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Volume 67,
Issue 10,
1986
Volume 67, Issue 10, 1986
- Review Article
- Animal
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Analysis of Foot-and-Mouth Disease Virus Type O1 Brugge Neutralization Epitopes Using Monoclonal Antibodies
More LessSummaryMonoclonal antibodies (MAbs) were elicited with inactivated, purified foot-and-mouth disease virus (FMDV) type O1 strain Brugge (140S) and with 12S protein subunits. Each MAb was tested for its capacity to bind to FMDV O1 Brugge 140S virions, 12S subunits and purified VP1 by radioimmunoassay (RIA) and to neutralize viral infectivity in mouse protection assays. Those MAbs which reacted only with 12S subunits in RIA did not neutralize infectious virus. One MAb, 12FE9.2.1, reacted with 140S, 12S and purified VP1 of FMDV O1 Brugge and neutralized infectious virus. Reactions with different biosynthetic FMDV type O1 Campos VP1 polypeptides localized the binding site of 12FE9.2.1 between amino acid residues 135 and 172. Eight MAbs reacted with both 140S virions and 12S subunits and neutralized infectious virus. Monoclonal antibodies that reacted only with 12S protein subunits or with 140S, 12S and VP1 did not compete in RIA with 140S/12S-reactive neutralizing MAbs for 12S binding sites. The ability of 140S/12S-reactive MAbs to compete for the 12S binding site defined by MAb 10GA4.2.2 was directly related to their capacity to neutralize infectious virus, suggesting that these MAbs were all affecting a single neutralization site. In mouse protection and plaque reduction neutralization assays, 10GA4.2.2 and 12FE9.2.1 were equally effective in neutralizing the homologous FMDV type O1 Brugge, and in cross-neutralization assays both exhibited high titres against four additional strains of type O1 FMDV.
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The Nucleotide Sequence of a Type 3 Poliovirus Isolated During a Recent Outbreak of Poliomyelitis in Finland
SummaryWe have cloned and sequenced the complete genome of a strain of poliovirus type 3 (23127) isolated during an outbreak of poliomyelitis in Finland. The genome is 7435 nucleotides long excluding the 3′ poly(A) stretch and is 95.5% homologous at the amino acid level to the previously sequenced type 3 strain, P3/Leon/37. The most striking feature of the presented sequence is the extent of amino acid substitutions relative to P3/Leon/37 and other type 3 strains in areas of known antigenic importance. The major antigenic determinant for virus neutralization (site 1), located at residues 89 to 100 of VP1, has three amino acid substitutions and there are six substitutions in site 3, a composite site made up of sequences from VP1 and VP3. The variation in these regions probably accounts for the observed unusual antigenicity and may explain why the virus was able to spread in a well-vaccinated community. Sequence comparisons imply that the virus is not derived from the currently used live attenuated vaccine.
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Live Lactate Dehydrogenase-elevating Virus (LDV) Induces Suppressor T Cells that Inhibit the Development of Delayed Hypersensitivity to LDV
T. Inada and C. A. MimsSummaryA strong delayed-type hypersensitivity (DTH) response to lactate dehydrogenase-elevating virus (LDV) in mice was induced by immunization with u.v.- or heat-inactivated virus by the intravenous, intraperitoneal and especially the subcutaneous route. The peak DTH response was seen 7 days after immunization. Live virus, in contrast, did not induce a DTH response in young (1- to 3-month-old) mice irrespective of inoculation route, except after pretreatment with cyclophosphamide (CY), when the response peaked at 14 days. Live virus, however, induced a significant DTH response in old mice (>8 months) without CY. The DTH response to inactivated virus was reduced when live virus was given intraperitoneally at the same time, but was partially restored when Ia-positive peritoneal cells were also given intravenously. The DTH response induced in infected mice pretreated with CY was suppressed by injection of spleen cells from infected or from normal 1- to 3-month-old mice. Suppression by normal spleen cells was abolished by treatment with anti-Thy1.2, anti-Lyt1 and anti-Lyt2 plus complement, whereas suppression by spleen cells from infected mice was prevented by anti-Thy1.2 and anti-Lyt2 plus complement. It is concluded that suppression of the DTH response was associated with induction of suppressor T cells and that severe depletion of Ia-positive cells may also have played a part.
