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PubMed1982

Instability of the viral M protein in BHK-21 cells persistently infected with Sendai virus.

The study of viral protein expression in BHK cells persistently infected with Sendai virus showed that the viral M protein was greatly reduced in amount or absent in these cells. Pulse-chase experiments demonstrated that the M protein was synthesized at a normal rate, but was unstable compared to the other viral proteins. The M protein instability was independent of temperature and could account for part of the reduction in viral production by persistently infected cells. When a virus stock was grown in embryonated chicken eggs from viruses produced by persistently infected BHK cells, the M protein of this stock presented a restored stability in BHK cells.

Animals↗
PubMed1993

Induction of mucosal disease in cattle persistently infected with noncytopathic bovine viral diarrhea-mucosal disease virus by superinfection with cytopathic bovine viral diarrhea-mucosal disease virus.

Three head of cattle persistently infected with noncytopathic bovine viral diarrhea-mucosal disease virus (ncBVD-MDV) were superinfected naturally or experimentally with cytopathic bovine viral diarrhea-mucosal disease virus (cBVD-MDV). In the naturally superinfected case, one animal manifested pyrexia and severe diarrhea, and died without developing antibodies to cBVD-MDV. However, another animal survived with only continual slight anorexia and pyrexia, and developed strong resistance to the superinfected strain. In the experimental cases, induction of MD was unsuccessful in two persistently infected cattle when superinfected with cBVD-MDV antigenically heterologous for persistently infected ncBVD-MDV. They also developed antibodies to the cBVD-MDV strain with which they had been infected. After 6 months, these cattle were infected again with a cBVD-MDV strain different from that used in the previous experiment. One animal infected with this strain, which was antigenically homologous to the persistently infected strain, died after developing MD symptoms without developing antibodies to the infecting strain. It is suggested that the antigenic relationship between the persistent ncBVD-MDV and the superinfected cBVD-MDV was an important factor in developing MD.

Animals↗
PubMed1991

Defective function of leukocytes from cattle persistently infected with bovine viral diarrhea virus, and the influence of recombinant cytokines.

Cattle persistently infected with bovine viral diarrhea (BVD) virus have decreased neutrophil and lymphocyte functions. We reevaluated these functions and further characterized the inhibition of persistent BVD virus infection in neutrophils, using sensitive kinetic assays. In addition, the influence of in vitro incubation of neutrophils with recombinant bovine interferon gamma (rBoIFN gamma) and in vitro incubation of lymphocytes with recombinant bovine interleukin-2 was evaluated. Significant (P less than 0.05) decrease in random migration under agarose, Staphylococcus aureus ingestion, cytochrome-C reduction, iodination, antibody-independent cell-mediated cytotoxicity, oxidant production, and cytoplasmic calcium flux were observed in neutrophils from cattle persistently infected with BVD virus, compared with noninfected control cattle. Incubation of neutrophils from noninfected controls with rBoIFN gamma significantly (P less than 0.05) decreased random migration under agarose, cytochrome-C reduction, and cytoplasmic calcium flux. Neutrophils from cattle persistently infected with BVD virus also had decreased random migration under agarose after incubation with rBoIFN gamma; in addition, antibody-independent cell-mediated cytotoxicity, elastase release, and cytoplasmic calcium flux were significantly enhanced. The rBoIFN gamma induced significantly (P less than 0.05) different effects on chemotaxis, cytochrome-C reduction, iodination, and cytoplasmic calcium flux of neutrophils from infected and control cattle. The rBoIFN gamma was more effective at improving the function of neutrophils from cattle persistently infected with BVD virus, compared with neutrophils from controls. Lymphocytes from infected cattle had decreased blastogenesis in response to phytohemagglutinin, concanavalin A, and pokeweed mitogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Human glioblastoma cells persistently infected with simian virus 40 carry nondefective episomal viral DNA and acquire the transformed phenotype and numerous chromosomal abnormalities.

