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Foot-and-mouth disease virus strains isolated from persistently infected cell cultures are attenuated for mice and cattle.

It was previously reported that during serial passage of a BHK-derived cell line persistently infected with foot-and-mouth disease virus, (termed C1-BHK-Rc1) the virus became increasingly more virulent for BHK-21 cells (de la Torre et al 1988). Virus strains isolated from different cell passage levels were tested for virulence in mice and cattle. The results showed that in the course of persistence in BHK cells, FMDV became progressively less virulent for mice and cattle.

Animals↗

Isolation and characterization of circulating feline leukemia virus-immune complexes from plasma of persistently infected pet cats removed by ex vivo immunosorption.

IgG and circulating IgG immune complexes (CIC) were purified from plasma of three pet cats persistently infected with feline leukemia virus (FeLV) by adsorption to, and elution from, Staphylococcus aureus Cowan I. CIC were then separated from free IgG by sucrose gradient ultracentrifugation and were analyzed for the presence of FeLV structural proteins and corresponding specific antibodies. Radioimmunoprecipitation analysis indicated that FeLV envelope (gp70) and major core (p30) proteins, along with cat IgG heavy and light chains, were present in the CIC from all three cats. Further analysis of the CIC from one of the cats also revealed the presence of FeLV core proteins p15 and p12. IgG purified from isolated CIC was also shown to bind specifically to purified FeLV gp70, p30, and p15. These data provide direct evidence for FeLV-specific CIC in the plasma of persistently viremic pet cats, and suggest these animals are immunologically response to the virus even though free antibodies against the major structural proteins cannot be demonstrated in standard assays.

Animals↗

Viral ribonuclei acid synthesis by Newcastle disease virus mutants isolated from persistently infected L cells: effect of interferon.

The synthesis of different viral ribonucleic acid (RNA) species was studied in chick embryo (CE) and mouse L-cell cultures infected with the Herts strain of Newcastle disease virus (NDV(o)) and a mutant isolated from persistently infected L cells (NDV(pi)). In CE cell cultures, both viruses synthesized significant amounts of 54, 36, and 18S RNA. However, in L cells, synthesis of 54S virion RNA was markedly reduced. From these results, it seems likely that the low yield of infective virus in L cells is due to a deficient synthesis of 54S RNA in this host. On this basis, however, it is apparent that the "covert" replication of NDV(o) in L cells is due to factors other than viral RNA synthesis. When low concentrations of interferon were used to pretreat CE cells, a differential effect on the synthesis of various RNA species was observed. The 18S RNA of NDV(o) was more sensitive to interferon action than the 36 and the 54S RNA species. In contrast, the 18S RNA of NDV(pi) was less sensitive than the 36S and the 54S RNA. The inhibition of 54S RNA synthesis correlated with the reduction of viral yield and explained the greater sensitivity of NDV(pi) to interferon.

Animals↗

Herpes simplex virus-specific hybridoma cells cannot be persistently infected with this virus.

Mouse hybridoma clones were examined for their ability to support replication of herpes simplex virus (HSV). Infection of hybridoma clone 1 cells producing an antibody not specific for HSV resulted in a persistent infection with a continuous production of infectious virus, whereas infection of the parental myeloma cells X63-Ag8.653 led to an abundant virus production and extinction of the culture. In contrast, infection of hybridoma cells producing HSV-specific antibodies was restricted to a few weeks. Infectious virus was isolated for a maximum of 10 days and, afterwards, viral antigens were detected by immunofluorescence for a maximum of 18 days. The neutralizing capacity of the antibodies was not essential since the pattern of infection in clone III E8 cells, producing a non-neutralizing antibody, did not differ from that observed in clones 2c and VI A6, which produced highly and weakly neutralizing antibodies, respectively. After loss of viral antigen, HSV DNA was no longer detected by Southern blot hybridization in hybridoma clone 2c cells. Since no difference other than the specificity of the produced antibodies is suspected between the hybridoma clones, the results suggest that the presence of HSV-specific antibodies in the B-lymphoid cell cultures is responsible for virus elimination from the cells.

