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1.
Obert LA  Hoover EA 《Journal of virology》2002,76(12):6311-6322
To identify the early target cells and tissues in transmucosal feline immunodeficiency virus (FIV) infection, cats were exposed to a clade C FIV isolate via the oral-nasal or vaginal mucosa and multiple tissues were examined by virus isolation coculture (VI), DNA PCR, catalyzed tyramide signal-amplified in situ hybridization (TSA-ISH), and immunohistochemistry between days 1 and 12 postinoculation (p.i.). FIV RNA was detected in tonsil and oral or vaginal mucosa as early as 1 day p.i. by TSA-ISH and in retropharyngeal, tracheobronchial, or external iliac lymph nodes and sometimes in spleen or blood mononuclear cells by day 2, indicating that regional and distant spread of virus-infected cells occurred rapidly after mucosal exposure. By day 8, viral RNA, DNA, and culturable virus were uniformly detected in regional and distant tissues, connoting systemic infection. TSA-ISH proved more sensitive than DNA PCR in detecting early FIV-infected cells. In mucosal tissues, the earliest demonstrable FIV-bearing cells were either within or subjacent to the mucosal epithelium or were in germinal centers of regional lymph nodes. The FIV(+) cells were of either of two morphological types, large stellate or small round. Those FIV RNA(+) cells which could be colabeled for a phenotype marker, were labeled for either dendritic-cell-associated protein p55 or T-lymphocyte receptor antigen CD3. These studies indicate that FIV crosses mucous membranes within hours after exposure and rapidly traffics via dendritic and T cells to systemic lymphoid tissues, a pathway similar to that thought to occur in the initial phase of infection by the human and simian immunodeficiency viruses.  相似文献   

2.
通过基因组定量研究猪瘟病毒在细胞中的增殖特性   总被引:1,自引:0,他引:1  
应用间接免疫荧光、Real-time PCR和病毒感染滴度(TCID50)测定技术,分别从病毒抗原、病毒基因组RNA复制水平和病毒感染滴度变化3个方面,研究了猪瘟病毒(CSFV)在PK-15细胞中增殖的特点,用猪瘟病毒石门株感染96孔板培养的细胞,1×102个TCID50/孔,间接免疫荧光检测结果显示感染后8h能检测到被荧光抗体染色的感染细胞,随感染时间的延长,出现荧光的细胞数量逐渐增多,在感染后72h,几乎所有细胞均能出现荧光。Real-time PCR结果显示在细胞感染初期的8~24h,病毒的基因组RNA复制呈加速趋势,其拷贝数在感染后72h达到高峰。此外,在感染后8h能检测到病毒基因组负链RNA转录,不过负链RNA在病毒增殖过程中维持在较低的水平。TCID50测定结果表明CSFV的感染滴度增加趋势与基因组类似,在病毒感染8h后能检测到具有感染性的子代病毒,感染滴度在8~20h之间逐渐增长,24~48h之间增长速度稍减慢,在感染后48~52h达到高峰,能在72h之内维持较高的感染滴度。  相似文献   

3.

Background

Measurement of HIV DNA-bearing cells in cerebrospinal fluid (CSF) is challenging because few cells are present. We present a novel application of the sensitive droplet digital (dd)PCR in this context.

Methods

We analyzed CSF cell pellets and paired peripheral blood mononuclear cells (PBMC) from 28 subjects, 19 of whom had undetectable HIV RNA (<48 copies/mL) in both compartments. We extracted DNA from PBMC using silica-based columns and used direct lysis on CSF cells. HIV DNA and the host housekeeping gene (RPP30) were measured in CSF and PBMC by (dd)PCR. We compared HIV DNA levels in virally-suppressed and-unsuppressed subgroups and calculated correlations between HIV DNA and RNA levels in both compartments using non-parametric tests.

Results

HIV DNA was detected in 18/28 (64%) CSF cell pellets, including 10/19 (53%) samples with undetectable HIV RNA. HIV DNA levels in CSF cell pellets were not correlated with RPP30 (p = 0.3), but correlated positively with HIV RNA in CSF (p = 0.04) and HIV DNA in PBMC (p = 0.03). Cellular HIV DNA in CSF was detected in comparable levels in HIV RNA-suppressed and unsuppressed subjects (p = 0.14). In contrast, HIV DNA levels in PBMC were significantly lower in HIV RNA-suppressed than in unsuppressed subjects (p = 0.014). Among subjects with detectable HIV DNA in both compartments, HIV DNA levels in CSF were significantly higher than in PBMC (p<0.001).

