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Acta virologica

Volume 47 / 2003 / number 3

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Letter-to-the Editor

Sequence analysis of infectious bursal disease virus isolates FROM India: Phylogenetic relationships


1Department of Animal Biotechnology, 2Faculty of Basic Sciences, Madras Veterinary College, Chennai 600 007, India

Received February 14, 2003; accepted June 30, 2003

Summary. – Prevalence of infectious bursal disease (IBD) among chickens in different parts of Tamil Nadu, India, has been studied by collection of bursal samples from suspected flocks and by performing reverse transcription–polymerase chain reaction (RT-PCR) for amplification of a specific product of 474 bp from the variable region of the VP2 gene. Among 53 bursal samples examined by RT-PCR, 40 showed a positive reaction. The amplified products were subjected to nucleotide sequencing and the obtained sequences were compared with those of IBD virus (IBDV) vaccine strain Georgia, the classical virulent strain 52/70 and the very virulent Japanese OKYM strain. Nucleotide homology data indicated that all the Tamil Nadu isolates showed homology ranging from 91 to 99.6% among themselves. When compared with the very virulent Japanese OKYM strain, four isolates grouped with that strain. Majority of the isolates clustered with the very the virulent OKYM strain as evident from phylogenetic analysis performed using the MEGA program. Comparison of the deduced amino acid sequences of IBDV isolates with those of the vaccine strain Georgia, the classical virulent strain 52/70 and the very virulent strain OKYM also revealed the presence of conserved serine-rich heptapeptide sequence in most of the isolates. Results of this study indicate that majority of the IBDV isolates are very virulent, which is evident from heavy mortality that has been reported in few flocks of poultry in spite of regular vaccination.
Key words: Infectious bursal disease virus; sequence and phylogenetic analysis; Tamil Nadu

Acta virologica 47: 131– 135, 2003

Acute diarrhea in paraguayan children population: Detection of rotavirus electropherotypes


1Instituto de Investigaciones en Ciencias de la Salud, Universidad Nacional de Asunción, Asunción, Paraguay; 2Hospital de Clínicas, Asunción, Paraguay; 3Sección Virología, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay

Received January 13, 2003; accepted July 8, 2003

Summary. – Group A rotavirus infections were detected in 93 of 410 fecal samples from children with acute diarrhea, admitted in three main hospitals of Asunción, Paraguay, from August 1998 to August 2000. Most of the rotavirus-infected patients were admitted during the winter season in the three epidemic years. The rotavirus infection rate was highest in infants from 6 to 23 months of age. In the 93 samples examined, 10 different rotavirus electropherotypes were recognized, but two of them largely predominated. Only one sample showed a short electropherotype pattern, thus indicating a minor involvement of the rotavirus subgroup I in rotaviral acute diarrhea in the area and the time during which the survey was carried out.
Key words: group A rotavirus; electropherotypes; infantile diarrhea

Acta virologica 47: 137 – 140, 2003

Protection of mice against experimental japanese encephalitis virus infections by neutralizing anti-glycoprotein e monoclonal antibodies


National Institute of Virology (NIV), Indian Council of Medical Research, 20-A, Dr. Ambedkar Road, P.O.B. No. 11, Pune 411001, India

Received February 12, 2003; accepted July 30, 2003

Summary. – Neutralizing monoclonal antibodies (MAbs) to glycoprotein E (gpE) of Japanese encephalitis (JE) virus given intraperitoneally (i.p.) (0.1 ml of immune ascitic fluid (AF) diluted 1:10 per mouse) to about 4-week-old Swiss mice 1 day prior or 2 days after the virus challenge (100 LD50 of JE virus administered intracerebrally (i.c.)) resulted in a decreased mortality along with an increased survival of the animals as demonstrated by the HAI-positive virus-specific (Hs) MAbs. The protective effect produced by four Hs MAbs was maximum when given 1 day prior the virus challenge, while other, namely HAI-positive flavivirus cross-reactive (Hx) and HAI-negative virus-specific (NHs) MAbs did not produce any effect. Interestingly, one of the two NHs MAbs, namely NHs-1 showed a reduced survival of mice given the MAb 2 days after the virus challenge. Administration of combinations of two or more Hs MAbs may be recommended due to their possible enhanced protection against JE virus infections in mice.
Key words: Japanese encephalitis virus; monoclonal antibodies; protection; Swiss mice

Acta virologica 47: 141 – 145, 2003

A rapid and effective rna release procedure for virus detection in woody plants by reverse transcription–polymerase chain reaction

