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Acta virologica

Volume 47 / 2003 / number 2

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Short Communication

Letter-to-the Editors

Chloride concentration discriminates between Foot-and-Mouth disease virus IRES-dependent translation and classical scanning translation: New aspects of the picornavirUS shutoff mechanism


Max-Planck-Institute for Marine Microbiology, Celsiusstr. 1, 28359 Bremen, Germany

Received January 17, 2003; accepted April 2, 2003

Summary. – Some picornaviruses might use the general increase of ionic strength in the host cell that occurs successively after infection to induce shutoff of host protein synthesis and to stimulate viral protein synthesis. In order to investigate this discrimination mode on a molecular level, in vitro experiments under different salt conditions comparing the Foot-and-mouth disease virus (FMDV) internal ribosome entry site (IRES)-dependent translation with the translation via the classical scanning mechanism were performed. For classical mRNA optimum concentrations of all investigated salts ranged between 70 and 100 mmol/l. However, for FMDV IRES-dependent translation the optima depended strongly on the anion used. While acetates caused only a weak stimulation of translation efficiency with maxima ranging between 150 and 180 mmol/l, chlorides lead to a strong stimulation with maxima ranging between 120 and 150 mmol/l. Competition experiments revealed that the concentration of chlorides had a greater influence on the discrimination between cellular and viral RNA translation than the total ionic strength. Taken together, the data support a model in which a specific increase in the chloride concentration rather than a general increase in the ionic strength is responsible for the shutoff effect induced by some picornaviruses.
Key words: in vitro translation; initiation; elongation; picornavirus; shutoff; IRES

Acta virologica 47: 65 – 72, 2003

Sequence analysis of segment A of a field virus isolate from an outbreak of infectious bursal disease in India

M.V. Bais, R.S. Kataria, A.K. Tiwari*, Indervesh, V.V S. Suryanarayana, S.K. Das

National Biotechnology Center, Indian Veterinary Research Institute, Izatnagar, 243122 UP, India

Received November 15, 2002; accepted April 3, 2003

Summary. – Sequence analysis of genome segment A of an Indian Infectious bursal disease virus (IBDV) field isolate (KT1/99) revealed total 95 nucleotide substitutions, resulting in 17 amino acid changes. Of these, five amino acid changes, namely F60S, T137I, I374V, V519I and E682D were unique to the KT1/99 isolate. The amino acid change P222A and the proposed hot mutation spot 680Y, reported to be present in very virulent IBDV isolates were also found in KT1/99. This isolate had nucleotide divergence of 1.1% to 4.95% from the other reported serotype 1 IBDV isolates and 19.6% from serotype 2 strain OH in polyprotein gene sequence, while divergence at amino acid level was 0.6% to 2.9% and 11.4%, respectively. Based on both nucleotide and amino acid sequence analysis, KT1/99 was grouped phylogenetically with the reported Bangladesh isolate BD3/99 in one cluster along with other reported very virulent isolates in same lineage.
Key words: Infectious bursal disease virus; nucleotide sequence; amino acid sequence; segment A

Acta virologica 47: 73 – 77, 2003

RNA-dependent RNA polymerase activity of classical swine fever virus NS5B protein expressed in natural host cells

M. Xiao1,2, J. Chen2, B. Li2*

1Biology Department, College of Life and Environment Sciences, Shanghai Teachers' University, Shanghai, 200234 P.R. China;
2Key Laboratory for Biodiversity Science and Ecological Engineering of Ministry of Education,
The Institute of Biodiversity Science, Fudan University, Shanghai, 200433 P.R. China

Received January 28, 2003; accepted April 4, 2003

Summary. – The NS5B gene, cloned from Classical swine fever virus (CSFV) genome, was expressed in porcine kidney cells PK-15, natural host of CSFV. In purifying cytoplasmic extracts from these cells by means of different concentrations of salt, glycerol and detergent four fractions, namely crude supernatant (SC) and different purified supernatants (S1, S2 and S3) were obtained. Using Western blot analysis the NS5B protein was found in all these fractions, showing that it was soluble in both higher and lower concentrations of salt, glycerol and detergent. The NS5B protein present in the four different fractions exhibited RNA-dependent RNA polymerase (RdRp) activity, but it was unable to complete the whole process of RNA synthesis. Site-directed mutation analysis showed that Thy216 and Cyt228 were essential for RNA synthesis while Cyt219 was not, suggesting that CSFV RdRp was template-specific. We conclude that initiation of RNA synthesis by CSFV RdRp includes also template priming.
Key words: NS5B gene; Classical swine fever virus; RNA-dependent RNA polymerase