A stable, persistent infection of A172 human glioblastoma cells with simian virus 40 (SV40) was readily established after infection at an input of 450 PFU per cell. Only 11% of the cells were initially susceptible to SV40, as shown by indirect immunofluorescent staining for the SV40 T antigen at 48 h. However, all cells produced T antigen by week 11. In contrast, viral capsid proteins were made in only about 1% of the cells in the established carrier system. Weekly viral yields ranged between 10(4) and 10(6) PFU/ml. Most of the capsid protein-producing cells contained enormous aberrant (lobulated or multiple) nuclei. Persistent viral DNA appeared in an episomal or "free" state exclusively in Southern blots and was indistinguishable from standard SV40 DNA by restriction analysis. Viral autointerference activity was not detected, and yield reduction assays did not indicate defective interfering particle activity, further implying that variant viruses were not a factor in this carrier system. Interferon was also not a factor in the system, as shown by direct challenge with vesicular stomatitis virus. Persistent infection resulted in cellular growth changes (enhanced saturation density and plating efficiency) characteristic of SV40 transformation. Persistent infection also led to an increased frequency of cytogenetic effects. These included sister chromatid exchanges, a variety of chromosomal abnormalities (ring chromosomes, acentric fragments, breaks, and gaps), and an increase in the chromosome number. Nevertheless, the persistently infected cells continued to display a bipolar glial cell-like morphology with extensive process extension and intercellular contacts.

Antigens, Viral, Tumor↗
PubMed1998

Persistently infected cattle stabilise bovine viral diarrhea virus leading to herd specific strains.

Animals persistently infected with BVDV are important in the epizootiology of the Bovine Viral Diarrhea (BVD) because they are a permanent source of contamination within a herd. These animals produce large quantities of virus and have, therefore, been proposed as responsible for generating antigenic variability. However, limited studies have failed to detect antigenic or genetic changes in viruses isolated at different time from persistently infected animals. One hypothesis to account for this stability is that the immunotolerance is accompanied by a selection against antigenic change. The presence of an immunotolerant persistently infected (IPI) animal in a herd would in turn lead to herd specific strains. To verify this hypothesis, we compared 17 BVDV strains isolated from IPI animals from 3 herds of Eastern Belgium. The comparison was based on the sequence of a 389 bp fragment of E2--a gene encoding for a highly variable glycoprotein. Sequences were strongly conserved within herds but were quite different between herds, indicating that BVDV herd-specific strains do exist and are associated with the presence of IPI animals.

Animals↗
PubMed1981

Cytophotometric analysis of lytically and persistently infected tissue culture cells with measles virus.

Lytically and persistently infected VERO and LUB cells were cytophotometrically characterized with the aid of computer analysis. Images scanned at 260 and 280 nm were processed by computer algorithms. With this approach, infected cells could be segmented and differentiated from uninfected cells. Lytically and persistently infected cells could be distinguished by distinct differences in nuclear and cytoplasmic optical densities. These findings are supported by biologic data based on the analysis of virus-specific proteins and nucleic acids. The applied computer aided cytophotometry provides a new approach in the study of virus cell interaction.

Animals↗
PubMed1997

The murine coronavirus mouse hepatitis virus strain A59 from persistently infected murine cells exhibits an extended host range.

