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The Th1/Th2 balance does not account for the difference of susceptibility of mouse strains to Theiler's virus persistent infection.

Theiler's virus causes a persistent infection with demyelination that is studied as a model for multiple sclerosis. Inbred strains of mice differ in their susceptibility to viral persistence due to both H-2 and non-H-2 genes. A locus with a major effect on persistence has been mapped on chromosome 10, close to the Ifng locus, using a cross between susceptible SJL/J and resistant B10.S mice. We now confirm the existence of this locus using two lines of congenic mice bearing the B10.S Ifng locus on an SJL/J background, and we describe a deletion in the promoter of the Ifng gene of the SJL/J mouse. We studied the expression of IFN-gamma, IL-2, IL-10, and IL-12 in the brains of SJL/J mice, B10.S mice, and the two lines of congenic mice during the first 2 wk following inoculation. We found a greater expression of IFN-gamma and IL-2 mRNA in the brains of B10.S mice compared with those of SJL/J mice. Also, the ratio of IL-12 to IL-10 mRNA levels was higher in B10.S mice. However, the cytokine profiles were the same for the two lines of resistant congenic mice and for susceptible SJL/J mice. Therefore, the difference of Th1/Th2 balance between the B10.S and SJL/J mice is not due to the Ifng locus and does not account for the difference of susceptibility of these mice to persistent infection.

Animals↗

Measles virus persistent infection: modification of the virus nucleocapsid protein.

The synthesis of intracellular measles virus proteins in persistently infected human cell cultures was studied. The virus-induced proteins were analysed after radioimmunoprecipitation by one- and two-dimensional polyacrylamide gel electrophoresis. The measles virus-induced nucleoprotein (NP) synthesized in persistently infected cells had a reduced binding capacity with measles virus antibodies (human convalescent serum) compared to the NP protein induced by the virus used to initiate the infection. In contrast, monospecific rabbit serum prepared against the original virus NP, or monoclonal anti-NP antibodies, precipitated NP proteins from acutely and persistently infected cells with equal efficiency. When the NP in acutely or persistently infected cells was labelled with either 14C- or 3H-amino acids and subjected to two-dimensional gel analysis, significant charge differences were observed between the virus proteins. When measles virus-infected cells were examined for virus protein synthesis at 40 degrees C, although no change was found in acutely infected cells, NP was not detected in the persistent infection.

Antibodies, Viral↗

Initiation and maintenance of persistent infection by respiratory syncytial virus.

Propagation of cells infected with temperature-sensitive (ts) mutants of respiratory syncytial (RS) virus at nonpermissive temperature (39 degrees C) resulted in cytolytic, abortive, or persistent infection, depending on the mutant used to initiate infection. Five mutants from complementation group B produced cytolytic or abortive infections, whereas a single mutant (ts1) from group D and a noncomplbmenting mutant produced persistent infections. The persistently infected culture initiated by mutant ts1 (RS ts1/BS-C-1) has been maintained in serial culture for greater than 100 transfers, and infectious-center assays and immunofluorescent staining indicated that all cells harbored the RS virus genome. RS ts1/BS-C-1 cultures were resistant to superinfection by homologous and some heterologous viruses, and interferon-like activity against some heterologous viruses was present in the culture medium. Small amounts (0.002 to 0.2 PFU/cell) of infectious virus were present in the culture fluid, but autointerfering defective particles were not detected. This released virus formed small plaques and produced persistent infection of BS-C-1 cells at 37 degrees C. The RS ts1/BS-C-1 cells contained abundant RS virus antigen internally, but little at the surface, although the cells showed enhanced agglutinability by concanavalin A. Nucleocapsids and the 41,000-molecular-weight nucleoprotein were present in extracts of both nucleated and enucleated cells. No infectious RS virus was obtained by transfection of DNA from RS tsl/BS-C-1 cells to susceptible BS-C-1 or feline embryo cells under conditions allowing efficient transfection of a foamy virus proviral DNA. It was concluded that persistent infection was maintained in part by a non-ts variant of RS virus partially defective in maturation. The karyotype of the RS ts1/BS-C-1 culture differed from that of unifected cells.

Antigens, Surface↗

Persistent infection of mouse fibroblasts (L cells) with Chlamydia psittaci: evidence for a cryptic chlamydial form.

