46 research outputs found
Functional analysis of the Bunyamwera orthobunyavirus Gc glycoprotein
The virion glycoproteins Gn and Gc of Bunyamwera orthobunyavirus (family Bunyaviridae) are encoded by the M RNA genome segment and have roles in both viral attachment and membrane fusion. To investigate further the structure and function of the Gc protein in viral replication, we generated 12 mutants that contain truncations from the N terminus. The effects of these deletions were analysed with regard to Golgi targeting, low pH-dependent membrane fusion, infectious virus-like particle (VLP) formation and virus infectivity. Our results show that the N-terminal half (453 residues) of the Gc ectodomain (909 residues in total) is dispensable for Golgi trafficking and cell fusion. However, deletions in this region resulted in a significant reduction in VLP formation. Four mutant viruses that contained N-terminal deletions in their Gc proteins were rescued, and found to be attenuated to different degrees in BHK-21 cells. Taken together, our data indicate that the N-terminal half of the Gc ectodomain is dispensable for replication in cell culture, whereas the C-terminal half is required to mediate cell fusion. A model for the domain structure of the Gc ectodomain is proposed
Lyophilisation of influenza, rabies and Marburg lentiviral pseudotype viruses for the development and distribution of a neutralisation-assay based diagnostic kit
Pseudotype viruses (PVs) are chimeric, replication-deficient virions that mimic wild-type virus entry mechanisms and can be safely employed in neutralisation assays, bypassing the need for high biosafety requirements and performing comparably to established serological assays. However, PV supernatant necessitates -80°C long-term storage and cold-chain maintenance during transport, which limits the scope of dissemination and application throughout resource-limited laboratories. We therefore investigated the effects of lyophilisation on influenza, rabies and Marburg PV stability, with a view to developing a pseudotype virus neutralisation assay (PVNA) based kit suitable for affordable global distribution. Infectivity of each PV was calculated after lyophilisation and immediate reconstitution, as well as subsequent to incubation of freeze-dried pellets at varying temperatures, humidities and timepoints. Integrity of glycoprotein structure following treatment was also assessed by employing lyophilised PVs in downstream PVNAs. In the presence of 0.5M sucrose-PBS cryoprotectant, each freeze-dried pseudotype was stably stored for 4 weeks at up to 37°C and could be neutralised to the same potency as unlyophilised PVs when employed in PVNAs. These results confirm the viability of a freeze-dried PVNA-based kit, which could significantly facilitate low-cost serology for a wide portfolio of emerging infectious viruses
Virus nomenclature below the species level : a standardized nomenclature for filovirus strains and variants rescued from cDNA
Specific alterations (mutations, deletions,
insertions) of virus genomes are crucial for the functional
characterization of their regulatory elements and their expression products, as well as a prerequisite for the creation
of attenuated viruses that could serve as vaccine
candidates. Virus genome tailoring can be performed either
by using traditionally cloned genomes as starting materials,
followed by site-directed mutagenesis, or by de novo synthesis
of modified virus genomes or parts thereof. A systematic
nomenclature for such recombinant viruses is
necessary to set them apart from wild-type and laboratoryadapted
viruses, and to improve communication and collaborations
among researchers who may want to use
recombinant viruses or create novel viruses based on them.
A large group of filovirus experts has recently proposed
nomenclatures for natural and laboratory animal-adapted
filoviruses that aim to simplify the retrieval of sequence
data from electronic databases. Here, this work is extended
to include nomenclature for filoviruses obtained in the
laboratory via reverse genetics systems. The previously
developed template for natural filovirus genetic variant
naming,\virus name[(\strain[/)\isolation host-suffix[/
\country of sampling[/\year of sampling[/\genetic
variant designation[-\isolate designation[, is retained, but we propose to adapt the type of information added to each
field for cDNA clone-derived filoviruses. For instance, the
full-length designation of an Ebola virus Kikwit variant
rescued from a plasmid developed at the US Centers for
Disease Control and Prevention could be akin to ‘‘Ebola
virus H.sapiens-rec/COD/1995/Kikwit-abc1’’ (with the
suffix ‘‘rec’’ identifying the recombinant nature of the virus
and ‘‘abc1’’ being a placeholder for any meaningful isolate
designator). Such a full-length designation should be used
in databases and the methods section of publications.
Shortened designations (such as ‘‘EBOV H.sap/COD/95/
Kik-abc1’’) and abbreviations (such as ‘‘EBOV/Kik-abc1’’)
could be used in the remainder of the text, depending on
how critical it is to convey information contained in the
full-length name. ‘‘EBOV’’ would suffice if only one
EBOV strain/variant/isolate is addressed.http://link.springer.com/journal/705hb201
Practical methods for proving program termination
Abstract. We present two algorithms to prove termination of programs by synthesizing linear ranking functions. The first uses an invariant generator based on iterative forward propagation with widening and extracts ranking functions from the generated invariants by manipulating polyhedral cones. It is capable of finding subtle ranking functions which are linear combinations of many program variables, but is limited to programs with few variables. The second, more heuristic, algorithm targets the class of structured programs with single-variable ranking functions. Its invariant generator uses a heuristic extrapolation operator to avoid iterative forward propagation over program loops. For the programs we have considered, this approach converges faster and the invariants it discovers are sufficiently strong to imply the existence of ranking functions