Coxsackie B1 virus infection enhances the bacterial invasiveness, the phagocytosis and the membrane permeability in HEp-2 cells

To analyze the effect of coxsackie B1 virus infection on bacterial invasiveness, phagocytosis and cytoplasma membrane permeability, we have studied invasiveness of Shigella flexneri, unspecific phagocytosis of latex particles and release of the non-metabolizible amino acid, alpha-aminoisobutyric acid (AIB). Virus infection enhanced invasiveness of S. flexneri and phagocytosis of latex beads and increased plasma membrane permeability as measured by release of AIB. The effect on all three functions increased with virus concentration, but the kinetics were different. During the early phase of virus infection there was no difference between the effect on invasiveness, phagocytosis and permeability in cell cultures pretreated with viable or with UV-inactivated virus. However, after 6 h, 5 h and 2 h respectively, there was an increased response in cell cultures pretreated with viable virus compared to cells inoculated with UV-inactivated virus. The results indicate that the virus effect on bacterial invasiveness is a function of several parameters, including phagocytosis and membrane function changes.     

Evidence that RNA 4 of alfalfa mosaic virus does not replicate autonomously

A mutant (Tbts7) of alfalfa mosaic virus, the coat protein of which is unable to activate the viral genome (the RNA species 1, 2, and 3, which need some coat protein for infectivity) at 30 degrees , can be rescued at this temperature by adding to the inoculum wild-type RNA 3 (the genome part that contains the coat protein cistron), but not adding wild-type RNA 4 (the subgenomic messenger for the coat protein). Unless RNA 3 of Tbts 7 has a second ts mutation at a site not occurring in RNA 4, it may be concluded from the above finding that RNA 4 does not replicate autonomously.     

Polypeptide synthesized in herpes simplex virus type 2-infected HEp-2 cells.

The proteins induced by herpes simplex virus type 2 in infected HEp-2 cells were studied by high-resolution polyacrylamide slab-gel electrophoresis. A total of 51 infected cell-specific polypeptides (ICSP) were detected, accounting for three-fourths of the coding capacity of the virus. The induced polypeptides were allocated to three groups based on their time of synthesis in the virus growth cycle. Cycloheximide treatment of infected cells during the early stage of virus growth was found to cause irreversible inhibition of protein synthesis. Herpes simplex virus type 2 polypeptides which underwent considerable posttranslational modification were detected.     

Norwalk virus does not replicate in human macrophages or dendritic cells derived from the peripheral blood of susceptible humans

Human noroviruses are difficult to study due to the lack of an efficient in vitro cell culture system or small animal model. Murine norovirus replicates in murine macrophages (MΦ) and dendritic cells (DCs), raising the possibility that human NoVs might replicate in such human cell types. To test this hypothesis, we evaluated DCs and MΦ derived from monocyte subsets and CD11c⁺ DCs isolated from peripheral blood mononuclear cells of individuals susceptible to Norwalk virus (NV) infection. These cells were exposed to NV and replication was evaluated by immunofluorescence and by quantitative RT-PCR. A few PBMC-derived DCs expressed NV proteins. However, NV RNA did not increase in any of the cells tested. These results demonstrate that NV does not replicate in human CD11c⁺ DCs, monocyte-derived DCs and MΦ, but abortive infection may occur in a few DCs. These results suggest that NV tropism is distinct from that of murine noroviruses.     

The replication of certain Coxsackie B virus strains in CHO cells

Cell surface molecules that can act as viral receptors may exert an important selective pressure on RNA viral quasi-species. Coxsackie-Adenovirus Receptor and Decay Accelerating Factor (DAF, CD55) have been identified as receptors for Coxsackie B virus. In studies of viral replication using different strains of Coxsackievirus serotype 4 (CBV-4), it was found that despite lack of expression of these cell surface molecules on Chinese Hamster Ovary (CHO) cells and despite their common use as negative controls in Coxsackie B virus receptor assays, two strains were able to replicate, one (V89-4557) without cytopathic effect (CPE), and the other (T318) with strong CPE. A weak signal was obtained using antibodies against enterovirus, visualized with FITC-conjugated antibodies, when the Coxsackievirus B4 strain V89-4557 was inoculated on CHO cells compared to no signal with the non-replicating Coxsackievirus B4 strain VD2921, indicating some degree of binding of the former to the cells.     

