Apparent Cytopathic Effect Research Articles

Activation of bovine herpesvirus 4 lytic replication in a non-permissive cell line by overexpression of BoHV-4 immediate early (IE) 2 gene

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with no clear disease association, it establishes persistent infections in its natural host, the bovine, and in an experimental host, the rabbit. BoHV-4 immediate early 2 (IE2) RNA is the less abundant, spliced, 1.8 kb RNA. The predicted amino acid sequence, of the IE2 protein, reveals that it could encode a 61 kDa protein with amino acid sequence homology to the Epstein-Barr virus (EBV) transactivator R protein and its homologues including, herpesvirus saimiri (HVS), equine herpesvirus 2 (EHV-2), murine herpesvirus 68 and Kaposi’s sarcoma-associated herpesvirus (KSHV). We examined recently the interaction of BoHV-4 with a human rhabdomyosarcoma cell line, RD-4, and found that although some infectious viruses can be produced, no cytopathic effect (CPE) was observed [J. Gen. Virol. 81 (2000) 1807]. Because IE2 could play a critical role in BoHV-4 productive infection and its overexpression in RD-4 cells could switch the non-permissive RD-4 status to a permissive one. RD-4 cells expressing stably BoHV-4 IE2 gene were generated. BoHV-4 IE2 induced an increased production of infectious viral particles sufficient to obtain an apparent cytopathic effect. It is concluded that BoHV-4 IE2 is a key factor in determining the outcome of BoHV-4 infection.

Establishment of Respiratory Syncytial Virus Persistence in Cell Lines: Association with Defective Interfering Particles

Defective interfering particles (DIP) have frequently been advanced to explain the persistence of virus in vitro. However, DIP involvement in the establishment of respiratory syncytial virus (RSV) persistence in cell lines has not been documented. We report establishment of RSV persistence in human epithelial larynx (HEp-2) and lung (H358) cell lines by infection with RSV enriched with DIP. Viral persistence in HEp-2 and H358 has been stably maintained for >3 years without apparent cytopathic effect or periodic crises. Persistent cultures resisted superinfection with the original virus and no cyclic variations in infectious virus and DIP yields were observed. During passages, low levels of infectious RSV and defined species of DIP were produced. Moreover, 80–90% of the cells exhibited viral antigen. Persistence in H358 cells was established with a buoyant density band containing DIP and infectious virus obtained from a H358 persistently infected culture. Thus, DIP are required for establishment of RSV persistence cultures in human epithelial cell lines; however, DIP per se are not able to establish persistence.

Construction and Characterization of Subgenomic Replicons of New York Strain of West Nile Virus

The lineage I strain of West Nile virus (WNV) frequently causes human epidemics, including the recent outbreak in North America (Lanciotti et al., 1999, Science 286:2333–2337). As an initial step in studying the replication and pathogenesis of WNV, we constructed several cDNA clones of a WNV replicon derived from an epidemic strain (lineage I) isolated from the epicenter of New York City in the year 2000. Replicon RNAs were in vitro transcribed from cDNA plasmids and transfected into BHK-21 cells. RNA replication in transfected cells was monitored by immunofluorescence analysis (IFA) and 5′ nuclease real-time RT-PCR (TaqMan). The replicon RNAs contained large in-frame deletions (greater than 92%) of the C-prM-E structural region yet still replicated efficiently in BHK-21 cells. 5′ nuclease real-time RT-PCR showed that a great excess of plus-sense replicon RNA over the minus-sense RNA was synthesized in transfected cells. Replication efficiency decreased upon insertion of a green fluorescent protein (GFP) reporter gene driven by an internal ribosomal entry site (IRES) in the upstream end of the 3′ untranslated region of the replicon. Strong GFP expression was detected in cells transfected with a replicon containing IRES-GFP positioned in the plus-sense orientation. IFA showed that GFP and viral proteins were exclusively coexpressed in transfected cells. In contrast, no GFP fluorescence was observed in cells transfected with a replicon containing IRES-GFP positioned in the minus-sense orientation, despite high levels of synthesis of viral proteins and RNA in the cells. Substitution of the GFP gene in the plus-sense GFP replicon with the neomycin phosphotransferase gene allowed selection of geneticin-resistant cells in which WNV replicons persistently replicated without apparent cytopathic effect. These results suggest that WNV replicons may serve as a noncytopathic RNA virus expression system and should provide a valuable tool to study WNV replication.