In murine 17 Cl 1 cells persistently infected with murine coronavirus mouse hepatitis virus strain A59 (MHV-A59), expression of the virus receptor glycoprotein MHVR was markedly reduced (S. G. Sawicki, J. H. Lu, and K. V. Holmes, J. Virol. 69:5535-5543, 1995). Virus isolated from passage 600 of the persistently infected cells made smaller plaques on 17 Cl 1 cells than did MHV-A59. Unlike the parental MHV-A59, this variant virus also infected the BHK-21 (BHK) line of hamster cells. Virus plaque purified on BHK cells (MHV/BHK) grew more slowly in murine cells than did MHV-A59, and the rate of viral RNA synthesis was lower and the development of the viral nucleocapsid (N) protein was slower than those of MHV-A59. MHV/BHK was 100-fold more resistant to neutralization with the purified soluble recombinant MHV receptor glycoprotein (sMHVR) than was MHV-A59. Pretreatment of 17 Cl 1 cells with anti-MHVR monoclonal antibody CC1 protected the cells from infection with MHV-A59 but only partially protected them from infection with MHV/BHK. Thus, although MHV/BHK could still utilize MHVR as a receptor, its interactions with the receptor were significantly different from those of MHV-A59. To determine whether a hemagglutinin esterase (HE) glycoprotein that could bind the virions to 9-O-acetylated neuraminic acid moieties on the cell surface was expressed by MHV/BHK, an in situ esterase assay was used. No expression of HE activity was detected in 17 Cl 1 cells infected with MHV/BHK, suggesting that this virus, like MHV-A59, bound to cell membranes via its S glycoprotein. MHV/BHK was able to infect cell lines from many mammalian species, including murine (17 Cl 1), hamster (BHK), feline (Fcwf), bovine (MDBK), rat (RIE), monkey (Vero), and human (L132 and HeLa) cell lines. MHV/BHK could not infect dog kidney (MDCK I) or swine testis (ST) cell lines. Thus, in persistently infected murine cell lines that express very low levels of virus receptor MHVR and which also have and may express alternative virus receptors of lesser efficiency, there is a strong selective advantage for virus with altered interactions with receptor (D. S. Chen, M. Asanaka, F. S. Chen, J. E. Shively, and M. M. C. Lai, J. Virol. 71:1688-1691, 1997; D. S. Chen, M. Asanaka, K. Yokomori, F.-I. Wang, S. B. Hwang, H.-P. Li, and M. M. C. Lai, Proc. Natl. Acad. Sci. USA 92:12095-12099, 1995; P. Nedellec, G. S. Dveksler, E. Daniels, C. Turbide, B. Chow, A. A. Basile, K. V. Holmes, and N. Beauchemin, J. Virol. 68:4525-4537, 1994). Possibly, in coronavirus-infected animals, replication of the virus in tissues that express low levels of receptor might also select viruses with altered receptor recognition and extended host range.

Animals↗
PubMed2004

Evaluation of antiviral activity and toxicity of recombinant human interferon alfa-2a in calves persistently infected with type 1 bovine viral diarrhea virus.

OBJECTIVE: To evaluate antiviral activity and toxicity of recombinant human interferon alfa-2a in calves persistently infected with noncytopathic type 1 bovine viral diarrhea virus (BVDV). ANIMALS: 5 Holstein heifers, 4 to 12 months of age. PROCEDURES: Calves persistently infected with noncytopathic type 1 BVDV were treated with recombinant human interferon alfa-2a every other day for 12 weeks. Viral loads were measured during the treatment period and compared with pre- and post-treatment values. Complete physical examinations were performed weekly, and calves were observed daily for signs of systemic illness. Complete blood counts and serum biochemical analyses were performed before, during, and after the treatment period. Because calves developed anemia during the treatment period, bone marrow biopsy specimens were collected. Antirecombinant human interferon alfa-2a antibody concentrations in serum samples obtained before, during, and after the treatment period were measured by use of an ELISA. RESULTS: Recombinant human interferon alfa-2a had no antiviral activity against noncytopathic type 1 BVDV in persistently infected calves. All calves developed microcytic anemia during the treatment period that persisted for up to 13 weeks after cessation of treatment. Anti-interferon antibodies were detected during the treatment period and persisted for at least 2 weeks after cessation of treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Because of lack of in vivo antiviral activity against BVDV, recombinant human interferon alfa-2a has little promise as a therapeutic agent for the treatment of BVDV infection, at least in persistently infected cattle. Furthermore, treatment was associated with adverse immunologic and hematologic effects.

Animals↗
PubMed1978

Establishment and characterization of a subacute sclerosing panencephalitis (measles) virus persistent infection in BGM cells.

