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PubMed1977

Antibody to influenza virus matrix protein detects a common antigen on the surface of cells infected with type A influenza viruses.

Antisera to the type-specific internal influenza virus matrix (M) protein of a type A influenza virus were produced in goats. In the presence of complement, anti-M serum was cytotoxic for target cells which were infected with a variety of serologically distinct type A influenza viruses, but did not react with type B influenza virus-infected cells. Absorption experiments indicated that anti-M serum detected a common antigen(s) on the surface of type A-infected cells. This serological cross-reactivity parallels the cross-reactivity observed for the cytotoxic T-cell response to type A viruses.

Antibodies, Viral↗
PubMed1993

Minor envelope proteins of duck hepatitis B virus are initiated at internal pre-S AUG codons but are not essential for infectivity.

In infected liver tissue three major and several minor duck hepatitis B virus (DHBV) envelope proteins are detectable by immunoblotting. Translation initiation at the second and the most distal internal ATG codon of the Pre-S/S gene is known to lead to synthesis of two major envelope proteins (P36 and P17) whereas the origin of a further major (P28) and other minor envelope proteins is not clear. Therefore, it was investigated whether translation initiation at pre-S ATGs leads to synthesis of these other envelope proteins and, if yes, whether they are components of viral particles and essential for infectivity. Each of the five ATG codons of the pre-S region of an infectious Chinese DHBV genome (DHBV26) was mutated separated by oligonucleotide-directed mutagenesis (point mutations or deletions) and the function of the corresponding mutant viruses were tested in vitro and in vivo. Immunoblot analysis of liver cell extracts or of extracts from hepatoma cells transfected with the DHBV genomes showed expression of minor pre-S proteins of about 35, 33, and 30 kDa. These proteins were not expressed when ATG codons at nucleotide positions 825, 882, and 957, respectively, were mutated. None of the ATG mutations abolished expression of the major P28 pre-S protein. In cell culture supernatants the minor pre-S proteins P35, P33, and P30 were identified as components of viral particles. With the exception of the DHBV genome containing the mutated ATG codon 801 (translation initiation codon for the major P36 pre-S protein) all forementioned DHBV mutant genomes were infectious. These data demonstrate that minor pre-S proteins are initiated at internal AUGs of the pre-S gene and are components of viral particles but are not essential for infectivity. In contrast to previously published speculations, the results also indicate that the major pre-S protein P28 is not initiated at AUG codon 957 but probably produced by proteolysis from larger pre-S proteins.

Animals↗
PubMed1989

The entry of African swine fever virus into Vero cells.

The entry of African swine fever virus into Vero cells has been investigated by both biochemical and morphological techniques. A quantitative electron microscopy analysis of the early steps of the infection has shown that African swine fever virus enters Vero cells by a receptor-mediated endocytosis mechanism. The internalization of virus particles is a temperature- and energy-dependent process, since it did not take place at 4 degrees or in the presence of NaF and 2,4-dinitrophenol. To determine the involvement of acidic intracellular vacuoles in the virus entry pathway we have tested the effect of lysosomotropic agents in the infection. Chloroquine, dansylcadaverine, amantadine, methylamine, and ammonium chloride inhibited African swine fever virus production in Vero cells. Dansylcadaverine and chloroquine did not inhibit virus adsorption and internalization; however, in the presence of these drugs, virus particles were retained in cytoplasmic vacuoles and early viral RNA and protein synthesis were not detected, indicating that these compounds inhibit an early step in the infectious cycle, probably the uncoating of the virus particle.

African Swine Fever Virus↗
PubMed1991

RNA--protein interactions within the internal translation initiation region of encephalomyocarditis virus RNA.

Various derivatives of the internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV) RNA have been used to analyze by UV-cross-linking its interaction with mRNA binding proteins from ascites carcinoma Krebs-2 cells. A doublet of proteins with Mr 58 and 60 kD bound to two regions of the IRES. One site is centered at nt 420-421 of EMCV RNA whereas the other is located between nt 315-377. Both sites form hairpin structures, the loops of which contain UCUUU motif, conserved among cardio- and aphthoviruses. The interaction of p58 and p60 with IRES is affected by the integrity of the stem-loop structure proximal to the start AUG codon (nts 680-787), although, under similar conditions, cross-linking of these proteins to this region was not detected. Deletions in the main recognition site of p58 strongly reduce the initiation activity of the IRES in vitro. However, elimination of p58 (p60) binding by these mutations does not completely abolish the ability of the IRES to direct polypeptide synthesis starting from the authentic AUG codon. The IRES can be assembled in vitro from two covalently unlinked transcripts, one containing the target site for p58 and the other encompassing the remaining part of the IRES fused to a reporter gene, resulting in considerable restoration of its activity. Implications of these findings for the mechanism of initiation resulting from internal entry of ribosomes are discussed.

