Ultrastructural study of viral replication in human fetal intestinal organ culture.
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Adenoviridae↗
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Results for “viral replication”
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IL-8 release from human neutrophils by the respiratory syncytial virus is independent of viral replication.
Elevated interleukin-8 levels and a massive accumulation of neutrophils (PMN) are the hallmark of a variety of severe lung diseases. The respiratory syncytial virus (RSV), an important respiratory pathogen, induces interleukin-8 (IL-8) release from human PMN, however, the mechanism is as yet unknown. We analyzed the role of virus uptake, intracellular virus replication, virus attachment, and of virus capsid proteins for the induction of IL-8 (protein + mRNA) in human PMN. Cell supernatants were analyzed for IL-8 release via enzyme-linked immunosorbent assay; cell pellets were analyzed for IL-8-specific mRNA expression and for RSV-specific genomic and RSV-specific mRNA by reverse transcriptase-polymerase chain reaction. Stimulation of human PMN with viable, heat-inactivated, or UV-inactivated RSV [at a multiplicity of infection (m.o.i.) from 0.01 up to 10] induced IL-8 production (protein + mRNA) to a similar degree. Maximal IL-8 release was observed at a m.o.i. of 5-10 after 18-24 h. RSV-specific genomic RNA was present inside PMN up to 24 h independent of whether viable or inactivated RSV was used. Withdrawal of extracellular viable or inactivated (heat, UV) RSV after infection of PMN (2 h) abolished IL-8 mRNA expression and IL-8 release; the intracellular persistence of RSV lasted for up to 24 h. Stimulation of human PMN with purified RSV G-protein, a major capsid protein, in a concentration range from 0.1 up to 2.5 microg/5 X 10(5) PMN resulted in an increased IL-8 release from human PMN but to a significantly lesser degree compared with the intact RSV. RSV G-protein concentration above 1 microg inhibited the RSV-induced IL-8 release by up to 90%. Our data contribute to the understanding of the pathomechanisms leading to IL-8 release from human PMN.
Base Sequence↗
Viral replication in mouse macrophages.
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Animals↗
[Detection of serum hepatitis B virus large envelope protein and its relationship with viral replication.].
BACKGROUND: To explore the significance of HBV large envelope protein (LHBs) in diagnosing HBV replication in chronic hepatitis B patients. METHODS: Serum HBV DNA was quantitively detected by using real-time polymerase chain reaction (RT-PCR), the LHBs and Pre-S1 were detected by using enzyme linked immunosorbent assay (ELISA) and HBV markers were detected by time differentiate immunofluorescence assay in 340 serum samples collected from chronic hepatitis B patients. RESULTS: Serum LHBs level was closely correlated with number of HBV DNA copies (r=0.899, P=0.0380). There was no significant difference between positive rate of LHBs and that of HBV DNA in different HBeAg pattern (P>0.05); the positive rate of LHBs was 83.15%, which was higher than that of Pre-S1 and HBeAg which were 50.54% and 54.48%, respectively. There was significant difference (P less than 0.05). CONCLUSION: The level of serum LHBs can be used to estimate the state of HBV replication and the sensitivity was superior to both Pre-S1 and HBeAg. So it may be used as a new serological marker to detect HBV replication.
DNA, Viral↗
The precore gene of the woodchuck hepatitis virus genome is not essential for viral replication in the natural host.
A number of naturally occurring hepatitis B virus mutants that cannot synthesize the virus precore protein have been identified. Such mutants have been associated with more severe forms of hepatitis, including fulminant hepatitis. The most common mutation observed is a substitution of G to A in the distal precore gene that converts a codon specifying Trp (TGG) to a termination codon (TAG). Using oligonucleotide-directed mutagenesis, we have produced the same point mutation in the precore gene of an infectious clone of woodchuck hepatitis virus (WHV). Transfection of mutant WHV DNA into the livers of adult woodchucks resulted in replication of the mutant in three of three susceptible animals. Levels of virus replication and transient elevations in liver enzymes in serum were similar to those of adult animals infected with wild-type WHV. Virions, found to possess mutant precore genes by polymerase chain reaction amplification and DNA sequencing, were recovered from the serum of one of the animals and inoculated subcutaneously into neonatal woodchucks. They produced infection in all five animals studied. The level of virus replication in neonatal animals infected with this mutant virus was comparable to that found in neonatal woodchucks infected with wild-type WHV, but none of five woodchucks infected with the precore mutant virus as neonates became chronic virus carriers. It was concluded that the precore gene of the WHV genome is not essential for virus replication in the natural host but may be important for chronic infection.
