ABSTRACT
The absence of extraneous agents (EA) in the raw material used for production and in finished products is one of the principal safety elements related to all medicinal products of biological origin, such as live-attenuated vaccines. The aim of this study was to investigate the applicability of the Lawrence Livermore Microbial detection array version 2 (LLMDAv2) combined with whole genome amplification and sequencing for screening for viral EAs in live-attenuated vaccines and specific pathogen-free (SPF) eggs. We detected positive microarray signals for avian endogenous retrovirus EAV-HP and several viruses belonging to the Alpharetrovirus genus in all analyzed vaccines and SPF eggs. We used a microarray probe mapping approach to evaluate the presence of intact retroviral genomes, which in addition to PCR analysis revealed that several of the positive microarray signals were most likely due to cross hybridization with the EAV-HPΔpol and ALV-E ev1, ev3 and ev6 loci sequences originating from the chicken genome. Sequencing of the vaccines on a MiSeq instrument verified the microarray findings and showed similar cross hybridization. Our results suggest that genomic microarrays and sequencing of avian attenuated vaccines may be applied in tests for EA.
Subject(s)
Endogenous Retroviruses/immunology , High-Throughput Nucleotide Sequencing/methods , Oligonucleotide Array Sequence Analysis/methods , Vaccines, Attenuated/immunology , Viral Vaccines/immunology , Animals , Chick Embryo , Chickens , Drug Contamination/prevention & control , Endogenous Retroviruses/genetics , Specific Pathogen-Free OrganismsABSTRACT
Emerging viruses are usually endemic to tropical and sub-tropical regions of the world, but increased global travel, climate change and changes in lifestyle are believed to contribute to the spread of these viruses into new regions. Many of these viruses cause similar disease symptoms as other emerging viruses or common infections, making these unexpected pathogens difficult to diagnose. Broad-spectrum pathogen detection microarrays containing probes for all sequenced viruses and bacteria can provide rapid identification of viruses, guiding decisions about treatment and appropriate case management. We report a modified Whole Transcriptome Amplification (WTA) method that increases unbiased amplification, particular of RNA viruses. Using this modified WTA method, we tested the specificity and sensitivity of the Lawrence Livermore Microbial Detection Array (LLMDA) against a wide range of emerging viruses present in both non-clinical and clinical samples using two different microarray data analysis methods.