Animals↗

Age-specific seroprevalence of Border disease virus and presence of persistently infected sheep in Basque dairy-sheep flocks.

Using p125/p80 antibody and antigen-ELISA tests, age-specific seroprevalence and presence of persistently infected (PI) sheep were investigated in six commercial latxa dairy-flocks, housed for variable periods. The flocks all had a recent history of Border disease (BD). Every flock included seropositive sheep and seven 0.5-3-year-old PI sheep were detected in two of four flocks tested. Age-specific antibody patterns differed according to the presence or absence of PI sheep in the flock. In flocks free of PI sheep, seroprevalence was 6-13% in 1-year-old sheep and 42-93% in older sheep. In contrast, seroprevalence was 67-99% in sheep raised with PI sheep for at least 1 year and 29-33% in replacement 0.5-0.6-year-old sheep (including a PI sheep) indicating that Border disease virus (BDV) transmission in Basque dairy-flocks can be relatively slow. Moderate seroprevalence in young replacement sheep should not discourage further testing to detect PI sheep, and our results highlight the risk of failing to achieve "natural vaccination" prior to pregnancy by mixing PI sheep with BDV-unexposed ewes.

Age Factors↗

Predictive values of serum and bulk milk sampling for the presence of persistently infected BVDV carriers in dairy herds.

The aim of this study was to examine whether it is possible to predict the presence of persistently infected (PI) animals with bovine viral diarrhoea virus on dairy farms in The Netherlands, based on a few blood samples of the herd, possibly in combination with a bulk milk test for antibodies. In 25 herds with, and 24 herds without, PI animal(s) the probabilities of obtaining at least x antibody positive animals out of a sample of n animals were calculated, with n varying from 3 to 7 and values for x that were considered were n, n - 1 to n - x. This probability, among animals 9-24 months old, ranged from 0.70 to 0.96 for herds with PI animals and from 0.13 to 0.37 for herds without. Using the result of bulk milk testing in addition did not add to the prediction. It was concluded that, due to the high percentage and large variation of antibody positive animals in herds without PI animals, it is not possible to predict the presence of PI animal(s) in dairy herds in The Netherlands using these methods.

Age Factors↗

Antigenic and genetic variation in influenza A (H1N1) virus isolates recovered from a persistently infected immunodeficient child.

Antigenic and genetic variations have been analyzed in eight consecutive isolates recovered from a child with severe combined immunodeficiency syndrome persistently infected with naturally acquired type A (H1N1) influenza virus over a 10-month period. Hemagglutination inhibition reactions and T1 oligonucleotide fingerprinting demonstrated that these viruses were related to strains causing outbreaks in the United States at that time (1983 to 1984) but that antigenic and genetic differences between consecutive isolates could be detected. This variation between isolates was examined further by sequencing the RNAs encoding the HA1 region of the hemagglutinin (HA) and the nucleoprotein (NP) in five of the consecutive isolates. Multiple point mutations were detected in both genes, and a deletion of one amino acid was detected in the HA. Depending on the isolates compared, 5.8 x 10(-3) to 17 x 10(-3) substitutions per nucleotide site per year were detected in the RNAs encoding the HA1, and 3.5 x 10(-3) to 24 x 10(-3) substitutions per nucleotide site per year were detected in the NP gene. Fifty-four percent of the base changes in the HA1 and 73% in the NP led to amino acid substitutions. A progressive accumulation of mutations over time was not observed, suggesting that the genetic diversity of these viruses may best be interpreted as the result of shifts in the population equilibrium (quasi-species) of replicating variant genomes.

Amino Acid Sequence↗

[Synthesis of RNA-containing virus-specific components in L cells persistently infected with SV5 virus].

An analysis of RNA-containing virus-specific structures was performed in the course of the establishment of persistent infection with SV5 virus in L cells. It was observed that up to the 11th passage in the LSV5 system, the same virus-specific structures were synthesized as in acute infection. At the level of the 18th passage synthesis of intracellular RNP was reduced and high 3H-uridine incorporation into light structures was observed. In the last passages examined (25th and 31st), the structures with rho 1.18--1.17 g/ml were predominant. The sedimentation RNA analysis (the 3rd, 6th, and 11th passages) revealed 50 S, 35--32 S, 24 S, and 18 S RNA. At later passages (25th and 31st), synthesis of 35 S, 24--22 S and lighter RNAs was dominant. The possibility of relationship between reduced 50 S RNA synthesis and low infectivity of intracellular virus is discussed.

