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PubMed1978

Isolation of a precipitating glycoprotein antigen from cell cultures persistently infected with bovine leukemia virus.

A procedure was developed to isolate a glycoprotein with precipitating antigen activity from fluids from fetal lamb kidney cell cultures persistently infected with bovine leukemia virus (BLV). The antigen was precipitated by ammonium sulfate and subjected to affinity chromatography on concanavalin A Sepharose. The glycoprotein was eluted with alpha-methyl-D-mannoside and was further purified by gel filtration over Sephadex G-100. Antigen activity was determined by agar gel immunodiffusion (AGID) reactions with serum from cattle infected with the virus. The major portion of the AGID activity was eluted from the Sephadex G-100 in the 60,000-dalton elution region. In some experiments, identical AGID activity was also found in the 18,000-dalton elution region. The larger protein was discovered to have a molecular weight of 58,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its designation as a glycoprotein was confirmed by carbohydrate-positive staining. The isolated BLV glycoprotein antigen did not contain ovine or bovine proteins as indicated by gel immunodiffusion.

Antigens, Viral↗
PubMed1988

Some observations on the semen of bulls persistently infected with bovine virus diarrhoea virus.

As a result of screening procedures employed for animals entering the AI service, two bulls were identified as being persistently infected with bovine virus diarrhoea virus by isolation of the virus from blood. Semen was collected on two occasions from these bulls; its quality as measured by density and motility was poor. Gross abnormalities of the sperm head, termed 'collapsed' heads, were seen in 28 to 45 per cent of sperm from one bull and in 1 per cent of sperm from the other. The collapsed heads were small and the whole head or its anterior part had the appearance of a dried pea. Electron microscopy showed the defect to consist of convoluted nuclear material with membrane-bound vacuoles and invaginations containing membranous debris and lamellar structures. In the 'high incidence' bull there was a corresponding increase in enlarged sperm heads. The 'low incidence' bull had sperm with heads of similar mean size to sperm from control bulls but with an increased variance. The semen was diluted in a lactose diluent, frozen and stored in liquid nitrogen. The distribution of viral antigen was determined and virus was isolated from several fractions of the semen, both before and after processing and cryopreservation. In one animal raw semen failed to yield virus but virus was recovered after processing, suggesting that raw semen may not be suitable for the efficient detection of the virus.

Animals↗
PubMed2005

Association between the existence of calves persistently infected with bovine viral diarrhea virus and commingling on pen morbidity in feedlot cattle.

OBJECTIVE: To determine the association between the existence of a calf persistently infected (PI) with bovine viral diarrhea virus (BVDV) and pen morbidity. ANIMALS: 5,041 calves in 50 pens at a feedlot in Iowa. PROCEDURE: In a longitudinal study, ear notches were collected from cattle and tested for BVDV antigen. Characteristics of each pen (owner, sex, disease rate, number of groups, and source) were recorded. The association between the existence of a BVDV-PI calf and morbidity in each pen was examined. RESULTS: Commingling was associated with an increase in respiratory tract disease (odds ratio [OR], 3; 95% confidence interval [CI], 2.5 to 3.6). Ten BVDV-PI calves (10/5,041 [0.2%]) were identified in 8 of 50 pens. A BVDV-PI calf was associated with reduced pen-level respiratory tract disease (OR, 0.7; 95% CI, 0.5 to 0.9). Disease prevalence (mean +/- SD morbidity, 7.9 +/- 3.1%) was lowest in pens containing single-source cattle and a BVDV-PI calf (4 pens containing 302 cattle), compared with single-source cattle with no BVDV-PI calf (mean morbidity, 11.89 +/- 9.7%; 31 pens containing 3,093 cattle), commingled cattle with no BVDV-PI calf (mean morbidity, 29.3 +/- 16.22%; 11 pens containing 1,127 cattle), and commingled cattle with a BVDV-PI calf (mean morbidity, 28.6 +/- 10.1%; 4 pens containing 519 cattle). CONCLUSIONS AND CLINICAL RELEVANCE: Commingling was the greatest risk factor associated with morbidity in each pen. A BVDV-PI calf in a pen was not associated with increased disease prevalence in commingled groups.

Animal Husbandry↗
PubMed1982

Experimental infection of rabbits with bovine herpesvirus-4: acute and persistent infection.

