Prevalence and molecular characterization of the polymerase gene of gibbon lymphocryptovirus

BACKGROUND: Lymphocryptovirus (LCV) is found in various non-human primates. As a herpesvirus naturally infecting gibbons it is closely related to human Epstein-Barr virus (EBV) with which it shares considerable genetic, biological and epidemiologic features. METHODS: We collected blood samples from 70 gibbons (51 Hylobates lar, 18 Hylobates pileatus and 1 Hylobates agilis) for further separation into serum and peripheral blood mononuclear cells (PBMC). RESULTS: Only 13 of 70 (18.6%) sera were serologically positive for human EBV IgG but 64 of 70 (91.4%) PBMCs yielded the partial LCV DNA polymerase gene by semi-nested PCR, which we subjected to direct sequencing. All sequences showed 84% nucleic acid and 91% amino acid identity to human EBV. Phylogenetic tree analysis demonstrated gibbon LCVs clustered separately from other gammaherpesvirinae but closely related to LCV of other species. CONCLUSIONS: Based on LCV DNA detection, we discovered a high prevalence of LCV infection among gibbons. Further characterization of non-human primate LCV might thus provide new insight into both evolution and pathogenicity of gammaherpesvirinae.     

Quantitative PCR in EBV-infected renal transplant patients

In this study we investigated the levels of Epstein Barr virus (EBV) DNA by quantitative polymerase chain reaction (Q-PCR) in serum, whole blood and peripheral blood mononuclear cells (PBMC) from anti-EA IgG seropositive or anti-EA IgG seronegative EBV infected renal transplant recipients. We compared serological data with the viral load to monitor the risk of developing post-transplant lymphoproliferative disorders (PTLD). All patients were asymptomatic and none of them developed PTLD at the time of the study. EBV DNA quantitation for each patient varied in whole blood and PBMC samples probably due to different numbers of mononuclear cells present in samples from which DNA was extracted (whole blood vs. purified PBMC). In 92% of the serum samples EBV DNA was undetectable probably due to absence of free genomes since the number of DNA copies detected in samples from whole blood and PBMC does not reach very high levels. The correlation between the presence of EA-antibody, considered serological evidence of EBV reactivation, and the viral load showed that 60% of EA-positive patients had quantifiable EBV DNA, whereas in 40% of EA-positive patients EBV DNA was undetectable, showing serological reactivity but no viral replication. Of the remaining EA-negative patients, EBV DNA could be detected in 71% of them, whereas 29% did not show EBV DNA, indicating no EBV replication. In conclusion, our results confirm that the presence of serum IgG anti-EA antibody is not a reliable marker of active EBV infection whereas the evaluation of the viral load in blood samples is a useful diagnostic tool to monitor and to better understand the course of EBV infection in immunocompromised renal transplant patients at risk of developing PTLD.     

Epstein-Barr virus types 1 and 2 differ in their EBNA-3A, EBNA-3B, and EBNA-3C genes.

The two Epstein-Barr virus (EBV) types, EBV-1 and EBV-2, are known to differ in their EBNA-2 genes, which are 64 and 53% identical in their nucleotide and predicted amino acid sequences, respectively. Restriction endonuclease maps and serologic analyses detect few other differences between EBV-1 and EBV-2 except in the EBNA-3 gene family. We determined the DNA sequence of the AG876 EBV-2 EBNA-3 coding region and have compared it with known B95-8 EBV-1 EBNA-3 sequences to delineate the extent of divergence between EBV-1 and EBV-2 isolates in their EBNA-3 genes. The B95-8 and AG876 EBV isolates had nucleotide and amino acid identity levels of 90 and 84%, 88 and 80%, and 81 and 72% for the EBNA-3A, -3B, and -3C genes, respectively. In contrast, nucleotide sequence identity in the noncoding DNA adjacent to the B95-8 and AG876 EBNA-3 open reading frames was 96%. We used the polymerase chain reaction to demonstrate that five additional EBV-1 isolates and six additional EBV-2 isolates have the type-specific differences in their EBNA-3 genes predicted from the B95-8 or AG876 sequences. Thus, EBV-1 and EBV-2 are two distinct wild-type EBV strains that have significantly diverged at four genetic loci and have maintained type-characteristic differences at each locus. The delineation of these sequence differences between EBV-1 and EBV-2 is essential to ongoing molecular dissection of the biologic properties of EBV and of the human immune response to EBV infection. The application of these data to the delineation of epitopes recognized in the EBV-immune T-cell response is also discussed.     