Infection of BGM cells with the Halle isolate of subacute sclerosing panencephalitis (SSPE) gave rise to a persistent infection (BGM/Halle), whereas infection of another African green monkey kidney cell line (Vero) under identical conditions led to a lytic infection. The BGM/Halle cells multiplied more slowly than the non-infected cells (even when the medium was changed daily). Under such conditions 10(7) to 10(8) p.f.u./ml/24 h of measles virus was released into the medium. It was established that the persistent infection was not due to the accumulation of thermosensitive mutants and that the virus was not modified as measured by several biological parameters. The virus released from BGM/Halle cells had, however, acquired an ability to give rise to a persistent infection in Vero cells. The quantity of virus released from persistently infected Vero cells was very low (10(2) to 10(3) p.f.u./ml). It was concluded that a host-cell factor plays a role in the restriction of virus replication.

Cell Division↗
PubMed1994

Phenotypic characteristics of dengue 2 virus persistently infected in a C6/36 clone of Aedes albopictus cells.

A C6/36 cell culture persistently infected by dengue 2 virus was established and retained for over 20 weeks. No CPE was observed at any stage of infection. However, some giant cells up to 2- or 3-fold the diameter of normal cells appeared in the established culture. Extracellular virus titers fluctuated with a periodic tendency while the antigen level remained constant, suggesting some viral particles evolved into noninfectious defective interfering particles. Plaque size tended to change during the passage of cultures. The passage 8 virus mainly produced plaques relatively smaller than those produced by the passage 1 virus. Nevertheless, the plaque sizes produced by the passages 18 virus were somewhat mosaic. A temperature-sensitive mutant was detected in the virus released from passages 8 and 18. In addition, passages 8 and 18 produced the lowest virus titers. When persistently infected viruses were grown in Vero cells at 37 degrees, at passage 1 there was a high titer which decreased 5 days postinoculation, whereas at passages 8 and 18 titers were undetectable until 4 days postinoculation. It seems that dengue 2 virus persistently infected in C6/36 cells could have been genetically altered. This will be tested by nucleic acid analysis of the viruses.

Aedes↗
PubMed2005

The fusion protein of wild-type canine distemper virus is a major determinant of persistent infection.

The wild-type A75/17 canine distemper virus (CDV) strain induces a persistent infection in the central nervous system but infects cell lines very inefficiently. In contrast, the genetically more distant Onderstepoort CDV vaccine strain (OP-CDV) induces extensive syncytia formation. Here, we investigated the roles of wild-type fusion (F(WT)) and attachment (H(WT)) proteins in Vero cells expressing, or not, the canine SLAM receptor by transfection experiments and by studying recombinants viruses expressing different combinations of wild-type and OP-CDV glycoproteins. We show that low fusogenicity is not due to a defect of the envelope proteins to reach the cell surface and that H(WT) determines persistent infection in a receptor-dependent manner, emphasizing the role of SLAM as a potent enhancer of fusogenicity. However, importantly, F(WT) reduced cell-to-cell fusion independently of the cell surface receptor, thus demonstrating that the fusion protein of the neurovirulent A75/17-CDV strain plays a key role in determining persistent infection.

Amino Acids↗
PubMed1990

[Patterns of acute and persistent infections in enteroviral heart diseases].

Enteroviruses are considered as the major etiologic agents of myocarditis in humans. Recent in situ hybridization studies on endomyocardial biopsies indicate that not only in acute myocarditis (pattern of acute infection), but also in chronic dilated cardiomyopathy enterovirus RNA can be detected (pattern of persistent infection). Our experimental studies on murine coxsackievirus B3 myocarditis provided evidence that persistent infection occurs also in mice. Quantitative in-situ hybridization and immunohistochemistry as well as electron microscopic in situ hybridization experiments were performed on ACA/SnJ mice three to thirty days after infection. The pattern of acute infection (days 3-9 p.i.) is characterized by rapid progression of myocardial lesions, an increasing number of inflammatory cells and a high number of infected myocytes. Hallmarks of the persistent pattern (day 15-30 p.i.) are reduced inflammation, reduced numbers of persistently infected cells and a slow progression of myocardial lesions. Infection is primarily restricted to degenerated, atrophic myocytes and to fibroblasts.