J.K. Kundu

Department of Virology, Research Institute of Crop Production, Drnovská 507, 161 06 Prague 6, Czech Republic

Received June 13, 2003; accepted August 19, 2003

Summary. – A rapid and effective RNA release procedure (RNA-RP) for detection of the Apple stem pitting virus (ASPV) and the Apple stem grooving virus (ASGV) in woody plants by reverse transcription–polymerase chain reaction (RT-PCR) was developed. RNA-RP released RNA into crude homogenate. RNA-RP was compared with classical phenol/chlorophorm extraction of RNA. The RT-PCR detection of ASPV and ASGV was shown to be similar by both the RNA preparation procedures. RNA-RP in contrast to the classical phenol/chloroform procedure represents a reliable and easy-to hand protocol convenient for routine virus detection. Leaf tissues, especially dormant bud leaves and leaves during blossom, were shown to be the most optimal material for detection of these viruses, irrespective of the used RNA preparation procedure.
Key words: ASPV; ASGV; RT-PCR

Acta virologica 47: 147 – 151, 2003

Polymerase chain reaction assay for cydia pomonella granulovirus detection in cydia pomonella population


1 Research Institute of Crop Production, Drnovská 507, Prague 6, 161 06, Czech Republic; 2ZD Chelčice, Libějovice, Czech Republic

Received May 26, 2003; accepted August 25, 2003

Summary. – The polymerase chain reaction (PCR) assay was successfully used to identify Cydia pomonella granulovirus (CpGV) in larvae of Cydia pomonella L. (codling moth). PCR with the primers CpGV-2A/CpGV-2B and CpGV-3A/CpGV-3B was found suitable for detection of CpGV. The primers Cp-I/Cp-II and Cp-III/Cp-IV were able to identify the transposable element TCp3.2 in C. pomonella larvae. The presence of CpGV in the larvae from orchards,which had been infected with CpGV was tested during 2 years post infection. (p.i.). CpGV was found in as many as 15% of the surviving larvae 1 year p.i. in one location. The virus was not detected in CpGV-infected orchards 2 years p.i. or in natural C. pomonella populations. This result suggests a poor persistence of CpGV in surviving C. pomonella individuals and its slow spread in a natural host population. One the other hand, the presence of a transposable element, transposon TCp3.2 may correlate with virus redistribution in this insect population.
Key words: Cydia pomonella; codling moth; Cydia pomonella granulovirus; PCR; transposon

Acta virologica 47: 153 – 157, 2003

Detection of immediate early protein icp27/ie63 and thymidine kinase in the course of reactivation of latent herpes simplex virus 1 infection


Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05 Bratislava 4, Slovak Republic

Received April 24, 2003; accepted August 19, 2003

Summary. – We followed the kinetics of reactivation of latent Herpes simplex virus 1 (HSV-1) infection established in rabbits by corneal route. The corresponding trigeminal ganglia (TG) were cultured and the culture medium was examined at daily intervals for release of infectious virus. Sections from the cultured TG fragments were stained with antisera against non-structural proteins such as the immediate early (IE) protein ICP27 and the early (E) proteins thymidine kinase (TK), the large subunit of ribonucleotide reductase (RR1), the ori-binding protein OBP and with a human serum obtained from volunteers immunized with an experimental subunit HSV-1 envelope (env) vaccine containing late structural proteins gB1, gC1, gD1 and gG1 (env antiserum). By indirect immunofluorescence (IF) test, ICP27 was detected in a few neurons from day 1 post explantation (p.e.), while TK was observed in neurons from day 2 p.e. Fluorescence with the human env antiserum was seen at day 3 p.e. The RR1 and OBP antisera stained productively infected Vero cells from 3 and 4 hrs post inoculation (p.i.), respectively. However, these sera showed no IF in cultured ganglion fragments at any interval examined. Our results showed the same cascade of HSV-1 IE and E protein expression during productive infection and reactivation in vitro.
Key words: HSV-1; ICP27; thymidine kinase; reactivation; latency; immunofluorescence; explantation technique

Acta virologica 47: 159 – 165, 2003

Sexually transmitted infections among prostitutes in bratislava, slovakia

M. Bystrická1, Z. Kriková1, M. Kŕčová1, K. Koncová2, E. Kováčová1, A. Vaculíková3, D. Staneková2, G. Russ1

1 Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05 Bratislava, Slovak Republic; 2 Institute of Preventive and Clinical Medicine, Bratislava, Slovak Republic, 3 Clinic of Infectious Diseases, Bratislava, Slovak Republic

