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PubMed1982

Development of protease activation mutants of HVJ (Sendai virus) in persistently infected cell cultures.

HVJ wild-type virus, in which the F protein is activated by trypsin but not by elastase, was spontaneously converted to a mutant with an F protein characterized by being activated by elastase alone. This spontaneous mutation generally occurred during serial passages of cells persistently infected with HVJ, even though the cells were first established by infection with plaque-purified wild-type virus. Multiple-cycle replication, plaque formation, haemolysis and SDS-polyacrylamide gel electrophoretic (SDS-PAGE) analysis showed that all the elastase-activated mutants isolated from HVJ carrier cells no longer required trypsin for F protein activation. At early passages, these protease activation mutants did not show temperature-sensitive (ts) growth, while at a later stage the mutants, together with the ts mutation, appeared dominant. The frequency of such a protease activation mutation during passage in the HVJ carrier cells seemed to depend on the cell species, but was increased when compared to lytic infections.

Animals↗

PubMed1995

Genotyping human papillomavirus type 16 isolates from persistently infected promiscuous individuals and cervical neoplasia patients.

Nucleotide sequence variation in the noncoding region of the genome of human papillomavirus type 16 (HPV16) was determined by direct sequencing and single-strand conformation polymorphism analysis of DNA fragments amplified by PCR. Individuals of diverse sexual promiscuity and/or cervicopathology were studied. In a group of 14 healthy, monogamous HPV16-positive females, only two HPV16 sequence variants could be documented. Among 17 females and 3 males with multiple sex partners and living in the same geographical region, nine sequence variants were found, whereas among 7 patients with cervical neoplasia from another region, five variants were detected. Although numbers are limited, in the group of individuals at high risk of acquiring a sexually transmitted disease or with cervical neoplasia, a larger number of HPV16 sequence variants was encountered (two types among 14 individuals versus nine types among 20; Fisher's exact test, P = 0.07). Seven of the individuals were sampled repeatedly over time. For these persistently infected women, no differences in HPV16 sequences were detected, irrespective of promiscuity, and persistence of a single viral variant, spread over multiple anatomic sites, for more than 2 years could be demonstrated. This indicates that viral persistence may be a common feature and that successful superinfection with a new variant may be rare, despite a potentially high frequency of viral reinoculation.

Base Sequence↗

PubMed2005

Indication of transmission of BVDV in the absence of persistently infected (PI) animals.

In a closed dairy herd all animals were tested serologically for BVD antibodies twice a year during a 6-year period. Seroconversions were detected every year. At the start of the 6-year monitoring period blood samples from all animals were examined by virus isolation. No persistently infected animals were identified. Entire-herd culturing for BVDV was repeated at the end of the third year. Samples from all newborn female calves were examined for BVDV at approximately 2 months of age and older. During the entire monitoring period BVDV was isolated in one newborn calf twice with an interval of 3 weeks. The mother had seroconverted during pregnancy. Five congenitally infected non-PI calves were identified, the mothers of which had seroconverted during late pregnancy; repeated sampling proved the calves to remain seropositive in a seronegative age cohort. Although direct and indirect introduction of BVDV from outside the herd can never be excluded it seems highly unlikely in this closed herd. The findings indicate that transmission of BVDV can take place over a long period of time in the absence of PI animals. This observation may have serious consequences for control programmes.

Animals↗

PubMed1999

Acute and persistent infection of human neural cell lines by human coronavirus OC43.