Acute Disease↗
PubMed1983

The relationship between prostaglandins and virus replication: endogenous prostaglandin synthesis during infection and the effect of exogenous PGA on virus production in different cell lines and in persistently infected cells.

African Green Monkey Kidney cells were shown to normally synthesize immunoreactive PGE1. Infection of these cells with Sendai virus did not alter rates of PGE1 synthesis, while it stimulated interferon production. PGAs, that we have previously shown to be potent inhibitors of Sendai virus replication in this system, at the same dose (4 micrograms/ml), also strongly inhibited the replication of this virus in HEp-2 cells and in VERO cells, a monkey kidney cell line that does not produce interferon. PGA1 was found to be effective in several cell and virus models, suggesting a broad spectrum of antiviral actions. Finally, we confirmed the observation that PGA1-treatment prevents the establishment of a "carrier state" by Sendai virus, and PGA1-cured cells did not show any sign of persistent infection for periods as long as 110 days after Sendai infection. Attempts to cure already established persistently infected cells were only partially successful.

Alprostadil↗
PubMed1983

Selection of a mutant S1 gene during reovirus persistent infection of L cells: role in maintenance of the persistent state.

LR-7 cells, variant L cells derived from a type 3 reovirus persistently infected (p.i.) carrier culture (R. Ahmed, W. M. Canning, R. S. Kauffman, A. H. Sharpe, J. V. Hallum, and B. N. Fields, Cell 25, 325-332, 1983) were used to define the viral genes critical for maintenance of the persistent state. A cloned viral isolate (L/C virus) derived from the p.i. culture replicated normally in LR-7 cells, while wild-type (wt) viruses of the three reovirus serotypes replicated less efficiently. To identify the viral gene(s) permitting enhanced replication of L/C virus in LR-7 cells, viral reassortants were prepared by mixed infection of L cells with L/C virus and type 1 wt. Study of the one-step growth curves and final yields of large numbers of reassortants in both L cells and LR-7 cells revealed that the presence of the S1 gene from L/C virus was critical for normal viral replication in LR-7 cells. However, this phenotype was suppressed by the simultaneous presence in reassortants of both the M2 and S4 genes from the type 1 wt parent. The critical change in the S1 gene occurred by passage 13 (63 days) after initiation of the carrier culture. Although multiple mutations are present in the viral population from p.i. cultures, certain specific mutations can be identified as critical for maintenance of the persistent state.

Animals↗
PubMed1977

Interferon production and response to exogenous interferon in two cell lines of mouse brain origin persistently infected with Sendai virus.

Two persistently infected cell lines established from C3H mouse brain cells infected in vivo with Sendai virus were shown to differ with respect to interferon (IF) production and response to exogenous IF. MB/Sen carrier cells contained 1-5 per cent antigen positive cells when examined by immunofluorescence, and virus was occasionally recovered from the culture medium. MB/SenAS carrier cells were maintained with 0.16 per cent Sendai antiserum in the supernatant medium. All MB/SenAS cells contained viral antigen and infectious virus was present in the culture medium. MB/Sen released IF spontaneously into the culture medium. Further IF production could be stimulated in MB/Sen by superinfection with Newcastle disease virus (NDV) or vesicular stomatitis virus (VSV). Exogenous IF provided good protection against VSV challenge. In contrast, MB/SenAS produced no IF spontaneously but could be stimulated by NDV and VSV to produce IF. Exogenous IF failed to reduce the amount of VSV released into the supernatant fluid. Replication of VSV was restricted in MB/SenAS as shown by a 2.3 log10 lower virus yield compared to MB/Sen.

Animals↗
PubMed1999

Persistent infection of human oligodendrocytic and neuroglial cell lines by human coronavirus 229E.