When monolayers of mouse fibroblasts (L cells) were infected with enough Chlamydia psittaci (strain 6BC) to destroy most of the host cells, 1 in every 10(5) to 10(6) originally infected cells gave rise to a colony of L cells persistently infected with strain 6BC. In these populations, the density of L cells and 6BC fluctuated periodically and reciprocally as periods of host cell increase were followed by periods of parasite multiplication. Successive cycles of L-cell and 6BC reproduction were sustained indefinitely by periodic transfer to fresh medium. Isolation of L cells and 6BC from persistent infections provided no evidence that there had been any selection of variants better suited for coexistence. Persistently infected populations consisting mainly of inclusion-free L cells yielded only persistently infected clones, grew more slowly, and cloned less efficiently. They were also almost completely resistant to superinfection with high multiplicities of either 6BC or the lymphogranuloma venereum strain 440L of Chlamydia trachomatis. These properties of persistently infected L cells may be accounted for by assuming that all of the individuals in these populations are cryptically infected with 6BC and that cryptic infection slows the growth of the host cell and makes it immune to infection with exogenous chlamydiae. According to this hypothesis, the fluctuations in host and parasite density occur because some factor periodically sets off the conversion of cryptic chlamydial forms into reticulate bodies that multiply and differentiate into infectious elementary bodies in a conventional chlamydial developmental cycle.

Animals↗

Interferon-mediated persistent infection of Saint Louis encephalitis virus in a reptilian cell line.

A persistent infection with Saint Louis encephalitis (SLE) virus in a poikilothermic cell line TH-1 (turtle heart cells) was studied. Infected TH-1 cells were subcultured weekly at 31 degrees C for 1 year and continued to produce low levels (10(2) to 10(3) p.f.u./ml) of virus without obvious cytopathic effects or marked cyclic events. Indirect fluorescent antibody and infectious centre assays indicated that less than 1% of the cells were producing detectable virus proteins or infectious virus. Defective-interfering particles, temperature-sensitive mutants and DNA provirus were not detected. Interferon (IFN) mediation of the persistent infection as considered since the persistently infected cells (PIC) and normal TH-1 cells were resistant to heterologous virus challenge after treatment with virus-free culture fluid from PIC. A direct relationship was found between the m.o.i. and the amount of IFN produced, plateauing at an m.o.i. of approx. 10. The reptilian IFN was physically and chemically similar to mammalian and avian IFN. Certain biological markers of the SLE virus changed during the persistent infection. It was less virulent for mice, showed distinct differences in cell culture host range and had increased thermal lability.

Animals↗

Growth of the Phlebovirus Toscana in a mosquito (Aedes pseudoscutellaris) cell line (AP-61): establishment of a persistent infection.

Toscana Virus, a sandfly-associated Phlebovirus, was adapted to grow in cultured Aedes pseudoscutellaris (AP-61) mosquito cell line. No evidence of virus growth was seen after primary infection of cell monolayers under maintenance conditions. On the contrary, persistent infections were established by subculturing infected cultures. Cytopathic effect was never observed. Significant titres of virus (10(3)-10(5) PFU/ml), as assayed in Vero cells at 37 degrees C, were released from persistent infected cells after several subcultures at 29 degrees C over a period of six months. The percentage of virus-producing cells in the persistently infected cultures varied from 0.003 to to 0.017 per cent, whereas from 10 to 50 per cent of cells were shown to retain viral antigens by immunofluorescence. The virus released from persistently infected cultures did not show changes in both plaque size and temperature sensitivity from the parental virus. The virus released from persistently infected cultures multiplied in AP-61 cell monolayers reaching relatively high titres (10(4) PFU/ml).

Aedes↗

Cell receptors for the mammalian reovirus: reovirus-specific T-cell hybridomas can become persistently infected and undergo autoimmune stimulation.

We have previously described the development of virus-specific helper T cell hybridomas which recognize structural determinants shared by type 1 and type 3 reoviruses that have been exposed to UV radiation. We have found that T-cell hybridomas become persistently infected with live type 3 reovirus used for the immunization. Persistently infected T-hybridoma cells were found to spontaneously produce interleukin 2 (IL-2). To analyze the mechanism of induction of IL-2 secretion of persistently infected T-cell hybridomas, we exposed T-cell hybridomas specific for UV-treated virus to replicating type 3 reovirus. The T-cell hybridomas became infected but did not produce IL-2 unless simultaneously exposed to syngeneic I-A+ antigen-presenting cells. In this situation, the persistently infected T-cell hybridomas produced IL-2 without being reexposed to virus. This process was not a consequence of nonspecific IL-2 gene activation, which occurs in cells persistently infected with reovirus, because reovirus infection did not activate IL-2 secretion in T-cell hybridomas with other antigenic specificities. Reovirus exposure also resulted in persistent infection of certain antigen-presenting B-cell tumor lines. The persistently infected B-cell tumor lines could stimulate reovirus-specific helper T cells but not T-cell hybridomas of other specificities. The data support the thesis that persistent infection of reovirus-specific T cells creates a mechanism in which the virus released from these cells is processed and then reexpressed by I-A+ antigen-presenting cells. The IA antigen and reovirus structures on the antigen-presenting cells then restimulate the T cells through their specific receptors, resulting in IL-2 synthesis and release. These observations may be relevant to mechanisms of autoimmunity induced by virus.

