Electronic Library of Scientific Literature - © Academic Electronic Press
Volume 44 / October 2000 / number 5
R.J. PHILLPOTTS, T.L. LESCOTT, S.C. JACOBS
D.E.R.A., Biological Sciences Department, Chemical and Biological Defence Sector, Porton Down, Wiltshire, SP4 0JQ, U.K.
Summary. – Vaccinia virus (VV) recombinants that contain the genes encoding the Venezuelan equine encephalitis virus (VEEV) structural gene region (C-E3-E2-6 K-E1) solidly protect mice against peripheral challenge with virulent VEEV, but provide only partial protection against airborne challenge. To improve upon these results we focussed on the principal antigens involved in protection. VV recombinants encoding the structural genes E3-E2-6 K-E1, E3-E2-6 K or 6 K-E1 were prepared and evaluated for their ability to protect Balb/c mice after a single dorsal scarification with 108 PFU against peripheral or airborne challenge with virulent VEEV. The antibody response was also examined. Our experiments provide new evidence that truncates of the VEEV structural region (E3-E2-6 K-E1, E3-E2-6 K), cloned and expressed in VV, protect against challenge with virulent virus. They also confirm the important role of E2 in protection. However, we were unable to improve upon previously reported levels of protection against airborne challenge. A substantial level of circulating antibodies and the presence of local IgA (not always induced by mucosal immunization) (Greenway et al., 1992) appear essential for protection against the airborne virus. Current VV-VEEV recombinants seem unable to elicit this level of immune response and further improvements are therefore required to increase the immunogenicity of VV-VEEV vaccines.
Key words: vaccinia virus; protective immunity; Venezuelan equine encephalitis virus
Acta virologica 44: 233 – 239, 2000
E.A. GOVORKOVA, A.S. GAMBARYAN, E.C.J. CLAAS, Y.A. SMIRNOV
The D.I. Ivanovsky Institute of Virology, Russian Academy of Medical Sciences, Gamaleya 16, 123098, Moscov, Russsia;
M.P. Chumakov Institute of Poliomyelitis and Viral Encephalitis, Russian Academy of Medical Sciences, Moscow, Russia;
Department of Virology, CKVL, Leiden University Medical Center, Leiden, The Netherlands
Summary. – Mouse-adapted (MA) variants of human and avian influenza A (H2) viruses were generated and characterized with respect to acquisition of virulence in mice. From the nucleotide sequence the amino acid sequence was deduced. The HA1 subunit of the hemagglutinin (HA) contained three amino acid substitutions in the A/black duck/New Jersey/1580/78-MA variant (Glu216->Asp, Lys307->Arg, and Thr318->Ile) and two substitutions in the A/JapanxBellamy/57-MA variant (Lys25->Thr and Ser203->Phe). In the M1 protein, there were two substitutions in the A/black duck/New Jersey/1580/78-MA variant (Asn30->Asp and Gln214->His) and a single substitution in the A/JapanxBellamy/57-MA variant (Met179->Lys). The M2 protein amino acid sequences of the parental virus and the MA variants differed by a single identical mutation (Asn93->Ser). The localization and atomic distances of the observed mutations on the three-dimensional (3D) structure of the HA protein were analyzed for influenza H2 viruses. The obtained results were similar to those published earlier on H1, H3 and H5 subtypes. The amino acid changes in the HA protein could be divided into two groups. In one group the substitutions were situated at the top of the molecule, while in the other group they were clustered in the stem area at the interface region between three HA monomers. The analysis revealed that the substitutions observed in the MA variants probably increase the flexibility of the HA molecule and/or weaken the interactions between monomers or subunits in the HA trimer. The relationships of the observed amino acid changes in the HA and M proteins to the biological properties of the respective viruses and possible mechanisms involved in the acquisition of viral virulence are discussed.
