Showing papers in "Virus Genes in 2003"

The evolution, distribution and diversity of endogenous retroviruses.

Abstract: The retroviral capacity for integration into the host genome can give rise to endogenous retroviruses (ERVs): retroviral sequences that are transmitted vertically as part of the host germ line, within which they may continue to replicate and evolve ERVs represent both a unique archive of ancient viral sequence information and a dynamic component of host genomes As such they hold great potential as informative markers for studies of both virus evolution and host genome evolution Numerous novel ERVs have been described in recent years, particularly as genome sequencing projects have advanced This review discusses the evolution of ERV lineages, considering the processes by which ERV distribution and diversity is generated The diversity of ERVs isolated so far is summarised in terms of both their distribution across host taxa, and their relationships to recognised retroviral genera Finally the relevance of ERVs to studies of genome evolution, host disease and viral ecology is considered, and recent findings discussed

Sequence Analysis of the Complete Genome of Rice Black-Streaked Dwarf Virus Isolated from Maize with Rough Dwarf Disease

Abstract: The complete nucleotide sequences of 10 genomic segments (S1-S10) from an isolate of rice black-streaked dwarf virus causing rough dwarf disease on maize (RBSDV-Hbm) in China were determined, a total of 29,142 base pairs (bp). Each segment possessed the genus-specific termini with conserved nucleotide sequences of (+) 5'-AAGUUUUU......CAGCUNNNGUC-3' and a perfect or imperfect inverted repeat of seven to eleven nucleotides immediately adjacent to the terminal conserved sequence. While the coding strand of most RBSDV-Hbm segments contained one open reading frame (ORF), there were two non-overlapping ORFs in S7 and S9, and one small overlapping ORF downstream of the major ORF in S5. Homology comparisons suggest that S1 encodes a RNA-dependent RNA polymerase (RdRp), with 63.5% and 32.6% identity to the putative RdRp encoded by Fiji disease virus (FDV) and Nilaparvata lugens reovirus (NLRV), respectively. The proteins encoded by S2, S3, and S4 showed various degrees of similarity to those encoded by the corresponding segments of FDV or NLRV. In S5 and S6, low identities were found to those of FDV only, but not to NLRV. Sequence analyses showed that RBSDV-Hbm had the most similarities in the genome organizations and the coding assignments with a RBSDV isolated from rice in China, in which each pair of the corresponding segments shared sequence identities of 93.8-98.9% and 93.5-100% at nucleotide or amino acid levels, respectively. In addition, phylogenetic analyses suggested that RBSDV-Hbm had the closest evolutionary relationship to RBSDV in Fijivirus.

Evolution of North American PVY(NTN) strain Tu 660 from local PVY(N) by mutation rather than recombination.

Abstract: A North American (NA) isolate of tobacco veinal necrotic strain of Potato virus Y (PVYN) (N-Jg) and a NA isolate of potato tuber necrotic strain of Potato virus Y (PVYNTN) (Tu 660) were tested for their phenotypes by inoculation to potato plants of three potato cultivars. Upon inoculation with Tu 660, tubers of the cultivars ‘Norchip’ and ‘Ranger Russet’ developed potato tuber necrotic ringspot disease (PTNRD) but not the tubers of ‘Russet Burbank’. N-Jg failed to induce PTNRD in the tested cultivars. The genomic RNAs of both strains were completely sequenced and analysed. High homology (98% and 99% identity on nucleotide and polyprotein, respectively) was found between Tu 660 and N-Jg. When polyproteins were compared with other isolates, high identity was observed between Tu 660 and an European (Eu) PVYN-605 (98%) and with an Eu-PVYNTN-H (96%). However, when individual mature proteins were compared, much lower identities (86.5–94%) were found between Tu 660 and PVYNTN-H compared to 98–99.5% between Tu 660 and PVYN-605 in the P3, 6K1 and CI regions. Further sequence analysis indicated that the PVYNTN-H is a hybrid molecule of the genomic RNA recombination of PVYO and Eu-PVYN as shown by Glais et al. (Arch Virol 147, 363–378), whereas NA-PVYNTN Tu 660 is free of recombination points. Phylogenetic analysis confirmed this observation, and suggested that, in light of high homology, the Tu 660 might have evolved from NA-PVYN by mutations rather than the genome recombinations. The non-recombinant nature of NA-PVYNTN Tu 660 strongly suggests that the recombinant structure of genome is not a necessary prerequisite for the PTNRD phenotype.

Evolution and diversity of HIV-1 in Africa--a review.

