Virus B3 Research Articles

Analysis of picornavirus 2A(pro) proteins: separation of proteinase from translation and replication functions

The poliovirus (PV) genome was manipulated by replacing its 2A-encoding sequence with the corresponding sequence of coxsackie B4 virus (CBV4) or human rhinovirus type 2 (HRV2). In vitro translation of the resulting chimeric PV genomes revealed a normal cis-cleavage activity for both heterologous 2A(pro) proteinases in the chimeric PV polyproteins. However, only the genome containing the 2A-encoding sequence of CBV4 (PV/CBV4-2A) yielded viable virus in transfected cells, producing a mixture of large and small plaques on HeLa cell monolayers. The large-plaque variants were found to contain single-amino-acid mutations at a specific site near the C terminus of the CBV4 2A(pro) protein. When the same single-amino-acid mutations were directly introduced into the parental PV/CBV4-2A genome, chimeric viruses with a large-plaque phenotype and a wild-type PV-like growth pattern were obtained upon transfection, an observation demonstrating that these point mutations alone had a drastic effect on the growth of the PV/CBV4 chimeric virus. On the other hand, the chimeric genome containing the 2A-encoding sequence of HRV2 (PV/HRV2-2A) produced a null phenotype in transfected HeLa cells, although low-level replication of this chimeric genome was evident. We conclude that only 2A(pro) of the more closely related enterovirus CBV4 is able to functionally substitute for that of PV in vivo, and a subtle genetic modification of the CBV4 2A(pro) protein results in a drastic improvement in the growth of the chimeric PV/CBV4-2A virus. In addition, this chimeric cDNA approach enabled us to dissect multiple biological functions encoded by the 2A(pro) proteins.

Foot-and-mouth disease virus Lb proteinase can stimulate rhinovirus and enterovirus IRES-driven translation and cleave several proteins of cellular and viral origin

Rhinovirus and enterovirus 2A proteinases stimulate translation initiation driven from the cognate internal ribosome entry segment (IRES) (S. J. Hambidge and P. Sarnow, Proc. Natl. Acad. Sci. USA 89:10272-10276, 1992; H.-D. Liebig, E. Ziegler, R. Yan, K. Hartmuth, H. Klump, H. Kowalski, D. Blaas, W. Sommergruber, L. Frasel, B. Lamphear, R. Rhoads, E. Kuechler, and T. Skern, Biochemistry 32:7581-7588, 1993). Given the functional similarities between the foot-and-mouth disease virus (FMDV) L proteinase and these 2A proteinases (autocatalytic excision from the nascent viral polyprotein and cleavage of eIF-4 gamma), we investigated whether the FMDV L proteinase would also be able to stimulate translation initiation. We found that purified recombinant FMDV Lb proteinase could stimulate in vitro translation driven from a rhinovirus or enterovirus IRES by 5- to 10-fold. In contrast, stimulation of translation initiation on a cardiovirus IRES by this proteinase was minimal, and stimulation of translation driven from the cognate FMDV IRES could not be evidenced. Studies using an inhibitor or a mutant Lb proteinase indicated that stimulation of IRES-driven translation is mediated via proteolysis of some cellular component(s). Our studies also demonstrated that the Lb proteinase is capable of stimulating initiation of translation on an uncapped cellular message. Unexpectedly, and in contrast to the 2A proteinases, the Lb proteinase specifically cleaved the products of the two reporter genes used in this study: Xenopus laevis cyclin B2 and influenza virus NS. Therefore, we also set out to investigate the requirements for substrate recognition by the Lb proteinase. Purified recombinant Lb proteinase recognized at least one mengovirus polypeptide and specifically cleaved human cyclin A and poliovirus replicase-related polypeptides. In the latter case, the site(s) of cleavage was located within the N-terminal part of polypeptide 3D. Sequence comparisons revealed no significant primary sequence similarities between the target proteins and the two sites already known to be recognized by the FMDV L proteinase.

