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PubMed1990

Evaluation of the infectivity, immunogenicity, and efficacy of live cold-adapted influenza B/Ann Arbor/1/86 reassortant virus vaccine in adult volunteers.

A cold-adapted (ca) influenza B reassortant that derived two genes encoding the hemagglutinin and neuraminidase from influenza B/Ann Arbor/1/86 wild-type virus and six internal RNA segments from ca influenza B/Ann Arbor/1/66 virus was evaluated in 66 adult volunteers having a serum hemagglutination inhibition antibody titer less than or equal to 1:8. The ca reassortant was attenuated and elicited the production of systemic and local antibodies; the 50% human infectious dose was 10(6.4) TCID50. Six weeks after vaccination, 12 unvaccinated volunteers and 13 recipients of ca virus (10(7.5) TCID50) were challenged experimentally with homologous wild-type influenza B virus. The ca vaccine completely protected against illness, and the magnitude of shedding was 50-fold less in vaccinees than in unimmunized controls, five of whom became ill. These findings indicate that the six internal RNA segments of the ca influenza B/Ann Arbor/66 donor virus confer desirable properties of a live virus vaccine to a reassortant derived from a virulent virus. Such reassortants may be suitable vaccines for healthy adults.

Adult↗

PubMed2006

Structure of the ribosome-bound cricket paralysis virus IRES RNA.

Internal ribosome entry sites (IRESs) facilitate an alternative, end-independent pathway of translation initiation. A particular family of dicistroviral IRESs can assemble elongation-competent 80S ribosomal complexes in the absence of canonical initiation factors and initiator transfer RNA. We present here a cryo-EM reconstruction of a dicistroviral IRES bound to the 80S ribosome. The resolution of the cryo-EM reconstruction, in the subnanometer range, allowed the molecular structure of the complete IRES in its active, ribosome-bound state to be solved. The structure, harboring three pseudoknot-containing domains, each with a specific functional role, shows how defined elements of the IRES emerge from a compactly folded core and interact with the key ribosomal components that form the A, P and E sites, where tRNAs normally bind. Our results exemplify the molecular strategy for recruitment of an IRES and reveal the dynamic features necessary for internal initiation.

Animals↗

PubMed1992

Multicenter evaluation of five commercial rubella virus immunoglobulin G kits which report in international units per milliliter.

In a multicenter study, the consistency of international units expressed by five commercially available rubella virus immunoglobulin G kits was evaluated. The linearity and within-run and between-run precision were determined for each kit. All kits demonstrated good linearity and had within-run and between-run precision coefficients of variation ranging from 5.1 to 21.7% and from 9.5 to 51.0%, respectively. To compare the international units expressed, the results from 40 samples tested in duplicate were compared with the results of a reference enzyme immunoassay calibrated with World Health Organization international standard serum and a hemagglutination inhibition test. The results of the kits were plotted against those of the reference tests, and linear regression analysis was applied. The Pearson correlation coefficient ranged from 0.64 to 0.75 when the commercial kit results were compared with those of the reference enzyme immunoassay, indicating only a moderate degree of correlation. Therefore, the international units expressed by the commercial kits are insufficiently consistent to be of practical use in diagnostic clinical microbiology.

Antibodies, Viral↗

PubMed2005

Virus-associated hemophagocytic syndrome in an international traveler as a differential diagnosis of SARS.

During the epidemic of severe acute respiratory syndrome in 2003, a 27-year- old Japanese woman presented a high fever and acute respiratory distress with pulmonary infiltrates after traveling to a high-risk area. An alternative diagnosis was made as Epstein-Barr virus-associated hemophagocytic syndrome, based on the proliferation of macrophages with hemophagocytosis in the bone marrow and Epstein-Barr viral marker profiles. Virus-associated hemophagocytic syndrome in an international traveler should be included in the differential diagnosis of severe acute respiratory syndrome.

Adult↗

PubMed1998

The 3'-untranslated region of hepatitis C virus RNA enhances translation from an internal ribosomal entry site.