Key words: influenza A viruses; H2 subtype; adaptation to mice; hemagglutinin (H2); M1 protein; M2 protein; amino acid sequence mutations
Acta virologica 44: 241 – 248, 2000
Laboratory of Molecular Biology and Virology, Research Institute of Viticulture and Enology, Matúškova 25, 833 11 Bratislava, Slovak Republic
Summary. – Three forms of anionic peroxidase (PRX) from hypersensitively reacting cucumber cotyledons were purified to homogeneity and different methods were used to analyze the nature of their carbohydrate chains. Immunoblot analysis with betaF1 antiserum showed that all three forms are highly glycosylated and contain asparagine N-linked glycans commonly found in other plant glycoproteins. Mobility shift analysis showed that chemical deglycosylation converted PRXs 1, 2 and 3 to the same-sized (35 K) products. Enzymatic deglycosylation with alpha-mannosidase converted PRX1 and PRX2 to immunoreactive products migrating in mobility shift polyacrylamide gels at the positions of PRX2 and PRX3, respectively. PRX3 treated with alpha-mannosidase yielded a product with Mr similar to that obtained with the chemical deglycosylation. Cleavage of the PRXs 1, 2 and 3 by formic acid at the Asp-Pro site resulted in peptide maps and the putative glycopeptide(s) were recognized using betaF1 antiserum. Only one glycopeptide was observed for each of the forms. Lectin-affinity blot analysis using biotin-conjugated lectins suggested that virus-inducible PRX contains complex-type N-glycosyl carbohydrate chain(s). These results indicate that heterogeneity of cucumber virus-inducible PRX is not caused mainly by differences in the terminal alpha-linked mannose residues.
Key words: cucumber; Cucumis; deglycosylation; glycopeptides; immunoblot analysis; lectin-affinity blot analysis; polyacrylamide gel electrophoresis; tobacco necrosis virus
Acta virologica 44: 249 – 257, 2000
R.S. KATARIA, A.K. TIWARI, G. BUTCHAIAH, J.M. KATARIA
National Biotechnology Centre; Division of Avian Diseases, Indian Veterinary Research Institute, Izatnagar, U.P., 243 122, India
Summary. – Two different radio-labeled nucleic acid probes, prepared from reverse transcription–polymerase chain reaction (RT-PCR) amplified variable region of VP2 and VP1 gene sequences of a highly virulent infectious bursal disease virus (IBDV), were tested for their ability to detect field isolates of IBDV directly in clinical bursal tissue specimens and vaccine strains of IBDV in tissue cultures. The VP2 gene probe was able to detect both field isolates and vaccine strains of IBDV under high as well as low stringency while the VP1 gene probe could differentiate under high stringency field isolates from vaccine strains, hybridizing only with RNA of field isolates. The sensitivity of both the probes was found to be 4 ng of purified viral RNA.
Key words: infectious bursal disease virus; detection; dot-blot hybridization; field strains; virulence; vaccine strains
Acta virologica 44: 259 – 263, 2000
R.D. SCHNAGL, N. BARTON, M. PATRIKIS, J. TIZZARD, J. ERLICH, F. MOREY
Department of Microbiology, La Trobe University, Bundoora, VIC 3083, Australia;
Department of Paediatrics,
and Pathology Laboratory, Alice Springs Hospital, Alice Springs, NT, Australia
Summary. – Norwalk-like viruses (NLVs) have now been found to be important causes of gastroenteritis amongst infants and young children as well as older children and adults. Although detected, such viruses appeared not to be a major cause amongst infants and young children hospitalized with gastroenteritis in Alice Springs, central Australia over the period January 1995–December 1997. Nine NLV-positive cases were identified amongst stools from 360 different patients. From the nine cases however, eight different NLV strains were identified from comparisons of the sequence of a section of the RNA polymerase gene, and a high degree of genomic diversity was evident amongst them. In general, these strains were more similar to those identified in other countries than to those identified in central Australia over the three year period. Of the strains identified,six (and most probably seven) were classified in genogroup I, while only one was classified in genogroup II. This predominance of genogroup I strains is in contrast to most of the more recent findings made elsewhere, including those made in other parts of Australia. Phylogenetic analysis indicated that the central Australian strains spanned a range of known representative NLV strains, with one of the genogroup I strains showing a 96% nucleotide identity to Saratoga virus.