Animals↗

Vesicular stomatitis virus can establish persistent infections in Syrian hamsters.

Persistent infections by vesicular stomatitis virus (VSV) of the Indiana serotype were readily established in adult Syrian hamsters following intraperitoneal injection of the virus. Plaque-forming virus, identified as VSV by serological and physical criteria, was isolated from brain homogenates of five hamsters that were tested 3 to 8 months after infection. Four of these animals had exhibited either transient or permanent paralysis, whereas the fifth appeared healthy, during the period of observation. At the time of sacrifice all hamsters had high titres of anti-VSV-neutralizing antibodies in their sera.

Animals↗

Absence of mycoplasmal gene in malignant mammalian cells transformed by chronic persistent infection of mycoplasmas.

Chronic persistent infections by mycoplasmas induced malignant transformation of C3H mouse embryo cells that normally had never been reported to undergo spontaneous transformation. This mycoplasma-mediated oncogenic process had a long latency (more than 7 weeks of continuous mycoplasmal infection) and showed a multistage progression characterized by reversibility (at least up to 11 weeks of mycoplasmal infection) and irreversibility of malignant properties upon removal of the mycoplasma from culture. Further prolonged infections (18 weeks) by Mycoplasma fermentans or M. penetrans resulted in permanent transformation of these C3H cells that no longer required the continued presence of the transformation-inducing mycoplasmas in cultures to retain their malignant properties. Previous studies of viral oncogenesis revealed that virus-transformed cells always had viral gene(s) present. Integration of viral gene(s) apparently played an important role in the process of oncogenesis. In this study, we examined if the continued presence of any mycoplasmal gene(s) in mammalian cells, in whatever form, was also crucial in causing malignant cell transformation. Representational difference analysis (RDA) was a recently developed powerful technique to compare differences between two complex genomes. In the RDA system, subtractive and kinetic enrichment was used to purify and isolate restriction endonuclease gene fragment(s) of mycoplasmal origin, presumably present only in mycoplasma-transformed C3H cells, but not in nonmycoplasma-exposed control C3H cells. After three rounds of subtractive hybridization following PCR enrichment for each of three different restriction enzymes DNA digests, no gene fragment of mycoplasmal origin was amplified or identified in the permanently transformed C3H cells. Differing from tumorigenesis in animal cells induced by most oncogenic viruses or in plant cells induced by Agrobacteria, mycoplasmas evidently did not cause malignant transformation by integrating their gene(s) into the mammalian cell genome.

Animals↗

Persistent infection with bovine herpesvirus-1 (infectious bovine rhinotracheitis virus) in cultured hamster cells.

Bovine herpesvirus-1 infection in hamster embryo cells was found to be dependent upon input multiplicity; productive infection was achieved at input multiplicities greater than one, while persistent infection was established when input multiplicities were about 0.5. This persistence was characterized by a noncyclic, minimal degree of cytopathic effect with a low level of released virus. Maintenance of the persistently infected cultures did not require external supportive measures. Subcultivation of the persistently infected cultures led to virus replication followed by CPE and then cell regrowth. With 3 to 4 weeks after subcultivation a persistent infection was re-established. The possible mechanism for the bovine herpesvirus persistence in hamster cells is discussed.

Animals↗

Isolation of highly fusogenic variants of simian virus 5 from persistently infected cells that produce and respond to interferon.

