Comparison of the genome sequences and the phylogenetic analyses of the GP78 and the Vellore P20778 isolates of Japanese encephalitis virus from India

The nucleotide sequence of the complete genomes of two Indian isolates of Japanese encephalitis virus were compared. One of these isolates, GP78 was obtained from northern India in 1978. The other, the Vellore P20778 isolate, was obtained from southern India in 1958. There was 4.40% nucleotide sequence divergence between the two Indian isolates that resulted in a 1.86% amino acid sequence divergence. Phylogenetic analyses showed that in evolutionary terms the north Indian GP78 isolate was close to the SA14 isolate from China whereas the south Indian Vellore P20778 isolate was close to the Beijing-1 isolate, also from China. The two Indian isolates, however, appear to have evolved independently

Sequence of Ovine Adenovirus Homologs for 100K Hexon Assembly, 33K, pVIII, and Fiber Genes: Early Region E3 Is Not in the Expected Location

AbstractOvine adenovirus OAV287 was previously isolated from sheep in Western Australia. As a first step in characterizing the genome of this virus we have determined the sequence of its genome between map units 65 and 81. This region was expected to contain the nonessential E3 region which, in other adenoviruses, lies between the genes encoding the pVIII and fiber proteins, although its size and complexity varies. OAV287 genes coding for the hexon assembly, 33K, pVIII, and fiber proteins were identified by their homologies with human Ad2. These genes lie in the same relative positions in the OAV287 genome, but the intergenic region between the pVIII and the fiber genes is only 197 nucleotides and these appear to be incapable of ceding for any protein. Thus, the ovine adenovirus E3 region is not present in the expected location. In addition, using cDNA synthesis, PCR amplification, and nucleotide sequencing we determined the location of splice junctions and transcription termination signals in mRNA species encoding these proteins. This showed that a family of variably spliced L4 RNAs is produced and that the region between the pVIII and the fiber genes contains several signals for RNA synthesis and processing. As the E3 region in human adenoviruses is nonessential for replication, in many instances it has been replaced with foreign DNA during the construction of recombinants. Because of this unexpected difference in the organization of the OAV287 genome further experimentation will be required to determine whether potential vaccine recombinants can be constructed for this adenovirus by making insertions into the pVIII/fiber intergenic region

Japanese encephalitis: pathogenesis, prophylactics and therapeutics

Japanese encephalitis (JE) is one of the most dreaded mosquito-borne viral encephalitis known to afflict humans. The Japanese encephalitis virus (JEV) is a neurotropic flavivirus that affects the CNS, causing extensive damage that may lead to fatality in about one third of patients. Half of the survivors suffer from severe neuropshychiatric sequelae. With nearly 3 billion people living under the current JE-endemic region, recurring incidents of epidemic are being reported at regular intervals. With no established antiviral therapies against JE available, vaccination has been the only way of preventing JE. Two types of JE vaccines are currently in vogue although the safety of administering them is questionable, in certain individuals. Thus, there is a need to develop a safe, affordable and potent JE vaccine and this review addresses the current efforts in this direction. This review also focuses on the pathophysiology of JE and efforts towards a possible breakthrough in anti-JEV therapy

The homologous region sequence (hr1) of Autographa californica multinucleocapsid polyhedrosis virus can enhance transcription from non-baculoviral promoters in mammalian cells

The Autographa californica multinucleocapsid polyhedrosis virus homologous region sequence hr1 enhances transcription from the viral polyhedrin promoter in Spodoptera frugiperda insect cells and independently functions as an origin of replication (ori) sequence. The binding of the host nuclear protein, hr1-binding protein (hr1-BP), is crucial for the enhancer activity (Habib, S., Pandey, S., Chatterji, U., Burma, S., Ahmad, R., Jain, A., and Hasnain, S. E. (1996) DNA Cell Biol. 15, 737-747 and Habib, S., and Hasnain, S. E. (1996) J. Biol. Chem. 271, 28250-28258). We demonstrate that hr1 can also enhance transcription from non-baculoviral promoters like cytomegalovirus and hsp70 in mammalian cells but does not support ori activity in these cells. Unlike insect cells, hr1 can also function in mammalian cells as an enhancer when present in trans. hr1 DNA sequence binds with high affinity and specificity to nuclear factors in the mammalian cells. The insect hr1-BP- and the hr1-BP-like proteins from mammalian cells (mhr1-BP) have different properties with respect to ion requirements, DNA groove binding, and molecular size. When mammalian cells are infected with a recombinant baculovirus containing two promoters, the baculovirus polyhedrin and Drosophila hsp70 gene promoter, the hsp70 gene promoter alone is active in these cells, and this activity is further enhanced by the presence of an additional hr1 in the recombinant virus. hr1 may thus also have a role in baculovirus-mediated gene delivery in mammalian cells

Efficacy of a monovalent human-bovine (116E) rotavirus vaccine in Indian children in the second year of life