Abstract: The HIV/AIDS pandemic represents a major development crisis for the African continent, which is the worst affected region in the world. Currently, almost 30 of the 42 million people infected with HIV worldwide live in Africa. AIDS in humans is caused by two lentiviruses, HIV-1 and HIV-2, which entered the human population by zoonotic transmissions from at least two different African primate species. Extensive phylogenetic analyses of partial and full-length genome sequences have helped to gain insights into the evolutionary biology and population dynamics of HIV. One of the major characteristics of HIV is its rapid evolution, which has resulted in substantial genetic diversity amongst different isolates, the majority of which are represented in Africa. Genetic variability of HIV and any consequent phenotypic variation poses a significant challenge to disease control and surveillance in different geographic regions of Africa. This review focuses on the origins and evolution of HIV, current classification and diversity of HIV isolates in Africa and provides an extensive account of the geographic distribution of HIV types, groups, and subtypes in each of the 49 African countries. Numerous epidemiological studies have provided a picture of HIV distribution patterns in most countries in Africa, and these show increasing evidence of the importance of HIV-1 recombinants. In particular, this review highlights that our current understanding of HIV distribution in Africa is incomplete and inadequately represents the diversity of the virus, and underscores the need for ongoing surveillance.

Quantitative analysis of human immunodeficiency virus type 1 DNA dynamics by real-time PCR: integration efficiency in stimulated and unstimulated peripheral blood mononuclear cells.

Abstract: We established a set of real-time PCR assay to accurately quantify human immunodeficiency virus type 1 (HIV-1) DNA in infected cells. Using this assay we were able to measure the strong-stop, full-length/ 1-LTR circle, 2-LTR circle, and integrated forms of viral DNA, and the data provided was quite consistent with the characteristics of mutant viruses in early phase of infection. Since our assay is particularly applicable to quantify the integrated DNA in small scale of samples, we measured the level of integrated DNA in wild-type virus (WT)- or Vpr-defective virus (deltaVpr)-infected peripheral blood mononuclear cells (PBMC), and examined whether quiescent condition of the PBMC influences integration step of HIV-1. Under stimulating condition approximately 25% of total viral DNA was in integrated form in either WT- or DeltaVpr-infected cells. In contrast, under unstimulated condition the level of integration efficiency was not significantly reduced in WT-infected cells, while this efficiency was severely impaired in the absence of vpr gene. This result clearly demonstrated a crucial role of the Vpr for nuclear localization and subsequent integration of viral DNA in nondividing cells. Therefore, our assay is useful for analyzing the events in early phase of HIV-1 infection under various conditions.

Molecular Epidemiology of SAT3-Type Foot-and-Mouth Disease

Abstract: VP1 gene nucleotide sequences of 51 SAT3-type foot-and-mouth disease (FMD) viruses from seven southern and eastern African countries were used to infer a gene phylogeny. Results obtained by phylogenetic analysis of the homologous 405 nt region corresponding to the C-terminal 128 amino acids of 1D and adjacent 7 amino acids of 2A indicate that there are six distinct virus lineages evolving independently in different geographical localities in accordance with the FMD topotype concept. Topotypes I-IV occur in southern Africa, whilst topotypes V and VI are unique to East Africa. Viruses of different topotypes differ from each other at 20% or more of the nucleotide sites, specified in this study. Despite the limited geographical distribution of this serotype, the level of intratypic variation is intermediate between that of SAT1 and SAT2, both of which are widely distributed in sub-Saharan Africa. Within SAT3, 37.3% and 47.4% of sites were completely conserved on nucleotide and amino acid levels, respectively. The locality-specific grouping of viruses permits accurate determination of the sources of outbreaks, whilst the high levels of variation within the immunodominant 1D protein has implications for the control of the disease through vaccination.

Snow Mountain virus genome sequence and virus-like particle assembly.

Abstract: Snow Mountain virus (SMV) belongs to the Norovirus genus of the Caliciviridae family. SMV is a genogroup II (GII) reference strain of human enteric caliciviruses associated with epidemic gastroenteritis. In this study, the positive sense RNA genome sequence of SMV was determined to be 7,537 nucleotides in length excluding the 3' polyadenylated tract. The genome is organized into three open reading frames typical of caliciviruses in the Norovirus genus. Pairwise sequence alignments showed SMV ORF1 is highly conserved with other genogroup II noroviruses, and most closely related to GII strains Melksham and Hawaii virus. In addition, comparative sequence analyses indicated that SMV is likely a recombinant norovirus. VP1/VP2 proteins self-assembled into virus-like particles (VLPs) when expressed in insect cells by a recombinant baculovirus. Characterization of one clone that expressed VP1, but failed to assemble into VLPs, identified histidine residue 91 as important for particle assembly under standard conditions of expression.

Immunological and regulatory functions of uninfected and virus infected immature and mature subtypes of dendritic cells--a review.