Acta virologica 47: 79 – 85, 2003

A sensitive and specific detection of human herpesvirus 8 by Polymerase chain reaction and dot blot hybridization

O. Kim1*, S.S. Kim2

1USDA-ARS, ADRU, 319 Bustad, WSU, Pullman, WA 99164, USA;
2Department of Viral Diseases, National Institute of Health, Seoul 122-701, Republic of Korea

Received February 27, 2003; accepted April 30, 2003

Summary. – Polymerase chain reaction (PCR) is a powerful technique of detecting Human herpesvirus 8 (HHV-8), but has a limited sensitivity and specificity. A new assay of HHV-8 based on combination of PCR with dot blot hybridization (DBH) was developed and evaluated for its sensitivity and specificity. An HHV-8-specific primer pair, ORF26out was used for amplification of target DNA. When the PCR product was detected visually the limit of detection was 0.1 ng DNA isolated from HHV-8-infected BC-3 cells. For DBH, the DNA amplified with the primer pair ORF26in specific for HHV-8 was labeled with digoxigenin (DIG). This DIG-labeled probe was capable of detecting 1.0 ng of DNA isolated from HHV-8-infected BC-3 cells. On the other hand, PCR combined with DBH (PCR/DBH) was more sensitive than PCR or DBH alone and also very specific. The sensitivity of PCR/DBH was higher than that of PCR and DBH alone. The PCR/DBH assay can be applied efficiently to confirm the presence of HHV-8 in clinical samples and to differentiate specifically HHV-8 infection from other viral infections.
Key words: polymerase chain reaction; dot blot hybridization; Human herpesvirus 8

Acta virologica 47: 87 – 90, 2003

Lytic replication of human herpesvirus 8 and induction of apoptosis

O. Kim1*, S.J. Yi2

1USDA-ARS, ADRU, 319 Bustad, WSU, Pullman, WA 99164, USA;
2College of Veterinary Medicine, Kyungpook National University, Republic of Korea

Received February 12, 2003; accepted May 6, 2003

Summary. – Although many viruses can induce apoptosis in infected cells, large DNA viruses, such as poxviruses, herpesviruses and adenoviruses, usually exhibit the ability to suppress the induction of apoptosis in the infected cells. We investigated the ability of Human herpesvirus 8 (HHV-8) to protect cells from apoptosis induced by the virus. HHV-8 has been shown to harbor genes with anti-apoptotic capacity. However, we demonstrate here that a lytic replication of HHV-8 resulted in induction of apoptosis using different techniques to detect apoptosis. Therefore, despite the presence of anti-apoptotic genes in its genome, HHV-8 could complete its cycle of productive infection while inducing apoptosis in infected cells. This finding might have implications for the pathobiology of HHV-8 and other gamma herpesviruses in vivo.
Key words: apoptosis; gammaherpesvirus; Human herpesvirus 8

Acta virologica 47: 91 – 95, 2003



1Programme in Infectious Diseases, Department of Microbiology, Faculty of Medicine, National University of Singapore, Kent Ridge 117597, Singapore;
2Department of Laboratory Medicine, National University Hospital, Kent Ridge, Singapore

Received March 26, 2003; accepted May 14, 2003

Summary. – To study the genetic variability and molecular epidemiology of Human respiratory syncytial virus (HRSV) occurring in Singapore, nucleotide sequencing of three membrane-associated genes (SH, G and F) of four local isolates was performed. Comparison of their nucleotide and amino acid sequences with those of the prototype strains A2 (subgroup A) and CH-18537 (subgroup B) indicated that the Singapore isolates belong to the subgroup A. Comparison of the Singapore isolates with the reference strain A2 showed that whereas the G protein was the most divergent with up to 15% difference, the F and SH proteins showed less diversity of only up to 4%. Each gene exhibited its distinct variable and conserved regions. The N- and O-glycosylation sites within the G protein of the isolates were analyzed to ascertain their potential implications on the antigenicity of the viral glycoprotein. Based on the second variable region of the G protein, phylogenetic analysis of the Singapore isolates with 91 previously identified genotypes of subgroup A revealed that more than one genotype (GA2 and GA5) may circulate in the local population at a given time. This epidemiological study reflects the pattern of genetic relationships between the HRSV isolates from Singapore to those from other parts of the world.
Key words: F, G, SH genes/proteins; genotypes; phylogeny; Human respiratory syncytial virus; sequence analysis