Animals↗

Dominant selection of an invariant T cell antigen receptor in response to persistent infection by Epstein-Barr virus.

To examine T cell receptor (TCR) diversity involved in the memory response to a persistent human pathogen, we determined nucleotide sequences encoding TCR-alpha and -beta chains from HLA-B8-restricted, CD8+ cytotoxic T cell clones specific for an immunodominant epitope (FLRGRAYGL) in Epstein-Barr virus (EBV) nuclear antigen 3. Herein, we show that identical TCR protein sequences are used by clones from each of four healthy unrelated virus carriers; a clone from a fifth varied conservatively at only two residues. This dominant selection of alpha and beta chain rearrangements suggest that a persistent viral infection can select for a highly focused memory response and indicates a strong bias in gene segment usage and recombination. A novel double-step semiquantitative polymerase chain reaction (PCR) procedure and direct sequencing of amplified TCR cDNA from fresh lymphocytes derived from three HLA-B8 individuals detected transcripts specific for the conserved beta chain in an EBV-seropositive donor but not in two seronegative donors. This report describes an unprecedented degree of conservation in TCR selected in response to a natural persistent infection.

Amino Acid Sequence↗

Cellular distribution of bovine leukemia virus proteins gp51SU, Pr72(env), and Pr66(gag-pro) in persistently infected cells.

Monoclonal antibodies (mAbs) against bovine leukemia virus (BLV) mature proteins and precursors were used to map the localization of these proteins in persistently infected non-lymphocytic cell lines using immunofluorescence assay (IFA) and immuno-electron microscopy. IFA staining was observed in the basolateral surface of live FLK-BLV cells. When using a mAb against Pr66(gag-pro), mottled pinpoint fluorescence was seen in the cell surface of polarized cells, but no reaction was observed in cells undergoing mitosis. However, a mAb against Pr72(env) stained only mitotic cells and cellular fragments. Additionally, in these dividing cells, this envelope (Env) precursor polyprotein was not evenly distributed but concentrated predominantly in only one daughter cell. To the best of our knowledge, this observation has not been reported previously, either for BLV or for other retroviruses. The results of immunogold electron microscopy confirmed the specificity of the mAbs in the intracellular level. In infected cells, Pr72(env) and gp51SU were seen in proximity at the plasma membrane in incipient budding sites. Additionally, the mAb against Pr72(env) also reacted with Env precursor polyproteins in the mitochondria of BLV-bat(2) ultrathin sections. These mAbs may be used as a tool for mapping virus excretion sites in the cell surface of naturally or in vitro infected cells in the different stages of the cell cycle.

Animals↗

[Development of the controlled model of persisting infection , caused by Pseudomonas aeruginosa and bacteria of the complex Burkholderia cepacia].

As the result of testing three different variants, the experimental models of persisting infection for P. aeruginosa and B. cepacia have been developed. These doses differ in the time of administration, doses of antibiotics and the infective doses of the microorganisms. The administration of the sub-inhibiting concentration of antibiotics for 5 days and the subsequent infection of laboratory animals (non-inbred mice) B. cepacia strains in a dose of LD50 leads to a considerable increase in the survival rate of mice and to a longer period (up to 20 days) of obtaining inoculative material from the spleen. The isolated cultures are characterized by a sharply slower growth on artificial culture media (up to 5-7 days as compared with 24-48 hours for the initial culture). The newly developed models have made it possible to control different stages of the infectious process in the induced increase or decrease of the virulent properties of the infective agent and in changes in the immune status of the host. As the result of these studies, in some mice (10%) infected with B. cepacia after the injection of gamma-hydroxybutyric acid lactone the infection has taken the acute form, while in the mice infected with P. aeruginosa no such effect has been observed. On the contrary, in the mice infected with P. aeruginosa and then receiving cyclophosphamide the transition of the infection into the acute form has been observed in 30% of the animals. In the mice infected with B. cepacia no such effect has been noted after the injection of this preparation. Different effects produced by cyclophosphamide and lactone are discussed from the positions of "quorum sensing" in pathogenic bacteria.