In subjects with antibody to hepatitis C virus a high serum level of interleukin-2 soluble receptor suggests activity of liver disease.

The hepatitis C virus (HCV) is an RNA virus without apparent cytopathic effects, and hepatocellular damage in chronic infection is generally believed to be immune-mediated by cytotoxic T lymphocytes. Activated T cells release the soluble form of the interleukin-2 (IL-2) receptor (sIL-2R) and its concentration is correlated with the degree of lymphocyte activation. We measured sIL-2R in 69 subjects: 24 healthy repeat blood donors (group I), 17 HCV carriers without liver damage (group II) and 28 patients with HCV-related chronic active hepatitis (group III). There was no significant difference between sIL-2R levels in patients of group I (36.5 +/- 14.6 U ml-1) and group II (46.8 +/- 17.4 U ml-1), and the levels for both of these groups were significantly lower than those observed in the patients with active HCV, group III (176.9 +/- 59.5 U ml-1). Hence, among HCV-infected subjects (HCV RNA positive) with persistently normal alanine aminotransferase (ALT) levels, the plasma levels of sIL-2R are normal, but, in patients (HCV RNA positive) with HCV-related chronic active hepatitis there are increased plasma levels of sIL-2R. We conclude that in HCV infection high levels of sIL-2R are related to activity of the disease rather than to virus replication. In patients with HCV-related chronic liver disease, the sIL-2R concentration may be a useful marker of disease activity.

Subgenomic replicons of the flavivirus Kunjin: construction and applications.

Several Kunjin virus (KUN) subgenomic replicons containing large deletions in the structural region (C-prM-E) and in the 3' untranslated region (3'UTR) of the genome have been constructed. Replicon RNA deltaME with 1,987 nucleotides deleted (from nucleotide 417 [in codon 108] in the C gene to nucleotide 2403 near the carboxy terminus of the E gene, inclusive) and replicon RNA C20rep with 2,247 nucleotides deleted (from nucleotide 157 [in codon 20] in C to nucleotide 2403) replicated efficiently in electroporated BHK21 cells. A further deletion from C20rep of 53 nucleotides, reducing the coding sequence in core protein to two codons (C2rep RNA), resulted in abolishment of RNA replication. Replicon deltaME/76 with a deletion of 76 nucleotides in the 3'UTR of deltaME RNA (nucleotides 10423 to 10498) replicated efficiently, whereas replicon deltaME/352 with a larger deletion of 352 nucleotides (nucleotides 10423 to 10774), including two conserved sequences RCS3 and CS3, was significantly inhibited in RNA replication. To explore the possibility of using a reporter gene assay to monitor synthesis of the positive strand and the negative strand of KUN RNA, we inserted a chloramphenicol acetyltransferase (CAT) gene into the 3'UTR of deltaME/76 RNA under control of the internal ribosomal entry site (IRES) of encephalomyelocarditis virus RNA in both plus (deltaME/76CAT[+])- and minus (deltaME/76CAT[-])-sense orientations. Although insertion of the IRES-CAT cassette in the plus-sense orientation resulted in a significant (10- to 20-fold) reduction of RNA replication compared to that of the parental deltaME/76 RNA, CAT expression was readily detected in electroporated BHK cells. No CAT expression was detected after electroporation of RNA containing the IRES-CAT cassette inserted in the minus-sense orientation despite its apparently more efficient replication (similar to that of deltaME/76 RNA); this result indicated that KUN negative-strand RNA was probably not released from its template after synthesis. Replacement of the CAT gene in the deltaME/76CAT(+) RNA with the neomycin gene (Neo) enabled selection and recovery of a BHK cell culture in which the majority of cells were continuously expressing the replicon RNA for 41 days (nine passages) without apparent cytopathic effect. The constructed KUN replicons should provide valuable tools to study flavivirus RNA replication as well as providing possible vectors for a long-lasting and noncytopathic RNA virus expression system.

The level of CD26 determines the rate of HIV entry in a CD4+ T-cell line.