Rotavirus gastroenteritis is one of the leading causes of diarrhea in Indian children less than 2 years of age. The 116E rotavirus strain was developed as part of the Indo-US Vaccine Action Program and has undergone efficacy trials. This paper reports the efficacy and additional safety data in children up to 2 years of age. In a double-blind placebo controlled multicenter trial, 6799 infants aged 6-7 weeks were randomized to receive three doses of an oral human-bovine natural reassortant vaccine (116E) or placebo at ages 6, 10, and 14 weeks. The primary outcome was severe (≥11 on the Vesikari scale) rotavirus gastroenteritis. Efficacy outcomes and adverse events were ascertained through active surveillance. We randomly assigned 4532 and 2267 subjects to receive vaccine and placebo, respectively, with over 96% subjects receiving all three doses of the vaccine or placebo. The per protocol analyses included 4354 subjects in the vaccine and 2187 subjects in the placebo group. The overall incidence of severe RVGE per 100 person years was 1.3 in the vaccine group and 2.9 in the placebo recipients. Vaccine efficacy against severe rotavirus gastroenteritis in children up to 2 years of age was 55.1% (95% CI 39.9 to 66.4; p<0.0001); vaccine efficacy in the second year of life of 48.9% (95% CI 17.4 to 68.4; p=0.0056) was only marginally less than in the first year of life [56.3% (95% CI 36.7 to 69.9; p<0.0001)]. The number of infants needed to be immunized to prevent one episode of severe RVGE in the first 2 years of life was 40 (95% CI 28.0 to 63.0) and for RVGE of any severity, it was 21 (95% CI 16.0 to 32.0). Serious adverse events were observed at the same rates in the two groups. None of the eight intussusception events occurred within 30 days of a vaccine dose and all were reported only after the third dose. The sustained efficacy of the 116E in the second year of life is reassuring

Single cell protein production by photosynthetic bacteria grown on the clarified effluents of biogas plant

Anaerobically digested cow dung was separated by centrifugation into solid residue and liquid supernatant fractions. Clarified supernatant fraction, rich in volatile fatty acids, supported the growth of photosynthetic bacteria. Single cell protein from different photosynthetic bacteria, grown on clarified supernatant, was found to be rich in essential and sulphur amino acids. Rhodopseudomonas capsulata produced the best single cell protein

High-level synthesis of Johnson grass mosaic virus coat protein in Escherichia coli and its auto-assembly to form virus-like particles

The coat protein (CP) of Johnson grass mosaic virus (JGMV) auto-assembles to form virus-like particles (VLPs) and hence could be useful for presenting small peptides to the immune system. We are therefore attempting to synthesize JGMV CP in large amounts in Escherichia coli. The JGMV CP-encoding DNA, cloned under the bacteriophage T7 promoter, showed only low levels of CP synthesis in E. coli. The predicted secondary structure of the CP mRNA showed that its translational initiation codon was part of a stable hairpin-loop structure. The initiation codon could be relieved of the hairpin-loop structure by substitution of three neighboring nucleotides. This resulted in a single amino acid change at the N-terminus of the protein. The modified RNA translated very efficiently, resulting in at least 16-fold higher CP accumulation in E. coli. The N-terminal amino acid substitution did not affect CP folding, as it auto-assembled in E. coli to form VLPs

Mov34 Protein from Mouse Brain Interacts with the 3′ Noncoding Region of Japanese Encephalitis Virus

The plus-sense RNA genome of Japanese encephalitis virus (JEV) contains noncoding regions (NCRs) of 95 and 585 bases at its 5′ and 3′ ends, respectively. The last 83 nucleotides of the 3′-NCR are predicted to form stable stem-loop (SL) structures. The shape of this 3′-SL structure is highly conserved among divergent flaviviruses even though only small stretches of nucleotide sequence contained within these structures are conserved. These SL structures have been predicted to function as cis-acting signals for RNA replication and as such may bind to viral and cellular proteins that may be involved in viral replication. We have studied the interaction of the JEV 3′-NCR RNA with host proteins using gel retardation assays. We show that the JEV 3′-SL structure RNA forms three complexes with proteins from the S100 cytoplasmic extract prepared from the neonatal mouse brain. These complexes could be obtained in the presence of 200 mM KCl, indicating that the RNA-protein interaction may be physiologically relevant. UV-induced cross-linking and Northwestern blotting analyses detected three proteins with apparent molecular masses of 32, 35, and 50 kDa that bound to the JEV 3′-SL structure RNA. Screening of the neonatal mouse brain cDNA library with the JEV 3′-SL structure RNA identified a 36-kDa Mov34 protein interacting with it. Competition experiments using the RNA extracted from JEV virions established that the 36-kDa Mov34 protein indeed bound to the JEV genome. Murine Mov34 belongs to a family of proteins whose members have been shown to be involved in RNA transcription and translation. It is, therefore, likely that the murine Mov34 interaction with JEV 3′-NCR has a role in RNA replication

A Japanese Encephalitis Virus Peptide Present on Johnson Grass Mosaic Virus-Like Particles Induces Virus-Neutralizing Antibodies and Protects Mice against Lethal Challenge

Protection against Japanese encephalitis virus (JEV) is antibody dependent, and neutralizing antibodies alone are sufficient to impart protection. Thus, we are aiming to develop a peptide-based vaccine against JEV by identifying JEV peptide sequences that could induce virus-neutralizing antibodies. Previously, we have synthesized large amounts of Johnson grass mosaic virus (JGMV) coat protein (CP) in Escherichia coli and have shown that it autoassembled to form virus-like particles (VLPs). The envelope (E) protein of JEV contains the virus-neutralization epitopes. Four peptides from different locations within JEV E protein were chosen, and these were fused to JGMV CP by recombinant DNA methods. The fusion protein autoassembled to form VLPs that could be purified by sucrose gradient centrifugation. Immunization of mice with the recombinant VLPs containing JEV peptide sequences induced anti-peptide and anti-JEV antibodies. A 27-amino-acid peptide containing amino acids 373 to 399 from JEV E protein, present on JGMV VLPs, induced virus-neutralizing antibodies. Importantly, these antibodies were obtained without the use of an adjuvant. The immunized mice showed significant protection against a lethal JEV challenge