Mutational analysis of the gene start sequences of pneumonia virus of mice

The transcriptional start sequence of pneumonia virus of mice is more variable than that of the other pneumoviruses, with five different nine-base gene start (GS) sequences found in the PVM genome. The sequence requirements of the PVM gene start signal, and the efficiency of transcriptional initiation of the different virus genes, was investigated using a reverse genetics approach with a minigenome construct containing two reporter genes. A series of GS mutants were created, where each of the nine bases of the gene start consensus sequence of a reporter gene was changed to every other possible base, and the resulting effect on initiation of transcription was assayed. Nucleotide positions 1, 2 and 7 were found to be most sensitive to mutation whilst positions 4, 5 and 9 were relatively insensitive. The L gene GS sequence was found to have only 20% of the activity of the consensus sequence whilst the published M2 gene start sequence was found to be non-functional. A minigenome construct in which the two reporter genes were separated by the F-M2 gene junction of PVM was used to confirm the presence of two alternative, functional, GS sequences that could both drive the transcription of the PVM M2 gene

Epidemiology of parainfluenza virus type 3 in England and Wales over a ten-year period

We have analysed data on respiratory syneytial (RS) and parainfiuenza type 3 (PF3) viruses reported to the Communicable Disease Surveillance Centre. London, over the period 1978–87. These confirm the annual winter epidemic of RS virus and show that, in England and Wales, PF3 is a summer infection with regular yearly epidemics

The host-range tdCE phenotype of Chandipura virus is determined by mutations in the polymerase gene

The emerging arbovirus Chandipura virus (CV) has been implicated in epidemics of acute encephalitis in India with high mortality rates. The isolation of temperature-dependent host-range (tdCE) mutants, which are impaired in growth at 39 °C in chick embryo (CE) cells but not in monkey cells, highlights a dependence on undetermined host factors. We have characterized three tdCE mutants, each containing one or more coding mutations in the RNA polymerase gene and two containing additional mutations in the attachment protein gene. Using reverse genetics, we showed that a single amino acid change in the virus polymerase of each mutant was responsible for the host-range specificity. In CE cells at the non-permissive temperature, the discrete cytoplasmic replication complexes seen in mammalian cells or at the permissive temperature in CE cells were absent with the tdCE mutants, consistent with the tdCE lesions causing disruption of the replication complexes in a host-dependent manner

Sequence variation in the haemagglutinin-neuraminidase gene of human parainfluenza virus type 3 isolates in the UK

The sequence variation in a 934 base-pair region of the gene encoding the haemagglutinin-neuraminidase of five human parainfluenza virus type 3 (HPIV3) isolates was determined together with that of a prototype UK strain. All of the clinical isolates were from the Manchester area of the UK and were obtained in 1990. 1991 and 1993. The gene segment was amplified by the polymerase chain reaction using HPIVB-specific oligonucleotide primers. The nucleotide homology of the strains was high, around 99% and specific differences in the UK sequences when compared with that of the US prototype strain were identified. In addition, a number of isolate-specific differences were seen. No correlation was detected between the observed nucleotide mutations and the year of isolation, which supports the hypothesis that HPIV3 shows cocirculation of a heterogeneous population of viruses rather than varying with time in a linear fashion. However, the data suggested that geographically-defined genetic lineages of HPIV3 may exist

Retrograde transport pathways utilised by viruses and protein toxins

A model has been presented for retrograde transport of certain toxins and viruses from the cell surface to the ER that suggests an obligatory interaction with a glycolipid receptor at the cell surface. Here we review studies on the ER trafficking cholera toxin, Shiga and Shiga-like toxins, Pseudomonas exotoxin A and ricin, and compare the retrograde routes followed by these protein toxins to those of the ER trafficking SV40 and polyoma viruses. We conclude that there is in fact no obligatory requirement for a glycolipid receptor, nor even with a protein receptor in a lipid-rich environment. Emerging data suggests instead that there is no common pathway utilised for retrograde transport by all of these pathogens, the choice of route being determined by the particular receptor utilised

Cloned defective interfering influenza RNA and a 7 possible pan-specific treatment of respiratory virus 8 diseases

Defective interfering (DI) genomes are characterised by their ability to interfere with the 3 replication of the virus from which they were derived and other genetically compatible 4 viruses. DI genomes are synthesized by nearly all known viruses and represent a vast 5 natural reservoir of antivirals that can potentially be exploited for use in the clinic. This review 6 describes the application of DI virus to protect from virus-associated diseases in vivo using 7 as an example a highly active cloned influenza A DI genome and virus that protects broadly 8 in preclinical trials against different subtypes of influenza A and against non-influenza A 9 respiratory viruses. This influenza A-derived DI genome protects by two totally different 10 mechanisms: molecular interference with influenza A replication and by stimulating innate 11 immunity that acts against non-influenza A viruses. The review considers what is needed to 12 develop DI genomes to the point of entry into clinical trials

Cellular mRNAs access second ORFs using a novel amino acid sequence-dependent coupled translation termination-reinitiation mechanism