Conclusions

Despite low mononuclear cell numbers in CSF, HIV DNA was detected in most virally suppressed individuals. In contrast to PBMC, suppressive ART was not associated with lower HIV DNA levels in CSF cells, compared to no ART, perhaps due to poorer ART penetration, slower decay of HIV DNA, or enrichment of HIV DNA-bearing mononuclear cells into the CSF, compared to blood. Future studies should determine what fraction of HIV DNA is replication-competent in CSF leukocytes, compared to PBMC.  相似文献   

4.
5.
6.
Previous studies using feline immunodeficiency virus (FIV) molecular clones lacking the putative transactivator gene (ORF-A/2) failed to address the issue of thymus pathogenesis or investigate the levels of viral replication in separate lymphoid compartments (Y. Inoshima, et al., J. Virol. 70:8518-8526, 1996; E. E. Sparger, et al., Virology 205:546-553, 1994). Using a highly pathogenic molecular clone of FIV, JSY3, and an ORF-A/2-deficient mutant, JSY3DeltaORF-A/2, we compared viral replication and the extent of thymic dysfunction as measured by the formation of lymphoid follicles and alteration of the thymocyte subsets. Viral replication was reduced in JSY3DeltaORF-A/2-infected cats as measured by lymphocyte coculture, immunohistochemistry, and quantitative PCR. Cell-associated viral load measured by lymphocyte coculture varied in a tissue-dependent manner with replication highest in lymphocytes isolated from the thymus, lower in those from the peripheral blood, and lowest in those from lymph node. Thymic proviral load and the number of viral p24 Gag-positive cells within the thymus detected by immunohistochemistry were also reduced. In addition, the onset of a reduced peripheral blood CD4/CD8 ratio was delayed in JSY3DeltaORF-A/2-infected cats. The formation and extent of thymic lymphoid follicular hyperplasia were similar in JSY3 and JSY3DeltaORF-A/2-infected cats as measured by anticytokeratin immunohistochemistry and flow cytometry for percent pan T-negative, immunoglobulin G-positive cells within the thymus. In contrast, comparison of thymocyte subpopulations demonstrated a reduced expansion of single-positive CD4(-) CD8(+) thymocytes in JSY3DeltaORF-A/2-infected cats. Level of viral replication, therefore, may not correlate with the formation of thymic lymphoid follicles but may correlate with the expansion of the single-positive CD4(-) CD8(+) thymocyte subpopulation.  相似文献   

7.
8.
The orf-A (orf-2) gene of feline immunodeficiency virus (FIV) is a small open reading frame predicted to encode a 77-amino-acid protein that contains putative domains similar to those of the ungulate lentiviral Tat protein. Orf-A is reported to be critical for efficient viral replication in vitro and in vivo. A series of FIV-pPPR-derived proviruses with in-frame deletions and point mutations within orf-A were constructed and tested for replication in feline lymphoid cells. Orf-A mutant proviruses were also tested for viral gene and protein expression, viral particle formation, and virion infectivity. Deletions within orf-A severely restricted FIV replication in feline peripheral blood mononuclear cells (PBMC) and interleukin-2-dependent T-cell lines. In addition, substitutions of alanines for leucines in the putative leucine-rich domain, for cysteines in the putative cysteine-rich domain, and for a tryptophan at position 43 in Orf-A restricted the replication of FIV mutants. Deletions and point mutations in orf-A imposed a small effect or no effect on FIV long-terminal-repeat-driven viral gene expression and had no effect on viral protein expression. However, release of cell-free, virion-associated viral RNA in supernatants from cells transfected with orf-A mutant proviruses was severely restricted but was rescued by cotransfection with a wild-type Orf-A expression vector. In addition, virions derived from orf-A mutant proviruses expressed reduced infectivity for feline PBMC. Our findings suggest that Orf-A functions involve multiple steps of the FIV life cycle including both virion formation and infectivity. Furthermore, these observations suggest that Orf-A represents an FIV-encoded analog more similar to the accessory gene vpr, vpu, or nef than to the regulatory gene tat encoded by the primate lentiviruses.  相似文献   