Translation of most eukaryotic mRNAs and many viral RNAs is enhanced by their poly(A) tails. Hepatitis C virus (HCV) contains a positive-stranded RNA genome which does not have a poly(A) tail but has a stretch of 98 nucleotides (X region) at the 3'-untranslated region (UTR), which assumes a highly conserved stem-loop structure. This X region binds a polypyrimidine tract-binding protein (PTB), which also binds to the internal ribosome entry site (IRES) in HCV 5'-UTR. These RNA-protein interactions may regulate its translation. We generated a set of HCV RNAs differing only in their 3'-UTRs and compared their translation efficiencies. HCV RNA containing the X region was translated three- to fivefold more than the corresponding RNAs without this region. Mutations that abolished PTB binding in the X region reduced, but did not completely abolish, enhancement in translation. The X region also enhanced translation from another unrelated IRES (from encephalomyocarditis virus RNA), but did not affect the 5'-end-dependent translation of globin mRNA in either monocistronic or bicistronic RNAs. It did not appear to affect RNA stability. The free X region added in trans, however, did not enhance translation, indicating that the translational enhancement by the X region occurs only in cis. These results demonstrate that the highly conserved 3' end of HCV RNA provides a novel mechanism for enhancement of HCV translation and may offer a target for antiviral agents.

3' Untranslated Regions↗

PubMed1989

Several rat cell lines share a common defect in their inability to internalize murine coronaviruses efficiently.

Infection of rat cells, Schwannoma RN2, hepatoma HTC or myoblast L6, with the murine coronavirus JHM strain results in a persistent infection characterized by the release of virus over an extended period of time with a limited cytopathology. Several stages of the viral replication cycle have been examined in these cells in comparison to those in mouse L2 cells, which are totally permissive to JHM infection. Although the rat cells bound as much virus as the mouse cells. Their ability to internalize it was 40-fold less efficient than the mouse cells. This lower internalization efficiency was not enhanced by pH shock of infected cells, but was by treatment with polyethylene glycol. In all cell types there appeared to be no major differences in the ability of the internalized virus to replicate the viral RNA as determined by slot-blot analysis with a radiolabelled viral cDNA. A similar genetic mechanism appears to be operative in the lines because somatic cell hybrids formed between these lines in various combinations were also deficient in the ability to internalize bound virus. Taken together these results imply that rat cell lines in general share a common deficiency in their inability to internalize murine coronaviruses efficiently. This low efficiency in viral internalization may explain in part the ability of these lines to sustain persistent infections.

Adhesiveness↗

PubMed2002

Internal initiation by the cucumber necrosis virus RNA-dependent RNA polymerase is facilitated by promoter-like sequences.

Tombusviruses, small positive sense RNA viruses of plants, are replicated by the viral-coded RNA-dependent RNA polymerase (RdRp) in infected cells. An unusual feature of the tombusvirus RdRp that is partially purified from cucumber necrosis virus (CNV)-infected plants is the ability to initiate complementary RNA synthesis from several internal positions on minus-strand templates derived from DI RNAs ( Nagy and Pogany, 2000 ). In this study, we used template deletion, mutagenesis, and oligo-based inhibition of RNA synthesis to map the internal initiation sites observed with the in vitro CNV RdRp system. Comparing sequences around the internal initiation sites reveals that they have either (i) similar sequences to the core minus-strand initiation promoter; or (ii) similar structures to the core plus-strand initiation promoter. In addition, we find similarities among the internal initiation sites and the subgenomic RNA initiation sites. These similarities suggest that the mechanism of internal initiation is similar to initiation from the terminal core promoters or the putative subgenomic promoter sequences. We propose that internal initiation on full-length RNA templates may be important in defective interfering (DI) RNA formation/evolution by producing intermediate templates for RNA recombination in tombusviruses. This may explain why tombusviruses are frequently associated with DI RNAs.

Base Sequence↗

PubMed2005

Real-time PCR quantitation of hepatitis B virus DNA using automated sample preparation and murine cytomegalovirus internal control.

Quantitation of circulating hepatitis B virus (HBV) DNA is important for monitoring disease progression and for assessing the response to antiviral therapy. Several commercial and 'in house' assays for HBV DNA quantitation have been described but many of these have limitations of relatively low sensitivity and limited dynamic range. This study describes the development and evaluation of a FRET-based real-time PCR assay designed to overcome these limitations and to provide accurate quantitation of DNA from all eight genotypes of HBV (A-H). The assay employs a fully automated nucleic acid extraction system permitting high-sample throughput with minimal 'hands-on' time and incorporates a murine cytomegalovirus (mCMV) internal control to prevent false negative results and under-reporting due to unrecognised problems with viral lysis, DNA purification or PCR amplification. Sensitivity, assessed by Probit analysis at the 95% detection level, was 24.4 IU/ml, associated with an extremely wide dynamic range (approximately 9 log10). Coefficients of variation were low for both intra-assay and inter-assay variability (CV%, 7-11%) and quantitative data correlated well (R2 = 0.97) with the Digene hybrid capture assay. This assay provides an ideal system for therapeutic monitoring and for studying the relationship between HBV viral load and stage of disease.

