The potential role of a late gene expression factor, lef2, from Bombyx mori nuclear polyhedrosis virus in very late gene transcription and DNA replication

Several late gene expression factors (Lefs) have been implicated in fostering high levels of transcription from the very late gene promoters of polyhedrin and p10 from baculoviruses. We cloned and characterized from Bombyx mori nuclear polyhedrosis virus a late gene expression factor (Bmlef2) that encodes a 209-amino-acid protein harboring a Cys-rich C-terminal domain. The temporal transcription profiles of lef2 revealed a 1.2-kb transcript in both delayed early and late periods after virus infection. Transcription start site mapping identified the presence of an aphidicolin-sensitive late transcript arising from a TAAG motif located at -352 nucleotides and an aphidicolin-insensitive early transcript originating from a TTGT motif located 35 nucleotides downstream to a TATA box at -312 nucleotides, with respect to the +1 ATG of lef2. BmLef2 trans-activated very late gene expression from both polyhedrin and p10 promoters in transient expression assays. Internal deletion of the Cys-rich domain from the C-terminal region abolished the transcriptional activation. Inactivation of Lef2 synthesis by antisense lef2 transcripts drastically reduced the very late gene transcription but showed little effect on the expression from immediate early promoter. Decrease in viral DNA synthesis and a reduction in virus titer were observed only when antisense lef2 was expressed under the immediate early (ie-1) promoter. Furthermore, the antisense experiments suggested that lef2 plays a direct role in very late gene transcription.

A 3-hour quantitative comparison of glucose-based versus rice-based oral rehydration solution intake by children with diarrhoea in Port Moresby General Hospital

Measurements were made of the intake of a WHO/UNICEF glucose-based and a rice cereal-based oral rehydration solution (ORS) by children with diarrhoea. Twenty children who presented to the Children's Outpatient Department at Port Moresby General Hospital with acute diarrhoea and mild dehydration were randomly assigned to an ORS and measurements were taken over the following 3 hours. For data analysis, the patients were paired by weight. Testing the means of the paired samples by t test showed that there was no significant difference between the amount of rice ORS and the amount of glucose ORS taken over 3 hours. The discovery of oral rehydration solution (ORS) for the treatment of diarrheal disease has been heralded as the most important medical discovery of the century. Cereal-based ORS is able to decrease stool output and the duration of diarrheal illness more than the standard glucose-based ORS, through the increased absorption provided by oligosaccharides without the imposition of a greater osmotic penalty. Moreover, the peptides in cereals enhance amino acid and water absorption, while providing nutritional benefits. UNICEF's glucose-based ORS is becoming more widely used in Papua New Guinea (PNG). 20 children aged 6-37 months (mean age, 15 months) who presented to the Children's Outpatient Department at Port Moresby General Hospital during September-October 1993 with acute diarrhea and mild dehydration were randomly assigned to receive either a rice-based ORS or standard glucose ORS, and measurements were taken over the following 3 hours. The patients were paired by weight for analysis. No statistically significant difference was found between the amount of rice ORS and the amount of glucose ORS taken over 3 hours.

Acceptability of a rice-based oral rehydration solution in Port Moresby General Hospital's Children's Outpatient Department

The guardians of children brought to the Port Moresby General Hospital's Children's Outpatient Department with a chief complaint of diarrhoeal disease were questioned regarding their preference of glucose-based vs rice-based oral rehydration solution (ORS) in order to determine the acceptability of a rice-based ORS. Of the 93 guardians interviewed, greater than 60% preferred the glucose-based solution in its mixability, appearance and taste, and 65% initially reported that their children preferred the taste of the glucose solution. However, after a 30-minute trial, only 58% of children still preferred the glucose solution. In a country where diarrhoeal disease is a leading cause of child death and guardians are the primary health care providers, the acceptability of an ORS is critical to the morbidity and mortality of Papua New Guinea's children. Killing an estimated 2.9 million children annually, diarrheal disease is the second leading cause of child mortality worldwide. Diarrheal disease is also the second leading cause of child mortality in Papua New Guinea (PNG), killing an average 193 inpatient children per year over the period 1984-90. However, despite the high level of diarrhea-related mortality and the proven efficacy of oral rehydration therapy (ORT) in managing diarrhea-related dehydration, standardized ORT has been underutilized in PNG. The current glucose-based oral rehydration solution (ORS) does not reduce the frequency or volume of a child's diarrhea, the most immediate concern of caregivers during episodes of illness. Cereal-based ORS, made from cereals which are commonly available as food staples in most countries, better address the short-term concerns of caregivers while offering a superior nutritional profile. A sample of guardians of children brought to the Port Moresby General Hospital's Children's Outpatient Department complaining of child diarrhea were asked about their preferences on glucose-based versus rice-based ORS in order to determine the acceptability of a rice-based ORS. More than 60% of the 93 guardians interviewed preferred the glucose-based solution for its mixability, appearance, and taste. 65% initially reported that their children preferred the taste of the glucose solution. However, after a 30-minute trial, only 58% of children still preferred the glucose solution.

