scholarly journals Establishment of a Rescue System for Canine Distemper Virus

2000 ◽  
Vol 74 (22) ◽  
pp. 10737-10744 ◽  
Author(s):  
Uta Gassen ◽  
Fergal M. Collins ◽  
W. Paul Duprex ◽  
Bert K. Rima
Keyword(s):  
Rna Polymerase ◽  
Measles Virus ◽  
Canine Distemper Virus ◽  
Reverse Genetics ◽  
Syncytium Formation ◽  
Full Length ◽  
Canine Distemper ◽  
Coding Region ◽  
T7 Promoter ◽  
Large Plaque

ABSTRACT Canine distemper virus (CDV) has been rescued from a full-length cDNA clone. Besides Measles virus (MV) andRinderpest virus, a third morbillivirus is now available for genetic analysis using reverse genetics. A plasmid p(+)CDV was constructed by sequential cloning using the Onderstepoort vaccine strain large-plaque-forming variant. The presence of a T7 promoter allowed transcription of full-length antigenomic RNA by a T7 RNA polymerase, which was provided by a host range mutant of vaccinia virus (MVA-T7). Plasmids expressing the nucleocapsid protein, the phosphoprotein, and the viral RNA-dependent RNA polymerase, also under control of a T7 promoter, have been generated. Infection of HeLa cells with MVA-T7 and subsequent transfection of p(+)CDV plus the helper plasmids led to syncytium formation and release of infectious recombinant (r) CDV. Comparison of the rescued virus with the parental virus revealed no major differences in the progression of infection or in the shape and size of syncytia. A genetic tag, consisting of two nucleotide changes within the coding region of the L protein, has been identified in the rCDV genome. Expression by rCDV of all the major viral structural proteins has been demonstrated by immunofluorescence.

scholarly journals The Hemagglutinin of Canine Distemper Virus Determines Tropism and Cytopathogenicity

2001 ◽  
Vol 75 (14) ◽  
pp. 6418-6427 ◽  
Author(s):  
Veronika von Messling ◽  
Gert Zimmer ◽  
Georg Herrler ◽  
Ludwig Haas ◽  
Roberto Cattaneo
Keyword(s):  
Vaccine Strain ◽  
Measles Virus ◽  
Canine Distemper Virus ◽  
Syncytium Formation ◽  
Genetic System ◽  
Reverse Genetic System ◽  
Canine Distemper ◽  
Large Plaque ◽  
Recombinant Viruses ◽  
H Protein

ABSTRACT Canine distemper virus (CDV) and measles virus (MV) cause severe illnesses in their respective hosts. The viruses display a characteristic cytopathic effect by forming syncytia in susceptible cells. For CDV, the proficiency of syncytium formation varies among different strains and correlates with the degree of viral attenuation. In this study, we examined the determinants for the differential fusogenicity of the wild-type CDV isolate 5804Han89 (CDV5804), the small- and large-plaque-forming variants of the CDV vaccine strain Onderstepoort (CDVOS and CDVOL, respectively), and the MV vaccine strain Edmonston B (MVEdm). The cotransfection of different combinations of fusion (F) and hemagglutinin (H) genes in Vero cells indicated that the H protein is the main determinant of fusion efficiency. To verify the significance of this observation in the viral context, a reverse genetic system to generate recombinant CDVs was established. This system is based on a plasmid containing the full-length antigenomic sequence of CDVOS. The coding regions of the H proteins of all CDV strains and MVEdm were introduced into the CDV and MV genetic backgrounds, and recombinant viruses rCDV-H5804, rCDV-HOL, rCDV-HEdm, rMV-H5804, rMV-HOL, and rMV-HOS were recovered. Thus, the H proteins of the two morbilliviruses are interchangeable and fully functional in a heterologous complex. This is in contrast with the glycoproteins of other members of the familyParamyxoviridae, which do not function efficiently with heterologous partners. The fusogenicity, growth characteristics, and tropism of the recombinant viruses were examined and compared with those of the parental strains. All these characteristics were found to be predominantly mediated by the H protein regardless of the viral backbone used.

scholarly journals Dual Promoters Improve the Rescue of Recombinant Measles Virus in Human Cells

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1723
Author(s):  
Soroth Chey ◽  
Juliane Maria Palmer ◽  
Laura Doerr ◽  
Uwe Gerd Liebert
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Measles Virus ◽  
Reverse Genetics ◽  
Hammerhead Ribozyme ◽  
Polymerase Activity ◽  
T7 Promoter ◽  
Gene Delivery Vectors ◽  
Cloned Cdna ◽  
Dual Promoters