Detection of a Novel Bovine Lymphotropic Herpesvirus

Degenerate PCR primers which amplify a conserved region of the DNA polymerase genes of the herpesvirus family were used to provide sequence evidence for a new bovine herpesvirus in bovine B-lymphoma cells and peripheral blood mononuclear cells (PBMC). The sequence of the resultant amplicon was found to be distinct from those of known herpesvirus isolates. Alignment of amino acid sequences demonstrated 70% identity with ovine herpesvirus 2, 69% with alcelaphine herpesvirus 1, 65% with bovine herpesvirus 4, and 42% with bovine herpesvirus 1. Phylogenetic analysis placed this putative virus within the tumorigenic Gammaherpesvirinae subfamily, and it is tentatively identified as bovine lymphotropic herpesvirus. This novel agent was expressed in vitro from infected PBMC, and cell-free supernatants were used to transfer infection to a bovine B-cell line, BL3. Analysis, with specific PCR primers, of DNA from bovine PBMC and lymphoma cells identified infection in blood of 91% of adult animals (n = 101), 63% of lymphomas (n = 32), and 38% of juveniles (n = 13). Of the adults, herpesvirus infection was present in 94% of animals that were seropositive for bovine leukemia virus (BLV) (n = 63) and in 87% of BLV-seronegative animals (n = 38). Of the seropositive group, 17 animals exhibited persistent lymphocytosis, and 100% of these were herpesvirus positive by PCR. A role for bovine lymphotropic herpesvirus as a cofactor in BLV pathogenesis is considered.     

DNA of herpesvirus pan, a third member of the Epstein-Barr virus-Herpesvirus papio group

The DNA of herpesvirus pan, a primate B-lymphotropic herpesvirus, shares about 40% well-conserved sequence relatedness with Epstein-Barr virus (EBV) and herpesvirus papio DNAs. Labeled cloned fragments from the EBV recombinant DNA library were cross hybridized to blots of EcoRI, XbaI, and BamHI restriction endonuclease fragments of herpesvirus pan DNA to identify and map homologous sequences in the herpesvirus pan genome. Regions of colinear homology were demonstrated between 6 x 10(6) daltons and 108 x 10(6) daltons in the DNAs. The structural organization of herpesvirus pan DNA was similar to the format of Epstein-Barr virus and herpesvirus papio DNAs. The DNA consists of two domains of largely unique sequence complexity, a segment US of 9 x 10(6) daltons and a segment UL of 88 x 10(6) daltons. US and UL are separated by a variable number of tandem repetitions of a sequence IR (2 x 10(6) daltons). There was homology between DNA which mapped at 26 to 28 x 10(6) daltons and 93 to 95 x 10(6) daltons in UL. The terminal reiteration component, TR, of herpesvirus pan DNA and sequences which mapped to the left of 6 x 10(6) daltons and to the right of 108 x 10(6) daltons had no detectable homology with the corresponding regions of Epstein-Barr virus DNA.     

Study of the viral infections and cytokines associated with recurrent aphthous ulceration