Immunogenic and protective properties of haemagglutinin protein (H) of Rinderpest virus expressed by a recombinant baculovirus

The hemagglutinin (H) protein of Rinderpest virus expressed by a recombinant buculovirus used as a vaccine produced high titres of neutralizing antibody to Rinderpest virus in the vaccinated cattle, comparable to the levels produced by live attenuated vaccine. The immunized cattle were protected against a vaccine-virus challenge, as demonstrated by the failure of development of antibodies to N protein of the vaccine virus. The lack of replication of vaccine virus in the immunized cattle indicated that they are capable of showing a protective response if challenged with a virulent virus.

Protection of rabbits against lapinized rinderpest virus with purified envelope glycoproteins of peste-des-petits-ruminants and rinderpest viruses

Haemagglutinin (HA) and fusion (F) proteins of peste-des-petits-ruminants virus (PPRV) and rinderpest virus (RPV) were purified by immunoaffinity chromatography. The purified proteins were characterized by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Rabbit hyperimmune sera were raised against the purified HA and F proteins and assayed by enzyme-linked immunosorbent assay (ELISA), haemagglutination-inhibition (HAI) and virus neutralization (VN) tests. The immunized animals were challenged with a virulent lapinized (rabbit-adapted) strain of RPV: Both HA and F proteins of PPRV protected rabbits against a lethal challenge with lapinized RPV. As expected, RPV HA and F proteins also conferred a similar protection against the homologous challenge. The postchallenge antibody responses were of a true anamnestic type.

Heat shock protein 70 on Neuro2a cells is a putative receptor for Japanese encephalitis virus

Japanese encephalitis virus (JEV) envelope (E) protein has been shown to play a critical role in attachment to cells. However, the receptor interacting with envelope protein has not been conclusively identified. Using mouse neuroblastoma (Neuro2a) cells and purified JEV-E protein in `Virus Overlay Protein Binding Assay' followed by MALDI-TOF analysis, we identified `heat shock protein 70' (Hsp70) as a possible receptor for JEV. Indirect immunofluorescence and flow-cytometry analysis demonstrated localization of Hsp70 on Neuro2a cell surface. Co-immunoprecipitation followed by Western blot analysis reconfirmed the interaction between Hsp70 and JEV-E protein. Further, anti-Hsp70 polyclonal-antibodies were able to block JEV entry into Neuro2a cells. Additionally, using the bioinformatic tool - FTDOCK, clocking between the proteins was performed. Amongst six interacting structural poses studied one pose involving RGD motif on JEV-E and leucine(539) on Hsp70 displayed stable interaction. These observations indicate that Hsp70 serves as putative receptor for JEV in Neuro2A cells.

Organization and bidirectional transcription of H2A, H2B and H4 histone genes in rice embryos

Our current understanding of the evolution of the histone gene family suffers from a lack of information on plant histone genes1. With a view to gathering some much needed information on these genes, we studied a rice genomic clone in pBR322 carrying H2A, H2B and H4 histone genes on a DNA fragment2 of 6.64 kilobases (kb). A restriction map of the insert was constructed and the organization of the three genes on this insert was determined. H2A and H2B histone genes were located at one end of the insert and H4 gene at the other with a 3.1 kb spacer in between. This cluster of three histone genes was found to be transcribed in a bidirectional fashion with H2A and H2B genes being encoded by one strand and the H4 gene by the other. These results indicate that plant histone gene organization differs from that of the sea urchin, but shows many similarities to the systems in other animals.