A strain of bovine herpesvirus-4 (BHV-4) isolated from bovine cases of mammary pustular dermatitis was used for experimental infection of rabbits. The strain is serologically indistinguishable from the group prototype Movar 33/63 and from the American isolate DN599. Groups of rabbits were inoculated by various routes. Intravaginal and conjunctival inoculations resulted in vulvovaginitis and conjunctivitis, respectively, and in shedding of virus. The rabbits seroconverted for the virus, with high titers of antibodies (indirect fluorescent antibody test) that persisted throughout the experiment. Treatment with dexamethasone, beyond the acute infection, did not produce recrudescence of disease or shedding of the virus. Rabbits were killed at various times, from 3 to 6 months post-infection, and the virus was recovered from explant cultures of spleen and by cocultivation of spleen cells with bovine lung cells. These results demonstrate the usefulness of the rabbit as a model for studying the pathogenesis of BHV-4 infection in cattle.

Animals↗
PubMed2003

Characterization of HeLa cells persistently infected with influenza virus B/Lee/40 with respect to telomerase activity and apoptosis.

OBJECTIVE: The purpose of this study was to examine telomerase activity and apoptotic changes in HeLa cells persistently infected with influenza viruses B/Lee/40 (He/Le cells). METHODS: He/Le cells were established as described previously [Intervirology 2002;45:67-70], and passaged twice a week. The existence of influenza virus genes was monitored by the reverse transcription polymerase chain reaction (RT-PCR). Telomerase activities in He/Le cells were assayed by Telochaser (stretch PCR method). Apoptotic changes in He/Le cells were examined using the Apoptotic DNA Ladder Kit and the In situ Cell Death Detection Kit, Fluorescence. RESULTS: In He/Le cells, (1) all eight influenza virus genes were detected by RT-PCR until 62 days post infection (p.i.); (2) only nucleoprotein gene remained detectable until 120 days p.i.; (3) telomerase activity of He/Le cells normalized to those of uninfected HeLa cells was remarkably decreased (16-55% of control) during the persistence of influenza and recovered up to 80% of control on day 168 p.i. when no influenza virus gene was detected by RT-PCR, and (4) no apoptotic changes were detected despite the continuous existence of influenza virus genes. CONCLUSION: In He/Le cells, telomerase activity was suppressed exclusively during the persistence of influenza, and no apoptotic changes were detected.

Apoptosis↗
PubMed1998

Molecular characterization of a nondemyelinating variant of Daniel's strain of Theiler's virus isolated from a persistently infected glioma cell line.

Wild-type Daniel's strain of Theiler's virus (wt-DA) induces a chronic demyelination in susceptible mice which is similar to multiple sclerosis. A variant of wt-DA (designated DA-P12) generated during the 12th passage of persistent infection of a G26-20 glioma cell line failed to persist and induce demyelination in SJL/J mice. To identify the determinants responsible for this change in phenotype, we sequenced the capsid coding sequence (nucleotides [nt] 2991 to 3994) and found three mutations in VP1: residues 99 (Gly to Ser), 100 (Gly to Asp), and 103 (Asn to Lys). To study the role of these mutations in neurovirulence and demyelination, we prepared a recombinant virus, DAP-1C-2A/DA, with replacement of wt-DA nt 2991 to 3994 with the corresponding region of DA-P12, and viruses with individual point mutations at VP1 residues 99(Ser), 100(Asp), and 103(Lys). DAP-1C-2A/DA and viruses with a mutation at VP1 residue 99 or 100 (but not 103) completely attenuated the ability of wt-DA to induce demyelination. Failure to induce demyelination was not due to a general failure in growth, since DA-P12 and other mutant viruses lysed L-2 cells in vitro as effectively as wt-DA. The change in disease phenotype was independent of the specific B- or T-cell immune recognition because a decrease in the neurovirulence of mutant viruses was observed in neonatal mice and immune-deficient RAG1 -/- mice. This difference in neurovirulence is not the complete explanation for the failure of DA-P12 to demyelinate, since virus with a mutation at residue 103(Lys) had decreased neurovirulence but did induce demyelination. Therefore, point mutation at VP1 residue 99 or 100 altered the ability of wt-DA to demyelinate, perhaps related to a disruption in interaction between virus and receptor on certain neural cells.

Animals↗
PubMed2005

Detection of BVDV persistently infected animals in Belgium: evaluation of the strategy implemented.

Until now, no official bovine virus diarrhea virus (BVDV) control program has been implemented in Belgium. The only legislation dealing with the detection of BVDV-infected animals concerns the purchase of animals. A strategy of control, based on the identification and elimination of persistently infected (PI) animals and the vaccination of cows before insemination has been designed in both the Northern and the Southern part of the country. The strategy of detection of PI animals relies on PCR testing of pools of blood. Individual blood samples corresponding to the positive pools are then tested by BVDV-antigen ELISA. A first evaluation of the measures already applied in Belgium is presented. Data obtained in 2003 are presented and discussed regarding the validation of the laboratory strategy, the prevalence of positive herds, the genotype of circulating viruses, the outcome of antigen positive animals and the need for improvement of the current legislation.