Animals↗
PCR analysis of HBV infected sera: relationship between expression of pre-S antigens and viral replication.
In order to determine the biological significance of the pre-S antigens in HBV infection, HBsAg sera were tested for the presence of pre-S1 and pre-S2. HBV DNA was detected by spot-hybridization and PCR. The data show a complete correlation between pre-S antigenemia and HBV DNA replication in anti-HBe positive cases. PCR but not spot-hybridization was adequately sensitive to also detect HBV DNA in roughly half of the preS negative sera as well. Thus PCR appears to be a valuable technique for detection of potentially infectious anti-HBe carriers.
DNA, Viral↗
Endocytosis in viral replication.
Although initially recognised as essential for the entry of certain viruses, endocytosis is now known to also play important roles in the replication of, and adaptation to, the host cell of a number of viruses. Here we consider several aspects of this association and recent results that have emerged to support this view.
Animals↗
Antigen-driven CD4+ T cell and HIV-1 dynamics: residual viral replication under highly active antiretroviral therapy.
Antigen-induced stimulation of the immune system can generate heterogeneity in CD4+ T cell division rates capable of explaining the temporal patterns seen in the decay of HIV-1 plasma RNA levels during highly active antiretroviral therapy. Posttreatment increases in peripheral CD4+ T cell counts are consistent with a mathematical model in which host cell redistribution between lymph nodes and peripheral blood is a function of viral burden. Model fits to patient data suggest that, although therapy reduces HIV replication below replacement levels, substantial residual replication continues. This residual replication has important consequences for long-term therapy and the evolution of drug resistance and represents a challenge for future treatment strategies.
Anti-HIV Agents↗
SUMOylation of the human cytomegalovirus 72-kilodalton IE1 protein facilitates expression of the 86-kilodalton IE2 protein and promotes viral replication.
The 72-kDa immediate-early 1 protein (IE1-72kDa) of human cytomegalovirus has been previously shown to be posttranslationally modified by covalent conjugation to the ubiquitin-related protein SUMO-1. Using an infectious bacterial artificial chromosome clone of human cytomegalovirus, we constructed a mutant virus (BADpmIE1-K450R) that is deficient for SUMOylation of IE1-72 kDa due to a single amino acid exchange in the SUMO-1 attachment site. Compared to wild-type virus, this mutant grew more slowly and generated a reduced yield in infected human fibroblasts, indicating that SUMO modification is required for the full activity of IE1-72 kDa. The lack of SUMOylation did not affect the intranuclear localization of IE1-72 kDa, including its ability to target to and disrupt PML bodies and to bind to mitotic chromatin. Likewise, SUMOylation-deficient IE1-72 kDa activated several viral promoters as efficiently as the wild-type protein. However, the failure to modify IE1-72 kDa resulted in substantially reduced levels of the IE2 transcript and its 86-kDa protein (IE2-86 kDa). These observations suggest that SUMO modification of IE1-72 kDa contributes to efficient HCMV replication by promoting the accumulation of IE2-86 kDa.
Cell Line↗
Jembrana disease virus Tat can regulate human immunodeficiency virus (HIV) long terminal repeat-directed gene expression and can substitute for HIV Tat in viral replication.
Jembrana disease virus (JDV) is a bovine lentivirus genetically similar to bovine immunodeficiency virus; it causes an acute and sometimes fatal disease in infected animals. This virus carries a very potent Tat that can strongly activate not only its own long terminal repeat (LTR) but also the human immunodeficiency virus (HIV) LTR. In contrast, HIV Tat cannot reciprocally activate the JDV LTR (H. Chen, G. E. Wilcox, G. Kertayadnya, and C. Wood, J. Virol. 73:658-666, 1999). This indicates that in transactivation JDV Tat may utilize a mechanism similar to but not the same as that of the HIV Tat. To further study the similarity of JDV and HIV tat in transactivation, we first tested the responses of a series of HIV LTR mutants to the JDV Tat. Cross-transactivation of HIV LTR by JDV Tat was impaired by mutations that disrupted the HIV type 1 transactivation response element (TAR) RNA stem-loop structure. Our results demonstrated that JDV Tat, like HIV Tat, transactivated the HIV LTR at least partially in a TAR-dependent manner. However, the sequence in the loop region of TAR was not as critical for the function of JDV Tat as it was for HIV Tat. The competitive inhibition of Tat-induced transactivation by the truncated JDV or HIV Tat, which consisted only of the activation domain, suggested that similar cellular factors were involved in both JDV and HIV Tat-induced transactivation. Based on the one-round transfection assay with HIV tat mutant proviruses, the cotransfected JDV tat plasmid can functionally complement the HIV tat defect. To further characterize the effect of JDV Tat on HIV, a stable chimeric HIV carrying the JDV tat gene was generated. This chimeric HIV replicated in a T-cell line, C8166, and in peripheral blood mononuclear cells, which suggested that JDV Tat can functionally substitute for HIV Tat. Further characterization of this chimeric virus will help to elucidate how JDV Tat functions and to explain the differences between HIV and JDV Tat transactivation.