Inhibition of avian myeloblastosis virus reverse transcriptase and virus inactivation by metal complexes of isonicotinic acid hydrazide

The cupric and ferric complexes of isonicotinic acid hydrazide (INH) inhibit the DNA synthesis catalysed by avian myeloblastosis virus (AMV) reverse transcriptase. The inhibition was to the extent of 95% by 50 μM of cupric-INH complex and 55% by 100 μM of ferric-INH complex. These complexes have been found to bind preferentially to the enzyme than to the template-primer. Kinetic analysis showed that the cupric-INH complex is a non-competitive inhibitor with respect to dTTP. The time course of inhibition has revealed that the complexes are inhibitory even after the initiation of polynucleotide synthesis. In vivo toxicity studies in 1-day-old chicks have shown that the complexes are not toxic up to a concentration of 500 μg per chick. Infection of the 1-day-old chicks with AMV pretreated with 150 μg of either of the complexes prevented symptoms of leukemia due to virus inactivation.

Analysis of human immunodeficiency virus type 1 drug resistance in children receiving nucleoside analogue reverse-transcriptase inhibitors plus nevirapine, nelfinavir, or ritonavir (Pediatric AIDS Clinical Trials Group 377)

In Pediatric AIDS Clinical Trials Group 377, antiretroviral therapy-experienced children were randomized to 4 treatment arms that included different combinations of stavudine, lamivudine (3TC), nevirapine (Nvp), nelfinavir (Nfv), and ritonavir (Rtv). Previous treatment with zidovudine (Zdv), didanosine (ddI), or zalcitabine (ddC) was acceptable. Drug resistance ((R)) mutations were assessed before study treatment (baseline) and at virologic failure. Zdv(R), ddI(R), and ddC(R) mutations were detected frequently at baseline but were not associated with virologic failure. Children with drug resistance mutations at baseline had greater reductions in virus load over time than did children who did not. Nvp(R) and 3TC(R) mutations were detected frequently at virologic failure, and Nvp(R) mutations were more common among children receiving 3-drug versus 4-drug Nvp-containing regimens. Children who were maintained on their study regimen after virologic failure accumulated additional Nvp(R) and 3TC(R) mutations plus Rtv(R) and Nfv(R) mutations. However, Rtv(R) and Nfv(R) mutations were detected at unexpectedly low rates.

Cloning Of Rice Dna And Identification Of Transfer-Rna Gene Clones

THE rapid development of recombinant DNA technology has brought forth a revolution in biology'>", it aids us to have a closer look at the 'way genes are organized, eS11 ecially in the complex eucaryotic genornes'<", Although many animal and yeast genes have been studied in detail using recombinant DNA technology, plant genes have seldom been targets for such studie., Germination is an ideal process to study gene expression .because it effects a . shift in the metabolic status of seeds from a state of 'dormancy to an active one. AJ;l understanding of gene organization and regulation darin.g germination can be accomplblted by molecular cloning of DNA from seeds lik.e rice. To study the status of histone, rRNA tRNA and other genes in the rice genome, a general method was developed to clone eucarvotic DNA in a' plasmid vector pBR 322. This essentially ~ involves the following steps. The rice embryo and plasmid pBR 322 DNAs were cut witll restriction endonuclease Bam Hi to generate stick.Y ends, The plasmid DNA was puosphatased, the DNA~ ware a~·tnealed and joined 'by T4 phage DNA ligase. The recombinant DNA molecules thus produced were transjerred into E. coli and colonies containing them Were selected by their sensitivity to tetracycline and resistance to ampicillin, Two clones were identified . 2S haVing tRNA genes by hybridization of the DNA in the clones \vitl1 32P-la.belled rice tRNAs.