Malignant lymphoma of the epididymis. A case report of bilateral involvement by a follicular large cell lymphoma

Malignant lymphoma of the testis involves the adjacent epididymis in approximately 60% of cases, but isolated epididymal lymphoma is rare. The file of a 34-year-old man was reviewed, and the biopsy material was studied by conventional morphology, immunohistochemistry, and polymerase chain reaction. The patient presented with painless bilateral epididymal enlargement. Sequential bilateral epididymal biopsies revealed follicular lymphoproliferation with minimal involvement of one testis. Immunoglobulin M antibodies to the Coxsackie B4 virus were increased, and the lesions were interpreted as an atypical reactive process. Chemotherapy was not pursued. Four years after presentation, there was no evidence of local disease, but a 3-cm lesion involving the skin of the forehead developed. An excision biopsy revealed a large cell follicular lymphoma with features resembling the epididymal lesion. Analysis by polymerase chain reaction revealed a t(14-18) translocation in skin and epididymal lesions confirming that the epididymal lesions were also lymphomatous. Staging revealed no other site of disease. To date, the patient, who was treated with methotrexate chemotherapy, remains well 28 months after treatment. Most lymphomas of the testis and epididymis occur in older men, have an intermediate or high grade, diffuse histology, disseminate early, and follow an aggressive clinical course. In contrast, this case is similar to the occasional reports of pseudolymphoma and follicular lymphoma that occur in younger men, are confined largely to the testis and epididymis, and have a good prognosis.

Detection of Transforming Growth Factor-.BETA.1 in Coxsackie B3 Virus-Induced Murine Myocarditis.

To clarify the role of TGF-β1 in myocardial healing process after virus-induced myocarditis, we have examined the time course of TGF-β1 expression and its localization in myocardial healing process after Coxsackie B3-induced murine myocarditis. TGF-β1 immunoreactivity increased in parallel with its mRNA level and reached a peak at 10 days after Coxsackie B3 virus inoculation. Immunohistochemically, TGF-β1 was localized at pre-necrotic area at an early phase (5 days); it increased and extended to the area around the necrotic foci when necrosis became manifest (10 days); it decreased when macrophages and fibroblasts migrated to the necrotic foci (15 days); and then it reached a normal level and localized a little in perivascular and calcified regions (30 days). To our knowledge, this is the first study which demonstrates that TGF-β1 mRNA increases in viral-induced myocarditis at acute inflammatory phase. This result suggests that TGF-β1 promotes the migration of macrophages and fibroblasts to necrotic foci and induces fibrosis.

Shigella flexneri adherence to and multiplication in coxsackie B1 virus-infected HEp-2 cells.

Coxsackie B1 virus infection enhances the susceptibility of in vitro cultured HEp-2 cells to invasiveness by Shigella flexneri. We have studied the effect of viral infection on two phases of the invasiveness. Only a minor part was mediated by enhanced bacterial adherence to the cells, and the intracellular multiplication was unaffected by the virus. Enhanced adherence was not dependent on the presence of the gene product of the 140 Md virulence associated plasmid. Our data indicate that enhanced invasiveness induced by viral infection is mediated by an effect on other phases of the invasiveness.

Complete nucleotide sequence of a strain of coxsackie B4 virus of human origin that induces diabetes in mice and its comparison with nondiabetogenic coxsackie B4 JBV strain

The E2 strain of coxsackie B4 virus (CB4), which is of human origin, can induce a diabetes-like syndrome in mice. The cDNA of the genome of the E2 strain was cloned and sequenced. The E2 viral genome was found to comprise 7,396 bases, which appear to encode a polyprotein of 2,183 amino acids with an overall similarity of 94.91% to nondiabetogenic CB4 prototype JBV strain. The E2 genome is organized like other enteroviruses. It has a 5' noncoding region of 744 nucleotides, a single long open translational reading frame starting at nucleotide 745 and extending to nucleotide 7293, a 3' noncoding region of 100 nucleotides, and a poly (A) tract. Genomic sequence comparison of the E2 and JBV strains showed 1,369 nucleotide substitutions in the genome of the E2 strain, most of which are single and silent. There were 111 resultant amino acid changes arising from some of these substitutions, including 82 amino acid changes in the noncapsid proteins, and 29 amino acid changes in the capsid proteins VP1, VP2, VP3, and VP4, which showed 11, 13, 4, and 1 substitution(s), respectively. Noncapsid protein P2-C showed eight amino acid substitutions. On the basis of the sequence comparison of E2 and JBV strains of CB4, we suggest that some of the amino acid changes in the capsid and noncapsid proteins of the E2 strain may be involved in the determination of its diabetogenicity.

