scholarly journals Population Variability Generated during Rescue Process and Passaging of Recombinant Mumps Viruses

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2550
Author(s):  
Anamarija Slović ◽  
Tanja Košutić-Gulija ◽  
Dubravko Forčić ◽  
Maja Šantak ◽  
Maja Jagušić ◽  
...  
Keyword(s):  
Viral Genome ◽  
Stop Codon ◽  
Consensus Sequence ◽  
Vero Cells ◽  
Population Variability ◽  
Noncoding Region ◽  
Host Cells ◽  
High Efficacy ◽  
Recombinant Viruses ◽  
Rescue System

Recombinant mumps viruses (MuVs) based on established vaccine strains represent attractive vector candidates as they have known track records for high efficacy and the viral genome does not integrate in the host cells. We developed a rescue system based on the consensus sequence of the L-Zagreb vaccine and generated seven different recombinant MuVs by (a) insertion of one or two additional transcription units (ATUs), (b) lengthening of a noncoding region to the extent that the longest noncoding region in MuV genome is created, or (c) replacement of original L-Zagreb sequences with sequences rich in CG and AT dinucleotides. All viruses were successfully rescued and faithfully matched sequences of input plasmids. In primary rescued stocks, low percentages of heterogeneous positions were found (maximum 0.12%) and substitutions were predominantly obtained in minor variants, with maximally four substitutions seen in consensus. ATUs did not accumulate more mutations than the natural MuV genes. Six substitutions characteristic for recombinant viruses generated in our system were defined, as they repetitively occurred during rescue processes. In subsequent passaging of primary rescue stocks in Vero cells, different inconsistencies within quasispecies structures were observed. In order to assure that unwanted mutations did not emerge and accumulate, sub-consensus variability should be closely monitored. As we show for Pro408Leu mutation in L gene and a stop codon in one of ATUs, positively selected variants can rise to frequencies over 85% in only few passages.

scholarly journals Hypoxic-response elements in the oncolytic parvovirus Minute virus of mice do not allow for increased vector production at low oxygen concentration

2006 ◽  
Vol 87 (5) ◽  
pp. 1197-1201 ◽  
Author(s):  
Charlotte Servais ◽  
Perrine Caillet-Fauquet ◽  
Marie-Louise Draps ◽  
Thierry Velu ◽  
Yvan de Launoit ◽  
...  
Keyword(s):  
Cell Metabolism ◽  
Consensus Sequence ◽  
High Titre ◽  
Host Cells ◽  
Low Oxygen ◽  
Minute Virus Of Mice ◽  
Minute Virus ◽  
Responsive Element ◽  
Low Oxygen Concentration

Vectors derived from the autonomous parvovirus Minute virus of mice, MVM(p), are promising tools for the gene therapy of cancer. The validation of their in vivo anti-tumour effect is, however, hampered by the difficulty to produce high-titre stocks. In an attempt to increase vector titres, host cells were subjected to low oxygen tension (hypoxia). It has been shown that a number of viruses are produced at higher titres under these conditions. This is the case, among others, for another member of the family Parvoviridae, the erythrovirus B19 virus. Hypoxia stabilizes a hypoxia-inducible transcription factor (HIF-1α) that interacts with a ‘hypoxia-responsive element’ (HRE), the consensus sequence of which (A/GCGTG) is present in the B19 and MVM promoters. Whilst the native P4 promoter was induced weakly in hypoxia, vector production was reduced dramatically, and adding HRE elements to the P4 promoter of the vector did not alleviate this reduction. Hypoxia has many effects on cell metabolism. Therefore, even if the P4 promoter is activated, the cellular factors that are required for the completion of the parvoviral life cycle may not be expressed.

scholarly journals Low-pH Endocytic Entry of the Porcine Alphaherpesvirus Pseudorabies Virus

