Electronic Library of Scientific Literature Electronic Library of Scientific Literature
Volume 43 / August 1999 / number 4
G. RATISH, S. VISWANATHAN, V.V.S. SURYANARAYANA
Indian Veterinary Research Institute, Hebbal, Bangalore 560 024, India
Summary. – Foot-and-mouth disease (FMD), one of the most contagious and economically important diseases of farm animals, is caused by a FMD virus (FMDV) which belongs to the family of Picornaviridae. The virus occurs as seven serotypes of which four (A, O, C and Asia 1) are prevalent in India. Immuno-prophylaxis supported by precise diagnosis is the prime requirement for achieving the success in controlling the disease. Recently, recombinant DNA technology is gaining importance for the production of cost-effective and safer diagnostics and immunogens. Based on this approach, cDNA of a part of gene for major variable immunogenic region, VP1, of FMDV of four serotypes (A22, O, C and Asia 1) was amplified by PCR and cloned into expression vector. The expression of the 16°K protein gene from the clones was induced with IPTG and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) and [35S]-methionine labeling. The immunoreactivity of the labeled proteins was assayed by immunoprecipitation with anti-FMDV type-specific sera. Since the proteins contain 6 His residues at the N-terminal end, their affinity purification was carried out using nickel nitrilo-tri-acetic acid (Ni-NTA) agarose matrix. The proteins were found to be immunoreactive and the useful in the FMD diagnosis.
Key words: PCR; diagnostics; FMDV; expressed protein; affinity purification
Acta virologica 43: 205 – 211, 1999
C. CHANYASANHA, F. HASEBE, R. MATIAS, A. IGARASHI
Department of Microbiology, Faculty of Public Health, Mahidol University, Bangkok,
Thailand;
Department of Virology, Institute of Tropical Medicine, Nagasaki University, Japan;
Institute of Biology, College of Science, University of the Philippines, Manila, Republic
of the Philippines;
Department of Molecular Epidemiology, Institute of Tropical Medicine, Nagasaki University,
Nagasaki, Japan
Summary. – Viral antigen production was examined in the culture fluid of Aedes albopictus clone C6/36 cell line incubated at 28°C and 37°C after infection with four strains of dengue type 4 (DEN-4) virus which were isolated from patients with different clinical severities. During the observation period from day 1 to day 18, the number of infected cells at each day for all four strains did not show any significant difference (P >0.05). Antigen production as determined by the hemagglutination (HA) test and sandwich enzyme linked immunosorbent assay (ELISA) was higher at 28°C than at 37°C for all four DEN-4 virus strains examined. The amount of viral antigen produced was highest for CT93-74 strain (dengue hemorrhagic fever syndrome (DHF) grade II) and was significantly different in comparison to other strains (P <0.001). This strain was followed by CT93-158 and CT93-129 strains (both of DHF grade I), and CT93-77 strain (dengue fever (DF)). The viral antigen production was apparently proportional to the clinical severity of the patient from whom the virus was isolated. These results show that CT93-74 strain could be used to produce DEN-4 virus antigen of sufficiently high titer in the C6/36 cell culture instead of classical extraction of this antigen from suckling mouse brains.
Key words: dengue type 4 virus; antigen; cell culture; incubation temperature;
HA test; sandwich ELISA
Acta virologica 43: 213 – 218, 1999
C. TOSH, R. VENKATARAMANAN, B. PATTNAIK, D. HEMADRI, A. SANYAL
Central Laboratory, All India Coordinated Research Project on Foot-and-Mouth Disease, Indian Veterinary Research Institute, Mukteswar-Kumaon, Nainital 263 138, India
Summary. – A set of five neutralizing monoclonal antibodies (MAbs) to an Indian strain (IND17/77) of type A (subtype A22) foot-and-mouth disease (FMD) virus (FMDV) was used in the study. Four of the MAbs (27S, 37S, 85S, and 143S) identified a trypsin-sensitive (TS) epitope(s) and were specific for VP1, while the remaining MAb (145S) reacted with a trypsin-resistant (TR) epitope and was specific for VP3 in Western blot analysis. Both the epitopes (TS and TR) were conformation-independent in nature. Results obtained in MAb-competition enzyme-linked immunosorbent assay (ELISA), and profiling of the (MAb) neutralization-escape mutants in ELISA and cross-neutralization test revealed two overlapping TS epitopes (27S/37S and 85S/143S) on the virus. Variation at both these epitopes was observed in some field isolates of serotype A. Comparison of deduced amino acid sequence in the VP1 region (aa 140-213) between the parent virus and the mutants identified Gly148 and Arg153 as critical for the formation of both the TS epitopes. Substitution of R153 by Gly or Ser was observed in mutants with no reactivity for the MAbs 85S/143S. However, these mutants maintained partial reactivity with MAbs 27S/37S, and substitution of Gly148 by Glu eliminated both the epitopes. No amino acid substitution was observed in the VP1 region of aa 200-213. Efficient neutralization of the MAb neutralization escape mutants (MAb-resistant (MAR) mutants) by bovine vaccinate serum (BVS) indicated involvement of other epitopes on the virion surface in eliciting neutralizing antibodies following vaccination.