Animals↗
PubMed1998

Persistent infection and vascular disease: a systematic review.

More than 150 epidemiological or clinical studies have reported on associations between vascular disease and the presence of certain persistent bacterial and viral agents, or of clinical conditions (e.g., periodontal disease) that are associated with persistent infection. This article provides a review of such studies, particularly in relation to Helicobacter pylori, Chlamydia pneumoniae, dental disease and cytomegalovirus (CMV), as well as references to possible mechanisms. The association between coronary heart disease and H. pylori or between heart disease and dental disease may be accounted for by residual confounding. Although markers of C. pneumoniae infection are around twenty times more common in atherosclerotic plaques than in disease-free blood vessels, the sequence of infection and disease is uncertain. For CMV, a limited number of patients with classic atherosclerotic coronary heart disease have been investigated in seroepidemiological studies. For all such agents, better and larger seroepidemiological and pathology-based studies are needed to resolve these uncertainties, as well as - at some stage - large-scale, randomised intervention studies.

Journal Article↗
PubMed1992

Age distribution of animals persistently infected with bovine virus diarrhea virus in twenty-two Danish dairy herds.

The objectives of this study were to compare the age distribution of animals persistently infected (PI) with bovine virus diarrhea virus (BVDV) in 12 herds with clinical BVD compared to ten herds without clinical BVD and to examine the incidence of PI calves born after the oldest PI animal. Blood samples from all animals were tested for bovine virus diarrhea virus and antibodies. In five herds, blood samples were obtained from calves born after the whole herd had been tested. All calves born by PI dams were also blood tested. In herds with clinical BVD the median age of PI animals was 248 days and in herds without clinical BVD the median age was 144 days. There was no significant difference between the age of PI animals in herds with clinical BVD compared to herds without clinical BVD (p = 0.48) suggesting similar epidemiology of the occurrences of PI animals in the two herd categories. Thereafter, all herds were used to study the incidence of PI animals. A total of 129 PI animals were found. In ten herds with 72 PI animals the age range of PI animals was more than six months. In these herds 26.3% of the PI animals were born within the first two months after birth of the oldest PI animal, no PI animals were born 2- less than 6 months, 52.7% were born 6- less than 14 months, 6.9% were born 14- less than 22 months and 13.9% (all born by PI dams) were born later than 22 months after the oldest PI animal.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1994

Reversal of the measles virus-mediated increase of phosphorylating activity in persistently infected mouse neuroblastoma cells by anti-measles virus antibodies.

To investigate the effect of persistent measles virus infection on signal transduction in cells of neuronal origin, the mouse neuroblastoma cell line NS20Y/MS, which is persistently infected with measles virus, was used. The results demonstrate an approximate 50% increase in total phosphorylation and a similar increase in protein kinase C (PKC) activity. Western blot analysis with anti-total PKC or anti-PKC-alpha antibodies revealed a significant increase in the level of an 80K immunoreactive PKC in NS20Y/MS cells. Following incubation of NS20Y/MS cells with polyclonal anti-measles virus antibodies, which down-regulate the level of measles virus proteins, total and PKC-mediated phosphorylation returned to the basal level of uninfected cells. This effect was reversible and removal of the antibodies resulted in restoration of the high level of total and PKC-mediated phosphorylation. The release of infectious measles virus was strongly inhibited by incubation of NS20Y/MS cells with the PKC inhibitor, 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H-7). These results demonstrate that measles virus induces elevation in cellular phosphorylation which is essential for measles virus production.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1985

Immunoglobulin A antibody against hepatitis B core antigen in the acute and persistent infection with hepatitis B virus.

Antibody to hepatitis B core antigen of immunoglobulin A class was determined in the serum of patients infected with hepatitis B virus by a sandwich-type solid-phase radioimmunoassay with monoclonal antibodies. The antibody, as defined by a sample to normal ratio greater than 2.1, was detected in all of 39 patients with acute hepatitis, with titers varying widely depending on the time of blood sampling. In persons with persistent infection, the antibody was detected in only 2 (4%) of 46 asymptomatic carriers of the virus, contrasting with the positivity in as many as 15 (41%) of 37 patients with chronic persistent hepatitis, in 45 (94%) of 48 patients with chronic active hepatitis, and in 40 (87%) of 46 patients with liver cirrhosis with or without hepatocellular carcinoma. The mean +/- SE titer of antibody in chronic persistent hepatitis (3.8 +/- 0.9) was significantly lower than those in chronic active hepatitis (13.8 +/- 3.2) and cirrhosis with or without carcinoma (25.6 +/- 6.1) (p less than 0.001). Based on the results obtained, the antibody may reflect hepatic injury in the persistent hepatitis B virus infection.