Acta virologica 47: 97 – 104, 2003


G. Raikhy, V. Hallan, S. Kulshrestha, M. Lal Sharma, R. Ram, A.A. Zaidi*

Plant Virology Laboratory, Floriculture Division, Institute of Himalayan Bioresource Technology, CSIR, Palampur, Himachal Pradesh, 176 061, India

Received April 29, 2003; accepted May 21, 2003

Summary. – Incidence of the Carnation etched ring virus (CERV), the only DNA virus reported to date on carnation, was investigated by a bioassay using a partially purified virus as inoculum and then by a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Out of 61 carnation cultivars analyzed 41 (67%) were found positive. The virus positivity was verified by polymerase chain reaction (PCR) and nucleotide sequencing. The amplified 1349 bp fragment was by about 98% and 96% identical with respect to coat protein (CP) and enzymatic polyprotein genes, respectively, as compared to the sequences available in the database. In terms of amino acid sequence similarity, the homology values were 99% and 97%, respectively. Comparison with other caulimoviruses revealed that CERV is most closely related to the Cauliflower mosaic virus (CaMV). High genetic stability of CERV may be attributed to the fact that it has evolved from the same initial sequence in an original host. Because of global market of cut flowers and vegetative propagation it has been dispersed around the world.
Key words: carnation; Carnation etched ring virus; coat protein; enzymatic polyprotein; Indian isolate; nucleotide sequencing

Acta virologica 47: 105 – 111, 2003


R.M. de Campos, D.F. Ferreira, V.F. da Veiga, M.A. Rebello, M.C.S. Rebello*

Department of Virology, Prof. Paulo de Góes Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, CEP 21941-590, RJ, Brazil

Received March 3, 2003 accepted May 29, 2003

Summary. – The effect of a cationic ionophore, monensin, on the replication of Mayaro virus in monkey kidney TC7 and Aedes albopictus cells has been studied. Treatment of these cells with 1 µmol/l monensin during infection did not affect the virus protein synthesis but inhibited severely the virus replication. Electron microscopy of the cells infected with Mayaro virus and treated with monensin revealed that the morphogenesis of Mayaro virus was impaired in TC7 but not in A. albopictus cells.
Key words: Mayaro virus; monensin; TC7 cells; Aedes albopictus cells; protein synthesis; virus morphogenesis

Acta virologica 47: 113 – 119, 2003

CD38 expression on CD8+ T cells in HUMAN IMMUNODEFICIENCY VIRUS 1-Positive Adults Treated WITH HAART

O. Beran1*, M. Holub1, J. Špála1, J. Kalanin2, M. Staňková1

1 3rd Department of Infectious and Tropical Diseases, First Faculty of Medicine, Charles University, Budínova 2, Prague 8, CZ-180 81, Czech Republic;
2 Department of Immunology, Institute of Clinical and Experimental Medicine; Prague, Czech Republic

Received February 14, 2003; accepted March 13, 2003

Summary. – The aim of this study was to assess whether the density of CD38 antigen expression on CD8+ T cells can be used as a marker of activation of the immune system in Human immunodeficiency virus 1 (HIV-1)-positive patients treated with highly active antiretroviral therapy (HAART). T cell subsets, expression of CD38 antigen on CD8+ T cells, HIV-1 viral load and stage of the disease were analyzed at baseline and after 12 months of HAART in 24 HIV-1-infected patients. Our data showed that the use of HAART is effective in reducing plasma viral load and in achieving a stable CD4+ count and percentage of CD8+/CD38+ cells. The percentages of CD8+/CD38+ cells in HIV-1-infected patients at baseline and after 12 months of HAART were significantly higher than those of controls. Analysis of the density of CD38 expression revealed that it was due to CD8+/CD38+ subsets with low and medium density of antigen expression. Absolute number of CD4+ T cells correlated negatively with the percentage of CD8+/CD38+ cells at baseline of the study. Persistent up-regulation of the CD38 expression on CD8+ T cells and its correlation with the decreased CD4+ count despite the reduction of plasma viral load may reflect residual replication of HIV-1 in reservoirs. Thus, this immunological parameter can serve as a biological marker of HIV-1 infection and might have utility in clinical management of HIV-1-infected persons.
Key words: HIV-1; CD38; CD8+; HAART; flow cytometry

Acta virologica 47: 121 – 124, 2003

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