Animals↗
PubMed2000

Improved version 2.0 qualitative and quantitative AMPLICOR reverse transcription-PCR tests for hepatitis C virus RNA: calibration to international units, enhanced genotype reactivity, and performance characteristics.

Version 2.0 qualitative and quantitative AMPLICOR reverse transcription-PCR tests for HCV were designed to improve on the performance of first version of the hepatitis C virus (HCV) tests. The new tests were calibrated in international units, the new commonly accepted standard unit of measurement for HCV RNA. The sensitivity of the qualitative tests was enhanced by modifying the specimen processing procedure to achieve a limit of detection 50 IU/ml. The limit of detection for the quantitative tests was 600 IU/ml. Modifications to the amplification reaction mixture and thermal cycling conditions enabled all genotypes to be amplified with similar efficiency. The quantitative tests exhibited a linear range extending from 500 to 500,000 IU/ml and excellent reproducibility, with coefficients of variation ranging from 18 to 39%, within the linear range. These data indicate that the version 2. 0 AMPLICOR HCV tests will improve diagnosis of HCV infection and will yield more-accurate titers for prognosis and for monitoring therapeutic efficacy, particularly at low viral loads. Furthermore, it will be possible to compare the performance characteristics and viral load measurements of AMPLICOR tests to those of other tests that adopt the international unit as the standard of measurement.

Calibration↗
PubMed1976

Detection, quantitation, and characterization of the major internal virion antigen of the bovine leukemia virus by radioimmunoassay.

The major internal polypeptide of the bovine leukemia virus (BLV) was purified to homogeneity with the use of gel filtration and affinity chromatography. Like previous results, the protein had a molecular weight of 25,000 daltons as determined by electrophoresis in polyacrylamide gels with sodium dodecyl sulfate. More than 90% of the 125I-labeled protein was precipitated by bovine sera that reacted in immunofluorescence tests with acetone-fixed BLV-infected cells. In contrast, minimal precipitation (less than 5%) was observed with sera from 36 cattle in leukemia-free herds; these sera, negative by immunofluorescence, included six samples that had high titers of antibodies to the foamy-like bovine syncytia virus (BSV). Antisera prepared against several other oncornaviruses or the Mason-Pfizer monkey virus (M-PMV) did not bind the BLV p25 protein. Conversely, the labeled p30 polypeptides of several oncornaviruses tested did not react with bovine sera that had high titers of antibodies to BLV p25. Competitive radioimmunoassay(s) (RIA) also failed to detect cross-reactions between BLV p25 protein and the internal polypeptides of other mammalian and avian oncornaviruses, M-PMV, or foamy-like BSV. The RIA for BLV p25 antigen was also highly sensitive and specific for the detection and quantitation of the antigen in virus preparations and cell homogenates.

Animals↗
PubMed1975

A morphological study of the internal component of influenza virus.

Rapid treatment of influenza virus directly on the microscope grid with non-ionic detergent had allowed better visualization of the internal component. Many micrographs show that this ribonucleoprotein (RNP) is present as a continuous stand of 6 nm diam. arranged in the form of a double coil or helix. In spite of the minimal treatment to which the virus was subjected most helices still showed signs of degradation. The findings that we have obtained lead us to suggest that the RNP component of influenza virus must be very sensitive to both chemical and physical manipulations, any of which could cause it to fracture from one continuous strand into several pieces, although such breakages could possibly occur at specific points along its length.

Animals↗
PubMed2000

Critical role for the cysteines flanking the internal fusion peptide of avian sarcoma/leukosis virus envelope glycoprotein.