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Polymorphonuclear Leukocyte Dysfunction Associated with Feline Leukaemia Virus Infection
More LessSummaryThe chemiluminescent characteristics of enriched (>95%) peripheral blood polymorphonuclear leukocyte populations (PMN) from normal and feline leukaemia virus (FeLV)-infected cats were investigated. FeLV-infected cats demonstrated a significantly lower (P < 0.001) PMN chemiluminescent response when compared to the response of normal age-matched controls. Normal PMN treated with FeLV-infected cat serum exhibited a depressed response in comparison to control cells. A titration of serum from infected cats supplemented with normal serum revealed a titratable suppression of chemiluminescence with increasing concentration of serum from the infected cats. However, PMN from FeLV-infected cats treated with normal serum displayed a slight increase in chemiluminescence over the same cells in autologous serum. The addition of inactivated FeLV to normal PMN caused a titratable decrease in chemiluminescence.
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Nuclear Accumulation of Measles Virus Nucleoprotein Associated with a Temperature-sensitive Mutant
More LessSummaryA measles virus (MV) Lec strain conditional-lethal (temperature-sensitive) mutant, designated MV ts38, has been isolated from 5-fluorouracil-mutagenized stock. The mutant has been characterized with regard to growth characteristics at 32 °C (permissive temperature) and 39 °C (non-permissive temperature). Virus-specific RNA transcription and/or translation appeared to be blocked at the non-permissive temperature as no virus-specific products could be detected by biochemical or immunological procedures. Following initiation of viral replication at 32 °C, with subsequent shift-up to 39 °C, presynthesized nucleoprotein (NP) was transported to, and accumulated in the cell nucleus whereas other viral proteins could not be detected there. A corresponding accumulation of NP in the nucleus is most often seen in association with MV neurotropic subacute sclerosing panencephalitis isolates in vivo and in vitro.
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Intranuclear Localization of Herpes Simplex Virus Immediate-Early and Delayed-Early Proteins: Evidence that ICP 4 Is Associated with Progeny Virus DNA
More LessSummaryThe localization of ICP 4, ICP 8, DNA polymerase and alkaline exonuclease within herpes simplex virus type 1 (HSV)-infected cells has been examined by immunofluorescence using specific antibodies to these proteins. Cells were simultaneously counterstained with the DNA-binding fluorochrome 4,6-diaminido-2-phenylindole (DAPI) to reveal the intranuclear distribution of DNA. These studies showed that in the absence of virus DNA replication ICP 4, ICP 8 and DNA polymerase were diffusely distributed throughout the nucleus but during virus DNA replication these proteins accumulated at specific foci within the nucleus. Initially these foci were near the nuclear membrane but with continuing virus DNA replication they increased in size until the whole of the nucleus became affected. The increase in size of these foci was coincident with a redistribution of nuclear DNA and margination of chromatin at the nuclear membrane, as revealed by DAPI staining. The number of foci initially present in an infected cell was dependent on the multiplicity of infection. The distribution of ICP 4, ICP 8 and DNA polymerase within the nucleus was altered by treating the cells with DNase. The majority of alkaline exonuclease was diffusely distributed throughout the nucleus during virus DNA replication and did not localize at specific foci within the nucleus. Autoradiographic examination of the incorporation of [3H]thymidine in cells infected with HSV showed that viral DNA replication occurred in restricted areas within the nucleus that were similar, in terms of number, location and size, to the foci where ICP 4, ICP 8 and DNA polymerase accumulated. Furthermore, in cells blocked in mitosis following infection with HSV, ICP 4, ICP 8 and DNA polymerase, but not alkaline exonuclease, localized in areas outside the condensed chromatin structures. DAPI staining revealed the presence of DNA in these areas and, as such structures were never seen when uninfected cells had entered mitosis, it is suggested that this extrachromosomal DNA is of viral origin. These studies therefore suggest that ICP 4 is associated with progeny virus DNA and that while its intranuclear localization is initially at non-viral sites, as DNA replication proceeds so ICP 4 is recruited into areas of virus DNA transcription and replication.