Effect of poly(I).poly(C12U) (Ampligen) on enteric virus (rotavirus, poliovirus and Coxsackie B3 virus) infections

The effects of poly(1)-poly(C 12 U) (Ampligen) on infections with enteric viruses (rotavirus, poliovirus and Coxsackie B3 virus) were studied in vitro. Ampligen exhibited antiviral activity against rotavirus, especially when treatment was performed prior to inoculation of the virus. It was partially effective against Coxsackie B3 virus, but not against poliovirus. It is suggested that the observed effects may be due to the production of interferon induced by Ampligen

Impaired Ca 2+ response to glucose in mouse β-cells infected with Coxsackie B or Echo virus

Five strains of Coxsackie B4 virus and one of Echo 11 virus were tested with regard to their ability to replicate in pancreatic mouse β-cells and interfere with the alterations of the cytoplasmic Ca 2+ concentration ([Ca 2+] i) induced by glucose. All strains except one both multiplied and caused cytopathic effect. In a control group 68% of the β-cells responded to 11 mM glucose with large amplitude oscillations of [Ca 2+] i. After inoculation with the infectious strains these oscillations appeared in only 5% of the β-cells, whereas the non-infectious strain did not modify the glucose effect on [Ca 2+] i. Despite the virus interference with the glucose response, [Ca 2+] i was increased after depolarization with excessive extracellular K + and the oscillations were induced in most β-cells when glucose was combined with the insulin-releasing sulfonylurea tolbutamide.

Identification of Serotype-Specific and Nonserotype-Specific B-Cell Epitopes of Coxsackie B Virus Using Synthetic Peptides

Coxsackie B viruses are thought to be involved in the induction of myocarditis. However, the diagnosis of acute infections by serology even today is practically impossible. The major problem is that no antigenic determinants of coxsackie B viruses have been identified or characterized which could be used as antigens in a rapid routine antibody screening test. Therefore, we synthesized overlapping peptides according to the sequence of the capsid protein VP1 of coxsackie B3 (CB3) virus in order to identify antigenic determinants located on VP1. Using sera raised against CB3 in mice, we were able to identify several antigenic determinants of CB3. Here we present a characterization of three epitopes found. We also tested the type-specificity of these antigenic determinants by using rabbit antisera against coxsackie viruses B1-B6. One antigenic determinant, peptide VP1-1, representing residues 1-15 of VP1, reacted highly type-specific for CB3. A second antigenic determinant (peptide VP1-3, residues 21-35 of VP1) reacted as well with the anti-CB3 sera as with the anti-CB4 sera. Therefore, the peptide VP1-3 seems to represent a non-type-specific antigenic determinant of coxsackie B viruses. Peptide VP1-24 (residues 229-243 of VP1) showed broad cross-reaction with anti-CB sera except with the anti-CB1 sera. This identification of type-specific and non-type-specific epitopes of coxsackie B viruses may provide the basis for the establishment of an effective and fast antibody screening test for coxsackie B viruses in patients with clinically suspected myocarditis.

157 Study of chinese herbal drugs against coxsackie B1, B3, B4 virus in cell culture: Tian-ming Zhang, Zhen-jiao Hu, Mei-ying Zhang. Virus Research Institute, Hubei Medical University, Wuhan, Hubei, People's Republic of China

157 Study of chinese herbal drugs against coxsackie B1, B3, B4 virus in cell culture: Tian-ming Zhang, Zhen-jiao Hu, Mei-ying Zhang. Virus Research Institute, Hubei Medical University, Wuhan, Hubei, People's Republic of China

Detection of coxsackie B4 virus RNA in infected mouse kidneys by in situ hybridization.

We describe a method for detecting virus-specific RNA sequences using a nonradioactive enzyme system, and analyze coxsackie B4 virus (cox.B4) RNA sequences in infected mouse kidney with pathological changes. Diffuse mesangial proliferation was observed transiently 24 h after inoculation of cox.B4 virus in glomeruli. OCT-treated thin sections of the same tissue were hybridized in situ using a digoxigenin-labeled oligonucleotide probe. The strongest signal was observed in the mesangial sample. These findings suggest that the transient proliferative mesangial changes were caused by direct injury following virus infection without immune complex.