A series of experiments were undertaken to examine how interferon and neutralizing antibodies influence the ability of simian virus 5 (SV5) (strain W3) to establish and maintain persistent infections in murine cells. In contrast to the rapid decline in SV5 protein synthesis observed in murine BALB/c fibroblasts (BF cells), which produce and respond to interferon, between 24 and 48 h postinfection there was no inhibition of virus protein synthesis in MSFI- cells, skin fibroblasts derived from alpha/beta-interferon receptor knockout BALB/c mice. Furthermore, the addition of anti-interferon antibodies to the culture medium of infected BF cells significantly reduced the observed decline in virus protein synthesis. Following infection of untreated BF cells, the majority replicated virus but survived the infection and eventually cleared the virus after 8 to 15 days. However, not all the cells were cured, and the cultures became persistently infected. Upon passage of persistently infected cultures, the virus fluxed between active and repressed states as a consequence of interferon production. This resulted in a balance being reached in which only 5 to 20% of the cells were infected at any one time. After 30 passages of the persistently infected cells, highly fusogenic virus variants arose (one of which was isolated and termed W3-f). W3-f remained as sensitive to interferon as the parental W3 isolate but, in the absence of interferon, spread much more rapidly than the parental W3 strain through BF cell monolayers. Sequence analysis revealed no deduced amino acid differences between the F proteins of W3 and W3-f. BF cell cultures persistently infected with W3-f were rapidly cleared of virus by the addition of virus-neutralizing antibodies to the culture medium. In contrast, neutralizing antibodies had little effect on the numbers of cells persistently infected with W3 over several passages. These results suggest that the ability of paramyxoviruses to cause cell-cell fusion may be selected for in vivo as a consequence of their adaptation to the interferon response rather than their need to escape from neutralizing antibodies. The significance of these observations with regard to persistent parainfluenza virus infections in vivo is further discussed.

Animals↗

Comparison of the agar gel immunodiffusion test and ELISA in the detection of bovine leukosis virus antibody in cattle persistently infected with bovine virus diarrhoea virus.

When six cattle persistently infected with bovine virus diarrhoea virus (BVDV) were inoculated with lymphocytes infected with bovine leukosis virus (BLV), a depressed antibody response to BLV was observed by ELISA which was due to a decrease in IgG1 synthesis. The ELISA was more sensitive and more reliable than the agar gel immunodiffusion (AGID) test in detecting BLV infection in cattle persistently infected with BVDV. Decreased antibody responses were manifested in the AGID test by negative, inconclusive or weakly positive reactions: only two of the six cattle developed antibodies that generated positive AGID reactions.

Animals↗

Prospects for dendritic cell vaccination in persistent infection with hepatitis C virus.

Although hepatitis C virus (HCV) is classified in the Hepacivirus genus in the family Flaviviridae, it is unlike most of the other members of this family due to its propensity to cause persistent infections. This persistent infection eventually results in chronic liver disease, cirrhosis and hepatocellular carcinoma in a proportion of infected individuals. It has been difficult to examine correlates of clearance or persistence because most acute phase HCV infections are subclinical or result in symptoms which are non-specific; consequently, acute infections are not generally recognised and patients often present many years later with persistent infection and accompanying chronic liver disease. Nevertheless, seminal studies, performed during the acute phase, have identified a number of factors which are likely to influence the outcome of infection, although it is possible that the mechanism is multifactorial. One of these factors is impairment of dendritic cell function by a mechanism resulting from expression of an HCV protein(s) in these cells. This may be a major factor in the failure of the immune response to expand after HCV infection, leading to persistence. Nevertheless, it may be possible to overcome this defect by autologous transfusion of HCV antigen-loaded, mature dendritic cells and the purpose of this review is to highlight the need and general approaches for developing dendritic cell-based immunotherapy for HCV infection.

Dendritic Cells↗

Use of serologic evaluation for antibodies against bovine viral diarrhea virus for detection of persistently infected calves in beef herds.

OBJECTIVE: To determine whether use of serologic evaluation of a sentinel sample of calves or cows for antibodies against bovine viral diarrhea virus (BVDV) would accurately predict whether an animal persistently infected with BVDV could be detected in beef herds. SAMPLE POPULATION: 27 cow-calf herds in which the status of persistently infected calves was not known and 11 herds known to have persistently infected calves. Procedure-Detection of persistently infected calves was determined through immunohistochemical testing of tissue obtained at necropsy of all calves that died during calving season and skin (ear notch) specimens obtained from all young stock in the fall of 2002. Serum samples were collected from 30 spring-born calves and 10 mature cows. RESULTS: Optimum serologic test performance at time of weaning was detected when 10 calves were evaluated. At least 3 of 10 randomly selected calves were likely to have a titer > 1:1000 against BVDV type I or II in 53% of herds in which a persistently infected calf was detected during that year (sensitivity, 53%). However, at least 3 of 10 randomly selected calves were also likely to have a titer > 1:1000 in 20% of herds that did not have a persistently infected calf detected during that year (specificity, 80%). CONCLUSIONS AND CLINICAL RELEVANCE: Despite the use of a number of various cutoff values and sample sizes, serologic evaluation of a small number of calves or cows could not be used to accurately predict the presence of persistently infected cattle in a herd.