The transmembrane subunit (TM) of the envelope glycoprotein (Env) of the oncovirus avian sarcoma/leukosis virus (ASLV) contains an internal fusion peptide flanked by two cysteines (C9 and C45). These cysteines, as well as an analogous pair in the Ebola virus GP glycoprotein, are predicted to be joined by a disulfide bond. To examine the importance of these cysteines, we mutated C9 and C45 in the ASLV subtype A Env (EnvA), individually and together, to serine. All of the mutant EnvAs formed trimers that were composed of the proteolytically processed surface (SU) and TM subunits. All mutant EnvAs were incorporated into murine leukemia virus pseudotyped virions and bound receptor with wild-type affinity. Nonetheless, all mutant EnvAs were significantly impaired ( approximately 1,000-fold) in their ability to support infectivity. They were also significantly impaired in their ability to mediate cell-cell fusion. Our data are consistent with a model in which the internal fusion peptide of ASLV-A EnvA exists as a loop that is stabilized by a disulfide bond at its base and in which this stabilized loop serves an important function during virus-cell fusion. The fusion peptide of the Ebola virus GP glycoprotein may conform to a similar structure.

Alpharetrovirus↗
PubMed1981

Optimum conditions for the radioimmunoprecipitation assay for the major internal protein of bovine leukemia virus.

The sensitivity of the radioimmunoprecipitation assay for the major internal protein (p24) of bovine leukemia virus (BLV) was examined. Conditions were varied for the assay by 1) using larger amounts of serum and 2) increasing the amount of second antibody. Sensitivity for the p24 radioimmunoprecipitation test was greatest when 10 microliter of test serum and 200 microliter of rabbit anti-bovine IgG were used. Under these conditions there were no discrepancies between the p24 radioimmunoprecipitation test and the radioimmunoprecipitation test for the surface glycoprotein (gp51) in 380 cattle from commercial dairy herds.

Animals↗
PubMed2006

Assessment of early virological response to antiviral therapy by comparing four assays for HCV RNA quantitation using the international unit standard: implications for clinical management of patients with chronic hepatitis C virus infection.

WHO International Standards for nucleic acid tests are used widely to compare the different assays used in HCV RNA quantitation. The aim of the study was to assess the impact of the international unit standard for measuring HCV RNA in the management of patients with chronic hepatitis C virus (HCV) infection. Twenty-seven naïve patients infected chronically by HCV were treated with ribavirin plus PEG-interferon-alfa-2b for 48 weeks. SVR was obtained for 16 patients (the other were non-responders). For HCV RNA quantitation, four assays were undertaken: Versant HCV RNA 3.0 (Bayer), Real time PCR (TaqMan, Roche), LCx HCV RNA (Abbott), and Cobas Amplicor-Monitor v2 (Roche). Considering a 2-log decline at Week 12 after the beginning of therapy, discordant results were found with the four HCV RNA methods in predicting SVR or non-response. At Week 4 and Week 12, significant differences were observed between Versant HCV RNA 3.0 versus PCR HCV Taqman, Versant HCV RNA 3.0 versus LCx HCV RNA, Cobas Monitor Amplicor HCV 2.0 versus LCx HCV RNA, and Cobas Monitor Amplicor HCV 2.0 versus PCR HCV Taqman (P < 0.001). The HCV RNA cutoff, given a 100% negative predictive value at Week 4 and Week 12, differed with the assays used to quantify HCV RNA, despite the use of the IU/ml units. Eighty-nine percent of serum values for HCV RNA were concordant by the IU standard. All assays, however, failed to detect HCV RNA in some cases. Despite the use of the IU standard HCV-infected patients might be monitored with only one assay.

Administration, Oral↗
PubMed1992

The sequence context of the initiation codon in the encephalomyocarditis virus leader modulates efficiency of internal translation initiation.