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Comparison of Restriction Site Polymorphisms among Clinical Isolates and Laboratory Strains of Human Cytomegalovirus
More LessSummaryWe have compared HindIII and EcoRI restriction sites in the long and short unique regions of the human cytomegalovirus (HCMV) genome among 20 low passage clinical isolates and four high passage laboratory strains (AD169, Davis, Towne, UW-1). This was done by hybridizing digested DNA on Southern blots with a series of subgenomic cloned fragments of AD169. Fourteen HindIII sites were conserved and three fragments (O, V, W) co-migrated among all strains. Nine HindIII sites found in AD169 were absent in one or more other strains. Eight additional HindIII sites were identified and three more hypothesized by the appearance of slightly smaller fragments. Sixteen EcoRI sites were conserved and six fragments (c, Y, S, W, B, R) co-migrated among all strains. Twelve EcoRI sites were absent or in altered locations and at least seven additional sites were identified in one or more strains. Although no two of these strains were identical throughout the genome, identical patterns of variation in a given region frequently occurred in multiple strains. Polymorphisms occurred throughout the entire genome, including the region specifying immediate early functions. All strains studied showed an identical fragment which hybridized to the transforming fragment of AD169. These restriction site polymorphisms may in the future serve as convenient markers for identification of functional variation among HCMV strains.
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Monoclonal Antibodies to Infectious Pancreatic Necrosis Virus: Analysis of Viral Epitopes and Comparison of Different Isolates
More LessSummaryA panel of five monoclonal antibodies (MAbs) produced against the West Buxton isolate of infectious pancreatic necrosis virus was used to investigate viral antigens and to compare different aquatic birnavirus isolates antigenically. Reciprocal blocking ELISA and neutralization assays indicated that these MAbs identified four, and possibly five, structurally and/or functionally different epitopes on the virion. Western immunoblot analysis demonstrated that one epitope was present on VP2, the large (51000 mol. wt., 51K) capsid protein, and another epitope was located on the two smallest structural proteins, VP3 (32K) and VP4 (30K). Three MAbs did not react with any of the solubilized viral proteins; the epitopes recognized by these MAbs may have been altered when the virion was solubilized with SDS. Comparison of reactivity patterns of the five MAbs with various aquatic birnaviruses in ELISA and neutralization tests demonstrated that 14 isolates tested from four serotypes represented a minimum of nine antigenically distinct viruses; i.e., distinct patterns of reactivity were shown among several viruses within the same serotype. Two MAbs identified different epitopes that were highly conserved among, and largely restricted to, members of the West Buxton (U.S.A.) serotype, whereas two other MAbs recognized an epitope(s) present only on some members of this serotype. The other MAb defined an epitope that was more widely distributed among the aquatic birnaviruses and found on all representatives tested from two serotypes.
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Glutamine Starvation of Murine Leukaemia Virus-infected Cells Inhibits the Readthrough of the gag-pol Genes and Proteolytic Processing of the gag Polyprotein
More LessSummaryThe production of Moloney murine leukaemia virus from chronically infected cells was inhibited after starvation of glutamine. While the rate of synthesis of the precursor of the core proteins, Pr65gag, was not affected in the starved cells, its proteolytic processing was blocked. Pulse-chase experiments indicated that glutamine was required during the synthesis of Pr65gag to facilitate its subsequent processing. In addition, the synthesis of Pr200gag-pol, the precursor of the protease, reverse transcriptase and endonuclease, was inhibited in the glutamine-starved cells. Starvation for other essential amino acids such as tyrosine and isoleucine affected neither the synthesis nor the processing of the virus proteins. These results suggest that the readthrough mechanism which enables synthesis of the Pr200gag-pol polyprotein is modulated in the chronically infected cells by glutamine levels. Since the viral protease is part of the pol gene, its synthesis may be inhibited in the glutamine-starved cells and Pr65gag is therefore not processed.
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Properties of Natural and Hybrid Murine Alpha Interferons
More LessSummaryFour natural murine interferon-α genes (MuIFN-α1, -α2, -α4 and -α6) and four hybrid genes (α1α4, α2α4, α4α1 and α4α2) were transiently expressed in monkey COS cells under the transcriptional control of the simian virus 40 early promoter. The proteins were labelled with [35S]methionine during a 16 h incubation and proteins secreted by the cells during this period were separated by polyacrylamide gel electrophoresis and subsequently visualized by fluorography. Under the conditions used, the IFNs represented 5 to 10% of the total amount of secreted proteins. All genes were found to encode biologically active IFN subspecies, including α4 which has a deletion of five amino acids. When the specific activities of the proteins were compared, it appeared that the specific antiviral activity of α4 on mouse cells was three- to sixfold higher than the activities of the other natural IFN subspecies. The specific activities of the hybrid proteins were similar to those of the natural proteins, except for the α2α4 hybrid which had a higher specific activity than the original proteins. The ability of the natural and hybrid subspecies to protect hamster cells against viral infection was determined using MuIFN-α1 as a standard. Large differences in activity were found, with α6 as the most and α4 as the least active subspecies.