Human coronaviruses (HuCV) cause common colds. Previous reports suggest that these infectious agents may be neurotropic in humans, as they are for some mammals. With the long-term aim of providing experimental evidence for the neurotropism of HuCV and the establishment of persistent infections in the nervous system, we have evaluated the susceptibility of various human neural cell lines to acute and persistent infection by HuCV-229E. Viral antigen, infectious virus progeny and viral RNA were monitored during both acute and persistent infections. The astrocytoma cell lines U-87 MG, U-373 MG, and GL-15, as well as neuroblastoma SK-N-SH, neuroglioma H4, and oligodendrocytic MO3.13 cell lines, were all susceptible to an acute infection by HuCV-229E. The CHME-5 immortalized fetal microglial cell line was not susceptible to infection by this virus. The MO3.13 and H4 cell lines also sustained a persistent viral infection, as monitored by detection of viral antigen and infectious virus progeny. Sequencing of the S1 gene from viral RNA after approximately 130 days of infection showed two point mutations, suggesting amino acid changes during persistent infection of MO3.13 cells but none for H4 cells. Thus, persistent in vitro infection did not generate important changes in the S1 portion of the viral spike protein, which was shown for murine coronaviruses to bear hypervariable domains and to interact with cellular receptor. These results are consistent with the potential persistence of HuCV-229E in cells of the human nervous system, such as oligodendrocytes and possibly neurons, and the virus's apparent genomic stability.

Astrocytoma↗
PubMed1990

Dengue-2 virus infection of human mononuclear cell lines and establishment of persistent infections.

Twenty three human mononuclear cell lines including ten myelomonocytic cell lines, eight B cell lines and five T cell lines, were examined to determine whether they could be infected with dengue-2 virus. All the cell lines were infected with dengue-2 virus as determined by immunofluorescent staining and by virus titration of culture supernatant fluids. K562, Jiyoye and Jurkat, respectively, showed the highest percentage of infected cells of these myelomonocytic, B and T cell lines. Antibody to dengue-2 virus at subneutralizing concentrations augmented dengue-2 virus infection of myelomonocytic cell lines, but not of B cell lines or of T cell lines. Persistent dengue-2 virus infection was established using a myelomonocytic cell line (K562), a B cell line (Raji), and a T cell line (HSB-2). These cell lines maintained a high percentage (more than 70%) of dengue-2 virus antigen-positive cells for at least 25 weeks. Very low titers of infectious dengue-2 virus were detected in the culture supernatant fluids of the persistently infected cells. Dengue-2 virus antigen-positive Raji cell clones were established from persistently-infected Raji cells using limiting dilutions and all of the cells in these clones were dengue-2 virus antigen-positive. These findings demonstrate that a variety of human mononuclear cell lines can be infected with dengue-2 virus and may be useful as models for the analysis of dengue virus-human cell interactions in dengue virus infections.

Antigens, Viral↗
PubMed1983

Virus-host cell interaction in rat C6 glial cell cultures persistently infected with herpes simplex virus.

The virus-host cell interaction in a rat C6 glial cell line persistently infected with herpes simplex virus type 1 (HSV1) was examined. The C6 cultures infected at an MOI of 10 or 0.01 exhibited virally induced cytopathology and produced infectious virus and viral antigens for 108 and 50 days, respectively. Infectious virus was continually present in the supernatant fluids of persistently infected cultures, with only a minority of the cells having viral CPE or viral antigens at any given time. There was no obvious pattern of periodic cyclical viral replication in these cultures; no predictable regularity in the fluctuations of virus production was evident. 'Cured' cultures were as susceptible to re-infection with HSV1 as previously uninfected cultures; thus, there was no selection of a subpopulation of C6 cells resistant to subsequent challenge with HSV1. The virus recovered from persistently infected cultures was more infectious for C6 cells relative to HeLa cells than was the original virus stock. Significant levels of interferon were not detected in HSV1-infected C6 cultures during either the acute or persistent phases of infection or in 'cured' cultures.

Animals↗