Differential accumulation of genetic and phenotypic changes in Venezuelan equine encephalitis virus and Japanese encephalitis virus following passage in vitro and in vivo.

The requirement to replicate in both vertebrate and invertebrate hosts is thought to limit the introduction of genetic changes into the genome of arboviruses. Serial passage under laboratory conditions will overcome this limitation allowing for genetic changes to be introduced and affecting the virulence of the virus for animals. In the studies detailed here, the consequence of removing the restriction of alternate replication was demonstrated to be different depending on the virus. Passing Venezuelan equine encephalitis virus in tissue culture cells, eggs or mice resulted in up to 11 nucleotide or amino acid changes but no significant change in the virulence of the virus for mice. Passing Japanese encephalitis virus (JEV) under the identical conditions resulted in as many as 22 nucleotide or amino acid changes that often resulted in improved survival probabilities. For JEV, most genetic changes along with the attenuated phenotype were selected within 5 passes.

Gas-permeable ethylene bags for the small scale cultivation of highly pathogenic avian influenza H5N1 and other viruses in embryonated chicken eggs.

BACKGROUND: Embryonated chicken eggs (ECE) are sometimes used for the primary isolation or passage of influenza viruses, other viruses, and certain bacteria. For small-scale experiments with pathogens that must be studied in biosafety level three (BSL3) facilities, inoculated ECE are sometimes manipulated and maintained in small egg incubators within a biosafety cabinet (BSC). To simplify the clean up and decontamination of an egg incubator in case of egg breakage, we explored whether ethylene breather bags could be used to encase ECE inoculated with pathogens. This concept was tested by determining embryo survival and examining virus yields in bagged ECE. RESULTS: Virus yields acceptable for many applications were attained when influenza-, alpha-, flavi-, canine distemper-, and mousepox viruses were propagated in ECE sealed within ethylene breather bags. CONCLUSIONS: For many small-scale applications, ethylene breather bags can be used to encase ECE inoculated with various viruses.

A real-time polymerase chain reaction assay to detect single nucleotide polymorphisms at codon 171 in the prion gene for the genotyping of scrapie susceptibility in sheep.

The objective of this study was to report a reliable real-time polymerase chain reaction assay compatible with the Roche LightCycler 2.0 capable of genotyping sheep for scrapie susceptibility at codon 171. The single nucleotide polymorphisms (SNPs) in the prion protein gene in sheep that may govern resistance to scrapie at codon 171 encode for lysine (K), histidine (H), glutamine (Q), and arginine (R). A modified proteinase K method for leukocytes or whole blood was used to isolate genomic DNA from sheep blood. Fluoresentric developed and optimized primers and probes for the codon 171 SNP assay. The assay was initially validated using 218 determinations from whole blood of known genotypes with 100% correct identity. The assay was further validated through a whole-blood check test provided annually by the National Veterinary Services Laboratory with a correct identification rate of 100%. From January 2005 to December 2006, 3,672 samples from blood were genotyped at codon 171. The genotypes were QR(171) (n = 1,838, 50.05%), RR(171) (n = 1,423, 38.75%), QQ(171) (n = 407, 11.08%), HR(171) (n = 2, 0.05%), and HQ(171) (n = 2, 0.05%). The combination of this simple extraction method and the novel Fluoresentric assay is very accurate, is capable of identifying all 4 SNPs at codon 171 in one reaction, and has proven to be a useful tool for producers in their selective breeding programs.