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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 (5NTR). 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 5NTR 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 5NTR 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.

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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.

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.

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.

Group B coxsackieviruses (CVBs) are a major cause of viral myocarditis and pancreatitis in humans and produce a similar pattern of disease in inbred strains of mice. As there are six strains of CVBs, individuals can be infected with multiple serotypes. This raises the possibility of antibody enhancement of infectivity (AEI) by cross-reactive but non-neutralizing antibody to a different strain from a prior infection. To determine whether AEI plays a role in coxsackievirus pathogenesis, an in vitro system using the murine macrophage cell line J774.1 was tested for enhanced infection when incubated with CVB3 plus anti-CVB2 antibody. Yields of virus were found to increase by 10-50-fold and the percentage of infected cells increased proportionately. The effect was Fcmediated as F(abh) 2 fragments of the antibody could not mediate the effect. To determine whether AEI could also be demonstrated in vivo CVB3 was injected into 5-week-old mice together with mouse polyclonal anti-CVB2. Controls included mice injected with PBS or CVB3 alone. Results showed that the titres of virus in tissues of animals injected with virus plus antibody were 1-2 logs higher than when virus was injected alone. This was accompanied by greater histopathological damage, particularly in the heart. These results have implications for human disease as infection with multiple strains likely occurs during the lifetime of an individual.

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.