Reverse genetics is a technology that allows the production of a virus from its complementary DNA (cDNA). It is a powerful tool for analyzing viral genes, the development of novel vaccines, and gene delivery vectors. The standard reverse genetics protocols are laborious, time-consuming, and inefficient for negative-strand RNA viruses. A new reverse genetics platform was established, which increases the recovery efficiency of the measles virus (MV) in human 293-3-46 cells. The novel features compared with the standard system involving 293-3-46 cells comprise (a) dual promoters containing the RNA polymerase II promoter (CMV) and the bacteriophage T7 promoter placed in uni-direction on the same plasmid to enhance RNA transcription; (b) three G nucleotides added just after the T7 promoter to increase the T7 RNA polymerase activity; and (c) two ribozymes, the hairpin hammerhead ribozyme (HHRz), and the hepatitis delta virus ribozyme (HDVrz), were used to cleavage the exact termini of the antigenome RNA. Full-length antigenome cDNA of MV of the wild type IC323 strain or the vaccine AIK-C strain was inserted into the plasmid backbone. Both virus strains were easily rescued from their respective cloned cDNA. The rescue efficiency increased up to 80% compared with the use of the standard T7 rescue system. We assume that this system might be helpful in the rescue of other human mononegavirales.

scholarly journals Probing Morbillivirus Antisera Neutralization Using Functional Chimerism between Measles Virus and Canine Distemper Virus Envelope Glycoproteins

Viruses ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 688 ◽  
Author(s):  
Miguel Angel Muñoz-Alía ◽  
Stephen J. Russell
Keyword(s):  
Neutralizing Antibodies ◽  
Measles Virus ◽  
Canine Distemper Virus ◽  
Reverse Genetics ◽  
Canine Distemper ◽  
Virus Envelope ◽  
Live Virus ◽  
Virus Challenge ◽  
Virus Attachment ◽  
Globular Head

Measles virus (MeV) is monotypic. Live virus challenge provokes a broadly protective humoral immune response that neutralizes all known measles genotypes. The two surface glycoproteins, H and F, mediate virus attachment and entry, respectively, and neutralizing antibodies to H are considered the main correlate of protection. Herein, we made improvements to the MeV reverse genetics system and generated a panel of recombinant MeVs in which the globular head domain or stalk region of the H glycoprotein or the entire F protein, or both, were substituted with the corresponding protein domains from canine distemper virus (CDV), a closely related morbillivirus that resists neutralization by measles-immune sera. The viruses were tested for sensitivity to human or guinea pig neutralizing anti-MeV antisera and to ferret anti-CDV antisera. Virus neutralization was mediated by antibodies to both H and F proteins, with H being immunodominant in the case of MeV and F being so in the case of CDV. Additionally, the globular head domains of both MeV and CDV H proteins were immunodominant over their stalk regions. These data shed further light on the factors constraining the evolution of new morbillivirus serotypes.

scholarly journals Antiviral Screen against Canine Distemper Virus-Induced Membrane Fusion Activity

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 128
Author(s):  
Neeta Shrestha ◽  
Flavio M. Gall ◽  
Jonathan Vesin ◽  
Marc Chambon ◽  
Gerardo Turcatti ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Small Molecule ◽  
High Throughput Screening ◽  
Measles Virus ◽  
Canine Distemper Virus ◽  
Rna Virus ◽  
Preventive Measure ◽  
Close Relative ◽  
Fusion Activity ◽  
Canine Distemper

Canine distemper virus (CDV), a close relative of the human pathogen measles virus (MeV), is an enveloped, negative sense RNA virus that belongs to the genus Morbillivirus and causes severe diseases in dogs and other carnivores. Although the vaccination is available as a preventive measure against the disease, the occasional vaccination failure highlights the importance of therapeutic alternatives such as antivirals against CDV. The morbilliviral cell entry system relies on two interacting envelope glycoproteins: the attachment (H) and fusion (F) proteins. Here, to potentially discover novel entry inhibitors targeting CDV H, F and/or the cognate receptor: signaling lymphocyte activation molecule (SLAM) proteins, we designed a quantitative cell-based fusion assay that matched high-throughput screening (HTS) settings. By screening two libraries of small molecule compounds, we successfully identified two membrane fusion inhibitors (F2736-3056 and F2261-0043). Although both inhibitors exhibited similarities in structure and potency with the small molecule compound 3G (an AS-48 class morbilliviral F-protein inhibitor), F2736-3056 displayed improved efficacy in blocking fusion activity when a 3G-escape variant was employed. Altogether, we present a cell-based fusion assay that can be utilized not only to discover antiviral agents against CDV but also to dissect the mechanism of morbilliviral-mediated cell-binding and cell-to-cell fusion activity.

scholarly journals Recovery of NanoLuc Luciferase-Tagged Canine Distemper Virus for Facilitating Rapid Screening of Antivirals in vitro

2020 ◽  
Vol 7 ◽  
Author(s):  
Fuxiao Liu ◽  
Qianqian Wang ◽  
Yilan Huang ◽  
Ning Wang ◽  
Youming Zhang ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Canine Distemper Virus ◽  
Reverse Genetics ◽  
Rapid Screening ◽  
Canine Distemper ◽  
Virus Propagation ◽  
The Family ◽  
Conventional Methods

