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At least 595 records · Page 33Linked to original sources
PubMed1987

Lymphoid cells in the spleens of woodchuck hepatitis virus-infected woodchucks are a site of active viral replication.

Lymphoid cells were purified from the spleens of 15 woodchucks and examined for the presence of woodchuck hepatitis virus (WHV). Lymphoid cells from the spleens of eight of eight chronically infected animals contained high levels of WHV RNA and DNA. A 100-fold lower level of WHV DNA was found in the spleen from one of five animals that had recovered from acute WHV infections 2 years before this analysis. No WHV nucleic acids were observed in either of two uninfected animals. WHV DNA patterns in the lymphoid cells from the spleens of the chronically infected animals, which included the presence of single-stranded DNA and RNA-DNA hybrid molecules, were identical to those observed in WHV-infected liver. WHV DNA in these cells was present in intact, 27-nm core particles which also contained the endogenous DNA polymerase activity. These results indicate that the spleen is a site of active WHV DNA replication and is most likely a major source of WHV-infected cells in the circulating lymphoid cell population.

Animals↗
PubMed1996

Conservation of a hairpin ribozyme sequence in HIV-1 is required for efficient viral replication.

We have previously described a hairpin ribozyme that targets a highly conserved sequence in the U5 region of HIV-1. To determine if escape mutations would compromise virus replication, we introduced critical mutations into the ribozyme target site of an infectious molecular clone of HIV-1MN. HIV-1 MNA has a substitution of A for G immediately 3' to the cleavage site and HIV-1 MNGC has two substitutions in the flanking sequences that are complementary to the ribozyme. In vitro studies confirmed that neither the MNA-nor the MNGC-mutated target sequence was cleaved by the ribozyme, and furthermore, the MNGC-mutated target sequence failed to bind the ribozyme. Compensatory GC substitutions in the substrate recognition domain of the ribozyme resulted in a switch of binding and cleavage specificity. Replication of both the MNA and MNGC mutant viruses was initially two to three logs lower than that of wild-type virus, but after 3 weeks, virus production rose sharply in both cultures. Nucleotide sequence of RT-PCR-amplified viral sequences obtained from virus produced at later time points revealed complete reversion of MNA or partial reversion of MNGC to wild-type genotypes. No additional mutations within the ribozyme target sequence were observed. These results indicate that mutations in this conserved ribozyme target sequence led to significant attenuation of HIV-1MN.

Animals↗
PubMed2005

Effects of indomethacin on viral replication markers in asymptomatic carriers of hepatitis B: a randomized, placebo-controlled trial.

OBJECTIVES: Previous studies have suggested some benefits of nonsteroidal antiinflammatory drugs (NSAIDs) use in patients with chronic viral hepatitis. We evaluated potential effects of indomethacin in asymptomatic carriers of hepatitis B surface antigen (HBsAg). DESIGN: Randomized, placebo-controlled, double-masked clinical trial. METHODS: One hundred and twelve patients who were confirmed to be HBsAg carriers for at least 6 months and had normal liver function tests, normal abdominal sonography, and no sign of cirrhosis were randomly assigned into two groups. One group (56 participants, mean age (+/-SD) 31.7 (+/-9.6) yr, 29 male, mean serum alanine aminotransferase (ALT) (+/-SD) 24.9 (+/-9.2)) received indomethacin capsules (25 mg) three times daily and the other group (56 participants, mean age (+/-SD) 33.8 (+/-10.2) yr, 33 male, mean serum ALT (+/-SD) 24.5 (+/-8.7)) took placebo capsules with identical package and appearance. All participants were under treatment for 6 months and were followed 3 months thereafter. Statistical analyses were performed both by intention-to-treat and on-treatment methods. RESULTS: Nine participants in the indomethacin group (16%) and 8 in the placebo group (14%) did not complete the trial. HBsAg seroconversion did not differ by treatment group (2 subjects in each group became seronegative). Hepatitis B virus DNA (HBV-DNA) became negative in sera of 7 participants in the indomethacin group but only in 1 in the placebo group (intention-to-treat p= 0.06; on-treatment p= 0.03). Seroconversion of HBeAg to anti-HBe occurred only in 5 participants in the indomethacin group (intention-to-treat p= 0.06; on-treatment p= 0.03). Adverse events included one case of hepatotoxicity and two cases of gastritis in the indomethacin group and one suspected gastritis in the placebo group. CONCLUSIONS: We suggest use of indomethacin only in the subgroup of asymptomatic HBsAg carriers who have detectable HBV-DNA or HBeAg in their sera.