Translation initiation on poliovirus and encephalomyocarditis virus (EMCV) mRNAs occurs by a cap-independent mechanism utilizing an internal ribosomal entry site (IRES). However, no unifying mechanism for AUG initiation site selection has been proposed. Analysis of initiation of mRNAs translated in vitro has suggested that initiation of poliovirus mRNA translation likely involves both internal binding of ribosomes and scanning to the first AUG which is in a favorable context for initiation. In contrast, internal initiation on EMCV mRNA may not utilize scanning, since ribosomes bind directly or very close to the initiation codon AUG-11. We have studied in vivo the sequence requirements for internal initiation around the EMCV initiation codon, both in monocistronic and in dicistronic mRNAs. Our studies show that the upstream AUG-10 is normally not used and that there is no specific sequence requirement for nucleotides between AUG-10 and AUG-11. However, the sequence context of AUG-11 does influence the efficiency of initiation at AUG-11. Efficient IRES-mediated internal initiation at AUG-11 exhibits a requirement for an adenine in the -3 position, similar to cap-dependent initiation. These results support a model for internal initiation on EMCV mRNA in which scanning starts at or near AUG-11. Although initiation primarily occurs at AUG-11, initiation at multiple downstream AUG codons can be detected. In addition, a poor sequence context around AUG-11 results in increased initiation at one or more downstream AUG codons, indicative of leaky scanning or jumping by the ribosome from AUG-11 mediated by the EMCV IRES.

Animals↗
PubMed1993

Varicella-zoster virus replication site in internal organs of an otherwise healthy child with varicella and sudden death.

Pathological findings of an otherwise healthy 17 month old boy who was exposed to Varicella-zoster virus (VZV) in his family and unexpectedly died 3 days after onset of varicella are reported. They showed a disseminated VZV infection with involvement of skin, lung, liver, spleen, gastrointestinal tract and other organs where VZV antigen was detected by the enzyme-immunoassay with monoclonal antibodies to VZV. Since the subject was the full-term product of an uncomplicated pregnancy, who grew and developed normally, and had no symptoms or laboratory findings suggestive of immunodeficiency until his death, these findings suggest that many organs are involved as major internal sites of viral replication before or during infection of skin with VZV in the immunocompetent host.

Antibodies, Monoclonal↗
PubMed1996

Two neutralizing anti-V3 monoclonal antibodies act by affecting different functions of human immunodeficiency virus type 1.

Monoclonal antibody (MAb) ICR41.1i (rat IgG2a) is specific for a conformation-dependent epitope of human immunodeficiency virus type 1 (HIV-1) V3 , and MAb F58 (mouse IgG1) recognizes the peptide IXXGPGR, at the tip of the V3 loop. Both MAbs neutralized HIV-1 strain IIIB in C8166 and HeLa-T4(CD4) cells. Neutralization by either MAb did not inhibit attachment of virus to target cells as determined by FACS analysis, ELISA or immunofluorescence, and such attachment was absolutely dependent on the availability of CD4 molecules. F58 inhibited virus-induced cell-cell fusion, and reduced internalization of virions in direct proportion to neutralization. In contrast, ICR41.li had no effect on HIV-1-mediated cell fusion or on internalization of virus. It was concluded that MAb F58 neutralized infectivity by inhibiting fusion of the virus with the cell and internalization of the viral core, and that ICR41.1i neutralized by inhibiting a post-fusion-internalization event. The possible mechanism by which a neutralizing antibody binds to the V3 loop and affects the function(s) of structures inside the virion is discussed. Lastly, postattachment neutralization (PAN) was investigated. F58 mediated PAN at 21 degrees C and 35 degrees C. However, ICR41.1i gave PAN at 21 degrees C but not at 35 degrees C, suggesting that a temperature-dependent event affecting the V3 loop had abrogated neutralization. Overall, it appears that antibodies to different epitopes within the V3 loop neutralize by affecting very different functions of the virus.

Animals↗
PubMed2005

[Origin of internal genes of two strains of swine influenza A (H1N1) virus].

BACKGROUND: Through the analysis of internal genes of viruses to understand whether the internal genes of two strains of swine influenza A(H1N1) virus contain the gene segment deriving from avian influenza A viruses, and whether the reassortment of the internal genes occurred between swine H1N1 and swine H9N2 viruses. METHODS: Viruses were passaged in embryonated hen eggs and virion RNA was extracted from allantoic fluids and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards, RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (version 1.03). RESULTS: All the six, but PB2 gene segment internal gene segments were the same between A/swine/Beijing/1/2002(H1N1) and A/Swine/Beijing/3/2002(H1N1) viruses. Whereas all the six internal gene segments in two trains of swine (H1N1) virus were similar to those of swine H1N1 viruses, but different from those of classical strain of swine (H1N1) virus. CONCLUSION: Two strains of Beijing swine H1N1 influenza A virus were not reassortant, All the six internal gene segments were closely related to swine influenza A (H1N1) viruses.

Animals↗