Cell biology of virus entry: a review of the 4th International Frederick Meeting.
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Animals↗
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Predicted secondary structure of the hepatitis G virus and GB virus-A 5'untranslated regions consistent with an internal ribosome entry site.
We describe comparative sequence analysis of 20 isolates of the recently discovered hepatitis G virus (HGV) and propose a model of the RNA secondary structure at the 3' end of the 5' untranslated region (UTR) of this virus. A single AUG starting at nucleotide position 552, which was in-frame and continuous with the putative polyprotein, was conserved in all 20 isolates and appeared to be the most likely site for the initiation of polyprotein synthesis. This consensus AUG was 14 amino acid residues upstream of a sequence with identity to the envelope protein E1 of hepatitis C virus (HCV), but the actual function of this N-terminal peptide of HGV is still not certain. None of the isolates encoded a sequence with similarity to the nucleocapsid protein of any known virus. The RNA secondary structure of the fragment under study was determined using thermodynamic modelling and validated using the results of comparative sequence analysis. Further support for the model was gained from the prediction of significant sequence and structural homology in the RNA secondary structure of the complete 5'-UTR of GB virus-A (GBV-A). A possible mechanism for translation initiation in HGV and GBV-A was suggested by the identification of features in common with the internal ribosome entry sites (IRESs) of HCV and picornaviruses.
Amino Acid Sequence↗
Molecular evolution of H6 influenza viruses from poultry in Southeastern China: prevalence of H6N1 influenza viruses possessing seven A/Hong Kong/156/97 (H5N1)-like genes in poultry.
The A/teal/Hong Kong/W312/97 (H6N1) influenza virus and the human H5N1 and H9N2 influenza viruses possess similar genes encoding internal proteins, suggesting that H6N1 viruses could become novel human pathogens. The molecular epidemiology and evolution of H6 influenza viruses were characterized by antigenic and genetic analyses of 29 H6 influenza viruses isolated from 1975 to 1981 and 1997 to 2000. Two distinct groups were identified on the basis of their antigenic characteristics. Phylogenetic analysis revealed that all H6N1 viruses isolated from terrestrial poultry in 1999 and 2000 are closely related to A/teal/Hong Kong/W312/97 (H6N1), and the nucleotide sequences of these viruses and of A/Hong Kong/156/97 (H5N1) were more than 96% homologous. The hemagglutinin (HA) of the 1999 and 2000 terrestrial viruses does not have multiple basic amino acids at the site of cleavage of HA1 to HA2; however, a unique insertion of aspartic acid in HA1 between positions 144 and 145 (H3 numbering) was found. The neuraminidase of these terrestrial H6N1 viruses has a deletion of 19 amino acids characteristic of A/Hong Kong/156/97 (H5N1). Evolutionary analysis suggested that these H6N1 viruses coevolved with A/quail/Hong Kong/G1/97-like H9N2 viruses and became more adapted to terrestrial poultry. These terrestrial 1999 and 2000 A/teal/Hong Kong/W312/97 (H6N1)-like viruses, along with the H9N2 viruses, could have been involved in the genesis of the pathogenic H5N1 influenza viruses of 1997. The presence of H6N1 viruses in poultry markets in Hong Kong that possess seven of the eight genes of the A/Hong Kong/156/97 (H5N1) virus raises the following fundamental questions relevant to influenza pandemic preparedness: could the pathogenic H5N1 virus reemerge and could the H6N1 viruses directly cross the species barrier to mammals?
Animals↗
A murine gammaherpesvirus.