DNA, Viral↗

PubMed2001

The vhs1 mutant form of herpes simplex virus virion host shutoff protein retains significant internal ribosome entry site-directed RNA cleavage activity.

The virion host shutoff (vhs) protein of herpes simplex virus (HSV) triggers global shutoff of host protein synthesis and accelerated turnover of host and viral mRNAs during HSV infection. As well, it induces endoribonucleolytic cleavage of RNA substrates when produced in a rabbit reticulocyte lysate (RRL) in vitro translation system. The vhs1 point mutation (Thr 214-->Ile) eliminates vhs function during virus infection and in transiently transfected mammalian cells and was therefore previously considered to abolish vhs activity. Here we demonstrate that the vhs1 mutant protein induces readily detectable endoribonuclease activity on RNA substrates bearing the internal ribosome entry site of encephalomyocarditis virus in the RRL assay system. These data document that the vhs1 mutation does not eliminate catalytic activity and raise the possibility that the vhs-dependent endoribonuclease employs more than one mode of substrate recognition.

Animals↗

PubMed1982

Isolation and comparative biochemical properties of the major internal polypeptides of equine infectious anemia virus.

We describe procedures for the large-scale production of equine infectious anemia virus (EIAV) and for the isolation of the four major non-glycosylated virion proteins, designated p26, p15, p11, and p9. Comparisons of the purified proteins by peptide mapping procedures and by enzyme-linked immunosorbent assays demonstrated the unrelatedness of the four proteins. The characteristic properties of each purified protein were examined by determining isoelectric points and amino acid compositions. We found that EIAV p26 and p9 focus at pI values of 6.2 and 5.0, respectively, and that these proteins contain no unusual amino acids. In contrast, EIAV p15 reproducibly displayed a heterogeneous isoelectric focusing pattern, with major pI values ranging from 5.7 to 8.3. This charge variation evidently correlated with different levels of phosphorylated serine or threonine or both, which could be detected by an amino acid analysis of purified p15. EIAV p11 apparently focused at a pI of greater than 10, reflecting its high content of basic amino acids. Moreover, localization experiments indicated that all four nonglycosylated proteins constitute the internal components of the virus, with all of the virion p11 closely associated with the viral RNA genome. Thus, our results demonstrated that EIAV, a lentivirus, contains structural polypeptides which are analogous to the structural polypeptides described previously in prototype C oncoviruses.

Amino Acids↗

PubMed2004

Hepatitis C virus targets DC-SIGN and L-SIGN to escape lysosomal degradation.

Hepatitis C virus (HCV) is a major health problem. However, the mechanism of hepatocyte infection is largely unknown. We demonstrate that the dendritic cell (DC)-specific C-type lectin DC-SIGN and its liver-expressed homologue L-SIGN/DC-SIGNR are important receptors for HCV envelope glycoproteins E1 and E2. Mutagenesis analyses demonstrated that both HCV E1 and E2 bind the same binding site on DC-SIGN as the pathogens human immunodeficiency virus type 1 (HIV-1) and mycobacteria, which is distinct from the cellular ligand ICAM-3. HCV virus-like particles are efficiently captured and internalized by DCs through binding of DC-SIGN. Antibodies against DC-SIGN specifically block HCV capture by both immature and mature DCs, demonstrating that DC-SIGN is the major receptor on DCs. Interestingly, internalized HCV virus-like particles were targeted to nonlysosomal compartments within immature DCs, where they are protected from lysosomal degradation in a manner similar to that demonstrated for HIV-1. Lewis X antigen, another ligand of DC-SIGN, was internalized to lysosomes, demonstrating that the internalization pathway of DC-SIGN-captured ligands may depend on the structure of the ligand. Our results suggest that HCV may target DC-SIGN to "hide" within DCs and facilitate viral dissemination. L-SIGN, expressed by THP-1 cells, internalized HCV particles into similar nonlysosomal compartments, suggesting that L-SIGN on liver sinusoidal endothelial cells may capture HCV from blood and transmit it to hepatocytes, the primary target for HCV. We therefore conclude that both DCs and liver sinusoidal endothelial cells may act as reservoirs for HCV and that the C-type lectins DC-SIGN and L-SIGN, as important HCV receptors, may represent a molecular target for clinical intervention in HCV infection.

Cell Adhesion Molecules↗

PubMed2003

H9N2 influenza viruses prevalent in poultry in China are phylogenetically distinct from A/quail/Hong Kong/G1/97 presumed to be the donor of the internal protein genes of the H5N1 Hong Kong/97 virus.