Key words: FMDV; serotype A; neutralization-escape mutant; trypsin-sensitive
epitope; trypsin-resistant epitope; 1D gene
Acta virologica 43: 219 – 225, 1999
V. REPKA, I. FISCHEROVÁ
Laboratory of Molecular Biology and Virology, Research Institute of Viticulture and Enology, Matúškova 25, 833 11 Bratislava, Slovak Republic
Summary. – Inoculation of cucumber (Cucumis sativus L. cv. Laura) cotyledons with tobacco necrosis virus (TNV) causes both qualitative and quantitative changes in the total and fractionated protein extracts as well as in amylolytic activity. Using a specific test it was demonstrated that the virus infection strongly enhances a major band (Rf 0.0645) of amylolytic activity, predominantly located in apoplast space. The accumulation of this extracellular amylolytic activity is regulated by a time-dependent manner and is correlated with the development of necrotic lessions. The amylolytic activity may be related to degradation of starch shown to be accumulated in the immediate vicinity of necrotic lesions associated with the hypersensitive response (HR). The possible biological function of the identified amylolytic activity in the term of “pathosmosis” is also discussed.
Key words: cucumber; tobacco necrosis virus; starch; electrophoresis;
immunoblot analysis
Acta virologica 43: 227 – 235, 1999
Y.A. SMIRNOV, A.S. LIPATOV, A.K. GITELMAN, Y. OKUNO, R. VAN BEEK, A.D.M.E. OSTERHAUS, E.C.J. CLAAS
The D.I. Ivanovsky Institute of Virology, Russian Academy of Medical Sciences, Gamaleya
16, 123098 Moscow, Russia;
Division of Virology, Osaka Prefectural Institute of Public Health, Osaka, Japan;
Department of Virology and WHO National Influenza Center, Erasmus University Rotterdam,
Rotterdam, The Netherlands;
Department of Virology, CKVL, Leiden University Medical Center, Leiden, The Netherlands
Summary. – The membrane-inserted hemagglutinin (HA) is the most variable protein of influenza viruses. Here we describe the characterization of a shared epitope in the HA of influenza A virus H1, H2, and H5 subtypes which were completely neutralized by a monoclonal antibody (MAb), directed against this epitope. This MAb (C179) also efficiently precipitated the HAs of these viruses. In addition, MAb C179 did not neutralize H6 subtype strains despite complete amino acid homology of the epitope regions. Furthermore, only the non-glycosylated form of the HA of one of the H6 subtype strains could be precipitated by the MAb. The conformational epitope may be masked by glycosylation, although it could not be excluded that differences in the primary amino acid sequence may cause the decreased accessibility of the epitope in H6 subtype strains.
Acta virologica 43: 237 – 244, 1999
R.S. KATARIA, A.K. TIWARI, G. BUTCHAIAH, J.M. KATARIA
National Biotechnology Center;
Division of Avian Diseases, Indian Veterinary Research Institute, Izatnagar, Bareilly, UP
243 122, India
Summary. – The techniques of reverse transcription-polymerase chain reaction (RT-PCR) and restriction analysis were used to differentiate highly virulent Indian field isolates of infectious bursal disease virus (IBDV) from vaccine strains. Primers were designed to amplify the variable region of VP2 gene coding for major virus neutralizing epitopes. The 552 bp PCR products generated from four vaccine strains and five field isolates were digested with restriction enzymes DraI, HhaI, MvaI, StuI, StyI, and TaqI, which could differentiate field isolates from vaccine strains. Based on restriction enzyme profiles derived from published sequences, Indian field isolates seem to be closely related to highly virulent Japanese, European, and Chinese strains of the virus.
Keywords: infectious bursal disease virus; VP2 gene; RT-PCR; restriction
analysis
Acta virologica 43: 245 – 249, 1999
P. KOCÁKOVÁ, V. HAJNICKÁ, M. SLOVÁK, P. A. NUTTALL, N. FUCHSBERGER
Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 842 46
Bratislava, Slovak Republic;
Institute of Preventive and Clinical Medicine, Bratislava;
Institute of Zoology, Slovak Academy of Sciences, Bratislava;
Institute of Virology and Environmental Microbiology, Oxford, United Kingdom
Summary. – In a previous study (Hajnicka, V. et al., Parasitology 116, 533–538, 1998), the infectivity titer of vesicular stomatitis virus (VSV) was shown to increase up to 10,000-fold when mouse L cells were treated with tick salivary gland extract (SGE) prior to infection. To examine this effect at the level of viral protein production, radiolabeled VSV-infected cells were analyzed by double-dimensional gel electrophoresis. A pre-treatment of cells with SGE from partially fed ticks in amounts corresponding to 1 or 3 salivary glands increased the level of both viral nucleocapsid (N) protein and phosphoprotein (P) in a dose-dependent manner. The effect was more pronounced for N protein and could account for the dramatic increase in infectious virus yield. Promotion of viral infectivity by arthropod saliva may support the arthropode-borne transmission cycle of VSV.