We have reported that CD26 could serve as a cofactor of CD4 in HIV entry. Recently, more evidence has been provided for the implication of CD26 in HIV entry, replication and cytopathic effect. Along with, we have demonstrated that the level of CD26 may determine the rate of HIV-envelope induced-apoptosis. The role of CD26 in HIV entry was further investigated using CEM T-cell line. Clones were established by transfection, expressing different levels of CD26. Entry, infection and cytopathic effect were monitored in several independent clones, and were found to be delayed in clones CD26-Low and CD26-SuperHigh compared to clones CD26-High. The delay was most significant in clones CD26-AntiSense, without any apparent cytopathic effect. These results demonstrate that relatively enhanced levels of CD26 contribute to an increased virus infection. Furthermore, they illustrate that CD26-SuperHigh clones manifest a phenotype similar to CD26-Low clones. This point out the critical role of CD26 in the rate of HIV entry and its cytopathic effect, two events which are initiated by the interaction of HIV envelope glycoproteins with cell-surface CD4.

Establishment and Characterization of Japanese B Encephalitis Virus Persistent Infection in the Sf9 Insect Cell Line

Abstract.The Sf9 cell line, commonly used for gene expression by recombinant baculoviruses, can be productively infected by Japanese B encephalitis virus (JEV). Two wild-type JEV strains (P3 and SA14) caused a cytopathic effect (CPE) in the Sf9 cells, while no apparent CPE was caused by an attenuated strain (SA14–14–2). The JEV viral antigens were expressed in the infected Sf9 cells and intracellular virus particles were found by electron microscopy as a result of infection with all three strains. Titres of cell-associated and cell-free supernatant virus remained stable for relatively long periods of cultivation, suggesting that both wild-type and attenuated JEV strains established productive and persistent infections of Sf9 cells. The JEV produced by the Sf9 cells could be neutralized by anti-JEV reference serum, but relatively smaller plaques were formed in BHK21 cells infected with JEV that had been cultivated long term in Sf9 cells. This system for virus propagation has a number of potentially important uses for enhancing progress in JEV study and control.

The Difference in gp160 and gp120 of HIV Type 1 in the Induction of CD4 Downregulation Preceding Single-Cell Killing

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is synthesized as a precursor glycoprotein, gp160, and is then processed into gp120 and gp41. In the present study, CD4+ cell clones expressing either gp160 or gp120 of HIV-1 under the transcriptional control of an inducible promoter were made in order to examine the effect of these env products on the downregulation of surface CD4 and cell injury. A complete disappearance of surface CD4 preceding single-cell death occurred in the cell clones expressing gp160, in which a complex between CD4 and gp160 was formed and then accumulated intracellularly. In contrast to this, the cell clones expressing gp120 neither exhibited any such depletion of surface CD4 nor showed any apparent cytopathic effect. Therefore, it is thought that gp160 but not gp120 plays a crucial role in both the downregulation of surface CD4 and the resultant cell death in the cells infected with HIV-1.

Replication of Border disease virus in ovine lymphocytes and monocytes in vitro

Adherent and non-adherent mononuclear cells obtained from the peripheral blood of normal sheep supported the in vitro replication of a non-cytopathic and a cytopathic strain of Border disease virus (BDV) with no apparent cytopathic effects. There was a significant rise in virus titres in adherent mononuclear cells (monocyte) and non-adherent (lymphocyte) cultures infected with both non-cytopathic and cytopathic strains of BDv 24 hours after inoculation. Peak virus titres of 5·36 log 10 TCID50 ml −1 were recorded in adherent samples incubated for 48 hours while peak titres of 6·17 log 10 TCID50 m1 −1 were recorded in lymphocyte culture after 72 hours of incubation. Both the non-cytopathic and the cytopathic strains of BDV produced significantly higher titres in nonadherent (lymphocyte) cultures than in adherent (monocyte) cultures (P<0·001) but the replication in adherent cells was faster than in nonadherent cell cultures. The addition of virus on both types of mononuclear cell cultures had no effect on cell viability but it had a significant inhibitory effect on the blastogenic responses of lymphocytes to phytohaemagglutinin.