Adolescent↗
PubMed2000

Production of recombinant snakehead rhabdovirus: the NV protein is not required for viral replication.

Snakehead rhabdovirus (SHRV) affects warm water fish in Southeast Asia and belongs to the genus Novirhabdovirus by virtue of its nonvirion gene (NV). Because SHRV grows best at temperatures between 28 and 31 degrees C, we were able to use the T7 expression system to produce viable recombinant SHRV from a cloned cDNA copy of the viral genome. Expression of a positive-strand RNA copy of the 11, 550-nucleotide SHRV genome along with the viral nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins resulted in the generation of infectious SHRV in cells preinfected with a vaccinia virus vector for T7 polymerase expression. Recombinant virus production was verified by detection of a unique restriction site engineered into the SHRV genome between the NV and L genes. Since we were now able to begin examining the function of the NV gene, we constructed a recombinant virus containing a nonsense mutation located 22 codons into the coding sequence of the NV protein. The NV knockout virus was produced at a concentration as high as that of wild-type virus in cultured fish cells, and the resulting virions appeared to be identical to the wild-type virions in electron micrographs. These initial studies suggest that NV has no critical function in SHRV replication in cultured fish cells.

Amino Acid Sequence↗
PubMed2005

HIV controllers: a homogeneous group of HIV-1-infected patients with spontaneous control of viral replication.

We identified a total 15 patients who have maintained undetectable plasma HIV RNA loads without antiretroviral treatment for >10 years from cohorts of 1300 and 1551 patients infected with human immunodeficiency virus (HIV). These 15 patients, whom we have referred to as "HIV controllers," are characterized by a low HIV DNA load in peripheral blood mononuclear cells and by a strong HIV-specific immune response.

Adult↗
PubMed2005

Ebola virus VP40 late domains are not essential for viral replication in cell culture.

Ebola virus particle formation and budding are mediated by the VP40 protein, which possesses overlapping PTAP and PPXY late domain motifs (7-PTAPPXY-13). These late domain motifs have also been found in the Gag proteins of retroviruses and the matrix proteins of rhabdo- and arenaviruses. While in vitro studies suggest a critical role for late domain motifs in the budding of these viruses, including Ebola virus, it remains unclear as to whether the VP40 late domains play a role in Ebola virus replication. Alteration of both late domain motifs drastically reduced VP40 particle formation in vitro. However, using reverse genetics, we were able to generate recombinant Ebola virus containing mutations in either or both of the late domains. Viruses containing mutations in one or both of their late domain motifs were attenuated by one log unit. Transmission and scanning electron microscopy did not reveal appreciable differences between the mutant and wild-type viruses released from infected cells. These findings indicate that the Ebola VP40 late domain motifs enhance virus replication but are not absolutely required for virus replication in cell culture.

Animals↗
PubMed1973

Growth characteristics of cytomegalovirus in human fibroblasts with demonstration of protein synthesis early in viral replication.

In high-multiplicity infection of human fibroblasts, human cytomegalovirus of WI-38 human diploid cells produced early cell rounding 6 to 24 h after inoculation. This early cell rounding was caused only by inoculation with infectious virions. Inhibitors of protein synthesis, but not DNA inhibitors, prevented this cytopathic effect. Apparently, a new protein is synthesized in infected fibroblasts from about 2 h postinoculation. Infectivity of cell-associated and supernatant infectious virus reached maximal levels at 5 to 7 and 10 days postinoculation, respectively. Synthesis of DNA, infectious virus, complement-fixing antigen, and precipitin antigen all began between 24 and 48 h, with the bulk of synthesis occurring 48 to 96 h postinoculation.

Amino Acids↗
PubMed1996

Mucosal and systemic immunity to bovine herpesvirus-1 glycoprotein D confer resistance to viral replication and latency in cattle.