Abstract: In 1868, dendritic cells (DCs) were discovered in human skin by Paul Langerhans using gold staining. These cells were named Langerhans cells (LCs) after their discoverer who, due to their dendrites, regarded them as neurons. One hundred and eleven years were to pass until it was discovered that in vertebrates these cells originate in the bone marrow as monocytes. In the 1980s, DC research was mostly carried out on DCs that are present in different tissues of mice and humans. These studies revealed that after interaction with foreign antigens, skin LCs/DCs migrate through the lymph vessels to the draining lymph nodes and induce the two arms of the immune response. The isolation of DCs from tissue cell suspensions opened the way to studies on the cells' surface proteins and their ability to stimulate immune responses. During the 1990s, studies revealed the role of DCs in the activation of naive T cells in the lymph nodes and the regulatory properties of DCs in lymph nodes, thymus, gut, and spleen. Part A of the review deals with the DC system of human and mice and immunological and regulatory functions of subsets of DCs in the skin with reference to migrating and stationary DCs, as well as the connection between DCs and the nervous system. Furthermore, the origin of both follicular DCs that are present in lymphoid tissues and thymic DCs are discussed. Part B is devoted to virus infections of DCs with an emphasis on infections caused by human herpes viruses. Part C presents the modulation of DC gene expression in response to the influenza virus. Contemporary research focuses on the role of DCs in the immune systems of vertebrates. Moreover, studies are being conducted on the regulatory functions of DCs by tissue cells in different organs of vertebrates.

High genetic diversity of the immunodominant region of the feline calicivirus capsid gene in endemically infected cat colonies.

Abstract: Feline calicivirus (FCV) is an important pathogen of domestic cats. In this study, we have determined the genetic diversity of FCV within four geographically separate colonies of endemically infected cats by sequencing the immunodominant and variable region E of the capsid gene. Comparison of isolates between colonies and between unrelated published sequences gave nucleotide distance values of 26-35% and 22-40%, respectively and suggested each colony was infected with a distinct virus strain. Comparison of isolates within individual endemically infected colonies showed nucleotide distance variability of 0-16%. This was greater than distances previously reported for epidemiologically related isolates from cases of acute disease (0-5%) and was consistent with the evolution of FCV from a single distinct ancestor sequence in each colony. The pattern of nucleotide substitutions generating the observed intra-colony diversity was associated with strong evidence for positive selection acting on immunodominant regions of the FCV capsid protein. We suggest that endemically infected colonies of cats may be important generators of genetic diversity for FCV and that this may ultimately lead to the generation of new strains.

Detailed computational analysis of a comprehensive set of group A rotavirus NSP4 proteins.

Abstract: Rotavirus infection causes diarrhea to humans, animals and birds. The NSP4 protein of Group A rotavirus has been recognized as a viral enterotoxin. This single protein plays important roles in viral pathogenesis and morphogenesis. Domains involved in structure and biologic functions have been proposed mainly based on the SA11 strain, a prototype of group A rotavirus. NSP4 has been classified into different genotypes based on sequence homology. These analyses are based on representative strains selected but not comprehensive. In this paper, we collected all NSP4 sequences in the GenBank and performed a detailed computational analysis. Our analysis of 176 NSP4 proteins in Groups A, B and C rotaviruses confirms that the recently published avian NSP4 sequences belong to a new genotype (Mori Y., Borgan M.A., Ito N., Sugiyama M. and Minamoto N., Virus Res 89, 145–151, 2002), besides the four known NSP4 genotypes of Group A mammalian rotaviruses. Significant differences were discovered in the physicochemical properties between the avian and mammalian NSP4 proteins. In particular, lack of a highly probable coiled-coil region in the avian sequences implies a diversion of the NSP4 quaternary structure from the latter, although the secondary and tertiary structures may be similar. Fourteen amino acids are found absolutely conserved in the Group A NSP4 sequences, regardless of genotype. Of the conserved residues, two are glycosylation sites, one is in the middle of the transmembrane segment, seven span the VP4 binding domain, and five are clustered in the middle of the toxic peptide region, indicating the functional importance of the conservation.

Phylogenetic relationships of West Nile viruses isolated from birds and horses in Israel from 1997 to 2001.

Abstract: In November 1997, an outbreak of a neuroparalytic disease caused by West Nile (WN) virus was diagnosed in young goose flocks. Domestic geese were similarly affected in the late summer and fall of 1998, 1999, 2000 and 2001. WN viruses were also isolated from migratory and wild birds and horses in 1998-2001. A 1278 bp sequence of the envelope gene of 24 Israeli WN virus isolates was compared with those of seven isolates from Africa, Europe and New York. As a result, the Israeli isolates could then be grouped into two clusters. The 15 avian and three equine from 1997-2001 in the first cluster of viruses were shown to be identical to WN-NY99, while the second cluster comprised one goose isolate from 1998 and two goose and two pigeon isolates from 2000. These closely resembled the most recent Old World isolates, and indicate that at least two WN genotypes were co-circulating in the region during this time.