Coxsackie B1 virus-induced murine myositis: Relationship of disease severity to virus dose and antiviral antibody response

Inoculation of Coxsackie B1 virus (CB1) in newborn CD1 Swiss mice induces a chronic myositis of proximal hindlimb muscles (CB1 myositis). To study the possible role of the virus dose, and of antiviral antibodies in the development of CB1 myositis, we infected groups of newborn mice with six CB1 doses, ranging from 30 to 10, 000 plaque forming units (pfu); after 4 and 8 weeks we determined morbidity and antiviral antibody titer, and quantified histopathological changes. At 4 weeks, morbidity and mononuclear cell infiltration differed significantly for the various groups, with the most prominent changes in 300 pfu animals. At 4 and 8 weeks diseased animals had significantly higher antibody titers than clinically unaffected animals, and at 4 weeks myopathic and infiltrative changes correlated positively with the serum antibody titer. Our data indicate that the virus dose plays a pathogenic role in CB1 myositis, and they suggest further study on the role of humoral immune mechanisms in the early phase of CB1 myositis.

Mapping of Antigenic Determinants of the Adenine-Nucleotide Translocator and Coxsackie B3 Virus with Synthetic Peptides: Use for the Diagnosis of Viral Heart Disease

Mapping of Antigenic Determinants of the Adenine-Nucleotide Translocator and Coxsackie B3 Virus with Synthetic Peptides: Use for the Diagnosis of Viral Heart Disease

Microinjection of HEp-2 cells with coxsackie B1 virus RNA enhances invasiveness of Shigella flexneri only after prestimulation with UV-inactivated virus.

Coxsackie B1 virus induces increased susceptibility to invasion by Shigella flexneri when HEp-2 cells are inoculated with the complete virus. When RNA from the same virus was microinjected into cells, virus RNA was synthesized and new virus particles were formed, but the transfected RNA had no effect on bacterial invasiveness. However, when the cells were prestimulated with UV-inactivated virus, the microinjected RNA induced an additional enhancement of bacterial invasiveness. Microinjected whole virus particles did not replicate and did not induce any change in bacterial invasiveness. The results indicate that an initial event in virus multiplication is necessary to achieve an effect of transfected viral RNA on invasion of S. flexneri.

Coxsackie B3 myocarditis induces a decrease in energy charge and accumulation of hyaluronan in the mouse heart

Interstitial oedema in chronic inflammation and ischaemia is related to an accumulation of hyaluronan (hyaluronic acid; HA) in the interstitium. As interstitial oedema will affect the oxygen transport in the interstitium we have evaluated whether accumulation of HA is related to signs of myocardial energy depletion in virus induced myocarditis. Myocarditis was induced in Balb/c mice by inoculation of Coxsackie B3 virus. The extractable HA content of the myocardium increased progressively from a baseline value of 153 +/- 26 micrograms g-1 dry weight to a maximum of 286 +/- 78 micrograms g-1 dry weight at day 7, whereafter there was a slight decline. Affinity histochemistry visualized HA in the endo-perimysium in healthy myocardium. At day 5 after Coxsackie B3 inoculation there was a general widening of the endomysium, which exhibited a positive staining for HA. In conjunction with focal inflammatory infiltrates the staining for HA was even more pronounced. The energy rich adenylates were reversibly affected by the Coxsackie B3 infection. There was a slight, but significant decline in EC from 0.74 +/- 0.05 in the control group to a minimum value of 0.64 +/- 0.10 at day 5, whereafter a restitution was observed at days 7 and 10. The total adenine nucleotide pool was similarly decreased from 27.8 +/- 2.9 mumol g-1 dry weight in controls to 24.6 +/- 2.1 micrograms g-1 dry weight at day 5, and normalized at days 7 and 10. The data suggest that virus induced myocarditis is associated with a local accumulation of HA in the myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)

Myocardite et rhabdomyolyse aiguë lors d'une infection à Coxsackie virus B4. Corrélation de l'état clinique avec les taux d'interféron gamma sécrété par les lymphocytes circulants

We report a case of myocarditis and acute rhabdomyolysis associated with Coxsakie B4 virus infection. Parallele evolution of clinical status and in vitro mononuclear cells gamma interferon secretion has been noted.