Animals↗
Characterization of West Nile viral replication and maturation in peripheral neurons in culture.
The North American West Nile virus (WNV), New York 1999 strain, appears to be highly neurotropic, and its neuroinvasiveness is an important aspect of human disease. The authors have developed an in vitro model to study WNV replication and protein processing in neurons. They compared WNV infection of the dorsal root ganglion (DRG) neurons (sensory neurons) and PC-12 cells (sympathetic neurons) to WNV infection of the mosquito cell line, C6/36, and Vero cells. WNV infection of both neuronal cell types and C6/36 cells was not cytopathic up to 30 days post infection, and continual viral shedding was observed during this period. However, WNV infection of Vero cells was lytic. Interestingly, WNV infection of neurons was not efficient, requiring a high multiplicity of infection of > or = 10. Indirect immunofluorescence assays using normal and confocal microscopy with flavivirus-reactive antibodies and WNV-infected neurons demonstrated viral antigen mostly associated with the plasma membrane and in the neurite processes. Treatment of WNV-infected C6/36, PC-12, or DRG cells with brefeldin A (BFA; a trans-Golgi inhibitor) or nocadazole (a beta-tubulin inhibitor) had little effect on viral maturation and secretion. Treatment of WNV-infected Vero cells with BFA resulted in a 1000-fold decrease in viral titer, but nocodazole had no effect. Our studies suggest that even though PC-12 and DRG neurons are mammalian cells, viral protein processing and maturation in these cells more closely resembles replication in C6/36 insect cells than in mammalian Vero cells.
Animals↗
Pre-S deletion mutants co-exist with wild-type virus at low viral replication stage in Singapore chronic hepatitis B virus carriers.
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Adult↗
Expression of full-length and truncated Rep genes from Mungbean yellow mosaic virus-Vigna inhibits viral replication in transgenic tobacco.
Mungbean yellow mosaic virus-Vigna (MYMV-Vig) is a bipartite geminivirus that causes a severe yellow mosaic disease in blackgram. An assay was developed to study MYMV-Vig replication by agroinoculation of tobacco leaf discs with partial dimers of the virus. This assay, in a non-host model plant, was used to evaluate pathogen-derived resistance contributed by MYMV-Vig genes in transgenic plants. Viral DNA accumulation was optimum in tobacco leaf discs cultured for 10 days after infection with Agrobacterium tumefaciens strain Ach5 containing partial dimers of both DNA A and DNA B of MYMV-Vig. Transgenic tobacco plants with MYMV-Vig genes for coat protein (CP), replication-associated protein (Rep)-sense, Rep-antisense, truncated Rep (T-Rep), nuclear shuttle protein (NSP) and movement protein (MP) were generated. Leaf discs from transgenic tobacco plants, harbouring MYMV-Vig genes, were agroinoculated with partial dimers of MYMV-Vig and analyzed for viral DNA accumulation. The leaf discs from transgenic tobacco plants harbouring CP and MP genes supported the accumulation of higher levels of MYMV-Vig DNA. However, MYMV-Vig accumulation was inhibited in one transgenic plant harbouring the Rep-sense gene and in two plants harbouring the T-Rep gene. Northern analysis of these plants revealed a good correlation between expression of Rep or T-Rep genes and inhibition of MYMV-Vig accumulation.
DNA Helicases↗
Lytic viral replication as a contributor to the detection of Epstein-Barr virus in breast cancer.