Expression of interferon inducible genes following Hantaan virus infection as a mechanism of resistance in A549 cells.

Abstract: Hantaan virus (HTN) is a causative agent of hemorrhagic fever with renal syndrome (HFRS). Little is known of its pathogenesis or the molecular mechanisms underlying resistance to HTN infection. In the present study, DNA microarray technology was used to monitor changes in mRNA levels after HTN infection, to elucidate resistance mechanisms to viral infection by understanding virus-host interactions. We found that several interferon (IFN)-inducible genes were up-regulated in host cells infected with HTN. According to previous available data, IFNs have been reported to be inhibitory, but their mode of action has not been yet clear. In this study, the 2',5'-oligoadenylated synthetase (OAS) and Mx1 genes, not a double-stranded RNA-dependent protein kinase R (PKR), of the IFN response pathways are associated with antiviral activity during HTN infection. Furthermore, A549 cells treated with IFN-alpha were protected against HTN infection. Taken together, these results confirmed that IFN plays a role in cellular defenses against HTN infection at an early stage of the infection and revealed the resistance mechanism for HTN infection.

ORF 5 of grapevine virus A encodes a nucleic acid-binding protein and affects pathogenesis.

Abstract: A previous functional analysis of the genome of grapevine virus A (GVA) was not conclusive as to the role of open reading frame 5 (ORF 5). This ORF encodes a 10-kDa protein (p10) carrying two distinct domains: a basic, arginine-rich domain and a zinc-finger domain. P10 was cloned and expressed in Escherichia coli, and was shown by northwestern assays to interact with nucleic acids. In-frame deletion of the basic region abolished P10's nucleic acid-binding capability, whereas substitution of cysteine residues by serine in the zinc-finger domain did not affect binding. These mutations were inserted into the full-length infectious clone. It has been shown that ORF 5 mutations do not affect replication of GVA-RNA. However, plants inoculated with the aforementioned mutations did not develop symptoms, and Western blot analysis revealed markedly reduced expression of the movement protein (the product of ORF 3).

Impact of Deletions within the Bam HI-L Fragment of Attenuated Marek's Disease Virus on vIL-8 Expression and the Newly Identified Transcript of Open Reading Frame LORF4

Abstract: Marek's disease (MD) in chickens is caused by MD herpesvirus (MDV), which induces T cell lymphomas. The early pathogenesis of MDV infection is characterized by a primary infection in B lymphocytes followed by infection of activated T lymphocytes. It has been speculated that a MDV-encoded homologue of interleukin-8 (vIL-8) may be important to attract activated T lymphocytes to infected B lymphocytes. Recently, more virulent strains of MDV have emerged, named very virulent plus (vv+)MDV, that cause earlier and more prolonged cytolytic infections compared to less virulent strains. In this report, it was found that vIL-8 mRNA expression in vivo was increased in very virulent (vv) and vv+MDV strains compared to mild (m) and virulent (v) strains, and could not be detected in two attenuated MDV strains examined using very sensitive real-time quantitative reverse transcription–polymerase chain reaction (qRT-PCR) assays. In order to identify potential mechanisms for the increased vIL-8 mRNA expression in more virulent strains, and lack thereof in attenuated strains, the vIL-8 gene and putative promoter sequences upstream of the vIL-8 gene were compared from 10 different MDV strains, including attenuated derivatives. Only the JM-16 strain (both non-attenuated and attenuated) and attenuated 584A (584Ap80C) encoded a predicted vIL-8 gene sequence different from all other strains examined. Within the putative vIL-8 gene promoter sequence, there was little difference among the non-attenuated strains; however significant deletions were identified in the attenuated JM-16/p71, Md11 (R2/23), and 584Ap80C strains. Additionally, these deletions were located within a previously hypothetical open reading frame (ORF) named LORF4. Rapid amplification of cDNA ends identified a full-length transcript of LORF4 in the MDV-transformed lymphoblastoid cell line MSB-1, and deletions within this ORF caused truncated predicted proteins in 4 out of 6 attenuated MDV strains examined.

Evolutionary history of hepatitis B virus genotype F: An in-depth analysis of Argentine isolates

Abstract: A revised analysis on the evolutionary history of hepatitis B virus (HBV) genotype F is herein presented with the incorporation of two new complete genomes from Argentina. The study of the phylogenetic-tree topology, genetic distances, and amino acid mutations confirmed with high reliability the existence of four different genetic clusters of this genotype. Argentine isolates were located in two groups of viruses that showed a great inner homogeneity but, interestingly, divergence between them was in the order of that existing among groups from different locations. Although the origin of these two viral populations is not clear, they do not seem to derive from each other, therefore the existence of at least two founder viral populations in Argentina is a more acceptable explanation.

Cloning and sequence analysis of the spike gene of porcine epidemic diarrhea virus Chinju99.