Human coronaviruses (HuCV) are recognized respiratory pathogens. Data accumulated by different laboratories suggest their neurotropic potential. For example, primary cultures of human astrocytes and microglia were shown to be susceptible to an infection by the OC43 strain of HuCV (A. Bonavia, N. Arbour, V. W. Yong, and P. J. Talbot, J. Virol. 71:800-806, 1997). We speculate that the neurotropism of HuCV will lead to persistence within the central nervous system, as was observed for murine coronaviruses. As a first step in the verification of our hypothesis, we have characterized the susceptibility of various human neural cell lines to infection by HuCV-OC43. 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, oligodendrocytic MO3.13, and the CHME-5 immortalized fetal microglial cell lines, were all susceptible to an acute infection by HuCV-OC43. Viral antigen and RNA and release of infectious virions were observed during persistent HuCV-OC43 infections ( approximately 130 days of culture) of U-87 MG, U-373 MG, MO3.13, and H4 cell lines. Nucleotide sequences of RNA encoding the putatively hypervariable viral S1 gene fragment obtained after 130 days of culture were compared to that of initial virus input. Point mutations leading to amino acid changes were observed in all persistently infected cell lines. Moreover, an in-frame deletion was also observed in persistently infected H4 cells. Some point mutations were observed in some molecular clones but not all, suggesting evolution of the viral population and the emergence of viral quasispecies during persistent infection of H4, U-87 MG, and MO3.13 cell lines. These results are consistent with the potential persistence of HuCV-OC43 in cells of the human nervous system, accompanied by the production of infectious virions and molecular variation of viral genomic RNA.

Cell Line↗

PubMed1984

Antigenic variation of HVJ (Sendai virus) HN glycoprotein detectable by monoclonal antibodies during persistent infection.

Three newly established monoclonal hybridoma antibodies to the haemagglutinin molecule of HVJ, designated A7, B3 and F11, recognize operationally non-overlapping antigenic determinants and have neutralizing activity. Using these antibodies, the frequencies of occurrence of neutralization-resistant antigenic variants were analysed in virus populations released from four cell lines persistently infected with HVJ, namely GM2-HVJ, LLCMK2-HVJ, Vero-HVJ and GEsl-HVJ at various passage stages. Antigenic variants were selected from culture fluids of these HVJ carrier cells at a total frequency of 10(-3.3), 10(-3.8) and 10(-3.6) by monoclonal antibodies A7, B3 and F11, respectively. These values were considerably higher than those of 10(-4.7) to 10(-5.2) detected in a stock preparation of wild-type virus with these antibodies. All the variant viruses isolated as above were negative in neutralization, haemagglutination inhibition and immunofluorescent staining tests with each monoclonal antibody used for their isolation, but were positive with the other antibodies.

Animals↗

PubMed1996

Establishment and characterization of Japanese B encephalitis virus persistent infection in the Sf9 insect cell line.

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.

Animals↗

PubMed2003

Latent tuberculosis: mechanisms of host and bacillus that contribute to persistent infection.

Most people infected with Mycobacterium tuberculosis contain the initial infection and develop latent tuberculosis. This state is characterised by evidence of an immune response against the bacterium (a positive tuberculin skin test) but no signs of active infection. It can be maintained for the lifetime of the infected person. However, reactivation of latent infection occurs in about 10% of infected individuals, leading to active and contagious tuberculosis. An estimated 2 billion people worldwide are infected with M tuberculosis--an enormous reservoir of potential tuberculosis cases. The establishment and reactivation of latent infection depend on several factors, related to both host and bacterium. Elucidation of the host immune mechanisms that control the initial infection and prevent reactivation has begun. The bacillus is well adapted to the human host and has a range of evasion mechanisms that contribute to its ability to avoid elimination by the immune system and establish a persistent infection. We discuss here current understanding of both host and bacterial factors that contribute to latent and reactivation tuberculosis.

Animals↗

PubMed2000

Borna disease virus persistent infection activates mitogen-activated protein kinase and blocks neuronal differentiation of PC12 cells.

Persistence of Borna disease virus (BDV) in the central nervous system causes damage to specific neuronal populations. BDV is noncytopathic, and the mechanisms underlying neuronal pathology are not well understood. One hypothesis is that infection affects the response of neurons to factors that are crucial for their proliferation, differentiation, or survival. To test this hypothesis, we analyzed the response of PC12 cells persistently infected with BDV to the neurotrophin nerve growth factor (NGF). PC12 is a neural crest-derived cell line that exhibits features of neuronal differentiation in response to NGF. We report that persistence of BDV led to a progressive change of phenotype of PC12 cells and blocked neurite outgrowth in response to NGF. Infection down-regulated the expression of synaptophysin and growth-associated protein-43, two molecules involved in neuronal plasticity, as well as the expression of the chromaffin-specific gene tyrosine hydroxylase. We showed that the block in response to NGF was due in part to the down-regulation of NGF receptors. Moreover, although BDV caused constitutive activation of the ERK1/2 pathway, activated ERKs were not translocated to the nucleus efficiently. These observations may account for the absence of neuronal differentiation of persistently infected PC12 cells treated with NGF.