In 1976, within a project on isolation of herpesviruses from small rodents in former Czechoslovakia, the mouse herpesvirus strain 68 (MHV-68) was isolated (Blaskovic et al., 1980). This virus was accepted by The International Committee on Taxonomy of Viruses (ICTV) as a new, so far unassigned species (member) of the Gammaherpesvirinae subfamily of the Herpesviridae family (Murphy et al., 1995). Besides MHV-68, four more isolates (MHV-60, MHV-72, MHV-76, and MHV-78) similar to MHV-68 were obtained in that field experiment in Slovakia. Later, three more isolates (MHV-Sumava from Bohemia and MHV-4556 and MHV-5682 from Slovakia) were obtained in other field experiments. All these isolates are in some properties similar but in others different from each other. Nevertheless, as their comparative genome sequence analysis is not yet available, we propose at present to regard all the abovementioned isolates as different isolates of the same virus and strain, i.e. MHV-68. It is not excluded that a more detailed characterization of these isolates in the future will lead to proposals of designating some of these isolates as new strains of the virus of concern. This review summarizes the up to date knowledge of various biological and physico-chemical properties of MHV-68. At least three isolates, MHV-68, MHV-72 and MHV-Sumava seem to be involved in malignant neoplasm development in mice. It should be stressed that the pathogenesis of the induced lymphoproliferative disease in mice is similar to that caused by Epstein-Barr virus (EBV) in humans.
Animals↗
Terminal and internal inverted repetitions in African swine fever virus DNA.
An electron microscopic analysis of the heteroduplexes formed by reannealing denatured terminal restriction fragments of African swine fever (ASF) virus DNA showed Y-shaped molecules with a 2.1-kilobase-pair-long double-stranded tail and two single-stranded arms. This indicated that ASF virus DNA has terminal inverted repetitions with a length of 2.1 kbp. In addition, under less restrictive hybridization conditions, most of the heteroduplexes showed a 0.13 kbp-long internal double-stranded region, separated from the long terminal repeat by a single-stranded asymmetric loop. These internal inverted repetitions did not match well, since the heteroduplexes melted under conditions where those of the terminal repetitions were stable. In the terminal fragments EcoRI-K' and D', the distance between the terminal and the internal inverted repetitions was 2.4 and 0.4 kbp, respectively.
African Swine Fever Virus↗
Conformational changes in the VP1-unique region of native human parvovirus B19 lead to exposure of internal sequences that play a role in virus neutralization and infectivity.
The unique region of the capsid protein VP1 (VP1u) of human parvovirus B19 (B19) elicits a dominant immune response and has a phospholipase A(2) (PLA(2)) activity, which is necessary for the infection. In contrast to the rest of the parvoviruses, the VP1u of B19 is thought to occupy an external position in the virion, making this region a promising candidate for vaccine development. By using a monoclonal antibody against the most-N-terminal portion of VP1u, we revealed that this region rich in neutralizing epitopes is not accessible in native capsids. However, exposure of capsids to increasing temperatures or low pH led to its progressive accessibility without particle disassembly. Although unable to bind free virus or to block virus attachment to the cell, the anti-VP1u antibody was neutralizing, suggesting that the exposure of the epitope and the subsequent virus neutralization occur only after receptor attachment. The measurement of the VP1u-associated PLA(2) activity of B19 capsids revealed that this region is also internal but becomes exposed in heat- and in low-pH-treated particles. In sharp contrast to native virions, the VP1u of baculovirus-derived B19 capsids was readily accessible in the absence of any treatment. These results indicate that stretches of VP1u of native B19 capsids harboring neutralizing epitopes and essential functional motifs are not external to the capsid. However, a conformational change renders these regions accessible and triggers the PLA(2) potential of the virus. The results also emphasize major differences in the VP1u conformation between natural and recombinant particles.
Antibodies, Monoclonal↗
Development of real-time detection direct test for hepatitis B virus and comparison with two commercial tests using the WHO international standard.