9.
Betanodavirus protein alpha induces cell apoptosis or secondary necrosis by a poorly understood process. In the present work, red spotted grouper nervous necrosis virus (RGNNV) RNA 2 was cloned and transfected into tissue culture cells (GF-1) which then underwent apoptosis or post-apoptotic necrosis. In the early apoptotic stage, progressive phosphatidylserine externalization was evident at 24h post-transfection (p.t.) by Annexin V-FLUOS staining. TUNEL assay revealed apoptotic cells at 24-72 h p.t, after which post-apoptotic necrotic cells were identified by acridine orange/ethidium bromide dual dye staining from 48 to 72 h p.t. Protein alpha induced progressive loss of mitochondrial membrane potential (MMP) which was detected in RNA2-transfected GF-1 cells at 24, 48, and 72 h p.t., which correlated with cytochrome c release, especially at 72 h p.t. To assess the effect of zfBcl-xL on cell death, RNA2-transfected cells were co-transfected with zfBcl-x(L). Co-transfection of GF-1 cells prevented loss of MMP at 24 h and 48 h p.t. and blocked initiator caspase-8 and effector caspase-3 activation at 48 h p.t. We conclude that RGNNV protein alpha induces apoptosis followed by secondary necrotic cell death through a mitochondria-mediated death pathway and activation of caspases-8 and -3.  相似文献   

10.
We have recently found that Moloney murine leukemia virus assembles within cytoplasmic vacuoles of chronically infected NIH/3T3 cells rather than at their surface (submitted for publication). In the present study we found that if these cells were treated with interferon (IF) for 24 to 48 h the intracellular virus particles accumulated at a two- to threefold-higher level than that observed in untreated cells. Nevertheless, despite this accumulation, no difference between IF-treated and untreated cells was observed in the amount of the total cytoplasmic viral RNA or in its 35S or 21S species. When cellular virions were sedimented from the cytoplasmic fraction, a markedly higher amount of viral RNA was detected in the viral pellet of IF-treated cells than was detected in untreated cells, whereas the amount of viral RNA left in the virus-free cytoplasm of IF-treated cells was much lower than that in the untreated cells. Furthermore, the amount of the cytoplasmic polyriboadenylic acid-containing viral RNA was also remarkably higher in the IF-treated cells. Viral polyribosomes appeared to be fully functional in IF-treated cells, since no effect of IF on viral protein synthesis could be detected. Analysis of the nuclear viral RNA showed no difference between IF-treated and untreated cells after 24 h of IF treatment. Both contained a comparable amount of 35S viral RNA. However, at 48 h a significant accumulation of viral RNA was observed in the nucleus of the IF-treated cells as compared with the untreated cells, although in both cases only 35S species were evident. This accumulation appeared to activate a degradation process which destroyed nuclear viral RNA, since a dramatic shift toward smaller-sized molecules of viral RNA and a remarkable reduction in its amount were observed after 72 h of IF treatment.  相似文献   

11.
Feline immunodeficiency virus (FIV) causes AIDS-like symptoms in infected cats. Concanavalin A (ConA)-stimulated peripheral blood mononuclear cells (PBMC) from chronically FIV strain PPR-infected cats readily expressed FIV. In contrast, when PBMC from these animals were stimulated with irradiated, autologous antigen-presenting cells (APC), at least a 10-fold drop in viral production was observed. In addition to FIV-specific cytotoxic T lymphocytes, anti-FIV activity was demonstrated in the cell-free supernatants of effector T lymphocytes stimulated with APC. The FIV-suppressive activity was induced from APC-stimulated PBMC of either FIV-infected or uninfected cats but not from ConA-stimulated PBMC. Suppression of FIV strain PPR replication was observed for both autologous and heterologous feline PBMC, was dose dependent, and demonstrated cross-reactivity and cell specificity. It was also demonstrated that the anti-FIV activity originated from CD8(+) T lymphocytes and was mediated by a noncytolytic mechanism.  相似文献   