Ten H9N2 influenza virus strains isolated from diseased chickens in different farms in China during 1995 to 1999 were antigenically and genetically characterized. The haemagglutinins of the isolates were not related to those of A/quail/Hong Kong/G1/97 (H9N2) (Qa/HK/G1/97), but were closely related to that of A/chicken/Hong Kong/G9/97 (H9N2) (Ck/HK/G9/97). The neuraminidase of these isolates had a deletion of three amino acid residues at positions 63 to 65 as compared with those of Ck/HK/G9/97, while that of Qa/HK/G1/97 lacked two amino acids at positions 38 and 39. The PB2 genes of the isolates were not related to those of Qa/HK/G1/97 or Ck/HK/G9/97, but showed some relationship to that of A/duck/Hong Kong/Y439/97 (H9N2) (Dk/HK/Y439/97). The PB1 genes of the isolates were not related to those of the three representative strains. The PA, NP, M, and NS genes of the isolates belonged to the same lineage as those of Ck/HK/G9/97, and were distinct from those of Qa/HK/G1/97 and Dk/HK/Y439/97. The present results indicate that H9N2 influenza viruses prevalent in chicken populations in China belong genetically to one lineage and are distinct from Qa/HK/G1/97, presumed to be the donor of the internal protein genes of the highly pathogenic H5N1 influenza virus in Hong Kong in 1997.

Amino Acid Sequence↗

PubMedDate not supplied

[The ultrastructural changes in guinea pig organs during the serial passage of the Ebola virus].

Morphological study of internal organs of guinea pigs inoculated with Ebola virus at 2-8 passages was carried out. In the course of these passages the number of infected cells and virus particles in the organs was shown to increase, and the destructive changes in organs became more pronounced. In the 1st-3rd passages Ebola virus replication was observed in macrophagal cells only but beginning from the 4th passage of virus reproduction was found also in hepatocytes, spongiocytes, and fibroblasts.

Animals↗

PubMed2004

Interregional transmission of the internal protein genes of H2 influenza virus in migratory ducks from North America to Eurasia.

H2 influenza virus caused a pandemic in 1957 and has the possibility to cause outbreaks in the future. To assess the evolutionary characteristics of H2 influenza viruses isolated from migratory ducks that congregate in Hokkaido, Japan, on their flyway of migration from Siberia in 2001, we investigated the phylogenetic relationships among these viruses and avian and human viruses described previously. Phylogenetic analysis showed that the PB2 gene of Dk/Hokkaido/107/01 (H2N3) and the PA gene of Dk/Hokkaido/95/01 (H2N2) belonged to the American lineage of avian virus and that the other genes of the isolates belonged to the Eurasian lineage. These results indicate that the internal protein genes might be transmitted from American to Eurasian avian host. Thus, it is further confirmed that interregional transmission of influenza viruses occurred between the North American and Eurasian birds. The fact that reassortants could be generated in the migratory ducks between North American and Eurasian avian virus lineage further stresses the importance of global surveillance among the migratory ducks.

Animals↗

PubMed1992

Construction of a bifunctional mRNA in the mouse by using the internal ribosomal entry site of the encephalomyocarditis virus.

Picornaviral mRNAs have been shown to possess special structures in their 5' nontranslated regions (5'NTRs) that provide sites for internal binding of ribosomes and thus direct cap-independent translation. The translational cis-acting elements for ribosomal internal entry into the 5'NTR of encephalomyocarditis virus (EMCV), a member of family Picornaviridae, have been named the internal ribosomal entry site (IRES). All of the published experiments regarding the IRES function of the picornavirus 5'NTR, however, were performed with cell extracts in vitro or with tissue culture cells in transient assay systems. In this study, we examined the IRES function of the EMCV 5'NTR in chimeric mouse embryos and demonstrated that this element does in fact work stably in mouse embryos as well as in embryonic stem (ES) cells. By using a dicistronic vector, pWH8, consisting of a promoter-driven neomycin resistance gene (neo) followed by the EMCV 5'NTR-lacZ sequence, we showed that more than half of the ES cells made G418 resistant by the vector stained positive for beta-galactosidase (beta-gal). On Northern (RNA) blots, all of the clones analyzed revealed a transcript of the expected size containing both the beta-gal and the neo cistrons. These results indicate that dicistronic mRNAs are produced from the stably integrated vector in those ES clones and that both of the cistrons are translated to produce functional proteins. The chimeric embryos derived from these ES clones also stained positive for beta-gal, suggesting that the bifunctional mRNAs are active in the embryos. This dicistronic vector system provides a novel tool by which to obtain temporally and spatially coordinated expression of two different genes driven by a single promoter in a single cell in mice.