AIMS: A highly reproducible and sensitive hepatitis B virus real-time detection direct (HBV RTD-direct) test using DNA extraction by magnetic beads coated with polyclonal anti-HBsAg, followed by the real-time detection polymerase chain reaction (PCR) method, was developed for the detection of HBV DNA. METHODS: The HBV DNA could be extracted from the HBsAg positive viral particles without resulting in viral DNA fragmentation. The HBV RTD-direct test was validated using a serial dilution panel of the WHO standard HBV DNA 97/746 I. RESULTS: The test had a dynamic range of 0.7-8.0 log10 international units (IU) per mL and the results were shown to be comparable to those obtained with two commercially available tests: the HBV DNA transcription-mediated amplification-hybridization protection assay and the Amplicor HBV Monitor test. In addition, the HBV RTD-direct test, based on magnetic extraction, successfully eliminated PCR inhibitors in clinical specimens. CONCLUSION: We conclude that the HBV RTD-direct test is an excellent alternative for monitoring patients undergoing antiviral treatment or for screening various clinical specimens.
DNA, Viral↗
A tyrosine-based motif in the cytoplasmic tail of pseudorabies virus glycoprotein B is important for both antibody-induced internalization of viral glycoproteins and efficient cell-to-cell spread.
Pseudorabies virus (PRV), a swine alphaherpesvirus, is capable of causing viremia in vaccinated animals. Two mechanisms that may help PRV avoid recognition by the host immune system during this viremia are direct cell-to-cell spread in tissue and antibody-induced internalization of viral cell surface glycoproteins in PRV-infected blood monocytes, the carrier cells of the virus in the blood. PRV glycoprotein B (gB) is crucial during both processes. Here we show that mutating a tyrosine residue located in a YXXPhi motif in the gB cytoplasmic tail results in decreased efficiency of cell-to-cell spread and a strong reduction in antibody-induced internalization of viral cell surface glycoproteins. Mutating the dileucine motif in the gB tail led to an increased cell-to-cell spread of the virus and the formation of large syncytia.
Amino Acid Motifs↗
Combinations of ganciclovir and antibody for experimental CMV infections.
The effect of combined treatment with ganciclovir and hyper immune serum (HIS) was evaluated in three animal models. It concerned a generalized CMV infection model, a meningo-encephalitis model and an interstitial lung disease (ILD) model in immunocompromised rats. In the generalized model, the ganciclovir and HIS had a moderate synergistic effect on survival and greatly decreased virus titers in internal organs. In contrast, in the meningoencephalitis model, combined treatment had no effect on the local virus titers and the histopathology. Combined treatment with ganciclovir and HIS, however, effectively abolished CMV-induced ILD.
Animals↗
An unusual internal ribosome entry site in the herpes simplex virus thymidine kinase gene.
We have investigated a herpes simplex virus mutant that expresses low levels of thymidine kinase (TK), a phenotype associated with drug resistance and pathogenicity, despite a single-base deletion in the gene. Using a dual-reporter system, a 39-nt sequence including the mutation was shown to direct expression of the downstream reporter gene in reticulocyte lysate. Translation of the downstream reporter was not impaired when the mRNA lacked a 5' cap or had a stable stem loop 5' of the upstream reporter and was relatively resistant to edeine, an antibiotic that prevents AUG codon recognition by the 40S-eIF2-GTP/Met-tRNAi complex. Twelve nucleotides were as active as the original sequence for translation of the downstream reporter. Surprisingly, this sequence lacks an AUG codon. Analysis of point mutations showed that a CUG codon in the sequence was important. However, many single-base changes had only limited effects, and introduction of AUG codons did not increase translation. A mutant virus containing both the single-base deletion and a mutation that reduced downstream translation in vitro had significantly less TK activity than a virus with the single-base deletion alone. Thus, a remarkably short internal ribosome entry site (IRES) that lacks an AUG codon resides in the viral tk gene. The IRES appears to be responsible for TK expression from a drug-resistant mutant that would otherwise express no TK, which may contribute to pathogenicity. Because we found numerous short sequences with IRES activity, there might be many hitherto unrecognized polypeptides expressed at low levels from eukaryotic mRNAs.
Animals↗
Radioimmunoassay of type D oncovirus from continuous J-96 cells.