Received July 24, 2003; accepted September 2, 2003

Summary. – Sera from 18 prostitutes from Bratislava were examined for the presence of antibodies to several sexually transmitted pathogens, namely Herpes simplex virus 2 (HSV-2), Human immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2), Hepatitis B and C viruses (HBV and HCV), Chlamydia trachomatis, and Treponema pallidum. Results of this screening indicated that 11 prostitutes (61%) carried 1 or more sexually transmitted infections. The most prevalent antibodies were directed against HSV-2 (9 cases, i.e. 50%), which represents the most common sexually transmitted infection agent.
Key words: Herpes simplex virus 2; Human immunodeficiency viruses 1 and 2; Hepatitis B virus; Hepatitis C virus; Chlamydia trachomatis; Treponema pallidum; prostitutes; antibodies

Acta virologica 47: 167 – 172, 2003

Isolates of infectious bursal disease virus from india are of very virulent phenotype

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

National Biotechnology Centre, Indian Veterinary Research Institute, Izatnagar-243 122, Bareilly, U.P., India

Received May 27, 2003; accepted September 3, 2003

Summary. – Four Indian field isolates, a classical virulent and an attenuated vaccine strains of Infectious bursal disease virus (IBDV) have been characterized by sequence analysis of part of the VP1 gene (from nucleotide 1538–1979) comprising one of viral RNA dependent RNA polymerase motifs. Sequence alignment of these viruses with reported viruses of other countries revealed Indian IBDV field isolates to be 100% similar to very virulent Japanese (OKYM), European (UK661) and Bangladesh (BD3/99) IBD viruses at amino acid level, whereas they had 0.2–0.9% divergence at nucleotide level. Out of the total 24 nucleotide changes found in the Indian field isolates, as well as reported very virulent viruses, only one resulted in amino acid change S-P at 562 position. The Indian field isolates displayed nucleotide divergence of 10.6–11.6% and amino acid divergence of 2.8–3.5% from the classical virulent and attenuated vaccine strains. The RNA dependent RNA polymerase motif from amino acid 528–541, present in the sequence analyzed, was conserved among all the viruses, irrespective of pathotype and serotype. In the phylogenetic tree, based on nucleotide sequence, Indian field viruses were grouped with reported very virulent viruses in one lineage whereas, classical virulent, attenuated vaccine and serotype 2 strains formed part of the second lineage. But in the phylogenetic tree based on amino acid sequence alignment, the serotype 2 strain OH grouped with Indian field isolates and reported very virulent viruses in one lineage and classical virulent and attenuated vaccine strains formed the second lineage.
Key words: Infectious bursal disease virus; VP1 gene; nucleotide sequence

Acta virologica 47: 173 – 177, 2003

Detection of egg drop syndrome 1976 virus by polymerase chain reaction and study of its persistence in experimentally infected layer birds


1 Division of Avian Diseases, Indian Veterinary Research Institute, Izatnagar 243 122, Bareilly, U.P., India; 2 Department of Research and Diagnosis of Poultry, Razi Vaccine and Serum Research Institute, Teheran, Iran

Received May 2, 2003; accepted September 9, 2003

Summary. – Polymerase chain reaction (PCR) assay was developed for the detection of Egg drop syndrome 1976 (EDS-76) virus in tissues, namely in the uterus, spleen and buffy coat. It was also used to study the persistence of the virus in tissues of experimentally infected layer birds. The PCR assay could detect as little as 10 fg of purified EDS-76 viral DNA. It also amplified the DNA of Fowl adenovirus serotypes 4 (FAV-4) and 8 (FAV-8). The virus persisted in the uterus up to day 21 post infection (p.i.). Detection of EDS-76 viral DNA in the buffy coat could be useful for studying the occurrence of the respective disease in layer bird flocks.
Key words: egg drop syndrome; Egg drop syndrome 1976 virus; polymerase chain reaction; diagnosis; buffy coat; persistence

Acta virologica 47: 179 – 184, 2003

Serological evidence for japanese encephalitis virus and west nile virus infections in water frequenting and terrestrial wild birds in kolar district, karnataka state, india. A retrospective study


National Institute of Virology, Indian Council of Medical Research, 20-A, Dr Ambedkar Road, Pune 411001, India