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Independent Regulation of the Antiviral States Induced by MuIFN-α/β and by MuIFN-γ
More LessSummaryThe stability profiles of the antiviral states induced in L-929 cells against mengovirus by murine interferons MuIFN-α/β and by MuIFN-γ were shown to be different. Treatment of cells with combinations of IFN-α/β and IFN-γ have previously been shown to result in a potentiated expression of the antiviral state. Here we report studies of the stabilities of the antiviral states induced by various combinations of IFN-α/β and IFN-γ and that the regulation of the antiviral states induced by IFN-α/β and IFN-γ were independent of each other.
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Dissociation of the Haemagglutination Inhibition and the Infectivity Neutralization in the Reactions of Influenza A/USSR/90/77 (H1N1) Virus Variants with Monoclonal Antibodies
SummaryVariants of influenza A/USSR/90/77 (H1N1) virus selected with monoclonal antibody HC 142 (Res 142-1 and Res 142-2) were resistant to this antibody in a virus-neutralization (VN) test, but were inhibited in a haemagglutination-inhibition (HI) test. A variant selected with HC 22 monoclonal antibody (Res 22) was resistant to HC 22 in both VN and HI tests but, like the variants selected with HC 142, was resistant to HC 142 only in VN tests. A mouse-adapted variant of A/USSR/90/77, shown previously to be resistant to HC 22, reacted with HC 142 in a manner similar to that of Res 142-1, Res 142-2 and Res 22. Another monoclonal antibody, HC 125, behaved similarly to HC 142. The addition of anti-immunoglobulin serum restored the ability of HC 142 and HC 125 (already bound to the virus) to neutralize the infectivity of the resistant variants. Polyacrylamide gel electrophoresis revealed differences in the mobility of haemagglutinin among the variants.
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Reactions of Sera from Patients with Rheumatoid Arthritis, Systemic Lupus Erythematosus and Infectious Mononucleosis to Epstein-Barr Virus-induced Polypeptides
More LessSummaryP3HR-1 and Ramos cells induced with sodium butyrate and 12-O-tetradecanoyl-phorbol 13-acetate were used in the protein immunoblot technique to identify Epstein-Barr virus (EBV)-specific antibodies present in sera from clinically normal individuals and patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and infectious mononucleosis (IM). Sixteen EBV-specific polypeptides were detected ranging in mol. wt. from 22000 (22K) to 140K. Many of the sera contained antibodies to different subsets of these antigens, and a high proportion expressed autoantibodies which reacted with cellular components from an EBV genome-negative cell line. About 50% of the sera from each category reacted with the 44K to 48K and 36K and 38K early antigen (EA) components. A high proportion of the SLE sera (64%) were found to contain anti-EA antibodies, suggesting an association between EBV and SLE. Almost all of the EBV-seropositive sera examined contained antibodies against a 22K late antigen, but none of the sera from IM patients reacted with this polypeptide.
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Monoclonal Antibodies against the Glycoproteins of Mumps Virus: Fusion Inhibition by Anti-HN Monoclonal Antibody
More LessSummarySix monoclonal antibodies (MAbs) against the haemagglutinin-neuraminidase (HN) protein and three against the fusion (F) protein of mumps virus were obtained. Anti-HN MAbs were classified into three antigen-specific groups in competitive binding assays. All of the anti-HN MAbs showed virus-neutralizing and haemolysis-inhibiting activities and inhibited fusion from without (FFWO). Three of them inhibited fusion from within (FFWI) and the cell-to-cell spread of infection. The most effective MAb in inhibiting FFWI possessed neuraminidase-inhibiting activity and little haemagglutination-inhibiting activity. All anti-F MAbs inhibited virus-induced haemolysis but none of them inhibited FFWO, FFWI or the cell-to-cell spread of infection.