Radioimmunoassay of J-96 virus and an extract of J-96 cells in the homologous and heterologous systems aimed at detecting antigenic determinants of p25 of Mason-Pfizer virus, as well as group-specific and interspecies antigenic determinants p30 of Rauscher leukaemia virus demonstrated that (1) J-96 virus contains a major internal protein immunologically identical with p25 protein of Mason-Pfizer virus based on the antigenic determinants detectable by the radioimmunoassay used; and (2) no interspecies antigenic determinants characteristic of the major internal protein of mammalian type C viruses were detectable in the J-96 virus or the J-96 cell extract.
Cell Line↗
Present status of classification of viruses of vertebrates.
The VIth Report of the International Committee on Taxonomy of Viruses (ICTV) was published in 1995. We have briefly characterized its contents and discussed the most important changes that have been made in the classification of viruses of vertebrates. The present line up of families and genera of viruses of vertebrates, and subviral agents and unassigned viruses is also provided. We propose an allocation of families of viruses of vertebrates according to a conjectural evolutional connection between the type and class of genomic nucleic acid. Finally the directions and intentions of the ICTV have been reviewed from the Virology Division News in Archives of Virology (1994/1995). These will be considered in the VIIth Report of the ICTV scheduled for publication after the XIth International Congress of Virology in Sydney in 1999.
Animals↗
A thymidine kinase-negative HSV-1 strain establishes a persistent infection in SCID mice that features uncontrolled peripheral replication but only marginal nervous system involvement.
A detailed knowledge of the pathogenesis of infections caused by thymidine-kinase (TK)-deficient herpes simplex virus type 1 (HSV-1) strains is important because such mutants can arise during treatment of HSV infections with acyclovir--especially in immunocompromised patients--and also because TK-negative mutants may become useful for the therapy of intracranial tumors. In this work, we studied the pathogenesis of a genetically engineered TK-negative HSV-1 strain dlsptk, in SCID mice (mice with severe combined immunodeficiency) after corneal infection. We found that dlsptk established a persistent infection that kills SCID mice within 80.2 +/- 21.3 days. The cause of death seemed to be related to uncontrolled viral replication in the superficial and deep facial tissues of the animals. Viremia probably did not occur, as judged by the inability to detect infectious virus and viral gene expression in various internal organs. However, the virus did reach the nervous system, most probably by axonal transport from the primary site of the infection. Virus-specific DNA reached low but detectable levels in the trigeminal ganglia and the brainstems by 7 days p.i. and remained at low levels for up to 50 days p.i. as determined by spot blot analysis. By in situ hybridization and immunostaining we determined that, in some of the neurons of the trigeminal ganglia infected by the virus, viral latency was established. However, our results suggested that in other infected neurons viral replication occurred and virus spread to surrounding nonneuronal cells and to the central nervous system. This work provides a new model in which the pathogenesis of infections caused by TK-deficient HSV strains in immunocompromised hosts can be effectively studied and which may also help to identify the potential side effects of the therapy of intracranial tumors with TK-negative HSV strains.
Animals↗
Cell biology of virus entry: a review of selected topics from the 3rd International Frederick meeting.
Following the first two Frederick meetings on virus entry in 1997 [Cell 91 (1997) 721](1) and in 2000 [Cell 101 (2000) 697](2), further developments in our understanding of the multifactorial and multistage process of virus entry, and possible biomedical implications were presented and discussed in a lively fashion by leading scientists from around the world at the third Frederick meeting on the Cell Biology of Viral Entry (May 7-10, Frederick, MD) organized by R. Blumenthal (NCI-Frederick, NIH, Frederick) and E. Hunter (University of Alabama, Birmingham). Unlike the previous two meetings, non-enveloped viruses were not discussed this time, and the focus was how envelope glycoproteins (Envs) mediate entry into cells. Major topics included Env structure, virus receptors, entry intermediates, membrane fusion, fusion kinetics, and rafts. Virus envelope structures will be described in more detail here because the other topics are extensively discussed in the other chapters of this volume.
