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

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

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

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

Understanding the SARS-CoV-2 virus neutralizing antibody response : lessons to be learned from HIV and respiratory syncytial virus

The SARS-CoV-2 pandemic commenced in 2019 and is still ongoing. Neither infection nor vaccination give long-lasting immunity and, here, in an attempt to understand why this might be, we have compared the neutralizing antibody responses to SARS-CoV-2 with those specific for human immunodeficiency virus type 1 (HIV-1) and respiratory syncytial virus (RSV). Currently, most of the antibodies specific for the SARS-CoV-2 S protein map to three broad antigenic sites, all at the distal end of the S trimer (receptor-binding site (RBD), sub-RBD and N-terminal domain), whereas the structurally similar HIV-1 and the RSV F envelope proteins have six antigenic sites. Thus, there may be several antigenic sites on the S trimer that have not yet been identified. The epitope mapping, quantitation and longevity of the SARS-CoV-2 S-protein-specific antibodies produced in response to infection and those elicited by vaccination are now being reported for specific groups of individuals, but much remains to be determined about these aspects of the host–virus interaction. Finally, there is a concern that the SARS-CoV-2 field may be reprising the HIV-1 experience, which, for many years, used a virus for neutralization studies that did not reflect the neutralizability of wild-type HIV-1. For example, the widely used VSV-SARS-CoV-2-S protein pseudotype has 10-fold more S trimers per virion and a different configuration of the trimers compared with the SARS-CoV-2 wild-type virus. Clarity in these areas would help in advancing understanding and aid countermeasures of the SARS-CoV-2 pandemic

Comparative modeling of CCRL1, a key protein in masked immune diseases and virtual screening for finding inhibitor of this protein

Human CCRL1 belongs to the family of silent chemokine receptors. This transmembrane protein plays a role in blunting function of chemokines through binding to them. This will attenuate immune responses. Interaction between CCRL1 and CCL21 determines this immune extinction. Thus inhibiting the action of this atypical chemokine seems to stimulate immune responses especially in the case of suppressed and immune deficient conditions. In this study we predicted 3D structure of CCRL1 using comparative modeling and Hiddebn Markov Model algorithm. Final predicted model optimized by Modeller v9.8 and minimized regarding energy level using UCSF chimera candidate version1.5.3. ClasPro webserver was used to find interacting residues between CCRL1 and CCL21. Interacting residues were used as target for chemical inhibitors by simulated docking study. For finding potential inhibitors, library of KEGG compounds screened and 97 obtained chemicals docked against interacting residues between CCRL1- CCL21 and MolDock was used as docking scoring function. Results indicated that Hexadecanal is a potential inhibitor of CCRL1- CCL21 interaction. Inhibition of this interaction will increase intercellular level of CCl21 and interaction between CCL21 and CCR7 causes immune potentiaiton

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