Coxsackie B virus antibodies in myocardial infarction

Evidence for the association between Coxsackie B virus infections and myocardial infarction was studied in a prospective follow-up examination. Using the micro neutralization test, 9 (15%) of 59 patients with acute myocardial infarction and 1 (2.6%) of 38 control patients showed a fourfold, or higher, antibody increase in paired serum samples against Coxsackie B1-5 viruses. This difference is significant (p less than or equal to 0.05). None of the patients or controls revealed symptoms of a viral infection during the blood sampling. Virus isolation from throat and feces was negative in all patients and controls. This finding agrees with some previous studies suggesting that the Coxsackie B group may in some cases have a causal role in myocardial infarction, or may act as a triggering factor.     

Myocarditis in mice infected with Coxsackie virus B3.

Perimyocarditis in the heart of BALB/c mice infected with Coxsackie virus group B type 3 (CB3) was studied to determine whether it is limited to the right perimyocardium and to show whether or not perimyocarditis or myocardial lesions are produced in both left and right ventricles. CB3 was recovered from the heart on days from 2 to 13 after inoculation, but thereafter no virus was isolated from any part of the heart. Histopathologically, from days 1 to 4, hyaline or granular degeneration and necrosis of the muscle fibres with or without calcium deposits and an inflammatory mononuclear cell infiltration was limited to the right perimyocardium. On days 6 to 18, however, degeneration and necrosis of the muscle fibres and an inflammatory mononuclear cell infiltration were found not only in the right perimyocardium, but also in both left and right ventricular wall, the left perimyocardium, both right and left endomyocardium and the septum. In the right ventricular lesions, the incidence and intensity of the histopathological changes in the perimyocardium were greater than those in the muscular layer or septum. In contrast, in left ventricular lesions, the incidence and intensity of the histopathological changes in the muscular layers were greater than those in the peri- and endo--cardium. It is inferred, therefore, that the right perimyocardium and left ventricular wall are more susceptible to CB3 infection than right ventricular wall or left peri- and endocardium. It is concluded that CB3 can produce not only right-sided perimyocarditis, but also both right and left ventricular lesions and endocardial or septal changes in the mouse heart.     

Calcium requirement for syncytium formation in HEp-2 cells by respiratory syncytial virus.

Respiratory syncytial virus (RSV) grown in HEp-2 cells in the absence of calcium did not induce cell fusion and syncytium formation. Although the infected cells contained viral antigens, the cytopathic effect (giant cell formation) typical for RSV was not observed in calcium-free cultures. Infectious virus yield was also slightly reduced (less than a one log10 reduction) in the absence of calcium. An analysis of viral proteins synthesized in both the presence and the absence of calcium revealed that the amount of fusion protein (F1) in calcium-free infected cultures was approximately one-third that in calcium-containing infected cultures. These results underscore the necessity of using calcium-containing growth medium for cell culture isolation and diagnosis of RSV.     

Experimental Coxsackie group B virus infections

Summary The ability of Coxsackie B3 virus to produce experimental infection in the mouse prenatally and post-natally has been examined. The embryos become infected only after intrauterine inoculation. The examined tissues show a negligible damage or none at all.Following post-natal infection, however, varied patterns of pathogenesis have been observed, depending upon the age of the host from suckling to adult.A comparative study has been carried out by using the other types of Coxsackie B viruses.presented byE. Irene Grodums Aided by a grant from The National Foundation and also by a Federal Public Health Research Grant of Canada.     

Correlation between Coxsackie B1 virus replication and enhanced invasiveness of Shigella flexneri.

Coxsackie B1 virus infection enhances the susceptibility of cultured HEp-2 cells to Shigella flexneri invasiveness. This can be reproduced partially with UV-inactivated virus, particularly the effect observed shortly after viral inoculation. The following phases of viral multiplication were correlated with the enhancing effect: uncoating of viral particles, synthesis of viral RNA and proteins, and assembly of newly produced virus particles. Uncoating of virus particles was completed within 60 min. This process was not correlated with the development of the early effect on invasiveness. Intact virus capsids seem to be necessary to enhance bacterial invasiveness in the early phase of virus infection. Separated capsid proteins had no effect either when applied to the cell surface or when microinjected into the cells. Virus protein synthesis was not required for the virus effect on bacterial invasiveness in the early infection phase, but it seems to be necessary in the late phase.     