Key words: VSV; nucleocapsid protein; saliva-activated transmission
Acta virologica 43: 251 – 254, 1999
Z. ŠUBR, J. MATISOVÁ
Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 842 46 Bratislava, Slovak Republic
Summary. – Diagnostic monoclonal and polyclonal antibodies against bean yellow mosaic virus (BYMV) and plum pox virus (PPV) were prepared, characterized and used for detection of these viruses in infected plants by enzyme-linked immunosorbent assay (ELISA), immunoblot analysis and tissue print immunoblot assay (TPIBA).
Key words: monoclonal antibodies; plum pox virus; bean yellow mosaic virus;
epitopes; capsid protein; ELISA; tissue print immunoblot assay; immunoblot analysis
Acta virologica 43: 255 – 257, 1999
Z. ŠUBR, M. GLASA
Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 842 46 Bratislava, Slovak Republic
Summary. – Slovak plum pox virus (PPV) isolates BOR-3 and KR-4 behaved atypically in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) of their capsid proteins (CPs). Whereas other tested PPV isolates could be sorted by SDS-PAGE into groups corresponding to the M or D strains, as controlled by restriction fragment lenght polymorphism (RFLP) analysis, the two abovementioned isolates behaved as the M strain according to RFLP analysis but not SDS-PAGE. Slight mobility differences were observed also among the isolates belonging to the D strain. SDS-PAGE of the CP thus cannot clearly distinguish between these two main PPV strains. BOR-3 isolate has been shown atypical also in its biological properties, and it reacted very weakly with a monoclonal antibody (MAb) recognizing well both M and D strains in immunoblot analysis.
Key words: plum pox virus; capsid protein; immunoblot analysis; proteolysis
Acta virologica 43: 259 – 262, 1999
L. NIKOLAEVA, A.S. GALABOV
Department of Virology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26 G. Bonchev Str., 1113 Sofia, Bulgaria
Summary. – When combined, enviroxime and disoxaril, two selective picornavirus inhibitors, exert a marked synergistic inhibitory effect on poliovirus type 1 replication in FL cells. The cytotoxicity of the compounds applied individually and in combination to the same cells was examined. The quantitative assay of the cytotoxic effect was made by determination of the growth curve of uninfected FL cells in the presence of increasing concentrations of the compounds applied alone and in combination. The obtained results indicate lack of a synergic cytotoxic effect of the combination of enviroxime and disoxaril. The previously established synergistic antiviral effect, and the lack of cross-resistance and synergic cytotoxic effect classify the combination of enviroxime and disoxaril as a very promising chemotherapeutic.
Key words: enviroxime; disoxaril; enterovirus; cytotoxicity; antivirals;
additive effects; synergism
Acta virologica 43: 263 – 265, 1999
H. TSUTSUMI, M. OHSAKI, K. SEKI, S. CHIBA
Department of Pediatrics, Sapporo Medical University School of Medicine, Chuoku S-1, W-16, Sapporo 060-8543, Japan
Summary. – The possible changes in transcriptional activities of the M1 muscarinic acetylcholine receptor (mAChR) and beta2-adrenergic receptor (AR) genes in respiratory syncytial virus (RSV)-infected human type 2 alveolar epithelial cells (A549 cells) were analyzed semiquantitatively by reverse transcription-polymerase chain reaction (RT-PCR). RSV enhanced M1 mAChR gene expression significantly at 4 hrs post infection (p.i.), and this enhancement persisted until 10 hrs, after peaking at 7 hrs. beta2-AR gene expression also increased significantly as early as at 1 hr p.i. and persisted for more than 10 hrs. These transcriptional enhancements were observed in cells treated with live but not with inactivated virus. Our observations suggest that RSV infection of human respiratory epithelial cells may enhance the expression of both parasympathetic and sympathetic receptors. The upregulated M1 mAChR gene in virus-infected cells may correlate with temporal airway hyperresponsiveness in subjects with RSV or other respiratory virus infection. The enhanced beta2-AR gene expression in peripheral lungs might explain the excessive mucus secretion observed during viral pneumonitis.
Key words: respiratory syncytial virus; human type 2 alveolar epithelial
cells; muscarinic acetylcholine receptor; beta2-adrenergic receptor
Acta virologica 43: 267 – 270, 1999
J. FRÁNOVÁ-HONETŠLEGROVÁ, M. ERBENOVÁ
Department of Plant Virology, Institute of Plant Molecular Biology, Academy of Sciences
of the Czech Republic, Branišovská 31, 370 05 České Budějovice, Czech Republic;
Research and Breeding Institute of Pomology Holovousy, 507 51 Holovousy, Czech Republic
Key words: strawberry crinkle rhabdovirus; strawberry mottle disease; Fragaria;
electron microscopy
Acta virologica 43: 271 – 272, 1999