Antibodies, Monoclonal↗
PubMed1990

Persistent infection of a glioma cell line generates a Theiler's virus variant which fails to induce demyelinating disease in SJL/J mice.

Theiler's murine encephalomyelitis virus (TMEV) induces demyelinating disease which is associated with persistent virus infection of the central nervous system. To study the interaction between TMEV and host cells, we infected the G26-20 glioma cell line in vitro, and this resulted in a lytic infection in which most, but not all, cells were killed. Surviving cells divided and formed a viable monolayer in which a small proportion of cells displayed viral cytopathic effects. Levels of virus produced by these cultures over a 6 month period fluctuated between 6 and 8 log10 p.f.u./ml as measured by viral plaque assay. Similarly, the percentage of cells producing both viral antigen and viral RNA, as measured by a simultaneous immunoperoxidase/in situ hybridization technique, varied between 5 and 30%. Although persistently infected cultures were susceptible to challenge by both vesicular stomatitis virus and herpes simplex virus, they were resistant to infection by homologous viruses. Interferon activity was not identified. TMEV isolated from passage 12 produced smaller plaques than wild-type Daniels strain virus (wt-DAV) on L-2 cell monolayers. In contrast to demyelination induced in SJL/J mice after intracerebral inoculation with wt-DAV, mice infected with the small plaque variant virus failed to develop viral persistence or chronic demyelination. However, following immunosuppression by total body irradiation, SJL/J mice infected with the small plaque variant developed viral persistence but no demyelination. Characterization of the biochemical and molecular determinants of the variant will lead to a better understanding of determinants important in viral persistence.

Animals↗
PubMed2003

Cathepsin B Is Inhibited in Mutant Cells Selected during Persistent Reovirus Infection.

Persistent reovirus infections of murine L929 (L) fibroblast cells select mutant (LX) cells that do not support proteolytic disassembly of reovirus virions within the endocytic pathway. To better understand the function and regulation of endocytic proteases, we conducted experiments to define the block to reovirus disassembly displayed by LX cells. In contrast to parental L cells, mutant LX cells harbor defects that interfere with the maturation and activity of cathepsin B and cathepsin L but not cathepsin H. The cDNAs encoding cathepsin B and cathepsin L in L cells are identical to those in LX cells, indicating that LX cells manifest an extrinsic block to the function of these enzymes. Mixed lysates of L cells and LX cells lack activity of both cathepsin B and cathepsin L, suggesting the presence of an inhibitor of cathepsin function in LX cells. A cathepsin B-green fluorescent protein (GFP) fusion protein expressed in L cells and purified by immunoprecipitation retains cathepsin B activity, whereas cathepsin B-GFP expressed in LX cells does not. However, activity of cathepsin B-GFP expressed in LX cells can be recovered by incubating the immunoprecipitate with L cell lysate followed by immunoprecipitation, providing further evidence that LX cells express a cathepsin inhibitor. Native-gel electrophoresis and gel filtration chromatography demonstrate that, in both cell lines, the double-chain form of cathepsin B is sequestered in a large molecular weight complex that renders this form of the enzyme inactive. Alteration of this sequestration complex appears to be responsible for inhibition of cathepsin B in LX cells. These findings suggest that cathepsins can be regulated within the endocytic pathway. Moreover, this regulation influences host cell susceptibility to intracellular pathogens.

Animals↗
PubMed1980

Cell-mediated immune response in pigs persistently infected with a Mycobacterium avium strain.

Following a persistent bacterial infection with a Mycobacterium avium strain in pigs, the lymphocyte stimulation (LS), leucocyte migration inhibition (LMI) and tuberculin skin reactivity responses were studied. The responses in the LS and LMI tests occurred within a period of 35 to 49 days after infection. Positive tuberculin skin responses were found 40 to 70 days after infection. No direct correlation was evident between the LS and LMI tests during the experimental period. The responses to purified protein derivatives (PPD) in the LMI test were of shorter duration than those seen in the LS test. The course of LS in pigs expressed high, lower and non-stimulatory responses to the tested PPDs. At the end of the experimental period, 173 days after infection, lymphocyte stimulatory reactivity to PPDs could be demonstrated in all pigs, whereas a positive tuberculin skin test was observed in only one animal. Lymphocyte responses to M avium PPD were generally higher than were the other tested PPDs.