Mouth ulcers are one of the most common oral complaints. However, the association between oral ulceration and viruses and cytokines is uncertain. We detected the presence of human papilloma virus (HPV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV)-1, HSV-2 and human herpesvirus (HHV)-8 DNA in oral tissues by polymerase chain reaction (PCR) and Southern hybridization techniques, and quantified the serum levels of cytokines including interleukin (IL)-2, IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), soluble Fas (sFas) and the Fas ligand (FasL) by enzyme-linked immunosorbent assay (ELISA) for 67 recurrent aphthous ulcer (RAU) patients and 72 normal individuals. Seven patient specimens were excluded from the study due to the negative PCR results for the beta-globin used as the internal control. Among the 32 (53.3%) virus-positive results from 60 patients' samples, 8 (13.3%) HPV, 4 (6.7%) HSV-1, 11 (18.3%) CMV, 9 (15.0%) EBV, and 16 (26.7%) HHV-8 samples proved to be positive. No HSV-2-positive samples were found. The percentage of single-virus infection (56.3%) was significantly greater than that of double-virus co-infection (31.3%) and the percentage of double-virus co-infection was significantly greater than the percentage of triple-virus co-infection (12.5%) (P < 0.05). In the 72 normal oral-tissue specimens, no viral DNA was detected. The mean serum cytokine level for patients was significantly (P < 0.05) greater than for controls for most of the separate age groups. The mean serum cytokine concentrations for the patient group demonstrated a diffuse pattern covering a wide range of serum concentrations, a very different result from the compact serum concentration pattern and lower mean serum cytokine concentrations revealed by the normal group. Overall association between viruses and recurrent aphthous ulceration is HHV-8 > CMV > EBV > HPV > HSV-1, regarding the frequency of prevalence (P < 0.05).     

作物数量遗传学基础 二、遗传力及其估算

两栖类染色体的研究,过去多使用以生殖 细胞和端蚌尾部上皮细胞为材料的水低渗压片 法^[6]。然而,生殖细胞和峪蚌组织受季节的限 制颇大,所得的中期分裂相亦不甚多。随着 低等脊稚动物组织培养技术^[1,2]与血液培养技 术^[3]的发展,近年来已更多的使用离体培养的 细胞来进行研究。     

Serological diagnosis of infection with human herpesvirus type 6.

OBJECTIVE--To identify clinical consequences of acute human herpesvirus type 6 infection by hypothesising that the virus will induce similar clinical syndromes to cytomegalovirus. DESIGN--Examination of consecutive serum samples from patients with illnesses compatible with acute cytomegalovirus infection or exanthem subitum by indirect immunofluorescence for the presence of antibodies to human herpesvirus type 6. An IgG absorption step was included to avoid false positive and negative results for IgM. The criterion standard for diagnosis of human herpesvirus type 6 infection was the presence of IgM human herpesvirus type 6 antibody (titre greater than 20) and a rising titre of IgG human herpesvirus type 6 antibody without serological evidence of alternative infection. SETTING--Routine viral diagnostic and reference laboratory in the largest teaching hospital in Sydney. PATIENTS--341 Consecutive serum samples were analysed from patients with hepatitis (147 samples); infectious mononucleosis-like illness (106); screens for toxoplasma, other viruses, rubella, cytomegalovirus, and herpesvirus (38); fever in an immunocompromised patient (eight); unusual neurological (nine) or haematological syndromes (14); splenomegaly (six); and rash in a child (13). RESULTS--Three cases of acute human herpesvirus type 6 infection were identified: in one patient aged 65 with a previous diagnosis of acute non-A non-B hepatitis, one aged 25 with a glandular fever-like illness, and one aged 6 with a glandular fever-like illness. All three illnesses resolved completely. 15 Further serum samples were positive for human herpesvirus type 6 antibody but were also diagnostic for acute infection with other viruses (cytomegalovirus (nine), Epstein-Barr virus (three), and HIV (one] or had a titre of IgM human herpesvirus type 6 antibody less than 20 (two). CONCLUSIONS--Acute human herpesvirus type 6 infection in immunocompetent patients may result in a mononucleosis-like illness or an acute but self limiting hepatitis.     

Sequence analysis of two exons from the murine dystrophin locus

We have isolated two genomic clones from the murine dystrophin locus, containing single exons encoding protein sequence from the putative actin-binding domain of the amino-terminus and the terminal portion of the triple helical domain. Using interspecific backcross progeny mice, both clones were shown to be X-linked. Sequence analysis indicated that the amino-terminal clone contains a 173 bp exon exhibiting 90% nucleotide sequence identity to human dystrophin exon 6, whilst the C-terminal clone contains a 61 bp exon with 93% nucleotide sequence identity to the human cDNA sequence.The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ nucleotide sequence databases under the accession numbers X58153 and X57745.     