12.
Aging is associated with altered immune function. We previously reported that splenocytes and thymocytes undergo apoptosis with aging in rats. In the present study, we examined the expression of genes associated with apoptosis in splenocytes and thymus in aging rats. We evaluated the expression of bax, interleukin 1-β-converting enzyme (ICE)/ced-3 protease family, caspase-3 and tumor suppressor gene p53. Rats in age groups of 6, 24, 48, and 96 weeks were sacrificed; thymocytes and splenocytes were isolated followed by lysis in a modified RIPA buffer containing protease inhibitors. Western blot analysis of proteins was performed by probing immunoblots with antibodies against p53, bax and PARP (poly ADP-ribose polymerase). Increased aging was associated with enhanced expression of bax, p53 and cleavage of PARP by Caspase-3. The expression of p53 and cleavage of PARP indicates the presence of damaged DNA; nevertheless, the cleavage of PARP or activation of caspase-3 may be playing an important role in the initiation of early events in apoptosis. These results suggest that aging of splenocytes and thymocytes is associated with the expression of cell death genes. The present study provides an insight into age-associated altered immune function.  相似文献   

13.
CTL fail to accumulate at sites of HIV-1 replication in lymphoid tissue   总被引:1,自引:0,他引:1  
The inability of HIV-1-specific CTL to fully suppress virus replication as well as the failure of administration of exogenous CTL to lower viral loads are not understood. To evaluate the hypothesis that these phenomena are due to a failure of CTL to localize at sites of HIV-1 replication, we assessed the distribution of HIV-1 RNA and HIV-1-specific CTL identified by HIV-1 peptide/HLA class I tetrameric complexes (tetramers) within lymph nodes of 14 HIV-1-infected individuals who were not receiving antiretroviral therapy. A median of 0.04% of follicular compared with 0.001% of extrafollicular CD4(+) cells were estimated to be producing HIV-1 RNA, a 40-fold difference (p = 0.0001). Tetramer-stained cells were detected by flow cytometry in disaggregated lymph node cells from 11 subjects and constituted a significantly higher fraction of CD8(+) cells in lymph node (mean, 2.15%) than in PBMC (mean, 1.52%; p = 0.02). In situ tetramer staining in three subjects' lymph nodes, in which high frequencies of tetramer-stained cells were detected, revealed that tetramer-stained cells were primarily concentrated in extrafollicular regions of lymph node and were largely absent within lymphoid follicles. These data confirm that HIV-1-specific CTL are abundant within lymphoid tissues, but fail to accumulate within lymphoid follicles where HIV-1 replication is concentrated, suggesting that lymphoid follicles may be immune-privileged sites. Mechanisms underlying the exclusion of CTL from lymphoid follicles as well as the role of lymphoid follicles in perpetuating other chronic pathogens merit further investigation.  相似文献   

14.
The objective of this study was to identify cellular and organ targets of acute feline immunodeficiency virus (FIV) infection in vivo. Tissues of FIV-infected cats were studied at eight time points during the first 3 months after experimental infection. FIV nucleic acids were first detected by in situ hybridization 21 days after infection, approximately 1.5 weeks after lymph node enlargement was first observed and 3 weeks before the primary acute flu-like illness. The majority of FIV-infected cells were present in lymphoid organs, though low numbers of infected cells were noted in nonlymphoid organs as well. Germinal centers harbored many of the FIV-infected cells within lymphoid tissues. The thymic cortex was also a major site of early infection. Combined in situ hybridization and immunohistochemistry revealed that T lymphocytes were the primary target of early FIV infection in tissues of cats before the onset of clinical signs of acute illness. An unidentified population of mononuclear cells and a few macrophages were also infected. During the ensuing acute flu-like illness, the proportion of FIV-infected macrophages in tissues increased dramatically. This early shift in the predominant cellular localization of FIV from T lymphocytes to macrophages may be important for establishing viral persistence.  相似文献   

15.
16.
Primary cultures of peripheral blood mononuclear cells (PBMC) from 51 HIV+ hemophiliac patients (HIV+ PBMC) were set up, allowing undisturbed cellular interaction in the absence of any exogenous stimuli. The optimum time for p24 detection was between 12 and 25 days. Infective virus was recovered from the culture supernatants (HIV+ SN) and the amount of p24 released ranged from 25 to 5300 pg/ml. Cells of the monocyte/macrophage (M/M) lineage were the main source of HIV in the HIV+ SN, as judged by intracellular staining of permeabilized cells with anti-p24 (KC57 monoclonal antibody) and flow cytometry analysis. M/M activation, differentiation, and proliferation occurred along the culture before the peak of in vitro HIV replication. Release of HIV p24 was highest in patients with >200 CD4+ T lymphocytes/mm3 who did not receive highly active antiretroviral therapy (HAART), but it was still detectable in 60-90% of patients who had responded to 1-2 years of HAART, reducing their plasma viral load to undetectable levels. It is proposed that this simple experimental system can be used to assess ongoing HIV infection of M/M with the patient's own viral variants.  相似文献   

17.