Biological Transport↗
Intracellular forms of pox viruses as shown by the electron microscope (Vaccinia, Ectromelia, Molluscum Contagiosum).
The intracellular development of three pox viruses has been studied with the electron microscope using thin sections of infected tissue. Cells infected with vaccinia, ectromelia, and molluscum contagiosum viruses all form developmental bodies preliminary to the production of mature virus. Developmental bodies, believed to be virus precursors, are round to oval, slightly larger than mature virus particles, less dense to electrons, and have a more varied morphology. It is suggested as a working hypothesis that the process of maturation of a virus particle takes place as follows. In the earliest form the developmental bodies appear as hollow spheres, imbedded in a very dense cytoplasmic mass constituting an inclusion body, or in a less dense matrix near the nucleus in cells without typical inclusion bodies. The spheres become filled with a homogeneous material of low electron density. A small, dense granule appears in each developmental body and grows in size at the expense of the low density material. Following growth of the granule, particles are found with the dimensions of mature virus and having complex internal structure resembling bars or dumbells. Mature virus is ovoid and very dense to electrons. An "empty" interior may be found within its thick walls.
Cytoplasm↗
Pathogenesis of simian foamy virus infection in natural and experimental hosts.
Inoculation of simian foamy virus type 1 into New Zealand white rabits resulted in an infection which was very similar to that observed in naturally infected nonhuman primates. Both intraperitoneal and intranasal inculations were found to be efficient procedures for the establishment of the infection in rabbits. Infection by the nasal route was found to be the best method, whereas no infection could be established by feeding virus in the drinking water. Once infection was established, virus persisted in the tissues and organs for as long as 264 days after inoculation, during which time the animals maintained significant levels of neutralizing antibody. Infectious virus was recovered from spleen, liver, lung, salivary gland, kidney, and, to a lesser extent, the brain. Virus was isolated from the blood only during early infection and never from the urine. A comparison of the distribution of foamy virus in naturally infected monkeys and baboon with experimentally infected rabbits showed that both groups harbored infectious virus in the same internal tissues and organs. Recovery of infectious virus from both groups of animals was accomplished by cultivation and/or co-cultivation of infected cells onto Vero cells.
Administration, Intranasal↗
Inhibition of Epstein-Barr virus-mediated capping of CD21/CR2 by alpha interferon (IFN-alpha): immediate antiviral activity of IFN-alpha during the early phase of infection.
Early events of human B-lymphocyte infection by Epstein-Barr virus involve the virus binding to CD21, capping, and subsequent internalization of the virus-receptor complex. We show here that alpha interferon (IFN-alpha) inhibits the capping of Epstein-Barr virus-CD21 complexes. Synthetic peptides with the CD21 binding motif of IFN-alpha mimic IFN-alpha activity, suggesting that this effect may be mediated by IFN-alpha-CD21 interaction. Our findings demonstrate a novel and immediate mechanism of IFN-alpha action.
B-Lymphocytes↗
An update on avian influenza in Hong Kong 2002.
An outbreak of highly pathogenic avian influenza caused by multiple genotypes of H5N1 virus occurred in Hong Kong, commencing in January 2002. Infection in local chicken farms was preceded by the detection of virus in multiple retail markets and the main poultry wholesale market. The first case of this disease on a local farm was detected on February 1, 2002. By February 9, 2002, 15 farms were infected, and by late March a total of 22 infected farms had been identified. Three main clusters of infected farms were seen, suggesting multiple incursions of virus, and subsequent limited lateral spread to neighboring firms. Control of this disease has been effected through a combination of quarantine, tightening of biosecurity measures, and depopulation of infected and contact farms. About 950,000 birds have been destroyed. Vaccination using a killed H5 vaccine was introduced in April 2002 to farms in one zone where infection has persisted. None of the viruses isolated contained the internal genes found in the 1997 H5N1 virus.
Animals↗