Sequence variability of simian immunodeficiency virus in a persistently infected rhesus monkey

A juvenile rhesus monkey that was inoculated intravenously with molecularly cloned SIVmac239 became persistently infected. A modified polymerase chain reaction (PCR) procedure was used to specifically amplify full-length envelope (env) gene sequences from DNA extracted from peripheral blood mononuclear cells (PBMC), lymph node tissue, and cells infected with recovered virus at 69 and 93 weeks post-infection. Extensive sequence variability accumulated in vivo in spite of infection with molecularly cloned virus. In the central portion of env. sequence variability was largely confined to three discrete regions.     

Phenotype, frequency, and EBV responsiveness of human marrow B and pre-B cells

We have purified subpopulations of B lineage cells from human adult (rib) bone marrow by cell sorting and panning. Limiting dilution analysis was then used for a clonal analysis of cells able to secrete IgG, IgA, or IgM spontaneously or after infection with EBV. Nonproliferating, high rate IgG or IgA producers occurred at frequencies of about one per 1000 marrow mononuclear cells. Their frequency and Ig production was unaffected by EBV, and they appeared not to express EBNA after exposure to EBV. These cells were Ia+, B1+, and over 85% expressed sIg of the IgM/D (up to 75%) and/or IgG/A isotypes (40 to 60%). B cells committed to the secretion of IgM represent 2 to 10% of marrow B lymphocytes. They were found to be Ia+/B1+/B2+/CALLA- and C3b receptor (CR3)-cells, and most (greater than 90%) required infection with EBV and proliferation to develop into IgM-producing lymphocytes. Thirty to 40% of these cells did not express Ig (H or L chain) on their surface, and therefore resembled pre-B cells at the beginning of the 4- to 5-wk culture period. Proliferating pre-B cells from adult human marrow have been described, but their conversion into IgM-producing cells has not been formally demonstrated. Although EBV induces IgM production, the expression of EBNA, and several rounds of cell division in these cells, the induction of stable (greater than 5 wk) growth transformation represents a rare event in these pre-B cells: in several thousand limiting dilution wells, not a single culture of sIg-cells showed stable growth transformation. The dichotomy between EBV-induced high-rate IgM responses and absent growth transformation discriminates activation and transformation as distinct aspects of EBV-induced B cell "responses", and suggests that cellular properties play critical roles for viral transformation. We propose a model in which cellular target genes for transforming sequences in the EBV genome are transiently expressed during B cell differentiation.     

The role of IgG avidity determination in diagnosis of Epstein-Barr virus infection in immunocompetent and immunocompromised patients

There is a high degree of variability in the serologic response to Epstein-Barr virus (EBV) infection, especially in viral capsid antigen (VCA)-IgM antibodies. Therefore, additional tests are needed to confirm primary infection. We evaluated the value of IgG avidity determination in diagnosis of EBV infection in immunocompetent and immunocompromised patients. A total of 236 serum samples from immunocompetent patients with symptoms suggestive of EBV infection were tested for the presence of VCA-IgM/IgG antibodies and IgG avidity. Using IgG avidity, acute primary infection was confirmed in 56.7% of the immunocompetent patients with positive and in 1.8% of patients with negative VCA-IgM. Recent primary infection was documented in 8.9% of the IgM positive and 3.5% of the IgM negative patients. In patients with indeterminate serology (equivocal IgM), 6.7% were classified by avidity index (AI) as acute primary infection, 10.0% as post-acute and 83.3% as past infection cases. Concerning the 32 immunocompromised patients, recent primary infection was documented in 3 of the 14 IgM positive patients. High AI was detected in 11 of these patients, indicating an IgM response due to reactivation. Determination of IgG avidity in combination with classical serologic markers seems to be a reliable method to confirm primary infection both in immunocompetent and immunocompromised patients. It may be especially useful to differentiate cases of primary infection in patients with undetectable VCA-IgM antibodies or indeterminate routine EBV serology.     

Antigens and DNA of a chimpanzee agent related to Epstein-Barr virus.