Apoptosis induced in CD4+ cells expressing gp160 of human immunodeficiency virus type 1.

In a previous study (Y. Koga, M. Sasaki, H. Yoshida, H. Wigzell, G. Kimura, and K. Nomoto, J. Immunol. 144:94-102, 1990), we demonstrated that the expression of gp160, a precursor form of envelope glycoprotein of human immunodeficiency virus type 1, in CD4+ cells causes the downregulation of surface CD4 and single-cell killing by forming intracellular gp160-CD4 complex. In the present study we investigated the events that lead to cell death in CD4+ cells expressing gp160. We found that apoptosis is induced in cells undergoing single-cell death. Moreover, even the cell clone, which expresses so little gp160 that it does not exhibit any apparent cytopathic effects, such as the inhibition of cell growth, was found to be highly susceptible to the apoptosis induction by the anti-Fas monoclonal antibody.

Interactions of Murine Gammaherpesvirus 68 with B and T Cell Lines

Murine gammaherpesvirus is a natural pathogen of wild rodents. We have established that in vivo the virus persists in B lymphocytes in a latent form and therefore has similar biological properties to Epstein-Barr virus and related gamma-I-herpesviruses. In this report we have established a persistent infection in mouse myeloma (B) cells (NSO cell line), but not in mouse thymoma (T) cells (BW 5147 cell line). The virus persists indefinitely in myeloma cells, without any apparent cytopathic effect, but with the production of infectious virus. We demonstrate that ACV abolishes the productive infection, but large numbers of cells harbor the virus in a latent form, as determined by an infectious center assay. Analysis of the viral DNA has shown that during a persistent infection linear virus genomes predominated, with low levels of circular DNA also present. Treatment of cells with ACV results in a significant reduction of linear genomes, but has no effect on the level of circular DNA molecules. These data provide further evidence to support our earlier observations on B cells as the site of latency and provides an in vitro model with which to study the molecular basis of MHV-68 latency/persistence.

A gene delivery/recall system for neurons which utilizes ribonucleotide reductase-negative herpes simplex viruses

We present data which show that ribonucleotide (RR)-negative herpes simplex virus type-1 (HSV-1) is a useful vector for gene delivery into neuronal cells. For these studies we used hrR3, a genetically engineered HSV-1 mutant which has an in-frame insertion of the bacterial lacZ gene into the HSV gene that encodes the large subunit (ICP6) of RR. After infection of rat primary sympathetic neuronal cultures with hrR3, the ICP6:: lacZ chimeric gene was expressed, as shown by blue staining of the cells upon exposure to X-Gal, a chromogenic β-galactosidase substrate. When the infection was performed in the presence of acyclovir, hrR3 appeared to become “latent”; neither infectious virus nor β-galactosidase activity was detectable in these neuronal cultures at 3 weeks after the acyclovir was removed. However, β-galactosidase activity was inducible in the “latent” cultures by superinfection with ICP6Δ (a RR-negative deletion mutant) without resulting in the “reactivation” of hrR3 and without apparent cytopathic effects. In contrast, superinfection with ICP6Δ + 3.1, a virus derived by marker rescue of ICP6Δ, resulted in the expression of lacZ, the release of hrR3 into the culture medium, and cytopathic effects. The introduction of a foreign gene into neuronal cells by a RR-negative herpes simplex virus, and the subsequent induction of gene expression by another noncomplementing virus, may constitute a prototype gene delivery/recall system for neurons.

Human immunodeficiency virus type 1vif, vpr, andvpu mutants can produce persistently infected cells

A series of human immunodeficiency virus type 1 (HIV-1) mutants in vif, vpr, vpu, and nef were constructed from an infectious plasmid (pNL 432) containing the full-length HIV-1 DNA by frameshift mutations. The capacities for replication and cell killing of these mutant viruses were examined in a clonal cell line (M 10) isolated from HTLV-I-transformed MT-4 cells. In all cases, the mutant viruses replicated, expressed HIV-1 antigens, and induced drastic cytopathic effects. However, some M 10 cells survived infection with vif, vpr, and vpu mutant viruses and became persistently HIV-1-infected, whereas no cells survived infection with the nef mutant as well as the wild-type virus. The HIV-1 particles produced from the surviving cells after infection with the vif, vpr, or vpu mutant viruses were fully replicative in M 10 cells without apparent cytopathic effects.