Pancreatic lesion in newborn mice caused by Coxsackie B1 virus]

Changes in the pancreatic gland manifested by necrosis of excretory cells and marked atrophy of insulae with the loss of beta-cells producing insulin were observed experimentally in suckling mice infected with Coxsackie B1 virus. It is suggested that the above changes of the pancreas may be a model of human diseases caused by Coxsackie viruses, including a model of development of some cases of diabetes.     

Coxsackie B virus infections in cardiology. Apropos of 66 cases

Coxsackie B virus infections are common and frequently asymptomatic. However in young people, they can cause primary congestive cardiomyopathy and complicate previous cardiovascular illnesses. Virus diagnosis is difficult and based mainly on the detection of significant rising or stating high neutralizing antibody titers. A clinical and epidemiological five years study investigated 3,856 sera. 30.2% of the patients had evidence of a significant antibody titer (greater than or equal to 64) to one of the group B Coxsackie viruses; this percentage reaches 34.6% in cardiology and fluctuates from year to year; Coxsackie B2 is predominant (55.9%) in cardiology; coxsackie B1 and B2 antibody responses were detected more frequently than B3, B4 and B5. Coxsackie B6 appears to be uncommon. From these, 66 patients have evidence of coxsackie B virus infections with 60.2% for Coxsackie B2; they include pericarditis (30.3%), acute and chronic congestive cardiomyopathies (19.7%). Moreover it has been suggested that Coxsackie B virus might be responsible for electrocardiographic abnormalities (9.1%), ischemic heart disease and myocardial infarction. Enzyme linked immunosorbent assay (ELISA) and radioimmunoassay with purified antigens (VP1 and VP4) did not appear better than microneutralisation for evaluation of IgM and IgG antibodies. To elucidate the mechanisms of cardiac injury it is refer to viral replication, virus specific and auto-reactive T cells cytotoxic activities.     

Coxsackie B virus infections in women with miscarriage

Serum samples from 1,765 consecutive Sardinian blood donors, negative for hepatitis B surface antigen (HBsAg) and for antibodies to human immunodeficiency virus (HIV) (anti-HIV), were evaluated for the presence of antibodies to hepatitis C virus (anti-HCV) by second-generation ELISA. Anti-HCV was detected in 25 (1.45%) of the 1,765 donors examined. Anti-HCV was found in 15 of the 1,690 (0.9%) donors with normal alanine aminotransferase (ALT) and in 10 of the 75 (13%) donors with elevated ALT (P < 0.0001). Of the 15 anti-HCV-positive donors with normal ALT, only five (33%) were confirmed to be positive by second-generation RIBA, six (40%) were indeterminate, while four (27%) were RIBA negative. HCV RNA, as detected by polymerase chain reaction (PCR) using a set of primers from the 5′-noncoding region, was found in six of the 15 (40%) donors with normal ALT, including five RIBA, positive and one indeterminant. Of the 10 anti-HCV-positive donors with elevated ALT, all were RIBA positive and eight (80%) had detectable HCV RNA. Thus, among ELISA-reactive donors, those with elevated ALT had a significantly higher probability of being positive for secondgeneration RIBA and HCV RNA compared to those with normal ALT levels (P = 0.028). None of the 65 donors with elevated ALT but negative for anti-HCV by ELISA had detectable serum HCV RNA, as compared to eight of 10 anti-HCV ELISApositive donors (P < 0.0001). However, although negative for HBsAg, 12 of the 65 (18%) had serum HBV DNA by PCR. This study demonstrates that the combined use of second-generation ELISA and RIBA anti-HCV assays is highly effective in identifying HCV infection, whereas the specificity of ELISA alone for the screening of blood donors with normal ALT values appears to be limited. In contrast, in donors with elevated ALT levels, there is a positive correlation between second-generation assays (ELISA and RIBA) and HCV viremia. The high proportion of inapparent HBV infection in blood donors with elevated ALT levels underlines the importance of this test for the prevention of transfusion-associated viral hepatitis.     

Improvement of respiratory syncytial virus replication in actively growing HEp-2 cells

The growth of respiratory syncytial (RS) and parainfluenza type III (PI3) viruses has been studied in actively growing versus relatively stationary HEp-2 cells. There was no effect on PI3 virus growth. RS virus synthesis was stimulated from 20- to 60-fold by growth in actively growing cultures when the cell density was approximately $\text{1}\text{3}$ that of a confluent culture. This stimulation was manifested by a greater yield of virus per cell, more virus-specific mRNA produced per cell, and a 50% decrease in the time before cytopathic changes appeared.