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Biomedical subjects

Y S Boriskin

Publications and source records attributed to Y S Boriskin.

At least 19 recordsLinked to original sources
PubMed2001

Quantitative nested PCR for human cytomegalovirus DNA: analysis using NucleoScan 2001.

A quantitative nested DNA PCR for human cytomegalovirus (CMV) was developed using PCR mimic DNA as an internal standard. A combination of ultra-thin gel and enhanced detection of ethidium bromide-stained PCR bands allowed us to achieve a 25-fold increase in sensitivity over the conventional gel electrophoreses, down to 0.1 ng of DNA per band. When the technique was applied to measuring CMV load in CMV positive leucocyte DNA preparations its sensitivity spanned the range from single to hundreds of CMV DNA copies in 0.1 microg of total white blood cell DNA.

Cytomegalovirus↗
PubMed2001

Human cytomegalovirus UL21.5 gene is expressed as an "early-late" gene in cultured human fibroblasts.

The human cytomegalovirus (CMV) UL21.5 gene encodes a secreted glycoprotein of unknown function. Both the UL21.5 protein and mRNA accumulate in abundance at late stages of infection making the RNA an attractive target for diagnosis of active CMV infection. The UL21.5 was originally described as a 'spliced late' gene (SLG) (Rawlinson and Barrell, J. Virol. 67, 5502 (1993)). However, we found that the the UL21.5 mRNA was detectable in CMV-infected patients before the onset of CMV DNA replication (Boriskin et al., J. Clin. Virol., in press). Here, we re-examined the UL21.5 mRNA kinetic class in CMV-infected human fibroblast culture using a RNAse protection assay and RT-PCR. The UL21.5 mRNA was detectable before the "true late" UL75 RNA, was resistant to a CMV DNA replication inhibitor but moderately sensitive to inhibitors of protein synthesis. In the presence of protein synthesis inhibitors the UL21.5 mRNA was detectable only by a nested reverse transcription - PCR (RT-PCR) with the bulk of it in unspliced form. This suggests that splicing factors for UL21.5 mRNA are encoded by the virus rather than by the cell. Our results indicate that UL21.5 should be defined as an "early-late" rather than a "late" (L) CMV gene.

Antigens, Viral↗
PubMed1999

Human cytomegalovirus genome sequences in lymph nodes.

Human cytomegalovirus (CMV) is a major cause of morbidity in heart and lung transplant patients, resulting from immunosuppression-mediated reactivation of latent CMV originating either from the transplanted tissue, or the recipient. We showed that out of eight donor/recipient pairs, the lymph nodes (LNs) of three donors and four recipients, all CMV seropositive, harboured CMV DNA at exceeding levels compared with those of matched blood samples, as well as CMV RNA otherwise undetectable in patients' blood. On follow-up, patients positive for CMV DNA and RNA in LNs developed viraemia 4 to 5 weeks earlier than those initially polymerase chain reaction-negative for CMV. Our results indicate that LN are a significant site for sequestration and persistence of CMV and that LN may be important in seeding of CMV-infected cells into the circulation.

Cytomegalovirus↗
PubMed1999

Viral loads in dual infection with HIV-1 and cytomegalovirus.

OBJECTIVE: A one year study of the relation between cytomegalovirus (CMV) and human immunodeficiency virus (HIV) viral loads in a cohort of children with vertically acquired HIV-1 infection. DESIGN: Comparative analysis of viral load measurements for CMV and HIV-1 in peripheral blood leucocytes (PBLs) of individual children in relation to age and clinical staging. METHODS: Nested polymerase chain reaction (PCR) was used to measure HIV-1 proviral DNA and CMV genomic DNA in PBLs of 56 children. RESULTS: The CMV load was highest in 0-2 year old HIV positive children with stage C disease (range, 1-7143 copies/100 ng DNA; median, 125) and was significantly lower in older children. Although higher in young children, HIV-1 viral load did not show the same marked reduction with age that is seen with CMV. Over a one year period, testing of serial samples for both viruses in a subgroup of children revealed a discordant relation between viral loads for CMV and HIV-1. CONCLUSIONS: CMV viral load falls much faster than HIV viral load in dually infected children. Screening for clinical CMV disease is most likely to be of benefit in children under 2 years of age with stage C disease. In the few children studied, levels of CMV and HIV replication appear to be independent.

Adolescent↗
PubMed1999

An abnormally long HIV-1 env DNA PCR product due to altered sequences of primer binding sites.

To investigate the generation of an abnormally long HIV-1 env PCR DNA product the latter was cloned and sequenced followed by sequence analysis of HIV-1 primer binding sites. We found that the formation of an abnormally long PCR product was due to HIV-1 env sequence alteration (a) in the reverse primer binding site resulting in faulty primer binding and (b) downstream from the forward primer sequence resulting in a new binding site with reverse complementary sequence with respect to the forward primer at the opposite end of the PCR product. Both changes led to amplification of a longer PCR product with forward primer alone. Our results indicate that the HIV-1 genetic diversity in the env gene can lead to amplification of a specific PCR product of unexpected size which can be disregarded in the absence of its cross-validation.