Animals↗

Characterization of vaccinia virus deletion mutants isolated from persistently infected Friend erythroleukemia cells.

Persistent viral infections in vitro are useful to study the evolution of virus populations in the absence of immunological pressure. Several deletion mutants have been isolated in this laboratory from Friend erythroleukemia cells persistently infected with vaccinia virus strain IHD-W, designated SQAvac. Two of the mutants, which remain stable after serial passage in L cells, have been characterized. The deletion which range between 20 to 22 kb, has been localized at the left terminus comprising HindIII fragments C and N. In addition, HindIII B fragment lost the sequences that hybridize to pAG5, a plasmid containing the 3.5 kb terminal sequences and acquired different restriction sites. Phenotypic characterization of these mutants revealed that they were not replication defective since they grew in all cell lines tested and produced plaques of the same size as the wild-type. However, they were less effective in suppressing host protein synthesis. The LD50 for the mutants titered in NIH Swiss female mice was greater than 10(9) PFU as compared to 10(6) PFU for the wild-type, indicating reduced virulence in vivo. These mutants, which display different properties to previously described mutants with deletions at the left terminus, provide another valuable system to study the molecular basis of virulence of vaccinia.

Animals↗

A variant of human cytomegalovirus derived from a persistently infected culture.

Infection of cells derived from an osteogenic sarcoma (HOS) with human cytomegalovirus (HCMV) resulted in persistent infection. It appears that persistent infection is due to a balance between release of virus and the growth of uninfected cells. Viruses derived from the persistently infected cultures were not temperature sensitive nor were they defective interfering particles. However, hybridization experiments using the Q-labeled probe from the XbaI Q fragment indicated that one copy of the repeat sequences contained in fragments Q and O of CMV, Towne DNA have been completely deleted from the virus DNA derived from the persistent culture. Thus the mechanism of persistent infection is probably due in part to a variant of CMV present in the cultures.

Base Sequence↗

Quasispecies evolution of a hypervariable region of the feline calicivirus capsid gene in cell culture and in persistently infected cats.

Feline calicivirus (FCV) is a respiratory pathogen of cats that is capable of causing persistent infections. This study examined the evolution of a hypervariable region of the FCV capsid gene both during 90 passages in cell culture and during replication in persistently infected cats. This region of the capsid protein is known to contain neutralization epitopes and may be a target for immune evasion during virus persistence in the host. Sequence analysis showed that FCV exists as a quasispecies which evolved both in cell culture and in persistently infected cats. Changes involved both loss of sequence present in the infecting isolate and a gain of both synonymous and non-synonymous nucleotide substitutions to generate sequences not detected within earlier isolates. Overall, these changes led to a reduction in population heterogeneity over time. Where virus populations were highly homogeneous allowing a consensus sequence to be determined, evolution rates for the consensus sequence ranged from 0.10-1.07 substitutions per nucleotide per year. Marked changes in virus neutralization profiles were seen in isolates obtained sequentially from a persistently infected cat. This was not the case with cell culture passaged virus, suggesting that the individual amino acid changes found only in virus from persistently infected cats may significantly alter the antigenic profile of FCV, and may be the result of immune selection.

Amino Acid Sequence↗

A study of measles virus antigens in acutely and persistently infected cells using monoclonal antibodies: differences in the accumulation of certain viral proteins.

Monoclonal antibodies were used to study measles virus (Hallé strain) infections. The distribution of virus antigens in acutely and persistently infected cells was examined by immunofluorescence, and the synthesis of the virus polypeptides in acutely infected cells was measured after immunoprecipitation and analysis in SDS-polyacrylamide gels. The virus nucleoprotein (NP) was detected 12 h after infection and was the major virus polypeptide synthesized throughout the virus cycle. The NP, matrix, and polymerase (L) polypeptides were first detected associated with the perinuclear membrane, but were later distributed throughout the cytoplasm. The hemagglutinin accumulated in the cell membrane in well-defined 'clusters'. In comparison with the acutely infected system, persistently infected cells presented important differences. There were only low levels of L polypeptides, and up to 50% of the cells contained intranuclear inclusions which stained specifically with NP monoclonal antibody. The relevance of these observations is discussed.

Animals↗