Abacá mosaic virus: a distinct strain of Sugarcane mosaic virus

Abacá mosaic virus (AbaMV) is related to members of the sugarcane mosaic virus subgroup of the genus Potyvirus. The ~2 kb 3′ terminal region of the viral genome was sequenced and, in all areas analysed, found to be most similar to Sugarcane mosaic virus (SCMV) and distinct from Johnsongrass mosaic virus (JGMV), Maize dwarf mosaic virus (MDMV) and Sorghum mosaic virus (SrMV). Cladograms of the 3′ terminal region of the NIb protein, the coat protein core and the 3′ untranslated region showed that AbaMV clustered with SCMV, which was a distinct clade and separate from JGMV, MDMV and SrMV. The N-terminal region of the AbaMV coat protein had a unique amino acid repeat motif different from those previously published for other strains of SCMV. The first experimental transmission of AbaMV from abacá (Musa textilis) to banana (Musa sp.), using the aphid vectors Rhopalosiphum maidis and Aphis gossypii, is reported. Polyclonal antisera for the detection of AbaMV in western blot assays and ELISA were prepared from recombinant coat protein expressed in E. coli. A reverse transcriptase PCR diagnostic assay, with microtitre plate colourimetric detection, was developed to discriminate between AbaMV and Banana bract mosaic virus, another Musa-infecting potyvirus. Sequence data, host reactions and serological relationships indicate that AbaMV should be considered a distinct strain of SCMV, and the strain designation SCMV-Ab is suggested.

First report of Tomato spotted wilt virus in chickpea (Cicer arietinum) in Australia

Tomato spotted wilt virus (genus Tospovirus) is recorded on chickpea (Cicer arietinum) in Australia for the first time. It caused shoot tip symptoms of wilting, necrosis, bunching and chlorosis, followed by premature death of plants.

First records of the papaya strain of Papaya ringspot virus (PRSV-P) in French Polynesia and the Cook Islands

The papaya strain of Papaya ringspot virus (PRSV-P), the cause of papaya ringspot disease, was confirmed in French Polynesia and the Cook Islands by double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA). In French Polynesia, the virus has probably been on the islands of Tahiti and Moorea for several years, but appears not to have spread to eight other islands. In contrast, PRSV-P has only recently appeared in the Cook Islands and is now the subject of an eradication campaign.

Nucleocapsid gene variability reveals two subgroups of Lettuce necrotic yellows virus

The complete nucleocapsid (N) genes of eight Australian isolates of Lettuce necrotic yellows virus (LNYV) were amplified by reverse transcription PCR, cloned and sequenced. Phylogenetic analyses of these sequences revealed two distinct subgroups of LNYV isolates. Nucleotide sequences within each subgroup were more than 96% identical but heterogeneity between groups was about 20% at the nucleotide sequence level. However, less than 4% heterogeneity was noted at the amino acid level, indicating mostly third nucleotide position changes and a strong conservation for N protein function. There was no obvious geographical or temporal separation of the subgroups in Australia.

Genetic variability of Tomato spotted wilt virus in Australia and validation of real time RT-PCR for its detection in single and bulked leaf samples

The potential for large-scale use of a sensitive real time reverse transcription polymerase chain reaction (RT-PCR) assay was evaluated for the detection of Tomato spotted wilt virus (TSWV) in single and bulked leaf samples by comparing its sensitivity with that of DAS-ELISA. Using total RNA extracted with RNeasy® or leaf soak methods, real time RT-PCR detected TSWV in all infected samples collected from 16 horticultural crop species (including flowers, herbs and vegetables), two arable crop species, and four weed species by both assays. In samples in which DAS-ELISA had previously detected TSWV, real time RT-PCR was effective at detecting it in leaf tissues of all 22 plant species tested at a wide range of concentrations. Bulk samples required more robust and extensive extraction methods with real time RT-PCR, but it generally detected one infected sample in 1000 uninfected ones. By contrast, ELISA was less sensitive when used to test bulked samples, once detecting up to 1 infected in 800 samples with pepper but never detecting more than 1 infected in 200 samples in tomato and lettuce. It was also less reliable than real time RT-PCR when used to test samples from parts of the leaf where the virus concentration was low. The genetic variability among Australian isolates of TSWV was small. Direct sequencing of a 587 bp region of the nucleoprotein gene (S RNA) of 29 isolates from diverse crops and geographical locations yielded a maximum of only 4.3% nucleotide sequence difference. Phylogenetic analysis revealed no obvious groupings of isolates according to geographic origin or host species. TSWV isolates, that break TSWV resistance genes in tomato or pepper did not differ significantly in the N gene region studied, indicating that a different region of the virus genome is responsible for this trait.