Animals↗
PubMed1997

Establishment of serial persistent infections with bovine viral diarrhoea virus in cattle and sheep and changes in epitope expression related to host species.

A pestivirus was transmitted by contact from a persistently infected (P.I.) bullock to pregnant sheep. This resulted in the birth of P.I. lambs, one of which in turn was able to transmit virus by contact to pregnant cattle. Two of these animals gave birth to P.I. calves, from one of which the virus was again transmitted by contact with pregnant sheep, leading to another generation of P.I. lambs. The expression of one or more epitopes on the E2 glycoprotein of the viruses isolated from this series of alternate cattle-sheep transmissions appeared to depend on the host species. Thus, several monoclonal antibodies which bound strongly to, and neutralised, viruses isolated from the bovine hosts, failed to bind or neutralise in the case of sheep isolates. The viral consensus sequences of the E2 gene as well as parts of the 5' untranslated region and of the Npro and capsid genes were compared between the different isolates. This revealed a high degree of genetic stability. However, a single codon change at amino acid position 9 of the E2 gene correlated with and was able to cause the loss of particular epitopes.

Animals↗
PubMed1976

Rhesus monkeys kidney cells persistently infected with Simian Virus 40: production of defective interfering virus and acquisition of the transformed phenotype.

Monolayer cultures of LLC-MK2 rhesus monkey kidney cells became persistently infected with simian virus 40 (SV40) when infected at a multiplicity of infection of 100 plaque-forming units/cell. A stable carrier state developed characterized by extensive viral proliferation without obvious cytopathic effect other than the slow growth of these cultures. By 11 weeks all cells produced the SV40 T antigen. In contrast, less than 5% of the cells produced V antigen. Virus-free clonal isolates were obtained by cloning in SV40 antiserum. Continuous cultivation in antiserum resulted in a temporary cure of unclone cultures. When virus did eventually reappear in the "cured" cultures the titers remained low. The virus produced by the carrier culture was defective at both 31 and 37% c, and it interfered with the growth of standard s40 during mixed infection of CV-1 green monkey kidney cells. All of the interfering activity in carrier culture homogenates could be sedimented by centrifugation at 109,000 x g for 3 h. These cultures were completely susceptible to vesicular stomatitis virus. Extensive viral deoxyribonucleic acid synthesis occurred in CV-1 cells infected with carrier culture virus. Carrier culture homogenates are only slightly less cytopathic to CV-1 cells than standard SV40. The carrier culture express several properties of SV40 transformation.

Animals↗
PubMed2004

Persistent infection with primate foamy virus type 1 increases human immunodeficiency virus type 1 cell binding via a Bet-independent mechanism.

We report that human T cells persistently infected with primate foamy virus type 1 (PFV-1) display an increased capacity to bind human immunodeficiency virus type 1 (HIV-1), resulting in increased cell permissiveness to HIV-1 infection and enhanced cell-to-cell virus transmission. This phenomenon is independent of HIV-1 receptor, CD4, and it is not related to PFV-1 Bet protein expression. Increased virus attachment is specifically inhibited by heparin, indicating that it should be mediated by interactions with heparan sulfate glycosaminoglycans expressed on the target cells. Given that both viruses infect similar animal species, the issue of whether coinfection with primate foamy viruses interferes with the natural course of lentivirus infections in nonhuman primates should be considered.

Animals↗
PubMed1979

Reproductive performance of apparently healthy cattle persistently infected with bovine viral diarrhea virus.

A Holstein-Friesian bull and three Holstein-Friesian cows were seronegative for bovine viral diarrhea (BVD) virus but were persistently infected with the virus. Virus was isolated from buffy coat cells and nasal and lacrimal secretions during their lifetime, and they remained free of clinical signs of BVD. The three cows were pregnant when purchased, and they gave birth to full-term calves. One calf lived only a few hours, one calf became ill and died within a few days, and one calf became ill and was euthanatized within a few weeks. One cow was then bred and became pregnant but aborted a 7-month fetus. A second cow was bred approximately 5 months after parturition but did not conceive. The third cow was necropsied 6 weeks after calving, because of loss of weight. Although the bull's semen contained BVD virus when seropositive cows were bred, normal calves were born. When seronegative heifers were bred, they became seropositive to BVD virus within two weeks, with higher titers in six weeks. On heifer conceived after one service but aborted a 6-month fetus. Three others continued to have estrous cycles until their titers rose to 1:128, then they conceived and gave birth to normal calves. Another heifer conceived on the first service, had a titer of 1:128 two weeks after breeding, and gave birth to a normal calf.

Animals↗