Animals↗

PubMed1998

Phylogenetic analysis of the entire genome of influenza A (H3N2) viruses from Japan: evidence for genetic reassortment of the six internal genes.

Nucleotide sequences of all eight RNA segments of 10 human H3N2 influenza viruses isolated during a 5-year period from 1993 to 1997 were determined and analyzed phylogenetically in order to define the evolutionary pathways of all genes in a parallel fashion. It was evident that the hemagglutinin and neuraminidase genes of these viruses evolved essentially in a single lineage and that amino acid changes accumulated sequentially with respect to time. In contrast, amino acid differences in the internal proteins were erratic and did not accumulate over time. Parallel analysis of the phylogenetic patterns of all genes revealed that the evolutionary pathways of the six internal genes were not linked to the surface glycoproteins. Genes coding for the basic polymerase-1, nucleoprotein, and matrix proteins of 1997 isolates were closest phylogenetically to those of earlier isolates of 1993 and 1994. Furthermore, all six internal genes of four viruses isolated in the 1995 epidemic season consistently divided into two distinct branch clusters, and two 1995 isolates contained PB2 genes apparently originating from those of viruses before 1993. It was apparent that the lack of correlation between the topologies of the phylogenetic trees of the genes coding for the surface glycoproteins and internal proteins was a reflection of genetic reassortment among human H3N2 viruses. This is the first evidence demonstrating the occurrence of genetic reassortment involving the internal genes of human H3N2 viruses. Furthermore, internal protein variability coincided with marked increases in the activity of H3N2 viruses in 1995 and 1997.

Amino Acid Sequence↗

PubMed2003

Multiplex RT-PCR detection of four aphid-borne strawberry viruses in Fragaria spp. in combination with a plant mRNA specific internal control.

The principal aphid-borne viruses infecting Strawberry (Fragaria spp.) Strawberry crinkle virus (SCV), Strawberry mild yellow edge virus (SMYEV), Strawberry mottle virus (SMoV) and Strawberry vein banding virus (SVBV) can cause serious crop losses. In this paper, a multiplex reverse transcriptase polymerase chain reaction (RT-PCR) method is described for the simultaneous detection of all four viruses in combination with a plant mRNA specific internal control which can be used as an indicator of the effectiveness of the extraction and RT-PCR. In total, 18 strawberry isolates infected naturally were analysed by this method. Every combination of RNA virus was able to be detected and a full complement of all four viruses were found together in three isolates, all taken from wild strawberry (Fragaria chiloensis (L.) Duch.) in Chile. The upper detection limit for the four viruses was at an extract dilution of 1/200. The broad applicability of the RNA specific internal control primers-which produced a PCR fragment of the expected size in 25 of 27 plant species tested-combined with improvements, made in extraction methods described provides potentially a standard method for comparable RT-PCR analyses in a wide variety of plant species.

Animals↗

PubMed2004

Structural and functional properties of an unusual internal fusion peptide in a nonenveloped virus membrane fusion protein.

The avian and Nelson Bay reoviruses are two of only a limited number of nonenveloped viruses capable of inducing cell-cell membrane fusion. These viruses encode the smallest known membrane fusion proteins (p10). We now show that a region of moderate hydrophobicity we call the hydrophobic patch (HP), present in the small N-terminal ectodomain of p10, shares the following characteristics with the fusion peptides of enveloped virus fusion proteins: (i) an abundance of glycine and alanine residues, (ii) a potential amphipathic secondary structure, (iii) membrane-seeking characteristics that correspond to the degree of hydrophobicity, and (iv) the ability to induce lipid mixing in a liposome fusion assay. The p10 HP is therefore predicted to provide a function in the mechanism of membrane fusion similar to those of the fusion peptides of enveloped virus fusion peptides, namely, association with and destabilization of opposing lipid bilayers. Mutational and biophysical analysis suggested that the internal fusion peptide of p10 lacks alpha-helical content and exists as a disulfide-stabilized loop structure. Similar kinked structures have been reported in the fusion peptides of several enveloped virus fusion proteins. The preservation of a predicted loop structure in the fusion peptide of this unusual nonenveloped virus membrane fusion protein supports an imperative role for a kinked fusion peptide motif in biological membrane fusion.

Amino Acid Sequence↗