Received May 22, 2003; accepted September 9, 2003

Summary. – In a serological survey of birds in a Japanese encephalitis (JE) endemic area of Kolar District, Karnataka State, India, 859 bird sera were tested by hemagglutination-inhibition test (HIT) for JE encephalitis and West Nile encephalitis (WNE) viruses. Only 2 (0.002%) and 178 (20.72%) sera were positive for JE virus (JEV) and WNE virus (WNV), respectively. Only 160 (18.63%) of 859 sera could be subjected to neutralizing test (NT). Of these, 20 (12.50%) and 62 (38.75%) were positive for JEV and WNV antibodies, respectively. These findings indicate that bird species such as Pond Herons and Little Egrets among ardeid birds and Grey Partridges and Quails among terrestrial birds are infected with JEV and WNV and play probably a role in the maintenance of these viruses in the abovementioned part of India.
Key words: ardeid birds; terrestrial wild birds; West Nile virus; Japanese encephalitis virus; hemaglutination-inhibition antibodies; hemagglutination-inhibition test; neutralizing antibodies; neutralizing test

Acta virologica 47: 185 – 188, 2003

Synergism between the nucleopolyhedroviruses of autographa californica and trichoplusia ni

J. Lara-Reyna1, M.C. Del Rincón-Castro2, J.E. Ibarra2*

1 Instituto de Fitosanidad, Colegio de Postgraduados, km. 35.5 Carr. México-Texcoco, 56230-Texcoco, Edo. de México; 2 CINVESTAV IPN, Apdo. Postal 629, 36500 Irapuato, Gto. Mexico

Received May 25, 2003; accepted September 11, 2003

Summary. – Previous observations on high virulence of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Trichoplusia ni single nucleopolyhedrovirus (TnSNPV) acting together led us to test possible synergism between these two nucleopolyhedroviruses (NPVs) on cabbage looper larvae. Because synergism between AcMNPV and the Trichoplusia ni granulovirus (TnGV) has been well established before, these two viruses were included in this study as a positive control. Each virus was assayed separately on first-instar cabbage looper and their LC50s were estimated at 2.33, 0.39 and 462 OB/mm2 diet for AcMNPV, TnSNPV and TnGV, respectively. LC50s of AcMNPV mixed with sub-lethal concentrations of TnSNPV and TnGV increased 8 and 10.7 times, respectively. Synergism between the viruses was analyzed by the ANOVA test for the LC50s, the Plackett and Hewlett's joint-action rate test, and the Tammes-Bakuniak graphic method. All three analyses corroborated the synergism between the viruses. The presence of a putative enhancin in the TnSNPV was analyzed by Southern blot hybridization, using a 1.5 kbp KpnI fragment from the TnGV vef gene as a probe. No hybridization was observed. The occurrence of a new putative synergistic factor in TnSNPV is discussed.
Key words: synergism; Trichoplusia ni single nucleopolyhedrovirus; Autographa californica multiple nucleopolyhedrovirus; joint-action ratio; ANOVA test; Tammes-Bakuniak graphic method; viral enhancin

Acta virologica 47: 189 – 194, 2003

First confirmed sheep scrapie with a136r154q171 genotype in slovakia


1 Institute of Neuroimmunology, Slovak Academy of Sciences and National Reference Laboratory for Transmissible Spongiform Encephalitis, Dúbravská cesta 9, 845 10 Bratislava, Slovak Republic; 2 State Veterinary and Food Administration, Bratislava, Slovak Republic; 3 University of Veterinary Medicine, Košice, Slovak Republic

Received June 16, 2003; accepted September 4, 2003

Summary. – The first confirmed evidence of scrapie in Slovakia was demonstrated in one sheep of the autochthonous Merino breed from the southeastern part of the country. The reported scrapie was diagnosed during compulsory transmissible spongiform encephalitis (TSE) screening of sheep over 9 months of age assigned for consumption. The positive ewe was 5-year-old, which did not show any clinical signs of scrapie. The presence of the proteinase-resistant prion protein (PrP) in brain was proved independently by two laboratories using two different immunochemical screening systems, namely the Prionics Check (Western blot analysis) and Enfer TSE enzyme-linked immunosorbent assay (ELISA). In addition, the genotyping analysis of PrP gene demonstrated the presence of PrP genotype from the high risk group R4. The affected sheep was homozygous for the allele PrPARQ (ARQ/ARQ) coding for alanine (A), arginine (R) and glutamine (Q) at three most relevant codons (136, 154 and 171, respectively. The healthy sister of the positive ewe was heterozygous in the PrP locus and carried alleles ARQ/ARR.
Key words: scrapie; prion; PrP gene; polymorphism; Slovakia

Acta virologica 47: 195 – 198, 2003

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