2018 ◽  
Vol 93 (2) ◽  
Author(s):  
Jonathan L. Miller ◽  
Darin J. Weed ◽  
Becky H. Lee ◽  
Suzanne M. Pritchard ◽  
Anthony V. Nicola
Keyword(s):  
Cell Lines ◽  
Pseudorabies Virus ◽  
Vero Cells ◽  
Low Ph ◽  
Host Cells ◽  
Multiple Model ◽  
Trade Restrictions ◽  
African Green Monkey Kidney ◽  
Entry Pathway ◽  
Model Cell

ABSTRACTThe alphaherpesvirus pseudorabies virus (PRV) is the causative agent of pseudorabies, a disease of great economic and welfare importance in swine. Other alphaherpesviruses, including herpes simplex virus (HSV), utilize low-pH-mediated endocytosis to enter a subset of cell types. We investigated whether PRV used this entry pathway in multiple laboratory model cell lines. Inhibition of receptor-mediated endocytosis by treatment with hypertonic medium prevented PRV entry. PRV entry into several cell lines, including porcine kidney (PK15) cells and African green monkey kidney (Vero) cells, was inhibited by noncytotoxic concentrations of the lysosomotropic agents ammonium chloride and monensin, which block the acidification of endosomes. Inactivation of virions by acid pretreatment is a hallmark of viruses that utilize a low-pH-mediated entry pathway. Exposure of PRV virions to pH 5.0 in the absence of host cell membranes reduced entry into PK15 and Vero cells by >80%. Together, these findings suggest that endocytosis followed by fusion with host membranes triggered by low endosomal pH is an important route of entry for PRV.IMPORTANCEPRV is a pathogen of great economic and animal welfare importance in many parts of the world. PRV causes neurological, respiratory, and reproductive disorders, often resulting in mortality of young and immunocompromised animals. Mortality, decreased production, and trade restrictions result in significant financial losses for the agricultural industry. Understanding the molecular mechanisms utilized by PRV to enter host cells is an important step in identifying novel strategies to prevent infection and spread. A thorough understanding of these mechanisms will contribute to a broader understanding of alphaherpesvirus entry. Here, we demonstrate PRV entry into multiple model cell lines via a low-pH endocytosis pathway. Together, these results provide a framework for elucidating the early events of the PRV replicative cycle.

scholarly journals The N Terminus of the Herpes Simplex Virus Type 1 Triplex Protein, VP19C, Cannot Be Detected on the Surface of the Capsid Shell by Using an Antibody (Hemagglutinin) Epitope Tag

2007 ◽  
Vol 81 (15) ◽  
pp. 8367-8370 ◽  
Author(s):  
Marieta Solé ◽  
Edward M. Perkins ◽  
Augusto Frisancho ◽  
Eugene Huang ◽  
Prashant Desai
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fluorescent Protein ◽  
Vero Cells ◽  
Amino Terminus ◽  
Epitope Tag ◽  
Recombinant Viruses ◽  
Capsid Shell ◽  
N Terminus ◽  
Simplex Virus

ABSTRACT The herpes simplex virus (HSV) triplex is a complex of three protein subunits, VP19C and a dimer of VP23 that is essential for capsid assembly. We have derived HSV-1 recombinant viruses that contain monomeric red fluorescent protein (mRFP1), a Flu hemagglutinin (HA) epitope, and a six-histidine tag fused to the amino terminus of VP19C. These viruses were capable of growth on Vero cells, indicating that the amino terminus of VP19C could tolerate these fusions. By use of immunoelectron microscopy methods, capsids that express VP19C-mRFP but not VP19C-HA were labeled with gold particles when incubated with the corresponding antibody. Our conclusion from the data is that a large tag at the N terminus of VP19C was sufficiently exposed on the capsid surface for polyclonal antibody reactivity, while the small HA epitope was inaccessible to the antibody. These data indicate that an epitope tag at the amino terminus of VP19C is not exposed at the capsid surface for reactivity to its antibody.