Abstract: The spike (S) gene of the porcine epidemic diarrhea virus (PEDV) Chinju99 which was previously isolated in Chinju, Korea was cloned and sequenced to aid in the development of genetically engineered vaccines and diagnostic reagents against PEDV. The nucleotide sequence encoding the entire S gene open reading frame (ORF) of Chinju99 was 4152 bases long encoding 1383 amino acids. It consisted of 1001 adenine (24.1%), 849 cytosine (20.4%), 877 guanine (21.1%) and 1425 thymine (34.3%) residues. The Chinju99 S ORF nucleotide sequence was 94.5% homologous with that of the Br1/87 and CV777 strains, respectively. The Chinju99 S protein had 92.8% amino acid identity with that of Br1/87 and CV777, respectively. The amino acid sequence contained 27 potential sites for asparagine (N)-linked glycosylation and there was a stretch of highly hydrophobic residues at position 1325–1350.

Phylogenetic analysis of neuraminidase gene of H9N2 influenza viruses prevalent in chickens in China during 1995-2002.

Abstract: The neuraminidase (NA) genes of 12 H9N2 influenza virus strains isolated from diseased chickens in different farms in mainland China during 1995–2002 were amplified and sequenced. Amino acids at hemadsorbing (HB) site of these isolates are different from those of A/quail/Hong Kong/G1/97-like viruses and A/chicken/Korea/96-like viruses. Neuraminidases of the 12 strains had a deletion of 3 amino acid residues at positions 63–65 as compared to that of A/turkey/Wisconsin/189/66, while those of Korea and Pakistan H9N2 isolates had no deletion. Phylogenetic analyses showed NA gene of these isolates belonged to that of A/duck/Hong Kong/Y280/97-like virus lineage. NA gene of the H9N2 viruses isolated in Korea and Pakistan belonged to lineage different from those of the 12 isolates. The present results indicate that the NA of H9N2 strains isolated in mainland China during the past 8 years were well preserved and the geographical distribution play a significant role in the evolution of the H9N2 influenza viruses.

Cloning and characterization of a totivirus double-stranded RNA from the plant pathogenic fungus, Helicobasidium mompa Tanaka.

Abstract: Virus-like particles (VLPs, named HmTV1-17), about 40 nm in diameter were found in the violet root rot fungus Helicobasidium mompa Tanaka strain No. 17, which had been isolated from an apple tree. Purified preparations of HmTV1-17 contained two species of double-stranded RNA (dsRNA), designated 17L and 17S. cDNAs were constructed from HmTV1-17 genomic dsRNAs purified using CF-11 cellulose column chromatography. The sequences of 17L and 17S cDNA comprised 5207 and 2096 bp, respectively. Although 17S has no large open reading flame (ORF) on either strand, 17L has two large overlapping ORFs. The 5′ located ORF1 encodes the coat protein (CP, 788 amino acids), whereas the gene product of ORF2, which is in the −1 frame relative to ORF1, shows the typical features of a RNA dependent RNA polymerase (RDRP, 845 amino acids). Phylogenetic analysis based on RDRP showed that HmTV1-17 is closely related to Sphaeropsis sapinea SsRV1, a member of the genus Totivirus from filamentous fungus S. sapinea.

Phylogenetic analysis of hemagglutinin and neuraminidase genes of H9N2 viruses isolated from migratory ducks.

Abstract: Genetic analysis indicated that the pandemic influenza strains derived from wild aquatic birds harbor viruses of 15 hemagglutinin (HA) and 9 neuraminidase (NA) antigenic subtypes. Surveillance studies have shown that H9N2 subtype viruses are worldwide in domestic poultry and could infect mammalian species, including humans. Here, we genetically analyzed the HA and NA genes of five H9N2 viruses isolated from the migratory ducks in Hokkaido, Japan, the flyway of migration from Siberia during 1997–2000. The results showed that HA and NA genes of these viruses belong to the same lineages, respectively. Compared with those of A/quail/Hong Kong/G1/97-like and A/duck/Hong Kong/Y280/97-like viruses, HA and NA of the migratory duck isolates had a close relationship with those of H9N2 viruses isolated from the chicken in Korea, indicating that the Korea H9N2 viruses might be derived from the migratory ducks. The NA genes of the five isolates were located in the same cluster as those of N2 viruses, which had caused a human pandemic in 1968, indicating that the NA genes of the previous pandemic strains are still circulating in waterfowl reservoirs. The present results further emphasize the importance of carrying out molecular epidemiological surveillance of H9N2 viruses in wild ducks to obtain more information for the future human influenza pandemics preparedness.

Sequence analysis of the 3' end of three Grapevine fleck virus-like viruses from grapevine.