Epstein-Barr virus (EBV) has an accepted association with the epithelial malignancy nasopharyngeal carcinoma and has also been reported in other more controversial carcinoma settings. Evaluation of EBV association with epithelial carcinomas such as breast cancer would benefit from a better understanding of the outcome of EBV infection of these cells. Cell-free preparations of a green fluorescent protein-expressing virus, BX1, were used to infect breast cancer cell lines, which were then examined for EBV gene expression and viral genome copy number. Reverse transcription-PCR analyses revealed that the cells supported a mixture of latency II and lytic EBV gene expression. Lytic Zta and BMRF1 protein expression was detected by immunohistochemistry, and DNA PCR analyses estimated an EBV copy number of 300 to 600 genomes per infected cell. Evidence for lytic EBV expression was also found in breast tissue, where reverse transcription-PCR analyses detected lytic Zta transcripts in 7 of 10 breast carcinoma tissues and 4 of 10 normal tissues from the same patients. Scattered cells immunoreactive for Zta protein were also detectable in breast carcinoma. Quantitative real-time PCR analysis of EBV-positive breast carcinoma tissues suggested that less than 0.1% of the cells contained viral genomes. We suggest that sporadic lytic EBV infection may contribute to PCR-based detection of EBV in traditionally nonvirally associated epithelial malignancies.
Antigens, Viral↗
Gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection.
Human and simian immunodeficiency virus (HIV and SIV) replicate optimally in activated memory CD4(+) T cells, a cell type that is abundant in the intestine. SIV infection of rhesus monkeys resulted in profound and selective depletion of CD4+ T cells in the intestine within days of infection, before any such changes in peripheral lymphoid tissues. The loss of CD4+ T cells in the intestine occurred coincident with productive infection of large numbers of mononuclear cells at this site. The intestine appears to be a major target for SIV replication and the major site of CD4+ T cell loss in early SIV infection.
Animals↗
Rotavirus genome segment 4 determines viral replication phenotype in cultured liver cells (HepG2).
One-step growth determinations were performed with five strains of rotavirus in HepG2, a cell line derived from human liver. Three virus strains (SA11-C13, SA11-C14, and RRV) replicated in HepG2 cells and attained yields 10- to 100-fold above input titers. Two virus strains (B223 and SA11-4F) failed to replicate above input titer. Analysis of reassortants that segregated the genes of parental virus pairs able and unable to replicate revealed that the HepG2 cell growth phenotype segregated with genome segment 4. Immunofluorescence analysis of infected HepG2 cells showed that the production of detectable antigen correlated with the growth phenotype and also segregated with genome segment 4. Thus, we conclude that (i) some virus strains were capable of replication in cultured liver cells while other strains could not replicate under identical conditions and that (ii) the inability of some virus strains to replicate resulted from a segment 4-associated block in replication before protein synthesis. These results are discussed in terms of what is known of the functions of VP4.
Antigens, Viral↗
Do high levels of viral replication contribute to fulminant hepatitis A?
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5' Untranslated Regions↗
A deletion in the proximal untranslated pX region of human T-cell leukemia virus type II decreases viral replication but not infectivity in vivo.
The function of untranslated (UT) nucleotide sequences in the proximal portion of the pX region of the human T-cell leukemia virus (HTLV) family of retroviruses remains enigmatic. Previous studies have shown that these sequences are not necessary for the expression of viral proteins or for the induction, transmission, or maintenance of the transformed cell type in vitro. To determine the effect of the UT region in vivo, separate groups of rabbits were inoculated with lethally irradiated, stable clones of the human B-lymphoblastoid cell line, 729, transfected with either a full-length wild-type HTLV-II clone (pH6neo) or a mutant clone containing a 324-bp deletion in the proximal UT portion of pX (pH6neo delta UT[6661-6984]), or nontransfected 729 cells. All rabbits inoculated with either wild-type or pX-deleted HTLV-II developed a similar profile and titer of serum antibodies against HTLV-II antigens, as determined by Western immunoblots, by 4 weeks postinoculation (PI). Antibody titers, as determined by enzyme immunoassay, were similar between the two groups of rabbits and increased over the 18-week period of study. All rabbits were killed at 18 weeks PI, and spleen, peripheral blood lymphocytes (PBMC), bone marrow, and mesenteric lymph node were assayed for HTLV-II tax/rex sequences by quantitative polymerase chain reaction. Virus was detected in all tissues tested from all rabbits inoculated with 729pH6neo cells containing wild-type HTLV-II, which contained between 1.4 and 0.3 mean copies of provirus per cell. In contrast, the distribution and number of provirus copies were more limited in rabbits inoculated with 729pH6neo delta UT(6661-6984) cells containing UT-deleted HTLV-II; in most tissues, there was a fivefold to sevenfold reduction in mean provirus copies per cell as compared with rabbits inoculated with wild-type HTLV-II. All rabbits inoculated with control 729 cells remained negative for HTLV-II infection, as determined by the same techniques. It was concluded that UT sequences in the proximal portion of HTLV-II are not necessary for infection but confer increased replicative capacity in vivo.
Animals↗