scholarly journals Widespread Distribution of a Group I Intron and Its Three Deletion Derivatives in the Lysin Gene of Streptococcus thermophilus Bacteriophages

2000 ◽  
Vol 74 (2) ◽  
pp. 611-618 ◽  
Author(s):  
Sophie Foley ◽  
Anne Bruttin ◽  
Harald Brüssow
Keyword(s):  
Stop Codon ◽  
Sequence Similarity ◽  
Consensus Sequence ◽  
Gc Content ◽  
Streptococcus Thermophilus ◽  
Variant Form ◽  
Bacterial Genomes ◽  
Stem Loop ◽  
Reading Frame ◽  
Group I

ABSTRACT Of 62 Streptococcus thermophilus bacteriophages isolated from various ecological settings, half contain a lysin gene interrupted by a group IA2 intron. Phage mRNA splicing was demonstrated. Five phages possess a variant form of the intron resulting from three distinct deletion events located in the intron-harbored open reading frame (orf 253). The predicted orf 253 gene sequence showed a significantly lower GC content than the surrounding intron and lysin gene sequences, and the predicted protein shared a motif with endonucleases found in phages from both gram-positive and gram-negative bacteria. A comparison of the phage lysin genes revealed a clear division between intron-containing and intron-free alleles, leading to the establishment of a 14-bp consensus sequence associated with intron possession. The conserved intron was not found elsewhere in the phage or S. thermophilusbacterial genomes. Folding of the intron RNA revealed secondary structure elements shared with other phage introns: first, a 38-bp insertion between regions P3 and P4 that can be folded into two stem-loop structures (shared with introns from Bacillusphage SPO1 and relatives); second, a conserved P7.2 region (shared with all phage introns); third, the location of the stop codon from orf 253 in the P8 stem (shared with coliphage T4 and Bacillus phage SPO1 introns); fourth, orf 253, which has sequence similarity with the H-N-H motif of putative endonuclease genes found in introns fromLactococcus, Lactobacillus, andBacillus phages.

scholarly journals Identification of a recently evolved goat embryonic beta-globin pseudogene which retains transcriptional activity in vitro.

1984 ◽  
Vol 4 (10) ◽  
pp. 2120-2127 ◽  
Author(s):  
S G Shapiro ◽  
J B Lingrel
Keyword(s):  
Transcriptional Activity ◽  
Stop Codon ◽  
Globin Gene ◽  
Consensus Sequence ◽  
Duplication Event ◽  
Beta Globin ◽  
Gene Sets ◽  
Sequence Elements ◽  
Group 5

A clone containing the entire goat epsilon V beta-globin gene, which lies downstream from the two tandemly duplicated four-gene sets containing the beta C and beta A genes in the linkage group 5'-epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-3', was isolated, and the sequence of the gene was determined. epsilon V is most homologous to the first gene in each of these sets, epsilon I and epsilon III, and appears to be a third duplicated copy of these genes, possibly the first gene in a third four-gene set. Homology of epsilon V to epsilon I is very high (93.2%) in coding regions, and all transcription, processing, and potential translation consensus sequence elements appear to be present, although the Hogness box of epsilon V is altered compared with that of epsilon I by the deletion of an A(AATAAAA----AATAAA). Nevertheless, epsilon V is clearly a pseudogene as a result of two deletions and one insertion (or insertion-deletion) in its coding sequence, the first of which produces an in-frame stop codon at amino acid 54. Unlike the more highly mutated goat beta-like pseudogene duplicates psi beta X and psi beta Z, epsilon V acquired its defects after the duplication event in which it was created. Its recently acquired defects have left the epsilon V promoter sufficiently conserved to retain transcriptional activity in vitro. The acquisition of defects by this gene may be related to the multiple gene duplications which have created at least five epsilon type genes in the goat beta-globin locus.

scholarly journals The F Gene of Rodent Brain-Adapted Mumps Virus Is a Major Determinant of Neurovirulence