Abstract: The 3' end of the genome of three Grapevine fleck virus-like viruses, i.e. Grapevine redglobe virus (GRGV), Grapevine asteroid mosaic-associated virus (GAMaV), and an unidentified virus from a Greek grapevine (accession GR8-19) was amplified from reverse transcribed total nucleic acid extracts from infected grapevine tissues and sequenced. The analysed genome portions differed in size and organization. The 3' ends of GAMaV (1852 nt) and of GR8-19 (1791 nt) resembled that of marafiviruses, as both encoded a single putative polyprotein containing the conserved "marafibox" sequence and lacked the stop codon between the replicase and coat protein genes. By contrast, the replicase and coat protein genes present in the terminal 2006 nt of GRGV genome were clearly separated and there was a 3'-proximal open reading frame encoding a putative proline rich protein with molecular mass of c. 17 kDa. The genome of all three viruses was polyadenylated. The organization of the 3' terminal genomic region and phylogenetic analysis of viral replicases and coat proteins suggest that GAMaV and the Greek virus GR8-19 belong in the genus Marafivirus, and GRGV in the genus Maculavirus, family Tymoviridae. Virus GR8-19 had molecular traits differing enough from GAMaV and other marafiviruses to be regarded as a new putative species in the genus Marafivirus, for which the name of Grapevine rupestris vein feathering virus is proposed.

Coat Protein Sequence Analysis Reveals Occurrence of New Strains of Sweet Potato Feathery Mottle Virus in Uganda and Tanzania

Abstract: The 3'-proximal part (1.8 kb) of the Sweet potato feathery mottle virus (SPFMV) genome was studied in four SPFMV isolates collected from farmers' fields in western Uganda (SPFMV-Bny), eastern Uganda (SPFMV-Sor) and Bagamoyo district, Tanzania (SPFMV-TZ1 and SPFMV-TZ2). Unlike the other three SPFMV isolates, SPFMV-Sor was not detected with the polyclonal antisera to SPFMV. It showed moderately high coat protein (CP) nucleotide (93.3-96.7%) and amino acid (93.6-96.8%) sequence identity to the isolates of the SPFMV strain group C. In contrast, identities (78.1-80.1%, and 79.9-83.1%) to isolates of the SPFMV strain groups O, RC, and the East African (EA) strain group were low. Similar to some isolates (SPFMV-CH2 and SPFMV-6) of strain group C, but different from other SPFMV isolates, SPFMV-Sor contained a deletion of 6 nucleotides in the CP-encoding region (CP amino acid positions 62-63). Phylogenetic analysis of the CP sequences indicated that SPFMV-Sor belongs to the SPFMV strain group C that has not been reported from Africa. Sequence data were obtained for the first time from Tanzanian SPFMV isolates in this study, and phylogenetic analysis indicated that they belong to the strain group EA, which is unique to East Africa.

High-resolution methylation analysis and in vivo protein-DNA binding at the promoter of the viral oncogene LMP2A in B cell lines carrying latent Epstein-Barr virus genomes.

Abstract: Latency protein LMP2A of Epstein-Barr virus (EBV) has been implicated in EBV related tumorigenesis. To understand the host cell dependent expression of the LMP2A gene, it is necessary to analyse the regulatory mechanisms of the LMP2A promoter (LMP2Ap). By transient transfection and in vitro binding analyses two CBF1 sites have previously been shown to be involved in the regulation of LMP2Ap. However, the promoter structure has not been examined at the nucleotide level in vivo. Therefore we undertook a comprehensive analysis of in vivo protein binding and of CpG-methylation patterns at LMP2Ap in a panel of B cell lines carrying latent EBV genomes. The presence of characteristic footprints on two CBF1 and further binding-sites, together with overall hypomethylation of CpG dinucleotides correlated well with promoter activity. In contrast, the absence of several genomic footprints, as well as the presence of patches of highly methylated CpG dinucleotides were characteristic of silent LMP2Aps.

Marek's disease virus latent protein MEQ: delineation of an epitope in the BR1 domain involved in nuclear localization.