Adolescent↗
PubMed1999

Is hepatitis C virus genotype 4 predominant in Saudi Arabia?

Hepatitis C virus (HCV) genotype 4 is believed to be predominant in the Middle East including Saudi Arabia (SA). We attempted to genotype 80 HCV isolates from different parts of SA by direct sequencing of a variable 222bp fragment from the NS5B region. The phylogenetic analysis of the NS5B sequences was complemented by direct sequence analysis of the conserved 5'-NCR region for HCV type-specific polymorphism. All 80 NS5B sequences separated into 3 clades which comprised 6 type 1b variants, 30 type 4 variants (24 of type 4a and 6 of type 4c or d) and 44 type 3 variants. Apart from two definitive type 3b variants the other 42 type 3 NS5B sequences formed 4 clusters with low similarity to type 3a-f HCV sequences from the database. The precise subtyping of these 42 type 3 variants awaits sequencing of longer HCV RNA stretches. Our results indicate that HCV type 4 may not be the only dominant genotype in SA.

Endemic Diseases↗
PubMed1997

Molecular evaluation of enteroviruses in the pathogenesis of idiopathic dilated cardiomyopathy.

BACKGROUND: There is great disparity in the literature as to the presence and relevance of enterovirus in heart tissue from patients with dilated cardiomyopathy (DCM). Published estimates of enteroviral positive tissue in DCM range from 0 to 50%. Very little sequence information is so far available on those samples which are positive. HYPOTHESIS: Re-examination of fresh biopsy material from patients previously tested, plus 13 new cases of DCM, and sequencing the products would yield information on the validity of the technique and on the type of virus being detected. METHODS: RNA from biopsy or explant tissue was tested for the presence of enterovirus using reverse transcriptase polymerase chain reaction (PCR). The nucleotide sequences of all positive PCR products were determined by direct sequencing. RESULTS: Positive PCR signals were found in 10% of samples from patients with DCM and in 16% of control tissues. Two DCM and 12 control samples gave the same nucleotide sequence, which was different from the CB3 used as a positive control. The other 4 DCM samples all produced multiple bands on sequencing. CONCLUSION: The results do not support a major role for enterovirus in DCM. There is need for some caution, however, as a review of the literature shows that studies using single biopsies, such as this one, produce consistently lower estimates for enterovirus than do those wherein multiple biopsies are examined.

Adolescent↗
PubMed1996

Human cytomegalovirus and acute rejection after heart transplantation are not directly associated.

Retrospective and prospective analyses of heart transplant recipients showed no significant association between acute rejection and the detection of cytomegalovirus (CMV) infection by culture or the polymerase chain reaction (PCR) for viral DNA, neither on grounds of the incidence of both conditions nor in relation to which was diagnosed first in the patient. Semiquantitative PCR of serial blood and endomyocardial biopsy specimens from individual patients revealed different patterns in the development of the viral DNA in the blood and the heart, also clear episodes of CMV infection in CMV antibody-negative recipients of hearts from CMV antibody-negative donors, none of whom went on to develop a CMV-specific antibody response. None of these findings was associated with the development of rejection in the patient. On the other hand, in those patients who did experience rejection, peak levels of CMV DNA in the blood and the heart were usually not reached until 6 weeks or more after transplantation, whereas in those in whom rejection was not detected at all during the period of observation, peak levels of CMV DNA were detected earlier, mainly within the first 6 weeks after transplantation. In several cases, the delayed increase in CMV DNA in those with rejection, albeit not the delay itself, was linked to treatment with steroids. These findings support the view that CMV infection and rejection are independent events, but that the timing of the infection, and whether or not rejection is detected, are indicative of the general status of the immune response in individual patients.

Cytomegalovirus↗
PubMed1996

A polymerase chain reaction to detect a spliced late transcript of human cytomegalovirus in the blood of bone marrow transplant recipients.

A reverse transcription (RT) nested polymerase chain reaction (PCR) procedure is described for detecting RNA to a spliced late gene (SLG) of human cytomegalovirus (CMV), the product of which (175 bp) is easily differentiated in agarose gels from the product when the target is unspliced viral RNA or DNA (258 bp). The SLG-RT-PCR has been compared against a semi-quantitative PCR for CMV DNA in buffy-coat specimens collected weekly after bone marrow transplantation from 3 patients and against the results of culturing these specimens for CMV both by conventional virus isolation, based on the detection of cytopathic effect, and by the early detection of infected cells by staining with virus-specific monoclonal antibodies. The detection of CMV RNA by SLG-RT-PCR correlated well with the detection of infective virus but only when the results of both culture methods were combined, in that neither culture method alone was as sensitive as the SLG-RT-PCR. The presence of SLG RNA in the circulation is of value as a marker of active CMV infection.