2007 ◽  
Vol 81 (15) ◽  
pp. 8293-8302 ◽  
Author(s):  
Ken Lemon ◽  
Bertus K. Rima ◽  
Stephen McQuaid ◽  
Ingrid V. Allen ◽  
W. Paul Duprex
Keyword(s):  
Reverse Genetics ◽  
Vero Cells ◽  
Mumps Virus ◽  
Recombinant Viruses ◽  
F Gene ◽  
Rodent Brain ◽  
Molecular Determinants ◽  
The Matrix ◽  
Hn Gene ◽  
Small Hydrophobic

ABSTRACT Prior to the introduction of live-attenuated vaccines, mumps virus (MuV) was the leading cause of virus-induced meningitis. Although vaccination has been effective at controlling the disease, the use of insufficiently attenuated strains has been associated with high rates of aseptic meningitis in vaccinees. The molecular basis of MuV attenuation is poorly understood, and no reliable molecular markers of virulence have been identified. In this study, reverse genetics has been used to identify molecular determinants of MuV neuropathogenesis. Recombinant viruses, containing the envelope-associated genes from the Kilham (MuVKH) rodent brain-adapted strain of MuV, were generated in the Jeryl Lynn 5 (MuVJL5) vaccine strain background. The syncytium phenotypes of the recombinant viruses on Vero cells differed depending on the source of the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins, with heterologous combinations showing either an increase or a decrease in the level of cell fusion compared to that of the homologous parental combinations. This was confirmed by transiently cotransfecting eukaryotic F and HN glycoprotein expression constructs. A Lewis rat model that discriminates between neurovirulent and nonneurovirulent MuV strains based on the extent of hydrocephalus induced in the rat brain after intracerebral inoculation was used to assess the phenotype of the recombinant viruses. Expression of the matrix (M), small hydrophobic (SH), or HN gene in isolation did not confer a neurovirulent phenotype. Expression of the F gene of the neurovirulent strain alone was sufficient to induce significant levels of hydrocephalus. Coexpression of the homologous HN gene led to a marginal increase in the level of hydrocephalus.

scholarly journals Specificity of Legionella pneumophila and Coxiella burnetii Vacuoles and Versatility of Legionella pneumophila Revealed by Coinfection

2005 ◽  
Vol 73 (8) ◽  
pp. 4494-4504 ◽  
Author(s):  
John-Demian Sauer ◽  
Jeffrey G. Shannon ◽  
Dale Howe ◽  
Stanley F. Hayes ◽  
Michele S. Swanson ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Coxiella Burnetii ◽  
Legionella Pneumophila ◽  
Close Association ◽  
Cell Types ◽  
Vero Cells ◽  
Host Cells ◽  
Mouse Bone Marrow ◽  
Wild Type ◽  
African Green Monkey Kidney

ABSTRACT Legionella pneumophila and Coxiella burnetii are phylogenetically related intracellular bacteria that cause aerosol-transmitted lung infections. In host cells both pathogens proliferate in vacuoles whose biogenesis displays some common features. To test the functional similarity of their respective intracellular niches, African green monkey kidney epithelial (Vero) cells, A/J mouse bone marrow-derived macrophages, human macrophages, and human dendritic cells (DC) containing mature C. burnetii replication vacuoles were superinfected with L. pneumophila, and then the acidity, lysosome-associated membrane protein (LAMP) content, and cohabitation of mature replication vacuoles was assessed. In all cell types, wild-type L. pneumophila occupied distinct vacuoles in close association with acidic, LAMP-positive C. burnetii replication vacuoles. In murine macrophages, but not primate macrophages, DC, or epithelial cells, L. pneumophila replication vacuoles were acidic and LAMP positive. Unlike wild-type L. pneumophila, type IV secretion-deficient dotA mutants trafficked to lysosome-like C. burnetii vacuoles in Vero cells where they survived but failed to replicate. In primate macrophages, DC, or epithelial cells, growth of L. pneumophila was as robust in superinfected cell cultures as in those singly infected. Thus, despite their noted similarities, L. pneumophila and C. burnetii are exquisitely adapted for replication in unique replication vacuoles, and factors that maintain the C. burnetii replication vacuole do not alter biogenesis of an adjacent L. pneumophila replication vacuole. Moreover, L. pneumophila can replicate efficiently in either lysosomal vacuoles of A/J mouse cells or in nonlysosomal vacuoles of primate cells.