Abstract: Marek's disease virus latent protein MEQ (MDV Eco Q) is abundantly expressed and consistently detected in MDV-induced tumors and cell lines. Deletion mutants were constructed to study the domain structure of MEQ. Four deletion mutants were obtained in the basic regions of MEQ, namely basic region 1 (DeltaBR1), basic region 2 (DeltaBR2), basic regions 1 and 2 (DeltaBR1 and 2), and the C-terminal (bZIP) domain. The BR1 and BR2 are nuclear localization signals and either is sufficient to cause transport of MEQ into the nucleus. In addition, the BR2 is also responsible for MEQ's nucleolar localization. A monoclonal antibody (Mab 23B46) was produced using recombinant fowlpox virus (rFPV) expressing MEQ (rFPV/MEQ) as a source of protein. The isotype of Mab 23B46 is IgG1 and immunoprecipitated a band in rFPV/MEQ infected cells with molecular weight of 60 kDa specific to MEQ protein. We detected abundant expression of MEQ in (rFPV/MEQ), recombinant baculovirus (rBac) (rBac/MEQ), and lymphoid tumors induced by MDV. In order to delineate the epitope of MEQ reactive with Mab 23B46, we used four deletion mutants from the basic and bZIP domains. We found the deletions in the N-terminal region including BR1 (DeltaBR1), and (DeltaBR1 and 2) completely abolished the specific binding with Mab 23B46 as shown by Western blot analysis and immunofluoresence test. Deletion of BR2 (DeltaBR2) and the C-terminal (bZIP) domain had no effect on antibody binding. These data provide direct evidence that monoclonal antibody reactive epitope is localized in the BR1 domain of the molecule. Since both BR1 and BR2 domains contain sequences important for nuclear entry, we now have reagent to further study and elucidate the mechanism of MEQ's involvement in nuclear and nucleolar localization.

Variants of human papillomavirus types 53, 58 and 66 identified in Central Brazil.

Abstract: The present study on molecular characterization of human papillomaviruses occurring in Central Brazil, describes two variants each of HPV-53 and HPV-58 and one variant of HPV-66 detected in samples from smears of women showing cervical intraepithelial neoplasia grade II (CIN II). Samples were assayed by PCR using MY09/ MY11 consensus primers, followed by restriction fragment length polymorphism typing. The five isolates showed atypical restriction fragment length profile and MY09/MY11 L1 PCR products were subsequently sequenced. Isolate Bsb-02 and Bsb-08 showed, respectively, 99% similarity to HPV-58 IS404 and 100% to HPV-58 IS417 previously described in the African Continent. Isolates Bsb-61 and Bsb-63 showed 98% similarity to HPV-53, and isolate Bsb-68, 97% similarity to HPV-66. Amino acid substitutions were found in two samples: one in Bsb-02 (T to N) at position 375 and the other in Bsb-61 (S to C) at position 343. Although all the substitutions in Bsb-68 proved to be silent, this sample showed the highest value of pairwise evolutionary distance (2.05%). In countries such as Brazil, where the virus prevalence is high and ethnicity, as well as socio-demographic characteristics, vary according to different regions, HPV variability must be wider and not yet clearly defined.

Identification and classification of the Spodoptera littoralis nucleopolyhedrovirus inhibitor of apoptosis gene.

Abstract: Baculoviruses possess two types of genes that suppressed apoptosis, p35 and inhibitor of apoptosis (iap). In this study we report the isolation and identification of an inhibitor of apoptosis gene Sliap in the genome of the Spodoptera littoralis nucleopolyhedrovirus (SINPV). The Sliap sequence predicted a 15 kDa polypeptide with only one BIR domain and a RING finger, both motifs characteristic of the IAP family of proteins, and a third specific acidic-rich motif. These characteristics, shared with the Spodoptera littura NPV IAP2/3, Epiphyas postvittana NPV IAP4, Lymantria dispar NPV IAP and Orgyia pseudotsugata NPV IAP4 (Orf 107) allowed us to classify them in a new homology group (IAP-4). Sliap was able to delay, but not to suppress, apoptosis induced by replication of a recombinant AcMNPV deficient in p35. In SINPV infected-SF9 cells Sliap was expressed earlier than sl-p49 suggesting that its role at the initiation of infection was to delay the apoptotic response of the host.

Cloning and sequencing of coat protein gene of an Indian potato leaf roll virus (PLRV) isolate and its similarity with other members of Luteoviridae.

Abstract: An Indian strain of potato leaf roll virus (PLRV) was purified to generate complementary DNA corresponding to the coat protein (CP) gene. Virus cDNA was synthesized from purified viral RNA using oligo (dT)-anchor primer and virus specific primers. The viral sequence encoding the coat protein was specifically amplified by polymerase chain reaction (PCR), using specific primers bordering the CP gene. The unique amplified product thus obtained was A-T cloned into the pGEM-T Easy vector and the authenticity of the cloned gene verified by dot blot hybridization and sequence analysis. Run-way-transcripts of the cloned CP gene could detect PLRV in tissue imprints and tissue dilution. The nucleotide sequences and the deduced amino acid sequences were compared with the other PLRV isolates and found to be 97-99% identical at both the nucleotide and amino acid sequence level of other isolates. Multiple sequence alignment of deduced amino acid sequences revealed considerable homology to other luteoviruses. A nuclear localization signal located close to the N-terminus of the CP gene was predicted. This is the first report of PLRV coat protein sequence from an Indian strain.

Matrix protein gene sequence analysis of avian paramyxovirus 1 isolates obtained from pigeons.

