than usual. We recommend that any child with epiphyseal stippling be assessed for peroxisomal disease and that the potential for spinal cord compression by dysplastic bone or cartilage be recognized. The association of peroxisomal dysfunction with congenital rubella has not been described previously. The interaction between rubella virus infection and peroxisomal function may need further investigation.

Interaction of rubella virus with human immune cells. I. Permissiveness of lymphocyte subpopulations.

The ability of rubella virus to infect and replicate in various lymphocyte subpopulations was examined. Purified populations of B-cells, CD4+ T-cells and CD8+ T-cells were found to support high levels of viral replication. In addition, when mixed PBMC were infected in vitro, viral antigen was shown to be expressed on the surfaces of CD4+ and CD8+ T-cells by flow cytometry indicating that RV does not have a selective tropism for a specific type of lymphoreticular cell.

The effect of antibody on rubella virus infection in human lymphoid cells.

The effect of rabbit anti-rubella virus (RV) serum on the course of RV infection of human lymphoid cells has been examined. The antibody was found to abolish viral replication such that no virus progeny could be detected either extracellularly (after removal of the antiserum) or intracellularly, in the treated cells. Viral protein synthesis was also found to be totally inhibited in the presence of anti-RV but resumed immediately on removal of the antibody block at levels suggesting that viral RNA had been accumulating in the cell. Infectious focus assays indicated that the effect could be explained in part by the restriction by antiviral antibody of cell-to-cell spread in the indicator monolayer. Thus by 48 h when 100% of lymphoid cells infected with virus alone were capable of forming infectious foci, only 40 to 50% of cells infected in the presence of antiviral antibody produced foci. However this restriction imposed by antibody did not adequately explain the total inhibition of viral protein synthesis that occurred under these conditions. Pretreatment of RV with excess neutralizing antiserum reduced the virus titre greater than 99.9% but did not eliminate infectious virus. The 'non-neutralized' fraction (10(2) to 10(3) p.f.u./ml) brought about a productive infection with final yields of 10 to 25% of control titres (greater than 10(5) p.f.u./ml). Incubation of virus with both anti-RV and goat anti-rabbit Ig eliminated viral infectivity, indicating that the 'non-neutralized' fraction was in the form of infectious immune complexes. The results suggest two major modes of action for anti-RV antibody, one in neutralizing extracellular virus, the second exerted on the infected cells, totally inhibiting viral translation and thus preventing the virus from completing its replicative cycle.

Rubella-associated arthritis. I. Comparative study of joint manifestations associated with natural rubella infection and RA 27/3 rubella immunisation.

Joint manifestations observed during the course of a prospective RA 27/3 rubella immunisation trial were compared with those observed during an intercurrent wild rubella epidemic in an outlying community. Among 44 rubella haemagglutination inhibition (HAI) negative females ranging in age from 17 to 33 years who received rubella vaccine, six (13.6%) developed acute polyarticular arthritis within two to four weeks postvaccine and two (4.5%) had continuing or recurrent arthropathy lasting longer than 18 months. In contrast, among 23 females ranging in age from 11 to 39 years undergoing wild rubella infection, 12 (52.2%) developed acute polyarticular arthritis and seven (30.4%) had recurrent arthropathy 18 months postinfection. Among 23 males ranging in age from 13 to 54 years undergoing wild rubella infection, only two (8.7%) developed acute arthritis and both individuals had continuing joint manifestations 18 months postinfection. Wild rubella infection in adult populations is associated with a higher incidence, increased severity, and more prolonged duration of joint manifestations than is seen after RA 27/3 rubella immunisation.

Persistent rubella virus infection associated with chronic arthritis in children.

We isolated rubella virus from lymphoreticular cells in 7 of 19 children with chronic rheumatic disease, including patients with systemic-onset juvenile rheumatoid arthritis (Still's disease) (1 of 5), polyarticular juvenile rheumatoid arthritis (2 of 2), pauciarticular juvenile rheumatoid arthritis (2 of 6), and seronegative spondyloarthritis (2 of 6). In contrast, rubella virus was not isolated from the control group, which included eight normal subjects and eight patients with other connective tissue diseases or traumatic joint effusion. In most members of the study group, mononuclear cells from both synovial fluid and peripheral blood were examined. Rubella virus was isolated from both cell populations in three patients, from only peripheral blood in one, and from only synovial fluid in two. In the children with systemic-onset juvenile rheumatoid arthritis, only peripheral blood was examined, and of the five samples analyzed, one was shown to have rubella virus. Virus was isolated on more than one occasion from four of seven persons. Persistence of rubella virus in lymphoreticular cells in 35 per cent of these cases of juvenile arthritis supports the view that the virus may be an etiologic agent in chronic human joint disease, but further work will be required to support this suggestion.

Postpartum rubella immunization: association with development of prolonged arthritis, neurological sequelae, and chronic rubella viremia.

Six women developed chronic long-term arthropathy after postpartum immunization against rubella. All individuals developed acute polyarticular arthritis within 12 days to three weeks postimmunization and have had continuing chronic or recurrent arthralgia or arthritis for two to seven years after vaccination. Acute neurological manifestations, consisting of carpal tunnel syndrome or multiple paresthesiae, developed postvaccination in three women. Two have developed continuing active or chronic recurrent episodes of blurred vision, paresthesiae, and painful limb syndromes together with recurrent joint symptoms. Chronic rubella viremia has been detected in peripheral blood mononuclear cell (MNC) populations in five of the six women up to six years after vaccination. In addition rubella virus was isolated from breast milk MNCs in one individual at nine months postvaccination and from peripheral blood MNCs in two of four breast-fed infants studied at 12-18 months of age. Immune responses to rubella virus studied at sequential intervals after vaccination correlated with development of rheumatologic and neurological manifestations.

Sequential studies on synovial lymphocyte stimulation by rubella antigen, and rubella virus isolation in an adult with persistent arthritis.

The response of synovial lymphocytes from a 65-year-old lady with persistent polyarthritis, to rubella antigen and a number of other microbial agents was studied over a period of 11 months by [3H]thymidine incorporation. The results were correlated with the ability to isolate rubella virus from both peripheral blood and synovial fluid during the same period. The patient showed initially a maximal stimulation index to rubella antigen assayed on five occasions over a five-month period. Rubella virus was detected in both peripheral blood and synovial fluid samples on three occasions during this period. Five months later the lymphoproliferative response of her synovial lymphocytes to rubella antigen had dropped to low levels, and virus could no longer be isolated from synovial exudates. At this time the patient's arthritis had become much less active, indicating that a good correlation existed between the presence of rubella virus, local lymphocyte sensitisation, and the inflammatory reaction.