Animals↗

PubMed1989

Antigenic and restriction enzyme analysis of isolates of Campylobacter fetus subsp venerealis recovered from persistently infected cattle.

Thirty-two isolates of Campylobacter fetus subsp venerealis were obtained from 1 bull and 4 heifers with experimentally induced infection. When whole-cell antigens of isolates were cross titrated with antisera to the infecting strain, isolates from 3 heifers had limited antigenic variation, whereas whole-cell antigens of isolates from 2 cattle (the bull and a heifer) differed serologically from those of the infecting strain. Changes were detected specifically in 6 heat-labile antigens. Of the 6 heat-labile factors evaluated, all were initially present on the infecting parent strain, but not on early isolates obtained from 4 of the 5 cattle. Restriction enzyme analysis revealed minor variation in the DNA fingerprints of isolates obtained from individual cattle, thus implying stability of the Campylobacter genome once persistent infection is established. Isolates with identical restriction enzyme patterns expressed different heat-labile antigens. Correlation could not be found between the DNA electrophoretic pattern and the expression of heat-labile antigens.

Animals↗

PubMed1989

Susceptibility of Coxiella burnetii to pefloxacin and ofloxacin in ovo and in persistently infected L929 cells.

The relative lack of efficacy of the antibiotic treatment of chronic Q fever endocarditis justifies the further evaluation of the susceptibility of Coxiella burnetii to the modern quinolone antibiotics. We evaluated the efficacies of pefloxacin and ofloxacin in controlling the Nine Mile isolate of C. burnetii by using an embryonated egg assay and persistently infected L929 cells in culture. Pefloxacin was effective in controlling the intracellular parasite at a concentration of 50 micrograms per egg and 1 microgram/ml in cultures of infected cells. Ofloxacin was effective at a concentration of 25 micrograms per egg and 0.5 microgram/ml in infected-cell cultures. In light of the fact that the concentrations of antibiotics used fall within physiological ranges used in humans, ofloxacin and pefloxacin may be useful in the clinical management of chronic Q fever, for which, to date, results have been poor.

Animals↗

PubMed2006

Superinfection of human immunodeficiency virus type 1 (HIV-1) to cell clone persistently infected with defective virus induces production of highly cytopathogenic HIV-1.

Superinfection with human immunodeficiency virus type 1 (HIV-1) in human subjects, defined as reinfection with a heterologous strain of HIV-1, has become a topic of great interest. To illustrate the significance of this occurrence, we performed HIV-1 superinfection of L-2 cells, which were isolated from MT-4 cells persistently infected with subtype B HIV-1 as a cell clone continuously producing defective HIV-1 particles. L-2 cells carrying provirus with a one-base insertion in the pol protease were superinfected with HIV-1 derived from primary isolates of subtype B or CRF01_AE. The kinetics of the superinfection in L-2 were very slow compared with those of primary infections in MT-4. Interestingly, L-2 shifted after superinfection to become a producer of highly cytopathogenic HIV-1. Molecular characterization revealed that superinfection occurred in only about 10% of the CRF01_AE-superinfected L-2, which carried provirus of both subtypes and produced viral particles containing genomic RNA of both subtypes. Surprisingly, such cytopathogenic HIV-1 showed predominantly the original subtype B phenotype. Thus, the mechanism of the production of cytopathic HIV-1 seemed to be mediated by trans complementation with pol products of superinfected CRF01_AE. These findings suggest the significance of long-lived infected cells as recipients for superinfection that could result in the generation of new HIV-1 variants with high virulence in patients who are off therapy or do not adhere to treatment, and may indicate the need for precautions against such superinfection.

Cell Line↗

PubMed2005

Relationship of bovine viral diarrhea virus persistent infection to incidence of diseases on dairy farms based on bulk tank milk test by RT-PCR.