In vitro replication of bovid herpesvirus-1 in macrophages derived from peripheral blood leucocytes of calves

Macrophages derived from peripheral blood leucocytes of calves from 1 to 100 days of age supported the replication of Bovid herpesvirus-1 (BHV-1) without apparent cytopathic effects. The replication of the virus was abortive, replication being restricted to limited numbers of passages. The abilities of macrophages to support replication of BHV-1 were similar for calves from 1 day to 100 days of age (P greater than 0.05). The replication of BHV-1 in macrophages did not significantly affect the capacity of macrophages to phagocytose and kill Staphylococcus aureus (P greater than 0.05).

HIV-1 Expression by T8 Lymphocytes After Transfection

Acquired immune deficiency syndrome (AIDS) is an immunosuppressive disease associated with the depletion of T4 lymphocytes. Recently, an HIV-1 genome was molecularly cloned and shown to be fully infectious in vitro by transfection experiments. In this study, we show that HIV-1 can be transfected into T4 and T8 lymphocyte subpopulations and viral proteins and infectious virus particles are observed in both short- and long-term cultures. In addition, transfected T8 cells can be maintained in culture for long periods without apparent cytopathic effect.

Nucleic acid synthesis during the focus formation by Shope fibroma virus on green monkey kidney cells

Nucleic acid synthesis during the focus formation on Shope fibroma virus (SFV)-infected cells was studied. When African green monkey kidney (AGMK) cells were infected with SFV, the “cell-focus” was observed as a local piling up of cells at 3 to 5 days after infection. The number of foci increased in proportion to the size of the SFV inoculum. In the SFV-AGMK cell system, however, the growth rate of virus was comparatively low and no apparent cytopathic effect was demonstrated as a result of virus infection. The incorporation of3H-thymidine into the nuclear fraction of infected cells was suppressed early in the cycle of virus replication and before focus formation. On the other hand, the rate of DNA synthesis increased markedly in the infected cytoplasm. Cytoplasmic RNA synthesis also tended to increase gradually accompanying the induction of the viral DNA synthesis. In addition, the sedimentation properties of the newly synthesized DNA were investigated by sucrose density gradient centrifugation.

PERSISTENT POLIOVIRAL INFECTION OF INTACT HUMAN AMNIOTIC MEMBRANE WITHOUT APPARENT CYTOPATHIC EFFECT.

Gresser, Ion (Children's Hospital Medical Center, Boston, Mass.), Charles Chany, and John F. Enders. Persistent polioviral infection of intact human amniotic membrane without apparent cytopathic effect. J. Bacteriol. 89:470-475. 1965.-Poliovirus type I multiplied in organ cultures of the intact amniotic membrane. The behavior of the virus in this system differed markedly from that observed in monolayer cultures of trypsinized amnion cells, since replication of new virus was demonstrable throughout a period as long as 3 months, extensive cellular destruction was not apparent, and only a small proportion of cells were found to be infected at a given time. It seems likely that the chronicity of infection is conditioned essentially by the persistent insusceptibility to polioviral infection of the large majority of cells (relative to monolayer cultures) rather than to interferon, which was present in the culture fluids. It was postulated that the greater suceptibility of cultured amnion cells to infection, as compared with cells of the intact membrane, is due in part to the enhancement of phagocytic activity attendant upon trypsinization and cultivation of the amnion cells in monolayers. Amniotic and chorionic membranes thoroughly washed free from blood contain and release in vitro antibody to poliovirus I and other viruses. Appreciation of this phenomen may be important in attempts to isolate viruses from infected tissues.

Virus resistance and sensitivity in cultured human synovial cells as a possible genetic marker

Cells from a permanent culture established from normal human synovial tissue demonstrate an ability to take up polio virus and permit it to reproduce without any apparent cytopathic effect. A group of these cells were permitted to reproduce in the presence of polio I virus for several passages until the offspring became completely resistant to polio I virus infection. When these cells were irradiated with ultraviolet light, measurable virus uptake and reproduction re-occurred after several passages.