Biological and biochemical studies of the herpesvirus of chimpanzees previously demonstrated to be antigenically related to human Epstein-Barr virus (EBV) indicated that the agent is similar to EBV in that: (i) leukocyte culture of chimpanzees whose sera contained antibody against EBV capsid antigen could yield long-term lymphoblastoid cell lines (Ch-LCL) with B-cell characteristics; (ii) the DNA of Ch-LCL contained sequences homologous to approximately 35 to 45% of human EBV; (iii) Ch-LCL contained an intranuclear antigen, Ch-NA, that could be identified with some chimpanzee or orangutan serum in anticomplimentary immunofluorescence assays; and (iv) treatment of Ch-LCL with iododeoxyuridine resulted in expression of new antigenic activity that reacted with EA+ but not EA- human sera. Two lines of evidence indicate that the chimpanzee agent, although related to human EBV, is a distinct agent: (i) Ch-NA was antigenically distinct from EBV-rebv infection although it cross-reacts of a limited extent with a minor component of EBNA; and (ii) Ch-LCL are missing 55 to 65% of the DNA sequences of human EBV.     

Detection of the markers of herpes simplex virus and cytomegalovirus in newborns and infants

A total of 111 children suspected for herpesvirus infection were examined. In blood and urine samples the infectious activity of herpes simplex virus (HSV) and cytomegalovirus (CMV) was detected by the rapid culture method (RCM) and the presence of virus DNA--by the polymerase chain reaction (PCR). HSV and/or CMV were detected by two laboratory methods in 57 examined children (51%). Of these, in 18 children (16.2%) both HSV and CMV were detected. The coincidence of the results of the detection of HSV and CMV by these two methods was observed in 72.4% and 75.2% of cases respectively. The comparative analysis of the detection of anti-CMV IgG and IgM was made with the use of commercial test systems produced bythe following manufacturers: "Vector-Best" and "Bioservice" (Russia), "HUMAN" and "Boehringer" (Germany). The effective detection of both anti-CMV (IgG and IgM) was ensured by the test systems "Boehringer". The test system "Vector-Best" for anti-CMV IgG proved to be not inferior as regards sensitivity and specificity. The German test systems demonstrated the highest specificity in the detection of low-avid antibodies. The data obtained in this study indicate that the detection rate of HSV and CMV markers in newborns and infants suspected for herpesvirus infection was, on the average, 20 - 40%. Reliable diagnostics in newborns and infants is possible only in the presence of the combination of at least 2 serological tests (the determination of antivirus IgM and IgG avidity) and 2 methods for the detection of direct herpesvirus markers (PCR and RCM).     

alpha-Interferon increases immunoglobulin production in cultured human mononuclear leukocytes

Interferons (IFN) are known to modulate immune responses in either an inhibitory or a stimulating manner. The present study was initiated to investigate the mechanisms by which alpha-IFN modulates Ig production of human peripheral blood mononuclear cells (PBMC). IgG and IgM production was measured in pokeweed mitogen- (PWM) stimulated 7-day cultures of PBMC. Significant enhancement of IgM and IgG production was observed when alpha-IFN was added. Overnight preincubation followed by washing also produced significant enhancement. The effect of alpha-IFN was not obtained in the absence of PWM or T cells. The effect of alpha-IFN on cultures of B and T cells was not altered by irradiation of T cells (2000 rad). alpha-IFN was not shown to enhance the production of helper factor but did increase the responsiveness of B cells to helper factor if the B cells were preincubated with alpha-IFN. Finally, alpha-IFN did not increase the Ig production of PBMC induced by Epstein Barr virus (EBV), and the outgrowth of EBV-infected PBMC was not affected. Overall, these results show for the first time that the effect of alpha-IFN on PBMC is due to an enhanced responsiveness of B cells to helper factors produced by radioresistant T cells.     

Detection of equine herpesvirus and differentiation of equine herpesvirus type 1 from type 4 by the polymerase chain reaction.