The prevalence of bovine viral diarrhea virus (BVDV) in dairy herds in Hokkaido, Japan, was estimated by reverse transcription polymerase chain reaction (RT-PCR) using bulk tank milk samples. Sixteen out of 265 dairy herds were identified as BVDV positive, and at least one persistently infected (PI) cattle was recognized in each of the positive herds except for two herds of which, owners did not agree to examine individual cows. The proportion of positive herds with a history of BVDV PI was significantly higher than that with no history of BVDV PI (odds ratio (OR) 4.25, 95% confidence interval (CI) 1.471-12.278, p = 0.004). The herds examined for BVDV were divided into two groups, high and low disease incidence groups based on the occurrence of diseases such as diarrhea, pneumonia or abortion in the past 1 year. The BVDV positive herds in the high disease incidence group were significantly more than that in the low disease incidence group (OR 2.92, CI 1.110-7.683, p = 0.024). It was observed that there were significantly (p = 0.008) more PI calves or heifers in farms of high disease incidence group than in farms of low disease incidence group. These results suggested that bulk tank milk test was available method for the detection of PI animals in dairy herds, and the existence of PI non-lactating cows in herd correlated with the incidence of diseases of the diarrhea or respiratory disorders.

5' Untranslated Regions↗

PubMed1998

Identification of persistent infection in experimental syphilis by PCR.

The studies described herein were designed to evaluate the usefulness of the PCR in detecting persistent syphilitic infection. Three groups of animals were used: a nonimmune group infected with Treponema pallidum (NI/TP), a nonimmune group injected with heat-killed treponemes (NI/HKTP), and an immune and reinfected group (I/TP). All animals were inoculated with similar numbers of organisms distributed at 10 sites on the clipped back and in both testes. The persistence of the treponemes was examined by PCR and the rabbit infectivity test (RIT). The kinetic studies and statistical analysis of their results demonstrated that the rate of bacterial clearance from the NI/TP group was very low and incomplete at 4 months after infection. It was significantly different from those of both the NI/HKTP (P < 0.001) and I/TP (P < 0.05) groups. No statistically significant differences in treponemal elimination were found between the NI/HKTP and I/TP groups. PCR can detect the DNA of dead organisms, but the latter are eliminated by the host relatively quickly (15 to 30 days) as compared to elimination of live treponemes (>120 days). PCR results correlated well with RIT results. These data suggest that PCR-positive specimens obtained from an untreated patient(s) or collected weeks after treatment indicate persistent infection. They also show that the process of elimination of T. pallidum from primary sites of infection is prolonged and incomplete.

Animals↗

PubMed2005

Transcriptional profiling of human herpesvirus type B (HHV-6B) in an adult T cell leukemia cell line as in vitro model for persistent infection.

Human herpesvirus 6 (HHV-6), which is present in more than 90% of the human, is known to cause infectious diseases in immuno-compromised patients, e.g., transplant patients. To clarify the possible role of the pattern of expression of HHV-6 genes in various types of HHV-6B infection, we sought to determine whether or not viral DNA microarray could be used for detailed characterization of viral transcription using a HHV-6B DNA microarray that contains 97 known open reading frames of HHV-6B. A subset of genes are preferentially expressed in persistent infection: U16 (IE-B, transactivator, US22 gene family), U18 (IE-B, homolog to HCMV IE glycoprotein), U20 (glycoprotein), U27 (DNA polymerase processivity transactivator), U82 (gL, gH accessory protein), U83 (chemokine), U85 (OX-2 homology, glycoprotein), U90 (IE-A), and U94 (transactivator), respectively. Although the function of each HHV-6B is not fully understood, our study suggests that comprehensive analysis of HHV-6B transcription is useful not only to clarify the pathogenesis of the virus but also to develop new strategies for anti-viral drugs.

Cell Line, Tumor↗

PubMed1985

Persistently infected cultures as a source of hepatitis A virus.