Canine distemper virus (CDV), belonging to the genus Morbillivirus in the family Paramyxoviridae, is a highly contagious pathogen, affecting various domestic, and wild carnivores. Conventional methods are too cumbersome to be used for high-throughput screening of anti-CDV drugs. In this study, a recombinant CDV was rescued using reverse genetics for facilitating screening of anti-CDV drug in vitro. The recombinant CDV could stably express the NanoLuc® luciferase (NLuc), a novel enzyme that was smaller and “brighter” than others. The intensity of NLuc-catalyzed luminescence reaction indirectly reflected the anti-CDV effect of a certain drug, due to a positive correlation between NLuc expression and virus propagation in vitro. Based on such a characteristic feature, the recombinant CDV was used for anti-CDV assays on four drugs (ribavirin, moroxydine hydrochloride, 1-adamantylamine hydrochloride, and tea polyphenol) via analysis of luciferase activity, instead of via conventional methods. The result showed that out of these four drugs, only the ribavirin exhibited a detectable anti-CDV effect. The NLuc-tagged CDV would be a rapid tool for high-throughput screening of anti-CDV drugs.

scholarly journals Canine Distemper Virus Uses both the Anterograde and the Hematogenous Pathway for Neuroinvasion

2006 ◽  
Vol 80 (19) ◽  
pp. 9361-9370 ◽  
Author(s):  
Penny A. Rudd ◽  
Roberto Cattaneo ◽  
Veronika von Messling
Keyword(s):  
Measles Virus ◽  
Canine Distemper Virus ◽  
Fluorescent Protein ◽  
Early Time ◽  
Olfactory Nerve ◽  
The Body ◽  
Target Cells ◽  
Canine Distemper ◽  
Invasion Routes ◽  
Green Fluorescent

ABSTRACT Canine distemper virus (CDV), a member of the Morbillivirus genus that also includes measles virus, frequently causes neurologic complications, but the routes and timing of CDV invasion of the central nervous system (CNS) are poorly understood. To characterize these events, we cloned and sequenced the genome of a neurovirulent CDV (strain A75/17) and produced an infectious cDNA that expresses the green fluorescent protein. This virus fully retained its virulence in ferrets: the course and signs of disease were equivalent to those of the parental isolate. We observed CNS invasion through two distinct pathways: anterogradely via the olfactory nerve and hematogenously through the choroid plexus and cerebral blood vessels. CNS invasion only occurred after massive infection of the lymphatic system and spread to the epithelial cells throughout the body. While at early time points, mostly immune and endothelial cells were infected, the virus later spread to glial cells and neurons. Together, the results suggest similarities in the timing, target cells, and CNS invasion routes of CDV, members of the Morbillivirus genus, and even other neurovirulent paramyxoviruses like Nipah and mumps viruses.

scholarly journals Canine Distemper Virus and Measles Virus Fusion Glycoprotein Trimers: Partial Membrane-Proximal Ectodomain Cleavage Enhances Function

2004 ◽  
Vol 78 (15) ◽  
pp. 7894-7903 ◽  
Author(s):  
Veronika von Messling ◽  
Dragana Milosevic ◽  
Patricia Devaux ◽  
Roberto Cattaneo
Keyword(s):  
Amino Acids ◽  
Protein Function ◽  
Measles Virus ◽  
Canine Distemper Virus ◽  
Transmembrane Domain ◽  
Fusion Pore ◽  
Canine Distemper ◽  
Furin Cleavage ◽  
F Protein ◽  
Membrane Processing

ABSTRACT The trimeric fusion (F) glycoproteins of morbilliviruses are activated by furin cleavage of the precursor F0 into the F1 and F2 subunits. Here we show that an additional membrane-proximal cleavage occurs and modulates F protein function. We initially observed that the ectodomain of approximately one in three measles virus (MV) F proteins is cleaved proximal to the membrane. Processing occurs after cleavage activation of the precursor F0 into the F1 and F2 subunits, producing F1a and F1b fragments that are incorporated in viral particles. We also detected the F1b fragment, including the transmembrane domain and cytoplasmic tail, in cells expressing the canine distemper virus (CDV) or mumps virus F protein. Six membrane-proximal amino acids are necessary for efficient CDV F1a/b cleavage. These six amino acids can be exchanged with the corresponding MV F protein residues of different sequence without compromising function. Thus, structural elements of different sequence are functionally exchangeable. Finally, we showed that the alteration of a block of membrane-proximal amino acids results in diminished fusion activity in the context of a recombinant CDV. We envisage that selective loss of the membrane anchor in the external subunits of circularly arranged F protein trimers may disengage them from pulling the membrane centrifugally, thereby facilitating fusion pore formation.

Infection of canine mononuclear leucocytes by measles virus: possible mechanism of protection from canine distemper

1981 ◽  
Vol 27 (10) ◽  
pp. 1128-1131 ◽  
Author(s):  
C. K. Ho ◽  
L. A. Babiuk
Keyword(s):  
Peripheral Blood ◽  
Measles Virus ◽  
Canine Distemper Virus ◽  
Lymphoid Tissues ◽  
Canine Distemper ◽  
Mononuclear Leucocytes

Measles virus was shown to be infectious to canine lymphocytes from peripheral blood as well as from different lymphoid tissues, and the same held true for canine macrophage cultures prepared from peripheral blood. The susceptibility of these leucocytes to measles virus was comparable with that of canine distemper virus. These observations supported the suggestion that interference with canine distemper virus by measles virus could be a possible mechanism of the heterotypic immunity observed in dogs.

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