Polycistronic transcripts are considered rare in the human genome. Initiation of translation of internal ORFs of eukaryotic genes has been shown to use either leaky scanning or highly structured IRES regions to access initiation codons. Studies on mammalian viruses identified a mechanism of coupled translation termination-reinitiation that allows translation of an additional ORF. Here, the ribosome terminating translation of ORF-1 translocates upstream to reinitiate translation of ORF-2. We have devised an algorithm to identify mRNAs in the human transcriptome in which the major ORF-1 overlaps a second ORF capable of encoding a product of at least 50 aa in length. This identified 4368 transcripts representing 2214 genes. We investigated 24 transcripts, 22 of which were shown to express a protein from ORF-2 highlighting that 3' UTRs contain protein-coding potential more frequently than previously suspected. Five transcripts accessed ORF-2 using a process of coupled translation termination-reinitiation. Analysis of one transcript, encoding the CASQ2 protein, showed that the mechanism by which the coupling process of the cellular mRNAs was achieved was novel. This process was not directed by the mRNA sequence but required an aspartate-rich repeat region at the carboxyl terminus of the terminating ORF-1 protein. Introduction of wobble mutations for the aspartate codon had no effect, whereas replacing aspartate for glutamate repeats eliminated translational coupling. This is the first description of a coordinated expression of two proteins from cellular mRNAs using a coupled translation termination-reinitiation process and is the first example of such a process being determined at the amino acid level

Episodic-Like Memory for What-Where-Which Occasion is Selectively Impaired in the 3xTgAD Mouse Model of Alzheimer’s Disease

Episodic memory loss is a defining feature of early-stage Alzheimer’s disease (AD). A test of episodic-like memory for the rat, the What-Where-Which occasion task (WWWhich), requires the association of object, location, and contextual information to form an integrated memory for an event. The WWWhich task cannot be solved by use of non-episodic information such as object familiarity and is dependent on hippocampal integrity. Thus, it provides an ideal tool with which to test capacity for episodic-like memory in the 3xTg murine model for AD. As this model captures much of the human AD phenotype, we hypothesized that these mice would show a deficit in the WWWhich episodic-like memory task. To test the specificity of any episodic-like deficit, we also examined whether mice could perform components of the WWWhich task that do not require episodic-like memory. These included object (Novel Object Recognition), location (Object Location Task, What-Where task), and contextual (What-Which) memory, as well as another three-component task that can be solved without reliance on episodic recall (What-Where-When; WWWhen). The results demonstrate for the first time that control 129sv/c57bl6 mice could form WWWhich episodic-like memories, wherea, 3xTgAD mice at 6 months of age were impaired. Importantly, while 3xTgAD mice showed some deficit on spatial component tasks, they were unimpaired in the more complex WWWhen combination task (which includes a spatial component and is open to non-episodic solutions). These results strongly suggest that AD pathology centered on the hippocampal formation mediates a specific deficit for WWWhich episodic-like memory in the 3xTgAD model

Intranasal immunisation with recombinant adenovirus vaccines protects against a lethal 2 challenge with pneumonia virus of mice

Pneumonia virus of mice (PVM) infection of BALB/c mice induces bronchiolitis leading to a fatal pneumonia in a dose-dependent manner, closely paralleling the development of severe disease during human respiratory syncytial virus infection in man, and is thus a recognised model in which to study the pathogenesis of pneumoviruses. This model system was used to investigate delivery of the internal structural proteins of PVM as a potential vaccination strategy to protect against pneumovirus disease. Replication-deficient recombinant human adenovirus serotype 5 (rAd5) vectors were constructed that expressed the M or N gene of PVM pathogenic strain J3666. Intranasal delivery of these rAd5 vectors gave protection against a lethal challenge dose of PVM in three different mouse strains, and protection lasted for at least 20 weeks post-immunisation. Whilst the PVM-specific antibody response isuch animals was weak and inconsistent, rAd5N primed a strong PVM-specific CD8+ T cell response and, to a lesser extent, a CD4+ T cell response. These findings suggest that protection induced by rAd5N was mediated by T-cells rather than serum antibody

Cloned defective interfering influenza virus protects ferrets from pandemic 2009 influenza A virus and allows protective immunity to be established

Influenza A viruses are a major cause of morbidity and mortality in the human population, causing epidemics in the winter, and occasional worldwide pandemics. In addition there are periodic outbreaks in domestic poultry, horses, pigs, dogs, and cats. Infections of domestic birds can be fatal for the birds and their human contacts. Control in man operates through vaccines and antivirals, but both have their limitations. In the search for an alternative treatment we have focussed on defective interfering (DI) influenza A virus. Such a DI virus is superficially indistinguishable from a normal virus but has a large deletion in one of the eight RNAs that make up the viral genome. Antiviral activity resides in the deleted RNA. We have cloned one such highly active DI RNA derived from segment 1 (244 DI virus) and shown earlier that intranasal administration protects mice from lethal disease caused by a number of different influenza A viruses. A more cogent model of human influenza is the ferret. Here we found that intranasal treatment with a single dose of 2 or 0.2 µg 244 RNA delivered as A/PR/8/34 virus particles protected ferrets from disease caused by pandemic virus A/California/04/09 (A/Cal; H1N1). Specifically, 244 DI virus significantly reduced fever, weight loss, respiratory symptoms, and infectious load. 244 DI RNA, the active principle, was amplified in nasal washes following infection with A/Cal, consistent with its amelioration of clinical disease. Animals that were treated with 244 DI RNA cleared infectious and DI viruses without delay. Despite the attenuation of infection and disease by DI virus, ferrets formed high levels of A/Cal-specific serum haemagglutination-inhibiting antibodies and were solidly immune to rechallenge with A/Cal. Together with earlier data from mouse studies, we conclude that 244 DI virus is a highly effective antiviral with activity potentially against all influenza A subtypes