Localized left ventricular aneurysms with normal global function caused by myocarditis

Left ventricular (LV) aneurysms usually result from coronary artery disease but may also occur in congenital, 1 traumatic 2,3 or infective heart diseases. LV aneurysms of infective origin are most often caused by Chagas's disease 4 and infective endocarditis. Regional wall motion abnormalities have been occasionally observed during viral myocarditis. 5 More recently Goudevenous et al 6 reported a case of LV aneurysm during Coxsackie B4 virus infection but without histologic evidence of myocarditis. This study reports 2 cases of severe lymphocytic myocarditis presenting with ventricular tachycardia and LV aneurysm.

Coxsackie B1 virus-induced changes in cell membrane-associated functions are not responsible for altered sensitivity to bacterial invasiveness.

To analyze the possible mechanisms by which coxsackie B1 virus infection affects the invasiveness of Shigella flexneri, we have studied the influence of intracellular levels of Na+ and K+, ATPase activity, cytoplasmic membrane potential, cAMP level and cell communication through gap junctions. 3h after adsorption of viable or UV-inactivated coxsackie B1 virus the Na(+)-K+ gradient of the cell collapsed, ATPase activity decreased, the cytoplasmic membranic potential-dependent tetraphosphonium ion uptake were reduced. No changes in cAMP or intercellular cell communication were observed. S. flexneri invasiveness in HEp-2 cell pretreated with viable or UV-inactivated coxsackie B 1 virus was enhanced, but bacterial invasiveness was unchanged in K(+)-depleted HEp-2 cells, cell cultures with high intracellular Na+ content or ouabain pre-treated cells compared to control cells. We found no correlation between the enhanced bacterial invasiveness in the early phase of coxsackie B 1 virus infection in HEp-2 cell cultures and intracellular K+ depletion, high intracellular Na+ content, inhibited Na(+)-K+ ATPase activity or membranic depolarization.

Adoptively transferred anti-idiotype pulsed B cells mediate autoimmune myocarditis.

Syngeneic mice receiving adoptively transferred enriched B cells or splenocytes pulsed in vitro with polyclonal anti-idiotypic antibodies (anti-Ids) against anti-CVB3 virus antibodies developed coxsackievirus B3 antigen-binding and virus-neutralizing antibodies during a 30-day period, in addition to overt expression of autoimmune myocarditis. Adoptive transfer of anti-Id pulsed T cells resulted in delayed appearance and transient expression of antiviral antibodies, and antiviral antibodies were marginal to absent in syngeneic animals receiving anti-Id pulsed macrophage populations. Proliferation analysis of recipient splenocytes indicated lack of proliferative capacity in response to monoclonal or polyclonal anti-Ids and only marginal proliferative capacity in response to coxsackievirus B3 virus antigen(s). In vitro assessment of delayed hypersensitivity in recipient animals demonstrated some specific immunity to anti-Ids in recipients receiving splenocytes or T cells. Anti-Ids expressing mimicry for heart-associated and/or viral antigen(s) interacting with B cells or other accessory cells suggest an autoantibody or anti-Id triggering of B-cell-mediated mechanisms involved in the development of myocarditis.

Successive infection of coxsackievirus B3 and encephalomyocarditis virus: an animal model of chronic myocarditis.

Successive infection of coxsackievirus B3 and encephalomyocarditis virus was investigated as a disease model of chronic myocarditis. Four-week-old C3H/He mice were inoculated with coxsackievirus B3 and then inoculated with encephalomyocarditis virus at 8 weeks old. The hearts were evaluated on histopathological changes compared with those of non-infected mice and mice infected with either virus alone. At 10 weeks old, the hearts of the mice infected successively with both viruses showed co-existence of fibrosis surrounding calcified lesions and marked cellular infiltration with myocardial necrosis. These findings resembled chronic active myocarditis in humans, unlike the lesions due to either virus alone. At 12 weeks old, the hearts of all the infected mice showed fibrosis with scarce cellular infiltration. The successively infected hearts also showed a significantly higher heart weight to body weight ratio than that of the non-infected control mice, and localized wall thinning in the damaged regions. Thus, we conclude that successive infection additively causes myocardial damage that resembles chronic myocarditis and may produce a heart condition similar to dilated cardiomyopathy.