scholarly journals Attachment and invasion ofToxoplasma gondiiandNeospora caninumto epithelial and fibroblast cell linesin vitro

Parasitology ◽  
2005 ◽  
Vol 131 (5) ◽  
pp. 583-590 ◽  
Author(s):  
YING LEI ◽  
M. DAVEY ◽  
J. T. ELLIS
Keyword(s):  
Cell Lines ◽  
Neospora Caninum ◽  
Fibroblast Cell ◽  
Cell Types ◽  
Vero Cells ◽  
Host Cells ◽  
Trypsin Treatment ◽  
Epithelial Cell Lines ◽  
Pre Treatment

Attachment and invasion ofToxoplasma gondiiandNeospora caninumto a cat and a dog fibroblast cell line and 2 epithelial cell lines (a cat kidney and Vero) were comparedin vitrousing fluorescence antibody methodology. In addition, trypsin treatment of tachyzoites was used to determine whether protein molecules were essential to the process of invasion. The results show that bothT. gondiiandN. caninuminvaded all 4 cell lines, and that pre-treatment ofT. gondiitachyzoites with trypsin caused an increase in the ability of the parasite to invade these host cells. FurthermoreT. gondii, in comparison toN. caninum, invaded all 4 cell lines at greater levels. The results here support the conclusion that bothT. gondiiandN. caninumhave the ability to invade a variety of cell types including both dog and cat cells, and questions the utility of Vero cells as an appropriate host cell forin vitrostudies on the biology of these taxa.

scholarly journals Recovery and Characterization of a Chimeric Rinderpest Virus with the Glycoproteins of Peste-des-Petits-Ruminants Virus: Homologous F and H Proteins Are Required for Virus Viability

2000 ◽  
Vol 74 (19) ◽  
pp. 9039-9047 ◽  
Author(s):  
Subash C. Das ◽  
Michael D. Baron ◽  
Thomas Barrett
Keyword(s):  
Clinical Signs ◽  
Vero Cells ◽  
Virus Genome ◽  
Peste Des Petits Ruminants ◽  
Live Virus ◽  
Domestic Ruminants ◽  
Rescue System ◽  
Ppr Virus ◽  
Rescue Procedure

ABSTRACT Rinderpest (RP) and peste-des-petits-ruminants (PPR) are two important diseases of domestic ruminants. To improve on currently available vaccines against PPR, we have created cDNA copies of the RP virus genome in which either the fusion (F) or hemagglutinin (H) gene, or both, was replaced with the corresponding gene from PPR virus. It was necessary to develop a modified rescue system in which the T7 RNA polymerase was provided by a recombinant fowlpox virus and the entire rescue procedure took place in Vero cells before we could obtain live virus from these chimeric constructs. No virus was recovered when only one of the glycoprotein genes was changed, but a chimeric virus containing both F and H genes from PPR virus was reproducibly rescued from cDNA, indicating that a virus-specific functional interaction takes place between the F and H proteins. The rescued virus expressing the PPR glycoproteins grew more slowly in tissue culture than either parental virus and formed abnormally large syncytia. Goats infected with the chimera showed no adverse reaction, as assessed by clinical signs, temperature, leukocyte count, virus isolation, and serology, and were protected from subsequent challenge with wild-type PPR virus.

Sign in / Sign up

Export Citation Format

Share Document