Abstract: The matrix protein gene was cloned and sequenced for several recent isolates of avian paramyxovirus type 1 (APMV-1). Specifically, isolates from pigeons and doves, members of the Columbidae family were examined. APMV-1 is the causative agent of Newcastle disease and the virus is associated with disease among a diverse number of avian species. Newcastle disease virus (NDV) isolates from pigeons have also been classified as pigeon paramyxovirus type 1 (PPMV-1). Matrix protein gene sequences for PPMV-1 isolates clustered together as a group relative to isolates from other species phylogenetically. However, there were also isolates from pigeons or doves that grouped with APMV-1 isolates from other species. This indicates that PPMV-1 may be circulating among Columbidae members as a distinct lineage, but that these avian species may also harbor other NDV strains as well. Of particular interest was a dove isolate from Europe that had an aberrant fusion protein cleavage site and was an outlying member phylogenetically between the two major groups of APMV-1 isolates.

Sequence analysis of a 5.1 kbp region of the Spodoptera frugiperda multicapsid nucleopolyhedrovirus genome that comprises a functional ecdysteroid UDP-glucosyltransferase (egt) gene.

Abstract: The ecdysteroid UDP-glucosyltransferase gene from the Spodoptera frugiperda multicapsid nucleopolyhedrovirus (SfMNPV) was identified using degenerate primers whose sequence were derived from conserved regions of the EGT proteins encoded by other baculoviruses Analysis of the gene sequence revealed the presence of an open reading frame (ORF) with potential to encode a polypeptide of 525 amino acids Promoter sequences typical of baculovirus genes were found in the 5' region of this ORF A polyadenylation signal was identified downstream the translation stop codon A transient expression assay showed that the product of this ORF was able to conjugate glucose from UDP-glucose with ecdysone confirming that the gene identified was indeed the SfMNPV egt gene The SfMNPV egt gene and the sequences of other baculovirus egt genes were used to infer a phylogenetic tree The nucleotide sequence of the entire BamHI fragment that contains the SfMNPV egt gene was determined Search of the available sequence databases suggested that, besides the egt gene, this region contains 5 ORFs similar to the baculovirus genes gp37 (fusolin), to ptp2 and to ORFs 28, 29, and 30 of Spodoptera exigua multicapsid nucleopolyhedrovirus Both the phylogenetic analysis of the egt genes and the gene order of the region that flanks the egt gene indicated that SfMNPV is closely related to the baculoviruses that infects S exigua and Mamestra configurata

Molecular analysis of Fiji Disease Fijivirus genome segments 1 and 3

Abstract: Fiji disease fijivirus (FDV) genomic segments 1 (S1) and 3 (S3) were completely sequenced. FDV S1 comprised 4532thinspnt and was predicted to encode a 170.6thinspkDa protein. FDV S3 comprised 3623thinspnt and was predicted to encode a 135.5thinspkDa protein. The terminal sequences of S1 and S3 were 5prime AAGUUUUU......CAGCUAGCGUC 3prime and 5prime AAGUUUUU......CAGCAGAUGUC 3prime, respectively, and located immediately adjacent to these sequences were 12thinspbp imperfect inverted repeats. The predicted translation product of FDV S1 showed highest similarity to Rice black-streaked dwarf virus (RBSDV) S1 and is thought to encode the viral RNA-dependent RNA polymerase (RdRp). The predicted translation product of FDV S3 was found to be most similar to RBSDV S4 which is thought to encode the 'B-spike' protein. The FDV sequence contained an ATP/GTP binding motif and a leucine zipper motif, but these motifs were not found in the RBSDV sequence. Phylogenetic analysis based on the amino acid sequences of the RdRp of FDV S1 and other reoviruses revealed that the fijiviruses form a cluster close to the oryzaviruses. The RdRp sequences were grouped into genera that were consistent with the current reovirus classification scheme that is based on physico-chemical and biological properties.

A carboxy-terminal region of the hepatitis B virus X protein promotes DNA interaction of CREB and mimics the native protein for transactivation function.

Abstract: Earlier we had shown that the conserved region E (residues 120-140) of HBV X protein (HBx) is crucial for transactivation. To investigate this region further, its oligomerisation was considered necessary to augment intracellular biochemical stability. Two to ten unit long tandem repeats of the E region (X16-n) were generated and their expression vectors constructed. Transient transfection of the E expression vectors along with different CAT constructs showed increase in the reporter activity. Interestingly a direct correlation was observed between the number of E repeat units in an expression vector and the level of transactivation. The transactivation levels with decameric X16 on different reporter constructs were comparable to those of the wild type HBx. Co-expression of X16 in a stable CHO-K1 cell line expressing the native HBx, showed co-operativity for transactivation. Further, X16 facilitated the binding of cAMP response element binding protein (CREB) to its responsive element just like the native HBx. The present study suggests that the C-terminal 'E' region of HBx represents its transactivation domain that acts by promoting the interaction of transcription factors to their cognate response elements.