Primary African green monkey kidney, continuous African green monkey kidney cell line BS-C-1, and buffalo green monkey kidney cultures were infected with a uniform inoculum of hepatitis A virus (HAV). Although both the cell line BS-C-1 and primary African green monkey kidney cultures produced useful amounts of virus, HAV was detected earlier and in greater quantities in primary African green monkey kidney cultures. A persistently infected primary African green monkey kidney culture was developed. The influence of incubation time (4 to 40 days) and concentration (2 to 15%) of fetal calf serum in the maintenance medium on production of HAV by this culture was examined. An incubation period of 24 to 28 days was found to be optimal; reducing this period led to decreased yields of HAV. No significant difference in the amount of HAV produced was observed with differing concentrations of fetal calf serum. Three different methods of extraction and the effect of multiple extractions on the recovery of HAV from cell lysates were examined. Sonication was a critical factor. Two extractions yielded more than 90% recoverable virus. Yields in excess of 10(11) physical particles of HAV per 850-cm2 roller bottle were routine. The total yield could be increased by concentrating the HAV present in spent maintenance medium by using bentonite or organic flocculation.

Animals↗

PubMed1995

Roles of immunoglobulin valency and the heavy-chain constant domain in antibody-mediated downregulation of Sindbis virus replication in persistently infected neurons.

Clearance of infectious Sindbis virus from neurons is mediated by antibody to the E2 glycoprotein. Properties of the antibody important for downregulation of Sindbis virus replication are unknown. Immunoglobulin isotypes and valency determine many biological properties of antibodies. An immunoglobulin G1 (IgG1) isotype switch mutant and F(ab')2 and Fab fragments of IgG3 monoclonal antibody 209 were prepared and tested for clearance of infectious virus from persistently infected rat dorsal root ganglion neurons in vitro. IgG1, IgG3, and IgG3-derived F(ab')2 fragments were similarly efficacious, while IgG3-derived Fab fragments had no effect on virus replication. Cross-linking of Fab with secondary antibodies restored antiviral activity. Therefore, we found no evidence that IgG subclass plays a role in control of intracellular Sindbis virus replication. However, bivalency appears to be crucial for the ability of E2-specific IgG molecules to mediate clearance of infectious virus from neuron cells, suggesting that cross-linking of E2 molecules is essential.

Animals↗

PubMed1991

Expression of the aphthovirus RNA polymerase gene in Escherichia coli and its use together with other bioengineered nonstructural antigens in detection of late persistent infections.

A plasmid has been constructed containing the DNA sequences that direct the expression of the aphthovirus RNA-dependent RNA polymerase (virus infection-associated antigen, VIAA) in its native form. The aphthovirus polypeptide was designed to contain only a single additional amino acid, the N-terminal methionine. The recombinant protein has been purified and used in enzyme-linked immunoelectrotransfer blots to detect aphthovirus-specific antibodies in the sera of persistently infected animals. Furthermore, studies were carried out to test the hypothesis that antibodies against other nonstructural antigens appear in the sera of these animals. It was established that antibodies against polypeptides 3A and 3B can serve as complementary markers for late aphthovirus-carrier state detection. The considerable potential of this approach to detect aphthovirus-specific antibodies, when the isolation of infectious virus is not possible, was demonstrated. Negative results were obtained in animals from virus-free areas and in vaccinated cattle. This assay has the added advantage that no infectious or noninfectious virus is involved during antigen production.

Animals↗

PubMed1990

Isolation of a specific cellular mRNA by subtractive hybridization in Theiler's virus persistent infection.

Viruses change the mRNA repertoire of the tissues they infect. They add viral mRNAs and they specifically alter the expression of some host genes. These events can play important parts in pathogenesis. In principle, it should be possible to isolate viral mRNAs and to identify changes in host gene expression using subtractive hybridization. We tested this approach in the persistent infection of mouse central nervous system by Theiler's virus. A cDNA library was constructed with poly A+ RNA from infected mouse spinal cords. The library was screened with a subtracted probe. We identified one mitochondrial gene, coding for subunit 1 of cytochrome oxidase, which is overexpressed in infected tissues whereas another mitochondrial gene, URF 2, is not. Subtractive hybridization should prove to be invaluable in studying the pathogenesis of chronic human central nervous system diseases of unknown etiology.

Animals↗