Although both equine herpesvirus type 1 (EHV-1) and equine herpesvirus type 4 (EHV-4) can be associated with respiratory disease, epizootics caused by EHV-1 are much more serious because the virus can cause abortions and paralysis. It is, therefore, important to identify the type of EHV involved in an outbreak by a test that is quick, sensitive, and reliable. We have adapted the polymerase chain reaction (PCR) to detect and distinguish between EHV-1 and EHV-4 in the same reaction. Primers for PCR were designed from the sequences of the glycoprotein B genes of EHV-1 and EHV-4. The PCR products derived from EHV-1 and EHV-4 were 135 and 326 base pairs, respectively, and could be readily separated by electrophoresis. The identity of the PCR products was confirmed by determining their nucleotide sequence, which agreed with the published sequence of the gB genes. The test could be performed directly on virus pelleted from small volumes (300 microL) of medium in which nasal swabs were transported and did not rely on the presence of infectious virus. The PCR was unaffected by conditions that reduced the infectivity of a virus preparation by 99%. The PCR detected EHV-4 in 5 of 10 nasal mucous samples taken from an outbreak of respiratory disease in race horses. Virus isolation in indicator cells was successful in detecting virus in four of the five samples positive by PCR.     

Modulation of Epstein-Barr virus-induced B-cell activation by concanavalin A

Direct addition of the T-cell mitogen, concanavalin A (Con A), to cultures of Epstein-Barr virus (EBV)-stimulated peripheral blood mononuclear cells (PBMC) resulted in a dose-dependent inhibition of immunoglobulin M (IgM) secreted in the supernatant, as measured by an enzyme-linked immunosorbent assay. Furthermore, Con A inhibited IgM secretion of isolated T-depleted cells stimulated with EBV, and both the proliferation and IgM secretion of EBV-driven lymphoblastoid cell lines. T-Enriched cells, precultured for 48 hr with Con A, were also able to suppress the IgM response of fresh autologous PBMC stimulated with EBV. This suppression was radiation sensitive (2000 rad), a procedure which resulted in enhancement of the IgM secretion of the responder cells in two out of three experiments. Studies on the long-term effects of Con A showed that the early suppression of IgM secretion was transient and that the mitogen prevented the development of the cytotoxic T-cell response normally seen with lymphocytes from EBV-seropositive donors after 5 weeks of culture. Thus, Con A appears to modulate human lymphocyte responses to EBV by multiple mechanisms.     

Correlation between human herpesvirus 6 and 7 infections after living related liver transplantation

Human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7) are closely related to each other. Interaction between the two viruses at the time of primary HHV-7 infection is suggested by in vivo and in vitro studies. However, interaction between the two viruses in organ transplant recipients has not been analyzed. We analyzed serially collected plasma samples obtained from 40 living related liver transplant recipients by serological assay (indirect immunofluorescence assay, IFA) and polymerase chain reaction (PCR). Significant increase or seroconversion of HHV-6 IgG and HHV-7 IgG antibody titers were observed in 45% and 58% of recipients respectively. Positive rate of IgM HHV-6 antibody increased up to 35% at 4 weeks after transplantation. However, no remarkable peak in the positive rate of HHV-7 IgM antibody was demonstrated. HHV-6 and HHV-7 DNA were detected in plasma in 15 (38%) and 16 (40%) of the 40 recipients respectively. HHV-6 DNA was detected in 10 (26%) of the 38 recipients at 2 weeks after transplantation. The positive rate of the virus genome in plasma gradually decreased after that time. HHV-7 DNA was detected in 5 (14%) of the 37 recipients at 2 weeks after transplantation; no obvious peak in the positive rate of HHV-7 DNA was demonstrated. Antibody responses involving both HHV-6 and HHV-7, including either a significant increase in IgG antibody titers or positive identification of IgM antibody were observed in 17 (43%) of the 40 recipients. Thirteen out of the 17 recipients demonstrated concurrent antibody response against both viruses. HHV-7 antibody response preceded the HHV-6 antibody response in 2 of the remaining 4 recipients, whereas the opposite was true in the other 2 recipients. Both HHV-6 and HHV-7 DNA were detected in 7 (18%) of the 40 recipients. In 4 of those 7 recipients, DNA from both viruses was concurrently detected, 3 of whom had HHV-7 DNA repeatedly detected after first detection of the virus DNA. The detection of HHV-7 DNA preceded the detection of HHV-6 DNA in 2 recipients, whereas HHV-6 DNA appeared first in 1 recipient.