Animals↗

Mycobacterium tuberculosis signal transduction system required for persistent infections.

It is estimated that nearly 2 billion people currently suffer from latent Mycobacterium tuberculosis infection. Although the key front-line antituberculosis drugs are effective in treating individuals with acute tuberculosis, these drugs are ineffective in eliminating M. tuberculosis during the persistent stages of latent infection. Consequently, therapeutics that directly target persistent bacilli are urgently needed. We have conducted a global analysis on a group of regulatory determinants that may play a role in M. tuberculosis virulence, and identified a two-component response regulator whose expression is required for entrance into and maintenance of persistent infection. Inactivation of this response regulator, Rv0981 (termed here mprA for mycobacterial persistence regulator), affected M. tuberculosis H37Rv growth in vivo in an organ- and infection stage-specific fashion. These results indicate that two-component systems are important for adaptation of the tubercle bacillus during stages of persistent infection.

Animals↗

[Modeling a persistent infection by tick-borne encephalitis virus strains in a cell culture of the clawed toad].

Differences in the ability of 12 tick-borne encephalitis virus strains to induce persistent infection in the culture of clawed frog cells incubated at 26-32 degrees C were shown. In 8 strains the process of persistence was accompanied by the stable preservation of the infectious virus in the culture medium during 10 passages, while in 4 strains the virus disappeared already in passages 3-8.

Animals↗

Persistent infection associated with residual cement after resection arthroplasty of the hip.

BACKGROUND: The danger of residual bone cement after resection of infected prosthetic components is controversial. PATIENTS AND METHODS: We analyzed 10 patients with infected total hip prosthesis who had been treated previously with resection arthroplasty and antibiotics and who had persistent infection with residual cement. In 9 patients, surgical debridement with resection of all the PMMA was performed, and adequate intravenous antibiotics were administered. 1 patient refused surgical treatment, but accepted antibiotics. RESULTS: At an average of 4 (1-18) years of follow-up, 8 patients had no signs or symptoms of recurrent infection. 1 severely immunodeficient patient died 2 years after the removal of residual cement, for reasons other than his hip. The patient who refused surgical treatment continues to have an active sinus 4 years after first consultation. INTERPRETATION: Residual cement may be responsible for chronic infection. At resection arthroplasty, as part of the treatment of an infected hip arthroplasty, all devitalized or foreign material must be removed.

Adult↗

Persistent infection of normal mice with human immunodeficiency virus.

In this article, we report the establishment of persistent HIV type 1 infection of normal Swiss mice after a single intraperitoneal injection with high-producing HIV-infected U937 cells. Anti-HIV antibodies were found more than 500 days after the original injection, and p24 antigenemia was detected in approximately 50% of the mice. By polymerase chain reaction (PCR) techniques, HIV-specific gag and env sequences were detected in DNA samples from peripheral blood mononuclear cells (PBMC) and peritoneal cells of seropositive mice 300 to 500 days after inoculation with HIV-infected cells. These DNA samples did not contain human DNA sequences, as determined by PCR analysis using primers and the probe for the HLA-DQ alpha gene. Low levels of p24 and detectable human reverse transcriptase activity were found in cultures of PBMC and peritoneal macrophages. Cocultivation of PBMC, peritoneal cells, and spleen cells with human uninfected U937 or CEM (a T lymphoma cell line) cells resulted in HIV infection of the target cells, as determined by PCR analysis and/or p24 assays. The intravenous injection of untreated Swiss mice with the PBMC from PCR-positive mice resulted in the development of an increasing antibody response to HIV in the recipient animals. Together these results indicate that cells from seropositive Swiss mice were persistently infected with HIV and were capable of producing infectious virus. The development of persistent HIV infection in an immunocompetent mouse may represent the starting point for further studies aimed at defining the host mechanisms involved in the restriction of virus replication, defining the pathogenesis of HIV infection, and testing antiviral compounds and vaccines.

Animals↗