Background

Infection with dengue viruses (DENV) causes a wide range of manifestations from asymptomatic infection to a febrile illness called dengue fever (DF), to dengue hemorrhagic fever (DHF). The in vivo targets of DENV and the relation between the viral burden in these cells and disease severity are not known.

Method

The levels of positive and negative strand viral RNA in peripheral blood monocytes, T/NK cells, and B cells and in plasma of DF and DHF cases were measured by quantitative RT-PCR.

Results

Positive strand viral RNA was detected in monocytes, T/NK cells and B cells with the highest amounts found in B cells. Viral RNA levels in CD14+ cells and plasma were significantly higher in DHF compared to DF, and in cases with a secondary infection compared to those undergoing a primary infection. The distribution of viral RNA among cell subpopulations was similar in DF and DHF cases. Small amounts of negative strand RNA were found in a few cases only. The severity of plasma leakage correlated with viral RNA levels in plasma and in CD14+ cells.

Conclusions

B cells were the principal cells containing DENV RNA in peripheral blood, but overall there was little active DENV RNA replication detectable in peripheral blood mononuclear cells (PBMC). Secondary infection and DHF were associated with higher viral burden in PBMC populations, especially CD14+ monocytes, suggesting that viral infection of these cells may be involved in disease pathogenesis.  相似文献   

18.
19.
Lin KC  Chang HL  Chang RY 《Journal of virology》2004,78(10):5133-5138
Japanese encephalitis virus (JEV) contains a single positive-strand RNA genome nearly 11 kb in length and is not formally thought to generate subgenomic RNA molecules during replication. Here, we report the abundant accumulation of a 3'-terminal 521- to 523-nucleotide (nt) genome fragment, representing a major portion of the 585-nt 3' untranslated region, in both mammalian (BHK-21) and mosquito (C6/36) cells infected with any of nine strains of JEV. In BHK-21 cells, the viral genome was detected as early as 24 h postinfection, the small RNA was detected as early as 28 h postinfection, and the small RNA was 0.25 to 1.5 times as abundant as the genome on a molar basis between 28 and 48 h postinfection. In C6/36 cells, the genome and small RNA were present 5 days postinfection and the small RNA was 1.25 to 5.14 times as abundant as the genome. The 3'-terminal 523-nt small RNA contains a 5'-proximal stable hairpin (nt 6 to 56) that may play a role in its formation and the conserved flavivirus 3'-cyclization motif (nt 413 to 420) and the 3'-terminal long stable hairpin structure (nt 440 to 523) that have postulated roles in genome replication. Abundant accumulation of the small RNA during viral replication in both mammalian and mosquito cells suggests that it may play a biological role, perhaps as a regulator of RNA synthesis.  相似文献   

20.
Abstract The peripheral blood mononuclear cells (PBMC) of woodchucks experimentally infected by woodchuck hepatitis virus (WHV) were examined simultaneously for the presence of membrane associated WHV antigens by cytofluorometry, and for WHV DNA and RNA sequences by the polymerase chain reaction (PCR). Four woodchucks were inoculated: two with a well-defined infectious inoculum and two with an inoculum obtained from an animal at the late incubation phase, which was positive for WHV DNA by PCR but still devoid of WHV markers. Infection was demonstrated in all four inoculated woodchucks by the appearance at different times of WHV DNA and WHV antigens in both leucocytes and serum. WHV DNA was first detected by PCR either in the serum (two cases) or in leucocytes (two cases). The mean percentage of cells positive for membrane associated WHsAg or WHcAg detected by cytofluorometry were 37%±25 and 17%±15 respectively. After 8 weeks, all inoculated animals were WHsAg positive in serum. These data suggest that PBMC are involved in the early events of hepadnavirus infection. They also show that sera which are positive by PCR for WHV DNA may transmit viral infection even while still seronegative for WHV markers and for WHV DNA by dot blot.  相似文献   

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