Base Sequence↗
PubMed1995

Activation and deactivation of membrane currents in human fibroblasts following infection with human cytomegalovirus.

The whole cell patch clamp technique was used to study the effects on membrane currents of infection of cultured human embryonic lung (HEL) fibroblasts with human cytomegalovirus (CMV). Four types of membrane currents were found in uninfected HEL cells, namely: Ca(2+)-activated potassium current, inward rectifier potassium current, delayed rectifier potassium current and voltage-dependent CMV. Voltage-dependent sodium current was detected in 30% of uninfected HEL cells whenever they were examined up to 72 h after seeding; however this current had completely disappeared by 18 h after infection with CMV. The delayed rectifier potassium current was detectable in 8% of uninfected HEL cells but, after infection, the proportion of cells expressing this current gradually increased from 20% at 18-24 h post-infection to 100% at 48 h and 72 h. Pharmacological agents known to regulate the activity of ion channels, via cellular secondary messengers, did not alter the frequency at which either current was detected in uninfected and infected cells. Phosphonoformate, an inhibitor of CMV DNA polymerase, caused 95% block of expression of CMV 'late' proteins in infected cells but did not prevent the switching off of the sodium current or the increased expression of the potassium current. The results indicate an association between the expression of CMV 'immediate-early' or 'early' proteins and the down-regulation of the sodium current and up-regulation of the potassium current.

Cells, Cultured↗
PubMed1994

Sequence variation within the carboxyl terminus of the nucleoprotein gene of mumps virus strains.

BACKGROUND: On rare occasions, clinically apparent mumps virus infection and meningitis can be caused by vaccine virus as well as wild type strains. This has been shown by nucleotide sequencing of short regions of the viral genome. OBJECTIVES: We wished to confirm and extended these findings by investigating for strain differences within the carboxyl terminus of the nucleoprotein (NP) gene, which is known to be highly variable amongst paramyxoviruses in general. STUDY DESIGN: A 576 bp carboxyterminal fragment of NP was amplified by reverse transcriptase (RT) PCR from several different strains and was sequenced directly. RESULTS: Three Urabe vaccine variants and five clinical isolates previously classified as Urabe strains revealed the same carboxyterminal pattern for NP. Three other isolates and the Leningrad-3 vaccine strain showed characteristic differences of up to 5% compared to the Urabe strains, with U/C substitutions being predominant. CONCLUSIONS: The carboxyterminal sequence of NP varies significantly between mumps virus strains such that it can be used for differentiating between vaccine and wild-type isolates.

Journal Article↗
PubMed1988

Salt-induced enhancement of measles virus yields in cultured cells.

Supplementation of culture medium with MgSO4 or Na2SO4 in millimolar concentrations caused an enhanced measles virus (MV) yield from cultured quail embryo cells. MgSO4 at 25-50 mM concentrations exhibited the most pronounced and consistent stimulatory effect. MV infectivity increases ranged from 2- to 200-fold; the effect was highly reproducible for stationary monolayer, roller or microcarrier-grown cell culture types. MgSO4 also improved MV plaque development and caused MV plaque size enlargement on Vero cell monolayers. At mM concentrations MgSO4 was not operative as a MV thermostabilizing agent; rather, salt-induced enhancement of MV yields appeared to be due to intracellular events, e.g., augmented viral protein synthesis.

Animals↗
PubMed1983

Myxoviruses do not induce non-specific alterations in membrane permeability early on in infection.

The permeability characteristics of cells infected with myxoviruses have been studied by measuring the concentrative uptake of nutrients, the concentration of intracellular K+, and the maintenance of the Na+ gradient across the plasma membrane. Cells either show no change at all (Sendai virus-infected BHK cells and measles virus-infected Vero cells) or they show a decreased ability to concentrate nutrients, while intracellular K+ and the Na+ gradient remain unchanged (Sendai and influenza virus-infected L-1210 cells, measles virus-infected lymphocytes and mumps virus-infected L-41 cells). In no case, therefore, was a change observed that resembles the non-specific increase in membrane permeability induced by haemolytic paramyxoviruses (35, 42) or the non-specific membrane leakiness postulated to take place in infected cells (8, 9). A preliminary account of some of these findings has been presented (39).

Animals↗
PubMed1981

Comparative study of rabies virus persistence in human and hamster cell lines.

Persistent infections by rabies virus in BHK-21/13S and HEp-2 cells were studied comparatively. No evidence of interferon production, selection of virus-resistant cells, or integration of the viral genome could be found. Persisting viruses replicated efficiently at 34, 36, and 40 degrees C. Both persistently infected cultures released defective interfering virus particles. A cyclical pattern of infection, which was not characteristic of the persistently infected HEp-2 system, was observed in persistently infected BHK cultures. The virus from persistently infected BHK cultures lost its virulence for mice, whereas the virus from persistently infected HEp-2 cultures retained mouse-killing capacity for more than 3 years.

Animals↗