Mucosal immunity in the respiratory tract directed against bovine herpesvirus-1 (BHV-1) glycoprotein B forms an effective barrier against BHV-1 replication in cattle. Here we investigated the ability of a second BHV-1 glycoprotein, gD, to engender specific antibodies in nasal secretion and serum and protect against infection. We expected gD to give greater protection than gB because anti-gD antibodies prevent viral penetration into cells at much lower concentrations than anti-gB antibodies. Calves vaccinated once subcutaneously and thrice intranasally with affinity-purified BHV-1 gD had mucosal antibodies and three of five were protected against intranasal challenge by 10(7) p.f.u. of BHV-1. Four of the five vaccinated calves were proven free of BHV-1 latency by the lack of viral shedding following immunosuppression. The putative mucosal adjuvant, cholera toxin B subunit (CTB), did not significantly enhance mucosal immunity or protection against challenge or latency (P0.5) since only 4 of 6 gD plus CTB immunized calves were completely protected. Taken together, these data suggest that BHV-1 gD may be useful in a mucosal vaccine against BHV-1 infection in cattle but is less than totally effective when used alone.

Animals↗
PubMedDate not supplied

Polymerase chain reaction to monitor repair of the HBV genome, the first step in viral replication.

A method based on the polymerase chain reaction (PCR) for evidencing repair of the hepatitis B virus (HBV) genome is described. Hepadnaviruses have a partially double-stranded relaxed circular genome (RC-DNA) which is converted into a covalently closed circular DNA (CCC-DNA) after entry of the virus into a target cell. Our aim was to set up a technique enabling us to determine whether possible in vitro replication of the virus in non-hepatic cells is initiated by formation of CCC-DNA. The relevant part of the strategy used for this PCR consisted of priming the HBV-DNA template with the same forward primer and with a reverse primer located either downstream or upstream from the minus strand gap. The CCC-DNA form was found, as expected, in cells in which the virus was known to be actively replicating; although most sera contained only the RC-DNA form, it was also possible to evidence the CCC form. Such PCR amplification led to detection of 50-500 copies of the viral DNA. The method described should be useful in studying the biological fate of HBV in non-hepatic cells (considered as non-permissive for virus replication), and in exploring the clinical significance of the presence of CCC-DNA in sera.

AIDS-Related Opportunistic Infections↗
PubMed1989

Transcriptional activation of homologous and heterologous genes by the hepatitis B virus X gene product in cells permissive for viral replication.

The potential of the hepadnavirus X gene product to activate gene expression in trans was tested through a series of cotransfections of X expression vectors with a variety of potential targets for transactivation. The X gene products from human hepatitis B virus (HBV), woodchuck hepatitis virus, and ground squirrel hepatitis virus are all equally active in augmenting the expression of a wide array of target promoters in both permissive and nonpermissive cells. Using the HBV genome itself as the source of X protein, we demonstrate that transactivation of HBV and heterologous genes occurs when X protein is expressed in its native state during productive infection of permissive cells. Run-on transcription analysis indicates that this transactivation occurs at the level of primary transcription.

Genes, Viral↗
PubMed1996

Ligation of CR1 (C3b receptor, CD35) on CD4+ T lymphocytes enhances viral replication in HIV-infected cells.

The present study provides evidence for a role of the C3b receptor, CR1 (CD35), in activation of HIV replication in CD4+ T lymphocytes. Ligation of CR1 with cross-linked anti-receptor MoAbs or with aggregated C3b enhanced transcription of viral genes and the release of p24 and RT activity from cultures of purified normal CD4+ T lymphocytes that had been infected with HIV-1 in vitro. No effect was observed upon ligation of CR2 (CD21), a C3 receptor that is also expressed on human CD4+ T cells. Cross-linking of CR1 also enhanced HIV replication in peripheral blood CD4+ lymphocytes isolated from HIV+ individuals. The enhancing effect of CR1 was not related to a mitogenic effect induced by stimulation of the receptor on T cells. The CR1 specificity of the enhancing effect was established by the observation that the addition of soluble recombinant CR1 to the cultures abolished the enhancement of p24 release induced by anti-CR1 MoAbs. Our results suggest that HIV replication may be triggered in resting HIV-infected CD4+ T lymphocytes through interaction with C3b-bearing immune complexes or particles.

Antibodies, Monoclonal↗
PubMed1997

Expression of interferon-gamma by a coronavirus defective-interfering RNA vector and its effect on viral replication, spread, and pathogenicity.