Key words: Australia; genomic variation; Norwalk-like viruses; prevalence
Acta virologica 44: 265 – 271, 2000
A. MIKULÁŠOVÁ, E. VAREČKOVÁ, E. FODOR
Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic;
Chemical Pathology Unit, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom
Summary. – The genome of influenza A virus consists of eight segments of negative-strand viral RNA (vRNA). During the replication cycle of the virus, the genomic vRNA is transcribed into positive-strand mRNA and complementary RNA (cRNA) in the cell nucleus. The promoter for the synthesis of mRNA molecules is located in a partially double-stranded RNA structure formed by the 5´- and 3´-terminal sequences of genomic vRNA segments. The virus encoded RNA-dependent RNA polymerase complex has to interact with both ends of the vRNA in order to generate capped RNA primers by endonucleolytic cleavage of cellular pre-mRNAs for the initiation of viral mRNA synthesis. Conserved sequence elements in the 5´-end, e.g. a polymerase binding site and a U5-7 sequence are required for polyadenylation of virus-specific mRNAs. Polyadenylation occurs by reiterative copying of the U5-7 sequence by the viral RNA polymerase, which is bound to the 5´end of the vRNA template. The U5-7 sequence acts directly as a template for the poly(A)-tail. During the replication cycle of the virus, a “switch” from mRNA to cRNA synthesis occurs, but the mechanism by which this switch occurs remains unclear. The viral nucleoprotein and its interaction with the polymerase proteins and vRNA might play a role in this process. In contrast to transcription, the process of replication – the synthesis of cRNA and vRNA, which are known to occur in the absence of primers – is poorly understood.
Key words: influenza A virus; transcription; replication; polyadenylation; RNA polymerase
Acta virologica 44: 273 – 282, 2000
R. KATYAL, S.V. RANA, K. SINGH
Department of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh 160 012, India
In 1976, John Rohde, highlighting the importance of diarrhea as prime killer of children in the developing world, beckoned the scientific community to “take science where the diarrhea is”. The World Health Organization estimates that one billion diarrheal episodes occur in infants annually resulting in 3.3 million deaths, making diarrheal disease a major contributor to infant mortality in developing world (Bern et al., 1992). The need for simple, effective and inexpensive intervention to treat diarrhea and to prevent its occurrence is urgent and abundantly clear. Among the etiological agents of acute infectious diarrhea rotaviruses account for nearly 25% of hospital admissions in India with vomitting and diarrhea followed by severe dehydration in very young children below 2 years of age (Broor et al., 1985). In developing countries, it has been estimated that more than 870,000 children die from rotavirus infection every year (Perez-Schael, 1996). The discovery of rotavirus by Bishop and colleagues in 1973 initiated a line of research that has progressed rapidly towards the goal of prevention of rotavirus diarrhea (Bishop et al., 1973).
Acta virologica 44: 283 – 288, 2000
Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany
Summary. – The present review focuses on recent development in our understanding of the molecular mechanisms of DNA replication of herpes simplex virus 1 (HSV-1). Progress made in the characterization of the early events of viral DNA synthesis and of virus-host cell interactions, especially in the context with the formation of viral DNA replication sites, is highlighted. An up-dated overview is presented on important stages of lytic infection cycle, such as virion entry, viral DNA synthesis, viral DNA cleavage and packaging into preformed capsids, and maturation (nucleocapsid envelopment) and egress of virions. Many novel interactions are discussed that extend not only our knowledge of the biology of this virus but may represent possible new targets for antiherpesviral therapy.
Key words: host shutoff; nuclear bodies; viral DNA recombination; viral DNA replication; viral DNA transcription; virion egress; virion entry; virion maturation
Acta virologica 44: 289 – 307, 2000