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Acyclovir Efficiently Inhibits Oropharyngeal Excretion of Epstein-Barr Virus in Patients with Acute Infectious Mononucleosis
More LessSummaryShedding of Epstein-Barr virus (EBV) into saliva was studied in 31 patients with verified acute infectious mononucleosis. The patients had been randomized for intravenous treatment with acyclovir (ACV) at 10 mg/kg body weight at 8 h intervals for 7 days, or placebo, in a double-blind trial. EBV in centrifuged throat washings was detected by transformation of umbilical cord lymphocytes and by immunofluorescence staining for EBV-associated nuclear antigen in fixed cell smears. Saliva samples were obtained before and during treatment, and after 4 weeks and 6 months, respectively. ACV effectively but transiently interrupted EBV production (P < 0.001), but virus shedding resumed at the initial level within 3 weeks of cessation of the treatment. Initially, 93.5% of the patients had detectable EBV in the saliva compared with 83% in the 4th week and 58% after 6 months.
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Chromosome Damage Induced by Herpes Simplex Virus Type 1 in Early Infection
More LessSummaryThe chromosome damage induced by herpes simplex virus type 1 (HSV-1) in vitro was examined up to 6 h after infection using HT-1080 cells. Initial damage occurring within 3 h was specific, involving uncoiling of chromosome 1q12-21 and to a lesser extent the pericentric regions of chromosomes 9, 16 and satellited chromosomes. For the initial unwinding, synthesis of the immediate early class of HSV proteins needed to occur as was demonstrated using HSV-1 temperature-sensitive mutants tsK and tsB7 and two viral inhibitors, β-propiolactone and psoralen plus long wavelength u.v. light. Later damage included chromatid breaks, acentric fragments and pulverization which did not take place until synthesis of delayed early proteins had begun.
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DNA Sequence of the Major Capsid Protein Gene of Herpes Simplex Virus Type 1
More LessSummaryThe DNA sequence of the region of the herpes simplex virus type 1 genome encoding the major capsid protein was determined. The predicted protein contains 1374 amino acid residues and has a molecular weight of 149075. Comparisons of the amino acid sequence of this protein with those predicted from the published DNA sequences of two other herpesviruses, varicella-zoster virus and Epstein-Barr virus, resulted in the identification of the major capsid protein gene in each genome.
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A Study of Rice Dwarf Virus in Vector Cell Monolayers by Fluorescent Antibody Focus Counting
More LessSummaryInfectivity assays of rice dwarf virus (RDV) were done by the fluorescent antibody focus counting technique on vector cell monolayers of the green rice leafhopper, Nephotettix cincticeps. The focus count method was shown to be an accurate and quantitative method for determining RDV infectivities. The optimal pH value for inoculation was about 6.0 in a solution containing 0.1 m-histidine HCl and 0.01 m-MgCl2. Below pH 5.5 and above 6.5, the infectivity of RDV dropped rapidly. The optimal adsorption period at 28 °C was dependent upon the RDV concentration. Optimal periods for adsorption with relative RDV concentrations of 10−3, 10−4 and 10−4.5 were about 60, 90 and 120 min, respectively. The period from virion adsorption to penetration into the cell was about 90 to 120 min. Infective progeny virions were first detected 12 h after the initial inoculation. From 12 to 20 h, the growth rate of the virus in the cells was exponential with a doubling time of about 96 min, and then from 20 to 28 h there was little or no further increase in infective virus. When the infectivities of the same inocula were compared by using the focus count and vector insect injection methods, the dilution endpoints were approx. 10−6 and 10−4, respectively. The focus count assay method was thus about 100 times more sensitive than vector injection.
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In vitro Translation of the Bipartite Genomic RNA of Pea Early Browning Virus
More LessSummaryTranslation of the two genomic RNAs of a British strain (SP5) of pea early browning virus (PEBV-B) in rabbit reticulocyte lysates produced three principal polypeptides of M r 165 × 103 (165K), 134K and 23K. The two largest polypeptides were produced by RNA-1 alone, and evidence is presented which suggests that the 165K polypeptide arises from the 134K product by a readthrough event. RNA-2 directed the translation of the 23K polypeptide, which was immunoprecipitated by anti-PEBV-B serum. A polypeptide of M r 24.3K, produced by translation of the genomic RNAs of the closely related broad bean yellow band virus, was also immunoprecipitated by anti-PEBV-B serum, confirming the serological relationship between the two viruses.
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