A defective-interfering (DI) RNA of the murine coronavirus mouse hepatitis virus (MHV) was developed as a vector for expressing interferon-gamma (IFN-gamma). The murine IFN-gamma gene was cloned into the DI vector under the control of an MHV transcriptional promoter and transfected into MHV-infected cells. IFN-gamma was secreted into culture medium as early as 6 hr posttransfection and reached a peak level (up to 180 U/ml) at 12 hr posttransfection. The DI-expressed IFN-gamma (DE-IFN-gamma) exhibited an antiviral activity comparable to that of recombinant IFN-gamma and was blocked by a neutralizing monoclonal antibody against IFN-gamma. Treatment of macrophages with DE-IFN-gamma selectively induced the expression of the cellular inducible nitric oxide synthase and the IFN-gamma-inducing factor (IGIF) but did not affect the amounts of the MHV receptor mRNA. Antiviral activity was detected only when cells were pretreated with IFN-gamma for 24 hr prior to infection; no inhibition of virus replication was detected when cells were treated with IFN-gamma during or after infection. Furthermore, addition of IFN-gamma together with MHV did not prevent infection, but appeared to prevent subsequent viral spread. MHV variants with different degrees of neurovirulence in mice had correspondingly different levels of sensitivities to IFN-gamma treatment in vitro, with the most virulent strain being most resistant to IFN-gamma treatment. Infection of susceptible mice with DE-IFN-gamma-containing virus caused significantly milder disease, accompanied by more pronounced mononuclear cell infiltrates into the CNS and less virus replication, than that caused by virus containing a control DI vector. This study thus demonstrates the feasibility and usefulness of this MHV DI vector for expressing cytokines and may provide a model for studying the role of cytokines in MHV pathogenesis.

Animals↗
PubMed1973

Measles virus ribonucleic acid and protein synthesis: effects of 6-azauridine and cycloheximide on viral replication.

Cycloheximide and 6-azauridine were employed to study the time course of measles virus protein and nucleic acid syntheses in AV3 cells. Synthesis of ribonucleic acid (RNA) essential for infectivity was first detected at 6 hr and increased concurrently with the formation of essential protein. Maximum levels of virus-specific RNA and protein were present by 18 hr, a time when only 5% of progeny virus was detected. Essential RNA and protein syntheses preceded the formation of infectious virus by at least 10 to 12 hr. The time course of RNA and protein syntheses essential for the formation of complement-fixing (CF) antigen and salt-dependent agglutinin (SDA) was also determined. RNA synthesis essential for the formation of SDA was first detected at 2 hr and was present maximally by 6 hr, whereas SDA-protein increased concurrently with the protein essential for infectivity. This suggested that the last protein essential for infectivity may be SDA. RNA synthesis essential for the formation of CF antigen was first detected at 4 hr, while CF-protein increased at 5 hr and preceded SDA-protein and protein essential for infectivity by approximately 3 hr. Reversal of inhibition of protein synthesis by cycloheximide indicated that early protein synthesis (1 to 3 hr) was required for the formation of infectious virus. The data suggest that the relatively long eclipse period observed with measles virus is related to a long maturation period rather than to late formation of early proteins, viral RNA, or structural proteins.

Amnion↗
PubMed1988

The sites of early viral replication in feline infectious peritonitis.

The sites of early replication of feline infectious peritonitis virus were studied following oral inoculation of specific-pathogen-free (SPF) cats with virus grown in cell cultures. Viral antigen was first detected by immunofluorescence in the tonsils and small intestine within 24 h of inoculation, and was later found in caecum, colon, mesenteric lymph nodes and liver. However, histological changes in the gut did not appear until relatively late in the course of infection. Virus was recovered from the oropharynx and the faeces from as early as the second or third day after inoculation, and shedding continued until euthanasia.

Animals↗
PubMed2006

Vaccinia virus K1L protein supports viral replication in human and rabbit cells through a cell-type-specific set of its ankyrin repeat residues that are distinct from its binding site for ACAP2.

Vaccinia virus (VV) K1L is a host-range gene and encodes a protein comprised of six ankyrin repeats (ANKs). We showed here that a large portion of the K1L protein, except ankyrin repeat 1 (ANK1) and C-terminal halves of ANK2 and ANK3, can be deleted or substituted with an unrelated ANK with no adverse effect on VV replication in human HeLa cells. In contrast, only ANK4 and ANK6 can be mutated without impairing VV replication in rabbit RK13 cells. The growth rate of VV in HeLa cells was reduced differentially by substituting phenylalanine 82 or serine 83 of ANK2 and abolished completely by substituting both residues. These substitutions, however, did not affect K1L's ability to bind ACAP2, a GTPase-activating protein for ARF6. Our data support the hypothesis that surface residues of a few consecutive K1L ANKs mediate the host-range function by interacting with protein factors that are distinct from ACAP2.

Amino Acid Sequence↗